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To determine the effectiveness of aerosol delivered methotrexate (AD-MTx) in a large-animal (porcine) model of proliferative vitreoretinopathy (PVR).
Prospective, randomized, interventional, double-masked, controlled, large-animal study with pre-determined clinical and histopathologic outcome criteria.
Half of the pigs were randomly assigned to receive an identical volume of aerosolized normal saline using identical delivery systems and treatment intervals.
PVR was surgically induced in 16 pigs (8 male:8 female), randomly assigned to receive two doses (Group A) or three doses (Group B) of either AD-MTx (1.6mg/0.4ml) or normal saline (AD-NS). Group A eyes were euthanized at week 2 (n=8), and Group B eyes were euthanized at week 3 (n=8). Masked clinical PVR score (0-6) from a vitreoretinal surgeon and histopathology PVR scores (0-8) from a masked ophthalmic pathologist determined outcomes.
Main Outcome Measures
The mean, combined clinical and histopathology score (both anterior and posterior) were used to determine the overall treatment effect between groups.
The mean masked score (± Standard Deviation) when combining all grading endpoints (clinical + histopathology), in the AD-MTx group was a mean of 8.0±2.3 versus a higher 9.9±2.0 with AD-NS control group (p=0.05). The clinical scoring in the AD-MTx was 3.88±1.2 versus 4.63±1.6 with AD-NS (p=0.16). The histopathology score for anterior PVR in the AD-MTx group was 2.5±0.8 versus 2.5±0.5 with AD-NS (p=0.50), and posterior PVR in the AD-MTx was 1.63±1.6 vs 2.75±1.3 with AD-NS (p=0.07). Comparing the methotrexate dosing frequency from group A (2 doses) to B (3 doses), the mean score was 8.75 vs 9.13 (p=0.38), suggesting an insignificant difference.
Following surgical induction of PVR in an aggressive, high-risk, large animal model, AD-MTx reduced posterior PVR formation as compared to AD-NS treated controls. Additional dosing at week 3 does not improve outcomes. No difference in anterior PVR formation was noted with intervention. This novel drug delivery system has implications for PVR reduction and warrants further investigation.
There are no effective pharmacotherapies to treat proliferative vitreoretinopathy (PVR). We demonstrated reduction in PVR using aerosolized methotrexate in the pig model. This technology should be studied in clinical trials of retinal detachment to reduce PVR.
Proliferative Vitreoretinopathy (PVR) is the leading cause of recurrent retinal detachment (RD), and occurs in approximately 10% of primary retinal detachment surgeries.
The rate of PVR increases when retinal detachments are associated with ocular trauma, open-globe injury, vitreous blood, intraocular foreign bodies, high myopia, trauma, inflammation, and in primary retinal detachments with large or multiple retinal tears.
Currently, PVR is managed surgically with pars plana vitrectomy, removal of surface membranes and occasionally removing or incising subretinal traction bands. Fibrosis is due to retinal pigment epithelial (RPE) epithelial-mesenchymal transition, glial cell proliferation, and inflammatory mediators.
Intraoperative intravitreal injections into a fluid filled eye may result in subretinal migration of MTx and inhibition of retinal break closure by preventing the desired formation of a chorioretinal adhesion. MTx injected into a silicone oil filled eye results in unpredictable pharmacologic drug distribution. Because MTx is hydrophilic molecule it will concentrate in the fluid phase, between the silicone-retina interface, potentially leading to local toxicity. Also, injecting the intravitreal MTx into silicone oil may lead to sequestration of the fluid component within the oil with unpredictable release kinetics. The hydrophilic MTx injections into a gas-filled eye will concentrate the drug by gravity in a position-dependent location, potentially lead to gravity-dependent, local retinal toxicity.
In 2007, we proposed a novel aerosolized, nanoparticle, intravitreal drug delivery to the retina and presented relevant pharmacokinetics using either a single-fill or flow-through delivery.
