This research was supported by the Israel Science Foundation grants 2670/23 and 1304/20.

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
<ol>
	<li>Prepare an anesthetic mixture of ketamine (80 mg/kg body weight in 0.8 mL) and xylazine (4 mg/kg body weight in 0.2 mL) and inject it intraperitoneally in a single injection to anesthetize the rats.</li>
	<li>Inject analgesic buprenorphine (0.03 mg/kg) intraperitoneally in a single injection.</li>
	<li>Apply topical ophthalmic anesthetic 0.4% oxybuprocaine to both eyes.</li>
</ol>
2. Creating a self-sealing corneal tunnel
<ol>
	<li>Stabilize the eye by holding the superior sclera at the vertical midline next to the corneoscleral junction with surgical ophthalmic forceps.</li>
	<li>Under a surgical microscope, place a sterile 0.8 mm, 31 G stiletto blade in the paracentral corneal region in the vertical midline (above the center of the pupil) in a flat position at an angle as close as possible to horizontal (Figure 2).</li>
	<li>In this position, puncture the cornea to make an incision and create a long tunnel (2-3 mm) until it penetrates into the central area of the anterior chamber. Avoid touching the lens (Figure 2). 
	NOTE: A successful tunnel will not induce leakage of the aqueous humor and shallowing of the anterior chamber.</li>
	<li>Apply topical 0.3% ofloxacin and 0.1% dexamethasone to the injected eye.</li>
	<li>Examine under slit microscopy as follows.
	<ol>
		<li>Observe the depth of the anterior chamber of the injected eye compared to the non-injected eye. 
		NOTE: The depth should be similar.</li>
		<li>Observe the lens of the injected eye compared to the non-injected eye. 
		​NOTE: The lens should be clear. Opacity may reflect lens damage during the surgical procedure.</li>
	</ol>
	</li>
</ol>
<img alt="Eye surgery procedure; labeled diagram showing lens, cornea, incision, anterior chamber, and blade." class="xfigimg" src="/files/ftp_upload/66662/66662fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Schematic representation of the blade and incision angle and position. <a href="https://www.jove.com/files/ftp_upload/66662/66662fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Option 1: Intracameral injection of trypan blue for assessing the successful injection into the anterior chamber
<ol>
	<li>Load 5 µL of trypan blue into a sterile 10 µL glass Hamilton syringe with a 34 G blunt needle. 
	NOTE: Injection of trypan blue is described as a means to evaluate the success of injection during the model calibration or setup stages. In the experimental settings, the syringe may be loaded with a solution of the compound of choice.</li>
	<li>Insert the loaded syringe needle through the tunnel created in section 2 into the anterior chamber.</li>
	<li>Inject and hold the needle in place after injection for 2-3 seconds until all fluid clears.</li>
	<li>Remove the needle by pulling it out gently and slowly to avoid leakage from the corneal tunnel.</li>
	<li>Examine under slit microscopy. Evaluate the depth of the anterior chamber to exclude shallowing and verify the presence of trypan blue in the anterior chamber.</li>
	<li>Repeat slit examination after 24 h, 48 h, and 72 h.</li>
</ol>
4. Option 2: Intracameral injection of Hoechst for assessing the bioavailability of injected material to the endothelial cell layer
<ol>
	<li>Load 5 µL of Hoechst into a sterile 10 µL glass Hamilton syringe with a 34 G blunt needle. 
	NOTE: Injection of Hoechst is described as a means to evaluate the bioavailability of injected material by uptake into the endothelial cell layer and is useful during the model calibration or setup stages. In the experimental settings, the syringe may be loaded with a solution of the compound of choice.</li>
	<li>Insert the loaded syringe needle through the tunnel created in section 2 into the anterior chamber.</li>
	<li>Inject and hold the needle in place after injection for 2-3 seconds until all fluid clears.</li>
	<li>Remove the needle by pulling it out gently and slowly to avoid leakage from the corneal tunnel incision.</li>
	<li>Approximately 15-20 min post-injection, euthanize the rats by intraperitoneal injection of 500 mg/kg sodium pentobarbitone.</li>
	<li>Enucleate both eyes and isolate corneas. Collect the non-injected cornea as a control.</li>
	<li>Stain both corneas with 0.5% Alizarin Red S according to manufacturer instructions to identify endothelial cells.</li>
	<li>Examine under a light microscope to image the alizarin-red staining of endothelial cells and under a fluorescent microscope to observe Hoechst staining, compared to the non-injected cornea as control.</li>
</ol>

