Long-term Effects On Hippocampus Caused By Seizures In Immature Rats And The Protective Effects Of Celecoxib

Objective:Lithium-pilocarpine was used to induce recurrent seizures model of rats. To investigate the changes of mossy fiber sprouting(MFS) in hippocampus of rats which suffered lithium-pilocarpine induced repeatedly seizures, and the correlation between MFS and spontaneous recurrent seizures(SRS).Methods:1. Preparation of the rats modelOne hundred and ninety immature rats were randomly divided into four groups: control group(n=48), EP1group(n=80), blank group(n=12), EP2group(n=50). Lithium-pilocarpine was used to induce epilepsy model of rats. Rats were given intraperitoneal injections of pilocarpine on PN21, PN25and PN29to induce recurrent seizures. The changes of MFS in hippocampus were examined on days1,3,7,14,20,30,45and60by Timm staining. Rats in EP2group were observed every day and were subdivided into EP2-SRS and EP2-nonSRS group, then the difference in MFS was compared between the two groups. The necrosis and apoptosis of the neurons in hippocampus were observed and the neurons in CA3and CA1areas were counted by Nissl staining, thus the effect of SRS on the hippocampal neurons could be assessed.2. Timm stainingThe sections were developed in the dark for45to60minutes in Timm solution. After washing, the slices were dehydrated in graded alcohol, cleared in xylene, and mounted on slides with Permount. The severities of MFS were calculated in inner molecular layer and CA3areas. 3. Nissl stainingAfter dewaxed, the slices were incubated for30minutes at60℃by thionine, dehydrated by gradient alcohol, and sealed after those processes. Then we observed the shape of hippocampal neurons and neuronal loss in each group.Results:1.The recurrent seizures rat model induced by the injection of lithium-pilocarpine was an ideal model. The model can partly reflect the recurrent seizures of clinical patients, and it could be used for experiments.2. Timm stainingCompared with control group, significant MFS phenomenon was detected from day14to day60after recurrent seizures in EP1group(P<0.05). There was no difference in MFS between EP2-SRS and EP2-nonSRS groups.3. Nissl stainingCompared with blank group, prominent neuron loss was observed in CA3and CA1regions of the rats with SRS(P<0.05).Conclusion:Recurrent seizures induced by lithium-pilocarpine in immature rats can cause an increase of MFS in the hippocampus, while there is a dissociation between SRS and MFS in the lithium-pilocarpine model of epilepsy. Objective:To explore cox-2inhibitors, celecoxib’s protective role in the attenuation of rats’ hippocampal neurons which were caused by lithium chloride-pilocarpine induced recurrent seizures and the mechanisms.Methods:1. Preparation of the rats modelTwenty immature rats were randomly divided into three groups:Control group (n=6), lithium-pilocarpine induced epilepsy group (n=12), Celecoxib treatment epilepsy group (Celecoxib group)(n=12). Lithium-pilocarpine was used to induce epilepsy model of rats. Rats were given intraperitoneal injections of pilocarpine on PN21, PN25and PN29to induce recurrent seizures. The rats in Celecoxib group were administered Celecoxib after epileptic challenges. They were fed with celecoxib (20mg/kg, o.p.) daily until45days after lithium-pilocarpine induced seizures.2. Morris water mazeAfter40days treatment with celecoxib, spatial learning and memorizing were tested by using Morris water maze experiment.3. Timm stainingThe sections were developed in the dark for45to60minutes in Timm solution. After washing, the slices were dehydrated in graded alcohol, cleared in xylene, and mounted on slides with Permount. The severities of MFS were calculated in inner molecular layer and CA3areas.4. Nissl stainingAfter dewaxed, the slices were incubated for30minutes at60℃by thionine, dehydrated by gradient alcohol, and sealed after those processes. Then we observed the shape of hippocampal neurons and neuronal loss in each group.5. Immunohistochemical experimentThe slices were dewaxed, repaired, sealed, add with the first antibody and second antibody on it, colored the target hippocampal neurons, dehydrated and sealed.Results:1. Morris water mazeCompared with control group, the escape latency of epilepsy group and celecoxib group is longer(P<0.05); the escape latency of celecoxib group is longer than epilepsy group(P<0.05).2. Timm stainingIn inner molecular layer and CA3area, the average Timm’s score of the epilepsy group and celecoxib group were significantly higher than that of the control rats(P<0.05); there was no difference in the average Timm’s score between celecoxib groupe and control group(P>0.05).3. Nissl stainingThere were obvious damaged neurons and neuronal loss in CA3and CA1areas at epilepsy group(P<0.05). Compared with epilepsy group, the loss of neurons in celecoxib group were reduced(P<0.05).4. Immunohistochemical experimentCompared with control group, the expression of NF-xBp65increased both in epilepsy group and celecoxib group(P<0.05); compared with epilepsy group, the expression of NF-κBp65in celecoxib group had an obviously decrease (P<0.05).Conclusion:Cox-2inhibitors, celecoxib exertes a neural protective role in the attenuation of rats’ hippocampal neurons which were caused by lithium chloride-pilocarpine induced recurrent seizures. The anticonvulsant mechanism of celecoxib may be that it reduces local inflammation reaction of hippocampal neurons. Celecoxib may cause damages to the spatial learning and memory ability.