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 border="0" cellpadding="0" cellspacing="0" width="647">
	<tbody>
		<tr height="46">
			<td height="46" style="height:46px;width:213px;">Zoletil</td>
			<td style="width:143px;">Virbac Laboratories, Carros Cedex, France</td>
			<td style="width:119px;"></td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:213px;">Xylazine hydrochloride&#160;</td>
			<td style="width:143px;">Fort Dodge Laboratories, Fort Dodge, IA</td>
			<td style="width:119px;"></td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:213px;">Proparacaine hydrochloride (Alcaine)</td>
			<td style="width:143px;">Alcon laboratories, Fort Worth, TX</td>
			<td style="width:119px;"></td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:213px;">Phenylephrine hydrochloride and tropicamide</td>
			<td style="width:143px;">Santen Pharmaceutical, Co., Osaka, Japan</td>
			<td style="width:119px;"></td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Recombinant Human VEGF 165</td>
			<td style="width:143px;">R&#38;D systems</td>
			<td style="width:119px;">293-VE-050</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Carbobate-Bicarbonate buffer</td>
			<td style="width:143px;">SIGMA</td>
			<td style="width:119px;">C3041-50CAP</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">NUNC MICROWELL 96F&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; W/LID NUNCLON D SI&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:143px;">Thermo SCIENTIFIC</td>
			<td style="width:119px;">167008</td>
			<td style="width:173px;">96 well plate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Bovine Serum Albumin (BSA) 25grams(Net)</td>
			<td style="width:143px;">BOVOGEN</td>
			<td style="width:119px;">BSA025</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Phosphate Buffered Saline (PBS) pH7.4 (1X), 500mL</td>
			<td style="width:143px;">gibco</td>
			<td style="width:119px;">10010-023</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">Sheep anti-Human IgG Secondary Antibody, HRP conjugate</td>
			<td style="width:143px;">Thermo SCIENTIFIC</td>
			<td style="width:119px;">PA1-28652</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Goat Anti-Human IgG Fc(HRP)</td>
			<td style="width:143px;">abcam</td>
			<td style="width:119px;">ab97225</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">Goat anti-Human IgG, Fab&#39;2 Secondary Antibody, HRP conjugate</td>
			<td style="width:143px;">Thermo SCIENTIFIC</td>
			<td style="width:119px;">PA1-85183</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">CelLytic MT&#160; Cell Lysis Reagent</td>
			<td style="width:143px;">SIGMA</td>
			<td style="width:119px;">C3228-50ML</td>
			<td style="width:173px;">lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">100 Scalpel Blades</td>
			<td style="width:143px;">nopa instruments</td>
			<td style="width:119px;">BLADE #15</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">100 Scalpel Blades</td>
			<td style="width:143px;">nopa instruments</td>
			<td style="width:119px;">BLADE #10</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">FEATHER SURGICAL BLADE STAINLESS STEEL</td>
			<td style="width:143px;">FEATHER</td>
			<td style="width:119px;">11</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">1-StepTM TMB-Blotting substrate solution, 250mL</td>
			<td style="width:143px;">Thermo SCIENTIFIC</td>
			<td style="width:119px;">34018</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Stable Peroxide Substrate Buffer (10X), 100mL</td>
			<td style="width:143px;">Thermo SCIENTIFIC</td>
			<td style="width:119px;">34062</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Softmax Pro</td>
			<td style="width:143px;">Molecular Devices</td>
			<td style="width:119px;">v.5.4.1</td>
			<td style="width:173px;">software for generating standard curve</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">SAAM II&#160;</td>
			<td style="width:143px;">Saam Institute, Seattle, WA</td>
			<td style="width:119px;"></td>
			<td style="width:173px;">software for pharmacokinetic modeling</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Phoenix WinNonlin</td>
			<td style="width:143px;">Pharsight, Cary, NC</td>
			<td style="width:119px;">v. 6.3</td>
			<td style="width:173px;">software for pharmacokinetic modeling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Avastin (bevacizumab)</td>
			<td style="width:143px;">Genentech</td>
			<td style="width:119px;"></td>
			<td style="width:173px;"></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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31.6K 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

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

Use of a Central Venous Line for Fluids, Drugs and Nutrient Administration in a Mouse Model of Critical Illness

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration to mimic the human clinical setting. Critically ill patients require intensive medical support in order to survive. While the majority of patients will recover within a few days, about a quarter of the patients need prolonged intensive care and are at high risk of dying from non-resolving multiple organ failure. Furthermore, the prolonged phase of critical illness is hallmarked by profound muscle weakness, and endocrine and metabolic changes, of which the pathogenesis is currently incompletely understood. The most widely used animal model in critical care research is the cecal ligation and puncture model to induce sepsis. This is a very reproducible model, with acute inflammatory and hemodynamic changes similar to human sepsis, which is designed to study the acute phase of critical illness. However, this model is hallmarked by a high lethality, which is different from the clinical human situation, and is not developed to study the prolonged phase of critical illness. Therefore, we adapted the technique by placing a central venous catheter in the jugular vein allowing us to administer clinically relevant supportive care, to better mimic the human clinical situation of critical illness. This mouse model requires an extensive surgical procedure and daily intensive care of the animals, but it results in a relevant model of the acute and prolonged phase of critical illness.

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration to mimic the human clinical setting.

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

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 ...

Self-Administration of Drugs in Mouse Models of Feeding and Obesity

Preclinical studies in mice often rely on invasive protocols, such as injections or oral gavage, to deliver drugs. These stressful routes of administration have significant effects on important metabolic parameters including food intake and body weight. Although an attractive option to circumvent this is to compound the drug in rodent food or dissolve it in water, these approaches also have limitations as they are affected by drug stability at room temperature for extended periods of time, the drug's solubility in water, and that the dosing is highly dependent on timing of food or water intake. The constant availability of the drug also limits translational relevance on how drugs are administered to patients. To overcome these limitations, drugs can be mixed with highly palatable food, such as peanut butter, allowing mice to self-administer compounds. Mice reliably and reproducibly consume the drug/peanut butter pellet in a short time frame. This approach facilitates a delivery approach with minimal stress compared with an injection or gavage. This protocol demonstrates the approach of drug preparation, animal acclimatization to placebo delivery, and drug delivery. The implications of this approach are discussed in studies related to timing of drug administration and the circadian rhythm.

The overall goal of this procedure is to describe a method for self-administration of drugs that can be used in mouse models of feeding and obesity.