The unique anatomy and physiology of the eye pose significant challenges for drug delivery. Both static barriers such as the cornea, sclera, blood-aqueous barrier, and blood-retinal barrier, and dynamic barriers such as choroidal and conjunctival blood flow, lymphatic clearance, and tear dilution, hinder effective drug penetration and distribution (Gaudana et al., 2010). Intravitreal injection is the most commonly used method for intraocular drug delivery. However, this approach primarily targets diseases of the posterior segment of the eye (Mansour et al., 2020; Rouvas et al., 2011; Stahl, 2020; Wu et al., 2019) and is less effective for anterior segment disorders. The treatment of anterior segment diseases typically relies on topical eye drops, systemic drug administration, or intracameral injection (Gaudana et al., 2010). Among these, intracameral injection directly delivers therapeutic agents to target tissues within the anterior chamber, bypassing various physiological barriers. To date, intracameral injection has been utilized for diverse applications. Calkins’ group pioneered the bead-induced ocular hypertension rodent model (Crish et al., 2007), and many subsequent studies have adapted this method to establish mouse models of glaucoma (Calkins et al., 2018; Cone et al., 2010; Johnson and Tomarev, 2010; Sappington et al., 2010; Zahavi et al., 2022). Additionally, intracameral injection is widely used to introduce viral vectors, such as adeno-associated virus (AAV) and lentiviruses, into the corneal endothelium and trabecular meshwork for gene therapy (Bogner et al., 2015; Pang et al., 2015; Zhang et al., 2024). Furthermore, intracameral injection has been employed to deliver cultured human corneal endothelial cells to treat endothelial dysfunction (Toda et al., 2019).

Intracameral injection is relatively straightforward in humans. However, it is particularly challenging in mice due to their small eye size and shallow the anterior chamber depth. In traditional intracameral injection techniques, the injection site is typically located at the corneal limbus or near the central cornea. Initially, sterile air or viscous agents are introduced into the anterior chamber, followed by the injection of the drug. The air bubbles or viscous agents act as a seal for the injection port, preventing drug leakage (Bogner et al., 2015; Cone et al., 2012; Frankfort et al., 2013; Liu and Ding, 2017). However, this approach is technically difficult and may lead to complications such as repeated corneal damage and increased intraocular pressure (IOP) due to the presence of air or viscous agents in the anterior chamber.

In this study, we introduce a modified intracameral injection technique for mice, which utilizes the ciliary sulcus behind the iris as the entry point to minimize the risk of drug leakage. This approach eliminates the need for injecting air or viscous agents and avoids corneal puncture. We further validated this method by using AAV transduction and microbeads injection, demonstrating that it is highly reproducible and associated with fewer complications.