Parkinsons & HBOT : simplebooklet.com

S-94Clinical and Experimental Rheumatology 20201Rheumatology Unit, University of Messina; 2Trauma and Orthopaedic Unit, Santissima Trinità Hospital, Cagliari; 3Department of Rheumatology, ASST Fatebenefratelli-Sacco, Milan; 4Department of Clinical Neurosciences, Hermanas Hospitalarias, Villa S. Benedetto Menni Hospital, Albese (Como); 5Humanitas Clinical and Research Centre, IRCCS, Rozzano, Italy.Fabiola Atzeni, MD, PhDIgnazio Francesco Masala, MDMariateresa Cirillo, MDLaura Boccassini, MDStefania Sorbara, MDAlessandra Alciati, MDPlease address correspondence to:Fabiola Atzeni, Dipartimento di Reumatologia Università di Messina, Via Consolare Valeria 1, 98100 Messina, Italy.E-mail: atzenifabiola@hotmail.com Received on October 2, 2019; accepted in revised form on December 16, 2019.Clin Exp Rheumatol 2020; 38 (Suppl. 123): S94-S98.© Copyright CLINICAL AND EXPERIMENTAL RHEUMATOLOGY 2020.Key words: Competing interests: none declared.ABSTRACTObjectivethe therapeutic mechanisms underlying hyperbaric oxygen therapy (HBOT), and reviews data concerning its effects     Methods. The studies included in this review all evaluated the effect of HBOT in patients with diseases involv-ing CNS. The PubMed databases were searched from 1980 to September 2019    -Results. HBOT is already indicated in various diseases and is the subject of continuous research and develop-ment. Data from models of PD show that it may play a neuroprotective role because of its ability to reduce oxida-tive stress and neurodegeneration, and protect against neuronal apoptosis. It is effective in improving the symptoms       --tivity in pain-related areas. Evidence from animal studies supports its use as an alternative treatment for other rheu-matic diseases as it alleviates pain and Conclusion. Data mainly from animal studies support the use of HBOT in the treatment of PD and rheumatic dis-clarify its therapeutic role in patients with these chronic disorders. Introduction                        --    --     ----Principles of hyperbaric oxygen therapy   -    --     -       -                                    Review

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Clinical and Experimental Rheumatology 2020Hyperbaric therapy in chronic diseases / F. Atzeni et al.-           -     -     -   -       --                         --             -                  -     -    -     -           -        -     -            -    -                 ---          -     --      -         -                   -     -            -                -       -                   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Clinical and Experimental Rheumatology 2020 --                 -          -                       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        -       -                            -      -                --et al.-                                   -      - et al.        i.e.                    -                                                      -                     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-Parkinson’s disease--                          -----    Hyperbaric therapy in chronic diseases / F. Atzeni et al.

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Clinical and Experimental Rheumatology 2020Hyperbaric therapy in chronic diseases / F. Atzeni et al.                 -        -                 -              -    -       -                               -  --       -            -    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 -    -                                                                          Conclusions                 -     References    J Intensive Care Med         J Vet Emerg Crit Care                          -Neurosci Lett      et al - Plos One     -     -   Plast Reconstr Surg       N Engl J Med    J Cardiovasc Surg            -Arch Surg      et al        -    -          - -     -Surgery  -Toxic ol A ppl Pha rmaco l     --Plastic Reconstr Surg         -Int Anesthesiol Clin     et al    Undersea Biomed Res--et al.     Aviat Space Environ Med--Acta Ophthalmol Scand              Eur Respir J      

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Clinical and Experimental Rheumatology 2020Hyperbaric therapy in chronic diseases / F. Atzeni et al.  Nat Rev Dis Primers      et al        Biochim Biophys Acta-    --Int J Neurol Neurosurg           -     J Formos Med Assoc-      -   -Clin BiochemVA N             -Resuscitation        Medicine     et al    -Isr Med Assoc J     et alJ Rheumatol      et al.-  Arthritis Rheum      et al  Ger Med Sci          Scand J Rheumatolet al    -et al         -     -         FrontPsychol     Set al    -   Front Psychol      et al    -    Clin Exp Rheumatol      et al    ---Clin Exp Rheumatol et al           -          Clin Exp Rheumatol --          Brain Res                         Arthri-tis Rheum     Br J Rheumatol              -J Painet alPain Physician     -      -          -J Appl Physiol--Clin Exp Immunol     et al    Clin Immunol-    J Rheuma-tol                 Int J Rheum Dis-             Ital J Pediatr             Clin Exp Dermatol

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Parkinson’sandHBOTParkinson’sdiseaseorPDisadisorderofthecentralnervoussystemthatimpairsmotorskills, speechandothermentalfunctions.PD  belongs toagroupcalledmovementdisorders.Thesedisorders typicallyproducemusclerigidity,tremorandageneralslowingofphysicalmovement. PDaffectsadultsofallagesbutisnotconsideredfatal.However,latestageParkinson’smayleadtochoking,pneumoniaorseriousfalls,allofwhichcancausedeathordisability.WhatHBOTcandoThesymptomsofParkinson’scanvaryintypeandseveritythereforeitcanbedifficulttopredictresults.OftenHBOThasbeen discoveredeffective by “accident” as in the case where a diabetic patient tried HBOT for a foot ulcer but foundthat the treatment vastly improved his Parkinson’s symptoms. After 50 years of HBOT treatment we do know thathyperbaricoxygenwillnotworsenPD.NumerousanimalstudieshaveshownthatHBOTworksasanantiinflammatory andmaybeusedinthiswaytoalleviatethesymptomsofPD.WhyisParkinson’sDiseaseamenabletooxygentherapy?Extensiveanimalresearchhasdemonstratedanonspecificchronicinflammatoryconditioninthesubstantianigraofthebrain.Hyperbaric Oxygen Therapy (HBOT) has been shownto be an antiinflammatory drug in many conditions.AnecdotalevidenceofmanypatientswithwellestablishedPDhavebeen treatedwithHBOTforotherconditionssuchasdiabetic foot ulcers.For example, a patient with advanced PD who is confined to a wheelchair may get up and walkacrosstheroomafteraseriesofHBOT.WhatbenefitscanIexpectfromoxygentherapyforParkinson’sDisease?Sinceeverypatientisdifferentitishardtopredicttheresultineachindividualcase.However,weknowfrom50yearsofexperience thatHBOTissafeandwillnotmakethepatientworse.Theusualcourseofoxygentherapyisoncedaily,fivedaysa week (MF)for eight weeks.Ifasignificantresponse is noted after40HBOTadditional treatments maybehelpful.OneTwoPunch:GlutathioneandHBOTJulianWhitaker,MDParkinson’s disease iscaused by thedegeneration of neurons in the area of the brain that manufactures dopamine, aneurotransmitter that affects movement. As dopamine production drops, characteristic tremors and speech, balance,and motor problems develop. The primary treatment for Parkinson’s is drugs that replace or mimic dopamine, andthoughthesemedsimprovesymptoms,theydonotslowdiseaseprogressionandtheirsideeffectsincreasewithlongtermuse.Although there’s a lot that medical science does not know about Parkinson’s, we do know that freeradical damagecontributestoitsprogressivenature.That’swhyweuseglutathione.Glutathione/HBOTtotheRescueGlutathioneisapowerfulnaturalantioxidant,andpatientswithParkinson’shavedangerouslylowlevelsofglutathioneintheaffectedareaofthebrain.Boostingstoresofthisprotectiveantioxida ntnotonlyguardsagainstfurtherdamage,italsoenhancesthefunctionofsurvivingneurons.

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Unfortunately, oral glutathione has a hard time crossing the bloodbrain barrier, so supplements aren’t very helpful.When glutathione isinfused intravenously, however, it hits its target.Most patients see dramatic improvementsafterjustahandfuloftreatments—andmanyperkupaftertheirfirstinfusion.Evenbetter,studiessuggestthatbenefitslast fortwotofourmonthsafteratreatmentcourse.Our patients with Parkinson’s disease are also treated with hyperbaric oxygen therapy. HBOT is highly beneficial forstroke, multiple sclerosis, and brain injuries. It floods the brain with oxygen, slows  neuronal degeneration, mobilizesrejuvenatingstemcel ls,andenhancesangiogenesis(thegrowthofnewbloodvesselsthatnurturedamagedareas).Itis thecombinationofthesetwotreatments,workingsynergistically,thatprovidessuchremarkableresults.SeriousCondition,SeriousInterventionParkinson’s is a serious condition that requires serious intervention. Coenzyme Q10, vitamin E, fish oil, curcumin,creatine, and vitamin D, along with Nacetylcysteine and vitamin C (both of which boost glutathione levels), showpromise in improving symptoms and even slowing progression. I certainly recommend taking them. However, thesesupplementsdonotcomeclosetoapproachingthetherapeuticpowerofIVglutathioneandHBOT.Sadly, very few medical facilities offer these therapies.In fact, many physicians don’t know a thing about them!Furthermore, although benefits are enduring, they don’t last forever, and maintenance treatments are required foroptimalfunction.Idon’tknowwhatit’sgoingtotaketogetconventionalphysicianstoembraceIVglutathioneandHBOT,butdon’tholdyour breath waiting for your docto come around. I stronglyurge you to find a treatmentcenter near you, andif youcan’t,considercomingtotheWhitakerWellnessInstitute .RecommendationsThesuggesteddailydosesofsupplementsforParkinson’sdiseasearecoenzymeQ10,aminimumof1,200IU;vitaminE,400–800IU;fishoil,6–8g;curcumin,1,000–2,000mg;creatine10g;vitaminD2,000–4,000IU;Nacetylcysteine1,200–1,800mg;andvitaminC,1,000–2,000mg.Lookfortheminyourhealthfoodstoreorcall(800)8106655toorder.Tak eindivideddoses.ReferencesHoggardM,etal.HyperbaricoxygentreatmentonaParkinson’sdiseasepatient:acasestudy.Proceedingsofthe14thInternationalCongressofHyperbaricMedicine,SanFr ancisco,CA,2002.Sechi G, et al. Reduced intravenous glutathione in the treatment of early Parkinson’s disease. ProgNeuropsychopharmacolBiolPsychiatry.1996Oct;20(7):1159–1170.ModifiedfromHealth&HealingwithpermissionfromHealthyDirections,LLC.Photocopying,reproduction,orquotationstrictlyprohibitedwithoutwrittenpermissionfromthepublisher.HyperbaricOxygenationDuringtheComplexTreatmentofParkinsonismV.YaNeretin,M.A.Lobov, S. V. Kotov,G.F.Cheskidova,G. S. Mo1chanova and0.GSafronova.Neurology Department(headed by Prof V. Ya. Neretin), M. F. Viadimirsky Regional Research Institute of Clinical Medicine (DirectorG. A.Onopriyenko),Moscow.Hyperbaric oxygenation (HBO2) was used to treat 64 patients suffering from parkinsonis m of diverse etiology. HBO2sessionswereprovideddaily)812percourse;treatmentpressurerangedfrom1.3to2atmandexposuretimeranged

