PURPOSE:Microcystoid macular degeneration (MMD) is a condition where cystoid vacuoles develop within the inner nuclear layer of the retina in humans in a variety of disorders. Here we report the occurrence of MMD in non-human primates (NHPs) with various retinal ganglion cell (RGC) pathologies and evaluate the hypothesis that MMD does not precede RGC loss but follows it. METHODS:Morphological studies were performed of the retinas of NHPs, specifically both rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis), in which MMD was identified after induction of experimental glaucoma (EG), hemiretinal endodiathermy axotomy (HEA), and spontaneous idiopathic bilateral optic atrophy. In vivo imaging analyses included fundus photography, fluorescein angiography (FA), optical coherence tomography (OCT), adaptive optics scanning laser ophthalmoscopy (AOSLO), light microscopy, and electron microscopy. RESULTS:MMD, like that seen on OCT scans of humans, was found in both rhesus and cynomolgus macaques with EG. Of 13 cynomolgus macaques with chronic EG imaged once with OCT six of 13 animals were noted to have MMD. MMD was also evident in a cynomolgus macaque with bilateral optic atrophy. Following HEA, MMD did not develop until at least 2 weeks following the RNFL loss. CONCLUSION:These data suggest that MMD may be caused by a retrograde trans-synaptic process related to RGC loss. MMD is not associated with inflammation, nor would it be an independent indicator of drug toxicity per se in pre-clinical regulatory studies. Because of its inconsistent appearance and late development, MMD has limited use as a clinical biomarker.

OBJECTIVES To evaluate the effect of QD or BID 0.02% netarsudil ophthalmic solution (Aerie Pharmaceuticals) on intraocular pressure (IOP) in normotensive dogs and to describe any adverse effects. ANIMALS STUDIED Normotensive Labrador retriever dogs were included in this study: 10 received netarsudil in one eye and artificial tears in the contralateral eye QD, and 10 received netarsudil in one eye and artificial tears in the contralateral eye BID. PROCEDURES Intraocular pressure curves were acquired over a 3-day acclimation period, 5-day dosing period (QD or BID-10 dogs/group), and 3-day recovery period. Toxicity was assessed daily using slit-lamp biomicroscopy and the semiquantitative preclinical ocular toxicology scoring system. RESULTS Once-daily dosing did not lower IOP over the entire 5-day dosing period (95% CI 0.1 to -0.9 mm Hg, P = .20) or on the last day of dosing (95% CI 0.4 to -0.9 mm Hg, P = .65). Twice-daily dosing resulted in a statistically significant, but clinically unimportant, IOP reduction over the entire 5-day dosing period (-0.6 mm Hg; 95% CI 0.05 to -1.1 mm Hg, P = .02) and on the last day of dosing (-0.9 mm Hg; 95% CI 0.2 to -1.5 mm Hg, P = .003). Adverse events were limited to transient mild-to-moderate conjunctival hyperemia during the dosing phase in eyes receiving netarsudil vs control (P < .0001). CONCLUSIONS Netarsudil 0.02% ophthalmic solution twice daily resulted in a small, statistically significant, but clinically unimportant, IOP reduction in normotensive dogs. Future studies should investigate efficacy in glaucomatous dogs.

OBJECTIVE To compare intraocular pressures (IOPs) estimated by rebound and applanation tonometry for dogs with lens instability. ANIMALS 66 dogs. PROCEDURES Medical records of dogs examined between September 2012 and July 2018 were reviewed for diagnoses of anterior (ALL) or posterior (PLL) lens luxation or lens subluxation. RESULTS Estimates of IOP obtained with rebound and applanation tonometry significantly differed from each other for all types of lens instability considered collectively (mean ± SE difference between tonometric readings, 8.1 ± 1.3 mm Hg) and specific types of lens instability considered individually (mean ± SE difference between tonometric readings: ALL, 12.8 ± 2.5 mm Hg; PLL, 5.9 ± 1.7 mm Hg; subluxation, 2.8 ± 0.8 mm Hg). Median (range) differences between rebound and applanation tonometer readings for dogs with ALL was 5 mm Hg (-9 to 76 mm Hg), with PLL was 3 mm Hg (-1 to 19 mm Hg), and with lens subluxation was 3 mm Hg (-9 to 18 mm Hg). In eyes with ALL, rebound tonometer readings exceeded applanation tonometer readings on 44 of 60 (73%) occasions. CONCLUSIONS AND CLINICAL RELEVANCE Rebound tonometry yielded higher estimates of IOP than did applanation tonometry in eyes with ALL and with all types of lens luxation considered collectively. Estimates of IOP in eyes with lens instability should ideally be obtained with both rebound and applanation tonometers. Veterinarians with only one type of tonometer should interpret results for dogs with lens instability concurrent with physical examination findings.

