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



HYPERBARIC OXYGEN THERAPY HYPERBARICS HELPS BY INCREASING BLOOD FLOW

HYPERBARIC OXYGEN THERAPY HYPERBARICS HELPS BY INCREASING BLOOD FLOW

HYPERBARIC OXYGEN THERAPY HYPERBARICS HELPS BY INCREASING OXYGEN REACH

HYPERBARIC OXYGEN THERAPY HYPERBARICS HELPS BY INCREASING OXYGEN REACH

HYPERBARIC OXYGEN THERAPY HYPERBARICS PROVIDES MEASUREABLE IMPROVEMENTS

HYPERBARIC OXYGEN THERAPY HYPERBARICS PROVIDES MEASUREABLE IMPROVEMENTS

HYPERBARIC OXYGEN THERAPY HYPERBARIC THERAPY STIMULATES STEM CELLS

HYPERBARIC OXYGEN THERAPY HYPERBARIC THERAPY IMPROVES THESE AREAS

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Restorative Neurology and Neuroscience 38 (2020) 93–107DOI 10.3233/RNN-190959IOS Press93Hyperbaric oxygen therapy improvesneurocognitive functions of post-strokepatients – a retrospective analysisAmir Hadannya, b,c,e,∗, Mor Rittblatb, Mor Bittermanb, Ido May-Razb, Gil Suzinb,Rahav Boussi-Grossb, Yonatan Zemelb, Yair Bechorb, Merav Catalognaband Shai Efratib,d,e,faNeurosurgery Department, Galilee Medical Center, Naharyia, IsraelbSagol Center for Hyperbaric Medicine and Research, Assaf Harofeh Medical Center, Zerifin, IsraelcGalilee Faculty of Medicine, Bar Ilan University, IsraeldResearch and Development Unit, Assaf Harofeh Medical Center, Zerifin, IsraeleSackler School of Medicine, Tel-Aviv University, Tel-Aviv, IsraelfSagol School of Neuroscience, Tel-Aviv University, Tel-Aviv, IsraelAbstract.Background: Previous studies have shown that hyperbaric oxygen therapy (HBOT) can improve the motor functions andmemory of post-stroke patients in the chronic stage.Objective: The aim of this study is to evaluate the effects of HBOT on overall cognitive functions of post-stroke patients inthe chronic stage. The nature, type and location of the stroke were investigated as possible modifiers.Methods: A retrospective analysis was conducted on patients who were treated with HBOT for chronic stroke (>3 months)between 2008-2018. Participants were treated in a multi-place hyperbaric chamber with the following protocols: 40 to 60daily sessions, 5 days per week, each session included 90 min of 100% oxygen at 2 ATA with 5 min air brakes every 20minutes. Clinically significant improvements (CSI) were defined as > 0.5 standard deviation (SD).Results: The study included 162 patients (75.3% males) with a mean age of 60.75 ± 12.91. Of them, 77(47.53%) had corticalstrokes, 87(53.7%) strokes were located in the left hemisphere and 121 suffered ischemic strokes (74.6%).HBOT induced a significant increase in all the cognitive function domains ( p < 0.05), with 86% of the stroke victims achiev-ing CSI. There were no significant differences post-HBOT of cortical strokes compared to sub-cortical strokes (p > 0.05).Hemorrhagic strokes had a significantly higher improvement in information processing speed post-HBOT (p < 0.05). Lefthemisphere strokes had a higher increase in the motor domain (p < 0.05). In all cognitive domains, the baseline cognitivefunction was a significant predictor of CSI (p < 0.05), while stroke type, location and side were not significant predictors.Conclusions: HBOT induces significant improvements in all cognitive domains even in the late chronic stage. The selectionof post-stroke patients for HBOT should be based on functional analysis and baseline cognitive scores rather than the stroketype, location or side of lesion.Keywords: HBOT , stroke, cognitive function, hyperbaric oxygen∗Corresponding author: Amir Hadanny, Sagol Center forHyperbaric Medicine and Research, Assaf Harofeh Medi-cal Center, Zerifin, Israel. Tel.: +972 8 9779395; E-mail:Amir.had@gmail.com.1. IntroductionStroke is the second-most cause of mortality andthe third leading cause for disability, worldwide(Langhorne, Bernhardt, & Kwakkel, 2011; Lozanoet al., 2012; Ojaghihaghighi, Vahdati, Mikaeilpour, &Ramouz, 2017; Ottenbacher & Jannell, 1993; Powers0922-6028/20/$35.00 © 2020 – IOS Press and the authors. All rights reservedThis article is published online with Open Access and distributed under the terms of the Creative Commons Attribution Non-Commercial License (CC BY-NC 4.0).

94 A. Hadanny et al. / HBOT cognitive effect in post stroke patientset al., 2018). When strokes transpire, whether theyare ischemic or hemorrhagic, the injured brain regioncorrelates with its related loss of function which maybe visual, motor, sensory or cognitive impairments.Most stroke studies focus on motor functions. How-ever, it is estimated that nearly half of the survivorssuffer from different degrees of cognitive dysfunction(Kelly-Hayes et al., 2003; Lee, Joshi, Wang, Pashos,& Christensen, 2007; Yoneda et al., 2005).The two leading subtypes of stroke are ischemicstroke, in 68% of the cases, and the less frequenthemorrhagic stroke, in 32% of the cases (Caplan,1989; Krishnamurthi et al., 2013; Powers et al., 2018;Zhang, Lo, Mychaskiw, & Colohan, 2005). Eventhough the two pathophysiological processes are dia-metrically opposed during the initiation phase, inthe subacute chronic phase they culminate in com-prised blood supply and subsequent brain ischemia(Caplan, 1989; Krishnamurthi et al., 2013; Powers etal., 2018). When the insult results in cognitive dys-function, usually more than one cognitive domain isinvolved such as memory, attention and visual spatial(VS) (Al-Qazzaz, Ali, Ahmad, Islam, & Mohamad,2014; Cumming, Marshall, & Lazar, 2013). Thesignificant factors that affect the cognitive impair-ments’ severity are older age, previous history ofstroke, and the pre-injury global cognitive function(GCF) (Ballard, Rowan, Stephens, Kalaria, & Kenny,2003; Mok et al., 2004; Patel, Coshall, Rudd, &Wolfe, 2003; Rasquin, Verhey, van Oostenbrugge,Lousberg, & Lodder, 2004). It has been shown thathemorrhagic strokes cause significantly more cogni-tive impairments compared to ischemic strokes, andare more associated with cognitive deficits acrossmultiple domains (Cumming et al., 2013). Cor-tical strokes were found with higher proportionsof cognitive impairments in the memory domainthan subcortical ones (Lange, Waked, Kirshblum, &DeLuca, 2000; Nys et al., 2007; Schouten, Schie-manck, Brand, & Post, 2009). Yet, higher corticalfunctions such as expressive aphasia were signifi-cantly impaired in subcortical stroke patients as wellas lower performances in the information processingspeed (IPS) domain compared with cortical strokepatients (Lange et al., 2000; Nys et al., 2007; T. Wag-ner & A. Cushman, 2017). With respect to dominantvs. non-dominant hemispheric lesion, there is evi-dence of a more severe cognitive impairments andan overall higher incidence of dementia following aninsult in the dominant hemisphere (Censori et al.,1996; de Oliveira, Correia Marin Sde, & FerreiraBertolucci, 2013; Tatemichi et al., 1993).Reducing the impact of post-stroke cognitiveimpairment is an important goal due to the highermortality and institutionalization rates of thosepatients (Pasquini, Leys, Rousseaux, Pasquier, &Henon, 2007; Tatemichi et al., 1994). Rehabilita-tion includes a multidisciplinary approach whichincludes physiotherapy, speech and language ther-apy, cognitive rehabilitation therapy, medicationsand more. However, these programs have lim-ited success (Hebert et al., 2016; Prvu Bettger &Stineman, 2007; Roine, Kajaste, & Kaste, 1993;Williams, Jiang, Matchar, & Samsa, 1999). Cog-nitive recovery after stroke occurs mainly withinthe first 30 days, with some post-stroke patientscontinuing to gain progress up to three monthsfrom injury, yet even with domain specific inter-ventions, improvement is minimal (Langhorneet al., 2011; Maulden, Gassaway, Horn, Smout,& DeJong, 2005; Ovbiagele & Nguyen-Huynh,2011).Hyperbaric oxygen therapy (HBOT), the appli-ca-tion of hyperbaric pressure in conjunction withincreased oxygen content, has been shown in sev-eral clinical studies to have the capacity to induceneuroplasticity even years after an acute insult(Boussi-Gross et al., 2013; Boussi-Gross et al., 2015;Efrati & Ben-Jacob, 2014; Efrati et al., 2013; Efratiet al., 2015; Hadanny & Efrati, 2016; Hadanny, Fish-lev, Bechor, Meir, & Efrati, 2016; Hadanny et al.,2015a, 2015b; Tal et al., 2015a, 2015b; Tal, Hadanny,Sasson, Suzin, & Efrati, 2017; Yildiz et al., 2004).The elevated oxygen concentration in the blood andinjured tissue during treatment (Calvert, Cahill, &Zhang, 2007; Niklas, Brock, Schober, Schulz, &Schneider, 2004; Reinert et al., 2003) helps supply theenergy needed to regenerate damaged brain tissue. Ithas been shown that HBOT induced neuroplasticityis mediated by stimulating cell proliferation (Mu etal., 2013), neurogenesis of endogenous neural stemcells (Yang et al., 2008), regeneration of axonal whitematter (Chang et al., 2009), improved maturation andmyelination of injured neural fibers (Haapaniemi,Nylander, Kanje, & Dahlin, 1998; Vilela, Lazarini,& Da Silva, 2008), and stimulation of axonal growth,thus increasing the ability of neurons to function andcommunicate with each other (Bradshaw, Nelson,Fanton, Yates, & Kagan-Hallet, 1996; Mukoyama,Iida, & Sobue, 1975). A retrospective analysis ofpost-stroke patients in the late chronic stage revealedthat HBOT can significantly improve the mem-ory domain (Boussi-Gross et al., 2015). However,the overall neurocognitive effects of HBOT and its

A. Hadanny et al. / HBOT cognitive effect in post stroke patients 95relation to the different stroke types and anatomicallocations were not investigated yet.The aim of the current study is to investigate theeffects of HBOT on the overall cognitive domainsof post-stroke patients in the late chronic stage. Thenature, type and location of the stroke as possiblemodifiers of HBOT effects were also investigated.2. Methods2.1. ParticipantsA retrospective study including post-strokepatients, more than three months post-injury, treatedwith HBOT between January 2008 and December2017. The study was approved by our InstitutionalReview Board (approval number: 0206-17-ASF).Inclusion criteria: stroke more than three monthsprior to their first cognitive evaluation, completionof 40 or 60 hyperbaric oxygen sessions and at leasttwo cognitive evaluations, 1–3 weeks prior to the firstHBOT session to and 1–3 weeks after last HBOTsession.Exclusion criteria: insufficient details of strokenature, history of a potential additional brain injury(traumatic brain injury, anoxic brain injury, subarach-noid hemorrhage, etc.), lack of pre or post-HBOTcognitive evaluations.2.2. Study designThe data were collected retrospectively frompatients’ medical records and included age, gen-der, level of education, handedness, stroke details(type, injured hemisphere, location of stroke, timefrom injury to HBOT, symptoms prior to treatment),number of HBOT sessions, chronic medical condi-tions (diabetes mellitus type II (DM II), hypertension(HTN), dyslipidemia, ischemic heart disease (IHD),previous stroke, smoking status), and chronic pre-scribed medications (anti-aggregation (AA)), statins,hypoglycemic medications, HTN medications). Dataof the HBOT protocol, and adverse events were alsocollected.The main analysis was to compare the stroke nature(hemorrhagic and ischemic) including all stroke loca-tions: cortical, subcortical, atypical locations (i.e.cerebellum or brain stem) and multiple locations.A second analysis (i.e. the location analysis) com-pared the two main stroke locations, cortical andsubcortical. To minimize unknown hemisphere dom-inance in left handed patients, a third analysis (i.e. thedominance analysis) included only the right-handedpatients for evaluating the effect of the injured hemi-sphere.2.3. Stroke subsetsPatients were divided into different groups basedon their stroke prerequisites, retrieved from originalimaging and medical records: by anatomical location:cortical (i.e. frontal, temporal, parietal and occipitalcortex) or subcortical (i.e. basal ganglia (BG), cere-bellum, pons, internal capsule and thalamus), by theinjured hemisphere: right or left, and by stroke type:ischemic or hemorrhagic (See Fig. 1).2.4. Hyperbaric oxygen treatmentParticipants were treated in a multi-place hyper-baric chamber (Haux-Life-Support GmbH, Ger-many) with the following protocols: 40 to 60 dailysessions, 5 days per week, each session included90 min of 100% oxygen at 2 ATA with 5 min airbreaks every 20 minutes.2.5. Cognitive evaluationAll the patients were inspected using theNeuroTrax computerized cognitive testing battery(NeuroTrax Corporation, Bellaire, TX). The Neu-roTrax system and a detailed description of thetests included were detailed in previous publica-tions(Achiron et al., 2013; Thaler et al., 2012; Zur,Naftaliev, & Kesler, 2014) and are also available onthe NeuroTrax website. In brief, NeuroTrax tests eval-uate multiple aspects of brain cognitive functionsincluding: memory, executive function (EF), visu-ospatial skills (VS), verbal function (VF), attention,information processing speed (IPS) and motor skills(MS). Cognitive domain scores were normalized forage, gender and education-specific levels.The participants completed two validated alternatetest forms of the NeuroTrax test battery at baselineand post-HBOT, to allow for iterative administrationswith minimal learning effects. Test-retest reliabilityof the tests were found to be high in both normaland injured populations, without significant learningeffects except in the VF & VS domains (Dwolatzky etal., 2003; L. Melton, 2005). Due to the low test-retestreliability of these domains, they were not evaluatedin the current study.

96 A. Hadanny et al. / HBOT cognitive effect in post stroke patientsFig. 1. Flowchart of the patients included in the study.2.6. Statistical analysisData were expressed as mean ± SD for paramet-ric variables and frequencies, and percentages fornonparametric variables. Parametric variables wereanalyzed by paired-sample T tests for intra-groupcomparison and independent-sample t-tests for inter-group comparison, whereas nonparametric variableswere analyzed by Pearson Chi-square test or Fisher’sexact test (where suitable) to identify significantvariables. Normal distribution for all continuous vari-ables was tested using the Kolmogorov-Smirnovtest.Clinically significant improvement (CSI) wasdefined as an absolute increase of 7.5 points of thenormalized score (0.5 of one standard deviation) inat least one cognitive domain. The cut-off for CSIwas determined by previous studies (Fischer et al.,2000; Schwid, Goodman, Weinstein, McDermott, &Johnson, 2007).Multiple linear regression models were per-formed to determine independent predictors for thepost-treatment cognitive score. Multivariate logis-tic regression models were performed to control forpotential confounders and to determine independentpredictors for CSI. Models included the followingcovariates: age, sex, stroke type, location of strokealong with side of injured hemisphere, time frominjury to HBOT, chronic medical conditions (DM II,HTN, dyslipidemia, IHD, active smoking), numberof HBOT sessions and baseline score before HBOTtreatment.

A. Hadanny et al. / HBOT cognitive effect in post stroke patients 97The alpha level was set to 0.05 (p-Value < 0.05).The data were statistically analyzed using SPSS ver-sion 22 software.3. Results3.1. Participants’ characteristicsOf the 351 patients who were assed for eligibil-ity, a total of 162 met the inclusion criteria and wereincluded in the final analysis (Fig. 1). The patients’average age was 60.75 ± 12.91 years old (23–83)and 122 (75.3%) were males. The average time fromthe stroke to HBOT was 2.78 ± 3.3 years. Regardingthe stroke type, 121 patients (74.69%) suffered froman ischemic stroke while 41 (25.31%) had a hemor-rhagic stroke. In 50 patients (30.86%), the stroke wasin the subcortical level, while 77 patients (47.53%)had a stroke in the cortical level and the remaining35 patients (21.6%) were affected in atypical loca-tions or multiple locations. With respect to the sideof injury, 87 strokes (53.7%) were located in the righthemisphere, and 62 strokes (38.3%) were in the lefthemisphere. Baseline participants characteristics aresummarized in Table 1.3.2. Cognitive function changesBasic analysis results revealed statistically signifi-cant improvements of all the cognitive domains afterHBOT by 2.34-20 (p < 0.05, see Table 2). The mem-ory domain had the most prominent improvements ofmean absolute change (MAC) (6.19 ± 20, p = 0.0004,see Table 2). CSI was achieved in 86% of the patientsin the entire cohort (see Fig. 4). The effects of theHBOT on the cognitive scores is summarized inTable 2.3.2.1. Ischemic vs. hemorrhagicAt baseline, there were significant differencesin baseline characteristics between patients withischemic compared to patients with hemorrhagicstroke which included age, presence of comorbidities,Table 1Patients’ baseline characteristicsAnalysis Entire cohort Location analysis Dominance analysis(n = 162) (n = 127) (n = 110)Age (years) 60.75 ± 12.91 60.86 ± 12.57 61.23 ± 12.3Sex – Males 122 (75.3%) 97 (76.4%) 78 (70.9%)Dominant hand – Right 120 (74.1%) 94 (74%) 110 (100%)Time from injury 2.78 ± 3.3 2.53 ± 2.95 2.63 ± 3.18Num. of HBOT sessions 40 sessions 26 (16%) 22 (17.3%) 20 (18.2%)60 sessions 136 (84%) 105 (82.7%) 90 (81.8%)Type of stroke Ischemic 121 (74.69%) 98 (77.17%) 85 (77.3%)Hemorrhagic 41 (25.31%) 29 (22.8%) 25 (22.7%)Location of injury Subcortical 54 (33.3%) 50 (39.4%) 36 (32.7%)Cortical 80 (49.4%) 77 (60.6%) 58 (52.7%)Atypical & multiple locations 28 (17.3) – 16 (14.5%)*Side of injury Right 62 (38.3%) 53 (41.7%) 56 (50.9%)Left 87 (53.7%) 74 (58.3%) 54 (49.1%)Bilateral 13 (8%) – –Symptoms Cognitive 77 (47.5%) 60 (47.2%) 49 (44.5%)Motor 132 (81.5%) 104 (81.9%) 90(81.8%)Speech 65 (40.1%) 54 (42.5%) 43 (39.1%)CN 67 (41.4%) 54 (42.5%) 46 (41.8%)Ataxia 57 (35.2%) 39 (30.7%) 34 (30.9%)Comorbidities DM II 48 (29.6%) 37 (29.1%) 28 (25.5%)HTN 107 (66%) 82 (64.6%) 74 (67.3%)Dyslipidemia 107 (66%) 82 (64.6%) 75 (68.2%)IHD 39 (24.1%) 30 (23.6%) 28 (25.5%)Previous stroke 18 (11.1%) 12 (9.4%) 10 (9.1%)Smoker 29 (17.9%) 23 (18.1%) 15 (13.6%)Medications AA 105 (64.8%) 78 (61.4%) 70 (63.6%)Statins 104 (64.2%) 79 (62.2%) 72 (65.5%)DM II medications 37 (22.8%) 27 (21.3%) 20 (18.2%)HTN medications 107 (66%) 84 (66.1%) 74 (67.3%)Data are expressed as means ± standard deviation. *Cerebellum insult only. HBOT – hyperbaric oxygen treatment, CN – cranial nerves, DMII – diabetic mellitus type 2, HTN – hypertension, AA – anti-aggregates.

98 A. Hadanny et al. / HBOT cognitive effect in post stroke patientsTable 2Cognitive domains – mean absolute changes of the entire cohortPre score Post score Pre-Post MAC P-value*GCF 87.48 ± 12.26 91.14 ± 12.10 3.53 ± 7.68 <0.0001Memory 82.09 ± 19.32 88.29 ± 19.15 6.12 ± 15.46 <0.0001EF 88.61 ± 14.15 91.09 ± 12.65 2.54 ± 10.37 0.003Attention 85.19 ± 17.08 87.83 ± 15.75 2.95 ± 12.63 0.04IPS 83.54 ± 15.45 86.34 ± 17.07 2.34 ± 9.28 0.005MS 91.91 ± 17.13 95.21 ± 15.89 3.96 ± 14.27 0.001Data are expressed as means ± standard deviation. *Significant by two-tailed paired t-test. Bold text marks statistical significance (P < 0.05).GCF – global cognitive function, EF – executive function, IPS – information processing speed, MS – motor skills.Table 3Baseline characteristics comparison of patients with ischemic and hemorrhagic strokesMain analysis Ischemic (n = 121) Hemorrhagic (n = 41) P-value*Age (years) 62.78 ± 12.3 54.77 ± 12.97 0.001Sex – males 90 (74.4%) 32 (78%) 0.64Dominant hand – right 91 (75.2%) 29 (70.7%) 0.575Time from injury 2.82 ± 3.52 2.61 ± 2.61 0.71Location of injury Subcortical 38 (31.4%) 16 (39%) 0.138Cortical 65 (53.7%) 15 (36.6%)Atypical & multiple locations 18 (14.9) 10 (24.4)Side of injury Right 66 (54.5%) 21 (51.2%) 0.524Left 47 (38.8%) 15 (36.6%)Bilateral 8 (6.6%) 5 (12.2%)Symptoms Cognitive 53 (43.8%) 24 (58.5%) 0.104Motor 98 (81%) 34 (82.9%) 0.784Speech 45 (37.2%) 20 (48.8%) 0.193CN 52 (43%) 15 (36.6%) 0.476Ataxia 40 (33.1%) 17 (41.5%) 0.333Comorbidities DM II 40 (33.1%) 8 (19.5%) 0.078HTN 85 (70.2%) 22 (53.7%) 0.068Dyslipidemia 90 (74.4%) 17 (41.5%) <0.0004IHD 35 (28.9%) 4 (9.8%) 0.003Previous stroke 15 (12.4%) 3 (7.3%) 0.374Smoker 25 (20.7%) 4 (9.8%) 0.071Medications AA 92 (76%) 13 (31.7%) <0.0001Statins 86 (71.1%) 18 (43.9%) 0.003DM II medications 31 (25.6%) 6 (14.6%) 0.113HTN medications 85 (70.2%) 22 (53.7%) 0.068Data are expressed as means ± standard deviation. *Significant by two-tailed paired t-test. Bold text marks statistical significance (P < 0.05).CN – cranial nerves, DM II – diabetic mellitus type 2, HTN – hypertension, AA – anti-aggregates.dyslipidemia and IHD, and medications prescribed(AA and statins) (p < 0.05, see Table 3). In addi-tion, the memory domain mean score of the ischemicstroke patients was significantly higher at baseline,compared to hemorrhagic stroke patients (83.87 vs76.82, p = 0.043, Table 4).Post-HBOT, the IPS domain had a significantlyhigher MAC in the hemorrhagic stroke patients com-pared to the ischemic stroke patients (5.39 vs. 1.36,p = 0.035, see Fig. 2). There were no other significantdifferences in the surplus of the cognitive domains(p > 0.05, see Fig. 2). In addition, there were no signif-icant changes in the CSI between hemorrhagic strokepatients compared to ischemic stroke patients (94.6%vs. 83.33%, p > 0.05, see Fig. 4).3.2.2. Cortical vs. subcorticalAt baseline, there were significant differences inspeech symptoms and presence of HTN betweenpatients with subcortical strokes compared to corticalstroke (p < 0.05, see Table 5).Compared to cortically located strokes, the EF& attention domains at baseline were significantlyhigher in the subcortically located strokes (92.37vs. 85.19, p = 0.009, 88.44 vs. 80.78, p = 0.012,respectively, see Table 4). There were no other signif-icant differences in cognitive domains (p > 0.05, seeTable 4).Post-HBOT, there were no significant differencesbetween patients with subcortical strokes comparedto cortical strokes (p > 0.05, see Supplementary

A. Hadanny et al. / HBOT cognitive effect in post stroke patients 99Table 4Pre-hyperbaric oxygen treatment cognitive domains scoresIschemic/hemorrhagic analysis Location analysis Dominance analysisIschemic Hemorrhagic P -Value* Subcortical Cortical P -Value* Rt. Injury Lt. Injury P -Value*GCF 88.18 ± 12.52 85.42 ± 11.36 0.214 88.76 ± 11.26 84.95 ± 13.53 0.1 88.81 ± 11.77 87.8 ± 14.28 0. 686Memory 83.87 ± 18.45 76.82 ± 21.06 0.043 81.68 ± 18.4 80.72 ± 20.18 0.788 87.15 ± 18. 9 81.96 ± 18.94 0.153EF 88.6 ± 14.26 88.63 ± 13.98 0.992 92.37 ± 13.61 85.19 ± 15.11 0.009 89.68 ± 14.69 90.29 ± 15.14 0.834Attention 85.65 ± 17.54 83.8 ± 15.73 0.56 88.44 ± 14.02 80.8 ± 19.57 0.012 84.93 ± 17.57 86.33 ± 18.33 0.685IPS 84.68 ± 15.7 79.66 ± 14.09 0.106 83.55 ± 14.22 82.7 ± 16.61 0.28 85.24 ± 16.99 81.21 ± 14.07 0.2MS 91.56 ± 17.79 92.93 ± 15.15 0.667 92.79 ± 16.25 89.69 ± 19.58 0.362 91.9 ± 15.48 90.18 ± 18.59 0.608Data are expressed as means ± standard deviation. *Significant by two-tailed paired t-test. Bold text marks statistical significance (P < 0.05). GCF – global cognitive function, EF – executivefunction, VS – visual spatial, IPS – information processing speed, MS – motor skills.Fig. I). Moreover, there were no significant changes inthe CSI between subcortical strokes compared to cor-tical strokes (90% vs. 87.23%, p > 0.05, see Fig. 4).3.2.3. Dominant vs. non-dominant hemisphereIncluding only right-handed patients, at baseline,there were significant differences in speech andmotor symptoms between patients with left dominanthemisphere strokes compared to right non-dominanthemisphere strokes (p < 0.05, see Table 6). Therewere no significant differences at baseline cognitivefunction between the dominant and non-dominanthemisphere strokes (p > 0.05, see Table 4).Post-HBOT, there were significantly largerincreases in MAC in the motor domains for patientswith left hemisphere strokes compared to righthemisphere strokes (8.02 vs. 1.42, p = 0.023, seeFig. 3). There were no other significant differencesfor the surplus cognitive domains (p > 0.05, seeFig. 3).There were no significant changes in the CSIbetween left dominant hemisphere strokes comparedto right non-dominant hemisphere stroke patients(90.57% vs. 76.47%, p > 0.05, see Fig. 4).3.3. Cognitive scores outcome predictorsForward stepwise multivariate linear regressionmodels were performed on the entire cohort as wellas on the location and dominance cohorts. The onlymajor statistically significant predictor on the post-HBOT score in all of the domains and analyses wasthe baseline cognitive domain score. Age, gender,handedness, stroke details (type, injured hemisphere,location, time from injury to HBOT), number ofHBOT sessions, chronic medical conditions (DM II,HTN, dyslipidemia, IHD, previous stroke), smokingstatus, chronic prescribed medications (AA, statins,DM II medications, HTN medications) had no effectin most domains.HTN was a significant predictor on post-HBOTscore in the GCS for the dominance analysis only,and the number of HBOT sessions was a significantpredictor on post-HBOT in the EF domain for allanalyses.Forward stepwise multivariate logistic regres-sion models were performed on the three differentanalyses, to evaluate significant predictors for CSIpercentage. Low baseline cognitive memory domainscore was the only statistically significant predictoron the CSI prevalence in the main and loca-tion analyses (OR = 0.94 ([0.909–0.972], p < 0.0003),

100 A. Hadanny et al. / HBOT cognitive effect in post stroke patientsFig. 2. Hemorrhagic/ischemic stroke MAC comparison of cognitive scores post-HBOT. Only the IPS domain was significantly increasedafter HBOT for the hemorrhagic stroke patients compared to ischemic strokes. Statistical significance (p < 0.05) is marked by *. Bars representmeans+standard deviation. Abbreviations: MAC – mean absolute change, HBOT – hyperbaric oxygen treatment, GCS – global cognitivescale, EF – executive function, IPS – information processing speed, MS – motor skills.Table 5Baseline characteristics comparison of patients with cortical and subcortical strokesLocation analysis Subcortical (n = 50) Cortical (n = 77) P-value*Age (years) 62.31 ± 11.28 59.91 ± 13.32 0.296Sex – males 42 (84%) 55 (71.4%) 0.091Dominant hand – right 36 (72%) 58 (75.3%) 0.679Time from injury 2.58 ± 2.87 2.51 ± 3 0.895Type of stroke Ischemic 35 (70%) 63 (81.8%) 0.121Hemorrhagic 15 (30%) 14 (18.2%)Side of injury Right 22 (44%) 31 (40.3%) 0.676Left 28 (56%) 46 (59.7%)Symptoms Cognitive 23 (46%) 37 (48.1%) 0.823Motor 43 (86%) 61 (79.2%) 0.321Speech 15 (30%) 39 (50.6%) 0.019CN 20 (40%) 34 (44.2%) 0.647Ataxia 20 (40%) 19 (24.7%) 0.077Comorbidities DM II 19 (38%) 18 (23.4%) 0.087HTN 38 (76%) 44 (57.1%) 0.026Dyslipidemia 34 (68%) 48 (62.3%) 0.518IHD 10 (20%) 20 (26%) 0.443Previous stroke 7 (14%) 5 (6.5%) 0.192Smoker 9 (18%) 14 (18.2%) 0.979Medications AA 27 (54%) 51 (66.2%) 0.175Statins 31 (62%) 48 (62.3%) 0.97DM II medications 13 (26%) 14 (18.2%) 0.311HTN medications 36 (72%) 48 (62.3%) 0.097Data are expressed as means ± standard deviation. *Significant by two-tailed paired t-test. Bold text marks statistical significance (P < 0.05).CN – cranial nerves, DM II – diabetic mellitus type 2, HTN – hypertension, AA – anti-aggregates.OR = 0.948 ([0.912–0.985], p = 0.007), respectively).In the dominance analysis, the low baseline cogni-tive memory domain score and shorter times thatpassed since the event to HBOT were the statisti-cally significant predictors on the post-HBOT score(OR = 0.949 ([0.912–0.986], p = 0.008), OR = 0.82([0.692–0.972], p = 0.022), respectively).3.4. SafetyThere were twelve (7.4%) side effect reports in theentire cohort. Eight experienced barotrauma (8/162,4.93%). Barotraumas were mild and all patients fullyrecovered after a few days. In addition, three patients(1.85%) reported minor otalgia without objective

A. Hadanny et al. / HBOT cognitive effect in post stroke patients 101Table 6Baseline characteristics comparison of patients with dominant and non-dominant strokesRt. handed analysis Non-dominant (n = 56) Dominant (n = 54) P-value*Age (years) 60.73 ± 13.78 61.75 ± 10.65 0.668Sex – males 38 (67.9%) 40 (74.1%) 0. 477Time from injury 2.57 ± 2.59 2.7 ± 3.72 0.827Type of stroke Ischemic 44 (78.6%) 41 (75.9%) 0.741Hemorrhagic 12 (21.4%) 13(24.1%)Location of injury Subcortical 20 (35.7%) 16 (29.6%) 0.72Cortical 29 (51.8%) 29 (53.7%)Atypical Locations 7 (12.5%) 9 (16.7%)Symptoms Cognitive 21 (37.5%) 28 (51.9%) 0.132Motor 50 (89.3%) 40 (74.1%) 0.04Speech 13 (23.2%) 30 (55.6%) 0.0004CN 20 (35.7%) 26 (48.1%) 0.19Ataxia 17 (30.4%) 17 (31.5%) 0.9Comorbidities DM II 14 (25%) 14 (25.9%) 0.912HTN 37 (66.1%) 37 (68.5%) 0.787Dyslipidemia 37 (66.1%) 38 (70.4%) 0.632IHD 13 (23.2%) 15 (27.8%) 0.587Previous stroke 2 (3.6%) 8 (14.8%) 0.044Smoker 10 (17.9%) 5 (9.3%) 0.19Medications AA 35 (62.5%) 35 (64.8%) 0.803Statins 35 (62.5%) 37 (68.5%) 0.511DM II medications 12 (22.2%) 8 (14.3%) 0.286HTN medications 34 (60.7%) 40 (74.1%) 0.137Data are expressed as means ± standard deviation. *Significant by two-tailed paired t-test. Bold text marks statistical significance (P < 0.05).CN – cranial nerves, DM II – diabetic mellitus type 2, HTN – hypertension, AA – anti-aggregates.Fig. 3. Dominant/non-dominant MAC comparison of cognitive scores post-HBOT. The motor domain was significantly increased afterHBOT at the dominant (i.e. left sided) stroke patients compared to non-dominant strokes. Statistical significance (p < 0.05) is marked by*. Bars represent means+standard deviation. Abbreviations: MAC – mean absolute change, HBOT – hyperbaric oxygen treatment, GCS –global cognitive scale, EF – executive function, IPS – information processing speed, MS – motor skills.barotrauma. One patient (0.06%) reported a mildheadache during recompression. In addition, twopatients with histories of known seizures prior toHBOT suffered seizures after a few sessions ofHBOT. The seizures did not occur while in the hyper-baric chamber and once the patients reported aboutthem, their anti-epileptic drugs were modified, andthey resumed HBOT shortly.4. DiscussionIn the current study, the effect of HBOT on post-stroke patients in the late chronic stages was analyzed.Even though the patients were treated after a medianof 1.5 ± 3.3 years post-stroke, there were significantcognitive improvements in all the cognitive domainswhich were measured using objective computerized

102 A. Hadanny et al. / HBOT cognitive effect in post stroke patientsFig. 4. Clinically significant improvement comparisons of hem-orrhagic vs. ischemic, cortical vs. sub-cortical and dominant vs.non-dominant stroke patients. Scores were not significantly differ-ent in all the domains (p > 0.05). Bars represent percentages.tests. Moreover, clinical significant improvements(CSI) were achieved in 86% of patients, with the mostsignificant measurable improvements gained in thedominant hemisphere stroke patients. Low baselinememory score was the significant predictor for CSI.Hemorrhagic stroke patients had significantly higherimprovement in IPS, but no other differences werefound compared to ischemic strokes. There were nosignificant differences in HBOT effects on subcorti-cal compared to cortical strokes. Patients with strokeslocated in the dominant hemisphere had significantlylarger improvement in the MS domain.In the current study, there were significantimprovements in all the cognitive domains whichreconfirms previous studies that evaluated the ther-apeutic effect of HBOT in the chronic late stageof post-stroke patients (Boussi-Gross et al., 2015;Hadanny et al., 2015a; Emily R. Rosario et al., 2018;Vila, Balcarce, Abiusi, Dominguez, & Pisarello,2005). In a previous study, there were significantimprovements in the neurological functions, testedby the National Institutes of Health stroke scale(NIHSS), activities of daily living (ADL) and qual-ity of life (Efrati et al., 2013). However, cognitivedomains were not reported. A later retrospectivestudy reported significant improvements in the mem-ory domain after HBOT. Yet, the other cognitivedomains were not explored and the stroke nature wasnot evaluated as a possible confounder (Boussi-Grosset al., 2015). Churchill published a prospective study(Churchill et al., 2013) that included 22 patients atleast one year after stroke. HBOT induced improve-ment in symptoms reports (51% memory, 51%attention/concentration, 48% balance/coordination,45% endurance, 20% sleep). However, on standard-ized evaluations of cognition and questionnaires nosignificant changes were reported. Another smallprospective study on seven patients showed verbalmemory and executive function improvements inaddition to sleep and quality of life changes (E. R.Rosario et al., 2018).The differences between hemorrhagic andischemic strokes were mild but evident in the highcognitive function domain (i.e. the IPS) whichcorrelates with the usually more severe outcomes ofpost-hemorrhagic stroke patients and the cognitivedeficits across multiple domains (Cumming et al.,2013). This domain is more sensitive than the othercognitive domains to an insult due to its integratingrole on other domains and its influence on down-Fig. 5. Cortical/subcortical (i.e. BG) MAC comparison of cognitive scores post HBOT. Scores MAC were not significantly different in allthe domains (p > 0.05). Bars represent means+standard deviation. Abbreviations: MAC – mean absolute change, HBOT – hyperbaric oxygentreatment, GCS – global cognitive scale, EF – executive function, IPS – information processing speed, MS – motor skills.

A. Hadanny et al. / HBOT cognitive effect in post stroke patients 103stream processes, which is manifested in the domainsscore. Nevertheless, hemorrhagic stroke patientsshowed significant improvements post-HBOT andthe low baseline cognitive domain score remainedthe major predictor for the post-HBOT domain score.The lack of any significant differences after HBOTbetween cortical and subcortical strokes is surpris-ing. Similar to our study, previous studies showedsubcortical stroke patients have higher post-strokecognitive scores compared to cortical stroke patients(Gottesman & Hillis, 2010; Kalaria & Ballard, 2001).However, post–HBOT, there were no significant dif-ferences between the two types. Even though it isexpected that subcortical strokes will have lower pro-portions of memory impairments (and converselyfor the IPS domain), no such differences were seenafter HBOT treatment. Our results indicate that theexcess oxygen from HBOT treatments functions onall ischemic areas regardless of their anatomical area.As expected, the higher improvements in the MSdomain seen in the dominant stroke patients, lies inthe basic functionality of the dominant side.The lack of any significant difference regard-ing HBOT’s beneficial effects to the stroke’s originand location could be explained by the com-mon pathophysiological final path of injury, i.e.ischemic/metabolic dysfunctional cells in injured nonnecrotic brain regions. As seen in previous stud-ies, stroke patients may have chronic penumbra evenyears after the insult, which can be identified usingSPECT imaging (Churchill et al., 2013; Jacobs, Win-ter, Alvis, & Small, 1969). Oxygenation improvesenergy metabolism in the border zones of focalcerebral ischemia represented by significant reduc-tion of areas with tissue acidosis and areas withATP depletion (Sun, Marti, & Veltkamp, 2008; Sun,Strelow, Mies, & Veltkamp, 2011).HBOT can alsodecrease the post ischemic inflammatory responseby reducing blood-brain-barrier damage (Veltkampet al., 2005), inflammatory cytokines release (Yu,Xue, Liang, Zhang, & Zhang, 2015) and suppressesthe aggravated response of astrocytes and microglio-sis (Gunther et al., 2005). Recently, it was shownHBOT mitigates the inflammatory response of theneuronal cells through the transfer of mitochon-dria from astrocytes (Lippert & Borlongan, 2019).HBOT reduces apoptosis which enables to preservemore brain tissues and promote neurologic functionalrecovery (Yin et al., 2003). Opening of mitochon-drial ATP-sensitive potassium channel plays a rolein this antiapoptotic effect of early hyperbaric oxy-genation (Lou, Chen, Ding, Eschenfelder, & Deuschl,2006). The intermittent hyperoxic exposure duringHBOT can induce hypoxia inducible factor-1 alpha(HIF-1!) by the so called “Hyperoxic-Hypoxic para-dox”(Duan, Shao, Yu, & Ren, 2015; Milosevic et al.,2009; Poli & Veltkamp, 2009; Soejima et al., 2013).HIF-1! is transcriptional regulator of genes involvedin angiogenesis, energy metabolism, and neuronalcell proliferation induced by HBOT (Duan et al.,2015; Milosevic et al., 2009; Poli & Veltkamp, 2009;Soejima et al., 2013).In summary, HBOT induces neuroplasticity, bytwo main physiological effects: increasing tissueoxygenation – the rate limiting factor for all regen-erative mechanisms, and the repeated oxygen levelfluctuations which increases HIF-1! which in turntriggers the regenerative processes in the metabol-ically injured brain areas regardless of the strokeorigin (Efrati & Ben-Jacob, 2014; Efrati et al., 2013).Therefore, the selection of stroke patients for HBOTshould be based on functional imaging and baselinecognitive domain scores rather than stroke type, loca-tion or side of lesion.The current study presents the largest cohort ofpost-stroke patients treated with HBOT in the latechronic stage. However, it has several limitations,which are mostly related to the fact that data werecollected retrospectively. Still, the findings presentedhere are in agreement with previous prospectiveRCT’s in which the neuroplasticity effects of HBOTwere established [28, 37, 48]. These therapeuticeffects were seen in our study in the chronic stagewhen patients are not expected to improve. Anotherstudy limitation is the missing data on the treatment’slong-term effects. Further long-term prospectivestudies should be performed.Another important limitation relates to the HBOTprotocol which was inconsistent in the cohort, whereseveral patients received 40 sessions compared to60 sessions in most patients. Although signifi-cant neurotherapeutic effects were shown with boththese protocols, the optimal protocol, which inducesmaximal neuroplasticity with minimal side effects,remains unknown.5. ConclusionsHBOT was found, in the largest post-stroke popu-lation published, to induce significant improvementsin all cognitive function domains even at the latechronic stage. Patients selection for HBOT shouldbe based on functional imaging and baseline cogni-

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Diving and Hyperbaric Medicine Volume 47 No. 2 June 2017110Ischaemia-reperfusion injury and hyperbaric oxygen pathways: a review of cellular mechanismsAshish Francis, Richard BaynosaDivision of Plastic Surgery, Department of Surgery, University of Nevada School of Medicine, Las Vegas, USACorresponding author: Ashish Francis, Division of Plastic Surgery, Department of Surgery, University of Nevada School of Medicine, 1701 W Charleston Blvd, Suite 400, Las Vegas, NV 89102, USAafrancis@medicine.nevada.eduAbstract(Francis A, Baynosa R. Ischaemia-reperfusion injury and hyperbaric oxygen pathways: a review of cellular mechanisms. Diving and Hyperbaric Medicine. 2017 June;47(2):110-117. doi: 10.28920/dhm47.2.110-117)Ischaemia-induced tissue injury has wide-ranging clinical implications including myocardial infarction, stroke, compartment syndrome, ischaemic renal failure and replantation and revascularization. However, the restoration of blood ow produces a ‘second hit’ phenomenon, the effect of which is greater than the initial ischaemic event and characterizes ischaemia-reperfusion (IR) injury. Some examples of potential settings of IR injury include: following thrombolytic therapy for stroke, invasive cardiovascular procedures, solid organ transplantation, and major trauma resuscitation. Pathophysiological events of IR injury are the result of reactive oxygen species (ROS) production, microvascular vasoconstriction, and ultimately endothelial cell-neutrophil adhesion with subsequent neutrophil inltration of the affected tissue. Initially thought to increase the amount of free radical oxygen in the system, hyperbaric oxygen (HBO) has demonstrated a protective effect on tissues by inuencing the same mechanisms responsible for IR injury. Consequently, HBO has tremendous therapeutic value. We review the biochemical mechanisms of ischaemia-reperfusion injury and the effects of HBO following ischaemia-reperfusion.Key wordsHypoxia; Hyperoxia; Reperfusion injury; Free radicals; Nitric oxide; Ischaemic preconditioning; Review articleIntroductionTissue ischaemia represents the nal common pathway of various disease states that include myocardial infarction, stroke, amputations, compartment syndromes and failing tissue flaps and grafts. In these scenarios, emergent interventions are undertaken to restore blood ow to the affected areas, which in some cases may be life- and limb-saving on a global scale. However, this reperfusion is not without consequence; despite restoration of ow, further tissue and microcirculation injury still occur, even to a greater extent than the initial ischaemic insult. Tissue necrosis and microcirculatory collapse that occur because of reperfusion following prolonged ischaemia is referred to as ischaemia-reperfusion (IR) injury. Examples of IR injury can be seen in many settings: thrombolytic therapy for stroke, any cardiovascular invasive procedure (e.g., angioplasty of the popliteal artery to coronary artery bypass with assisted circulation), organ transplantation, and major trauma resuscitation. Reactive oxygen species (ROS) have been shown to be the principal mediators of this phenomenon. During IR injury, the blood-endothelial cell interface shows increased microcirculatory neutrophil adhesion that incites tissue necrosis and starts a feedback loop that results in further ROS production and injury. Given the potentially devastating clinical outcomes of IR injury, much investigation has been undertaken to better understand the molecular signals and changes that occur in the microcirculation. As our understanding of the mechanisms of IR injury has evolved, so too has interest in therapeutic interventions to reverse or prevent it, particularly with hyperbaric oxygen (HBO). The purpose of this article is to discuss the biochemical mechanisms of ischaemia-reperfusion injury and review the effects of HBO treatment.Ischaemia-reperfusion injury (Figure 1)OXIDATIVE STRESSROS, chiey oxygen free radicals, serve a cell-signalling role and are formed during cellular metabolism.1 While their role in normal homeostasis continues to be investigated, their involvement in mediating oxidative damage seen during IR injury has been documented extensively.2 The predominant ROS in the cell are superoxide and hydroxyl radicals. Their cellular toxicity results from lipid peroxidation and its associated membrane damage, direct DNA damage, and production of other free radical and reactive species.3 Due to the instability of free radical species, free radical scavengers have been utilized to indirectly prove the detrimental effects of ROS in IR injury. Using a mouse hind limb model, improvement in inammatory cell inltration in skeletal muscle after IR was shown when pretreating with edavarone, a synthetic free radical scavenger.4

Diving and Hyperbaric Medicine Volume 47 No. 2 June 2017 111In the normal physiologic state, multiple antioxidant mechanisms exist to counteract the effect of these ROS: superoxide dismutase (SOD), glutathione, and catalase.5 Once these systems are overwhelmed, as occurs during IR injury, excess ROS is produced and incites tissue damage. These antioxidant systems have also been utilized as physiologic free radical scavengers that provide improvements in skeletal muscle function after undergoing IR injury. As a model for limb replantation, rabbit tibialis anterior muscle was subjected to IR at ve-and eight-hour intervals and muscle function was examined after administration of the hydroxyl free radical scavenger dimethylsulfoxide (DMSO) and superoxide free radical scavenger SOD prior to reperfusion.6 Muscle treated with SOD had normal strength after ve hours of ischaemia but no protective effective after eight hours; conversely, DMSO had improved function after eight hours but no effect after ve hours compared to untreated controls.6During IR injury, two sources for ROS generation are xanthine oxidase and neutrophils. Xanthine oxidase, the conversion product of oxidative damage to xanthine dehydrogenase found in skeletal muscle endothelial cells, produces superoxide and hydrogen peroxide (H2O2) during purine metabolism.3 These ROS recruit neutrophils to the blood-endothelial cell interface, thereby initiating migration into the surrounding tissues. Neutrophils then produce a greater amount of ROS, further precipitating the effects of IR injury. Consequently, much research into IR injury has focused on the interaction between neutrophils at the blood-endothelial cell interface. Neutrophils generate a large amount of extracellular superoxide owing to the presence of membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Following dismutation to H2O2, subsequent reactions result in the formation of other toxic molecules: hydroxyl radical (reaction with ferritin) and hypochlorous acid (reaction with chloride via neutrophil myeloperoxidase). It appears that, in IR injury, xanthine oxidase may produce the initial liberation of ROS species, with further propagation coming from neutrophils, nally culminating in tissue injury.7,8Programmed cell death (apoptosis) has also been implicated in IR injury although the mechanisms remain unclear. ROS accumulation has been shown to induce apoptosis.9 More recently, nitric oxide (NO) has been suggested as a mediator of IR injury. NO is produced by nitric oxide synthase (NOS), which has three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS).10 The former two isoforms are expressed constitutively, whereas the latter requires protein synthesis. It should be noted that NO competes with oxygen in binding to terminal cytochrome c oxidase, which has a higher afnity for NO than for oxygen. At higher oxygen concentrations, NO is consumed by cytochrome c oxidase and thus simulates a hypoxic environment. Conversely in lower oxygen tension, NO is not consumed and is available to mediate its physiologic effects.11High levels of NO produced by iNOS may interact with superoxide to produce peroxynitrite, resulting in Figure 1Mediators of ischaemia-reperfusion injury; oxidative stress, microvascular dysfunction, and the neutrophil-endothelial cell interaction produce changes in cellular physiology that increase cell damage and tissue death; CAM – cellular adhesion molecule; EC – endothelial cell; PMN – polymorphonuclear neutrophil; ROS – reactive oxygen species

