Intervention Effects Of Physical Training And 3-Methyladenine On Brain Damage Of Neonatal Rat With Recurrent Seizures

Object: To compare the intervention effect and molecular mechanism of physical training and 3-Methyladenine on brain damage of neonatal rat with recurrent seizures.Methods: Seventy-fifth Sprague-Dawley neonatal rats (postnatal days6, P6) were randomly assigned to five experimental groups: the control group (CON, n=15), the control hydrotherapy group (HCON, n=15), the recurrent-seizure group (RS, n=15), the recurrent-seizure hydrotherapy group (HRS, n=15), the 3-MA treated-seizure group (3-MA, n=15). The volatile agent flurothyl was used to induce 30 min seizure attack. At postnatal day 6 (P6), recurrent seizures were induced once per day for consecutive 6 days in the RS group,HRS group and 3-MA group. Rats in the CON and HCON groups were placed into the container for an equal amount of time to their counterpart without exposure to flurothyl. In addition, rats in the 3-MA group were pretreated with 3-Methyladenine before exposure to flurothyl. In HCON and HRS groups, physical training (Bubble Bath Hydrotherapy) intervention was used after the end of the last seizure for 28 consecutive days. Inspections were made for maturation of physical characteristics such as weight,eye opening,incisor eruption and ear unfolding. Behavior changes of brain damage of neonatal rat with recurrent seizures were observed with double-blind method including swimming development (P12, P17), suspension behavior,negative trend behavior and open field behavior (P17, P26, P33, P40). Mossy fiber sprouting were determined by Timm staining at P42. Morris water maze test was performed to assess learning and memory ability during P43-P49. PRG-1,Bcl-2,LC3 and Beclin1 protein level in hippocampus and cerebral cortex were detected by western blot method at P50.Results: 1. Maturation of physical characteristics: body weights of the rats in the RS group were decreased significantly (P﹤0.05). Body weights of HRS rats were significantly increased than that of RS rats at P26,P33 and P40 (P﹤0.05). Body weights of 3-MA rats were significantly increased than that of RS rats at P12,P14 and P18 (P﹤0.05). There were no significant difference of physiological development phenomenon (incisor eruption, ear unfolding, eye opening) among the five groups (P﹥0.05).2. Neurobehavioral analysis: (1) The scores of swimming behavior in RS rats were decreased significantly compared with that of CON rats (P﹤0.05). There were no significant difference between HRS and RS rats on swimming behavior(P﹥0.05). But swimming behavior scores in 3-MA rats were increased significantly compared with that of CON rats (P﹤0.05). (2) There was no significant difference in the test of suspension behavior(P﹥0.05). (3) The scores of the negative trend experiment in RS rats were lower than that of CON rats at different days (P﹤0.05). Moreover, the scores were higher in 3-MA rats compared with that of RS rats (P﹤0.05). (4) The scores of open filed behavior in RS rats were decreased significantly compared with that of CON rats at P17,P26 and P33(P﹤0.05). The scores in HRS rats were increased significantly than that in RS rats at P26,P33 and P40(P﹤0.05). In addition, the scores in 3-MA rats were increased than that in RS rats at P17 and P26(P﹤0.05).3. Morris water maze test: (1) The average latencies of all rats were reduced by degree from d1 to d5. The escape latency was significantly longer in rats of RS group than that of CON group at d4 and d5(P﹤0.05). The escape latency was significantly shorter in rats of HRS group than that of RS group at d4(P﹤0.05). And the escape latency was significantly shorter in rats of 3-MA group than that of RS group at d4 and d5(P﹤0.05). (2) As far as search strategy is concerned, rats of RS group performed worse than those in CON,HCON and 3-MA groups at d4 and d5, with statistically significant difference(P﹤0.05). (3) The frequency of passing through the platform quadrant in rats had no significantly difference among the five groups(P﹥0.05).4. Timm staining showed that a lot of aberrant mossy fiber sprouting was seen in the inner molecular layer of the granule cells of dentate gyrus and the stratum pyramidale of CA3 subfield in RS group. In CA3 subfield of HRS group, aberrant mossy fiber density was obviously decreased than that of RS group(P﹤0.05). However, in CA3 subfield and dentate gyrus of 3-MA group, aberrant mossy fiber density was significantly decreased than that of RS group(P﹤0.05). But there were no significantly aberrant mossy fiber sprouting in hippocampus in CON and HCON groups.5. Western blot analysis: (1) The expression of PRG-1 in hippocampus and cerebral cortex of RS group were significantly higher than that of CON group(P﹤0.05). There were no significant difference between HRS and RS groups(P﹥0.05). Moreover, the level of PRG-1 protein in hippocampus and cerebral cortex of 3-MA group were significantly lower than that of RS group(P﹤0.05). (2) The level of Bcl-2 in hippocampus and cerebral cortex of RS group were significantly lower than that of CON group(P﹤0.05). The level of Bcl-2 in hippocampus and cerebral cortex of HRS group were significantly higher than that of RS group(P﹤0.05). In addition, the level of Bcl-2 in hippocampus of 3-MA group were significantly higher than that of RS group(P﹤0.05). (3) There were no significant difference of the ratio of LC3Ⅱto LC3Ⅰand the level of Beclin1 protein in hippocampus and cerebral cortex among all the five groups.Conclusions: 1. Maturation of physical characteristics, neurobehavioral and learning ability were damage following flurothyl-induced recurrent neonatal seizures, which might be associated with the aberrant mossy fiber sprouting, significantly increased level of PRG-1 protein and decreased level of Bcl-2 protein in hippocampus and cerebral cortex.2. Physical training (Bubble Bath Hydrotherapy) after seizures could significantly promote the body weights, improve performance emotion and cognitive function, which might be associated with the decreased aberrant mossy fiber sprouting, the increased level of Bcl-2 protein in hippocampus and cerebral cortex.3. Pretreatment with autophagy inhibitor 3-MA could significantly promote the body weights, improve neurobehavioral and cognitive capacity, which might be associated with the decreased aberrant mossy fiber sprouting, the decreased level of PRG-1 protein and the increased level of Bcl-2 protein in hippocampus or cerebral cortex. 4. The results indicate that physical training (Bubble Bath Hydrotherapy) as well as autophagy inhibitor 3-MA could play a positive neurorehabilitation role on neonatal seizure-induced brain damage. Moreover, the intervention effect of 3-MA might be more effective.