delivering intrathecal injections to neonatal mice

在新生小鼠中进行鞘内注射的综合方案

Intrathecal (IT) injection is a common clinical procedure used to administer medications, collect cerebrospinal fluid, and maintain intracranial pressure in both pediatric and adult patients in clinics1,2. The administration of medications via intrathecal injection is an effective approach for increasing medication concentrations in the central nervous system (CNS) while minimizing systemic exposure. Consequently, this method enhances therapeutic efficacy and reduces side effects, especially for temperature-sensitive and short half-life drugs3.
In preclinical studies testing new drugs and treatments using rodent models, it is imperative to employ a reliable method of drug administration that offers greater precision and result reproducibility4,5. For preclinical studies evaluating new treatments for neurogenetic and neurodevelopmental disorders, early treatment is crucial for initial proof-of-concept studies because earlier interventions are typically predicted to yield more favorable outcomes6,7,8.
Compared to conventional intracerebroventricular (ICV) injections, IT injections carry significantly lower risks since they obviate the need for direct penetration through the cerebral cortex. This advantage substantially reduces the potential damage to regional cortical tissue and surrounding nerves. Furthermore, IT injections allow for at least a fivefold increase in the administrable volume of medications through a single injection, greatly enhancing the feasibility of repeated administrations. However, due to the small size and fragile nature of newborn mice, performing intrathecal injections in newborn pups is technically challenging and requires specialized techniques, equipment, and meticulous handling.
This article provides a detailed protocol with step-by-step instructions for performing intrathecal injections in P1 newborn pups. The key considerations and good laboratory practices are emphasized here to ensure the consistency of administration and the safety and well-being of the animals during the procedure. By following this protocol, researchers can confidently conduct experiments with precision and reproducibility while minimizing any potential risks or discomfort to the animals.

作者聚焦： 鞘内注射 – 一种高效可靠的递送方法，用于测试新生小鼠大脑中基因编辑的功效

新生小鼠鞘内注射用于基因组编辑和药物递送

Our group employs an interdisciplinary approach involving genomic discovery and functional modeling to study genetics and epigenetic basis of neurodevelopmental disorder. We perform molecular, neurophysiological, and neurobehavioral analysis using patient-induced pluripotent stem cell, or iPSC, in the mutant mice to dissecting the pathophysiology of neurodevelopmental disorder. We aim to develop a gene-editing-based treatment for this disease.Intracerebroventricular, also called ICV, injection is only method to directly deliver treatment to the central nervous system of neonatal mice. However, it has to penetrate through the cerebral cortex. The intrathecal, IT, injection is a commonly employed procedure in clinics, serving as an effective means to administer treatments directly into the central nervous system.However, intrathecal injection in neonatal pups can pose significant technical challenges due to their small size and fragile nature. We are trying to develop an efficient and reliable delivery method to test the efficacy and the gene editing in mouse brain that is non-viral and non-nanoparticle. Compared to conventional ICV injection, intrathecal injection presents a significantly lower risk, as it avoid the need for directly penetration through the cerebral cortex.This add advantage to minimize potential damage to regional cortical tissue and the surrounding nerves. Intrathecal injections also enable at least a fivefold increase in the administrable volume of medications through a single injection and greatly enhance the feasibility of repeated administrations.

Watch this Scientific Journal Video about Delivering Intrathecal Injections to Neonatal Mice at JoVE.com

向新生小鼠提供鞘内注射

首先，在处理幼崽时将幼崽移到远离大坝的单独笼子中。在开始注射程序之前，使用解剖显微镜确认椎间隙，或至少确认幼犬椎管的中线。在 25 或 10 微升注射器中装入 10 微升所需的注射物质，如药物制剂、病毒制剂或控制人工脊髓液等。一旦动物被完全麻醉，如身体运动减少或不存在所证实的那样，轻轻地将幼崽放在显微镜下。用左手食指和拇指小心触诊位于双侧骨盆带之间的中线椎间隙。轻轻旋转尾巴的底部，以帮助识别脊柱的中线。小心地插入针头，在压痕相交处将其倾斜至 70 至 80 度角，同时确保它与中央矢状面对齐。当针头与骨骼接触时，逐渐将角度减小到大约 30 度，并将针头推进约 2 毫米进入椎间隙。在 50 至 60 秒内缓慢注入最多 10 微升体积。注射完成后，将针头保持在原位 10 到 20 秒。轻轻旋转取出针头，以免漏水。注射后，将幼犬放在加热垫上 10 到 15 分钟，让幼犬完全恢复并重新加热。在野生型新生儿中鞘内注射快速绿色立即导致给药溶液的广泛分布。YFP报告小鼠在鞘内注射基于CRISPR-Cas9的基因编辑10天后，在整个大脑中显示出广泛的YFP表达。

delivering-intrathecal-injections-to-neonatal-mice

Research

JoVE Journal

Neuroscience

Delivering Intrathecal Injections to Neonatal Mice

841 次观看.  Yale University School of Medicine. 首先，在处理幼崽时将幼崽移到远离大坝的单独笼子中。在开始注射程序之前，使用解剖显微镜确认椎间隙，或至少确认幼犬椎管的中线。在 25 或 10 微升注射器中装入 10 微升所需的注射物质，如药物制剂、病毒制剂或控制人工脊髓液等。一旦动物被完全麻醉，如身体运动减少或不存在所证实的那样，轻轻地将幼崽放在显微镜下。用左手食指和拇指小心触诊位于双侧骨?...

视频: 向新生小鼠提供鞘内注射

Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and treatments. By directly delivering medications and treatments into the cerebrospinal fluid of the central nervous system, this method achieves higher localized drug concentrations while reducing systemic side-effects compared to other routes such as intravenous, subcutaneous, or intramuscular injections. Its importance extends beyond clinical settings, as intrathecal injection plays a vital role in preclinical studies focused on treating neurogenetic disorders in rodents and other large animals, including non-human primates. However, despite its widespread application, intrathecal injection in young, particularly neonatal pups, poses significant technical challenges due to their small size and fragile nature. Successful and reliable administration of intrathecal injections in newborn mice requires meticulous attention to detail and careful consideration of various factors. Thus, there is a crucial need for a standardized protocol that not only provides instructions but also highlights key technical considerations and good laboratory practices to ensure procedural consistency, as well as the safety and welfare of the animals.
To address this unmet need, we present a detailed and comprehensive protocol for performing intrathecal injections specifically in newborn pups on postnatal day 1 (P1). By following the step-by-step instructions, researchers can confidently perform intrathecal injections in neonatal pups, enabling the accurate delivery of drugs, antisense oligos, and viruses for gene replacement or genome editing-based treatments. Furthermore, the importance of adhering to good laboratory practices is emphasized to maintain the well-being of animals and ensure reliable experimental outcomes. This protocol aims to address the technical challenges associated with intrathecal injections in neonatal mice, ultimately facilitating advances in the field of neurogenetic research that aims to develop potential therapeutic interventions.

The present protocol outlines step-by-step instructions for performing intrathecal injections in neonatal mice for gene editing and drug delivery.

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and ...