Name: use of rabbit eyes in pharmacokinetic studies of intraocular drugs
Uploaded: 2016-07-23T13:00:00.000+00:00
Duration: 10 min 2 s
Description: Watch this Scientific Journal Video about Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs at JoVE.com

We would like to thank Ms. Ji Hyun Park and Ji Yeon Park for their technical assistance in the animal experiments. This work was supported by a grant from the Seoul National University Bundang Hospital Research Fund (grant number: Grant No. 14-2014-022) and from a grant (CCP-13-02-KIST) from the Convergence Commercialization Project of the National Research Council of Science and Technology, Seoul, Korea.

Our protocol follows the guidelines of the Institutional Animal Care and Usage Committee (IACUC) of Seoul National University Bundang Hospital, which approved all of the animal procedures and animal care methods presented in this protocol. The IACUC is in full compliance with the eighth edition of Guide for the Care and Use of Laboratory Animals (2011). All procedures were performed with adherence to the guidelines of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research in animals. Individual cages were used for housing the rabbits. Additional surgery or preparation before carrying out this experiment (i.e., sterilization) may not be required.
1. Intraocular Injection of the Drug into Rabbit Eyes
<ol>
	<li>Anesthetize healthy New Zealand white rabbits weighing 1.5-2 kg with an intramuscular injection of a mixture of tiletamine hydrochloride and zolazepam hydrochloride (15 mg/kg) and xylazine hydrochloride (5 mg/kg). Verify anesthesia by monitoring the palpebral (blink) reflex or by ear pinch. 
	NOTE: If an intraocular drug binds to ocular pigments, the degree of binding might induce modification of the drug&#39;s ocular pharmacokinetics. For example, the half-life of a pigment-binding drug in the vitreous and aqueous humors of pigmented rabbits may be increased compared to that in albino rabbits14. In this case, the data obtained from pigmented rabbit eyes may be more similar and applicable to human eyes as human eyes have a different degree of pigmentation and albino rabbit eyes may not represent human counterparts. Thus, considering drug-pigment interaction, the use of pigmented or albino rabbits should be carefully considered and the results should be interpreted together with the strain (pigmentation) of rabbit. However, albino rabbits are recommended for use in studies comparing pharmacokinetic properties as the drug-pigment interaction may be a confounding factor in the comparison.
	<ol>
		<li>Apply a topical anesthetic with 1% proparacaine hydrochloride ophthalmic eye drops. Use vet ointment (2% hydroxypropyl methylcellulose) to prevent eye dryness until recovery from anesthesia. 
		NOTE: Never leave the rabbit unattended while anesthetized.</li>
	</ol>
	</li>
	<li>Dilate the pupil with one or two drops of phenylephrine hydrochloride and tropicamide.</li>
	<li>For surgical preparation before the intraocular injection, apply 5-10% povidone-iodine to the periocular skin with a cotton swab5. Place one drop of 5-10% povidone-iodine on the conjunctiva of the eye.</li>
	<li>Administer the intraocular drugs either intravitreally or intracamerally using aseptic techniques5. 
	NOTE: The drug absorbed in systemic circulation may also penetrate the fellow eye. When a drug is administered to both eyes, the drug concentration in one eye may be affected by drug injected into the other eye. If it is confirmed that the effect of the contralateral injection can be neglected as the drug in systemic circulation cannot penetrate the other eye, the use of both eyes for intraocular injections can be considered, as it is economical and minimizes the number of animals sacrificed for the pharmacokinetic studies. For pharmacokinetic study on systemic concentration following intraocular injection, the use of only one eye is appropriate.
	<ol>
		<li>For intravitreal injections, inject the drug intravitreally 1 mm behind the surgical limbus in the superotemporal quadrant using a 30 G needle and either commercial 1 ml syringes, insulin syringes, or glass syringes perpendicularly to the scleral surface5. 
		NOTE: The volume of drug used for intracameral and intravitreal injection varies depending on the drugs under investigation. For pharmacokinetic experiments on anti-VEGF agents using rabbit eyes, previous studies used 0.025, 0.05 (most common), or 0.1 ml of bevacizumab or ranibizumab for the intracameral or intravitreal injection. In our previous studies on intraocular pharmacokinetics of anti-vascular endothelial growth factor, 0.05 ml of bevacizumab15, 0.025 ml of ranibizumab, or 0.03 ml of VEGF-Trap16 were used. The largest safe volume without paracentesis is believed to be 0.1 ml, although there has been little evidence to support this17. If excessive volume is injected either intracamerally or intravitreally, intraocular pressure is greatly increased. In addition to optic nerve damage directly caused by increased intraocular pressure (IIOP), namely glaucomatous optic nerve damage, in extreme cases the IIOP leads to impaired ocular perfusion and central retinal artery occlusion, which is analogous to stroke in the brain.</li>
