| ObjectiveTo investigate the mechanism on the intervention of electroacupuncture combined with enriched rehabilitation training on nerve regeneration after cerebral ischemia by regulating Nogo receptor complexMethodsThe focal cerebral ischemia model in rats were established by thread embolism method. A total of 75 SD rats(males,clean grade) were randomly divided into 5 groups:sham operation group, model group, electroacupuncture group, enriched rehabilitation training group and electroacupuncture combined with enriched rehabilitation training group. Sham operation group and model group was not given any treatment, the electroacupuncture group was treated by electroacupuncture, the enriched rehabilitation group was treated by enriched environment and rehabilitation training(balance beam,rotating bar, net screen), the acupuncture-rehabilitation group was treated by electroacupuncture combined with enriched rehabilitation training, the animals sacrificed to gain materials at 3d,7d,14 d post operation. The Nerve function defect score was used to observe the rat's nerve function defect at 4h, 3d, 7d, 14 d in each group; The behavioral function score was used to observe the rat's behavior function of 4h, 3d,7d,14 d in each group; The TTC staining method was used to observe the rat's volume of cerebral infarction in each group; The transmission electron microscope was used to observe the ultrastructure of neurons and synapses of cerebral cortex and hippocampus in each group at 14 d post operation; The HE staining method was used to observe the pathological changes of cerebral cortex and hippocampus in each group at 14 d post operation; The immunohistochemical method was used to observe expression of Nogo receptor complex(Ng R, LINGO-1, p75 NTR, TROY) in the rat's cerebral cortex and hippocampus at 14 d post operation.Results1. Neurological deficits scores assessment: Neurological deficits scores was 0 in the sham operation group at postoperative each time; At all ponts post operation,compared with sham operation group, the neurological deficits scores in model group were increased significantly(P<0.01); At 4h, 3d post operation, compared with the model group, the neurological deficits scores in electroacupuncture group, enriched rehabilitation group and acupuncture-rehabilitation group were decreased(P>0.05); At7 d, 14 d post operation, compared with the model group, the neurological deficits scores inelectroacupuncture group,enriched rehabilitation group and acupuncture-rehabilitation group were decreased significantly(P<0.05 or P<0.01), and the scores in acupuncture-rehabilitation group is more obvious than electroacupuncture group and enriched rehabilitation group(P<0.05).2. Behavioral function score(1) Balance beam walking testAt all ponts post operation, compared with sham operation group, the balance beam walking test score in model group were increased significantly(P<0.01); At 4h, 3d post operation, compared with the model group, the balance beam walking test score in electroacupuncture group, enriched rehabilitation group and acupuncture-rehabilitation group were decreased(P>0.05); At 7d, 14 d post operation, compared with the model group, the balance beam walking test score in electroacupuncture group, enriched rehabilitation group and acupuncture-rehabilitation group were decreased significantly(P<0.01), compared with electroacupuncture group, the scores in enriched rehabilitation group and acupuncture-rehabilitation group were decreased significantly(P<0.05 or P<0.01),and acupuncture-rehabilitation group is more obvious than enriched rehabilitation group(P<0.05).(2) Rotating bar walkingAt all ponts post operation, compared with sham operation group, the rotating bar walking test score in model group were increased significantly(P<0.01); At 4h, 3d post operation, compared with the model group, the rotating bar walking test score in electroacupuncture group and enriched rehabilitation group were decreased(P>0.05),the scores in acupuncture-rehabilitation group was decreased(P<0.05); At 7d, 14 d post operation, compared with the model group, the rotating bar walking test score in enriched rehabilitation group and acupuncture-rehabilitation group were decreased significantly(P<0.01), compared with electroacupuncture group, the scores in enriched rehabilitation group and acupuncture-rehabilitation group were decreased significantly(P<0.05).(3) Net screen testAt all ponts post operation, compared with sham operation group, the net screen test score in model group were increased significantly(P<0.01); At 4h, 3d post operation,compared with the model group, the net Screen test score in electroacupuncture group,enriched rehabilitation group and acupuncture-rehabilitation group were decreased(P>0.05); At 7d, 14 d post operation, compared with the model group, the net screen test score in electroacupuncture group, enriched rehabilitation group and acupuncture-rehabilitation group were decreased significantly(P<0.01), compared with electroacupuncture group, the scores in enriched rehabilitation group and acupuncture-rehabilitation group were decreased significantly(P<0.01).3. The volume detection of cerebral infarction: sham operation group without infarction and infarction in model group obviously. After treatment, the volume of cerebral infarction reduced in the all intervention groups; At 3d post operation,compared with the model group, the volume of cerebral infarction in electroacupuncture group, enriched rehabilitation group and acupuncture-rehabilitation group were decreased(P>0.05); At 7d,14 d post operation, compared with the model group, the volume of cerebral infarction in electroacupuncture group, enriched rehabilitation group and acupuncture-rehabilitation group were decreased(P<0.05 or P< 0.01), and the volume of cerebral infarction in acupuncture-rehabilitation group is more obvious than electroacupuncture group and enriched rehabilitation group(P<0.05).4. The ultrastructure of neurons and synapses of cerebral cortex and hippocampus:The neuron morphology of ischemic region cortex and