Eus fragmentation (shown with star) and nucleolus disappearance.StatisticsThe results were expressed as mean 6 S.D. The differences between normal control group and SPS groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post-ER- Pathway is Title Loaded From File Involved in PTSD-Induced ApoptosisFigure 7. RT-PCR of Caspase-12 in the hippocampus of the SPS rats. Caspase-12 mRNA expression (A) and results from its quantitative analysis (B). Caspase-12 mRNA level in the hippocampus of rats subjected to SPS was higher than that in the hippocampus of control rats. *P,0.05 10457188 vs. the control group, #P,0.05 vs. the SPS 1 day group. doi:10.1371/journal.pone.0069340.gImmunohistochemical Detection of GRPGRP78 (ir) was distributed mainly in the cytoplasm of the hippocampal cells in the control group (Fig. 3A). At 16574785 1 day after SPS, increased expression of GRP78-ir was observed (Fig. 3B, 3E), and then peaked at 4 days after SPS (Fig. 3C, 3E). In contrast, decreased expression of GRP78 was observed at 7 days after SPS (Fig. 3D, 3E).GRP78 mRNA ExpressionThe mRNA expression level of GRP78 in the hippocampus of the SPS group was higher than that of the control group. At 4 day after SPS, mRNA expression of GRP78 peaked and then decreased at 7 days after SPS (Fig. 5A, 5B). The mRNA expression data were consistent with western blot data.Western Blot Analysis of Casapse-12 Western Blot Analysis of GRPWestern blots to GRP 78 showed a single band at 78 KD, and the band density mean value of the control group was set as 100 . Data were expressed as normalized optical density. b -actin protein level was used as the internal control at each time point. In the SPS group, the expression of GRP78 protein in the hippocampus increased at 1 day after SPS and peaked at 4 day after SPS. The extent of decrease in the GRP78 protein level at 7 days after SPS group was significantly greater than that at 4 days after SPS (Fig. 4). In order to understand more about casapse-12 cellular location following activation, we performed western blot on cell fractions purified by differential centrifugation. Figure 6A shows a panel verifying the quality of fractionation. In the SPS group, the expression of caspase-12 protein in the ER of the hippocampus exhibited thick band, increased at 1 day after SPS and peaked at 4 day after SPS. The expression of caspase-12 protein in cytoplasm and mitochondria of the hippocampal cells showed very tiny band and did not change between control group and SPS group (Fig. 6B).ER- Pathway is Involved in PTSD-Induced ApoptosisFigure 8. Change of intracellular Title Loaded From File calcium levels in the hippocampal cells of the SPS rats. Image of intracellular calcium levels in the control group (A), 1 day (B) and 4 days(C) after SPS and change in intracellular calcium (Ca2+) levels (nmol/l) from its quantitative analysis. *P,0.05 vs. the control group. doi:10.1371/journal.pone.0069340.gCaspase-12 mRNA ExpressionThe mRNA expression of caspase-12 was not detected in the control group. At 1 day after SPS, caspase-12 mRNA expression level was significantly increased, peaked at 4 days after SPS, and then disappeared completely at 7 days after SPS (Fig. 7).Free Ca2+ Concentration in the HippocampusThe intracellular free Ca2+ level in the hippocampus neurons was significantly higher in rats at 1 day after SPS than in control group and returned to normal levels 7 days after SPS (Fig. 8).mRNA Expression of CaM and CaMKIIaThe mRNA levels of CaM and CaMKIIa were normalized to the b-a.Eus fragmentation (shown with star) and nucleolus disappearance.StatisticsThe results were expressed as mean 6 S.D. The differences between normal control group and SPS groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post-ER- Pathway is Involved in PTSD-Induced ApoptosisFigure 7. RT-PCR of Caspase-12 in the hippocampus of the SPS rats. Caspase-12 mRNA expression (A) and results from its quantitative analysis (B). Caspase-12 mRNA level in the hippocampus of rats subjected to SPS was higher than that in the hippocampus of control rats. *P,0.05 10457188 vs. the control group, #P,0.05 vs. the SPS 1 day group. doi:10.1371/journal.pone.0069340.gImmunohistochemical Detection of GRPGRP78 (ir) was distributed mainly in the cytoplasm of the hippocampal cells in the control group (Fig. 3A). At 16574785 1 day after SPS, increased expression of GRP78-ir was observed (Fig. 3B, 3E), and then peaked at 4 days after SPS (Fig. 3C, 3E). In contrast, decreased expression of GRP78 was observed at 7 days after SPS (Fig. 3D, 3E).GRP78 mRNA ExpressionThe mRNA expression level of GRP78 in the hippocampus of the SPS group was higher than that of the control group. At 4 day after SPS, mRNA expression of GRP78 peaked and then decreased at 7 days after SPS (Fig. 5A, 5B). The mRNA expression data were consistent with western blot data.Western Blot Analysis of Casapse-12 Western Blot Analysis of GRPWestern blots to GRP 78 showed a single band at 78 KD, and the band density mean value of the control group was set as 100 . Data were expressed as normalized optical density. b -actin protein level was used as the internal control at each time point. In the SPS group, the expression of GRP78 protein in the hippocampus increased at 1 day after SPS and peaked at 4 day after SPS. The extent of decrease in the GRP78 protein level at 7 days after SPS group was significantly greater than that at 4 days after SPS (Fig. 4). In order to understand more about casapse-12 cellular location following activation, we performed western blot on cell fractions purified by differential centrifugation. Figure 6A shows a panel verifying the quality of fractionation. In the SPS group, the expression of caspase-12 protein in the ER of the hippocampus exhibited thick band, increased at 1 day after SPS and peaked at 4 day after SPS. The expression of caspase-12 protein in cytoplasm and mitochondria of the hippocampal cells showed very tiny band and did not change between control group and SPS group (Fig. 6B).ER- Pathway is Involved in PTSD-Induced ApoptosisFigure 8. Change of intracellular calcium levels in the hippocampal cells of the SPS rats. Image of intracellular calcium levels in the control group (A), 1 day (B) and 4 days(C) after SPS and change in intracellular calcium (Ca2+) levels (nmol/l) from its quantitative analysis. *P,0.05 vs. the control group. doi:10.1371/journal.pone.0069340.gCaspase-12 mRNA ExpressionThe mRNA expression of caspase-12 was not detected in the control group. At 1 day after SPS, caspase-12 mRNA expression level was significantly increased, peaked at 4 days after SPS, and then disappeared completely at 7 days after SPS (Fig. 7).Free Ca2+ Concentration in the HippocampusThe intracellular free Ca2+ level in the hippocampus neurons was significantly higher in rats at 1 day after SPS than in control group and returned to normal levels 7 days after SPS (Fig. 8).mRNA Expression of CaM and CaMKIIaThe mRNA levels of CaM and CaMKIIa were normalized to the b-a.
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