| Objective: Epilepsy is refers to the neurons spontaneous, repeated, rigiddischarge attack. Epilepsy is the most common neurodegenrative diseases,except stroke. The global incidence of epilepsy is about1%.It is reported thatabout half of the epilepsy patients with cognitive dysfunction.Epilepsy canlead to cognitive,emotional and behavioral abnormalities.Epilepsy lesion,seizure type and social psychological factors can affect cognition,in recentyears, the study found that antiepileptic drugs itself also has the side effects oflead to cognitive impairment.Therefore to deepen understanding of epilepsycan help new antiepileptic drug research and development, also provide abasis for rational clinical treatment.Oxidative stress is involved in manyphysiological and pathological process of neurodegenerative diseases,there isalso a recent study found that the oxidative stress in the pathogenesis ofepilepsy. Seizures process with increased formation of oxygen free radicals,thebrain is lipid peroxides sensitive organs,oxidative stress injury of hippocampalneurons is epileptic rats occurred,one of the important factors for thedevelopment of cognitive dysfunction. Alpha Lipoic acid is a powerfulantioxidant in vivo, has the potential for the prevention and treatment ofcognitive dysfunction caused by epilepsy. At present, alpha lipoic acid is usedfor prevention and treatment of diseases such as:diabetes and its complications,degradation of nervous system and liver function disorder symptoms.Andalpha lipoic acid in improving the role of cognitive impairment caused byepilepsy has not yet been reported.This experiment through establish chronicepileptic rat model kindled by amygdala stimulating,and give alpha lipoicacid intervention.We observed the alteration of behavior and the hippocampalneuronal injury in rats, the expression and distribution of CaMKⅡa and P-CaMKⅡa protein in hippocampus through the methods of Western Blot andImmunohistochemistry. We observed the alteration of hippocampal tissuemorphology through the methods of Nissel staining and tested the ability oflearning and memory of rats through the methods of Morris water maze. Forexploring the neuroprotective effects of alpha lipoic acid in cognition ofepilepsy and the potential mechanisms.Methods:72clean grade adult male Wistar rats (7-8weeks) wererandomly divided into six groups consisting of12animals each.They are thenormal control group (NC group), sham operated group(S group), epilepsygroup (E group), nomal α-lipoic acid group(ALA group),α-lipoic acid pluslow dosage group(（20mg/kg ALA+E group）and α-lipoic acid plus highdosage group(（40mg/kg ALA+E group）. The normal control group：withoutany treatment; sham operated group: The amygdala implanted electrodes;epilepsy group:120%seizure threshold stimulation once a day after theamygdala implanted electrods; The nomal α-lipoic acid group:The normal ratswithout any treatment was injected intraperitoneally every day in a dose of40mg/kg α-lipoic acid; α-lipoic acid plus low dosage group and α-lipoic acidplus high dosage group: The administration work was conducted between08:30-09:30AM, the rats were received ALA pretreatment, respectively,indoses of20mg/kg and40mg/kg, after ALA were given30min,120%seizurethreshold stimulation once a day, a total of15. Animals were observed for1hour, seizure activity was observed for scored according to Racine.Stage0: noresponse; Stage1: hyperactivity, vibrissae twitching; Stage2: head nodding,head clonus and myoclonic jerk; Stage3: unilateral forelimb clonus; Stage4:rearing with bilateral forelimb clonus; Stage5: generalized tonic-clonicseizure (GTCS) with loss of postural control.24h after the last administration,Morris water maze test was performed.Before the traning started, rats wereallowed to swim freely in the pool for120s without platform.(1)Visibleplatform trial: to exclude the sensorimotor or motivational factors in rats onlearning performance, we added the visible trial on the last day. Rats weregiven four trials per day similar to those described above for the hidden platform trial, but the escape platform was elevated above water surface2cm,the escape latency were recorded.(2)Place navigation test was to test the rats’learning ability. Rats were given two sessions per day for4days. Each sessioncomprised four trials, with an intertribal interval of60s, and the intersessioninterval was>2h. In each trail, the rat was gently placed into the pool at themiddle of the circular edge in a randomly selected quadrant, with the nosepointing toward the wall. If rats could not find escape to the platform within120s by themselves, they were placed on the platform by hand and allowed toremain there for30s and their escape latency was accepted as120s. Afterclimbing onto the platform, the animal remained there for30s before thecommencement of the next trial.