PRIMARY OBJECTIVE:To evaluate the effect of latex tip cover manufacturer on accuracy and repeatability of Tono-Pen Vet™ in canine eyes.ANIMAL STUDIED:Twelve enucleated globes from six dogs.PROCEDURES:The anterior chamber was cannulated and connected to a calibrated manometer. Intraocular pressure (IOP) measurements were obtained using the Tono-Pen Vet and TONOVET Plus at manometric IOP ranging from 5 to 80 mmHg. At each IOP, the Tono-Pen Vet was used with a new Ocu-Film™ latex tip cover (the only manufacturer-approved brand of cover) followed by a new Softips™ latex tip cover. For comparison, the TONOVET Plus was also used at each IOP with a new disposable rebound probe. Measured IOP values were analyzed by linear regression and intraclass correlation coefficient (ICC).RESULTS:Tono-Pen Vet accuracy was unaffected by tip cover manufacturer or by frequent change in cover. Using ICC analysis, repeatability of measurements using either tonometer was good to excellent at physiologic IOP levels but variably decreased with both devices at supraphysiologic IOP.CONCLUSIONS:Neither tip cover manufacturer nor frequent changes in tip cover adversely affect Tono-Pen Vet accuracy. Measurement repeatability with Tono-Pen Vet and TONOVET Plus is widely variable at supraphysiologic IOP. Therefore, minor changes in IOP >25 mmHg should not be used to make clinical decisions without considering this variability.

Canine glaucoma is a group of disorders that are generally associated with increased intraocular pressure (IOP) resulting in a characteristic optic neuropathy. Glaucoma is a leading cause of irreversible vision loss in dogs and may be either primary or secondary. Despite the growing spectrum of medical and surgical therapies, there is no cure, and many affected dogs go blind. Often eyes are enucleated because of painfully high, uncontrollable IOP. While progressive vision loss due to primary glaucoma is considered preventable in some humans, this is mostly not true for dogs. There is an urgent need for more effective, affordable treatment options. Because newly developed glaucoma medications are emerging at a very slow rate and may not be effective in dogs, work toward improving surgical options may be the most rewarding approach in the near term. This Viewpoint Article summarizes the discussions and recommended research strategies of both a Think Tank and a Consortium focused on the development of more effective therapies for canine glaucoma; both were organized and funded by the American College of Veterinary Ophthalmologists Vision for Animals Foundation (ACVO-VAF). The recommendations consist of (a) better understanding of disease mechanisms, (b) early glaucoma diagnosis and disease staging, (c) optimization of IOP-lowering medical treatment, (d) new surgical therapies to control IOP, and (e) novel treatment strategies, such as gene and stem cell therapies, neuroprotection, and neuroregeneration. In order to address these needs, increases in research funding specifically focused on canine glaucoma are necessary.

Objective The purpose of this study was to compare the leakage rates of perilimbal uniplanar and biplanar clear corneal incisions in dogs when subjected to increased intraocular pressure (IOP) both from within the eye and via external pressure. Procedure Uniplanar clear corneal incisions were created in eight freshly enucleated canine eyes using a 3.2 mm straight slit knife while 8 fellow eyes received a biplanar clear corneal incision consisting of an approximately 300 mu m deep groove followed by a 3.2 mm straight slit knife entry into the anterior chamber. Both wounds were reapposed using three simple interrupted 8-0 polyglactin 910 sutures. Eyes were cannulated with two 25 g needles: One connected to a pressure transducer, and the other connected to a reservoir of isotonic saline. The IOP at which the wound leaked was recorded when the intraocular pressure was increased internally by raising the height of the fluid bag, and again when the cornea was externally compressed. Kaplan-Meier survival curves compared incision types for each method of increasing IOP and were evaluated using Mantel-Cox log-rank analysis. Results Both wound types resisted leakage at IOP in the physiologically achievable range and no significant differences were observed between clear corneal incisions when pressure was applied externally (P = .353) or was increased from within the globe (P = .615). Conclusion Ex vivo uniplanar and biplanar clear corneal incisions in dogs are equally strong, with no significant differences in leakage rates when IOP is increased internally or externally.

