| The hypoxic-ischemic encephalopathy(HIE),a serious complication of neonatal asphyxia,is the major cause of severe neurodevelopment disability(cerebral palsy,mental retardation,epilepsy and learning disabilities)in children.Despite advances in technology allowing better obstetric and neonatal care and a deeper understanding of the pathophysiology of perinatal asphyxia,the current clinical management of HIE has still been limited to supportive measures.It is essential to derive new strategies to prevent or interrupt the process underlying brain injury and to promote regeneration of damaged brain tissue.Cell transplantation is effective in experimental and clinical researches of brain ischemia,brain trauma,epilepsy and Parkinson's disease and rekindled the hope of treatment of central nervous system diseases. Many studies of cell transplantation were based on the models of rats with middle cerebral artery occlusion(MCAO).In recent years,a few studies have demonstrated that the intracerebral transplantation of neural stem cells(NSCs)and bone marrow stromal cells(BMSCs)could reduce long-lasting neurologic function defects in neonatal rats with hypoxic-ischemic brain damage(HIBD).The appropriate time to transplant after HIBD is unknown.In the acute setting,release of excitotoxic neurotransmitters,free radicals,and proinflammatory mediators might threaten new tissue introduced into the damaged region.Furthermore,inflammation leading to microglial activation may inhibit endogenous neurogenesis and may thereby suppress the growth and survival of transplanted cells.On the other hand, it may be better to take advantage of local repair processes,including the release of neurotrophic factors from the intrinsic milieu and the host environment during the early recovery phase to facilitate implant growth, survival,differentiation,and/or integration.Delaying transplantation, however,poses the disadvantage of allowing the formation of scar tissue, which might adversely affect implanted cells.So,the choice of timing must be considered to get the best effect.The study in adult rats suggested that early intervention with intravenous administration of autologous mononuclear cells from bone marrow can reduce lesion size in the MCAO model and improve functional outcome.However, another study demonstrated that BMSCs given 1 week after spinal cord injury led to significantly larger numbers of surviving cells than immediate treatment and significant improvements of gait.The brain tissue of neonate rats,under developing process,are different from adult rats and have the more plasticity than that of adult ones.Up to date,little is about the optimal time of transplantation for the treatment of HIBD in neonate rats.It has been considered that BMSCs improve neurologic function of damaged brain tissue through the following mechanisms,(1)The transplanted BMSCs differentiated to new neurons and replaced the lost neurons caused by hypoxic-ischemic(HI)insult.BMSCs have the abilities of self-renewal and multilineage differentiation and can differentiate into osteocytes,chondrocytes,adipocytes,cadiocytes, neural cells and so on when exposed to specific experimental conditions. The neuron differentiation rate of BMSCs were different because of different induced ways,which suggest that environmental conditions are very important to neural differentiation of BMSCs.(2)Various growth factors produced by BMSCs,including nerve growth factor(NGF), brain-derived neurotrophic factor(BDNF),glial cell line-derived neurotrophic factor(GDNF),and neurotrophin-3(NT-3),play an important role in the transplantation treatment of HIBD.So,we employed the following experiment to investigate the therapeutic time window for intracerebral administration of BMSCs after HIBD in neonate rats and to provide the theoretical bases for further clinic treatment. Partâ… The effect of different time intracerebral transplantation of rat bone marrow stromal cells on behavior of neonate rats with hypoxic-ischemic brain damageObjective To investigate the effect of different time intracerebral transplantation of rat BMSCs on behavior of neonate rats with HIBD.Methods 7-day-old neonate rats were subjected to left carotid artery ligation and exposure to 2 hours of 8%oxygen.1×105 BMSCs were transplantation into the left hippocampus at 24 h,72 h and 7 d after induction of HIBD,respectively..T-maze,radial arm maze test and sensorimotor function were measured at 32-day-old,40-day-old and 42-day-old,respectively.Histological analysis was used to evaluated the neuron density of CA1 in the left hippocampus.Result Greater recovery of T-maze and radial arm maze testing were found in the 3 transplanted groups rats than those HIBD rats without cell transplantation(P＜0.01) The rats with BMSCs transplantation at 24 h after induction of HIBD had better behavioral improvement as compared with those rats with BMSCs transplantation at 72 h and 7 d after induction of HIBD(P＜0.05).Conclusion Intracerebral transplantation of BMSCs in rats with HIBD can improve long-lasting neurological function outcome and the time of 24 h after HIBD is optimal transplantation window. Partâ…¡Differentiation of rat bone marrow stromal cells into neural cells induced by hypoxic-ischemic brain tissue extracts of neonate ratsObjective To investigate the effect of brain tissue