(3)Spatial probe test: on the fifth day, a probetrial without the platform was assessed, and the crossing times was recorded.After the behavior test:(1) six rats from each group were perfused and thebrains were removed, Nissel staining was performed to observe the neurondamage in hippocampus in rats;The expression of P-CaMKⅡa protein in theCA1region of the hippocampus was determined through the methods ofImmunohistochemistry.(2)6rats in each group were sacrificed and thehippocampus were quickly separated. The expression of CaMKⅡa andP-CaMK Ⅱa protein in hippocampus was determined through the methods ofWestern Blot.Results:1Results of Morris water maze （:1）Visible platform trial:There was nosignificant difference among the groups (P>0.05).（2）Place navigation test：Comparisons were made among the mean escape latencies of each group oneach day. On day1. the mean escape latency of rats in E group was longerthan that in NC group;Compared with E group, the mean escape latencies ofthe rats among20mg/kg ALA+E group,40mg/kg ALA+Egroup,ALAgroup,Sgroup were shorter and the comparisons of the six groups were of nodifference in significance(P>0.05).On day2,the escape latency of each groupwas much shorter than the first day(P<0.05);The escape latency of the rats inE group was longer than that in NC group(P<0.05);Compared with E group, the mean escape latencies of the rats among20mg/kg ALA+E group,40mg/kgALA+Egroup,ALAgroup, S group were much shorter (P<0.05)，and comparedwith NC group have no statistical significance.On day3,the escape latency ofeach group was much shorter than the second day(P<0.05);The results amongNC group, E group,20mg/kg ALA+E group,40mg/kg ALA+Egroup,ALAgroup, S group consisted with the second day.On day4,the escape latency ofeach group was much shorter than the third day(P<0.05);The results amongNC group, E group,20mg/kg ALA+E group,40mg/kg ALA+Egroup,ALAgroup,S group consisted with the third day.(3)Spatial probe test: The numberof crossing times through the platform quadrant in E group decreasedobviously compared with that in NC group(P<0.05);Compared with E group,the numbers of crossing times through the platform quadrant in20mg/kgALA+E group and40mg/kg ALA+E group increased significantly,but Wereof no statistic significance compared with that in NC group,ALA group and Sgroup(P>0.05).2Results of Nissl staining:Neurons in NC group, ALA group and Sgroup were well-distributed karyotin,clear with normal nucleolus and richnissl bodies in kytoplasm, there was no significantly neuron loss. While in theE group, neuron loss was obviously, with shrunken plasma body and pyknoticnuclei. In the20mg/kgALA+E group and40mg/kgALA+E group, mostneurons cells were normal and only a few showed chromatin condensation.3Result of Immunohistochemistry:The P-CaMKⅡa of hippocampusimmunohistochemical products were brown and most of them were in theendochylema of the neuron. The number of immunopositive cells in E groupwas decreased obviously compared with NC group(P<0.05). The number ofimmunopositive cells in20mg/kg ALA+E group and40mg/kg ALA+E groupwas increased obviously compared with E group(P<0.05).Comparisons ofneuron expressing P-CaMKⅡa immunohistochemical products amongNC,ALA,S,20mg/kg ALA+E group and40mg/kg ALA+E groups were of nodifference in significance (P>0.05).4Result of Western blot: The expression level of CaMKⅡa protein in hippocampus in rats in each group were determined by CaMKⅡa/GAPDH.The expression of CaMKⅡa protein in E group was decreasedobviously compared with NC group (P<0.05). The expression of CaMK Ⅱaprotein in20mg/kg ALA+E group and40mg/kg ALA+E group was increasedobviously compared with E group (P<0.05). The expression of CaMKⅡaprotein among S,NC, ALA,20mg/kgALA+E group and40mg/kg ALA+Egroup were no significant difference (P>0.05).The expression level of P-CaMKⅡa protein in hippocampus in rats ineach group were determined by P-CaMKⅡa/GAPDH.The expression ofP-CaMKⅡa protein in E group was decreased obviously compared with NCgroup (P<0.05). The expression of P-CaMKⅡa protein in20mg/kg ALA+Egroup and40mg/kgALA+E group was increased obviously compared with Egroup (P<0.05). The expression of P-CaMKⅡa protein among S, NC,ALA,20mg/kgALA+E group and40mg/kgALA+E group were no significantdifference (P>0.05).Conclusions:1This study adopts the amygdala stimulating-kindled chronic epilepsy ratmodel,which can well simulate human cognitive dysfunction after epilepsy.Itis an ideal model to the study of cognitive impairment after seizures.2Epilepsy could make the expression level of CaMKⅡa and P-CaMKⅡaprotein reduced and damage the brain.The study suggested that CaMK Ⅱa andP-CaMK Ⅱa protein might be associated with cognitive impairment afterepilepsy.3ALA could obviously improve the impaired cognitive function afterepilepsy and increased the expression level of CaMKⅡa and P-CaMKⅡaprotein in the hippocampus. Accordingly, alpha lipoic acid could improvecognitive impairment after epileptic and have a certain protective effect onbrain tissue.To achieve this effect may be by adjusting CaMKⅡa and P-CaMK Ⅱa protein activity and resistance to oxidative stress effect. |
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