PurposeInvestigate a significant, dose-related increase in IOP, leading to glaucomatous damage to the neuroretina and optic nerve following intravitreal (ITV) administration of a bispecific F(ab')2 [anti-VEGF/Angiopoietins [ANGPT]F(ab')2] molecule in adult monkeys.MethodsITV ocular tolerability and investigation of anti-VEGF/ANGPT F(ab')2 (blocking both ANGPT1 and ANGPT2) was done in monkeys; mechanistic studies were done in neonatal mice.ResultsFollowing the second ITV dose of anti-VEGF/ANGPT F(ab')2, all 1.5- and 4-mg/eye treated monkeys developed elevated IOP, which eventually was associated with optic disc cupping and thinning of the neuroretinal rim. Histopathologic examination showed nonreversible axonal degeneration in the optic nerves of animals administered 1.5 mg/eye and higher that was considered secondary to high IOP. Anti-ANGPT Fab also caused elevated IOP in monkeys, but anti-VEGF Fab did not contribute to the IOP increase. In addition, an anti-ANGPT2-selective antibody did not change IOP. In mice simultaneous blockade of ANGPT1 and ANGPT2 impaired the expansion and formation of Schlemm's canal (SC) vessels, similar to genetic ablation of Angpt1/Angpt2 and their receptor TIE2. As previously reported, blocking ANGPT2 alone did not affect SC formation in mice.ConclusionsDual inhibition of ANGPT1/ANGPT2, but not ANGPT2 alone, leads to increased IOP and glaucomatous damage in monkeys. This confirms a role for TIE2/ANGPT signaling in the control of IOP in adults, a finding initially identified in transgenic mice. Dual pharmacologic inhibition of ANGPT1/ANGPT2 may affect aqueous drainage and homeostasis in adult monkeys and may be useful in developing novel models of glaucoma.

OBJECTIVE:To determine the effect of a bimatoprost sustained-release intracameral implant (Bimatoprost SR) on episcleral venous pressure (EVP) in normal dogs.METHODS:Normotensive beagle dogs were randomized to receive Bimatoprost SR 30 μg (n = 7) or sham injection (needle insertion only, n = 7) in one eye on day 1. EVP was measured with an episcleral venomanometer through day 65. Episcleral aqueous outflow vessels were identified using fluorescence imaging following intracameral injection of indocyanine green in one additional animal. A separate cohort of dogs that had been trained for conscious intraocular pressure (IOP) measurements received Bimatoprost SR 30 μg (n = 8) in one eye; IOP was evaluated through day 66.RESULTS:Baseline mean EVP was 10.0 mmHg in the Bimatoprost SR group and 10.4 mmHg in the sham group. Eyes treated with Bimatoprost SR exhibited a transient increase in mean EVP that peaked at day 8, followed by a decrease to levels below baseline. From day 29 to day 65, the change in mean EVP from baseline ranged from -2.4 to -3.9 mmHg (P < 0.05 vs. sham). Baseline mean IOP in eyes treated with Bimatoprost SR was 14.9 mmHg, and a steady IOP reduction was maintained through day 66. Bimatoprost SR-treated eyes exhibited a selective, sustained dilation of aqueous outflow vessels that was not observed in sham-treated eyes.CONCLUSIONS:In normal dogs, Bimatoprost SR was associated with a transient increase in EVP followed by a sustained decrease. Changes in EVP were accompanied by a sustained dilation of aqueous outflow vessels.

