Changes in nitric oxide and expression of nitric oxide synthase in spinal cord after acute traumatic injury in rats

The aim of this study was to observe the time course of NO production and NOS expression in the spinal cord following acute traumatic injury. Rat spinal cord was injured by extradural static weight-compression, which resulted in an incomplete transverse spinal cord lesion with paralysis of the lower extremities. Using this model, measurement of NO by microdialysis and Griess reaction and histological and immunohistochemical examinations using polyclonal antibodies to nNOS and iNOS were performed from immediately to 14 days after injury. In injured cord, the amount of NO markedly increased immediately after injury and gradually decreased between 1 and 12 h after injury. A second wave of increase in NO level was observed at 24 h and 3 days after injury. Histologically, hematomas and necrotic changes were observed after injury and demyelination of nerve fibers increased with time in the compressed segment. Immunohistochemically, the number of cells with expression of nNOS was increased immediately to 12 h after injury. Expression of iNOS was observed from 12 h to 3 days after injury. These findings suggested that the initial maximal increase of NO production might be caused mainly by nNOS and that the second wave of increase in NO might be due mainly to iNOS.     

大鼠脊髓损伤后一氧化氮合酶基因表达的变化

目的　探讨大鼠脊髓损伤后3种类型一氧化氮合酶（NOS）mRNA表达的变化规律。方法　成年SD大鼠36只,随机分为种类6组,每组6只大鼠。建立大鼠脊髓压迫伤模型,以逆转录-聚合酶链反应（RT-PCR）法测定伤段脊髓组织神经型（nNOS）、诱导型（iNOS）及内皮型（eNOS）一氧化氮合酶的mRNA表达情况。结果　脊髓压迫伤后nNOSmRNA及NOSRNA表达增强,伤后6h达到高峰0.633±0.012、1.236±0.207;iNOSmRNA表达亦增高,但在伤后24h才达到高峰1.043±0.049。结论　脊髓损伤后NOSmRNA的表达增强,但不同类型的NOSmRNA变化规律不同,增强或抑制不同NOSmRNA的表达可能减轻脊髓继发性损伤。     

白细胞介素-10对大鼠脊髓诱导型一氧化氮合酶基因表达的干预作用

目的　探讨诱导性一氧化氮合酶 (iNOS)mRNA在损伤脊髓组织中的表达规律及白细胞介素 (IL) 10的干预作用。方法　将 64只SD大鼠随机分为 3组 :单纯脊髓损伤 (SCI)组、IL 10组及假损伤组 (Sham组 )。除Sham组不致伤脊髓外 ,均以改良Allen(10 g× 5cm)打击法制作大鼠急性SCI模型 ,IL 10组于脊髓损伤后 3 0min腹腔注射IL 10溶液 10 μg ,分别于伤后 3、12、2 4、48及 72h处死 ,用逆转录 聚合酶链反应 (RT PCR)法测定损伤脊髓组织iNOSmRNA的表达情况 ,并与单纯SCI组和Sham组作对照。结果　脊髓无损伤时未发现iNOSmRNA表达 ,损伤后逐渐出现表达上调 ,于伤后 12h明显增强 ,2 4h达高峰 (0 .60 85± 0 .0 166) ;IL 10组中 2 4h亚组iNOSmRNA表达 (0 .5 70 5± 0 .0 2 0 3 )出现抑制性改变 ,差异有显著性 (P <0 .0 1)。结论　IL 10可明显抑制大鼠脊髓损伤后iNOSmRNA的高表达     

一氧化氮/一氧化氮合酶系统在脊髓继发性损伤中的作用

脊髓损伤(spinal cord injury,SCI)是临床亟待解决的一大难题,虽然SCI的实验性治疗在18世纪就已经开始,但迄今尚未取得有意义的突破。原因可能是对SCI后病理发展过程认识不足。目前认为急性脊髓损伤有两种损伤机制,即原发性机械损伤及迟发性结构损害所致的继发性损伤,后者产生的损伤程度更严重、范围更广。因此,虽然原发的脊髓横断伤较少,但由于继发的不可逆的结构的改变,损伤仍然非常严重,故阻断继发性损伤的发生成了SCI研究的焦点。一氧化氮(Nitric Oxide,NO)/一氧化氮合酶(Nitric Oxide Synthase,NOS)系统是近年来被认为在继发…     

Gene expression of inducible nitric oxide synthase in injured spinal cord tissue

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Rapid functional recovery after spinal cord injury in young rats

