The Intervention Effect Of Ketogenic Diet On Neurobehavioral Changes Induced By Recurrent Neonatal Seizures And The Expression Of Sprouting-related Genes

Objective: To explore the intervention effect of ketogenic diet (KD) in the preventionof neurobehavioral demages after flurothyl-induced recurrent neonatal seizures in rats andthe effect on the expression of hippocampal regenerative sprouting-related genes.Methods: Postnatal days8(P8) Sprague-Dawley (SD) rats (quantity:48) wererandomly divided into two groups: the non-seizure group (NS group, n=24) and therecurrent-seizure group (RS group, n=24).Rats were fed for one day in the currentenvironment. From P9, rats in RS group were subjected to recurrent seizures induced byvolatile flurothyl30min each day for consecutive8days. While rats in NS group wereplaced into the container for an equal amount of time to their counterpart withoutexposuring to flurothyl. At P28, according to whether rats were fed on KD or not, eachgroup was divided into two groups again: non-seizure and normal diet group(NS+NDgroup, n=12)、 non-seizure and ketogenic diet group(NS+KD group, n=12)、recurrent-seizure and normal diet group(RS+ND group, n=12)、recurrent-seizure andketogenic diet group(RS+KD group, n=12). NS+ND group and RS+ND group were fed onnormal diet while NS+KD group and RS+KD group were fed on ketogenic diet. Rats werefed for four weeks. We monitored levels of glucose and ketone in the blood at P28、P35、P42、P49. During the diet(P28～P49), we measured the weight for each rat every threedays. And We examined scores on neurological behavior by forelimb suspensionexperiment、 plane righting experiment、negative geotaxis reaction experiment、cliffavoidance test at P35、P42、P49. At P42and P49, rats were subjected to open field test, andexploratory behavior was assessed. During P50to P57, rats underwent testing in theMorris water maze to assess visual-spatial learning and memory. Zinc transporter3(ZnT3) and Metallothionein-3(MT3)、ApoE and Clusterin(CLU)、Acyl-coenzyme A:cholesterolacyltransferase-1(ACAT-1) protein levels in hippocampus and cerebral cortex weredetermined by western blot at P58. Mossy fiber sprouting in hippocampus was determinedby Neo-Timm’s staining at P58.Results:1. Weight analysis: Before the intervention of ketogenic diet, the weights ofrats in RS group were significantly lighter than NS group from P17to P27(P＜0.01). Afterthe intervention of ketogenic diet, there were some differences among four groups(P＜0.01). Compared with NS+ND group, the weights of rats in NS+KD group and RS+NDgroup were significantly lighter from P28to P49(P＜0.01). And the weights of rats inRS+KD group were lighter than RS+ND group from P31to P49(P＜0.01).2. Neurodevelopmental indicators analysis: Compared with NS+ND group, thetime of forelimb suspension、negative geotaxis reaction and cliff avoidance in RS+NDgroup was longer at P35、P42、P49(P＜0.05), while the time of plane righting in RS+NDgroup at P35、P42、P49was shorter (P＜0.05). Compared with RS+ND group, the time offorelimb suspension、negative geotaxis reaction and cliff avoidance in RS+KD group wasshorter at P35、P42、P49(P＜0.05), while the time of plane righting in RS+ND group atP42、P49was longer(P＜0.05). Compared with NS+ND group, the time of forelimbsuspension and negative geotaxis reaction in NS+KD group was longer at P35、P42、P49(P＜0.05).3. Open field test analysis: Compared with NS+ND group, the delay time in theRS+ND group was longer at P42、P49(P＜0.05), the scores in RS+ND group was lower atP42、P49(P＜0.05), the times of grooming in RS+ND group was more at P42、P49(P＜0.05)；the delay time in the NS+KD group was longer at P49(P＜0.05) and the times ofgrooming in NS+KD group was more at P42(P＜0.05). Compared with RS+ND group,the delay time in the RS+KD group was shorter at P42、P49(P＜0.05), the scores inRS+KD group was higher at P42、P49(P＜0.05), the times of grooming in the RS+KDgroup was fewer at P42、P49(P＜0.05).4. Morris water maze analysis:⑴T he escape latencyof four groups shortenedgradually in consecutive five days. The escape latency in RS+ND group was longer thanNS+ND group from D1to D5(P＜0.05), and the escape latency in RS+KD group was shorter than RS+ND group from D4to D5(P＜0.05).⑵The frequency of passing throughthe platform quadrant in RS+ND group was much less than NS+ND group(P＜0.05), andthe frequency of passing through the platform quadrant in RS+KD group was more thanRS+ND group(P＜0.05).5. Neo-Timm’s analysis：Aberrant mossy fiber sprouting in the stratum pyramidale ofCA3subfield and the inner molecular layer of the granule cells of dentate gyrus in RS+NDgroup was more than that in NS+ND group(P＜0.01). In the dentate gyrus and CA3subfield of the RS+KD group, aberrant mossy fiber density was significantly decreasedthan that of the RS+ND group(P＜0.05).6. Western blot analysis：⑴Zinc ion transport signaling pathway:Compared toNS+ND group, the level of ZnT3and MT3in cerebral cortex of the RS+ND group werehigher (P＜0.05) and the level of ZnT3and MT3in hippocampus of the RS+ND groupwere significantly higher (P＜0.01). And compared to RS+ND group, the level of ZnT3and MT3in hippocampus and cerebral cortex of the RS+KD group were lower (P＜0.05).But there were no significantly difference between NS+ND group and NS+KD group (P＞0.05) and there were no significantly difference between NS+ND group and RS+KD group(P＞0.05).⑵Lipid metabolism signaling pathway: Compared to NS+ND group, the levelof ApoE and ACAT-1in hippocampus and cerebral cortex of the RS+ND group weresignificantly higher (P＜0.01) and the level of CLU in hippocampus and cerebral cortex ofthe RS+ND group were higher (P＜0.05). And compared to RS+ND group, the level ofCLU and ACAT-1in hippocampus and cerebral cortex of the RS+KD group were lower (P＜0.05), the level of ApoE in hippocampus of the RS+KD group was significantly lower(P＜0.01) and the level of ApoE in cerebral cortex of the RS+KD group were lower (P＜0.05). But there were no significantly difference between NS+ND group and NS+KDgroup (P＞0.05) and there were no significantly difference between NS+ND group andRS+KD group (P＞0.05).Conclusions：1. Flurothyl-induced recurrent neonatal seizures may damage growthand development, neurobehavioral and learning ability and leaded to more aberrant mossyfiber sprouting in the stratum pyramidale of CA3subfield and the inner molecular layer ofthe granule cells of dentate gyrus. These changes may be related to up-regulation of zinc ion transport related molecules ZnT3and MT3and lipid metabolism related moleculesApoE、CLU and ACAT-1.2. Ketogenic diet after recurrent neonatal seizures could reduce body weight andimprove neurobehavior. These changes may be related to down-regulation of zinc iontransport related molecules ZnT3and MT3and lipid metabolism related molecules ApoE、CLU and ACAT-1.3. It showed that the neuroprotective effects of ketogenic diet on recurrent neonatalseizure-induced brain damage might be through the zinc ion transport signaling pathwayand lipid metabolism signaling pathway.