脊髓损伤模型大鼠软脊膜下注射2型腺相关病毒免疫荧光染色分析

Immunofluorescence staining of adeno-associated virus type 2 after subpial injection in rat models of spinal cord injury

背景:经软脊膜下注射腺相关病毒的方法可将病毒局限于以注射点为中心的较小范围内,全身毒副作用小,节省病毒的用量,但目前尚无将此方法应用于脊髓损伤大鼠的相关报道,对损伤部位星形胶质细胞和神经细胞的转染情况尚不明确。目的:探讨通过软脊膜下注射2型腺相关病毒对脊髓损伤部位的星形胶质细胞和神经细胞的转染效果及手术过程的技术要点。方法:实验方案经吉林大学动物实验伦理委员会批准。24只Wistar大鼠随机分为2组:腺相关病毒组和模型组,均使用钳夹法构建脊髓损伤模型。腺相关病毒组经软脊膜下注射60μL病毒溶液;模型组经软脊膜下注射等量5%右旋糖酐溶液。于术后7,14和21d取脊髓标本,进行免疫荧光染色,观察星形胶质细胞分布情况和2型腺相关病毒转染情况。结果与结论:①经软脊膜下注射病毒后,病毒溶液最终均匀分布于损伤部位上下约1 cm的范围内;②术后7,14 d仅有微量绿色荧光蛋白在脊髓损伤区域表达,直至术后21 d,在脊髓损伤空洞周围可见有大量反应性星形胶质细胞,其在脊髓空洞周边部表达最为强烈,出现明显的胶质界膜;同时发现,2型腺相关病毒可稳定转染星形胶质细胞和神经细胞,其中星形胶质细胞集中分布于脊髓空洞周边部,而神经细胞散在分布于病毒转染范围内,注射范围以外正常组织仅见微量2型腺相关病毒转染;③该方法具有能将病毒局限于损伤部位,可靶向性转染星形胶质细胞和神经细胞,具有注射量病毒量低,全身毒副作用小,成本节约等优点。

BACKGROUND: The method of injecting adeno-associated virus subpial can limit the virus to a small area centered on the injection point,with less systemic toxicity and side effects, and save the amount of virus. However, there are no reports on the application of this method in spinal cord injury rats, and the transfection of astrocytes and neurons at the injured site is still unclear.OBJECTIVE: To investigate the transfection effect of subpial injection of adeno-associated virus 2 on astrocytes and neurons in the spinal cord injury site and the technical points of the surgical procedure.METHODS: The study was approved by the Experimental Animal Ethics Committee of Jilin University. Twenty-four Wistar rats were randomly divided into two groups: adeno-associated virus group and model group. Rats in both groups were used to establish the spinal cord injury model using the clamp method. The adeno-associated virus group received 60 μL of virus solution by subpial injection, and the model group received an equal amount of 5% dextran solution. At postoperative 7, 14, and 21 days, spinal cord specimens were taken for immunofluorescence staining to observe the distribution of astrocytes and the transfection of adeno-associated virus 2.RESULTS AND CONCLUSION:(1) After subpial injection of the virus, the virus solution evenly distributed in the range of about 1 cm above and below the injured site.(2) At 7 and 14 days after surgery, only a small amount of green fluorescent protein was expressed in the spinal cord injury area. Until day 21, a large number of reactive astrocytes were observed around the spinal cord injury cavity. The strongest expression was in the periphery of the spinal cord cavity, with obvious glial boundary membrane. It was also found that adeno-associated virus 2 could stably transfect astrocytes and neurons, in which astrocytes were concentrated around the periphery of the spinal cord cavity,while neurons were scattered within the scope of virus transfection. Only trace adeno-associated virus 2 transfection was found in normal tissues outside the scope of injection.(3) The method has the advantages of confining the virus to the injured site, targeting astrocyte and nerve cell transfection, low viral dosage, low systemic toxicity and side effects, and low cost.