Therapeutic Study Effect Of Co-graft Of Schwann Cells With Neural Stem Cells On Animal Model With Parkinson's Disease

PD is one of extrapyramidal diseases , characterized by selective degeneration of dopaminergic neurons in substantia nigra projecting to neostriatum. The capacity of dopamine from substantia nigra to striatum is decreased and symptoms are induced including paralysis agitans, myotonia, dyskinesia and so on. Intrastriatal grafting of dopamine-secreting cells from neural and paraneural tissues, such as fetal mesencephalon or adrenal medulla, has been reported to result in amelioration of the motor syndrome in Parkinson' s patients and in animal models of PD. By now there have been more than 300 cases of PD patients with NSCs graft. However the curative effect is not sure. Although transplantation is considered a promising treatment for PD, its clinical use is still restricted to few cases. The major limiting factors of this therapy are difficulty in obtaining sufficient viable embryonic mesencephalic tissue and the controversial ethical and/or legal issues raised by the use of human fetal allografts. In the non-control clinical trials there is a certain curative effect in most PD patiets with neural stem cells transplantation. Autoplasical data have showed the surviving of dopaminergic neurons in graft. But there is prominent individual variance. Recently two group clinical double-blind control trials have demonstrated that there is distinct difference between groups of transplantation therapy in different years and severe degree of diseases. So the most ideal transplantation method is : the grafts can keep their viability in vivo for long time to produce dopamine and can secrete all kinds of trophic proteins to improve the circumstances in graft and prevent progressive deterioration of PD.PART One : The effect of Schwann cells on the developing striaturns in co-culture.Objective To explore the effect of Schwann cells(SCs) on the striaturnsgrowth in vitro.Methods SCs co-culture with striaturns coming from embryonic, new and grow-up SD rats in vitro. The neurites around the striaturns and cells move from the striaturns will be observed at 3d, 1, 2, 3, 4 week.Results In co-culture group, the neurites around the the striaturns of embryonic, new, grow-up rats emergenced at the 3d, 1, 2, week respectively. The cells moving from the striatums of embryonic, new, grow-up rats emergenced at the 1, 2, 3, week respectively. the cells moving from the striaturns of the grow-up rats were small, but it is still obvious compared to the control group(P<0.05).Conclusion SCs have the ability to promote the neurites growth of striaturns and cells moving from striaturns, its can be used for cell transplantation therapy to neurodegenerative diseases.PART Two : Magnetic resonance imaging of SPIO labeled neural stem cells in Parkinson rat model following transplantation.Objective: To investigate the changes in magnetic resonance (MR) imaging following the transplantation of superparamagnetic iron oxide (SPIO) labeled neural stem cells in the treatment of Parkinson disease.Methods: The mesencephalic NSCs were obtained from the brain of SD rats at embryonic day 14-16. Superparamagnetic iron oxide (SPIO) was transferred to NSCs by Lipofectamine transfection. SD rats received unilateral stereotaxic injections of 6-OHDA. 18 PD lesioned rats were selected for transplantation by estimation of rotational behavior in response to amphetamine and randomly assigned to three groups, i.e. sham group, PBS group and NSCs transplanted group, with 6 rats in each group. MR scanning was performed at 1, 2, 4, 6, 8 and 10 weeks following transplantation. At the meantime, rotational behavior was assessed in each group.Results: SPIO particles were clearly visible with Prissian blue staining in neurospheres and cells derived from NSCs. The rotation behavior of theNSCs transplanted group was remarkably improved compared with that of sham group and PBS group (p<0.05). In vivo MR tracking of NSCs showed that SPIO labeling led to a strong susceptibility change of signal 1 week after transplantation on T2 weighted images. And a large circular hypointense signal appeared in the transplanted area on T2* gradient echo images. At 10 weeks following transplantation, the hypointense signal on T2 weighted and T2* gradient echo images was still displayed.Conclusions: SPIO particles could label NSCs effectively, and MRI detection of SPIO labeled cells is a promising method and novel approach to analyzing the NSCs following transplantation in the treatment of PD.PART Three: Effect of co-graft of Schwann cells with neural stem cells on rats with Parkinson disease.Objective To explore the effect of Schwann cells and neural stem cells co-graft on PD rats.Methods 18 hemiparkinsonian rats with right nigra lesioned were selected for transplantation by estimation of rotational behavior in response to apomorphine and randomly assigned to three groups: control group, NSCs group and co-graft group, with 6 rats in each group. PBS, NSCs, SCs plus NSCs were transplanted into the unilateral 6-OHDA lesioned neostriatum of the PD rat respectively. The mesencephalic NSCs were obtained from the brain of SD rats at embryonic day 13-16 and SCs came from the sciatic nerves of neonatal rats at day 1-3.Results TH immunoreactive neurons emerged in the striatum of both NSCs group and co-graft group 10 weeks after transplantation. The volumes and nucleus of neurons in co-graft group were bigger than the ones of NSCs group and so do the number of the DA neurons (P<0.05). Compared with control group, the percent of apomorphine-induced rotation in both NSCs group and co-graft group was decreased significantly (P<0.01) , but no difference between them(P>0. 05) .Conclusion Schwann cells and neural stem cells co-graft is effective in treating PD rats. It was suggested that not only dopaminergic neurons differentiated from NSCs functioned in the unilateral 6-OHDA lesioned neostriatum. but also SCs improved the survival of NSCs by secreting many neurotrophic factors. Our results encourage the use of co-graft in cell-replacement therapy for Parkinson's disease. PART Four: Effect of co-graft of Schwann cells with neural stem cells on rhesus monkey with Parkinson's diseaseObjective To further explore effect of co-graft of Schwann cells with neural stem cells on rhesus monkey with Parkinson's disease.Methods 3 PD rhesus monkeys were assigned to control monkey, NSCs monkey and co-graft monkey according to their grafts. PBS, NSCs, SCs plus NSCs were transplanted into the right striatum of the PD rhesus monkeys respectively. The mesencephalic NSCs were obtained from the brain of human embryo at embryonic week 6-8, SCs come from the sural nerve of PD rhesus monkey itself. The effect of cell transplantation was estimated by monkey's behavior, PET and immunohistochemnistry staining of brain tissue.Results Monkey behavior study: 1 month after graft, there were significantly improvements in co-graft monkey, while there were no changes in other 2 monkeys; 2 month after graft, there were significantly improvements in co-graft monkey and NSCs monkey, while there were no changes in control monkey; 4 month after graft, monkey behaviors were recovered to normal, while there were no changes in control monkey. ¹⁸F-FP-β-CIT PET study: 2 month after graft, DAT binding uptake could be observed in bilateral striaturn of co-graft monkey, while only in the left normal side of striaturn of 2 other monkeys. 4 month after graft, DAT binding uptake could be observed in bilateral striaturn of co-graft monkey and NSCs monkey, while only in the left normal side ofstriaturn of the control monkey. Immunohistochemical staining of brain tissue: there was no tyrosine hydroxylase positive cells in control monkey while many positive ones in the NSCs monkey and the co-graft monkey. The tyrosine hydroxylase positive neurons in the co-graft monkey took on an appearance of plump body and multiple processes, there was more cells contrast to the NSCs monkey.Conclusion This part of study further proved Schwann cells and neural stem cells co-graft is effective in treatment of PD rats. The Schwann cells can be exenterated easily from the peripheral nerves tissues and offer potential clinical advantages without transplant rejection due to autograft and ethical and/or legal controversies. Our results encourage the use of co-graft of NSCs and SCs in cell-replacement therapy for Parkinson's disease.