		<li>For intracameral injections, inject the drug into the anterior chamber by inserting a 30 G needle through the corneoscleral limbus with the bevel up and advancing it parallel to the iris plane to minimize the risk of trauma to the iris or lens. 
		NOTE: Depending on the drug formulation, needles larger than 30 G can be used. The conditions requiring larger-gauge needle include drugs containing microspheres, large-protein drugs, and high viscosity formulations.</li>
		<li>After injection, compress the injection site with a sterile cotton-tip applicator to promote wound healing. 
		NOTE: Normally, 30 sec&#160;is enough for wound healing, when a 30 G needle is used for intraocular injection. However, if a larger gauge needle is used for intraocular injection, longer time would be appropriate for wound sealing to minimize the leakage from the sclerotomy wound. To check for sclerotomy wound leakage is important to ensure that the amount of intraocular drug used immediately after the intraocular injection is the same as the injected amount, especially if a larger gauge needle is used.</li>
	</ol>
	</li>
	<li>Observe the treated rabbits daily for one week, and once a week afterwards, for any sign of severe intraocular inflammation (conjunctival injection and hypopyon) until euthanasia. 
	NOTE: Analgesics after injection are not required as intraocular injection using a 30 G needle is a minimally invasive procedure that is not accompanied by post-surgical pain. Do not return an animal that has undergone surgery to the company of the other animals until fully recovered.</li>
</ol>
2. Sample Preparation
<ol>
	<li>For enucleation, euthanize the rabbits at various time points (e.g., 1 hr or 1, 2, 5, 9, 14, or 30 days) after the intraocular drug injection. 
	NOTE: These time points depend on the drug of interest and the known pharmacokinetic profiles. For each time point, use at least two eyeballs13. For reliable data quality, sampling time should be chosen carefully, at least four time-points with balanced sampling for at least a time-span of two half-lives of the drug13. We believe that 6-8 time-points with balanced sampling may be appropriate. For small molecules (&#8804;1,000 Da)18, in particular, more than 1 time-point during the first 24 hr is crucial. Accordingly, for macromolecules (&#62;1,000 Da)18, a sampling period such as 1 hr and 1, 2, 5, 9, 14, and 30 days19 can be a good option. For small molecules (&#8804;1,000 Da), 1, 2, 4, and 8 hr and 1, 3, and 7 days can be one option20. Depending on the molecular weight, the sampling period can be further modified.
	<ol>
		<li>For euthanasia, administer 10 ml of 15% intravenous KCl rapidly after anesthetizing rabbits with an intramuscular injection of a mixture of tiletamine hydrochloride, zolazepam hydrochloride (15 mg/kg), and xylazine hydrochloride (5 mg/kg).</li>
	</ol>
	</li>
	<li>Open the palpebral fissure with an eyelid retractor. Make a 360&#176; conjunctival incision 2-3 mm posterior to the limbus and extend it posteriorly by dissecting the conjunctiva and Tenon&#39;s capsule from the globe.</li>
	<li>Cut the extraocular muscles close to their insertion into the globe. Clamp the optic nerve with curved forceps and then cut the nerve between the forceps and the globe. Remove the eyeball itself while leaving the surrounding tissues intact. After enucleation, immediately freeze the eyeballs and store them at -80 &#176;C. 
	NOTE: There may be two options for immediate freezing. If the drug concentration is measured at a very early phase (i.e. less than 1 hr), liquid nitrogen may be more appropriate to ensure better timely freezing of the eyeball. However, for subsequent periods, ice can be used to cool the eyeball immediately, and then deep freezer can be used to store the eyeballs at -80 &#176;C.</li>
	<li>After obtaining the eyeballs for all of the time points, separate the frozen eyeballs into three compartments, the vitreous, the aqueous humor, and the retina/choroid. Separate these compartments before defrosting. 
	NOTE: The most important point for the separation into three compartments is the speed of the procedure. The eyeball&#160;should be separated very quickly before defrosting and the procedure can be performed on the ice to delay thawing.
	<ol>
		<li>To open the globe, make an incision at the corneal limbus (300&#176; or greater) with a scalpel blade. Obtain the frozen compartment in front of the iris, the aqueous humor.</li>
		<li>Remove the frozen iris and lens by pulling and excoriating using tissue forceps. When the vitreous becomes accessible, obtain the frozen vitreous by separating it from the remaining tissues (retina/choroid/sclera).</li>
		<li>Using a No. 15 scalpel blade, separate the retina/choroid tissue from the underlying sclera.</li>
	</ol>
	</li>