Gas-phase, aerosol drug delivery (AD) to the neurosensory retinal surface, combined with a long-acting gas tamponade has several advantages. First, it allows for a predictable and targeted drug distribution of MTx to the entire retinal surface. Second, as opposed to a fluid-filled eye, a gas-filled eye will limit MTx diffusion through open retinal breaks and localize the drug on the retinal surface, where PVR typically forms. Gas bubble surface tension occludes the retinal breaks and access of MTx into the subretinal space, where it would inhibit chorioretinal adhesion formation that is needed to close retinal break(s). And third, residual MTx aerosol phase drug will stratify, in a gradient manner, into the fluid phase and potentially optimize a beneficial, regional, local drug concentration inferiorly (or positionally dependent) where PVR is more common without compromising retinal drug delivery to the remainder of the retinal surface. Therefore, using this technique, we anticipate a lower risk of local retinal drug toxicity and a more predictable drug delivery to the entire retinal surface.
In this study, we examined the combination of MTx, an antiproliferative agent, delivered using an AD system in a large animal, porcine model of PVR. The advantage of the porcine model is that standard, human vitrectomy instrumentation can be employed. Importantly, the gas-phase of drug allows for tamponade of the neurosensory retina with a long-acting perfluorocarbon gas to seal retinal breaks. Thus, this laboratory scenario closely resembles surgical care that is common practice in current vitreoretinal surgical reattachment procedures as well as re-creates a relevant environment to study PVR formation and inhibition.
This study involved animal subjects and all animals were cared for according to both the ARVO Statement for the Use of Laboratory Animals in Ophthalmic and Vision Research. The Mayo Clinic Institutional Animal Care and Use Committee (IACUC) approved the study design (#A00005162-20) and ethical use of animals. The study design was a prospective, randomized (block), double masked, and controlled study.
Sixteen pigs (Sus scrofa), eight females and eight males, weighing between 25-35 kg were divided equally into 2 groups: Aerosol delivery (AD) of Methotrexate (AD-MTx) or AD of normal saline (AD-NS). Eight eyes were initially treated (four per group) during the first half of the study. A two-disc diameter (2-DD) retinal tear was created to specifically cross the border of the posterior vitreous detachment interface, thus increasing the likelihood to induce PVR and RD. The eight eyes in the second half of the study had a one-disc diameter (1-DD) tear to induce PVR. We reduced the tear size (2-DD to 1-DD) based on the observed PVR severity from the first eight eyes during the first half of the study. The smaller retinal tear was intended to reduce PVR severity. There was an equal ratio of male:female pigs in the AD-MTx and the AD-NS in both groups. Also, there was equal ratio of male:female in the 1-DD and the 2-DD PVR induction groups.
Surgical Preparation: Animals were fasted, sedated and induced for anesthesia using telazole (5 mg/kg) and xylazine (2 mg/kg), both via intramuscular injection. Once sedated, only the right eye was operated, prepared with topical proparacaine (1-2 drops), atropine 1% (x 3 every 5 minutes) and phenylephrine 2.5% (x3 every 5 minutes). An intravenous line delivered lactated ringers. Animals were intubated and general anesthesia maintained using inhalational 1.5-3.0% isofluorane vaporized in oxygen. Body temperature was maintained using warming blankets and monitored using a rectal temperature probe. The animal was positioned on the left side (left eye protected). The right eye was prepared by trimming the eyelashes and applying topical betadine 5% solution with sterile cotton tip applicators to remove eyelid debris, clean the fornices, and periorbital skin. The surface was dried with sterile 4x4 gauze sponge. A sterile drape was used to cover the eye and a lid speculum was inserted to expose the ocular surface.
Surgical Procedure: A lateral cantholysis was performed after clamping the tissue with a forceps to allow for vitreoretinal surgical access. A localized conjunctival peritomy was performed to facilitate 1) the infusion line, 2) an illuminated vitreoretinal pick, and 3) the 23-gauge, high-speed vitreous cutter. Once the infusion line was inserted through the pars plana (2 mm posterior to the surgical limbus), the tip was visually confirmed prior to starting the intraocular infusion of balanced salt solution (BSS, Alcon Laboratories, Ft. Worth TX). A pars plana lensectomy was performed, removing most of the lenticular capsule, and creating a unicameral eye. The posterior hyaloid was elevated using a combination of a soft-tip aspiration cannula and an illuminated pick. Once the hyaloid was elevated (visually confirmed) and detached nearly to the equator of the eye where a visible line was observed as the posterior vitreous face separated from the neurosensory retina (Figure 1). A sub-total pars plana vitrectomy was performed with the vitreous cutter. Since we separated the posterior hyaloidal face to the mid-equatorial region, the PVD line was an important landmark used as a bisection site for the retinal tear, and this helped to ensure PVR and RD development (100%) by creating fibrosis (tissue reaction) at the defined vitreoretinal interface border.
