This protocol describes a technique for intracameral injection in rats using a central corneal incision and a long tunnel into the anterior chamber. This injection method minimizes the risk of inducing inadvertent tissue damage and thereby improves precision and reproducibility.
Intracameral injection is a standard administration routine in ophthalmology. The application of intracameral injection in rodents for research is challenging due to the limiting dimensions and anatomy of the eye, including the small aqueous humor volume, the lens curvature, and lens thickness. Potential damage during intracameral injections introduces adverse effects and experimental variability. This protocol describes a procedure for intracameral injection in rats, allowing precision and reproducibility.
Sprague-Dawley rats were used as experimental models. Since the lens position in rats protrudes into the anterior chamber, injecting from the periphery, as done in humans, is unfavorable. Therefore, an incision is created in the central corneal region using a 31 gauge 0.8 mm stiletto blade to form a self-sealing tunnel into the anterior chamber. An incision at an angle close to the flat allows to create a long tunnel, which minimizes the loss of aqueous humor and shallowing of the anterior chamber. A 34 gauge nanoneedle is inserted into the tunnel for injection. This enables penetration with minimal friction resistance and avoids touching the lens. Injection of trypan-blue allows visualization by slit microscopy the presence of the dye in the anterior chamber and exclude leakage. Bioavailability to the corneal endothelial layer is demonstrated by injection of Hoechst dye, which stained the nuclei of corneal endothelial cells after injection.
In conclusion, this protocol implements a procedure for accurate intracameral injection in rats. This procedure may be used for intracameral delivery of various drugs and compounds in experimental rat models, increasing the efficiency and reproducibility of ophthalmic research.
The bioavailability of compounds delivered by topical administration to the surface of the eye is greatly limited, typically <5%1. Compounds administered by eye drops are mainly eliminated by drainage, induced lacrimation, tear fluid turnover, and conjunctival absorption. In addition, the permeation of compounds through the ocular surface is highly restricted by the cornea-conjunctiva barrier1,2,3. The cornea is composed of three main layers: the outermost epithelium, the intermediate stroma, and the innermost endothelium. The superficial corneal epithelium is interconnected by strong tight junctions and creates high paracellular resistance, which is the main barrier to substance permeability. Multiple epithelium layers further limit the permeation of hydrophilic and large molecules through the intercellular spaces of the cornea epithelium. Succeeding the epithelium, the stroma is composed of collagen fibers and contains aqueous pores. In contrast to the corneal epithelium, the stroma allows the movement of hydrophilic drugs; however, it is greatly impermeable to lipophilic compounds1,2,3. Together, the corneal epithelium and stromal layers present major tissue barriers that limit drug absorption. The corneal endothelium is not considered to restrict drug transport.
Alternative to the corneal delivery route is the conjunctival route. The conjunctiva is a multi-epithelium layer that covers the inner side of the eyelids and the anterior part of the sclera. The conjunctiva is characterized by fewer tight junctions than the corneal epithelium, allowing better permeability of hydrophilic drugs. However, vascularization of the conjunctiva results in systemic absorption of a large fraction of the administered molecules, again greatly limiting the bioavailability of delivered compounds to the anterior chamber1,2. An efficient way to bypass the outer ocular permeability barriers is to deliver the drug directly into the region of interest. For example, intravitreal injection is common for delivery into the vitreous humor4. Likewise, intracameral injection is utilized for delivery into the anterior chamber5. Establishing an efficient concentration at the anterior chamber is critical to various clinical situations, such as the treatment of infection by intracameral injection of antibiotics and postoperative anti-inflammatory treatments in cataract surgeries. Despite the advantage of improved substance bioavailability granted by intracameral injection, there are major safety concerns that should be considered. For example, intracameral drug injection may induce increased intraocular pressure, toxic anterior segment syndrome, and toxic endothelial cell destruction syndrome5,6. It is, therefore, essential to carefully assess in pre-clinical studies the efficacy and safety of drugs delivered by intracameral injections to maximize treatment efficiency and minimize potential adverse effects in patients.
