Study On The Expression Of Brain Damage Markers And The Intervention Of Leptin By Using Two-hit Seizure Model

Objective:To study the effect of acute leptin intervention on seizure-induced brain injury in neonatal rats by using two-hit seizure model with trifluoroethylether and penicillin.Methods:Forty postnatal Days 6 Sprague-Dawley?SD?rats were divided into four groups according to the random number table?10 rats in each group?including blank control group?Control group?;Leptin group?Leptin group?;simple seizure group?RS group?and convulsive plus leptin group?RS+Leptin group?.Rats were fed for 1 day before the study.,from P7,RS+Leptin group and RS group were treated with trifluoroether repeatedly to induce seizure,5 times a day for 7 days?P7-P13?,and the model of trifluoroethylether seizure in neonatal rats was established successfully.Leptin group and RS+Leptin group were given leptin?4mg/kg?intraperitoneally after 1 hour of daily seizures.Neurobehavioral tests?negative reflexes,cliff avoidance reflexes,forelimb suspension reflexes,open field experiments,and planar reflexes?were performed at P27and P34;swimming experiments were performed at P14 and P20;water maze test was performed on P27-P33.The body weight of the rats was weighed to determine the body weight changes.In P35,rats were injected intraperitoneally with penicillin,and the behavioral changes were observed,the seizure threshold and survival rate was recorded.Subsequently,each group randomly received 5,a total of 20,decapitated brain,using Timm staining method to observe the hippocampal moss fiber abnormal germination.The expression of MAP-2,S100B and GFAP protein in the hippocampus and cerebral cortex of each group were detected by immunohistochemistry.Results:1.The effect on weight:There were significant differences in body weight between the four groups and the difference was statistically significant?p<0.05?.Furthermore,the body weight of RS+Leptin group and RS group were significantly lower than those of control group at each time point of P12-P34?P<0.05?,and the difference was statistically significant?p<0.05?.There was no significant difference between Control group and Leptin group?p>0.05?.2.Neurological score:?1?Negative reflections:P27 and P34,the difference was statistically significant[F_P27?3,16?=44.674,p<0.05;F_P34?3,16?=149.179,p<0.05],compared with the control group,the negative time of RS group was significantly prolonged?p<0.05?,RS+Leptin group compared with RS group,the negative time of RS+Leptin group was significantly shortened?p<0.05?.?2?forelimb suspension reflex:P27and P34,the difference was statistically significant[F_P27?3,16?=26.642,p<0.05;F_P34?3,16?=76.687,p<0.05],compared with the control group,the suspension time of RS group was significantly shortened?p<0.05?,RS+Leptin group compared with RS group,the suspension time of RS+Leptin group was significantly prolonged?p<0.05?.?3?cliff avoidance reflex:P27 and P34,the difference was statistically significant[F_P27?3,16?=140.972,p<0.05;F_P34?3,16?=21.026,p<0.05],compared with the control group,the avoidance time of RS group was significantly prolonged?p<0.05?,RS+Leptin group compared with RS group,the cliff avoidance time of RS+Leptin group was significantly shortened?p<0.05?.?4?plane righting reflex:P27 and P34,the difference was statistically significant[F_P27?3,16?=41.552,p<0.05;F_P34?3,16?=40.143,p<0.05],compared with the Control group,RS group was significantly longer?p<0.05?,RS+Leptin group compared with RS group,the plane correction of RS+Leptin group was significantly shortened?p<0.05?.?5?swimming experime nt:P14 and P20,the difference was statistically significant[F_P14?3,16?=14.762,p<0.05;F_P20?3,16?=7.935,p<0.05],compared with the Control group,the swimming score of RS group was significantly lower?p<0.05?,compared with RS group,RS+Leptin swimming group showed a significant increase in swimming experiment score?p<0.05?.3.Open field experiment:?1?delay time:P27 and P34,the difference was statistically significant[F_P27?3,16?=30.899,p<0.05;F_P34?3,16?=9.730,p<0.05],compared with the control group,the delay time of RS group was significantly prolonged?p<0.05?,RS+Leptin group compared with RS group,the delay time of RS+Leptin group was significantly shortened?p<0.05?.?2?opening score:P27 and P34,the difference was statistically significant[F_P27?3,16?=50.143,p<0.05;F_P34?3,16?=17.408,p<0.05],compared with the Control group,the opening score of RS group was significantly decreased?p<0.05?,RS+Leptin group compared with RS group,the opening score of RS+Leptin group increased significantly?p<0.05?.?3?level Score:P27 and P34,the scores of the four groups were different and the difference was statistically significant[F_P27?3,16?=48.868,p<0.05;F_P34?3,16?=13.564,p<0.05].compared with the Control group,the level score of RS group was significantly decreased?p<0.05?,RS+Leptin group compared with RS group,RS+Leptin group significantly increased the level of score?p<0.05?.?4?vertical score:P27 and P34,the difference was statistically significant[F _P27?3,16?=10.719,p<0.05;F_P34?3,16?