Either a 2-DD (animals 1-8) or a 1-DD (animals 9-16) full-thickness retinal break near the equator (Figure 2A and 2B) was created using the illuminated pick, bisecting the posterior hyaloid separation interface, and also purposefully including the rupture of a small retinal blood vessel, leading to a small, localized vitreous hemorrhage (Figure 2A and 2B). The extent of the hemorrhage was recorded and modified for consistency using intraocular pressure tamponade. A soft-tip cannula was inserted through the full-thickness retinal break and retinal pigment epithelial (RPE) cells were gently aspirated into a 3-cc syringe primed with BSS. An air-fluid exchange was performed for a complete air-fill. Ten minutes were allowed for dehydration of the vitreous base and all residual fluid was aspirated. The 3-cc syringe containing the solution of aspirated RPE cells along with retinal elements (presumably containing glial elements) was then reinjected into the vitreous space (approximately 0.2 cc of solution: Figure 3).
Aerosolized Methotrexate delivery (AD-MTx): The surgeon was masked regarding the drug or control solution selected at this point in the surgery (after PVR induction) and animals were randomly assigned a treatment (author CGA). Once assigned, masking was no longer applicable and either AD-NS (clear solution) or AD-MTx (yellow solution) was applied in an umasked fashion. In the MTx eyes, MTx solution containing 1.6mg/0.4ml was applied to the retinal surface using an aerosolized spray device and delivered across the entire retinal surface. Delivery time was approximately 1 minute. Note that the particle size was approximately 10 micrometers (phase doppler particle analyzer, TSI, Inc., Shoreview, MN), and are affected by gravity so we would anticipate a gravity-dependent drug gradient. Next, the intraocular air was exchanged using a fluid-air-gas exchange technique with a total exchange volume of 50 ml of 15% perfluropropane (C3F8) gas passing through the eye to leave a final, intraocular concentration of 15% C3F8 gas. Sclerotomies were closed using 7-0 mattress style vicryl suture and the conjunctiva closed using interrupted 6-0 plain gut suture. A retrobulbar block containing 1 ml of 0.75% bupivacaine, 25 mg. of ceftazidime, and 40 mg of triamcinolone
was given at the conclusion of the case into the subtenon space using a blunt cannuale. The peribulbar block provided post-operative anesthesia, a corticosteroid, and an antibiotic. Topical atropine drops and antibiotic ointment were applied. Animals were observed until they were able to stand, eat, and drink. Post-operative pain control (if needed) was managed with buprenorphine (0.12 mg/kg) delivered with subcutaneous injections and as directed by the veterinary staff. The surgical procedures were well tolerated, and the need to supplement the post-operative pain medication was uncommon.
Group A (2 doses, 2 week-survival): At one-week post-operative, group A eyes were re-induced (as outlined above), sedated, but not intubated. The eyes were examined, and intraocular pressure measured. Next, a fluid-air-gas exchange was performed to allow the AD-MTx to be delivered to an eye filled with air. The technique was as follows: 1) animals were positioned with the right cornea in a dependent position, 2) a 30-gauge needle on a tuberculin syringe with the plunger removed was inserted trans-corneal, into the vitreous chamber to drain collected fluid, 3) a second 30-gauge needle on a 50-ml syringe (sterile air) was inserted trans-corneal to maintain intraocular pressure during the drainage of intraocular fluid. Once the fluid was drained, the same prior assignment of either AD-MTx (n=4 animals) or AD-NS (n=4 animals) was sprayed diffusely across the retinal surface. Once the animal was randomized, they remained in the same treatment group (MTx or NS), and the designated agent was re-applied for each drug delivery interval. To facilitate the 23-gauge cannula of AD delivery, a 23-gauge MVR blade was inserted tangentially through the superior cornea, creating a beveled, self-sealing corneal incision. Since the eyes were all aphakic, trans-corneal delivery to the vitreous provided sufficient access to re-treat the entire neurosensory retina. The angle of AD spray was intended to cover the entire retina. Next, the fluid-air-gas exchange technique with 15% perfluropropane (C3F8) gas was performed (as described above). At post-operative week-2, animals were sedated (as above) and euthanized (pentobarbital-fatal dose). The right eye of each animal was enucleated and fixed in 10% formalin.
