Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

Dongxiao Li; Yundu Li; Yunyun Tian; Zuoshang Xu; Yansu Guo

doi:10.3791/58565

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

重组腺相关病毒在成年小鼠中的直接注射

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08:17 min

February 15, 2019

DOI: 10.3791/58565-v

Dongxiao Li*¹, Yundu Li*², Yunyun Tian¹, Zuoshang Xu³, Yansu Guo^1,4

¹Department of Neurology,Second Hospital of Hebei Medical University, ²No.2 Middle School of Shijiazhuang, ³Department of Biochemistry and Molecular Pharmacology,University of Massachusetts Medical School, ⁴Key Laboratory of Hebei Neurology

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Overview

This article presents a direct intrathecal injection technique utilizing 1% lidocaine hydrochloride in a viral solution to enhance the delivery of adeno-associated virus to small animals, specifically targeting central nervous system diseases such as ALS. It establishes a scoring system that correlates the degree of transient weakness induced by lidocaine with transduction efficiency.

Key Study Components

Area of Science

Neuroscience
Gene Therapy
Intrathecal Delivery

Background

Adeno-associated viruses are commonly used for gene delivery to the nervous system.
Methods for effective delivery in small animal models are critical for advancing therapeutic strategies.
Lidocaine has been employed for its transient paralysis effects, which indicate successful intrathecal injections.
Optimizing delivery methods can improve outcomes in experimental treatment of central nervous system diseases.

Purpose of Study

To demonstrate a reliable technique for intrathecal delivery of viral solutions in awake small animals.
To create a predictive scoring system for evaluating transduction efficiency.
To improve the understanding of the effectiveness of adeno-associated virus delivery mechanisms.

Methods Used

The technique involves direct intrathecal injection using a 27 gauge needle attached to a 25 microliter Hamilton syringe.
Small animals, specifically mice aged 30 to 70 days, are used as the biological model.
Important procedural steps include preparing the syringe, aligning the needle for injection, and monitoring transient weakness as an indicator of success.
Post-injection, mice undergo surgical procedures to extract brain and spinal cord tissues for analysis.

Main Results

The study finds a robust correlation between the magnitude of transient limb weakness and the extent of spinal cord transduction.
Significant eGFP immunostaining results indicate varying levels of transduction across different nervous system regions based on the weakness scores.
Key structures showing strong transduction include the olfactory bulb, hippocampus, and anterior horns of the spinal cord.

Conclusions

This study establishes a new method for effective adeno-associated virus delivery to the central nervous system in small animals.
The transient weakness score serves as a reliable measure of injection efficacy.
Findings enhance the understanding of gene therapy approaches and their optimization in treating neurological diseases.

Frequently Asked Questions

What is the advantage of using lidocaine in the injection?

Lidocaine induces transient paralysis, providing a clear visual indicator for the success of the intrathecal injection.

How is the injection performed on the animal?

The animal is positioned prone, and the intervertebral space is palpated to guide the needle insertion for intrathecal delivery.

What types of outcomes can be measured from this method?

Outcomes include the extent of viral transduction, as indicated by eGFP immunostaining and assessment of transient limb weakness.

How can this method be adapted for other studies?

The technique may be modified for different viral vectors or other small animal models, facilitating broader applications in gene therapy research.

What are some limitations of this technique?

Successful implementation requires sufficient practice to minimize complications and ensure consistent delivery across trials.

What biological regions were effectively transduced?

Key areas include the olfactory bulb, hippocampus, and spinal cord, with transduction intensity correlating with the weakness score of the mice.

Why is spinal cord tissue extraction important?

Extracting and analyzing spinal cord tissue helps evaluate the efficacy of the viral vector and provides insight into therapeutic impacts on the nervous system.

在这里, 我们提出了一个直接鞘内注射技术使用1% 利多因盐酸在病毒溶液中, 以确保有效的腺病毒传递给小动物, 并建立评分系统, 以预测在中央的传导效率神经系统根据利多卡因引起的短暂虚弱程度。

这种方法有助于在清醒的小动物中有效传递与腺相关的病毒转基因，用于实验性治疗中枢神经系统疾病，如ALS。这种方法的主要优点是，病毒溶液包括1%利多基盐酸盐，在注射成功后诱导暂时性瘫痪。虽然此方法为判断内部自动化情况的成功率提供了视觉指标，但足够的实践对于提高交付的成功率至关重要。

