This research was supported by the Australian Research Council through the ARC Centre of Excellence in Vision Science (CE0561903), the National Health and Medical Research Council (1049990), and Progetto regionale speciale multiasse 'Reti per l'Alta Formazione' - PO FSE Abruzzo 2007-2013 - Azione 4.II.iii.

All experiments conducted were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and with the approval of the University of L&#8217;Aquila Animal Ethics Committee and the Australian National University Animal Experimentation Ethics Committee. Adult rats (P100-200) were used throughout this study.
1. Prepare the Copolymer Resin Pellets
<ol>
	<li>Place 20 Elvax 40W pellets into a small glass beaker in the fume cupboard.</li>
	<li>Fill the beaker with 100% ethanol ensuring to cover all pellets.</li>
	<li>Cover the beaker with Parafilm, and soak the pellets in 100% ethanol at room temperature for 7-10 days. NOTE: From this step onwards, do not use metal or plastic to handle or store the resin.</li>
</ol>
2. Prepare the Drug Solution for Loading
<ol>
	<li>Dissolve the drug of choice in 0.1% dimethyl sulfoxide (DMSO). Make the drug solution up to 40 &#181;l in volume. Load the resin with three times the concentration of the drug that would be given in a single dose.</li>
</ol>
3. Prepare and Load the Drug-resin Complex
<ol>
	<li>Transfer the 20 washed pellets to another small glass beaker. Dissolve the pellets in 4 ml dichloromethane for approximately 45 min. Cover the beaker with Parafilm while the pellets are dissolving.</li>
	<li>Prepare a solution of Fast Green FCF by dissolving it in 0.1% DMSO to a concentration of 0.001 mg/ml.</li>
	<li>Take one pipette, and draw up 40 &#181;l of the drug solution prepared earlier. Load another pipette with 40 &#181;l of Fast Green solution.</li>
	<li>Add the two solutions simultaneously into the beaker. Mix rapidly using a glass stirring rod until the green dye is uniformly distributed throughout the mixture.</li>
	<li>Promptly transfer the beaker onto dry ice for 10 min to fast freeze the resin.</li>
	<li>Set up an &#8220;evaporative chamber&#8221; to evaporate the solvent. Fill a larger container up to one-third with calcium sulfate pebbles. Create a well in the pebbles, and carefully place the beaker in the well so that the pebbles reach up to half of the beaker, to stabilize the beaker in the chamber.</li>
	<li>Cover the larger outside container with Parafilm.</li>
	<li>Transfer the evaporative chamber to the freezer. Keep at -20 &#176;C for 2-3 weeks.</li>
</ol>
4. Prepare the Drug-resin Complex for Surgical Implantation
<ol>
	<li>Remove the evaporative chamber from the freezer. Transfer the solid drug-resin block to a glass dish kept on ice.</li>
	<li>With the aid of a microscope or loupe, cut a piece approximately 0.05 mm in diameter and 0.1 mm in length from the block using a trephine or punch tool.</li>
	<li>Wrap the remainder of the block in freeze-proof material (e.g., aluminum foil) and keep at -20 &#176;C until required.</li>
</ol>
5. Surgically Implant the Drug-resin Complex into the Rat Eye
<ol>
	<li>Prepare the animal for surgery. Anesthetize using an intraperitoneal injection of a mixture of ketamine (100 mg/kg body weight) and xylazine (12 mg/kg body weight). Observe the animal until full anesthesia is achieved. NOTE: The animal should have a loss of movements (except breathing), and a loss of corneal and toe pinch reflexes.</li>
	<li>Transfer the fully anesthetized animal to the surgery table. Use mydriaticum (e.g.,&#160;atropine sulphate), and local anesthetic (e.g.,&#160;tetracaine hydrochloride) drops on the eye(s) undergoing surgery. Apply artificial tears to keep the cornea moist during surgery. Use disposable needles and sterile instruments throughout the surgery.</li>
	<li>Stabilize the eye using a pair of blunt curved forceps after the pupil is fully dilated. Use an operating microscope to make a full depth puncture wound to reach the vitreous, approximately 2 mm from the limbus using a 25 G needle.</li>
	<li>Insert the prepared piece of the drug-resin complex into the puncture wound using a pair of fine, pointy forceps (e.g., tweezers) that are able to penetrate the puncture site.</li>
	<li>As prophylaxis for bacterial infection, apply antibiotic ointment to the eye, to prevent inflammation as well as preventing corneal dryness while the animal is recovering.</li>
	<li>Observe the animal until full recovery. Do not leave the animal unattended until it has regained consciousness. Do not return the animal to the company of other animals until fully recovered. If the animal shows signs of stress or discomfort, analgesics should be used according to the approved ethics protocol.</li>
	<li>Follow the usual experimental procedures for collection and analysis of the tissue.</li>
	<li>At the completion of experiments, euthanize the animal with an intraperitoneal injection of a pentobarbital (60 mg/kg body weight) 2% lidocaine hydrochloride solution.</li>
</ol>
NOTE: Lignocaine Hydrochloride minimizes local discomfort.