Group B (3 doses, 3 week-survival): Animals in group B were treated as outlined above for Group A. However, at post-operative week-2, the animals were treated with a third dose of either AD-NS or AD-MTx, respectively (n=4 per group) according to pre-assigned treatment. Then, at post-operative week 3, the animals were euthanized and right eyes enucleated (as above).
Enucleated eyes were transported in 10% buffered neutral formalin and fixed for a minimum of 48 hours. The globes were sectioned horizontally using a 3-9 o’ clock orientation (pupil-optic nerve), exposing both anterior and posterior portions of the globe. Gross photographs documented the representative retinal appearance (Figure 4). A calotte from each eye was submitted for histopathological preparation. Calottes were processed by dehydration in an ascending series of ethanol (70%- 100%), cleared with xylene and infiltrated in paraffin under vacuum with agitation. The calottes were then embedded in paraffin and 10 sections, 5 μm thick, were cut from each paraffin block and mounted on Superfrost Plus, charged microscope slides (Fisher Scientific, Waltham, MA). Slides were dried at 60 degrees for 30 minutes, cooled and then stained. Nine sections were stained with Mayer’s hematoxylin (Sigma-Aldrich, St. Louis, MO) followed by Eosin/Phloxine solution (Richard-Allan Scientific, Kalamazoo, MI) and one section was stained with periodic acid Schiff (PAS) stain. The sections were then prepared using mounting media (Permount, Thermo Fisher Scientific, Fair Lawn, NJ) and a coverslip was placed to cover the tissue section.
All grading analysis was performed by masked graders (CGA masked the images and TWO graded the images without knowledge of the treatment). Gross sections were analyzed first, following sectioning of the enucleation globes. This allowed for a clear, unobstructed view of the retinal tissues. In our porcine model, the cornea uniformly became opaque due to 1) the unicameral status of the eye, 2) the predominant gas-phase during the study, 3) modestly elevated intraocular pressures, 4) using the trans-corneal route for delivering drug, and 5) potentially toxicity from AD-MTx. The mechanism of elevated intraocular pressures in most eyes was likely due to anterior rotation of the iris, pupillary block and partial angle closure. The subsequent corneal opacification limited an adequate view to assess the in-vivo RD or PVR status of the neurosensory retina. Post-enucleation, globes were sectioned in an anterior-posterior manner, allowing a clear view of the posterior segment for the masked examiner to provide an accurate clinical assessment. A masked histopathology analysis (TWO masked the images and DRS graded the images without knowledge of the treatment). was performed using separate and combined scores from light microscopic analysis of both the anterior and posterior PVR. All grading was performed in a masked manner as the pathologist was not aware of assigned treatment. Grading criteria are outlined in Table 1.
Table 1Clinical and Histopathology Grading of Enucleated Globes
We used a technique for one-sided p-value analysis that compares the groups with the study powered to have an 80% chance of detecting a 2.5-point difference (predetermined by estimate from prior experience in the pig model) in total PVR score (scale = 0-14) assuming a standard deviation (SD) of ±1.8 in each study group at a 5% alpha (type 1 error) or 95% confidence level (minimum sample size n= 8 per group). We assume that the total score represents a continuous variable with an assumption of a normal distribution. We chose a simple, one-sided t-test analysis with a ‘success-failure’ outcome of our desired goal (to assess if our intervention reduced PVR formation) based on our power calculations to determined animal numbers and adhere to the ethical reduction in numbers of animals required for experimental therapies.
We detected a statistically significant reduction in overall PVR formation in the AD-NS control group total score (9.88±2.0) as compared to the AD-MTx group total score (8.00±2.3; p=0.05; Table 2). Overall, we found moderate to severe PVR in all animals tested with total PVR scores ranging from a low of four to a maximum of 14. Since total PVR scores in the first eight animals ranged from seven to 13 (mean 9.75±3.8), we reduced overall PVR severity in the next group by decreasing the retinal tear size from 2-DD to 1-DD. In the second eight animals, the total PVR score trended less, ranging from four to 12 (mean 8.13±4.8; p=0.08).
