Protective Effects of Sodium (±)-5-Bromo-2-(α-Hydroxypentyl) Benzoate in a Rodent Model of Global Cerebral Ischemia

Abstract

The aim of the current study was to explore the protective effects of sodium (±)-5-bromo-2-(α-hydroxypentyl) benzoate (brand name: brozopine, BZP) in a rat model of global cerebral ischemia. The rat model was established using a modified Winocur's method; close postoperative observation was conducted at all times. Neurological function was detected through prehensile traction and beam-walking test. BZP reduced mortality and prolonged the survival time of rats with global cerebral ischemia, within 24 h. There was a decreased survival rate (60%) in the Model group, while the survival rate of the BZP (3 and 12 mg/kg) remarkably increased the survival rate (to 80 and 90%, respectively), in a dose-dependent manner. Compared with the Model group (survival time: 18.50 h), the administration of BZP (0.75, 3, and 12 mg/kg) prolonged the survival time (to 20.38, 21.85, and 23.90 h, respectively), particularly in BZP 12 mg/kg group (P < 0.05). Additionally, the BZP (12 mg/kg) group exhibited an improvement in their motor function (P < 0.05). The BZP groups (0.75, 3, and 12 mg/kg) displayed significantly reduced necrosis and the percentage of apoptotic cells (P < 0.05 and P < 0.01, respectively). Compared with Model group, BZP (0.75, 3, and 12 mg/kg) increased the NeuN optical density values (P < 0.01). Rats with global ischemia had a high expression of Cyt-c, caspase-3, and the Bax/Bcl-2 ratio compared with sham group (P < 0.01). BZP (0.75, 3, and 12 mg/kg), however, reduced the expression of Cyt-c, caspase-3, and the Bax/Bcl-2 ratio, in a dose-dependent manner (P < 0.01). There was low expression of p-Akt and PI3K in Model group, compared with the sham group (P < 0.01). Meanwhile, BZP (0.75, 3, and 12 mg/kg) increased the expression of p-Akt and PI3K in a dose-dependent manner (P < 0.01). We also found the expression of Cyt-c, caspase-3, Bax/Bcl-2 ratio, PI3K, p-Akt, and comprehensive score were directly related. In conclusion, BZP had therapeutic potential and prevented stroke in rat model of global cerebral ischemia. The underlying mechanisms may be related to the inhibition of apoptosis and activation of the survival-signaling-pathway.

Keywords: apoptosis; brozopine; global cerebral ischemic rat; neurological function; survival.

FIGURE 1

The study design.

FIGURE 2

The effect of BZP, within 24 h, on survival rate and survival time in a rat model of global cerebral ischemia. The x-axis represents the hour of administration.

FIGURE 3

The neurological deficits score on pre-ischemic treatment with BZP in rats with global ischemia. (A) Prehensile traction test. (B) Beam-walking test. The x-axis represents the hour of administration, while the y-axis represent the prehensile traction score and beam-walking score. N^∗ is the number of survival animals after 24 h.

FIGURE 4

The effect of BZP on morphologic changes in the CA1 region of the hippocampus in rats with global ischemia. The total number of necrosis cells was counted in five distinct areas of the hippocampus under the microscopic field (magnification ×200) in each group. (A,B) The different groups are as follows: (a) Sham, (b) Model group, (c) NBP-K (9.6 mg/kg), (d) Br-NBP (10.5 mg/kg), and (e–g) BZP (0.75, 3, and 12 mg/kg, respectively). Data were presented as mean ± standard deviation. A one-way analysis of variance was used to determine if the differences between the groups were statistically significant. ^∗∗P < 0.01 vs Sham group; ^#P < 0.05, ^##P < 0.01 vs Model group; P < 0.05, P < 0.01 vs NBP-K group; P < 0.05, P < 0.01 vs Br-NBP group. Arrows show that nerve cells sparse, irregular, even disappeared, nucleus shrinkage with neuronal necrosis.

