The neuroprotective effect of methanol extract of gagamjungjihwan and fructus euodiae on ischemia-induced neuronal and cognitive impairment in the rat

Abstract

Gagamjungjihwan (GJ), a decoction consisting of five herbs including ginseng, Acori Graminei Rhizoma, Uncariae Ramulus et Uncus, Polygalae Radic and Frustus Euodiae (FE), has been widely used as herbal treatment for ischemia. In order to investigate the neuroprotective action of this novel prescription, we examined the influence of GJ and FE on learning and memory using the Morris water maze and studied their affects on the central cholinergic system in the hippocampus with neuronal and cognitive impairment. After middle cerebral artery occlusion was applied for 2 h, rats were administered GJ (200 mg kg(-1), p.o.) or FE (200 mg kg(-1), p.o.) daily for 2 weeks, followed by training and performance of the Morris water maze tasks. Rats with ischemic insults showed impaired learning and memory of the tasks. Pre-treatment with GJ and FE produced improvement in the escape latency to find the platform. Pre-treatments with GJ and FE also reduced the loss of cholinergic immunoreactivity in the hippocampus. The results demonstrated that GJ and FE have a protective effect against ischemia-induced neuronal and cognitive impairment. Our results suggest that GJ and FE might be useful in the treatment of vascular dementia.

Figure 1

Time to escape on the platform during acquisition trials of the Morris water maze test. Four trials per day over 6 days were performed for the acquisition test. Rats were treated with GJ (200 mg kg⁻¹, p.o., GJ + ISCH group, n = 5) and FE (200 mg kg⁻¹, p.o., FE + ISCH group, n = 5) for 2 weeks after induction of cerebral ischemia. The sham-operated control group (SHAM, n = 6) and ischemia group (SAL + ISCH, n = 6) were not given any drug for 2 weeks after induction of ischemia. Significance with Tukey's test following an one-way ANOVA is indicated as *P <  .05, **P < .001 (Sham-operated versus SAL + ISCH, SAL + ISCH versus GJ + ISCH and SAL + ISCH versus FE + ISCH). Vertical lines indicate S.E.M (N = 5-6).

Figure 2

Time spent around the platform on the water maze test. The task was performed with four daily trials on the seventh day without the platform for the retention test. Rats were treated with GJ (200 mg kg⁻¹, p.o., GJ + ISCH group, n = 5) and FE + ISCH (200 mg kg⁻¹, p.o., FE + ISCH group, n = 5) for 2 weeks after cerebral ischemia. The sham-operated control group (SHAM, n = 6) and ischemia group (SAL + ISCH, n = 7) were not given any drug for 2 weeks after induction of ischemia. Significance with Tukey's test following an one-way ANOVA is indicated as *P < .05, **P <  .01 (Sham-operated versus SAL + ISCH, SAL + ISCH versus GJ + ISCH). Vertical lines indicate S.E.M (N = 5-6).

Figure 3

Swim distance to escape on the platform during acquisition trials of the Morris water maze test. Four trials per day over 6 days were performed for the acquisition test. Rats were treated with GJ (200 mg kg⁻¹, p.o., GJ + ISCH group, n = 5) and FE (200 mg kg⁻¹, p.o., FE + ISCH group, n = 5) for 2 weeks after induction of cerebral ischemia. The sham-operated control group (SHAM, n = 6) and ischemia group (SAL + ISCH, n = 6) were not given any drug for 2 weeks after induction of ischemia. Significance with Tukey's test following an one-way ANOVA is indicated as *P < .05, **P < .01, ***P < .001 (Sham-operated versus SAL + ISCH, SAL + ISCH versus GJ + ISCH and SAL + ISCH versus FE + ISCH). Vertical lines indicate SEM (N = 5-6).

Figure 4

Swim distance traveled after removal of the platform on the water maze test. The task was performed with four daily trials on the seventh day without the platform for the retention test. Rats were treated with GJ (200 mg kg⁻¹, p.o., GJ + ISCH group, n = 5) and FE + ISCH (200 mg kg⁻¹, p.o., FE + ISCH group, n = 5) for 2 weeks after cerebral ischemia. The sham-operated control group (SHAM, n = 6) and ischemia group (SAL + ISCH, n = 7) were not given any drug for 2 weeks after induction of ischemia. Significance with Tukey's test following an one-way ANOVA is indicated as *P < .05 (Sham-operated versus SAL + ISCH, SAL + ISCH versus GJ + ISCH and SAL + ISCH versus FE + ISCH). Vertical lines indicate S.E.M (N = 5-6).

Figure 5

Photographs showing the distribution of ChAT immunoreactive cells in the hippocampus of SHAM (a), SAL + ISCH (b), GJ + ISCH (c) and FE + ISCH (d) groups. Rats after water maze learning task. Sections were cut coronally at 30 μm and the scale bar represents 50 μm (100 × 100).

Figure 6

The percentage (±SE) values of quantities of choline acetyltransferase (ChAT) immunostained nuclei in different hippocampal areas of the experimental groups after the water maze learning task. Immunohistochemical data of ChAT were analyzed by performing separate one-way ANOVA of neurons among groups followed by the Tukey test. *P < .05, **P < .01, ***P < .001 (Sham versus SAL + ISCH, SAL + ISCH versus GJ + ISCH and SAL + ISCH versus FE + ISCH). Vertical lines indicate SEM (N = 15–18).

Figure 7

The percentage (±SE) of SHAM values of density of acetylcholinesterase (AchE) stained nuclei in different hippocampal areas of the experimental groups after the water maze learning mask. The results of AchE-reactivity were analyzed by performing separate one-way ANOVA of neurons among the groups followed by the Tukey test. *P < .05, **P < .01, ***P < .001 (Sham versus SAL + ISCH, SAL + ISCH versus GJ + ISCH and SAL + ISCH versus FE + ISCH). Vertical lines indicate SEM (N = 15–18).