	<li>For immunoassays, defrost the aqueous humor samples, and measure the volume of each sample. Measure the weight of the frozen samples by subtracting the weight of an empty tube from that of the tube containing the frozen sample. 
	NOTE: Because the specific gravity of the frozen samples is approximately 1, the weight of each sample can be used to calculate the volume of the sample.</li>
	<li>Weigh the vitreous samples, defrost the samples, and solubilize them in 1.0 ml of phosphate-buffered saline containing 1% bovine serum albumin on a rotator overnight at 4 &#176;C. Then, centrifuge the samples at 387 x g for 10 min21.</li>
	<li>Weigh the frozen retina/choroid samples for homogenization. Add protein extraction reagent with a ratio of tissue to reagent of 1:10 (1 g of tissue/10 ml of reagent). Homogenize the tissue using a pre-chilled microhomogenizer. Centrifuge the lysed sample for 10 min at 12,000-20,000 x g and transfer the supernatant to a chilled EPP tube.</li>
</ol>
3. Immunoassay
NOTE: Several analytical methodologies can be used for the measurement of protein concentration. Choose an appropriate quantitative method, depending on the detection range. Briefly, the selected ion monitoring mode of HPLC can detect picogram levels of molecule, whereas LC-MS/MS can detect nanogram and picogram levels of protein for profiling with MRM/PRM mode, respectively. The detection limit of ELISA is considered to be at the picogram level.
<ol>
	<li>For the indirect enzyme-linked immunosorbent assays (ELISAs)&#160;to measure concentrations of anti-VEGF agents, use ELISA kits in 96-well plates to detect the drug of interest and generate a standard curve of known drug concentrations.
	<ol>
		<li>Dilute the vitreous, the aqueous humor, and the retina/choroid samples with 0.1% bovine serum albumin in 1x phosphate-buffered saline (PBS) to concentrations that are within the linear range, and use these for the assay.</li>
	</ol>
	</li>
	<li>Divide the samples into aliquots on the plate at 100 &#181;l/well. Incubate overnight at 4 &#176;C and then wash the plate three times using 200 &#181;l of the washing solution of 0.05% Tween20 in 1x PBS. 
	NOTE: The anti-VEGF present in the sample acts as the primary antibody for ELISA; therefore, additional use of a primary antibody is not necessary.</li>
	<li>Dilute the secondary antibody to 1:20,000 in 0.1% BSA in 1x PBS, and add 100 &#181;l of the diluted-solution per well. After incubating the plate with a diluted secondary antibody overnight at 4 &#176;C, measure the optical density at 450 nm wavelength. Subtract the average zero standard optical density from the average of duplicate readings for each standard and sample.</li>
	<li>Create a standard curve based on the relative light signal from solutions of the drug with known concentrations by reducing the data using computer software capable of generating a four-parameter logistic (4-PL) curve fit, such as SoftMax Pro. For creating a standard curve, the 4-PL curve fit can be obtained by clicking on [4-parameter] in [Standard Curve] - [Fit].</li>
	<li>Calculate the drug concentration in the samples from the standard curve. 
	NOTE: The limits of detection (LOD) of anti-VEGF drugs were investigated in our experiment. The LOD of bevacizumab was 0.024-3.125 ng/ml and that of aflibercept was 0.039-10 ng/ml.</li>
</ol>
4. Pharmacokinetic Analysis Methods
NOTE: For PK analysis, one can use either compartmental or non-compartmental analysis. In compartmental analysis, disposition behavior of molecules can be explained by an equation (model). Thus, compartmental PK analysis can predict the concentration at any time t whereas the non-compartmental model cannot visualize or predict concentration-time profiles for other dosing regimens. However, fitting of compartmental models can be a complex and lengthy process. In contrast, assumptions are less restrictive in non-compartmental model. The non-compartmental method is simple and used commonly to calculate pharmacokinetic parameters such as half-life, clearance, and volume of distribution. We chose compartmental models for pharmacokinetic studies on anti-VEGF agents.
<ol>
	<li>Analyze the drug concentration data with the compartmental models using modeling software such as Phoenix WinNonlin software.
	<ol>
		<li>Click [PK model] in [WNL5 classic modeling] and map the observation time, administered dose, and drug concentration in the [Setup] menu.</li>
		<li>Select compartmental model (e.g., number of compartments) in the [model selection] tab, and click the [Execution] button to calculate model parameters.</li>
	</ol>
	</li>
	<li>After the analyses, select the final compartment model that best describes the drug concentration data based on the following criteria: (1) Akaike Information Criterion, (2) precision of parameter estimates (e.g., standard error), and (3) graphical analysis (e.g., goodness of fit plots).</li>
	<li>Calculate pharmacokinetic parameters of interest, such as half-life (T1/2) and area under the time-concentration curve (AUC), from the model parameters and equations driven by the compartmental model.</li>
</ol>

