EffectofanIntravenousAdministrationofAdipose-Tissue-DerivedAutologousStem CellsontheSkinTemperatureofParalyzedLimbsofStrokePatients

1. Abstract Background: Cold or warm feelings were reported to be common symptoms of stroke patients. We recently reported that intravenousadministrationofautologousAdipose-Tissue-Derived Stem Cells (ADSCs) significantly improves neurological functions, including a skin temperature increase, shortly after treatment. Aim:ToconfirmtheeffectofADSCsontheskintemperature of stroke patients, we measured skin temperatures immediately before and shortly afterADSC therapy and evaluated the clinical effect on stroke patients in relevance with the skin temperature. Patients and Methods: The skin temperature of 16 stroke patientswasevaluated.Theskintemperatureof9healthysubjects without ADSC treatment was measured as a control. Skin temperaturewasmeasuredonhealthyandparalyzedlimbsofstroke Citation: IchihashiM,EffectofanIntravenousAdministrationof Adipose-Tissue-Derived Autologous Stem Cells on the SkinTemperatureofParalyzedLimbsofStrokePatients. AnnClinMedCaseRep.2023;V10(13):1-9 patients, immediately before, during and shortly after treatment withASDCsat8skinlocation.BeforeADSCstreatment,patients and their families are fully explained of safety, efficacy and pos- sible side effects of cell therapy, and patients and their families are explained about possible presentation and publication of their clinical data under the privacy protection of subjects. Results: The mean skin temperature of healthy big toes was lowest among the 8 measured locations. The mean skin temperatureofparalyzedbigtoeswaslowercomparedtothehealthyside, andincreasedsignificantlyshortlyafterADSCinfusion.Skintemperature of patients with large cranial tissue damage did not increasein4of5cases.PatientswithNIHstrokescalerecoveryand skin temperature increase of paralyzed limbs suggested common recovery mechanism.thattheskintemperatureofthetoeofstrokepatientssignificantly elevatedshortlyafterADSCtherapy.Further,ourstudyalsoindicated that skin temperature regulation by ADSCs may correlate with the recovery of other skin functions, such as tactile. In conclusion,wespeculatethatADSCsmayexertbeneficialtherapeutic effectsatleastpartlyviaanincreasedbloodcirculationimmediate- ly afterADSC treatment, although the detailed mechanism of the skintemperatureincreaseanditseffectonotherneuronalfunctions remains to be clarified.

2. Key words Acoustic cells; Amplification; Protein molecules; Receptor; Stapedotomy; Ionic canals; Transforming and transmitting auditory information

2. Introduction Autologous mesenchymal stem cells have been reported to be effectiveintherecoveryonsensoryandmotorfunctionaloutcomes mostly at subacute stages and rarely in chronic stages of stroke patients[1-3].Further,humanadultmesenchymalstemcells are known to be highly resistant to spontaneous transformation, strongly indicating that mesenchymal stem cell transplantation maybeapromisingandsafetherapeuticmodalityforstrokes[4,5]. Toourknowledge,mostclinicalstudiesonmesenchymalstemcell therapy of strokes evaluated the efficacy one week to a year after stem cell therapy. So, there is little information available about theearlyeffectsofmesenchymalstemcellsduringorimmediately after intravenous transplantation. We recently reported the characteristics of the recovery of motor, sensory and cognitive functions of 21 stroke patients who were observed during and shortly after Adipose-Tissue-Derived Stem Cells (ADSCs) transplantation therapy. Further, we suggested a possible role for biological small molecules secreted from stem cells suspended in 200 ml saline during the 90-minute-drip-infusion.6 In addition, we experienced several cases who exhibited visible reddish changes of paralyzed limb skin during or immediately afterADSC infusion, suggesting an increase of peripheral blood flow and skin temperature of the paralyzed extremities. Recently, extracellular vesicles derived from mesenchymal stem cells were suggested to mediate the cell therapeutic effects on stroke by facilitating intercellular communications in a paracrine fashion,andtoregulateintrinsiccellfunctions,soexosomeshave been extensively studied and reported to be major mediators of stem cell therapy in stroke and other disorders including skin diseases[7-12].WespeculatethatinadditiontomiRNAs,othersmall molecules, such as cytokines, growth factors and other factors released fromADSCs, may play a pivotal role inADSC-induced early functional recovery of stroke patients, since small trophic molecules can travel throughout the body within a minute and affect the biology of both proximal and distant responder cells. Tobetterunderstandtheefficacyoftherapyofstrokepatientswith ADSCs, we studied the relationship between early clinical recoveryofmotor/sensoryfunctionsandbloodcirculationofparalyzed limbs of stroke patients after intravenousADSCs administration, by measuring the skin temperature of healthy and of paralyzed limbs before, during and 30 min after ADSC therapy. The mean temperature of the paralyzed big toes of 16 stroke patients significantly increased immediately after ADSC therapy comparedtobeforetreatment,whereasthetemperatureofthecontralateral healthy big toes showed a tendency to increase, but not at a statistically significant level. Ourstudyforthefirsttimeintheworlddemonstratedthattheskin temperature of the big toe of paralyzed leg significantly elevat-ed during and shortly after a single intravenous drip infused of ADSC. In addition, the study also indicated that the homeostatic recoveryofskintemperaturebyADSCtherapymaycorrelatewith the recovery of other skin functions, such as tactile and temperaturesensationsofparalyzedlimbs.Further,ourstudysuggestthat treatment with ADSCs may exert early therapeutic beneficial effectsatleastpartlyviaincreasedbloodcirculation,possiblydueto small bioactive molecules secreted fromADSCs during and after ADSC administration. In addition, we speculate that small molecules may be key effectors that exert their biological functions quickly, within several hours and even one month after ADSC therapy,althoughthedetailedmolecularmechanismsoftheeffect of ADSCs on skin temperature and other functional recovery remains to be elucidated

