Effects of Asiatic Acid on Spatial Working Memory and Cell Proliferation in the Adult Rat Hippocampus

Abstract

Asiatic acid is a pentacyclic triterpene from Centella asiatica. Previous studies have reported that asiatic acid exhibits antioxidant and neuroprotective activities in cell culture. It also prevents memory deficits in animal models. The objective of this study was to investigate the relationship between spatial working memory and changes in cell proliferation within the hippocampus after administration of asiatic acid to male Spraque-Dawley rats. Control rats received vehicle (propylene glycol) while treated rats received asiatic acid (30 mg/kg) orally for 14 or 28 days. Spatial memory was determined using the novel object location (NOL) test. In animals administered asiatic acid for both 14 and 28 days, the number of Ki-67 positive cells in the subgranular zone of the dentate gyrus was significantly higher than in control animals. This was associated with a significant increase in their ability to discriminate between novel and familiar object locations in a novel object discrimination task, a hippocampus-dependent spatial memory test. Administration of asiatic acid also significantly increased doublecortin (DCX) and Notch1 protein levels in the hippocampus. These findings demonstrate that asiatic acid treatment may be a potent cognitive enhancer which improves hippocampal-dependent spatial memory, likely by increasing hippocampal neurogenesis.

Keywords: cell proliferation; hippocampus; spatial memory..

Figure 1

Mean exploration times of the animals exploring each object during the familiarization (A) and choice (B) trials of the novel object location test after treatment. There were no significant differences in exploration times of either object for any group in the familiarization (10 animals per group). In the choice trial, all groups spent a significantly longer time exploring the object in the novel location when compared with the familiar location (p < 0.05). Moreover, exploration times of the novel location in animals that received asiatic acid for 28 days was significant higher than 14 days (p < 0.05). Statistical assessment was by paired Student t-test. NOL: Novel Object Location.

Figure 2

The preference index (PI) showed a significant difference from 50% chance in all groups (A; p < 0.05). The animals received asiatic acid for 28 days, but they had a significantly higher PI compared to controls (* p < 0.05); The total exploratory time (B) in familiarization and choice trials in all groups was not significantly different; a two-way Analysis of variance with Fisher's Least Significant Difference post hoc test was used to compare between all groups.

Figure 3

The number of Ki-67 positive cells in the SGZ (subgranular zone) of the hippocampal dentate gyrus. Ki-67 positive cells are stained green in the SGZ of the dentate gyrus (section counterstained with red nuclear dye, PI; (A) = control 14 day; (B) = asiatic acid 14 day; (C) = control 28 day and (D) = asiatic acid 28 day). The level of proliferating cells in animals receiving asiatic acid for both 14 and 28 days were significantly higher than controls (* p < 0.05) (E). A two-way Analysis of variance with Fisher's Least Significant Difference post hoc test was used to compare between all groups.

Figure 3

The number of Ki-67 positive cells in the SGZ (subgranular zone) of the hippocampal dentate gyrus. Ki-67 positive cells are stained green in the SGZ of the dentate gyrus (section counterstained with red nuclear dye, PI; (A) = control 14 day; (B) = asiatic acid 14 day; (C) = control 28 day and (D) = asiatic acid 28 day). The level of proliferating cells in animals receiving asiatic acid for both 14 and 28 days were significantly higher than controls (* p < 0.05) (E). A two-way Analysis of variance with Fisher's Least Significant Difference post hoc test was used to compare between all groups.

Figure 4

Photographs of DCX (Stem Cells and Doublecortin) and Notch1 protein expressions. DCX expression in the hippocampus of asiatic acid-treated animals showed a significant increase compared to the control animals in both time periods (A, B; * p < 0.05, ). Animals treated with asiatic acid for both time periods had significantly higher Notch1 expressions than controls (C, D; * p < 0.05). Levels of Notch1 and DCX were normalized to GAPDH (Glyceraldehyde 3-phosphate dehydrogenase). Two-way ANOVA with LSD post hoc test was used to compare between all groups.

Figure 4

Photographs of DCX (Stem Cells and Doublecortin) and Notch1 protein expressions. DCX expression in the hippocampus of asiatic acid-treated animals showed a significant increase compared to the control animals in both time periods (A, B; * p < 0.05, ). Animals treated with asiatic acid for both time periods had significantly higher Notch1 expressions than controls (C, D; * p < 0.05). Levels of Notch1 and DCX were normalized to GAPDH (Glyceraldehyde 3-phosphate dehydrogenase). Two-way ANOVA with LSD post hoc test was used to compare between all groups.