Regulation Of Ketogenic Diet On Recurrent Neonatal Seizuresinduced Brain Damage And The Signaling Pathways

Objective: To investigate the regulation of ketogenic diet on recurrent neonatal seizures- induced brain damage and the signaling pathways.Methods: 48 Sprague-Dawley(SD) rats were assigned to two groups randomly:recurrent seizures group(RS group) and non-seizures group(NS group). The rats in RS group were subjected to recurrent seizures during the consecutive 8 days, from P9 to P16,and seizures continued for 30 min each day.According to the diet intervention, at P21, the rats were further divided into four groups randomly: non-seizure without KD group(NS+ND), non-seizure with KD group(NS+KD), recurrent seizure without KD group(RS+ND) and recurrent seizure with KD(RS+KD). Neurobehavioral parameters(plane righting test, cliff avoidance test, forelimb suspension test and negative geotaxis test) were observed at P28 and P35. The open field test was performed at P35 to assess the exploratory behavior and emotion. The Morris water maze test was performed from P37 to P43 to assess the learning and memory of the rats. All the rats were sacrificed on P43. The expression of Cathepsin E, CLU, Beclin-1, Bcl-2, LC3Ⅱ/LC3Ⅰ, p62 and c PLA2 were detected by western blot. Mossy fiber sprouting in hippocampus was detected by Neo-Timm’s staining. Monitor the weight, blood glucose and blood ketone of the rats during the experiment.Results: 1. Weight analysis: There were significant differences [F(3,33)=383.9,P<0.0001] among the four groups. Post-hoc test,The weight of the rats in RS+ND group was droped significantly compared to NS+ND group from P21 to 43(P < 0.05). Both in NS+KD group and RS+KD group, the ketogenic diet can decreased the weight of rats significantly from P25 to P43.(P<0.05).2. Neurobehavioral parameters analysis:(1) plane righting test:There were no significant differences among the four groups(P >0.05) both at P28 and P35.(2) cliffavoidance test: There were no significant differences among the four groups at P28(P>0.05);There were significant differences among the four groups at P35(P<0.05). Post-hoc test,compared to NS+ND, the time in RS+ND group was longer(P<0.05), and compared to RS+ND group the time was shorter in RS+KD group(P < 0.05).(3) forelimb suspension test: There were no significant differences among the four groups at P28(P>0.05);There were significant differences among the four groups at P35(P<0.05). Post-hoc test,compared to NS+ND, the time in RS+ND group was shorter(P<0.05), and compared to RS+ND group the time was longer in RS+KD group(P<0.05).(4) negative geotaxis test :There were significant differences among the four groups at both P28 and P35(P<0.05). Post-hoc test,compared to NS+ND group, the time in RS+ND group was longer(P<0.05), and compared to RS+ND group, the time in RS+KD group was shorter(P<0.05)both at P28 and P35;(5) the open field test: compared to NS+ND group, the delay time in RS+ND group was longer(P<0.05), the score of horizontal activity, vertical activity and locomotor activity was significantly reduced in RS+ND group(P<0.05); compared with RS+ND group, the delay time was shorter in RS+KD group(P<0.05), the score of vertical activity and locomotor activity was significantly increased in RS+KD group(P<0.05),but there were no significant differences between RS+KD and RS+ND group about the score of horizontal.There no significant differences among the four groups about gloom times.3. Morris water maze analysis:(1) Repeated two-way ANOVA of escape latencies revealed significant group difference [F(3,33)=11.79, P < 0.0001], training day difference[F(4,44)=13.34, P < 0.0001] and interaction [F(12,132)=2.661, P=0.0031]between training day factor and group factor. Subsequent comparision, escape latency was longer significantly in RS +ND group than that in NS+ND group at day3,day 4 and day5(P < 0.05), escape latency was longer significantly in RS+KD group than that in NS+ND group only at day 4(P<0.05).(2)The frequency of passing through the platform in RS+ND group was much less than that in NS+ND group(P < 0.05).However, the frequency in RS+KD group was much more than that in RS+ND group(P<0.05).3. Neo-Timm’s analysis: Campared to NS+ND group, aberrant mossy fiber sprouting was significantly increased in the hippocampus CA3 and dentate gyrus(DG) areas inRS+ND group(P < 0.05). KD can significantly decreased aberrant mossy fiber which induced by neonatal recurrent seizures(P<0.05).4.Western blot analysis: Neonatal recurrent seizures induced significantly high expression of Cathepsin E, CLU, p62 in both hippocampus and cerebral cortex and Beclin-1 in hippocampus(P < 0.05). In addition Neonatal recurrent seizures induced significantly down-regulated expression of LC3Ⅱ/LC3Ⅰ in hippocanmpus and cerebral cortex and c PLA2 in hippocanmpus(P<0.05). Ketogenic diet can regulate the abnormal expression of the above mentioned proteins. There are no significant differences in the expression of Bcl-2 and Beclin-1/ Bcl2 among the four groups.Conclusions: 1. Inhalated flurothyl-induced neonatal recurrent seizures can demage the growth and development and lead to neurobehavioral and cognitive deficit, ketogenic diet improve neurobehavioral and cognitive deficit which due to neonatal recurrent seizures.2. Ketogenic diet can block aberrant mossy fiber sprouting in hippocampus CA3 and DG areas induced by neonatal recurrent seizures.3. Neonatal recurrent seizures induced significantly high expression of Cathepsin E,CLU, p62 and Beclin-1(only in hippocampus) and low expression of LC3Ⅱ/LC3Ⅰ and c PLA2(in hippocanmpus). Ketogenic diet can regulate the abnormal expression of the above mentioned proteins.4. It indicated that neonatal recurrent seizures induced changes of autophagy and lipid metabolism pathway related proteins in long-term hippocampus and cortex. Ketogenic diet might play neuroprotective effect through autophagy and lipid metabolism signaling pathway.