Amantadine alleviates postoperative cognitive dysfunction possibly by increasing glial cell line-derived neurotrophic factor in rats

Abstract

Background: Postoperative cognitive dysfunction is a clinical entity that is associated with poor outcome. We determined the effectiveness of amantadine in reducing surgery-induced cognitive impairment and the role of glial cell line-derived neurotrophic factor (GDNF) in this effect.

Methods: Four-month old male Fischer 344 rats were subjected to right carotid exposure under intravenous anesthesia. Some rats received intraperitoneal injection of 25 mg/kg/day amantadine for 3 days with the first dose at 15 min before the surgery or intracerebroventricular injection of GDNF or an anti-GDNF antibody at the end of surgery. One week later, rats were started to be tested by Barnes maze and fear conditioning. Hippocampus was harvested at 6 h, 24 h or 10 days after the surgery for biochemical analysis. C8-B4 cells, a microglial cell line, were pretreated with 1 ng/ml GDNF for 30 min before being exposed to 5 ng/ml lipopolysaccharide for 2 h.

Results: Surgery increased the time to identify the target box in the Barnes maze when tested 1 day [22 (median) (11-66) (interquartile range) of control group vs. 158 (29-180) of surgery group, n = 15, P = 0.022) or 8 days after the training sessions and reduced context-related freezing behavior in the fear conditioning test. These effects were attenuated by amantadine (25 (14-90), n = 15, P = 0.029 compared with surgery group at 1 day after the training sessions in Barnes maze) and intracerebroventricular GDNF. Amantadine increased GDNF that was co-localized with glial fibrillary acidic protein, an astrocytic marker, in the hippocampus. Intracerebroventricular injection of an anti-GDNF antibody but not the denatured antibody blocked the effects of amantadine on cognition. Surgery induced neuroinflammation that was inhibited by amantadine. Lipopolysaccharide increased interleukin 1β production from C8-B4 cells. This effect was inhibited by GDNF.

Conclusions: Our results suggest that amantadine attenuated surgery-induced learning and memory impairment. This effect may be mediated by GDNF via inhibition of neuroinflammation.

Fig. 1. Amantadine attenuated surgery-induced learning and memory impairment

Four-month old Fischer 344 rats were subjected to right carotid exploration under intravenous anesthesia with or without the treatment of amantadine. Barnes maze training sessions started 1 week after the surgery. A: Barnes maze training sessions. Results are mean ± S.D. (n = 15). * P < 0.05 compared with the corresponding data on day 1. B: Barnes maze memory phase. C: fear conditioning. Results in panels B and C are in box plot format (n = 15). ●: lowest or highest score (the score will not show up if it falls in the 95th percentile); between lines: 95th percentile of the data; inside boxes: 25th to 75th percentile including the median of the data. & P < 0.05 compared with the control group, ^ P < 0.05 compared with surgery alone group.

Fig. 2. Glial cell line-derived neurotrophic factor (GDNF) attenuated surgery-induced learning and memory impairment

Four-month old Fischer 344 rats were subjected to right carotid exploration under intravenous anesthesia. Some rats received intra-cerebroventricular injection of GDNF. Barnes maze training sessions started 1 week after the surgery. A: Barnes maze training sessions. Results are mean ± S.D. (n = 8). * P < 0.05 compared with the corresponding data on day 1. B: Barnes maze memory phase. C: fear conditioning. Results in panels B and C are in box plot format (n = 8). Inside boxes: 25th to 75th percentile including the median of the data.

Fig. 3. Amantadine inhibited surgery-induced ionized calcium binding adapter molecule 1 (Iba-1) expression at one day after the surgery

Four-month old Fischer 344 rats were subjected to right carotid exploration under intravenous anesthesia with or without the treatment of amantadine. Hippocampus was harvested at 24 h after the surgery. A: representative immunostaining images for Iba-1. Scale bars = 30 µm. B: graphic presentation of the percentage area that is Iba-1-postive staining in the CA3 and dentate gyrus (DG). Values are presented as mean ± S.D. (n = 6). * P < 0.05 compared with the control group, ^ P < 0.05 compared with surgery alone group.

Fig. 4. Amantadine inhibited surgery-induced interleukin (IL)-6 and IL-1β expression

Four-month old Fischer 344 rats were subjected to right carotid exploration under intravenous anesthesia with or without the treatment of amantadine. Hippocampus was harvested at 6 h or 24 h after surgery. A: representative Western blot images of IL-1β. B: graphic presentation of IL-1β abundance. C: representative Western blot images of IL-6. D: graphic presentation of IL-6 abundance. Values are expressed as fold changes over the mean values of control rats and are presented as mean ± S.D. (n = 6). * P < 0.05 compared with the control group, ^ P < 0.05 compared with corresponding surgery only group. Ama: amantadine, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, Sur: Surgery.