FIGURE 5

Images of the expression of neuronal nuclei and apoptosis, following pretreatment with BZP, in ischemia-induced rats. Total number of apoptotic cells and neuronal nuclei was counted in five distinct areas of the ischemic penumbra under a microscopic field (magnification × 200), in each group. (A) TUNEL-staining. (B) Neuronal nuclei staining. The different groups are as follows: (a) Sham, (b) Model group, (c) NBP-K (9.6 mg/kg), (d) Br-NBP (10.5 mg/kg), and (e–g) BZP (0.75, 3, and 12 mg/kg, respectively). Data were presented as mean ± standard deviation. A one-way analysis of variance was used to determine if the differences were statistically significant. ^∗∗P < 0.01 vs Sham group; ^#P < 0.05, ^##P < 0.01 vs Model group; P < 0.05, P < 0.01 vs NBP-K group; P < 0.05, P < 0.01 vs Br-NBP group. Arrows in (A) show that cell body shrinkage, nuclear pyknosis, nuclear fragmentation, and the formation apoptotic bodies. Arrows in (B) show that mature neurons loss.

FIGURE 6

Immunohistochemical staining analysis of Cyt-c, caspase-3, the Bax, and Bcl-2 ratio in the CA1 hippocampal region in rats with global cerebral ischemia, after pretreatment with BZP, NBP-k, or Br-NBP. The total number of positive cells was counted in five distinct areas of the ischemic penumbra under the microscopic field (magnification ×200) in each group. (A) Cyt-c. (B) Caspase-3. (C) Bax. (D) Bcl-2. (E) The Bax/Bcl-2 ratio. The different groups are as follows: (a) Sham, (b) Model group, (c) NBP-K (9.6 mg/kg), (d) Br-NBP (10.5 mg/kg), and (e–g) BZP (0.75, 3, and 12 mg/kg, respectively). Data were presented as mean ± standard deviation. A one-way analysis of variance was used to determine if the differences between groups were statistically significant. ^∗∗P < 0.01 vs Sham group; ^#P < 0.05, ^##P < 0.01 vs Model group; P < 0.05, P < 0.01 vs NBP-K group; P < 0.05, P < 0.01 vs Br-NBP group. Arrows show that the expression of Cyt-c, caspase-3, Bax, Bcl-2, p-Akt and PI3K positive cell.

FIGURE 7

Immunohistochemical staining analysis of p-Akt and PI3K in the CA1 hippocampus region of rats with global cerebral ischemia, after pretreatment with BZP, NBP-K, or Br-NBP. The total number of positive cells was counted in five distinct areas of ischemic penumbra under the microscopic field (magnification ×200) in each group. (A) p-Akt. (B) PI3K. The groups were as follows: (a) Sham, (b) Model group, (c) NBP-K (9.6 mg/kg), (d) Br-NBP (10.5 mg/kg), and (e–g) BZP (0.75, 3, and 12 mg/kg, respectively). Data were presented as mean ± standard deviation. A one-way analysis of variance was used to determine if the differences between the groups were statistically significant. ^∗∗P < 0.01 vs Sham group; ^#P < 0.05, ^##P < 0.01 vs Model group; P < 0.05, P < 0.01 vs NBP-K group; P < 0.05, P < 0.01 vs Br-NBP group. Arrows show that the expression of Cyt-c, caspase-3, Bax, Bcl-2, p-Akt and PI3K positive cell.

FIGURE 8

The correlation between the expression of apoptosis-related factors and comprehensive score in Model and BZP (0.75, 3, and 12 mg/kg) groups in rats with global ischemia. Correlation between the expression of apoptosis-related factors and comprehensive score in (A) Model group, (B) BZP 12 mg/kg group, (C) BZP 3 mg/kg group, and (D) BZP 0.75 mg/kg group. r², correlation coefficient of the expression of apoptosis-related factors and comprehensive score. Statistical significant denoted by P < 0.001.