The Intervention Effect Of Leptin In Acute Phase On Neonatal Recurrent Seizures Induced Brain Damage And The Mechanism

Objective:To explore the intervention effect and mechanism of leptin(LEP) in acute phase on neurobehavioral、learning and memory demages induced by flurothyl- induced long-term recurrent neonatal seizures in rats.Methods:On postnatal day 7(P7), 40 Sprague-Dawley rats were randomly assigned to four groups:(n=10/each group): the control group(CON group)、the normal control plus LEP-treated group(LEP group)、the flurothyl- induced recurrent seizures group(RS group), the recurrent seizures group plus LEP-treated group(RS+LEP group). The rats of CON group were given normal feeding daily. The rats of RSgroup were feeding as CON group, and flurothyl- induced seizures lasted 30 min per day from P7 to P13.The rats of RS+MEL group were treated as RS group, in addition,the intraperitoneal injection of leptin(4mg/kg) were given to rats 1h later after the convulsion.The rats of CON+LEP group were treated as RS+LEP group without flurothyl.We observed these neurobehavioral results respectively at P23 and P30,such as plane righting test,cliff avoidance test,negative geotaxis reaction test and forelimb suspension test.The open field test was performed at P23 and P30. Morris water maze test was performed during P27 to P33. All the rats were sacrificed at P34.The mossy fiber sprouting was detected by Timm staining,and the protein levels of Beclin-1, Beclin-1/Bcl-2,Cathepsin E and P53 in cerebral cortex andhippocampus were detected by western blot analysis.Results : 1. Neurobehavioralanalysis : The time of plane righting,cliff avoidance,negative geotaxis reaction in RS group was longer than CON group at P23 and P30(P<0.05), while the time of plane righting,cliff avoidance,negative geotaxis reaction in RS+LEP group was trend to normal,and was shorter than CON group at P23 and P30(P<0.05).The time of forelimb suspension in RS group was shorter than CON group at P23 and P30(P<0.05), while the time of forelimb suspension in RS+LEP group was trend to normal,and was longer than RS group at P23(P<0.05).2. Open-field behavior testanalysis:(1)The delay time of the RS group was longer than CON group at P23 and P30(P<0.05),while the delay time of the RS+LEP group was trend to normal,and was shorter than RS group at P23 and P30(P<0.05).(2)The level scores of RS group were higher than CON group(P<0.05), while the level scores of the RS+LEP group was trend to normal,and was lower than RS group at P23 and P30(P<0.05).(3)The vertical scores of RS group were higher than CON group(P<0.05), while the vertical scores of the RS+LEP group was trend to normal,and was lower than RS group at P23 and P30(P<0.05).(4)The locomotor scores of RS group were higher than CON group(P<0.05), while the locomotor scores of the RS+LEP group was trend to normal,and was lower than RS group at P23 and P30(P<0.05).3.Morris water maze analysis:(1) Escape latency :the average latency of four groups was reduced by degree from P28 to P32. The escape latency of RS group was longer than CON group at P29, P30,P31 and P32(P<0.05),while the escape latency of RS+LEP group was trend to normal,and was shorter than RS group at P29, P30,P31 and P32(P<0.05).(2) The probe test:the frequency of passing through the platform of RS group was much less than CON group at P34(P<0.05),while the frequency of passing through the platform of RS+LEP group was trend to normal,and was much more than RS group at P34(P<0.05)4. Neo-Timm’s analysis:Aberrant mossy fiber sprouting in the hippocampal CA3 region and dentate gyrus of RS group was much more than that in CON group(P<0.05),while the aberrant mossy fiber sprouting in the hippocampal CA3 region and dentate gyrus of RS+LEP group was trend to normal,and was much less than that in RS group(P<0.05),5. Western blot analysis :(1)The protein level of Beclin-1 、Beclin-1/Bcl-2、 Cathepsin E、P53 in cerebral cortex of the RS group were higher than CON group(P <0.05);while the protein level of Beclin-1、Beclin-1/Bcl-2、Cathepsin E、P53 in cerebral cortex of the RS+LEP group were trend to normal,were lower than RS group(P <0.05).(2)The protein level of Beclin-1、Beclin-1/Bcl-2、Cathepsin E、P53 in hippocampal of the RS group were higher than CON group(P <0.05);while the protein level of Beclin-1、Beclin-1/Bcl-2、Cathepsin E in hippocampal of the RS+LEP group were trend to normal,were lower than RS group(P<0.05).Conclusions : 1.The intervention of leptin in acute phase after seizures can significantly impare the deficit of neurobehavioral and learning ability caused by long-term recurrent neonatal seizures in rats,it also can significantly suppress the aberrant mossy fiber sprouting in the hippocampal dentate gyrus and CA3 region.It shows that leptin plays a role of neuroprotection.2.The intervention of leptin in acute phase after neonatal seizures can suppress theabnormal expression of autophagy signaling pathway protein such as Beclin-1, Beclin-1/Bcl-2,Cathepsin Eand P53.3. It shows that the intervention of leptin in acute phase after seizures may play neuroprotective effect through autophagy signaling pathway.