Analysis showed that the chop was barely detected in sham group. In brains of CAL-120 ischemia group, it was increased 6 hour after 15 MedChemExpress (-)-Indolactam V minutes of ischemia and gradually decreased thereafter; however, the degree of increase was much smaller in the hypothermia brains. (c) Quantitative analysis of Western blotting showed that hypothermia after ischemia significantly decreased chop after 15 minutes of ischemia (P,0.05 compared with ischemia brains at the same time points. 6 rats from each group at every time points were used for analysis). doi:10.1371/journal.pone.0053431.gthe same time points, 6 rats from each group at every time points were used for analysis). Immunohistochemistry showed the chop was barely detected in sham group (A). The expression of chop in hypothermia group (B) is much weaker than that in ischemia group (C) at reperfusion 24 hours. /4006visual field. Western blot analysis showed that the chop was barely detected in sham group. In brains of ischemia group, it was increasedhour after 15 minutes of ischemia and gradually decreased thereafter; however, the degree of increase was much smaller in the hypothermia brains. Quantitative analysis of Western blotting showed that hypothermia after ischemia significantly decreased chop after 15 minutes of ischemia (P,0.05 compared with ischemia brains atHypothermia Attenuating Apoptosis through CHOPthe same time points. 6 rats from each group at every time points were used for analysis).DiscussionIt has been reported that hypothermia blocked the TNF pathway of apoptosis or extrinsic Fas-mediated apoptosis pathway and the mitochondria pathway of apoptosis. However, whether hypothermia can block endoplasmic reticulum mediated apoptosis is never known. This study confirms that mild hypothermia for 3 h after global ischemia protects hippocampus neurons from a global ischemic insult, and demonstrates that this protection following global ischemia is associated with reduced apoptosis as assessed by TUNEL staining. Furthermore, we showed for the first time that mild hypothermia was associated with increased GRP78 expression and decreased chop expression suggesting a mechanism for the observed neuroprotection. ER is the site for protein synthesis and folding, and also involved in calcium homeostasis and lipid biosynthesis. Cerebral ischemia causes severe ER stress that results in ER function disruption and3.3 TUNELTUNEL positive staining was absent in the sham groups (Fig. 4), and there were few TUNEL positive cells among the hypothermia group (Fig. 4). However, many positive cells could be observed in the ischemia group (Fig. 4). Detection of apoptosis in hippocampus CA1 pyramidal neurons was carried out using TUNEL staing. The sham group showed a large number of neurons and almost no TUNEL-positive cells (5.161.2) (A). In ischemia (C) and hypothermia (B) groups, the number of neurons was decreased and substantial TUNEL-positive cells were detected. The number of TUNEL-positive cells in hypothermia group (34.464.2) (B) is more than in ischemia group (40.565.7) (C), /4006visual field.Figure 4. Neuronal apoptosis in CA1 region of hippocampus induced by global cerebral ischemia. Detection of apoptosis in hippocampus CA1 pyramidal neurons was carried out using Tunel staing. The sham group showed a large number of neurons and almost no TUNELpositive cells (5.161.2) (A). In ischemia (C) and hypothermia (B) groups, the number of neurons were decreased and substantial TUNEL-positive cells were detected.Analysis showed that the chop was barely detected in sham group. In brains of ischemia group, it was increased 6 hour after 15 minutes of ischemia and gradually decreased thereafter; however, the degree of increase was much smaller in the hypothermia brains. (c) Quantitative analysis of Western blotting showed that hypothermia after ischemia significantly decreased chop after 15 minutes of ischemia (P,0.05 compared with ischemia brains at the same time points. 6 rats from each group at every time points were used for analysis). doi:10.1371/journal.pone.0053431.gthe same time points, 6 rats from each group at every time points were used for analysis). Immunohistochemistry showed the chop was barely detected in sham group (A). The expression of chop in hypothermia group (B) is much weaker than that in ischemia group (C) at reperfusion 24 hours. /4006visual field. Western blot analysis showed that the chop was barely detected in sham group. In brains of ischemia group, it was increasedhour after 15 minutes of ischemia and gradually decreased thereafter; however, the degree of increase was much smaller in the hypothermia brains. Quantitative analysis of Western blotting showed that hypothermia after ischemia significantly decreased chop after 15 minutes of ischemia (P,0.05 compared with ischemia brains atHypothermia Attenuating Apoptosis through CHOPthe same time points. 6 rats from each group at every time points were used for analysis).DiscussionIt has been reported that hypothermia blocked the TNF pathway of apoptosis or extrinsic Fas-mediated apoptosis pathway and the mitochondria pathway of apoptosis. However, whether hypothermia can block endoplasmic reticulum mediated apoptosis is never known. This study confirms that mild hypothermia for 3 h after global ischemia protects hippocampus neurons from a global ischemic insult, and demonstrates that this protection following global ischemia is associated with reduced apoptosis as assessed by TUNEL staining. Furthermore, we showed for the first time that mild hypothermia was associated with increased GRP78 expression and decreased chop expression suggesting a mechanism for the observed neuroprotection. ER is the site for protein synthesis and folding, and also involved in calcium homeostasis and lipid biosynthesis. Cerebral ischemia causes severe ER stress that results in ER function disruption and3.3 TUNELTUNEL positive staining was absent in the sham groups (Fig. 4), and there were few TUNEL positive cells among the hypothermia group (Fig. 4). However, many positive cells could be observed in the ischemia group (Fig. 4). Detection of apoptosis in hippocampus CA1 pyramidal neurons was carried out using TUNEL staing. The sham group showed a large number of neurons and almost no TUNEL-positive cells (5.161.2) (A). In ischemia (C) and hypothermia (B) groups, the number of neurons was decreased and substantial TUNEL-positive cells were detected. The number of TUNEL-positive cells in hypothermia group (34.464.2) (B) is more than in ischemia group (40.565.7) (C), /4006visual field.Figure 4. Neuronal apoptosis in CA1 region of hippocampus induced by global cerebral ischemia. Detection of apoptosis in hippocampus CA1 pyramidal neurons was carried out using Tunel staing. The sham group showed a large number of neurons and almost no TUNELpositive cells (5.161.2) (A). In ischemia (C) and hypothermia (B) groups, the number of neurons were decreased and substantial TUNEL-positive cells were detected.
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