| Objective:To explore the effect of cluster needling of scalp acupuncture(CNSA) combined with enriched environment(EE) on neuronal apoptosis and the expression of BDNF/TrkB signaling pathway associated proteins in the hippocampus CA1 region of neonatal rats with hypoxic-ischemic brain damage(HIBD).Methods:Referring to Rice-Vannucci method, hypoxic-ischemic brain damage (HIBD) model in rat was established. Rats were randomly divided into sham control(SC) group, hypoxia-ischemia (HI) group, cluster needling of scalp acupuncture (CNSA) group, enriched environment (EE) group and CNSA+EE group, then at 14d,28d post HIBD, each group was divided into 2 subgroups. Rats in group HI, CNSA, EE, CNSA+EE were made into the model with HIBD. Rats in SC and HI group without any intervention. CNSA group was treated by cluster needling of scalp acupuncture therapy, EE group was treated by enriched environmental stimulation, and CNSA+EE group was treated by cluster needling of scalp acupuncture combined with enriched environmental stimulation.14d,28d post HIBD, we used Morris water maze to determine ability of spatial learning and memory; the pathological changes in the hippocampus CA1 region was observed by HE staining; TUNEL assay to detect cell apoptosis level in hippocampal CA1 region; Western Blot was used to measured the expression of Caspase-3、Bcl-2、BDNF、TrkB、p-ERK and p-Akt in the hippocampus CA1 region.Results:1. Spatial learning and memory function(1) Place navigation test (PNT):At 14d,28d post HIBD, mean escape latency(MEL) in HI group, compared to SC group, was significantly longer (P<0.05); MEL in all three intervention groups, compared to HI group, was all significantly shorter (P<0.05); compared to CNSA or EE group, MEL in CNSA+EE group was shorter (P<0.05). (2) Spatial probe test (SPT):At 14d,28d post HIBD, times of platform crossing(TPC) in HI group, compared to SC group, was significantly reduced (P<0.05); TPC in all three intervention groups, compared to HI group, was significantly increased (P<0.05); compared to CNSA or EE group, TPC in CNSA+EE group was increased (P<0.05).2. Pathological changes of hippocampus CA1At 14d,28d post HIBD, no pathological changes of hippocampus CA1 was obseverd in SC group, the structure of brain tissue was organized, tightly packed cells, clear cell outline, shape integrity, centered nucleus, prominent nucleoli, abundant cytoplasm, no neuronal loss. While, neurons in hypoxic-ischemic hippocampus CA1 region were disorder, blurred or disappeared, cell gap significantly increased, neuronal cell degeneration, necrosis, cell body shrinkage, condensation or fragmentation of nucleus, a lot of neuronal loss. Each treatment group at each time point, pathological damage has been improved in three intervention groups compared to HI group, and CNSA+EE group was better than CNSA or EE group.3. Cell apoptosis level of hippocampal CA1At 14d,28d post HIBD, the number of TUNEL-positive cells in HI group, compared to SC group, was significantly increased(P<0.05); compared with HI group, the number of TUNEL-positive cells in all three intervention groups was significantly reduced(P<0.05); compared to CNSA or EE group, the number of TUNEL-positive cells in CNSA+EE group was reduced (P<0.05).4. The expression of Caspase-3、BDNF、TrkB、p-ERK and p-Akt in hippocampal CA1 region(1) Caspase-3:At 14d,28d post HIBD, comopared to SC group, the Caspase-3 express in HI group was obvious increase(P<0.05); the Caspase-3 express in all three intervention groups, compared with HI group, was lower(P<0.05); compared to CNSA or EE group, the Caspase-3 express in CNSA+EE group was lower(P<0.05).(2)Bcl-2:At 14d、28d post HIBD, comopared to SC group, the Bcl-2 express in HI group was obvious increase(P<0.05); the Bcl-2 express in all three intervention groups,compared with HI group, was higher(P<0.05); compared to CNSA or EE group, the Caspase-3 express in CNSA+EE group was higher(P<0.05).(3) BDNF:At 14d、28d post HIBD, comopared to SC group, the BDNF express in HI group was obvious up-regulated (P<0.05); the BNDF express in all three intervention groups, compared with HI group, was higher(P<0.05); compared to CNSA or EE group, the BDNF express in CNSA+EE group was higher(P<0.05).(4) TrkB:At 14d、28d post HIBD, comopared to SC group, the TrkB express in HI group was obvious up-regulated (P<0.05); the TrkB express in all three intervention groups, compared with HI group, was higher(P<0.05); compared to CNSA or EE group, the TrkB express in CNSA+EE group was higher(P<0.05).(5) p-ERK:At 14d、28d post HIBD, comopared to SC group, the p-ERK express in HI group was obvious up-regulated (P<0.05); the p-ERK express in all three intervention groups, compared with HI group, was higher(P<0.05); compared to CNSA or EE group, the p-ERK express in CNSA+EE group was higher(P<0.05).(6) p-Akt:At 14d、28d post HIBD, comopared to SC group, the p-Akt express in HI group was obvious up-regulated (P<0.05); the p-Akt express in all three intervention groups, compared with HI group, was higher(P<0.05); compared to CNSA or EE group, the p-Akt express in CNSA+EE group was higher(P<0.05).Conclusion:1. CNSA combined with EE can improve spatial learning and memory function, and mitigate the pathological injury in hippocampus CA1 region of HIBD rats, exert neuroprotective effect.2. CNSA combined with EE can mitigate cell apoptosis, down-regulate the Caspase-3 express, and up-regulate the Bcl-2 express in the hippocampus CA1 region of HIBD rats.3. CNSA combined with EE can enhance BDNF、TrkB、p-ERK、p-Akt express in the hippocampus CA1 region of HIBD rats, which suggested that the neuroprotective effect of CNSA combined with EE may be mediated by BDNF/TrkB signal pathway. |
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