Changes Of Arc/Arg3.1 And GluR1 In Hippocampus Of Chronic Epileptic Rats And The Impact Of Memantine

Objectives: Clinical investigations have shown that many epileptic patients have deleterious cognitive and behavioral consequences at the same time of seizures, which have greatly affect the quality of life. As well as the pathogen of epilepsy and the side effects of antiepileptic drugs, the epilepsy itself is one of the main factors for cognitive impairment in patients with epilepsy. Arc/Arg3.1 is an immediate early gene (IEG) which is rapidly and transiently induced in neurons in response to physiological and pathological stimuli, and plays an important role in synaptic plasticity and memory storage. AMPA receptors are ionotropic glutamate receptors involved in synaptic plasticity and synaptic transmission, and related with long-term potentiation. We used pentylenetetrazole to establish a stable animal model of chronic epilepsy, and applied NMDA receptor antagonist-memantine to intervene the chronic epileptic rats. We also observed the learning and memory changes of epileptic rats by Morris water maze, as well as applied Western blot and RT-PCR to detect Arc/Arg3.1 and the AMPA receptor-GluR1 protein and transcription levels, to study the role of memantine on epileptic rats, and to explore the molecular mechanism of the cognitive impairment of epilepsy.Methods: Adult male Sprague–Dawley (SD) rats were selected through Y-Maze tests and then divided into 4 groups randomly. The 20 rats in the normal control group (NC) were injected saline intraperitoneally (i.p) by 3.5ml/kg for consecutive 44 days. The 35 rats in PTZ group received intraperitoneal injection of 1% pentylenetetrazol (PTZ) by 35mg/kg for consecutive 44 days. The 35 rats in PTZ+MMT group received intraperitoneal injection of 1% pentylenetetrazol (PTZ) by 35mg/kg for consecutive 30 days. From the 31st day on,the rats in PTZ+MMT group were injected memantine intraperitoneally by 10 mg/kg 15 minutes before PTZ in the rest 14 days. The 20 Rats in MMT group were injected intraperitoneally saline by 3.5ml/kg for consecutive 30 days. From the 31st day on, the rat in MMT group were injected MMT intraperitoneally by 10 mg/kg 15 minutes before saline in the rest 14 days. Then those rats were tested with Morris Water Maze (MWM) for the measurement of learning and memory abilities. The expression of Arc/Arg3.1 and the AMPA receptor GluR1 transcription and protein levels were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot.Results: Beginning from day 6 on, rats in PTZ and PTZ+MMT groups had seizures induced by repetitive PTZ, which were characterized by head shaking, squealing and crawling. Moreover, loss of righting reflex and generalized tonic–clonic convulsions were seen on day 18~day 24, which indicated reaching the kindling standard. Rats were regarded as fully kindled when they exhibited seizure stagesⅣ~Ⅴin 3 times continuously. On day 30, the numbers of the rats being fully kindled in PTZ group, PTZ+MMT group were 25, 27 respectively. No seizures were found in the NC group and MMT group. On day 31~day 44, the performance of PTZ group rats was like the former, as well as the PTZ+MMT group still had seizures stagesⅣ~Ⅴ, but the lasting time of seizures and the recovering time after seizures were shorter than before.Place navigation results in MWM test: Comparisons were made among the mean escape latencies on each day. On day 1, comparisons of the four groups were no difference in significance(P>0.05).On day 2, the mean escape latencies of rats in PTZ group(18.6±6.13)s and MMT group (14.4±4.37)s were much longer (P<0.01) than that in NC group (7.61±6.13)s. Moreover, the mean escape latency of rats in PTZ+MMT group (8.40±1.96) s was shorter than that of rats in PTZ group (P<0.05). On day 3, the mean escape latency of the rats in PTZ group (16.1±3.19) s was obvious different from that in NC group (6.37±1.91) s (P<0.01). The mean escape latencies of the rats in PTZ+ MMT group (7.50±2.57) s was shorter than that in PTZ group (P<0.05). The comparison of mean escape latency of the rats in MMT group (10.4±3.15) s was longer than that in NC group. On day 4, the mean escape latency of the rats in PTZ group(11.4±2.51)s was obvious different from that in NC group(4.17±1.88)s(P<0.05).The mean escape latency of the rats in PTZ+ MMT group (5.80±1.87)s was shorter than that in PTZ group(P<0.05).Spatial probe test: The number of crossing times through the platform quadrant within 120 s in PTZ group(7.01±1.13) decreased obviously compared with that in NC group(13.5±1.77)(P<0.01). The number of crossing times in PTZ+MMT group(11.83±1.03) increased obviously compared with that in PTZ group(P<0.01). The number of crossing times in MMT group (9.27±1.41) decreased compared with that in NC group (P<0.05). There were no significant difference of the swimming time spent in the platform quadrant of the rats in four groups (P>0.05).The expression of Arc mRNA in the hippocampus: The expression level in PTZ group(0.291±0.032) reduced obviously compared with that in NC group(0.776±0.039)(P<0.01).Compared with that in NC group, the Arc mRNA expression levels in PTZ+MMT (0.701±0.038) and MMT group (0.781±0.077) were of no statistic significance(P>0.05). The Arc mRNA expression levels in PTZ+MMT increased obviously compared with that in PTZ group (P<0.01).The expression of GluR1 mRNA in the hippocampus: The expression level in PTZ group(1.535±0.056) increased obviously compared with that in NC group(0.934±0.039)(P<0.01).Compared with that in NC group, the GluR1 mRNA expression levels in PTZ+MMT (0.953±0.047) and MMT group (0.962±0.052) were of no statistic significance(P>0.05). The GluR1 mRNA expression levels in PTZ+MMT reduced obviously compared with that in PTZ group (P<0.01).The expression of Arc protein in the hippocampus: The expression level in PTZ group(0.131±0.043) reduced obviously compared with that in NC group(0.352±0.061)(P<0.05).Compared with that in NC group, the Arc protein expression levels in PTZ+MMT (0.347±0.058) and MMT group (0.324±0.049) were of no statistic significance(P>0.05). The Arc protein expression levels in PTZ+MMT increased obviously compared with that in PTZ group (P<0.05). The expression of GluR1 protein in the hippocampus: The expression level in PTZ group(4.922±0.289) increased obviously compared with that in NC group(2.451±0.143)(P<0.01).Compared with that in NC group, the GluR1 protein expression levels in PTZ+MMT (1.967±0.259) and MMT group (2.319±0.280) were of no statistic significance(P>0.05). The GluR1 protein expression levels in PTZ+MMT reduced obviously compared with that in PTZ group (P<0.01).Conclusion: The epileptic model kindled by PTZ in the experiment did have impairments in learning and memory abilities.The reducing expression of Arc mRNA and protein but increasing expression of GluR1 mRNA and protein in the hippocampus could show that Arc and GluR1 might be involved in the pathogenesis of cognitive dysfunction after epilepsy. Memantine might improve spatial learning and memory of pentylenetetrazole-kindled rats via regulating Arc/Arg3.1 and GluR1 expression.