The Intervention Effect Of Melatonin On Neurobehavioral And Pathological Changes Induced By Recurrent Neonatal Seizures

Objective：To explore the intervention effect of melatonin(MEL) in the prevention ofneurobehavioral demages after flurothyl-induced recurrent neonatal seizures in rats and theeffect on the expression of hippocampal regenerative sprouting-related genes..Methods：6-day-old (P6) Sprague-Dawley (SD) rats (quantity:72) were randomlydivided into four groups(n=18/each group): the control group (CON group)、the normalcontrol after MEL-treated group (MEL group)、the recurrent neonatal seizure group (RSgroup), the MEL-treated seizure group (RS+MEL group). Rats in RS and RS+MELgroups had five seizures per day for nine consecutive days, with a minimum of30minbetween seizures. And the seizures were induced by using flurothyl. Each rat in MEL andRS+MEL groups was pretreated with MEL (55mg/kg) before seizures were induced. Inaddition, rats in CON and MEL groups were treated without flurotlyl. At3h after the lastconvulsion, Beclin1and Bcl-2protein level in hippocampus and cerebral cortex weredetected by western blot method. We examined scores on neurological behavior by planerighting experiment、negative geotaxis reaction experiment、cliff avoidance test、forelimbsuspension experiment at P24、P31. During P28to P35, rats underwent testing in theMorris water maze to assess visual-spatial learning and memory. At P35, rats weresubjected to open field test, and exploratory behavior was assessed. Zinc transporter3(ZnT3)，CaMKII， ACAT-1and Cathepsin E protein levels in hippocampus and cerebralcortex were determined by western blot method at P35. Mossy fiber sprouting inhippocampus was determined by Neo-Timm’s staining at P35.Results：1.Western blot analysis：⑴Compared to CON group, the level of Beclin-1in hippocampus and cerebral cortex of the RS group were higher (P＜0.05).And comparedto RS group, the level of Beclin-1in hippocampus and cerebral cortex of the RS+MELgroup were lower (P＜0.05). But there were no significantly difference between RS+MELgroup and CON group (P＞0.05) and there were no significantly difference between CON group and MEL group (P＞0.05).⑵Compared to CON group, the level of Bcl-2inhippocampus and cerebral cortex of the RS group were significantly lower (P＜0.05). Andcompared to RS group, the level of Bcl-2in hippocampus and cerebral cortex of the RS+MEL group were higher (P＜0.05). But there were no significantly difference betweenRS+MEL group and CON group (P＞0.05) and there were no significantly differencebetween CON group and MEL group (P＞0.05).2. behavior analysis：(1) Compared with CON group, the time of negative geotaxisreaction and cliff avoidance in RS group was longer at P24，P31(P＜0.05), while the timeof plane righting and forelimb suspension in RS group at P24，P31was shorter (P＜0.05).Compared with RS group, the time of negative geotaxis reaction and cliff avoidance inRS+MEL group was shorter at P24，P31(P＜0.05), while the time of plane righting andforelimb suspension in RS+MEL group at P24，P31was longer(P＜0.05).(2) In theOpen-field behavior test: Compared with CON group, the delay time in the RS group waslonger (P＜0.05);the activities of RS group were markedly reduced than CON group inthe horizontal movements(P＜0.05), while RS+MEL group were significantly increasedthan RS group (P＜0.05).(3) Morris water maze test:〈1〉The average latencies of all ratswere reduced by degree from D1to D5. The escape latency was significantly longer in RSgroup than that in CON group at D2,D4,D5(P＜0.05).And the escape latency wassignificantly shorter in RS+MELgroup than that in RS group at D2,D4,D5(P＜0.05).〈2〉The frequency of passing through the platform quadrant in RS group was much less thanCON group(P＜0.05).3. Neo-Timm’s analysis：Aberrant mossy fiber sprouting in the stratum pyramidaleof CA3subfield and the inner molecular layer of the granule cells of dentate gyrus in RSgroup was more than that in CON group(P＜0.01). In the dentate gyrus and CA3subfieldof the RS+MEL group, aberrant mossy fiber density was significantly decreased than thatof the RS group(P＜0.05).4. Western blot analysis：⑴Compared to CON group, the level of ZnT3，ACAT-1and Cathepsin E in hippocampus and cerebral cortex of the RS group were higher(P＜0.05).And compared to RS group, the level of ZnT3and Cathepsin E in hippocampusand cerebral cortex of the RS+MEL group were lower (P＜0.05). And compared to RS But there were no significantly difference between RS+MEL group and CON group (P＞0.05) and there were no significantly difference between CON group and MEL group (P＞0.05).⑵Compared to CON group, the level of CaMKII in hippocampus and cerebralcortex of the RS group were significantly lower (P＜0.05). And compared to RS group, thelevel of CaMKII in hippocampus and cerebral cortex of the RS+MEL group were higher(P＜0.05). But there were no significantly difference between RS+MEL group and CONgroup (P＞0.05) and there were no significantly difference between CON group and MELgroup (P＞0.05).Conclusions：1. Neurobehavioral and learning ability were damage followingflurothyl-induced recurrent neonatal seizures, which leaded to the aberrant mossy fibersprouting in the CA3subfield and dentate gyrus, and resulted in the significantly increasedlevel of Beclin-1, ZnT3,ACAT-1and Cathepsin E protein and decreased level of Bcl-2、CaMKII protein in hippocampus and cerebral cortex.2. MEL used before seizures could significantly improve impared neurobehavioraland learning ability after recurrent seizures.3. MEL used before seizures could suppressed the aberrant mossy fiber sprouting inthe CA3subfield and the dentate gyrus after recurrent seizures and downregulate theexpression of Beclin-1，ZnT3，ACAT-1and Cathepsin E in hippocampus and cerebralcortex.Additionally, MEL could upregulate the expression of Bcl-2and CaMKII inhippocampus and cerebral cortex.4. MEL has significant protective function on recurrent neonatal seizure-induced braindamage through decreasing the expression of Beclin-1, ZnT3, ACAT-1and Cathepsin Eand increasing the expression of Bcl-2, CaMKII, ACAT-1and Cathepsin E protein inhippocampus and cerebral cortex.