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The authors have nothing to disclosure.

This paper demonstrated the use of a drug-resin complex for the slow-release delivery of drugs to the retina.&#160;We aimed to present a method which is relatively inexpensive and easy to apply in a small animal model.
Given that the function of APB is to act as a glutamate analogue, it will block the retinal response in the eye. The results show that APB caused the blockage of the retinal response by one week postimplantation. This indicates that APB was successfully released into the vitreous and retina, and that its effect was limited to the eye that the drug-resin complex was implanted into. The full effect of the drug became obvious one week after implantation (Figure 1B). Figure 1A&#160;shows that at 1 day after implantation, a significant reduction of the b-wave was only present at the 106&#966; intensity in the implanted eye. This may indicate that at this early time point, only the cone&#160;pathway, which responds&#160;&#160;to higher light intensities, was affected.&#160;To determine the length of time that the resin can deliver the APB to the retina, further time points should be investigated. It is proposed that recordings at further time points within the 0-7 day span should be taken, for example, at 3 and 5 days after implantation, to determine exactly when the retinal response is fully abolished. Additionally, recordings should be taken beyond 7 days, to determine how long the APB is released for. This would be evident as the retinal response would continue to be abolished if APB is continually being released slowly into the vitreous.
In this study, the drug-resin complex was found to be safe for delivery to the retina. APB was dissolved in 0.1% DMSO. At concentrations equal to or higher than 0.6%, DMSO can cause retinal toxicity. However, 0.1% DMSO has been found to be a safe vehicle for the delivery of compounds to the rat eye35, and this concentration was used throughout all experiments in this study. The implants were inserted transclerally into the vitreous cavity at the ora serrata. The implants were observed to remain at the ora serrata in cryosections of the rat eye, which was collected after the ERG recordings were made. This positioning of the implant guarantees that the drug-resin complex does not cause vision impairment. This method involving the drug-resin complex has been tested with other compounds (publication in preparation), and it was found that no structural or functional damage to the eye was observed up to two months after implantation. A total of 60 animals were implanted, and in 90% of cases the implant position remained unchanged.
Several modifications can be made to the technique if necessary. In these experiments, three times the concentration of a single dose of APB was loaded into the resin (50 &#181;M x 3 = 150 &#181;M), and this should be used as a guideline for preparing the drug solution for loading. However, the effective concentration of the drug in the resin is variable, and should be tested and modified accordingly. The time points where analysis is conducted can also be modified, to determine how long the resin is effective at releasing the drug. In these experiments, it was shown that the stable effective dose of the drug is delivered through the drug-resin complex for at least one week, so it is recommended that analysis of the tissue is performed at least one week after the implantation. During the drug loading step, Fast Green was used to ensure that the mixture had homogenized. Other biologically safe color dyes can be used as an alternative to Fast Green, to visualize the uniformity of the drug within the resin. A limitation of this method is that the Fast Green dye is the only measure of uniform mixing. This technique could also be tested using alternative animal models and drugs, with only minor adjustments to the method. If a higher amount of the drug needs to be delivered, a larger size implant may be necessary. If successful in these models, this copolymer resin could be considered as a therapeutic option for delivering drugs slowly to the retina to have a continued effect over time.
There are several critical steps within the protocol where extra care should be taken. Plastic and metal instruments should not be used throughout the synthesis and handling of the resin. This prevents contamination of the polymer, as dichloromethane can dissolve plastic and corrode metal. Proper dryness of the drug-resin block should be achieved before using it for surgical implantation. Ensure that the beaker containing the drug and resin is fully exposed to the calcium sulfate in order to evaporate the solvent. Alternatively, a lyophilizer can be used to freeze dry the drug-resin. The drug-resin block is ready for use when the solvent has completely evaporated, and only a solid bluish block remains in the beaker. When cutting a piece from the block for implantation, each piece should only be prepared immediately before the surgery as the pieces are very small and are difficult to store. A trephine or punch tool should be used to cut pieces of equal size, ensuring that each piece contains approximately the same amount of the drug. Finally, when inserting the piece into the puncture wound made by the 25 G needle, use fine, pointy tweezers to carefully guide the piece into the wound. An operating microscope or high power loupe should be used as a visual aid.
Only hydrophilic drugs and compounds have been tested using this method. The drug-resin complexes have not shown any undesirable precipitation or byproduct formation, when the drug was mixed with the polymer and the dichloromethane. It is suggested that with each new drug, the concentration, half-life of effect, and any possible reactions with the resin mixture should be tested by the user to determine if the method is suitable.