Diving and Hyperbaric Medicine Volume 47 No. 2 June 2017112mitochondrial cytochrome c release and caspase activation, and ultimately apoptosis.12,13 Rat intestinal mucosa subjected to IR injury demonstrated iNOS, NO, and apoptosis.14 The data regarding NO and apoptosis is conicting as other studies have demonstrated a protective role for NO against apoptosis.15,16 Further investigation is required to clarify this relationship. Therefore, it appears that the secondary products of superoxide radical interactions (i.e., hydroxyl radical, hypochlorous acid, peroxynitrite) are the primary mediators of the toxic effects of IR injury rather than superoxide itself. Moreover, there is a growing body of evidence demonstrating a cellular signalling role for superoxide and other oxidative species.17MICROCIRCULATION DYNAMICSIn studies investigating reperfusion following myocardial infarction in dogs, reperfusion results in improved ow through the large vessels. However, the microcirculation demonstrates obstruction, circulatory collapse, and myocardial function still suffers.18 These ndings suggest that IR injury involves changes in the microcirculation. An early study found signicant increases in vasoconstricting thromboxanes compared to vasodilating prostaglandins in a rat hind-limb model of the no-reow state, a phenomenon describing complete microcirculatory failure, compared to ischaemic limbs with reow.19 This study also found decreased venous outow with absent vascular thrombosis, suggesting a mechanism of excess thromboxane release producing microcirculatory vasoconstriction in the impending no-reow state.19 Similar ndings regarding vasoconstriction in IR injury were observed in a rat skeletal muscle utilizing intravital microscopy to examine arteriolar and venule diameters.20 It was found that reperfusion resulted in initial vasodilation followed by severe vasoconstriction after one hour. Distant arteriolars were spared from this effect, suggesting an inuence of the local environment damaged by neutrophils.20Another study demonstrated that vasoactive substances exert a greater effect on arteriolar smooth muscle than on the endothelium in the microcirculation.21 It was concluded that there is a barrier to diffusion of water-soluble vasoactive substances between the smooth muscle and endothelium. During IR injury, the post-capillary venule endothelial cells are damaged by neutrophils. This might provide a putative explanation as to why the vasoconstrictive effect of IR injury is conned to the immediately adjacent microarterioles. Other vasoactive substances have been implicated in modulating microarteriolar vasoconstriction, including serotonin and leukotrienes.22,23 While much of the focus has been on neutrophil-induced injury following reperfusion, other cell lines may be involved. Skeletal muscle IR injury results in adenosine-regulated mast cell degranulation that initiates arteriolar vasoconstriction.24 Therefore, it is likely that the dynamic microvascular changes occurring during IR injury reect the complex interplay between multiple cell lines and vasoactive molecules. Additional research is necessary to better understand these interactions.NEUTROPHIL-ENDOTHELIAL CELL ADHESIONIt is interesting to note that tissue damage from ischaemia differs microscopically from that from IR injury. With ischaemia, tissue architecture is preserved and there is a relative acellularity. In contrast, IR injury is characterized by tissue necrosis and leukocyte inltration, especially neutrophils. Neutrophils are recruited to the ischaemic site following reperfusion and, prior to extravasation, adhere to the endothelium. Given the role of neutrophils in mediating IR injury and ROS production, investigation of the neutrophil-endothelial cell interface has increased for its potential therapeutic targetting.Neutrophil adhesion to the microvascular endothelium is dependent on interactions between receptors and ligands on the surface of the neutrophil and endothelium called cell adhesion molecules (CAMs). Examples include P-selectin, E-selectin, and ICAM-1. Expression of these molecules varies depending on the local tissue conditions and on cytokine release. P-selectin is expressed on the surface of endothelial cells within 15 minutes of middle cerebral artery occlusion, where it can bind to its respective neutrophil ligand (P-selectin glycoprotein ligand-1).25 P-selectin is contained within Weibel-Palade bodies and is secreted onto the endothelial cell surface after these storage granules are exocytosed.26Whereas P-selectin expression occurs acutely following IR injury, other CAM molecules (e.g., E-selectin and ICAM-1) expression occurs in a delayed fashion, likely because of increased CAM molecule expression via transcription and translation.27 E-selectin and ICAM-1 then bind to neutrophil ligands to produce the neutrophil-endothelial cell adhesion. Using monoclonal antibody against CD18 to block neutrophil-endothelial cell adherence, a study of rat skeletal muscle showed that ICAM-1 interacts with neutrophil cell-surface CD18 molecules during IR injury.28 Microarteriolar vasoconstriction was also blocked, suggesting that neutrophil CD18 plays a key role in altering the microcirculation during IR injury. Using phorbol-12 myristate 13-acetate (PMA) to simulate endothelial cell injury, it has been shown that ischaemia-reperfusion also upregulates neutrophil CD18.29 In another in vitro study of neutrophil adherence, confocal microscopy demonstrated neutrophil capping, which refers to the change in polarity of surface CD18 molecules by concentrating them in one area, thereby increasing the likelihood of adhesion to endothelial ICAM-1 after IR injury.30Protection against IR injury has been shown by blocking neutrophil-endothelial cell adhesion in vivo. Use of anti-CD18 monoclonal antibody in baboons decreased cerebral infarction.31 Additionally, a knock-out mice model

Diving and Hyperbaric Medicine Volume 47 No. 2 June 2017 113for ICAM-1 exhibited a six-fold decrease in infarction size compared to wild-type.32 Interestingly, neutrophil recruitment was similar between the groups, suggesting that the extent of inammatory cell response is less crucial than the interaction between neutrophils and the endothelium.32Hyperbaric oxygen (Figure 2)HALLMARK STUDIES The initial ischaemic insult deprives tissues of oxygen and results in cellular injury, so it follows that restoration of oxygenation through the microcirculation would halt or perhaps reverse the tissue necrosis. IR therefore represents a paradoxical process because reperfusion produces a greater degree of tissue damage, dependent on ROS. In addition, it was thought that a hyperoxic tissue environment provided by administration of HBO following IR injury would increase ROS production and worsen the extent of tissue necrosis.Unexpectedly, after subjecting an axial skin ap model to eight hours of ischaemia to determine the effects of HBO during reperfusion, skin-ap survival was signicantly improved following HBO.33 It had been anticipated that HBO would increase free radical liberation, worsen IR injury and decrease skin-ap survival. Subsequent ndings of increased perfusion of ischaemic skin aps treated with HBO using laser Doppler analysis conrmed these results.34 Similarly, HBO provided a three- to six-fold improvement in free skin-ap survival in rats after microvascular anastomosis.35 The authors posited that 24 hours was the threshold for irreversible ischaemic damage.35 Consequently, we now understand that HBO ameliorates the detrimental effects of IR injury by improving tissue microcirculation.IMPROVEMENT IN OXIDATIVE DAMAGE AND CELL DEATHSince ROS are a key mediator in IR injury, studies have investigated the effects of HBO on the generation of these toxic molecules. Carbon monoxide (CO) simulates ischaemic conditions, a concept that was utilized in a rat model of brain injury.36 Lipid peroxidation mediated by CO was inhibited by preventing xanthine oxidase formation, presumably decreasing superoxide radical and hydrogen peroxide levels.36 Free radical scavenging systems, specifically SOD, may also be upregulated following HBO treatment.35 HBO has also been shown to upregulate Figure 2Physiologic effects following hyperbaric oxygen therapy for ischaemia-reperfusion; hyperbaric oxygen results in interference with neutrophil-endothelial cell interactions, promotes arteriolar vasodilation, and ameliorates cellular damage; solid arrows represent stimulatory pathways; dotted arrows represent inhibitory pathways; ECM − extracellular matrix; eNOS − endothelial nitric oxide synthase; HBO − hyperbaric oxygen; ICAM − intercellular adhesion molecule; iNOS − inducible nitric oxide synthase; NO − nitric oxide; PMN − polymorphonuclear neutrophil; ROS − reactive oxygen species; SOD − superoxide dismutase; tPA − tissue plasminogen activator; uPA − urinokinase-like plasminogen activator; VEGF− vascular endothelial growth factor

Diving and Hyperbaric Medicine Volume 47 No. 2 June 2017114antioxidant gene expression in human endothelial cells which may protect against oxidative damage seen in IR injury.37 As a result, HBO appears to produce its benecial effects on ischaemic tissue by both decreasing production of ROS and increasing their degradation. Rat studies of renal IR injury treated with HBO prior to ischaemia demonstrated decreased oxygen radical-induced lipid peroxidation.38 A more recent study demonstrated HBO-induced inhibition of apoptosis and improvement in cellular proliferation following renal IR injury.39Additionally, utilizing a validated ischaemic ap model in rats, it was demonstrated that HBO improves ischaemic wound healing compared to untreated and N-acetylcysteine-treated (a non-specic free radical scavenger) groups. The mechanisms for this appeared to be by down-regulation of hypoxia-inducible factor-1 alpha (HIF-1 alpha) and p53- and caspase-3-mediated apoptosis.40 In addition, the inammatory response, as demonstrated by VEGF, cyclooxygenase-2, and neutrophil counts, were reduced in the HBO group.40 Subsequently it was demonstrated that HBO increased antioxidant enzyme expression (SOD, catalase, and glutathione peroxidase) and decreased pro-oxidant enzyme expression (iNOS and gp91-phox) in ischaemic wounds.41 These ndings show that HBO does not exacerbate ROS-mediated tissue injury.INTERFERENCE WITH NEUTROPHIL-ENDOTHELIAL CELL ADHESIONThe potential mechanisms of HBO on neutrophil adhesion have also been of great interest. In a rat gracilis model investigating neutrophil adherence following HBO, adherence was signicantly decreased during and after four hours of ischaemia. When HBO was initiated one hour after reperfusion, however, leukocyte adhesion was reduced to a lesser degree.20 In the absence of ischaemia, HBO had no observable effect on the neutrophil-endothelial cell interaction. The same study also reviewed the effects on microvascular vasoconstriction. Vasoconstriction was inhibited when HBO was initiated during ischaemia, immediately after reperfusion, and one hour after reperfusion, with maximal effect during ischaemia.20 These ndings suggest that the maximal benecial response to HBO occurs during the ischaemic phase and may be time-dependent. A rodent model of renal IR injury demonstrated decreased neutrophil infiltration following HBO and associated improvements in blood urea nitrogen and creatinine levels.42A study at 284 and 304 kPa reported on the inhibition of human neutrophil adherence to injured endothelium via beta-2-integrin (CD18) function.43 CD18-mediated neutrophil adhesion was inhibited but expression was not affected. Moreover, this study demonstrated that this function is cyclic GMP (cGMP)-regulated. HBO inhibited the function of guanylate cyclase and neutrophil adhesion was restored directly by cGMP incubation and also by increasing guanylate cyclase activity with N-formyl-Met-Leu-Phe (FMLP).43 cGMP production was altered by inhibition of the membrane-bound guanylate cyclase, but free intracellular guanylate cyclase was unaffected. Therefore, it appears that HBO inhibits CD18 activity via impaired cGMP production. The nding that CD18 expression is not decreased by HBO treatment has been conrmed by others in a skeletal muscle rat model.29 Neutrophil capping and CD18 surface polarization were inhibited by HBO, providing another plausible mechanism for the reversal of IR injury.44The role of the endothelial cell cannot be overlooked in IR injury and other investigators have sought to focus on endothelial cell CAM (E-selectin and ICAM-1) expression after HBO. Ischaemia-reperfusion was simulated with hypoxia and hypoglycaemia exposure and demonstrated increased adhesion of neutrophils to endothelium.45 HBO signicantly reduced ICAM-1 expression and neutrophil adhesion.45 Similar results were noted in in vivo experiments of rat skeletal muscle aps.46 HBO was administered at253 kPa for 90 minutes and down regulated ICAM-1 expression with a resultant improvement in ap survival.THE ROLE OF NITRIC OXIDENO has been shown to regulate many processes in IR injury including the microcirculation through its vasodilatory properties and reversal of leukocyte adhesion. Loss of the protective effect of NO resulted in increased CAM expression.47 Indirect evidence has been found for increased survival and decreased neutrophil-endothelial adhesion after infusion of L-Arginine, a NO precursor, into ischaemic muscle.48 eNOS inhibition promotes neutrophil adherence in the endothelium. In a previously mentioned study, HBO induced expression of eNOS; additionally, inhibition of NOS attenuated inhibition of ICAM-1 after HBO.45 Another study of isolated rat neutrophils showed that NO inhibited CD18 activity by decreasing guanylate cyclase activity, corroborating the nding that NO decreases neutrophil adhesion.49 Rat studies have demonstrated a favorable effect of HBO following IR injury on intestinal mucosa and hepatic cell apoptosis which may be mediated through decreased iNOS activity with a resultant decrease in peroxynitrite.50,51The NO-dependent effect of HBO on CD18 polarization also has been examined.52 Following administration of a NO scavenger, CD18 polarization and adherent neutrophils increased signicantly compared to untreated controls. Furthermore, NOS inhibitors given before HBO restored neutrophil adhesion and capping via CD18. Together these ndings represent a NO-regulated mechanism underlying the benecial effects of HBO after IR injury and its associated CD18 polarization.52 More recently, two important ndings on the effect of HBO on NOS activity and expression in IR injury in rats have been demonstrated. First, eNOS was increased in pulmonary tissues, which supports the theory that the benecial effects of HBO therapy occur systemically, not locally. Additionally, a temporal relationship of HBO-mediated NOS effects exists with an early phase increase

Diving and Hyperbaric Medicine Volume 47 No. 2 June 2017 115in eNOS enzymatic activity and a subsequent late-phase increase in protein expression, the delay accounted for by transcription and translation.53 Unpublished data from our laboratory suggests that the NO-dependent effect of HBO on CD18 polarization occurs through the plasmin-mediated release of membrane-bound vascular endothelial growth factor (VEGF).The role of VEGF has recently been an area of investigative focus. HBO improves wound angiogenesis by increasing VEGF transcription and protein production.54 In contrast to the acute changes seen after HBO following IR injury, the effects on VEGF represent long-term effects. VEGF can be bound to the extracellular matrix and released by the activity of various proteases, including plasmin.55 Plasminogen is activated to yield plasmin by tissue or urokinase-like (tPA or uPA) plasminogen activators, the expression of which is increased with HBO.56 The enhanced plasmin activation results in release of VEGF from the extracellular matrix and increased NO production.57 IR injury increases alpha2-antiplasmin, thereby decreasing the amount of plasmin available to release stored VEGF.58 Part of the benecial effect of HBO after IR injury may be to increase levels of tPA and uPA, increasing plasmin beyond the inactivating capability of alpha2-antiplasmin present from the initial ischaemic event.HYPERBARIC OXYGEN PRECONDITIONINGPreconditioning refers to the administration of HBO to limit the effects of subsequent ischaemia. The putative mechanism appears to be from the induction of antioxidant intracellular systems including catalase and SOD.59,60 It has been suggested that the cardio-protective effects of HBO therapy in a rat model were NOS-regulated.61 Much of the recent literature regarding HBO and IR injury has focused on this phenomenon. There is evidence that preconditioning rat skin flaps with HBO prior to IR injury resulted in improved survival and microcirculatory perfusion.62 This benecial response was the result of increased expression of anti-apoptotic factor B-cell lymphoma-2 (Bcl-2) and inhibition of apoptotic factors phosphorylated apoptosis signal-regulating kinase 1 (pASK-1), phosphorylated c-Jun N-terminal kinase (pJNK) and Bcl2-associated K protein (Bax). A diminution of the inammatory cytokine cascade has also been advocated.63The preconditioning effect of HBO was confirmed in an hepatic IR study in rats. HBO not only resulted in improvements in serum alanine aminotransferase and aspartate aminotransferase levels, but also demonstrated improvements in mitochondrial respiration and swelling.64 The relationship between mitochondrial injury, cytochrome c release, and apoptosis suggests that the improvement in mitochondrial function revealed by this study may result in inhibition of apoptosis. HBO preconditioning provides systemic and local tissue benets; however, the response appears to be dependent on the timing of HBO exposure with the exact window remaining unknown.65 HBO preconditioning may be useful for limiting the well-documented neurological complications following elective cardiac surgery and carotid endarterectomy. It may also prove useful in complex reconstructive procedures requiring composite tissue allotransplantation, such as face and hand transplants, where ischaemia times can be extremely prolonged and multiple tissue types are involved including muscle and bone.LimitationsThe studies mentioned above, and others, have provided a great deal of clarity in this challenging area. However, it should be noted that much of our understanding of IR injury and HBO derives from experimental studies in animal models or cell culture. While these experimental ndings are promising, it is unclear whether the physiologic outcomes correlate to clinically relevant ndings. The clinical data regarding this topic are mostly from retrospective studies and are insufciently powered with small sample sizes, and thus, the clinical impact remains debatable.ConclusionsStudies of the microcirculation, neutrophil adhesion, and ROS reect the complex interactions between various cell signalling molecules, intracellular pathways, and cell types in IR injury. HBO can ameliorate the cytotoxic effects of reperfusion injury in a dose-dependent manner. However, the critical issue at the centre of IR injury is the duration of ischaemia as the primary factor in determining the outcome from injury. The adage ‘time is muscle/nerve’ emphasizes the well-established fact that irreversible damage is observed in ischaemia-sensitive tissues such as muscle and nerve tissue after six hours of ischaemia. The window of reversible changes presents a valuable opportunity not only for promptly initiating the appropriate interventions to reverse the ischaemic aetiology, but also to institute adjunctive therapies such as HBO to limit the extent of tissue damage after IR injury.Our understanding of IR and the protective effects of HBO continues to evolve. The evidence from rat skeletal muscle and skin flap studies provides insight into the potential for HBO in the treatment of IR injury following crush injuries, and failing grafts and aps. Other studies of animal heart, brain, kidney, intestine and liver demonstrate the positive effects of HBO in the context of myocardial infarction, stroke, ischaemic renal failure and solid organ transplantation. HBO preconditioning has promising therapeutic value and its applications are under investigation. Additionally, standardized protocols for HBO have not been dened. It is apparent that questions remain for these processes and therefore more basic-science and clinical research is required to elucidate them.

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J Reconstr Micro. 1989;5:343.34 Zamboni WA, Roth AC, Russell RC, Smoot EC. Effect of hyperbaric oxygen on reperfusion of ischaemic axial skin aps: a laser doppler analysis. Ann Plast Surg. 1992;23:339.35 Kaelin CM, Im MJ, Myers RA, Manson PN, Hoopes JE. The effects of hyperbaric oxygen on free aps in rats. Arch Surg. 1990;125:607-9.36 Thom SR. Functional inhibition of leukocyte B2 integrins by hyperbaric oxygen in carbon monoxide-mediated brain injury in rats. Toxicol Appl Pharmacol. 1993;123:248-56.37 Godman CA, Joshi R, Giardina C, Perdrizet G, Hightower LE. Hyperbaric oxygen treatment induces antioxidant gene expression. Ann N Y Acad Sci. 2010;1197:178-83.38 Gurer A, Ozdogan M, Gomceli I, Demirag A, Gulbahar O, Arikok T, et al. Hyperbaric oxygenation attenuates renal ischaemia-reperfusion injury in rats. Transplant Proc. 2006;38:3337-40.

Diving and Hyperbaric Medicine Volume 47 No. 2 June 2017 11739 Migita H, Yoshitake S, Tange Y, Choijookhuu N, Hishikawa Y. Hyperbaric oxygen therapy suppresses apoptosis and promotes renal tubular regeneration after renal ischaemia/reperfusion injury in rats. Nephrourol Mon. 2016;8:e34421. doi: 10.5812/numonthly.34421.40 Zhang Q, Chang Q, Cox RA, Gong X, Gould LJ. Hyperbaric oxygen attenuates apoptosis and decreases inammation in an ischaemic wound model. J Invest Dermatol. 2008;128:2102-12.41 Zhang Q, Gould LJ. Hyperbaric oxygen reduces matrix metalloproteinases in ischaemia wounds through a redox-dependent mechanism. J Invest Dermatol. 2014;134:237-46.42 Solmazgul E, Uzun G, Cermik H, Atasoyu EM, Aydinoz S, Yildiz S. Hyperbaric oxygen therapy attenuates renal ischaemia/perfusion injury in rats. Urol Int. 2007;78:82-5.43 Thom SR, Mendiguren I, Hardy K, Bolotin T, Fisher D, Nebolon M, Kilpatrick L. Inhibition of human neutrophil beta2-integrin-dependent adherence by hyperbaric O2. Am J Physiol. 1997;272:C770-7.44 Khiabani KT, Bellister SR, Skaggs SS, Stephenson LL, Wang WZ, Zamboni WA. Reperfusion induced neutrophil CD18 polarization: effect of hyperbaric oxygen. J Surg Res. 2008;150:11-6.45 Buras JA, Stahl Gl, Svoboda KK, Reenstra WR. Hyperbaric oxygen downregulates ICAM-1 expression induced by hypoxia and hypoglycemia: the role of NOS. Am J Physiol Cell Physiol. 2000;278:C292-302.46 Hong JP, Kwon H, Chung YK, Jung SH. The effect of hyperbaric oxygen on ischaemia-reperfusion injury: An experimental study in a rat musculocutaneous ap. Ann Plast Surg. 2003;51:478-87.47 Lefer AM, Lefer DJ. The role of nitric oxide and cell adhesion molecules on the microcirculation in ischaemia-reperfusion. Cardiovasc Res. 1996;32:743-51.48 Meldrum DG, Stephenson LL, Zamboni WA. Effects of L-NAME and L-Arginine on ischaemia-reperfusion injury in rat skeletal muscle. Plast Reconstr Surg. 1999;103:935-40.49 Banick PD, Chen Q, Xu YA, Thom SR. Nitric oxide inhibits neutrophil beta 2 integrin function by inhibiting membrane-associated cyclic GMP synthesis. J Cell Physiol. 1997;172:12-24.50 Bertoletto PR, Fagundes DJ, Simoes MJ, Oshima CT, Montero EF, Simoes RS, Fagundes AT. Effects of hyperbaric oxygen therapy on the rat intestinal mucosa apoptosis caused by ischaemia-reperfusion injury. Microsurgery. 2007;27:224-7.51 Chaves JC, Fagundes DJ, Simoes MJ, Bertoletto PR, Oshima CT, Taha MO, et al. Hyperbaric oxygen therapy protects the liver from apoptosis caused by ischaemia-reperfusion injury in rats. Microsurgery. 2009;29:578-83.52 Jones SR, Carpin KM, Woodward SM, Khiabani KT, Stephenson LL, Wang WZ, Zamboni WA. Hyperbaric oxygen inhibits ischaemia-reperfusion-induced neutrophil CD18 polarization by a nitric oxide mechanism. Plast Reconstr Surg. 2010;126:403-11.53 Baynosa RC, Naig AL, Murphy PS, Fang XH, Stephenson LL, Khiabani KT, et al. The effect of hyperbaric oxygen on nitric oxide synthase activity and expression in ischaemia-reperfusion injury. J Surg Res. 2013;183:355-61.54 Sheikh AY, Gibson JJ, Rollins MD, Hopf HW, Hussain Z, Hunt TK. Effect of hyperoxia on vascular endothelial growth factor levels in a wound model. Arch Surg. 2000;135:1293-7.55 Keyt BA, Berleau LT, Nguyen HV, Chen H, Heinsohn H, Vandlen R, Ferrara N. The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency. J Biol Chem. 1996;271:7788-95.56 Tjarnstrom J, Holmdahl L, Falk P, Falkenberg M, Arnell P, Risberg B. Effects of hyperbaric oxygen on expression of brinolytic factors of human endothelium in a stimulated ischaemia/reperfusion situation. Scand J Clin Lab Invest. 2001;61:539-46.57 Hood JD, Meininger CJ, Ziche M, Granger HJ. VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells. Am J Physiol. 1998;H1054-8.58 Bayes-Genis A, Guindo J, Oliver A, Badimon L, Fiol M, Mateo J, et al. Elevated levels of plasmin-alpha2 antiplasmin complexes in unstable angina. Thromb Haemost. 1999;81:865-8.59 Kim CH, Choi H, Chun YS, Kim GT, Park JW, Kim MS. Hyperbaric oxygenation pretreatment induces catalase and reduces infarct size in ischaemic rat myocardium. Pugers Arch. 2001;442:519-25.60 Nie H, Xiong L, Lao N, Chen S, Xu N, Zhu Z. Hyperbaric oxygen preconditioning induces tolerance against spinal cord ischaemia by upregulation of antioxidant enzymes in rabbits. J Cereb Blood Flow Metab. 2006;26:666-74.61 Cabigas BP, Su J, Hutchins W, Shi Y, Schaefer RB, Recinos RF, et al. Hyperoxic and hyperbaric-induced cardioprotection: role of nitric oxide synthase 3. Cardiovasc Res. 2006;72:143-51.62 Xiao YD, Liu YQ, Li JL, Ma XM, Wang YB, Liu YF, et al. Hyperbaric oxygen preconditioning inhibits skin ap apoptosis in a rat ischaemia-reperfusion model. J Surg Res. 2015;199;732-9.63 Kang N, Hai Y, Liang F, Gao CJ, Liu XH. Preconditioned hyperbaric oxygenation protects skin ap grafts in rats against ischaemia/reperfusion injury. Mol Med Rep. 2014;9:2124-30.64 Losada DM, Chies AB, Feres O, Chaib E, D’Albuquerque LA, Castro-e-Silva O. Effects of hyperbaric oxygen therapy as hepatic preconditioning in rats submitted to hepatic ischaemia/reperfusion injury. Acta Cir Bras. 2014;29:61-6.65 Losada DM, Jordani ME, Jordani MC, Piccinato MA, Fina CF, Feres O, et al. Should preconditioning hyperbaric oxygenation protect the liver against ischaemia-reperfusion injury? An experimental study in a rat model. Transplant Proc. 2014;46:56-62.Submitted: 21 September 2016; revised 10 January 2017Accepted: 03 February 2017View publication statsView publication stats

Clinical StudyThe Effect of Hyperbaric Oxygen Therapy onFunctional Impairments Caused by Ischemic StrokeEmily R. Rosario ,StephanieE.Kaplan,SepehrKhonsari,GarrettVazquez,Niyant Solanki, Melanie Lane, Hiriam Brownell, and Sheila S. RosenbergCasa Coli n a Hos pital and Centers for Healthca re, Po m ona CA, 255 East Bonita Avenue, Pom ona, CA 91767, USACorrespondence should be addressed to Emily R. Rosario; erosario@casacolina.orgAcademic Editor: Herbert BrokCopyright © 2018 Emily R. Rosarioet al.is is an openaccess articledistributed under the CreativeCommons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Backgrou n d. While research suggests a benet of hyperbaric oxygen therapy (HBOT) for neurologic injury, controlled clinical trialshave not been able to clearly dene the benets. Objective. To investigate the eect s of HB OT on physi cal and cognitive impairmentsresulting from an ischemic stroke. Methods. Using a within-subject design a baseline for current functional abilities was establishedover a 3-month period for all subjects (n=7). Each subject then received two 4-week periods of HBOT for a total of 40 90-minutetreatments over a 12-week period. Subjects completed a battery of assessments and had blood drawn six times over the 9-month totalduration of the study. Results. We found improvements in cognition and exec utive function as well as physical abilities, specically,improved gait. Participants reported improved sleep and quality of life following HBOT treatment. We also saw changes in serumlevels of biomar kers for inammation and neura l recovery. In the functional domains where improvement was observed followingHBOT treatment, the improvements were maintained up to 3 months following the last treatment. However, the physiologicalbiomarkers showed a pattern of more transient changes following HBOT treatment. Conclusions. Findings from this study supportthe idea of HBOT as a p otential intervention following stroke.1. IntroductionEach year over 795,000 Americans will suer a stroke result-ing in death or signicant disa bility. While considerable func-tional gains are oen made, signicant assistance in daily lifeis still required in approximately one-third of stroke survivors[1]. Following an ischemic stroke, in w hich cerebral bloodow is impaired, irreversible neural injury occurs withinminutes (for review s ee [2, 3]). Of particular therapeuticin terest are the regions surrounding the focal site of injurywhere the tissue is at risk but not facing irreparable damage,and the potential to salvage these neurons still exists [4–6].Imaging has shown that those at risk regions may persist in adysfunctional state for mon ths to years aer the inj ury [7, 8].Cell death and reduced neuronal activity resulting f rom anischemic event can be at tributed to excitotoxicity, oxidativestress, inammation, and apoptosis, which are all pathwayswhere hypoxia plays a key role (For review see [5]). Decreasedoxygenation to the damaged area including blood vesselsfurther prevents tissue repair and the generation of newsyna ptic connections [8, 9]. Consequently, increased oxygenhas been considered as a potential treatment for stroke forseveral decades [10].Hyperbaric oxygen can be dened as the breathing of100% oxygen at a pressure higher tha n atmospheric pressure.Initially, hyperbaric oxygen therapy (HBOT) was used totreat decompression sickness in divers; however, over theyears its far-reaching potential was recognized, and it hasbeen approved for a variety of purposes including woundrepair , carbon monoxide poisoning, anemia, thermal burns,delayed radiation injuries, osteomyelitis, and actinomycosis(for review see [ 11]). In addition to these conditions, therehas been a great deal of interest in the use of HBOT forbrain injury, stroke, and cerebral palsy. e use of HBOTfor brain injury is based on the hypothesis that injur ed orinactive neurons would benet from increased blood owand oxygen delivery, which would act to metabolically orelectrically reactivate the cells [8, 12–15]. However, whileHBOT is approved for several clinical indications [16], theeects of HBOT in the brain have yet to be clearl y dened.        

2 Neurology Research InternationalSupporting a role for the use of HBOT in stroke patientsis a wealth of experimental studies in a number of dierentanimal models [17–22]. Specically , in a variety of animalmodels including young and aged rats, mice, rabbits, and dogsanumberofdierentexperimentalparadigmshavebeenused to induce stroke-like neural injury. In these dierentmodels, HBOT has been shown to decrease intracranial pres-sure, reduce blood-brain-barrier permeab ility and cerebraledema [23], increase cellular metabolism, stabilize levels ofglutamate, glucose, and pyruvate, attenuate inammatoryresponse [24], and increase antia poptotic bcl family genes[6, 17, 20, 25–28]. HB OT has also b een shown to be ecaciousin improving learning and memory decits in rats [29–31],an eect we predict may be correlated with the cellularand molecular eects contributing to neuronal viability andplasticity [15]. Although observed eects of HBOT varydepending on the experimental conditions (e.g., animalmodel, type of injury, timing of treatment, duration of treat-ment HBOT, etc.), animal studies suggest numerous benetsand an underlying mechanism, which warrants continuedresearch in both animals and humans to fully elucidate thebenets of HBOT.Despite the seemingly overwhelming potential of HBOTas dened by basic research and the underlying mechanisticrationale, clinical investigations have largely not produced theexpected results. While research has provided some favorableevidence for HB OT in both acute strokes and p oststroke[21, 22, 32–39, 39, 40], methodological issues have limited thein terpretation and generalizability of the results [2, 6]. etiming of HBOT aer stroke and duration of treatmen tremain critical questions to be answered. In this preliminarystudy we attempted to address some of these issues byin vestigating the use of HBOT as a therapeutic interventionfor stroke patien ts in the chronic stage of their illness using awithin subjects design. While the sample was small and notblinded to treatment we predicted that HBOT would resultin numero us benets on a variety of functional impairmentsthat may occur following an ischemic stroke. We assessed alarge number of dependent variables to attempt to capture allpossible changes within the range of poststroke impairments.Of co urse, so me participants did not show decits in certaindomains prior to treatment; therefore we would not expect achan ge in those areas.2. Methods2.1. Participant. Seven subjects (4 females) were enrolled inthis study; 6 com pleted the study. Subjects eligible for thisstudy were male or female and any age between 18 and 80years who had suered an ischemic stroke at least 12-monthago (to minimize the chance for spon taneous recovery) andexhibited some functional impairments. Of the participants50% were 1 year aer stroke when they enrolled in thestudy and the other 50% were 2 yea rs aer stroke (Table 1).Astablebaseline(i.e.,noclinicallymeaningfulfunctionalimpr ovement over a 3-month period o n measures of cogni-tive or motor behavior ) was established for each participantbefore he or she could move forward in the study. Signicantimprovemen t was noted in some quality of life domains overthe 3-month baseline period however, these subjective patientreport domains were not enough to prevent moving forwardwith the study when there were no cognitive or physicalfunctional changes. Subjects were excluded from the study ifthey satised any of the following conditions: hydrocephalus,recurrent stroke, neurologic condition that aect motor orcognitive ability (i.e., Alzheimer’s disease, Parkinson’s disease,ALS, multiple sclerosis), history of seizures, were receivin gthrombolytics, COPD with CO2 retention, pneumothorax,bowel obstruction, sickle cell disease, cardiac arrhythmia,claustrophobia, active alcohol or dr ug abuse, current par-ticipation in physical, occupational, or speech therapy, orextremely severe cognitive impairment.2.2. Study Design. To carr y out this study we used a within-subject design in which each subject provided his or her ownbaseline (i.e., pretreatment) comparison. A stable baselinewas conrmed by the absence of functional recovery over theprevious 3-month s. Each subject was tested on a battery ofoutcome measures (cognitive, physical, speech, and quality oflife measures) twice during a 3-month period to determinethe baseline and 4 more times throughout the 9-monthstudy to assess the eect of HBOT. I f a steady baseline wasestablished (based on the variables measured), the subjectwas eligible to begin the rst round of HBOT treatment.HBOT consisted of 20 treatments of 100% O2 at 2.0 ATAfor 60 minutes each day M onday through Friday for a to talof 4 weeks. Aer this rst treatment period ended andfollowing four weeks without HBOT (labeled as O belowand in Figures 1–3), data were collected for the same outcomebattery. e second round of HBOT trea tment was identicalto the rst in all conditions. Following the second 4 weeksof HBOT outcome, data were collected again. Finally , follow-up testing on all experimental endpoints was completed foreach subject 3-months aer completion of the second roundof HBOT. A physician monitored each subject throughoutthe 9-month study dura tion to assess any health issues andpotential complications.2.3. Experimental Endpoints. e experimental endpointsfor this study included sp eech measures, neuropsychologicalmeasures, physical measures, quality of life measures, andphysiological biomarkers.2.3.1. Language Measures. To assess changes in aphasia,communication ability, language, and verbal uency theBoston Naming Test (BNT) and Reading ComprehensionBattery for Aphasia (RCBA) with latencies were used. ePorch Index of Communication Ability (PICA) scoring wasused for all tests, which provides a multidimen sional scoringsystem for communication ability that describes accuracy,responsiveness, completeness, promptness, and eciency ofeach response.2.3.2. Neuropsychological Measures. Cognitive and behav-ioral impairments (e.g., memory, att ention/concentration,verbal uency , and depression) were assessed using the Mini-Mental Status Exam (MMSE), California Verbal Learningtest (CVLT-II), Grooved Pegboard test (GP), Trails A and B,

Neurology Research International 3Table 1: Subject demographics.Subject ID Gender Age Time from Injury Ty pe of Stroke1 Male 79 2 years Ischemic R MCA2 Male 61 1year Ischemic R MCA3 Female 57 1year Ischemic R MCA5 Female 57 2years Ischemic R MCA6 Female 59 2years Ischemic R MCA7 Female 31 1year Ischemic R MCA01530456075Mean score (T score)California Verbal Learning Test: verbal memoryBaseline Treatment ∗(a)020406080California Verbal Learning Test: verbal memoryMean score (T score)BaselineBaselineTreatment 1OTreatment 2Follow up(b)Baseline03691215Mean score (scaled score)Wechsler Memory Scale: non-verbal memoryTreatment ∗(c)05101520Wechsler Memory Scale: non-verbal memoryMean score (scaled score)BaselineBaselineTre atment 1OTre atment 2Follow up(d)Figure 1: Eect of HBOT on memory: we observed a signicant eect of HBOT on verbal and nonverbal memory using the CVLT (a &b), which measures verbal memory, and the WMS (c & d), which measures nonverbal me mory. Graphs (a) and (c) represent the dierencebetween baseline and treatment and (b) and (d) show all individual data points.Controlled Oral Word Association test (COWAT), SemanticFluency (SF, animals), Wechsler Abbreviated Scale of Intelli-gence (WASI) block design, Wechsler Memory Scale (WMS)Visual reproduction, and Delis Kaplan Executive FunctionSystem (DKEFS).2.3.3. Physical Measures. Physical abilities including, gait,balance, and upper extremity function were assessed usingthe Upper Extremity Fugl My er (UEFM), Berg Balance test,and GaitRite computerized system. Gait velocity, step length,and step time were measured with the GaitRite system.

4 Neurology Research InternationalBaseline TreatmentUpper Extremity Fugl Meyer015304560Mean score∗(a)Upper Extremity Fugl Meyer020406080Mean scoreBaselineBaselineTre atment 1OTre atment 2Follow up(b)Baseline Tre atment Gait Velocity020406080100% Normalized Gait Velocity∗(c)Gait Velocity0255075100125% Normalized Gait VelocityBaselineBaselineTreatment 1OTreatment 2Follow up(d)Figure 2: Eect of HBOT on Gait and UE mobility: we observed signicant imp ro v ement in ph ysical abilities as measured with the UpperExtremity Fugl Meyer (UEFM, (a & b) and gait velocity (c & d). Graphs (a) and (c) represent the dierence between baseline and treatmentand (b) and (d) show all individual data points.2.3.4. Quality of Life and Stroke Recovery. Health status fol-lowing a stroke was assessed for this study using the StrokeImpact Scale, which measures 8 dierent domains: strength,hand function, ADL/IADL, mobility, communication, emo-tion, memory/thinking, and participation [41, 42]. In addi-tion to the Stroke Impact Scale, items from the NIH fundedPatient-Reported Outcomes measurement Information Sys-tem (PROMIS) were used to measure pain, fatigue, sleep, andsa tisfaction with participation in social roles and activities[43–45]. e B eck Depression Inventory-2 (BDI) was used toassess depression.2.3.5. Biomarkers. Potential biomarkers for treatment and re-covery were assessed using ELISA’s for astrogliosis (GFAP,Aplco Immunoassays, Salem, NH), astrocytic damage(S100𝛽,AplcoImmunoassays,Salem,NH),neuronaldam-age (Neuro n specic enolase, Aplco Immunoassays, Salem,NH), and neuroinammation (IL-6, TNF- 𝛼,R&DSystems,Minneapolis, MN). ese factors have been shown in animaland human studies to be regulated by neural injury andrecovery and measurable in plasma or serum [46–48]. esepotential biomarkers were chosen on the basis that pre-vious studies have observed an eect of HBOT on neuro-protection and inammation (for review see Background, or[6, 17, 20, 27, 28]).2.4. Data Analysis. First, we established the stable baselineby comparing Baseline 1 and Baseline 2. Only the SIS globaland sleep measures (part of Quality of Life) revealed reliableimpr ovement across the two baseline assessments. Next,Cohen’s d eect sizes were calculated for each measure bycombining the two baseline data points and the two treatmentdata points: Cohen’s d = (MTre a tme nt1,2-MBaseline1,2)/SD.Inaddition, the dependent means t test was reported for each

Neurology Research International 5Baseline Tre atment Sleep0204060Mean score (T score)∗(a)Sleep020406080Mean score (T score)BaselineBaselineTre atment 1OTre atment 2Follow up(b)Baseline Tre atment Stroke Impact Scale: Global Recovery020406080Mean score (0 - 100)∗(c)Stroke Impact Scale: Global Recovery020406080100Mean score (0 - 100)BaselineBaselineTreatment 1OTreatment 2Follow up(d)Figure 3: Eect of HBOT on Quality of Life: participants reported signicant improvement in sleep (a & b) and overall global recovery (c&d)followingHBOT.Graphs(a)and(c)representthedierencebetweenbaselineandtreatmentand(b)and(d)showallindividualdatapoints.eect size. All t tests were two-tailed and based on 5 degreesof freedom (df) except for gait velocity , which was basedon 4 df. In domains where the eect size was greater than0.5, which is typically considered a large eect, additionalcontrasts based on repeated measures ANOVA were usedto test patterns in the data. Consistent with the t tests,we compared (Baseline 1 and Baseline 2) with (Treatment1andTreatment2)todeterminewhetherimprovementisassociated with the intervention. Next, to determine thelongevity of treatment eects we compared (Treatment 1 andTreatment 2) with (O and Follow-Up), which represent 1month withou t treatment and 3 months without treatment,respectively. Finally, O and Follow-Up were contrastedagainst each other to examine fading of gains over time. Toassure that the eects observed are the result of the physio-logic intervention and not another factor such as a practiceeect, we calculated a linear trend score to reect steadyimprovement over Treatment 1 (contrast coecient, -3), O(coecient score, -1), Treatment 2 (contrast coecient, +1),and Follow-U p (contrast coecient, +3), as would be expect-ed due to practice. Perfect maintenance of gains wouldresult in a at function; con tin uing improvement even in theabsence of treatment would suggest a positive contrast score.We did not assess the linear trend over the entire study be-cause that set of contrast coecients correla t ed too highlywith a pure trea t ment eect. Further, we did not include thebiomarkers in this analysis because previous analyses hadalready demonstrated that practice could not account forthose pat terns. All analysis was com p leted with JMP soware.3. ResultsTo examine the eect of HBOT following an ischemic strokewe utilized a within design where each subject provided his

6 Neurology Research Internationalor her own baseline. A total of 7 subjects were enrolled inthis study. One pa tient withdrew due to ear pain associatedwith the HBOT, but this was the only adverse event notedduring this study. One participant did not return for the 3-month follow-up visit. All subjects experienced an ischemicstroke in the right hemisphere of the brain (Table 1). Of theremaining 6 participan ts, 4 were female and 2 were male withages ranging from 31 years to 79 years.3.1. Language Domains. To investigate the potential eect ofHBOT on aphasia, communication ability, language, andverbal uency the RCBA and BNT were used. We did notidentify signicant decits at baseline in these speech and lan-guage domains; therefore, as we would not exp ec t dierenceswhen comparing treatment to baseline no further analysiswas completed.3.2. Neuropsychological Doma ins. An extensive neuropsy-chological battery was used to measure changes in cognitiveimpairments (e.g., memory, attention/concentration, andverbal uency). U sing the MMSE we did not observe impair-men ts in general cognition for any participant (a verage scorebefore treatment = 28.6 out of 30 possible points). erefore,we did not complete further analysis on the MMSE. Overall,mild to moderate impairments were observed in the variouscognitive domains measured. e baseline was stable for allthe cognitive assessments. We observed a signicant eect oftreatment on verbal and nonverbal memory. Specically, weobserved a signicant eect of HBOT on the CVLT, whichmeasures verbal memory, and the WMS, which measuresnonverbal memory (Figure 1, Tables 2 and 3). We conrmedthe treatment eect as compared with the pretreatmentbaseline and were able to determine that this eect was main-tained through the 3-month follow-up (Table 3). A lineartrend score of 3.2 (t=0.13) was identied for the CVLT and -1.4(t=-0.17) for the WMS suggesting no practice eect. Althoughthe eect size of 0.8 suggests a potential eect on processingspeed and executive function, as measured with Trails A a ndthe DKEFS, we did not observe a signican t treatment eectin these domains or on language or visuospatial abilities inthese participants (Tables 2 and 3).3.3. Physical Funct ion. Gait, balance, and upper extremitymovement were compared before and aer HBOT treatment.We observed signicant improvement in physical abilities asmeasured with the Upper Extremity Fugl Meyer (UEFM) andgait velocity. Gait velocity, as reported by the %-normalizedto the general population increased nearly 20%, this eectwas maintained at follow-up (Figure 2, Tables 2 and 3).Other aspects of gait were measured such as step length andcycle time but we only observed signicant changes w henexamining gait velocity. We also measured balance using theBerg Balance scale but did not observe any changes at anyof the testing points throughout the study. e linear trendscores for all the physical domains were all near zero againsuggesting little to no practice eect (t = -0.133 - .33).3.4. Quality of Life. Using the PROMIS QOL measure, par-ticipants reported signicant improvement in sleep followingHBOT and at the 3-m onth follow-up (Figure 3, Tables 2 and3). Signicant improvement in ov erall recovery as measuredwith the SIS was also reported following HBOT and at 3months following treatment ( F igure 3, Tables 2 and 3). Alinear trend score of -11 (t= -1.1) was identied for sleep whilea mean linear score of 41 (t=1.8) was noted for global recovery.In all but 1 patient the linear contrast for global recovery waspositive suggesting this eect due to more than the treatment.While we observed a decrease in depression levels this eectdid not reach signicance. No other signicant dierenceswere noted as a result of treatment on individual domainsrelated to QOL or recovery using the PROMIS or SIS, suchas hand function, satisfaction, or activities of daily living.3.5. Biomarkers. To examine physiological biomarkers fortreatment and recovery neural, glial, and inammatory mark-ers were measured. e strengths of these relationships aredisplayed with the eect size, which range from .69 to 1.5.Asignicanttreatmenteectwasobservedfor3ofthephysiological biomarkers measured, NSE, TNF-alpha, andIL-6 (Tables 2 and 3). While these biomarker values appearedto be returning to baseline levels at both the 1 month o and3-month follow-up, a signicant eect was only observed forNSE and TNF-a (Table 3).4. Discu ssione purpose of this study was to investigate the role ofHBOT as a therapeutic intervention for stroke patients. Astroke may result in a variety of functional decits includingphysical, cognitiv e, and behavio ral impairments. Using awithin-subject design, we measured the impact of HBOTacross a num ber of functional doma ins including speech,lan guag e, cognition, physical function, emotional / behaviorimpairments, and quality of life. In this preliminary study, ourapproach was to identify eects that were strong enough toemerge with a very small sample and, then, to examine thena ture of those eects. For example, whether or ho w quicklythey fade and whether they can be at tributed to practice.is a pproach is likely to underestimate the potential forhyperbaric treatment due to the low statistical power foriden tifying eects strong enough for examination. However,the consistency of impro vement noted over repeated assess-men ts spread out over months argues against any accountbased on statistical uke. Signicant improvements followingHBOT were observed with cognition (including, memory,and processing speed), gait velocity, upper extremity mobility,sleep, and overall recovery, as measured wi th the SIS. esetreatment eects were maintained when examined at 3months following treatment with the potential exception ofthe UEFM. We also observed a signicant change in neuraland inammatory biomarker expression levels in responseto HBOT. e pattern observed for the biomarkers wasdierent than all the functional measures suggesting transientphysiological responses but sustained functional chan ge.Although we observed signicant improvements in cog-nition and gait velocity, there are limitations to in t erpreta tion.For example, while there was an increase in gait velocityfrom baseline to treatment other gait kinematics such as