Responses to traumatic injury in the immature spinal cord may be different from those in adults. We modified an adult model of weight-drop injury to characterize the histopathology and functional recovery after spinal cord injury (SCI) in rat pups at postnatal day 14-15. A 10-g weight was dropped from 2.5 or 5.0 cm at T8-T9. Hindlimb function was evaluated at 24 h and 1, 2, 3, and 4 weeks after injury using the Combined Behavioral Score that estimates overall hind limb sensorimotor function, and the BBB scale for open field locomotion. Histopathology was examined at 15 min, 24 h, and 4 weeks after SCI. The initial hemorrhagic lesion was similar to that seen in adults, but the time course of secondary loss of ventral horn motor neurons was extended. By 4 weeks, only a partial rim of white matter surrounding a central cavity was seen. The 5.0 cm injury group exhibited significantly less recovery of function at 4 weeks than the 2.5 cm group. In the latter, the degree of hindlimb deficit at 4 weeks was similar to that previously described for adults with 10 g x 2.5 cm SCI. However, pups in both injury groups exhibited a significantly faster rate of recovery than adults. Recovery was maximal by 1 week after SCI in pups as compared to 3-4 weeks in adults. The more rapid functional recovery observed in the pups suggests that this new model may be useful for studying mechanisms of functional plasticity after SCI.     

Effects of nerve growth factor on neuronal nitric oxide production after spinal cord injury in rats

OBJECTIVE: To explore the protective effects of nerve growth factor (NGF) on injured spinal cord. METHODS: The spinal cord injury (SCI) model of Wistar rats was established by a 10 gx2.5 cm impact force on the T(8) spinal cord. NGF (60 microg/20 microl) was given to the rats of the treatment group immediately and at 2, 4, 8, 12, 24 hours after SCI. The level of neuronal constitutive nitric oxide synthase (ncNOS) and the expression of ncNOS mRNA in the spinal cord were detected by the immunohistochemistry assay and in situ hybridization method. RESULTS: Abnormal expression of ncNOS was detected in the spinal ventral horn motorneuron in injured rats. The levels of ncNOS protein in the NGF group were significantly lower than those in the normal saline group (P<0.05 ). The ncNOS mRNA expression was found in the spinal ventral horn motorneuron in injured rats and the expression in the NGF group was significantly decreased compared with that in the normal saline group (P<0.01). CONCLUSIONS: NGF can protect the injured tissue of the spinal cord by prohibiting abnormal expression of nitric oxide synthase and the neurotoxicity of nitric oxide.     

The role of constitutive nitric oxide synthase in pathogenesis of secondary lesion after spinal cord injury. Preliminary results

BACKGROUND: Secondary lesion (SL) is an early phenomenon of cellular death following spinal cord injury (SCI). Nitric oxide (NO) could be involved in its pathogenesis. NO is a gaseous metabolite produced by 2 constitutive isoforms of NO synthase (cNOS), constantly active, and by 1 inducible isoform (iNOS), synthesized during inflammation and able to produce large amount of NO. High concentrated NO is toxic for cells; therefore, NO concentration is strictly and finely regulated. We suppose that major inhibitory effect on the iNOS expression is represented by the same physiological concentration of NO, synthesized by cNOS. The aim of this study is to assess the role of the 2 cNOS in pathogenesis of SL after SCI in rat. METHODS: A dorsal SCI has been performed on rats (n=5) by a vascular clip (50 g/mm(2) for 15"). Fifteen minutes after trauma, activity of nNOS and eNOS has been measured (U/mg) in the cervical, dorsal and lumbar segments of spinal cord. Uninjured rats (n=5) served as control group. m-RNA for iNOS in untreated rats (n=2) has been also investigated by Northern blotting. RESULTS: In injured rats nNOS activity has shown a reduction in dorsal and lumbar segments, compared to the control group. eNOS activity, highly variable in the control group, has not been detectable in injured spinal cord. i-NOS mRNA has not been found in spinal cord of uninjured rats. CONCLUSIONS: These results would be in line with our hypothesis and provide the bases for other investigations. New therapeutic strategies for SL prevention, based on the modulation of cNOS, will be evaluated.     