Optimized Rat Intracameral Injection Technique for Reduced Tissue Damage

<ol>
	<li>Ramsay, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+and+conjunctival+drug+permeability:+Systematic+comparison+and+pharmacokinetic+impact+in+the+eye.">Corneal and conjunctival drug permeability: Systematic comparison and pharmacokinetic impact in the eye.</a> Eur J Pharm Sci. 119, 83-89 (2018).</li><li>Cholkar, K., Dasari, S. R., Pal, D., Mitra, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Eye: Anatomy, Physiology and Barriers to Drug Delivery. Ocular Transporters and Receptors. , (2013).</li><li>Prausnitz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+of+cornea,+sclera,+and+conjunctiva:+A+literature+analysis+for+drug+delivery+to+the+eye.">Permeability of cornea, sclera, and conjunctiva: A literature analysis for drug delivery to the eye.</a> J Pharm Sci. 87 (12), 1479-1488 (1998).</li><li>Varela-Fern&#225;ndez, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+delivery+to+the+posterior+segment+of+the+Eye:+Biopharmaceutic+and+pharmacokinetic+considerations.">Drug delivery to the posterior segment of the Eye: Biopharmaceutic and pharmacokinetic considerations.</a> Pharmaceutics. 12 (3), 269 (2020).</li><li>Gautam, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracameral+drug+delivery:+A+Review+of+agents,+indications,+and+outcomes.">Intracameral drug delivery: A Review of agents, indications, and outcomes.</a> J Ocul Pharmacol Ther. 39 (2), 102-116 (2023).</li><li>Shah, T. J., Conway, M. D., Peyman, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracameral+dexamethasone+injection+in+the+treatment+of+cataract+surgery+induced+inflammation:+design,+development,+and+place+in+therapy.">Intracameral dexamethasone injection in the treatment of cataract surgery induced inflammation: design, development, and place in therapy.</a> Clin Ophthalmol. 12, 2223-2235 (2018).</li><li>Perlman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+human+disease:+An+evolutionary+perspective.">Mouse models of human disease: An evolutionary perspective.</a> Evol Med Public Health. 2016 (1), 170-176 (2016).</li><li>Chawla, S., Jena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Anatomy and Physiology of Laboratory Rat.Essentials of Laboratory Animal Science: Principles and Practices. , (2021).</li><li>Lundstr&#246;m, M., Wejde, G., Stenevi, U., Thorburn, W., Montan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endophthalmitis+after+cataract+surgery:+a+nationwide+prospective+study+evaluating+incidence+in+relation+to+incision+type+and+location.">Endophthalmitis after cataract surgery: a nationwide prospective study evaluating incidence in relation to incision type and location.</a> Ophthalmology. 114 (5), 866-870 (2007).</li><li>Fine, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clear+corneal+incisions.">Clear corneal incisions.</a> Int Ophthalmol Clin. 34 (2), 59-72 (1994).</li><li>Herretes, S., Stark, W. J., Pirouzmanesh, A., Reyes, J. M. G., McDonnell, P. J., Behrens, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflow+of+ocular+surface+fluid+into+the+anterior+chamber+after+phacoemulsification+through+sutureless+corneal+cataract+wounds.">Inflow of ocular surface fluid into the anterior chamber after phacoemulsification through sutureless corneal cataract wounds.</a> Am J Ophthalmol. 140 (4), 737-740 (2005).