The Study On Infusion Of Placenta-derived Mesenchymal Stem Cells On Hypoxic-ischemic Brain Damage In Rats

BackgroundHypoxic-ischemic brain injury (hypoxic-ischemic brain damage, HIBD) is a kind of newborn brain damage caused by perinatal asphyxia and hypoxic ischemic during perinatal period. Perinatal asphyxia can cause3-5/1000severe hypoxic-ischemic encephalopathy in live births, and leading to neonatal death and legacy of some neurological disorders such as cerebral palsy, epilepsy and mental retardation. Among them, cerebral palsy is the most important motor function in childhood disability disease in children which can cause lifelong disability. The high incidence and morbidity of HIBD have caused a great spiritual and economic burden to the children himself, families and society.The pathogenesis of HIBD is a very complex pathological process caused by a series of chain reactions involved in a variety of mechanisms integrated, and the exact pathogenesis is not very clear. At present, the common method used in treatment of neonatal HIBD is hyperbaric oxygen therapy, nerve cell nutrition drugs, physical rehabilitation et al. These methods have some effect in minor damaged children. But for the central nervous system dysfunction caused by severe encephalopathy, these methods are ineffective. So it has a very important clinical significance to find an effective method to reduce the mortality and disability rate of HIBD children. In recent years, studies have shown that mesenchymal stem cells (MSCs) therapy have got a great progress in treatment of HIBD animal model and preclinical trials. Bone marrow (BM) is the most common source of MSCs. But the proportion of MSCs in marrow is scarce. The proliferation and differentiation potential is also weak in BM-MSCs. The invasive procedures and increased probability of viral infection limited the further clinical application of BM-MSCs. The present study demonstrated that MSCs can be isolated from the mice. Placenta-derived mesenchymal stem cells (PD-MSCs) were consistent with international standards of MSCs and may become an ideal seed cells in allogeneic cell therapy.The pathogenesis of HIBD involves inflammation, oxidative stress injury, brain energy metabolism disorder and so on. Inflammatory cells can penetrate into the brain parenchyma and release a variety of neurotoxic substances to cause inflammatory cascade reaction. A variety of cytokines and helper T cells17(Th17) and regulatory T cells (Treg) have played a crucial role in the occurrence and development of HIBD. In addition, oxidative stress injury is also the key pathological process in hypoxic-ischemic induced brain injury. The keapl-Nrf2/IS signaling pathway is one of the important chemical pathway in the body’s defense mechanism to against oxidative stress injury. Nrf2regulates the expression of HO-1enzyme, which located downstream of heme oxygenase, through anti-oxidation, anti-apoptotic and anti-inflammatory properties.Mechanism of cell transplantation in the treatment of brain injury is considered to be the result of alternative nerve cells before. But now we consider it is a multifaceted joint comprehensive repair mechanism. Not only the transplanted cells can directly replace damaged cells, but also through the promotion of endogenous neural stem cells repair, secretion of trophic factors, and the improvements of local microcirculation. Thus this study explores the mechanism and pathogenesis of PD-MSCs based HIBD therapy from both of anti-inflammatory and anti-oxidative stress response mechanism.ObjectivesPart I:To assess the effect of PD-MSCs based rat HIBD model therapy through the growth and development of rats, neurological behavior, and pathological changes. Part II:To discuss the molecular mechanism of PD-MSCs based HIBD treatment from three aspects:the inhibition of the inflammatory response, immune regulation, and anti-oxidative stress.Methods1PD-MSCs were isolated and cultured from the placenta of pregnant rats. Flow cytometry was used in cell phenotype identifications. Induced differentiation was also used to prove the multi-differentiation ability of these cells. GFP-transfected PD-MSCs were used to locating the distribution of MSCs infused.2Healthy wistar rats with7-day-old (postnatal day7, P7) were select to produce neonatal HIBD rats in accordance with the Modified Rice method. Behavioral changes observed using Bederson score functional rating scale. The standard is0:no symptoms of nerve injury;1point:catch big mouse tails raised, rats cannot fully extend the contralateral paw;2points:Large mouse forelimb decline of resistance to the contralateral thrust;3points:the emergence of the phenomenon to thecontralateral circling in rats. After48h, HIBD rats were randomly divided into four groups:control group (Control), HIBD group (HIBD), PD-MSCs treated group (HIBD+PD-MSCs) and fibroblasts treated group (HIBD+Fibroblasts). PD-MSCs or fibroblasts were injected in to brain using stereotactic guidance.3After cell therapy, skin appearance, body weight and any abnormal growth and behavior performance were observed in every experimental rat.4Hanging wire test and Vertical pole test evaluation exercise behavior of rats; and Morris water maze test was used to evaluate the cognitive function of rats. Hanging wire test and Vertical pole test were completed in P10, P16, P22and P28. Morris water maze test was completed at P24-P28.5. The proportion of CD4+CD25+T lymphocytes was analyzed5days after cell therapy.6The expression of TNF-a, IFN-γ, IL-10, IL-17mRNA were detected at3h,6h,24h,3d and5d after