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 Title Loaded From File 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 Title Loaded From File 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.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.