</li><li>Masket, S., Belani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proper+wound+construction+to+prevent+short-term+ocular+hypotony+after+clear+corneal+incision+cataract+surgery.">Proper wound construction to prevent short-term ocular hypotony after clear corneal incision cataract surgery.</a> J Cataract Refract Surg. 33 (3), 383-386 (2007).</li><li>Taban, M., Rao, B., Reznik, J., Zhang, J., Chen, Z., McDonnell, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+morphology+of+sutureless+cataract+wounds+-+Effect+of+incision+angle+and+location.">Dynamic morphology of sutureless cataract wounds - Effect of incision angle and location.</a> Surv Ophthalmol. 49, S62-S72 (2004).</li><li>Belforte, N., Sande, P. H., de Zaval&#237;a, N., Dorfman, D., Rosenstein, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+benefit+of+radial+optic+neurotomy+in+a+rat+model+of+glaucoma.">Therapeutic benefit of radial optic neurotomy in a rat model of glaucoma.</a> PLoS One. 7 (3), e34574 (2012).</li><li>Moreno, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+experimental+model+of+glaucoma+in+rats+through+intracameral+injections+of+hyaluronic+acid.">A new experimental model of glaucoma in rats through intracameral injections of hyaluronic acid.</a> Exp Eye Res. 81 (1), 71-80 (2005).</li><li>Belforte, N., Sande, P., de Zaval&#237;a, N., Knepper, P., Rosenstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+chondroitin+sulfate+on+intraocular+pressure+in+rats.">Effect of chondroitin sulfate on intraocular pressure in rats.</a> Invest Ophthalmol Vis Sci. 51 (11), 5768-5775 (2010).</li><li>Matsumoto, Y., Kanamori, A., Nakamura, M., Negi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+chronic+glaucoma+model+induced+by+intracameral+injection+of+microbeads+suspended+in+sodium+sulfate-sodium+hyaluronate.">Rat chronic glaucoma model induced by intracameral injection of microbeads suspended in sodium sulfate-sodium hyaluronate.</a> Jpn J Ophthalmol. 58 (3), 290-297 (2014).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+rat+model+of+ocular+hypertension+by+a+single+intracameral+injection+of+cross-linked+hyaluronic+acid+hydrogel+(Healaflow&#174;+).">A novel rat model of ocular hypertension by a single intracameral injection of cross-linked hyaluronic acid hydrogel (Healaflow&#174; ).</a> Basic Clin Pharmacol Toxicol. 127 (5), 361-370 (2020).</li><li>Bowen, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+the+safety+and+efficacy+of+intracameral+cefuroxime,+moxifloxacin+and+vancomycin+at+the+end+of+cataract+surgery:+a+meta-analysis.">Comparative analysis of the safety and efficacy of intracameral cefuroxime, moxifloxacin and vancomycin at the end of cataract surgery: a meta-analysis.</a> Br J Ophthalmol. 102 (9), 1268-1276 (2018).</li><li>Kato, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prophylactic+antibiotics+for+postcataract+surgery+endophthalmitis:+a+systematic+review+and+network+meta-analysis+of+6.8+million+eyes.">Prophylactic antibiotics for postcataract surgery endophthalmitis: a systematic review and network meta-analysis of 6.8 million eyes.</a> Sci Rep. 12 (1), 17416 (2022).</li><li>Wang, M., Liu, Y., Dong, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cefuroxime+intracameral+injection+antibiotic+prophylactic+on+postoperative+endophthalmitis+wound+post-cataract:+A+meta-analysis.">Effect of cefuroxime intracameral injection antibiotic prophylactic on postoperative endophthalmitis wound post-cataract: A meta-analysis.</a> Int Wound J. 20 (5), 1376-1383 (2023).