Neurology Research International 7Table 2: Ee ct of HBOT on func tional impairments: means and eect size.DomainBaseline(mean+SD)HBOT(mean+ SD)Paired t testB1,2 vs T1,2Eect SizeCognitiveCVLT-II49 .6 +10.4 59 + 11.9 0.01∗1.56GP39 .4 +9.5 44.5 + 15.5 0.130.38Trails A34.1 +8.6 39 .2 + 7. 4 0.100.79Trails B37.9 +12.6 40.8 + 8.4 0.510.28COWAT38.1 +8.3 41.7 + 9.6 0.090.81SF - animals42.9 +10.8 44.1 + 15.2 0.760.13WASI44.1 +12.5 44.6 + 9.5 0.780.11WMS10.4 +0.9 12.2 + 2.1 0.03∗1.11DKEFS9.7 +2.9 11.1 + 1.6 0.110.76PhysicalUEFM37.7 +23.1 42.0 + 22.2 0.03∗0.85Berg48.7 +9.7 50.7 + 9.8 0.260.5Gait Velocity68.7 +8.7 83.5 + 8.4 0.01∗1.5Step length3.79 +1.5 2.36 + 1.9 0.33-0.39Step time0.05 +0.06 0.04+ 0.02 0.36-0.36Quality of LifeBDI14.5 +10.8 10.0 + 7. 7 0.09-0.82SIS Global52.1 +26.5 61.7 +18.4 0.04∗0.73SIS Strength49 .0 +13.6 49.5 + 13.5 0.750.04SIS Memory85.9+17.7 90.9 + 12.8 0.590.34SIS Emotional74.4 +15.8 82.6 + 13.8 0.140.54Communication84.8 +20.1 86.8 + 12.4 0.650.28SIS ADL’s72.3 +19 .3 72.5 + 19.9 0.950.02SIS mobility71.2 +10.9 78.3 + 15.8 0.320.38Hand Function58.9 +32.5 60.6 + 31.8 0.570.23Participation50.6 +13.8 64.6 + 30.1 0.110.54Physical Comp.56.0 +25.8 61.1 + 24.4 0.090.81Sleep41.2 +7. 7 48.5 + 9.8 0.04∗1.17Satisfaction46.2 +6.1 53.8 + 12.2 0.160.68BiomarkersNSE2.2 +0.4 2.9 + 0.5 0.005∗2.1GFAP10.1 +18.9 17.3 + 27.5 0.130.74IL-66.3 +6.8 3.9 + 4.9 0.05∗-1.0TNF-a7. 0 +2.0 4.7 + 1.4 0.01∗-1.5stride length and step length which compa re symmetry ofthe le to right side did not show signicant dierences; theparticipants were just walking at a faster speed maintainingtheir biomechanical decits. Furthermore, there were nosignicant dierences on the Berg Balance test, which mayindicate the increases in gait velocity may not be due toimprovements of motor con t rol of the paretic limb. Ingeneral the testers suggested that participants might havebeen trying to perform better at each testing interval, as theywere not blinded to treatment. However, there is evidencesuggesting a minimal practice eect with the CVLT andWMS [49, 50]. Further, a linear trend score was calculatedfor each signicant eect to dierentiate between a treatmenteect and other confounding variables such as a practiceeect. Continuing improvement even in the absence oftreatment was signied by a positi ve contrast score, whichwe only observed for global recovery. erefore, the statisticalimprov ement in quality of life demonstrated by the SIS mayhave been in part due to the treatment but may also havebeen an eect of increased attention paid by clinical sta oranother indirect result of participation in this study . Socialisolation is very common with a long-term disability alongwith signicant loss of self-worth, income, indep endence,and many other domains, thus a sustained increase in global

8 Neurology Research InternationalTable 3: Eec t of HBOT on fun ct iona l impair ment s: repeat ed measures ANOVA.Domain B1 versus B2 B1,2 vs T1,2 T1,2 vs O and Follow-up O vs Follow-upCognitiveCVLT-II F(1,5)=0.13, p=0.74 F(1,5)=15.7, p=0.01 F(1,4)=0.13, p=0.74 F(1,4)=2.78, p=0.19Trails A F(1,5)=0.01, p=0.77 F(1,5)=3.9, p=0.10 F(1,4)=1.27, p=0.34 F(1,4)=2.95, p=0.18COWAT F(1,5)=2.59, p=0.17 F(1,5)=4.0, p=0.09 F(1,4)=0.31, p=0.61 F(1,4)=0.003, p=0.96WMS F(1,5)=0.29, p=0.61 F(1,5)=7.74, p=0.03 F(1,4)=1.0, p=0.39 F(1,4)=1.0, p=0.39DKEFS F(1,5)=0.11, p=0.78 F(1,5)=3.6, p=0.11 F(1,4)=0.07, p=0.81 F(1,4)=0.11, p=0.76PhysicalUEFM F(1,5)=0.65, p=0.46 F(1,5)=9.6, p=0.03 F(1,4)=6.5, p=0.06 F(1,4)=0.24, p=0.64Gait Velocity F(1,4)=3.7, p=0.12 F(1,4)=24.9, p=0.01 F(1,3)=0.13, p=0.75 F(1,3)=0.25, p=0.65Quality of LifeBDI F(1,5)=0.16, p=0.71 F(1,5)=6.7, p=0.09 F(1,4)=1.4, p=0.32 F(1,4)=0.66, p=0.48SIS Global F(1,5)=8.4, p=0.03 F(1,5)=7.2, p=0.04 F(1,4)=2.0, p=0.23 F(1,4)=1.67, p=0.27SIS Emotional F(1,5)=0.56, p=0.28 F(1,5)=3.0, p=0.14 F(1,4)=0.83, p=0.41 F(1,4)=0.51, p=0.51SIS Participation F(1,5)=0.005, p=0.9 F(1,5)=3.7, p=0.11 F(1,4)=0.56, p=0.50 F(1,4)=0.08, p=0.80SIS Physical F(1,5)=2.8, p=0.17 F(1,5)=4.2, p=0.09 F(1,4)=0.96, p=0.38 F(1,4)=1.0, p=0.36Sleep F(1,5)=6.5, p=0.06 F(1,5)=8.2, p=0.04 F(1,4)=1.5, p=0.28 F(1,4)=0.001, p=0.97Satisfaction F(1,5)=0.06, p=0.82 F(1,5)=2.7, p=0.16 F(1,4)=6.8, p=0.06 F(1,4)=0.11, p=0.75BiomarkersNSE F(1,5)=0.01, p=0.85 F(1,5)=22.8, p=0.005 F(1,4)=16.0, p=0.01 F(1,4)=0.24, p=0.64GFAP F(1,5)=0.06, p=0.81 F(1,5)=3.27, p=0.13 F(1,4)=2.97, p=0.16 F(1,4)=0.13, p=0.74IL-6 F(1,5)=0.02, p=0.89 F(1,5)=6.0, p=0.05 F(1,4)=5.3, p=0.08 F(1,4)=1.1, p=0.36TNF-a F(1,5)=0.04, p=0.85 F(1,5)=14.2, p=0.013 F(1,4)=18.4, p=0.01 F(1,4)=0.34, p=0.59improv ement, participation, and emotional wellbeing are ofparticular note despite the cause. Changes in the physiologi-cal biomarkers suggest measureable dierences are occurringfollowing HBOT however in this current study we are unableto clearly dene the link between these biomark ers andfunctional changes. However, one remarkable feature of theseresults is that the biomarker measures are so sensitive tothe presence and absence of hyperbaric treatment, even forpatients in the chronic stages of their illness.e use of HBO as a treatment following stroke was rstraised 40–50 years ago [32, 51]. Despite decades of interestin HBOT previous studies investigating the eects of HBOTfollowing a stroke have produced mixed results [10, 11, 18,19, 32, 40, 52–60]. ere are a number of variables thatmay account for the incongruous literature including severalrelated to the study design including the treatment protocol,study population, inclusion of and type of contro l group, out-comes measured, and timing of treatment. An update from a2014 Cochrane review reported that when taken together theexisting literature does not nd HBOT a n eective interven-tion in the acute phase following an ischemic stroke [2, 10].However, statistically signicant improvement was noted infunctional outcomes in some of the studies [10]. Our ndingsfall in line with the literature as we did observe signicanteects on some domains but there were also a number ofareas where no functional change was observed. Consistentwith our obser vation that HBOT improves memory is arecent retrospective study tha t also observed signicantimpr ovement in verbal and no nverbal memory [39]. Despitedierences in the time from stroke onset and treatmentprotocol as well as dierences in the measures for v erbal andnonverbal memory, the percent change, around 20%, wassimilar between both studies. Using single-photon emissioncomputed tomography (SPECT) they posit that their memorychanges correla te with metabolic changes in the brain [39].Arecentrandomizedcontrolledtrialwithpatients1yearaer stroke also used SPECT to begin to elucidate potentialmechanisms of action of HBOT [40]. Specically, the authorsreported changes in brain metabolism observed with SPECTthat correlated with quality of life, the NIHSS, and activities ofdaily living [40]. Comparably, we observed changes in overallglobal recovery and some aspects related to quality of life,such as sleep in our similar study population with participantsthat were on average 1.5 years aer stroke. We also endeavoredto look at mechanism by looking at blood biomarkers forneural activity (GFAP, NSE) and inammation (TNF-a, IL-6). We found transient changes in the expression levels ofthese markers suggesting the potential role of the oxygenin mo dulating neural and inammatory signaling cascades,which may lead to the sustained functional changes weobserved. Animal models hav e outlined multiple possibilitiesfor the role of HBOT on antia poptotic and anti-ina mmatorysignaling pathways including Nogo-A, bcl-2 for plasticityand TNF, IL-1, IL-6, and COX-2 for inammation [15, 17,30, 61–73]. However, with blood based markers that arefound throughout the body we are unable to make denitivesta tements about mechanism at this time.Adding to the studies investigating the eects of HBOTfollo wing stroke, with their mixed results, there has been astrong recent interest in the eectiveness of HBOT following

Neurology Research International 9aTBI,duetotheincreaseinbraininjuriessustainedduringrecent military combat conicts. e Department of Defenseis implementing many facilities whose purpose is to usehyperbaric oxygen therapy to help veterans recover from TBI.AnumberofcasestudieshavesupportedtheuseofHBOTfollo wing a TBI, suggesting benecial eects even y ears aerinjury [12]. In a large single center double blind randomizedsham con tro lled prospective trial at the US Air Force Schoolof Aerospace medicine the eects of 2.4 a tmospheres of HBOwas assessed in 50 military service members [74]. e controlgroup received 1.3 ATA HB O. While some measures wereimprov ed following HBOT, they were improved in both the2.4 and 1.3 ATA groups, which has resulted in discussionregarding the appropriateness of 1.3 ATA as a control or rathershould it be seen as a low dose of HBOT [12]. Interestingly,in a study of 60 service members at the Naval medicineoperational training center in Naval Air station Pensacola nodierences were found when comparing oxygen at 1.5 and2.0 ATA for individuals with mild TBI [75]. In our study weused 2.0 ATA and found similar results on cognition andaspects of recovery and QOL aer a strok e as these studieswith did with varying doses of HBOT following TBI. Forexample, 1.5 ATA of oxygen was used for individuals withmild TBI and signican t changes were noted in cognition andQOL [76]. However this study, similar to ours, did not have acontrol group that was blinded to condition. e limitationsregarding dierent types of controls groups contin ue to makeit challenging to clearly ascertain the role of HBOT followingneurologic injury.Other confounding variables including the type andsensitivity of outcome measures or domains assessed, and thetiming of HBOT may all play a role in the incongruous andinconsistent ndings in the literat ure. Due to the nature ofischemic injury one may conclude that HBOT would be mosteective during acute injury when neuron s can be rescuedin addition to modulating plasticity and synaptic changesin new or existing neurons to compensate. Due to normalrecovery during the initial 6–12 mon ths following injury it isdicult to assign responsibility to one inter vention. Despitethe preponderance of evidence for an acute timeframe, Boussiet al. suggest neuroplasticity is possible in patients as faras 5 years aer trauma tic brain injury [76]. Based on thechanges we observe in biomarker expression levels, we alsosupport the idea that neural chan g es are occurring yearsfollowing an in j ury, as well as inammato ry changes, whichmay lead to downstream signaling cascades [76]. Still, we areunable to denitely link changes in physiological biomarkersto functional chan ges, which may be more evident if HBOTis used acutely similar to pharmacologic interventions thatcomplement to the rehabilitative process.We appreciate that there are several limitations to thedesign of this study, some of which have been discussedabove. While the within-subject design eliminates some ofthese issues previously discussed with RCT, the small samplesize and lack of blinding a nd controls limit the generalizabil-ity of the results. Prior research has demonstrated the sig-nicant subject and observer/researcher bias inherent in thistype of research, specically with HBOT, and thus interpreta-tion of the results its overall contribu tion to the scien tic arenarrow. P otentially the grea test limitation is only includingsubjects who have experienced a plateau in their recov ery.is time frame places our study at risk of missing a criticaltherapeutic window to rescue cells before they are no longerviable, suggestin g tha t the eects we hope to observe fromHBOT treatment would be due to other mechanisms, i.e., notclassical neuroprotection pathways. However, this time frameis necessary for this study design as th e baseline must be stableto compare treatment eects. is is where other observa-tional studies and even controlled clinical trials have fallenshort and why the results from those studies are ultimatelyineectual. Ho wever, despite the limitation s that can befound in most experimental design, the growing body ofliterature prov ides new and reliable data helping us to betterunderstand the eects of HBOT on impairments resultingfrom ischemic strokes.5. Summaryis stud y investigated the impact of HBOT as a therapeuticintervention following stroke across a number of functionaldomains including speech, language, cognition, physicalfunction, and quality of life. We found a benecial eect ofHBOT on memory, processing speed, gait velocity, upperextremity mobility, sleep, and overall recovery. We alsoobserved signicant transient changes in neural and inam-matory biomark ers in response to HBOT that may result inthe sustained functional changes that were observed. Despi tethese encouraging results further research is needed to moreclearly dene the mechanism and potential role of HBOTfollowing stroke.Data Availabilitye data from this study is not an archived dataset. It was asmall case series study.Disclosureis wo rk was presented at the American Congress forRehabilitative Medicine and thus the abstract has beenpreviously published in the Archives of Physical Medicineand Rehabilitation.Conflicts of Intereste authors declare that they have no conicts of interest.Acknowledgmentsis research was funded solely by the Casa C olina Foun-dation. e authors would like to thank Dr. Loverso, theCasa Colina Board of Directors, and the Casa Colina Foun-dation for supporting this research. We would also like tothank Kerry Gott, MD , Laura Seibert, PhD; Cindy Sendor,MA,CCC-SLP, Jose Fuentes, PhD, Felice Loverso, PhD, Lau-ren Meeks, BS., Elizabeth Cisneros, PhD , Adeel Po palzai, DO,

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Improvement of Memory Impairments in Poststroke Patients byHyperbaric Oxygen TherapyRahav Boussi-Gross, Haim Golan, Olga Volkov,and Yair BechorAssaf Harofeh Medical Center, Zerifin, IsraelDan HoofienHebrew University of JerusalemMichal Schnaider BeeriIcahn School of Medicine at Mount SinaiEshel Ben-JacobTel Aviv University and Rice UniversityShai EfratiAssaf Harofeh Medical Center, Zerifin, Israel and Tel-Aviv UniversityObjective: Several recent studies have shown that hyperbaric oxygen (HBO2) therapy carry cognitive andmotor therapeutic effects for patients with acquired brain injuries. The goal of this study was to addressthe specific effects of HBO2on memory impairments after stroke at late chronic stages. Method: Aretrospective analysis was conducted on data of 91 stroke patients 18 years or older (mean age !60 years)who had either ischemic or hemorrhagic stroke 3–180 months before HBO2therapy (M " 30 –35months). The HBO2protocol included 40 to 60 daily sessions, 5 days per week, 90 min each, 100%oxygen at 2ATA, and memory tests were administered before and after HBO2therapy using NeuroTrax’scomputerized testing battery. Assessments were based on verbal or nonverbal, immediate or delayedmemory measures. The cognitive tests were compared with changes in the brain metabolic state measuredby single-photon emission computed tomography. Results: Results revealed statistically significantimprovements (p # .0005, effect sizes medium to large) in all memory measures after HBO2treatments.The clinical improvements were well correlated with improvement in brain metabolism, mainly intemporal areas. Conclusions: Although further research is needed, the results illustrate the potential ofHBO2for improving memory impairments in poststroke patients, even years after the acute event.Keywords: hyperbaric oxygen, memory impairment, strokeSupplemental materials: http://dx.doi.org/10.1037/neu0000149.suppStroke is a major cause of adult disability and mortality, giving riseto severe, long-term impairments in the physical, emotional, andcognitive state of the survivors (Zhang, Chapman, Plested, Jackson, &Purroy, 2012). Cognitive impairments after stroke are very common(Edwards, Jacova, Sepehry, Pratt, & Benavente, 2013; Tatemichi etal., 1994); the overall prevalence stands on 22%, 3 months after onset,with no further improvement in most cases (Douiri, Rudd, & Wolfe,2013). Cognitive impairments commonly involve memory deficitsthat lead to a decline in everyday functioning and in social function-ing, life satisfaction and quality of life of patients and caregivers(Baumann, Couffignal, Le Bihan, & Chau, 2012; Hochstenbach,Mulder, van Limbeek, Donders, & Schoonderwaldt, 1998; Snaphaan&deLeeuw,2007;Tatemichietal.,1994).Therapyandrehabilitationprograms are valuable for improving memory deficits early after braininjury, but they usually provide only partial relief (Cicerone et al.,2005; Fish, Manly, Emslie, Evans, & Wilson, 2008; Jennett & Lin-Rahav Boussi-Gross, The Institute of Hyperbaric Medicine, AssafHarofeh Medical Center, Zerifin, Israel; Haim Golan and Olga Volkov,Nuclear Medicine Institute, Assaf Harofeh Medical Center; Yair Bechor,The Institute of Hyperbaric Medicine, Assaf Harofeh Medical Center; DanHoofien, Department of Psychology, Hebrew University of Jerusalem;Michal Schnaider Beeri, Department of Psychiatry, the Icahn Schoolof Medicine at Mount Sinai, The Joseph Sagol Neuroscience Center, ShebaMedical Center, Tel-Hashomer, Israel; Eshel Ben-Jacob, School of Physicsand Astronomy, The Raymond and Beverly Sackler Faculty of ExactSciences, Sagol School of Neuroscience, Tel-Aviv University and Centerfor Theoretical Biological Physics, Rice University; Shai Efrati, The In-stitute of Hyperbaric Medicine, Research and Development Unit, AssafHarofeh Medical Center, Sackler School of Medicine and, Sagol School ofNeuroscience, Tel-Aviv University.The study was supported by a grant from the research fund of AssafHarofeh Medical Center, Israel. Eshel Ben-Jacob was supported by agrant from the Tauber Family Funds and the Maguy-Glass Chair inPhysics of Complex Systems. We thank NeuroTrax corporation for theuse of their software, Dr. Glen Doniger in particular for his great helpin administering the software’s data and results. The authors expressspecial thanks to Michal Ben-Jacob for her significant help in editingthe manuscript.Correspondence concerning this article should be addressed to ShaiEfrati, The Institute of Hyperbaric Medicine, Assaf Harofeh MedicalCenter, Zerifin, Israel. E-mail: efratishai@013.netThis document is copyrighted by the American Psychological Association or one of its allied publishers.This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.Neuropsychology © 2014 American Psychological Association2014, Vol. 28, No. 6, 000 0894-4105/14/$12.00 http://dx.doi.org/10.1037/neu00001491

coln, 1991; Nair & Lincoln, 2007; Rohling, Faust, Beverly, & De-makis, 2009). There are currently no efficient memory rehabilitationprograms available for late chronic stages.Hyperbaric oxygen (HBO2)treatmenthasbeenrecentlyshowntoinduce neuroplasticity in brain injured patients, even at chronic stages(Boussi-Gross et al., 2013; Efrati & Ben-Jacob, 2014a, 2014b; Efratiet al., 2013). HBO2is the inhalation of 100% oxygen at pressuresexceeding 1 atm absolute (ATA) to enhance the amount of oxygendissolved in the blood and body fluids. As is explained in detail by(Efrati & Ben-Jacob, 2014b), the diverse and powerful innate repairmechanisms activated by HBO2are associated both with the elevatedlevel of dissolved oxygen and the elevated pressure.There is previous evidence for cognitive improvements after HBO2in different neurological conditions (Barrett et al., 1998; Boussi-Grosset al., 2013; Hardy et al., 2002; Jacobs, Winter, Alvis, & Small, 1969;Rossignol, Rossignol, James, Melnyk, & Mumper, 2007; Tapeantong&Poungvarin,2009).However,thereisanongoingdebateregardingthe efficacy of HBO2in victims of traumatic brain injury (TBI) andstroke. Although it is widely agreed, and confirmed by almost all thestudies, that HBO2treatments lead to significant improvements, thedebate is related, mostly to the control group issue and the minimaleffective dosages (the minimal pressure that does not have any phys-iological effect on the central nervous system [CNS]) (Churchill et al.,2013; Efrati & Ben-Jacob, 2014a; Golden, Golden, & Neubauer,2006; Harch et al., 2012; Mukherjee et al., 2014; Wolf, Cifu, Baugh,Carne, & Profenna, 2012). For detailed assessment of the debate see(Efrati & Ben-Jacob, 2014a, 2014b). Recently, in a prospective ran-domized trial with mild TBI patients, it was found that HBO2couldsignificantly improve cognitive impairments and metabolic brain def-icits even at chronic stages of the injury (Boussi-Gross et al., 2013).In addition, in another study with poststroke patients, it was reportedthat HBO2could lead to significant motor function improvements alsoat the late chronic stage (Efrati et al., 2013). These findings imply thatHBO2might induce neuroplasticity processes in the injured brain inregions where anatomical or metabolic mismatch is detected usingcomputed tomography (CT) or single-photon emission computedtomography (SPECT). In light of these findings, we decided toinvestigate whether HBO2had beneficial effects on impaired memoryfunction in poststroke patients. The idea was to perform retrospectiveanalysis of the specific effects of HBO2on memory impairments inpoststroke patients through its effect on metabolic dysfunction inbrain regions involved in memory processes. The retrospective anal-ysis was performed on previously chronically disabled poststrokepatients who had received HBO2at the hyperbaric unit of AssafHarofeh Medical Center. Notably, to reveal the metabolic changesresponsible for the memory functions we compared the changes in thememory functions with changes in the brain metabolic state as in-ferred by SPECT brain imaging data.MethodParticipantsThis study is a retrospective analysis of changes in memoryfunctions and their related brain activity of 91 stroke patients afterhyperbaric oxygen therapy. Patients were treated at the hyperbaricunit of Assaf Harofeh Medical Center, Israel, between the years2008 –2012. The study was approved by the local ethics commit-tee. Inclusion criteria: Participants who have completed at least 40sessions of HBO2, and two cognitive evaluations, one before andone after HBO2treatments.Materials and ProcedureHyperbaric oxygen treatment. The following HBO2proto-col was practiced: 40 –60 daily sessions, 5 days per week, 90 mineach, 100% oxygen at 2ATA. The difference in treatments number(40 or 60) were because of the fact that some of the patients hadparticipated in a prospective randomized controlled study on phys-ical and quality of life improvement in stroke patients after HBO2(Efrati et al., 2013) and had 40 treatment sessions according to thatstudy’s protocol. The rest of the patients had 60 treatment sessionsbecause of later considerations regarding the length and benefit ofthe treatments, based on our prolonged experience with thosepatients and the treatment outcome.Memory evaluation. All patients underwent baseline andposttreatment evaluations 1–3 weeks before and after HBO2.Memory was assessed using memory test scores of NeuroTraxcomputerized cognitive testing (previously known as “Mind-Streams”) of NeuroTrax Corp., Houston, TX. A detailed descrip-tion of the tests in the battery can be found on NeuroTrax Web site(www.neurotrax.com) and in Doniger (2014b). NeuroTrax testingbattery has been previously used in several studies of poststrokepopulation (Kliper et al., 2013; Shopin et al., 2013; Weinstein,Goldbourt, & Tanne, 2013), including the Tel Aviv Brain AcuteStroke Cohort (TABASCO) study, which is an ongoing, prospec-tive cohort study with !1,125 consecutive stroke patients designedto evaluate the association between predefined demographic, psy-chological, inflammatory, biochemical, neuroimaging, and geneticmarkers, measured during the acute phase, and the long-termoutcome (Ben Assayag et al., 2012).Although there are several cognitive tests in the battery, thechanges in memory functions were evaluated by analysis of thememory tests scores of the following five memory measures:Immediate verbal memory (IVM): Ten pairs of words werepresented, followed by a recognition test in which the firstword of a previously presented pair appeared together with alist of four words from which the patient chose the othermember of the pair. There were four immediate repetitionsand a total score of all four was calculated.Delayed verbal memory (DVM): Delayed repetition of thesame 10 previously learned pairs after 10–15 min.Immediate nonverbal memory (INVM): Eight pictures ofsimple geometric objects were presented, followed by a rec-ognition test in which four versions of each object werepresented, each oriented in a different direction. There werefour immediate repetitions, and a total score of all four wascalculated.Delayed nonverbal memory (DNVM): Delayed repetitionof the previously learned eight figures after 10 –15 min.Total memory index (TMI): Calculated mean score of allfour scores described above.This document is copyrighted by the American Psychological Association or one of its allied publishers.This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.2BOUSSI-GROSS ET AL.

Construct validity of the memory tests was reported in severalstudies (Abramovitch, Dar, Hermesh, & Schweiger, 2012; Doniger& Simon, 2014; Doniger et al., 2006; Dwolatzky et al., 2003;Elstein et al., 2005); for example, verbal memory tests were foundto well correlate with several familiar paper-and-pencil verbalmemory tests, such as logical memory subtest in Wechsler Mem-ory Scale-Third Edition (WMS-III), California verbal learning test(CVLT), Hopkins verbal learning test (HVLT), and others (rs "0.59 –0.73) (Doniger & Simon, 2014). The nonverbal memory hadstrong psychometric properties, including good reliability (Cron-bach’s alpha " 0.65– 0.71) and was found to correlate with severalsubtests of the WMS-III, the Rey Auditory Verbal Learning Test,and the Rey Osterreith Complex Figure Test in cohorts of clinicaland healthy participants (rs " 0.52– 0.77) (Abramovitch et al.,2012; Doniger & Simon, 2014).After administration, NeuroTrax data was automatically andblindly uploaded to the NeuroTrax central server, and outcomeparameters were calculated using custom software blind to diag-nosis or testing site. To account for the well-known effects of ageand education on cognitive performance, each outcome parameterwas normalized and fit to an IQ-like scale (M " 100, SD " 15)according to the patient’s age and education. Normative data,provided by NeuroTrax, consisted of test data of cognitivelyhealthy individuals in controlled research studies at more than 10clinical sites (Doniger, 2014a).Notably, the patients were given two different tests versions ofthe NeuroTrax test battery before and after HBO2therapy, to allowrepeated administrations with minimal learning effect. Test–retestreliability for those versions was evaluated and found high, with nosignificant learning effect (Dwolatzky et al., 2003; Melton, 2005).Assessment of brain activity. Brain activity was assessedusing SPECT 1–3 weeks before and after HBO2therapy. Theimaging was conducted using 925–1,110 MBq (25–30 mCi) oftechnetium-99methyl-cysteinate-dimmer (Tc-99m-ECD) at 40 – 60min postinjection. A dual detector $ camera (ECAM or Symbia T,Siemens Medical Systems, Malvern, PA) equipped with highresolution collimators was used and the data was acquired inthree-degree steps and reconstructed iteratively with Changmethod (%"0.12/cm) attenuation correction (Jaszczak, Chang,Stein, & Moore, 1979). Pre- and post-HBO2treatments werecompared. Brain perfusion analysis was performed first by fusingpre- and posttreatment SPECT studies to pretreatment brain CT.Both SPECT studies were normalized to maximum brain activityin the entire brain. SPECT images were then reoriented intoTalairach space using NeuroGam (Segami Corporation) to identifyBrodmann cortical areas and to compute the mean perfusion ineach Brodmann area (BA). In addition, volume rendered brainperfusion images were reconstructed and normalized to entirebrain maximal activity. All SPECT analyses were done by studyteam members who were blinded to the laboratory and clinicaldata. SPECT scans were performed at late-morning to mid-day. Onthe day of the SPECT scan, patients were treated only with theirchronic medications and were instructed not to smoke.Change in perfusion in all BA for each subject was determinedby calculating the percentage of difference between postperiod andpreperiod divided by the preperiod perfusion. An average of theseperfusion changes for each BA was calculated.Statistical AnalysisThe memory tests data was statistically analyzed using SPSS soft-ware (version 16.0). Continuous data was expressed as means & SDsor SEs.Preliminary analysis. Analysis of variance (ANOVA) wasused to evaluate the effect of HBO2therapy on patients’ memoryperformance. The dependant variables were scores in the differentmemory measures, the independent variables were time of testing(before or after HBO2) and group. Because most of the initialcomplaints were regarding motor and physical rather than cogni-tive disabilities, we subdivided the patients into two groups ac-cording to their baseline memory scores (low\high): “low baselinescore” (LBS) (baseline score #85, i.e., 1 SD below average), and“high baseline score” (HBS) (baseline score '85). Main effects(time) and interactions (Time ( Group) were examined and effectsizes were calculated using )2.Main analysis. Paired t tests were used for intragroup com-parisons. Effect sizes for main comparisons were calculated usingCohen’s d measure. The percentages of relative changes aftertreatment were also calculated for each memory measure, for theLBS groups only (see results of preliminary analysis), by subtract-ing the pretreatment score from the posttreatment score, and di-viding by the pretreatment score.In addition, Jacobson and Truax’s analysis was performed todetermine the clinical significance of the results (Jacobson &Truax, 1991). This is a relative common method used for calcu-lating clinical significance by establishing cutoff scores forparticipants’ classification into one of the four categories: re-covered, improved, unchanged, or deteriorated. Reliable changeindex (RCI) was calculated to assess clinical change signifi-cance i n the LBS groups. RCI enables to determine whether themagnitude of change from baseline to posttreatment (either posi-tive or negative) is significantly larger than arbitrary changesbecause of the instrument measurement error (Jacobson & Truax,1991). An RCI larger than &1.96 would be unlikely to occurwithout actual change (p # .05). Symbols associated with thederivation of RCI scores are presented in Table 1. The formulaTable 1Symbols and Explanations of the Jacobson-Truax’s ClinicalChange AnalysisSymbol ExplanationX1 Patient’s baseline score in one of the memory measuresX2 Patient’s posttreatment score in one of the memory measuresS1 SD of patients’ baseline scores in one of the memorymeasuresM1 Mean of patients’ baseline scores in one of the memorymeasuresS0 SD of normal population scores in one of the memorymeasures ("15)M0 Mean of normal population scores in one of the memorymeasures ("100)rxxTest–retest reliability coefficient for NeuroTrax memoryindexSE The SE of measurement for memory measuresSdiff The SE of difference for memory measuresThis document is copyrighted by the American Psychological Association or one of its allied publishers.This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.3IMPROVEMENT OF MEMORY IMPAIRMENTS IN POSTSTROKE

used in RCI calculations is RC !X2"X1Sdif f, where Sdiffis the SE ofthe differences, defined as the spread of distribution of changesbecause of arbitrary time variability of the score value, and is equalto!2"SE#2. SE is the standard error of measurement and is foundby S1!1"rxx. The value of rxxused in our study was r " .84, avalue of a test–retest reliability coefficient for NeuroTrax totalmemory index in a previous study of 57 healthy volunteers with amedian test–retest interval of 4.84 weeks (Schweiger, Doniger,Dwolatzky, Jaffe, & Simon, 2003).To assess the statistical significance of the patients’ clinicalchanges we calculated, the Jacobson-Truax’s RCI and categorizedfor each patient (positive change, no change, or negative change).Jacobson and Truax’s “cutoff score” was also calculated foreach memory measure. The cutoff score is the score in the post-treatment assessment, above which the patient is classified as“recovered,” that is, having higher probability to be part of thenormal population distribution than of the impaired one. Thiscutoff score was set with consideration of the means and SDs ofboth the normal and impaired populations:S0M1#S1M0S0#S1.ResultsPreliminary analysis results revealed significant improvement inalmost all memory measures after HBO2therapy, as well as strongand significant cross time effect (before-after HBO2therapy) andcross group effect (LBS-HBS) with medium to large effect sizes inall memory measures (see Table 2). The LBS groups exhibitedsignificantly larger improvement whereas the HBS did not showimprovements. Figure 1 represents a visual example of the crosstime and cross group in the TMI scores. These observations, alongwith the distribution of subjects (mean and SD, see Table 2) led tothe possibility of a ceiling effect in the performance of the HBSpatients who were initially less impaired in their memory function.Therefore, in the rest of the analyses we focused on the LBSgroups of patients and their potential for improvements after HBO2treatments. The number of patients in each group ranged from n "41 to n " 48, with an overlap of patients between the differentmeasures (see Figure 2A for the detailed distribution).Table 2Descriptive Statistics of Memory Scores Before and After HBO2Therapy and ANOVA Results of All Patients, and Memory LowBaseline Score and High Baseline Score SubgroupsMemorymeasure NMean (SD)Fp-value Effect size ()2)Before AfterTMIAll 91 83.06 (18.71) 89.38 (18.10) Time 20.93 0.00001 0.19 MediumImpaired 48 68.66 (13.41) 80.45 (19.62) Time ( Group 19.35 0.00002 0.17 MediumUnimpaired 43 99.13 (6.90) 99.36 (8.83)IVMAll 91 76.31 (25.80) 88.09 (24.69) Time 36.51 0.00000 0.29 LargeImpaired 47 55.34 (18.12) 73.84 (25.83) Time ( Group 13.25 0.00045 0.13 MediumUnimpaired 44 98.72 (6.85) 103.31 (10.66)DVMAll 91 81.74 (26.07) 90.03 (24.78) Time 14.56 0.00025 0.14 MediumImpaired 43 58.62 (18.58) 80.59 (27.07) Time ( Group 30.22 0.00000 0.25 LargeUnimpaired 48 102.4 (8.12) 98.49 (19.17)INVMAll 91 87.71 (18.83) 90.16 (16.69) Time 5.53 0.02089 0.05 SmallImpaired 41 71.10 (11.73) 82.14 (16.14) Time ( Group 32.43 0.00000 0.27 LargeUnimpaired 50 101.33 (10.90) 96.74 (14.19)DNVMAll 91 86.36 (19.39) 89.33 (18.47) Time 3.15 0.07944 0.03 SmallImpaired 45 70.88 (14.15) 81.04 (19.05) Time ( Group 17.18 0.00008 0.16 MediumUnimpaired 46 101.51 (9.09) 97.44 (13.84)Note. p value derived from ANOVAs main and interaction effects. TMI " total memory index; IVM " immediate verbal memory; DVM " delayedverbal memory; INVM " immediate nonverbal memory; DNVM " delayed nonverbal memory.Figure 1. Total memory index scores of all patients, divided to lowbaseline score (LBS) and high baseline score (HBS) groups. There is asignificant improvement in the LBS group after HBO2therapy and nochange in the HBS group.This document is copyrighted by the American Psychological Association or one of its allied publishers.This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.4BOUSSI-GROSS ET AL.

Participants’ ProfilePatients were 18 years or older (mean age !60 years) who hadeither ischemic or hemorrhagic stroke 3–180 months before HBO2(M " 30 –35 months, median " 19 –23 months). Basic demo-graphics and injury characteristics of all patients in the differentgroups are presented in Table 3.Memory Improvement EvaluationStatistical significance. There was a significant improve-ment in all memory scores of LBS patients after HBO2therapycompared with baseline (TMI: t(47)" 5.47, p # .000005; IVM:t(46)" 5.42, p # .000005; DVM: t(42)" 5.31, p # .000005;INVM: t(40) " 4.98, p # .00005; DNVM: t(43)" 3.99, p #Figure 2. (A) Number of poststroke patients analyzed in this study, assort to the different memory measures(TMI " total memory index; IVM " immediate verbal memory; DVM " delayed verbal memory; INVM "immediate nonverbal memory; DNVM " delayed nonverbal memory). (B) Number of patients with availableSPECT data, assorted to the different memory measures.aPatients assorted to the different memory measuresaccording to their baseline performance; only patients with low baseline scores (#85, i.e., 2 SD below normalaverage) were included in the main analysis of the different memory measures, with an overlap of patientsbetween the measures.bSPECT data of 56 out of 91 patients was available (61% of the patients).Table 3Basic Demographic and Injury Characteristics of Low Baseline Score (LBS) Groups (Baseline Score #85)TMI IVM DVM INVM DNVMNa48 47 43 41 45Age 60.6 60.4 60.5 62.4 62.1Months since injury (mean/median) 35.2/19.2 32.4/18.9 34.8/24 30.0/23.0 31.2/23.0Years of education 14.1 14.4 14.3 13.9 14.1Gender–male 42 (87.5%) 42 (89.3%) 38 (88.3%) 36 (87.8%) 38 (84.4%)Side of injuryRight 15 (34.9%) 15 (31.9%) 13 (30.3%) 14 (34.1%) 14 (31.1%)Left 29 (60.4%) 28 (53.1%) 26 (60.4%) 24 (58.5%) 27 (60%)Both 4 (8.3%) 4 (8.5%) 4 (9.3%) 3 (7.4%) 4 (8.9%)EtiologyIschemic 35 (72.9%) 33 (70.3%) 31 (72.1%) 31 (75.7%) 34 (75.5%)Hemorragic 12 (27.1%) 14 (29.7%) 12 (27.9%) 10 (24.3%) 11 (24.5%)No. of treatments60 25 (52.1%) 27 (57.5%) 25 (58.1%) 19 (46.3%) 22 (48.8%)40 23 (47.9) 20 (42.5%) 18 (41.9%) 22 (53.7%) 23 (51.2%)Note. TMI " total memory index; IVM " immediate verbal memory; DVM " delayed verbal memory; INVM " immediate nonverbal memory;DNVM " delayed nonverbal memory.aWith overlap between patients in the different memory measures groups.This document is copyrighted by the American Psychological Association or one of its allied publishers.This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.5IMPROVEMENT OF MEMORY IMPAIRMENTS IN POSTSTROKE

.0005). Effect sizes of all results were medium to large (Co-hen’s d " 0.69, 0.82, 0.94, 0.78, and 0.61, respectively). Theresults are presented in Figure 3 and in Table 4.Relative changes. The percentages of relative change for eachmemory measure are presented in Figure 4. The change percentages werefound to be as follows: 18% for the TMI measure, 43.5% for IVM, 48.6%for DVM, 17.5% for INVM and 18% for DNVM.Clinical significance. There were 35– 46% of the patientswho achieved significant clinical improvement in the differentmemory measures, out of which a very high percentage (78.9–100%) recovered, that is, passed the cutoff score differentiatingbetween impaired and unimpaired populations in their posttreat-ment assessment. More specifically, as summarized in Table 4and in Figure 5, in the TMI, 35% of the patients fell in thepositive change category after HBO2(out of which 94% passedthe recovery cut-off score), 60% remained with no change, and4% were in the negative change category; in the IVM, 38%demonstrated positive change (out of which 100% passed therecovery cut-off score), 57% had no change and 4% withnegative change; in the DVM, 44% demonstrated positivechange (out of which 94% passed the recovery cut-off score),51% had no change and 4.6% had negative change; in theINVM, 46% demonstrated positive change (out of which 79%passed the recovery cut-off score), 49% had no change and4.5% had negative change; and finally, in the DNVM, 43%demonstrated positive change (out of which 79% passed therecovery cut-off score), 52% had no change and 4.5% hadnegative change.Figure 3. Memory scores (mean * SE) before and after HBO2therapy. All improvements are significant at alevel of p # .0005.Table 4Mean & SD of Memory Tests Scores Before and After HBO2TherapyMemory measure NMean & SDp value%Before HBO2After HBO2Positive change(recovereda)Negativechange No changeTMI 48 68.06 & 13.93 80.2 & 19.92 #0.000005 35.42 (94.12) 4.17 60.42IVM 47 54.80 & 18.56 72.61 & 26.43 #0.000005 38.33 (100) 4.25 57.40DVM 43 57.64 & 19.34 78.71 & 28.07 #0.000005 44.19 (94.74) 4.65 51.16INVM 41 70.55 & 11.96 82.73 & 16.96 #0.00005 46.34 (78.95) 4.88 48.78DNVM 45 70.60 & 14.29 80.78 & 19.29 #0.0005 43.18 (78.95) 4.55 52.27Note. p value derived from a paired one-tailed t test comparison of the means. Percentage of clinical change was calculated according to Jacobson andTruax’s reliable change index (RCI) for each memory measure.aPercent of patients out of the positively changed passing the cutoff score, that is, with higher probability to be part of the normal population distributionthan the impaired one.This document is copyrighted by the American Psychological Association or one of its allied publishers.This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.6BOUSSI-GROSS ET AL.

SPECT ResultsCortical analysis: BAs. Fifty-six patients had availableSPECT imaging before and after HBO2therapy (Figure 2B).Patients in the four memory groups were divided into “im-proved” versus “not improved” according to the clinical signif-icance analysis and RCI score. All patients presenting positivechange according to this analysis were considered improvedand the rest were classified as not improved. The analysis wasconducted specifically for the delayed memory measures(DVM; DNVM), because these are “single-task” scores thatdescribe most accurately episodic memory deficits and have thepotential to correlate best with distinct brain area functions. Themean percentages of relative change in activation of cortical BAfor clinically improved versus not-improved patients are pre-sented in Figure 6. Several BAs were found to present higherrelative change in activation in the improved group versus thenot improved. In the DNVM measure, the most significantchange in activation was in right BA36 (part of the perirhinalcortex in the rhinal sulcus), followed by right BA28 (part of theenthorhinal area in the medial temporal lobe) and other BAssuch as right and left BA20 (in the inferior temporal gyrus),BA21 (the middle temporal area), and BA38 (the temporopolararea). In the DVM measure, the most significant change inactivation was in left BA36, followed by right BA46 (themiddle frontal area) and BA36, left BA23 (the ventral posteriorcingulate area), right BA9 (part of the dorsolateral and medialprefrontal cortex), and BA6 (part of the precentral gyrus) andleft BA4 (primary motor cortex), BA8 (anterior to the premotorcortex), and BA6 (Figure 7).DiscussionThis study presents analysis of the effects of HBO2treatmentson memory impairments in poststroke patients during the latechronic, unremitting stage. The analysis revealed statistically sig-nificant improvement in memory functions in most patients. Morespecifically, up to 45% of the patients had positive change in allfour measures of memory function up to a level of recovery. Theseclinical changes were found to be in good agreement with meta-bolic brain changes assessed by SPECT brain imaging.The results are consistent with previously reported HBO2in-duced neuroplasticity effects at late chronic stage, months to yearsafter the acute insult (Boussi-Gross et al., 2013; Efrati et al., 2013).Together with reported evidence of significant cognitive improve-ment in mild TBI patients with HBO2(Boussi-Gross et al., 2013),they suggest that HBO2may serve as an effective intervention forcognitive impairments in patients presenting brain metabolic dys-function because of acute damage.SPECT analysis was used to identify the brain regions associ-ated with the memory impairments and improvements. We foundthat the perirhinal cortex (BA36) and its activation correlated withclinical improvement in the delayed memory measures. Patientswho demonstrated improvement in their verbal and nonverbaldelayed memory abilities after HBO2had the highest percentage ofrelative change in activation in the left and right perirhinal cortex(PrC), respectively. The PrC is known to play a crucial role inrecognition memory, the PrC and the hippocampus often functionas interacting components of an integrated recognition memorysystem (Brown & Aggleton, 2001; Buffalo, Reber, & Squire,1998). Because our memory assignments in the cognitive testswere indeed recognition tasks, the involvement of the PrC in thisFigure 4. Mean percentages and SEs of relative changes in the different memory measures after HBO2.This document is copyrighted by the American Psychological Association or one of its allied publishers.This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.7IMPROVEMENT OF MEMORY IMPAIRMENTS IN POSTSTROKE

process was quite predictable. Other brain areas that showedsignificant change in activation in the clinically improved groupwere part of the enthorinal cortex (BA28), which has been foundto be affected in Alzheimer’s disease and to play a role in theprocess of spatial memory (Khan et al., 2014; Suthana et al., 2012).The temporal pole (BA38), is among the earliest affected byAlzheimer’s disease and the earliest involved at the start of tem-poral lobe seizures (Ding, Van Hoesen, Cassell, & Poremba,2009). Studies in monkeys revealed a role for the temporal pole ina variety of functions, visual discrimination of two-dimensionalpictures, and the mnemonic functions of matching and learning(Dupont, 2002).More important, the main lesion area, the “chronic penumbra”or “stunned brain” was identified, an area characterized by criti-cally reduced cerebral blood flow (CBF), abolished synaptic ac-tivity but preserved structural integrity (Furlan, Marchal, Viader,Derlon, & Baron, 1996). These areas of the brain that are damagedbut not dead after stroke offer the promise that, with propertherapy, their function could be restored (Lo, 2008). HBO2isexpected to induce improvement of recognition memory deficitsmainly in patients who had the potential for improvement of thePrC activation (i.e., this area was not anatomically disrupted, onlydysfunctional, as in the case of penumbra tissue). The abovepossibilities require further investigation and research as well asinvestigation of the anatomical damage localization and volume. Adetailed discussion of the metabolic processes, possible innaterepair mechanisms and neuroplasticity activated by HBO2treat-ments at late chronic stages of stroke in the injured brain ispresented in (Efrati & Ben-Jacob, 2014b; Efrati et al., 2013).Being a retrospective analysis of previously published data, theanalysis lacks a control group. However, the findings presentedhere are in agreement with and reinforce similar findings fromprevious prospective controlled trials in which the neurotherapeu-tic effects of HBO2in stroke and TBI patients were tested (Boussi-Figure 5. Patients scores before (X axis) and after (Y axis) HBO2in: (A) total memory index (TMI) and (B)other four memory measures. The colors represent the clinical change of each patient according to RCI (red "improved, blue " no change, green " deteriorated). The horizontal line represents the cut-off score; improvedpatients above this score are considered “recovered” according to the explanation in the method part in the text.This document is copyrighted by the American Psychological Association or one of its allied publishers.This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.8BOUSSI-GROSS ET AL.

