Verapamil Inhibits Mitochondria-Induced Reactive Oxygen Species and Dependent Apoptosis Pathways in Cerebral Transient Global Ischemia/Reperfusion

Abstract

The prefrontal cortex is the largest lobe of the brain and is consequently involved in stroke. There is no comprehensive practical pharmacological strategy for ameliorating prefrontal cortex injury induced by cerebral ischemia. Therefore, we studied the neuroprotective properties of verapamil (Ver) on mitochondrial dysfunction and morphological features of apoptosis in transient global ischemia/reperfusion (I/R). Ninety-six Wistar rats were allocated into four groups: control, I/R, I/R+Ver (10 mg/kg twice 1 hour prior to ischemia and 1 hour after reperfusion phase), and I/R+NaCl (vehicle). Animals were sacrificed, and mitochondrial dysfunction parameters (i.e., mitochondrial swelling, mitochondrial membrane potential, ATP concentration, ROS production, and cytochrome c release), antioxidant defense (i.e., superoxide dismutase, malondialdehyde, glutathione peroxidase, catalase, and caspase-3 activation), and morphological features of apoptosis were determined. The results showed that mitochondrial damage, impairment of antioxidant defense system, and apoptosis were significantly more prevalent in the I/R group in comparison with the other groups. Ver decreased mitochondrial damage by reducing oxidative stress, augmented the activity of antioxidant enzymes in the brain, and decreased apoptosis in the I/R neurons. The current study confirmed the role of oxidative stress and mitochondrial dysfunction in I/R progression and indicated the possible antioxidative mechanism of the neuroprotective activities of Ver.

Figure 1

ROS formation in isolated prefrontal cortex mitochondria. The data are presented as mean ± SD (n = 6). ^∗P < 0.001 vs. the control group, ^∗∗P < 0.001 vs. I/R+NaCl and I/R groups.

Figure 2

MMP decrease in the prefrontal cortex mitochondria isolated from rats. The data are presented as mean ± SD (n = 6). ^∗P < 0.001 vs. control group, ^∗∗P < 0.01 vs. I/R+NaCl and I/R groups.

Figure 3

Progressive mitochondrial swelling in the prefrontal cortex mitochondria isolated from experimental rats. The data are presented as mean ± SD (n = 6). ^∗P < 0.001 compared with the control group, ^∗∗P < 0.01 vs. I/R+NaCl and I/R groups.

Figure 4

Quantitative real-time polymerase chain reaction (PCR) analysis of relative expression mRNAs in the prefrontal cortex. The p53 (a), Bax (b), Bcl-2(c), caspase-8 (d), and cytochrome c (e). Verapamil treatment could significantly reduce the expression of proapoptotic mRNAs (Bax, p53, cytochrome c, and caspase-8) and improved antiapoptotic (Bcl-2) mRNAs. Data are presented as means ± SD of the normalized PCR product concentrations for each dilution step. ^∗P < 0.01 compared with the control group, ^∗∗P < 0.01 vs. I/R+NaCl and I/R groups.

Figure 5

Effects of verapamil on transient global I/R-induced apoptotic neurodegeneration. Representative photomicrographs of TUNEL staining and cell counting. (a) Representative images of the TUNEL-positive cell (arrows) were obtained from sections prepared from the animals in control, I/R, I/R+NaCl, and I/R+Ver groups. (b) DAPI nuclear staining was indicated overall in the cellular morphology of the TUNEL sections. (c) Treatment rats with verapamil significantly reduced the TUNEL-positive cell compared to I/R and I/R+NaCl groups. Data are represented as mean ± SD. Scale bars 100 μm, magnification ×400. ^∗P < 0.005 vs. control group, ^∗∗P < 0.005 vs. I/R and I/R+NaCl groups.

Figure 6

Nissl staining of rats' prefrontal cortex following transient global I/R and cell counting. The prefrontal cortex of control animals (a) did not contain any damaged neurons. However, the cortex of rats in the I/R (b) and I/R+NaCl (c) groups was characterized by fewer Nissl stain neurons (thin arrows) than that in the I/R+Ver group (d) and markedly contained shrunken, intensely stained, and dystrophic neurons (thick arrows). As shown in the graph (e), treatment rats with verapamil significantly reduced neuronal cell loss in the prefrontal cortex. Data are represented as mean ± SD. Scale bars 100 μm, magnification ×400. ^∗P < 0.001 vs. control group, ^∗∗P < 0.001 vs. I/R+NaCl and I/R groups.