Effects And Mechanisms Of Long-term Administration Of Ketogenic Diet And Melatonin On Neonatal Seizure-induced Brain Damage In Two-hit Seizure Model

Objective: To observe the seizure activity and record the seizure threshold and survival rate after flurothyl and penicillin-induced two-hit seizure model in rats.Methods: Sprague-Dawley(SD) rats(number=120) at postnatal days 8(P8) were randomly divided into two groups: the non-seizure group(n=60) and the recurrent-seizure group(n=60). Rats were fed for 1 day before the study. From P9, rats in the recurrent-seizure group were subjected to recurrent seizures induced by volatile flurothyl five times each day for consecutive 13 days, with a interval time of 30 minutes once. While rats in the non-seizure group were placed into the container for an equal amount of time to their counterpart without exposuring to flurothyl. At P63, penicillin was dissolved in saline and administered intraperitoneally(i.p.) to all rats at a dose of 5.1×106U/kg/d, after which rats were closely observed for about 1 h. We observed seizure activity recorded the seizure threshold and survival rate.Results: Compared to the non-seizure group(31.46±7.13 min), the seizure threshold in the recurrent-seizure group was significantly smaller(13.67±5.65 min, P<0.01). There are 75% survival rate in the non-seizure group and 55% in the recurrent-seizure group.Conclusions:1. This research has established a two-hit seizure model successfully,which means penicillin was administered intraperitoneally to flurothyl-induced recurrent-seizure rats.2. After penicillin intraperitoneally, the seizure threshold in flurothyl-induced recurrent-seizure rats was smaller and had a low survival rate.Objective: To explore the effects and mechanisms of long-term administration of ketogenic diet on neurobehavioral changes and mossy fiber sprouting induced by recurrent neonatal seizures.Methods: Sprague-Dawley(SD) rats(number=60) at postnatal days 8(P8) were randomly divided into four groups: the non-seizure and normal diet group(NS+ND group,n=15) 、 the non-seizure and ketogenic diet group(NS+KD group, n=15) 、 the recurrent-seizure and normal diet group(RS+ND group, n=15)、and the recurrent-seizure and ketogenic diet group(RS+KD group, n=15). Rats were fed for 1 day before the study.From P9, rats in the RS+ND and RS+KD group were subjected to recurrent seizures induced by volatile flurothyl five times each day for consecutive 13 days, with a interval time of 30 minutes once. While rats in the NS+ND and NS+KD group were placed into the container for an equal amount of time to their counterpart without exposuring to flurothyl.At P23, rats in NS+ND and NS+KD group were fed on normal diet, while rats in RS+ND and RS+KD group were fed on ketogenic diet. Rats were fed for 30 days. We observed neurological behavior by neurological reflex test at P33、P43、P53. Morris water maze were performed from P54 to P60. Open field test were performed at P61 and P62. At P63,penicillin was dissolved in saline and administered intraperitoneally(i.p.) to all rats at a dose of 5.1×106U/kg/d, after which rats were closely observed for about 1 h. We observed seizure activity recorded the seizure latency and survival rate. We monitored levels of glucose and ketone in the blood at P63. ZIP7、Apo A1、c PLA2、P53、P62 protein levels in hippocampus and cerebral cortex were determined by western blot at P63. Mossy fiber sprouting in hippocampus was determined by Timm staining at P63.Results: 1. Weight analysis: After the intervention of ketogenic diet, there were some differences among four groups(P<0.01). Compared with NS+ND group, the weights of rats in RS+ND and RS+KD group were smaller significantly from P23 to P53(P<0.01),the weights of rats in NS+KD group were smaller significantly from P26 to P53(P<0.01).Compared with RS+ND group, the weights of rats in RS+KD group were smaller than RS+ND group from P29 to