| Objectives:1. To develop an animal model for nonbacterial chronic prostatitis in rats with the use of intraprostatic injection of3%λ-carrageenan, and to assess the validity and duration of this model;2. To investigate the role of increased oxidative stress and oxidative damage of spinal cord in nonbacterial chronic prostatitis in rats induced by intraprostatic λ-carrageenan injection, and to explore its possible mec-hanism;3. To investigate the effect of N-acetylcysteine(NAC) on nonbacterial chronic prostatitis induced by intraprostatic λ-carrageenan injection in rats and its possible mechanism.Methods:Male Sprague-Dawley rats weighing250-350g were used for the experiments. In experimental groups(n=40),50ul3%λ-carrageenan was injected into the ventral prostate in SD rats under the condition of anesthesia and sterile.50ul of sterile normal saline was injected into the control group. At different time points(before and after24h,7d,14d and30d of injection), radiant heat and von Frey filaments (mechanical stimuli) were applied to the scrotum of rats. The escape latency (s) from radiantheat, and the bending force (g) of the filament to which the animal responded by moving were taken as measures of heat and mechanical thresholds respectively. After determination of these thresholds, the prostate was removed. The expression of cyclooxygenase-2(COX2) in prostate was determined by Western-blot. Evans blue(50mg/kg) was also injected intravenously to assess for plasma protein extravasation at different time points after injection of λ-carrageenan.50ul3%carrageenan was injected into both right and left ventral lobes of the prostate gland in SD rats(n=20). At different time points(before and after24h,7d,14d and30d of injection), after measures of heat and mechanical thresholds, rats were sacrificed and the L6-S1spinal cord were assayed. The level of8-isoprostane(8-epi PGF2a) and superoxide dismutase(SOD) in spinal cord were examined by enzyme linked immunosorbent assay(ELISA) and xanthine oxidase respectively and caspase-3expression was evaluated by immunohistochemistry.10%NAC(300mg/kg) was intraperitoneal injection before the surgery and once a day after the surgery for seven days. At24h after the injection of carrageenan and one hour after the last NAC injection, the pain thresholds were examined and prostate and L6-S1spinal cord were removed. The expression of cyclooxygenase-2(COX2),8-isoprostane, SOD and caspase-3were assessed by the same methods.Results:After injection of λ-carrageenan, compared to control group, inflamed animals showed a significant reduction in mechanical threshold (mechanical allodynia)at24h and7d(p=0.022,0.046, respectively), and a significant reduction in heat threshold (thermal hyperalgesia) at24h,7d and14d(p=0.014,0.018,0.002, respectively) in the scrotal skin. Significant increase of inflammatory cell accumulation, COX2expression and Evans blue extravasation were observed at24h,7d and14d after injection. Intensified extent of oxidative stress of spinal cord was found as the pain thresholds decreased. The level of8-isoprostane in L6-S1spinal cord of inflamed animals increased and activity of SOD declined significantly at7d after injection(p=0.008, p=0.001respectively). Significant difference still remained by the14th day but it returned to normal by the30day. L6-S1spinal cord caspase-3fluorescence staining in the experimental group showed much more intensity than control group by the7th and14th day after injection while no substantial difference was found by the24th hour and the30th day. Compared with the simple model group, the heat and mechanical thresholds in NAC intervention group increased significantly by the24th hour and the7th day after intraprostatic injection of3%λ-carrageenan, and COX2expression in prostate was significantly reduced(P=0.046, P=0.027). Level of8-epi PGF2a in spinal cord was decreased with no statistical significance by the24th hour but significant lower(P=0.035) by the7th day. Caspase-3fluorescence staining in intervention group was less intensive than that in model group at7d after injection.Conclusion:1. It is safe and effective to develop the animal model for nonbacterial chronic prostatitis in rats with the use of intraprostatic injection of3%λ-carrageenan, and its duration is more than2weeks;2. There exist increased oxidative stress and oxidative damage in L6-S1spinal cord of the animal model induced by intraprostatic λ-carrageenan injection, which suggest oxidative stress in spinal cord is closely associated with nonbacterial chronic prostatitis;3. Neurons apoptosis in L6-S1spinal cord caused by ROS may result in the nonbacterial chronic prostatitis pain of this model rats;4. Systemic NAC administration suppresses the inflammatory in prostate and the increased oxidative stress in spinal cord induced by intraprostatic λ-carrageenan injection. NAC may be a promising drug for treatment of CP/CPPS. |
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