Background

The early post-natal injection of substances into rodents is required for several purposes, including the testing of pre-clinical therapeutics in disease models (Hua et al., 2011), delivering recombination-inducing substances such as tamoxifen (Pitulescu et al., 2010), and performing mechanistic studies with viruses or toxins (MacDonald et al., 2021). Several delivery routes into neonates are available, each with advantages and pitfalls. For instance, injection into the cerebral lateral ventricles (i.e., intracerebroventricular) can bypass the blood–brain barrier and deliver substances throughout the central nervous system via the cerebrospinal fluid, but this injection paradigm will not result in systemic delivery, is not suitable for repetitive treatments, and is technically challenging (Passini and Wolfe 2001). Alternatively, intravenous injections permit greater volumes (~50–100 μL) and result in a more systemic effect; however, this route is tricky to master (Sands and Barker 1999; Kienstra et al., 2007) and becomes more difficult in pups older than two days due to vein visualisation issues (Gombash Lampe et al., 2014). Subcutaneous injections are straightforward and enable larger dosing volumes if needed, but rates of substance absorption are slower compared to other delivery routes (Turner et al., 2011). Intraperitoneal neonatal injections also allow body-wide substance administration via transfer to the circulatory system (thus rapid absorption) and require less expertise and technical ability but are limited by injection volume and are not frequently used in a clinical setting (Al Shoyaib et al., 2019).

Nevertheless, different injection routes into neonatal rodents can result in distinct tissue targeting (Ohshima et al., 2015) and varied temporal pharmacodynamics post-injection (Statler et al., 2007; Foust et al., 2008); Therefore, it is important to master a variety of administration strategies. Indeed, depending on the dosing and targeting requirements, combining injection paradigms in pups is possible and can provide a superior therapeutic effect compared to single-route injections (Nizzardo et al., 2014).

Several published methods for the delivery of substances into neonates via intravenous (temporal/facial vein, jugular vein, or retro-orbitally) and intracerebroventricular routes are available (Sands and Barker 1999; Kienstra et al., 2007; Glascock et al., 2011; Yardeni et al., 2011; Gombash Lampe et al., 2014). However, a similar, detailed protocol for the intraperitoneal injection of mouse neonates is lacking, although approaches have been presented in brief (Ostermann et al., 2013; Xu et al., 2018; MacDonald et al., 2021).

Here, we describe a stepwise method for accurate injections into the peritoneal cavity of mouse pups, which has been used to successfully deliver adeno-associated viruses to drive transgene expression in 1–3-day-old mice (MacDonald et al., 2021; Sleigh et al., 2023). Intraperitoneal injections are highly reproducible and easy to master and can be used for repeated treatments. Success rates will depend on experimenter experience and the substances being injected, but our recent experiments suggest that a near 100% success rate is achievable.