</li><li>Katz, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracameral+cefuroxime+and+the+incidence+of+post-cataract+endophthalmitis:+an+Israeli+experience.">Intracameral cefuroxime and the incidence of post-cataract endophthalmitis: an Israeli experience.</a> Graefes Arch Clin Exp Ophthalmol. 253 (10), 1729-1733 (2015).</li><li>Lipnitzki, I., Ben Eliahu, S., Marcovitz, A. L., Ezov, N., Kleinmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraocular+concentration+of+moxifloxacin+after+intracameral+injection+combined+with+presoaked+intraocular+lenses.">Intraocular concentration of moxifloxacin after intracameral injection combined with presoaked intraocular lenses.</a> J Cataract Refract Surg. 40 (4), 639-643 (2014).</li><li>Colleaux, K. M., Hamilton, W. K., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+prophylactic+antibiotics+and+incision+type+on+the+incidence+of+endophthalmitis+after+cataract+surgery.">Effect of prophylactic antibiotics and incision type on the incidence of endophthalmitis after cataract surgery.</a> Can J Ophthalmol. 35 (7), 373-378 (2000).</li><li>Libre, P. E., Della-Latta, P., Chin, N. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracameral+antibiotic+agents+for+endophthalmitis+prophylaxis:+A+pharmacokinetic+model.">Intracameral antibiotic agents for endophthalmitis prophylaxis: A pharmacokinetic model.</a> J Cataract Refract Surg. 29 (9), 1791-1794 (2003).</li><li>Carino, N. S., Slomovic, A. R., Chung, F., Marcovich, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topical+tetracaine+versus+topical+tetracaine+plus+intracameral+lidocaine+for+cataract+surgery.">Topical tetracaine versus topical tetracaine plus intracameral lidocaine for cataract surgery.</a> J Cataract Refract Surg. 24 (12), 1602-1608 (1998).</li><li>Minakaran, N., Ezra, D. G., Allan, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topical+anaesthesia+plus+intracameral+lidocaine+versus+topical+anaesthesia+alone+for+phacoemulsification+cataract+surgery+in+adults.">Topical anaesthesia plus intracameral lidocaine versus topical anaesthesia alone for phacoemulsification cataract surgery in adults.</a> Cochrane Database Syst Rev. 7 (7), (2020).</li><li>Dan-Ni, W., Li-Dian, H., Zhi-Guo, P., Qiang, W., Lei, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracameral+anti-VEGF+injection+for+advanced+neovascular+glaucoma+after+vitrectomy+with+silicone+oil+tamponade.">Intracameral anti-VEGF injection for advanced neovascular glaucoma after vitrectomy with silicone oil tamponade.</a> Int J Ophthalmol. 14 (3), 129-135 (2021).</li><li>Bhagat, P. R., Agrawal, K. U., Tandel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+effect+of+injection+bevacizumab+through+various+routes+in+neovascular+glaucoma.">Study of the effect of injection bevacizumab through various routes in neovascular glaucoma.</a> J Curr Glaucoma Pract. 10 (2), 39-48 (2016).</li><li>Al-Qaysi, Z. K., Beadham, I. G., Schwikkard, S. L., Bear, J. C., Al-Kinani, A. A., Alany, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+release+ocular+drug+delivery+systems+for+glaucoma+therapy.">Sustained release ocular drug delivery systems for glaucoma therapy.</a> Expert Opin Drug Deliv. 20 (7), 905-919 (2023).</li><li>Eghrari, A. O., Gottsch, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fuchs'+corneal+dystrophy.">Fuchs' corneal dystrophy.</a> Expert Rev Ophthalmol. 5 (2), 147-159 (2010).</li></ol>

