Quantitative analysis of experimental animals
Sixty male Sprague-Dawley rats were randomly divided into five groups: sham-surgery group, 90-minute ischemia group, ischemia and reperfusion groups (90-minute ischemia followed by 1-, 3-, 24-hour reperfusion), with 12 rats in each group. In addition, a control group (n = 6) was also included. Six rats in each group were used for the detection of real- time fluorescence quantitative PCR, while the remaining six rats in each group were used for immunohistochemistry. In total, 60 Sprague-Dawley rats were involved in the final analysis, without any dropout or loss.
Successful middle cerebral artery occlusion (MCAO) model confirmed by 2,3,5-triphenyltetrazolium chloride staining
2,3,5-Triphenyltetrazolium chloride staining revealed that transient MCAO after 24-hour ischemia (no reperfusion) produced large apparent ischemic lesions in the basal ganglia and adjacent cerebral cortex (Figure 1).
Figure 1 The infarction region visualized by 2,3,5- triphenyltetrazolium chloride staining.
Photographs are shown at 24 hours after cerebral ischemia. Arrow indicates the region of cerebral infarction.
mRNA levels of SPCA2 Ca2+ pump in the cerebral cortex decreased after ischemia/reperfusion injury
Quantitative PCR was used to analyze SPCA2 Ca2+ pump mRNA levels. SPCA mRNA expression was detected in areas with ischemia/reperfusion injury using reverse transcription- PCR (Figure 2). Compared with the sham-surgery group, the level of SPCA2 mRNA expression was not different after 90 minutes of ischemia with no reperfusion (P > 0.05). However, cells in the cortex reacted to ischemic injury as observed by a reduction in SPCA2 gene expression in the reperfusion period. mRNA levels of SPCA2 decreased in a time-dependent manner, with a decrease of 44% when compared to the sham control after 1 hour reperfusion (P < 0.05), a decrease of 33% after 3 hours of reperfusion (P < 0.05), and a decrease of 31% after 24 hours of reperfusion (P < 0.05). In the ischemia and of reperfusion groups, SPCA2 mRNA expression levels gradually decreased as reperfusion proceeded, and the minimum value was observed at 24 hours of reperfusion (P < 0.05; Figure 2).
Figure 2 Expression of secretory pathway Ca2+- ATPases 2 (SPCA2) mRNA in the rat brain after 120 minutes of ischemia followed by 0 (ischemia only), 1, 3, and 24 hours of reperfusion.
aP < 0.05, vs. sham-surgery group (C group). Data are expressed as mean ± SEM of six rats in each group. One- way analysis of variance followed by Fisher’s least significant difference test was used for statistical analysis. Isc: Ischemia; Rep: reperfusion; h: hours.
Protein levels of SPCA2 Ca2+ pump in the cortex and hippocampus decreased after ischemia/reperfusion injury
Immunohistochemical analysis was used to evaluate whether mRNA expression changes induced by ischemia also affected SPCA2 protein levels. Expression of SPCA2 protein in the cerebral cortex and hippocampus appeared similar to that of mRNA levels and were not different compared with the sham-surgery group (P > 0.05). SPCA2 protein expression also decreased in the later reperfusion period, with a significant decrease of 30% compared with the sham-surgery group after 1 hour of reperfusion (P < 0.05), a 38% decrease after 3 hours of reperfusion (P < 0.05), and a 33% decrease after 24 hours of reperfusion (P < 0.05). A small but not significant increase was observed during reperfusion in the 3-hour group (P > 0.05). In the ischemia and reperfusion groups, the level of SPCA2 protein expression gradually decreased as reperfusion proceeded (P < 0.05; Figures 3, 4).
Figure 3 Protein levels of secretory pathway Ca2+-ATPases 2 (SPCA2) in the rat brain cortex (A) and hippocampus (B) after 90-minute ischemia and 0, 1, 3, and 24 hours of reperfusion.
aP < 0.05, vs. sham-surgery group (C group). Data are presented as mean ± SEM of six rats in each group. One- way analysis of variance followed by Fisher’s least significant difference test was used for statistical analysis. Isc: Ischemia; Rep: reperfusion; h: hours.
Figure 4 Expression of secretory pathway Ca2+-ATPases 2 in the cortex and hippocampus of the rat brain following cerebral ischemia reperfusion (DAB staining, light microscope, × 200).
(A, A') Sham-surgery group; (B, B') 2-hour ischemia group; (C, C') ischemia + 1 hour of reperfusion, (D, D') 3 hours of reperfusion; (E, E') 24 hours of reperfusion. Upper: Brain cortex; lower: hippocampus. Secretory pathway Ca2+- ATPases 2 protein expression decreased during the reperfusion period compared with the sham-surgery group.