Basic Research On Transplantation Of Neural Stem Cells In The Parkinsonian Rat Model

Parkinson's disease (PD) is a progressive neuro degenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and the consequent loss of projecting nerve fibers in the striatum produces the classical symptoms of resting tremor, bradykinesia, rigidity and postural instability etc. PD is one of the most common neuro degenerative disorders and has significantly the incidence of the upward trend with the extension of average life expectancy and population aging. Conventional pharmacological therapy and deep-brain stimulation (DBS) only temporarily reduce or alleviate the symptoms, but can not stop the process of dopaminergic neurons loss, long-term decline in efficacy, side effects increase. Neural stem cells (NSCs) provide attractive prospects for transplantation in neuro degenerative diseases. NSCs transplantation can not only complement the dopaminergic neurons loss, but also secrete neurotrophic factor to inhibit the loss process, long-term effective in improving symptoms of PD patients.At the first part of the study, we use the ways of our laboratory set up to separate and cultivate the embryonic 14 d (E14) rats NSCs which were obtained from the forebrain subventricular zone (SVZ). The SVZ pieces were incubated in DMEM/F12 culture medium consisting of some neurotrophic factors without serum. The neurospheres were passaged weekly after dissociation and the cells were grown and expanded in flasks for several weeks. They demonstrated typical characteristics of stem cells including clonality, nestin-positivity and the ability to differentiate into any neural cell type in the CNS. The NSCs were labeled by enhanced green fluorescent protein (EGFP) using recombinant adeno-associated virus (rAAV₂) containing the marker gene of EGFP and still have a proliferation capacity after infection by AAV₂-EGFP. After differentiation, the EGFP labeled cells show a better morphological integrity from the cell body to neurite endings with a green fluorescent marker and in vitro the ratio of the cells with EGFP marker was about 65%. From this part, we successfully set up the method of isolation, culture, identification and marking tracer of NSCs.PD without spontaneous tendency in animals, it is required to establish the appropriate animal models to carry out experimental study. A stable animal model PD with analog of human PD pathology and behavioral changes is of great significance to the study of pathogenesis and treatment. PD rat becomes the most widely used experimental research model because of its economic, easy to operate and the easy testing of its behavior change etc. The methods of PD model production are now more sophisticated and commonly made by 6-hydroxydopamine (6-OHDA) administrtion. At the second part of our study, the adult Sprague-Dawley(SD) rats were used to establish PD models by unilateral microinjection of 6-OHDA which damaged dopaminergic neurons in the substantia nigra. Stereotaxic injections of 6-OHDA were performed in the right MFB of 105 adult SD rats. After 4 weeks, 57 rats were selected as successful PD models based on the presence of apomorphine-induced rotational behavior (>7 rpm). The success rate for production of the 6-OHDA-induced PD model was 54%. The effects of the PD models' stability were evaluated during the first, second, and fourth months. There was no improvement in rotational scores in any PD rats at any time point(P >0.05). The extent of the 6-OHDA lesions in the MFB was verified by immunohistochemistry using an anti- tyrosion hydroxylase (TH) antibody. Brain sections from both (lesioned and unlesioned) sides of the SNc were compared simultaneously. The neurons are in larger quantities, larger cell body and obvious neurite in the non-lesioned area. A significant reduction more than 90% in TH-positivity compared with the non-lesioned area (left side) was seen in the lesioned area (right side). This phenomenon was also observed four months later and proved the model's success and stability. The NSCs labeled by EGFP were transplanted into the SNc or striatum of PD rats or normal rats. PD rats grafted with NSCs in the SNc or striatum showed significant reductions in rotational behavior compared with the pre-transplantation period with the grafed cells survival in the host (P <0.05, P<0.01).With NSCs transplantation for the treatment of PD, the transplanted cells, whether or not to have the capacity of migration and anchoring at the injury site specifically or survival and differentiation to dopaminergic neurons in SN, are the key issues to the effect of the treatment of PD. At the third part of this study, we explored the behavior of cells after transplantation. The EGFP labeled cells are found in brain slices at any time point. EGFP tracer as recorded in the cells is long-term expression and exogenous NSCs can survive in host brain for long term. The immunohisto-chemistry results showed that grafted cells can differentiate into neurons, oligodendrocytes and glial cells in brain slices at any time point. There was regularity about the migration of grafted cells. The grafted cells in the SN migrate farther the distance with time lengthens, but the overall are at the SN region migration, only a small number of cells migrated to the region outside the SN. The cells transplanted into the striatum were cord-like arrangement from the transplant site to the ventral posterior medial SN part through a more extensive long-distance migration and there were some scattered grafted cells at SN. These results suggest that transplanted NSCs can survive well in vivo and possess the ability to migrate and differentiate, with a high degree of plasticity. The migration and differentiation patterns of transplanted NSCs may be partially regulated by the injured brain.In summary, we have reached the following conclusions:1. The cultured NSCs origin from SVZ of embryonic 14 d SD rats had the characteristics of proliferation and passage ability. They demonstrated typical characteristics of stem cells including clonality and the ability to differentiate into any neural cell type in the central nervous system (CNS) . The NSCs were successfully labeled by EGFP using rAAV₂ containing the marker gene of EGFP and still had a proliferation capacity after infection. Successfully established the methods of culture and identification of NSCs from SVZ of embryonic SD rats. 2. To establish PD models by unilateral microinjection of 6-OHDA at MFB which damaged dopaminergic neurons in the substantia nigra. The effectiveness of the 6-OHDA lesion was confirmed by apomorphine-induced rotational behavior and verified by immunohistochemistry. The results demonstrated that methods of making PD models were successful. The PD rats grafted with EGFP labeled NSCs in the SNc or striatum showed significant reductions in rotational behavior compared with the pre-transplantation period.3. To observe the differentiation and migration characteristics of grafted NSCs, grafed cells were traced and detected at various time periods after transplantation. The grafted NSCs had the ability to differentiate into any neural cell type in the host brain and there was regularity in migration. The grafted cells in SN migrated farther the distance with time lengthens, but the overall were at the SN region. The grafted cells in striatum migrated through a more extensive long-distance to SN and there were some scattered grafted cells at SN. The results suggested that the lesion microenvironment in the adult brain may be one of the factors which improve the survival, differentiation and migration of grafted cells in CNS.