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

The experiments in the protocol were approved by&#160;the National Permit Committee - for animal science and comply with the ARVO Statement&#160;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. 
		&#8203;NOTE: The lens should be clear. Opacity may reflect lens damage during the surgical procedure.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66662/66662fig02.jpg" /> 
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&#160;injection into the anterior chamber
<ol>
	<li>Load 5 &#181;L of trypan blue into a sterile 10 &#181;L 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 &#181;L of Hoechst into a sterile 10 &#181;L 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&#160;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

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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. 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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. 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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 &#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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alizarin Red&#160;</td>
			<td>Alpha Aesar</td>
			<td>042040.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine&#160;</td>
			<td>Richter pharma</td>
			<td>102047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone 0.1%&#160;</td>
			<td>Fisher Pharmaceutical</td>
			<td>393102-0413</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton glass syringe 10 &#956;L&#160;</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%&#160;</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&#160;</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&#160;</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&#160;</td>
			<td>615648</td>
			<td></td>
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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.

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

Intracameral Injection in Rats with Low Risk of Adverse Effects

Intracameral injections allow to bypass the outer ocular barrier and directly deliver compounds to the aqueous humor. However, conducting intracameral injection in rodent present technical challenges that may result in adverse effect and impact the experimental outcome. We, therefore, design the protocol considering the red-eye anatomy to allow intracameral injection with minimal risk of adverse effects.Intracameral injections in rodents involve the risk of tissue damage due to the small eye dimensions, limited aqueous humor of volume, and lens position. Tissue damage during manipulation can lead to complication, like anterior chamber shallowing, lens injury, endothelial cell damage, and inflammation. All of this cause experimental variability and influence findings.The length and angle of the incision are critical. We make the incision in the central corneal area where the anterior chamber is the deepest at an angle close to flat. This generates a long tunnel, which helps in reducing the loss of aqueous humor.Injecting through the tunnel improves precision and reduces the likelihood of touching the lens. The described intracameral injection method can be utilized in various experimental settings to generate disease models or assess different treatments. We expect that the precision and reproducibility of this injection technique will be a valuable tool in experimental ophthalmology.

Sprague Dawley rats were intracamerally injected with 5 &#181;L 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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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 ...

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

Research

JoVE Journal

Biology

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

To begin, prepare the animal for surgery and apply 0.3%ofloxacin and 0.1%dexamethasone to the animal's eye. Using surgical ophthalmic forceps, hold the superior sclera at the vertical midline next to the corneoscleral junction to stabilize the eye. Under a surgical microscope, place a sterile 0.8 millimeter 31-gauge stiletto blade in the paracentral corneal region on the vertical midline.In this position, puncture the cornea to make an incision and create a long tunnel until it penetrates into the central area of the anterior chamber. Then apply topical 0.3%ofloxacin and 0.1%dexamethasone to the eye. Next, under a slit microscope, observe the depth of the anterior chamber of the injected eye compared to the non-injected eye.Also observe the lens of the injected eye compared to the non-injected eye.

Intracameral Injection of Trypan Blue in Rat Eyes to Assess Delivery into the Anterior Chamber

Begin by loading five microliters of trypan blue into a sterile 10 microliter glass Hamilton syringe, equipped with a 34 gauge blunt needle. Then insert the loaded syringe needle through the self-sealing corneal tunnel into the anterior chamber. Inject the solution in the eye and hold the needle in place for two to three seconds until all fluid clears.Pull out the needle gently. 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.Using this protocol, the corneas of Sprague-Dawley rats were injected with trypan blue. Immediate slit lamp examination post-injection revealed that the anterior chamber was visibly stained with trypan blue, confirming the successful delivery of the substance to the target area.

Intracameral Injection of Hoechst to Assess the Bioavailability of Dye to the Corneal Endothelial Cell Layer

To begin, load five microliters of hoechst solution into a sterile 10 microliter glass Hamilton syringe, attached to a 34 gauge blunt needle. Insert this syringe needle into the anterior chamber through the self-sealing corneal tunnel, and inject the solution. Hold the needle in place for two to three seconds, until all fluid clears.Pull out the needle slowly to remove it, avoiding leakage. Next, enucleate both eyes. To isolate the corneas, cut the sclera around the optic nerve, and make four additional cuts from the optic nerve towards the cornea.Drain the excess fluid with the Whatman filter paper. Spread out the cut sclera, and separate the retina from the sclera. Pull out and discard the lens and retina.Next, remove the iris. Cut and discard the sclera around the cornea. And carefully lift up the cornea.Placing the corneas on a glass slide, stain them with 0.5%Alizarin Red to identify endothelial cells. After removing the excess stain, wash the cornea three times, and place a cover slip on it. Examine the corneas under a light microscope to image the Alizarin Red staining of endothelial cells.Also, evaluate the corneas under a fluorescent microscope to observe hoechst staining, and compare it to the non-injected cornea as control. Hoechst dye, a DNA binding cell permeable fluorescent marker, was utilized to assess drug bioavailability through intracameral injection. Corneal endothelial cell uptake of hoechst was examined 15 minutes post-injection by fluorescent microscopy.Alizarin Red staining highlighted the intercellular borders of the endothelial layer. Also, positive hoechst staining within corneal endothelial cell nuclei confirmed the successful delivery of the dye via this method.

392 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.

Optimized Rat Intracameral Injection Technique for Reduced Tissue Damage

Summary