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from40to60minutes.Amarkedbeneficialeffectwasnotedin55patients.HBO2treatmentproducedbetterresultsinthepresenceofvascillar parkinsonism,inpatientsunder65yearsofage,andwhenthehistoryofdiseaserangedfrom1to 5 years. The akineticorigid syndrome regressed to a greater extent, with HBO2 proving to be less effective whentremblinghyperkinesiswas present Su bmittedtotheeditorialofficeon03March1988 HyperbaricOxygenTreatmentonaParkinson’sDiseasePatient:ACaseStudyHoggardML,JohnsonKEandShirachiDY.Chico Hyperbaric Center, Chico, CA 95926 and Department of Physiology and Pharmacology, T. J. Long School ofPharmacy,UniversityofthePacific,Stockton,CA95207,USA.INTRODUCTIONParkinson’s Disease (PD) is a chronic neurodegener ative disorder, which is characterized by the loss of dopaminergicneurons whose cell bodies are located in the substantia nigra pars compacta (SNpc) and project to the striatum. Theinitiation of this neuronal degeneration is not known, however the process of neuronal loss is suggested to occur viaapoptosisratherthanbynecrosis(1).Withtheonsetoftheneurodegenerationoftheseneuronsistheassociatedlossofthe neurotransmitter, dopamine  (DA), from its nerve endings and its subsequent release in the striatum. The majorsymptomswhichareobservedduetotheprogressivelossinfunctionofthenigrostriataldopaminergicneuronsmaybeone or more of the following: resting tremor, rigidity, bradykinesia and/or postural instability. The actual clinicalmanifestationofthediseaseinanyonepatientishighlydependen tuponthedegreeofseverityoftheneuronalloss,ageof the patient and the length of time passed between the onset of the symptoms and the time of diagnosis. Earlydetectionisimportantinordertoinstitutea therapeuticstrategytorelievethesymptomsand/ordelaytheprogressionofthediseasestate.ThemajortreatmentstrategycurrentlyusedistoaffectthefunctionofDA.BecausesystemicallyadministeredDAdoesnotcrossthebloodbrainbarrier;Levodopa(prodrug)isadministered,whichistakenupintothebrain.SinceLevodopaismetabolizedbothperipherallyandcentrallytoDAbyaDOPAdecarboxylase,carbidopaaninhibitorofthisenzymeisadministeredincombinationwithLevodopatodecreaseitsmetabolismperipherallyincreasingitsuptakeintothebrain.DA agonists andmonamine oxidaseB(MAOB) inhibitors are also administered asa mono therapy or as an adjunct toLevodopacarbidopa(Sinemet)therapy,dependingupontheclinicalcondition.TakingaverydifferentapproachinthetreatmentofPD,Borromeietal.in1996showedthathyperbaricoxygen(HBO)therapyappearedtobeeffectiveinamelioratingmanyofthebehavioralandmotordeficitsobservedinPDpatients(2).The objective of this study was to determine whether HBO therapy might enhance the effects of an antiparkinsontreatmentinaPDpatientasanadjuncttherapeuticmodality.METHODSBriefpatienthistory:A72yearoldmalewasdiagnosedwithidiopathicPDandplacedonSinemet(10/100)threedoses3timesdaily.Oneyearafterdiagnosis forPDthepatientwasdiagnosedwithtotalocclusionoftherightcoronaryartery.Asuccessful total occlusion angioplasty was performed and he was placed on Lopressor and Lipitor 10 mg daily. Therewere no complications from this surgical procedure. Eighteen months after being diagn o sed  as a PD patient he wastreated with hyperbaric oxygen (HBO) at 1.9 ATA for 90 min. The patient was treated daily 5 times each week for 5weeks(25treatments).DuringthetreatmentthepatientgraduallyreducedhisdoseofSinemetuntilhewascompletelyoff of this medication between the 3rd and 4th week of HBO treatment. At this point his physician placed him onselegiline10mgtwicedaily.

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Clinicaltesting:Thepatient’svoiceandspeechwereevaluatedby aspeechlanguagepathologist, andtheJebsenTaylorhand function test was performed by an occupational therapist prior to and after the end of the HBO therapy. Thepatientwasinformedofallaspectsofhyperbaricoxygentherapy,includingallrisksofadverseeffectsaccordingtotheDeclarationofHelsinki.Thepatientalsosignedaninformedconsentformdetailingthetreatmentandtherightsofthepatient.RESULTSVoice and speech. There was little change in the overall evaluation of voice and speech after HBO therapy.Communicationstatuschangedverylittle.Heappearedtobetalkingmoreandhisratewassomewhatimproved.Hestill haddifficultyprojectinghisvoice.JebsenTaylorHandFunctionTest.Theresultsofthis testareshowninTable1.IntestingthedominanthandthereweresmallincrementsofimprovementafterHBO.Thetotalimprovementwasmorethan10%,whiletheimprovementinthenondominanthandwasnearly32%.During the treatment period, the patient voluntarily reduced his Sinemet doses until he was completely off the drugafter34weeksofHBOtherapy,whichwasanunexpectedresult.HehascontinuedtoremainoffofSinemettherapy.Nocomplicationsoradversesideeffectssuchasmyopiawereobserved.ThelongtermexposureofHBOwastoleratedwellbythepatient.DISCUSSIONPDischaracterizedbytheloss ofdopaminergi c neuronsofthe nigrostriatalpathway.Itisnotclearhowthisneuronaldegeneration is initiated, but there appears to be a number of potential ways in which this might occur in any oneindividual, including genetics, disease,  drugs or other chemicals, oxidative stress and/or other environmental factors. However, once it is initiated there seems to be agreement that the degenerative process involves apoptosis and notnecrosis.TheresultsofthisstudysuggestthatHBOmightbeapossiblenewmodalityoftreatmentforPDbecauseitappearedtobeabletoreplaceSinemetasatherapeuticregimen.ThemechanismbywhichtheHBOeffectmightbeoccurringmaybepartlyduetoanantiapoptoticeffect.IthasbeenshownthatHBOincreasedtheexpressionofBcl2protein,amajorantiapoptoticprotein,intreatingforebraincerebralischemiaingerbils(3).TheBcl2proteinhasalsobeenelevatedbyrepeated HBO treatment in normal gerbils (4). So itis possible thatHBOinthisstudyinhibited the apoptotic pathwayinvolvedintheprogressiveneuronaldegenerationbystimulatingtheexpressionoftheBcl2proteins.OtherpossibleHBOeffectsshouldnotbediscountedsuchasimprovedoxygenperfusionduetoincreasedextravascularoxygen diffusion and to possible angiogenesis (5). Axonal repair and regeneration and/or synaptogenesis could occurdue to increased expression of neurotrophin(s), since HBO has been shown to increase vascular endothelial growthfactor(6)andactsynergisticallywithplateletderivedgrowthfactorandtransforminggrowthfactorbeta(7).The results of this case study agree with much of the results observed in the clinical studyby Borromei and hiscoworkers. It is not clear from their study whether some of their patients were concurrently being treated with antiparkinsondrugs.Inourstudy,HBOreplacedtheSinemettherapyandappeare dtoimprovetheclinicalcondition.Thus,results from this case study suggest that HBO therapy might be a potential therapeutic modality in treating patientssufferingfromPDwithoutcausinguntowardsideeffectssuchasdyskinesiaobservedinlongtermSinemettherapy.Inconclusion,wesuggestthatHBOtherapymightbeneuroprotective innaturetothenigrostriatalneuronsbyactingasan antiapoptotic process. This couldstabilizeneuronal function, thereby pote ntially decreasing the progression of theneurodegenerationobservedinParkinson’sDisease.

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Hyperbaric oxygen treatment for Parkinson’sdisease with severe depression and anxietyA case reportJin-Jin Xu, MD, Si-Tong Yang, MD, Ying Sha, MD, Yuan-Yuan Ge, MD, Jian-Meng Wang, MD∗AbstractRationale: Patients with Parkinson’s disease (PD) frequently suffer from psychiatric disorders, and treating these symptomwhereas managing the motor symptoms associated with PD can be a therapeutic challenge.Patient concerns: We report a case of PD patient with severe depression and anxiety that refused to be treated withdopaminagonists or SSRIs, the most common treatments for PD patients suffering from psychiatric symptoms.Diagnoses: Parkinson’s disease with severe depression and anxiety.Interventions: This man was treated with hyperbaric oxygen treatment for 30 days.Outcomes: Clinical assessment scores for depression and anxiety, including Uniﬁed Parkinson’s Disease Rating ScaleI (UPDRS I),UPDRS II, Hanmilton Depression Rating Scale, and Hamiliton Anxiety Rating Scale, were improved following the hyperbaric oxygentreatment.Lessons: Hyperbaric oxygen treatment may be a potential therapeutic method for PD patient suffering from depression andanxiety. Further research is needed to validate this ﬁnding and explore a potential mechanism.Abbreviations: MRI = magnetic resonance imaging, PD = Parkinson’s disease, SSRIs = selective serotonin reuptake inhibitors,UPDRS = Uniﬁed Parkinson’s Disease Rating Scale I.Keywords: anxiety, depression, hyperbaric oxygen, Parkinson’s disease1. IntroductionPatients with Parkinson’s disease (PD) frequently suffer fromnonmotor symptoms, with up to 40% to 60% of PD patientssuffering from psychiatric symptoms.[1]Psychiatric symptomscan exacerbate the neuromotor symptoms of PD, complicatingthe care of patients with PD.[2,3]Previous research has suggestedthat psychiatric symptoms comorbid with PD decreases thequality of life for the patient and increases the burden on thecaregiver.[4]However, the treatment options for psychiatricsymptoms of patients with PD are limited. Currently, the maintreatment strategies for these patients involve the use of dopamineagonists or selective serotonin reuptake inhibitors (SSRIs). Thiscase report presents a PD patient with severe depression andanxiety who showed marked psychiatric improvement usinghyperbaric oxygen treatment after refusing conventional phar-maceutical treatments.2. Case reportThe study was approved by the ethical committee of FirstHospital of Jilin University, China. Written informed consentwas obtained.A 45-year-old male patient initially presented 1.5 years agowith resting tremor and bradykinesia in the left upper limb.Symptoms subsequently developed in the left lower limb andright limbs. He was diagnosed with PD, and anti-Parkinsonianagents were prescribed. The exact details of his prescription areunknown, as the patient refused any medication because hebelieved the drug treatment was ineffective.Three months ago, the resting tremor and bradykinesiaprogressively intensiﬁed. Additionally, he began to presentpsychiatric symptoms, including a loss of interest in daily life,unwilling to communicate with others, and often having suicidalthoughts. He was diagnosed with severe depression and anxietyassociated with PD at another hospital, and prescribedcitalopram and pramipexole. However, again the patient refusedto accept the drug treatment. At admission to our hospital, he hada poor diet, extremely inadequate sleep (about 2–3 hours per dayon average), and weight loss of about 20 kg from the onset of PD.He had no previous history of medical illness. His mother hadsuffered from PD, with an initial onset around 40 years old. Theneurological examination on admission found slightly clumsyspeech patterns, masking face, and increased muscle tone andhyperactive deep tendon reﬂexes in the limbs. All otherparameters of the neurological examination were within normallimits. In addition, nonenhanced cranial magnetic resonanceimaging (MRI) scan and Doppler ultrasound of the head andneck did not identify any evidence of pathology. Depression andanxiety were assessed clinically using several rating systems,Editor: N/A.The authors declare no conﬂicts of interest.Department of Geriatrics, the First Hospital of Jilin University, Changchun, China.∗Correspondence: Jian-Meng Wang, Department of Geriatrics, First Hospital ofJilin University, Changchun 130021, China (e-mail: 2444862570@qq.com).Copyright © 2018 the Author(s). Published by Wolters Kluwer Health, Inc.This is an open access article distributed under the Creative CommonsAttribution-NoDerivatives License 4.0, which allows for redistribution, commercialand non-commercial, as long as it is passed along unchanged and in whole, withcredit to the author.Medicine (2018) 97:9(e0029)Received: 29 December 2017 / Accepted: 6 February 2018http://dx.doi.org/10.1097/MD.0000000000010029Clinical Case ReportMedicine®OPEN1

