| Objective:To investigate the effect of leptin on long-term neurobehavioral and seizure threshold of secondary strike experiment in neonatal seizure rats induced by lithium chloride-pilocarpine and its intervention mechanism.Methods:A total of 50 postnatal day5(P5)Sprague-Dawley(SD)rats were randomly divided into four groups:blank control group(Control group),simple leptin group(Leptin group),simple seizure group(RS group),convulsions with leptin group(RS+Leptin group).Control group and leptin group were 10 respectively,RS group and RS+Leptin group were 30 rats in total.All the rats in RS group and RS+Leptin group were injected intraperitoneally with lithium chloride(5mEq/kg)in P6.Pilocarpine(320 mg/kg)was administered intraperitoneally 30 minutes after scopolamine methyl chloride(1mg/kg)injection at P7 to block peripheral effect of pilocarpine.The rats from the successful model were randomly assigned to the RS group and RS+Leptin group(n=10/group).The intraperitoneal injection of leptin(4mg/kg)were given to rats in Leptin group and RS+Leptin group from P8 to P14.The neurological reflex(planar correction experiment,forelimb suspension test,negative trend test,cliff avoidance experiment)and cognitive emotion test(open field test)were performed at P23 and P30.Morris water maze was performed on P28~P33.All the animals were intraperitoneally injected with penicillin(5.1×10~6U/kg/d)and at P34 and the first time of seizure was recorded in 1 hour.The mossy fiber sprouting was detected by Timm staining.Western blot was performed to detect the expression of Zinc transporter 3(ZnT3)and Calbindin D28k(CB-D28k)in the cortex and hipeppocampus.Results:1.Neurobehavioralanalysis:At P23 and P33,there were significant differences in the time of forelimb suspension among four groups(P<0.05).The time of forelimb suspension in RS group was shorter than that in Control group(P<0.05),while the time of forelimb suspension in RS+Leptin group was longer than that in RS group(P<0.05).There were significant differences in the time of plane righting,negative geotaxis and cliff avoidance reaction among four groups at P23 and P30(P<0.05).The time of plane righting,negative geotaxis and cliff avoidance reaction in RS group was longer than that in Control group(P<0.05),while the time of plane righting,negative geotaxis and cliff avoidance reaction in RS+Leptin group was shorter than that in RS group(P<0.05).2.Open-field behavior testanalysis:(1)At P23 and P33,there were significant differences in the delay time among four groups(P<0.05).The delay time of the RS group was longer than that in Control group(P<0.05),while the delay time of the RS+Leptin group was shorter than that in RS group(P<0.05).(2)There were significant differences in the locomotor scores among four groups at P23 and P30(P<0.05).The locomotor scores of RS group were higher than that in Control group(P<0.05),while the locomotor scores of the RS+Leptin group was lower than that in RS group(P<0.05).3.Morris water maze analysis:(1)Place navigation test:there were significant differences among day[F(3,12)=53.70,P<0.0001],group[F(4,12)=84.98,P<0.0001],interaction[F(12,80)=1.95,P<0.05]by repeated two-way ANOVA analysis from P28 to P32.The escape latency of RS group was longer than Control group at P29 to P32(P<0.05),while the escape latency of RS+Leptin group was shorter than RS group(P<0.05).(2)The probe test:the frequency of passing through the platform of RS group was much less than Control group at P34(P<0.05),while the frequency of passing through the platform of RS+Leptin group was much more than RS group(P<0.05).4.Seizure threshold:There were significant differences in the seizure threshold among four groups at P34(P<0.05).The seizure threshold of RS group were lower than that in Control group(P<0.05),while the seizure threshold of the RS+Leptin group was higher than that in RS group(P<0.05).5.Neo-Timm's analysis:There were significant differences in the aberrant mossy fiber sprouting in the hippocampal CA3 region and dentate gyrus among four groups(P<0.05).Aberrant mossy fiber sprouting in the hippocampal CA3 region and dentate gyrus of RS group was much more than that in Control group(P<0.05),while the aberrant mossy fiber sprouting in the hippocampal CA3 region and dentate gyrus of RS+Leptin group was much less than that in RS group(P<0.05),6.Western blot analysis:(1)There were significant differences in the protein level of ZnT3 among four groups(P<0.05).The protein level of ZnT3 in cerebral cortex and hippocampal of the RS group was higher than Control group(P<0.05),while The protein level of ZnT3 of the RS+Leptin group was lower than RS group(P<0.05).(2)There were significant differences in the protein level of CB-D28k among four groups(P<0.05).The protein level of CB-D28k in cerebral cortex and hippocampal of the RS group was lower than Control group(P<0.05),while The protein level of CB-D28k of the RS+Leptin group was higher than RS group(P<0.05).7.The analysis of linear correlation:(1)There was negative correlation between the expression of ZnT3 and CB-D28K in the cortex(r=-0.682,P=0.003<0.05).(2)There was negative correlation between ZnT3 and CB-D28K expression in the hippocampus too(r=-0.552,P=0.012<0.05).(3)There was negative correlation between the total expression of ZnT3 in cortex and hippocampus and the total expression of CB-D28k(r=-0.596,P=0.00005<0.05).Conclusions:1.Leptin can improve the long-term neurobehavioral and hippocampal mossy fiber germination in rats with neonatal seizure induced by lithium chloride-pilocarpine.2.The intraperitoneal injection of penicillin at P34 on the basis of Lithium chloride-pilocarpine can build a second strike model in rats,and leptin can improve the seizure threshold of seizure rats.3.The intervention of leptin after seizure can reduce the expression of ZnT3 and increase the expression of CB-D28k in the cortex and hippocampal.There was a significant negative correlation between the expression levels of the two molecules.4.This study suggests that the neuroprotective effect of leptin against lithium chloride-pilocarpine in neonatal seizures rats may be achieved by reducing the expression of ZnT3 and increasing the expression of CB-D28k in brain. |
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