Fig. 5. Amantadine inhibited surgery-induced ionized calcium binding adapter molecule 1 (Iba-1) expression at ten days after the surgery

Four-month old Fischer 344 rats were subjected to right carotid exploration under intravenous anesthesia with or without the treatment of amantadine. Hippocampus was harvested at 10 days after the surgery. A: representative immunostaining images. Scale bars = 200 µm. B: graphic presentation of the percentage area that is Iba-1-postive staining in the CA3 and dentate gyrus (DG). Values are presented as mean ± S.D. (n = 6). * P < 0.05 compared with the control group, ^ P < 0.05 compared with surgery alone group. AMA: amantadine.

Fig. 6. Amantadine inhibited surgery-induced nuclear translocation of p65

Four-month old Fischer 344 rats were subjected to right carotid exploration under intravenous anesthesia with or without the treatment of amantadine. Hippocampus was harvested at 6 h or 24 h after surgery. A: representative immunostaining images of the hippocampal CA3 region at 24 h after the surgery. Scale bars = 30 µm. B: representative Western blot images. C: graphic presentation of p65 abundance in nuclei. Values are expressed as fold changes over the mean values of control rats and are presented as mean ± S.D. (n = 6). * P < 0.05 compared with control group, ^ P < 0.05 compared with corresponding surgery group. Ama: amantadine, Iba-1: ionized calcium binding adapter molecule 1, NF-κB: nuclear factor κ-light-chain-enhancer of activated B cells, Sur: Surgery.

Fig. 7. Amantadine increased glial cell line-derived neurotrophic factor (GDNF) expression

Four-month old Fischer 344 rats were subjected to right carotid exploration under intravenous anesthesia with or without the treatment of amantadine. Hippocampus was harvested at 6 h or 24 h after surgery. A, B and C: representative immunostaining images of hippocampus harvested at 24 h after surgery. Scale bars = 30 µm. D: representative Western blot images. E: graphic presentation of GDNF abundance. Values are expressed as fold changes over the mean values of control rats and are presented as mean ± S.D. (n = 6). ^ P < 0.05 compared with corresponding surgery alone group. F: graphic presentation of the percentage area that is glial fibrillary acidic protein (GFAP)-positive staining in the CA3 and dentate gyrus (DG) at 24 h after the surgery. Values are presented as mean ± S.D. (n = 6). * P < 0.05 compared with the control group. Ama: amantadine, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, Iba-1: ionized calcium binding adapter molecule 1, Sur: Surgery.

Fig. 8. Glial cell line-derived neurotrophic factor (GDNF) inhibited lipopolysaccharide (LPS)-induced microglial activation

C8-B4 cells were pretreated with 1 ng/ml GDNF for 30 min before being exposed to 5 ng/ml lipopolysaccharide. A: representative immunostaining images of the cells after being exposed to lipopolysaccharide for 1 h. Nuclei were stained by Hoechst 33342. Scale bars = 50 µm. B: graphic presentation of the percentage cells that are positive staining of intra-nuclear p65 after being exposed to lipopolysaccharide for 1 h. C: interleukin (IL)-1β concentrations in culture medium that was harvested at 24 h after the cells were incubated with lipopolysaccharide for 2 h. Values are presented as mean ± S.D. (n = 6). * P < 0.05 compared with the control group, ^ P < 0.05 compared with the group treated with lipopolysaccharide only.

Fig. 9. Anti-glial cell line-derived neurotrophic factor (GDNF) antibody blocked the effects of amantadine on learning and memory after surgery

Four-month old Fischer 344 rats were subjected to right carotid exploration under intravenous anesthesia with or without the treatment of amantadine. Some rats received intra-cerebroventricular injection of an anti-GDNF antibody. Barnes maze training sessions started 1 week after the surgery. A: Barnes maze training sessions. Results are mean ± S.D. (n = 8). * P < 0.05 compared with the corresponding data on day 1. B: Barnes maze memory phase. C: fear conditioning. Results in panels B and C are in box plot format (n = 8). Inside boxes: 25th to 75th percentile including the median of the data. & P < 0.05 compared with the control group, ^ P < 0.05 compared with surgery plus amantadine plus boiled antibody group.