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including the Uni ﬁed Parkinson’s Disease Rating Scale I(UPDRSI), UPDRS II, Hamilton Depression Rating Scale(HAM-D), and Hamilton Anxiety Rating Scale (HAM-A). Theresults are summarized in Table 1.After admission, the patient again refused conventional drugtreatment. He was instead treated with hyperbaric oxygentreatment as an alternative to pharmaceutical therapy. Theprotocols of hyperbaric oxygen treatment were as follows: thepatient inhaled pure oxygen through a mask in 2 sessions of 40minutes, separated by a 10 minute break, in hyperbaric chamber.The pressure was set at 2.0 ATA (atmosphere absolute). After 4days of hyperbaric oxygen treatment, the patient had signiﬁcantlyimproved sleepingquality, increasing sleeping duration from 2 to 3hours prior to admission to about 5 hours per night. Coincidental-ly, his overall mood improved. The patient continued to receivehyperbaric oxygen treatment for 1 month. The treatment coursewas smooth and without complications. After ending treatment,the sleeping time recovered to within normal limits, with theduration of 8 to 10hours, and body weight increased by about 10kg. Additionally, the resting tremor and bradykinesia improvedsigniﬁcantly. Table 1 summarizes the results of the clinicalassessments performed both before and after the end of hyperbaricoxygen treatment. Although the scores after hyperbaric oxygentreatment decreased signiﬁcantly, they remained in the abnormalrange. Follow-up 1 month after discharge indicated that theimprovements in the patient’s sleep and mood persisted, and he didnot need assistance in his daily life.3. DiscussionPD occurs in 0.3% of the general population and about 1% to2% of individuals older than 60 years.[5]Patients with PD oftenalso suffer from depression and anxiety, which are the mostcommon psychiatric complications for these patients. In fact,these 2 psychiatric disorders are more prevalent in PD patientsthan in the general population, with a lifetime risk of developingdepression or anxiety of about 60%, with the cross-sectionalprevalence for each disorder only being 30% to 40%.[1].Idiopathic Parkinson disease is the most common cause ofParkinsonism, which has four cardinal manifestations includingresting tremor, bradykinesia, rigidity, and gait disturbance.[6]Parkinson disease also causes important nonmotor symptoms,such as sleep disturbance, mood disorder, cognitive impairment,and autonomic dysfunction. The patient’s initial symptomspresented in this paper were resting tremor and bradykinesia.Over time, he developed nonmotor symptoms, including sleepdisturbance, and mood disorder. Affective disorders have a strongeffect on quality of life and increase PD patient disability, somanagement of anxiety and depression for this patient, particu-larly because of his refusal of other drug treatments, was a priority.Several medications are available for treatment of both themotor symptoms and nonmotor symptoms of PD. The choice oftreatment for an individual patient is based on their speciﬁcsymptoms, age, disease stage, functional disability, and generallevel of physical activity and productivity. In the clinic, levodopaor dopamine agonists, such as pramipexole, are the 2recommended effective drugs for the treatment of the motorsymptoms of PD.[7]Several randomized controlled trials haveshown that dopamine agonists provide effective monotherapy inearly PD, and in patients with more advanced disease who displaysuboptimal responses to levodopa.Adverse effects can accompany the treatment with levodopa ordopamine agonists. For example, nausea, vomiting, andorthostatic hypotension may occur early in the treatment processof PD, whereas dyskinesia, visual hallucinations, and psychiatricsymptoms sometimes occur after treatment in more advancedstages of the disease.[8]These adverse effects signiﬁcantlydecrease the treatment compliance in the clinic, and more effortsshould be made to address them. Although motor function mayworsen, stopping or reducing the levodopa or dopamine agoniststo control the nonmotor symptoms, especially psychiatricsymptoms, may be a better choice for the overall well-being ofthe patient. Additionally, selective serotonin reuptake inhibitors(SSRIs) may be effective in controlling the psychiatric symptoms.Psychiatric symptoms can exacerbate the debility of PD andcreate complexities in treating both the motor and nonmotorsymptoms of patients with PD.In the present study, the patient refused currently accepteddrug therapies to treat his symptoms of PD. To treat this patient,we were compelled to explore other potentially effectivetreatment strategies. In recent years, hyperbaric oxygen treatmenthas emerged as a potential treatment for neurological disorders,including PD, by providing neuroprotective effects.[9,10]Severalmechanisms underlying the neuroprotective effects of hyperbaricoxygen treatment in PD have been explored in the previousstudies. One possibility is that hyperbaric oxygen treatment couldreduce oxidative stress and inﬂammation, which are knownfactors in the pathogenesis of PD.[11]For example, in a rat modelof middle cerebral artery occlusion, hyperbaric oxygen treatmentdramatically reduced the formation of hydroxyl free radicals inthe striatum, which was associated with increased superoxidedismutase and catalase activity and the reduction of malondial-dehyde and lipid peroxidation levels.[12]Additionally, hyperbaricoxygen treatment is associated with reduced myeloperoxidaseactivity and inhibition of neutrophil inﬁltration, biomarkersindicating inﬂammation.[13,14]Although the potential mecha-nisms accounting for the effectiveness of hyperbaric oxygentreatment were not addressed for this patient, further studiesshould be conducted to verify the effectiveness of this treatment inother patients, as well as to understand the underlying molecularmechanism.Studies have shown that hyperbaric oxygen treatmentcombined with other therapies is effective in improving thenonmotor symptoms of Parkinson’s Disease.[15,16]However, afew report is available that evaluating the effects of hyperbaricoxygen treatment using speciﬁc scientiﬁc scales includingUPDRSI, UPDRS II, HAM-D, and HAM-A as described in thepresent study. Further well-designed randomized controlled trialsusing abovementioned scientiﬁc scales are needed to evaluate thetreatment efﬁcacy.In conclusion, this report suggests that hyperbaric oxygentreatment may be a potential therapeutic method for treatingpsychiatric symptoms in patients with PD. However, furtherresearch is needed to validate its effects and explore itsmechanism.Table 1Patient scores on clinical tests before and after hyperbaric oxygentreatment.UPDRS I UPDRS II HAM-D HAM-ABefore treatment 38 15 31 32After 1 month treatment 20 8 19 17HAM-A = Hamilton Anxiety Ratin g Scale, HAM-D = Hamilton Depression Rating Scale, UPDRS =Uniﬁed Parkinson ’ s Disease Rating Scale.Xu et al. Medicine (2018) 97:9 Medicine2

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Effects of high-intensity intervaltraining with hyperbaric oxygenMiguel Alvarez Villela1†, Sophia A. Dunworth1,2†,Bryan D. Kraft1,3†, Nicole P. Harlan1,3, Michael J. Natoli1,Hagir B. Suliman1and Richard E. Moon1,2,3*1Center for Hyperbaric Medicine and Environmental Physiology, Duke University Medical Center,Durham, NC, United States,2Department of Anesthesiology, Duke University Medical Center, Durham,NC, United States,3Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine,Duke University Medical Center, Durham, NC, United StatesHyperbaric Oxygen (HBO2) has been proposed as a pre-conditioning method toenhance exercise performance. Most prior studies testing this effect have beenlimited by inadequate methodologies. Its potential ef ﬁcacy and mechanism ofaction remain unknown. We hypothesized that HBO2could enhance aerobiccapacity by inducing mitochondrial biogenesis via redox signaling in skeletalmuscle. HBO2was administered in combination with high-intensity intervaltraining (HIIT), a potent redox stimulus known to induce mitochondrialbiogenesis. Aerobic capacity was tested during acute hypobaric hypoxiaseeking to shift the limiting site of whole body V_O2 from convection todiffusion, more closely isolating any effect of improved oxidative capacity.Healthy volunteers were screened with sea-level (SL) V_O2peak testing.Seventeen subjects were enrolled (10 men, 7 women, ages 26.5±1.3 years,BMI 24.6±0.6 kg m−2,V_O2peak SL = 43.4±2.1). Each completed 6 HIIT sessionsover 2 weeks randomized to breathing normobaric air, “HIIT+Air” (PiO2=0.21 ATM) or HBO2(PiO2= 1.4 ATM) during training, “HIIT+HBO2”group. Training workloads were individualized based on V_O2peak SL test.Vastus Lateralis (VL) muscle biopsies were performed before and after HIITin both groups. Baseline and post-training V_O2peak tests were conducted in ahypobaric chamber at PiO2 = 0.12 ATM. HIIT signiﬁcantly increased V_O2peak inboth groups: HIIT+HBO231.4±1.5 to 35.2±1.2 ml kg−1·min−1and HIIT+Air29.0±3.1 to 33.2±2.5 ml kg−1·min−1(p = 0.005) without an additional effect ofHBO2(p = 0.9 for interaction of HIIT x HBO2). Subjects randomized toHIIT+HBO2displayed higher skeletal muscle mRNA levels of PPARGC1A,aregulator of mitochondrial biogenesis, and HK2 and SLC2A4, regulators ofglucose utilization and storage. All other tested markers of mitochondrialbiogenesis showed no additional effect of HBO2to HIIT. When combinedwith HIIT, short-term modest HBO2(1.4 ATA) has does not increase whole-body V_O2peak during acute hypobaric hypoxia. (ClinicalTrials.gov Identiﬁer:NCT02356900; https://clinicaltrials.gov/ct2/show/NCT02356900).KEYWORDShigh-intensity interval training, hyperbaric oxygenation, mitochondrial turnover, high-altitude, oxygen consumptionOPEN ACCESSEDITED BYGiuseppe De Vito,University of Padua, ItalyREVIEWED BYGraham Ripley McGinnis,University of Nevada, United StatesLynn Hartzler,Wright State University, United States*CORRESPONDENCERichard E. Moon,richard.moon@duke.edu†These authors have contributed equallyto this work and share ﬁrst authorshipSPECIALTY SECTIONThis article was submittedto Exercise Physiology,a section of the journalFrontiers in PhysiologyRECEIVED 07 June 2022ACCEPTED 27 July 2022PUBLISHED 19 August 2022CITATIONAlvarez Villela M, Dunworth SA, Kraft BD,Harlan NP, Natoli MJ, Suliman HB andMoon RE (2022), Effects of high-intensity interval training withhyperbaric oxygen.Front. Physiol. 13:963799.doi: 10.3389/fphys.2022.963799COPYRIGHT©2022AlvarezVillela,Dunworth,Kraft,Harlan, Natoli, Suliman and Moon. Thisis an open-access article distributedunder the terms of the CreativeCommons Attribution License (CC BY).The use, distribution or reproduction inother forums is permitted, provided theoriginal author(s) and the copyrightowner(s) are credited and that theoriginal publication in this journal iscited, in accordance with acceptedacademic practice. No use, distributionor reproduction is permitted which doesnot comply with these terms.Frontiers in Physiology frontiersin.org01TYPE Original ResearchPUBLISHED 19 August 2022DOI 10.3389/fphys.2022.963799

