Intervention And Mechanisms Of A Calpain Inhibitor On Developmental Seizure-induced Brain Damage

Object: To explore the long-term effects of penicillin-induced developmental seizureson mossy fiber sprouting (MFS), the intervention and mechanisms of E-64d ondevelopmental seizure-induced brain damage.Methods: Eighty-sixth Sprague-Dawley rats(postnatal days P21)were randomlydivided into four groups:the control group (CONT group, n=19),the E-64d treated-controlgroup(ECONT group,n=19),the recurrent-seizure group (RS group,n=24) and the E-64dtreated-seizure group (ERS group,n=24).The penicillin was used to induce seizure attack.Rats in RS group were injected i.p. with penicillin(5.1×106U·kg-1·d-1)every other day ineleven consecutive days from postnatal day21to induce seizure; Rats from the CONTgroup were injected equal amount of normal sodium(NS) for the same time, while ratsfrom the ECONT group were only injected E-64d(4μg,1μg/μl);In E-64d group, E-64d(4μg,1μg/μl)was injected each day before seizure was induced. Selected randomly12ratsthat up to standard from each group. At21d after the last convulsion, ZnT-3,ApoE andCLU protein level in hippocampus and cerebral cortex were detected by western blotmethod. At the same time, mossy fiber sprouting were determined by Timm staining.Results:1. Neo-Timm's analysis: In CA3subfield and dentate gyrus of RS group,aberrant mossy fiber density was significantly increased than that of CONT group (P﹤0.01). In CA3subfield and dentate gyrus of ERS group, aberrant mossy fiber density wassignificantly decreased than that of RS group(P﹤0.01). However, there were nosignificantly difference between CONT and ECONT groups(P>0.05).2. Western blot analysis:(1)The expression of ZnT-3in hippocampus and cerebralcortex of RS group was significantly higher than that of CONT group and ERS group(P<0.05).There were no significant difference between ERS and CONT group(P>0.05).There were also no significant difference between ECONT and CONTgroup(P>0.05).(2)The expression of ApoE protein in hippocampus and cerebral cortex ofRS group were significantly higher than that of CONT group (P<0.01). The expression ofApoE in hippocampus and cerebral cortex of ERS group were lower than that of RS group,but there were no significant difference. There were no significant difference between ERSand CONT group (P>0.05). There were also no significant difference between ECONT andCONT group (P>0.05).(3) The expression of CLU protein in hippocampus and cerebralcortex of RS group were significantly higher than that of CONT group (P<0.01). Theexpression of CLU in hippocampus and cerebral cortex of ERS group were lower than thatof RS group, but there were no significant difference. There were no significant differencebetween ERS and CONT group (P>0.05). There were also no significant differencebetween ECONT and CONT group (P>0.05).Conclusion:1. Aberrant mossy fiber sprouting was found in the CA3subfield anddentate gyrus following penicillin-induced developmental seizures.The protein level ofZnT-3,ApoE,CLU were significantly increased in hippocampus and cerebral cortex in thelong-term period following penicillin-induced developmental seizures, which wereassociated with the aberrant mossy fiber sprouting.2. Pretreatment with E-64d could significantly inhibit the aberrant mossy fibersprouting in the CA3subfield and the dentate gyrus after penicillin-induced recurrentdevelopmental seizures and decrease the abnormal expression of ZnT-3,ApoE and CLUprotein in hippocampus and cerebral cortex.3. These results showed that the effects of E-64d on recurrent developmentalseizure-induced brain damage might be through decreasing the expression of ZnT-3,ApoEand CLU protein in hippocampus and cerebral cortex.