hippocampal was irregular,synapses in the neuropil were significantly reduced, cell boundary was unclear or disappeared, cell vacuolar degeneration, mitochondria swelled and cristae distorted or even fracture, dilation of rough endoplasmic reticulum, pre and post synaptic membrane is not clear, presynaptic terminal is not clear, the number of synaptic vesicles decreased significantly in model group. The can make The ischemic cortex and hippocampus neuron cell membrane was integrity in each intervention group, the number of synaptic neuropil increased, improve the structure of mitochondria, rough endoplasmic reticulum,improve the neuronal apoptosis and necrosis. And the protective of neurons and synaptic ultrastructure in acupuncture-rehabilitation group was more obvious.5. Histopathological observation: In the sham operation, the level of brain tissue was distinct, and the morphology and structure of nerve cells were normal in sham operation group; In the model group, the brain edema was obvious, the structure was loose, the cell arrangement was disordered, the vacuole like change was obvious, the nuclear condensation and deep staining, the disappearance of the nucleus and other pathological changes. At 14 d post operation, the pathological changes of ischemic cortex and hippocampus were degrees reduced in each intervention group. At 14 d post operation, the inflammatory infiltration were significantly reduced, the number of neurons increased, the deformation and necrosis of the neurons decreased, and the injury was significantly reduced in the ischemic cortex and the hippocampus of the rats in the acupuncture-rehabilitation group.6. The changes of Nogo receptor complex((Ng R, LINGO-1, p75 NTR, TROY))(1) NgR: The number of NgR positive cells in the ischemic cortex and hippocampus after cerebral ischemia was significantly increased. A small amount of Ng R positive expression was found in the sham operation group. Compared with the sham operation group, the number of Ng R positive cells in the model group increased significantly(P<0.01). Compared with the model group, the Ng R positive cells in the lectroacupuncture group, enriched rehabilitation group and acupuncture-rehabilitation group were significantly reduced(P<0.01), and the Ng R positive cells in acupuncture-rehabilitation group was more reduced than electroacupuncture group and enriched rehabilitation group(P<0.05).(2) LINGO-1: The number of LINGO-1 positive cells in the ischemic cortex and hippocampus after cerebral ischemia was significantly increased. A small amount of LINGO-1 positive expression was found in the sham operation group. Compared with the sham operation group, the number of LINGO-1 positive cells in the model group increased significantly(P<0.01). Compared with the model group, the LINGO-1positive cells in the ectroacupuncture group, enriched rehabilitation group and acupuncture-rehabilitation group were significantly reduced(P<0.01), and the LINGO-1 positive cells in acupuncture-rehabilitation group was more reduced than electroacupuncture group and enriched rehabilitation group(P<0.01).(3) p75NTR: The number of p75 NTR positive cells in the ischemic cortex and hippocampus after cerebral ischemia was significantly increased. A small amount of p75 NTR positive expression was found in the sham operation group. Compared with the sham operation group, the number of p75 NTR positive cells in the model group increased significantly(P<0.01). Compared with the model group, the p75 NTR positive cells in the ectroacupuncture group, enriched rehabilitation group and acupuncture-rehabilitation group were significantly reduced(P<0.01), and the p75 NTR positive cells in acupuncture-rehabilitation group was more reduced than electroacupuncture group and enriched rehabilitation group(P<0.01).(4) TROY: The number of TROY positive cells in the ischemic cortex and hippocampus after cerebral ischemia was significantly increased. A small amount of TROY positive expression was found in the sham operation group. Compared with the sham operation group, the number of TROY positive cells in the model group increased significantly(P<0.01). Compared with the model group, the TROY positive cells in the ectroacupuncture group, enriched rehabilitation group and acupuncture-rehabilitation group were significantly reduced(P<0.01), and the TROY positive cells in acupuncture-rehabilitation group was more reduced than electroacupuncture group and enriched rehabilitation group(P<0.01).Conclusion1. Electroacupuncture combined with enriched-rehabilitation therapy can promote the rat's nerve function reconstruction after focal cerebral ischemia, which is better than simple electroacupuncture treatment and abundant rehabilitation therapy.2. Electroacupuncture combined with enriched rehabilitation therapy can improve cerebral ischemia balance, walking and grasping ability and increase the muscle strength of suffering from limb is better than the simple electroacupuncture treatment,and improving the balance ability is better than the simple enriched rehabilitation treatment in.3. Electroacupuncture combined with enriched rehabilitation therapy can reduce the volume of cerebral infarction, which is better than simple electroacupuncture treatment and enriched rehabilitation treatment.4. Electroacupuncture combined with enriched rehabilitation therapy can improve the neurons, synaptic ultrastructure and pathological changes of the cerebral cortex and hippocampus in rats, which is better than the simple electroacupuncture treatment and rich rehabilitation treatment.5. Electroacupuncture combined with enriched rehabilitation therapy can down regulate the expression of Ng R, LINGO-1, p75 NTR and Troy in the ischemic cortex and hippocampus of rat, reduce Nogo receptor protein complex formation, preventing axonal growth cone collapse and promote nerve growth, which is the probably mechanism of electroacupuncture combined with enriched rehabilitation therapy can promote nerve function remodeling after cerebral ischemia. |
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