| Objective:Blood spinal cord barrier (BSCB) is usually destructed after spinal cord injury. Thereis currently little to know about the correlation of BSCB and spinal injury-induced pain(SIP). Recent studies showed that gliacyte inside the spinal cord and inflammatory factorplayed an important role in the generation and development of SIP. In this study, we built apain model of spinal cord injury, so as to observe the changes of pain ethology in rats,detected the content of the Evans blue in the injury segment of the spinal cord to appraisethe changes of the permeability of the BSCB.We observed the changes of the correlatedinflammatory factors (IL-1β,IL-6,TNF-α) and the morphology and the expression of someproteins of the microglia/macrophages to investigate the influence of medium molecularweight hydroxyethyl starch130/0.4(HES130/0.4), low molecular weight hydroxyethylstarch40(HES40) and saline on the blood spinal cord barrier and the changes of painethology after spinal cord injuries. We preliminarily discussed the potential mechanismsand wished to find a new way for the treatment and prevention of the secondary pain afterspinal cord injury.Method:1.72SD adult male rats (weight250±10g) were divided into4groups by randomnumber method:saline group, low molecular weight HES group (HES40group), mediummolecular weight HES group (HES130/0.4group) and sham operation group, and for18ineach group. The rats in sham operation group will be only operated with laminectomy, andthe rest in the other3groups were prepared to pattern rats of moderate spinal cord (T10)contusion by NYU spinal cord impact instrumentⅠ. After the operating they will beinjected with saline, HES40and HES130/0.4two times per day as they divided. Afteroperation,all of them will be given bladder artificial massage2-3times per day to help themto urinate until they rebuilt the automatic micturition reflex. 2. Each group of the rats were tested for the changes of PWPT(paw withdraw pressurethreshold)and PWL (paw withdraw latency) at the point of1d before the operation, and10d,14d,20d,30d,60d after it, and compared with the ethological performance simultaneously.3.3days after operations, each group will be measured by the relative amount ofEvans blue in the injury segments of the myeloid tissue to estimate the conditions of theBSCB.4.10days after operations, the activation and expression of Ca-binding protein,protein labeling of Iba1and ED1on the lysosomal membrane in the macrophage/microgliawere investigated by immunofluorescence histochemical method and western blot.5.The changes of expressions of IL-1β, IL-6and TNF-α in injured segments of themyeloid tissue were surveyed by ELISA.6. Statistics software SPSS17.0was used for data analysis, and P<0.05would beconsidered as statistical significant.Result:1.3days after operations, compared to sham operation group, the relative amount ofEvans blue in myeloid tissue were: saline group:(2.58±0.29), HES40group:(2.38±0.47),HES130/0.4group:(1.38±0.34). The relative amount of Evans blue in HES130/0.4groupwas notably less than that in saline group and HES40group (P<0.05).2. PWPT and PWL of the rats in operation groups were significantly decreased(P<0.05) at the time point of10d,14d,20d,30d,60d after operations compared to thosebefore operations. They shew notably higher value (P<0.05) of PWPT and PWL inHES130/0.4group than in saline group and HES40group after operations.3.10days after the operations, immunofluorescence histochemical results shew thatIba1and ED1were all in active state in HES130/0.4group, HES40group and saline group,but they were significantly lower active in scope and quantity in HES130/0.4group than insaline group and HES40group.4.10days after operations, the relative expressions of Iba1and ED1detected byWestern blot. The grayscale value analysed by software “quantity one” was notably higherin HES130/0.4group, HES40group and saline group than in sham operationgroup(P<0.05). The relative grayscale of Iba1were following: saline group:(0.36±0.034),HES40group:(0.35±0.029), HES130/0.4group:(0.27±0.047), sham operation group: (0.12±0.024); the relative grayscale of ED1were following: saline group:(0.54±0.044),HES40group:(0.55±0.028), HES130/0.4group:(0.38±0.054), sham operation group:(0.06±0.015). The relative grayscales of Iba1and ED1in HES130/0.4group weresignificantly lower than in saline group and HES40group (P<0.05).5.10days after operations, the relative expressions (compared to sham operationgroup) of IL-1β, IL-6and TNF-α in injured myeloid tissue of rats detected by ELISA ineach operation group were following: IL-1β: saline group:(1.46±0.10), HES40group:(1.42±0.14), HES130/0.4group:(1.18±0.11); IL-6: saline group:(1.32±0.12), HES40group:(1.33±0.08), HES130/0.4group:(1.12±0.09);TNF-α: saline group:(1.41±0.10), HES40group:(1.39±0.11), HES130/0.4group:(1.19±0.12). All of the3inflammatory cytokines inHES130/0.4group were significantly lower expressed than those in saline group andHES40group (P<0.05).Conclusion:1. The pain model of rats prepared by NYU spinal cord impact instrument have greatvalues in repeatability, stability and clinical correlativity and can meet the demands for SIPexperiment.2. After spinal cord injury (SCI), the BSCB of rats were destructed, microglia in injurysegment of myeloid tissue was activated, the expressions of inflammatory factors increasedand the pain threshold of rats decreased notably.3. HES130/0.4can decrease the permeability of BSCB efficiently, lighten theinflammatory reaction represented by the activation of microglia/macrophagocyte on theinjury area, down regulate the expressions of IL-1β, IL-6and TNF-α and increase the painthreshold of rats to a certain extent, though there was no significant differences in ethology. |
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