Effects Of POSTN, OPC Or Hypothermia On Neonatal Rats After Hypoxic-ischemic Brain Injury

Part I. The effect of POSTN on the proliferation and differentiation of neural stem cells in neonatal rats after hypoxic-ischemic brain injuryObjective:The purpose of this study was to investigate whether POSTN could provide neuroprotection against hypoxic-ischemic neonatal rat brain injury. To provide a new therapeutic strategy for neonatal brain injury.Methods:7-day-old neonatal rat pups had the left carotid artery ligation, and exposed to8%oxygen for2h to induce HI, then POSTN protein was injected through lateral ventricles. The effect of POSTN on neural stem cells proliferation and differentiation was evaluated using bromodeoxyuridine (BrdU) label and cell-specific immunofluorescence. Spatial cognitive function was assessed by Morris water maze test.Results:Treatment with POSTN significantly increased the number of new cells (BrdU+cells) in the subventricular zone (SVZ) and hippocampus dentate gyrus (DG). The new neural stem cells (BrdU+/Nestin+cells) were peaked at7d after operation, while the new neurons (BrdU+/Map-2+cells) and astrocytes (BrdU+/GFAP+cells) were peaked at14d. Treatment with POSTN improved the spatial learning deficits in navigation trials (shorter escape latency time and less swimming distance) and the memory deficits in probe trials (more number of platform crossings and percentage of time in the target quadrant) in Morris water maze performance.Conclusions:POSTN showed a neuroprotection role by promoting neural stem cell proliferation and differentiation after neonatal hypoxic-ischemic rat brain injury. POSTN also improved the function of spatial learning and memory, which maybe helpful for the neural repair after brain injury. Part II. The experimental effect of OPC transplantation on the treatment of neonatal rats after hypoxic-ischemic brain injuryObjective:To study whether cultured Olig2+-GFP+-OPC could differentiate into oligodendrocytes in vitro and the transplanted cells could survive in vivo, we transplanted Olig2+-GFP+-OPC into neonatal rats after hypoxic-ischemic (HI) brain injury. To explore the feasibility of OPC transplantation on the treatment of periventricular leukomalacia (PVL) in premature infants.Methods:Olig2+-GFP+-mES cells were induced to differentiated into OPC by embryonic body (EB)-mediated neural differentiation methods, including all-trans retinoic acid (RA) induced and five step methods. Using immunocytochemical staining, we observed the differentiation and maturation of OPC in vitro by contrast phase microscopy and fluorescence microscopy.3-day-old neonatal rat pups had the left carotid artery ligation, and exposed to6%oxygen(37℃) for2.5h to induce HI. To evaluate animal model by haematoxylin-eosin(HE) staining. To observe the transplanted cells through lateral ventricles injection surviving by immunofluorescence staining.Results:Olig2+-GFP+-mES formed EB in embryonic stem cell culture medium, and gradually developed into neural precursor cell balls in RA (0.5μM) and SHH (100ng/ml) neural differentiation culture medium. Olig2+-GFP+-positive cells were appeared in EB4d, and peaked in EB12d. Olig2+-GFP+-positive cells could differentiate into OPC in vitro, and then differentiate and mature to myelinating oligodendrocytes. There were cerebral edema widely; neuronal cells necrosis and focal cerebromalacia formation in HI group. GFP expressed in the lateral ventricle surrounding and nearby brain areas, that confirmed OPC transplantation successfully and migration to the surrounding.Conclusions:Olig2+-GFP+-mES could be induced to differentiated into a highly concentrated and functional Olig2+-GFP+-OPC by EB-mediated neural differentiation methods in vitro. Olig2+-GFP+-OPC could differentiate and mature to myelinating oligodendrocytes. The transplanted cells through lateral ventricles injection on the treatment of neonatal rats after hypoxic-ischemic brain injury could survive. Part Ⅲ. Effects of hypothermia on the activation and proliferation of hippocampal astrocytes in hypoxic-ischemic neonatal rat brainObjective:To study the effects of post hypoxic-ischemic hypothermia on newborn rat hippocampal cells surviving and astrocyte activation and proliferation, exploring neuroprotection mechanisms of hypothermia on hypoxic-ischemic injured brain.Methods:Hippocampal slices were prepared from3-day-old neonatal rat pups and cultured using transwell plate for4days prior to experimentation. Slices were randomly divided into hypothermia (33℃,24h) and normothermia (37℃) groups after oxygen-glucose deprivation (OGD). Hippocampal slices were cultured for7days (37℃) in control groups and not treated with OGD. Hippocampal cells surviving and astrocyte proliferation were detected by propidium iodide (PI) and immunofluorescence staining. In vivo animal experiments,7-day-old neonatal rat pups were subjected to left common carotid artery ligation followed by8%02+92%N2for2h. Animals were randomly divided into normothermia groups (rectal temperature36-37℃for24h) and hypothermia groups (rectal temperature32-33℃for24h) after hypoxia-ischemia. Control groups were not treated with hypoxia-ischemia. Animal brain sections were perfused and obtained at3days and7days after hypoxia-ischemia. Hippocampal tissue cell surviving and astrocytic proliferation were detected by DAPI (4’-6-Diamidino-2-phenylindole) and immunohistochemical staining.Results:There was an obvious decrease of cell surviving at3d after hypoxic-ischemia or OGD in normothermia groups compared with hypothermia groups. Glial fibrillary acidic protein (GFAP) for astrocyte staining result showed an obvious increase of astrocyte cells number in the normothermia group in cultured hippocampal tissue at3d after OGD compared with that in the hypothermia group. There was a significant difference between these two groups (P<0.05). In vivo, the number of astrocytes of hypothermia groups significantly decreased (P<0.05) at3days and7days after hypoxia-ischemia, consistent with in vitro study.Conclusions:Hypothermia can decrease the activation and proliferation of hippocampal astrocytes in the neonatal rat brain after hypoxic-ischemic brain injury.