许旺细胞源神经营养因子对脊神经根性撕脱所致神经元死亡的干预

背景:许旺细胞源神经营养因子是从许旺细胞胞浆中分离纯化出的一种相对分子质量为58000的神经营养物质,具有明显的神经营养活性,能对抗一氧化氮神经毒性物质。目的:建立根性撕脱伤动物模型,观察许旺细胞源神经营养因子对脊神经根性撕脱所致脊髓前角运动神经元死亡的保护作用。设计:重复观察测量。单位:深圳市第二人民医院骨三科,中山大学附属第一医院显微外科。材料:实验于2003-03/05在中山大学医学院实验动物中心完成。选用清洁级3～4月龄SD大鼠20只,随机分为许旺细胞源神经营养因子组、生理盐水对照组,各10只。两组动物均以左侧为正常侧,右侧为损伤侧。方法:①两组大鼠均建立颈6,7神经根性撕脱伤动物模型。②许旺细胞源神经营养因子组将一小块预浸有质量浓度为1g/L的许旺细胞源神经营养因子40μL的明胶海绵覆盖于损伤侧硬膜囊表面,生理盐水对照组将一小块预浸有等量生理盐水的明胶海绵覆盖于损伤侧硬膜囊表面。③明胶海绵表面置硅胶管,硅胶管一端与明胶海绵缝扎,另一端用凡士林封闭固定于皮下,关闭切口后伤口局部肌肉注射青霉素预防感染。术后两组动物分别通过硅胶管定期注射许旺细胞源神经营养因子或生理盐水20μL,1次/周,3周后取材。④切取颈6,7脊髓节段,观察损伤侧脊髓前角运动神经元的存活率和形态学变化以及一氧化氮合酶的表达。主要观察指标:①脊髓运动神经元存活情况和形态学变化。②脊髓运动神经元一氧化氮合酶表达的变化。结果:实验选用20只大鼠,全部进入结果分析。①脊髓运动神经元存活情况和形态学变化:颈6,7神经根性撕脱术后3周,生理盐水对照组损伤侧68.6%的运动神经元死亡,存活率31.4%,明显低于正常侧(P<0.01),且存活的神经元胞体严重萎缩;许旺细胞源神经营养因子组损伤侧脊髓运动神经元的死亡率较生理盐水对照组减少35%(P<0.01),存活率为66.4%,且存活的神经元胞体代偿性增大,基本与正常侧相似(P>0.05)。②脊髓运动神经元一氧化氮合酶表达的变化:正常情况下,脊髓中的一氧化氮合酶阳性神经元主要见于后角浅层,中央导水管周围、中间外侧柱和脊髓背根神经节中的内脏传入神经元,前角运动神经元不表达一氧化氮合酶。颈6,7神经根性撕脱术后3周,生理盐水对照组损伤侧脊髓运动神经元一氧化氮合酶表达明显增多,而其正常侧和许旺细胞源神经营养因子组损伤侧脊髓运动神经元均未见一氧化氮合酶表达增加。结论:脊神经根性撕脱可引起脊髓前角运动神经元的死亡和一氧化氮合酶表达,许旺细胞源神经营养因子则对受损的神经元有明显的保护作用和抑制一氧化氮合酶表达。提示许旺细胞源神经营养因子的神经元保护作用可能是通过改变神经元的一些细胞分子例如一氧化氮合酶而实现的。

Protection of motoneurons from spinal root avulsion induced cell death by Schwann cell derived neurotrophic factor

BACKGROUND: Schwann cell derived neurotrophic factor, which is isolated and purified from the kytoplasm of Schwann cell with the relative molecular mass of 58000, is a kind of neurotrophic substance possessing obvious neurotrophic activity. It can be against neurovirulent substance of nitrogen monoxidum.OBJECTIVE:To create root avulsion animal models and observe the protective effects of Schwann cell derived neurotrophic factor (SDNF) on motoneurons of spinal anterior horn from spinal root avulsion induced cell death.DESIGN: Repeated observation and measure.SETTING: Third Department of Orthopaedics, Second People's Hospital of Shenzhen; Department of Micro-surgery , First Hospital Affiliated to Sun Yat-sen University.MATERIALS: This experiment was conducted at the Experimental Animal Center of Medical College of Sun Yat-sen University from March to May 2003. Twenty Sprague-Dawley rats with the age of 3-4 months, of clean degree, were selected and divided randomly into experimental group of Schwann cell derived neurotrophic factor and control group of normal saline with 10 rats in each group. The right side was injured, and the left side was intact served as normal control side.METHODS : ①A rat model of C6,7 spinal root avulsion induced motoneuron degeneration was established. ② A small piece of gelfoam presoaked in 40 μL SDNF solutions (1 g/L) was placed in contact with the injured spinal cord in the animals of the experimental group. Normal saline was added as the same way as above in the animals of the control group. ③ A silica pipe was put on the surface of gleform, one end of the silica was sutured to the glefoam , and the other end wasfixed subcutaneously with vaselinum. Local intramuscular injection of penicillinum was performed on the wound following closing the incision. All rats received an injection (20 μL) of either SDNF or normal saline solution at the lesion site through the silica pipe sutured to the glefoam once a week after the surgery. All the animals were killed by the end of the third weeks. ④The spinal region of C6,7 level was dissected out for observing survival rate and morphological change of motoneurons of spinal anterior horn as well as the expression of nitricoxide synthase(NOS).MAIN OUTCOME MEASURES: ① Survival and morphological change of spinal motor neurons. ②Change of nitricoxide synthase expression of spinal motor neurons.RESULTS: Totally 20 rats were enrolled in the experiment, and all of them entered the stage of result analysis. ① Survival and morphological changeof spinal motor neurons: 68.6% motoneurons of injured side of the control group died at 3 weeks after surgery. The survival rate was 31.4%,which was significantly lower than that of the intact side (P ＜ 0.01), and the survived neurons was shrinked significantly; the death rate of spinal motor neurons of injured side of experimental group was decreased by 35%as compared with control group (P＞ 0.05). The survival rate was 66.4%,and the survived neuron body was increased, similar to the intact side (P ＞ 0.05). ② Change of nitricoxide synthase expression of spinal motor neurons: In normal spinal cord, NOS positive neurons were shown in dorsal horn, surrounding the central canal and in the intermediolateral column.NOS was not seen in the anterior horn motonurons. At the end of the third week after C6,7 spinal root avulsion, increased NOS expression was not found at the injured side in the Schwann cell derived neurotrophic factor group and the intact side in the control side, while the significantly increased NOS expression of spinal motoneurons was found at the injured side of the control group.CONCLUSION: Degeneration of spinal motoneuron and increased expression of NOS can be induced by spinal root avulsion. SDNF has a significant effect in protecting spinal motoneurons from spinal root avulsion induced cell death and inhibiting the expression of NOS. These results suggest that the effects .of SDNF on motoneuron survival may be achieved by modifying the expression of certain cellular molecule such as NOS.