| Purposes:To study the relationship between learning and memory impairments in temporal lobe epilepsy (TLE) rats and the immunity activity of choline acetyltransferase (ChAT) in prefrontal cortex (PFC), and to discuss the neuromechanism of epilepsy with learing and memory impairments, thus to provide clinical proofs in the therapy of epilepsy with learning and memory impairments. To discuss the protection and therapy effects of Scorpion Venom heat-resistance protein (SVHRP) on Kainic acid (KA) made TLE model rats with defects in special learning and memory, and to study the protection ability of SVHRP on ChAT immunity positive neurons in PFC.Methods:50 healthy SD (Sprague-Dawley) rats were chosen and 10 rats were selected randomly as the saline control group (NS+NS). Other 40 rats were injected with epilepsy-caused dose (10mg/kg, 5mg/ml) of KA, and 34 rats behaving epilepsy in degree 4-5 were selected. The 34 rats were divided into 2 groups on the average randomly. One group was KA+SVHRP, the other was KA+NS. In the next 10 days, intraperitoneal injection was executed in KA+SVHRP rats with SVHRP (0.25mg/kg, 50μg/ml, 5ml/kg), meanwhile, NS+NS and KA+NS rats were injected with the same dose of normal saline in the same way. Injection was performed for 10 days, and then all the rats were injected with subthreshold dose of KA (5mg/kg,5mg/ml) subcutaneously for the selection of TLE model animals after the test of sensibility of seizure.KA+NS rats which were alive and acted in degree 4-5 seizure were chosen, KA+SVHRP rats which were alive and didn't act in degree 4-5 seizure were chosen, Morris water maze (MWM) was used to test the ability of learning and memory in 3 groups. The rats were than executed and brains were taken out to test the number of ChAT positive neurons in PFC with immunohistochemistry (ABC method), and the number was used to show thequantity of acetylcholine (Ach) indirectly.Results:No degree 4-5 seizure was detected in NS+NS rats after injection of subthreshold dose of KA; 10 KA+NS rats behaved as degree 4-5 seizure, among which 5 as degree 5, 5 as degree 4, and 1 as degree 3, 1 as degree 2, 0 in degree 1 and 0; comparatively, in SVHRP treated rats there was 0 rat behaved as degree 5, 1 as degree 4, 2 as degree 3, 2 as degree 2, 4 as degree 1, 5 as degree 0. Rank sum test of ordered variables was used to test seizure degrees and found that the seizure degrees in KA+SVHRP rats were less than those in KA+NS rats. There was statistical significance (P<0.05) between the 2 groups. It suggested that SVHRP could restrain the function of KA and to reduce epilepsy sensibility.Results of MWM showed statistical significance (F=166.193, P<0.05) in time factor (day), suggesting that latency has the tendency of changing with time. Statistical significance(F=23.706, P<0.05)in the interaction between day and group (day×group) suggested that time factor changed according to difference groups. Group factor contributed (F=237.926,P<0.05) in 3 groups, suggesting latency difference in 3 groups. In place test in 5 days, compared with NS+NS (n=10) group, KA+NS (n=10) group has longer latency in day 1, day 2, day3, day4, day5, and there was difference (P<0.05) in the results; KA+NS group has shorter latency in day 1, day 2, day3, day4, day5 than KA+SVHRP (n=13) group, and there was difference(P<0.05), but there was no marked difference between NS+NS and KA+SVHRP group. It suggested that compared with saline control rats, the ability of spacial learning ability in TLE rats have been decreased, and SVHRP could improved the demolished learning ability caused by KA.Results of occupied percentage of time in target quadrant (TR) and opposite quadrant (OP) in spatial probe test showed a classic advantage search strategy in saline group compared with a classic edge strategy in TLE group. TLE rats crossed the platform for 0.70±0.67 times, saline control and SVHRP treated rats were 3.30±1.64 and 2.54±0.97 times respectively. F=13.727, P<0.05 in 3 groups, suggesting marked difference among 3 groups. TLE group crossed the platform less time than saline group and the result has statistical significance (P<0.05), implying that KA caused TLE rats have descended memory; and SVHRP treated group crossed more times than TLE group, implying that SVHRP treated rats have improved memory ability than KA caused TLE rats; but there was no marked difference between saline group and SVHRP treated group.Results in immunohistochemistry showed that NS+NS rats had 19.70±3.75 ChATimmunity positive cholinergic neurons, and the number was 11.80±4.31 in KA+NS rats and 12.94±3.40 in KA+SVHRP rats within the area of 3120.4μm2. KA caused TLE rats had less number of ChAT immunity positive cholinergic neurons compared with saline rats (P<0.05); and SVHRP treated TLE rats had more ChAT neurons than TLE rats, but no statistical significance was presented. It suggested that KA caused TLE could descend the number of ChAT immunity positive cholinergic neurons in PFC of rats but SVHRP could not improve the impaired number of ChAT immunity positive cholinergic neurons caused by KA effectively.Conclusions : Learning and memory ability in KA caused TLE rats was significantly decreased and SVHRP could improve learning and memory ability, but it could not improve the decreased number of ChAT positive neurons caused by TLE. It suggested that the decreased spacial learning and memory ability was correlated with descended number of ChAT immunity positive neurons and damage of acetylcholine system in PFC. SVHRP may has no significant therapy capacity in improving the damaged cholinergic neurons in PFC, suggesting that SVHRP could improve ChAT positive neurons in some other brain domains except PFC to improve learning and memory ability of rats in this experiment. Further study was expected in the clarification of the function of cholinergic neurons in some brain areas besides PFC. It is significantly meaningful that the experiment will illuminate the pathogenesis of epilepsy and it will act as a guide in the clinical therapy of epilepsy with learning and memory impairments. |
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