Ocular diseases reported in frogs include uveitis and glaucoma, which are associated with changes in intraocular pressure (IOP). The objectives of this study were to characterize the normal IOP for White's tree frogs ( Litoria caerulea ) using two types of rebound tonometers, and to assess whether time of day or method of restraint affected IOP. Eighteen conscious, unrestrained, ophthalmologically normal frogs were used to measure IOP using TonoVet® and TonoLab® tonometers, at three time points during the day. In a subset of 12 frogs, IOP was measured while under manual restraint using the TonoVet. Anesthesia was induced in 9 frogs using two different concentrations of MS-222 (0.5 g/L and 2 g/L) in order to evaluate for changes in IOP with the TonoVet. Mean (± SD) IOP values for the TonoLab (16.8 ± 3.9 mm Hg) were significantly higher than TonoVet values (14.7 ± 1.6 mm Hg; P < 0.01). TonoVet IOP values did not significantly change with time of day. TonoLab values were significantly lower in the evening (1600-1800; 14.5 ± 3.1 mm Hg), compared with morning and midday measurements (0800-1000 and 1200-1400; 18.0 ± 3.8 mm Hg; P < 0.01). Manually restrained frogs had significantly lower IOP (13.4 ± 1.5 mm Hg) compared with unrestrained frogs (15.3 ± 1.2 mm Hg; P < 0.01). Chemical restraint did not cause significant changes in IOP. Intraocular pressure can be measured with both types of rebound tonometers in White's tree frogs, but time of day and manual restraint can affect IOP values.

OBJECTIVE:The pig has an increasingly important role in ocular drug delivery models, but the most accurate tonometer in this species is unknown. The purpose of this study was to evaluate the accuracy of TonoVet and Tono-Pen Vet tonometers in the ex vivo porcine eye.PROCEDURE:Four freshly enucleated normal porcine eyes were cannulated with two 25-gauge needles; one connected via tubing to a mercury manometer calibrated continuous physiologic recorder and the other connected to a reservoir of lactated Ringer's solution on an adjustable stand. Triplicate IOP readings were taken with the TonoVet and then the Tono-Pen Vet at 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80 mmHg.RESULTS:Linear regression showed strong linear trends for both the TonoVet (r2 = 0.969) and Tono-Pen Vet (r2 = 0.983). The TonoVet slightly underestimated IOP at lower pressures and slightly overestimated IOP at higher pressures (y = 1.092x - 4.0, where y = tonometer reading, x = manometer reading, and 4.0 = intercept). The Tono-Pen Vet consistently underestimated IOP (y = 0.773x - 2.1). These differences were statistically significant (P = <0.001, one-way repeated-measures ANOVA).CONCLUSION:As in other species, both the TonoVet and Tono-Pen Vet tonometers do not measure true IOP in the porcine eye; however, the TonoVet more closely approximated true IOP in the normal porcine eye than the Tono-Pen Vet and may be the tonometer of choice for this species.

Laser-induced experimental glaucoma (ExGl) in non-human primates (NHPs) is a common animal model for ocular drug development. While many features of human hypertensive glaucoma are replicated in this model, structural and functional changes in the unlasered portions of trabecular meshwork (TM) of laser-treated primate eyes are understudied. We studied NHPs with ExGl of several years duration. As expected, ExGl eyes exhibited selective reductions of the retinal nerve fiber layer that correlate with electrophysiologic measures documenting a link between morphologic and elctrophysiologic endpoints. Softening of unlasered TM in ExGl eyes compared to untreated controls was observed. The degree of TM softening was consistent, regardless of pre-mortem clinical findings including severity of IOP elevation, retinal nerve fiber layer thinning, or electrodiagnostic findings. Importantly, this softening is contrary to TM stiffening reported in glaucomatous human eyes. Furthermore, microscopic analysis of unlasered TM from eyes with ExGl demonstrated TM thinning with collapse of Schlemm's canal; and proteomic analysis confirmed downregulation of metabolic and structural proteins. These data demonstrate unexpected and compensatory changes involving the TM in the NHP model of ExGl. The data suggest that compensatory mechanisms exist in normal animals and respond to elevated IOP through softening of the meshwork to increase outflow.

