| Objective:Through experimental study determined the early intervention time points for Jiaji electro-acupuncture combined with MP treatment of ASCI, and observe the Jiaji electropuncture combined with MP early intervention of ASCI rats micro environment NMDAR/calpain signaling pathway and apoptosis effect and try to reveal the mechanism of Jiaji electro acupuncture combined with MP treatment of ASCI.Methods:The experimental study was divided into three parts.Experiment 1:Observation of the earliest time of apoptosis after acute spinal cord injury (ASCI), clear early intervention time point.48 female Wister rats were randomly divided into sham operation group (Sham) and acute spinal cord injury group (SCI), each group was divided into four subgroups:lh,3h,6h and 8h. The rats were sacrificed at the corresponding time point after the model was made. HE staining was used to observe the pathological changes of the spinal cord in rats. TUNEL staining was used to detect the apoptosis of rats. The earliest time point of apoptosis appeared, and the best time of early intervention was determined.Experiment 2:Comparative Analysis of Jiaji electroacupuncture (EA) and methylprednisolone (MP) treatment of the ASCI.144 female Wister rats were randomly divided into Sham group, SCI group, EA group, MP group,EA+MP group and MDL28170group, each divided into 1 day,3 days,7 days,14 days four subgroups. BBB score was used to observe the motor function of ASCI rats; HE staining was used to observe the influence of different treatment methods on ASCI rat spinal cord tissue pathology; TUNEL staining of different treatment methods for detection of cell apoptosis in rats with effect; comparison of EA group, MP group and EA+MP group of different curative effect to select ASCI optimal treatment program.Experiment 3:Effect of Jiaji electro-acupuncture on NMDAR/Calpain pathway and cell apoptosis after ASCI.120 female Wister rats were randomly divided into normal group (Normal), Sham group, SCI group, EA+MP group and MDL28170 group. Each group was divided into 1 days,3 days,7 days,14 days four subgroups. Nissl staining was used to observe the changes of each rat spinal cord neurons apoptosis, Immunohistochemistry and Western blot detected in the spinal cord tissue NMDAR/calpain pathway related factor NMDA-NR1 and calpain2, cleaved caspases-3 expression, in order to identify the impact of changes after ASCI passage of each factor on apoptosis.Result:Experiment 1:(1) HE staining showed that after SCI when rats 1h loose organizational structure, there are hematoma, cells did not change significantly; after 3h start, mild swelling of the cell, the cell membrane and the nucleus did not change significantly; after 6h start cell edema, the gap becomes larger cell shrinkage, cell becomes small, apoptosis occurs; after 8h loose tissue, may have vacuolization.(2) TUNEL assay showed postoperative 6h started, SCI group began to apoptosis, with statistical significance (P<0.05) and the Sham group differences. After 8h, a further increase in apoptotic cells, some cells apoptotic bodies appear, with statistical significance (P<0.05) and the Sham group differences.Experiment 2:(1) BBB scores showed that, after 3d, compared with SCI group, EA group, MP group, EA+MP group,MDL28170 group limb motor function in rats was significantly increased, significant difference (P< 0.05). After 14d, compared with EA group, MP group, MDL28170 group limb motor function of rats in group EA+MP were increased significantly (P< 0.05).(2) HE staining showed that the SCI Id group, bleeding apparently, visible changes at an early stage of apoptosis; 3d cells destroy the most obvious, apoptotic cells, the nucleolus disappeared, karyopyknosis; 7d after operation, the nerve cell apoptosis has been reduced, nerve cell necrosis, reduce the number of normal neurons; 14d when apoptosis is not obvious, visible vacuoles. The rest 4 treatment groups, the cell damage was significantly reduced as compared with the ASCI group, especially in EA+MP group of cells is well preserved.(3) TUNEL detection showed that after 3d,compared with the SCI group, EA group, MP group,MDL28170 group apoptosis of EA+MP group decreased significantly after surgery (P< 0.05). After surgery 14d,compared with the EA group, MP group,MDL28170 group apoptosis of the EA+MP group decreased significantly, the difference was significant (P< 0.05).Experiment 3:(1) The Nissl staining results can be seen, SCI group 1 d neuronal cell body when the swelling deformation, Nissl body swollen and broken, the decrease in the number of neurons; 3 d neuronal cell body damage is apparent, nucleolus disappeared, damaged neurons increased obviously; 7 d when neurons austerity, it become visible nucleoli, nissl body dissolved, decreased; 14 d neurons squeeze smaller, deep into cytoplasm. The rest of the four treatment groups neurons damage significantly reduce in the SCI group, EA, MP, EA+MP, MDL28170 treatment can reduce the neuron damage degree, improve nerve cells form, prevent nissl body broken dissolved, increase the quantity, especially in the EA+MP group curative effect is distinct.(2) Immunohistochemical results showed that 3 days after operation, compared with SCI group, EA+MP group and MDL28170 group NMDA-NR1 and calpain-2, caspases-3 expression decreased, a statistically significant difference (P< 0.05); at 7d and 14d,compared with MDL28170 group,EA+MP group NMDA-NR1 and calpain-2, caspases-3 expression decreased, the difference is statistically significant (P< 0.05).(3) Western blot results showed that 3 days after operation, compared with SCI group, EA+MP group and MDL28170 group NMDA-NR1 and calpain-2, caspases-3 protein expression decreased, the difference has statistical significance (P< 0.05); at 7d and 14d, compared with MDL28170 group, EA+MP group NMDA-NR1 and calpain-2, caspases-3 expression decreased, the difference is statistically significant (P< 0.05).Conclusion:(1) Apoptosis occurs in 6h after acute spinal cord injury, before this time point may be the time for early intervention of ASCI.(2) The combination of EA and MP can improve the ASCI rats BBB scales,inhibition of neuronal apoptosis and protect the injured neurons.(3) Acute Spinal Cord Injury activation of excitatory amino acid toxicity, inhibition ASCI microenvironment calpain-2 expression can reduced neuronal apoptosis, suggesting that NMDAR/Calpain pathway mediated apoptosis after ASCI.(4) The combination of EA and MP can cut NMDA-NR1, calpain-2 expression, inhibition of excitatory amino acid toxicity thereby reduced expression of cleaved caspases-3 and inhibit neuronal apoptosis, improve micro-environment, reduce spinal cord secondary damage. |
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