The Relationship Between Epileptic Seizures And Inflammatory Reaction And Its Influence On Cognitive Function In Rats

Objective To elucidate the relationship between the epileptic seizure and inflammatory reaction and its influence on learning and memory function in rats.Methods 110 healthy male Sprague Dawley rats were randomly divided into three group according to random number table method:control group(n=10),celecoxib pretreatment group(EP-C,n=20)and epilepsy group(EP,n=80).Epilepsy model group according to observation time is further divided into:0h ? 1h ? 3h ? 12 h ? 24 h ? 10 d and 30 d.Intraperitoneal injection Lithium-pilocarpine(127mg/kg/20mg/kg,Li-Pilo)to establish Status Epilepticus(SE)model.Observe the behavioral changes in rats with epilepsy.To monitor and record the characteristics of acute and chronic epileptic seizures in rats,including onset time of epileptic seizures,eizure frequency and duration.Nissl staining was used to detect the neuronal damage;Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of TNF-alpha and IL-1beta protein;Streptavidin-biotin-peroxidase complex immunohistochemical method was used to detect the expression levels of TNF-alpha,IL-1beta,GABAAR,NMDAR2 B,GFAP,Iba1 and NGB;Timm staining was used to detect the degree of mossy fiber sprouting(MFS)in dentate gyrus and CA3 area.In addition.Randomly selected 30 Sprague Dawley rats,RMT-100 rat eight arm maze test was performed at 5d?10d?15d?25d?35d after the status epilepticus,observe and record working memory error(WME),reference memory error(RME),the total test time,the total number into the arm and the average time to explore.Results1.The time of peripheral cholinergic response is 5.72±2.43 min after i.p.pilocarpine,and the time of unilateral forelimb clonus and forelimb clonus alternating symptoms i.p.Pilocarpine is8±2.50 min,the time of reached the seizure grade? and ?after i.p.pilocarpine is25.76±8.35 min,such as bilateral forelimb clonus,hind stand,dumping imbalance and other symptoms.The control group during the experiment is always no seizure perfprmance.2.After SE,the hippocampal neurons were damaged severely.with the progress of epileptic seizures,the number of surviving neurons in CA1 and CA3 regions showed a near linear decline.Among them,the number of surviving neurons in(12h,24 h,10d,30d)CA1 area,(0h,12 h,24h,10 d,30d)CA3 area and(12h,24 h,10d,30d)DG area were more less than the control group significantly(p<0.05~0.001).3.Hippocampus in normal control group,the TNF-alpha and IL-1beta protein levels were at a low level.After SE,these two proteins were gradually increased significantly.the time-to-peak of the former is 24 h,and then a mild decline,but continued higher than the control group.Including3 h,12h,24 h,10d after SE,the IL-1beta protein were significantly higher than the control group(p<0.01),While the time-to-peak of the latter is 1h,and then a mild decline to the lowest in 3h after SE,and then showed a rebound increase trend,but its still higher than the control group.Among them,the TNF-alpha protein level in 1h was significantly higher than the control group(p<0.05).In addition,immunohistochemistry further confirmed that the time-to-peak of IL-1beta expession in each regions of hippocampus both were 12 h after SE,then a mild decline,but still higher than the control group.Including(12h,24h)CA1,(12h,24h)CA3 and 12 h DG region were significantly higher than the control group(p<0.05~0.001).However,the time-to-peak of TNF-alpha expression in hippocampus CA1?CA3?DG area respctively were 3h?1h?12h after SE,and then gradually decline,but continued higher than the control group.Including 3hCA1,(1h,3h,24 h,10d)CA3and(0h,1h,3h,12h)DG region were significantly higher than the control group(p<0.05~0.001).4.After SE,the expression levels of NR2 B and GABAAR were increased in each district of hippocampus.the time-to-peak of the former in CA1?CA3 and DG were respectively 24h?10d and12 h after SE,followed by a downward trend,but continued higher than the control group,(12h,24 h,30d)CA1,(10d,30d)CA3 and 12 h DG region expression levels were higher than the control group,the differences were statistically significant(p<0.05~0.001).However,the latter expression levels were increased slower than the NR2 B,and CA1,CA3 and DG expression levels were gradually increased to 24h?24h and 12 h after SE respectively showed a slight downward trend,both decreased to the lowest level in 10 d after SE,followed by a rebound trend,(24h,10 d,30d)CA1,30 d CA3 and 30 d DG were significantly higher than the control group,the differences were statistically significant(p<0.05~0.001).5.After SE,the GFAP and IBA1 expression level in each region of hippocampus in different degree rise.the time-to-peak of GFAP in CA1 ? CA3 and DG were 10 d ? 