A Study On Cotransplantation Of Bone Mesenchymal Stem Cells And Salvia Mihiorrhiza Into The Neonatal Rats With Hypoxic-ischemic Encephalopathy

Objective: To explore the migration and differentiation of bone mesenchymal stem cells(MSCs) implanted in the brain of neonatal rat with hypoxic-ischemic encephalopathy(HIE). To observe the result of Salvia Miltiorrhiza injection inducing mesenchymal stem cells(MSCs)to differentiate into neuron-like cells in vitro and the influence on neural differentiation ratio of MSCs implanted in the brain of neonatal rat with hypoxic-ischemic encephalopathy(HIE).Methods: 1. MSCs were isolated and purified by adhering to the culture glassware wall and prelabeled with BrdU for 72 hours before transplantation. 2. 7-day-old SD neonatal rats(n=36)were divided randomly into normal control group(n=8),HIE group (n=8),cellular transplant of MSCs group(n=10),cotransplantation of MSCs and salvia miltiorrhiza group(n=10). Animal models of HIE were established in neonatal rats of HIE group,cellular transplant of MSCs group,cotransplantation of MSCs and salvia miltiorrhiza group as described by Rice. At 48hours,14days,21 days,28days after the operation, taking 2 brains of neonatal rats in HIE group respectively to identify the result,meanwhile treating the rats in normal control group with the same method as normal control. 3.72 hours after hypoxia-ischemia, approximate 10μl(1x10~6) MSCs suspension were injected into the brain of each neonatal rat of cellular transplant of MSCs group and cotransplantation of MSCs and salvia miltiorrhiza group through the left side bregma,and 2～3hours after injection the neonatal rats in cotransplantation of MSCs and salvia miltiorrhiza group were injected intraperitoneally with Salvia Miltiorrhiza injection(1ml/kg.day)for 2 weeks . 4.18 days after cellular transplant, all rats of both groups were sacrificed and their cerebra were made in paraffin sections. Immunohistochemical staining with chromogen diaminobenzidine(DAB) was used to detect and count the cells derived from MSCs, to explore the distribution and migration of bone mesenchymal stem cells(MSCs) implanted in the brain of neonatal rat with hypoxic-ischemic encephalopathy(HIE);To determine the neural differentiation ratio of the BrdU positive cells entering the brains , Immunofluorescence double labeling for BrdU and neural cells specific antigens(Nestin,NSE,GFAP) was performed,then compare the ratios between two groups.5. The MSCs were induced to neural differentiation by free-FBS culture medium contained Salvia Miltiorrhiza (200g/L) for 8 hours,then the expression of Nestin,NSE,GFAP were detected by immunohistochemical staining with chromogen diaminobenzidine(DAB).Results: 1. After the operation of hypoxia-ischemia, all animals appeared neurobehavior abnormal with varying degree,HE staining showed cerebral edema,degeneration,and necrosis of the neurons in the left cerebral hemisphere were obvious. 2. MSCs were distributed throughout the cerebra in both groups 18days after transplantation. More BrdU positive cells were observed in the left cerebral cortex, and some focus aggregations of MSCs were found. To compare with cellular transplant of MSCs group, MSCs distributed more extensively, and more MSCs migrated to the right cerebral hemisphere in cotransplantation of MSCs and salvia miltiorrhiza group. There was a significant difference(P<0.05)of the MSCs count between two groups in each different brain section and both cerebral hemisphere. 3.Majority of MSCs located at the cerebral cortex, hippocampus of left cerebral hemisphere should differentiated into neuronal-like cells, expressed Nestin,NSE and GEAP. There was a significant difference(P < 0.05)in the neural differentiation ratio(Nestin,NSE)of MSCs between two groups,but no difference in GFAP..4. The MSCs were induced to neural differentiation by free-FBS culture medium contained Salvia Miltiorrhiza (200g/L) for 8 hours,thcn the expression of Nestin,NSE,GFAP were detected by immunohistochemical staining with chromogen diaminobenzidinc(DAB).The MSCs were induced by free-FBS culture medium contained Salvia Miltiorrhiza (200g/L) in vitro, 2 hours later MSCs began to differentiate into neural-like cells. Neuron -like cells which expressed Nestin,NSE were detected by immunohistochemical staining ,and they didn't express GFAP.Conclusion: 1 .During this experiment,we succeeded in establishing hypoxic -ischemic encephalopathy model of neonatal rat according to Rice method. 2.18 days after cellular transplant, MSCs could survive and migration throughout the cerebra in both groups,and distribute more extensively by using Salvia Miltiorrhiza at the same time. 3. Majority of MSCs located at the cerebral cortex, hippocampus of left cerebral hemisphere should differentiated into neuron -like cells, expressed Nestin,NSE and GFAP. Using Salvia Miltiorrhiza in addition should promote MSCs to express Nestin,NSE, but have no significant influence on GFAP. .4. MSCs could be induce to differentiate into neuron-like cells with Salvia Miltiorrhiza in vitro ,expressNestin,NSE.