The treatment of chronic diseases such as diabetes and high blood pressure presents many challenges, as these diseases generally require treatment for extended periods of time, often for life. This has called for the development of slow release drug delivery systems, which reduce the need for frequent dosing. The effectiveness of these slow release methods has been demonstrated through the development of insulin pumps, to reduce the number of insulin injections needed to treat diabetes. Chronic diseases of the eye, particularly those affecting its inner layers, require the frequent administering of drugs through invasive procedures. One such chronic disease affecting the human eye is age related macular degeneration (AMD). It affects the central retina, which is a layer of neural tissue situated at the back of the eye responsible for initiating vision. AMD is the leading cause of blindness in the Western World1. A particular challenge with treating retinal diseases is that the drug is required to reach this deep layer in the eye, which often requires invasive methods of delivery. Drugs are usually administered to the vitreous chamber and retina using intravitreal injections. However, with each injection there is a risk of postinjection complications, including endophthalmitis, retinal detachment, cataract and vitreous hemorrhaging2. This risk is multiplied with every reinjection of the drug.
Reducing the need for multiple injections would be a major benefit in the treatment of AMD. To treat the wet form of AMD, where new vessel growth is a hallmark, the established therapeutic strategy is to target the endothelial growth factors (VEGF) using VEGF inhibitors3. At present, these are delivered through repeated intravitreal injections. Similarly, in the treatment of macular oedema, a common complication of diabetic retinopathy, corticosteroids are delivered through repeated injections4. Delivery of these drugs through slow release methods could indeed reduce the risk of postinjection complications.
The notion of a slow release drug delivery system was first described using a silicone rubber vehicle to deliver small molecules into animal tissues5. Since then, other slow release methods have been developed to deliver larger molecules, several of which have been tested in the eye. Particle carriers, such as biodegradable microspheres, poly-lactide-co-glycolide (PLGA) nanoparticles and phospholipid vesicles (liposomes) can be useful as delivery vehicles6,7. PLGA nanoparticles and liposomes have been compared in an in vitro environment for their ability to deliver anti-cancer agents across the sclera over time7. Both vehicles were effective in slowly releasing the drugs. However, the study was only conducted in an in vitro environment. Bochot et al. (2002)8 tested the efficacy of liposomes to deliver molecules to the retina in vivo. They have demonstrated that liposomes successfully deliver small oligonucleotides to the rabbit retina. The authors suggested that liposomes could be beneficial in the treatment of retinal diseases8. However, the nature of these vesicles to float in the vitreous means that they will likely blur or impair vision9.
Okabe et al. (2003)10 used non biodegradable polymer discs made up of 33% ethylene-vinyl acetate to apply beta-methasone in rabbits. They implanted discs into a scleral pocket and demonstrated the effective release of the drug into the vitreous and retina for up to one month10. However, in this particular protocol, the implant was relatively large and rigid, and required a more complex surgical procedure including a large scleral incision and suturing.
An earlier study investigated the tissue response to various polymer vehicles by implanting them into the cornea of rabbit eyes, and found that ethylene-vinyl acetate copolymers washed in alcohol did not cause inflammation or irritation. These complexes were shown to sustain the delivery of larger compounds into animal tissues for extended periods of time, some exceeding up to 100 days dependent on the drug11. One such type of copolymer resin has been developed industrially in the form of Elvax 40W (40% by weight ethylene-vinyl acetate comonomer content with a &#8216;W&#8217; amide additive to improve pellet handling). This copolymer resin is an inert substance which is stable at both room and body temperature. It has not been shown to cause allergies or toxicity in biological tissue. This resin has effectively delivered a large variety of drugs in different experimental models that investigate the functions of various systems, such as the mammary ductal system12, the primary auditory cortex13,14, and the frog visual system15. This resin has also been used in the eye to deliver drugs to developing turtle16,17, chicken embryo18,19, and adult ferret retinas20. In the rat central nervous system, the resin has only been used in the brain21-23, but its use in the rat eye has not been documented.
The advantages of using this copolymer resin to deliver drugs slowly to the retina over other methods, is that it is a stable compound that does not cause inflammation or irritation in the eye. Unlike particle carriers, the drug-resin complex would not impair vision after implantation, as it usually remains at the site of delivery instead of floating in the vitreous. It would only require a simple implantation process into the vitreous cavity close to the limbus of the eye, and would not require suturing after implantation. Recently, there has been an emergence in several novel delivery systems, such as encapsulated cell technology (ECT)24,25, hydrogels26, and microfilms27. However, the method used in the current study for preparing and delivering the drug-resin complex is both easy to follow and inexpensive, thereby being more advantageous for use in a basic research environment. The challenge of using this complex to deliver long-term drug treatments is to determine the optimal concentration of the drug that will maximize the therapeutic benefits of having fewer intravitreal injections.
This paper aims to demonstrate the use of the drug-resin complex for long-term treatment of the adult rat retina. The efficacy of this mode of delivery is tested using the glutamate analogue 2-amino-4-phosphonobutyrate (APB) as the drug. APB blocks the light response of the ON bipolar cells by mimicking glutamate, an endogenous neurotransmitter in the retina28. When APB competes with glutamate for its receptor, it blocks the light response. APB has been used in physiological studies to control retinal function and measure its effect using electrophysiological methods such as electroretinography (ERG). In previous studies, APB has been used for both short-term29 and long-term treatment of the developing retina; the latter involved giving a single dose via intraocular injection daily for 30 days30. The same amount of APB (0.092 mg in sterile saline, at a concentration of 50 &#181;M) was used for all injections as suggested in previous work28,29,31. We chose APB to test the drug-resin complex as a slow-release vehicle to deliver drugs into the eye. The methods outlined in this study are similar to previously described methods involving the preparation of the drug-resin complex16,32; however we also detail its use specifically in the adult rat eye. After surgical implantation of the APB-loaded resin into the eye, ERG was performed to establish whether APB abolishes the retinal light response, and therefore whether APB has been successfully released into the vitreous and retina.

