| Objective:To explore the intervention effect of CBI, an autophagy inhibitor, in theprevention of neurobehavioral and pathological demages after flurothyl-induced recurrentneonatal seizures in rats.Methods:6-day-old (P6) Sprague-Dawley (SD) rats (quantity:88) were randomlydivided into four groups(n=22/each group): the control group (CONT group),the normalcontrol after CBI-treated group (CCBI group),the recurrent neonatal seizure group (RSgroup), the CBI-treated seizure group (CBI group). Rats in RS and CBI groups had fiveseizures per day for nine consecutive days, with a minimum of30min between seizures.And the seizures were induced by using flurothyl. Each rat in CCBI and CBI groups waspretreated with CBI (2μl,0.5g·L-1) before seizures were induced. In addition, rats inCONT and CCBI groups were treated without flurotlyl. We examined development indexes(weight, ear separation, incisor eruptio),neuralbehavioral deficits during postnatal day1today35. Mossy fiber sprouting in hippocampus was determined by Neo-Timm's staining atP35. Zinc transporter3(ZnT3) and Clusterin protein levels in hippocampus and cerebralcortex were determined by western blot method at P35.Results:1. behavior analysis:(1) The weights of rats in RS group were lighter thanCONT and CBI group from P10to P14(P＜0.01).(2) In the Open-field behavior test: theactivities of RS group and CBI group were markedly reduced than CONT group in thehorizontal movements(P＜0.05), the RS group were significantly decreased than theCONT group in the vertical motions (P＜0.05).(3) Morris water maze test〈:1〉The averagelatencies of all rats were reduced by degree from D1to D5. The escape latency wassignificantly longer in RS group than that in CONT group at D5(P＜0.01).And the escapelatency was significantly longer in RS group than that in CBI group at D2and D5(P＜0.05).〈2〉As far as search strategy is concerned, rats of RS group performed worse than those in CONT and CBI groups (P＜0.05).〈3〉The frequency of passing through theplatform quadrant in rats had no significantly difference among the four groups (P＞0.05).2. Neo-Timm's analysis:Timm staining showed that a lot of aberrant mossy fibersprouting was seen in the inner molecular layer of the granule cells of dentate gyrus andthe stratum pyramidale of CA3subfield in RS group. In the CA3subfield and dentategyrus of the RS group, aberrant mossy fiber density was significantly increased than that ofCONT group and CBI group(P﹤0.01).In CA3subfield of CBI group, aberrant mossy fiberdensity was obviously increased than that of CONT group(P﹤0.01). However, there wereno significantly difference between CONT group and CCBI group(P＞0.05).3. Western blot analysis:Protein expression is consistent in hippocampus and cortex.ZnT3and Clusterin expression in RS group was significantly higher than that in CONTgroup and CBI group at P35(P＜0.05).But there was no significant difference of betweenCONT group and CBI group at P35(P＞0.05).And there was no significant differencebetween CONT group and CCBI group at P35(P＞0.05).Conclusions:1. Maturation of physical characteristics, neurobehavioral and learningability were damage following flurothyl-induced recurrent neonatal seizures, which leadedto the aberrant mossy fiber sprouting in the CA3subfield and dentate gyrus, and resulted inthe significantly increased level of ZnT3and Clusterin protein in hippocampus andcerebral cortex.2. CBI used before seizures could significantly improve growing development,emotional function and cognitive ability, and reduce the neurobehavioral injury afterrecurrent seizures.3. CBI used before seizures could downregulate the aberrant mossy fiber sprouting inthe CA3subfield and the dentate gyrus after recurrent seizures and downregulate theexpression of ZnT3and Clusterin protein in hippocampus and cerebral cortex at P35.4. CBI has significant protective function on recurrent neonatal seizure-induced braindamage through ZnT3/Clusterin pathway. |
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