The Effect And Mechanism Of Leptin On Neonatal Rat With Brain Injury Induced By Recurrent And Prolonged Seizure

Objective: To investigate the effect and molecular mechanism of leptin on brain injury induced by ecurrent and prolonged seizures in neonatal rats.Methods: 40 postnatal days6(P6) Sprague Dawley(SD) rats, were randomly divided into four groups(10 rats in each group): blank control group(Control group), simple leptin group(Leptingroup), simple seizure group(RS group) and convulsions with leptin group(RS+Leptin group). group of RS and RS+Leptin rats were subjectedto flurothyl induced recurrentseizures 30 minutes per day for 7 days(P6-P12).At P13 Leptingroup and RS+Leptin group ratswere injected with leptin(2mg/kg/day) for 10 days. Neurobehavioral test(negative geotaxis reflex, forelimb suspension reflex,cliff avoidance reflex,plane righting reflex and open field test)were implemented on P23 and P30;Morris water maze escape latency and exploring experiment were implemented on P27-P33; regularly check the weight changes of the rats.On P34 all rats were sacrificed.The expression of zinc channel proteins: Zn T1, ZIP7; membrane injury marker protein: c PLA2; autophagy signaling pathway associated protein: Beclin-1, Bcl-2, LC3Ⅱ / LC3Ⅰ, Cathepsin E; molecular memory: Ca MKⅡα and Ca MKⅡβwere detected by western blot. Mossy fiber sprouting were detected by Neo-Timm’s staining.Results: 1.Weight analysis: The weight of the four groups were significantly difference [F(27, 360) = 3.099, P <0.05].Post-hoc test: at each time point during the P12-P33, compared with Control group,RS group and RS + Leptin group were significantly decreased(P <0.05).There wereno significant differences among Leptin group and Control group.2. Neurobehavioral text:(1)cliff avoidance reflex:on P23 and P30, the four groups were statistically significant differences [FP23(3, 16) = 168.1, P <0.05; FP30(3, 16) = 24.84, P <0.05].Further comparison: the cliff avoidancetime of RS group weresignificantly longer than those in Control group(P <0.05), RS + Leptin group were trend to normal andwere significantly shortercompared with the RS group(P <0.05);(2)forelimb suspension reflex:on P23 and P30, the four groups were statistically significant differences [FP23(3, 16) = 37.15, P<0.05;FP30(3, 16) = 4.68, P<0.05].Further comparison: the forelimb suspensiontime of RS group were significantly shorter than those in Control group(P <0.05), RS + Leptin group were trend to normal and were significantly longer compared with the RS group(P <0.05);(3)cliff avoidance reflex:on P23 and P30, the four groups were statistically significant differences[FP23(3, 16) = 136.2, P<0.05;FP30(3, 16) = 12.47,P<0.05].Further comparison: the cliff avoidancetime of RS group were significantly longer than those in Control group(P <0.05), RS + Leptin group were trend to normal and were significantly shorter compared with the RS group(P <0.05);(4)plane righting reflex:on P23 and P30, the four groups were statistically significant differences[FP23(3, 16) = 12.63, P<0.05; FP30(3, 16) = 16.04,P<0.05].Further comparison: the plane rightingtime of RS group were significantly longer than those in Control group(P <0.05), RS + Leptin group were trend to normal and were significantly shorter compared with the RS group(P <0.05).3.Open field test:(1)delay time:on P23 and P30, the four groups were statistically significant differences[FP23(3, 16) = 15.50, P<0.05;F P30(3, 16) = 15.50,P<0.05]. Further comparison: the delay time of RS group were significantly longer than those in Control group(P <0.05), RS + Leptin group were trend to normal and were significantly shorter compared with the RS group(P <0.05);(2)locomotor score :on P23 and P30, the four groups were statistically significant differences[FP23(3, 16) = 182.10, P<0.05; FP30(3, 16) = 8.80,P<0.05]. Further comparison: the locomotor score of RS group were significantly reduced than those in Control group(P <0.05), RS + Leptin group were trend to normal and were significantly higherthan the RS group(P <0.05).4.Morris water maze:(1)Place navigation text: two-way ANOVA analysis showedthat the difference of escape latency of four groups werestatistically significant [F(12, 48) = 17.52, P <0.05]; Further comparison: on 2-4 days of the test, the escape latency of RS group was significantly longercompared with Control group(P <0.05), RS + Leptin group were trend to normal andthe latency were significantly shorter than the RS group(P <0.05);(2) probe test: the difference was statistically significance [F(3, 16) = 17.71, P <0.05], further comparison: RS group significantly reduced the number of crossing the platform than the Control group(P <0.05), RS + Leptin group were trend to normal and significantly increased the number of crossing the platform than the RS group(P <0.05).5.Neo-Timm’s staining:Four groups of rats hippocampal CA3 region and dentate gyrus abnormal mossy fiber sprouting semiquantitative score were statistically significant different [F CA3(3, 16) = 21.69, P <0.05; F dentate gyrus(3, 16) = 84.51, P <0.05], further comparison: RS group germination significantly increased than in the Control group(P <0.05), RS + Leptin group were trend to normal and germination decreased than the RS group(P <0.05).6.Western blot analysis: Zn T1,ZIP7,c PLA2,Beclin-1,LC3Ⅱ/LC3Ⅰand Cathepsin E were significantly increased and Bcl-2 and Ca MKⅡαwere significantly decreased in RS group than Control group(P <0.05).Leptin can block the abnormal expression of the above mentioned protines.Conclusions:1.Prolonged leptin treatment can significantly improve prolonged recurrent neonatal seizures contributeed neurobehavioral and cognitive impairment and hippocampal mossy fiber sprouting abnormalities.2.Prolonged recurrent neonatal seizures canupregulatecortical and hippocampal zinc channel proteins, membrane protein damage marker and autophagy pathway proteins and downregulatethe memory-related molecules.3. The present study shows that Zn Ts / c PLA2 / Atg and functional molecules Ca MKⅡα neuroprotective effects of leptin may be relevant.