Effects of grape seed proanthocyanidin extract on pentylenetetrazole-induced kindling and associated cognitive impairment in rats

Abstract

Numerous studies have demonstrated the antioxidant effects of grape seed proanthocyanidin extract (GSPE). The generation of free radicals and the ensuing apoptosis may contribute to the pathogenesis of epilepsy; therefore, in the present study, we examined the effects of GSPE on cognitive impairment and neuronal damage induced by chronic seizures in rats. Seizures were induced by a daily intraperitoneal (i.p.) injection of pentylenetetrazole (PTZ; 35 mg/kg/day, 36 days). Two other groups were treated with GSPE (100 or 200 mg/kg/day, orally) for 24 days and then for 36 days prior to each PTZ injection. After the final PTZ injection, hippocampus-dependent spatial learning was assessed using the Morris water maze (MWM). The rats were then sacrificed for the measurement of hippocampal malondialdehyde (MDA, a measure of lipid peroxidation) and glutathione (GSH, a measure of endogenous antioxidant capacity) levels, and for the expression of pro-apoptotic factors [cytochrome c (Cyt c), caspase‑9 and caspase‑3]. The mitochondrial generation of reactive oxygen species (ROS), degree of mitochondrial swelling, neuronal damage and mitochondrial ultrastructure were also examined. Performance in the MWM was markedly impaired by PTZ-induced seizures, as evidenced by longer escape latencies during training and fewer platform crossings during the probe trial. This cognitive decline was accompanied by oxidative stress (MDA accumulation, ROS generation, reduced GSH activity), an increased expression of pro-apoptotic proteins, as well as damage to CA1 pyramidal neurons and the mitochondria. Pre-treatment with GSPE dose‑dependently reversed PTZ-induced impaired performance in the MWM, oxidative stress, mitochondrial ROS generation, the expression of pro-apoptotic proteins and neuronal and mitochondrial damage. Thus, GSPE may reverse the hippocampal dysfunction induced by chronic seizures, by reducing oxidative stress and preserving mitochondrial function.

Figure 1

Effect of grape seed proanthocyanidin extract (GSPE) pre-treatment on behavior in rats with pentylenetetrazole (PTZ)-induced seizures. All animals showed a progressive aggravation of seizures after the injection with PTZ. The aggravation of seizures in the PTZ group was more marked and more rapid than in the other groups. These abnormal results were effectively reversed by high-dose GSPE pre-treatment. Values are expressed as the means ± SEM.

Figure 2

Effect of grape seed proanthocyanidin extract (GSPE) pre-treatment on performance in the Morris water maze (MWM) in pentylenetetrazole (PTZ)-treated epileptic rats. (A) Escape latencies in MWM training trials. (B) Number of platform crossings during the probe trial. (C) Time spent in the target quadrant. Values are expressed as the means ± SEM (n=6/group). ^*P<0.05 compared to the control (CON) group. ^#P<0.05, ^&P<0.01 compared to the PTZ group.

Figure 3

Grape seed proanthocyanidin extract (GSPE) rescues CA1 pyramidal neurons from seizure-induced damage as revealed by Nissl staining. (A) Control group; (B) pentylenetetrazole (PTZ) group; (C) PTZ + 100 mg/kg GSPE group; (D) PTZ + 200 mg/kg GSPE group; (E) GSPE alone group. Photomicrographs showing sample CA1 subfields (magnification, ×400) in the coronal plane for each treatment group. A damaged cell body is indicated by the black arrow. These signs of neural damage were reduced by GSPE pre-treatment. (F) Photomicrograph shows the whole hippocampal sample (magnification, ×40) in the coronal plane. CA1 subfield is indicated by the black frame. n=3/group.

Figure 4

Effect of grape seed proanthocyanidin extract (GSPE) pre-treatment on the quantity of Nissl-positive CA1 pyramidal neurons in the hippocampus. The number of surviving pyramidal cells was counted under a light microscope and expressed as cells per mm linear length of the rat CA1 subfield. Data are shown as the means ± SEM. ^*P<0.05 compared to the control (CON) group; ^#P<0.05 compared to the pentylenetetrazole (PTZ) group.

Figure 5

Pre-treatment with grape seed proanthocyanidin extract (GSPE) reverses pentylenetetrazole (PTZ)-induced ultrastructural damage to mitochondria. Photomicrographs (magnification, ×40,000) showing mitochondrial ultrastructure in the CA1 area of the hippocampus. (A) Control group; (B) PTZ group; (C) PTZ + 100 mg/kg GSPE group; (D) PTZ + 200 mg/kg GSPE group; (E) GSPE alone group. In (B), a mitochondrial vacuole is indicated by a thin black arrow and one damaged mitochondria by a thick black arrow. In the PTZ group, mitochondria ridge disorder and vacuoles were commonly observed. These abnormal changes were reversed by GSPE pre-treatment (C and D). n=3/group.

Figure 6

Effects of seizures and grape seed proanthocyanidin extract (GSPE) pre-treatment on the levels of malondialdehyde (MDA), glutathione (GSH) and mitochondrial reactive oxygen species (ROS) generation in the hippocampus. (A) MDA accumulation was increased by pentylenetetrazole (PTZ) and reduced by GSPE pre-treatment. (B) GSH was reduced by PTZ and restored by GSPE pre-treatment. (C) Mitochondrial ROS production, as detected by DCFH-DA fluorescence by flow cytometry, was higher in hippocampal tissue from the PTZ-treated rats and reversed by GSPE pre-treatment. (D) The degree of mitochondrial swelling was also higher in the PTZ-treated rats and markedly suppressed by GSPE pre-treatment. Bars represent the mean ± SEM (n=6/group in MDA and GSH, n=4/group in ROS and degree of mitochondrial swelling). ^*P<0.05 compared to the control (CON) group; ^#P<0.05, ^&P<0.01 compared to the PTZ group.

Figure 7

Effects of grape seed proanthocyanidin extract (GSPE) pre-treatment on the expression of apoptosis-related proteins in the hippocampus. (A) Control group; (B) pentylenetetrazole (PTZ) group; (C) PTZ + 100 mg/kg GSPE group; (D) PTZ + 200 mg/kg GSPE group; E) GSPE alone group. Relative densities of cytochrome c (Cyt c)/β-actin, caspase-9/β-actin and caspase-3/β-actin are shown in cytosolic fraction (A); relative densities of Cyt c/β-actin are shown in mitochondrial fraction (B). Bars represent the mean ± SEM (n=6/group). ^*P<0.05 compared to the control (CON) group; ^#P<0.05 compared to the PTZ group.