|
Object:The aim of this study was to observe the effect of MEK inhibitors (U0126) combined with chondroitin sulfate ABC on the composition of glial scars following spinal cord injury (SCI) in a rat model, explore the relationship between the different components of glial scar and the neurological function recovery, primarily investigate the influence of combined use of MEK inhibitors and chondroitin sulfate ABC on the neurological function recovery, and make further investigation for the treatment of spinal cord injury.Methods:A total of96adult female Sprague-Dawley(SD) rats were used in these studies. All rats were randomly divided into four groups as follows:Group Ⅰ (SCI control, saline treatment, n=24),Group Ⅱ (ChABC treatment, n=24), Group Ⅲ (U0126treatment, n=24) and Group Ⅳ (U0126+ChABC treatment). Each group was sub-divided for postinjury1-day,1-week,2-week and4-week, each subgroup has6rats.All SCI rats were performed with advanced Allen’s method, and a PE-0402tube was placed into cavitas subarachnoidealis postinjury. After ASCI30min, rats were received diverse treatments. Control rats (group I) were injection with6μl saline through the tube everyday for1week. For rats in group Ⅱ:6μ1(1U/ml) ChABC were first injected through the tube everyday for1week. For rats in group Ⅲ:2μL (lOmg/ml) U0126were first injected through the tube everyday for1week. For rats in group Ⅳ:U0126and ChABC in combination were injected by their respective ways and times.The behavior of each group were observed before operation and3d,7d,14d, and28d after operation and taken BBBscore. Then5randomly selected rats in each group were sacrificed at scheduled time points. Spinal cord materials were obtained, followed by HE staining, Nissl staining, and double immunofluorescence staining of GFAP/CSPG, observation and calculation of positive cells or positive area. Protein integral absorbance were analyzed by software. The experimental data was analyzed by SPSS17.0statistical software.Results:①BBB scores:BBB scores of preinjury is21.One day later after SCI, the BBB scores is0. The activity of hind-leg exhibited different degrees of recovery after SCI with the time past. One week later after SCI, the rats could start to scramble with two hind legs towing and acrotarsium on the ground, the scores of group Ⅰ is (4.33±0.88), the scores of group Ⅱ is (10.05±0.99), the scores of group Ⅲ is (7.69±1.24), the scores of group IV is (11.10±0.88), significantly difference for the scores between group Ⅰ and Ⅱ、Ⅲ、Ⅳ, P<0.05, significantly difference for the scores between group Ⅲ and Ⅱ、Ⅳ, P<0.05, there were no significantly difference for the scores among group Ⅱ and Ⅳ, P>0.05.Two weeks later after SCI, the activity of hind-leg were much power, the scores of group Ⅰ is (4.37±0.91), the scores of group Ⅱ is (14.00±0.75), the scores of group Ⅲ is (11.76±1.35), the scores of group Ⅳis (15.37±1.20), significantly difference for the scores between group Ⅰ and Ⅱ、Ⅲ、 Ⅳ, P<0.05, significantly difference for the scores between group Ⅲand Ⅱ、Ⅳ, P<0.05, significantly difference for the scores between group Ⅱ and Ⅳ, P>0.05. Four weeks later after SCI, the rats were able to stand with the hind-leg, the scores of group I is (4.83±1.05), the scores of group Ⅱ is (16.27±1.28), the scores of group Ⅲ is (14.21±2.47), the scores of group Ⅳ is (18.21±0.87), significantly difference for the scores between group Ⅰ and Ⅱ、Ⅲ、Ⅳ,P<0.05, significantly difference for the scores among group Ⅱ、Ⅲ、Ⅳ, P<0.05.(2) histological observation:①HE staining:3days later after SCI, large areas of necrotic tissue and hemorrhage were observed. Neuronal necrosis, dissolution, and a large number of red particles eosinophilic were observed.7days later after SCI, partial disappearance of hemorrhage, inflammatory cell infiltration, necrotic areas with unclear boundaries around, inflammatory cell of group Ⅱ、Ⅲ、Ⅳ were less than group Ⅰ, and glial scar formation was observed.14days later after SCI, hemorrhage was most absorbed, inflammatory cell infiltration was still observed, but less than earlier stage, cysts and cavity formation were observed, glial cells proliferated and glial scar formation.28days later after SCI, there were obvious cavity formation with clear boundary. Group Ⅰ was more serious than group Ⅱ、Ⅲ about these lesions, group Ⅱ、Ⅲ were at approximately equal extent, and the lesion in group Ⅳ was slightest.②Nissl staining:1days later after SCI, Nissl bodies decomposed into sand granular, fine dust-like, or even disappear.14days later after SCI, Nissl bodies gradual recovery, from granular to coarse granular.28days later after SCI, the most neurons were observed in group Ⅳ,and also the cell bodies were the biggest, followed by group Ⅱ,and then group Ⅲ.③GFAP/CSPGs double immunofluorescence staining:the GFAP expression in the spinal cord injury site was increased, and CSPGs expression was observed. The GFAP expression focused on the edge of the region of spinal cord injury, however, the CSPGs expression focused on the centre of the region of spinal cord injury,14days later after SCI, the fluorescence intensity values of GFAP of groups Ⅰ,Ⅱ,Ⅲ and Ⅳ were (0.64±0.12),(0.51±0.08),(0.35±0.12) and (0.32±0.09), respectively. And the fluorescence intensity of GFAP in groups Ⅲ and Ⅳ was significantly lower than that in groups Ⅰ and Ⅱ (P<0.05). The fluorescence intensity values of CSPGs of groups Ⅰ,Ⅱ,Ⅲ and Ⅳ were (0.0732±0.0112),(0.0493±0.0082),(0.0613±0.0096) and (0.0327±0.0072), respectively. Significant difference among group Ⅱ、Ⅲ and Ⅳ (P<0.05).28days later after SCI, the fluorescence intensity values of GFAP of Ⅰ,Ⅱ,Ⅲ and Ⅳ were(0.66±0.12),(0.40±0.11),(0.27±0.10) and (0.13±0.05) respectively. The expression of GFAP of group Ⅰ was significantly more than that of the other3groups (P<0.05). The fluorescence intensity values of CSPGs of groups Ⅱ、Ⅲ and Ⅳ compare to group Ⅰ were (0.0683±0.0072),(0.0460±0.0051),(0.0555±0.0114) and (0.0207±0.0055), respectively. Significant difference among group Ⅱ、Ⅲ and Ⅳ (P<0.05).④GAP-43immunohistochemistry:7days later after SCI, the integral absorbance of GAP-43of groups Ⅱ and Ⅳ were higher than that of group Ⅲ (P<0.05);14 days later after SCI, The expression of GAP-43in group Ⅳ was higher than that of group Ⅱ. The expression of GAP-43in group Ⅱ and Ⅳ were higher than that of group Ⅲ, and the integral absorbance of GAP-43of groups Ⅱ and Ⅳ were higher than that of group Ⅲ (P<0.05)Conclusion:1. U0126can inhibit the activation of astrocyte which regard GFAP as the marker protein, Chondroitinase ABC can destroy CSPGs specificity, inhibit the formation of glial scar, promote axon growth, indirect promote the regeneration and recovery of spinal cord injury.2. Combined U0126and Chondroitinase ABC can inhibit the glialscar formation following spinal cord injury more evidently than otherindividually treatment groups, the two drugs work well together insynergy to inhibit the glial scar formation.3. The inhibition of axonal regeneration of chemical barriers glial scar might stronger than that of physical barriers.
|