Gross et al., 2013; Efrati et al., 2013). These studies providedconvincing evidence that HBO2can induce neuroplasticity atchronic stages in areas with metabolic dysfunction. If an area ofmetabolic dysfunction relevant to memory function in the brain issuch that can improve after HBO2, one can expect clinical im-provement in memory, as was demonstrated in this study. Inaddition, the correlation found between the clinical measures ofmemory improvement and brain imaging in areas relevant to thesame clinical improvement also strengthens the findings. Finally,most of the patients in this study were in the chronic stage whereno spontaneous improvement was expected. Clearly, further, largerprospective randomized trials on the effect of HBO2on cognitiveimpairment in poststroke patients should be conducted.The study has several limitations: the first is that the dataanalyzed was done retrospectively so there is a lack of follow-updata or data regarding long-term effects of the treatments. Thesignificant improvements found in this study mean that it is im-portant to perform future studies to replicate these findings as wellas study whether the effects persist over time.Another study limitation relates to the focus on recognitionmemory in the selected tests. There is a differentiation betweenrecall and recognition memory abilities, as well as evidence fordifferent brain structures and networks (Cabeza et al., 1997;Eichenbaum, Yonelinas, & Ranganath, 2007; Staresina & Dava-chi, 2006). The current research deals only with recognition abil-ities because of the limitation of the test battery used, althoughthese were shown to highly correlate with other recall paper basedmemory tests (Doniger et al., 2006; Dwolatzky et al., 2003; Elsteinet al., 2005). However, future studies should widen the explorationof HBO2effect on the different memory abilities, including recallmemory, as well as other cognitive impairments.Additional study limitation relates to the HBO2treatment pro-tocol. Even though a significant beneficial effect is notice withHBO2treatment, the exact HBO2protocol needed to induce max-imal neuroplasticity with minimal side effects was behind thescope of this study. It is well recognized that HBO2can inducesignificant neurophysiological effect even at lower pressures and itis also possible that additional HBO2sessions could have bringFigure 6. Mean percent of relative change in SPECT activation. This figure presents the mean relative changein SPECT activation in (A) the delayed nonverbal memory (DNVM) group, and (B) the delayed verbal memory(DVM) group. In each group, the mean percent of relative change is presented for both the clinically improvedgroup (blue-to-red bars) and the not-improved group (green bars). In (A), improved group: n " 10; not-improvedgroup: n " 17; in (B), improved group: n " 12; not-improved group: n " 12.This document is copyrighted by the American Psychological Association or one of its allied publishers.This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.9IMPROVEMENT OF MEMORY IMPAIRMENTS IN POSTSTROKE

further improvement (Efrati & Ben-Jacob, 2014a, 2014b; Efrati etal., 2013). Future pharmacodynamics studies, using differentHBO2protocols, are needed to optimization of the HBO2treatmentprotocol.In conclusion, this study demonstrates, for the first time, signif-icant improvements in memory functions of poststroke patientsafter HBO2at the late chronic stage. Further prospective, random-ized controlled trials, including more extensive cognitive exami-nations, are needed to specify the patients who might benefit themost from this treatment.ReferencesAbramovitch, A., Dar, R., Hermesh, H., & Schweiger, A. (2012). Com-parative neuropsychology of adult obsessive-compulsive disorder andattention deficit/hyperactivity disorder: Implications for a novel execu-tive overload model of OCD. Journal of Neuropsychology, 6, 161–191.http://dx.doi.org/10.1111/j.1748-6653.2011.02021.xBarrett, K. F., Masel, B. E., Harch, P. G., Ingram, F., Corson, K. P., &Mader, J. T. (1998). Cerebral blood flow changes and cognitive im-provement in chronic stable traumatic brain injuries treated with hyper-baric oxygen therapy. Neurology, 50, 178 –179.Baumann, M., Couffignal, S., Le Bihan, E., & Chau, N. (2012). Lifesatisfaction two-years after stroke onset: The effects of gender, sexoccupational status, memory function and quality of life among strokepatients (Newsqol) and their family caregivers (Whoqol-bref) in Lux-embourg. BMC Neurology, 12, 105. http://dx.doi.org/10.1186/1471-2377-12-105Ben Assayag, E., Korczyn, A. D., Giladi, N., Goldbourt, U., Berliner, A. S.,Shenhar-Tsarfaty, S.,...Bornstein, N. M. (2012). Predictors forpoststroke outcomes: The Tel Aviv Brain Acute Stroke Cohort(TABASCO) study protocol. International Journal of Stroke, 7, 341–347. doi:10.1111/j.1747-4949.2011.00652.xBoussi-Gross, R., Golan, H., Fishlev, G., Bechor, Y., Volkov, O., Bergan,J., . . . Efrati, S. (2013). Hyperbaric oxygen therapy can improve postconcussion syndrome years after mild traumatic brain injury–randomized prospective trial. PLoS ONE, 8, e79995. http://dx.doi.org/10.1371/journal.pone.0079995Brown, M. W., & Aggleton, J. P. (2001). Recognition memory: What arethe roles of the perirhinal cortex and hippocampus? Nature ReviewsNeuroscience, 2, 51– 61. http://dx.doi.org/10.1038/35049064Buffalo, E. A., Reber, P. J., & Squire, L. R. (1998). The human perirhinalcortex and recognition memory. Hippocampus, 8, 330 –339. http://dx.doi.org/10.1002/(SICI)1098-1063(1998)8:4#330::AID-HIPO3'3.0.CO;2-LCabeza, R., Kapur, S., Craik, F. I. M., McIntosh, A. R., Houle, S., &Tulving, E. (1997). Functional neuroanatomy of recall and recognition:A PET study of episodic memory. Journal of Cognitive Neuroscience, 9,254 –265. http://dx.doi.org/10.1162/jocn.1997.9.2.254Churchill, S., Weaver, L. K., Deru, K., Russo, A. A., Handrahan, D.,Orrison, W. W., Jr.,...Elwell, H. A. (2013). A prospective trial ofhyperbaric oxygen for chronic sequelae after brain injury (HYBOBI).Undersea & Hyperbaric Medicine, 40, 165–193.Cicerone, K. D., Dahlberg, C., Malec, J. F., Langenbahn, D. M., Felicetti,T., Kneipp, S.,...Catanese, J. (2005). Evidence-based cognitiverehabilitation: Updated review of the literature from 1998 through 2002.Archives of Physical Medicine and Rehabilitation, 86, 1681–1692.http://dx.doi.org/10.1016/j.apmr.2005.03.024Ding, S. L., Van Hoesen, G. W., Cassell, M. D., & Poremba, A. (2009).Parcellation of human temporal polar cortex: A combined analysis ofmultiple cytoarchitectonic, chemoarchitectonic, and pathological mark-ers. The Journal of Comparative Neurology, 514, 595– 623. http://dx.doi.org/10.1002/cne.22053Doniger, G. M. (2014a). Guide to normative data. Retrieved from http://www1.neurotrax.com/docs/norms_guide.pdfDoniger, G. M. (2014b). NeuroTrax computerized cognitive tests: Testdescriptions.Retrievedfromhttp://www1.neurotrax.com/docs/TestDescriptions.pdfDoniger, G. M., & Simon, E. S. (2014). Construct validity of NeuroTrax:Comparison with paper-based tests.Retrievedfromhttp://www1.neurotrax.com/docs/construct_validity.pdfDoniger, G. M., Simon, E. S., Okun, M. S., Rodriguez, R. L., Jacobson,C. E., Weiss, D.,...Fernandez, H. H. (2006). Construct validity of aFigure 7. Activated Brodmann areas (BAs) of patients that had cognitive improvement after HBO2treatment.The colored BAs represent areas with significant increase metabolism in the cognitive improved patients: (A) thedelayed nonverbal memory (DNVM) group, and (B) the delayed verbal memory (DVM) group.This document is copyrighted by the American Psychological Association or one of its allied publishers.This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.10BOUSSI-GROSS ET AL.

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CA S E R E P O R T Open AccessHyperbaric oxygen therapy after acuteischemic stroke with large penumbra: acase reportOmar Hussein1, Khalid Sawalha2*, Ahmed Abd Elazim1, Diana Greene-Chandos1and Michel T. Torbey1AbstractBackground: Hyperbaric oxygen therapy (HBOT) for the treatment of acute stroke has been under the radar for along time. Previous studies have not been able to prove efficacy. Several factors might have contributed to suchinconsistent results. The timing of delivering the hyperbaric oxygen in relation to the stage of stroke evolution maybe an important factor. This was not taken into account in the previous studies as there was no feasible andstandardized method to assess the penumbra in the acute phase. Now with the perfusion scan appearing as a keyplayer in the acute stroke management, precise stroke patient selection for hyperbaric oxygen therapy deserves asecond chance similar to mechanical thrombectomy.Case presentation: A 62-year-old female patient who presented with acute large vessel stroke was not eligible forchemical or mechanical thrombectomy. There was a large penumbra on imaging. She got treated with severalsessions of hyperbaric oxygen over a 2-week period immediately after stroke. The patient showed significantimprovement on the follow-up perfusion imaging as well as some clinical improvement. The more impressiveradiological improvement was probably due to the presence of relatively large core infarction at baseline affectingfunctional brain areas. The patient continued to improve clinically on her 6-month follow up visit.Conclusion: Our case demonstrates immediate stroke-related penumbra improvement associated with HBOT.Based on that, we anticipate a potential role for HBOT in acute stroke management considering precise patientselection. Future randomized controlled trials are needed and should take that in consideration.Keywords: Hyperbaric oxygen therapy, Acute ischemic stroke, HBOT, Penumbra, Precision medicineBackgroundThe ischemic penumbra is the region of the brain thatsurrounds the core infarcted tissue. This region is notapoptotic yet but is at risk of progressing to an expandedcore infarction unless an intervention is taken. Contrar-ily, cerebral oligemia is the region of the brain tissue thatis temporary ischemic following a stroke but is like ly torecover spontaneously and thus considered benign. In anutshell, a core infarction is an unsalvageable tissue, apenumbra is a potentially salvageable tissue but withintervention, and finally oligemia is a spontaneouslysalvageable tissue [1].There have been remarkable advances in brain imagingin the last decade specifically in the perfusion studies. In1980, Dr. Leon Axel has set the ground for perfusionscans to be used in mapping the cerebral blood flow byhis theoretical analysis at the time [2]. With multiple tech-niques and softwares arising (e.g., RAPID software [3, 4])since then, differentiation between the core, penumbra,and oligemia was made possible [5]. With such advances,vascular neurologists and neuroradiologists are able to de-termine with high precision the region of ischemic brainthat is salvageable. This has opened the way to reinvesti-gate the usefulness of mechanical thrombectomy again© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.* Correspondence: Khalid.Sawalhamd@baystatehealth.org2Department of Internal Medicine, University of Massachusetts MedicalSchool-Baystate Campus, Worcester, USAFull list of author information is available at the end of the articleThe Egyptian Journal of Neurology, Psychiatry and NeurosurgeryHussein et al. The Egyptian Journal of Neurology, Psychiatry and Neurosurgery (2020) 56:93 https://doi.org/10.1186/s41983-020-00225-9

after failing in prior studies (IMS III [6], SYNTHESIS EX-PANSION [7], and MR RESCUE [8]). Failure of thesetrails was attributed to lack of precision in patientselection due inconsistency in neuroimaging as well asusing the “Merci device” instead of the “stent retriever” formechanical thrombectomy. From 2015 through 2018,some breakthrough clinical trials (MR CLEAN [9], REVASCAT [10]), ESCAPE [11], SWIFT PRIME [12],EXTEND-1A [13], DIFFUSE 3 [14], and DAWN [15]) hasreintroduced mechanical thrombectomy in the manage-ment of acute ischemic stroke within a window of up to24 h from symptoms onset respectively with remarkablesuccess. Of note, the first three trials have relied on non-contrast CT head and CT angiography while the later fourhave relied on the perfusion scans (RAPID software).Consequently, mechanical thrombectomy for acute strokemanagement has made its way in the 2015 and 2018AHA/ASA updated guidelines [16]. A large multicentermeta-analysis done by the HERMES group [17] showedthat favorable outcomes were seen in 46% of patientsenrolled in the intervention arm as compared to 27% inthe control arm when pooled data from the first 5 trialswere analyzed. On the other hand, When Diffuse 3 andDAWN trials were co-analyzed, 47% of the interventiongroup versus 15% of the control group showed favorableoutcomes despite of the large window gap for interventionbetween the two analyses. In another analysis by theHERMES group, they showed that the estimated ischemiccore volume was independently associated with functionalindependence and functional improvement [18]. Thisindicates that mechanical thrombectomy relies heavily onthe perfusion imaging results and thus precise patientselection is warranted.Recent human and animal studies showed that thepenumbra can extend beyond 48 h and are associatedwith worse outcomes if not treated [19–21]. In a DAWNtrial subanalysis, 21 patients who were enrolled into thestudy actuall y met the perfusion imaging criteria but didnot meet the time window which extended beyond 24 h.The average time from last known well to mechanicalthrombectomy was 54.5 h (range 24.1–155.7 h). Theresults of this subanalysis were similar to the mainDAWN trial. It showed that the benefit from mechanicalthrombectomy extends beyond the 24-h time window. Itcame to the conclusion that the tissue window based onthe perfusion scans is a more accurate parameter thanthe time window [22].Likewise, HBOT for acute cerebral ischemia hasfailed in the past. A recent 2014 Cochrane rev iew[23] concluded that HBOT is not superior to conven-tional tr eatment in terms of 6-month mortali ty rate(prior to t he mechanical thrombectomy era). Despitethat few studies showed some functional and clinicalimprov ement; o n the larger scale, these results wereinconsistent. Logically, like me chanical thrombectomy,achieving reper fusion and increasing oxygen tensionhelp improving oxygenation to ischemic areas. Unlikemechanical thrombectomy, HBOT is not expected toreach the core infarction due to blocke d blood supplyand thus expectedly may have less chance for reperfusioninjury. Nonetheless, mechanical thrombectomy achievesreperfusion whereas HBOT delay tissue demise untilrevascularization is achieved or a collateral circulation ismaintained. Again, precision in patient selection is a key.We present a case of acute symptomatic ICA occlusionwho was not a candidate for mechanical or chemicalthrombectomy. After obtaining the family consent, thepatient received several sessions of HBOT (Fig. 1) over 2weeks immediately after the stroke. Remarkable improve-ment on serial perfusion scans was evident.Fig. 1 Hyperbaric chamber. Image of the used hyperbaric chamber. Model 3300, company’s name: Sechrist, Anaheim, California, USA, year ofmanufacture: 2010Hussein et al. The Egyptian Journal of Neurology, Psychiatry and Neurosurgery (2020) 56:93 Page 2 of 7

Case presentationA 62-year-old fully functioning woman with history ofhypertension and obesity presented with sudden onsetright-sided weakness and global aphasia. Her last-known-well was > 24 h and thus not eligible for chemical ormechanical thrombectomy. Her initial NIH stroke scale(NIHSS) was 22. CT-Head showed left-MCA hyperdensesign and watershed hypodensity in the left subcorticalarea. CT-Angiography showed a long segment occlusionof left internal carotid artery just cranial to bifurcationextending cranially to the level of carotid terminus. Therewas opacification of left-A1 segment via ACOM, traceopacification of the left ophthalmic artery and left MCAbranches by left posterior communication artery, withminimal reconstitution peripherally by the leptomeningealcollaterals (Fig. 2). The initial CT-perfusion (CBF < 30%and T-max > 6.0 s) showed an ischemic tissue size of 147ml (Fig. 3). The patient received the conventional stroketherapy for secondary prevention including aspirin 81 mgdaily and a lipid lowering medication along with physicaltherapy and speech therapy. The patient required thera-peutic hypertension due to blood pressure-dependentmental status and aphasia fluctuations indicating unstablecollaterals. Therapeutic hypertension using vasopressorswas maintained for 2 days post-stroke. Systolic blood pres-sure was maintained between 160 and 180 mmH2O.However, the patient received five sessions of hyperbaricoxygen therapy (HBOT; hyperbaric chamber, model 3300,company’s name: Sechrist, Anaheim, CA, USA, year ofmanufacture: 2010) over a 2-week period that started inpost-stroke day 2 (after stopping therapeutic hyperten-sion). HBOT was performed in post-stroke day 2 (PSD-2),PSD-4, PSD-7, PSD-10, and PSD-15. During that period,permissive hypertension was allowed. Systolic bloodpressure was running between 100 and 140 mmH2O.Each HBOT session consisted of 2-h 100% O2at 2.5 ATA.Fig. 2 Computed tomography angiogram. Presenting computed tomography-angiographic study showing complete occlusion of left intracranialportion of the internal carotid artery at the level of carotid terminus (red arrow) (a). Compared to the patent right middle cerebral artery (MCA)(b), there was trace opacification of left MCA branches (blue arrow) by left posterior communication artery (red arrow) (c), with minimalreconstitution peripherally by the leptomeningeal (red arrows) (d)Hussein et al. The Egyptian Journal of Neurology, Psychiatry and Neurosurgery (2020) 56:93 Page 3 of 7

Serial CT-perfusion studies were done after the HBOTsessions (Fig. 3). The first follow-up CT-perfusion doneafter 2 sessions of HBOT showed reduction of the ischemicarea to 77 ml. Her NIHSS improved to 16. The secondfollow-up CT-perfusion after 2 more sessions of HBOTshowed further reduction of the ischemic area to 38 ml.Her NIHSS improved to 12. The final CT-perfusion doneafter fifth session of HBOT showed near stabilization of theischemic area at 48 ml. Her NIHSS improved to 10. Herglobal aphasia improved (only motor aphasia partiallypersisted), and there was no clinical deterioration despitenormalization of the blood pressure after 2 days due toevidence of small intracerebral hemorrhage. Her 6-monthfollow-up visit showed improvement of her aphasia andmotor functions. Her modified ranking scale was four asshe still needs assistance with walking.DiscussionThe patient showed significant improveme nt on thefollow-up perfusion imaging as well as some clinicalimprovement. The lack of full-clinical improvement wasprobably due to the presence of relatively large coreinfarction at baseline affecting functional brain areas.The proposed beneficial mechanism of HBOT includescounteracting hypoxia by inducing hyperoxemia whichleads to improved perfusion and oxygenation of the pen-umbra and the brain microcirculation [24]. While thisaction seems exciting, it is not specific for hyperbaricoxygen and reports of normobaric oxygen therapy(NBOT) delivery in the acute stroke presumptively leadto the same effect [25]. While NBO is feasible, quick,and easy to use, it lacks an equivalent neuroprotectiveeffect as HBOT. In an animal study that comparedFig. 3 CT perfusion: serial computed tomography-perfusion studies over 2 weeks using the RAPID software. a The initial CT-perfusion onpresentation showing a core infarction of 58 ml, ischemia of 147 ml, mismatch volume of 89 ml and mismatch ratio of 2.5. b–d Follow-up CT-perfusion studies after starting hyperbaric oxygen therapy (HBOT) presented in order showing ischemia reduction (b 77 ml, c 38 ml, and d 48 ml).Although there is a slight increase in the penumbra after the fifth session of HBOT, a clear trend of penumbra reduction is seen through thefollow-up studies compared to the initial study. Although the amount of clinical improvement is less than the radiological improvement, therelatively large core infarction and its location might be the determinant of that perfusion/clinical mismatched improvementHussein et al. The Egyptian Journal of Neurology, Psychiatry and Neurosurgery (2020) 56:93 Page 4 of 7

HBOT, NBOT, and normobaric air, the HBOT only hadsignificantly smaller infarct size with no difference in thebrain oxygenation indicating the presence of neuropro-tective effect in the HBOT group [26]. The neuroprotec-tive effect of HBOT comes from the antioxidant [27]and anti-inflammatory effects associated with it. Theantioxidant effect is paradoxical and controversial as ithas been also reported to induce an oxidative stressinjury in the brain. However, this might be related towhether revascularization prior to HBOT was achievedor not. In other words, prior revascularization might in-crease the risk of oxidative injury. However, preservingHBOT for strokes that are not eligible to revasculariza-tion might sound reasonable. The anti-inflammatoryeffect is maintained through inhibition of leucocyteactivation, regulate abnormal cellular metabolites, recov-ering the blood–brain barrier and thus reducing thecerebral edema [24, 28–32]. Other factors leading toneuroprotection with HBOT have been described. Theseinclude mitochondrial regulation [33], decreased corticaland hippocampal caspase-3 [34], increased growthfactors [35], and reduction in hypoxia-inducible factor-1[36]. A major risk of HBOT, as mentioned before, is theworsened oxidative stress with HBOT leading to glutamateinducted excitotoxic cell death [37]. Similar effect wasreported with NBOT [38]. Another potential risk of HBOTis that it might theoretically lead to a steal phenomenonwhich can be detrimental. This means routing of the bloodfrom the ischemic tissue to the normal brain tissue due tovasodilatation of the vessels on the normal side. Thisoccurs when there is a persistent arterial occlusion thatlead to hypercarbia in the ischemic region that leads todecreased flow velocities in that region at the expectedtime of normal brain vasodilatation induced by HBOT [39]leading to blood shift to the normal non ischemic areas.This usually leads to worsening of the patient’smanifesta-tions after an initial improvement [40]. While there hasnot been reports associating the steal phenomenon withHBOT, it remains a potential risk of the treatment.The timing of HBOT for acute stroke treatment isanother controversial topic. While in the past, it wasbelieved that the earlier the treatment was starte d, thebetter the outcomes will be [41]. More recently, it isbelieved that later onset, whether acutely 2–5 days post-stroke or chronically, and longer course HBOT hassignificant effect on neurogeneration [42–44].As previou sly mentioned, the previous 2014 Cochranereview was conducted before the recent breakthroughtrials for validatio n of mechanical thrombectomy guidedby the perfusion scans (precise selection of patients).Despite that the authors found no good evidence toshow that HBOT improves clinical outcomes whenapplied during acute presentation of ischemic stroke,this was attributed to insufficient evidence by 11 RCTs.The possibility of clinical b enefit was not e xcluded.Similar reviews discussing failure of mechanicalthrombectomy as a treatment for stroke exist b eforethose breakthrough tria ls. Ho wever, they kep t thedoor opened f or further modifications [45, 46]. None-theless, reemergence of mec hanical th rombectomyafter t he establishment of reliable brain perfusionscanning pursued [47, 48].In the new era of precision medicine [49]andperfusion-guided therapy for acu te ischemic stroke,HBOT as an alternative therapy for acute ischemicstroke, for which the traditional repe rfusion methodsare difficu lt to achieve , should be readdressed. This isespecially true if a significant penumbr a exists. Condi-tions, like sym ptomatic critical carotid stenosis andcritical vertebral and/or basilar arteries stenosis orocclusion, ar e also eligible especially if the surgical op-tion is contraindicated, unac hie vable, or will be delayed.While an alt ernative therapy like therapeutic hyperten-sion is always an option, it might have significant sideeffects like bleeding and/or organ damage like myocar-dial i nfarction.Based on that, we anticipate a larger potential role forHBOT in acute stroke management in the future consid-ering precise patient selection. Follow-up randomizedcontrolled trials (RCT) should take that in consideration.LimitationThe intention of this report, like any other case report,is never to creat e an attribution, association, or correl-ation but to report an observation and draw attentionfor the need for such RCT. Case reports are importanttools to report observations. Most major discoveries ortrials were based on observations from case reports orcase series. We obtained a CT perfusion scan immedi-ately after each hyperbaric therapy whic h showedconsistent improvement. This is the observation that weare reporting. This might be the result of naturalresolution and thus we recommend that a randomizedcontrol trial should be conducted. Withou t the report ofobservations, we will never be able to conduct suchtrials. Nonetheless, the persistence of penumbra beyond48 h is not so common. While this might be unexpectedby some, it strengthen our argument that HBOT mightbe useful in the prevention of demise of the penumbra.Mechanical t hrombectomy itself as a treatment forstroke showed initial failure (IMS III, SYNTHESISEXPANSION, and MR RESCUE). Without persistentclinicians reportin g association of benefit after preciseselection of patients based on the relatively ne wlydeveloped CT perfusion imaging, they would havenever been able to re-conduct trials like (MR CLEAN,REVASCAT, ESCAPE, SWIFT PRIME, EXTEND-1A,DIFFUSE 3, and DAWN) that have proven the correlation.Hussein et al. The Egyptian Journal of Neurology, Psychiatry and Neurosurgery (2020) 56:93 Page 5 of 7

Another limitation is the presence of a confoundingfactor for HBOT which is the induced therapeutichypertension. This only lasted for 2 days. While thiscould certainly contribute to the initial improvement,the continued improvement over the pursuing 2 weekswhile on HBOT might indicate a potential role for suchintervention.ConclusionHBOT, as a salvage therapy for the cerebral ischemicpenumbra, is potentially beneficial and deserves a secondchance of evaluation after precise selection of patientswith large vessel occlusion (LVO) strokes. Although it car-ries potential risks like oxidative injury, this is theoreticallyless significant than those associated with mechanicalrevascularization as the dead tissue is not subject to suchstress. HBOT, if proven effective, can be an alternative, ad-junctive, or back-up therapy for the treatment of patientssuffering of acute LVO stroke associated with large pen-umbra and with anticipated delayed or contraindicatedmechanical thrombectomy. Randomized controlled trialsare needed to prove such hypothesis.AbbreviationsHBOT: Hyperbaric oxygen therapy; NIHSS: National institution of health strokescale; NBOT: Normobaric oxygen therapyAcknowledgementsNot applicableMachine usedHyperbaric chamber, model 3300, company’s name: Sechrist, Anaheim, CA,USA, year of manufacture: 2010MRI: Siemens Skyra 3 Tesla, Siemens Healthineers AG, Germany, 2014CT scan: Siemens Force, Siemens Healthineers AG, Germany, 2014Authors’ contributionsAll authors contributed significantly to the manuscript writing and design.“All authors have read and approved the manuscript”. OH: writingmanuscript and design the case report. KS: writing manuscript and designthe case report. AA: literature review. DG: literature review, reviewingmanuscript. MT: reviewing and approving the manuscript. Criticizing thecontent and structure of manuscrip t.FundingAll authors have no funding disclosure.Availability of data and materialsData is limited to one patient in this case report. Data is available wheneverrequestedEthics approval and consent to participateCase report s are exempted from institutional board review and ethicalapproval.Consent for publicationA written informed consent was obtained from the participantCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Neurology, The New Mexico University Health SciencesCenter, Albuquerque, USA.2Department of Internal Medicine, University ofMassachusetts Medical School-Baystate Campus, Worcester, USA.Received: 11 May 2020 Accepted: 31 August 2020References1. Wu L, Wu W, Tali ET, Yuh WT. Oligemia, penumbra, infarction:understanding hypoperfusion with neuroimaging. Neuroimaging Clin NAm. 2018;28(4):599– 609.2. Axel L. 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A Dual Role for Hyperbaric Oxygen in Stroke Neuroprotection: Preconditioning of the Brain and Stem CellsGrant M. Liska, Trenton Lippert, Eleonora Russo, Norton Nieves, and Cesar V. Borlongan11Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FLAbstractStroke continues to be an extremely prevalent disease and poses a great challenge in developing safe and effective therapeutic options. Hyperbaric oxygen therapy (HBOT) has demonstrated significant pre-clinical effectiveness for the treatment of acute ischemic stroke, and limited potential in treating chronic neurological deficits. Reported benefits include reductions in oxidative stress, inflammation, neural apoptosis, and improved physiological metrics such as edema and oxygen perfusion, all of which contribute to improved functional recovery. This pre-clinical evidence has failed to translate into an effective evidence-based therapy, however, due in large part to significant inconsistencies in treatment protocols and design of clinical studies. While the medical community works to standardize clinical protocols in an effort to advance HBOT for acute stroke, pre-clinical investigations continue to probe novel applications of HBOT in an effort to optimize stroke neuroprotection. One such promising strategy is HBOT preconditioning. Based upon the premise of mild oxidative stress priming the brain for tolerating the full-blown oxidative stress inherent in stroke, HBOT preconditioning has displayed extensive efficacy. Here, we first review the pre-clinical and clinical evidence supporting HBOT delivery following ischemic stroke and then discuss the scientific basis for HBOT preconditioning as a neuroprotective strategy. Finally, we propose the innovative concept of stem cell preconditioning, in tandem with brain preconditioning, as a promising regenerative pathway for maximizing the application of HBOT for ischemic stroke treatment.Keywordspreconditioning; ischemia; neuroprotection; neurodegeneration; cell therapy; regenerative medicine1.0 IntroductionStroke is defined as a sudden loss of blood supply to brain tissue resulting from either hemorrhagic or ischemic pathology causing severe neurological deficit (Jickling et al., 2014). In the United States, stroke is the fifth leading cause of death, with an occurrence rate of roughly 800,000 per year (George et al., 2017). Stroke can be broadly classified into Correspondence should be addressed to Dr. Cesar V. Borlongan (cborlong@health.usf.edu). Conflicts of Interest: The authors declare that they have no conflicts of interest.HHS Public AccessAuthor manuscriptCond Med. Author manuscript; available in PMC 2018 August 03.Published in final edited form as:Cond Med. 2018 June ; 1(4): 151–166.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

hemorrhagic and ischemic stroke, with the latter accounting for 87% of all strokes (Go et al., 2014). Hemorrhagic stroke is overwhelmingly fatal, and thus provides a smaller therapeutic opportunity. Thrombolytic tissue plasminogen activator (TPA) is currently the only Food and Drug Administration (FDA)-approved intervention available for ischemic stroke, and is approved for use only within the first 4.5 hours following onset; delivery beyond this window is associated with a stark increased occurrence of severe hemorrhagic transformation (Knecht et al., 2017). Someone in the U.S suffers from stroke every 4 seconds, and with limited therapeutic options, new and effective stroke treatments are an urgent necessity.Ischemic stroke pathology is characterized by abrupt blood vessel occlusion, causing ischemic damage to the area of the brain supplied by the occluded artery. During the acute phase, this primary anoxic environment induces a cascade of excitotoxicity, oxidative stress, and microglial activation throughout the infarcted region, resulting in extensive neural death (Stonesifer et al., 2017). Reactive oxygen species (ROS) weaken vasculature and create ionic imbalances, which lead to an abnormal increase of water movement into the intracellular space, resulting in edema (Stokum et al., 2016). In the subacute phase, cytokines, chemokines, and matrix metalloproteases (MMPs) are released, contributing to neuroinflammation (Lakhan et al., 2009). Elevated expression of MMPs increases blood-brain barrier (BBB) permeability, allowing migratory waves of leukocytes into the infarct area and exacerbating inflammatory activity (Lakhan et al., 2009). Multiple cell phenotypes which comprise the neurovascular unit (Lo et al., 2009) within the penumbra – the area of brain tissue surrounding the infarcted core – are susceptible to the abovementioned pathological mechanisms as well. These cells, however, are salvageable, and their protection is of great interest in rescuing the motor and cognitive functional deficits which follow stroke (Wetterling et al., 2016).Hyperbaric oxygen treatment (HBOT) is a non-invasive therapy performed in a pressurized chamber where pure oxygen is administered at above-normal atmospheric pressure. This condition improves oxygenation from lungs to systemic organs, enhancing biomolecular processes specifically in ischemic conditions (Thom, 2011). Under increased oxygenation in the infarct core and penumbra, HBOT can reduce secondary brain injury effects such as apoptotic pathway initiation, oxidative stress, and rampant inflammation (Ostrowski et al., 2017). HBOT has shown promising restorative effects in a wide range of pathological contexts, including within TBI, SCI, stroke and other non-neurological maladies such as carbon monoxide poisoning, gangrene, and arterial gas embolism (Baratz-Goldstein et al., 2017). Among the neurological pathologies, in general, few effective treatment options are offered and severe debilitation and/or death are common outcomes. Current HBOT experimental studies have demonstrated improvement in facilitation of cerebral oxygenation, metabolism, angiogenesis, and reduction in inflammation in stroke and non-stroke disease states (Zhai et al., 2016). HBOT studies aim to effectively attenuate ischemic-related damage, as well as utilize various other mechanisms, all in an effort to improve the quality of life of affected patients. Here, we review the current status of HBOT for ischemic stroke, present scientific support and rationale for HBOT preconditioning in the stroke brain, and finally propose HBOT priming of stem cell transplantation as a promising and novel paradigm for stroke therapy.Liska et al.Page 2Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

2.0 HBOT in Ischemic Stroke: Potential Benefits and LimitationsEarly investigations began 50 years ago on the results of hyperoxia following stroke; however, HBOT was viewed as more dangerous than beneficial, and the application of its therapeutic value was abandoned. In the last 20 years, this tapered view of HBOT has shifted towards a favorable assessment of its potential application in stroke as further research has revealed the ability of HBOT to reduce the severity of infarct volume if administered during the reperfusion window (Hu et al., 2017). This, however, creates a very limited opportunity for effective treatment as determination of reperfusion will require imaging modalities to inform the appropriate course of action, and by the time such assessment is completed the narrow effective timeframe for HBOT has likely passed (Chang et al., 2000). Other studies showed that delayed HBOT past the effective window of treatment resulted in worse outcomes versus the normobaric groups, potentially due to the role of ROS in glutamate-induced excitotoxic cell death (Singhal et al., 2002). Thus, indiscriminate use of HBOT is inadvisable, as treatment with excess oxygen can cause additional harm, such as obstructive pulmonary disease (Singhal et al., 2002).Normobaric oxygen has also been critically evaluated as a potential treatment for stroke, with therapeutic efficacy being both reported and refuted (Padma et al., 2010; Singhal et al., 2005; Shi et al., 2016). While normobaric oxygen therapy does increase dissolved O2 available within the blood, it has not been demonstrated to exert other neuroprotective effects which are central to HBOT effectiveness both post- and pre-stroke, and is thus less intriguing when exploring preconditioning paradigms (Padma et al., 2010; Singhal et al., 2005; Shi et al., 2016).To date, two primary methods of utilizing HBOT have been employed -- post-stroke and preconditioning. Following stroke, the goal of HBOT is to induce hyperoxia during the ischemic and reperfusion periods or expose the subject to repeated treatments once past the initial early treatment window (Zhai et al., 2016). Current preconditioning treatments focus on exposing the individual (albeit endogenous cells) to a mild stressor, therefore increasing tolerance to future stressors (Godman et al., 2010).2.1 Preclinical functional outcomes of post-stroke HBOTA typical HBOT treatment is conducted at 2.5 atmospheres (ATM) for a period of 60–90 minutes (Ostrowski et al., 2016), although protocols vary outside of this framework. If the pressure exceeds 2.5 ATM during HBOT, oxygen toxicity as well as increased oxidative stress can present throughout the body. Moreover, as the pressure increases above the recommended level, the risk of seizure activity increases drastically (Zhai et al., 2016). The primary goal of acute HBOT treatment is to increase oxygen levels in the ischemic region during stroke occlusion, in pursuit of minimizing hypoxic damage. Once past the initial treatment window, repeated HBOT treatments over several days have been shown to promote stimulation of endogenous repair processes (Wang et al., 2008).The potency of HBOT appears to wane significantly as treatment initiation is delayed. Following an ischemic event, several studies have demonstrated effectiveness of HBOT when administered within 30 to 60 minutes of stroke, as evidenced by reduced infarct Liska et al.Page 3Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

volume and improved behavioral scores (Chang et al., 2000; Hu et al., 2017). While useful as a proof-of-concept, this highly acute treatment timeframe provides little translational value. Importantly, though, investigations have found effectiveness with HBOT at less restrictive time points, such as HBOT (2.5 ATA for 2 hours) at 6 hours after reperfusion (Yin et al., 2003), and HBOT (3 ATA, 1 hour) at 3 and 6, but not 12, hours after reperfusion (Lou et al., 2004). While single-session HBOT has been reported as effective when initiated up to 18 hours (Xue et al., 2008) and 48 hours (Mu et al., 2013) after stroke, therapeutic effects with HBOT at more extended timepoints (i.e. 12+ hours after reperfusion) will generally necessitate more extensive and tailored treatment protocols. Delayed repeat treatment, with HBOT (2.5 ATA, 2 hours) initiated at 6 or 24 hours following stroke and delivered for 6 consecutive days, resulted in amelioration of infarct size and neurological deficits (Yin et al., 2005). Another report found significant chronic improvement in behavioral and histological outcomes following MCAO when exposed to an aggressive regimen of 15 HBOT sessions (2.5 ATA, 90 min) 5 times per week over 3 weeks, initiated 24 hours after stroke (Lee et al., 2013). Scarce evidence exists for the effectiveness of HBOT when initiated more than 48 hours post-stroke; a single study using a severe model of focal ischemia found that one session of HBOT (3 ATA, 1 hour) exhibited significant neuroprotective effects when delivered up to 72 hours post-stroke (Veltkamp et al., 2005).2.2 Clinical results of HBOT in strokeThere have been only a handful of randomized controlled clinical trials involving HBOT to date, resulting in inconclusive results (Bennett et al., 2014). Many different factors likely contribute to the varying clinical results, including the unstable clinical status of acute stroke patients, preventing them from receiving HBOT during the early effective treatment window of 3–5 hours in humans (Sanchez, 2013). Moreover, wide variations in treatment protocols and stage of patient enrollment (i.e. more acute, sub-acute and chronic) likely inhibit the ability to draw reliable conclusions and compare outcomes across trials. Nonetheless, clinical success for HBOT has been achieved; for example, the use of HBOT has been shown to reduce levels of cerebral and myocardial biomarkers and reduce the length of intensive care unit stays (Li et al., 2011). Although clinical trials for HBOT have not been deemed overwhelmingly successful, the potential clinical significance of HBOT cannot be ignored, and further trials to elucidate the best methods should be pursued (Hu et al., 2016). With an increasing number of clinical trials being conducted, cerebral plasticity has been identified as a benefit of HBOT; however, these trials lack the necessary results to unequivocally conclude efficacy (Zhai et al., 2016). In a recent clinical study, the efficacy of HBOT in restoring memory function for chronic stroke patients was examined, revealing significant memory improvement following HBOT treatment, which was accompanied by an increased brain metabolic rate (Ploughman et al., 2015). Another clinical study utilized HBOT 5 days per week for patients with chronic stroke, and resulted in significant improvements in memory and attention testing scores (Hadanny et al., 2015). The timing of HBOT application has been examined through several clinical studies. Consistent with the preclinical studies discussed above, these studies found that the earlier HBOT is initiated in relation to the initial ischemic event, the greater its therapeutic effectiveness (Chang et al., 2000; Ding et al., 2014). Once past 12 hours post-ischemia, the benefits of single treatment HBOT are drastically reduced, although the application of repetitive HBOT use in the sub-Liska et al.Page 4Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

acute stroke brain has documented neurogenic effects (Hu et al., 2014). Apparent from the completed clinical trials of HBOT for stroke is the consistent safety of this therapeutic strategy, despite inconsistent efficacy. These studies elucidate the clinical potential of using HBOT for chronic stroke patients, but the data are far more varied in acute stroke patients.3.0 Unpacking the Mechanisms of Action of HBOT in Stroke3.1 Physiological and metabolic effectsNeurons are particularly susceptible to oxygen deprivation, and thus the primary mechanism of acute HBOT delivery is to increase perfusion and oxygenation of at-risk tissue. Studies have consistently demonstrated that HBOT therapy can enhance arterial oxygen saturation and increase tissue oxygen content through enhanced cerebral microcirculation (Matchett et al., 2009; Zhai et al., 2016). Moreover, HBOT enhances BBB stability through various molecular mechanisms including MMP regulation (Ding et al., 2014), decreases intracranial pressure, and relieves cerebral edema (Zhai et al., 2016). Secondary effects of HBOT in the stroke brain may result from metabolic regulation such as reducing extracellular glutamate levels, which contribute to excitotoxic death and neural dysfunction (Gao-Yu et al., 2011).3.2 Antioxidant effectsAs will be discussed later, introducing high O2 levels can actually induce oxidative stress; however, in an apparent paradox, HBOT has been consistently shown to confer oxidative protection against stroke-induced ROS and nitrosative species (Li et al., 2008). HBOT treatment following stroke has been shown to reduce the levels of pro-oxidative enzymes such as malondialdehyde and to increase the antioxidant activity of CAT and SOD (Li et al., 2008). Other studies have found reduced stroke-generated ROS, such as hydroxyl free radicals in the striatum following HBOT therapy (Yang et al., 2010). Complex effects of HBOT on nitric oxide synthase have also been implicated in its antioxidant protective properties (Zhou et al., 2012). The counterintuitive effects of HBOT on reducing oxidative damage may result both from our incomplete understanding of HBOT mechanisms and from variations in experimental pressure and duration of treatment sessions. Moderate treatment protocols may simultaneously induce a degree of oxidative stress yet compensate for this through other antioxidant mechanisms. Regardless, additional studies into the intricacies of HBOT’s effects on oxidative pathways are necessary.3.3 Anti-inflammatory effectsRunaway inflammation is recognized as a cornerstone of stroke secondary cell-death pathology, and its sequestration is known to improve functional outcomes (Borlongan et al., 2012). General markers of inflammation, such as tumor necrosis factor alpha, have been observed to decrease in HBOT-treated animals following stroke (Yu et al., 2015), while specific subpopulations of immune cells, such as CD40+ microglia, have also been demonstrated to decline following HBOT (Lavrnja et al., 2015). Inhibition of leukocyte accumulation within the ischemic area was found in HBOT-treated animals and was attributed to a reduction in the levels of inflammatory chemokines (Rink et al., 2010). Similarly, a study found that HBOT reduced myeloperoxidase activity – an indirect measure of inflammatory response – and inhibited neutrophil infiltration (Miljkovic-Lolic et al., Liska et al.Page 5Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

2003). The COX-2 signaling pathway has been proposed as a possible underlying mechanism of the HBOT-mediated reduction in inflammation (Cheng et al., 2011).3.4 Additional neuroprotective mechanismsA plethora of molecular pathways have been shown to be modulated by HBOT, which contribute to reduction in apoptosis and preservation of neural tissue. Many of these are intimately connected with the mechanisms described in this section, as neuronal apoptosis is an endpoint outcome commonly resulting from ROS, metabolic restriction, and inflammatory response; however, discrete pathways may also contribute to the protective effects of HBOT. Among those reported are a reduction in HIF-1(Sun et al., 2008), decreased cortical and hippocampal caspase-3 (Calvert et al., 2003), increased growth factors such as GDNF and NGF (Zhang et al., 2012), and mitochondrial regulation (Lou et al., 2006). Finally, direct effects on glial cells have been suggested to assist in preserving susceptible neurons (Gunther et al., 2005).4.0 Implications of HBOT in Other Neurological and Non-neurological Conditions4.1 HBOT in acute and chronic TBIA large number of studies have demonstrated the safety and effectiveness of HBOT in diverse models of traumatic brain injury (TBI), especially within the acute phase (Zhang et al., 2012; Lim et al., 2013; Wee et al., 2015; Lim et al., 2017). One such study evaluated HBOT (2.8 ATA, 45 min, twice a day for three consecutive days) initiated 3 hours after a dynamic cortical deformation model of TBI, and revealed significant histopathological alterations indicating HBOT effectiveness (Vlodavsky et al., 2006). In another study, mice with moderate closed head weight drop traumatic brain injury (mTBI) treated with HBOT (2 ATA, 60 min, four consecutive days) initiated at either 3 hours or 7 days post-injury exhibited significant recovery of learning and cognitive abilities compared to non-treated controls, displaying performance comparable to sham mice (Baratz-Goldstein et al., 2017). Despite wide-ranging variations in pre-clinical and clinical HBOT protocol, a meta-analysis of its clinical application in acute TBI revealed legitimate and consistent effectiveness in conferring neuroprotection (Wang et al., 2016). Clinical evaluations have revealed the potential of HBOT to confer neurorestorative effects in the chronic TBI brain as well; a recent study which initiated HBOT at 6 months to 27 years post-injury in human patients found upregulated angiogenesis and cerebral perfusion associated with improvement in memory, executive functions, information processing speed, and global cognitive scores as measured by an objective computerized exam (Tal et al., 2017). Additional studies of delayed HBOT for chronic TBI have supported these findings (Harch et al., 2012; Boussi-Gross et al., 2013; Tal et al., 2015; Harch et al., 2017), while other studies have provided discrepant results, such as HBOT (1.5 ATA, 90 min) given 60 days post-TBI for 15 consecutive days resulting in no beneficial effects (Yang et al., 2014).Liska et al.Page 6Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