Table 2Clinical and Pathology Analysis of Proliferative Vitreoretinopathy (PVR) Scores
None of the individual PVR subgroup analysis scores were statistically significant, while both clinical and posterior pathology score demonstrated trends toward significance. The anterior pathology scores were nearly identical (Table 2). A comparison of group A (2 doses) versus group B (3 doses) was not significantly different. The total PVR score in group A was 9.13±1.7 versus group B at 8.75±2.87; p=0.38. Data analysis was performed using SAS (version 9.4; SAS Institute Inc., Cary, North Carolina).
Overall, the animals tolerated the surgical procedure and there were no severe adverse events or systemic complications. Animals recovered rapidly and ate without observable behavior changes, gained weight throughout the study, and none required any unexpected post operative pain management intervention. All animals developed corneal edema that precluded a meaningful poster segment evaluation. Several animals (n=3 of 16) had a small or limited hyphema. Intraocular pressures (IOPs) were uniformly elevated at the first week post-PVR induction with IOPs ranging from 15 to 38 mm of Hg as tested using applanation with a mean pressure of 27 mm of Hg. However, by post-PVR induction week two, the IOPs ranged from 5 to 25 mm of Hg with a mean pressure of 14 mm of Hg. In the eight animals that were maintained through post-PVR induction week three, the IOPs ranged from 5 to 15 mm of Hg with a mean of 11 mm of Hg. This trend likely represents the influences of intraocular gas tamponade, reduction in pupillary block, and also by progressive RD development with the associated reduction in IOP.
Representative clinical (Figure 4A-D), anterior histopathology (Figure 5) and posterior histopathology (Figure 6A-D) represents the variations in the outcome assessment for this large animal model PVR.
The pig model is an aggressive and highly reactive model to study drug delivery and surgical methodology relevant to PVR development and prevention. In the pig model, we have replicated a human disease process by inducing an iatrogenic retinal tear near a vitreous attachment site, thus simulating the posterior vitreous base insertion origin common in human retinal tear formation. We also purposefully included a small retinal blood vessel to simulate small retinal hemorrhages that occur in human retinal tears. And, we aspirated and re-injected both RPE and small glial cells from the neurosensory retina. Thus, we’ve created a large animal model, with high-risk for PVR formation, in an eye that has a size similarity to human eyes. This represents a viable, pre-clinical testing environment to study novel therapeutic technologies.
Current vitreoretinal surgical instrumentation is easily adapted to the pig eye. Other advantages are based on similarities with human globe size, scleral thickness,
vitreous structure, retinal vascular pattern (holangiotic), both the RPE cell and glial cell mediated healing response, choroidal blood flow, and lens structure. Other authors have employed other methods to induce PVR in the pig model,
yet none have been able to generate a consistent tissue response. The methodology presented in this study allows for an aggressive, reproducible (100%) generation of moderate (1 disc diameter retinal tear) to severe PVR (2-disc diameter retinal tear) that is measured (graded) in a masked fashion and thus reduces bias, both clinically and with histopathologic analysis.
By applying the aerosolized methotrexate, we have demonstrated a reduction of PVR formation in our large animal model. While this response seems modest in this animal model, the implications for humans may differ. This study has implications for translational progression of this technology from the pig to human studies and forms a solid scientific basis for moving this technology from the lab into the clinical arena. By using gas-tamponade in the study design, we mirror current clinical standards of care that are used to manage eyes at risk for PVR (gas-phase tamponade). Logically, if a surgeon used a single AD-MTx application, there could potentially also be a silicone oil filled eye for tamponade. Such a study arm was not included in this study, yet would represent a common clinical scenario that should be studied. We have shown that AD drug delivery methodology has known and reproducible in-vivo tissue delivery kinetics.
