The Experimental Study Of HNSCs Transplantation Efficacy Promoted By HBO On Neonatal Wistar Rats With Hypoxic Ischemic Brain Damage

Objectives:To observe the effect of hyperbaric oxygenation(HBO) to the survival,migration and differentiation of implanted human neural stem cells(hNSCs)on hypoxic ischemic brain damage(HIBD)in neonatal Wistar rats.Methods:Postnatal day 7(d7)Wistar rat pups were randomly divided into HIBD(n=20),HIBD+transplantation(n=24),HIBD+HBO (n=20)and HIBD+transplantation+HBO(n=24)groups.HIBD animal model was duplicated by Rice method.Implanted hNSCs suspension was prepared by cell culture method and transplanted into the ventricles by microinjection method on d10.HBO intervention was started within 15min after the transplantation and once daily for consecutively 10 days. TUNEL staining and cell counting of the positive cells in cortex and hippocampus were done on d11,d12,d13 and d20.HE staining and Nisel staining were done for histopathological study on d20,and meanwhile Fluorescent Immunohistochemical(FIHC)staining was done for assessing the survival,migration and differentiation of implanted hNSCs in HIBD+transplantation and HIBD+transplantation+HBO groups.Results:1.Primary hNSCs could be prepared after being cultured for 12 days. FIHC showed positive Nestin staining feature.These primary hNSCs could be induced to differentiated into cells with NF or GFAP positive staining features,indicating the stem cell characteristics of the primary hNSCs.2.TUNEL staining and cell counting:TUNEL positive cells could be observed at cerebral cortex and hippocampus in each group.On d11, compared to HIBD group,the number of TUNEL positive cells in HIBD+HBO groups was not significantly different,while that in HIBD+transplantation and HIBD+transplantation+HBO groups were significantly decreased(P＜0.01).On d20,compared to HIBD group,the number of TUNEL positive cells in HIBD+HBO,HIBD+transplantation and HIBD+transplantation+HBO groups were all significantly decreased(P＜0.01).The number of TUNEL positive cells in HIBD+transplantation+HBO was significantly decreased than HIBD+transplantation group(P＜0.01)on both d11 and d20.3.In HIBD+transplantation and HIBD+transplantation+HBO groups:on d20,FIHC staining showed that implanted hNSCs extensively migrated from ventricles to periventricular brain parenchyma,thalamus and hippocampus.The distribution and style of migration was not significantly different.But the number of survived cells in HIBD+transplantation+HBO group was significantly more than that in HIBD+transplantation group(P＜0.01).There were neurons and astrocytes differentiated from hNSCs,and undifferentiated hNSCs in both groups.The differentiated neurons and astrocytes were morphologically similar to that of the host cells.Conclusions:1.After being transplanted into the brain of neonatal Wistar rats,hNSCs could survive,migrate and differentiated into neurons and astrocytes.2.To some extent,hNSCs transplantation had neuroprotective effect to HIBE in neonatal Wistar rats.3.HBO therapy may enhance the above neuroprotective effect of hNSCs transplantation via promoting graft cell survival rate,migration and differentiation.