3. PatientsandMethods PatientsandSkinTemperatureMeasurement We report an unblinded clinical study of 16 stroke patients who wanted to be treated with ADSCs, expecting some motor and sensory functional recovery of stroke symptoms which remained after common therapy for strokes. The mean age of the subjects was 54.6 (range: 37~80 years) at the time of their stroke and 57.5 (37~84 years) at the time of ADSC treatment. Demographic and clinical characteristics of the patients are shown in Table 1. AutologousADSCs were administered to treat chronic stroke patients who had a stroke onset more than 7 months and less than 9 years earlier. In the present clinical study, 16 cases of 3 ischemic and 13 hemorrhagic stroke patients were included. WeanalyzedtheeffectsofADSCtherapyonskintemperatureand functional recovery of 16 chronic stroke patients who had severe to moderate neurological abnormalities of hemi-paralysis that remained after common therapy including rehabilitation for acute, subacute and chronic stages, by comparing the effects shortly before and immediately after ADSC therapy. For skin temperature measurements, we used a combined instrumentofLT-8seriesandLT-ST08-12(GramCorporation,Saitama, Japan),whichisdesignedtomeasureskintemperatureatanaccuracyof±0.01℃.Wemeasuredtheskintemperatureofeachpatient lying on their back on a bed at a room temperature of 24±0.5℃. Theseverityandfunctionalevaluationofeachpatientwasdetect- ed by the NIH Stroke Scale (NIHSS), and skin temperature was measuredatthemiddlefingerandbigtoeofhealthyandparalyzed limbsofeachpatient.WeevaluatedtheNIHSSandmeasuredskin temperature shortly before and immediately afterADSC therapy, sinceweexperiencedandreportedacasewhohadaquiterapidrecoveryofmorethan3NIHSSscorewithintwohoursafterADSC Briefly, ADSCs were prepared from the subcutaneous fat tissueof the abdominal skin of each patient. Patients were treated witha local anesthetic patch and injection in the skin approximately10 cm to the side of the umbilicus, and 2~3 rice-sized pieces of subcutaneous fat tissues were surgically obtained from a 0.7 cm incision.Thefattissueswerecutinto15-20smallpiecesandwere placedonascaffoldofnonwovenfabricpaintedwithhydroxyap- atite (BioMiraiKobou, Tokyo, Japan) in culture dishes and were cultured at 5% CO2 and 37℃ in medium supplemented with 4% autologous serum for 11 to 13 days. They were then trypsinized (0.25%trypsin,BioMiraiKobo,Tokyo,Japan)andreseededinT75 flasks and further cultured for approximately 3 days in medium containing 2% serum, after which they were re-trypsinized and cultured inT300 flasks (BM Equipment,Tokyo, Japan), and then trypsinized again and cultured for 3 days in HyperFlasks (Corning Japan, Tokyo, Japan), before the final cell preparation for the treatment.Atthedayoftransplantation,cellsweretrypsinizedand washed 4 times with saline, then passed through two filters (40 µm and 100 µm) and an average number of 1.0 x 108 (0.6~1.4 x 108)ADSCswerepreparedandresuspendedin200mlsalineand administered intravenously to the patients. The cells were further filteredthrougha180µmporesize-meshduringthedripinfusion to remove clusters of cells. Stem cell characteristics of collected cellswereconfirmedwithflowcytometryusingCD73,CD90,and CD105 for positive antibodies, and CD45 for negative antibody against stem cells, respectively. StatisticalAnalysis Aprobabilityvalueof0.05isconsideredstatisticallysignificant. All values are presented as means ± SD. Comparisons between groups were made using one-way analysis of variance (ANOVA, nonparametric) with statistical software. therapy.6ThestudywasconductedaccordingtotheDeclaration Table1:Skintemperatureof9healthy subjects. ofHelsinkiPrinciplesusingaprotocolethicallyreviewedandapprovedbytheArts-GinzaClinicEthicsCommittee.Informedconent to participate in this study was obtained from each subject or family member before commencement of the studyBriefly, ADSCs were prepared from the subcutaneous fat tissueof the abdominal skin of each patient. Patients were treated witha local anesthetic patch and injection in the skin approximately10 cm to the side of the umbilicus, and 2~3 rice-sized pieces of subcutaneous fat tissues were surgically obtained from a 0.7 cm incision.Thefattissueswerecutinto15-20smallpiecesandwere placedonascaffoldofnonwovenfabricpaintedwithhydroxyap- atite (BioMiraiKobou, Tokyo, Japan) in culture dishes and were cultured at 5% CO2 and 37℃ in medium supplemented with 4% autologous serum for 11 to 13 days. They were then trypsinized (0.25%trypsin,BioMiraiKobo,Tokyo,Japan)andreseededinT75 flasks and further cultured for approximately 3 days in medium containing 2% serum, after which they were re-trypsinized and cultured inT300 flasks (BM Equipment,Tokyo, Japan), and then trypsinized again and cultured for 3 days in HyperFlasks (Corning Japan, Tokyo, Japan), before the final cell preparation for the treatment.Atthedayoftransplantation,cellsweretrypsinizedand washed 4 times with saline, then passed through two filters (40 µm and 100 µm) and an average number of 1.0 x 108 (0.6~1.4 x 108)ADSCswerepreparedandresuspendedin200mlsalineand administered intravenously to the patients. The cells were further filteredthrougha180µmporesize-meshduringthedripinfusion to remove clusters of cells. Stem cell characteristics of collected cellswereconfirmedwithflowcytometryusingCD73,CD90,and CD105 for positive antibodies, and CD45 for negative antibody against stem cells, respectively. StatisticalAnalysis Aprobabilityvalueof0.05isconsideredstatisticallysignificant. All values are presented as means ± SD. Comparisons between groups were made using one-way analysis of variance (ANOVA, nonparametric) with statistical software.