Experimental animal models are indispensable in pre-clinical studies to investigate new treatments. Small rodents, such as mice and rats, are the most commonly utilized laboratory animals for such purposes. These animals exhibit numerous similarities to human anatomy and physiology, providing valuable insights. Moreover, their use is economically advantageous due to their small size, ease of maintenance, fast gestation, and ability to produce large numbers of offspring7.
Despite the widespread use of small rodents in eye disease models, their unique eye dimensions and anatomy pose significant challenges during experimental manipulations. For instance, procedures like intracameral injections, which are relatively straightforward in humans, become technically demanding in mice and rats. The challenges stem from factors such as the small volume of aqueous humor, the relatively large and inflexible lens, and the obstructive positioning and curvature of the lens within the rodents' eyes (Figure 1)8. These challenges increase the risk of damage during intracameral injections in rodents, leading to potential adverse effects and introducing experimental variability that can impact the validity of study conclusions. In our research, we have successfully developed a procedure for safe intracameral injection in rats. The technique involves creating a long, flat, self-sealing tunnel in the cornea into the anterior chamber. This method not only ensures precision but also enhances experimental reproducibility, addressing the issues associated with injection techniques in small rodents.
Figure 1: Schematic representation of the anatomical anterior segment features of rat and human eyes. Please click here to view a larger version of this figure.
The experiments in the protocol were approved by the National Permit Committee – for animal science and comply with the ARVO Statement the use of animals in ophthalmic and vision research. Female Sprague-Dawley rats, aged 8-10 weeks, were used for the present study and were exposed to 12/12 h light-dark cycles. The animals were obtained from a commercial source (see Table of Materials).
1. Animal preparation
2. Creating a self-sealing corneal tunnel
Figure 2: Schematic representation of the blade and incision angle and position. Please click here to view a larger version of this figure.
3. Option 1: Intracameral injection of trypan blue for assessing the successful injection into the anterior chamber
4. Option 2: Intracameral injection of Hoechst for assessing the bioavailability of injected material to the endothelial cell layer
Sprague Dawley rats were intracamerally injected with 5 µL of trypan blue according to the protocol described above. Slit lamp examination immediately after injection demonstrated that the chamber was stained with trypan blue, indicating that the injected material reached the anterior chamber (Figure 3). Furthermore, the anterior chamber depth was intact, suggesting that the injection did not cause leakage of aqueous humor and shallowing of the chamber.
Figure 3: Intact anterior chamber following intracameral injection. Trypan blue was injected into the rat's anterior chamber. Slit microscopy examination demonstrates the presence of trypan blue without leakage or shallowing of the anterior chamber. Scale bar = 1 mm. Please click here to view a larger version of this figure.
Next, Hoechst, a cell-permeable fluorescent dye that binds DNA and stains cell nuclei, was injected to evaluate the bioavailability of drugs delivered by the described intracameral injection route. The uptake of Hoechst by endothelial cells was evaluated 15 min post-injection by isolating corneas and observing under a fluorescent microscope. To identify endothelial cells, the corneas were stained with Alizarin Red S, which stains the intercellular borders of the endothelium cell layer. As a control, we examined the non-injected eye from the same rat. Our results demonstrate that the endothelial cell layer was intact following injection, supporting that the described procedure does not cause damage to the endothelium. Furthermore, endothelial cells were positive for Hoechst nuclear staining, demonstrating the uptake of the injected Hoechst following intracameral injection (Figure 4).
Figure 4: Bioavailability of intracamerally injected material to the endothelial cell layer. Hoechst was injected into the rat's anterior chamber. The cornea was isolated 15 min post-injection, stained with Alizarin Red S to observe endothelial cells, and imaged under a fluorescent microscope to observe Hoechst staining. Overlay images demonstrate nuclear Hoechst staining in corneal endothelial cells. Scale bars = 50 µM. Please click here to view a larger version of this figure.
Pre-clinical research models should provide a controlled and reproducible environment to ensure the reliability and applicability of findings. In ophthalmology research, eye injection models are commonly used in diverse research aspects ranging from establishing disease models, testing new treatments, and assessing tissue reactions and potential adverse effects.
