The Study Of Effect And Molecular Mechanism Of Activated Microglia After Acute Cerebral Infarct

BackgroundCerebrovascular disease (CVD), served as a common disease of central neurvous system (CNS), is a major issue of public health. Cerebral infarct, also named ischemic stroke, is the disease that the death of hypoxic and ischemic brain tissue resulting from the disorder of blood supply leads to the symptom of neurological deficits. Cerebral infarct, which is the most common type of CVD and accounts for80%of the incidence, is another high mortility disease followed ischemic heart disease. Even so some patiens survives from the disease, they remains severel disabled, thus imposing a heavy burden to fomily and community. In addition, the incidence is predicted to grow with the increasing age of the population. Therefore, finding a safe and effective therapeutic method has been major purpose of medicine. At present, most studies indicated that brain injury attributed to those factors such as microglial activity under cerebral hypoxia and ischemical condition, release of proinflammar factors and excitory amino acids and generation of oxygen free radicals. However, the exact mechanisms remain unclear. MG are major immunocytes and play neurotrophic, neuroprotective and repaireing neuron cells role. Rest MG provide immuno-minitor role but the effect of activated MG in central nervous disease (CND) is still debated. Previous in vitro studies displayed that activated MG provide a neuroprotective role in CND through releasing neurotrophic factors or inhibit neurons apoptosis. Therefore, identifying the effect of activated MG after cerebral infarct would contribute to explain the pathological process of cerebral infarct and to find new therapeutic method. Therefore, it is necessary that further studying role of MG in CND in order to reveal the exact effect and mechanism of activated MGExtracellular signal-regulated kinase1/2(ERK1/2), one of the best-characterized members of the mitogen-activated protein kinase (MAPK) family, mediates a range of activity from metabolism, motility, and inflammation to cell death and survival. It is phosphorylated and activated through a three-tiered MEK mode via cell surface receptors stimulated by growth factors or cytokines. Most phosphorylated ERK1/2translocates to cell nucleus and induces genic transduction and expression and is closely associated with cell proliferation and differentiation. It is well documented that phosphor-ERK1/2mainly expresses in neurons of cortex in peri-infarct sites after acute cerebra ischemia. It is also reported that ERK1/2located in MG is phosphorylated in response to acute cerebral infarct and phosphor-ERK1/2promotes MG to synthesize and secrete brain-derived neurotrophic factor (BDNF).BDFN is a member of neurotrophic factor family and generally locals in CNS, sensory neurons and the neurons of spinal cord motor. Recently, numerous studies from world have confirmed that BDNF not only play a critical role in neuron surviva, differentiation and sustaining normal physiological function but protecting neurons against injury. Meanwhile, it also promotes neuron regeneration. However, more views supported that the neuroprotective roles of BDNF are due to inhibiting cytotoxity of excitory amino acids and stabling Ca2+concentration of cytoplasm. Some studies have showed that BDNF prevented Ca2+from entering into cell and secreting from cytoplasm. The BDNF treatment increases the number of hippocampus neuron with calcium binding protein (CBP). The neurons with CBP show more significant ability in resisting glumatic acid cytotoxity and reducing Ca2+concentration in cytoplasm than neurons not with CBP. Therefore, BDNF could stable Ca2+concentration in cytoplasm through CBP role.Calbindin-D28k is a major member of CBP family. Recently, many experts have done numerous in vivo and in vitro studies to explore the effect of calbindin-D28k. although calbindin-D28k locates in neurons, it is not linked with neurotransmitter at all. It indicated that calbindin-D28k role in mediating Ca2+concentration in brain is associated with the some phosiological repense of neurons with CBP. Calbindin-D28k plays a role in synaps linking and maybe prevents neurons subjecting to injury due to excessive Ca2+concentration-depended on voltage difference. Calbindin-D28k stabilizes Ca2+concentration in cytoplasm through stably binding Ca2+and buffering and transporting Ca2+to arrest over Ca2+accumulation. Calbindin-D28k can activate Ca2+/Mg2+/-ATP enzyme activation and arrest Ca2+excessive accumulation in cytoplasm. It is a critical factor in sustaining Ca2+balance that calbindin-D28k bufferes and transports excessive Ca2+in neuronal plasm. Many studies have revealed that calbindin-D28k can remarkly reduce neurons injury induced by hypoxia and ischemia. In addition to this, the neuroprotective role of Calbindin-D28k maybe be associated with inhibiting calpain activation. Excessive elevation of cytosolic Ca2+levels after acute cerebral infarct activates calpain. Activated calpain can cleave a-spectrin into two protein fragments as molecular weight of145and150KDa and neurons necrosis is increased.Since MG play a critical role in ischemic cerebral injury and the effect and molecular mechanisms of atctivated MG in response to cerebral infarct especially actue cerebral infarct remain unclear, it is necessary to further study the role of activated microglia under in vivo condition after acute cerebral ischemia. The present study maybe provide new method and potential target for treating acute cerebral infarct.Objective:To provide transplanting microglia for detecting the effect of activated microglia after acute cerebral ischemia.Methods:Mixed glial cells were obtained from cortex of SPF Kunming mouse embryo for14days. Traditional McCarthy and modulated McCarthy method i.e trypsin predigesting combinated with slight tapping were employed to extract and purify MG Then immunocytochemistry was used to detect the antigenic markers of cell and flow cytometry was employed to determined cultured cell purity. Finally, purity cell was cultured or subcultured.Results:Primary cultured results showed that more cells were obtained by modified McCarthy method than traditional McCarthy method. Immunocyto chemistry determining showed that the surface