The Study On The Mechanism Of Hypothermic Therapy For Neonatal Hypoxic-ischemic Brain Injury

Hypoxic-ischemic brain injury (HIBI) is an important mortality and disables cause in neonates. Multicentre clinical trial has demonstrated that hypothermia is an effective therapy for hypoxic ischemic brain injury in neonates. However, the underlying mechanisms of hypothermic therapy on the HIBI are incomplete clear. In this study, we investigated the effects of hypothermia on neural stem cells, oligodendroglia cells and astrocyte in HIBI, to explore the mechanism of brain protection of hypothermic therapy. This study is divided into three parts.Part I:The effects of hypothermia on neural stem cells in HIBIObjectives:In this part of the study, we investigated the effects of hypothermia on the proliferation and apoptosis of neural stem cells in HIBI, to explore the mechanisms of hypothermia on neurogeneis and nerve regeneration.Metholds:7-day-old rats were subjected to left carotid artery ligation followed by8%oxygen for2h. They were divided into hypothermia (rectal temperature,32-33℃) and normothermia (rectal temperature,36-37℃) groups immediately after hypoxia-ischemia. The animals were sacrificed at7d and14d for immunohistochemical or immunofluorecent staining to study the neuroprotective effects of hypothermia on hypoxic-ischemic brain injury in immature rat brain.Results:1. Nestin-labeled neural stem cells did not change greatly, but immature neuron (Tuj-1) increased significantly in the striatum in the hypothermia compared to normothermia group at1and2weeks after HI. Newborn neural progenitors (BrdU+-Nestin+) did not change dramatically, but newborn immature (BrdU+-Tuj-1+) and mature (BrdU+-Map-2+) neurons increased significantly in the hypothermia compared with normothermia group. Meanwhile, the apoptosis rate of neural precursors, immature and mature neurons, assessed by double labeling of active Casp-3with nestin/Tuj-1/Map-2, decreased noticeably in the hypothermia compared with normothermia group respectively at1and2weeks after HI. In addition, hypothermia significantly increased expression of Bcl-2, which coexisted with nestin/Tuj-1/Map-2. Inhibition of Bcl-2expression reversed the decreased apoptosis rate of neural precursors and neurons in hypothermia animal striatum of neonatal rat brain.2. Neuron numbers significantly increased but neuborn cells (BrdU+) reduced greatly in SGZ in the hypothermic group compared with the normothermic group. Hypothermia did not change the number of neural stem cells (Nestin1+). However, the differentiational rate of newly generated cells into neuron (BrdU+-Map-2+/BrdU+or BrdU+-NeuN+/BrdU+) were dramatically increased in hypothermia compared with normothermia at2w and6w after HI. In contrast, the differentiation rate of newly generated cells into astrocyte(BrdU+-O4+/BrdU+) and oligodendrocyte (BrdU+-GFAP+/BrdU+) were significantly reduced in hypothermia compared with normothermia.Conclusion1. Hypoxic-ischemic hypothermia increased newborn immature and mature neuron numbers.2. Bcl-2involved in reduced apoptosis of neural stem cells, immature and mature neuron in the striatal region after HI.3. Hypoxic-ischemic hypothermia promoted newborn cells differentiate into neuron, and suppressed them differentiate into astrocyte and oligodendrocyte.Part II:The effects of hypothermia on differentiation and maturation of oligodendrocyte precursor cells in HIBIObjectives:White and central gray matter injuries are both detected in neonatal brain injury. White matter injury mainly correlated with damage to cells of the oligodendroglia lineage via necrotic and apoptotic death. In this part of the study, we used of HIBI animal model and oligodendrocyte lineage cells cultured in vitro to investigate the effects of hypothermia on the proliferation, differentiation and maturation of oligodendrocyte precursor cells, as well as myelin sheath repair.Metholds:The HIBI animal model preparation and hypothermia intervention were the same as "part I". The animals were sacrificed at1d,3d,7d,14d and42d for immunohistochemical or immunofluorecent staining and western blotting