<table><tbody><tr><td>Elvax 40W Pellets</td><td>Du Pont, DE, USA</td><td></td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich Co. LLC., MO, USA</td><td></td><td></td></tr><tr><td>2-amino-4-phosphonobutyric acid (APB)</td><td>Sigma-Aldrich Co. LLC., MO, USA</td><td>A1910</td><td></td></tr><tr><td>Dichloromethane</td><td>Sigma-Aldrich Co. LLC., MO, USA</td><td>34856</td><td></td></tr><tr><td>Fast Green FCF</td><td>Sigma-Aldrich Co. LLC., MO, USA</td><td></td><td></td></tr><tr><td>Drierite, calcium sulfate</td><td>Sigma-Aldrich Co. LLC., MO, USA</td><td>238910</td><td></td></tr><tr><td>Ketamine, Ilium Ketamil</td><td>Troy Laboratories Pty. Ltd., NSW, Australia</td><td></td><td></td></tr><tr><td>Xylazine, Ilium Xylazil-20</td><td>Troy Laboratories Pty. Ltd., NSW, Australia</td><td></td><td></td></tr><tr><td>Atropine sulphate, Minims eye drops</td><td>Bausch &amp;amp; Lomb Pty. Ltd., NSW, Australia</td><td></td><td></td></tr><tr><td>Tetracaine hydrochloride, Minims eye drops</td><td>Bausch &amp;amp; Lomb Pty. Ltd., NSW, Australia</td><td></td><td></td></tr><tr><td>Chloramphenicol, Chlorsig ointment</td><td>Aspen Pharma Pty. Ltd., NSW, Australia</td><td></td><td></td></tr><tr><td>Pentobarbital, Lethabarb</td><td>Virbac Australia Pty. Ltd., NSW, Australia</td><td></td><td></td></tr><tr><td>Lidocaine hydrochloride, Ilium Lignocaine-20</td><td>Troy Laboratories Pty. Ltd., NSW, Australia</td><td></td><td></td></tr><tr><td>Uni-Core Punch Tool</td><td>World Precision Instruments Inc., FL, USA</td><td></td><td></td></tr><tr><td>Curved Forceps</td><td>World Precision Instruments Inc., FL, USA</td><td></td><td></td></tr><tr><td>Operating Microscope, Zeiss OPMI 99</td><td>Zeiss, West Germany</td><td></td><td></td></tr><tr><td>25 G Insulin needle</td><td>Terumo Corp., Tokyo, Japan</td><td></td><td></td></tr><tr><td>Dumont tweezers</td><td>World Precision Instruments Inc., FL, USA</td><td></td><td></td></tr></tbody></table>