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IntroductionHyperbaric oxygen (HBO2) has been proposed as a methodfor pre-conditioning to enhance exercise performance. Manyathletes have admitted to using HBO2(for instance, 1.3 -2.8 atmospheres absolute [ATA] for 50 - 90 minutes) basedon the principle that short exposures to HBO2immediatelybefore maximal exercise testing facilitates “oxygen loading”(Cabric et al., 1991; McGavock et al., 1999; Rozenek et al.,2007; Kawada et al., 2008). While tissue oxygen levelsnormalize almost immediately after removal from thehyperoxic environment, in one study, HBO2therapy led toincreased peak oxygen uptake (V_O2peak) that was linked toincreased skeletal muscle mitochondrial mass (Hadanny et al.,2022).Hyperbaric oxygen has also been studied in combinationwith exercise training, albeit with mixed results. Whenadministered in combination with moderate continuoustraining in athletes, it resulted in no additional improvementson V_O2peak compared with training under normoxic conditions(Burgos et al., 2016). However, in a more recent study, HBO2wasadministered in combination with high-intensity intervaltraining (HIIT) and led to increased peak work rate and peakminute ventilation compared with air controls, but no differencesin V_O2peak (DeCato et al., 2019). The underlying hypothesis isthat the mitochondrial bioenergetic program is synergisticallyactivated by these two stimuli, potentially via redox signaling.The mitochondrial biogenesis program is cytoprotective andgenerates and distributes healthy mitochondria throughout thecell in response to oxidant and other stresses (Piantadosi andSuliman, 2012). Given the link between mitochondrial mass andaerobic capacity (Weibel et al., 1991), we proposed that inductionof the mitochondrial biogenesis program would result indetectable improvements in V_O2peak.The co-activator and binding partner peroxisomeproliferator-activated rece ptor gamma coactivator 1-alpha(PGC-1α)isareliablemarkerforactivationofthisbigenomic program in humanskeletalmuscle.Bothendurance and interval exercise training consistentlyincrease its expression (Pilegaard et a l., 2003; Calvo et al.,2008; Gibala et al., 2009; Olesen et al., 2010), and there isevidence that the r elationship between PGC-1 α and exercisemay be bidirectional, as overexpression of this molecule intransgenic mice increases mitochondrial content and, in turn,improves exercise performance (Calvo et al., 2008). Activationof skel etal muscle P GC-1α is also seen with low-dosehyperbaric exposure (1.25 ATA with FiO20.36) (Takemuraand Ishihara, 2017 )andotherstudieshavefoundHBO2canactivate mitoc hondrial biogenesis in the brain (Gutsa eva et al.,2006; Hsu et al., 2022 ). Hence, HIIT and HBO2together couldsynergistically increase PGC -1α and improve exercisecapacity. However, HBO2showed a neutral effect onV_O2peak compared to HIIT alone (DeCato et al., 2019).In the present study we directly examined the combinedeffect of HBO2and HIIT on skeletal muscle bioenergetics as wellas the attendant changes on V_O2peak during acute hypobarichypoxia. We chose a HIIT regimen similar to that employed byDeCato, et al. (DeCato et al., 2019), and measured changes inexercise capacity in a hypobaric hypoxic environment becausehypoxic conditions (reduced oxygen supply) were believed toemphasize the role of peripheral determinants of V_O2(i.e.capillary surface area and mitochondrial capacity) more sothan central determinants (i.e. lung capacity, heart function,and oxygen carrying capacity) (di Prampero, 2003). Thisstrategy therefore allowed us focus as much as possible on themitochondrial contributions of VO2and concurrently measurechanges in the skeletal muscle mitochondrial biogenesisprogram.Materials and methodsSubjects and study enrollmentAfter institutional review board (IRB) approval and writteninformed consent, healthy non-smoker volunteers, ages 18 to35 years, were screened. Subjects were excluded who had chroniccomorbidities, pregnancy, sickle cell heterozygosity (African-American candidates were screened), physicaldeconditioning (sea-level V_O2peak below 35 mL.kg-1.min-1formen or 30 mL.kg-1.min-1for women) (Gill et al., 2014; Bennett-Guerrero et al., 2017; Bennett-Guerrero et al., 2021), or wereunable to provide written informed consent in English.Study design overviewTo determine study eligibility, potential subjects underwent abaseline medical history, physical exam, and sea-level (SL)graded maximal exercise test on a cycle ergometer. If eligiblefor participation, subjects were enrolled and underwent a vastuslateralis (VL) muscle biopsy followed 48 hours later by a gradedmaximal exercise test to exhaustion during acute hypobarichypoxia (HH) in a hypobaric chamber decompressed to abarometric pressure of 429 mmHg (PiO2=0.12 atmospheres[ATM]) (Figure 1). Participants were then randomized 1:1 toa supervised, 6 session, high-intensity interval training programwhile breathing normobaric air, “HIIT+Air” group(PiO2=0.21 ATM), or 100% oxygen in a hyperbaric chamber“HIIT+HBO2” group (PiO2=1.4 ATM). Selection of the PO2wasbased on a balance between the likelihood of a positive effect andan acceptable risk of central nervous system oxygen toxicityduring exercise (Koch et al., 2013). One day after the last trainingsession, a VL biopsy was obtained from the contralateral leg and amaximal exercise test during acute HH (PiO2=0.12 ATM) wasrepeated after a two-day recovery period. Subjects wereFrontiers in Physiology frontiersin.org02Alvarez Villela et al. 10.3389/fphys.2022.963799

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groups using identical cycle ergometer setups and two-way non-rebreathing valve systems. Continuous ECG and periodic bloodpressure monitoring were performed during each session.Subjects in the HIIT+Air group acted as controls training inour physiology laboratory at sea-level with the on-demandbreathing system in direct communication with surroundingambient air. Subjects randomized to train in the HBO2groupwere pressurized in a dry hyperbaric chamber to a pressure of1.4 ATA over 1 minute, or slower depending upon subjectcomfort. The described on-demand breathing system wasconnected to a pure oxygen source (FiO2=100%). A studyinvestigator acted as a tender inside the chamber throughouteach training session. Subjects were attached to a safety harnessto avoid trauma in the event of an oxygen-induced convulsion.The chamber was decompressed back to sea-level pressure(1 ATA) over 2 minutes. Each training session consisted of atotal of 30 minutes of exercise: Three minutes of warm-up,followed by six 30-second high-intensity interval periodsinterspersed with six 4-minute active recovery periods(Figure 2). Prescribed workloads for each period werecalculated as percentages of the subject’s own peak workload(Watts) achieved during the screening SL maximal exercise test:60% of peak output for the warm-up period, 120% for the high-intensity intervals and 50% for the recovery periods (Figure 2).During training, subjects were encouraged to maintain a cadenceof 75 rpm while the pedaling resistance was adjusted by thesupervising investigator to the prescribed workload for eachperiod.Acute hypobaric hypoxia maximal exercise testing: Maximalexercise tests were performed 1 week before and 1 week after thelast HIIT session (Figure 1) in a hypobaric chamber at asimulated atmospheric pressure of 429 mmHg and FiO2=21%,(PiO2=0.12 ATM). Workload increments at altitude were 25Wevery 3 minutes. For each test, the chamber was depressurized at2,000 ft per minute for 7.5 minutes to 15,000 ft. Recalibration ofthe metabolic cart was performed at target atmospheric pressure.A study investigator was present inside the chamber throughoutcompletion of the test and the subject was attached to a safetyharness to avoid trauma in the event of hypoxia-inducedsyncope. In addition to inability to maintain cadence, subjectrequest and muscle cramping, tests were also terminated forsevere hypoxemia (persistent O2saturation of <70% on pulse-oximetry).Skeletal muscle biopsiesVastus lateralis (VL) muscle biopsies were performed understerile technique, using local anesthesia and a University CollegeHospital Muscle Biopsy Needle (120 mm, 5.0 mm OD, Popper &Sons, New Hyde Park, NY). Samples were obtained (total weight200 mg), snap frozen in liquid nitrogen, and stored at -80°C.Western blot studiesMuscle proteins were separated by SDS-PA GE andidentiﬁed by Western blot analysis (Suliman et al., 200 7a).Membranes were incubated with validat ed antibodies againstmitofusin-2 (Mfn2, 1:800, sc- 50331), dynamin-1-like protein(Drp1, 1:1000, sc-271583), citrate synthas e (1:2000,ab129095), ATP synthase subunit a (ATPase6, 1:1000,ab219825), cyt ochrome c oxidase (COI, 1: 1000, PA 5-26688), NADH de hydrogenase subunit 1 (ND1, 1:1000,ab181848), catalase (1:1500, ab209211), heme oxygenase-1(HO-1, 1 :1000, BML-HC3001), and superoxide dismutase-2(SOD2, 1:2000, sc-137254), using GAPDH (1:5,000, G9545 ) orporin (1:5,000, PC548) ant ibody (Sigma, St. Louis, MO) as aloading control. After three washes in Tris-buffered salinewith Tween, membranes were incubated with the appropriatehorseradish peroxidase-conjugated secondary antibody (anti-goat, sc-2354, or anti-rabbit sc-2357 antibodies, both 1:10,000). Blots were developed with enhancedchemiluminescence (Wes tern Blot ting Luminol Reagent, sc-2048). Proteins we re quantiﬁed by densitometry on digitizedimages from the mid dynamic range using Quantity One (Bio-Rad) and expressed relative t o GAPDH or porin.Polymerase chain reaction studiesTotal RNA was extracted from the muscle samples andanalyzed by RT-PCR as previously described (Pecorella et al.,2015). RNA (1 μg) was reverse-transcribed by u sing randomhexamer primers and a Superscript enzyme (Invitrogen) andreal-time PCR was performe d with a StepOne plus and geneexpression master mix (Applied Biosystems, Fost er City, CA).Primers and probes from Applied Biosystems were used fornuclear respiratory factor-2 (NRF2), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A),mitochondrial transcription factor A (TFAM ), DNApolymerase subunit gamma-1 (POLG), mitochondrialDNA-directed RNA polymerase (POLRMT), glucosetransporter 4 (SLC2A4), glucose transporter 1 (SLC 2A1),and hexokinase-2 (HK2 ). 18S rRNA was used as aninternal control. For the quantitative RT-PCR (qPCR)reaction, a difference of 1.0 in Ct value represents atwofold difference in transcript level. QRT-PC R wasperformed in triplicate. Quantitative PCR was then used tomeasure mtDNA copy number. Total mtDNA was isolatedfrom muscle samples and determined by real-time PCR asdescribed (Pecorella et al., 2015). Brieﬂ y, RT-PCR wasperformed using SYBR green qPCR Super Mix UDG(Invitrogen). The StepOne plus sequence detector s ystem(Applied Biosystems) was used to record and analyzeﬂuorescence intensities during PCR.Frontiers in Physiology frontiersin.org04Alvarez Villela et al. 10.3389/fphys.2022.963799

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Statistical analysesBased on previous studies by our laboratory measuring PGC-1αprotein expression in healthy subjects after exercise training, wecalculated that a sample size of at least 18 subjects (9 in each group)was required for 80% power to detect a signiﬁcant change (alpha0.05) in molecular markers of mitochondrial biogenesis followingour intervention. The pre-speciﬁed primary outcomes includedchange (pre-HIIT vs. post-HIIT) in V_O2peak, V_O2 at VT,mitochondrial mass, Tfam expression, and PGC-1α expression.Grouped data are reported as means ± SEM unless otherwiseindicated, and analyzed via repeated-measures two-way ANOVA(or if missing values, by mixed-effects analysis) with Fisher’sleastsigniﬁcant difference post-hoc testing (GraphPad Prism v8, SanDiego, CA). p <0.05 was considered statistically signiﬁcant .ResultsEnrollment and baseline characteristicsTwenty-ﬁve subjects were screened, four were excluded prior torandomization: one for below cut-off sea-level V_O2peak, one due tosyncope during ﬁrst VL biopsy, and two because of training sessionscheduling conﬂicts. Twenty-one subjects completed the studyprotocol. Four were excluded from the data analysis due tosubmaximal effort on sea-level or altitude testing (RER<1.0), ordue to technical failures related to calorimetric gas analysis ataltitude. Therefore, 17 subjects (10 men, 7 women) wereincluded in the data analysis. Nine were in the air group and8intheHBO2group. Ages ranged 19–35 years, BMI24.6±0.6 kg m−2,sea-level(SL)V_O2peak 43.4±9 ml kg−1·min−1andbaseline hypobaric hypoxia (HH) V_O2peak 30.1±1.7 ml kg−1·min−1(Table 1). Baseline characteristics are summarized in Table 1.Subjects in both groups were s imil ar in age, BMI and ﬁtnesslevel (V_O2peak SL HIIT+HBO2group = 45.2±2.69 ml·kg·min−1vs. HIIT+Air 41.7±3.30 ml kg−1·min−1; p =0.31).Effects of acute hypobaric hypoxiaMaximal exercise testing during acute HH (barometricpressure = 429mmHg; PiO2=0.12ATM)resultedinanequal(non-statistically signiﬁcant) Δ V_O2peak from sea-level in bothgroups, -13.5±4.6 ml kg−1·min−1in the HIIT+HBO2group (48%decrease from baseline) and -13±3.9 ml kg−1·min−1in the HIIT+Airgroup (56% decrease from baseline). A linear correlation wasobserved between Δ V_O2peak with acute altitude exposure andsea-level V_O2peak values, with the largest drops occurring in theﬁttest subjects (R = 0.8; p <0.001). This was in keeping with priorreports on the effect of acute hypoxia upon peak oxygenconsumption (Lawler et al., 1988).Physiological effects of high-intensityinterval training at sea-level or hyperoxic-hyperbaric environmentHeart rates were measured for each subject during the ﬁrst,third and sixth recovery periods of the 1stand 6thtraining sessionsbut were not statistically signiﬁcant between the two groups(152 ± 13 in the HIIT+Air group and 142 ± 21 in theHIIT+HBO2group). Average workload (Watts) performedduring each training session was also similar between bothgroups, 736±53.7 W in the HIIT+Air group and 688±64 W inthe HIIT+HBO2group (p = 0.4). Physiologic and hemodynamicTABLE 1 Baseline Subject Characteristics including all subjects. All measurements performed before trainin g at sea-level (SL) and during hypobarichypoxia (HH) (mean ± SEM). †p-value represents comparison between groups. HH testing performed in a hypobaric chamber, PiO2=0.14.Abbreviations: BMI, body mass index; HR, heart rate; PiO2,pressureofinspiredoxygen;RER,respiratoryexchangeratio;SBP,systolicbloodpressure;SL, sea-level; V_O2peak, peak oxygen consumption.HIIT+Air HIIT+HBO2All p-Value†N 9 8 17Age (years) 27.8 ± 1.9 25.0 ± 1.7 26.5 ± 1.3 0.3Sex (M/F) 5/4 5/3 10/7BMI (kg/m2) 25.4 ± 0.8 23.7 ± 0.8 24.6 ± 0.6 0.14Body Fat (%) 19.1 ± 3.1 13.8 ± 2.3 16.6 ± 2.0 0.18Resting HR SL (bpm) 76.9 ± 4.3 72.0 ± 3.2 74.6 ± 2.7 0.37Resting SBP SL (mmHg) 121.2 ± 3.7 118.6 ± 4.2 120.1 ± 2.7 0.64V_O2peak SL (mL·kg−1·min−1) 41.7 ± 3.3 45.2 ± 2.7 43.4 ± 2.1 0.42RER SL 1.21 ± 0.03 1.24 ± 0.01 1.23 ± 0.02 0.3Peak Work SL (Watt) 253.9 ± 18.4 237.4 ± 22.0 246.2 ± 13.9 0.57Baseline V_O2peak HH (mL·kg−1·min−1) 29.0 ± 3.1 31.4 ± 1.5 30.1 ± 1.7 0.49RER HH 1.32 ± 0.07 1.34 ± 0.06 1.33 ± 0.05 0.84Frontiers in Physiology frontiersin.org05Alvarez Villela et al. 10.3389/fphys.2022.963799

