Lumbar Intrathecal Injection in Adult and Neonatal Mice

Abstract This article describes a step‐by‐step process of lumbar intrathecal injection of Evans blue dye and AAV9‐EGFP in adult (2‐month‐old) and neonatal (postnatal day 10) mice. Intrathecal injection is a clinically translatable technique that has already been extensively applied in humans. In mice, intrathecal injection is considered a challenging procedure that requires a trained and experienced researcher. For both adult and neonatal mice, lumbar intrathecal injection is directed into the L5‐L6 intervertebral space. Intrathecally injected material enters the cerebrospinal fluid (CSF) within the intrathecal space from where it can directly access the central nervous system (CNS) parenchyma. Simultaneously, intrathecally injected material exits the CSF with pressure gradient and enters the endoneurial fluid and ultimately the peripheral nerves. While in the CSF, the injectable material also enters the bloodstream and systemic circulation through the arachnoid villi. A successful lumbar intrathecal injection results in adequate biodistribution of the injectable material in the CNS, PNS, and peripheral organs. When correctly applied, this technique is considered as minimally invasive and non‐disruptive and can be used for the lumbar delivery of any solute. © 2024 Wiley Periodicals LLC. Basic Protocol 1 : C57BL/6 adult and P10 mice lumbar intrathecal injection Basic Protocol 2 : Tissue collection and preparation for evaluating Evans blue dye diffusion Basic Protocol 3 : Tissue collection and preparation for immunohistochemistry staining Basic Protocol 4 : Tissue collection and vector genome copy number analysis