Preliminary Study On The Neuroprotective Mechanisms Of Exogenous Neuroglobin

Part I Exogenous Ngb protects the hippocampal neurons in vivo hypoxia/reoxygenation and ischemia-reperfusion injury1. The establishment of the model of cerebral hypoxia in miceObjective:To elucidate the effect of hypoxia/reoxygenation (H/R) in the injury of hippocampus neurons, and the neuroprotection of Ngb on hypoxia-induced hippocampus neurons injury in mice. Methods:Mice were exposed to hypoxia (8% O2) in a special box for 6h, mice under nonnoxic condition were used for controls. After hypoxia, recombinant Ngb (B,17KD) and modified-recombinant Ngb (A,20.3KD; E,18KD) were injected respectively (intraperitoneally (i.p.),5mg/kg, once time/day) for 3 consecutive days during 72h reoxygenation in normal room air, nonnoxic and hypoxia control groups were injected respectively with saline in sane manner. NeuN stain was used to detect the loss of hippocampus neurons after H/R injury. Results:Immunohistochemistry test demonstrated that the number of NeuN positive neurons in CA1 and CA2 was significantly decreased after hypoxia 6h/reoxygenation 72h, as compared to the normoxia group, but the administration of Ngb reduced the loss of hippocampal neurons. Conclusion:Under 6h hypoxia and 72h reoxygenation, the injury of hippocampus in mice is severe, but the administration of Ngb reduces the loss of hippocampal neurons.2. The establishment of the model of cerebral ischemia in miceObjective:To study the effect of ischemia-reperfusion (I/R) in the injury of hippocampus neurons, and the neuroprotection of Ngb on ischemia-induced hippocampus neurons injury in mice. Methods:The bilateral common carotid arteries occluding (15min) was selected as the model of global ischemia. The group of sham-operated mice underwent all of the procedures, except that their arteries were not occluded, which was used for control. After ischemia, recombinant Ngb and modified-recombinant Ngb were injected respectively (intraperitoneally (i.p.),5mg/kg, once time/day) for 3 consecutive days during 72h reperfusion in normal room air, sham and ischemia control groups were injected respectively with saline in sane manner. NeuN stain was used to detect the loss of hippocampus neurons after I/R injury. Results:Exposure to ischemia for 15 min followed by 72h reperfusion resulted in a marked loss of neurons in hippocampal regions, including CA1, CA2 subfields (p<0.01), but the administration of Ngb reduced the loss of hippocampal neurons, as compared with ischemia control groups. Conclusion:I/R injury induces the loss of hippocampal neurons, however the administration of Ngb reduces the injury of hippocampal neurons.3. The effects of exogenous Ngb on neurotrophic factors in vivo H/R and I/R injuryObjective:To explore the effects of exogenous Ngb on neurotrophic factors and the astrocytes in vivo H/R and I/R injury. Methods:Semi-quantitative RT-PCR was used to analyze the mRNA expression of neurotrophic factors in the brain homogenate, and the levels of G-CSF protein were measured by ELISA. At the same time, GFAP stain was used to detecte the activation of astrocytes in hippocampal DG area. Results:H/R and I/R injury promoted the expression of many neurotrophic factors mRNA, and the intervention of Ngb further increased the expression of G-CSF, BDNF, GDNF, CNTF mRNA; The levels of G-CSF protein in Ngb-treated mice were also elevated compared with saline-injected mice; Meanwhile we also found that the number of GFAP positive astrocytes in DG area increased significantly in Ngb-treated mice. Conclusion:Ngb can elevated the expression of G-CSF, BDNF, GDNF, CNTF mRNA and the levels of G-CSF protein in the brain homogenate of H/R injury and I/R injury. Simultaneously, Ngb can also activate the astrocytes in DG area.PartⅡThe protective effects of exogenous Ngb in vitro1. The protective effects of exogenous Ngb on the neuronsObjective:To confirm the effect of exogenous Ngb on the proliferation and G-CSF secretion in the normal culture neurons; And to observe the role of Ngb in anti-apoptosis and G-CSF secretion via the neuron OGD model. Methods:In normal culture conditions, we detected the proliferation of the neurons that were treated with different concentrations of different Ngb for 24h,48h and 72h by MTT assay, and the levels of G-CSF in supernatants by ELISA. In the OGD model, we tested the survival of the neurons that were exposed to OGD for 3h followed by treating with different concentrations of different Ngb and reoxygenating for Oh,3h,6h,12h,24h using MTT and LDH assays. Simultaneously, the levels of G-CSF in supernatants were measured by ELISA. Results: In normal culture conditions, treatment with Ngb significantly enhanced the proliferation of neurons, as compared with the control group. The proliferation of neurons intervented by Ngb for 48h reached a peak, at this time point the