4. ResultsSkinTemperatureoftheExtremitiesofHealthySubjects Ninehealthysubjectswereenrolledinthestudyandtheskintemperature of the extensor surface of the elbow, middle forearm, hand joint, middle finger, thigh, lower leg, foot joint and big toe wasmeasured(Table2).Themeantemperatureofthebigtoeskin, including both the right and left big toe of each subject, was the lowest among the skin locations measured, and the mean temperature of the middle fingers of all 9 subjects was higher than all other locations (Figure 1). Based on these results, we selected the middlefingerandthebigtoeofeachhealthyandparalyzedsideof stroke patients to evaluate the effects ofADSC administration on skin temperature. Figure1:Skintemperatureofupperandlowerextremitiesof9healthysubjects. Skin temperature was measured at 8 locations: extensor surface of the upper arm, forearm, hand joint, middle finger, upper leg, lower leg, foot joint and big toe.The middle finger temperature was higher than the other 7 locations, and the big toe temperature was statistically the lowest among the 8 locations (except the upper arm). Statistical analysis was conducted using Tukey’s test; ** indicates p0.01, * indicates p0.05 EffectofADSCsontheSkinTemperatureoftheParalyzed ExtremitiesofStrokePatients Sevenofthe16strokepatientsshowedaskintemperatureincrease at the big toe on the paralyzed side, and the mean skin tempera- ture of the big toe on the paralyzed side of 16 stroke patients was significantlylowerthanthehealthysidebeforetreatmentwithADSCs, but it increased to a significantly higher level comparable to the non-treated healthy toe skin temperature after treatment with ADSCs (Figure 2). The mean skin temperature of the paralyzed middlefinger,forearmandlowerlegofeachstrokepatient,however, did not increase significantly afterADSC therapy. Interestingly, the skin temperature of the big toe on paralyzed side didnot decrease more than 1.0℃ in any patient afterADSC therapy, whereas,theskintemperatureoftheparalyzedandhealthymiddle fingers of some stroke patients decreased more than 1.0℃, evento3.0℃,afterADSCtherapy,althoughthemechanismoftheADC-induced skin temperature change remains to be clarified. The mean skin temperature of the middle finger and the big toeon the healthy sides of stroke patients beforeADS Skin temperature measured at the middle finger and the big toe of healthy (H) and paralyzed (P) limbs of 16 stroke patients, before and afterADSC administrationareshownasfilledcirclesandtheaverageofeachgroupisshownbyathickbar(―).TemperatureafterADSCadministrationincreased significantly compared to the temperature beforeADSC treatment only at the big toe on the paralyzed side.The average skin temperature of pretreatment paralyzed big toes was significantly lower compared to healthy limbs. **indicatesasignificantdifferencebetweenthemeanvaluesofhealthyandparalyzedbigtoeskintemperaturebeforeADSC treatment. *indicatesasignificantdifferencebetweenthemeanbigtoeskintemperaturebetweenpre-andpost-ADSC therapy. Statisticalanalysisofskintemperaturebeforeandaftertreatmentwasperformedusingt-test,andstatisticalanalysisofskintemperatureonthehealthy sideandontheparalyzedsideatthebeginningandtheendofthetreatmentwasperformedusingaPairedt-test.Ap-valueoflessthan0.05isconsidered statistically significant (**:p 0.01, *: p 0.05). Meanskintemperatureofthemiddlefinger(A)andthebigtoe(B)onthehealthysideofstrokepatientswasstatisticallyatthelevelofhealthysubjects, whereasthetemperatureofthebigtoeontheparalyzedsideofstrokepatientswasstatisticallylowerthanhealthysubjectsandthehealthysideofstroke patients. Statistical analysis was conducted according to Tukey’s test. StatisticalanalysisofskintemperatureonthehealthysideandparalyzedsidewasperformedusingaPairedt-test,andstatisticalanalysisofskintem- perature of healthy subjects, healthy side and paralyzed side, was