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parameters during maximal exercise testing before and aftertraining are detailed in Table 2. The addition of HBO2toHIIT did not result in an additional effect on V_O2at VT orduring peak exercise compared to the effect of HIIT alone (p =0.0045 for training effect, p = 0.9 for training x exposure effect).VT as V_O2peak % remained unchanged in both groups aftertraining (air, 73.2±2.5 to 70±2.0% and HBO2, 66.2±2.7 to67±2.8%). There was no signiﬁcant effect of training or HBO2exposure on systolic blood pressure (SBP) or heart rate (HR) atrest and at peak exercise. There was, however, a signiﬁcant effectof HBO2on attenuating pulse pressure rise during exercise (p =0.05 for interaction, post-hoc comparison).Safety of training and hypobaric andhyperbaric exposuresNo adverse events occurred during training. In the HBO2group, there were no complications related to chambercompression/decompression and no oxygen-inducedconvulsions or symptoms suggestive of oxygen toxicity, suchas seizures or reductions in forced vital capacity. All acute high-altitude exposures were well tolerated, with no syncopal or pre-syncopal events. No tests were stopped due to hypoxemia(deﬁned as SpO2<70%). One subject suffered vasovagalsyncope during baseline muscle biopsy with uneventfulrecovery and one other subject declined repeat biopsy due todiscomfort.Effects of high-intensity interval trainingand hyperbaric oxygen on skeletal muscleSkeletal muscle biopsies from a subset of subjects(HIIT+Air, n = 7; HIIT+HBO2, n = 6) were analyzed forchanges in protein and gene expression. We found increasedprotein expres sion in mitofusi n-2 (Mfn2), citrate synthase,ATPase 6 , cytochr ome c oxidase (COI), and ND1 fromexercise traini ng alone, but the addition of HBO2had noeffect (Figure 3). Neither intervention altere d express ion ofDrp1. Quantitative PCR analysis showed signiﬁcantly increasedmuscl e expression of NRF2 and TFAM,twogenescriticaltoregul ation of mitochondrial biogenesis, from exercise trainingalone but not from HBO2(Figure 4). We also found thatexercise training but not HBO2increased gene expression ofthe poly meras es POLG and POLRMT, which are necessary formitochondrial DNA replication and transcription, respectively.Moreover, compared to baseline values, there was sig niﬁcantlyhigher PPARGC1A expression and mtDNA copy number inmuscle samples taken after training. However, withHIIT+HBO2, PPARGC1A expression was further augmentedcompa red with t he HIIT+Air group (p =0.005).Because both HIIT and HBO2stimulate ROS production thatmay activate anti-oxidant responses, we measured expression ofselect mitochondrial anti-oxidant proteins (Figure 5). We foundsigniﬁcantly increased expression of HO-1 and SOD2 withexercise training in both groups but no effect from HBO2.However, compared to baseline and to the HIIT+Air Group,the addition of HBO2to HIIT signiﬁcantly increased skeletalmuscle catalase expression (p <0.005).Finally, we investigated whether there were changes inmarkers of substrate (glucose) utilization (Figure 6). Wefound that exercise training alone signiﬁcantly increased geneexpression of the glucose transporters SLC2A1 and SLC2A4;however, compared with air treated subjects, the addition ofHBO2further increased SLC2A4 expression (p = 0.0007).Additionally, while neither HIIT nor HBO2altered HK1expression, exercise training signiﬁcantly increased HK2expression that was further increased by addition of HBO2(p = 0.02).TABLE 2 Physiologic effect of training in all subjects. N = 17. All measurements done during acute hypobaric hypoxia (HH), 429 mmHg barometricpressure (PiO2= 0.14) before and after training (mean ± SEM). *p <0.05. Abbreviations: HH, hypobaric hypoxia; HR, heart rate; PP, pulse pressure;SBP, systolic blood pressure; V_O2peak, peak oxygen uptake; VT, ventilatory threshold by modiﬁed V-slope method.HIIT+Air HIIT+HBO2p-Value Training Effect p-ValueInteractionTrainingx ExposurePre Post Pre PostV_O2peak HH (mL·kg−1·min−1) 29.0 ± 3.1 33.2 ± 2.5 31.4 ± 1.5 35.2 ± 1.2 0.0045* 0.9VT HH (mL·kg−1·min−1) 21.3 ± 2.4 23.6 ± 2.0 18.6 ± 1.8 21.8 ± 2.0 0.016* 0.7Peak Workload HH (W) 214 ± 13 217 ± 22 191 ± 13 200 ± 16 0.32 0.6Resting SBP HH (mmHg) 118 ± 4 120 ± 4 125 ± 7 117 ± 4 0.14 0.6Resting HR HH (bpm) 95 ± 3 92 ± 2 98 ± 9 88 ± 6 0.15 0.3Peak SBP HH (mmHg) 148 ± 5 154 ± 6 141 ± 7 139 ± 7 0.8 0.4Peak HR HH (bpm) 178 ± 5 176 ± 4 174 ± 3 178 ± 3 0.6 0.6Increase in PP Rest to Peak HH Exercise (mmHg) 29 41 28 23 0.7 0.05Frontiers in Physiology frontiersin.org06Alvarez Villela et al. 10.3389/fphys.2022.963799

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such as citrate synthase and ATPase6, which are measures ofmitochondrial volume density, or NRF2 and TFAM expression,which are measures of mitochondrial biogenesis activation. Thereasons for this are not clear, but could possibly relate todifferences in peak expression after HBO2, insufﬁcient dose ofHBO2to fully activate mitochondrial biogenesis, or lack ofbiological effect by HBO2. These ﬁndings contrast with recentwork by Hadanny, et al. that found increased V_O2peak in healthymaster athletes after HBO2treatment that was linked to increasedskeletal muscle mitochondrial mass (Hadanny et al., 2022).However, we are skeptical of these molecular ﬁndings, asMitoTracker Green ﬂuorescence was measured in ﬁxedtissues, but is only validated and speciﬁc in live cells, and thePG1-alpha protein measurements were not stated as the nuclear(activated) fraction. Moreover, no changes were seen in MFN1/2 or OPA1, which are markers for mitochondrial quality andintegrity. Our study also differed from the Hadanny study, as itincluded younger subjects, fewer HBO2sessions, lower doses ofHBO2, and added the HIIT intervention. This latter differencemay be most relevant, as we found mitochondrial biogenesis(using appropriate techniques) was activated by HIIT alone,leading to increased mitochondrial mass. In fact, themitochondrial biogenesis program may have been maximallyactivated by HIIT and may have reduced our ability to detectfurther effects from HBO2. HIIT alone activated mitochondrialbiogenesis (evidenced by increased TFAM, NRF1, andPPARGCA1 gene expression), increased mitochondrial qualityand integrity (increased MFN2 protein expression), andincreased mitochondrial mass (increased citrate synthase andATPase 6 protein expression). Furthermore, to our knowledge,this is the ﬁrst study to translate from rodents to humans thatinterval exercise training increases gene expression of POLG, themitochondrial DNA polymerase that repairs mtDNA damage(Clark-Matott et al., 2015) postulated to explain some of thehealth beneﬁts of regular exercise (Garatachea et al., 2015). Wealso report for the ﬁrst time in any species the exercise-inducedgene expression of mitochondrial DNA-directed RNApolymerase (POLRMT), which transcribes mtDNA to RNA,and may be a novel biomarker for mitochondrial exerciseresponse.Breathing hyperbaric oxygen signiﬁcantly raises tissueoxygen levels and increases production of reactive oxygenspecies (ROS), predominantly at the mitochondrial electrontransport chain, where molecular oxygen is reduced to formsuperoxide (Jamieson et al., 1986). Superoxide is then convertedby superoxide dismutase (SOD) to hydrogen peroxide (H2O2), aless toxic intermediate that is converted to water by catalase. Wefound that exercise but not HBO2signiﬁcantly increasedmitochondrial SOD (SOD2) expression. However, onlyexercise and HBO2together increased catalase expression. Weare not sure why attendant increases in SOD2 after HBO2werenot seen, as HBO2does increase superoxide formation (Jamiesonet al., 1986; Groger et al., 2009), but possible explanations includemissed peak expression, or reduced signal due to the highlyefﬁcient nature of the SOD system. It is also possible that theHBO2host response is more dependent on cytosolic SOD3,which we did not measure. Regardless, the higher catalaseexpression seen after training with HBO2likely reﬂectselevated tissue levels of H2O2. Hydrogen peroxide can activatePGC-1α expression (St-Pierre et al., 2006) as well as other cellularredox sensors (e.g. Akt) (Suliman et al., 2007b; Piantadosi andSuliman, 2012), providing other plausible mechanisms for thehigher PPARGC1 mRNA levels seen after HBO2.Glucose is a critical energy source during exercise, and itsmetabolism is regulated by insulin-dependent cellular uptake viaglucose transporters and step-wise enzymatic catabolism. Wefound evidence this system was upregulated by exercise,consistent with prior studies (Pilegaard et al., 2003; Littleet al., 2010), and perhaps further augmented by addition ofHBO2. Speciﬁcally, compared to sea-level training, HIIT+HBO2subjects displayed signiﬁcantly higher gene expression of SLC2A4(GLUT4), the dominant glucose transporter for skeletal muscle,and hexokinase-2 (HK2), a rate-limiting enzyme in glycolysisthat also couples glucose metabolism to oxidativephosphorylation (Roberts and Miyamoto, 2015). Thesechanges could be consistent with improved musclebioenergetic efﬁciency, and if so, parallel those seen withexogenous CO administration (Rhodes et al., 2009).Our study was limited by a number of factors. First, it ispossible that maximal exercise testing is not the best test toexamine the effects of changes in mitochondrial function, evenunder hypoxic conditions. Exercise efﬁciency during steady-stateexercise or time to exhaustion during constant speed exertionhave been proposed as more accurate methods for this purpose,and should be considered in future studies (Broskey et al., 2015).Secondly, our study design was based on the assumption that therate-limiting step in V_O2peak in healthy humans is oxygensupply. This was based on the concept of symmorphosis,which stipulates that biological structure is matched tophysiological functional capacity. In the case of oxygentransport (except for lung capacity which exceeds demand),symmorphosis predicts matching of oxygen supply anddemand in peripheral tissues at each step of the oxygencascade (Weibel et al., 1991). Hence, we proposed that anacute reduction in ambient oxygen tension during maximalexercise would allow for a more accurate assessment ofperipheral V_O2components, and more speciﬁcally, ofmitochondrial oxidative capacity upon oxygen consumptionrate (di Prampero, 2003). However, recent data calls intoquestion the validity of symmorphosis in trained individuals,ﬁnding that V_O2peak is limited by mitochondrial O2demandrather than supply (Gifford et al., 2016). However, that study wasconducted under different conditions (normoxia) and indifferent participants (endurance-trained athletes) than ourstudy. Nevertheless, it is possible that the use of acutehypobaric hypoxia in our study to isolate mitochondrial O2Frontiers in Physiology frontiersin.org10Alvarez Villela et al. 10.3389/fphys.2022.963799

