Experimental Study On Intracerebral Transplantation Of Neural Stem Cells As The Treatment For Parkinson's Disease

Parkinson's disease (PD) is a severe neurological disease characterized by dysfunction of the dopaminergic nigrostriata system. The major neurochemical manifestation of this disorder is the loss of the neurotransmitter dopamine (DA) in the striatm as a result of the progressive degeneration of the dopaminergic neurons in the substantia nigra. The most common therapy is a pharmacological treatment. Although chronic peripheral administration of dopa consistently alleviates the symptoms of the disease in its early stages, its efficacy decreases with clinical progression, and adverse motor complications develop in the long-term.In recent years, many scholars have deeply studied on Neural Stem Cells (NSCs) and have made tremendous progress. NSCs are multipotent cells able to self-renew and generate immature and differentiated cell populations by asymmetric division. They can be used to repair damage of nervous system, Therefore, implantation with NSCs may be an ideal therapy. In this study, it is necessary to establish good rat models of PD and isolate NSCs from the E14 rat ventral mesencephalons for further cultivation, differentiation and identification. It is providing evidence for NSCs transplantation as the treatment for Parkinson's disease.Content and methodsNSCs were isolated from the E14 rat ventral mesencephalons and mechanically dissociated into single cell suspension. Then, cultured the cells in serum-free(DMEM/F12 medium containing B27 and bFGF)and the effection of culture density and passage time on the NSCs growth were investigated; Through the specific color of nestin, we can identify primary and cultured NSCs; To evaluate the potential differentiation ability of NSCs, the cells were cultured in medium containing 5%fetal bovine serum (FBS) and 2%B27 for 7d(37℃,5%CO₂, fully humidified atmosphere), and then the GFAP (special marker of astrocytes)and NSE (special marker of neurons)immunocytochemistry were carried out to determine the character of the spreading cells.6-hydroxy dopamine (6-OHDA) is a neurotoxin that can be competitively damage the dopamine neurons.6-OHDA was injected into Caudate Putamen Uni(tCPU)of adult SD rats(180-200g) to make PD models with stereotactic technique. The following two methods to evaluate the models:â‘ with apomorphine(APO)to induce rotation in rats, rotation≥7r/min are regarded as successful PD models.â‘¡assess the PD models by immunohistochemical method of nigral dopaminergic neurons in morphological observation.ResultsThe average survival rate of the NSCs By mechanical separation was 89.14%. Early separated cells were round, large, and have clear boundaries which have strong refractive index and smooth without protrusions. after inoculation of 3 days, it can form typical neurospheres, large and small; passage after culture 6 days, the number of nerve ball cells reach 100-200 again, Neurospheres formation, time and form similar to the original culture, Nestin positive identification as to prove that the NSCs have the proliferation capacity; NSCs cultured in differentiation medium 36h, around the ball extended a number of neural processes; Cultured 7 days later, stem cell balls adjacent develop to mutual cross-linked cells. There are neurons cells and glial cells from the view of Morphology, identified by immunocytochemistry, GFAP-positive cells were astrocytes, positive for NSE were neurons, which prove that NSCs have the differentiation potential; In addition, NSCs were able to differentiate into TH-positive dopaminergic neurons, which provide the basis for the treatment of PD by NSCs transplantation.To 6-OHDA striatal injection of PD rat model, after two weeks the APO-induced rats appeared obvious rotation, followed by a slow rise up to five weeks and maintain stable state, the success rate reached to 62.1 percent; And rats in the eight periods after induced, the rotational behavior can occur, so it can provide a reliable animal model with the cell transplantation for the treatment of many neurodegenerative diseases. In this experiment, the NSCs can be cultured in vitro after transplantation to the striatum, with behavior detection, we have preliminary proof that NSCs transplantation to the striatum can relieve the symptoms of PD.