The Role Of CAMP Signal Pathway In Learning And Memory Dysfunction Induced By Chronic Manganese Exposure In Rats

[Objective] To observe the effects of chronic manganese exposure on learning and memory, manganese levels in several body tissues and the proteins related to cAMP pathway in rat. Then exploring the mechanism of the relationship between Mn-induced cognitive impairment and cAMP pathway in the hippocampus of Mn-treated rats, as well as analyzing the feasibility of different tissues used as the biomarkers to evaluate Mn exposure and neurotoxicity in the body.[Methods] Thirty-two male Sprague-Dawley rats were divided into 4 groups, which received MnCl2·4H2O at a dose of 0,5,10 or 20 mg/kg/day in the injection volume of 2ml/kg by intraperitoneal injection for 5 days a week for 18 weeks. At the 6th,12th and 18th week, learning and memory of rats were assessed using the Morris water maze test. Treatment 24 hours after the last infected in 18th week, the rats were killed and the tissues samples were collected, then to measure the organ weight and organic coefficient in rats. Hippocampus, plasma, serum, teeth and hair Mn concentrations were measured using graphite furnace atomic absorption spectrometry. Plasma BDNF and hippocampus cAMP, pCREB and BDNF levels were assessed using ELISA. Hippocampus cPKA levels were assessed using western blot. In the end, correlation analysis was used to determine linear relationship in different experimentation indexes.[Results](1) The heart, liver, spleen and kidney organic coefficient increased significantly along with the dose of Mn, whereas decreased in testis organic coefficient (P<0.05). The heart organic coefficient of high-dose group was significantly higher than the low-dose group and control group (all P<0.05). The liver, spleen and kidney organic coefficient of medium-and high-dose group were significantly higher than the low-dose group and control group (all P< 0.05). On the contrary, the testis organic coefficient of medium-and high-dose group was significantly lower than the low-dose group and control group (all P <0.05).(2) The hippocampus, plasma, serum, teeth and hair Mn concentrations increased significantly along with the dose of Mn in rats (P< 0.05). Hippocampus Mn concentrations were higher in the medium-and high-dose group than the control group, and the Mn concentrations of the high-dose were significantly higher than the low-dose group’s (all P<0.05). Plasma Mn concentrations were higher in the medium-and high-dose group than the control group, and the Mn concentrations of the medium-and high-dose were significantly higher than the low-dose group’s (aMP<0.05). Mn concentrations in teeth and hair were higher in the medium-and high-dose group than in the low-dose group and control group. Moreover, the Mn concentrations of high-dose were significantly higher than the medium-dose group’s (all P<0.05).(3) In place navigation test of MWM test, mean escape latency was longer in the high-dose group than in the control group in the 5th day of the 6th week, the 4th and 5th day of the 12th week as well as the 5th day of the 18th week (all P <0.05). In additional, mean escape latency in the high-dose group was longer than in the control group on all days in the 18th week (all P<0.05). These results suggested that Mn exposure impaired spatial learning in rats.(4) hi spatial probe test of MWM test, the numbers of platform crossings were lower in all Mn-treated groups than in the control group in week 12 and 18 (all P<0.05). Moreover, there were significantly fewer platform crossings in the high-dose group than the low-dose group in week 18 (P<0.05). These results suggested that Mn exposure impaired spatial memory in rats.(5) The plasma BDNF, hippocampus cAMP, cPKA, pCREB and BDNF levels decreased significantly along with the dose of Mn in rats (P<0.05). The expression levels of hippocampus cAMP and pCREB were lower in all Mn-treated groups than in the control group, and lower in the medium-and high-dose groups than in the low-dose Mn group (all P<0.05). The expression levels of hippocampus cPKA were lower in all Mn-treated groups than in the control group, and lower in the high-dose Mn groups than in the low-dose Mn group (all P<0.05). The hippocampus BDNF levels decreased significantly along with the dose of Mn in rats, and the levels difference among each groups are statistically significant (all P<0.05). Moreover, The expression levels of hippocampus BDNF were lower the medium-and high-dose Mn groups than in the control group, and lower in the high-dose Mn groups than in the low-dose Mn group (all P<0.05). Taken together, these results suggested that chronic manganese exposure could affect the expression of the proteins related to cAMP pathway.(6) The results of correlation analysis were list as follow:① Hippocampal BDNF levels were negatively associated with plasma Mn levels (r=-0.872, P< 0.01), hippocampal Mn levels (r=-0.719, P< 0.01) and escape latency (r=-0.581, P< 0.01), but positively associated with number of platform crossings (r= 0.485, P< 0.01) and hippocampal cAMP levels (r= 0.817, P< 0.01).② Plasma BDNF levels were negatively associated with plasma Mn levels (r=-0.483, P< 0.01), hippocampal Mn levels (r=-0.682, P< 0.01) and escape latency (r=-0.568, P< 0.01), but positively associated with number of platform crossings (r= 0.516, P< 0.01), hippocampal cAMP and BDNF levels (r= 0.419, r= 0.477, P< 0.01, respectively).③ Hippocampal cAMP levels were negatively associated with plasma Mn levels (r=-0.759, P< 0.01), hippocampal Mn levels (r=-0.592, P< 0.05) and escape latency (r=-0.406, P< 0.01), but positively associated with number of platform crossings (r= 0.495, P< 0.01).④ Hippocampal Mn levels were negatively associated with number of platform crossings (r=-0.734, P< 0.01), but positively associated with escape latency (r= 0.862, P< 0.01).⑤ Plasma Mn levels were positively associated with hippocampal Mn levels (r= 0.669, P< 0.01) and escape latency(r= 0.541, P<0.01), but negatively associated with hippocampal cAMP levels (r=-0.759, P<0.01), hippocampal BDNF levels (r=-0.872, P<0.01) and plasma BDNF levels (r=-0.483, P< 0.01). However, no statistically significant correlation was found between plasma Mn concentrations and number of platform crossings (r=-0.290, P>0.05).⑥ Teeth Mn levels were positively associated with hippocampal Mn levels (r= 0.760, P< 0.01) and escape latency (r= 0.716, P<0.01), but negatively associated with number of platform crossings (r=-0.514, P<0.01), hippocampal cAMP levels (r=-0.693, P<0.01), hippocampal BDNF levels (r=-0.812, P<0.01) and plasma BDNF levels (r=-0.617, P< 0.01).(7) Hair Mn levels were positively associated with hippocampal Mi levels (r= 0.839, P< 0.01) and escape latency (r= 0.862, P<0.01), but negatively associated with number of platform crossings (r=-0.566, P<0.01), hippocampal cAMP levels (r=-0.665, P<0.01), hippocampal and plasma BDNF levels (r=-0.797, r=-0.669, P< 0.01, respectively).[Conclusions](1) Chronic manganese exposure could increase hippocampus Mai concentrations and resulting in decline in spatial learning and memory ability of rats, perhaps by inhibiting the cAMP signalling pathway in the hippocampus.(2) Plasma BDNF levels could reflect the hippocampus BDNF levels in rats.(3) Chronic manganese exposure could increase Ma levels in the teeth, hair* and plasma, which might reflect hippocampus Mn levels.(4) Teeth Mn, hair Mn and plasma BDNF might be the potential biomarker to evaluate Mn exposure and neurotoxicity in the body.