hypoxic-ischemic injury and3d after cell treatment in rats.7The expression of HO-1and Nrf2mRNA were detected at6h,24h,48h,72h, and5d after hypoxic-ischemic injury and3d after cell treatment in rats.8Five days after cell therapy, western blot were used to evaluate the expression of Foxp3in spleen tissues and HO-1, Nrf2in hippocampus.9Three days after cell therapy, TNF-a, IFN-γ, IL-10, and IL-17’s levels in peripheral serum were detected using ELISA.10. MDA content was detected6h,24h,48h, and72h after hypoxic-ischemic injury and3d after cell treatment in rats.11. HE staining and Nissl staining were performed after P28movement experiment.Results1MSCs can be isolated and cultured from the placenta of pregnant rats. The results of flow cytometry and induced differentiation proved that our cells accords with the international standards of mesenchymal stem cells. 2Newborn rats appeared irritability and cyanosis at the beginning of hypoxia, and then gradually turned suppression, and even convulsions. Compared with the control group, HIBD group lost weight significantly. HIBD rats showed significant motor function backward in Hanging wire test and Vertical pole test. According morris water maze navigation test, the escape latency was significantly longer in HIBD group. At the sixth days of space exploration experiments, HIBD group significantly reduced the frequency of the original platform. Brain swelling and pale was observed in early period, while atrophy, infarction, liquefaction were proved in late period. Injured side cortex, hippocampal neurons, cerebellar Purkinje cell disorder, degeneration and necrosis were confirmed using H&E staining. Nissl bodies subsided, eosinophilic variant of neurons and glial cells macrophages phenomenon were also observed. From growth and development, motor function assessment, pathological changes, and many others, we think the HIBD model is successful.3. PD-MSCs migrated in brain:Six hours after transplantation, GFP-positive PD-MSCs were found mainly in the injection site. Six days after transplantation, green fluorescent signal was significantly weakened. GFP-positive cells were found in the ischemic side vaccinated, while the contralateral cortex was found no GFP-positive cells. PD-MSCs mainly focus in the needle tract, and/or injection site. Then a large number of cells moved out of the injection site, spread throughout the cortex and periventricular region.4Flow cytometry results showed, the CD4+CD25+T cells were11.4%in HIBD group, slightly more than the normal control group (8.69%). After cell therapy, CD4+CD25+T cells were15.1%in fibroblasts treated group, while CD4+CD25+T cells reaching18.0%in PD-MSCs treatment group. The Foxp3protein of spleen was higher in HIBD group than the normal control group, but without significant difference. After PD-MSCs treatment, the expression Foxp3protein in rat spleen increased significantly compared to HIBD group with statistical significance.5After hypoxic-ischemic injury, the expression of IFN-y, TNF-a, IL-17mRNA in brain tissue dynamically increased in HIBD group. The TNF-a mRNA expression increased3h after injure, and reached the peak at24h. The increase of IFN-y and IL-17mRNA were much later than the expression of TNF-amRNA, both reach the peak at3d after HI injury. The expression of IL-10mRNA began to increase at6h, reached the peak after the3d.6. Compared to HIBD and fibroblasts group, the expression of IFN-y, TNF-a, and IL-17mRNA were significantly lower in PD-MSCs group. The expression of IL-10mRNA was significantly up-regulated in PD-MSCs group, HIBD group, and fibroblast treatment group, especially in PD-MSCs treatment group. In ELISA experiments, HIBD serum IFN-y, TNF-a, IL-17and IL-10levels were higher than the normal control group. The serum IFN-y, TNF-a, and IL-17levels were down-regulated, while the IL-10level was increased significantly in PD-MSCs treatment group.7. In HIBD group, the expression of HO-1and Nrf2began to increase at6h and peaked at48h HIBD. Then the expression of HO-1and Nrf2decresed slightly, but still remained at a high level in72h and5d. In PD-MSCs group, the expression of HO-1and Nrf2mRNA were higher than normal controls group after treatment. HO-1and Nrf2expression in PD-MSCs group were higher than fibroblast treatment group and HIBD group. Western blotting results confirmed these results. 8. Six hours after HI, MDA levels were significantly higher in brain tissue and reached the peak level at72h, followed by MDA levels decrease. Five days after HI, MDA level was still higher than the normal level. MDA levels were significantly reduced in PD-MSCs treated group compared to hypoxic-ischemic group and fibroblasts group.Conclusions1From growth and development, motor function assessment, pathological changes, and many other data, we successfully established the HIBD model.2Tissue explants adherent method can be successfully used in rat PD-MSCs culture.3. PD-MSCs injection has not only improved neuromotor function of HIBD rats, but also improved long-term learning and memory function of HIBD rats.4. An imbalance among pro-inflammatory cytokines IFN-y, TNF-a, IL-17and anti-inflammatory cytokines IL-10is one of the pathogenesis of HIBD. There also exists dysfunction of CD4+CD25+Treg cells.5. An imbalance between antioxidant (Nrf2/HO-1) and oxidation (MDA) levels is one of the pathogenesis of HIBD.6. Regulation of Th17/Treg cell imbalance may be one effective mechanism in PD-MSCs based HIBD treatment.7Up-regulation of Keapl-Nrf2/ARE/HO-1pathway may be one effective mechanism in PD-MSCs based HIBD treatment.