P53(P<0.01).2. Neurodevelopmental indicators analysis: ⑴ Plane righting reflex test: At P33,there were no significant differences among the four groups[F(3, 16) =0.51,P=0.682>0.05]. At P43, there were significant differences among the four groups[F(3, 16) =19.95,P<0.0001]. Post-hoc test, compared with NS+ND group, the time in RS+ND group was shorter(P<0.05).There were no significant differences among the other groups(P>0.05).At P53, there were significant differences among the four groups[F(3, 16) =7.23,P=0.003<0.01]. Post-hoc test, compared with RS+ND group, the time in RS+KD group was longer(P<0.05). There were no significant differences among the other groups(P>0.05).⑵Forelimb suspension test: At P33, there were significant differences among the four groups[F(3, 16) =6.3,P=0.005<0.01]. Post-hoc test, compared with NS+ND group,the time in RS+ND group was shorter(P<0.05). There were no significant differences among the other groups(P>0.05).At P43, there were no significant differences among the four groups[F(3, 16) =0.34, P=0.797 > 0.05]. At P53, there were no significant differencesamong the four groups[F(3, 16) =1.5, P=0.252 > 0.05]. ⑶ Negative geotaxis reflex test: At P33, there were significant differences among the four groups[F(3, 16)=10.68, P=0.0004 < 0.01]. Post-hoc test, compared with NS+ND group, the time in RS+ND and RS+KD group was longer( P < 0.05).At P43, there were significant differences among the four groups[F(3, 16) =52.06, P < 0.0001]. Post-hoc test,compared with NS+ND group, the time in RS+ND、RS+KD and NS+KD group was longer(P<0.05).While compared with RS+ND group, the time in RS+KD group was shorter(P<0.05).At P53, there were significant differences among the four groups[F(3, 16)=13.98, P < 0.0001]. Post-hoc test, compared with NS+ND group, the time in RS+ND group was longer(P<0.05).While compared with RS+ND group, the time in RS+KD group was shorter( P < 0.05). ⑷ Cliff avoidance test: At P33, there were significant differences among the four groups[F(3, 16) =28.09,P<0.0001]. Post-hoc test, compared with NS+ND group, the time in RS+ND group was longer(P<0.05).While compared with RS+ND group, the time in RS+KD group was shorter(P<0.05).At P43, there were significant differences among the four groups[F(3, 16) =43.68,P<0.0001]. Post-hoc test,compared with NS+ND group, the time in RS+ND group was longer(P<0.05).Whilecompared with RS+ND group, the time in RS+KD group was shorter(P<0.05).At P53,there were significant differences among the four groups[F(3, 16) =31.07, P < 0.0001].Post-hoc test, compared with NS+ND group, the time in RS+ND and NS+KD group was longer(P<0.05).While compared with RS+ND group, the time in RS+KD group was shorter(P<0.05).3. Morris water maze analysis: ⑴ Place navigation test: there were significant differences among group[F(3, 12) = 156.6, P < 0.0001] 、 day[F(4, 16) = 63.33, P <0.0001] 、 interaction[F(12, 48) = 4.650, P < 0.0001] by repeated two-way ANOVA analysis. From 1 day to 5 day, compared with NS+ND group, the escape latency in RS+ND group was longer(P<0.05). From 2 day to 5 day, compared with RS+ND group,the escape latency in RS+KD group was shorter(P<0.05).In addition, the escape latency in NS+KD group was longer than that in NS+ND group(P<0.05). ⑵Spatial probe test:there were significant differences among the four groups[F(3, 16) = 12.41, P =0.0002].Post-hoc test, compared with NS+ND group, the frequency of passing through the platform in RS+ND and NS+KD group was much less(P<0.05).While compared with RS+ND group, the frequency of passing through the platform in RS+KD group was much more(P<0.05).4. Open field test analysis: ⑴Immobility time: there were significant differences among group[F(3, 12) = 101.7,P < 0.0001]、interaction[F(3, 12) = 7.116,P = 0.0053] by repeated two-way ANOVA analysis. However, there were no difference among day[F(1, 4)= 5.213,P = 0.0845]. Compared with NS+ND group, the immobility time in RS+ND and NS+KD group(P61、P62)was longer(P<0.05).While compared with RS+ND group,the immobility time in RS+KD group(P61、P62)was shorter(P<0.05).