performed using a Paired Tukey’s test.Ap-value of less than 0.05 is considered to indicate a statistically significant difference (**: p 0.01, *: p 0.05). EffectofADSCTreatmentonSkinTemperatureandother Functional Recovery of Stroke Patients in Relevance to Brain Lesion Characteristics Six of the 16 stroke patients showed a functional recovery by NIHSS assay shortly after ADSC therapy, and in 3 of those 6 patients, the skin temperature of the big toe on the paralyzed side was significantly elevated (Table 1). These results suggest thatthe skin temperature increase of paralyzed toes immediately after ADSC therapy may be correlated with other functional recoveries of stroke patients. 3 of those 7 had putamen bleeding, while theremaining3and1caseshadthalamicbleedingandrupture at the anterior cerebral artery, respectively. The skin temperature of the 3 putamen bleeding cases who showed increased toe skin temperaturedecreased(Table1).Theseresultssuggestthatinjured locations of the brain, putamen or thalamic areas may affect via a common route the skin temperature alteration of paralyzed limbs after ADSC therapy. Effect of Patient’s Clinical Characteristics, Age, Sex and BrainLesionVolumeonSkinTemperatureafterADSCTreatment The effect of clinical characteristics of stroke patients on immediate skin temperature changes after ADSC therapy was studied focusing on age, sex, ischemia or hemorrhage, and brain lesion volume examined by MRI and CT at stroke onset. The age, sex andbrainlocationsofpatientswithparalysishadnoeffectonskin temperature change after ADSC therapy. Interestingly, the skin temperatureofpatientswithbrainlesionvolumeslargerthan5cm indiametershowednoincreaseofskintemperatureatanylocation afterADSCtreatment,butthetemperaturedecreasedat4ofthe5 paralyzed middle fingers. The mechanism of the temperature decreaseofthemiddlefingerontheparalyzedsideofstrokepatients havinglargebraindamagevolumeremainstobeclarified(Table 1). SkinTemperatureAlterationatDifferentLocationsof Stroke Patient’s Extremities after ADSC Treatment Themiddlefingerof6patientsandthebigtoeof7patientsonthe paralyzedsideofthe16patientsshowedanapparentskintemperatureincreaseduringorimmediatelyafterADSCtreatment,but2 middlefingersand3bigtoesonthehealthysideofthosepatients also showed a temperature increase, respectively (Table1). Two of the 6 patients with paralyzed-middle finger temperature increaseand3ofthe7patientswithparalyzed-bigtoetemperature increase showed skin temperature increases at both the paralyzed and healthy middle fingers and the paralyzed and healthy big toes, respectively. These results suggested that the skin temperatureincreaseelicitedbyADSCtherapytakesplacesimultaneously or independently at the paralyzed and healthy limbs. Further, the temperature increased only in 4 of 16 cases at both the middle finger and the big toe of the paralyzed limbs (Table1). These resultssuggestthattheeffectofADSCsonskintemperatureincrease may be initiated by biological factors acting not only at the brain lesionpenumbraalone,butalsoatthecontra-lateralbrainarearesponsible for skin temperature homeostasis, although the detailed mechanismoftheeffectofADSCsonskintemperatureremainsto be clarified. EffectoftheTimeLagBetweenStrokeOnsetandADSCs Therapy The skin temperature of both groups treated earlier than a 1-year lag time, and later than a 1-year lag time showed similar increase ratios,3of6and6of10,respectively(Table1).Basedontheseresults,weconcludedthattheshortertimelagbetweenstrokeonset andADSCtherapymaynotexertastrongeffectontheskintem- peraturechangeoftheparalyzedlimbs.Weneedtostudytheeffect of lag between stroke onset andADSC administration on the skin temperatureusingmorestrokecases,sinceADSCtherapyshorter thanaone-yearlagtimeshowedbetterNIHSSrecoverycompared to a longer than one year lag time in our previous study [6].

