Effects of apoptosis-related proteins caspase-3, Bax and Bcl-2 on cerebral ischemia rats

Abstract

Neuron apoptosis is known to mediate a change of ethology following cerebral ischemia-reperfusion injury in rats. Additionally, Bcl-2, Bax and caspase-3 proteins may exert a significant effect on neuron injury. The aim of this study was to investigate the role, mechanism of action and clinical significance of these proteins in neuron apoptosis and functional impairment following cerebral ischemia-reperfusion injury in rats. Sixty male healthy adult Wistar rats were randomly assigned into control (n=6), sham operation (n=6) and experimental (n=48) groups. The model of rat cerebral ischemia-reperfusion injury was set up according to the method of Zea-Longa. Eight subsets of 6 rats-subset were designed according to time points (at 3, 6, 12, 24 and 48 h and at 3, 7 and 14 days). Nerve functional injury was evaluated and graded using nerve function score, balance, coordination function detection and measurement of forelimb placing. The neurons expressing caspase-3, Bax and Bcl-2 in the cortical area, CA3, CA1, stratum lucidum (Slu) and molecular layer of the dentate gyrus (MoDG) of the hippocampus were detected using immunohistochemistry or the TUNEL method. The expression of caspase-3, Bax and Bcl-2 genes was detected by the reverse transcriptase polymerase chain reaction (RT-PCR). The results indicated that, compared to the sham operation group, the score of nerve function and balance beam walking were distinctly higher (P<0.01) and the percentage of rat foreleg touching the angle or margin of the table was significantly lower in the experimental rat group (P<0.01) at 3 h following reperfusion. The expression of TUNEL-positive neurons was high in the cortical area and the CA3 region of the hippocampus (P<0.01), caspase-3 was at peak value in the cortical area and the CA1 region of the hippocampus (P<0.01), Bax was increased in the cortical area and the Slu of the hippocampus (P<0.01) and Bcl-2 was low in the cortical area and the MoDG of the hippocampus (P<0.01) in the experimental group at 48 h following reperfusion. In conclusion, cerebral ischemia/reperfusion injury may cause neurological impairment and lead to a change of ethology, and neuron apoptosis may be associated with the activation of caspase-3 and Bax and the downregulation of Bcl-2.

Keywords: Bax; Bcl-2; caspase-3; cerebral ischemia/reperfusion injury; neurological impairment; neuron apoptosis.

Figure 1

TUNEL staining of apoptotic neurons in (A) the cerebral cortex and (B) the hippocampal CA3 region. Apoptotic neurons are round, with intensely stained buff-coloured nuclei and shrunk, heavily stained cell membranes. Negative cells show deeply stained nuclei, lightly stained cytoplasm and maintained cell membrane intergrity. Magnification, ×200.

Figure 2

Reverse transcriptase polymerase chain reaction (RT-PCR) results for caspase-3, Bax, Bcl-2 and β-actin mRNA in each group at various time points. (a) Marker; (b) Sham operation group; (c) 3 h; (d) 12 h; (e) 24 h; (f) 48 h; (g) 7 days and (h) 14 days following ischemia/reperfusion in the experimental group.

Figure 3

(a) Expression of caspase-3, Bax and Bcl-2 in the cerebral cortex at 12 h following reperfusion. (A–C) Sham operation group; (D–F) ischemia-reperfusion group. (A) The number of caspase-3-positive neurons was distinctly increased compared to (D); (B) the number of Bax-positive neurons was higher compared to (E); (C) The number of Bcl-2-positive neurons was lower compared to (F). Immunohistochemichal staining, A,B,D and E: magnification, ×200; E and F: magnification, ×100. (b) Expression of caspase-3, Bax and Bcl-2 in the hippocampus at 12 h following reperfusion. (A–C) Sham operation group; (D–F) Ischemia-reperfusion group. (A) The number of caspase-3-positive neurons was distinctly increased compared to (D); (B) the number of Bax-positive neurons was higher compared to (E); (C) the number of Bcl-2-positive neurons was lower compared to (F). Immunohistochemichal staining, magnification, ×200.