⑵The frequency of locomotor activity: there were significant differences among group[F(3, 12) = 35.72,P< 0.0001] 、 day[F(1, 4) = 74.17, P =0.001] by repeated two-way ANOVA analysis.However, there were no difference among interaction[F(3, 12) = 3.41, P = 0.0531].Compared with NS+ND group, the frequency of locomotor activity in RS+ND and NS+KD group(P61、P62)was much less(P<0.05).While compared with RS+ND group,the frequency of locomotor activity in RS+KD group(P61、P62)was much more(P<0.05).5. Seizure latency and survival rate:⑴Seizure latency: Compared with NS+ND group, the seizure latency in RS+ND group was much shorter(P<0.05).While compared with RS+ND group, the seizure latency in RS+KD group was much longer(P<0.05). ⑵survival rate: NS+ND group(73.3%)、NS+KD group(60%)、RS+ND group(46.7%)、RS+KD group(73.3%)。6. Timm’s analysis:Compared with NS+ND group, aberrant mossy fiber sprouting in CA3 subfield and dentate gyrus in RS+ND group was much more(P < 0.05).While compared with RS+ND group, aberrant mossy fiber sprouting in CA3 subfield and dentate gyrus in RS+KD group was much less(P<0.05).7. Western blot analysis:⑴ In hippocampus: Compared with NS+ND group, the level of ZIP7、Apo A1、P53、P62 in RS+ND group was higher(P<0.05), while the level of c PLA2 in RS+ND group was lower(P<0.05). Compared with RS+ND group, the level of ZIP7 、Apo A1、P53、P62 in RS+ND group was lower(P<0.05), while the level of c PLA2 in RS+ND group was higher(P < 0.05). ⑵ In cerebral cortex: Compared with NS+ND group, the level of ZIP7、Apo A1、P53、P62 in RS+ND group was higher(P<0.05), while the level of c PLA2 in RS+ND group was lower(P < 0.05). Compared with RS+ND group, the level of ZIP7、Apo A1、P53、P62 in RS+ND group was lower(P<0.05),while the level of c PLA2 in RS+ND group was higher(P<0.05).Conclusions: 1. Long-term administration of ketogenic diet may ease neurobehavioral and cognitive damage, inhibit aberrant mossy fiber sprouting in the stratum pyramidale of CA3 subfield and the inner molecular layer of the granule cells of dentate gyrus, raise the seizure threshold and survival rate.2. Long-term administration of ketogenic diet could significantly down-regulation of ZIP7、Apo A1、P53、P62 and up-regulation of c PLA2.3. It showed that the neuroprotective effects of long-term administration of ketogenic diet on neonatal seizure-induced brain damage might be through the zinc-lipid peroxidation-autophagy signaling pathway.Objective: To explore the effects and mechanisms of long-term administration of melatonin on neurobehavioral changes and mossy fiber sprouting induced by recurrent neonatal seizures.Methods: Sprague-Dawley(SD) rats(number=60) at postnatal days 8(P8) were randomly divided into four groups: the control group(CONT group, n=15)、the control and MEL-treated group(MEL group, n=15)、the recurrent-seizure group(RS group, n=15)、and the recurrent-seizure and MEL-treated group(RS+MEL group, n=15). Rats were fed for 1day before the study. From P9, rats in the RS and RS+MEL group were subjected to recurrent seizures induced by volatile flurothyl five times each day for consecutive 13 days,with a interval time of 30 minutes once. While rats in the CONT and MEL group were placed into the container for an equal amount of time to their counterpart without exposuring to flurothyl. At P23, MEL was dissolved in 5% ethanol and administered intraperitoneally(i.p.) to rats in MEL and RS+MEL group at a dose of 10mg/kg for 30 days, while rats in CONT and RS group were intraperitoneally(i.p.) 5% ethanol for 30 days. We observed neurological behavior by neurological reflex test at P33 、 P43 、 P53.Morris water maze were performed from P54 to P60. Open field test were performed at P61 and P62. At P63, penicillin was dissolved in saline and administered intraperitoneally(i.p.) to all rats at a dose of 5.1×106U/kg/d, after