急性脊髓损伤后促红细胞生成素受体的表达及其意义

目的探讨急性脊髓损伤后促红细胞生成素(EPO)及其受体(EPO-R)在脊髓内的表达。方法Wistar大鼠69只,随机分为三组：正常对照组(5只,不手术,作为后两组的对照)、假手术组(仅行椎板切除术)和脊髓损伤组。根据术后时间点不同后两组又分为1h、6h、12h、24h、3d、7d、14d和28d八个亚组(每组4只)。采用RT-PCR、Western blot免疫印迹法和免疫组织化学染色法检测EPO及EPO-R的表达。结果正常对照组、假手术组和脊髓损伤组在各时相点均未发现有EPO表达。正常对照组、假手术组未发现EPO-R的表达,脊髓损伤组在伤后1h未见EPO-R mRNA和蛋白的表达,6h开始有表达,12h有明显的表达,至24h达到高峰,3d和7d时仍维持在高表达水平,未见减弱,14d开始下降,至28d时仍有EPO-R蛋白的表达。免疫组化显示EPO-R阳性细胞主要位于神经元、少突胶质细胞、血管内皮细胞和脊髓中央管内室管膜细胞。结论大鼠急性脊髓损伤后脊髓内大量表达EPO-R,这是外源性EPO与EPO-R结合产生神经保护作用的分子基础。     

神经病理性疼痛大鼠脊髓背角一氧化氮合酶各亚型的表达及活性变化

目的 比较神经病理性疼痛大鼠模型脊髓背角中神经型(nNOS),诱导型(iNOS)和内皮型(eNOS)三种一氧化氮合酶(NOS)的表达及活性变化.方法 雌性SD大鼠20只,150～200 g,随机均分为坐骨神经保留性损伤(SNI)组和对照组.SNI组大鼠于左后肢制备SNI模型,对照组大鼠暴露坐骨神经后立即缝合手术切口.术后观测疼痛的变化,于术后第14天断头处死大鼠,截取L4～6段脊髓,分离左右侧脊髓,于-80℃储存.SNI组和对照组各取5只大鼠脊髓,RT-PCR技术扩增nNOS、eNOS和iNOS片段,以β-actin作为内参照,比较SNI组与对照组NOS表达的相对差异.取两组另外5只大鼠脊髓冰上迅速匀浆,均取40 μg的蛋白,分别检测组成型NOS(cNOS,即nNOS和eNOS)和iNOS的活性.结果 SNI组手术侧nNOS、iNOS的mRNA表达均高于SNI组非手术侧和对照组手术侧(P<0.05),而eNOS的表达在SNI组手术侧、非手术侧和对照组手术侧之间差异并无统计学意义.NOS亚型催化活性检测显示SNI组手术侧iNOS和cNOS的活性均高于SNI组非手术侧和对照组手术侧(P<0.05).结论 神经病理性疼痛大鼠模型脊髓背角中iNOS的表达及其合成NO的活性均增加,可能促进了疼痛的发生,nNOS在疼痛中可能具有较强的调节能力.     

急性脊髓损伤后诱生型一氧化氮合酶的表达、分布及作用

目的：研究大鼠急性脊髓损伤（spinal cord injury,SCI)后不同时间脊髓内诱生型一氧化氮合酶（inducble nitric oxide synthase,iNOS)的表达及其在不同类型细胞中的分布、血清中一氧化氮（nitric oxide,NO)含量的变化及iNOS特异性抑制剂氨基胍（aminoguanidine,AG)对大鼠后肢功能的影响。方法：Allen′s模型致伤后，分别于损伤后1、2、3、5、7d取伤段脊髓，作蛋白质印迹分析。在SCI后3d，用免疫荧光和免疫组化顺序标记方法分析iNOS在脊髓神经元、小胶质细胞和星形细胞中的表达与分布。应用比色法测定SCI后血清硝酸盐和亚硝酸盐的含量。观察腹腔注射20mg/kg、200mg/kg AG后SCI大鼠后肢功能的变化。结果：SCI后1d可见iNOS表达，3d达高峰，7d明显降低。在SCI后，神经元、星形细胞和小胶质细胞中均可iNOS表达，其中以神经元表达为主。SCI后血清硝酸盐和亚硝酸盐的含量于伤后1h和3-7d时出现两个高峰。损伤后腹腔注射20mg/kg、200mg/kg AG均能显著抑制血清硝酸盐和亚硝酸盐含量的升高，同时大鼠后肢功能显著恢复。结论：SCI后脊髓内iNOS表达增高，并分布在以神经元为主的多种细胞中。iNOS产生的NO加重了继发性脊髓损伤，对iNOS的抑制可能有助于脊髓损伤后神经功能的恢复。     