4.2 HBOT in spinal cord injurySeveral studies have demonstrated that spinal cord injuries (SCIs) are exacerbated more by secondary injury response mechanisms than by the primary insult (Geng et al., 2015). Secondary cell death mechanisms initiate a cascade of biomolecular events inducing reactive oxidative damage, astrocytic glial scarring, and infiltration of glia, lymphocytes, activated monocytes, and phagocytic macrophages, which may be amenable to HBOT (Oyinbo, 2011). Interestingly, HBOT experimental studies have shown positive therapeutic effects in SCI by providing a neuroprotective microenvironment that decreases anoxic conditions and enhances neuronal regeneration (Wang et al., 2014). For example, a recent report found significant positive alterations in oxidative pathway enzymes, apoptotic markers, and inflammatory mediators (Shams et al., 2017). Other studies have found preservation of BBB integrity following SCI and reversal of motor deficits (Sun et al., 2017). Patients with SCI treated with HBOT exhibited significant improvement of neurological function compared to control group patients, as well as preservation of various neuron physiological functions, such as evoked potential amplitude and conduction velocity (Tan et al., 2018). Postulated mechanisms of action of HBOT in SCI mirror those to be discussed later, but include increased expression of vascular endothelial growth factor (VEGF), axonal regeneration, and decreased apoptosis (Fu et al., 2017).4.3 HBOT in other pathological contextsVarious disease pathologies that involve molecular cascades accompanied by oxidative stress, inflammation, and ischemia have found therapeutic effects from HBOT (Daruwalla et al., 2006; McDonagh et al., 2007; Danesh-Sani et al., 2012; Borab et al., 2017). Patients with maladies involving tissue hypoxia, including diabetic ulcers, have been examined as HBOT candidates (Londahl, 2012; Londahl, 2013), and acute coronary syndrome benefits from HBOT (Shuvy et al., 2013; Bennett et al., 2015). In addition, disease-specific mechanisms can underlie HBOT effectiveness, such as the antimicrobial properties of HBOT for necrotizing soft tissue infections (Bhutani et al., 2012) or increased gas-dissolution for air embolisms (Perez et al., 2017). Psychological symptoms, such as post-traumatic stress disorder (PTSD) and post-concussive syndrome, which may develop secondary to brain injury, have also displayed favorable responses to HBOT (Boussi-Gross et al., 2013). One study found a reduction in PTSD symptoms following 40 sessions of HBOT (1.5 ATA, 60 min) over the course of 30 days (Harch et al., 2009; Harch et al., 2012). Treated subjects experienced improvements in cognitive function, decreased anxiety/depression, and improved cerebral vascular blood flow in the white matter region (Harch et al., 2017). Finally, spontaneous physiological abnormalities including autism spectrum disorders have been reported as amenable to HBOT (Rossignol et al., 2006), although the evidence for these claims is controversial (Xiong et al., 2016). In summary, apparent from the extensive research into HBOT for non-stroke disorders is its consistent safety profile when delivered within normal protocols for appropriate disease indications.5.0 Pre-clinical Findings with HBOT Preconditioning for StrokeA growing body of evidence indicates the therapeutic potential of HBOT delivered prior to ischemic onset (Hu et al., 2016). The introduction of hyperoxia to a healthy brain induces Liska et al.Page 7Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

mild oxidative stress, enabling endogenous cells to develop a greater tolerance to a future insult. Concerns regarding the efficacy of HBOT following stroke have been raised due to oxygen’s role in pathways associated with worse outcomes following stroke, such as glutamate-induced excitotoxic cell death (Singhal et al., 2002), as well as general lack of consistent and effective clinical HBOT treatment protocols. However, HBOT preconditioning could circumvent these concerns as mild oxidative stress is not coupled with more significant cerebrovascular events that initiate various apoptotic pathways; rather, HBOT conditions the cells to withstand future cerebrovascular events and their accompanying oxidative damage (Ostrowski et al., 2016). Patient populations at a particularly high risk for stroke (such as those with combinations of key factors such as morbid obesity, hypertension, low physical activity, diabetes mellitus, smoking, history of stroke, etc.) may benefit from preemptive HBOT. Similarly, patients who are diagnosed with carotid artery plaque are considered high-risk patients and could be candidates for preconditioning strategies to minimize damage from potential future strokes (Zhang et al., 2017). Indeed, as the ability of clinicians to accurately predict probable stroke cases advances via imaging techniques, such as magnetic resonance T2 mapping (Chai et al., 2017), preconditioning therapies, in particular HBOT, will undoubtedly increase in value.The first published pre-clinical study of HBOT preconditioning in ischemic stroke reported that this strategy conferred ischemic tolerance and prevented neuronal death within the hippocampus of the gerbil brain following forebrain ischemia inflicted 48 hours after the final session (Wada et al., 1996). Subsequent studies showed that HBOT was protective against transient, but not permanent, stroke in a dose-dependent manner, and that a regimen of 5 treatments (2.5 ATA, 1 hour) over consecutive days was more effective than 3 treatments over consecutive days in rescuing functional deficits when initiated 24 hours before transient middle cerebral artery occlusion (MCAO) (Xiong et al., 2000). Independent laboratories have supported these findings, with one study, for example, demonstrating that HBOT (2.5 ATA. 1 hour, twice daily) conferred neurological and histopathological protection from MCAO inflicted 24 hours after the final session (Li et al., 2008). Other investigators have opted for more aggressive treatment protocols – such as 3.5 ATA for 1 hour, five consecutive days – finding significant histopathological signals of neuroprotection following forebrain ischemic stroke inflicted 12 hours after the final HBOT session (Yamashita et al., 2009). The preemptive “therapeutic window” of HBOT has also been explored, with HBOT delivered 24 hours before injury bestowing neuroprotection, but not at 72 hours (Hirata et al., 2007). Importantly, the length of this window may be a function of the intensity and number of HBOT sessions delivered prior to injury.The mechanisms underlying the effects of HBOT undoubtedly involve several pathways that work in parallel, or independently, to induce preconditioning in the brain (Francis et al., 2017). While the mechanisms of post-injury HBOT were mentioned previously, in the following sections we present a detailed overview of the preclinical studies describing the mechanisms of action proposed to confer therapeutic protection for HBOT delivered before injury onset.Liska et al.Page 8Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

5.1 Preparation for oxidative stressThe chief mechanism of action for HBOT implicated to confer neuroprotection appears to be its action as an oxidative preconditioning agent (Gao et al., 2017). Prolonged hypoxia contributes to oxidative stress, antioxidant system imbalance, and eventual tissue injury. Preconditioning with HBOT can exert a protective role by priming brain tissue for oxidant stress, making it less susceptible to stroke-induced injury mechanisms. It is widely accepted that increased production of ROS and reactive nitrogen species (RNS), such as peroxynitrite or NO2, makes a major contribution to the development of CNS oxygen toxicity. Cells have evolved several defense strategies against ROS involving antioxidant enzymes, including superoxide dismutase (SOD) to scavenge superoxide, catalases (CAT) and peroxidases to break down hydrogen peroxide, and glutathione S-transferase to neutralize lipid peroxides, as well as their auxiliary enzymes, glutathione reductase (GRX) and glucose-6-phosphate dehydrogenase (G6PD). Low levels of ROS stimulate adaptive responses by increasing the cellular activity of these enzymatic antioxidants, while pathological levels of ROS, such as those produced under conditions of hyperoxia, can overwhelm the antioxidative capacity of the cells and cause oxidative injury. This oxidative stress manifests as protein oxidation, DNA damage with increased mutational rates, and lipid peroxidation, resulting in membrane damage, metabolic perturbation and death (Pisoschi et al., 2015). HBOT produces an elevated O2 partial pressure and increased mitochondrial generation of H2O2, elevating ROS production (Hu et al., 2016). However, several lines of investigation have shown that HBOT induces an initial oxidative stress that acts as a trigger mechanism prompting anti-oxidative responses (Gao et al., 2017).In a model of focal cerebral ischemia, HBOT preconditioning induced an increase in the activity of SOD and CAT in the brain tissue associated with decreased mortality rate, improved neurological recovery, and lessened neuronal injury (Li et al., 2008). In addition, malondialdehyde (MDA) content, a marker of lipid peroxidation and oxidative stress, decreased in the ischemic penumbra and hippocampus (Li et al., 2008). Since HBOT preconditioning stimulates ROS production, one possible explanation is that modest levels of ROS stimulate compensatory increases of CAT and SOD, which scavenge excessive ROS and attenuate the lipid peroxidation following stroke (Li et al., 2008). Moreover, in a spinal cord ischemia animal model, HBOT preconditioning similarly increased CAT and SOD activities, with the administration of a CAT inhibitor (3-amino-1,2,4-triazole) before ischemia, attenuating the spinal cord ischemic tolerance induced by HBOT preconditioning (Nie et al., 2006). In addition, administration of a free radical scavenger, dimethylthiourea, before preconditioning reversed the increased activities of both enzymes in spinal cord tissue (Nie et al., 2006). The results indicate that an initial oxidative stress upregulates the antioxidant enzyme activities, playing an important role in the formation of the tolerance against ischemic injury by HBOT preconditioning.Repeated exposure to non-convulsive HBOT provides protection against central nervous system (CNS) oxygen toxicity by decreasing levels of GRX and G6PD, and conferring a significant increase in glutathione peroxidase (GSH-Px) activity, as well as by increasing glutathione S-transferase (GST) activity (Arieli et al., 2014). G6PD catalyzes the oxidation of glucose to generate NADPH from NADP+ while NADPH-oxidase catalyzes the Liska et al.Page 9Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

production of superoxide by oxidation of NADPH (Arieli et al., 2014). Hence, by downregulating G6PD activity, HBOT may indirectly reduce oxidative stress. In addition, the decrease of G6PD activity is associated with a downregulation in the activity of GRX and an increase in GSH-Px, indicating that HBOT may upregulate antioxidants and downregulate pro-oxidant enzymes.In the healthy brain, HBOT induces an increase of heat shock proteins (HSPs), which play important roles in cellular repair and protective mechanisms. HBOT preconditioning has been shown to upregulate HSP70 specifically (Ni et al., 2013), which exerts protective effects including prevention of protein aggregation, refolding of partially denatured proteins, reduction of inflammatory responses, and inhibition of apoptosis (Brown, 2007). Moreover, in vitro studies have shown that HBOT preconditioning protects neurons against oxidative injury and oxygen-glucose deprivation (OGD) by upregulating HSP32 expression (Li et al., 2008; Huang et al., 2014). HSP32, also named heme oxygenase-1, degrades heme into three products: carbon monoxide (CO), ferrous iron, and biliverdin. Free heme is produced mainly through the oxidation of hemoproteins, including hemoglobin, myoglobin, and neuroglobin. In the center of heme is a Fe atom that can react with H2O2 and gives rise to toxic hydroxyl radicals. Catalysis of heme by HSP32 produces ferritin release, and its accumulation provokes iron sequestration and thus may provide protection against oxidative damage (Li et al., 2008; Huang et al., 2014). In addition, ROS and NO are two well-established inducers of HSP32; of note, HBOT-induced HSP32 expression is mediated via the ROS/p38 MAPK/Nrf2 pathway and by MEK1/2/Bach1-mediated negative regulation (Huang et al., 2016). Oxidative stress resulting in free radical generation should encourage HSP expression, as these studies confirm. However, a study showed no induction of HSP72 expression within peripheral blood mononuclear cells (PBMC) following a single HBOT exposure in healthy males, indicating the importance of cell-specific response to HBOT (Vince et al., 2010).Another protective effect of HBOT preconditioning against oxidative stress may involve expression of many Nrf2-regulated antioxidant genes. The Nrf2 signaling pathway has the potential to activate over 200 antioxidant and cytoprotective genes (Srivastava et al., 2013). HBOT preconditioning was shown to increase the levels of Nrf2 and enhance some of its target genes such as key proteins for intracellular GSH synthesis and transit (GST, GCL, cGT and MRP1), molecular chaperones (HSP32 and HSPA1A), and anti-oxidative enzymes (SOD1, GST) (Xu et al., 2014; Huang et al., 2016; Perdrizet, 2016; Xue et al., 2016; Zhai et al, 2016). The neuroprotective mechanism of HBOT preconditioning is also mediated by upregulating SirT1 expression in at least three different ways: (1) upstream regulation for fasting-induced activation of the Nrf2 pathway by affecting the activity of the PPAR-/PGC1-1 complex that binds to Nrf2 promoter and activates its expression; (2) inhibition of apoptosis by increasing the protein expression of anti-apoptotic Bcl-2, decreased pro-apoptotic cleaved caspase-3, deacetylating p53; (3) upregulation of FoxO, promoting the expression of SOD and CAT in response to oxidative stress (Zeng et al., 2012; Yan et al., 2013; Bian et al., 2015; Xue et al., 2016; Ding et al., 2017).Exposure to HBOT is associated with increased levels of nitric oxide (NO) (Goldstein et al., 2006; Liu et al., 2008; Arieli et al., 2014). NO acts as an important neurotransmitter and plays a dual role in both neuroprotection and neurotoxicity depending on the NO synthase Liska et al.Page 10Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

(NOS) isoform, the cell type by which it is produced, as well as the temporal stage after ischemic onset (Chen et al., 2017). Immediately after brain ischemia, NO release from endothelial NOS (eNOS) is protective mainly by promoting vasodilation; however, after ischemia develops, NO produced by overactivation of neuronal NOS (nNOS) and expression of iNOS both contribute to brain damage. While nNOS-derived NO decreases neurogenesis, NO produced by eNOS and iNOS seems to stimulate it (Sawada et al., 2009). NO, as a vasodilator of cerebral vessels, can increase tissue oxygenation, yet may also increase the delivery of ROS to tissue. In addition, NO can combine with oxygen radicals to form the potent oxidant peroxynitrite and induce nitrosative stress. However, it was shown that HBOT preconditioning has a protective effect involving alterations in the enzymatic activity of the antioxidant system and lower levels of peroxynitrite mainly in the hippocampus (Arieli et al., 2014).The increase of eNOS and nNOS mRNA/proteins in hypothalamus and hippocampus, as well as the elevated NO, may enhance the sensitivity to convulsions after repeated HBOT exposures, potentially leading to seizures during the subsequent oxygen exposures (Liu et al., 2008). Interestingly, the increase of Mn-SOD, CAT and Bcl-2 and the reduction of apoptosis seem to be mediated by NO, because the neuroprotective effect of HBOT preconditioning is abolished by a nonspecific NOS inhibitor, L-NAME (Wang et al., 2009). These results suggest that NO after HBOT preconditioning exerts both neuroprotection and neurotoxicity, indicating that further studies are needed to better understand the role of NO in HBOT preconditioning.5.2 Reduction of apoptosis, activation of autophagy, and promotion of cell survivalROS can react with macromolecular components and induce the cells to undergo necrosis or apoptosis. Inhibition of thioredoxin reductases (TrxR), one of the major redox systems in cells involved in the control of cellular redox balance, has been shown to result in generation of ROS and induced cell apoptosis in neuronal cell lines (Seyfried et al., 2007). In a post-traumatic stress disorder (PTSD)-induced rat model, HBOT preconditioning upregulated the expression of TrxR-1 and TrxR-2 mRNA in the hippocampus concurrent with a reduction in neuronal apoptosis and preserved viable neurons (Peng et al., 2010). Further evidence demonstrated that HBOT preconditioning lessens apoptosis via mitochondrial pathway modulation. In particular, a decrease in the activity of capase-3 and -9, and reduced cytoplasm cytochrome c levels were shown to upregulate the ratio of Bcl-2 and Bax proteins associated with reduced brain edema, decreased infarction volume, and improved neurological recovery (Li et al., 2008; Li et al., 2009; Wang et al., 2010; Lu et al., 2012; Lu et al., 2013). Moreover, the reduction in apoptosis was associated with Mn-SOD and CAT increase and elevated NO after HBOT preconditioning (Wang et al., 2009). Additionally, HBOT preconditioning reduces early apoptosis and apoptosis progression through induction of BDNF and suppression of p38/MAPK phosphorylation (Ostrowski et al., 2008; Yamashita et al., 2009). As mentioned above, HBOT preconditioning can limit apoptosis through the upregulation of SirT1 expression, which increases expression of anti-apoptotic Bcl-2, decreases pro-apoptotic cleaved caspase-3, and deacetylates p53 (Yan et al., 2013).Liska et al.Page 11Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

Interestingly, ROS also regulate starvation-induced autophagy, which is clearly a survival mechanism, partly through the class III phosphoinositide 3-kinase pathway (Wang et al., 2010). It was shown that HBOT preconditioning significantly increases the level of protein expression of LC3-II and Beclin 1 and induces autophagosome formation in the ischemic penumbra following ischemia in rat brain (Yan et al., 2011). Therefore, autophagy can be activated by ROS and may confer neuroprotection. Another mechanism by which HBOT preconditioning can promote cell survival is by reducing matrix MMP-9 activity/tissue expression, which plays a deleterious role after global cerebral ischemia. HBOT preconditioning suppressed post-ischemic MMP-9 activity, CA1 cell damage, and improved functional performance (Ostrowski et al., 2010).Finally, HBOT preconditioning can promote proliferation and counter cell loss through different mechanisms including activation of the Wnt signaling pathway, secretion of vascular endothelial growth factor (VEGF) and upregulation of HIF-1 (Wang et al., 2007). It was shown that the levels of VEGF, VEGFR2, Raf-1, MEK1/2, and phospho-extracellular signal-regulated kinase (ERK) 1/2 protein were boosted by HBOT, which was associated with NSC proliferation and migration to the lesion area, and improved neurological function (Yang et al., 2017).5.3 Immunosuppression and immunopreparationReducing and preventing aberrant inflammation is another prominent mechanism of HBOT preconditioning (Hu et al., 2016). HBOT preconditioning was shown to reduce the expression of neurotoxicity microglia alongside a decrease in TNF- and neuronal degeneration, all of which is connected with reduced cerebral edema and amelioration of motor dysfunction after intracerebral hemorrhage (Yang et al., 2015). Moreover, HBOT preconditioning reduced cyclooxygenase-2 (COX-2) expression (which is involved in post-ischemic neuroinflammation), increased the level of surviving neurons in the CA1, attenuated post-operative brain edema, and improved neurological outcomes after global ischemia and surgical brain injury (Jadhav et al., 2009; Cheng et al., 2011). Preconditioning with HBOT also regulates the expression of Osteopontin (OPN), which was shown to reduce the expression of interleukin (IL)-1/nuclear factor--gene binding (NFB) and to augment protein kinase B (Akt) (Hu et al., 2015). HBOT preconditioning could significantly mitigate cognitive impairment and preserve other physiological functions via reduction of systemic and hippocampal pro-inflammatory cytokines and caspase-3 activity (Gomez et al., 2012; Sun et al., 2014).5.4 Preservation of blood-brain barrier, edema minimization, and angiogenesisHBOT preconditioning has been shown to attenuate brain edema and improve neurological outcomes following surgical brain injury (SBI), ischemic and hemorrhagic stroke, TBI and high altitude exposure (Qin et al., 2007; Hu et al., 2008; Jadhav et al., 2010; Lin et al., 2012; Soejima et al., 2012; Soejima et al., 2013, Fang et al., 2015; Hu et al., 2015; Guo et al., 2016). HBOT preconditioning may reduce edema and protect the BBB by suppressing the inflammatory response. It was shown that HBOT preconditioning decreased both infarction and hemorrhage volumes, and improved neurobehavioral function through reduced expression of the NLRP3 inflammasome and its downstream targets after hemorrhagic Liska et al.Page 12Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

transformation (Guo et al., 2016). Moreover, HBOT preconditioning reduces cerebral vasospasm and stabilizes the blood–brain barrier (BBB) by increasing OPN expression in the brain, inhibiting IL-1/NFB, and suppressing MMP-9 (Hu et al., 2015). In support of this, some studies showed that HBOT preconditioning improved neurological deficits and reduced hemorrhagic volume via decreasing HIF-1 and its downstream MMP-2 and MMP-9 (Hu et al., 2008; Ostrowski et al., 2008; Soejima et al., 2013). In addition, it was shown that HBOT preconditioning may depend on the induction of MMP-9 in the pre-ischemic phase and may be in part mediated by exhaustion of MMP-9 stores in cerebral tissues (Ostrowski et al., 2008).HBOT preconditioning-induced HSP-70 overexpression in the hippocampus can significantly attenuate brain edema, cognitive deficits, and hippocampal oxidative stress (Lin et al., 2012). It was indicated that HBOT preconditioning can induce cytoprotective effects on human microvascular endothelial cells via upregulation of Nrf2 and HSP32 or heme oxigenase-1 (Godman et al., 2010). However, more recently it was shown that HBOT improved neurological deficits, infarction volume, BBB disruption, and hemorrhagic transformation without including the activation of these proteins in a focal cerebral ischemia model (Soejima et al., 2012).Reduced intracerebral edema may also be achieved with HBOT preconditioning by downregulating the expression of aquaporin 4 (AQP-4), a key factor that effects the water and electrolyte balance in the CNS, thus impeding intracerebral hemorrhage and protecting neural tissue (Fang et al., 2015). On the other hand, it was shown that levels of both AQP-4 and VEGF were significantly increased in cultured astrocytes after HBOT preconditioning (Wang et al., 2016). These findings suggest that HBOT preconditioning is also able to promote transient and regulated BBB opening, which may contribute to the induction of cerebral ischemic tolerance as well as representing a possible strategy for promoting drug transport into the CNS (Wang et al., 2016). In addition, it was shown that preconditioning with HBOT protects against brain edema formation following intracerebral hemorrhage by activation of the p44/42 MAPK pathway, whose activation has been linked to ischemic tolerance (Qin et al., 2007). Changes in tight junction protein (TJP) expression can lead to the loss of BBB integrity and BBB breakdown. Interestingly, it was shown that HBOT preconditioning protected the integrity of the BBB in an in vitro model through modulation of occludin and ZO-1 expression under hypoxic conditions (Hao et al., 2016).Finally, HBOT preconditioning may exert protective effects on energy metabolism and tissue perfusion by 1) stabilizing the glucose level; 2) decreasing the lactate/pyruvate ratios and glycerol in the peri-infarct area; 3) inhibiting the increase of the glutamate level; and 4) upregulating Ang-2, which is associated with increased microvessel density in the penumbra, reduced brain injury, and improved neurological function after focal cerebral ischemia (Gao-Yu et al., 2011; Duan et al., 2015).5.5 Considerations for HBOT preconditioning protocolsThe constituent components of HBOT (hyperoxia and hyperbaricity) are critical for the induction of tolerance against ischemic injury since simple hyperbaricity (2.5 ATA, 21% O2) did not induce ischemic tolerance (Dong et al., 2002). Interestingly, there is a close link Liska et al.Page 13Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

between hyperoxia and hypoxia (Mik, 2011). HBOT and hypoxic preconditioning elicit similar preconditioning efficacy in neonatal brain, but invoke different defenses against oxidative stress (Freiberger et al., 2006). Usually, the HBOT preconditioning is carried out at 2–3 ATA and the duration of each exposure ranges from 60 to 90 minutes (Theodoraki et al., 2011; Liu et al., 2012; Losada et al., 2014). The interval of 24 hours is the most commonly applied in HBOT preconditioning (Theodoraki et al., 2011; Liu et al., 2012). Serial exposition for 3 or 5 days at HBOT preconditioning can induce ischemic tolerance in a dose-dependent manner (Xiong et al., 2000; Dong et al., 2002). Interestingly, HBOT-induced neuroprotection against ischemic injury appears to have a time window, showing protection at 6 h, 12 h and 24 h pre-stroke, but not at 72 h (Hirata, 2007). However, neuroprotection induced by preconditioning consists of biphasic time windows defined by immediate and delayed preconditioning effects (Yokobori et al., 2013; Hu et al., 2016). Immediate preconditioning is observed within 1 h after the preconditioning stimulus and is characterized by transient changes in activity of ion channels, secondary messengers, and enzyme activity (Yokobori et al., 2013). Delayed preconditioning consists of intracellular changes that develop more slowly and manifest as long-lasting alterations in gene expression and protein expression profiles (Yokobori et al., 2013).6.0 HBOT-primed Stem Cells as a Promising TherapyPresent within discrete niches of the adult brain, stem cells exert neuroprotective and neurorestorative effects following stroke by migrating to infarcted and damaged regions whereby they utilize multi-pronged therapeutic tactics to confer their benefits (Sullivan et al., 2015). Among the mechanisms employed by these undifferentiated cells are bystander effects, such as secretion of trophic factors, anti-inflammatory molecules, apoptotic pathway regulators, and angiogenic factors (Napoli et al., 2018) and, to a much lesser extent, direct cell replacement of damaged neural tissue (Stonesifer et al., 2017). Unfortunately, the ability of unaided endogenous stem cells to initiate robust recovery following stroke is extremely limited. In this light, therapeutic modalities that enhance the brain’s intrinsic reparative capacity are highly attractive. A growing body of literature supports enhancing the effects of HBOT on endogenous stem cells as a prominent mechanism of action in the post-stroke brain (Thom et al., 2006). This knowledge – in addition to supporting the previously discussed concepts of HBOT as a standalone therapy for stroke or prophylactic treatment – indicates a possible role for HBOT in conditioning of stem cells prior to transplantation. The following sections will discuss the effects of HBOT on stem cells, and how these effects may be lent favorably to a stem cell preconditioning paradigm.6.1 HBOT effects on endogenous and transplant stem cell populationsHBOT exerts dynamic effects on various populations of stem cells in vivo. Perhaps most prominently, HBOT enhances the total number of available endogenous stem cells by increasing the quantity of circulating stem cells in a pressure-sensitive manner (Thom et al., 2006; Dhar et al., 2012; Heyboer et al., 2014) and upregulating neural stem cell proliferation within the neurogenic niches of the adult brain (Yang et al., 2008). This phenomenon has been demonstrated in multiple injury models including heightened sub-ventricular zone (SVZ) and sub-granular zone (SGZ) proliferation following ischemic brain damage (Yang et Liska et al.Page 14Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

al., 2008; Wei et al., 2015), increased neural stem cell proliferation in the piriform cortex in the vascular dementia brain (Zhang et al., 2010), and upregualtion of both neural stem cell proliferation (Yang et al., 2017) and circulating stem cells (Shandley et al., 2017) following TBI. Moreover, this effect has been observed in non-oxy-injured animals (Heyboer et al., 2014), insinuating the potential for therapeutic effectiveness independent of pathological idiosyncrasies, as well as for pre-injury applications. In addition to encouraging proliferation of neural stem cells, HBOT promotes their migration to areas of injury, as demonstrated in a rat model of TBI (Yang et al., 2017). Furthermore, of the various stem cells which are released into circulation following HBOT, particular subsets – such as vasogenic endothelial progenitor cells (EPCs) – may enact particularly potent effects in the stroke brain (Thom et al., 2011). Paired with the mechanisms described in Sections 3 and 5, the presence of heightened stem cell numbers in the periphery and brain likely contribute to the therapeutic benefits of HBOT observed in the stroke brain.Of great interest are the mechanisms by which HBOT increases available stem cells within the body. The circulating stem cells released following HBOT originate from the bone marrow, and their departure into circulation is largely attributed to heightened nitric oxide synthesis by eNOS (Heyboer et al., 2014). Experiments using eNOS-knockout mice and nitric oxide synthase inhibitors support this concept (Thom et al., 2006). The mechanisms underlying HBOT-induced upregulation of cerebral neurogenesis, however, are more elusive and likely multi-factorial. A number of key signaling molecules and growth factors modulated by HBOT have been identified and may be responsible for the increase (Mu et al., 2011). Among these, HBOT is known to exert a stabilizing effect on HIF-1, a transcription factor specifically activated by hypoxia, slowing its degradation by the prolyl hydroxylase pathway (Milosevic et al., 2009) and allowing for increased activation of the Wnt/-catenin signaling pathway, which has been directly linked to neural stem cell proliferation (Qi et al., 2017). Supporting this, Wnt-3 expression was observed to increase substantially in HBOT-treated ischemic-hypoxia neonate rats in parallel with elevated SVZ neurogenesis (Wang et al., 2007). Particularly interesting, studies have reported that high levels of ROS within proliferative NSCs may serve secondary messenger functions which contribute to their self-renewal, and that pharmacological inhibition of ROS reduces their proliferative capacity (La Belle et al., 2011). Thus, an HBOT-induced increase in ROS levels may increase NSC levels by enhancing signaling pathways which are intrinsically linked to NSC survival and proliferation.HBOT may also promote neural stem cell proliferation via upregulation of key regulatory molecules such as vascular endothelial growth factor (VEGF) (a downstream target of HIF-1), its receptor (VEGFR2) (Yang et al., 2017), ERK (Jiang et al., 2015), and CREB (Zhu et al., 2004; Mendoza-Paredes et al., 2008) – all of which play roles in neurogenic pathways (Lu et al., 2011). HIF-1 induces the expression of hundreds of gene products in response to hypoxia or ischemia involved in processes such as angiogenesis, glycolysis, inflammation, proliferation and growth, which collectively promote cell survival and neuroprotection after brain injury (Milosevic et al., 2009; Hu et al., 2016). It was shown that erythropoietin (EPO), another target gene of HIF-1, was upregulated in the cerebral cortex and hippocampus and prevented changes to BBB permeability, decreased brain edema, reduced infarction volumes, and improved neurobehavioral outcome after HBOT (Gu et al., Liska et al.Page 15Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

2008; Peng et al., 2008). Conversely, it has also been shown that a systemic reduction in EPO levels by HBOT promotes bone marrow homing and engraftment after allogeneic umbilical cord blood (UCB)-derived hematopoietic stem/progenitor cell (HSPC) transplantation (Aljitawi et al., 2016). In addition, the increased nuclear expression of HIF-1 following HBOT is associated with increased expression of CXCR4 (Chen et al., 2017). A recent study showed that the HIF-1-CXCR4 pathway can promote proliferation of neural crest stem/progenitor cells (NCSCs) by decreasing nuclear expression of the cyclin-dependent kinase inhibitor CDKN1A (p21CIP1/WAF1) and by SDF-1/CXCL12 signaling mediated by receptor CXCR4 under hypoxia (Chen et al., 2017). Moreover, after HBOT, increased cytoplasmic expression of TPM1 and decreased nuclear expression of TP53 and CDKN1A resulted in decreased apoptosis and increased proliferation of NCSCs (Chen et al., 2017). Finally, HBOT-induced vascularization may increase neurogenesis by providing metabolic support to under-perfused neurogenic areas (Tal et al., 2017).6.2 HBOT and exogenous stem cellsA careful analysis of combining HBOT with stem cell transplantation offers insight into potential underlying mechanisms and interplay between the two treatments. This combinatorial approach has been studied in a number of neurological and non-neurological contexts including TBI (Zhou et al., 2016), SCI (Geng et al., 2015) and diabetes mellitus (Estrada et al., 2008). One study demonstrated that HBOT improves graft survival within the bone marrow, peripheral blood, and spleen following umbilical cord blood stem cell transplantation in a rodent model of whole-body irradiation injury (Aljitawi et al., 2014). Similar results were found in a model of SCI, with greater MSC graft survival found in HBOT+cell therapy animals than in cell therapy alone (Geng et al., 2015). Animals receiving the combination therapy were also observed to have an assuaged inflammatory response, with lower levels of pro-inflammatory mediators TNF-, IL-6, and IFN- at various time points when compared to animals receiving cell therapy alone (Geng et al., 2015). In a traumatic nerve crush model, transplantation of human amniotic fluid mesenchymal stem cell (AFS) delivered with adjunctive HBOT displayed synergistic effects (Pan et al., 2009). Among the improvements reported in this study were a suppressed inflammatory response in combination therapy animals, upregulated nerve regeneration metrics such as neurofilament production and s-100 expression, and, interestingly, a dramatic decrease in expression of the apoptosis marker TUNEL in the transplanted AFS (Pan et al., 2009).6.3 Effects of HBOT in vitro: potential for stem cell primingIn light of the broad effects of HBOT on endogenous and transplanted stem cells, further investigations into the effects of HBOT on stem cells in vitro are warranted. Importantly, many of the molecular signaling pathways which are discussed above (i.e. Wnt/-catenin, VEGF/VEGFR2, and CREB) are likely mediated by non-stem cell host tissue secretions. Thus, while the knowledge obtained from studies of HBOT on stem cells in vivo guides our scientific inquiries, the mechanisms, both stem cell- and non-stem cell-mediated, must be considered. Moreover, potential adverse effects of HBOT on stem cells should also be examined. Indeed, in vitro study of HBOT on stem cells has found unique effects on stem cell cultures. In contrast to the paradigm described previously of HBOT-mediated increase in Liska et al.Page 16Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

stem cell proliferation, HBOT was shown to decrease cell survival in mesenchymal stem cell (MSC) cultures (Schulze et al., 2017). The enhanced oxygen tension delivered by HBOT increases the formation of ROS and exerts oxidative stress on cells (Thom, 2009; Cheung et al., 2018). Similar results have been reported with other stem cell populations, including umbilical cord blood stem cells (Cheung et al., 2018).The therapeutic potential of HBOT priming stem cells for subsequent transplantation will also require careful evaluation, as exposure to oxidative stress in vitro may improve stem cell resiliency to oxidative stress upon transplantation (employing a similar conceptual framework as described for HBOT preconditioning in Section 5). With this in consideration, we propose that in vitro HBOT priming may have genetic, molecular, or transcriptomic effects on stem cells which increase their therapeutic potential, and further, that preliminary HBOT-induced oxidative stress may improve the resiliency of these stem cells to the harmful microenvironment of the post-stroke brain upon transplantation.This strategy of HBOT-primed stem cells has been employed in a limited fashion in other pathological contexts; human adipose-derived stem cells were demonstrated to have improved extracellular matrix-secreting capabilities following transplant into a rabbit cartilage defect model when primed with 2.5 ATA HBOT for 1 hour (Dai et al., 2015). In another study, increased O2 exposure in vitro prompted the maturation and differentiation of human embryonic stem cell-derived pancreatic progenitor cells into the desired -cell lineage (Cechin et al., 2014).Precedent evidence on priming exists for this therapeutic approach in stroke as well. Hypoxic preconditioning of stem cells (5% O2 for 24 hours) has been shown to increase graft survival post-transplantation in a hemorrhagic stroke mouse model (Wakai et al., 2016) and an ischemic stroke model (23031629), as well as to enhance stem cell migratory/homing ability (Hu et al., 2011; Lee et al., 2013; Wei et al., 2013). By preparing stem cells for the metabolic challenges posed by the microenvironment of damaged tissue, post-transplantation cell survival and function are bolstered (Wei et al., 2012). Hypoxia is a major contributor to low graft survival following post-stroke transplantation and serves as the premise for hypoxic preconditioning, yet it is not the only prevalent challenge posed to cells within the post-stroke brain. Rampant oxidative stress contributes significantly to both endogenous cell and graft cell death (Chen et al., 2011; Xing et al., 2012). As such, exposing stem cells to mild/moderate oxidative stress before transplantation may extend their survival time in a similar manner to hypoxic precondition by permitting their genetic and phenotypic acclimation to a state of oxidative stress.HBOT may also alter and enhance the stem cell secretome, boosting its effectiveness within the stroke brain. In vitro experiments have revealed that HBOT exerts a notable effect on the secretion profile of stem cells, including a number of proteins implicated in: 1) the oxidative stress response such as vasorin, thrombospondin-4, thioredoxin, heat shock protein (HSP) 90, HSP70, and gamma-glutamylcyclotransferase; and 2) various neuroprotective pathways such as peroxiredoxin, cystatin C, laminin, syndecan and thymosin-beta (Schulze et al., 2017). The increased cellular nitric oxide recorded following HBOT consequently upregulates expression of certain growth factors including VEGF and transforming growth Liska et al.Page 17Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

factor-beta 1 (TFGb1) (Venetsanou et al., 2012). Moreover, using MSC cultures, HBOT was shown to increase expression of placental growth factor (PlGF) in a dose-dependent manner, which was associated with increased MSC tubule formation and enhanced migratory ability (Shyu et al., 2008). Interestingly, HBOT has been shown to prevent differentiation of stem cells in culture (Cheung et al., 2018). Depending on the treatment strategy of transplantation (i.e. direct cell replacement paradigms such as with oligodendrocyte progenitor cell transplantation, or immunomodulation paradigms such as with MSC transplantation), the ability to promote or maintain naïve stem cell states may be an additional benefit of HBOT priming.Interestingly, HBOT-preconditioned stem cells may be an effective adjunctive to HBOT-preconditioning of the host. Given the extensive evidence supporting the neuroprotective capacity of HBOT-preconditioning on the healthy brain (see Section 5), stem cell preconditioning could also be executed in tandem to offer a readily available cell therapy option following stroke. This could present a potent dual therapy approach for patients at an exceptionally high risk for stroke, utilizing both a preemptive neuroprotective modality as well as a post hoc neurorestorative biologic.In summary, the ability of HBOT to prolong graft survival via oxidative stress conditioning, prevent premature differentiation, enhance migratory capacity, promote injury homing, upregulate trophic factors within the secretome, and encourage anti-inflammatory mediation all indicate the great potential of HBOT to advance the therapeutic efficacy of stem cells transplanted in the stroke brain alone, or in synchrony with host preconditioning therapies.7.0 Future Directions and ConclusionPreclinical studies have demonstrated the efficacy of HBOT in preserving vulnerable neural tissue and improving functional outcomes in various stroke models. A wide range of HBOT regimens have been reported as effective, with 1) pressures ranging from 2.0 ATA-3.0ATA, 2) durations lasting from 45 min-2 hrs, and 3) time of initiation ranging from immediately after stroke to 48+ hours. Unfortunately, the wide range of effective parameters has prevented a consensus regimen from being employed in clinical trials, slowing the progress of HBOT translation. Compounding this issue, human effectiveness has not been well proven due to a lack of high-quality multicenter randomized controlled trials. The successful translation of clinical HBOT will largely rest on the field’s ability to organize and implement standardization and rigorous methodology in its clinical trials. Nonetheless, the basic science field has continued to advance our understanding of the mechanisms of HBOT in the injured and healthy brain, and this has paved the way for the emergence of HBOT preconditioning paradigms. While preconditioning strategies are innovative and may hold real benefits for certain patient populations, obvious limitations exist regarding the ability to preemptively deliver stroke therapy; strokes are acute events which are unpredictable even in patients with extensive predispositions.In light of these challenges – namely, the difficulties in translating canonical HBOT therapy, and limited application of preconditioning – we propose here a novel application of HBOT as a preconditioning mechanism for stem cell transplantations (Figure 1). Based on the Liska et al.Page 18Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

extensive literature, we speculate that oxidative preconditioning of stem cell grafts via HBOT may be an effective means of increasing graft survival and optimizing graft function within the post-ischemic environment. Advancing this concept will require due diligence in verifying the genetic, epigenetic, secretome, and functional influence that HBOT exerts on specific stem cell populations, and characterizing the in vivo response of HBOT-primed versus non-primed stem cells. This therapeutic strategy may be able to harness the promising laboratory findings that have been widely reported for both HBOT and stem cell transplantation following cerebrovascular accident, offering a hybrid approach of either standalone or combinatorial pre-conditioning strategies for conferring neuroprotection in the stroke brain.AcknowledgmentsCVB is funded by NIH R01NS071956, NIH R01 NS090962, NIH R21NS089851, NIH R21 NS094087, and VA Merit Review I01 BX001407.ReferencesAljitawi OS, Paul S, Ganguly A, Lin TL, Ganguly S, Vielhauer G, Capitano ML, Cantilena A, Lipe B, Mahnken JD, Wise A, Berry A, Singh AK, Shune L, Lominska C, Abhyankar S, Allin D, Laughlin M, McGuirk JP, Broxmeyer HE. Erythropoietin modulation is associated with improved homing and engraftment after umbilical cord blood transplantation. Blood. 2016; 128(25):3000–3010. [PubMed: 27760758] Aljitawi OS, Xiao Y, Eskew JD, Parelkar NK, Swink M, Radel J, Lin TL, Kimler BF, Mahnken JD, McGuirk JP, Broxmeyer HE, Vielhauer G. Hyperbaric oxygen improves engraftment of ex-vivo expanded and gene transduced human CD34(+) cells in a murine model of umbilical cord blood transplantation. Blood Cells Mol Dis. 2014; 52(1):59–67. [PubMed: 23953010] Arieli Y, Kotler D, Eynan M, Hochman A. Hyperbaric oxygen preconditioning protects rats against CNS oxygen toxicity. Respir Physiol Neurobiol. 2014; 197:29–35. [PubMed: 24675062] Baratz-Goldstein R, Toussia-Cohen S, Elpaz A, Rubovitch V, Pick CG. Immediate and delayed hyperbaric oxygen therapy as a neuroprotective treatment for traumatic brain injury in mice. Mol Cell Neurosci. 2017; 83:74–82. [PubMed: 28690173] Bennett MH, Lehm JP, Jepson N. Hyperbaric oxygen therapy for acute coronary syndrome. Cochrane Database Syst Rev. 2015; (7):CD004818. [PubMed: 26202854] Bennett MH, Weibel S, Wasiak J, Schnabel A, French C, Kranke P. Hyperbaric oxygen therapy for acute ischaemic stroke. Cochrane Database Syst Rev. 2014; (11):CD004954. [PubMed: 25387992] Bhutani S, Vishwanath G. Hyperbaric oxygen and wound healing. Indian J Plast Surg. 2012; 45(2):316–324. [PubMed: 23162231] Bian H, Hu Q, Liang X, Chen D, Li B, Tang J, Zhang JH. Hyperbaric oxygen preconditioning attenuates hemorrhagic transformation through increasing PPARgamma in hyperglycemic MCAO rats. Exp Neurol. 2015; 265:22–29. [PubMed: 25542160] Borab Z, Mirmanesh MD, Gantz M, Cusano A, Pu LL. Systematic review of hyperbaric oxygen therapy for the treatment of radiation-induced skin necrosis. J Plast Reconstr Aesthet Surg. 2017; 70(4):529–538. [PubMed: 28081957] Borlongan CV, Glover LE, Sanberg PR, Hess DC. Permeating the blood brain barrier and abrogating the inflammation in stroke: Implications for stroke therapy. Curr Pharm Des. 2012; 18(25):3670–3676. [PubMed: 22574981] Boussi-Gross R, Golan H, Fishlev G, Bechor Y, Volkov O, Bergan J, Friedman M, Hoofien D, Shlamkovitch N, Ben-Jacob E, Efrati S. Hyperbaric oxygen therapy can improve post concussion syndrome years after mild traumatic brain injury - randomized prospective trial. PLoS One. 2013; 8(11):e79995. [PubMed: 24260334] Liska et al.Page 19Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

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Figure 1. A summary of prominent mechanisms of hyperbaric oxygen therapy for pre-stroke, post-stroke, and stem cell preconditioning applications. BBB - blood brain barrier; ICP - intracranial pressure.Liska et al. Page 30Cond Med. Author manuscript; available in PMC 2018 August 03.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

Biomolecules 2020, 10, 1279 2 of 26the intracellular space occurs during the acute phase and results in edema [6]. In the subacute phase,aberrant neuroinflammation heightens the presence of matrix metalloproteases (MMPs), which causean increase in blood-brain barrier (BBB) permeability, consequently allowing leukocytes to migrateto the damaged area of the brain and increase inflammatory activity [7]. The expression of reactiveoxygen species-generating enzymes, like NADPH oxidases (NOXs), is also implicated in increasedBBB permeability by promoting inflammation and dysfunction of endothelial tissue [5]. Other areasof the brain tissue surrounding the infarct area are vulnerable to the same exacerbating damage, butother cell phenotypes can be protected and serve as an area of focus towards monitoring the motorand cognitive functional deficits that precede a stroke [8].Hyperbaric oxygen therapy (HBOT) serves as a potential non-invasive therapy where pure oxygencan be administered in a pressurized chamber at high levels of atmospheric pressure. With increasedoxygenation, HBOT can be used to improve oxygen flow from lungs to systemic organs and can reducesecondary brain injury e↵ects, including apoptotic pathway initiation, oxidative stress, and rampantinflammation [9]. By restoring oxygen tension, HBOT has been shown to restore cellular energyproduction, stabilize cellular calcium, decrease NOXs expression, and attenuate oxidative stress [10].Mechanisms reached with hyperbaric oxygen can also be achieved at lower or normal oxygenconcentrations [11]. During the recovery stages, where oxygen levels are close to normal, HIF-1 alphasynthesis increased, and a relatively hypoxic environment existed [11]. In this setting, MMP, a hypoxicstimulus [12], and EPO production elevated as well, which was also observed in healthy humansubjects. This suggests that similar mechanisms of HBOT can be demonstrated with lower tensions ofoxygen. Additionally, HBOT has also proven beneficial in the treatment of other pathological diseases,including traumatic brain injury (TBI), spinal cord injury (SCI), and stroke [13]. Currently, thereare few treatment options available for many neurological diseases; however, experimental studiesutilizing HBOT demonstrate promising results. HBOT studies target ameliorating ischemic-relateddamage to improve the quality of life of a↵ected patients. In this review, the use of HBOT for ischemicstroke will be covered in-depth, including information on the application of HBOT preconditioning inthe stroke brain and the potential of HBOT priming for stem cell transplantation. Here, we providethe pre-clinical bases for advancing the use of HBOT as a promising target for stroke therapy.2. HBOT in Ischemic Stroke: Potential Benefits and LimitationsIn early investigations on hyperoxia following a stroke, HBOT was seen as a dangerous apparatuswith few beneficial properties and was deemed to have no therapeutic value. Nevertheless, in the last20 years, expansive research has demonstrated the ability of HBOT to reduce the severity of infarctvolume and serve as a potential treatment option [14]. However, HBOT must be administeredduring the small reperfusion window, which creates a limited opportunity for e↵ective treatment.This has been a reoccurring problem with HBOT because of the difficulty associated with determiningthe reperfusion window and extensive imaging to determine the appropriate course of action [15].Evidently, some studies indicate that delayed HBOT past the e↵ective window of treatment actuallycauses worse outcomes. This is suggested to be due to the role of ROS in glutamate-induced excitotoxiccell death [16]. Therefore, further research on the use of HBOT must be conducted and carried outsafely to prevent treatment with excess oxygen that could cause additional harm, including obstructivepulmonary disease [16].Acute ischemic stroke (AIS) serves as a primary cause of death worldwide and is characterized byan occlusion of the cerebral artery. The therapeutic treatment plans for AIS are, however, limited to date.HBOT treatment serves as an option to improve AIS-induced brain tissue hypoxia [17]. The primarygoal of acute HBOT treatment is to increase oxygen levels in the ischemic region during stroke occlusion,in pursuit of minimizing hypoxic damage. Studies have demonstrated the ability of HBOT to beused to enhance the arterial partial pressure of oxygen [18], increase the oxygen content [19], stabilizethe blood-brain barrier [20], decrease the intracranial pressure, and relieve cerebral edema. Moreover,HBOT serves as a safe practice for the treatment of acute ischemic stroke moving forward.

