VEGF189-Secreting Human Adipose-derived Mesenchymal Stem Cell Modulates Pain Hypersensitivity By Negative Control Of TRPV1 In A Mice Model Of Parkinson's Disease

Part 1 Establishment of PD mouse model and assessment of pain behaviorObjective: To develop a unilateral Parkinson's disease(PD)mouse model by microinjecting 6-hydroxydopamine(6-OHDA)into the left striatum.Then assess the pain behaviors in such semi PD mouse model by thermal hyperalgesia,mechanical hyperalgesia,and spontaneous pain test.Methods: Twenty C57BL/6 mice were randomly divided into PD group and sham group(10 per group).Experimental parkinsonism was achieved through unilateral injection of 6-OHDA into left striatum or sham was achieved through unilateral injection of equivalent volume saline into left striatum.Apomorphine(APO)was used to monitor ipsilateral rotations in mice.The ipsilateral rotations were counted for 1 h at 1 and 2 weeks post 6-OHDA.Paw withdrawal latency(PWL),Von Frey test,and Conditioned place preference(CPP)were conducted at 2 weeks after 6-OHDA.Western blot analysis and immunostaining was performed with anti-tyrosine hydroxylase(TH)to estimate the damage degree of the nigra-striatum system.Results:1.Compared to sham mice,6-OHDA-lesioned mice exhibited ipsilateral rotation after APO at 1 week(622.60±21.94)and 2 week(657.40±24.03)post 6-OHDA injection.The protein level of TH was also reduced in the left STR,compared with that in the right STR.In conclusion,we demonstrated that unilateral injection of 6-OHDA induce semi PD mice model.2.With PWL testing,6-OHDA-lesioned mice exhibited impaired right hind paw thermal hyperalgesia,compared with sham animals(6-OHDA right vs.sham right: 4.912±0.409 vs.12.425±0.584;P<0.005).However,there was no significant difference on PWL in the left hind paw between 6-OHDA-lesioned mice and sham animals.Along with changes in thermal sensitivity,6-OHDA-lesioned animals exhibited significant decreases in EF50 s of both right hind paw(6-OHDA right vs.sham right: 0.785±0.071 vs.1.843±0.123;P<0.005)and right orofacial area(6-OHDA right vs.sham right: 0.642±0.046 vs.1.546±0.107;P<0.005),which contrast with the sham group.Moreover,the EF50 in left hind paw and left orofacial area was lower in 6-OHDA-lesioned mice than that in sham mice.However,6-OHDA induced semi PD mice model did not induce spontaneous pain by CPP test.Conclusion: Unilateral injection of 6-OHDA into left striatum can induce semi PD mice model.Such semi PD mice model display thermal and mechanical pain hypersensitivity.Part 2 Sensation of TRPV1 by 5-HT/5-HT3A in pain in 6-OHDA-lesioned miceObjective: To study the mechanism of TRPV1 involved in pain in 6-OHDA-lesioned mice,and the relationship with 5-HT dependent descending facilitation from rostral ventromedial medulla(RVM).Methods: A semi PD mice model was conducted by microinjection of 6-OHDA into left striatum.APO induced rotation was performed at 1 week after surgery,and Von Frey test on oral area was performed at 2 weeks after surgery,after which,20 semi PD mice were selected.To determine the changes of descending 5-HT positive fibers in the Vc in 6-OHDA-lesioned mice,we examined the density of 5-HT positive fibers in the Vc via immunostaining.Intra-RVM gene transfer of a plasmid encoding a sh RNA targeting neuronal Tph-2,a key enzyme in the synthesis of 5-HT,was performed to verify whether 5-HT dependent descending facilitation from RVM is involved in PD related pain.The density of 5-HT3AR and TRPV1 in Vc in 6-OHDA-lesioned mice was also examined.The Von Frey test was performed before and after 5-HT3AR agonist(SR57227),5-HT3AR antagonist(Y25130),and TRPV1 antagonist(AMG9810)to evaluate the effects of 5-HT3A and TRPV1 involved in PD related pain.Results: 1.There was a dramatic increase in 5-HT immunoreactivity in the right Vc compared with the left Vc in 6-OHDA-lesioned mice(p<0.05).After Tph2-sh RNA gene transfer in RVM,a significant decrease of 