which rats were closely observed for about 1 h. We observed seizure activity recorded the seizure latency and survival rate. We monitored levels of glucose and ketone in the blood at P63. Zn T1、ZIP7、Apo A1、c PLA2、P53、P62、Beclin1、Bcl-2、LC3Ⅱ/Ⅰ、Cathepsin E protein levels in hippocampus and cerebral cortex were determined by western blot at P63. Mossy fiber sprouting in hippocampus was determined by Timm staining at P63.Results: 1. Neurodevelopmental indicators analysis: ⑴ Plane righting reflex test:At P33, there were no significant differences among the four groups[F(3, 16) =2.518,P=0.095>0.05]. At P43, there were significant differences among the four groups[F(3, 16)=8.241,P=0.0015<0.01]. Post-hoc test, compared with NS+ND group, the time in RS andRS+MEL group was shorter(P<0.05).At P53, there were significant differences among the four groups[F(3, 16) =4.622,P=0.0164<0.05]. Post-hoc test, compared with CONT group, the time in RS group was shorter(P<0.05). ⑵Forelimb suspension test: At P33,there were significant differences among the four groups[F(3, 16) =9.238, P=0.0009 <0.01]. Post-hoc test, compared with CONT group, the time in RS group was shorter(P<0.05).At P43, there were significant differences among the four groups[F(3, 16) =8.544,P=0.0013 < 0.01]. Post-hoc test, compared with CONT group, the time in RS group was shorter(P<0.05). At P53, there were significant differencesamong the four groups[F(3,16) =7.642,P=0.0022<0.01]. Post-hoc test, compared with CONT group, the time in RS group was shorter(P<0.05). While compared with RS group, the time in RS+MEL group was longer(P < 0.05). ⑶ Negative geotaxis reflex test: At P33, there were significant differences among the four groups[F(3, 16) =27.07,P<0.0001]. Post-hoc test, compared with CONT group, the time in RS and RS+MEL group was longer( P < 0.05).While compared with RS group, the time in RS+MEL group was shorter(P<0.05).In addition,compared with MEL group, the time in RS and RS+MEL group was longer(P<0.05).At P43, there were significant differences among the four groups[F(3, 16) =73.44, P <0.0001]. Post-hoc test, compared with CONT group, the time in RS and RS+MEL group was longer(P<0.05).While compared with RS group, the time in RS+MEL group was shorter(P<0.05).In addition, compared with MEL group, the time in RS and RS+MEL group was longer(P<0.05).At P53, there were significant differences among the four groups[F(3, 16) =154.2,P<0.0001]. Post-hoc test, compared with CONT group, the time in RS and RS+MEL group was longer(P<0.05).While compared with RS group, the time in RS+MEL group was shorter(P<0.05).In addition, compared with MEL group, the time in RS and RS+MEL group was longer(P<0.05).⑷Cliff avoidance test: At P33, there were significant differences among the four groups[F(3, 16) =175.8,P<0.0001]. Post-hoc test, compared with CONT group, the time in RS and RS+MEL group was longer( P <0.05). In addition, compared with MEL group, the time in RS and RS+MEL group was longer(P<0.05).At P43, there were significant differences among the four groups[F(3,16) =61.89,P<0.0001]. Post-hoc test, compared with CONT group, the time in RS andRS+MEL group was longer( P < 0.05).While compared with RS group, the time in RS+MEL group was shorter(P<0.05).In addition, compared with MEL group, the time in RS and RS+MEL group was longer(P<0.05).At P53, there were significant differences among the four groups[F(3, 16) =114.6,P<0.0001]. Post-hoc test, compared with CONT group, the time in RS and RS+MEL group was longer(P<0.05).While compared with RS group, the time in RS+MEL group was shorter(P<0.05).In addition, compared with MEL group, the time in RS and RS+MEL group was longer(P<0.05).2. Morris water maze analysis: ⑴ Place navigation test: there were significant differences among group[F(3, 12) = 71.29, P < 0.0001] 、 day[F(4, 16) = 153.7, P <0.0001] 、 interaction[F(12, 48) = 4.258, P =0.0001] by repeated two-way ANOVA analysis. From 