5. DiscussionAsfarasweknow,ourstudyshowsforthefirsttimeintheworld that ADSCs administered intravenously increaseatatistically significantlytheskintemperatureofthebigtoeontheparalyzedside of stroke patients at the chronic phase shortly after drip infusion. Basedontheseresults,wesuggestthatthereisanincreaseofblood flow at the paralyzed limbs shortly after the systemic infusion of ADSCs. Further, the results show that the mean skin temperature ofthebigtoeonthehealthysideofstrokepatientswasstatistically at the same level of healthy subjects, and the mean skin temperatureontheparalyzedsideofstrokepatientswasstatisticallylower than that on the healthy side (Figures 2,3). Studies of skin temperatures on hemiplegic limbs reported previouslyareconflicting[13,14].Earlierstudiesclaimedanincreased temperatureofthehemiplegicarm,andonestudysuggestedatwostage theory of initial warmth of the paralyzed limb at the acute phase and coldness in the chronic phase. Most previous studies were conducted based on the perception of coldness or warmness bystrokepatients.Wanklynetal.showedthatstrokepatientswith coldness had a lowered skin temperature and a markedly lower blood flow in the paralyzed limbs compared to the unaffected limbs[15].Naveretal.reportedthat43%ofstrokepatientshada sensationofcoldnessintheiranalysisof37casesandfurtherthey reported that no patients with right cortical hemispheric lesions had noticed coldness in the contralesional side [16]. In our study, weobservedpatientswithcoldnesswhohadacorticalhemispher- ic lesion even on the right side. The detailed mechanism causing the coldness of limbs of stroke patientsisstillunknown,butitmaybecausedbydisturbedsympatheticallymediatedvasomotortone,leadingtoreducedbloodflow, initially triggered by damaged brain lesions. The signaling systems that modulate the structural and functional plasticity ofAutonomicNervousSystem(ANS)arelargelyunknown.TheANSis easily influenced by Central Nervous System (CNS) and by neuroendocrine system involved in stress response and, also greatly influenced by neurotrophic factors [17]. Therefore, we speculate that ADSCs-induced skin temperature increase could be caused by some neurotrophic factors, such as BDNF (brain derived neurotrophic factor), NGF (nerve growth factor), IGF-1 (insulin-like growth factor), and CNTF (ciliary neurotrophic factor) which are expectedtobesecretedfromADSCsintothemediumduringdrip infusion. The present study also suggests that a skin temperature increase observed at the paralyzed limbs of many stroke patients, particularlyofthebigtoe,afterADSCtherapymaycontributetorecovering the warm sensation of cold extremities of stroke patients. Fingercoldnesswasreportedtobeamajorsignofstrokepatients inapreviousstudy[14],butweobservedamoresevereskintemperature decrease of the big toe compared to the finger. We proposethatthebigtoeskinmaybethebestlocationfortemperature measurement of stroke patients, at least those at the chronic stage. It is not yet clear enough from our study that the skin temperature increase afterADSC therapy may play a role in the recovery of other neuronal functions of stroke patients since we identified only3caseswhoshowedatemperatureincreaseoftheirparalyzed limbs among the 6 cases who had recovery of their NIHSS. In addition, our study indicates that the effect of ADSC therapy on the skin temperature of paralyzed limbs may be affected by the characteristicsoftheinjuredbrainincludinglesionvolume,which may cause a severe circulation disturbance of brain tissue at the chronicstageofstroke,sincetheskintemperatureofthebigtoeon theparalyzedsidedidnotincreaseaftertreatmentwithADSCsin patientshavingbrainlesionvolumeslargerthan5cmindiameter. Thepresentstudyalsosuggeststhattheskintemperatureincrease observedattheparalyzedlimbsshortlyafterADSCstherapymay be correlated with the functional recovery of skin tactile sensations,butitisnotclearhowADSCtherapymaycorrelatewithotherfunctionalrecoveries,sincethenumberofpatientsweexamined was too small to reach a conclusion about that. Importantly, our results strongly indicate that extracellular small molecules secreted from stem cells suspended in 200 ml saline, begin to circulate during the 90 minutes of drip infusion and may play a pivotal role in the recovery of neuronal and circulatory functions following ADSC therapy, since most of the stem cells administered are considered to be trapped in the lungs at least for the first 24 hours, and only a few percent of cells are expected to recirculate to other organs including the brain, after a 1 to 2 day lag time [18-20].We speculate that biological molecules secreted fromADSCscomposedofmiRNAs,growthfactors,cytokinesand other small molecules, contribute directly and/or indirectly to the functional recovery shortly after ADSC infusion. Further, based onthepresentstudy,wespeculatethatmiRNAsmaynotbeama- jor contributor to the skin temperature increase immediately after drip-infusion, since the biological effects of miRNAs may appear at least a few hours after drip infusion to exert clinical functional recovery. Our study also contributes to the promotion of the application of ADSC therapy for hemorrhagic strokes, since clinical reports of mesenchymal stem cell therapy of hemorrhagic strokes are still limited [21-27] compared to studies of ischemic strokes. Further, preclinical studies showed the functional effects of systemically administered mesenchymal stem cells on traumatic and hemorrhagic strokes [28-32], although the pathophysiology and mechanisms of recovery are reported to differ between ischemic and hemorrhagicstrokes[33].fore penumbra with intracerebral hemorrhage in contrast to ischemic stroke [34]. In addition, a review of mesenchymal stem cell therapy for ischemic strokes that analyzed 13 clinical trials between 2014and2020basedonsearchingthePubMeddatabaseconcludedthatmoreevidenceregardingthesafetyandefficacyofmesenchymal stem cell therapy of stroke is needed [35]. Infuturestudies,weaimtoconfirmtheeffectofADSCsontheskin temperature of stroke patients and its relationship to other neurologicalfunctionalrecoveriesbyanalyzingmoreclinicalcases.To use skin temperature as a non-invasive biomarker evaluating the effects ofADSC therapy of chronic stroke patients, it is required toshowhowskintemperaturecorrelateswithotherbiomarkersof strokepatientsandbraininjury[36-40].Further,weaimtounder- stand the mechanism of the effects of ADSCs on stroke patients using in vitro and in vivo studies, specifically focusing on small molecules that are released from ADSCs during treatment that might play an important role in the temperature increase and in the functional recovery of strokes even during and shortly after the therapy.