Intracameral injections serve as a common technique in experimental ophthalmology, facilitating the direct delivery of compounds to the aqueous humor while bypassing the outer ocular tissue barriers5. This targeted approach through intracameral injection ensures that an optimal concentration of the drug reaches the intended site of action, such as the lens, trabecular meshwork, or corneal endothelium, thereby maximizing therapeutic benefits.
Performing intracameral injections in small rodents presents several technical challenges that can impact experimental reproducibility and success. First, the small dimensions of the rodent eye pose difficulties in access and manipulation without causing damage to ocular structures. Rats, in this context, offer some advantages over mice due to their larger eyes. Second, the positioning of the lens in the confined anterior chamber can obstruct the path to the injection site. Compared to other mammals, including humans, rats have a lens known to be protruding or prominent, contributing to its relatively large size relative to the eye (Figure 1). Additionally, the rat lens lacks the flexibility seen in the human lens. As a result, careful maneuvering of the injection needle is required to navigate around the lens without causing damage.
Tissue damage during manipulation can lead to various complications, including shallowing of the anterior chamber, increased intraocular pressure, inflammation or anterior uveitis, damage to endothelial cells, cataract formation, other structural changes or deformities, and the risk of infection. These challenges highlight the need for precise techniques and careful consideration of anatomical differences when performing intracameral injections in small rodent models.
The technique for intracameral injection described in this protocol was designed to minimize the risk of damaging ocular tissues during the injection process. The method involves establishing a self-sealing tunnel into the anterior chamber, facilitating entry with minimal friction resistance. A well-sealed incision is crucial to reduce the risk of postoperative hypotony and fluid leakage and prevent infection by microorganisms from the lids and lashes. The angle and length of the incision are both critical to the wound dynamics, the development of adverse effects, and the subsequent recovery. Incisions that are too large may opacify the visual axis and induce corneal striae or edema, while short incisions can destabilize the anterior chamber and induce iris prolapse. Performing an incision at an angle close to the corneal plane axis allows for the generation of a long uniplanar tunnel without the associated risks. Importantly, penetration of the needle into the anterior chamber through a guided long tunnel improves precision, reducing the likelihood of inadvertently touching the lens. In addition, in contrast to injection in humans, where peripheral incisions are preferred, the tunnel in rats is performed at the central cornea, where the anterior chamber is deepest. Experimental validation confirmed that this method allows for injections without experiencing leakage of aqueous humor or shallowing of the anterior chamber and without touching the lens.
Previous studies described various methods for injection into the anterior chamber in rats. For instance, the Rosenstein group described in several studies the injection to the anterior chamber through the corneoscleral limbus. Injection of 20 µL liquid progressively increased the chamber's depth and thereby separated the needle from the iris, which supports avoiding contact of the needle with the lens. Side effects of the injection were reported as transient corneal edema and approximately a 5% incidence of cataracts14,15,16. Matsumoto et al. described an intracameral microbead injection technique in rats to produce a glaucoma model. The authors utilized a single-step incision, creating a sclerocorneal tunnel using a 34 G needle, which was inserted bevel-up into the anterior chamber. The authors reported avoiding hitting the cornea or iris and noted no inflammatory incidents, with only a few cases (<7% of examined animals) of damaging the endothelial layer during injections. However, the authors described that this modified injection alone was not sufficient to prevent significant leakage out of the anterior chamber and reflux into the subconjunctival space. To overcome this, a 20 µL mixture of microbeads with dispersive ophthalmic viscosurgical device (OVD; Viscoat) was injected. This resulted in significantly elevated intraocular pressure (IOP) levels up to 4 weeks after injection17. Similarly, Liu et al. described intracameral injection using a 32 G needle inserted into the anterior chamber parallel to the iris along the limbus, creating a self-sealing corneal tunnel18. The authors injected 3 µL of sodium hyaluronic acid hydrogel, and a cotton swab was used to compress the injection site to minimize leakage out of the anterior chamber. The high molecular weight gel was described to be retained in the anterior chamber and block the outflow of aqueous humor through the trabecular meshwork18. It is noteworthy that the modified intracameral injection method described here tolerates injection without viscoelastic agents and is therefore applicable to a wide range of research applications. Nevertheless, as this study was limited to the injection of liquid solutions in a volume of no more than 5 µL, a precise comparison of injection methods and injected materials should be performed for specific research purposes to determine the most suitable technique.