Marcovich A. L. holds patents in Steba Biotech, Yeda Weizmann, EyeYon Medical, and Mor Isum and is a consultant for EyeYon Medical and Johnson &#38; Johnson. All other authors have no competing interests.

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 t...

The bioavailability of compounds delivered by topical administration to the surface of the eye is greatly limited, typically &lt;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.
<img alt="Rat and human eye cross-section diagram comparing anatomical structures: iris, lens, cornea, pupil." class="xfigimg" src="/files/ftp_upload/66662/66662fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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>

<table><tbody><tr><td>Alizarin Red&amp;nbsp;</td><td>Alpha Aesar</td><td>042040.5</td><td></td></tr><tr><td>Buprenorphine&amp;nbsp;</td><td>Richter pharma</td><td>102047</td><td></td></tr><tr><td>Dexamethasone 0.1%&amp;nbsp;</td><td>Fisher Pharmaceutical</td><td>393102-0413</td><td></td></tr><tr><td>Hamilton glass syringe 10 &amp;mu;L&amp;nbsp;</td><td>Hamilton Co.</td><td>721711</td><td></td></tr><tr><td>Hoeschst</td><td>Merck</td><td>B2261</td><td></td></tr><tr><td>Ketamine</td><td>Bremer pharma GMBH (medimarket)</td><td>17889</td><td></td></tr><tr><td>Ofloxacin 0.3% eye drops</td><td>Allergan</td><td>E92170</td><td></td></tr><tr><td>Oxybuprocaine Hydrochloride 0.4%&amp;nbsp;</td><td>Fisher Pharmaceutical</td><td>N/A</td><td></td></tr><tr><td>Pentobarbital sodium 200 mg/mL</td><td>CTS</td><td>N/A</td><td></td></tr><tr><td>Slit microscope&amp;nbsp;</td><td>Haag-streit bern</td><td>b-90019115</td><td></td></tr><tr><td>Sprague-Dawley Rats</td><td>Envigo</td><td>N/A</td><td></td></tr><tr><td>Stiletto blade 31 G 0.8 mm&amp;nbsp;</td><td>Tecfen medical (skymed)</td><td>QKN2808</td><td></td></tr><tr><td>Surgical microscope</td><td>Zeiss</td><td>OPMI-6 CFC</td><td></td></tr><tr><td>Trypan Blue</td><td>Sartorius</td><td>03-102-1B</td><td></td></tr><tr><td>Xylazine</td><td>Eurovet Animal Health&amp;nbsp;</td><td>615648</td><td></td></tr></tbody></table>

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.

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.
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Author Spotlight: A Novel Protocol for Intracameral Injections to Enhance Precision in Rodent Ophthalmology

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 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 ...

intracameral-injection-in-rats-with-low-risk-of-adverse-effects

Research

JoVE Journal

Biology

688 Views.  Kaplan Medical Center. 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.

Video: Author Spotlight: A Novel Protocol for Intracameral Injections to Enhance Precision in Rodent Ophthalmology

Intravitreous Injection for Establishing Ocular Diseases Model

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as direct application of local treatment. This video introduces how to use simple and inexpensive tools to finish the intravitreous injection procedure. Use of a 1 ml syringe, instead of a hemilton syringe, is used. Practical tips for how to make appropriate injection needles using glass pipettes with perfect tips, and how to easily connect the syringe needle with the glass pipette tightly together, are given.
To conduct a good intravitreous injection, there are three aspects to be observed: 1) injection site should not disrupt retina structure; 2) bleeding should be avoided to reduce the risk of infection; 3) lens should be untouched to avoid traumatic cataract. In brief, the most important point is to reduce the interruption of normal ocular structure. To avoid interruption of retina, the superior nasal region of rat eye was chosen. Also, the puncture point of the needle was at the par planar, which was about 1.5 mm from the limbal region of the rat eye. A small amount of vitreous is gently pushed out through the puncture hole to reduce the intraocular pressure before injection. With the 45&deg; injection angle, it is less likely to cause traumatic cataract in the rat eye, thus avoiding related complications and influence from lenticular factors. In this operation, there was no cutting of the conjunctiva and ocular muscle, no bleeding. With quick and minor injury, a successful intravitreous injection can be done in minutes. 
The injection set outlined in this particular protocol is specific for intravitreous injection. However, the methods and materials presented here can also be used for other injection procedures in drug delivery to the brain, spinal cord or other organs in small mammals.

Intravitreous injection is a widely used technique in visual sciences research for ocular diseases or as direct application of local treatment. This video demonstrated a protocol for intravitreous injection using a 1ml syringe with glass pipette. Useful tips about avoiding massive bleeding and lens damage are given.

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as ...

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. Several drugs are administered into the anterior chamber or vitreous, and the intraocular injection has been effective in curing various intraocular diseases. Rabbit eyes have been widely used for ophthalmic research, as the animal is easy to handle and economical compared to other mammals, and the size of a rabbit eye is similar to that of a human eye. Using a 30 G needle, drugs can be injected into the intracameral and intravitreal spaces of rabbit eyes. The eyeballs are then frozen until analysis, and can be divided into the aqueous humor, vitreous, and retina/choroid. The vitreous and retina/choroid samples can be homogenized and solubilized before analysis. Then, immunoassays can be performed to measure the concentrations of intraocular drugs in each compartment. Appropriate pharmacokinetic models can be used to calculate several parameters, such as the half-life and maximum concentration of the drug. Rabbit eyes can be a good model for pharmacokinetic studies of intraocular drugs.

