creating a self-sealing corneal tunnel for intracameral injection in rats

<meta charset="utf8"></head><body>Azaltılmış doku hasarı için optimize edilmiş sıçan intracameral enjeksiyon tekniği

The bioavailability of compounds delivered by topical administration to the surface of the eye is greatly limited, typically &#60;5%1. Compounds administered by eye drops are mainly&#160;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&#160;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&#39; 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66662/66662fig01.jpg" /> 
Figure 1: Schematic representation of the anatomical anterior segment features of rat and human eyes. <a href="https://www.jove.com/files/ftp_upload/66662/66662fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<meta charset="utf8"></head><body>Yazar Spotlight: Kemirgen Oftalmolojisinde Hassasiyeti Artırmak için İntrakameral Enjeksiyonlar için Yeni Bir Protokol

Yan Etki Riski Düşük Olan Sıçanlarda İntrakameral Enjeksiyon

Sıçanlarda intrakameral enjeksiyon için kendinden sızdırmaz bir kornea tüneli oluşturulması

creating-self-sealing-corneal-tunnel-for-intracameral-injection

Research

JoVE Journal

Biology

Creating a Self-Sealing Corneal Tunnel for Intracameral Injection in Rats

36 Görüntüleme. Başlamak için, hayvanı ameliyata hazırlayın ve hayvanın gözüne% 0.3 ofloxacin ve% 0.1 deksametazon uygulayın. Cerrahi oftalmik forseps kullanarak, gözü stabilize etmek için superior sklerayı korneoskleral bileşkenin yanındaki dikey orta hatta tutun. Cerrahi mikroskop altında, dikey orta hattaki parasantral kornea bölgesine steril bir 0,8 milimetre 31 gauge stiletto bıçağı yerleştirin.Bu pozisyonda, bir kesi yapmak için korneayı ...

Video: Sıçanlarda intrakameral enjeksiyon için kendinden sızdırmaz bir kornea tüneli oluşturulması

Intracameral Injection in Rats with Low Risk of Adverse Effects

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&#160;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&#160;visualization&#160;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.

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.