The clinical application of intracameral injections is diverse and extensive. For example, intracameral administration of corticosteroids and antibiotics, such as cefuroxime and moxifloxacin, is common during cataract surgeries as a prophylactic measure to prevent postoperative infection and endophthalmitis19,20,21,22,23,24,25. Intraocular delivery is also employed for local anesthesia and mydriasis during routine cataract surgeries26,27. Furthermore, the treatment of glaucoma involves the use of various intracamerally delivered drugs that reduce intraocular pressure, such as prostaglandin analogs or muscarinic agonists, which increase aqueous humor outflow, antifibrotic agents such as mitomycin C to prevent scarring and fibrosis, and anti-vascular endothelial growth factor (anti-VEGF) agents for neovascular glaucoma28,29,30. This diverse range of intracameral treatments reflects their crucial role in addressing various ophthalmic conditions and enhancing patient outcomes.
The method of intracameral injection described here will be advantageous for varied pre-clinical studies aimed at advancing the extensive clinical applications of intracameral injections. For example, using intracameral injection in rat models to determine the efficacy, optimal dosages, timing, and long-term outcomes of various medications is a crucial initial step before advancing to clinical trials. In such studies, improving precision and reproducibility of the injection technique will be key to successful achievements and study progression.
Furthermore, the described method of intracameral injection may be employed to generate experimental models of different ocular pathologies by delivering vectors or chemicals to the anterior chamber. Such experimental models are key to investigating disease mechanisms and developing new treatments. For instance, Fuchs' endothelial dystrophy (FED), a progressive corneal disease causing gradual vision loss, lacks a cure31. Primary management involves monitoring and providing symptom relief, often through topical medications for temporary edema alleviation or eye lubrication. In cases of disease progression or severe vision impairment, corneal transplantation (endothelial keratoplasty) becomes a necessary intervention. Research aimed at developing new treatment options for FED, potentially offering alternatives to corneal transplantation, is highly warranted. Given the limited bioavailability of drugs delivered through the cornea or conjunctiva1,2,3, intracameral administration emerges as an advantageous route for treating the endothelium layer. Experimental models of intracameral injection play a crucial role in developing novel therapeutic approaches for FED. Using the approach described herein for intracameral injection can induce the uptake of injected material into the corneal endothelial layer. Implementing this method in intracameral injection allows for the creation of experimental models for FED treatment with high precision and reproducibility. This not only contributes to advancing our understanding of FED but also opens avenues for developing targeted and effective therapeutic interventions.
In summary, described here is an optimized procedure for intracameral injection in rats with low risk of adverse effects, which would be valuable for improving pre-clinical ophthalmology research and contributing to the development of new therapeutic possibilities for ocular pathologies.
The authors have nothing to disclose.
This research was supported by the Israel Science Foundation grants 2670/23 and 1304/20.
Alizarin Red | Alpha Aesar | 042040.5 | |
Buprenorphine | Richter pharma | 102047 | |
Dexamethasone 0.1% | Fisher Pharmaceutical | 393102-0413 | |
Hamilton glass syringe 10 μL | Hamilton Co. | 721711 | |
Hoeschst | Merck | B2261 | |
Ketamine | Bremer pharma GMBH (medimarket) | 17889 | |
Ofloxacin 0.3% eye drops | Allergan | E92170 | |
Oxybuprocaine Hydrochloride 0.4% | Fisher Pharmaceutical | N/A | |
Pentobarbital sodium 200 mg/mL | CTS | N/A | |
Slit microscope | Haag-streit bern | b-90019115 | |
Sprague-Dawley Rats | Envigo | N/A | |
Stiletto blade 31 G 0.8 mm | Tecfen medical (skymed) | QKN2808 | |
Surgical microscope | Zeiss | OPMI-6 CFC | |
Trypan Blue | Sartorius | 03-102-1B | |
Xylazine | Eurovet Animal Health | 615648 |
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