extracts of neonate rats with hypoxic-ischemic brain damage(HIBD)on the differentiation of bone marrow stromal cells(BMSCs)into neural cells Methods Eighteen 7-day-old neonate rats were induced HIBD by left carotid artery ligation and followed 2 hours of 8%oxygen exposure,and another 15 the same day old neonate rats were served as normal rats.The left and fight brain tissue extracts of the normal and HIBD rats were prepared at 8-day-old(24 h after HIBD),10-day-old(72 h after HIBD) and 14-day-old(7 d after HIBD),respectively(n=5).The rat BMSCs of passage 3～5 were cultured in medium with or without previous brain tissue extracts.The expressions of neuron specific encolase(NSE),glial fibrillary acidic protein(GFAP)and O4 marked oligodendrocyte were detected after 3 days by immunocytochemistry.Results The expressions of NSE,GFAP and O4 of BMSCs cultured in medium with left or fight brain tissue extracts of different day old normal rats were higher than those of BMSCs cultured without extracts,respectively(P＜0.01),and the expressions of NSE,GFAP and O4 of BMSCs cultured in medium with left brain tissue extracts of 8-day-old and 10-day-old HIBD rats were higher than those of BMSCs cultured with fight brain tissue extracts of the same day HIBD rats and BMSCs cultured with left or fight brain tissue extracts of the same day normal rats(P＜0.01 or 0.05). The expressions of NSE,GFAP and 04 of BMSCs cultured in medium with left brain tissue extracts of 8-day-old HIBD rats were higher than those of BMSCs cultured with left brain tissue extracts of 10-day-old and 14-day-old HIBD rats(P＜0.01 or 0.05).Conclusions The brain tissue extracts of normal and HIBD rats can induce BMSCS into neural cells and the damaged brain tissue extracts of 8-day-old HIBD rats is the best inductor.Partâ…¢Protective effects of different time intracerebral transplantation of rat bone marrow stromal cells in neonate rats with hypoxic-ischemic brain damageObjective Cell replacement,synthesis of various growth factors, through which further reducing HI induced neural cells apoptosis,white matter injury and promoting endogenous neurogenesis,are suggested to responsible for protective effects of BMSCs transplantation.This study was designed to investigate the effect of different time BMSCs transplantation on such mechanisms and to determine the optimal therapeutic time window of cell transplantation on neonate rats with HIBD.Methods Eighty 7-day-old Sprague-Dawley rats were divided into 5 groups:normal control group(n =15),model control group(n = 17), 24 h transplantation group(n=17),72 h transplantation group(n=16),7 d transplantation group(n=15).The rats of the previous four groups (except the normal control group)were established HIBD model,1×105 of passage 3～5 BMSCs stained with Hochest 33324 were transplanted into the left hippocampus at 24 h(24 h transplantation group),72 h(72 h transplantation group)and 7 d(7 d transplantation group)after induction of HIBD.Cell apoptosis was detected using TUNEL kits at 21-day-old (n=5).The neuron density of the left hippocampus CA1 area and cortex was analyzed by Nissl staining and myelin basic protein(MBP) expression in the ipsilateral corpus callosum,subcortical white matter and the number of oligodendrocyte positively stained O4 in the left periventricular area and subcortical white matter were evaluated by immunohistochemistry.Results HI insult resulted in cell apoptosis,loss of neuron and O4 positive oligodendrocytes,and hypomyelination as indicated by decreases MBP immunostaining.Transplantation of BMSCs after HIBD effectively prevented the HI induced previous brain damage. BMSCs transplantation at 24 h after induction of HIBD led to significantly larger numbers of neuron and oligodendrocytes and significantly reduced the numbers of TUNEL positive cells as compared with BMSCs given at 72 h and 7 d after induction of HIBD,and BMSCs transplantation at 24 h and 72 h greatly improved MBP immunoreactivity than treatment at 7 d after HIBD,respetively.(P＜0.05 or P＜0.01).The labeled BMSCs survived and migrated from the injection site and were found mainly in the left hemisphere,and some of them differentiated into neural cells,expressing neuronal,glial cell and oligodendrocyte precursor markers.The NSE expression of BMSCs in the left hemisphere of rats with cell transplantation at 24 h after induction of HIBD was higher than those rats with cell transplantation at 72 h and 7 d (P＜0.01),respectively.Conclusion BMSCs transplantation at 24 h after induction of HIBD have the most effects in reducing cell apoptosis,loss of neuron and O4 positive oligodendrocytes and in increasing the neuron differentiation rate,and the time of 24 h after HIBD is maybe the optimal therapeutic window.In short,these results indicated that BMSCs intracerebral transplantation at 24 h,72 h and 7 d after HIBD in neonate rats can effectively prevent the HI induced brain damage by differentiating into neural cells,reducing cell apoptosis,loss of neurons and attenuating white matter injury and improve long-lasting neurological function outcome,and the early damaged ipsilateral brain tissue extracts of HIBD rats have the most effects in inducing BMSCs into neural cells,and the time of 24 h after HIBD is maybe the optimal therapeutic window.
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