Biomolecules 2020, 10, 1279 3 of 26In regard to chronic ischemic stroke, HBOT demonstrates limited potential in treating chronicneurological deficits. Nonetheless, in a recent study, the efficacy of HBOT in restoring memory functionfor chronic stroke patients revealed significant memory improvement and increased brain metabolicrate [21]. Additionally, patients experienced significant improvement in memory and attentiontesting scores [22]. Studies have demonstrated that the earlier HBOT is initiated, the greater itstherapeutic e↵ectiveness [23]. Clinical trials have demonstrated consistent safety, but inconsistentefficacy. Limited studies have demonstrated the efficacy of HBOT for the treatment of chronic ischemicstroke, and the data is still unreliable for further use in patients.Another potential treatment for stroke that has been explored is the use of normobaric oxygen,which its therapeutic efficacy has been both reported and refuted [16,24,25]. The normobaric oxygenparadox (NOP) was introduced to describe a potent mechanism where Hypoxia Inducible Factor1 alpha (HIF-1 alpha) expression is regulated by oxygen [26]. Because HIF-1 alpha is responsiblefor erythropoietin (EPO) and VEGF expression, normobaric oxygen may be correlated to EPOproduction [27]. Studies hypothesize that sudden return to normoxia after a small exposure tonormobaric hyperoxia would result in a relative hypoxia state [27,28]. This, in turn, would elevateEPO production because hypoxia is a natural trigger for EPO production [26,28]. Additionally,relative hypoxia induced by oxygen gradient and NBO may induce reticulocyte production [28].Normobaric oxygen therapy increases dissolved oxygen levels within the bloodstream, but displayscontroversial neuroprotective e↵ects. Specifically, critical information regarding the optimal therapeutictime frame of NBO and its long-term e↵ects are still unclear Therefore, it does not serve the samee↵ectiveness as the HBOT for post- and pre-stroke conditions and is less of an intriguing optionfor preconditioning [16,24,25]. Nevertheless, the synergistic use of normobaric oxygen treatmentaccompanied by HBOT shows promise in reducing stroke mortality [2,29]. Recentin vivoinvestigationshave shown that HBOT and normobaric oxygen treatment may widen the limited window forthrombolytic therapy, and various neuroprotective medications [29,30].Currently, HBOT has been used as a post-stroke therapy as well as a preconditioning method.The goal of HBOT is to stimulate a hyperoxia environment during ischemic and reperfusion periodsfollowing a stroke. Additionally, HBOT may expose a patient to recurrent treatments after the initialearly treatment time-frame has passed [31]. Preconditioning treatments have focused on exposing anindividual to a mild stressor, which is supposed to enhance the brain’s resistance to future stressors [32].2.1. Preclinical Functional Outcomes of Post-stroke HBOTTypically, HBOT treatment is carried out at 2.5 atmospheres (ATA) for 60–90 min [33].The atmospheric pressure must not exceed the given 2.5 ATA during HBOT, or it could cause oxygentoxicity, increase oxidative stress throughout the body, or increase the risk of seizure activity [31].The goal of HBOT treatment is to increase the oxygen concentration in the ischemic region of the brainto minimize hypoxic damage. If administered after the initial treatment window, continuous HBOTtreatments could promote the stimulation of endogenous repair processes [34].HBOT is highly e↵ective when administered 30 to 60 min after stroke, and the potency of treatmentlessens if initiation is delayed any further. HBOT exhibits therapeutic potential in its ability to reduceinfarct volume and improve behavioral scores in patients [14,15]. Research has demonstrated di↵erenttime points at which HBOT is e↵ective, such as HBOT (2.5 ATA for 2 h) at 6 h after reperfusion [35],and HBOT (3 ATA, 1 h) at 3 and 6 h after reperfusion [36]. Additionally, single-session HBOT isreported to be e↵ective up to 18 h and 48 h after stroke [37]. In order to determine the therapeutice↵ects with HBOT at various timepoints, extensive and tailored treatment protocols are required.On the other hand, delayed treatment with HBOT (2.5 ATA, 2 h) at 6 or 24 h after stroke results in anincrease of infarct size and causes neurological deficits [38]. Little evidence has proven the e↵ectivenessof HBOT that is given more than 48 h post-stroke. One study has used one session of HBOT (3 ATA,1 h) and exhibited significant neuroprotective e↵ects when delivered 72 h post-stroke [20]. Therefore,it is imperative to deliver HBOT within the given time-frame to ensure the best outcomes.

Biomolecules 2020, 10, 1279 4 of 262.2. Clinical Results of HBOT in StrokeMany clinical trials have explored the use of HBOT, but have revealed inconclusive results [39].Various factors, including the unstable clinical status of the acute stroke patients, could be the cause forvarying results and prevent the patients from the given e↵ective treatment window that is required3–5 h after reperfusion [40]. Additionally, many treatment protocols could be di↵erent and take awide variety of patients that could hinder the ability to draw reliable conclusions and comparisonsbetween groups. It is imperative to acknowledge the fact that clinical success for HBOT has beenachieved, and the use of HBOT has been shown to lessen levels of cerebral and myocardial biomarkersand reduce the length of stay in an intensive care unit [41]. Moreover, the clinical significance of HBOTshould not be overlooked, and further research should be carried out [42]. A growing number ofclinical trials have been performed and demonstrate cerebral plasticity, as well as the ability of HBOTto restore memory function and improvement following a chronic stroke [21]. The timing of the HBOTapplication is critical. Conclusive studies have found that if HBOT is initiated earlier, it leads to greatertherapeutic e↵ectiveness [15,23]. After 12 h post-ischemia, the benefits of single treatment HBOT arereduced. However, repetitive applications of HBOT in the sub-acute stroke brain document neurogenice↵ects [43]. Studies have even demonstrated a consistent safety and the potential of using HBOT forchronic stroke patients, a breakthrough finding that was thought of only as detrimental previously.Nevertheless, contradicting results in the use of HBOT for acute and chronic stroke patients warrantfurther exploration to clear up the inconsistencies in studies.3. Unpacking Mechanisms of Action of HBOT in Stroke3.1. Physiological and Metabolic E↵ectsThe goal of HBOT is to increase perfusion and oxygenation of at-risk tissue. HBOT therapy can beused to enhance arterial oxygen saturation, and augment tissue oxygen content via enhanced cerebralmicrocirculation [18,31]. Additionally, HBOT serves a vital role in enhancing BBB stability throughMMP regulation [23], and can also decrease intracranial pressure and relieve cerebral edema [31].Secondary e↵ects of HBOT in the ischemic brain may be prevalent, due to the reduction of extracellularglutamate levels, causing neural dysfunction and excitotoxic death [44].3.2. Antioxidant E↵ectsHBOT demonstrates the ability to provide oxidative protection against stroke-induced ROSand nitrosative species [45]. This startling discovery reverts the fact that introducing high levels ofoxygen can actually induce oxidative stress and exhibits the efficacy of HBOT. Following a stroke,HBOT has been proven to reduce levels of pro-oxidative enzymes, including malondialdehydeand increase the activity of CAT and SOD [45]. Additionally, other studies have found reducedstroke-generated ROS in the striatum after HBOT therapy [46]. HBOT also plays an e↵ect on nitricoxide synthase and provides a ntioxidant protective properties [47]. However, various experimentaldesigns and di↵erent durations of treatment sessions may hinder a complete understanding of the roleof the HBOT for reducing oxidative damage. It is important to note that HBOT treatment may causeoxidative stress, but can be balanced by antioxidant mechanisms. Further studies are required todetermine the e↵ects of HBOT on oxidative pathways.3.3. Anti-Inflammatory E↵ectsAberrant inflammation is a key player in the pathogenesis of str oke, and catalyst for secondarycell-death in the brain. Interestingly, HBOT has been linked to anti-inflammatory effects in the settingof ischemic stroke. Experimental stroke studies have shown decreases in markers of inflammation,such as tumor necrosis factor-alpha and CD40+ micr oglia in HBOT-treated animals [48,49]. Althoughthe mechanism underlying the anti-inflammatory effects of HBOT has not been fully elucidated, pre-clinicalstudies showed reduced secretion of inflammatory chemokines, which inhibit the accumulation of

Biomolecules 2020, 10, 1279 5 of 26leukocytes at the ischemic ar ea [17]. Similarly, another study showed HBOT r educed myeloper oxidaseactivity and inhibited neutrophil infiltration of the injured tissue [50].3.4. Additional Neuroprotective MechanismsHBOT has been linked to many pathways that preserve neural tissue and reduce apoptosis asdisplayed in Table 1. Mechanisms mentioned previously in this section many be heavily related to theseas neuronal apoptosis is prompted via metabolic restriction, inflammation, and ROS. Undiscoveredpathways may also contribute to HBOTs efficacy. Notable findings include a lower concentrationof cortical and hippocampal caspase-3 [51], reduced HIF-1↵[52], augmented growth factor levelsof GDNF and NGF [53], and regulation of mitochondria [54]. Furthermore, HBOT has been seen todirectly modulate glial cells, consequently contributing to the protection of vulnerable neurons [55].4. Implications of HBOT in Other Neurological and Non-Neurological Conditions4.1. HBOT in Acute and Chronic TBIHBOT has been shown to be a safe and e↵ective treatment for TBI, specifically when conductedduring the acute phase [53,56–58]. Utilizing a rodent model of TBI, HBOT initiated at 3 h after corticaldeformation induction attenuated symptoms after histopathological analysis [59]. Another murinemodel of TBI o↵ered evidence that HBOT-induced cognitive and learning regenerative abilities werepreserved from 3 h after injury to 7 days [13]. While experimental protocols, such as therapeuticwindows, may vary, HBOT consistently provides neuroprotection for acute TBI [60]. Nonetheless,HBOT may also translate to functional benefits in chronic TBI as well. A human TBI-HBOT studyindicated increased cerebral perfusion and angiogenesis and ameliorated memory ability, executivefunction, information processing speed, and global cognitive scores up to 27 years’ post-injury [61].Although many studies have found similar therapeutic e↵ects of HBOT in TBI [62–65], other studieshave not supported these findings [66].4.2. HBOT in Spinal Cord InjuryOverwhelming evidence has indicated that cell death responses induced by secondary injurycontribute more significantly to SCIs than primary trauma [67]. The following mechanisms areassociated with secondary cell damage and can potentially be ameliorated by HBOT: Reactive oxidativeinjury, astrocytic glial scarring, glial penetration, and lymphocyte/activated monocyte/phagocyticmacrophage generation [68]. Importantly, HBOT engenders neuroprotection in SCI via attenuationof anoxia and heightened rehabilitation of neurons [69]. HBOT correlates with decreased oxidativeenzymes, apoptotic factors, and inflammatory agents [70]. In addition, HBOT has been shown tosafeguard BBB function and improve motor dysfunction post-SCI [71]. Notably, SCI patients whounderwent HBOT, demonstrated substantial amelioration of neurological injury and maintainedneuronal functions, such as induced potential amplitude and conduction velocity, more e↵ectivelythan the control group [72]. The mechanisms behind HBOT-induced neuroprotection may involvethe upregulation of vascular endothelial growth factor (VEGF) expression, restoration of axons,and inhibition of apoptosis [73].4.3. HBOT in Other Pathological ContextsSeveral studies have elucidated that HBOT bears curative potential in a multitude of disorders thatmanifest in oxidative stress, inflammation, and ischemic injury [74–77]. HBOT has been explored as atherapeutic regimen in patients su↵ering from diseases that entail hypoxia of tissues, such as diabeticulcers [78,79] and acute coronary syndrome [80,81]. Evidently, the therapeutic abilities of HBOT mayencompass tissue repair processes specific to the disease. For instance, HBOT imparts antimicrobialcapacity in necrotizing tissue contamination [82] and enhances gas dissolution in air embolisms [83].In addition, HBOT has shown promising results in psychological pathologies, such as post-traumatic

Biomolecules 2020, 10, 1279 6 of 26stress disorder (PTSD) and post-concussive malady, that may arise following brain damage [63].After 40 HBOT treatments (15 ATA, 60 min) conducted within 30 days, PTSD manifestations weresignificantly alleviated [62,84]. HBOT therapy ameliorated cognitive deficits, diminished anxietyand depression, and augmented blood circulation via the cerebrovasculature into the white matter [65].Additionally, spontaneous physiological irregularities like autism spectrum illnesses may be potentialtherapeutic targets for HBOT [85]; however, HBOT’s neuroprotective e↵ects in these disorders warrantfurther investigation [86]. In light of the current COVID-19 pandemic, clinical trials have exploredHBOT in infected patients experiencing extreme respiratory distress. COVID-19 infection generatesa potent cytokine storm that spurs the onset of progressive hypoxia, leading to severe respiratoryillness [2,87]. Furthermore, HBOT’s capacity to enhance oxygen flow and attenuate inflammation maybear curative e↵ects in COVID-19 patients [87]. In Wuhan, HBOT was administered to critically illCOVID-19 patients and resulted in ameliorated hypoxia, increased blood flow, and mitigated immunesystem deficits. Remarkably, these patients demonstrated substantial recovery and were released fromhospital care after 3–8 HBOT sessions [2]. In another clinical trial, COVID-19 patients demonstratedalleviated shortness of breath [87], though a significant reduction in mortality rate was not observed.Therefore, the potential of HBOT in COVID-19 treatment warrants further clinical investigation.As copious evidence suggests, HBOT similarly a↵ords a stable safety profile in non-stroke diseaseswhen it is administered properly.5. Pre-Clinical Findings with HBOT Preconditioning for StrokeHBOT preconditioning has been shown to provide therapeutic benefit in neurological diseases likeischemic stroke [42,88]. The mechanism behind the protection is attributed to the introduction of mildoxidative stress, which builds tolerance in endogenous cells to future insult. Patient populations with ahigher risk for ischemic strokes, such as those with comorbidities (e.g., obesity, diabetes, hypertension,atherosclerosis, etc.), may benefit from preemptive HBOT, and advancements in imaging techniquesthat allow for more accurate prediction of stroke risk may increase the value of preconditioningtherapies [89].The first pre-clinical study evaluating the e↵ects of HBOT preconditioning on a gerbil ischemicstroke model showed that the therapy conferred tolerance to ischemia and prevented neuronal death [90].The following studies using other animal models showed that HBOT was protective against transient,not permanent, stroke, and protection was conferred in a dose-dependent manner [91]. A treatment offive sessions (2.5 atmosphere absolute [ATA], 1 h) over consecutive days was more e↵ective than threesessions at rescuing functional deficits in rats after middle cerebral artery occlusion (MCAO) 24 h afterthe last session [91]. These results have been replicated by other studies, including one that showed thatfour sessions of HBOT (2.5 ATA, 1 h, twice a day) o↵ered neurological and histopathological protectionfrom MCAO 24 h after the last session [92]. More aggressive treatments (3.5 ATA, 1 h, five consecutivedays) have also provided significant histopathological signs of neuroprotection [93]. Other pre-clinicalstudies have explored the therapeutic window for HBOT, which suggests neuroprotection can beachieved by treatment 24 h before ischemia, but not 72 h [94]. However, it is important to note thatintensity and number of sessions may play a bigger role in treatment e↵ects, and the therapeutic windowmust be further investigated. In the following sections, we will discuss the potential mechanismsunderlying neuroprotection provided by HBOT preconditioning.5.1. Preparation for Oxidative StressHBOT primarily generates neuroprotection through its interactions with an oxidativepreconditioning factor [95]. Long-term subjection to hypoxic environments engenders drastic oxidativestrain, the disarray of the antioxidant network, and ultimate cell damage. Initial exposure of braintissue to non-lethal hypoxia via HBOT preconditioning can shield neurons from future ischemicinjury by fortifying tissue against oxidative stress. As evidence suggests, oxidative toxicity maybe spurred by ROS and reactive nitrogen species (RNS) (e.g., peroxynitrite and NO2) upregulation

Biomolecules 2020, 10, 1279 7 of 26in the CNS. Fortunately, cells can combat ROS escalation through defense mechanisms inducedby antioxidant enzymes. Superoxide dismutase (SOD) sequesters superoxide, catalase/peroxidasesneutralize hydrogen peroxide, and glutathione S-transferase o↵ set lipid peroxides. Auxiliary enzymes,such as glutathione reductase (GRX) and glucose-6-phosphate dehydrogenase (G6PD), also contributeto brain tissue defense. Although sparse numbers of ROS can be beneficial, as they promote antioxidantenzyme pathways that make up the adaptive cellular response, lethal amounts of ROS, engenderedby hyperoxia, surpass the cellular antioxidative potential and generate oxidative damage. Ischemicinjury in the brain induces oxidation of proteins, lipid peroxidation and augmented DNA mutation,leading to cell membrane impairment, disturbances in metabolism, and tissue death [96]. ROS levelsare higher with HBOT, due to increased oxygen partial pressure and upregulated H2O2producedby mitochondria [42]. Moreover, non-lethal oxidative stress spurred by HBOT serves as a protectiveprocedure, stimulating antioxidative activity [95].Anin vivoinvestigation utilizing a focal cerebral ischemia model found that HBOT preconditioningelevated SOD and CAT mechanisms in cerebral tissue, leading to increased survival rates, as wellas ameliorated neurological function and cell damage [92]. Notably, the stroke-a✏icted penumbraand hippocampus demonstrated diminished levels of lipid peroxidation and oxidative stress biomarker,malondialdehyde (MDA) [92]. In the same way, HBOT preconditioning with a spinal cord ischemiaexperimental model upregulated SOD and CAT processes. However, activation of the CAT inhibitor,3-amino-1, 2, 4- triazole, prior to ischemic stroke, abolished the beneficial e↵ects of HBOT, like spinalcord resilience to oxidative stress decreased significantly [97]. When dimethylthiourea, a free radicalscavenger, was delivered to the spinal cord ahead of HBOT, the elevated SOD and CAT activitywas eliminated [97]. Moreover, HBOT preconditioning spurs preliminary oxidative stress thatprompts antioxidative mechanisms from enzymes, leading to increased resistance of tissue to futureischemic damage.By suppressing GRX and G6PD and elevating glutathione peroxidase (GSH-Px) and glutathioneS-transferase (GST) pathways, frequent non-lethal HBOT preconditioning imparts neuroprotectionagainst oxidative damage in the central nervous system [98]. Therefore, in an indirect manner,HBOT attenuates oxygen toxicity through the repression of G6PD mechanisms. Importantly, HBOTpreconditioning bolsters antioxidative processes and dilutes enzymatic activity of pro-oxidants,as HBOT-induced diminishment of G6PD can be correlated with the truncation of GRXand the augmentation of GSH-Px activity.Under normal conditions, HBOT promotes neuronal rehabilitation and neuroprotection byupregulating heat shock proteins (HSPs), particularly HSP70 [99]. HSP70 inhibits protein build-up,restores slightly denatured proteins, attenuates inflammation, and hinders apoptosis, all of whichcontribute to neuroprotection [100]. In addition, as displayedin vitro,HBOTfortifiestheexpressionof HSP32, shielding neuronal tissue from oxidative damage, and oxygen-glucose deprivation(OGD) [92,101]. HSP32 or heme oxygenase-1 breaks down heme into carbon monoxide (CO), biliverdin,and ferrous iron. Hemoprotein oxidation, such as hemoglobin, myoglobin, and neuroglobin oxidation,engender the formulation of free heme. An iron atom lies in the middle of the heme molecule and caninteract with H2O2to form deleterious hydroxyl radicals. HSP32 catalyzes heme molecules, leading tothe generation and build-up of ferritin release, which in turn, engenders the removal of iron, therebysafeguarding tissue from oxidative injury [92,101]. Importantly, HSP32 is known to be incited by ROSand nitric oxide (NO), as the ROS/p38/MAPK/Nrf2 pathway and MEK1/2Bach1- regulated negativefeedback modulate HSP32 activity [102]. Moreover, free radical production spurred by oxidative stressshould promote HSP mechanisms. At very low concentrations, free radicals produced by mitochondriaand NOXs serve a regulatory ole in cellular activity—their physiological role in cellular activity tiesin with the use of HBOT. Nonetheless, evidence points to the idea that response to HBOT may becell-specific. For instance, one HBOT subjection in healthy males demonstrated no elevation in HSP72activity in peripheral blood mononuclear cells (PBMC) [103].

Biomolecules 2020, 10, 1279 8 of 26Additionally, HBOT may impart neuroprotection against oxygen toxicity via an increase inNrF2-mediated antioxidant gene expression. Notably, more than 200 antioxidants and cytoprotectivegenes can be turned on by the Nrf2 pathway [104]. HBOT not only promotes Nrf2 activity, but alsoupregulates essential proteins involved with intracellular GSH production and transport (e.g., GST,MRPI, GCL, cGT), the assembly/disassembly of macromolecules (e.g., HSPA1A, HSP32), and antioxidantenzymes (e.g., SOD1, GST), which are all target genes of Nrf2 [31,102,105–107]. In addition, by bolsteringthe expression of SirT1 in more than three various ways, HBOT preconditioning imparts neuroprotection.Firstly, Sirt1 expression can be increased by mediating the fasting-induced initiation of Nrf2 signalingupstream through the regulation of the PPAR-   01A0  \Ohorn\textrighthorn{O}     01A1  \ohorn\textrighthorn{o}     01A4  \m{P}\textPhook     01A5  \m{p}\texthtp\textphook     01A9  \ESH\textEsh   01AA  \textlooptoprevesh\textlhtlongi    01AB  \textpalhookbelow{t}\textlhookt      01AC  \m{T}\textThook     01AD  \m{t}\texthtt\textthook     01AE  \M{T}\textTretroflexhook      01AF  \Uhorn\textrighthorn{U}     01B0  \uhorn\textrighthorn{u}     01B1  \textupsilon\m{U}   01B2  \m{V}\textVhook     01B3  \m{Y}\textYhook     01B4  \m{y}\textyhook     01B5  \B{Z}\Zbar     01B6  \B{z}      01B7  \m{Z}\EZH\textEzh   01B9  \textrevyogh     01BA  \textbenttailyogh      01BB  \B{2}\textcrtwo    01BE  \textcrinvglotstop       01BF  \wynn   01C0  \textpipe\textpipevar\textvertline   01C1  \textdoublepipe\textdoublepipevar   01C2  \textdoublebarpipe\textdoublebarpipevar   01C3  \textrclick    01C4  \v{\DZ}      01C5  \v{\Dz}          01C6  \v{\dz}      01C7  \LJ    01C8  \Lj        01C9  \lj    01CA  \NJ    /PGC1-1↵complex that attaches to the Nrf2 promoter,stimulating expression. Secondly, SirT1 expression is mediated via repression of apoptosis, spurredby the upregulation of protein anti-apoptotic Bcl-2 expression, depletion of cleaved caspase-3, whichis pro-apoptotic, and the removal of acetyl groups from p53. Thirdly, expression of SirT1 can bemodulated through the augmentation of FoxO, which in turn, elevates SOD and CAT activity underoxidative stress [107–110].Indeed, HBOT preconditioning can be linked to the inflation of nitric oxide, [111,112] as shownin Figure 1. Serving as a key neurotransmitter, NO is generated by NO synthase (NOS) and is acritical agent of neuroprotection and neurotoxicity [113]. Following cerebral ischemia, endothelialNOS (eNOS) secretion of NO is beneficial, as it stimulates vasodilation. Conversely, once ischemiaevolves further, NO generated by hyperactivity of neuronal NOS (nNOS) and iNOS expressionlead to cerebral injury. NO released by eNOS and iNOS promote synaptic plasticity and neuronaldevelopment, whereas NO secreted by nNOS has the opposite e↵ect, attenuating neurogenesis [114].Since NO improves the vasodilation of the cerebrovasculature, it may fortify the oxygenation oftissues. Furthermore, NO possesses the capacity to favor or impair apoptosis. NO may also regulatecellular metabolism in the presence of dysfunctional mitochondria. On the other hand, it may escalatethe transit of ROS to tissues as well. Additionally, NO may react with free radicals to generate toxicoxidant peroxynitrite and engender nitrosative injury. Importantly, preconditioning with HBOTelevates antioxidant enzymatic activity and represses peroxynitrite primarily in the hippocampus,demonstrating HBOT’s protective capabilities [98].Biomolecules 2020, 10, x 9 of 27 Figure 1. Hyperbaric oxygen therapy (HBOT) effects on Antioxidants and NO. As low amounts of NO displays beneficial effects after stroke, high concentrations of NO produced via iNOS or eNOS may augment neuroinflammation and neurotoxicity. NO provides these negative effects through various mechanisms, including cGMP, cAMP, G-protein, JAK/STAT, and MAPK dependent pathways. Moreover, NO is also believed to modulate specific gene expression, further exacerbating inflammation, and toxicity [115]. Aside from NO’s capacity to cause inflammation, HBOT-induced upregulation of eNOS and nNOS mRNA and protein, along with increased NO in the hypothalamus and hippocampus, may amplify convulsion susceptibility following consecutive HBOT subjections, which may exacerbate the risk of seizures in successive HBOT exposures [112]. Notably, the nonspecific NOS inhibitor, L-NAME, eliminated HBOT-induced neuroprotection, indicating that elevations in Mn-SOD, CAT, and Bcl-2, as well as apoptosis inhibition, may be regulated by NO [69]. Furthermore, following HBOT preconditioning, NO bears both neuroprotective and neurotoxic effects, and thus, further investigation into the mechanisms of NO after HBOT pre-treatment is warranted. 5.2. Reduction of Apoptosis, Activation of Autophagy, and Promotion of Cell Survival ROS molecules possess the ability to react with molecular components to initiate apoptosis or necrosis. Inhibiting major redox systems, such as thioredoxin reductases (TrxR), results in the production of ROS and increased cell apoptosis [116]. PTSD models in rats revealed the upregulation of TrxR-1 and TrxR-2 mRNA in the hippocampus in addition to decrease levels of apoptosis of neurons after HBOT [117]. Additionally, HBOT preconditioning reduced cellular necrosis by modulating mitochondrial pathways. Specifically, cytoplasm cytochrome c levels, as well as capase-3 and capase-9 activity were reduced, upregulating Bcl-2 and Bax proteins linked with improved brain recovery [93,118–121]. Inducing BDNF and inhibiting p38/MAPK phosphorylation also reduced the early onset of apoptosis and apoptosis progression [93,122]. Therefore, HBOT preconditioning in stroke evidently limits apoptosis progression by promoting anti-apoptotic activity and protein expression. In addition to initiating apoptosis, ROS also moderates starvation-induced autophagy via class III phosphoinositide 3-kinase pathway, which sabotages the survival mechanism. ROS-induced autophagy was demonstrated when HBOT preconditioning upregulated protein expression levels of LC3-II and Beclin 1, causing autophagosomes to form in the ischemic penumbra post-ischemia in rat brain models [123]. Additionally, HBOT preconditioning enhanced cell survival by downregulating Figure 1. Hyperbaric oxygen therapy (HBOT) e↵ects on Antioxidants and NO.As low amounts of NO displays beneficial e↵ects after stroke, high concentrations of NO producedvia iNOS or eNOS may augment neuroinflammation and neurotoxicity. NO provides these negativee↵ects through various mechanisms, including cGMP, cAMP, G-protein, JAK/STAT, and MAPK

Biomolecules 2020, 10, 1279 9 of 26dependent pathways. Moreover, NO is also believed to modulate specific gene expression, furtherexacerbating inflammation, and toxicity [115].Aside from NO’s capacity to cause inflammation, HBOT-induced upregulation of eNOS and nNOSmRNA and protein, along with increased NO in the hypothalamus and hippocampus, may amplifyconvulsion susceptibility following consecutive HBOT subjections, which may exacerbate the riskof seizures in successive HBOT exposures [112]. Notably, the nonspecific NOS inhibitor, L-NAME,eliminated HBOT-induced neuroprotection, indicating that elevations in Mn-SOD, CAT, and Bcl-2,as well as apoptosis inhibition, may be regulated by NO [69]. Furthermore, following HBOTpreconditioning, NO bears both neuroprotective and neurotoxic e↵ects, and thus, further investigationinto the mechanisms of NO after HBOT pre-treatment is warranted.5.2. Reduction of Apoptosis, Activation of Autophagy, and Promotion of Cell SurvivalROS molecules possess the ability to react with molecular components to initiate apoptosis or necrosis.Inhibiting major r edox systems, such as thioredoxin reductases (TrxR), results in the pr oduction of ROSand increased cell apoptosis [116]. PTSD models in rats r evealed the upregulation of TrxR-1 and TrxR-2mRNA in the hippocampus in addition to decrease levels of apoptosis of neurons after HBOT [117].Additionally, HBOT preconditioning reduced cellular necr osis by modulating mitochondrial pathways.Specifically, cytoplasm cytochrome c levels, as well as capase-3 and capase-9 activity were reduced,upregulating Bcl-2 and Bax proteins linked with improved brain r ecovery [93,118–121]. Inducing BDNFand inhibiting p38/MAPK phosphorylation also reduced the early onset of apoptosis and apoptosisprogr ession [93,122]. Therefor e, HBOT preconditioning in stroke evidently limits apoptosis progressionby promoting anti-apoptotic activity and protein expression.In addition to initiating apoptosis, ROS also moderates starvation-induced autophagy viaclass III phosphoinositide 3-kinase pathway, which sabotages the survival mechanism. ROS-inducedautophagy was demonstrated when HBOT preconditioning upregulated protein expression levels ofLC3-II and Beclin 1, causing autophagosomes to form in the ischemic penumbra post-ischemia in ratbrain models [123]. Additionally, HBOT preconditioning enhanced cell survival by downregulatingMMP-9 expression, inhibited CA1 cell damage, and promoted healthy functional performance [122].Furthermore, preconditioning HBOT can activate Wnt signaling pathway, upregulate HIF-1, and secretevascular endothelial growth factor (VEGF) to mitigate cell loss. HBOT increased levels of VEGF,VEGFR2, MEK1/2, Raf-1, and phosphor-extracellular signal-regulated kinase (ERK)12protein thatfurther improved neurological functions [123].5.3. Immunosuppression and ImmunopreparationInterestingly, HBOT preconditioning has been observed to reduce and even prevent aberrantinflammation by lowering neurotoxicity microglia activity, TNF-↵expression, and neuronaldegeneration [42], resulting in improvement in motor function after intracerebral hemorrhage [123].Additional mechanisms include downregulation of expressions associated with post-ischemicneuroinflammation, such as cyclooxygenase-2 (COX-2), and alleviates cognitive impairmentsand physiological dysfunctions by restricting pro-inflammatory cytokines and caspase-3 activityin the hippocampus [55,124]. Based on these findings, HBOT preconditioning also alleviates cognitiveimpairment and protects brain functions by modulating pro-inflammatory cytokines and caspase-3pathways [55,124].5.4. Preservation of Blood-Brain Barrier, Edema Minimization, and AngiogenesisHBOT demonstrated preservation of the blood-brain barrier (BBB) and minimizes edema afterthe onset of surgical brain injuries (SBI), stroke (either ischemic or hemorrhagic), and TBI [42,125–129].These protective mechanisms exist due to the suppression of inflammatory responses by loweringhemorrhage volumes and reducing NLRP3 inflammasome expression to recover cognitive functions.Furthermore, HBOT preconditioning also relieved neurological dysfunctions and reduced blood

Biomolecules 2020, 10, 1279 10 of 26volumes by reducing HIF-1↵, MMP-2, and MMP-9 [42,129]. However, HBOT preconditioning mayoverexpress HSP-70 in the hippocampus, which could lead to cognitive deficits and oxidative stress [130].Preconditioning of HBOT could bring protective e↵ects of microvascular endothelial cell protection byincreasing Nrf2 and HSP32 activity. Recent studies, however, reveal the therapeutic e↵ects of HBOTon infarction volume, BBB, and transformed hemorrhage in the absence of the mentioned proteins inthe focal cerebral ischemia model [129].HBOT preconditioning may reduce edema via downregulation of aquaporin 4 (AQP-4) expression,which possess the mechanism to hinder hemorrhage and preserve neural tissue [131]. Culturedastrocytes post-HBOT revealed an increase in AQP-4 and VEGF, demonstrating the ability to modulateBBB openings. This mechanism may introduce a possible treatment option for drug transportation intothe CNS [132]. Additionally, HBOT promotes the p44/42 pathway to help prevents the development ofbrain edema post-intracerebral hemorrhage; the activation of the pathway correlates to the cerebralischemic tolerance that was observed [125].In vitromodels highlight the protective abilities ofHBOT for BBB integrity when occluding and ZO-1 activities were regulated in hypoxic settings [133].Alongside protecting BBB integrity and minimizing edema, HBOT may also protect energy metabolismand tissue perfusion by stabilizing glucose levels, preventing glutamate levels from increasing, loweringlactate/pyruvate ratios, and increasing Ang-2 activity. Protecting energy metabolism gave therapeutice↵ects, including increased microvessel density, reduced brain injury, and alleviated post-ischemicneurological deficits [44,134].5.5. Considerations for HBOT Preconditioning ProtocolsHyperbaricity of 2.5 ATA and 21% O2 is not enough to promote ischemic tolerance. Therefore,understanding the components of HBOT in both hyperoxia and hyperbaricity scenarios is crucial toinduce tolerance against ischemic injuries [135]. Both preconditioning of HBOT and hypoxia possesssimilar efficacies in the neonatal brain. However, the unique defense mechanisms are used duringoxidative stress [136]. Regular HBOT preconditioning consists of 2–3 ATA with 60–90 min of exposurewith 24 h intervals [137–139]. E↵ective ischemic tolerance can be developed when dosed with HBOTfor 3–5 days [91,135]. Although, HBOT also induced neuroprotection against brain injuries, due toischemia during a certain time frame [94], neuroprotection was achieved through biphasic time framescharacterized by instant and delayed preconditioning e↵ect [42,140]. Instantaneous preconditioningwas evident within the first hour after treatment, demonstrating alterations in ion channel activities,enzyme activity, and secondary messengers [140]. On the other hand, delayed preconditioninginvolved cellular changes that progressed slower and developed enduring changes in gene and proteinexpression [140].6. HBOT-Primed Stem Cells as a Promising TherapyStem cells located within niches of the matured brain possess protective and restorativecapabilities for the brain post-stroke via migration to damaged sites. Stem cells are armed withmechanisms, such as secretion of angiogenic factors, trophic factors, regulation of cell death pathway,anti-inflammatory molecules, and replacement of damaged neuronal tissues [3]. However, limitationsexist that prevent stem cells from engaging unaided stroke recovery, making therapeutic strategies thatpromote the brain’s reparative capabilities appealing. Enhancement of HBOT e↵ects on stem cells isbecoming more evident in post-stroke recovery studies [141], proposing a potent role of HBOT in stemcell conditioning before cell transplantation.6.1. HBOT E↵ects on Endogenous Stem CellsIn vivostudies reveal the profound e↵ects of HBOT on stem cell populations as shown in Table 2.Most prominent examples include increased amounts of endogenous stem cells via enhancement ofstem cells in a pressure-sensitive environment [141,142] and multiplication of neural stem cells inadult brain niches [143], which have been observed in various models of TBI-induced injuries [144]

Biomolecules 2020, 10, 1279 11 of 26and non-oxygen-induced injuries [145]. Alongside neural stem cell proliferation, HBOT possessesthe ability to target damaged sites [123], proposing HBOT therapeutic e↵ects that are not dependenton disease pathologies. During circulation, vasogenic endothelial progenitor cells (EPCs) and otherspecific stem cells released post-HBOT may demonstrate therapeutic e↵ects on the stroke-injuredbrain [146]. Stem cells located locally or peripherally in the brain may induce treatment benefits whencombined with mechanisms of HBOT preconditioning and HBOT mechanisms in stroke.An accumulation of studies has demonstrated growing interest in the mechanisms of HBOT thatenhance bone marrow-derived stem cell circulation [145]. Investigations experimented with nitricoxide synthase inhibitors and mice administered with eNOS [141], which revealed the elusive nature ofHBOT mechanisms in cerebral neurogenesis upregulation. Among the various signaling and growthmolecules, HIF-1 alpha was stabilized by HBOT, inhibiting activation of hypoxia [147], slowing downprolyl hydroxylase-induced degradation [148], and enhanced signaling of Wnt/beta-catenin pathwaysthat raised levels of active neural stem cells [149]. High levels of ROS during NSCs proliferation mayalso induce their own renewal, suggesting drug inhibition of ROS may downregulate ROS reproductiveactivity [150] and enhancement of pro-NSC signaling pathways to promote NSC survival.Vascular endothelial growth factor (VEGF), another highly important regulatory molecule, and itsreceptors, VEGFR2, ERK, and CREB, may be involved in HBOT-induced NSC proliferation, due totheir significance in neurogenic pathways [151]. Targeting VEGF downregulates HIF-1 alpha, which isresponsible for promoting hypoxia- and ischemia-related genes and expressions via inflammation,proliferation, glycolysis, and angiogenesis [42,148]. Past studies have demonstrated nontherapeutice↵ects of upregulating erythropoietin (EPO), another HIF-1 alpha gene target, in the hippocampusand cerebral cortex, including BBB permeability prevention, brain edema reduction, decreased infarctionvolume, and post-HBOT neurobehavioral improvement [152,153]. On the other hand, reduced EPOlevels via HBOT promoted homing and engraftment mechanisms of transplantation of stem cellsderived from the umbilical cord blood [154]. Furthermore, an increase in HIF-1 alpha expressionwas correlated to CXCR4 upregulation after HBOT [113], promoting neural crest stem/progenitorcells (NCSCs) via inhibition of hypoxia-involved signaling receptors. Cytoplasmic activity of TPM1increased, and TP53 and CDKN1A, a cyclin-dependent kinase inhibitor, decreased following HBOT,which led to lower rates apoptosis and higher rates of NCSCs reproduction [113].6.2. HBOT and Exogenous Stem CellsThe combination of HBOT and stem cell transplantation may reveal underlying mechanismsamong these treatments. This concept has been researched in neurological and non-neurologicalsettings, including TBI [155], SCI [156], and diabetes mellitus [157]. HBOT has been found to promotegraft survival in the bone marrow, peripheral blood, and spleen. These results were discovered afteran umbilical cord blood stem cell transplantation present in a rodent model of whole-body irradiationinjury [158]. In a murine SCI model, enhanced MSC graft survival was revealed in animals withcombined HBOT and cell therapy [156]. These animals also displayed an alleviated inflammatoryresponse, including decreased levels of pro-inflammatory mediators, TNF-↵, IL-6, and IFN-↵[156].Results from another study show a suppressed inflammatory response along with an upregulatednerve regeneration and a decrease in expression of TUNEL, an apoptosis marker, in combinationtherapy animals [159].6.3. E↵ects of HBOT In Vitro: Potential for Stem Cell PrimingMolecular signaling pathways, including Wnt/-catenin, VEGF/VEGFR2, and CREB, arepotentially mediated by non-stem cell host tissue secretions. While information gained fromin vivostudies guidein vitroresearch, both stem cell and non-stem cell-mediated mechanisms must beanalyzed. The negative e↵ects of HBOT on stem cells must also be considered. HBOT reduced cellsurvival in mesenchymal stem cell (MSC) cultures [160]. The augmented oxygen tension expressed byHBOT subsequently enhances the formation of ROS and releases oxidative stress on cells [161,162].

Biomolecules 2020, 10, 1279 12 of 26Careful evaluation of HBOT priming stem cells for transplantation must be conducted,since exposure to oxidative stressin vitromay advance stem cell resiliency to oxidative stress aftertransplantation.In vitroHBOT priming potentially has genetic, molecular, or transcriptomic e↵ects onstem cells, which enhanced their therapeutic viability. HBOT-induced oxidative stress may augmentthe resiliency of stem cells to the harmful post-ischemic brain environment.Hypoxic preconditioning of stem cells has also shown promising results. It has been found toenhance graft survival after transplantation in a hemorrhagic stroke mouse model [163]. Results alsorevealed an increase in stem cell migratory and homing ability [164–166]. Cell survival and functionare also boosted after transplantation [164]. Hypoxia contributes to the low graft survival occurringpost-stroke transplantation. Rampant oxidative stress is also a significant contributor to graftand endogenous cell death [167,168]. Exposing stem cells to oxidative stress before transplantation hasthe potential to expand the survival time by allowing for genetic and phenotypic acclimation in anoxidative stress environment.HBOT can also potentially increase stem cell secretome.In vitroexperiments demonstrate thatHBOT a↵ects the secretion profile of stem cells. This involves proteins implicated in the oxidativestress response and proteins involved in neuroprotective pathways [160]. After HBOT, there isincreased cellular nitric oxide which allows for the upregulation of growth factors, including VEGFand TFGb1 [160]. MSC cultures also demonstrated HBOT having the ability to enhance the expressionof placental growth factor (P1GF). This was also correlated with increased MSC tubule formationand increased migratory ability [169]. HBOT has also revealed its ability to inhibit the di↵erentiationof stem cells in culture [162]. Depending on the form of transplantation administered, the ability topromote stem cells may become a potential benefit of HBOT priming.Table 1. Chronological Reports of the Mechanisms Regulating HBOT-Induced Neuroprotection.Study DiscoveryJadhav et al., 2009In surgical brain injury (SBI) mice, HBOT preconditioning ameliorated neurologicalfunction and cerebral edema; these neuroprotective e↵ects seemed to be regulated byCOX-2 mechanisms, as HBOT attenuated SBI-induced elevation of hypoxia-induciblefactor-1alpha and COX-2 activity [170].Mu et al., 2013In permanent MCAO animal models, daily HBOT conditioning at 48 h post-surgerydiminished infarct volume and improved neurological function, which correlatedwith elevated CREB protein expression in the hippocampus and peri-infarct area,boosting cell multiplication. Regarding acute pMCAo models, HBOT increasedcerebral PP1- expression, alleviating CREB phosphorylation and ubiquitinationspurred by ischemia. Moreover, HBOT’s regenerative e↵ects against ischemic strokecan be associated with CREB and PP1- mechanisms [37].Lu et al., 2014In transient MCAO rat models, HBOT spurred an increase in ERK1/2 signaling due tohigher levels of ROS, leading to the attenuation of autophagy. When U0126,an inhibitor of the ERK1/2 pathway, was applied, infarct size and autophagy wereameliorated [171].Xue et al., 2016MCAO rats subjected to HBOT preconditioning exhibited diminished infarct size,improved neurological behavior, and upregulated Sirt1, Nrf2, HO-1, and SOD1expression, as well as reduction of MDA. Blocking of Sirt1 or Nrf2 abolishedHBOT-induced protective e↵ects, as Nrf2, HO-1, and SOD1 were repressed.Moreover, the protective actions of Sirt1, spurred by HBOT, may consist ofthe Nrf2/antioxidant defense mechanism [172].Guo et al., 2016Following successive HBOT pre-treatment over five days, rats underwenthyperglycemic MCAO. Preconditioning with HBOT significantly amelioratedhemorrhagic transformation induced by the Nod-like receptor protein 3 signalingand reduced infarct size, altogether rehabilitating neurological performance.HBOT’s neuroprotective e↵ects could be linked to the ROS/thioredoxin-interactingprotein/Nod-like receptor protein 3 mechanism [126].Yang et al., 2017HBOT ameliorated neurological impairment in TBI rats via upregulation of VEGF,VEGFR2, Raf-1, MEK1/2, and ERK1/2, stimulating proliferation of neural stem cells(NSC) and homing of these cells to the lesion site. The examination of HBOT’sprotective e↵ects in vitro showed similar results, as HBO drastically amplified NSCproliferation and VEGF/ERK signaling [123].

Biomolecules 2020, 10, 1279 13 of 26Table 1. Cont.Study DiscoveryHu et al., 2017In hyperglycemia MCAO rats, exposure to two atmospheres of HBO for an hourimmediately after dextrose administration ameliorated depleted ATPand nitcotinamide adenine dinucleotide levels, which in turn elevated silent matingtype information regulation 2 homolog 1, alleviating cerebral infarct and neurologicaldysfunction, along with repressing hemorrhagic transformation [14].He et al., 2019Mice models of acute TBI demonstrated escalated levels of apoptotic neuronsand caspase-3 activity, along with attenuation of signaling pathways that regulateapoptosis in neurons (e.g., pAkt/Akt, pGSK3/GSK3, and -catenin).By eliminating the TBI-induced alterations in these pathways, HBOT suppressedneuronal apoptosis [173].Ying et al., 2019BDNF/TrkB signaling has been shown to influence rehabilitation after SCI. In vivo,SCI rat models were exposed to HBOT, and both dendritic/synaptic deteriorationand apoptosis were ameliorated, which could be linked to higher levels of BDNFand TrkB activity. When ANA-12, an inhibitor of the BDNF/TrkB pathway,was administered, HBOT’s neuroprotective e↵ects were reversed, indicating thatHBOT’s therapeutic benefits are mediated by BDNF/TrkB signaling [174].Zhou et al., 2019Following HBOT, Sprague-Dawley rats with spinal cord injury (SCI) displayedameliorated motor function and attenuated secondary injuries, such as inflammationand glial scar production. By blocking AKT and NF-kB signaling, HBOT repressedmolecules associated with inflammation (iNOS and COX-2) and glial scar generation(GFAP and NG2) [175].Table 2. Milestone Studies on the Use of HBOT for Stem Cells.Study DiscoveryYang et al., 2008Rats were subject to unilateral carotid artery ligation and then 2 h of hypoxia.HBO2 was then administered following the hypoxic-ischemic event. The HBOTwas found to upregulate neural stem cell proliferation in neurogenicenvironments within the adult brain [143].Li et al., 2008A murine model subjected rats to common carotid artery ligation and hypoxia for90 min. HBOT was administered 24 h prior to the hypoxic-ischemic injury.Results revealed that rats preconditioned with HBOT had an increased survivalrate, and the infarct ratio was decreased. This indicates that HBOT can providebrain protection via the inhibition of neuronal apoptosis pathways [45].Li et al., 2009HBOT preconditioned rats where investigated to determine if apoptoticinhibition through a mitochondrial pathway was correlated with neuroprotectionin the ischemic injury in the rat brain. Preconditioning was conducted four times,followed by brain evaluation. Results indicated that HBO-PC significantlyreduced brain edema and decreased infarction volume and improvedneurological recovery [117].Rink et al., 2010Transient MCAO rodents outline the therapeutic potential of normobaricand hyperbaric oxygen treatments during ischemia and after ischemia.HBOT-treated rodents revealed inhibited leukocyte accumulation in the ischemicarea due to a reduction in levels of inflammatory chemokines [50].Cechin et al., 2014This study allowed pancreatic progenitor cells to mature in aperfluorocarbon-based culture device that could adjust the levels of pO2.Enhanced O2 exposure in vitro led to maturation and di↵erentiation of humanembryonic stem cell-derived pancreatic progenitor cells [176].Hadanny et al., 2015Patients with cardiac arrest-induced chronic cognitive impairments where treatedwith sessions of HBOT and analyzed. After administering HBOT five days perweek to chronic stroke patients, patients had significant improvements inmemory and attention testing [22].Dai et al., 2015A rabbit model seeded human adipose-derived stem cells on agelatin/polycaprolactone sca↵old to determine the functional and histochemicalimprovement of tissue-engineered cartilage after HBOT. The humanadipose-derived stem cells were found to have improved extracellularmatrix-secreting abilities after transplantation into a rabbit cartilage defect modelwhen primed with HBOT [177].Yang et al., 2017This study investigated the mechanism of HBOT that promote NSC proliferationand recovery following TBI. The study used 24 rats split into a sham group, a TBIgroup, and an HBO treated TBI group to determine the neurological di↵erences.Neurological function was evaluated and monitored throughout the week. HBOTwas found to promote neural stem cell migration to areas of injury withinthe brain in rat models of TBI that were preconditioned with HBO [123].