The authors have no conflicts of interest to disclose.

With the increasing use of intraocular drugs, such as anti-vascular endothelial growth factor (VEGF) agents, for the treatment of diverse ocular diseases, knowledge of the tissue distribution and clearance of the drug after the intraocular injection is important. Understanding the pharmacokinetics of intraocular drugs is important for understanding the efficacy and safety of drugs, determining the optimal dosage of the drugs, and minimizing systemic or intraocular complications. However, detailed pharmacokinetic studies ...

Before the advent of intraocular drug delivery, the main concern of medical therapy for intraocular diseases was the efficiency with which the drug could penetrate into the eye. The blood-ocular barrier prevents many substances, including drugs, from diffusing into the eye. Therefore, concentrations of drugs that are above therapeutic levels may not be easily obtained. The intraocular drug administration method, including intracameral and intravitreal injections, can directly bypass the blood-ocular barrier1-3, so that therapeutic concentrations of drugs can be achieved in the eye4,5.
Accordingly, intravitreal drug delivery has become a popular method of treatment for several intraocular diseases5,6. For example, intravitreal injection is widely performed for age-related macular degeneration, diabetic retinopathy, retinal vein occlusions, and intraocular infections7-10. In particular, since the introduction of anti-VEGF medications, the frequency of intravitreal injections has remarkably increased for the treatment of retinal diseases. Therefore, it is important to understand the intraocular pharmacokinetics of such drugs for evaluating the efficacy and safety of the medical therapy.
Although the intraocular administration of drugs is considered a major breakthrough in medical therapy for ocular diseases, monitoring the drug concentration within the eyeball is technically demanding. Because human eyes contain only small amounts of aqueous humor (about 200 &#181;l) and vitreous (about 4.5 ml, Table 1), it is technically difficult to obtain sufficient amounts of ocular fluid to measure the drug concentration. Furthermore, methods that are used to obtain the eye fluid, such as vitreous tapping or anterior chamber paracentesis, may damage the ocular tissue and result in serious complications, such as cataracts, endophthalmitis, or retinal detachment11,12. Accordingly, animal models are used in pharmacokinetic studies of commonly used intraocular drugs13. Among these animal models, rabbits or monkeys are the most frequently used animals.
Rabbits, which are small mammals of the order Lagomorpha in the family Leporidae, are found in several parts of the world. Because rabbits are not aggressive, they are easy to handle, use in an experiment, and observe. Lower cost, ready availability of the animal, similar eye size to humans, and a large database of information for comparison favor performing pharmacokinetic studies using rabbit eyes. In this paper, a protocol for pharmacokinetic studies of intraocular drugs in rabbit eyes is described.

