Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery

Summary

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

Abstract

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.

Introduction

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 clinics^1,2. 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.

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Protocol

The described procedures and protocols were in compliance with the guidelines outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Additionally, the procedures received approval from the Animal Care and Use Committee at Yale University School of Medicine. Newborn wild-type (WT) C57BL/6J male and female mice were used for presented study. The animals were obtained from a commercial source (see Table of Materials).

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Discussion

Described is a step-by-step procedure for intrathecal injection in neonatal mice (P1), resulting in widespread drug distribution in their brains. In comparison to the common intracerebroventricular injection method for delivering medication to neonatal mice, which involves piercing the cerebral cortex¹¹, intrathecal injection avoids direct injury to the neonatal mouse brain due to needle penetration. Due to minimal invasiveness, intrathecal injection can be performed repeatedly when necessary, sim.......

Materials

Name	Company	Catalog Number	Comments
Balance	Ohaus Corporation	30253017
C57BL/6J mice	The Jackson Laboratory	000664
Digital Microscope	RWD	DOM-1001
DPBS	ThermoFisher	14190144
Fast Green	Sigma	F7252-5G
Heating pad	RWD	69020
Needles	Hamilton	6PK (34/0.375”/4/12DEG)S
Syringe	Hamilton	1702RN
Syringe Filters	Sigma	SLGVM33RS