5-HT immunoreactivity in the Vc was detected.The mechanical hypersensitivity after local gene transfer was significantly reversed.Compared to sh Ctrl mice,PD mice received Tph-2 exhibited significant increased EF50s(p<0.05).In conclusion,5-HT dependent descending facilitation plays an important role in 6-OHDA induced semi PD mice model.2.Immunostaining test showed that the density of 5-HT3AR in right Vc was increased compared with that in left Vc.Microinjection of Y25130 in Vc can alleviated the hyperalgesia in PD mice model.Interestingly,the sham animals received SR57227 exhibited significant decreases in EF50 s compared with sham animals received vehicle.However,SR57227 induced hypersensitivity can be blocked by Y25130.3.Immunostaining and Western blot test showed that the density of TRPV1 in right Vc was increased compared with that in left Vc.Microinjection of AMG9810 in Vc can alleviated the hyperalgesia in PD mice model.Moreover,AMG9810 can alleviate hypersensitivity induced by SR57227.In conclusion,TRPV1 plays a vital role in pain modulation in PD.Conclusion: Sensation of TRPV1 potentiated by 5-HT/5-HT3AR is essential for inducing hyperalgesia in 6-OHDA-lesioned mice?Part 3 VEGF-Secreting Human Adipose-derived mesenchymal stem cell modulates pain hypersensitivity in 6-OHDA-lesioned miceObjective: To conduct a VEGF-Secreting Human Adipose-derived mesenchymal stem cell(hAMSCs)system.And evaluate the safety and efficiency of hAMSCs-VEGF-GFP in PD.Methods: The primary hAMSCs were isolated by collagenase digestion,cultured and passaged in vitro.The lentivirus carrying VEGF189+GFP gene and Vector+GFP gene were transferred into the hAMSCs respectively.Western blot method was used to detect the expression of VEGF189 of hAMSCs-VEGF-GFP and hAMSCs-Vector-GFP.A semi PD mice model was conducted by microinjection of 6-OHDA into left striatum.APO induced rotation was performed at 1 week after surgery,and Von Frey test on oral area was performed at 2 weeks after surgery,after which,20 semi PD mice were selected to receive either hAMSCs-GFP-VEGF or hAMSCs-Vector-GFP.All the animals were imaged using an In vivo imaging system(IVIS)from 1 week to 6 weeks after transplantation.Mechanical hyperalgesia was tested at 1 week and 6 weeks,and the change of TRPV1,TH+ neuron and tumor related factors were evaluated by immunostaining at 6 weeks after transplantation.Results: 1.Immunostaining and Western blot test showed that hAMSCs-VEGF-GFP can secrete VEGF189 while hAMSCs-Vector-GFP cannot.The level of VEGF189 of two cells was significantly different(p<0.05).And VEGF189 can downregulate TRPV1 in Vc in vivo.The GFP positive ratio of hAMSCs-VEGF-GFP was not significantly different from that of hAMSCs-Vector-GFP.2.The survival of transplanted hAMSCs-VEGF-GFP or hAMSCs-Vector-GFP remained at 6 week post-transplantation assessing by IVIS,and the survival rate did not differed between two cells.Immunostaining test showed that there was no tumor related factors expression at 6 weeks after transplantation,which demonstrated that such system is oncologic safety in mice models.3.TRPV1 hyperactivity in Vc was almost completely blocked by hAMSCs-VEGF-GFP,but nor by hAMSCs-Vector-GFP.Consistent with the imaging results,mechanical hyperalgesia in the facial territories after transplantation was significantly attenuated by hAMSCs-VEGF-GFP versus before transplantation compared with hAMSCs-Vector-GFP(p<0.05).Immunostaining test showed that hAMSCs-VEGF-GFP cannot affect the nigral dopamine neurons,which means that hAMSCs-VEGF-GFP modulates mechanical hypersensitivity via direct effect on TRPV1 instead of on dopamine neurons.Conclusion: The hAMSCs infected with LV-VEGF189 could express VEGF189 steadily and efficiently.And the hAMSCs-VEGF-GFP is oncologic safety in mice models.hAMSCs-VEGF-GFP transplanting for 6-OHDA-lesioned mice models can alleviate mechanical hyperalgesia by downregulating TRPV1 instead of DA neuron.