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demand as a determinant of V_O2peak may not have worked asexpected. Third, it is possible that subjects in the HBO2grouptrained at workloads that, although equivalent at sea-level to theAir group, represented a lower level of effort under theirhyperoxic training condition. Rate of perceived exertionduring training was not captured in our study. Fourth, thelack of morphological analysis of our muscle biopsies did notallow us to assess the effect of our intervention upon capillarydensity and muscle ﬁber remodeling, also important factorsaffecting the diffusion component of whole-body oxygenuptake. Fifth, we did not measure mitochondrial function(due to logistical and technical considerations) which mayhave uncovered underlying differences between the groupsthat were not otherwise evident. Finally, we cannot excludethe possibility of a beneﬁcial training effect of higher PO2(e.g.2 ATA) when combined with HIIT; however, this was not studiedto the greater risk of central nervous system oxygen toxicity.In conclusion, our study found no beneﬁcial effects of addingmodestly-dosed HBO2to HIIT for improving aerobic ﬁtness andno deﬁnite molecular evidence of activation of skeletal musclemitochondrial biogenesis. Future studies could investigatewhether similar HBO2doses improve glucose utilization andstorage after HIIT and whether these accelerate recovery.Data availability statementThe raw data supporting the conclusion of this article will bemade available by the authors, without undue reservation.Ethics statementThe studies involving human participants were reviewed andapproved by Duke University Institutional Review Board. Thepatients/participants provided their written informed consent toparticipate in this study.Author contributionsMV, SD, and BK drafted the manuscript which was approvedby all authors. All authors collected, analyzed and/or interpretedthe data. RM conceived the overall study design.FundingThis study was funded by the Duke Center for HyperbaricMedicine and Environmental Physiology.AcknowledgmentsThe authors are grateful to Claude Piantadosi for hisguidance on study design and analysis. We are also gratefulfor the technical assistance of Eric Schinazi, Aaron Walker, andKenny Parker, and for the collaborations of Mona Parikh andBrye Roberts.Conﬂict of interestThe authors declare that the research was conducted in theabsence of any commercial or ﬁnancial relationships that couldbe construed as a potential conﬂict of interest.Publisher’s noteAll claims expressed in this article are solely those of theauthors and do not necessarily represent those of their afﬁliatedorganizations, or those of the publisher, the editors and thereviewers. Any product that may be evaluated in this article, orclaim that may be made by its manufacturer, is not guaranteed orendorsed by the publisher.ReferencesBennett-Guerrero, E., Lockhart, E. L., Bandarenko, N., Campbell, M. L., Natoli,M. J., Jamnik, V. K., et al. (2017). A randomized controlled pilot study of VO2 maxtesting: a potential model for measuring relative in vivo efﬁcacy of different redblood cell products. 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Citation: Hsu, H.-T.; Yang, Y.-L.;Chang, W.-H.; Fang, W.-Y.;Huang, S.-H.; Chou, S.-H.; Lo, Y.-C.Hyperbaric Oxygen TherapyImproves Parkinson’s Disease byPromoting Mitochondrial Biogenesisvia the SIRT-1/PGC-1↵ Pathway.Biomolecules 2022, 12, 661. https://doi.org/10.3390/biom12050661Academic Editors: VladimirN. Uversky and Liang-Jun YanReceived: 22 February 2022Accepted: 28 April 2022Published: 30 April 2022Publisher’s Note: MDPI stays neutralwith regard to jurisdictional claims inpublished maps and institutional afﬁl-iations.Copyright: © 2022 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/).biomoleculesArticleHyperbaric Oxygen Therapy Improves Parkinson’s Disease byPromoting Mitochondrial Biogenesis via theSIRT-1/PGC-1↵ PathwayHung-Te Hsu1,2,*, Ya-Lan Yang3, Wan-Hsuan Chang3,4, Wei-Yu Fang3, Shu-Hung Huang4,5,6,7,Shah-Hwa Chou8,9and Yi-Ching Lo3,4,10,11,*1Faculty of Anesthesiology, School of Medicine, College of Medicine, Kaohsiung Medical University,Kaohsiung 80708, Taiwan2Department of Anesthesia, Kaohsiung Medical University Chung-Ho Memorial Hospital,Kaohsiung 80756, Taiwan3Department of Pharmacology, School of Medicine, College of Medicine, Kaohsiung Medical University,Kaohsiung 80708, Taiwan; tajen400221162@gmail.com (Y.-L.Y.); u109800003@kmu.edu.tw (W.-H.C.);tunafung@gmail.com (W.-Y.F.)4Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University,Kaohsiung 80708, Taiwan; huangsh63@gmail.com5Division of Plastic Surgery, Department of Surgery, Kaohsiung Medical University Chung-Ho MemorialHospital, Kaohsiung Medical University, Kaohsiung 80756, Taiwan6Department of Surgery, School of Medicine, College of Medicine, Kaohsiung Medical University,Kaohsiung 80708, Taiwan7Hyperbaric Oxygen Therapy Center, Kaohsiung Medical University Chung-Ho Memorial Hospital,Kaohsiung Medical University, Kaohsiung 80756, Taiwan8Department of Medicine, Faculty of Medicine, College of Medicine, Kaohsiung Medical University,Kaohsiung 80708, Taiwan; shhwch@kmu.edu.tw9Department of Chest Surgery, Kaohsiung Medical University Chung-Ho Memorial Hospital,Kaohsiung 80756, Taiwan10Department of Medical Research, Kaohsiung Medical University Chung-Ho Memorial Hospital,Kaohsiung 80756, Taiwan11School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan* Correspondence: hdhsu@kmu.edu.tw (H.-T.H.); yichlo@kmu.edu.tw (Y.-C.L.)Abstract:Hyperbaric oxygen therapy (HBOT) has been suggested as a potential adjunctive therapyfor Parkinson’s disease (PD). PD is a neurodegenerative disease characterized by the progressive lossof dopaminergic neurons in the substantia nigra pars compacta (SNpc). The aim of this study wasto investigate the protective mechanisms of HBOT on neurons and motor function in a 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD and 1-methyl-4-phenylpyridinium(MPP+)-mediated neurotoxicity in SH-SY5Y cells on the potential protective capability.In vivo: maleC57BL/6 mice were randomly divided into three groups: control, MPTP group and MPTP+HBOTgroup. The MPTP-treated mice were intraperitoneally received MPTP (20 mg/kg) four times at 2 hintervals within a day. The day after MPTP treatment, MPTP+HBOT mice were exposed to hyperbaricoxygen at 2.5 atmosphere absolute (ATA) with 100% oxygen for 1 h once daily for 7 consecutive days.In vitro: retinoic acid (RA)-differentiated SH-SY5Y cells were treated with MPP+for 1 h followed byhyperbaric oxygen at 2.5 ATA with 100% oxygen for 1 h. The results showed that MPTP induced asigniﬁcant loss in tyrosine hydroxylase (TH)-positive neurons in the SNpc of mice. HBOT treatmentsigniﬁcantly increased the number of TH-positive neurons, with enhanced neurotrophic factor BDNF,decreased apoptotic signaling and attenuated inﬂammatory mediators in the midbrain of MPTP-treated mice. In addition, MPTP treatment decreased the locomotor activity and grip strength ofmice, and these effects were shown to improve after HBOT treatment. Furthermore, MPTP decreasedmitochondrial biogenesis signaling (SIRT-1, PGC-1↵and TFAM), as well as mitochondrial markerVDAC expression, while HBOT treatment was shown to upregulate protein expression. In cellexperiments, MPP+reduced neurite length, while HBOT treatment attenuated neurite retraction.Conclusions: the effects of HBOT in MPTP-treated mice might come from promoting mitochondrialBiomolecules 2022, 12, 661. https://doi.org/10.3390/biom12050661 https://www.mdpi.com/journal/biomolecules

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Biomolecules 2022, 12, 661 2 of 14biogenesis, decreasing apoptotic signaling and attenuating inﬂammatory mediators in the midbrain,suggesting its potential beneﬁts in PD treatment.Keywords: Parkinson’s disease; hyperbaric oxygen therapy; mitochondrial biogenesis1. IntroductionParkinson’s disease (PD) is a progressive, neurodegenerative, motor disorder, whichaffects⇠1% of the population aged 60 years and over [1]. PD is characterized by thedegeneration of dopaminergic neurons within the substantia nigra pars compacta (SNpc),leading to symptoms of bradykinesia, resting tremor and muscle rigidity. The disease canalso present with non-motor symptoms, such as sleep dysfunction, cognitive impairmentand depression [2]. Although a variety of possible pathogenic mechanisms have beenproposed over the years, including the excessive release of oxygen free radicals duringenzymatic dopamine breakdown, damage to mitochondrial function, loss of nutritionalsupport, and kinase activity abnormalities, destruction of calcium homeostasis and proteindegradation dysfunction, detailed pathogenesis is still uncertain [3–5].Hyperbaric oxygen therapy (HBOT) is a treatment that places patients in a hyperbaricchamber above 1.4 atmospheres absolute [6], allowing them to breathe pure oxygen natu-rally [7]. Compared with normal pressure, HBOT improves single-task or multi-task sportsand cognitive performance in healthy humans [8]; however, the mechanism of this uniquetreatment is not yet fully understood. Research on the neuroprotective effects of HBOThave shown that hyperbaric oxygen can inhibit inﬂammation, reduce hypoxia and improvenervous system microcirculation [9]. There have been case reports of HBOT being used inthe treatment of psychiatric symptoms in PD patients [10]. In addition, it has been shown ina Parkinson’s mouse model that HBOT can effectively inhibit the decrease in dopaminergiccells in the substantia nigra [11]. Although these research results show the potential ofHBOT in the treatment of PD, the detailed neuroprotective mechanism remains unclear.It is known that PD is associated with defects in mitochondrial respiration. This hypothesisarose in the late 1970s, when accidental exposure to1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP), a contaminant from the synthesis of 1-methyl-4-phenyl-4-propionoxy-piperidine (MPPP),was found to cause parkinsonism and DA neurodegeneration [12]. There has also been strongrecent evidence implicating mitochondrial dysfunction as a primary pathogenic pathway leadingto the demise of dopaminergic neurons in PD [13]. The underlying mechanism may be theinhibition of complex I of the electron transport chain (ETC), which, in turn, increases reactiveoxygen species [14,15]. The substantia nigra, compared to other brain regions, is more prone tomitochondrial complex I dysfunction, which results from the generation of ROS in the substantianigra dopaminergic neurons during DA metabolism [16].Therefore, mitochondrial function, particularly its preservation or promotion, is ofspecial interest as a potential therapeutic target of PD and other neurodegenerative dis-orders. Mitochondrial biogenesis is a process by which new mitochondria are formed bythe growth and division of preexisting mitochondria. It involves synthesis of the innerand outer mitochondrial membranes and mitochondrial-encoded proteins, synthesis andimports of nuclear-encoded mitochondrial proteins, and replication of mitochondrial DNA(mtDNA). Mitochondrial biogenesis is tightly regulated by several cell-signaling pathways.The sirtuin-1 (SIRT-1) and peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1↵) is one such major pathway, and is considered to be the master regulator.PGC-1↵is responsible for activating mitochondrial transcription factor A (TFAM) and bind-ing to promoter regions of nuclear genes that encode subunits of the ﬁve complexes in themitochondrial ETC, thereby increasing the assembly of the respiratory apparatus and regu-lating genes involved in heme biosynthesis, the import of nuclear-encoded mitochondrialproteins, and mtDNA replication and transcription [17].