to study the proliferation, apoptosis, differentiation and maturation of oligodendrocyte precursor cells. We use electron microscopy and open field or rotarod to test myelinated axons and animals motor function. In addition, we cultured neural stem cells from E14rats brain, and induced them to differentiate into oligodendrocyte precursor cells. Double immunofluorescent staining and western-blotting were used to verify the proliferation, differentiation and maturation of oligodendrocyte precursor cells after oxygen glucose deprivation (OGD).Results:1. Neuronal loss and axonal injury (SMI32+NF200) were significantly decreased in hypothermia compared with normothermia in the hippocampal CA1region after HI. Early OL progenitors (A2B5+) were elevated, but preOLs (04+) and active caspase-3were dramatically reduced in the hypothermic rat brain. Further study showed that the apoptotic rate of pre-OLs (caspase-3+-O4+/O4+) was markedly attenuated by hypothermic treatment compared to normotheermic animals. Mature OLs (CNPase+and GST-pi+) and MBP protein expression were significantly increased in the hippocampus of hypothermia-treated rat brain. Furthermore, axonal myelination also increased in hypothermic animals in contrast to normothermic rats.2. Hypothermia successfully enhanced early OL progenitors (NG2+) and its proliferation in the CC at different time points after HI. Late OL progenitors (04+) accumulation decreased accompanied with increased OL maturation in hypothermia compared to normothermia. Using an in vitro model-OGD,we demonstrated that hypothermia decreased preOLs accumulation and promoted OPC differentiation and maturation. Further data indicated that the proliferation of oligodendrocyte lineage cells did not changed greatly between hypothermia and nomothermia, but the apoptotic rate was significantly suppressed by hypothermia at0,4,8and12days after OPC differentiation in vitro. The myelinated axons and animal behavior both markedly increased in hypothermic-compared to normothermic-animals after HI.Conclusion Hypoxic-ischemic hypothermia elevated proliferation of early oligodendrocyte precursor cells, reduced accumulation and apoptosis of late oligodendrocyte precursor cells, promoted differentiation and maturation of oligodendrocyte, increased myelinated axons and animals behavior in immature rat brain after HI Part Ⅲ:The effects of hypothermia on astrocyte activation and inflammatory reaction in HIBIObjectives:Astrocytes are the most numerous cells type in the central nervous system (CNS). They provide structural, metabolic and trophic support to neurons and modulate synaptic activity. The aim of this study was to test hypothermic effects on astrocyte activation and its related inflammatory reaction in the hippocampus after HI in the immature rat brain.Metholds:The HIBI animal model preparation and hypothermia intervention were the same as "part I". The animals were sacrificed at Oh,12h, Id,2d,3d and7d for immunohistochemical or immunofluorecent staining to detect astrocyte activation and apoptosis, GDNF and IL-6protein expression in the hippocampus. Real-time PCR were used to study GFAP, GDNF, IL-6and TNF-α gene expression.Results:Hypothermia significantly reduced glial fibrillary acidic protein (GFAP) mRNA and immunoreactivity in the hippocampus CA1region than the normothermia group. No significant difference in glial cell line derived neurotrophic factor (GDNF) mRNA expression in the hippocampus in the two groups at various time points after HI. However, GDNF protein level was significantly increased in the hypothermia group. Further study showed that the apotosis of astrocyte (Casp-3+-GFAP+) obviously reduced in the hypothermic rat hippocampus compared with normothermia at7days after HI. Besides, astrocyte related inflammatory factors tumor necrosis factor alpha (TNF-alpha) and interleukin-6(IL-6) gene and protein expression were all dramatically decreased in the hypothermia compared with the normothermia group.Conclusion Hypothermia reduced activation and apoptosis of astrocyte, enhanced expression of GDNF and inhibited IL-6and TNF-alpha levels in neonatal rat brain.