1 day to 5 day, compared with CONT group, the escape latency in RS+ND group was longer(P<0.05). From 2 day to 5 day, compared with RS group, the escape latency in RS+MEL group was shorter(P<0.05).In addition, at day 2, the escape latency in RS+MEL group was longer than that in CONT group(P<0.05). ⑵Spatial probe test:there were significant differences among the four groups[F(3, 16) = 28.53,P <0.0001].Post-hoc test, compared with CONT group, the frequency of passing through the platform in RS and RS+MELgroup was much less(P<0.05).While compared with RS group, the frequency of passing through the platform in RS+MEL group was much more(P<0.05).3. Open field test analysis: ⑴Immobility time: there were significant differences among group[F(3, 12) = 117.6,P < 0.0001]、day [F(1, 4) = 1184,P < 0.0001] by repeated two-way ANOVA analysis. However, there were no difference among interaction [F(3, 12)= 0.5514, P = 0.6568 > 0.05]. Compared with CONT group, the immobility time in RS group(P61、P62)was longer(P<0.05).While compared with RS group, the immobility time in RS+MEL group(P61、P62)was shorter(P<0.05).⑵The frequency of locomotor activity: there were significant differences among group[F(3, 12) = 238.3,P < 0.0001]、interaction [F(3, 12) = 4.866, P = 0.0193] by repeated two-way ANOVA analysis.However, there were no difference among day [F(1, 4) = 0.045, P =0.8416 > 0.05].Compared with CONT group, the frequency of locomotor activity in RS and RS+MEL group(P61、P62)was much less(P<0.05).While compared with RS group, the frequencyof locomotor activity in RS+MEL group(P61、P62)was much more(P<0.05).4. Seizure latency and survival rate: ⑴ Seizure latency: Compared with CONT group, the seizure latency in RS and RS+MEL group was much shorter(P < 0.05).While compared with RS group, the seizure latency in RS+MELgroup was much longer(P <0.05). In addition, compared with MEL group, the seizure latency in RS and RS+MEL group was much shorter(P<0.05). ⑵survival rate: CONT group(80%)、NS+MELgroup(78%)、RS group(46.7%)、RS+MEL group(53.3%)。5. Timm’s analysis:Compared with CONT group, aberrant mossy fiber sprouting in the supra granular region of the dentate gyrus and CA3 subfield of the hippocampus in RS group was much more(P < 0.05).While compared with RS group, aberrant mossy fiber sprouting in the supra granular region of the dentate gyrus and CA3 subfield of the hippocampus in RS+MEL group was much less(P<0.05).6. Western blot analysis:⑴ In hippocampus: Compared with CONT group, the level of ZIP7、Apo A1、P53、P62、 Beclin1、Beclin1/ Bcl-2、LC3Ⅱ/Ⅰ、Cathepsin E in RS group was higher(P<0.05), while the level of Zn T1、 c PLA2 、Bcl-2in RS group was lower(P<0.05). Compared with RS group, the level of ZIP7、Apo A1、P53、P62、Beclin1、Beclin1/ Bcl-2、LC3Ⅱ/Ⅰ、Cathepsin E in RS+MELgroup was lower(P<0.05), while the level of Zn T1、 c PLA2、Bcl-2 in RS+MEL group was higher(P<0.05). ⑵ In cerebral cortex: Compared with CONT group, the level of ZIP7、Apo A1、P53、P62 in RS group was higher(P<0.05), while the level of Zn T1、 c PLA2 in RS+ND group was lower(P<0.05).Compared with RS group, the level of ZIP7、Apo A1、P53、P62 、Beclin1、Beclin1/ Bcl-2、LC3Ⅱ/Ⅰ、Cathepsin E in RS+MEL group was lower(P<0.05), while the level of Zn T1、c PLA2 、Bcl-2 in RS+ND group was higher(P<0.05).Conclusions: 1. Long-term administration of melatonin may ease neurobehavioral and cognitive damage, inhibit aberrant mossy fiber sprouting in the stratum pyramidale of CA3 subfield and the inner molecular layer of the granule cells of dentate gyrus, raise the seizure threshold and survival rate.2. Long-term administration of melatonin could significantly down-regulation of ZIP7、Apo A1、P53、P62 、Beclin1、Beclin1/ Bcl-2、LC3Ⅱ/Ⅰ、Cathepsin E and up-regulationof Zn T1、 c PLA2、Bcl-2.3. It showed that the neuroprotective effects of long-term administration of melatonin on neonatal seizure-induced brain damage might be through the zinc-lipid peroxidation-autophagy signaling pathway.