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Biomolecules 2022, 12, 661 10 of 14Biomolecules 2022, 12, x 11 of 15 Figure 6. Effects of HBOT on the protein expressions of mitochondrial biogenesis in the midbrain tissue of MPTP-treated mice. Protein expression of (A) SIRT-1, (B) PGC-1, (C) TFAM and (D) VDAC were detected by Western blotting. All data are expressed as mean ± SEM (n = 3–4). # p < 0.05, ## p < 0.01 and ### p < 0.001 compared with the control group; * p < 0.05 and *** p < 0.001 com-pared with the MPTP group. 4. Discussion In the present study, we examined the effects of HBOT treatment on locomotor ac-tivities and dopaminergic neurons in the substantia nigra of MPTP-treated mice. Accord-ing to our results, locomotor activities, as quantified by the total distance traveled, grip strength, and the time on rotarod, were improved in the MPTP+HBOT mice (Figure 2). Previous studies suggested that exposure to hyperbaric oxygen enhances the oxida-tive capacity of skeletal muscle fibers and the spinal motoneurons innervating them and, thus, has implications for locomotor activity. Our results appear to be concordant with these previous findings [22,23]. On the other hand, the selective degeneration of dopamin-ergic neurons and the reduced expression of TH in the SNpc, along with the decreased levels of DA transporter (DAT) and DA in the striatum, are the main pathological hall-marks of PD and correlate with motor dysfunction [24,25]. In our study, we observed that HBOT attenuated the decrease in dopaminergic cells and the reduced expression of TH in the midbrain of MPTP-treated mice (Figure 1). Therefore, HBOT appears to be a promis-ing treatment for PD, with exciting clinical potential. In addition, there are also case re-ports of the use of HBOT in the treatment of psychiatric symptoms in PD patients [10]. Although our experiments did not examine the emotional effects of HBOT in the animal model of Parkinson’s disease, the potential interference of motoric impairment and αA. B. C. D. Figure 6.Effects of HBOT on the protein expressions of mitochondrial biogenesis in the midbraintissue of MPTP-treated mice. Protein expression of (A) SIRT-1, (B) PGC-1↵,(C) TFAM and (D) VDACwere detected by Western blotting. All data are expressed as mean±SEM (n = 3–4). # p < 0.05,## p < 0.01and ### p < 0.001 compared with the control group; * p < 0.05 and *** p < 0.001 comparedwith the MPTP group.4. DiscussionIn the present study, we examined the effects of HBOT treatment on locomotor activi-ties and dopaminergic neurons in the substantia nigra of MPTP-treated mice. According toour results, locomotor activities, as quantiﬁed by the total distance traveled, grip strength,and the time on rotarod, were improved in the MPTP+HBOT mice (Figure 2).Previous studies suggested that exposure to hyperbaric oxygen enhances the oxidativecapacity of skeletal muscle ﬁbers and the spinal motoneurons innervating them and, thus,has implications for locomotor activity. Our results appear to be concordant with theseprevious ﬁndings [22,23]. On the other hand, the selective degeneration of dopaminergicneurons and the reduced expression of TH in the SNpc, along with the decreased levelsof DA transporter (DAT) and DA in the striatum, are the main pathological hallmarks ofPD and correlate with motor dysfunction [24,25]. In our study, we observed that HBOTattenuated the decrease in dopaminergic cells and the reduced expression of TH in themidbrain of MPTP-treated mice (Figure 1). Therefore, HBOT appears to be a promisingtreatment for PD, with exciting clinical potential. In addition, there are also case reports ofthe use of HBOT in the treatment of psychiatric symptoms in PD patients [10]. Althoughour experiments did not examine the emotional effects of HBOT in the animal model ofParkinson’s disease, the potential interference of motoric impairment and locomotor expres-sion in mood disorders are related to PD (anxiety, depression), which clearly demonstrated

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Biomolecules 2022, 12, 661 11 of 14their impact on motor performance in the OF test [14,26]. Therefore, we cannot rule out thepossibility that HBOT can improve the results of locomotor behaviors by improving theanimal model’s psychiatric symptoms, and this requires further experiments to prove.Although HBOT has an effect on the preservation of dopaminergic cells and motorfunction in MPTP-treated mice, the exact mechanism is still unclear. Neuroinﬂammation isknown to be one of the most important processes involved in the pathogenesis of PD. MPTPis a potent neurotoxin used to mimic PD in a wide range of organisms, including non-human primates, guinea pigs, mice, dogs and cats [27]. Previous studies have demonstratedthat mice treated with MPTP have signiﬁcantly increased inﬂammatory mediators, such asiNOS, COX-2, and TNF-↵in the SNpc [28,29]. In our study, we demonstrated that HBOTprotects against MPTP-induced neuroinﬂammation in PD by inhibiting the NF-B signalingpathway in the midbrain of MPTP-treated mice (Figure 3), indicating that HBOT has theeffect of suppressing neuroinﬂammation.In addition, the anti-apoptotic effect of HBOT has been demonstrated by mediatingthe enhancement of Bcl-2 expression and increasing intracellular oxygen bio-availability.Both of these may contribute to the preservation of mitochondrial integrity and reduce theactivation of the mitochondrial pathway of apoptosis [19]. At the same time, HBOT alsoattenuates the decrease in the Bcl-2/Bax ratio, and reduces the expression of cytochrome cand the rising level of cleaved caspase 3 in the midbrain of MPTP-treated mice (Figure 4).These results indicate that HBOT has the anti-apoptotic effect to counter the apoptosis ofDA neurons in PD.Neurogenesis is deﬁned as the generation of neurons within the brain. It has beensuggested that HBOT exerts neuroprotective effects through the activation of cellulartranscription factors [30]. The CREB is an intracellular protein that regulates the expressionof genes that are important in dopaminergic neurons [31]. The activity of CREB in neuronshas been correlated with various intracellular processes, including differentiation, survival,and neurogenesis [32]. Activated CREB promotes the expression of BDNF, which belongsto a family of neurotrophic that have a crucial role in neuronal survival and enhanced nervetransmission via long-term potentiation. This combination of neurogenesis and optimizedneuronal function is believed to prevent neurogenerative diseases [33]. According toour results, HBOT may promote the activation of CREB and increase the expression ofBDNF in the substantia nigra of MPTP-treated mice (Figure 5). The increase in neuriteoutgrowth is observed in the MPP+-treated SH-SY5Y cells with HBOT (Figure 5). Theabove results suggest that HBOT may promote neurogenesis via the activation of theCREB/BDNF pathway.Mammalian sirtuins (SIRT-1–SIRT-7) have been implicated in a number of cellularand physiological processes, including gene silencing, apoptosis, mitochondrial function,energy homeostasis, and longevity [34]. Stimulation of SIRT-1 has been shown to increasethe expression and activity of PGC-1↵, a master regulator involved in mitochondrialbiogenesis [35]. In cellular disease models, the activation of PGC-1↵ blocks dopaminergicneuron loss induced by mutant↵-synuclein or the pesticide rotenone [36–38]. It acts asa coactivator for transcription factors, such as TFAM, which, in turn, increase mtDNAand mitochondrial biogenesis. In the substantia nigra of transgenic mice, inactivation ofTFAM alleles induces respiratory chain deﬁciency in dopaminergic neurons and aggravatesPD [39]. In addition, Voltage-Dependent Anion Channels (VDACs) also serve as dockingsites for misfolded or mutated proteins, associated with many neurodegenerative disorders,including PD. Interactions with these abnormal proteins alter the physiological activity ofVDAC, contributing to mitochondrial dysfunction typical of these pathologies [40,41]. TheVDAC proteins represent the most important pore-forming proteins of the mitochondrialouter membrane, directly involved in metabolism and apoptosis regulation [42]. Therefore,the SIRT-1/PGC-1↵signaling pathway plays a critical role in PD development and may bea potential target for PD therapy. In the present study, we demonstrated that HBOT causeda signiﬁcant up-regulation of SIRT-1 expression, along with PGC-1↵and its downstreamtranscription factor TFAM and mitochondrial marker VDAC (Figure 6). The increased

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Biomolecules 2022, 12, 661 12 of 14mitochondrial biogenesis has been further conﬁrmed by the increased expression of TH,the key enzyme in DA synthesis, in the brain tissue of MPTP+HBOT-treated mice. This alsoprovided evidence for the hypothesis that HBOT treatment can improve mitochondrialbiogenesis by activating the SIRT-1 dependent PGC-l↵ signaling cascade.5. ConclusionsCurrently, the cause of PD in humans remains unclear and despite continuous progressin research, most of the treatment options are symptomatic and non-curative. In the presentstudy, we provide preliminary evidence that HBOT treatment may decrease neuroinﬂam-mation by inhibiting the NF-kB signaling pathway and promote neurogenesis via theactivation of the CREB/BDNF pathway. Furthermore, this therapy may improve mito-chondrial biogenesis via the SIRT-1/PGC-1↵-dependent signaling cascade and have theability to improve dopaminergic functions by increasing tyrosine hydroxylase expression.Hence, our results suggest that HBOT has potential as an adjunctive treatment, protectingdopaminergic neurons in the clinical treatment of PD.Author Contributions:Conceptualization, H.-T.H., S.-H.H. and Y.-C.L.; methodology, Y.-L.Y.,W.-H.C.and W.-Y.F.; investigation, Y.-L.Y., W.-H.C. and W.-Y.F.; resources, H.-T.H., S.-H.H. and Y.-C.L.; datacuration, H.-T.H.; writing—original draft preparation, H.-T.H. and Y.-L.Y.; writing—review andediting, W.-Y.F., Y.-C.L. and S.-H.C.; project administration, H.-T.H.; funding acquisition, H.-T.H. andY.-C.L. All authors have read and agreed to the published version of the manuscript.Funding:This work was supported by the Ministry of Science and Technology of Taiwan (grantnumber: MOST109-2314-B-037-013) and Kaohsiung Medical University Chung-Ho Memorial Hospital(grant number: KMUH110-0M78) to Hung-Te Hsu.Institutional Review Board Statement:Experimental procedures were carried out in accordancewith the Guide for the Care and Use of Laboratory Animals and were approved by the InstitutionalAnimal Care and Use Committee (IACUC) of Kaohsiung Medical University (IACUC ApprovalNo: 180267).Informed Consent Statement: Not applicable.Data Availability Statement: Data available upon request from the corresponding authors.Acknowledgments: The authors thank Jadzia Tin-Tsen Chou for her help with language editing.Conﬂicts of Interest: The authors declare no conﬂict of interest.References1. Tysnes, O.B.; Storstein, A. Epidemiology of Parkinson’s disease. J. Neural Transm. 2017, 124, 901–905. [CrossRef][PubMed]2. Kalia, L.V.; Lang, A.E. Parkinson’s disease. Lancet 2015, 386, 896–912. [CrossRef]3.Siciliano, M.; Trojano, L.; Santangelo, G.; De Micco, R.; Tedeschi, G.; Tessitore, A. Fatigue in Parkinson’s disease: A systematicreview and meta-analysis. Mov. Disord. Off. J. Mov. Disord. Soc. 2018, 33, 1712–1723. [CrossRef][PubMed]4. Dick, F.D. Parkinson’s disease and pesticide exposures. Br. Med. Bull. 2006, 79–80, 219–231. [CrossRef]5.Block, M.L.; Hong, J.S. Microglia and inﬂammation-mediated neurodegeneration: Multiple triggers with a common mechanism.Prog. Neurobiol. 2005, 76, 77–98. [CrossRef]6.Nambu, H.; Takada, S.; Fukushima, A.; Matsumoto, J.; Kakutani, N.; Maekawa, S.; Shirakawa, R.; Nakano, I.; Furihata, T.;Katayama, T.; et al. Empagliﬂozin restores lowered exercise endurance capacity via the activation of skeletal muscle fatty acidoxidation in a murine model of heart failure. Eur. J. Pharmacol. 2020, 866, 172810. [CrossRef]7.Lu, Z.; Ma, J.; Liu, B.; Dai, C.; Xie, T.; Ma, X.; Li, M.; Dong, J.; Lan, Q.; Huang, Q. Hyperbaric oxygen therapy sensitizes nimustinetreatment for glioma in mice. Cancer Med. 2016, 5, 3147–3155. [CrossRef]8.Vadas, D.; Kalichman, L.; Hadanny, A.; Efrati, S. Hyperbaric Oxygen Environment Can Enhance Brain Activity and MultitaskingPerformance. Front. Integr. Neurosci. 2017, 11, 25. [CrossRef]9. Sanchez, E.C. Hyperbaric oxygenation in peripheral nerve repair and regeneration. Neurol. Res. 2007, 29, 184–198. [CrossRef]10.Xu, J.J.; Yang, S.T.; Sha, Y.; Ge, Y.Y.; Wang, J.M. Hyperbaric oxygen treatment for Parkinson’s disease with severe depression andanxiety: A case report. Medicine 2018, 97, e0029. [CrossRef]11.Kusuda, Y.; Takemura, A.; Nakano, M.; Ishihara, A. Mild hyperbaric oxygen inhibits the decrease of dopaminergic neurons in thesubstantia nigra of mice with MPTP-induced Parkinson’s disease. Neurosci. Res. 2018, 132, 58–62. [CrossRef][PubMed]