slow-release drug delivery through elvax 40w to the rat retina: implications for the treatment of chronic conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

Full-field electroretinography (ERG) was used to detect the effect of APB on the retina. For details on how the ERG was performed, refer to the following studies33,34. In brief, after anesthetizing the animal, a gold electrode was placed on the cornea of the eye, and the reference electrode was placed in the anterior scalp between the eyes, to record the electrical activity of the retina. Stimuli were generated using an electronic flash unit and the intensity ranged from 10 to 106 photoisomerization, which represents the number of photosensitive molecules activated per rod. In these experiments, ERG recordings were taken at 1 and 7 days post procedure. The right eye of each animal was implanted with the drug-resin complex, whereas the fellow left eye was used as a non treated internal control.
Figure 1 shows the changes in the amplitude of the b-wave with an increase in photoisomerization per rod (&#966;). In the control eye, the amplitude of the b-wave increases as the flash intensity increases. In Figure 1A, retinal response recordings are given 1 day after implantation. The implanted eye showed a similar amplitude pattern to the control eye. The amplitude difference between the two eyes became significantly different at 106 &#966; (p &#60;0.0001). This indicates that at this early stage, the drug-resin complex does not have an effect on retinal response until reaching higher flash intensities. Figure 1B&#160;demonstrates changes in the ERG response at 7 days after implantation. The b-wave is abolished at all levels of flash intensity in the implanted eye, when compared to the control eye, which has amplitudes comparable to the 1 day recordings. This is significantly different to all amplitudes for the implanted eye at 1 day (p &#60;0.0001). This shows that the retinal response has been abolished by (one week) after the implantation of the drug-resin complex into the eye.
<img alt="Figure 1" fo:content-width="4in" src="/files/ftp_upload/51563/51563fig1highres.jpg" width="400" /> 
Figure 1.&#160;The amplitude of the b-wave at 1 day and 1 week after the implantation of the drug-resin complex. For both panels, the control eye is shown in orange (squares), whereas the implanted eye is shown in green (circles). The symbol * represents significance at this point between the two eyes, where p &#60;0.0001. For all groups, n = 5. (A) The amplitude of the b-wave 1 day after the implantation of the drug-resin complex into the eye. The amplitude of the b-wave in relation to the flash intensity (photoisomerization per rod &#966;) is an indicator of retinal response. There is no significant difference in the b-wave amplitude between the eyes at all flash intensities except at 106&#966;, where the implanted eye has a lower amplitude. (B) The amplitude of the b-wave 1 week (7 days) after implantation.&#160;The amplitudes recorded from both eyes are significantly different at all flash intensities. In the implanted retina,&#160;amplitude of b-wave was not measurable at any stimulus intensities.