<table><tbody><tr><td>Zoletil</td><td>Virbac Laboratories, Carros Cedex, France</td><td></td><td></td></tr><tr><td>Xylazine hydrochloride&amp;nbsp;</td><td>Fort Dodge Laboratories, Fort Dodge, IA</td><td></td><td></td></tr><tr><td>Proparacaine hydrochloride (Alcaine)</td><td>Alcon laboratories, Fort Worth, TX</td><td></td><td></td></tr><tr><td>Phenylephrine hydrochloride and tropicamide</td><td>Santen Pharmaceutical, Co., Osaka, Japan</td><td></td><td></td></tr><tr><td>Recombinant Human VEGF 165</td><td>R&amp;amp;D systems</td><td>293-VE-050</td><td></td></tr><tr><td>Carbobate-Bicarbonate buffer</td><td>SIGMA</td><td>C3041-50CAP</td><td></td></tr><tr><td>Nunc Microwell 96F w/lid 
Nunclon D Si</td><td>Thermo SCIENTIFIC</td><td>167008</td><td>96 well plate</td></tr><tr><td>Bovine Serum Albumin (BSA) 25 g(Net)</td><td>BOVOGEN</td><td>BSA025</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS) pH 7.4 (1x), 500 ml</td><td>gibco</td><td>10010-023</td><td></td></tr><tr><td>Sheep anti-Human IgG Secondary Antibody, HRP conjugate</td><td>Thermo SCIENTIFIC</td><td>PA1-28652</td><td></td></tr><tr><td>Goat Anti-Human IgG Fc(HRP)</td><td>abcam</td><td>ab97225</td><td></td></tr><tr><td>Goat anti-Human IgG, Fab&#039;2 Secondary Antibody, HRP conjugate</td><td>Thermo SCIENTIFIC</td><td>PA1-85183</td><td></td></tr><tr><td>CelLytic MT&amp;nbsp; Cell Lysis Reagent</td><td>SIGMA</td><td>C3228-50ML</td><td>lysis buffer</td></tr><tr><td>100 Scalpel Blades</td><td>nopa instruments</td><td>BLADE #15</td><td></td></tr><tr><td>100 Scalpel Blades</td><td>nopa instruments</td><td>BLADE #10</td><td></td></tr><tr><td>Feather surgical blade stainless steel</td><td>FEATHER</td><td>11</td><td></td></tr><tr><td>1-StepTM TMB-Blotting substrate solution, 250 ml</td><td>Thermo SCIENTIFIC</td><td>34018</td><td></td></tr><tr><td>Stable Peroxide Substrate Buffer (10x), 100 ml</td><td>Thermo SCIENTIFIC</td><td>34062</td><td></td></tr><tr><td>Softmax Pro</td><td>Molecular Devices</td><td>v.5.4.1</td><td>software for generating standard curve</td></tr><tr><td>SAAM II&amp;nbsp;</td><td>Saam Institute, Seattle, WA</td><td></td><td>software for pharmacokinetic modeling</td></tr><tr><td>Phoenix WinNonlin</td><td>Pharsight, Cary, NC</td><td>v. 6.3</td><td>software for pharmacokinetic modeling</td></tr><tr><td>Avastin (bevacizumab)</td><td>Genentech</td><td></td><td></td></tr></tbody></table>

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.

The procedure that is used to conduct intravitreal injections of a drug of interest in rabbit eyes with sterile techniques is shown in Figure 1. The treated eyes are enucleated at a scheduled time and stored at -80 &#176;C. For the analysis, three compartments, the aqueous humor, the vitreous, and the retina/choroid, are separated from the frozen rabbit eyes, as demonstrated in Figure 2. Samples of the compartments are prepared for the ELISA. After incuba...

Watch this Scientific Journal Video about Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs at JoVE.com

Use of Rabbit Eyes in 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. ...

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31.8K Views.  Seoul National University Bundang Hospital. 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.

Video: Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.

This manuscript describes the novel setup and operating procedure of a photoacoustic microscopy and optical coherence tomography dual-modality system for noninvasive, label-free chorioretinal imaging of larger animals, such as rabbits.

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into ...

Engineering

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal abnormalities. In addition, pathological myopia is a common irreversible cause of visual impairment and blindness1-3. Our study demonstrates the effect of scleral crosslinking using riboflavin and ultraviolet-A radiation on the development of axial myopia in a rabbit model. The axial length of the eyeball was measured by A-scan ultrasound in New Zealand white rabbits aged 13 days (male and female). The eye then underwent 360&#176; conjunctival peritomy with scleral crosslinking, followed by tarsorrhaphy. Axial elongation was induced in 13 day-old New Zealand rabbits by suturing their right eye eyelids (tarsorrhaphy). The eyes were divided into quadrants, and every quadrant had two scleral irradiation zones, each with an area of 0.2 cm&#178; and a radius of 4 mm. Crosslinking was performed by dropping 0.1% dextran-free riboflavin-5-phosphate onto the irradiation zones 20 sec before ultraviolet-A irradiation and every 20 sec during the 200 sec irradiation time. UVA radiation (370 nm) was applied perpendicular to the sclera at 57 mW/cm&#178; (total UVA light dose, 57 J/cm&#178;). Tarsorrhaphies were removed on day 55, followed by repeated axial length measurements. This study demonstrates that scleral crosslinking with riboflavin and ultraviolet-A radiation effectively prevents occlusion-induced axial elongation in a rabbit model.

