| Objective: To explore the intervention effect of ketogenic diet(KD) in the prevention of neurobehavioral demages after flurothyl-induced recurrent neonatal seizures in rats and the effect on the expression of related genes.Methods: Postnatal days 8(P8) Sprague-Dawley(SD) rats(quantity: 24) were randomly divided into two groups: the control group(NS group) and the recurrent-seizure group(RS group), each group has twelve rats. From P9, rats in RS group were subjected to recurrent seizures induced by volatile flurothyl 30 min each day for consecutive 8 days. While rats in NS group were done the same expect exposuring to flurothyl. At P21, according to whether rats were fed on KD or not, each group was divided into two groups again: non-seizure and normal diet group(NS+ND group)、non-seizure and ketogenic diet group(NS+KD group) 、 recurrent-seizure and normal diet group(RS+ND group) 、recurrent-seizure and ketogenic diet group(RS+KD group), each group has six rats. NS+ND group and RS+ND group were fed on normal diet, while NS+KD group and RS+KD group were fed on ketogenic diet. Rats were fed for ten days. Blood glucose and beta-hydroxybutyric acid were detected by cutting rats’ vena caudalis on the 0 day、7 day and 10 day of ketogenic diet intervention. The rats’ weight were recorded every other day from P9. Neurobehavioral tests include plane righting experiment、cliff avoidance test、forelimb suspension experiment、negative geotaxis reaction experiment were implemented twice respectively on P27 and P31, While Open Field Test were implemented on P31. On P32, the totally 24 rats were sacrificed and Western blot were applied to detect the related protein expression levels of Cathepsin E, ZIP7, MT3, CLU in hippocampus and cerebral cortex.Results: 1. Weight analysis: Before the intervention of ketogenic diet, the weights of rats in RS group were significantly lighter than NS group from P17 to P20(P<0.05). After the intervention of ketogenic diet, there were some differences among four groups(P<0.05). Compared with NS+ND group, the weights of rats in NS+KD group were significantly lighter from P25 to P31(P<0.05). And the weights of rats in RS+KD group were lighter than RS+ND group from P23 to P31(P<0.05).2. Neurobehavioral test:(1) Plane righting test: Compared with NS+ND group, the time of plane righting in RS+ND group was shorter(P<0.05); while compared with RS+ND group, the time of plane righting in RS+KD group was longer(P<0.05).(2) Cliff avoidance test: Compared with NS+ND group, the time of cliff avoidance in RS+ND group was longer(P<0.05); while compared with RS+ND group, the time of cliff avoidance in RS+KD group was shorter(P<0.05).(3) Forelimb suspension test: Compared with NS+ND group, the time of forelimb suspension in RS+ND group was shorter(P<0.05); while compared with RS+ND group, the time of forelimb suspension in RS+KD group was longer(P<0.05).(4) Negative geotaxis reaction test: Compared with NS+ND group, the time of negative geotaxis reaction in RS+ND group was longer(P<0.05); while compared with RS+ND group, the time of negative geotaxis reaction in RS+KD group was shorter(P<0.05).3. Open field test analysis: Compared with NS+ND group, the delay time in the RS+ND group was longer(P<0.05), the scores in RS+ND group was lower(P<0.05), the times of grooming in RS+ND group was more(P<0.05). Compared with RS+ND group, the delay time in the RS+KD group was shorter(P<0.05), the scores in RS+KD group was higher(P<0.05), the times of grooming in the RS+KD group was fewer(P<0.05).4. Western blot analysis:(1) the level of Cathepsin E, MT3 and CLU in both hippocampus and cerebral cortex in RS+ND group was significantly higher than those in NS+ND group(P<0.05); While the proteins level in RS+KD group was significantly lower than those in RS+ND group(P<0.05).(2) ZIP7 protein level of both hippocampus and cerebral cortex in RS+ND group was significantly lower than those in NS+ND group(P<0.05); While the protein level in RS+KD group was significantly higher than those in RS+ND group(P<0.05).Conclusions: 1. Flurothyl-induced recurrent neonatal seizures may damage growth and development, neurobehavioral and cognition of emotion, and resulted in significantly increased level of Cathepsin E, MT3, CLU protein and decreased level of ZIP7 protein in hippocampus and cerebral cortex.2. Ketogenic diet after recurrent neonatal seizures could reduce body weight and improve neurobehavior and cognition of emotion. These changes may be related to down-regulation of Cathepsin E, MT3, CLU and up-regulation of ZIP7.3. It showed that the neuroprotective effects of ketogenic diet on recurrent neonatal seizure-induced brain damage might be through the zinc ion transport signaling pathway and lipid metabolism signaling pathway.4. Ketogenic diet can cause weight loss, but no link between weight loss and damage neurobehavior. |
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