Rabbits are widely used to study the pharmacokinetics of intraocular drugs. We describe a method for conducting pharmacokinetic studies of intraocular drugs using rabbit eyes.

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. ...

Medicine

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

Neuroscience

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Performing Intracerebroventricular Injection in Mice Using a Free-Hand Approach

Free-Hand Intracerebroventricular Injections in Mice

The investigation of neuroendocrine systems often requires the delivery of drugs, viruses, or other experimental agents directly into the brains of mice. An intracerebroventricular (ICV) injection allows the widespread delivery of the experimental agent throughout the brain (particularly in the structures near the ventricles). Here, methods for making free-hand ICV injections in adult mice are described. By using visual and tactile landmarks on the heads of mice, injections into the lateral ventricles can be made rapidly and reliably. The injections are made with a glass syringe held in the experimenter&#39;s hand and placed at approximate distances from the landmarks. Thus, this technique does not require a stereotaxic frame. Furthermore, this technique requires only brief isoflurane anesthesia, which permits the subsequent assessment of mouse behavior and/or physiology in awake, freely behaving mice. Free-hand ICV injection is a powerful tool for the efficient delivery of experimental agents into the brains of living mice and can be combined with other techniques such as frequent blood sampling, neural circuit manipulation, or in vivo recording to investigate neuroendocrine processes.

Author Spotlight: Insights, Innovations, and Future Avenues in Reproductive Neurocontrol 

Here, a simple and rapid approach for performing intracerebroventricular injections in mice using a free-hand approach (that is, without a stereotaxic device) is described.

The investigation of neuroendocrine systems often requires the delivery of drugs, viruses, or other experimental agents directly into the brains of mice. ...

A Neonatal Rodent Model of Retroorbital Vein Injection

Intravenous (iv) injection is the most used route of drug administration in neonates in the clinical setting. Therefore, retroorbital vein injection is an important method for compound administration in research, where successful proof-of-concept studies can progress into much-needed neonatal clinical trials. Most intravenous studies in neonatal rodents use the superficial temporal/facial vein. However, retroorbital injection becomes unreliable in neonatal rodents older than 2 days after the skin darkens and the vein is no longer visible. In the present protocol, we describe the retroorbital injection of the venous sinus in both the neonatal mouse and rat at ages when the superficial temporal vein is no longer visible, but the eyes have not opened yet. Eye-opening facilitates retro-orbital injection by enabling the researcher to clearly see that they are not perforating the eye when inserting the needle. We demonstrate that this technique can be performed in a reliable and reproducible manner without adverse effects. Additionally, we show that it can be used in many studies, such as administering compounds to study neonatal brain injury.

This protocol aims to demonstrate a reproducible venous administration route that can be used in rats and mice throughout the neonatal period. This procedure is important for preclinical rodent studies that wish to mirror drug administration in neonatal care units primarily using intravenous administration.

Intravenous (iv) injection is the most used route of drug administration in neonates in the clinical setting. Therefore, retroorbital vein injection is an ...

Lumbar Puncture Delivery of AAV9 in Juvenile Rats for CNS Gene Therapy

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a toxic gene, or provide a gene whose product can modulate the biology of the disease. The delivery method for the therapeutic vector can take many forms, ranging from intravenous infusion for systemic delivery to direct injection into the target tissue. For neurodegenerative disorders, it is often desirable to skew transduction towards the brain and/or spinal cord. The least invasive approach to target the entire central nervous system involves injection into the cerebrospinal fluid (CSF), allowing the therapeutic to reach a large fraction of the central nervous system. The safest approach to deliver a vector into the CSF is the lumbar intrathecal injection, where a needle is introduced into the lumbar cistern of the spinal cord. This technique, also known as a lumbar puncture, has been widely used in neonatal and adult rodents and in large animal models. While the technique is similar across species and developmental stages, subtle differences in size, structure, and elasticity of tissues surrounding the intrathecal space require accommodations in the approach. This article describes a method for performing lumbar puncture in juvenile rats to deliver an adeno-associated serotype 9 vector. Here, 25-35 &#181;L of vector were injected into the lumbar cistern, and a green fluorescent protein (GFP) reporter was used to evaluate the transduction profile resulting from each injection. The benefits and challenges of this approach are discussed.

