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

doi:10.3969/j.issn.2095-4344.1481

Abstract

Abstract:

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.

Key words: adeno-associated virus 2, spinal cord injury using the clamp method, subpial injection, astrocyte, neurocyte, green fluorescent protein, glial fibrillary acidic protein, neurofilament-H, immunohistochemistry, immunofluorescence, glial scar

CLC Number:

Bai Qiushi1, 2, Su Sheng1, 2, Wang Liangjia1, 2, Zheng Yangyang2, Kang Juanjuan2, Xu Jinying2, Chi Guangfan2. Immunofluorescence staining of adeno-associated virus type 2 after subpial injection in rat models of spinal cord injury[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(31): 4979-4985.

Figures/Tables 1

2.1 实验动物数量分析实验24只Wistar大鼠均进入结果分析，中途无脱失。 2.2 脊髓损伤模型评价完成钳夹脊髓损伤的大鼠术后均出现截瘫、尿潴留，部分出现血尿、肠梗阻。术后因泌尿系统感染及膀胱破裂死亡3只，其中腺相关病毒组1只，模型组2只，另外有不明原因死亡2只，腺相关病毒组1只，模型组1只，均用同体质量雄性Wistar大鼠补充。 2.3 病毒溶液分布情况在聚乙烯导管插入脊髓损伤上方0.3 cm并开始注射后，可观察到无色透明病毒溶液借助流速将导管上方约0.2 cm的脊髓组织与软脊膜分离开并均匀分布包裹整个脊髓组织。随导管缓慢向外抽出，病毒溶液最终均匀分布于损伤部位上下各0.5 cm的范围内。在聚乙烯导管拔出后观察到极少量病毒溶液附于导管壁上随拔出过程带出，导管拔出后仅观察导管遗留空洞处，并无明显溢出。最终可见以损伤部位为中心上下共约1 cm范围内软脊膜轻微隆起，其内为无色透明样液体。模型组与腺相关病毒组所见相同。 2.4 脊髓组织病理变化模型组与腺相关病毒组均在剥离脊髓标本时发现与周围组织粘连严重，且损伤部位与未损伤脊髓部位的白色相比颜色呈淡黄色。2组粘连情况与颜色无明显区别。 2.5 免疫荧光法观察胶质纤维酸性蛋白在脊髓损伤区域的表达模型组和腺相关病毒组脊髓纵切面可见钳夹处较正常部位脊髓横径略短，脊髓结构排列混乱，灰白质界限不清，形成脊髓空洞。在脊髓空洞周围可见有大量表达胶质纤维酸性蛋白阳性细胞，胞体呈星形，向周围发出许多长的突起，其在脊髓空洞周边部表达最为强烈，出现明显的胶质界膜。见图2。 2.6 2型腺相关病毒转染脊髓星形胶质细胞与神经细胞情况与模型组比较发现，术后7，14 d仅有微量绿色荧光蛋白在脊髓损伤区域表达，至21 d在低倍镜下可见2型腺相关病毒携带的绿色荧光蛋白集中分布于脊髓损伤周边部，而远处仅可见微量绿色荧光蛋白表达。经与胶质纤维酸性蛋白表达情况比较发现，发现较多红色荧光与绿色荧光重合区域集中分布于脊髓空洞周边部，即受2型腺相关病毒转染的星形胶质细胞。见图3。经与NF-H表达情况比较发现，发现较多红色与绿色荧光重合区域散在分布于病毒转染范围内，即受2型腺相关病毒转染的神经细胞。见图4。 "

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