and effectively inhibits PVR formation. However, a predictable pharmacokinetic delivery using intravitreal injections of MTx into fluid filled eyes does not match the clinical scenario for patient care. Eyes at risk for PVR formation require an intravitreal tamponade, most commonly using perfluoro-octane (C3F8), sulfur hexafluoride (SF6), or silicone oil. Intravitreal fluid injections directly given by using a syringe and needle into a tamponade agent result in unpredictable drug distribution to the neurosensory retina due to gravity-dependent fluid dynamics and drug partitioning from silicone oil. For example, some areas of fluid concentration could deliver toxic doses where injected fluid is concentrated, while other areas would go un-treated with little or no drug delivery. A key advantage of gas-phase (AD) is that drug delivery is more evenly, reliably, and predictably distributed. Gas-phase AD could be performed at the time of vitreoretinal surgery for retinal detachment and potentially supplemented in the clinic with air-fluid, reapplication of AD-MTx. Intravitreal injections are much better tolerated and managed in current practice than in the past, due to the current anti-vascular endothelial growth factor and steroid implant pars plana injections delivered in current practice. Furthermore, modifying current in-office intravitreal injections would require a practice adjustment to accommodate gas-fluid exchange procedures. If silicone oil is used as a tamponade, AD-MTx could only be re-applied when the Silicone oil is removed, thus limiting the frequency of delivery. The clinical goal of intravitreal, AD-MTx is to reduce or eliminate PVR formation in the early stages (prevention). Also, a theoretic advantage of AD-MTx, is that the gas tamponade secludes drug from entering the subretinal space, and this avoids inhibition of a desired healing (chorioretinal adhesion) of the retinal break(s). Prior studies suggest that nanoparticle drug may well penetrate the neurosensory retina and could also inhibit the chorioretinal adhesion.
Thus, we believe that AD-MTx has many advantages for reducing PVR, especially in high-risk surgical cases of primary retinal detachment. To the best of our knowledge, a direct comparison of intravitreal versus aerosol delivery has not been conducted, yet such a study would be of high value.
High-risk, primary retinal detachment cases may represent an ideal population to apply AD-MTx in a human clinical trial. Such cases include retinal detachments with large or multiple clock-hour retinal tear(s), vitreous blood, or a history of PVR. While all animals in our study received either two or three doses of AD-MTx, and we did not detect a significant difference between these dosing groups, drug dosing would need to be carefully evaluated in preparation for human studies. After further, more expanded pre-clinical studies, a dose escalation study may be the most appropriate method to effectively introduce this technology into the clinical arena.
The multiple variables in our model of PVR include: 1) the length of the retinal tear (one- or two-disc diameters in this study), 2) amount of vitreous hemorrhage from the torn retinal blood vessel, 3) aspiration of RPE cells and elements of torn retina that are then re-injected to simulate RPE cell dispersion and glial healing responses, 4) dosing strategies, and 5) length of follow-up. Our PVR model is very aggressive with a goal to determine a clinically relevant, measurable, and meaningful reduction in PVR formation. We did not detect significant differences between two or three separate AD-MTx dosing intervals. We conclude that most of the PVR response in the pig occurs early in the post-PVR induction period (1-2 weeks). The healing response phase may be longer in human PVR development, and has been noted to peak from six to twelve weeks post-operative.
In summary, we present compelling evidence for a novel therapeutic technology and drug delivery methodology to reduce or prevent PVR formation. Our conclusion is based on data from a large-animal, double-masked, placebo controlled pre-clinical trial. Further refinement of dosing, dosing intervals, and a safety study are needed. The AD-MTx may reduce or help prevent PVR formation in humans when used early in the formative stages of proliferation. Data presented herein forms the basis for consideration of future human clinical trials using aerosolized methotrexate to reduce proliferative vitreoretinopathy, the leading cause of recurrent retinal detachment.
Clinical risk factors for proliferative vitreoretinopathy after retinal detachment surgery.
Financial interest conflict disclosure: This work was supported by NIH/NEI EY030819 (Naqwi is study PI, Olsen is research site-PI). All studies were carried out at Mayo Clinic, Rochester, MN. Amir Naqwi is owner of Abbe Vision Inc. and this manufactures the aerosol generation system. Timothy W. Olsen is an equity owner, Kathy Wabner and Jenn Schmit are part-time employees of iMacular Regeneration, LLC. This entity has no technology described or included in this manuscript. Collin Asheim is a medical student, participating in research. Portions of this work were presented at the University of North Dakota Frank Low Research Day (April 21, 2022).
Ethics Statement: The Mayo Clinic Institutional Animal Care and Use Committee approved the ethical use of animals for this study (#A00005162-20; Mayo Clinic IAUCUC, Rochester, Mn).
We demonstrate a reduction in proliferative vitreoretinopathy in a prospective, double-masked, randomized, large-animal surgical model using intravitreal, aerosolized methotrexate in an animal aggressive model to study experimental proliferative vitreoretinopathy with histopathologic validation.