| Treatment of spinal cord injury (SCI) has puzzled researchers worldwide for a long period of time. More and more researchers are focusing their eyes on the development in this field. Effective treatment of those SCI cases takes a great significance in medical management and expenditure of society. Multiple cytokines participate the secondary damage after primary injury, which take an important role in the process of apoptosis and affect the outcome of neural functional recovery. Management in the early stage can prevent or reverse those secondary pathological damages, reduce apoptosis neural cells, and greatly improve the neural functional recovery of the injured spinal cord. Interleukin-10 (IL-10), which is a potential anti-inflammatory agent, can inhibit the cytokine production from Th1 cells and suppress the activity of those cytokines. Moreover, cytokines such as chemotactic factor, cell adhesion factor, active oxygen and nitrogen medium et al, can also be suppressed by IL-10. Early experiment on brain injury revealed that IL-10 could function as an anti-inflammation factor to protect neural cells. Whether IL-10 could be used in SCI for neural protection purpose, few literature has been reported abroad and no related literature was found domestically.Objective In the present study, rat SCI model was established by operation. IL-10 solution was delivered to animal models at the early phase of SCI. At different time point after the injury, TNF- a mRNA and iNOSmRNA, activity of NOS, NO concentration, neural apoptosis and morphological studies were carried out. Functional evaluation was also performed to observe the influence of IL-10 on the outcome of neural functional recovery. The possible mechanism of neural protection of IL-10 was discussed. The purpose of this study is to discuss the possible mechanism of neuro-protective effect of IL-10 after SCI and provide theoretical basis for the future clinical application of IL-10 on SCI cases.Methods Modified Allen impaction methods were used in the present study to establish SCI models in Sprague-Dawley rats. After anesthesia by sodium pentobarbital through intraperitoneal injection, spinal segment of T10 was exposed by operation. Then the SCI injury was reduced at T10 by adjusting the height of the weight drop (10.0g) to 5 cm above the exposed spinal cord to create spinal cord injury. All the rats in the experiment were assigned into IL-10 group, control group and sham operation groupat random. At 30 min after the impaction, IL-10 water solution was delivered intraperitoneally in IL-10 group, while same volume of normal saline was given to serve as control. In sham operation group, the spinal cord were only exposed, but not injured. In experiment one, tilt plate test and BBB evaluation were performed at 12h, 24h, 48h, 4d, 8d, 16d and 24d respectively. At 24d after the injury, samples of the injured spinal cord were harvested for light microscope and electronic microscope observation to evaluate the degree of injury. In experiment two, at lh,6h,12h,24h and 48h after spinal cord injury, samples of the injured spinal cord were harvested, the expression of TNF- a mRNA was determined with reverse transcription polymerase chain reaction (RT-PCR) and concentration of TNF- a was determined with ELISA; In experiment three, at 3h, 12h, 24h, 48h and 72h after the injury, the expression of iNOSmRNA was determined with RT-PCR, and activity of NOS and concentration of NO was determined with chemical analysis methods. In experiment four, apoptosis cell labeled with terminal deoxynu-cleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL method) were carried out and apoptosis cell number was counted at 12h, 24h, 48h, 4d, 8d, 16d and 24d respectively after SCI. Results1. Different degrees of neural functional recovery were found in the two groups during the 24 days'observation. On 16, 20 and 24days after the injury, there existed a striking difference between IL-10 group and control group when the results of parallel tilt plate and BBB evaluation were taken into consideration (PO.05). Specimens from the IL-10 group showed less structure destruction under both light and electronic microscope observation.2. It was revealed by TNF- a RT-PCR test that expression of TNF- a mRNA got an elevation at lh after SCI, it kept at high level until 12h time point. Then it declined to baseline level at 48h. In IL-10 group, the expression of TNF- a mRNA also showed an elevation, but in a delayed peak value manner. It kept at high level within a relative short time, at 12h after SCI it fall back to baseline. In the comparison among subgroups (lh,6h,12h,24h and 48h) between control group and IL-10 group, expression of TNF- a mRNA in 24h subgroup between IL-10 group and its counterpart in control group exist a statistic difference (PO.05). There exist a significant statistic difference in expression of TNF-a mRNA in lh, 6h and 12h subgroups between IL-10 group and their counterparts in control group (PO.01).Concentration of TNF- a got an elevation at 1 h after SCI, and reached its peakvalue at 6h. At 48h, it declined to normal baseline. In IL-10 group, concentration of TNF- a was also higher, but in a lower amplitude and in a delayed peak time manner, compared to their counterparts in control group (P<0.05 or PO.01).3. It was revealed by the agarose electrophoresis results that no iNOS mRNA was expressed in sham operation group. However, in injured spinal cord tissue, expression of iNOS mRNA was elevated, and reached its peak value at 24h time point. In comparison among subgroups (3h, 12h, 24h, 48h and 72h) between control group and IL-10 group, expression of iNOS mRNA in 12h and 48h subgroups between IL-10 group and control group exist statistic difference (PO.05), and there exist a significant statistic difference in 24h subgroups between IL-10 and control group (PO.Ol).Both control group and IL-10 group showed an elevated activity of NOS and concentration of NO. However, those values are lower in IL-10 group than in control (P<0.05 or PO.01). In the comparison of activity of NOS, there exist a statistic difference in 3h and 72h subgroup between IL-10 group and control group (pO.05), and there exist significant statistic difference in 12h, 24h and 48h subgroup between IL-10 group and control group (pO.Ol). In the comparison of concentration of NO, there exist a statistic difference in 48h and 72h subgroup between IL-10 group and control group (p<0.05), and there exist significant statistic difference in 12 and 24h subgroup between IL-10 group and control group (pO.Ol).4. No apoptosis neural cells was found in sham operation group. Sporadically distributed apoptosis cells were found as early as 12h after SCI, more TUNEL positive cells were found at 24h, and reached its peak value at 48h. At the same time point, less apoptosis cells were found in IL-10 group than control group. In the comparison of TUNEL positive cells number, there exist a statistic difference in 24h and 4d subgroup between IL-10 group and control group (pO.05), and there exist significant statistic difference in 48h subgroup between IL-10 group and control group (pO.Ol). Conclusions1. After SCI, the expressions of TNF-a mRNA and iNOSmRNA were elevated, synthesis and concentrition of TNF- a and NO and activity of NOS were increased in the injured spinal cord. The elevated inflammation cytokines gravitated the secondary injury and induce great quantities of neural cell apoptosis, which result in severe loss of neural function.2. IL-10, as a potent anti-inflammation factor, could suppress the synthesis of TNF- a , lowered the concentration of TNF- a , inhibit the expression of iNOS mRNAand activity of NOS, reduce the concentration of NO. Therefore, IL-10 block the cell apoptosis signal pathway and reduced the number of apoptosis neural cell. The early administration of IL-10 could significantly improve the neural functional recovery of the injured spinal cord. IL-10 may become a promising neural protection agent in the treatment of SCI.
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