6. ConclusionOurstudyindicatedthatasingleintravenousdripinfusionofADSCsrecoverstheskintemperatureandotherfunctionsofparalyzed limbofstrokepatientsshortlyafteradministration,andapossible pivotal role of trophic factors and other small molecules secreted from stem cells in ADSCs therapy, although the precise role of ADSCs remains to be clarified by further studies.

7. AcknowledgmentsWe gratefully acknowledge Yumiko Kimura at Arts Ginza Clinic who gratefully contributed to evaluating the effect of stem cell treatmentinthisstudy.WealsothanktoDr.VinceHearingforhis careful and critical reading and editing of our manuscript.

References References

1. Bang OY, Lee JS, Lee PH, Lee G.Autologous mesenchymal stemcell transplantation in stroke patients.Ann Neurol. 2005; 57: 874-82.

2. RaJC,ShinIS,KimSH,KangSK,KangBC,LeeHY,etal.Safetyofintrav enousinfusionofhumanadiposetissue-derivedmesenchy-mal stem cells in animal and humans. Stem Cells Dev. 2011; 20:1297-308.

3. BhasinA,SrivastavaMV,BhatiaR,MohantyS,KumaranSS,BoseS..A utologousintravenousmononuclearstemcelltherapyinchron-ic ischemic stroke. J Stem Cells Regener Med. 2012; 3: 181-9.

4. Lee JS, Hong JM, Moon GJ, Lee PH,Ahn YH, Bang OY.Alongtermfollow-upstudyofintravenousautologousmesenchymalstemcell transplantation in patients with ischemic stroke.Stem Cells.2010; 28: 1099-106.

v5. von Bahr L, Batsis I, Moll G, Hägg M, Szakos A, Sundberg B, etal.Analysis of tissues following mesenchymal stromal cell therapyin humans indicates limited long-term engulfment and no ectopic tissue formation. Stem Cells. 2012; 30: 1575-8.