Biomolecules 2020, 10, 1279 14 of 26Research supports the evidence that HBOT-preconditioning on the healthy brain providesneuroprotective capacity. Stem cell preconditioning could also be used as a viable cell therapy strategypost-stroke. This could also be a viable and potent dual therapy technique administered to patientswith a high risk for stroke. This strategy uses both a neuroprotective and neurorestorative approach.The capability of HBOT to extend graft survival through oxidative stress conditioning,inhibit premature di↵erentiation, augment migratory capacity, enhance injury homing, promoteanti-inflammatory mediation, and upregulate trophic factors in the secretome demonstrate the potentialof HBOT. HBOT is a viable therapeutic strategy that can work alone or combined with otherpreconditioning strategies to enhance the therapeutic efficacy of transplanted stem cells inthe stroke brain.6.4. Recent Literature on HBOT and StrokeRecent literature has expanded knowledge of HBOT and its underlying mechanisms, all of whichfurther elucidate its potential as a therapy for stroke, stroke-related symptoms, and other diseases.As an efficient and feasible treatment, HBOT has elicited neuroprotective e↵ects before the stroke,displayed regenerative e↵ects during the acute phase of stroke, and even alleviated symptoms duringthe chronic phase of the stroke. Furthermore, many diseases that mimic stroke pathology have foundHBOT similarly e↵ective.6.4.1. PreconditioningPretreatment with HBOT has been a focal point of research over recent years. Anin vitrostudyexamined and found that HBOT preconditioning of primary rat neuronal cells (PRNCs) mitigates celldeath via mitochondrial transfer from astrocytes. PRNCs were subject to HBOT before exposure to tumornecrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS) injury to induce stroke-like cell death.Upon examination, cell viability and mitochondrial transfer were both observed at augmented levelscompared to the non-HBOT treated group. The ability to ameliorate both stroke-induced inflammationand cell death through preconditioning and mitochondrial transfer bolster this as a prophylactictherapy to prevent the devastating e↵ects associated with stroke [178]. Another investigation elucidatedthe e↵ects of HBOT on a rat model of permanent MCAO. HBOT lowered infarct volume and improvedneurological scores in injured rats. An autophagy marker, Beclin-1, was seen at decreased levelsafter treatment. Expression of fodrin1 ceased and necrosis marker, PI-positive cells, were seen atdecreased levels. TUNEL-positive cells, an indicator of apoptosis, were observed at reduced levels,and caspase-3 was downregulated. Taken together, this data indicate that HBOT may amelioratethe detrimental e↵ects associated with ischemia through mitigating autophagy activity, apoptosis,and necrosis [179]. Another murine model featuring intracerebral hemorrhage investigated HBOT’sability to attenuate edema, inflammation, and microglia activation. Pre-conditioning with HBOTwas conducted for five days before ICH induction. MMP9 and brain edema were both less inthe HBOT group when compared to control. Neuronal cell death and neurological deficits wereminimized in the HBOT preconditioned group. Notably, the expression of M1 markers was reduced,consequently inhibiting microglia polarization and inflammatory pathways. This was apparentwhen measuring the concentration of pro-inflammatory cytokines, TNF-alpha, and IL-1, with anindication that levels were downregulated. Furthermore, phosphorylation of JNK and STAT1 weresignificantly decreased in the HBOT group [180]. Lastly, a preconditioning combination therapybetween melatonin (Mel) and HBOT was found to provide more favorable e↵ects in the protectionof ischemic injury-induced cognitive dysfunction and compromised parenchymal integrity in rats.Brain infarct area was lower in rats treated with HBOT-Mel than in rats treated with either HBOTor Mel monotherapy. Apoptotic, autophagy, and inflammatory markers indicated that combinationtherapy was more e↵ective. The additional benefits provided by HBOT-Mel therapy warrant furtherinvestigation in the prevention of detrimental outcomes as a result of ischemic injury [181].

Biomolecules 2020, 10, 1279 15 of 266.4.2. Post-Stroke TreatmentOn top of providing robust neuroprotective e↵ects before a stroke, HBOT has also indicated efficacyin treating stroke patients’ post-ischemia. A study elucidated the e↵ects of HBOT on chronic strokepatients who each underwent 40–60 sessions of HBOT therapy. Notably, 86% of patients displayedclinically significant improvements in cognitive function. When comparing cortical and hemorrhagicstroke victims, cortical stroke patients displayed heightened improvements in information processing.Data also suggested that baseline cognitive function should be contemplated rather than stroke typeand location when predicting the magnitude of clinical improvements. [182]. Furthermore, upper limbmotor dysfunction is a common debilitation after su↵ering a stroke. HBOT therapy, in combinationwith upper limb exercise and mental imagery (EMI), has shown promising results in clinical trialsin improving outcomes of chronic stroke patients. When HBOT-EMI patients were compared toEMI patients alone, there were no statistically significant di↵erences. However, HBOT-EMI patientsshowed an upward trend of improved motor function in upper limbs compared to the EMI group.Although not many di↵erences were observed, data indicate that HBOT is a safe and practicaltherapy for chronic stroke patients, and this combination therapy should be explored further inthe future [183]. Other than physical dysfunction, mental illness may manifest after stroke. A linkhas been found between HBOT and post-stroke depression (PSD), a common symptom many strokepatients experience. This is a devastating consequence of stroke, especially in other countries where itusually goes untreated. A clinical trial revealed an increased response rate and decreased depressionscores post-HBOT. Furthermore, HBOT, in conjunction with antidepressants, was significantly moree↵ective than each respective monotherapy. HBOT is a safe and feasible treatment to treat PSD;however, further elucidation is imperative [184].6.4.3. Diseases Resembling Stroke Pathology and HBOTHBOT has been explored in treating diseases with pathological links to stroke. For example,TBI often presents with neuronal apoptosis, resembling stroke pathology. HBOT was investigatedon a mouse model of TBI. Induction of TBI on mice resulted in activation of caspase-3, decreasedlevels of pGSK3/GSK3, pAkt/Akt, and-catenin, and increased the prevalence of apoptotic neuronsprior to HBOT. HBOT administration during the acute stage of TBI decreased apoptosis, possiblythrough attenuating the Akt/GSK3/-catenin pathway. Further investigation is necessary to fullycomprehend the capacity of mediating this pathway and its implications within controlling TBI-inducedapoptosis [173]. Cerebral air embolism, a phenomenon that complicates many medical proceduresand can provide life-threatening symptoms, may be attenuated via HBOT. Cerebral air embolismpathology resembles that of stroke, often presenting with acute cerebral ischemia-induced edema.A patient undergoing a right internal jugular catheter procedure soon presented with fixed gazepalsy and left-sided hemiparesis upon removal of the wire. Imaging supported intraparenchymalair and a bubble in the right internal jugular vein. Soon after the manifestation of these symptoms,the patient underwent HBOT. Highly significant neurological improvements were seen over the courseof the next week, indicating functional recovery. HBOT therapy may elicit ameliorative e↵ects oncentral air embolism complications, and further investigation is warranted [185]. Lastly, HBOT efficacywas investigated in patients with hypoxic-ischemic encephalopathy (HIE). The subset of patientsthat received HBOT within nine months after the injury displayed the most significant results.This group showed improvements in the disorder of consciousness, as well as a more favorable comarecovery scale-revised score. Overall, HBOT within nine months after HIE may facilitate a functionalrecovery [186].6.4.4. Optimizing TreatmentRecent research advances have provided insightful information pertaining to HBOTand conducting the most efficient therapeutic strategy. Patients contemplating HBOT therapy may

Biomolecules 2020, 10, 1279 16 of 26undergo SPECT/CT imaging as a predictor of HBOT efficacy. A patient analysis supported a linkbetween large penumbra size and significant benefit from HBOT. This size was decreased substantiallyduring therapy, and further supported by improved clinical neurologic status and better qualityof life. Conversely, patients with small penumbra size did not significantly improve from HBOT,and the change penumbra size was negligible. This data suggests that patients presenting with largepenumbra size may display more significant improvements than others. This imaging-based methodof prediction may allow the ability to efficiently select patients that could benefit more from HBOTtherapy [187]. Moreover, HBOT for prolonged periods of time can cause oxygen toxicity. It is importantto evaluate e↵ective protocols to ensure that this phenomenon does not occur during therapy. A murinemodel exhibited that intermittent hyperbaric oxygen exposure (IE-HBO) is linked to protection againstoxygen toxicity. Continuous exposure (CE) of HBO increased concentrations of Peroxiredoxin 6 (Prdx6)protein, an endogenous antioxidant, indicating a relationship between the two. However, the IE-HBOgroup displayed higher amounts of Prdx6 in the rat brains and lungs compared to the CE-HBO group.IE-HBO also enhances NSGPx and GSH activity while mitigating oxidant formation in the lungsand brain. Taken together, IE-HBO mediated Prdx6 expression can suppress oxidative damage inthe brain and lungs and overall protect against oxygen toxicity [188]. These findings allow for a saferand more e↵ective treatment plan when utilizing HBOT to treat stroke and other diseases.In all, HBOT presents with a multitude of treatment possibilities ranging from preconditioningto treating chronic disease. Moving forward, research should continue to explore this prophylactictherapy to better understand the underlying mechanisms that make it so e↵ective. Similarly, treatmenttiming, session amount, and treatment duration should all be elucidated to establish optimal conditionsto incorporate the best results.7. Future Directions and ConclusionsHBOT has the ability to preserve vulnerable neural tissue and improve outcomes in stroke models,as seen in pre-clinical studies. However, further research needs to be conducted to find the moste↵ective regimen of HBOT. Randomized controlled trials need to be administered in order to testhuman e↵ectiveness. Even though further research regarding HBOT needs to be studied, currentresearch has advanced our understanding of the mechanisms of HBOT, specifically in the injuredand healthy brain. This knowledge has paved the way for the development of HBOT preconditioningstrategies. Although HBOT preconditioning is a viable and innovative strategy that contains benefitsfor certain patients, limitations are present involving the ability to deliver stroke therapy.HBOT has the ability to be applied as a preconditioning mechanism for stem cell transplantation.Research indicates that oxidative preconditioning of stem cell grafts through HBOT may be a viablestrategy to promote graft survival and optimize graft function during the post-ischemic environment.In order to explore this concept, further research will be necessary regarding the genetic, epigenetic,secretome, and functional influence that HBOT exerts on stem cell populations. This potentialtherapeutic strategy would o↵er a hybrid approach of combining preconditioning strategies forneuroprotection in the ischemic state of the brain.Author Contributions:Conceptualization, B.C., N.S., B.G.-P., M.S., J.C., Y.J.P. and C.V.B.; literature analysis,B.C., N.S., B.G.-P., M.S., J.C., Y.J.P. and C.V.B.; resources, C.V.B.; writing—original draft preparation, B.C., N.S.,B.G.-P., M.S., J.C., Y.J.P. and C.V.B; writing—review and editing, B.C., N.S., B.G.-P., M.S., J.C., Y.J.P. and C.V.B.;supervision, C.V.B.; project administration, C.V B.; funding acquisition, C.V.B. All authors have read and agreed tothe published version of the manuscript.Funding:Cesar V. Borlongan is funded by National Institutes of Health (NIH) R01NS090962, NIH R01NS102395and NIH R21NS109575.Acknowledgments:The authors thank the entire sta↵ of the Borlongan Neural Transplantation Laboratory forcritical discussion of this manuscript.Conflicts of Interest: The authors declare no conflict of interest.

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www.PRSGlobalOpen.com 1Disclosure: The authors have no nancial interest to declare in relation to the content of this article. From the *Division of Plastic & Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, Calif.; †Advanced Wound Care Center, Stanford University Medical Center, Redwood City, Calif.; and ‡University Center for Plastic, Reconstructive, Aesthetic and Hand Surgery, University Hospital Regensburg and Caritas Hospital St. Josef, Regensburg, Germany.Received for publication January 13, 2020; accepted July 31, 2020.Drs. Hajhosseini and Kuehlmann contributed equally to this work.Copyright © 2020 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.DOI: 10.1097/GOX.0000000000003136Hyperbaric oxygen therapy (HBOT) serves as a “pri-mary” or “adjunctive” therapy in a wide range of pathologies. It is considered the mainstay of man-agement for potentially life-threatening conditions such as carbon monoxide poisoning, decompression illness, and gas embolisms.1–3 Additionally, HBOT has been utilized for decades as an adjunctive therapy in a variety of medical disciplines, including chronic wounds.4–9 A 2017 report by Kaiser Health News estimated that nearly 1,300 hospitals in the United States have installed hyperbaric facilities.10Chronic cutaneous wounds are dened as “wounds that have failed to proceed through an orderly and timely series of events to produce a durable structural and functional closure.”11 Major etiologies that exhibit such wounds include diabetes, pressure, venous insufciency, and peripheral arterial disease. Chronic wounds pose a signicant burden of disease, affecting approximately 6.5 million Americans, with the care costs in the United States alone exceeding $50 billion annually.12 Those aficted experience decreased quality-of-life, pain, restricted mobility, loss of limb, and even loss of life. The incidence of chronic wounds is on the rise due to an increasing elderly population and growing prevalence of obesity and diabetes.In general, chronic wounds are characterized by hypoxia, impaired angiogenesis, and prolonged inam-mation, all of which may theoretically be ameliorated by HBOT (Fig. 1). Nonetheless, the cellular, biochemical, and physiological mechanisms by which HBOT achieves benecial results in chronic wounds are not fully under-stood, and there remains skepticism regarding its efcacy. This review provides a comprehensive overview of HBOT and discusses the developmental history of HBOT, its mechanisms of action, and recent ndings regarding its efcacy as a treatment option for chronic wounds. This article digs deep into the roots of controversy surround-ing the effectiveness of this treatment modality and offers future directions to address existing challenges.Babak Hajhosseini, MD*†Britta A. Kuehlmann, MD*‡Clark A. Bonham, BS* Kathryn J. Kamperman, DNP*Geoffrey C. Gurtner, MD*† Summary: Hyperbaric oxygen therapy (HBOT) serves as “primary” or “adjunctive” therapy in a wide range of pathologies. It is considered the mainstay of manage-ment for potentially life-threatening conditions such as carbon monoxide poison-ing, decompression illness, and gas embolisms. Moreover, HBOT has been utilized for decades as an adjunctive therapy in a variety of medical disciplines, including chronic wounds, which affect approximately 6.5 million Americans annually. In general, chronic wounds are characterized by hypoxia, impaired angiogenesis, and prolonged inammation, all of which may theoretically be ameliorated by HBOT. Nonetheless, the cellular, biochemical, and physiological mechanisms by which HBOT achieves benecial results in chronic wounds are not fully understood, and there remains signicant skepticism regarding its efcacy. This review article pro-vides a comprehensive overview of HBOT, and discusses its history, mechanisms of action, and its implications in management of chronic wounds. In particular, we dis-cuss the current evidence regarding the use of HBOT in diabetic foot ulcers, while digging deeply into the roots of controversy surrounding its efcacy. We discuss how the paucity of high-quality research is a tremendous challenge, and offer future direction to address existing obstacles. (Plast Reconstr Surg Glob Open 2020;8:e3136; doi: 10.1097/GOX.0000000000003136; Published online 25 September 2020.)Hyperbaric Oxygen Therapy: Descriptive Review of the Technology and Current Application in Chronic WoundsREVIEW ARTICLE

PRS Global Open • 20202METHODSThe data outlined in this article have been extracted from Systematic Reviews published in English from January 1, 2000 to July 1, 2019, extracted from The National Library of Medicine’s MEDLINE database, using the search terms “Hyperbaric,” “Hyperbaric Oxygen,” “Hyperbaric Oxygen Therapy,” and “Chronic Wound.”Historical NotesHBOT is not a novel concept, as the rst reports of its use date back to 1662 when the British physician Henshaw rst utilized compressed air for hyperbaric therapy in a chamber called a “Domicilium” (Fig.2).13 In 1789, toxic effects of oxygen were rst reported, thereby increasing a reluctance to use HBOT.13A wide-spread use of HBOT was not adopted until the 20th century. In 1928, a Kansas City physician, Cunningham, built a large hyperbaric chamber spanning 5 stories, which was capable of accommodating up to 40 patients at a time (Fig.3).13 Ite Boerema, recognized as the father of modern hyperbaric medicine, published the rst clinical paper on HBOT in 1956 at the University of Amsterdam, describing the intraoperative use of hyperbaric oxygen to prolong safe operating times during cardiac surgery (Fig.4). Boerema later reported on HBOT’s benecial effects as a treatment for necrotizing infections and ischemic leg ulcers.14Kulonen rst reported use of HBOT in chronic wounds in 1968. As research has begun to elucidate the oxygen-dependent cellular processes involved with tissue repair, such as collagen production by broblasts and the microbicidal activity of macrophages, the utilization of HBOT in the treatment of chronic wounds has become commonplace. This was followed by the decision by the Centers for Medicare & Medicaid Services to initiate reim-bursement for HBOT for the treatment of diabetic foot ulcer (DFU) in 2002.Overview and Description of the TechnologyHBOT entails full body exposure and breathing of 100% oxygen while inside a hyperbaric chamber pressur-ized to greater than sea level (“sea level” is dened as 1 atmosphere absolute [ATA]).15,16 Typically, treatments involve pressurization to between 2.0 and 2.5 ATA, which would be equivalent to ~250 kPa/inch2, approximately the pressure at a depth of ~15 m of water. Treatment duration varies from 45 to 300 minutes depending upon the indication for which HBOT has been prescribed, with most treatment sessions lasting from 90 to 120 min-utes.17 Therapy for acute indications may require only 1 or 2 treatment sessions, whereas chronic medical condi-tions may warrant up to 30 or more treatment sessions. Patients may receive up to 3 treatment sessions per day depending on the medical indication. Chambers are either single-occupant (mono-place) or multiple-occu-pant (multi-place).18Mechanisms of ActionMost therapeutic benets of HBOT can be attributed to the relationships between gas concentration, volume, and pressure. We know from Henry’s law that the amount of an ideal gas dissolved in a solution is directly propor-tional to its partial pressure (Fig.5). Therefore, increasing partial pressure of oxygen in arterial blood during HBOT would improve the cellular delivery and supply of oxygen. This is the primary principle behind the effectiveness of HBOT in treating conditions in which oxygen delivery has been compromised, such as carbon monoxide poisoning and ischemia.Another major effect of HBOT can be explained by Boyle’s law, which indicates that the volume of a gas bubble is inversely related to the pressure exerted upon it (Fig.6); this is the central concept underlying the benecial prop-erties of HBOT in management of conditions such as decompression illness and intravascular embolism.18Several other therapeutic mechanisms of HBOT have been described in recent literature. It has been demon-strated that HBOT enhances neovascularization, and plays a role in improving the immune response, activating broblasts, downregulating inammation, upregulating synthesis of growth factors, potentiating antibiotics and antibacterial processes, enhancing antioxidant response, and ameliorating ischemia-reperfusion injury.2,9,18–22Fig. 1. Pathology of chronic wounds. Chronic wounds are characterized by hypoxia, impaired angio-genesis, and prolonged inammation.

Hajhosseini et al. • Hyperbaric Oxygen Therapy3Contraindications and Adverse EffectsAlthough hyperbaric oxygen therapy remains rela-tively safe, several adverse side effects have been observed. Reversible myopia has occurred as a direct result of oxygen’s effects on the eye’s lens, whereas others have experienced barotrauma in the ears and sinuses and in rare cases, the teeth, and lungs.23 Middle ear barotrauma is among the most common side effects of HBOT, affect-ing up to 2% of the patients undergoing therapy. This can be prevented and managed by autoination techniques and inserting tympanostomy tubes, respectively. Other observed side effects include chest tightness, coughing, fatigue, headaches, vomiting, and a burning sensation in the chest.2,24 Although undesirable, these effects are reversible and nonfatal, leaving HBO therapy as a safe adjunctive treatment method for approved morbidities.Oxygen toxicity is among the more serious complica-tions associated with HBOT and can be associated with neurologic (eg, convulsions and psychological changes) and/or pulmonary (eg, pulmonary edema and respiratory failure) symptoms. Decompression sickness may occur in patients breathing compressed air that contains nitrogen. Fire hazard is considered the most common fatal compli-cation of HBOT.9,18,21,25,26HBOT may not be suitable for some individuals due to their current health or treatment regimen. “Absolute” con-traindications for HBOT include untreated pneumothorax Fig. 2. 1662: Henshaw’s Domicilium.Fig. 3. 1928: Cunningham’s steel ball hospital.Fig. 4. Ite Boerema operating in pure oxygen.Fig. 5. Henry’s Law: The concentration of a dissolved gas equals the pressure times the solubility coecient of that gas.Fig. 6. Boyle’s Law: Elevating hydrostatic pressure increases partial pressure of gases and causes a reduction in the volume of gas-lled spaces.

PRS Global Open • 20204and concomitant use of certain chemotherapeutics such as doxorubicin or cisplatin. Additionally, there are several “relative” contraindications that warrant extreme caution; these include poorly controlled seizure disorder, hyper-thyroidism, congestive heart failure with ejection fraction less than 30% (it is important to note that oxygen is a vaso-constrictor, and as a result HBOT may increase cardiac afterload), severe chronic obstructive pulmonary disease, asymptomatic pulmonary blebs, or bullae incidentally found on chest radiograph, active upper respiratory or sinus infections, recent ear or thoracic surgery, history of pneumothorax, uncontrolled fever, claustrophobia, and inability to equalize pressure in the middle ear.18,21Indications and Clinical UseHBOT serves as a “primary” therapy for a number of medical conditions. There exists an indisputable level of evidence that supports HBOT as the standard of care for the potentially fatal conditions of carbon monoxide poi-soning, decompression illness, and arterial and venous gas embolisms.1–3 As such, HBOT has been approved by the Undersea and Hyperbaric Medical Society (UHMS) for 13 illnesses, including decompression sickness and arterial gas embolisms, though others propose it as a treatment for conditions outside of this list.27 (Please see Tables1 and 2 for the full list of HBOT indications currently approved by “Undersea & Hyperbaric Medical Society” and “Centers for Medicare & Medicaid Services”, respectively.)Albeit not as strong as the available evidence for its “primary” use, research has shown HBOT to be benecial as an “adjunctive” therapy in the case of a diverse range of other pathologies including but not limited to those of neurology, oncology, orthopedic, rheumatology, cardio-vascular, genitourinary, gastrointestinal, and hepatobiliary origin, as well as acute and chronic wounds. Moreover, some studies have displayed HBOTs favorable impact on radiation-induced injuries where brotic deposition, diminished vascularity, and tissue hypoxia play role in the disease pathogenesis.18,28–32 Although there appears to be a correlation between the use of HBOT and an improved outcome, causation has yet to be denitively established. Conditions such as diabetic foot ulcers, ischemic stroke, sports injuries, and multiple sclerosis are common dis-eases that are treated with HBOT but a lack of strong sup-port from peer-reviewed research, with many studies being underpowered. As such, HBOT has been described as “a therapy in search of disease.”27,33 Further studies need to be performed that are properly randomized, controlled, and conducted so that its proper uses may be identied.Over the past decade, Cochrane Reviews has assessed potential “adjunctive” indications for HBOT. The results of these Systematic Reviews are summarized in Table 3. It is important to point out that the authors have unani-mously taken note of the fact that the majority of the trials included in these Systematic Reviews suffered from small sample sizes, methodological deciencies, and/or poor reporting outcomes, concluding that the results should be interpreted “cautiously.” The one common consensus in these Systematic Reviews was that “appropriately Powered trials of high methodological rigor is required to dene which patients, if any, can be expected to benet most from HBOT.”4,34–47Financial CostCost-effectiveness is a central issue in modern healthcare. The cost of a full course of HBO treatment for diabetic foot ulcers varies by location and depends upon several factors, such as setup costs and ongoing costs, reimbursement sys-tems, and the number of patients treated per center. Costs also differ geographically. In the United States, charges are typically between $200 and $1,250 per treatment session, with a full course of treatment averaging 50–60 hours in the HBO chamber and costing from $50,000 (Medicare) to $200,000 (private pay).21,48 In 2011, a full HBO treatment in the Netherlands was about €6,920 (equaled $7,762), display-ing the cost differential outside the United States.49According to market research, the global HBOT devices market size was estimated at USD 2.21 billion in 2016.50 A rising number of university and private compa-nies funded clinical trials indicates an ongoing adoption of the technique and contributes to propel growth of Table 1. Indications for Hyperbaric Oxygen Therapy per Undersea and Hyperbaric Medical SocietyIndications for HBOT per Undersea and Hyperbaric Medical SocietyAir or gas embolismCarbon monoxide poisoningCyanide poisoningClostridial myositis and myonecrosis (gas gangrene)Crush injury, compartment syndrome, and other acute traumatic ischemiasDecompression sicknessArterial inefciencies: central retinal artery occlusionArterial inefciencies: enhancement of healing in selected problem woundsSevere anemiaIntracranial abscessNecrotizing soft–tissue infectionsOsteomyelitis (refractory)Delayed radiation injury (soft tissue and bony necrosis)Compromised grafts and apsAcute thermal burn injuryIdiopathic sudden sensorineural hearing lossTable 2. Indications for Hyperbaric Oxygen Therapy per Centers for Medicare and Medicaid ServicesIndications for HBOT per Centers for Medicare and Medicaid ServicesAcute carbon monoxide intoxicationDecompression illnessGas embolismGas gangreneAcute traumatic peripheral ischemiaCrush injuries and suturing of severed limbsAcute peripheral arterial insufciencyProgressive necrotizing infectionsPreparation and preservation of compromised skin graftsChronic refractory osteomyelitisOsteoradionecrosisSoft-tissue radionecrosisCyanide poisoningActinomycosisDiabetic wounds of the lower extremity with type 1 or 2 diabetes, a Wagner Grade 3 or higher ulcer, and failure of adequate course of standard wound therapy

Hajhosseini et al. • Hyperbaric Oxygen Therapy5the HBOT market. Additionally, technological develop-ment in the eld of hyperbaric oxygen therapy devices is expected to push/increase the demand over the next years and further impel their growth. As HBO can be used to treat several conditions noninvasively, market research found that nearly 90% (1800 out of 2000) of hospitals and 71% (500 out of 700) of clinics are already offering hyper-baric oxygen therapies for many of the diseases previously detailed, including chronic wounds.Efcacy in Chronic WoundsHBOT has been used as an “adjunctive” therapy for chronic wounds since the mid 1960s. The mechanisms by which HBOT may augment healing in chronic wounds are not fully understood, though several rationales have been proposed throughout years. It has been demonstrated that HBOT can modulate the local and systemic effects wit-nessed in both acute and chronic injuries. In general, the common denominators in chronic wounds are hypoxia, prolonged inammation, and impaired angiogenesis, all of which may potentially be ameliorated by HBOT.18,51The data on efcacy of HBOT in chronic wounds are often inconsistent and inconclusive.51–56 Among various eti-ologies involved in the development of chronic wounds, the highest number of studies and the bulk of HBOT literature have been devoted to the subject of DFUs. A 2004 Cochrane Review evaluated the role of HBOT in chronic wounds, concluding that HBOT may reduce the risk of major ampu-tation in DFU patients and may improve healing at 1 year. Unfortunately, many of the studies reviewed suffered from limited sample sizes and methodological aws. The same study evaluated the role of HBOT in chronic wounds of venous, arterial, and pressure etiology, and concluded that the routine utilization of HBOT for these indications was not justied based on the evidence (Table3).34In 2015, an updated Cochrane Review was conducted. The evidence from this study revealed that HBOT may improve the healing rate of DFU in the short term (ie, 6 wks), but not the long term (ie, 1 y). The authors further found no signicant difference in major amputation rate in DFU population, while once again emphasizing the various aws in the study design and reporting outcomes of the trials included (Table 3).4 Löndahl et al57 conducted a random-ized, double-blinded, placebo-controlled clinical trial in 2010 evaluating 94 patients with Wagner Grade 2, 3, or 4 DFUs. They concluded that adjunctive HBOT facilitates healing in selected patients.57 A 2017 report by Lam et al demonstrated that HBOT may improve healing and decrease amputation in “ischemic” DFUs; however, there was limited evidence on its effect on nonischemic DFUs and nondiabetic arterial ulcers.51Zhao et al58 conducted a meta-analysis on DFUs in 2017 studying 9 randomized clinical trials. They found that although HBOT was associated with a greater reduction in the wound size compared with the standard therapy, no Table 3. Cochrane Review Results on Potential Indications for Hyperbaric Oxygen TherapyCochrane Study TitlePublication Year Authors’ ConclusionsHBOT for chronic wounds42015 In diabetic foot ulcers, HBOT signicantly improved healing in the short term, but not in the long term.HBOT for chronic wounds342004 In diabetic foot ulcers, HBOT signicantly reduced the risk of major amputation and may improve the chance of healing at 1 year.The routine management of chronic wounds associated with other pathologies with HBOT is not justiedHBOT for late radiation tissue injury352016 For LRTI affecting tissues of the head, neck, anus, and rectum, HBOT is associated with improved outcome. HBOT appears to reduce the chance of osteoradionecrosis following tooth extraction in an irradiated eld. No evidence of important clinical effect on neurological tissues.HBOT for autism spectrum disorder362016 No evidence that HBOT improves symptoms of ASDHBOT for necrotizing fasciitis372015 Failed to support or refute the effectiveness of HBOTHBOT for acute coronary syndrome382015 There is some evidence from small trials to suggest that HBOT is associated with a reduction in the risk of death, the volume of damaged muscle, the risk of major adverse cardiac events, and time to relief from ischemic pain. The routine application of HBOT cannot be justied.HBOT for migraine and cluster headache392015 There was some evidence that HBOT was effective for the termination of acute migraine in an unselected populationHBOT for acute ischemic stroke402014 No good evidence to show that HBOT improves clinical outcomes, but the possibility of clinical benet has not been excludedHBOT for malignant otitis externa412013 No clear evidence to demonstrate the efcacy of HBOT when compared with antibiotics and/or surgeryHBOT for acute surgical and traumatic wounds422013 No high-quality evidence. Although 2 small trials suggested that HBOT may improve the outcomes of skin grafting and trauma, these trials were at risk of bias.HBOT for bony fractures432012 No evidence to support or refute the effectiveness of HBOT for the management of delayed or nonunion bony fracturesHBOT for idiopathic sudden sensorineural hearing loss and tinnitus442012 For people with acute ISSHL, the application of HBOT signicantly improved hearing, but the clinical signicance remains unclear.No evidence of a benecial effect of HBOT on chronic ISSHL or tinnitusHBOT for traumatic brain injury452012 Although the addition of HBOT may reduce the risk of death and improve the nal GCS, there is little evidence that the survivors have a good outcome. The routine application of HBOT to these patients cannot be justied.HBOT for vascular dementia462012 Insufcient evidence to support HBOT as an effective treatmentCCS, Glasgow Coma Scale; ISSHL, Idiopathic Sudden Sensorineural Hearing Loss; LRTI, late radiation tissue injury.

PRS Global Open • 20206differences existed with respect to the rate of complete heal-ing, amputation risk, and adverse events.58 The following year, in 2018, Ennis et al53 conducted a retrospective study of over 600,000 Wagner Grades 3 and 4 DFUs concluding that HBOT may be of benet in the case of “advanced” ulcers. Most recently, in 2019, Golledge and Singh59 carried out a systematic review and meta-analysis of 9 clinical trials in the eld of DFUs. Authors concluded that HBOT improves the healing of DFUs and reduces the amputation rate.59In contrast, 2 recent studies by Fedorko et al52 and Santema et al55 found that HBOT did not offer any signi-cant advantages toward complete wound healing in DFUs associated with lower-limb ischemia. However, these stud-ies too have been subject to criticism due to several meth-odological errors.60–62While denitive proof for HBOT as a therapeutic has yet to be established, it appears that by and large, among the potential indications for HBOT in the eld of chronic wounds, the strongest favorable evidence exists for isch-emic, infected (ie, Wagner Grade 3 or worse) DFUs.51–56Why the Controversy?As we have highlighted in this article, much contro-versy exists with regard to the adjunctive therapeutic effects of HBOT on chronic wounds. There are several culprits for the existing discord. First and foremost, a comprehensive mechanistic understanding of the tech-nology is lacking. HBOT acts through diverse and not-fully-understood mechanisms to promote angiogenesis and decrease inammation. Moreover, many of the ini-tial HBOT studies that demonstrate favorable outcomes were performed in the inpatient/hospital setting, which ensured proper patient, physician, and staff compliance; it is not completely unexpected to see that these results have not fully translated to the reality of the outpatient/clinic setting. Also, there are inherent impediments to an ideal study design investigating HBOT; as an example, the unique environment of hyperbaric chambers generates signicant challenges to ideal blinding of both patients as well as investigators.7 Finally, trials investigating HBOT are faced with the same challenges such as “procedural varia-tions” and others that are almost impractical to account for, which have plagued clinical studies in this particular eld for decades.63–66To make the matter even worse, similar to the ef-cacy trials, there have been contradictory reports on eco-nomics and cost-effectiveness of HBOT in the eld of chronic wounds. The cost of diabetic foot disease in the United States in 2007 was $30 billion, of which $19 bil-lion was due to foot ulceration and $11 billion to amputa-tions. It was estimated in 2007 that effective diabetic foot ulcer and amputation prevention could realistically save the US healthcare system up to $21.8 billion annually.67 Unfortunately, studies have failed to prove unanimously that HBOT has the potential to lower the costs of care for DFUs. The 2008 Study by Canadian Agency for Drugs and Technologies in Health reported that adjunctive HBOT for DFUs is cost-effective compared with standard care alone.68 The more recent study conducted in 2017 by Health Quality Ontario indicated that adjunctive HBOT for DFUs may lower costs due to reduced amputation rate, but overall authors concluded that “there is uncertainty” regarding cost-effectiveness.69This overall environment of uncertainty has inevitably led to discrepancies between “accepted,” “covered,” and “off-label” indications for HBOT. This has brought several stakeholders with differing motivations into play, paving the way for the utilization of HBOT for unregulated and unwarranted indications, whereby little to no supportive evidence exist.70 Not surprisingly, the skepticism ensued has made it even more challenging to vindicate this poten-tial therapy or to see its merits.CONCLUSIONSCompressed air and hyperbaric oxygen have been uti-lized in medicine for centuries. HBOT is now considered the mainstay of treatment for a number of life-threatening conditions such as carbon monoxide poisoning, decom-pression illness, and gas embolism.1–3,71 Moreover, HBOT has the distinctive ability to remedy tissue hypoxia, reduce inammation, and alleviate ischemia-reperfusion injury.7 The current evidence in the eld of chronic wounds suggests that HBOT may have favorable effects on isch-emic, infected (ie, Wagner Grade 3 or worse) DFUs.51–56 Despite many studies highlighting the potential benets of HBOT, much controversy remains with regard to its efcacy in wound healing.15 The paucity of high-quality randomized controlled trials makes it difcult to properly assess the efcacy of HBOT. To accurately validate the potential benets of HBOT, more vigorous investigations with adequately powered sample sizes are warranted.Geoffrey C. 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biomoleculesReviewHyperbaric Oxygen Treatment—From Mechanisms toCognitive ImprovementIrit Gottfried1, Nofar Schottlender1,2and Uri Ashery1,2,*!"#!$%&'(!!"#$%& 'Citation: Gottfried, I.; Schottlender,N.; Ashery, U. Hyperbaric OxygenTreatment—From Mechanisms toCognitive Improvement. Biomolecules2021, 11, 1520. https://doi.org/10.3390/biom11101520Academic Editor: Vladimir N.UverskyReceived: 14 September 2021Accepted: 13 October 2021Published: 15 October 2021Publisher’s Note: MDPI stays neutralwith regard to jurisdictional claims inpublished maps and institutional affil-iations.Copyright: © 2021 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).1School of Neurobiology, Biochemistry and Biophysics, Life Sciences Faculty, Tel Aviv University,Tel Aviv 6997801, Israel; iritgo@tauex.tau.ac.il (I.G.); schottlender@mail.tau.ac.il (N.S.)2Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel* Correspondence: uria@tauex.tau.ac.il; Tel.: +972-3-6409827Abstract:Hyperbaric oxygen treatment (HBOT)—the medical use of oxygen at environmentalpressure greater than one atmosphere absolute—is a very effective therapy for several approvedclinical situations, such as carbon monoxide intoxication, incurable diabetes or radiation-injurywounds, and smoke inhalation. In recent years, it has also been used to improve cognition, neuro-wellness, and quality of life following brain trauma and stroke. This opens new avenues for theelderly, including the treatment of neurological and neurodegenerative diseases and improvementof cognition and brain metabolism in cases of mild cognitive impairment. Alongside its integrationinto clinics, basic research studies have elucidated HBOT’s mechanisms of action and its effects oncellular processes, transcription factors, mitochondrial function, oxidative stress, and inflammation.Therefore, HBOT is becoming a major player in 21st century research and clinical treatments. Thefollowing review will discuss the basic mechanisms of HBOT, and its effects on cellular processes,cognition, and brain disorders.Keywords:hyperbaric oxygen treatment (HBOT); cognition; brain disorders; neuroprotection;neuroinflammation; Alzheimer’s disease1. Hyperbaric Oxygen Treatment (HBOT): The ConceptHBOT—the medical administration of 100% oxygen at environmental pressure greaterthan one atmosphere absolute (ATA)—is used clinically for a wide range of medicalconditions. One of HBOT’s main mechanisms of action is elevation of the partial pressure ofoxygen in the blood and tissues as compared to simple oxygen supplementation [1,2]. Thisallows five to ten times more oxygen to enter the blood plasma and to reach tissues sufferingfrom low oxygen supply (following, e.g., brain injury, stroke, or vascular dysfunction).Therefore, it is not surprising that HBOT has been used for over 50 years for wounds (non-healing diabetic foot ulcers), air embolisms or decompression sickness, burned tissue repair,carbon monoxide intoxication, peripheral arterial occlusive disease, smoke inhalation,radiation injury, and promoting recovery from serious illness [3–10]. Nevertheless, today,there are only 13 FDA-approved HBOTs [11]; however, in parallel, there are a growingnumber of “off-label” uses, which have not been cleared by the FDA, such as treatmentfor stroke patients or patients suffering from Alzheimer’s disease (AD) [12,13], and eventreatment of COVID-19 patients, which have shown very promising results [14–19]. Furtherclinical trials that are currently in progress, and additional basic scientific studies aimed atunderstanding HBOT’s mechanisms of action, will most probably expand the use of HBOTto other areas.2. Cognitive Improvement2.1. Cognitive Improvement Following Brain InjuriesAlthough the use of HBOT in cases of brain-related disorders is pending FDA ap-proval, there are numerous studies showing improved cognitive assessment followingBiomolecules 2021, 11, 1520. https://doi.org/10.3390/biom11101520 https://www.mdpi.com/journal/biomolecules

Biomolecules 2021, 11, 1520 2 of 11treatment for several brain injuries [20]. For example, post-stroke patients suffer fromreduced cognitive performance, and in particular, memory difficulties. HBOT for strokepatients at late chronic stages has shown significant improvements in all memory measures.These clinical improvements are well-correlated with improvements in brain metabolism,mainly in temporal areas. High oxygen (92%) alone was also shown to positively affectthe working memory of individuals with intellectual and developmental disabilities, atleast in the short-term [21]. Similar improvement was seen in a large cohort of post-strokepatients who underwent 40 HBOT sessions (2 ATA), leading to significant neurological andcognitive improvements, even at the late chronic phase after stroke [22,23]. Mechanistically,in preclinical studies, HBOT has been suggested to reduce oxidative stress, inflammation,and neural apoptosis, thereby improving functional recovery from stroke [24]. It was alsosuggested that HBOT in rats suffering from ischemic stroke stimulates the expression oftrophic factor and neurogenesis, and the mobilization of bone marrow stem cells to theischemic area, which can enhance cell repair [25]. In addition, HBOT elevates cerebral bloodflow (CBF), associated with restoration of physical abilities and cognitive functions [26,27].The improvement in cognition and executive functions, as well as in physical abilities, gait,sleep, and quality of life in these stroke patients continued for up to three months afterthe last treatment, which was the follow-up period in that study [27]. These encouragingresults suggest the occurrence of long-term changes, lasting the order of months. Simi-larly, in patients with mild traumatic brain injury (TBI), HBOT improved hippocampalCBF [28] and facilitated recovery during the rehabilitation phase [29]. Moreover, growingevidence suggests that HBOT can induce neuroplasticity and improve cognitive functionin patients suffering from chronic neurocognitive impairment due to TBI, stroke, andanoxic brain damage [22,23,30–32]. These changes were associated with the induction ofcerebral angiogenesis, increased CBF and volume, and improved cerebral white and graymicrostructures [33].Other teams have investigated whether HBOT can improve brain function and cogni-tion in neurodegenerative diseases such as AD and vascular dementia (VD), and if HBOTcan also affect healthy people or improve cognitive decline in the elderly who are sufferingfrom cognitive impairments.2.2. Cognitive Improvement Following HBOT in AD and VDRecent human studies have shown that HBOT can improve cognitive functions inpatients with mild cognitive impairment (MCI), AD, and VD [13,20,34–38], and amelioratethe reduced brain metabolism in MCI and AD [34,35]. Similarly, cerebrovascular diseasepatients showed improvement in motor and cognitive performance compared to a controlgroup following HBOT [38]. Interestingly, improvements in cognitive function assessed byMini-Mental State Exam (MMSE) and Mini-Cog test were reported in AD patients even onemonth after the end of the last HBOT, and for up to three months in amnestic MCI patients.In addition, HBOT ameliorated the reduced brain glucose metabolism in some of the ADand amnestic MCI patients [34]. These are very promising results, because they suggestthat even with severe cognitive deterioration in progressive neurodegenerative braindisorders, relatively short-duration HBOT (40 min once a day for 20 days) can improveconditions for one to three months. In a more severe case of AD, a longer treatment ofeight weeks (1.15 ATA) reversed the patient’s symptomatic decline and PET scan showedan increase in brain metabolism [35]. Nevertheless, the current belief is that HBOT cannotrevert severe cases with major neuron loss and therefore should be considered mainlyat early disease stages, when only minimal cognitive deficiency is detected. It shouldbe noted that the elevation of pressure by itself was also suggested to regulate AD [39].However, further research in this direction should explore the exact effect. A largergroup of VD patients who received 12 weeks of HBOT (2 ATA) showed improvementin MMSE scores and elevated serum humanin levels [36]. Humanin is a unique humanmitochondrion-derived peptide that has neuroprotective effects [40–42] and, together withfindings of increased brain metabolism, this suggests an important role for improving

Biomolecules 2021, 11, 1520 3 of 11mitochondrial function as part of HBOT’s mechanism of action. As HBOT use in the clinicis considered to be safe and well-tolerated, it should be considered and recommendedas an alternative therapeutic approach for AD and VD [37], as well as in early stages ofMCI. Hence, HBOT improves several aspects of brain activity including an improvementin cerebral blood flow, brain metabolism, and brain microstructure, and this leads toimprovement in cognitive functions and physical functions, sleep, and gait leading to anoverall improved performance (Figure 1). Nevertheless, it is also clear that although theeffects of HBOT last, in some studies, for several months, when treating patients withprogressive neurodegenerative diseases such as AD, maintenance HBO treatments willprobably be needed.Biomolecules 2021, 11, x FOR PEER REVIEW 3 of 11 [39]. However, further research in this direction should explore the exact effect. A larger group of VD patients who received 12 weeks of HBOT (2 ATA) showed improvement in MMSE scores and elevated serum humanin levels [36]. Humanin is a unique human mi-tochondrion-derived peptide that has neuroprotective effects [40–42] and, together with findings of increased brain metabolism, this suggests an important role for improving mi-tochondrial function as part of HBOT's mechanism of action. As HBOT use in the clinic is considered to be safe and well-tolerated, it should be considered and recommended as an alternative therapeutic approach for AD and VD [37], as well as in early stages of MCI. Hence, HBOT improves several aspects of brain activity including an improvement in cer-ebral blood flow, brain metabolism, and brain microstructure, and this leads to improve-ment in cognitive functions and physical functions, sleep, and gait leading to an overall improved performance (Figure 1). Nevertheless, it is also clear that although the effects of HBOT last, in some studies, for several months, when treating patients with progressive neurodegenerative diseases such as AD, maintenance HBO treatments will probably be needed. Figure 1. HBOT improves brain function. HBOT has been shown to improve cerebral blood flow, brain metabolism, and brain microstructure, leading to improved cognitive functions, physical func-tions, sleep, and gait. 2.3. Cognitive Improvement in Healthy Individuals Over the last few decades, several studies have examined the possible contribution of HBOT to cognitive performance in both young and elderly populations. In one of the first studies examining the effects of HBOT on the elderly [43], it was found to improve cognitive function in elderly patients with cognitive deficits. In a more recent study with a cohort of healthy young adults, HBOT increased spatial working memory and memory quotient, and this was correlated with changes in regional homogeneity as measured by resting-state functional MRI [44]. In another prospective study, double-blind randomized healthy volunteers were asked to perform a cognitive task, a motor task and a simultane-ous cognitive–motor task (multitasking) while in a functional HBO chamber. Compared to the performance under normobaric conditions, single cognitive and motor task, and Figure 1.HBOT improves brain function. HBOT has been shown to improve cerebral blood flow,brain metabolism, and brain microstructure, leading to improved cognitive functions, physicalfunctions, sleep, and gait.2.3. Cognitive Improvement in Healthy IndividualsOver the last few decades, several studies have examined the possible contributionof HBOT to cognitive performance in both young and elderly populations. In one of thefirst studies examining the effects of HBOT on the elderly [43], it was found to improvecognitive function in elderly patients with cognitive deficits. In a more recent study with acohort of healthy young adults, HBOT increased spatial working memory and memoryquotient, and this was correlated with changes in regional homogeneity as measured byresting-state functional MRI [44]. In another prospective study, double-blind randomizedhealthy volunteers were asked to perform a cognitive task, a motor task and a simultane-ous cognitive–motor task (multitasking) while in a functional HBO chamber. Comparedto the performance under normobaric conditions, single cognitive and motor task, andmultitasking performance scores were significantly enhanced by the HBO environment,supporting the hypothesis that oxygen is a rate-limiting factor for brain activity [45]. Theseresults were further validated by two recent studies that examined the effects of HBOT onhealthy young [46] and old [47] adults. In these studies, HBOT resulted in an improvedlearning curve and higher resilience to interference of episodic memory in the healthyyoung adults [46], and induced cognitive enhancements in healthy aging adults, which