Watch this Scientific Journal Video about Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions at JoVE.com

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

slow-release-drug-delivery-through-elvax-40w-to-rat-retina

Nanyang Technological University

Research

JoVE Journal

Medicine

11.5K Views.  University of L'Aquila. This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

Video: Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

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

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

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

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

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

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

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

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

Neuroscience

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

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

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

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

Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, typically via intravitreal injections. Encapsulated cells expressing the biologic of choice are becoming a tool for local protein production and release (e.g., via long-term drug delivery). In addition, encapsulation systems utilize permeable materials that allow diffusion of nutrients, waste, and therapeutic factors into and out of cells. This occurs while masking the cells from the host immune response, avoiding the need for suppression of the host immune system. This protocol describes the use of alginate as a polymer in microencapsulation coupled with the electrospray method as a microencapsulation technique. ARPE-19 cells, a spontaneously arising human RPE cell line, has been used in long-term cell therapy experiments due to its lifetime functionality, and it is used here for encapsulation and delivery of the capsules to mouse eyes. The manuscript summarizes the steps for cell microencapsulation, quality control, and ocular delivery.

Presented here is a protocol for the use of alginate as a polymer in microencapsulation of immortalized cells for long-term delivery of biologics to rodent eyes.

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, ...

Bioengineering

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 ...

Evaluating Calcium Activity in Retinal Ganglion Cells Layer

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

Author Spotlight: An Innovative Approach to Neural Electrical Stimulation Using Calcium Imaging 

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...

Intravitreal Injections in the Ovine Eye

There are several methods for the delivery of therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or topical administration. IVT drug delivery involves an injection into the vitreous humor of the eye, a gelatinous substance that fills the posterior chamber of the eye and maintains the shape of the eye globe. Although the IVT route is less specifically targeted than subretinal delivery, it is much less invasive and is widely used in clinical settings for a range of ocular diseases. 
We previously demonstrated the efficacy of intravitreal delivery of an adeno-associated virus (AAV)-mediated gene therapy product (AAV9.CLN5) in sheep with a naturally occurring CLN5 form of neuronal ceroid lipofuscinosis (NCL). Affected sheep received IVT gene therapy in one eye, with the other untreated eye serving as an internal control. Retinal structure and function were maintained in the treated eye up to 15 months after treatment, while the untreated eye displayed progressively declining function and severe atrophy during postmortem examination. Based on the sheep studies, the CLN5 gene therapy product was cleared as a candidate investigational new drug (IND) by the United States Food and Drug Administration in September 2021. This paper details the surgical protocol for IVT delivery of a therapeutic viral vector to the ovine eye.

Intravitreal injections were performed in the sheep eye with the aim of delivering viral-mediated gene therapy to the retina.

There are several methods for the delivery of therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or ...

Intraoperative Visualization of Subretinal Injection and Retinal Detachment in Rats

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

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

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

Transconjunctival Approach for Injection into the Rat Optic Nerve

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

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

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

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Summary

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Chapters in this video

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

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

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

Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye

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

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Intravitreal Injections in the Ovine Eye

Intraoperative Visualization of Subretinal Injection and Retinal Detachment in Rats

Transconjunctival Approach for Injection into the Rat Optic Nerve