6. IchihashiM,TanakaM,IizukaT,NagoeN,SatoY,TakahashiH,etal.The rapeuticeffectofintravenouslyadministeredautologousadi-posederivedstemcellsonchronicstagestrokepatients.IntJStemCell Res Ther. 2020; 7: 1-9.

7. Dabrowska S, Andrzejewska A, Lukomska B, Janowski M. Neuroinflammation as a target for treatment of stroke using mesenchymal stem cells and extracellular vesicles.J Neuroinflammation.2019; 16: 1-17.

. DoeppnerT,Herz J,GorgensA, SchlechterJ,LudwigAK, RadtkeS,et al. Extracellular vesicles improve post-stroke neuroregenerationand prevent postischemic immunosuppression. Stem Cells TranslMed. 2015; 4: 1131-43.

9. Xin H, Buller B, Kataklowaski M, Katakowski M, ZhangY,WangX, et al. Exosome-mediated transfer of miR-133b from multipotentmesenchymalstromalcellstoneuralcellscontributestoneur iteout-growth. Stem Cells. 2012; 30: 1556-64.

10. Assuncao-Silva RC, Mendes-Pinheiro B, Patricio P, Patrício P,BehieLA,TeixeiraFG,etal.Exploitingtheimpactofthesecretomeof MSCs from different tissue sources on neuronal differentiationand axonal growth.Biochimie. 2018; 155: 83-91.

11. VizosoFJ,EiroN,CotaL,EsparzaP,LandinM,DiazRodriguezP,etal.Mesenchymalstemcellsinhomeostasisandsystemicd iseases:Hypothesis,evidences,andtherapeuticopportunities.IntJMolS ci.2019; 20: 3738.

12. PinhoAG,CibraoJR,SilvaNA,etal.Cellsecretome:Basicinsightsandth erapeuticopportunitiesforCNSdisorders.Pharmaceuticals.2020; 12: 31.

13. HerbaultAG, Cole J, Sedgwick E.Acerebral hemisphere influenceon cutaneous vasomotor reflexes in humans. J Neurol NeurosurgPsychiatry. 1990; 53: 118-20.

14. Korpelanien JT, Sotaniemi KA, Myllyla VV. Asymmetrical skintemperature in ischemic stroke. Stroke. 1995; 26: 2543-547.

15. WanklynP,IlsleyDW,GreensteinD,HamptonIF,RoperTA,KesterRC, et al. The cold hemiplegic arm. Stroke. 1994; 25: 1765-70.

16. NaverH,BlomstrandC,EkholnS,JensenC,KarlssonT,Wallin G.Autonomicandthermalsymptomsanddysfunctionafterstroke.Strok e. 1995; 26: 1379-85.

17. Mattson MP, Wan R. Neurotrophic factors in autonomic nervoussystemplasticityanddysfunction.NeuronalMed.2008;10:157 -68.

18. EggenhoferE,LukF,DahlkeMH,HoogduijnMJ.Thelifeandfateof mesenchymal stem cells. Front Immunol. 2014; 5: 148.

19. De Witte SFH, Luk F, Parraga JMS, Gargesha M, Merino A, Korevaar SS, et al. Immunomodulation by therapeutic mesenchymalstromal cells (MSC) is triggered through phagocytosis of MSC bymonocytic cells. Stem Cells. 2018; 36: 602-15.

20. Niyibizi C, Wang S, Mi Z, Robbins PD. The fate of mesenchymalstem cells transplanted into immunocompetent Volume10Issue13-2023 CaseReport http://www.acmcasereports.com/ 9 neonatal mice: Im-plications for skeletal gene therapy via stem cells. Mol Ther. 2004;9: 955-63.

21. TurnbullMT, ZubairAC, MeschiaJF, FreemanWD.Mesenchym. 22. LiZ-A,ZhangZ-T,GuoC-J,GengFY,QiangF,WangLX.Autolo-gous bone marrow mononuclear cell implantation for intracerebralhemorrhage―a prospective clinical observation. Clin Neurol Neu-rosurg. 2013; 115: 72-76.

23. Brunet M-C, Chen SH, Khandelwal P, Hare JM, Starke RM, Peterson EC, et al. Intravenous stem cell therapy for high-grade aneurysmalsubarachinoidhemorrhage:casereportandliteraturereview.Wo rld Neurosurg. 2019; 128: 573-5.

24. Rosado-de-CastroPH,deCarvalhoFG,deFreitasGR,Mendez-OteroR,PedroMorenoPimentelCoelhoPM.Reviewofpreclinicalandclinicalstudies of bone marrowderived cell therapies for intracere-bral hemorrhage. Stem Cell Int. 2016; 461783.