12 h and 24 h respectively,and then showed a mild declne,but still higher than the control group.Including(3h,12 h,24h,10d)CA1,(12h,24 h,10d)CA3 and(12h,24h)DG were significantly higher than the control group(p<0.05~0.001).However,the expression level of IBA1 in CA1?CA3 continued torise,both time-to-peak were 30 d after SE.However,the expression level of IBA1 in DG area was slightly decreased,and time-to-peak was 24 h,and then a mild decline,but still higher than the control group.Including(10d,30d)CA1,(24h,10 d,30d)CA3 and(12h,24h)DG region were higher than the control group significantly(p<0.05~0.001).6.The time to the fourth grade of epileptic seizure and the onset of spontaneous recurrent seizures for the first time in chronic phase in EP-C group were longer than that of EP group(p<0.05),and the number of spontaneous recurrent seizures in EP-C group was significantly less than that of EP group,the differences were statistically significant(p<0.05).7.The number of hippocampal neuron survival in EP-C group were significantly more than that of EP group,and the number of CA1 and CA3 neurons in the hippocampus of these two groups were less than that of control group,and the number of DG neurons of EP group were less than that of control group,the differences both were statistically significant(P<0.001).8.The Timm particles in CA3 area of hippocampus in group EP was significantly more than those in group EP-C,and the Timm particles of CA3 and DG in two groups were significantly more than that of control group,the differences both were statistically significant(p<0.05~0.001).9.After SE,the expression level of neuroglobin(NGB)in hippocampal CA1,CA3 and DG regions were increased.and the time-to-peak of CA1 and DG regions both were 24 h after SE,but was still higher than the control group.And the CA3 area showed a continue rising trend.Among them,CA1(24h,10 d,30d),CA3(24h,10 d,30d)and DG(12h,24 h,10d,30d)were higher than that of control group significantly(p<0.05~0.001).10.After SE,with the progress of epileptic seizures,the number of WME and RME increased gradually.the number of WME(15d,25 d,35d),RME(10d,15 d,25d,35d)and total number of into arms(10d,15 d,25d)were significantly more than before(p<0.05~0.001).The total test time at each time point and the average exploration time of fifth day after SE were longer than before,the differences were statistically significant(p<0.05).11.There was a significant negative correlation between the number of RME and the number of neuron survival in hippocampal CA1 and CA3 area(r=-0.579 p=0.000;r=-0.454 p=0.002).There also was a significant negative correlation between the number of WME and the number of surviving hippocampal CA1 and CA3 area neurons(r=-0.470 p=0.001;r=-0.507 p=0.000).Conclusion:1.There is a complex network relationship between inflammation reaction and epileptic seizures,anti-inflammatory treatment can delay the onset of epilepsy and reduce the number of spontaneous seizures,it is suggested that the anti-inflammatory treatment may have anti-epileptic effect,which may provide some reference for the development of new antiepileptic drugs bytargeting some inflammation related pathways.2.Epileptic seizures seriously affects the learning and memory function of rats,with the progress of epileptic seizures,the more serious cognitive impairment.in adddition,there was a significant negative correlation between the severity of cognitive impairment and the number of surviving neurons in hippocampus,it is suggested that the cognitive dysfunction associated with epilepsy may be related to the pathological changes of hippocampus.in addition,the relative hypoxia of brain tissue caused by epileptic seizures may be one of the causes of cognitive dysfunction associated with epilepsy.