PURPOSE:To present a semiquantitative ocular scoring system comprising elements and criteria that address many of the limitations associated with systems commonly used in preclinical studies, providing enhanced cross-species applicability and predictive value in modern ocular drug and device development.METHODS:Revisions to the ocular scoring systems of McDonald-Shadduck and Hackett-McDonald were conducted by board-certified veterinary ophthalmologists at Ocular Services On Demand (OSOD) over the execution of hundreds of in vivo preclinical ocular drug and device development studies and general toxicological investigations. This semiquantitative preclinical ocular toxicology scoring (SPOTS) system was driven by limitations of previously published systems identified by our group's recent review of slit lamp-based scoring systems in clinical ophthalmology, toxicology, and vision science.RESULTS:The SPOTS system provides scoring criteria for the anterior segment, posterior segment, and characterization of intravitreal test articles. Key elements include: standardized slit lamp settings; expansion of criteria to enhance applicability to nonrabbit species; refinement and disambiguation of scoring criteria for corneal opacity, fluorescein staining severity, and aqueous flare; introduction of novel criteria for scoring of aqueous and anterior vitreous cell; and introduction of criteria for findings observed with drugs/devices targeting the posterior segment. A modified Standardization of Uveitis Nomenclature (SUN) system is also introduced to facilitate accurate use of SUN's criteria in laboratory species.CONCLUSIONS:The SPOTS systems provide criteria that stand to enhance the applicability of semiquantitative scoring criteria to the full range of laboratory species, in the context of modern approaches to ocular therapeutics and drug delivery and drug and device development.

PurposePoly(lactic-co-glycolic) acid (PLGA) inserts have been successfully developed for the treatment of posterior eye disease as a means of reducing injection frequency of intravitreally administered therapeutics. PLGA microspheres are also of interest for the delivery of intravitreal drugs, since they offer the advantage of being easily injected without surgical procedures or large injectors.MethodsIn the current study, the toxicity of PLGA microspheres and rods was investigated in nonhuman primates (NHPs) and rabbits. An in vitro assessment of cytokine responses to PLGA in peripheral blood mononuclear cells (PBMCs) and macrophages was also performed.ResultsIntravitreal administration of 3, 10, or 12.5 mg/eye of PLGA microspheres in NHPs resulted in a severe immune response characterized by a foreign body response. Follow-up studies in the rabbit confirmed this finding for PLGA microspheres ranging in size from 20 to 100 μm. In contrast, administration of PLGA rod implants with a similar PLGA mass did not elicit a significant immune response. In vitro assays in PBMCs and macrophages confirmed proinflammatory cytokine release upon treatment with PLGA microspheres but not PLGA rods.ConclusionsThese data demonstrate a lack of tolerability of PLGA microspheres upon intravitreal injection, and suggest that the size, shape, and/or surface area of PLGA depots are critical attributes in determining ocular toxicity.

Purpose: To characterize the inflammatory response and determine the no-observable-effect level (NOEL) in cynomolgus monkey eyes after intravitreal (ITV) injection of endotoxin. Methods: The inflammatory response to endotoxin was assessed in a single-dose study in monkeys at doses of 0.01 to 0.51 endotoxin units (EU)/eye. Tolerability was assessed by clinical ophthalmic examinations, intraocular pressure measurements, fundus color photography, optical coherence tomography, and anatomic pathology. Results: ITV injection of endotoxin at ≥0.04 EU/eye resulted in a dose-related anterior segment inflammatory response. No aqueous flare or cell was noted in the 0.01 EU/eye dose group. A more delayed posterior segment response characterized by vitreous cell was observed beginning on day 5, peaking on day 15, and decreasing in some groups. Microscopic findings of mononuclear cell infiltrates in the vitreous were observed in eyes given ≥0.21 EU/eye. Conclusion: The NOEL for ITV endotoxin in cynomolgus monkeys was 0.01 EU/eye, suggesting that this species is as sensitive as rabbits to the effects of endotoxin. The vitreous cavity also appears more sensitive to endotoxin than the anterior segment/aqueous chamber. Overall, the magnitude of the inflammatory response at ≥0.04 EU/eye suggests that dose-response curve in monkeys is steeper than in rabbits. These data highlight the importance of assessing endotoxin level in ITV formulations, as levels as low as 0.04 EU/eye may confound the safety evaluations of ITV therapeutics in cynomolgus monkeys.