Author Spotlight: Assessing Intrathecal Gene Therapy Efficacy in Juvenile Rats

A surgical procedure is described to perform injections into the lumbar cistern of the juvenile rat. This approach has been used for the intrathecal delivery of gene therapy vectors, but it is anticipated that this approach can be used for a variety of therapeutics, including cells and drugs.

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a ...

Intraoperative Visualization of Subretinal Injection and Retinal Detachment in Rats

Subretinal injection, the delivery of a solution between the photoreceptors and the retinal pigment epithelium (RPE), creates a subretinal space where components are in direct contact with photoreceptors and RPE cells. This delivery method allows for targeted treatment of these cells. Therapeutics for subretinal injection have been developed and approved, particularly for inherited retinal diseases. In animals, subretinal injection procedures can be challenging due to the lens size, especially in rodents. This article describes methods for subretinal injection in rats, enabling intraoperative visualization and control of both the injection site and the size of the detached area, as performed in humans. The procedure is conducted under general and local anesthesia and requires pupil dilation. Using an ophthalmic microscope, the subretinal injection is performed through a 30 G scleral channel, with the cannula tip gently applied to the retina to create a retinotomy. Volumes ranging from 10-25 &#181;L can be delivered, corresponding to two-fifths to half of the rat retina. Immediate postoperative examinations using fundus photography and optical coherence tomography confirm successful delivery into the subretinal space with visible subretinal fluid. The major risks of this procedure include lens damage (cataract), detachment failure, intravitreal hemorrhage, subretinal hemorrhage, and postoperative keratitis. In addition to delivering therapeutics into the subretinal space, this technique is used to induce short-term or long-term retinal detachment using aqueous or viscous products, respectively. Unlike the transscleral approach, this method enables precise intraoperative positioning of the retinal detachment.

This article describes methods for subretinal injection in rats, utilizing intraoperative visualization to control both the injection site and the area of retinal detachment.

Subretinal injection, the delivery of a solution between the photoreceptors and the retinal pigment epithelium (RPE), creates a subretinal space where ...

Transconjunctival Approach for Injection into the Rat Optic Nerve

The rat serves as an important model for studying disorders of the optic nerve, including heritable, traumatic, neoplastic, and autoimmune conditions. Accessing the rat optic nerve for experimental manipulations and injections is challenging due to the small size of the orbit and the vascularity of the surrounding orbital tissues. Previous techniques have involved a cutaneous incision, which carries a higher risk of infection and requires wound closure. This study aims to describe a unique, less invasive, and potentially more efficient approach to accessing the rat optic nerve. A transconjunctival incision, along with a rat spinal cord hook, is used to isolate the optic nerve for experimental injections. In these experiments, an adenoviral vector is injected into the optic nerve with the goal of using this technique for direct gene therapy delivery in the future. This technique can also be applied by investigators requiring access to the rat optic nerve for other experimental studies, including drug delivery and optic nerve injury research.

This protocol describes a unique technique using a transconjunctival approach to access the rat optic nerve for injections.

The rat serves as an important model for studying disorders of the optic nerve, including heritable, traumatic, neoplastic, and autoimmune conditions. ...

Compound Administration III

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
There are many commonly used routes for compound administration in laboratory mice and rats. However, certain protocols may require the use of less commonly used routes, including intradermal, intranasal, and intracranial injections. Specialized training is essential for these procedures to be performed successfully. Justification for these routes may need to be provided to gain Institutional Animal Care and Use Committee (IACUC) approval.

Compound Administration in Rodents- Intradermal, Intranasal &amp; Intracranial Routes

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
There are many commonly used routes for compound ...

Intracameral Injection in Rats with Low Risk of Adverse Effects

Summary

Explore More Videos

Intravitreous Injection for Establishing Ocular Diseases Model

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Free-Hand Intracerebroventricular Injections in Mice

A Neonatal Rodent Model of Retroorbital Vein Injection

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

Intraoperative Visualization of Subretinal Injection and Retinal Detachment in Rats

Transconjunctival Approach for Injection into the Rat Optic Nerve

Compound Administration III