异丙酚对慢性神经痛大鼠脊髓诱导型一氧化氮合酶表达的影响

目的 研究不同剂量异丙酚对慢性神经痛大鼠触诱发痛痛阈及其脊髓组织诱导型一氧化氮合酶(iNOS)mRNA的影响。方法 Wistar大鼠40只,随机分为四组,Ⅰ组为空白对照;Ⅱ、Ⅲ、Ⅳ组结扎左侧坐骨神经。术后第7天,Ⅰ、Ⅱ组腹腔注射生理盐水50 ml·kg-1,Ⅲ、Ⅳ组腹腔注射异丙酚50 ml·kg-1或75ml·kg-1,每天一次共6 d。在术后第6、10和12天,使用Von Frey法分别测定各组大鼠触诱发痛痛阈,比较不同剂量的异丙酚对大鼠痛阈的影响。术后第12天取L4-5和L5-6节段脊神经节和脊髓组织,采用半定量RT-PCR法,对各组大鼠脊髓组织iNOS mRNA表达进行检测。结果 术后第10和12天,Ⅲ、Ⅳ组大鼠双侧的痛阈值高于Ⅱ组(P<0.05);术后第12天,Ⅲ、Ⅳ组大鼠脊髓组织iNOS mRNA表达低于Ⅱ组(P<0.05)。结论 异丙酚通过抑制脊髓组织iNOS mRNA转录,降低其表达,而起到一定的抗伤害作用。     

诱导型一氧化氮合酶、一氧化氮在脑积水大鼠脑组织及脑脊液中的含量变化

目的探讨诱导型一氧化氮合酶(iNOS)和一氧化氮(NO)在实验性大鼠脑积水中的病理生理作用。方法实验组SD大鼠36只,随机平分为3个组,用显微外科技术向枕大池内注入 25％白陶土混悬液,分别在第2、3、4周后行核磁共振检测,鉴定脑积水形成情况,之后24 h内取脑脊液和脑组织,用免疫组织化学方法测脑组织不同部位iNOS表达情况,硝酸还原酶法测脑脊液中 NO含量。对照组12只大鼠于枕大池内注入生理盐水,用同样方法检测。结果实验组共25只大鼠成功诱发脑积水。与对照组相比,实验组脑组织中iNOS表达率均升高,尤其是白陶土注射后第 4周脑室周围白质更为明显(40．65±4．06)％;实验组脑脊液中NO含量亦均增加,其中白陶土注射后第4周最高(51．37±6．48)μmol／L。结论 iNOS和NO可能参与了脑积水的病理生理过程。     

Endothelial nitric oxide synthase expression in ischemia-reperfusion injury after living related-donor renal transplantation

Ischemia-reperfusion injury during renal transplantation has been linked to early graft dysfunction and late graft failure. Nitric oxide (NO), produced by NO synthase (NOS), participates in the recovery from ischemia. We correlated the intensity of graft immunoreactivity for the endothelial NOS isoform (eNOS) during early reperfusion with graft function in 25 children receiving grafts from related donors. Renal allograft biopsy specimens were obtained before transplantation, 1 h after renal artery reperfusion, and 1 year after transplantation. Immunohistochemical staining for eNOS occurred mainly within the endothelium of glomerular capillaries and peritubular capillaries as well as in tubule cells. The mean intensity score for eNOS staining (0-9) was 3.0+/-1.4 before transplantation, 4.5+/-1.9 at 1 h, and 3.3+/-1.9 at 1 year (baseline vs 1 h, P<0.05). Creatinine clearance (ml/min) in patients with a 1-h eNOS score of below 5 and of at least 5, respectively, was 77.1+/-28.4 vs 104.3+/-25.3 at 1 month, 78.7+/-33.4 vs 105.2+/-24.4 at 3 months, 64.7+/-30.1 vs 100.1+/-25.3 at 1 year, 58.2+/-31.3 vs 84.7+/-18.8 at 3 years, and 71.2+/-19.7 vs 78.3+/-23.1 at 5 years ( P<0.05 for 1 month, 1 year, and 3 years). We concluded that elevated eNOS expression after reperfusion in living related-donor renal transplantation enhances the recovery from renal ischemia and, consequently, reduces late graft deterioration.     

大鼠胚胎脊髓移植后增加损伤脊髓生长抑素mRNA的表达

[目的] 研究大鼠胚胎脊髓移植后是否能够影响生长抑素（SOM）mRNA的表达。[方法]将动物分为单纯半切洞损伤组（A组）、胚胎脊髓移植组（B组）。术后1、3、7、14、28d，采用原位杂交方法观察SOM mRNA的表达，采用计算机图像分析技术，进行定量分析。[结果] 胚胎脊髓移植组SOM mRNA蛋白表达明显多于单损伤组。[结论] 作者的研究表明胚胎脊髓移植后可使损伤脊髓SOM mRNA表达增多。