=6.646,p<0.05).P27,compared with the Control group,the vertical score of RS group was significantly decreased?p<0.05?,RS+Leptin group compared with RS group,RS+Leptin group significantly increased the vertical score?p<0.05?;P34,compared with the Control group,the vertical score of RS group was significantly decreased?p<0.05?,there was no significant difference between RS+Leptin group and RS group?p>0.05?.4.Morris water maze:?1?place navigation test:two-way ANOVA analysis showed that there was a significant diffierence in the interaction between the intergroup factors and the number of training days in the four groups of water maze escape[F?12,48?=9.186,p<0.05],the significant difference in training days[F?4,16?=794.3,p<0.05)],significant difference between groups[F?3,12?=335.0,p<0.05];Further comparison:on 1-5 days of the test,compared with the control group,the escape latency of RS group was significantly longer than that of control group?p<0.05?.on 2-5days of the test,The escape latency of the RS+Leptin group?the seizure group after leptin acute intervention?was significantly shorter than that of the RS group?p<0.05?.?2?Spatial probe test:There were significant differences in the number of cross-platform between the four groups[F?3,16?=17.600,p<0.05],compared with the control group,the number of crossing the platform in the RS group was significantly lower than that in the Control group?p<0.05?,and the number of the RS+Leptin group was significantly higher than that of the RS group?P<0.05?.5.Seizure latency and survival rate:?1?seizure latency:there was significant difference between the four groups[F?3,36?=8.650,p<0.05],compared with the Control group,the seizure latency of RS group was significantly decreased,RS+Leptin group compared with RS group,RS+Leptin group seizure latency was significantly increased?p<0.05?.?2?survival rate:Control group?80%?,Leptin group?90%?,RS group?40%?,RS+Leptin group?70%?.6.Neo-Timm staining analysis:There was significant difference in the semi-quantitative score of aberrant mossy fiber sprouting in CA3 subfield and dentate gyrus between the four groups[F_CA3?3,16?=73.457,p<0.05;F_{dentate gyrus}?3,16?=80.690,p<0.05],compared with the Control group,the germination of RS group was significantly increased?p<0.05?,RS+Leptin group compared with RS group,RS+Leptin group significantly reduced the germination?p<0.05?.7.Immunohistoche mical staining analysis:?1?MAP-2 protein:The number of MAP-2 positive cells in hippocampal CA3?CA1 region and cerebral cortex of four groups was different and the difference was statistically significant[F_CA3?3,16?=98.509,p<0.05;F_{CA 1}?3,16?=40.616,p<0.05;F _{cerebral cortex}?3,16?=120.180,p<0.05],compared with Control group,the expression of MAP-2 protein in RS group was significantly decreased?p<0.05?,RS+Leptin group compared with RS group,RS+Leptin group showed significant increase in MAP-2 protein cells?p<0.05?,The number of MAP-2positive cells in hippocampal dentate gyrus o f four groups was not statistically significant[F_{dentate gyrus}?3,16?=0.917,p>0.05].?2?S100B protein:The number of S100B positive cells in hippocampal CA3?CA1?dentate gyrus and cerebral cortex of four groups was different and the difference was statistically significant[F_{CA 3}?3,16?=191.422,p<0.05;F_CA1?3,16?=141.788,p<0.05;F_{dentate gyrus}?3,16?=419.310,p<0.05;F_{cerebral cortex}?3,16?=465.197,p<0.05),compared with the Control group,the expression of S100B cells in RS group was significantly increased?p<0.05?,RS+Leptin group compared with RS group,the expression of S100B protein in RS+Leptin group was significantly decreased?p<0.05?.?3?GFAP protein:The number of GFAP-positive cells in hippocampal CA3?CA1?dentate gyrus and cerebral cortex of four groups was different and statistically significant[F_CA3?3,16?=313.358,p<0.05;F_CA1?3,16?=49.256,p<0.05;F_{dentate gyrus}?3,16?=59.773,p<0.05;F_{cerebral cortex}?3,16?=156.166,p<0.05],compared with the Control group,the expression of GFAP cells in RS group was significantly increased?p<0.05?,RS+Leptin group compared with RS group,the expression of GFAP protein in RS+Leptin group was significantly decreased?p<0.05?.Conclusions:1.Flurothyl-induced recurrent neonatal seizures damaged growth?development and neurobehavioral,decreased seizure threshold and survival rate,leaded to more aberrant mossy fiber sprouting in the stratum pyramidale of CA3 subfield and the inner molecular layer of the granule cells of dentate gyrus,increased the numbe r of positive cells of S100B and GFAP in the CA3 region,CA1 region and dentate gyrus of the hippocampus and cerebral cortex,decreased the number of positive cells of MAP-2 in the CA3 region and CA1 region of the hippocampus and cerebral cortex.2.The use of leptin in the acute phase of seizures can significantly improve the neurological behavior and cognitive impairment in rats,reduce aberrant mossy fiber sprouting in the stratum pyramidale of CA3 subfield and the inner molecular layer of the granule cells of dentate gyrus,improve the seizure threshold and survival rate,reduce the number of positive cells of S100B and GFAP in the CA3 region,CA1 region and dentate gyrus of the hippocampus and cerebral cortex,increase the number of positive cells of MAP-2 in the CA3 region and CA1 region of the hippocampus and cerebral cortex.3.This study shows that leptin plays a neuroprotective role following neonatal seizure-induced brain injury.