Treatment With Bone Marrow Stromal Cells And Reduced Glutathione In A Rat Parkinson' Disease Model

Idiopathic Parkinson's disease (PD) is a progressive neurodegenerative disorder which is primarily characterized by degeneration of the dopaminergic neurons of the nigrostriatal pathway. Although levodopa is still considered as the gold standard of antiparkinsonian drug therapy, chronic levodopa treatment is associated with the development of adverse events in the majority of patients, such as motor fluctuations, dyskinesias, and neuropsychiatric problems. Nowadays, most of the PD research has been focused on cell transplantation, which could be a potential therapeutics. Bone Marrow Stromal Cells (BMSCs) are stem cells with the characteristics of self renewing, multi-potency and easily expanding in vitro. Therefore, BMSCs are ideal target cells for cell transplantation. Under specific conditions, BMSCs are capable to differentiate into other cell types, such as neural stem cells, neural cells and glial cells. They also are able to secret brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and etc. A study has shown that there was a short-term (35 days) functional recovery with BMSC transplantation in a PD animal model. However, the long-term functional recovery has not been studied. As we know, it has not been confirmed if BMSCs are able to differentiate into neural stem cells, neural cells and glial cells and dopamine cells when they are transplanted into caudate putamen of PD rats. In PD, the underlying mechanisms of dopamine cells death in the zona compacta of substantia nigra remain unclear. The possible pathogenesis includes oxidative stress, excitotoxicity, and immunogenicity mitochondrial dysfunction. The consistent findings of decreased levels of the major antioxidant glutathione in substantia nigra in PD patients has provided most of the basis for the oxidative stress hypothesis of the etiology of PD. Several studies have demonstrated a deficiency in reduced glutathione (GSH) in the nigra of patients with PD. In particular, the magnitude of reduction in GSH seems to be paralleled to the severity of the disease. In untreated early-staged PD patients, treatment with GSH has shown some efficacy and the progression of the disease is delayed. Recent studies proved that BMSCs can differentiate into neural stem cells, neurons and glial cells induced by some antioxidants. GSH is an antioxidant, but so far there is no report stating that BMSCs is able to differentiate into neurons induced by it. Sufficient number of BMSCs will secrete GDNF and also differentiate into neurons. GSH not only has the potential the treatment of PD, but also can induce BMSCs to differentiate into neurons. So treatment with both BMSCs and GSH in Parkinson's Disease has incredible potential. Our research was focused on: 1) observing the in vitro culture condition of Bone Marrow Stromal Cells (BMSCs) from SD rats and transplanting BMSCs to treat Parkinson`s disease in SD rats. 2) Investigating the differentiation of rat bone marrow stromal cells (rBMSCs) into neuron-like cells by L-DMEM/ reduced glutathione. 3) Establishing 6-HODA-PD model with simple method. 4) The effect and possible mechanisms of treatment with both BMSC transplantation and reduced glutathione in 6-HODA-PD Rats. Part I: In Vitro Culture of BMSCs from SD Rats Objective To develop the in-vitro culture condition of BMSCs from SD rats differentiate into neural stem cells. Methods SD rats (weight 60~90g) were sacrificed. BMSCs were isolated from their bone marrows. Cultured BMSCs were observed daily under phase-contrast microscope and the cell purity were further confirmed by FACS analysis. Results 92.9% of the passage 3 cells were CD90 (one of the BMSCs'surface markers) positive. On the other hand, CD45 + cells were dropped from 73.4% in primary culture to 2.3% in the passage 3. Conclusion The passage 3 cells could be used to cell induce and differentiation and cell transplantation because of its high purity of BMSCs.Part II: The Differentiation of Rat BMSCs into Neural Cell by the Reduced Glutathione. Objective To investigate the differentiation of rat BMSCs into neuron-like cells. Methods BMSCs were isolated from bone marrow of femur, shin bone and humerus. Primary cells were expanded in L-DMEM culture medium supplemented with 10% FCS. The differenctiation of BMSCs would be detected after 24 hours treatment with reduced glutathione in L-DMEM medium. As a control, the