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CO MM EN TA RY Open AccessHyperbaric oxygen therapy promotesneurogenesis: where do we stand?Jun Mu1,2, Paul R Krafft1and John H Zhang1,2,3*AbstractNeurogenesis in adults, initiated by injury to the central nervous system (CNS) presents an autologous repairmechanism. It has been suggested that hyperbaric oxygen therapy (HBOT) enhances neurogenesis whichaccordingly may improve functional outcome after CNS injury. In this present article we aim to reviewexperimental as well as clinical studies on the subject of HBOT and neurogenesis. We demonstrate hypotheticalmechanism of HBOT on cellular transcription factors including hypoxia-inducible factors (HIFs) and cAMP responseelement binding (CREB). We furthermore reveal the discrepancy between experimental findings and clinical trials inregards of HBOT. Further translational preclinical studies follo wed by improved clinical trials are needed toelucidate potential benefits of HBOT.IntroductionNeurogenesi s is defined as generation of neurons withinthe brain. In adults, neurogenesis occurs primarily in twobrain regions: the subventricular (SVZ) and the subgranu-lar z one (SGZ) of the hippocampal dentate gyrus (DG).Injury to the central nervous system (CNS) includingtrauma, cerebral ischemia and epileptic seizures have beenreported to induce neurogenesis, and surviving cells maybe functionally integrated into existing neural circuits [1].Consequently, further endoge nous promotion of neuro-genesis may hold promise for restoration of cerebral func-tions after CNS injury.Hyperbaric oxygen therapy (HBOT) refers to the medi-cal use of oxygen at a level higher than atmospheric pres-sure. Initially, indicated fordecompressionillnessithasbeen further applied to clinical conditions includingcrush injury, diabetic foot, skin grafts, thermal bur ns andto several neurological diseases [2]. Elevation of partialoxygen pressure in the body, leads to increased oxygentransport capacity of erythrocytes, facilitating peripheralregeneration processes (e.g. angiogenesis).It has been suggested that HBOT exerts neuroprotec-tive effects through a variety of mechanisms, includingthe activation of cellular transcription factors [3]. How-ever, due to inconsistent results and few clinical trials,HBOT for neurologic disorders has not yet beenapproved by the FDA. Further preclinical studies areneeded to clarify the effect of HBOT on neurogenesisand to ensure a successful translation to clinical trials.Literature ReviewPublications were identified by PubMed/Medline andWeb of Science, using the following keywords: neurogen-esis, hyperbaric oxygen, ischemia, proliferation and BrdU.All p ublications, languages and subsets were explored.Results from previous studies were summarized into thefollowing four categories: hypoxic-ischemic encephalopa-thy (HIE) (Table 1), vascular dementia ( Table 2), perma-nent middle cerebral artery occlusion (MCAo) (Table 3)and human mesencephalic neural progenitor cells(hmNPCs) (Table 4).Regarding HBOT, most preclinical studies were per-formed using a rat model of HIE. Wang et al. i nitiated 7days of HBOT (2.0 ATA, 100% oxygen, 1 hour daily) start-ing 3 hours after experimental HIE in rats. Results showeda significantly increased amount of BrdU+/nestin+ cells inthe SVZ with a peak at 7 days after HIE [4]. 21 days later,more BrdU+/b-tubulin+ cells were observed in the cortexof treated rats, suggesting that HBOT promotes the prolif-eration, differentiati on and migration of newly generatedcells [5].Our preliminary data shows that HBOT decreases theinfarct size with a significantly increased number of BrdU(+) cells in the peri-infarct area 2 week after experimental* Correspondence: johnzhang3910@yahoo.com1Department of Physiology, Loma Linda University School of Medicine,11021 Campus Street, Loma Linda, CA 92354, USAFull list of author information is available at the end of the articleMu et al. Medical Gas Research 2011, 1:14http://www.medicalgasresearch.com/content/1/1/14MEDICAL GAS RESEARCH© 2011 Mu et al; licensee Bi oMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses /by/2.0 ), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

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HIE. We treated operated animals with 1.5 ATA HBO,100% oxygen once a day for 3 consecutive days. BrdU,dissolved in saline, was injected intraperitoneally (50 mg/kg) 24 hours after HIE, once a day for a total of 7 days.Furtherm ore in vit ro studies suggest that HBOT pro-motes neural stem cells differ entiation into neurons oroligodendrocytes, while inhib iting those stem cells fromdifferentiating into astrocytes [6,7]. HBOT also enhancesthe proliferation of other supporting cells, including glialcell line-derived neurotrophic nerve growth factor(GDNF) [8] and vascular endothelial growth factor(VEGF) positive cells [8] as well as epithelial cells [9]and human microvascular endothelia l cells (HMEC-1)(Table 5) [10].Hypothetical mecha nismsNumerous in vivo and in vitro studies confirm thatHBOT induces neurogenesis [5-7,10- 13] however, under-lying mechanisms remain unknown. Activation of severalsignaling pathways and t ranscription factors have beensuggested to play an important role in HBOT inducedneurogenesis, inc luding W nt, hypoxia-in ducible factors(HIFs) and cAMP response element-binding (CREB).HIF-1 is a he terodimeric transcriptional complex com-posed of an inducible HIF-1a subunit and a constitutiveHIF-1b subunit. HIF- 1a is the principal mediator of cel-lular hypoxia adaptations [14]. Therefore activated byhypoxia, HIF-1a cause s the transcription of its regulateddownstream genes, including erythropoietin (EPO) andVEGF which are known to promote neurogenesis [15].However accumulation of HIF-1a induces expression ofp53 [16] and BNIP3 [17], leading to neuronal cell death.Thus neuroprotection may occur shortly after cerebralischemia at balanced level s of HIF-1a. In the presence ofoxygen and iron, HIF-1a is rapidly degra ded via the pro-lyl hydroxyla se pathway . Javorin a et al. discovered thatHBOT exposure stabilizes HIF-1a levels in hmNPCs andfurthermore induces neurogenesis in vitro [7]. We sug-gest that HBOT prevents the accumulation of HIF-1aand therefore exerts its neuroprotective effect (Figure 1).Wnt sig nal ing h as be en su gge sted to play a n imp or-tant role in the regulation of cell proliferation and dif-ferentiation during the stage of CNS development. Wnt-3isthestartingproteinofthispathway.Wangetal.confirmed inc reased level of W nt-3 in HBOT rats 3days after HIE induction, which was positively correlatedwith the proliferation of stem cells [4]. The authors sug-gest that cell proliferati on via Wnt pathway is regulatedthrough b-catenin. Furthermore, in vitro studies demon-strated that b-catenin siRNA decrease s the amount ofnewly generated neurons by repressing the Neurogenin1(NGN1) gene, which can be reversed by HBOT [6]. Ithas been recently reported that HIF-1a modulates Wnt/b-catenin signaling in hypoxic embryonic stem cells(ESC) by enhancing b-catenin activation, and expressionof the downstream effectors lymphocyte enhancer fac-tor-1 (LEF-1) and T-cell factor-1 (TCF-1) [18].It has been implicated that Hif-1a deletion reducesWnt/b-catenin signaling in the SGZ , causing impairedWnt-dependent processes, including neural stem cell pro-liferation, differentiation and neuronal maturation [18].We conclude that activation of the Wnt pathway mayoccur via HBOT induced control of HIF-1a (Figure 1).CREB plays a well-documented role in neuronal plasti-city and formation of lon g-term memory, mainly throughup-regulation of its downstream genes including brainderived neurophic factor (BDNF), Bcl-2, c-fos and VGF.Activation of CREB increases neurogenesis in the DGafter focal cerebral ischemia in rats, and protects aga insthypoxic brain injury [19]. Application of 100% oxygenincreased CREB expression in striatum and hippocampusin a neonatal p iglet model of intermittent apnea [20].HBO preconditioning furthermore increased the ratio ofBcl-2 and Bax e xpressio n in a MCAo/reperfusion modelTable 2 Vascular dementiaAnimal model/Cell lineHBO therapy Method/DurationNeurogenesis InfarctsizeNeurobehavioraltestingMechanism Institute Referencevascular dementia(ligation ofBilateral CCA)100%,2 ATA,2 h/d × 10d/ piriform cortex(Pir)↑DCX+, Nestin+/ Shuttle boxtesting/ ThirdMilitary MedicalUniversityZhang et al[12]Table 3 Permanent MCAoAnimalmodel/CelllineHBO therapy Method/DurationNeurogenesis InfarctsizeNeurobehavioraltestingMechanism Institute ReferencePermanentMCAo100%,2.5 ATA,1.5 hFrom 15 min, 1.5 h, 3h after MCAo/ D7↑GFAP+↓ garcia / University ofLeipzig, GermanyGunther,et al [13]Mu et al. Medical Gas Research 2011, 1:14http://www.medicalgasresearch.com/content/1/1/14Page 3 of 7

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to establish HBOT as a prevention and/or treatmentmodality for neurological diseases.AcknowledgementsWe thank Robert P. Ostrowski for his valuable contributions. This work issupported by a grant from National Nature Science Foundation of China(No.30570657) and 973 project (2009CB918300).Author details1Department of Physiology, Loma Linda University School of Medicine,11021 Campus Street, Loma Linda, CA 92354, USA.2Department ofNeurology, The First Affiliated Hospital of Chongqing Medical University, 1 YiXue Yuan Road, Chongqing 400016, China.3Department of Neurology, LomaLinda University School of Medicine, 11234 Anderson Street, Loma Linda, CA92354, USA.Conflicts of Interests /DisclosuresThe authors declare that they have no competing interests.Received: 6 April 2011 Accepted: 27 June 2011 Published: 27 June 2011References1. Alvarez-Buylla A, Lim DA: For the long run: maintaining germinal nichesin the adult brain. Neuron 2004, 41(5):683-6.2. 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Atzeni et al.-               -     -     -   -       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     -     -    -     -           -        -     -            -    -                 ---          -     --      -         -                   -     -            -                -       -                            -                                            --        -             -          -     ----                 

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