Asiatic acid protects against cognitive deficits and reductions in cell proliferation and survival in the rat hippocampus caused by 5-fluorouracil chemotherapy

Abstract

The chemotherapy drug, 5-fluorouracil (5-FU), has been reported to cause cognitive impairments in cancer patients. The drug also reduces cell proliferation and survival in the brain. Asiatic acid (AA) is a triterpene compound found in Centella asiatica that can protect against reduction of neurogenesis in the hippocampus and memory deficits induced by valproic acid (VPA). In the present study, we investigated the preventive effects of AA on the deficits in spatial working memory and cell proliferation and survival caused by 5-FU chemotherapy in a rat model. Male Sprague Dawley rats received 5-FU (5 i.v. injections, 25 mg/kg) on day 8, 11, 14, 17 and 20 of the study. This was co-administered with AA (30 mg/kg, oral gavage tube) either 20 days before receiving 5-FU (preventive), after receiving 5-FU (recovery), or for the entire period of the experiment (throughout). Spatial working memory was determined using the novel object location (NOL) test and hippocampal cell proliferation and survival of dividing cells were quantified using immunohistochemistry. Rats in the 5-FU alone and recovery groups showed memory deficits in the NOL test and reductions in cell proliferation and cell survival in the subgranular zone (SGZ) of the hippocampal dentate gyrus. Rats in the control, AA alone, and both preventive and throughout co-administration groups, however, did not exhibit these characteristics. The results showed that 5-FU chemotherapy impaired memory and reduced cell proliferation and cell survival in the SGZ of the hippocampal dentate gyrus. However, these impairments in the animals receiving 5-FU chemotherapy were restored to control levels when AA was co-administered before and during 5-FU treatment. These data demonstrate that AA can prevent the spatial working memory and hippocampal neurogenesis impairments caused by 5-FU chemotherapy.

Fig 1

Mean exploration times (mean ± SEM) of animals during the familiarization (A) and choice (B) trials of the NOL test after treatment. In the familiarization trial, animals from all groups showed no significant difference in the total exploration time of the two objects (p>0.05). In the choice trial, the control, AA, 5-FU+AA (preventive), and 5-FU+AA (throughout) groups spent significantly longer exploring the object in the novel location compared with that in the familiar location (*p<0.05), whereas the 5-FU and 5-FU+AA (recovery) groups did not (p>0.05).

Fig 2. Exploration times of animals in NOL test.

The preference index (PI) were significantly higher than 50% (chance) in the control, AA, 5-FU+AA (preventive), and 5-FU+AA (throughout) groups (*p<0.05, A), whereas the PI in the 5-FU and 5-FU+AA (recovery) groups did not deviate significantly from chance (p>0.05). Total exploration time of the familiarization and choice trials (B) did not differ significantly among the various groups (p>0.05).

Fig 3. Effect of 5-FU and AA on proliferating cell counts in the dentate gyrus.

Representative images of Ki-67 positive cells in the dentate gyrus in each group (A-F). Ki-67 positive cells were stained green in the subgranular zone (SGZ) of the dentate gyrus. Section were counterstained with red nuclear dye, propidium iodide: PI. Arrowheads indicate Ki-67 positive cells in the the dentate gyrus (scale bars: 100 μm). Inserted images show Ki-67 immunostaining under high magnification (scale bar: 50 μm). Mean Ki-67 positive cell counts of the 5-FU group were significantly lower than the control, AA, and 5-FU+AA (preventive and throughout) groups (*p<0.05, G). Inaddition, Ki-67 positive cell number in the 5-FU+AA (recovery) group were significantly lower than in the control group (#p<0.05, G). ML: molecular layer, GCL: granule cell layer.

Fig 4. Effect of 5-FU and AA on cell survival in the dentate gyrus.

Representative images of BrdU positive cells in the dentate gyrus in each group (A-F). BrdU positive cells were stained green in the subgranular zone (SGZ) of the dentate gyrus. Sections were counterstained with red nuclear dye, propidium iodide: PI. Arrowheads indicate BrdU positive cells in the the dentate gyrus (scale bars: 100 μm). Inserted images show BrdU immunostaining under high magnification (scale bar: 50 μm). Mean BrdU positive cell counts in the 5-FU group were significantly lower than the control, AA, and 5-FU+AA (preventive and throughout) groups (*p<0.05, G). Furthermore, BrdU positive cell counts in the 5-FU+AA (recovery) group were significantly less than those in the control group (#p<0.05, G). ML: molecular layer, GCL: granule cell layer.