在开始手术之前，将 27 量针连接到 25 微升汉密尔顿注射器上，然后将针头的斜端与注射器上的体积刻度对齐。然后小心地将8微升的4×10到病毒溶液的第10个基因组拷贝放入注射器中，注意避免气泡。接下来，将一只清醒的30至70天12至30克的老鼠放在生物安全罩易发位置的床上，用无菌纱布覆盖上半身，使老鼠平静下来，避免被咬伤。

用拇指一侧紧紧抓住鼠标的骨盆腰带，另一侧用食指和中指，用拇指和食指将皮紧绷在双边骨盆腰带之间。然后剃掉双边骨盆腰带之间的毛皮，用碘基磨砂和70%乙醇对裸露的皮肤进行消毒。对于腺相关病毒溶液的直接内部传递，在双边骨盆腰围之间的中线上对椎间空间进行点心，并使用指甲压入皮肤，以指示L5至L6椎间空间。

轻轻旋转尾巴底部，露出脊柱的中线，并调整针斜向动物头部。将鼠标牢固固定到位，沿脊柱中线对齐针头，然后轻轻地垂直地将针头插入缩进中心，使注射器处于中央下垂平面中。当针头与骨骼接触时，慢慢将角度减小至约 30 度，然后将针头滑入椎间空间。

突然的尾部轻拂是成功进入硬膜内空间的标志。当针头进入椎间空间时，尖端会感觉被牢牢地夹住。注入向量溶液，并在硬膜内空间内保持针头一分钟。

然后缓慢地旋转地提取针头，以尽量减少泄漏。立即对小鼠四肢的短暂无力进行评分，以评估注射质量，并返回小鼠的笼子，从瘫痪中恢复。在适当的实验终点点，将注射小鼠的四肢和头部固定到泡沫盒盖上的易发位置，并使用剪刀将皮肤从头部剥离到囊中。

将头骨夹在眼睛之间，沿着头骨的中线和小脑上方的水平线切割，并打开两侧的头骨。使用钳子取出腹骨，并使用眼科剪刀双边打开脊柱管。切开两侧的肋骨，小心地取下椎骨的上部。

使用弯曲的钳子抬起大脑，切断头骨基的神经。然后小心提取整个大脑和脊髓，将组织修复为4%的甲醛24小时。在固定期结束时，在4摄氏度下，在30%蔗糖溶液中冷冻保护大脑、颈椎和腰椎，然后嵌入最佳切割温度化合物中，然后捕捉到液氮中的冷冻。

然后使用低温计温器获取每个组织25微米厚的部分，将冷冻部分储存在0.01摩尔PBS中，在4摄氏度时。对于组织的免疫组织化学分析，用1%过氧化氢预处理自由浮动部分10分钟，然后用PBS清洗10分钟。接下来在PBS中孵育含有5%血清和0.3%非离子洗涤剂的阻断溶液中的样品1小时，随后在4摄氏度下用适当的原抗体进行过夜标记。

第二天，用3个10分钟的PBS加Tween洗涤部分，并在最后一次洗涤后，用适当的相应的生物基化二次抗体孵育幻灯片，持续1小时。在孵育结束时，在新鲜 PBST 中清洗部分 3 次，正如刚刚演示的，用无限生物素过氧化物酶复合物孵育样品 40 分钟，然后用适当的色度剂染色。将标记部分安装到玻璃显微镜幻灯片上，并在安装部分完全干燥后将幻灯片浸泡在无水乙醇中 5 分钟。

接下来，将幻灯片浸泡在二甲苯中 10 分钟，然后用适当的安装介质密封。然后用配备充电耦合装置的光学显微镜以 100、200 和 400 倍的放大倍率对幻灯片进行成像。eGFP对颈椎和腰椎组织部分进行免疫损伤，发现弱分为0的小鼠腰椎脊髓很少或没有转导，弱分为1的小鼠的转导率略有增强，弱分为4或5的小鼠的转导率则略有增强。

显示不同程度瞬态肢体弱点的小鼠脊髓组织部分的GGFP染色强度的定量表明，病毒溶液注射后虚弱的严重程度与脊髓转导的程度密切相关。在大脑中，在嗅球、多侧前额皮质、凹陷陀螺和海马、小脑皮质和脑干边缘区域（包括面部核、胆管丛和阴性上皮细胞）的CA3区域检测到强大的eGFP信号。前角的运动神经元在脊髓的不同水平上也强烈转导。

此外，在皮层中，检测到包括金字塔细胞在内的GP正神经元以及各种胶质细胞类型，包括微胶质、星形细胞和橄榄细胞。这项技术使神经学领域的研究人员能够探索小觉醒动物直接在内分娩对中枢神经系统疾病的治疗效果。

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