We demonstrate the effect of scleral crosslinking with riboflavin and UVA on an axial elongation rabbit eye. Axial elongation was induced in 13 day-old New Zealand rabbits (male and female) by suturing their right eye eyelids (tarsorrhaphy).

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

Bioengineering

A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for quality of life and health care costs. The lack of informative animal models that recapitulate its key features impedes the search for new therapeutic agents for DED. Available DED animal models have limited reproducibility and efficacy. A model is presented here in which DED is induced by injecting the mitogen concanavalin A (Con A) into the orbital lacrimal glands of rabbits. Innovative aspects of this model are the use of ultrasound (US) guidance to ensure optimal and reproducible injection of Con A into the inferior lacrimal gland; injection of Con A into all orbital lacrimal glands that limits compensatory production of tears; and use of periodic repeat injections of Con A that prolong the state of DED at will. DED and its response to test agents are monitored with a panel of parameters that assess tear production, the stability of the tear film, and the status of the corneal and conjunctival mucosa. They include tear osmolarity, tear break-up time, Schirmer&#39;s tear test, rose bengal staining, and tear lactoferrin levels. The induction of DED and the monitoring of its parameters are described in detail. This model is simple, robust, reproducible, and informative. This animal model is suitable for the study of tear physiology and of the pathophysiology of DED as well as for the assessment of the efficacy and safety of candidate agents for the treatment of DED.

This article describes the development of a method to induce acute or chronic dry eye disease in rabbits by injecting concanavalin A to all portions of the orbital lacrimal gland system. This method, superior to those already reported, generates a reproducible, stable model of dry eye suitable for the study of pharmacological agents.

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Neuroscience

Rabbit Eye Dissection and Cryosectioning for Immunohistochemistry Studies

Obtaining High-Quality Cryosections of Whole Rabbit Eye

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed in the fixative. Then, the cornea and iris are removed and the eye is left overnight for additional fixation at 4 &#176;C. Following fixation, the lens is removed. The eye is then placed in a cryomold and filled with an embedding medium. By removing the lens, the embedding medium has better access to the vitreous and leads to better retinal stability. Importantly, the eye should be incubated in embedding medium overnight to allow complete infiltration throughout the vitreous. Following overnight incubation, the eye is frozen on dry ice and sectioned. Whole retinal sections may be obtained for use in immunohistochemistry. Standard staining protocols may be utilized to study the localization of antigens within the retinal tissue. Adherence to this protocol results in high-quality retinal cryosections that may be used in any experiment utilizing immunohistochemistry.

Author Spotlight: Optimizing Retinal Tissue Processing for Immunohistochemistry in Rabbit Models

This protocol describes a reliable method for obtaining high-quality cryosections of whole rabbit eyes. It details rabbit eye dissection, fixation, embedding, and sectioning procedures, which may be easily adapted for use in any study utilizing immunohistochemistry in larger eyes.

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed ...

Biology

Optimized Rat Intracameral Injection Technique for Reduced Tissue Damage

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

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 ...

Performing Injection into Palpebral Portion of the Superior Lacrimal Gland: A Technique for Inducing Dry Eye Disease in Rabbit Model

Source: Honkanen, R. A., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/59631">A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies.</a> J. Vis. Exp. (2020).
In this video, we show a procedure to induce aqueous-deficient dry eye disease in a rabbit model by injecting the palpebral portion of the superior lacrimal gland with Concanavalin A. This model is useful for pharmacokinetic and biodistribution assays of drugs for treating dry eye disease.

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

Summary

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A Step by Step Protocol for Subretinal Surgery in Rabbits

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Obtaining High-Quality Cryosections of Whole Rabbit Eye

Intracameral Injection in Rats with Low Risk of Adverse Effects

Performing Injection into Palpebral Portion of the Superior Lacrimal Gland: A Technique for Inducing Dry Eye Disease in Rabbit Model