antigen of most puriftied cells was CD68and CDllb. the results of flow cytometry technique showed the number of CD11b positive cells was over92%of total cells number by modilated method but80%by McCarthy and there was significant difference between modilated and traditional McCarthy methods in the number of CDllb positive cells (p<0.05). Therefore, these cells by isolating and puritfy were microglia and can be employed to the studies of microglial function.Conclusions:Modified McCarthy method improves microglial yield and purity. Objective:To determine effect of activated microglia on neurological function and infarct volume of mouse after acute cerebral ischemia in vitro.Methods:First, MG were marked by CFDA-SE with green fluorescence and the signal intension of fluorescence was observed under fluorescence microscope at12,24and72h as well as7day after marking. In addition, MTT was employed to detect the effect of CFDA-SE on the growth and proliferation of MG. Secondly, Once the signal intension reached the experiment criterion, MG marked by CFDA-SE were injected into permenant middle cerebral artery occlusion (pMCAO) mice body through subclavian vein, and the brains were disassociated at12,24and72h. The disassociated brains were sliced by frost slice techniques and the number of MG with fluorescence was observed under fluorescence. Finally,66mice were divided randomly into4groups:A group (MCAO+MG transplanting), B group (MCAO+MG medium), C group (MCAO group) and D group (sham operation+MG transplanting). Triphenyltetrazolium chloride (TTC) was employed to detect infarct volume and Zea-longa and cylinder test were used to evaluate neurological deficits at12,24and72h after transplanting.Results:The results derived from cell marking showed that the flueroscence intension of marked cells was significant and unchanged at at12,24and72h after treatment. However, the signal intension of attached cells was partially attenuated at7day after treatment. MTT test revealed that CFDA-SE did not affect the growth and proliferation of microglia. The results derived from cell tracing showed that few cells marked with flueroscence was observed at12h after transplanting and the number of marked cells was increased at24and further increased at72h after treatment. In addition, the marked cells mainly scattered in peri-infarct site at72h after treatment. Compared with B and C groups, mice in A group had smaller infarct volume, lower Zea-longa score and more frequencies touching bottle wall with hemi-paretic forepaws at24and72h after transplanting (P<0.05).Conclusions:CFDA-SE marking liquid is a good liquid marking MG and dose not affect MG growing and proliferation. CFDA-SE sustains intensive flueroscene signal for7days and it has met the criterion of present experiment. Primary cultured MG can enter the peri-infarct site of MCAO mice through blood brain barrier and the number of MG in brain is increased at72h after transplanting. Activated microglia play a neuroprotective role generally after acute cerebral ischemia. Objective:To detect whether activated microglia influence neuronal death and releasing of cytokines after acute cerebral ischemia in tissue and molecular levels.Methods:Two hundred sixty four adult male SPF kunming mouse weighing25-35g were used in present section. The animal grouping and treating were similar with Part Ⅱ. Nectrotic and apoptosic neuron cells, survival neurons and proinflammar and neurotrophic factors, such as TNF-a, INL-1β,GDNF and BDNF, in brain of mice in4groups were determined by HE, TUNEL, immunohistochemistry at12,24and72h after transplanting. Duable flueronscence staining was used to detect phosphorylated ERK1/2expression on MG, astrocytes and neurons. Western blot was used to test the expression levels of cleaved a-spectrin.Results:Compared with B and C groups, mice in A group showed less HE positive cells but similar number in TUNEL positive cells in peri-infarct at24and72h after transplanting (P<0.05). There were no difference among the three groups in TNF-a, IL-1β and GDNF positive cells number at24and72h after treatment (P>0.05). However, the number of BDNF positive cells was remarkly increased compared with B and C groups (P<0.05). Duable flueroscene staining showed that pERKl/2mainly located in neuronal nuclear in the brain of MCAO or sham-operation mice. Compared with B, C and D group, the number of pERK1/2positive cells was remarkly increased at24and72h after transplantion. In adddition, there was no difference between B and C groups in the number of pERKl/2positive cells (P>0.05).Conclusions:Activated microglia inhibit neuronal cells necrosis and promote BDNF and p-ERK1/2expressions after acute cerebral ischemia. Objective:To determine the effect of activated microglia on the expression of p-ERK1/2and significance of the effect after acute cerebral ischemia, and to provide theory evidence.Methods:Two hundred and sixteen mice were divided randomly into4groups: A group (MCAO+MG transplanting), B group (MCAO+MG transplanting+U0126, inhibitor group), C group (MCAO group) and D group (sham operation). Western blot was used to test the expression levels of pERKl/2, BDNF, calbindin-D28k and cleaved a-spectrin at12,24and72h after acute cerebral infarct. Immunohistochemistry and RT-PCR were used to detect the number of BDNF positive cells and the expression level of BNDF and calbindin-D28k mRNA respectively at the three time points.Results:Compared with B and C groups, expression levels of pERK1/2protein and the number of BDNF positive cells of mice in A group were increased at24and72h after transplanting (P<0.05). The expression levels of BDNF mRNA and protein were increased at24and72h after transplanting and the increase in expression level of calbindin-D28k protein was at72h (P<0.05). In addition, the expression level of cleaved a-spetrin protein in A group was remarkably increased compared with B and C group at72h after treatment (P<0.05). However, there was no difference between B and C groups in the expression of pERKl/2, BDNF, calbindin-D28k and cleaved a-spectrin protein and mRNA expressions of BDNF and calbindin-D28k (P>0.05).Conclusions:Activated microglia up-regulate expression of BDNF through ERK1/2signal pathway. BDNF inhibit neuronal necrosis through promoting expression of Calbindin-D28K after acute cerebral ischemia.