| Part 1:Establishment and Stability Measurement of Specialized SpinalInjury Model in RatsObjective: To design a simple and stable Allens rat spinal impactor; and evaluate the parameters and stability of the spinal cord injury model in rats made by the impactor; thus reducing the threshold of modeling spinal cord injury in rats.Methods: This study includes two parts: impulse screening and stability evaluation. First, 30 male SD rats(BW: 220-250g) were selected and assigned, with 6 rats in each group. Weight Dropping method was used on the rats. Firing pin was used to drop from 0cm, 3cm, 6cm, 9cm and 12 cm to impact the rats. 0cm group was the sham-operative group, in which the vertebral plate was only opened. After impacting, the height(6 cm and 9 cm) that caused significant incomplete spinal cord injury was determined through behavior score and pathological sections. Another 18 SD rats(6 in sham-operative group, 6 cm group and 9 cm groups) were selected for stability and effect assessment.Results: HE staining and BBB scores in sham-operative group were in accordance with normal rats. Postoperative BBB scores of spinal cord injury group were reduced for different degrees compared with preoperative scores. The differences of impact heights between each group were statistically significant(P<0.05). Through HE staining, it could be seen that the grey and white structures were incomplete, and large focal necrosis, cell swelling and erythrocyte aggregation were visible in damaged area. For the maximum injury area ratio, the differences(P>0.05) between the 6 cm group and 9 cm group as well as the consistence in each of the two groups had statistical significance(P<0.05); in addition, the maximum injury area ratio was closely negatively related to the BBB scores(r=-0.851,p=0.000).Conclusion: The designed specialized rat spinal impactor can successfully establish acute spinal cord injury models in rats with different damage degrees. The models were with high stability and good repeatability. This method could provide effective support for the further research on rat spinal cord injury. Part 2:The Influence of Blocking JAK2/STAT3 Signal Pathway in Apoptosis after AcuteSpinal Cord Injury in RatsObjective: 1. To explore the number of apoptotic cells occurred after the spinal cord injury of rats by using HE staining and TUNEL apoptosis detection methods, and the impact of different doses of JAK2/STAT3 signal transduction pathway blocker AG490 on the number of spinal cord morphology and apoptotic cells. 2. To detect the impact of different doses of AG490 on the expression amount of P-STAT3(phosphorylated signal transduction and transcription activator 3) P-JAK2(phosphorylated protein tyrosine kinase 2)by the measure of westernblot.. 3. To explore the impact of different levels of JAK2/STAT3 signal transduction pathway blockage on the apoptosis of acute spinal cord injury(ASCI) and their relevance.Methods: 60 healthy adult male SD rats weighing 220-250 g were adopted. They were divided into five groups: sham operation group(E group, n = 12), spinal cord injury group(A group, n = 12), spinal cord injury + AG490 intervention group: 5mg/kg weight group(B group, n = 12), 10mg/kg weight group(C group, n = 12) and 15mg/kg weight group(D group, n = 12). “Spinal beater special for rats” that is developed on the basis of ALLEN'S weight drop method was used to make the acute spinal cord injury model of rats, but sham operation group only resected equant lamina while not damaging the spinal cord. Preoperative BBB rating and the rating at postoperative 24 h were recorded. The rats were put to death at 24 h after the operation, 6 rats from each group were embedded in paraffin for the detection of HE staining and TUNEL apoptosis, and another 6 rats were taken fresh tissues to conduct westernblot assay for detecting the expression changes of P-JAK2 and P-STAT3. The relation scheme between the levels of P-JAK2 and P-STAT3 and the number of apoptosis was drawn.Results: A stable ASCI model was successfully established and the satisfactory sample of spinal cord tissue was obtained. HE staining showed that the degree of spinal cord injury was of no significant difference among the groups(P>0.05); TUNEL assay showed few apoptotic cells in the control group and most apoptotic cells in the injury group, as well as that the number of apoptotic cells in the injury group intervened by AG490 was increased along with the reduction of AG490 dose; westernblot detection showed that the levels of P-JAK2 and P-STAT3 in group A, B, C and D decreased with the increasing doses of AG490, with significant differences among the groups(p<0.05), and the data differences within the was set smaller without o statistical significance(P>0.05).Conclusion: After the acute spinal cord injury of rats, the application of AG490 was of no impact on the observation of cord morphology, but the levels of P-JAK2 and P-STAT3 and the number of apoptotic cells had all reduced with the increasing dose of AG490. |
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