G-CSF production also increased, in which the levels of G-CSF increased most obviously in the group of high concentration of Ngb; In the OGD model, the LDH release of neurons treated with Ngb was lower than the control group at 12h and 24h after reoxygen. Simultaneously, treatment with Ngb increased the survival of OGD-neurons, and the effects of Ngb were better at 24h after reoxygen. After OGD 3h and reoxygenation 24h, treatment with Ngb significantly enhanced the G-CSF production of neurons, as compared with the control group, in which the levels of G-CSF increased most obviously in the group of high concentration of Ngb. Conclusion:Ngb induces the proliferation and promotes the survival in cultured neurons. At the same time, Ngb enhances the levels of G-CSF in normal neurons and OGD-neurons.2. The protective effects of exogenous Ngb on the astrocytesObjective:To confirm the effect of exogenous Ngb on the G-CSF secretion in the normal culture astrocytes; And to observe the role of Ngb in anti-apoptosis and G-CSF secretion via the astrocytes OGD model. Methods:In normal culture conditions, we tested the levels of G-CSF in the supernatants of astrocytes that were treated with different concentrations of different Ngb for 24h,48h and 72h by ELISA. In the OGD model, we detected the survival of the astrocytes that were exposed to OGD for 3h followed by treating with different concentrations of different Ngb and reoxygenating for Oh,3h,6h,12h,24h using MTT assays. Simultaneously, the levels of G-CSF in supernatants were measured by ELISA. Results:In normal culture conditions, treatment with Ngb significantly increased the G-CSF production of astrocytes, as compared with the control group. The G-CSF production was maximum in the group of high concentration of Ngb-B, Ngb-A at 48h after intervention and Ngb-E at 72h after intervention; In the OGD model, the survival of astrocytes treated with Ngb was higher than the control group at 12h and 24h after reoxygen, and the effects of Ngb were better at 24h after reoxygen. After OGD 3h and reoxygenation 24h, treatment with Ngb significantly enhanced the G-CSF production of astrocytes, as compared with the control group, in which the levels of G-CSF augmented most obviously in the group of high concentration of Ngb. Conclusion:Ngb reduces the OGD-induced injury and enhances the survival in cultured astrocytes. At the same time, Ngb elevates the levels of G-CSF in normal astrocytes and OGD-astrocytes.3. Preliminary study on the neuroprotective mechanisms of exogenous NgbObjective:To elucidate the underlying neuroprotective mechanism of exogenous Ngb via the neuron and astrocyte OGD model. Methods:Astrocytes were exposed to OGD for 3h, followed by treating with different concentrations of Ngb and reoxygenation for 24h. The supernatants which contained the most G-CSF production acted as astrocyte-conditioned medium (ACM-Ngb), the supernatants under PBS intervention were used for controls(ACM-PBS). And then ACM-Ngb and ACM-PBS were respectively added to primary cultured neurons that underwent OGD for 3h. After reoxygenating for 24h, the survival of neurons were measured by MTT and LDH assays; To determine the effect of G-CSF in the Ngb-mediated neuroprotection, inhibiting G-CSF/G-CSFR signaling in neuron cultures by adding anti-G-CSF receptor antibodies, affinity-purified Isotype IgG was used for control. And then the neurons underwent OGD for 3h, followed by treating with ACM-Ngb and high concentration of different Ngb. After reoxygenating for 24h, the survival of neurons were measured by MTT and LDH assays. Results:ACM-Ngb significantly reduced the levels of LDH release and increased the survival of neurons compared with ACM-PBS. As expected, LDH assay showed that the rate of LDH release of neurons were significantly increased, while the viability of neurons was reduced after blocking the G-CSF receptor in the neurons. Conclusion:In vitro the OGD model demonstrated that Ngb-induced neuroprotection is mediated partially through production of G-CSF which is autocrined by neurons and paracrined by astrocytes. Conclusion1. After hypoxia 6h/reoxygenation 72h and ischemia 15min/reperfusion 72h, the injury of hippocampus in mice is severe, but the administration of Ngb reduces the loss of hippocampal neurons.2. Ngb can elevated the expression of G-CSF, BDNF, GDNF, CNTF mRNA and the levels of G-CSF protein in the brain homogenate of H/R injury and I/R injury. Simultaneously, Ngb can also activate the astrocytes in DG area in vivo.3. Ngb induces the proliferation of normal neurons, and reduces the OGD-induced injury and enhances the survival in cultured astrocytes and neurons in vitro.4. In the normal and OGD conditions, Ngb elevates the levels of G-CSF in astrocytes and neurons. Receptor blocking clearly demonstrated that Ngb-induced neuroprotection is mediated partially through endogenous G-CSF.