25. ChangZ,MaoG,SunL,AoQ,GuY,LiuY.Celltherapyforcerebralhemmo rhage: five year follow-up report. Exp Ther Med. 2016; 12:3535- 40.

26. RowartP,ErpicumP,DetryO,WeekersL,GrégoireC,LechanteurC, et al. Mesenchymal stromal cell therapy in ischemic/reperfusioninjury. J Immunol Res. 2015; 602597.

27. JiangY, Zhu W, Zhu J, Wu L, Xu G, Liu X. Feasibility of delivering mesenchymal stem cells via catheter to the proximal end of thelesion artery in patients with stroke in the territory of the middlecerebral artery. Cell Transplant. 2013; 22: 2291-8.

28. Kim J-M, Lee S-T, Chu K, Jung KH, Song EC, Kim SJ, et al. Systemic transplantation of human adipose stem cells attenuated cerebralinflammationanddegenerationinahemorrhagicstrokemodel.Brai n Res. 2007; 1183: 43-50.

29. Huang P, Gebhart N, Richelson E, Brott TG, Meschia JF. Mechanism of mesenchymal stem cell-induced neuron recovery and an-tiinflammation. Cytotherapy. 2014; 16: 1336-44.

30. Xie J, Wang B, Wang L, Dong F, Bai G, Liu Y. Intracerebral andintravenous transplantation represents a favorable approach for ap-plication of human umbilical cord mesenchymal stromal cells inintracerebral hemorrhagic rats. Med Sci Monit. 2020; 22: 3552- 61.

31. Duan S,Wang F, Cao J,Wang C. Exosomes derived from microRNA-146a-5p-enrichedbonemarrowmesenchymalstemcellsallevi-ate intracerebral hemorrhage by inhibiting neuronal apoptosis andmicrogliaM1polarization.DrugDesDevTher.2020;14:3143-58.

32. ChenK-H,ShaoP-L,LiY-C,ChiangJY,SungP-H,ChienH-W,etal. Human umbilical cord-derived mesenchymal stem cell therapyeffectivelyprotectthegrainarchitectureandneurologicalfuncti oninrat after acute traumatic brain injury. CellTransplant. 2020; 29: 1-15.

33. XiG,KeepRF,HoffJT.Mechanismsofbraininjuryafterintracere-bral hemorrhage. Lancet Neurol. 2006; 5: 53-63.

34. QureshiAI,WilsonDH,HanleyDF,TraysmanRJ.Noevidenceforanisc hemicinmassiveexperimentalintracerebralhemorrhage.Neu-rology. 1999; 52: 266-72.

35. GautamJ,AlarefA,HassanA,KandelRS,MishraR,JahanN.Safetyandefficacyofstemcelltherapyinpatientswithischemicstroke.Cureus. 2020; 12: e9917. Volume10Issue13-2023 CaseReport http://www.acmcasereports.com/ 11

36. WisemanS,MarlboroughF,DoubalF,WebbDJ,WardlawJ.Bloodm arkers of coagulation, fibrinolysis, endothelial dysfunction andinflammation in lacunar stroke versus non-lacnar stroke and non-stroke: systemic review and meta-analysis. Cerebrovasc Dis. 2014;37: 64-75.

37. Altendahl M, Maillard P, Harvey D, Cotter D, Walters S, Wolf A,et al. An IL-18-centered inflammatory network as a biomarker forcerebral white matter injury. PLOS ONE. 2020; doi.org/10.1371.

38. Denes A, Pinteaux E, Rothwell NJ, Allan SM. Interleukin-1 andstroke: biomarker, harbinger of damage, and therapeutic target.Cerebrovasc Dis. 2011; 32: 517-27.

39. GlushakovaOY,GlushakovAA,WijesingheDS,ValadkaAB,Hayes RL,GlushakovAV.Prospectiveclinicalbiomarkersofcaspase-mediated apoptosis associated with neuronal and neurovascular damagefollowingstrokeandotherseverebraininjuries:implicationsforc hronic neurodegeneration. Brain Circ. 2017; 3: 87-108.

40. Gutierrez-Fernandez,M, Rodrigues-Frutos B, Ramos-Cejudo J,Vallejo-Cremades MT, Fuentes B, Cerdán S, et al. Effects of in-travenous administration of allogenic bone marrow-and adiposetissue-derived mesenchymal stem cells on functional recovery andbrainrepairmarkersinexperimentalischemicstroke.StemCellR esTher. 2013; 4: 11

IchihashiM. EffectofanIntravenousAdministrationofAdipose-Tissue-DerivedAutologousStem CellsontheSkinTemperatureofParalyzedLimbsofStrokePatients. Annals of Clinical and Medical Case Reports 2023