BMSCs were cultured in L-DMEM only. Neuron-specific enclose (NSE) was determined by immunocytochemistry method. Results At 6 h and 24h treatment of reduced glutathione , BMSCs showed neuronal morphologies and formed extensive networks under microcope. NSE positive cells were 64%±4.3%, 71%±5.0% respectively. Conclusion The reduced glutathione may induce BMSCs to differentiate into neuron –like cells expressing NSE in vitro. Part III Study on Comparing Different Means of Injection 6-OHDA into Medial Fore Brain Bundles and Establish the Rat Model with Parkinson's Disease Objective To establish a simple and effective rat model in Parkinson`s disease. Methods In two groups, 6-OHDA was injected into two points of medial forebrain bundles on the right sides at the same dosage. In first group, two burr holes were made with a dental drill. two injection points are –1.8mm antero-posterio to and –2mm lateral to the bregma, -8mm ventral to the dura mater and –1.8mm antero-posterio to and –3mm lateral to the bregma, -7.5mm ventral to the dura mater at stereotactic coordinates. In second group, one burr hole was made, two injection points are –1.8mm antero-posterio toand –2.5mm lateral to the bregma, -8mm ventral to the dura mater and –1.8mm antero-posterio to and –2.5mm lateral to the bregma, -7.5mm ventral to the dura mater. After 6 weeks, apomorphine-induced rotation test was performed to examine the disease progress in this rat model. Results In group 51% of them were proved to be successful after by apomorphine induction. In group 2, the successful ratio is 77%. There was significant difference (P<0.05) between the two groups. Conclusions This rat model in PD can be created in high successful ratio by two simple injections with 6-OHDA Part IV Treatment with BMSCs and Reduced Glutathione in the Established Rat Model in PD Objective To examine the effect of treatment with BMSCs and reduced glutathione in Parkinson's disease rat model. Methods Fifty one PD rats were used and randomly divided into two groups: 6-OHDA total lesion model group and partial lesion model group. Total lesion model group were further divided into five sub-groups: (1) BMSCs sub-group; (2) GSH sub-group; (3) BMSCs and GSH sub-group; (4) sham treatment sub-group; (5) untreated sub-group. Partial lesion model group included: (1) GSH sub-group; (2) untreated sub-group. Functional outcome measurements were performed using the apomorphine induced rotation test at 2,4,6 and 8 weeks after treatment. Caudate putamen tissue was prepared for immunofluorescent staining to detect whether the BrdU-labeled BMSCs or DAPI-labeled BMSCs expressed the neural stem cells marker-nestine,the astrocytic marker-GFAP(glial fibriliary acidic protein) ,the neuron marker-NSE or the dopaminergic neuron marker-TH. Results A decrease of per minute numbers of apomorphine induced rotation test was noted in rats of BMSCs sub-group and BMSCs and GSH sub-group at every time point. The decrease was more significant in BMSCs and GSH sub-group than in BMSCs sub-group. GSH can reduce the per minute numbers ofapomorphine induced rotation test in rats of partial lesion model group, but can not in rats of total lesion model group. Some of the BrdU-labeled BMSCs or DAPI-labeled BMSCs were reactive for nestine, GFAP, NSE or TH. Conclusions Treatment with BMSCs and reduced glutathione may improve neurological outcome in 6-OHDA total lesion PD rat model. The effectiveness is better in BMSCs and GSH sub-group than in BMSCs sub-group. GSH may improve neurological outcome of 6-OHDA partial lesion model rats.After tranplantatation,some of the BMSCss expressed nestine,GFAP,NSE or TH at caudat putmen. Overall Conclusions: 1. The passage 3 BMSCs could be used as transplantation cells due to its high purity. 2. The reduced glutathione may induce BMSCs into neuron-like cells expressing NSE in vitro. 3. Rat models of PD can be established successfully by two easy injection with 6-OHDA into rat medial forebrain bundles which are –1.8mm antero-posterio to and –2.5mm lateral to the bregma, -8mm ventral to the dura mater and –1.8mm antero-posterio to and –2.5mm lateral to the bregma, -7.5mm ventral to the dura mater. 4. Treatment with both BMSCs and reduced glutathione may improve neurological outcome of 6-OHDA total lesion induced PD model in rats. The effectiveness is better in BMSCs and GSH sub-group than in BMSCs sub-group. GSH may improve neurological outcome of 6-OHDA partial lesion model rats.