| Background and objective:Stroke is characterized by high incidence, high mortality and high disability, whereas ischemic stroke has been the most common type and severely harmful to human health. The molecular mechanism of neuronal death is extremely complex under cerebral ischemia, three main cellular pathways of which has been found including over activation of glutamate receptor, oxidative stress and apoptosis. The LIM proteins are characterized by 2 or more LIM domains. LMO4 is a recently discovered transcription co-factor that belongs to LIM family. LMO4 could regulate the function of other proteins through activating or suppressing the corresponding transcription factors, playing crucial roles in gene expression, cell differentiation and development, and the formation of cytoskeleton. Both pCREB and pStat3 belongs to nuclear transcription factors also, partial studies have demonstrated that both contribute to protection of injured neurons in cerebral ischemia. In the cell models of cerebral ischemia, LMO4 can facilitate survival of hypoxic neurons in vitro by regulating the IL-6/Stat3 signal pathway, and pCREB mediates the transcriptional activity of LMO4 as an upstream molecular element. In this study we will speculate the temporal expression and localization of LMO4, pCREB and pStat3 in animal models of cerebral ischemia-reperfusion, and explore the relation of LMO4 with pCREB and pStat3 and the role of LMO4 in ischemic neuronal survival, hence providing new molecular target for introducing drugs of ischemic stroke.Methods:84 Sprague-Dawley rats of healthy adult male were randomly divided into seven of sham operation group, and ischemic-reperfusion 1h,3h,6h,12h,24h,48h group. Each group had 12 rats, of which 6 were used for molecular biology study and the rest for histology study. Suture methods were used to make the focal middle cerebral artery occlusion model and the ischemic brain was reperfused 2 hours after occlusion. The neurological deficit score was rated by Longa scale. Animals were killed at various reperfusion intervals. Routine HE staining was used to speculate the histopathological change. Nissle staining was used to evaluate the neuronal survival. MAP2 staining was used to detect the dendrite. TUNEL methods was used to detect the apoptotic cell. Immunoflurence was used to detect the quantity and location of,LMO4,pCREB,pStat3 on the penumbra cortex. Double immunoflurence was used to speculate the colocalization between LMO4 and MAP2, pCREB, pStat3, TUNEL positive cells respectively and between NEUN and pCREB, pStat3 respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the LMO4 mRNA level of penumbra tissue in sham group and ischemic subgroups. Western-blot was used to detect the LMO4, pCREB and pStat3 protein level of penumbra tissue in sham group and ischemic subgroups.Results:①the qualified rate was 76.60% for model inclusion and the 48h mortality rate was 13.83%. Death was mainly caused by hemorrhage and brain edema. TTC staining of the brain tissue revealed cortex and caudate putamen infarction in the qualified rat models and no infarction in the sham group rats.②HE staining revealed typical necrosis, neuronal loss, tissue edema in ischemic models, number of total cells decreased in infarction area, whereas cells in the sham group kept intact.③Nissle staining revealed that cells swelled and nissle body of the neuronal cells disappeared, the number of surviving neuron decreased with perfusion time on the penumbra cortex (P<0.05), reached the lowest level at 48h (P<0.01), whereas cells in the sham group kept intact.④The length and diameter of the dendrites decreased, OD signal of the MAP2 positive cells decreased with perfusion time on the penumbra cortex in ischemic group, reached the lowest level at 48h (P< 0.01), whereas the dendrites in the sham group kept intact.⑤TUNEL positive cells were mainly located in the peri-infarction area, the number of apoptotic cells increased at 6h after reperfusion (P< 0.05), peaked at 24h, and persisted at 48h (P<0.01).⑥Compared to the sham group, LMO4 mRNA level of the penumbra cortex in ischemic group increased at 3h after reperfusion, peaked at 24h, and obviously declined at 48h (P<0.05 or P<0.01);⑦Compared to the sham group, LMO4 protein level of the penumbra cortex in ischemic group increased at 3h after reperfusion, peaked at 24h, and obviously declined at 48h, pCREB and pStat3 protein level increased at 3h after reperfusion, peaked at 24h, and gradually declined at 48h (P<0.05 or P<0.01);⑧Compared to the sham group, the number of LMO4, pCREB, pStat3 positive cells in ischemic group increased at 6h after reperfusion, peaked at 24h, and gradually declined at 48h (P<0.05 or P<0.01);⑨Double immunoflurence indicated that both LMO4 and pCREB were only expressed in neurons, whereas pStat3 was expressed in neurons and glial cells; LMO4 was mainly localized in nucleus, a little part in cytoplasm, both pCREB and pStat3 were only localized in nucleus; the expression of LMO4 was segregated from the TUNEL positive cells in the penumbra cortex in ischemic group; LMO4 was completely coexpressed with pCREB, whereas was partially coexpressed with pStat3.Conclusions:①Cerebral ischemia reperfusion induces the overexpression of LMO4, pCREB and pStat3, with a dynamic profile, which may be an endogenous adaptive protective mechanism of neuronal cells triggered by ischemia reperfusion injury.②LMO4 may contribute to ischemic neuronal survival through interacting with pCREB and regulating the activity of pStat3. Background and objective:Edaravone is a kind of newly synthesized radical scavenger and anti-oxidant, and can improve the neurological deficit after cerebral ischemia. The neuroprotective role of edaravone in cerebral ischemia has been proved by many studies, however, the exact molecular mechanism for which remains unclear till now, especially the anti-apoptotic mechanism. Apoptosis is a kind of programmed cell death highly regulated by genes. LMO4 can mediate the expression of many other genes as a transcription co-factor. The LMO4 deficient mouse presents with all kinds of development deficits, suggesting its anti-apoptotic effect on epithelial cell and neuronal cell. This study was to further explore the molecular mechanism of edaravone protecting the ischemic neurons and the effect of edaravone on LMO4 expression.Methods:36 SD rats of healthy adult male were randomly divided into three of sham control group (n=12), ischemic model group (n=12) and edaravone treated group (n=12). Focal cerebral ischemia model was made by suture occluding one side of middle cerebral artery. Blood flow was restored after 2 hours of occlusion and the animals were killed at 24h after perfusion. Rats received intraperitoneal injections of edaravone for the drug group and same volume of physiological saline for the model group upon reperfusion. Routine HE and Nissle staining was used to speculate the histopathological change and evaluate the neuronal survival. TUNEL methods was used to detect the apoptotic cell. RT-PCR, western-blot and immunoflurence were used to detect the levels of LMO4 protein and mRNA on the penumbra cortex in the three groups, the final data were compared among different groups.Results:①Cellular morphology was normal in the sham group. Necrotic cell was seen both in the model group and the drug group, but less severe in the latter, with morphologically normal cells more common in the latter (P<0.01); Nissle staining revealed that the number of surviving neurons on the penumbra cortex was significantly higher in the drug group than in the model group (P<0.05);②No TUNEL positive cell was seen in the sham group, but was seen both in model group and the drug group, with the number much less in the latter (P<0.01);③The levels of LMO4 protein and mRNA were low in the sham group, however, that of the penumbra cortex were high in the model group and significantly higher in the drug group (compared to the model group, P<0.05 or P<0.01);④A few LMO4 positive cells were seen in the sham group, large number of LMO4 positive cells were seen on the peri-infarction cortex in the model group, and much larger were seen in the drug group (compared to the model group, P<0.01).Conclusions:Edaravone can enhance the overexpression of LMO4, which may be one of the mechanisms underlying the neuroprotection of this drug.
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