Biomolecules 2021, 11, 1520 4 of 11were associated with regional improvement in CBF [47]. Similarly, in a recent paper, agroup of elderly patients with memory loss at baseline to HBOT showed improved cog-nitive performances following 60 daily HBOT sessions (2 ATA) and this was associatedwith an increase in CBF [48]. Interestingly, when HBOT was applied for a short time (only15 consecutive days), there was no improvement in cognitive impairment in the elderly [49],suggesting that a longer treatment is necessary. Indeed, current protocols are extendingthe treatment to two to three months (40–60 daily sessions, 5 days per week, 2–3 ATA) andpromise to yield more significant and long-lasting effects [12].In summary, it is clear that the HBO environment, in and of itself, improves cognitiveperformance, and that this can be attributed directly to the elevated oxygen levels, suggest-ing that oxygen is a rate-limiting factor for brain activity [45]. However, repeated exposureto HBOT for longer periods of time is needed to achieve long-lasting effects that lead tochanges in vascular, neuronal, and cellular activity, as detailed in Figure 2 [12].Biomolecules 2021, 11, x FOR PEER REVIEW 4 of 11 multitasking performance scores were significantly enhanced by the HBO environment, supporting the hypothesis that oxygen is a rate-limiting factor for brain activity [45]. These results were further validated by two recent studies that examined the effects of HBOT on healthy young [46] and old [47] adults. In these studies, HBOT resulted in an improved learning curve and higher resilience to interference of episodic memory in the healthy young adults [46], and induced cognitive enhancements in healthy aging adults, which were associated with regional improvement in CBF [47]. Similarly, in a recent paper, a group of elderly patients with memory loss at baseline to HBOT showed improved cog-nitive performances following 60 daily HBOT sessions (2 ATA) and this was associated with an increase in CBF [48]. Interestingly, when HBOT was applied for a short time (only 15 consecutive days), there was no improvement in cognitive impairment in the elderly [49], suggesting that a longer treatment is necessary. Indeed, current protocols are extend-ing the treatment to two to three months (40–60 daily sessions, 5 days per week, 2–3 ATA) and promise to yield more significant and long-lasting effects [12]. In summary, it is clear that the HBO environment, in and of itself, improves cognitive performance, and that this can be attributed directly to the elevated oxygen levels, sug-gesting that oxygen is a rate-limiting factor for brain activity [45]. However, repeated ex-posure to HBOT for longer periods of time is needed to achieve long-lasting effects that lead to changes in vascular, neuronal, and cellular activity, as detailed in Figure 2 [12]. Figure 2. HBOT affects multiple cellular and molecular pathways. HBOT affects several molecular and cellular pathways that are important for cellular and neuronal recovery including neuroprotec-tion via SIRT1, oxidative stress via SIRT1 and Nrf-2, apoptosis via SIRT1, neurogenesis via Wnt3. Green frames represent proteins and processes that are upregulated; red frames represent proteins and processes that are downregulated. Abbreviations: nuclear factor erythroid 2-related factor 2 (Nrf-2), nuclear factor kappa B (NF-B), Hypoxia-Inducible Factor 1-alpha (HIF1a), heme oxygenase 1 (HO-1), superoxide dismutase 1 (SOD1), malondialdehyde (MDA), B-cell lymphoma 2 (Bcl2), Bcl-2-associated X protein (Bax), vascular endothelial growth factor (VEGF-A), Glutathione-S-transfer-ases (GST), Glutathione Peroxidase (GPx), tumor necrosis factor alpha (TNFa),Wnt Family Member 3 (Wnt3). Figure 2.HBOT affects multiple cellular and molecular pathways. HBOT affects several molecular and cellular pathwaysthat are important for cellular and neuronal recovery including neuroprotection via SIRT1, oxidative stress via SIRT1 andNrf-2, apoptosis via SIRT1, neurogenesis via Wnt3. Green frames represent proteins and processes that are upregulated;red frames represent proteins and processes that are downregulated. Abbreviations: nuclear factor erythroid 2-relatedfactor 2 (Nrf-2), nuclear factor kappa B (NF-B), Hypoxia-Inducible Factor 1-alpha (HIF1a), heme oxygenase 1 (HO-1),superoxide dismutase 1 (SOD1), malondialdehyde (MDA), B-cell lymphoma 2 (Bcl2), Bcl-2-associated X protein (Bax),vascular endothelial growth factor (VEGF-A), Glutathione-S-transferases (GST), Glutathione Peroxidase (GPx), tumornecrosis factor alpha (TNFa), Wnt Family Member 3 (Wnt3).3. Mechanistic Explanation for the Effects of HBOT on CognitionWhat are the cellular and molecular pathways that contribute to the long-term neuron,function- and cognition-enhancing effects of HBOT? A series of studies using animalmodels for brain injuries and brain diseases showed an improvement in the animals’cognitive performance and provided a mechanistic understanding of some of HBOT’s

Biomolecules 2021, 11, 1520 5 of 11effects. Not surprisingly, these effects are not mediated by a single pathway, but werefound to be mediated by several pathways, including inhibition of apoptosis, improvementof mitochondrial function, stem cell proliferation, enhancement of antioxidant defenseactivity, reduction in neuroinflammation, and neuroprotection (Figure 2). The “normobaricoxygen paradox” or “hyperoxic–hypoxic paradox” has been suggested to play a key rolein HBOT’s effects [12,50–52]. It is based on the fact that during HBOT sessions, oxygenlevel is increased from 21 to 100% (or less in some cases) and at the end of each treatment,oxygen level is reduced back to 21%. Such fluctuations activate several factors: elevation ofoxygen can activate nuclear factor erythroid 2-related factor 2 (Nrf-2), while the reductionto 21% can be interpreted as a hypoxic signal and activate Hypoxia-Inducible Factor 1-alpha(HIF1a) [50,51]. HIF1a belongs to a family of proteins that are involved in angiogenesisand vascular remodeling, erythropoiesis, glycolysis, iron transport, and survival [53–55].Nrf2 is involved in several cellular defense mechanisms, and it mediates the repair anddegradation of damaged proteins [51,55,56], and activates the antioxidant pathways andthe detoxification of endogenous and exogenous products [57]. Under high hyperoxia,nuclear factor kappa B (NF-B), which is usually activated under oxidative stress andinflammation, is also activated [51], and mediates inflammatory and immune responses.NF-B is also involved in synaptic plasticity and in the antiapoptotic pathway by activatingBcl-2 [58]. Some of these effects are discussed below. It should be noted that the optimalconditions for achieving best results from the “Hyperoxic–Hypoxic Paradox” requireadditional research in the coming years.3.1. Mitochondrial FunctionMitochondria consume roughly 85 to 90% of the oxygen that we breathe and are themajor source of ATP production. It is therefore likely that the main molecular target ofHBOT is the mitochondrion. As already noted, humanin, a neuroprotective mitochondrion-derived peptide in humans, was elevated in VD patients following HBOT [36], suggestinga major role for mitochondrial activity in HBOT’s mechanisms of action. Recent studieshave suggested the therapy’s direct effects on neurons were mediated by mitochondrialtransfer from cell to cell. HBOT was shown to facilitate the transfer of mitochondria fromastrocytes to neuronal cells, making the latter more resilient to neuroinflammation [59].This neuroglial crosstalk may facilitate recovery and explain some of the mechanismsinduced by HBOT [50]. In TBI rats, HBOT for 4 h (1.5 ATA) led to an increase in ATP levelsand neuron survival, both of which were associated with improved cognitive recovery [60].Furthermore, in a rat model for AD, HBOT reduced mitochondria-mediated apoptosissignaling by increasing Bcl-2, which is anti-apoptotic, and decreasing Bcl-2-associated Xprotein (Bax), which is pro-apoptotic [61].3.2. Neurogenesis and AngiogenesisAn additional avenue for cognitive improvement might be stem cell proliferation.Stem cell proliferation has been documented on various occasions following HBOT [62–64],and evidence for neuronal cell proliferation has emerged in the last two decades. In an earlystudy, HBOT for hypoxic ischemic neonatal rats promoted neurogenesis of endogenousneuronal stem cells, as measured by an increase in both 5-bromo-20-deoxyuridine (BrdU)and doublecortin, in the subventricular zone (SVZ) and the hippocampal dentate gyrus(DG)—an area involved in spatial navigation [65]. Accordingly, HBOT improved spatiallearning and memory abilities in rats with TBI [66]. This was associated with an increase inhippocampal neuronal activity.These results were further supported by another study in which HBOT induced neu-ronal cell proliferation, as revealed by an increase in nestin and BrdU in the hippocampalDG area [67] and elevation of Wnt-3 and nestin in the SVZ [68]. In a study aimed atexamining the mechanistic contribution of HBOT to recovery from TBI, it was found thatHBOT increases neuronal stem cell proliferation and migration to the lesion area, as wellas the levels of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2, Raf-1,

Biomolecules 2021, 11, 1520 6 of 11Mitogen-activated protein kinase (MEK1/2), and phospho-extracellular signal-regulatedkinase (ERK) 1/2 protein [69]. Accordingly, it was suggested that HBOT promotes neu-ronal stem cell proliferation and possibly angiogenesis through VEGF/ERK signaling [69].Moreover, in a rat model for VD, HBOT also stimulated neurogenesis in the piriform cortexand improved blood supply [70]. HBOT was also shown to enhance mobilization of bonemarrow stem cells to an ischemic area and the release of trophic factors that can promotebrain and neuronal recovery and enhance neurogenesis [25]. Interestingly, in patients withdelayed encephalopathy after acute carbon monoxide poisoning, HBOT mobilized, circulat-ing stem cells in the peripheral blood, which was associated with improved cognition [71].In a TBI rat model, HBOT stimulated angiogenesis as evidenced by a higher numberof BrdU- and VEGF-positive cells, and an increase in the number of BrdU- and NeuN-positive cells, suggesting enhanced neurogenesis [72]. These findings provide support forimprovement of human brain cognition associated with changes in cerebral angiogenesisand neuronal growth and proliferation improving CBF and brain activity [33]. Indeed, arecent study showed that HBOT improves blood flow in an AD mouse model by mitigatingthe blood vessel constriction that occurs in these AD mice under the regular course ofthe disease but without HBOT. This was associated with an improved performance ofthe AD mice [48]. Moreover, in elderly patients with significant memory loss at baseline,HBOT increased CBF and improved cognitive performance [48]. It would be interesting toexamine whether HBOT also restores neurogenesis in neurodegenerative diseases such asAD, and whether it will affect neurogenesis and angiogenesis [73] in wild-type mice andhealthy humans.3.3. NeuroinflammationAnother important effect of HBOT in several brain dysfunctions is reduced neuroin-flammation. TBI is usually associated with increased inflammation, apoptosis and gliosis,neuronal cell death, and cognitive and motor dysfunction. In a TBI rat model, HBOTwas shown to reduce neuroinflammation and increase levels of the anti-inflammatorycytokine interleukin (IL)-10; these changes were associated with improvements in cogni-tive deficit [72]. In an AD mouse model, HBOT reversed hypoxia and ameliorated brainpathology, and improved the animals’ behavioral performance [74,75]. This improvementwas also associated with a reduction in proinflammatory cytokines such as IL-1b, IL-6, andtumor necrosis factor alpha (TNF↵), and an increase in anti-inflammatory cytokines such asIL-4 and IL-10, leading to reduced neuroinflammation. HBOT also significantly improvedrecovery from sepsis following cecal ligation and puncture; the treatment was associatedwith a reduction in the inflammatory response, including decreased expression of TNF↵,IL-6, and IL-10 [17,76]. Changes in cytokines following exposure to oxygen have also beenreported in humans. A low-intensity exercise program in combination with exposure tomild hyperoxia (30%) elevates the proinflammatory IL-6 that contributes to host defenseduring infection and tissue, while at both mild (30% oxygen) and high hyperoxic state(100% oxygen), the anti-inflammatory cytokine IL-10 was elevated significantly [52]. In arat model for MCI, HBOT had a protective effect on early cognitive dysfunction that wasmediated by ERK. These animals performed better in the Morris water maze, and showedless apoptosis and better hippocampal cell morphology [77]. In a rat model for AD thatwas induced by injections of amyloidpeptide into the hippocampus, HBOT improvedanimal behavior, and reduced neuronal damage, astrocyte activation, and dendritic spineloss. This was associated with a reduction in hippocampal p38 mitogen-activated proteinkinase (MAPK) phosphorylation [78], which occurs in the early stage of the disease and isassociated with increased neuroinflammation, cytoskeletal remodeling, and tau phospho-rylation [79,80]. These papers suggest that the MAPK/ERK pathways, which are involvedin cell proliferation and plasticity, are also a target for HBOT.

Biomolecules 2021, 11, 1520 7 of 113.4. Neuroprotective, Antioxidant, and Antiapoptotic ActivitiesHBO preconditioning induced tolerance to cerebral ischemia [81]. This was mediatedby an increase in SIRT1, a class III histone deacetylase, which has been suggested to beinvolved in neuroprotection [82]. The neuroprotective effect of preconditioning HBOT wasassociated with a reduction in lactate dehydrogenase and was attenuated by a reductionin SIRT1 activity or expression by either the SIRT1 inhibitor EX527 or SIRT1 knockdown.Interestingly, the neuroprotective effect was mimicked by resveratrol, a SIRT1 activator.Changes in SIRT1 level were also associated with elevation in B-cell lymphoma 2 (Bcl-2)expression and a decrease in cleaved caspase 3 level, suggesting that some of the effectsmight be mediated via inhibition of apoptosis [82]. Moreover, expression of SIRT1 in thebrain was associated with increased expression of the nuclear factor erythroid 2-relatedfactor 2 (Nrf-2), heme oxygenase 1 (HO-1), and superoxide dismutase 1 (SOD1), whereasthe level of malondialdehyde (MDA) decreased, supporting the notion that HBOT enhancesthe antioxidant defense pathway, thereby assisting in neuroprotection [83]. Indeed, HBOpreconditioning increased the expression of SIRT1, Nrf-2, and HO-1 and amelioratedmemory dysfunction in additional models of cognitive decline [84], and SIRT1 was alsoshown to play a role in recovery after middle cerebral artery occlusion in rats. Therefore,this might serve as the mechanism for HBOT’s effects in cases of acute ischemic stroke [85].A combination of HBOT and Ginkgo biloba extract following induction of toxicity withamyloid(Afragments) demonstrated enhanced SOD and glutathione levels, whilelevels of MDA and Bax, and activity of caspases 9 and 3 were reduced in rat hippocampaltissue, suggesting both antioxidant and antiapoptotic activity [61,86]. In a mouse modelfor mild TBI, HBOT improved learning abilities and prevented astrocyte activation andneuronal loss, suggesting a neuroprotective effect [87]. Additional involvement in apoptoticpathways was demonstrated in an AD rat model that showed improved cognitive andmemory abilities following HBOT, which were associated with NF-B pathway activationand reduced hippocampal neuron loss [88]. Further animal model studies may revealadditional mechanisms underlying the effects of HBOT, thus facilitating the development ofmore efficient HBOT protocols. Taken together, HBOT has a multifaceted neuroprotectiveeffect on the brain that involves the immune, neuronal and vascular systems, leading toenhancement and recovery of cognitive performance.4. HBOT—The Next LeapHBOT has been used for centuries to treat a variety of symptoms and syndromes, andin recent years, it has been shown to improve many brain disorders. Nevertheless, it is stillnot fully established clinically, and additional basic research and clinical trials are necessary.Notably, in recent years, numerous such clinical trials have been supported by the NIH.Over 230 clinical trials examining HBOT have been reported (https://clinicaltrials.gov/,accessed on 4 September 2021). Of these, 50 clinical trials are examining the effects of HBOTon brain-related injuries and disorders. Current and future clinical trials will provideadditional validated information for a wider range of disorders, while basic researchwill expand our mechanistic understanding and help optimize treatment conditions byallowing for more accurate determinations of treatment length, frequency of treatments,and the exact protocol. This will reduce cost, time, and complications. Overall, HBOT isbecoming a central player in the 21st century healthcare system with the ability to improveboth personal performance and cognition.Author Contributions:All authors were involved in writing—original draft preparation, review,and editing. All authors have read and agreed to the published version of the manuscript.Funding:N.S. is supported by a Scholarship from the Tel Aviv University Center for CombattingPandemics. U.A. was supported by The Aufzien Family Center for the Prevention and Treatment ofParkinson’s Disease at Tel Aviv University.Conflicts of Interest: The authors declare no conflict of interest.

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Review Article Access this article online Quick Response Code Website http www braincirculation org DOI 10 4103 bc bc_31_19 Hyperbaric oxygen therapy A new look on treating stroke and traumatic brain injury Bella Gonzales Portillo Trenton Lippert Hung Nguyen Jea Young Lee Cesar V Borlongan Abstract Although hyperbaric oxygen therapy HBOT is common as a treatment for injuries this study aimed to research the ability of HBOT in preconditioning to diminish any potential damage The hypothesis stated that HBOT preconditioning alleviated the death of cells in primary rat neuronal cells PRNCs by transferring mitochondria from astrocytes In this experiment PRNCs were given an HBOT treatment before a tumor necrosis factor alpha or lipopolysaccharide injury which resembled cell death associated with stroke and traumatic brain injury TBI After being examined the study found more cell viability in the PRNCs that had received HBOT precondition and a mitochondrial transfer The mitochondrial transfer was visualized by a series of images showing the transfer after the HBOT treatment This study demonstrated the ability of HBOT preconditioning as a treatment for inflammation in stroke and TBI with the transfer of mitochondria from astrocytes to PRNCs reducing cell death Along with discussion of the study this review also focuses on different stroke treatments in comparison with HBOT Keywords Hyperbaric oxygen therapy lipopolysaccharide mitochondria preconditioning primary rat neuronal cells stroke traumatic brain injury tumor necrosis factor alpha Introduction Status of Stroke and Traumatic Brain Injury D Department of Neurosurgery and Brain Repair College of Medicine University of South Florida Morsani Tampa FL USA Address for correspondence Dr Cesar V Borlongan College of Medicine University of South Florida Morsani Tampa FL 33612 USA E mail cborlong health usf edu Submission 27 05 2019 Revised 30 08 2019 Accepted 02 09 2019 iseases associated with the central nervous system incorporate a diverse set of pathologies 1 However the two most dominant adult neurodegenerative diseases in the United States namely stroke and traumatic brain injury TBI affect a large array of people 1 The American Heart Association found that stroke is responsible for 130 000 deaths each year making it the fifth leading cause of death in the United States Stroke is the number one cause for long term disability in the United States making the associated health care costs surpass 33 billion per year 2 In 2013 alone This is an open access journal and articles are distributed under the terms of the Creative Commons Attribution NonCommercial ShareAlike 4 0 License which allows others to remix tweak and build upon the work non commercially as long as appropriate credit is given and the new creations are licensed under the identical terms For reprints contact reprints medknow com 2019 Brain Circulation Published by Wolters Kluwer Health Medknow TBI led to 2 8 million emergency room visits hospitalizations and deaths while approximately 3 1 billion people were living with this disease with the health care cost of 76 5 billion in 2012 3 4 Cell Death Stroke and TBI utilize similar pathologies for the primary and secondary cell death mechanism which results from recurring neuroinflammation 5 An important pathological feature includes the creation of a necrotic tissue core which becomes irreparable after stroke and TBI 6 8 Secondary cell death of stroke and TBI have been linked to the breakdown of the blood brain barrier BBB which allows for inflammatory cytokines to cross the BBB and ultimately increase inflammatory response thus damaging the outcomes 9 Additional How to cite this article Gonzales Portillo B Lippert T Nguyen H Lee JY Borlongan CV Hyperbaric oxygen therapy A new look on treating stroke and traumatic brain injury Brain Circ 2019 5 101 5 101

Gonzales Portillo et al Hyperbaric oxygen therapy for neurodegenerative diseases neurodegeneration following the damaged BBB has been found due to factors such as oxidative stress apoptosis and mitochondrial dysfunction 10 13 Hyperbaric Oxygen Therapy A Novel Therapeutic Approach Hyperbaric oxygen therapy HBOT has been presented as a possible treatment for TBI and stroke 14 15 This method uses a pressurized chamber of 2 3 absolute atmospheres which results in hyperoxygenation of tissues thus inducing angiogenesis and the recruitment of progenitor cells to the damaged regions 16 18 HBOT can be used for patients with open wounds from burns or diabetic ulcers 19 20 The chronic stages of stroke are a possible window for HBOT to be used as the acute stage is more difficult and in need of higher technology to successfully accomplish this therapy 14 This therapy works to alleviate impairments associated with strokes such as memory loss language and comprehension deficits 14 21 The unpredictable occurrence of TBI impedes the usage of HBOT as treatment for brain trauma patients 22 Secondary cell death has become the main target for HBOT treatment as reduction in the levels of inflammatory cytokines has been associated with limiting peri infarct peri impact tissue loss 23 24 There is still more to investigate surrounding HBOT and the sequestration of inflammation 25 The Effectiveness of Hyperbaric Oxygen Therapy The role of the preconditioning paradigm is yet to be explored regarding HBOT Studies have demonstrated preclinical efficacy of HBOT preconditions for neuronal cell loss but rarely any mechanism based assessments have explained the function of HBOT 26 29 Alternative therapies to treat stroke and TBI have been investigated further 30 The mitochondria is an important point of investigation to further develop the understanding of HBOT preconditioning 31 33 Functioning extracellular mitochondria were transferred from astrocytes to neurons after neuronal cell death due to stroke 34 The current study looked at HBOT preconditioning in the limitation of neuronal cell death after the inflammatory response which imitated secondary cell death associated with stroke and TBI The possibility of transferring mitochondria as the treatment of neuronal cells was also explored in this study 35 40 The hypothesis states that when astrocytic mitochondria were transferred into neurons after HBOT preconditioning neuronal cell viability after inflammatory response would be improved Transferring Mitochondria This study illustrated the effects of HBOT preconditioning as a therapy treatment against cell 102 death that is associated with neurodegenerative diseases specifically stroke and TBI The study found that when primary neuronal cells were subjected to HBOT preconditioning before an inflammatory insult there was a reduced number of cell deaths On further investigation the study discovered an increase in the number of astrocytic mitochondria found in the primary neuronal cells These findings indicate that HBOT can reduce the inflammatory response of the neuronal cells through the transfer of mitochondria The function of mitochondria is important in stroke and TBI since it plays a role in the secondary injury mechanism 41 42 However using HBOT preconditioning as a treatment for these diseases has provided mixed results ranging from therapeutic to harmful 14 Reducing Cell Death The use of HBOT as therapeutic treatments has only been discovered recently Studies have proposed a variety of mechanisms that allow for HBOT s positive effects such as reducing inflammation and stabilizing the BBB 43 46 These mechanisms are associated with the destruction of mitochondria which led the current study to research the role of mitochondria as a target of HBOT 47 48 After stroke the transfer of mitochondria was observed lining up with the research 34 This discovery forms the possibility of an HBOT treatment for individuals at a high risk of the diseases by providing a way to reduce secondary cell death The current study was able to detect an increase in astrocytic mitochondria in the primary rat neuronal cells of the HBOT treated group using Mitotracker labeling Resisting Inflammatory Response Researchers also found that injured neurons who had been exposed to tumor necrosis factor alpha TNF alpha or lipopolysaccharide LPS increased the astrocytic mitochondrial transfer It was found that HBOT preconditioning in common conditions aided astrocytic mitochondrial transfer in comparison to the TNF alpha and LPS groups HBOT preconditioning combined with the inflammatory response increased the transfer of mitochondria This indicates that neurons with more astrocytic mitochondria are more likely to survive an inflammatory response than neurons with less astrocytic mitochondria This information supports the idea that astrocytic mitochondria are more resistant to inflammatory response than neuronal mitochondria 35 49 Hyperbaric Oxygen Therapy Induces Neuroprotection The study demonstrated that the HBOT treatment was tolerated because the cells remained viable marking this a safe and effective procedure Figure 1 It was also Brain Circulation Volume 5 Issue 3 July September 2019

Gonzales Portillo et al Hyperbaric oxygen therapy for neurodegenerative diseases Figure 1 Stroke impairs the mitochondria in cultured cells which is repaired by hyperbaric oxygen therapy found that the mitochondrial transfer occurred almost immediately after the HBOT treatment and lasted for around 20 min after the treatment ceased Using HBOT treatment for a short period induces neuroprotection and protects the cells from the effects of prolonged HBOT 50 51 This study had some limitations The images that were collected were taken after the HBOT which prevented the researchers from accurately detecting the beginning of the mitochondrial transfers Applying Hyperbaric Oxygen Therapy to Clinics Although this study demonstrated a single HBOT treatment multiple short HBOT treatments may also provide a functional result due to the neurological deficits caused by stroke and TBI Additional studies will continue to occur exploring both post and pre injury HBOT To begin using this treatment at the clinic a trial will need to take place with a population of individuals who are at a higher risk of these cerebrovascular injuries HBOT treatments will need to be observed in in vivo disease models To find the safest and effective treatment of HBOT various trials under different conditions will need to be tested 52 Recent studies have been able to test HBOT treatments on rodent models allowing them to find successful protocols 53 The Potential of Hyperbaric Oxygen Therapy Following the Food and Drug Administration regulations HBOT will be implemented in clinics once treatments are revised for human 19 20 HBOT preconditioning provides a possible treatment for inflammation associated with many cerebrovascular diseases HBOT can be an alternative method to other treatments for TBI and stroke such as invasive procedures such cell transplants 54 The astrocytic mitochondrial transfer to neurons acts as a mechanism of HBOT to provide protections against inflammation This ability to limit damage done by cerebrovascular injuries in high risk individuals may reduce the burden of these diseases on our economy Brain Circulation Volume 5 Issue 3 July September 2019 Current Stroke Treatments Revascularization has been explored as a stroke therapy method however new research points to nondrug neuroprotective therapies such as oxygen therapy as a way to prevent brain damage that results from a stroke 55 An ischemic stroke occurs when the blood supply to the brain is blocked therefore depriving the brain of sufficient oxygen 56 Studies have shown that HBOT can minimize neurological impairment caused due to a stroke by increasing oxygen supply therefore reducing ischemia injury 56 HBO preconditioning performed on rats has also been found to enhance an enzyme that protects against MCAO 57 Along with HBO normobaric oxygen NBO therapy has been explored In contrast to HBO NBO administers 100 oxygen at one atmosphere Studies have shown that NBO counteracts hypoxic conditions induced by an ischemic event 58 NBO protects the BBB from damage by inhibiting an NADPH oxidase enzyme complex that is unregulated during a stroke 59 HBO therapies have been administered following an ischemic event Results from a study show that these therapies had neuroprotective effects by facilitating the BBB integrity 60 63 In a different study focused on neuroprotection mediated by hormetic mechanisms hormetic dose responses were observed as decreasing the amount of damage caused by stroke and TBI 61 Along with these stroke therapies one study found that transplanting amniotic fluid stem cells may help reduce the damage caused due to a stroke by promoting neurogenesis 64 Stem cells are a viable option for stroke treatment since they can initiate regenerative processes in the brain 65 Mitochondrial dysfunction has been discovered to play a role in the neural damage that results from an ischemic event There is evidence that transferring healthy mitochondria from stem cells to replace ischemic injured cells is a viable method for treating damaged cells 66 Financial support and sponsorship Dr Borlongan is funded by National Institutes of Health NIH R01NS090962 NIH R01NS102395 NIH R21NS109575 and Veterans Affairs Merit Review I01 BX001407 103

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Received 21 January 2019 Revised 28 February 2019 Accepted 7 March 2019 DOI 10 1111 cns 13124 ORIGINAL ARTICLE Prophylactic treatment of hyperbaric oxygen treatment mitigates inflammatory response via mitochondria transfer Trenton Lippert1 2 Cesario V Borlongan1 1 Department of Neurosurgery and Brain Repair Center of Excellence for Aging and Brain Repair USF Morsani College of Medicine Tampa Florida 2 University of South Florida Honors College Tampa Florida Correspondence Cesario V Borlongan Department of Neurosurgery and Brain Repair Center of Excellence for Aging and Brain Repair USF Morsani College of Medicine Tampa FL Email cborlong health usf edu Funding information Center for Scientific Review Grant Award Number R01NS102395 Summary Aims Hyperbaric oxygen therapy HBOT has been widely used as postinjury treat ment however we investigate its ability to mitigate potential damage as a precondi tioning option Here we tested the hypothesis that HBOT preconditioning mitigates cell death in primary rat neuronal cells PRNCs through the transfer of mitochondria from astrocytes Methods Primary rat neuronal cells were subjected to a 90 minute HBOT treatment at 2 5 absolute atmospheres prior to either tumor necrosis factor alpha TNF alpha or lipopolysaccharide LPS injury to simulate the inflammation plagued secondary cell death associated with stroke and traumatic brain injury TBI After incubation with TNF alpha or LPS the cell viability of each group was examined Results There was a significant increase of cell viability accompanied by mitochon drial transfer in the injury groups that received HBOT preconditioning compared to the injury alone groups 44 5 2 vs 68 4 48 n 20 P

LIPPERT and BORLONGAN 816 chronic neuroinflammation 5 A key common pathological feature is neuronal cells was also examined 35 We hypothesized that a transfer the formation of a necrotic tissue core which is unrecoverable fol of astrocytic mitochondria into neurons following HBOT precondi lowing stroke and TBI 6 8 The onset and progression of secondary tioning would improve neuronal cell viability following inflammatory cell death of both diseases has been linked to the blood brain barrier insults BBB breakdown allowing various inflammatory cytokines to per meate the BBB infiltrate the brain and upregulate the inflammatory response 9 altogether worsening the disease outcomes In addition several other exacerbating factors such as oxidative stress apopto sis and mitochondrial dysfunction have been shown to contribute to additional neurodegeneration following BBB damage 10 13 2 M ATE R I A L S A N D M E TH O DS 2 1 Cell culture The research procedures involving animals were approved by the Hyperbaric oxygen therapy HBOT has been a treatment of in USF Institutional Animal Care and Use Committee IACUC Primary terest for stroke as well as TBI over the past decade 14 15 HBOT uti rat neuronal cells PRNCs were dissected from cerebral cortices of lizes a pressurized chamber usually 2 3 absolute atmospheres ATA E18 Sprague Dawley rat embryos Cells were seeded on poly D ly resulting in hyperoxygenation of tissues inducing local angiogenesis sine Fisher Scientific ICN10269480 coated glass coverslips in 6 in damaged regions of the body and recruitment of progenitor cells well plates Fisher Scientific 0877124 and cultured in Dulbecco s to the damaged regions 16 18 The common FDA approved uses of Modified Eagle Media DMEM Fisher Scientific 10567 014 con HBOT include treating patients with open wounds often resulting taining 4 5 g L glucose l glutamine 25 mmol L HEPES 10 fetal 19 20 The ability for a patient to bovine serum FBS Fisher Scientific SH3007103 and 1 antibiotic undergo HBOT during the acute stage of stroke has been difficult to antimycotic at a density of 1 5 106 cells per well After 24 hours accomplish due to the overall timeline of the ischemic event and a the medium was changed to Neurobasal medium Fisher Scientific limited number of facilities having the necessary equipment there 21103049 supplemented with B 27 Fisher Scientific 17504044 fore the chronic stages of stroke have been targeted as a possible Cells were cultured in incubator at 37 C with 5 CO2 Cells were from burn injuries or diabetic ulcers therapeutic window for HBOT 14 This regimen seeks to ameliorate utilized for experiments 7 days after seeding Astrocytes derived cognitive impairments synonymous with stroke such as memory from U 87 MG Astrocyte Cell Line Sigma 89081402 The cells loss language and comprehension deficits 14 21 Similarly the unfore were passaged six times for growth in T 175 flasks Fisher Scientific seen occurrence of TBI presents as a logistical hurdle in introducing 12562001 in DMEM with 10 FBS and 1 penicillin streptomycin HBOT as an acute treatment regimen for brain trauma patients 22 Fisher Scientific 15140122 One day prior to coculture the astro Mechanistically the secondary cell death with wider therapeutic cytes were seeded on a 0 4 m mesh plate insert Fisher Scientific window characterized by inflammation has become the main target 0877115 in DMEM with 10 FBS and 1 penicillin streptomycin of HBOT treatment research as reducing the levels of inflammatory at a density of 3 105 cells per well On day 7 postseeding the cytokines has been linked to limiting peri infarct peri impact tissue astrocyte mesh inserts were placed in coculture with the PRNCs loss 23 24 However the therapeutic mechanism of HBOT mediating Figure 1 the sequestration of inflammation is not fully understood 25 A key unexplored research theme in the use of HBOT as a treat ment for stroke and TBI involves investigations into the role of the 2 2 HBOT preconditioning paradigm Several studies have shown the preclini Hyperbaric oxygen therapy was administered using an OxyCure cal efficacy of HBOT preconditioning for attenuating neuronal cell 3000 hyperbaric incubator OxyHeal Health Group National City loss following an ischemic or traumatic event but little mechanism CA USA The HBOT regimen consisted of a single treatment at 2 5 based assessment has elucidated the therapeutic pathways solicited absolute atmospheres lasting 90 minutes with a 10 minute ascent by HBOT in these studies 26 29 The importance of alternative thera and descent period Figure 2 Following HBOT the cultures were pies for stroke and TBI patients has become evident 30 To this end returned to an incubator at 37 C and 5 CO2 Cultures remained in cognizant that mitochondrial dysfunction closely approximates a incubation for 24 hours prior to insult pathologic inflammatory response resulting from ischemia and trau matic insult evaluation of the mitochondria poses as a logical target of investigation in order to begin to understand the mechanism of 2 3 Injury HBOT preconditioning 31 33 Of note healthy extracellular mitochon To recreate the secondary cell death of inflammation observed in dria have been demonstrated to transfer from astrocytes into neu stroke and TBI we employed two established inflammation induc rons following stroke resulting in reduced neuronal cell death 34 ing agents 36 38 The tumor necrosis factor alpha TNF alpha Fisher In this study we explored the ability of HBOT preconditioning to Scientific 210TA020CF only and HBOT plus TNF alpha cocultures limit neuronal cell death following inflammatory insults mimicking were treated with 50 ng mL of TNF alpha RD for 24 hours39 in the the secondary cell death associated with ischemic stroke and TBI incubator at 37 C and 5 CO2 The Lipopolysaccharide LPS Fisher More importantly the potential role of HBOT in transferring mito Scientific NC0202558 injury and HBOT plus LPS groups were chondria as a therapeutic mechanism in facilitating survival of the treated with 100 ng mL of LPS and returned to the incubator 40 Each

817 LIPPERT and BORLONGAN F I G U R E 1 Experimental design Timeline of experiment including cell culture and immunocytochemistry Hyperbaric oxygen preconditioning in vitro treatment Astrocytes stained with Mitotracker are cocultured on top of E18 primary cortical neurons PRNCs in a growth medium The cocultured plates were then placed in a hyperbaric oxygen chamber for treatment Mitotracker of astrocytes was found in the primary E18 cortical neurons F I G U R E 2 Hyperbaric oxygen therapy course Pressure graph of hyperbaric oxygen preconditioning During the first 10 min there is a constant increase of pressure until the chamber reaches 49 5 feet of seawater FSW This pressure is sustained for 70 min followed by a constant depressurization of the chamber over a 10 min period The total length of the preconditioning treatment is 90 min insult used the same medium as previously described for neurons and astrocytes The control plates remained untreated images were captured per well in randomly selected regions to determine cell viability at 10 Analysis of cell intensity was per formed by ImageJ NIH During cell counting 30 25 cm2 regions 2 4 Measurement of cell viability After the 24 hour incubation with TNF alpha and LPS all groups were counted using ImageJ and calculated to determine the number of cells per 1 cm2 Analysis of data was conducted using GraphPad Prism 6 GraphPad Software La Jolla California USA were assessed for cell viability using calcein AM Fisher Scientific 4892010K After treatment the astrocyte mesh inserts were re moved and the remaining 6 well dish was incubated with 1 mol L 2 5 Immunocytochemistry calcein AM for 30 minutes in the incubator at 37 C with 5 CO2 Prior to coculturing neurons and astrocytes each was labeled with Bright green fluorescence was retained within living cells Five mitotracker in order to visualize mitochondria belonging to each

LIPPERT and BORLONGAN 818 cell type Astrocytes were labeled with Mitotracker Deep Red FM Fisher Scientific M22426 for 30 minutes in DMEM with 10 FBS and 1 penicillin streptomycin Fisher Scientific 15 140 122 with 500 nmol L of Mitotracker Deep Red Neurons were labeled with Mitotracker Green FM Fisher Scientific M7514 for 30 min utes in Neurobasal with B 27 supplement at 37 C and 5 CO2 Following HBOT and insult cells were rinsed with Dulbecco s phosphate buffered saline with calcium and magnesium DPBS Fisher Scientific 14080055 and then fixed with 4 paraform aldehyde PFA for 20 minutes at room temperature Cells were rinsed again with DPBS Cultures were permeabilized with 0 3 Triton X for 5 minutes at room temperature Plates were rinsed with DPBS before blocking with 5 goat serum at room tem perature Mouse anti MAP2 1 g mL was added to each well and incubated overnight at 4 C After rinsing several times with DPBS Alexa Fluor 488 goat anti mouse 1 g mL was added and incubated for 60 minutes at room temperature Plates were then rinsed with DPBS and coverslips were placed on slides using Vectashield with 4 6 diamidino 2 phenylindole DAPI Fisher Scientific NC9524612 Immunostaining images were captured using an Olympus FV1200 Spectral Inverted Laser Scanning Confocal Microscope Colocalizations of astrocytic mitochondria with PRNCs as well as with endogenous mitochondria from PRNCs were assessed 3 R E S U LT S 3 1 Hyperbaric oxygen treatment rescues cell viability Calcein AM cell viability staining was performed and imaged using a fluorescent inverted microscope Figure 3 Cell viability of E18 cortical neurons cocultured with U 87 astrocytes was measured via cell counting of neurons and intensity of calcein staining Control E18 cortical neurons had an average of 7 11 cells per 1 cm2 and an average intensity of 4290 E18 cortical neurons exposed only to HBOT had an average of 7 15 cells per 1 cm2 and an average inten sity of 3744 Cortical neurons administered TNF alpha had an aver age of 5 60 cells per 1 cm2 and an average intensity of 1507 while cortical neurons administered LPS had an average of 3 56 cells per 1 cm2 and an average intensity of 2025 The cortical neurons exposed to HBOT and then administered TNF alpha had an aver age of 5 34 cells per 1 cm2 and an average intensity of 3045 The cortical neurons exposed to HBOT and then received LPS had an average of 5 64 cells per 1 cm2 and an average intensity of 3192 A One way ANOVA was conducted to analyze the effect of HBOT on each condition There was a statistically significant effect of HBOT on viable cell count per 1 cm2 for all six conditions F5 167 33 18 P

819 LIPPERT and BORLONGAN F I G U R E 4 Mitochondrial transfer to primary cortical neurons Migration of astrocyte mitochondria was tracked using Mitotracker staining of astrocytes prior to coculture with primary rat neuronal cells PRNCs Cocultures were subjected to the same preconditioning setting as previously described Transfer of astrocyte mitochondria cyan was quantified by counting the number PRNCs containing cyan and dividing by the total number of PRNCs marked with MAP2 red DAPI was utilized to mark PRNC nuclei P

LIPPERT and BORLONGAN 820 F I G U R E 5 Live imaging of primary cortical neurons undergoing mitochondrial transfer Rat E18 neuronal cells were harvested and seeded in poly D lysine coated 100 g mL 6 well plates at 1 5 106 cells well in Dulbecco s Modified Eagle Media high glucose with 1 antibiotic antimycotic for 24 h The media was changed every 3 d and the cells were subcultured at 90 confluency as needed Twenty four hours prior to the preconditioning U87 astrocytes were stained with MitoTracker Deep Red FM 500 nmol L according to manufacturer s protocol and seeded into coculture inserts at 0 5 106 cells well On the day of the experiment rat E18 neuronal cells were stained with MitoTracker Green FM 200 nmol L according to manufacturer s protocol The neuronal cells were then cocultured with U87 astrocytes for 3 h prior to Hyperbaric oxygen therapy HBOT administration The cells were subjected to 70 min of HBOT at 2 5 ATA with 10 min pressurization and depressurization at a rate of 0 07 atm min for a total of 90 min Directly following HBOT treatment the cocultured astrocytes were removed and the confocal z stacks live images were captured at 180 every 5 min for 30 min Primary rat neuronal cell PRNC mitochondria Green Astrocyte mitochondria Red The scale bar corresponds to 20 m 4 D I S CU S S I O N TBI providing a method to reduce the Inflammation plagued sec ondary cell death The present study demonstrated the therapeutic effects of HBOT We found a substantial increase of astrocytic mitochondria in the preconditioning in protecting against the secondary cell death as primary neurons particularly in the HBOT preconditioned groups via sociated with cerebrovascular events specifically stroke and TBI Mitotracker labeling Although there appears to be a natural trans Primary neurons that were exposed to HBOT at 24 hours prior to fer of mitochondria from astrocytes to neurons as seen in the con an inflammatory insult exhibited a significant reduction in cell death trol cells grown under ambient cell culture condition the presence compared to the injury only groups Further analysis revealed sub of an injury to the neurons with exposure to TNF alpha or LPS sig stantial increase of astrocytic mitochondria in the inflammation in nificantly increased this astrocytic mitochondrial transfer Figure 4 sulted primary neurons particularly in the HBOT preconditioned Additionally HBOT preconditioning under ambient condition facil groups Altogether these results suggest HBOT reduces the del itated the astrocytic mitochondrial transfer when compared to the eterious inflammatory response potentially through the transfer of TNF alpha and LPS only groups Figure 4 The combination of pre mitochondria from astrocytes to neurons highlighting a highly inno conditioning and inflammatory insult further increased the astrocytic vative mechanistic pathway mediating the therapeutic efficacy of mitochondrial transfer Figure 4 suggesting that neurons primed HBOT preconditioning with a surplus of astrocyte mitochondria were better metabolically Mitochondrial dysfunction stands as a therapeutic target equipped to survive an inflammatory insult compared to neurons with in stroke and TBI due to its role in the secondary injury mecha only a small number of astrocytic mitochondria That astrocytic mito nism 41 42 However HBOT as a treatment for cerebrovascular chondria may be more resistant to insults than neuronal mitochondria diseases has yielded mixed results 14 The initiation of HBOT may partially supports the notion that astrocytes in general do not easily dictate the therapeutic or detrimental outcomes To date the succumb to cell death after stroke compared to neurons 35 49 modality of HBOT preconditioning has been largely neglected Of equal translational importance we also demonstrated that ad HBOT s use as a prophylactic treatment for cerebrovascular dis ministration of HBOT at 2 5 ATA for 90 minutes was well tolerated as eases has only recently come into the spotlight for stroke and TBI evidenced by maintained neuronal cell viability highlighting the safety research Recent studies have postulated various mechanisms un of the HBOT as a prophylactic treatment Such initiation of HBOT derlying HBOT s neuroprotective effects including stabilizing the before injury led to a significant neuroprotective effect Figure 3 In BBB and reducing inflammation 43 46 That BBB breakdown and in addition we found that the transfer of mitochondria occurred imme flammatory response are closely associated with mitochondrial im diately within a short period ie 5 minutes following HBOT Figure 6 pairment provided the impetus in the present study to examine the and persisted at least up to 20 minutes post HBOT Figure 6 This ob role of mitochondria as a therapeutic target of HBOT 47 48 Indeed servation of effective transfer of mitochondria even with acute HBOT the transfer of mitochondria from astrocytes into neurons was ob prior to injury supports the use of a short bout of HBOT at low ATA as served after stroke 34 Here we showed that astrocytic mitochon a powerful approach to induce neuroprotection which circumvents dria also transferred to neurons under ambient condition or when reported adverse effects of prolonged HBOT at high ATA 50 51 exposed to an inflammatory insult but such transfer was more ro While not significantly detracting from our conclusions there bustly recognized when treated with HBOT prophylactically These are limitations to this investigation The live imaging obtained findings form the basis for prophylactic HBOT for individuals who was conducted after completion of HBOT exposure thereby pre are at high risk of cerebrovascular events specifically stroke and venting us from accurately pinpointing the onset of mitochondria

821 LIPPERT and BORLONGAN F I G U R E 6 Live imaging of primary cortical neurons undergoing mitochondrial transfer Rat E18 neuronal cells were harvested and seeded in poly D lysine coated 100 g mL 6 well plates at 1 5 106 cells well in Dulbecco s Modified Eagle Media high glucose with 1 antibiotic antimycotic for 24 h The media was changed every 3 d and the cells were subcultured at 90 confluency as needed Twenty four hours prior to the preconditioning U87 astrocytes were stained with MitoTracker Deep Red FM 500 nmol L according to manufacturer s protocol and seeded into coculture inserts at 0 5 106 cells well On the day of the experiment rat E18 neuronal cells were stained with MitoTracker Green FM 200 nmol L according to manufacturer s protocol The neuronal cells were then cocultured with U87 astrocytes for 3 h prior to Hyperbaric oxygen therapy HBOT administration The cells were subjected to 70 min of HBOT at 2 5 ATA with 10 min pressurization and depressurization at a rate of 0 07 atm min for a total of 90 min Directly following HBOT treatment the cocultured astrocytes were removed and the confocal z stacks live images were captured at 180 Primary rat neuronal cell PRNC mitochondria Green Astrocyte mitochondria Red White arrows indicate the movement of astrocyte mitochondria into the PRNC during 5 min intervals The scale bar corresponds to 20 m transfers Moreover although a single bout of HBOT was shown Florida Morsani College of Medicine Tampa Florida USA All re here as safe and effective in promoting neuroprotection repeated search materials including data reported in this study can be easily short HBOT exposures may provide more stable and long lasting accessed by contacting Dr Cesario Borlongan functional outcomes considering the devastating neurological deficits acutely and chronically after a cerebrovascular event The combination of pre and postinjury HBOT will also warrant addi tional studies In order to translate this preconditioning paradigm C O N FL I C T O F I N T E R E S T The authors declare no conflicts of interest to the clinic identification of a candidate population of individuals who are at an increased risk of cerebrovascular injury will be key to the successful enrollment of patients The observed in vitro HBOT results definitely will require validation in in vivo disease models ORCID Cesario V Borlongan https orcid org 0000 0002 2966 9782 In the end testing a variety of HBOT conditions in clinically rele vant models are critical to achieving the optimal safe and effective regimen of mitochondria transfer mediated neuroprotection 52 REFERENCES Recent studies have established successful protocols for testing 1 Borlongan CV Burns J Tajiri N et al Epidemiological survey based formulae to approximate incidence and prevalence of neuro logical disorders in the United States a meta analysis PLoS One 2013 8 10 e78490 2 Benjamin EJ Blaha MJ Chiuve SE et al Heart disease and stroke statistics 2017 update a report from the American 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opportunities for therapeutic intervention Brain Behav Immun 2012 26 8 1191 1201 HBOT in rodent models for both single and multiple treatments 53 Due to prior FDA approved indications HBOT has an established infrastructure in clinics allowing for it to be quickly implemented once treatments are optimized for humans 19 20 HBOT preconditioning poses as a prophylactic treatment for sequestration of inflammation which is a pathological condition rampant in many cerebrovascular diseases HBOT may be a leading alternative treatment for TBI and stroke modalities as steers away from invasive procedures such as exogenous cell transplantation fol lowing a major cerebrovascular event 54 Mitochondrial transfer from astrocytes to neurons is a potential primary mechanism of action of HBOT to conferring neuroprotective effects against inflammation The ability to limit the severity of cerebrovascular injury in identified at risk individuals may reduce the health burden and socioeconomic load of these diseases on our healthcare system and economy AC K N OW L E D G E M E N T S This research was supported by Dr Cesario Borlongan at the Center of Excellence for Aging and Brain Repair at the University of South

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