Determination of trypan blue efficacy in the mitigation of ex vivo canine PCO formation.

PURPOSE: To determine whether trypan blue (TB) reduces canine lens epithelial cell (LEC) or corneal endothelial cell (CEC) viability in vitro; if cell death is noted, to subsequently evaluate the molecular mechanism. METHODS: Cellular viability was determined using a lactate dehydrogenase (LDH) assay. In TB-treated LECs, caspase 3/7 activity was assessed to evaluate apoptosis; autophagy was evaluated using immunoblotting against LC3 and p62. To evaluate the effects of TB on ex vivo posterior capsule opacification (PCO), following mock cataract surgery, lens capsules were treated with TB and subsequently maintained in culture to determine LEC migration and proliferation. RESULTS: Following acute exposure, TB did not significantly reduce LEC or CEC viability at any of the concentrations tested. Increased caspase 3/7 activity was found in LEC cultures treated with TB for an extended period of time; no change in LC3 or p62 expression was noted. Ex vivo PCO formation was not significantly altered by TB treatment. CONCLUSIONS: Acute exposure to TB did not reduce LEC or CEC viability, and only longer exposure to TB was able to initiate apoptosis. Treatment with intraocular TB at the time of cataract surgery is likely safe to the CECs but will not prevent PCO formation.

MG53 promotes corneal wound healing and mitigates fibrotic remodeling in rodents.

The cornea plays an important role in transmitting light and providing protection to the eye, but is susceptible to injury and infection. Standard treatments for corneal wounds include topical lubricants, antibiotics, bandage contact lens, and surgery. However, these measures are often ineffective. Here we show that MG53, a protein with an essential role in cell membrane repair, contributes to the corneal injury-repair process. Native MG53 is present in the corneal epithelia, tear film, and aqueous humor, suggesting its potential function in corneal homeostasis. Knockout of MG53 in mice causes impaired healing and regenerative capacity following injury. Exogenous recombinant human MG53 (rhMG53) protein protects the corneal epithelia against mechanical injury and enhances healing by promoting migration of corneal fibroblasts. Using in vivo alkaline-induced injury to the rat cornea, we show that rhMG53 promotes re-epithelialization and reduces post-injury fibrosis and vascularization. Finally, we show that rhMG53 modulates TGF-ß-mediated fibrotic remodeling associated with corneal injury. Overall, our data support the bi-functional role of MG53 in facilitating corneal healing and maintaining corneal transparency by reducing fibrosis and vascularization associated with corneal injuries.

Effects of grape seed extract, lutein, and fish oil on responses of canine lens epithelial cells in vitro.

OBJECTIVE To determine the effects of grape seed extract (GSE), lutein, and fish oil containing omega-3 fatty acids on oxidative stress, migration, proliferation, and viability of lens epithelial cells (LECs). SAMPLE Lens capsules or cultured LECs obtained from canine cadavers. PROCEDURES An antioxidant reductive capacity assay was used to determine reducing capability of each substance. The LECs were cultured and incubated with various substances, including N-acetyl cysteine (NAC), when appropriate, and dimethyl sulfoxide (DMSO) as positive and vehicle control substances, respectively. A dichlorofluorescein assay was used to evaluate reactive oxygen species (ROS) production, and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine cell viability. Ex vivo posterior capsule opacification (PCO) was used to evaluate LEC migration and proliferation. RESULTS Antioxidant reductive effects of GSE surpassed those of NAC, lutein, and fish oil containing omega-3 fatty acids. The GSE reduced ROS production in LECs, compared with the DMSO vehicle control, whereas lutein was pro-oxidative. All test substances reduced cell viability. Ex vivo PCO was not altered by GSE, was decreased by lutein, and was increased by fish oil containing omega-3 fatty acids, compared with results for the DMSO vehicle control. CONCLUSIONS AND CLINICAL RELEVANCE Only GSE had significant antioxidant capabilities and reduced ROS production; however, no effect on ex vivo PCO was detected. Fish oil containing omega-3 fatty acids increased ex vivo PCO. No conclusions could be made regarding antioxidant effects of these substances on LECs. These findings suggested that the substances will not decrease PCO.

Cyclosporine A prevents ex vivo PCO formation through induction of autophagy-mediated cell death.

The purpose of this study was to determine the Cyclosporine A (CsA) dose and minimum drug delivery time needed to prevent posterior capsule opacification (PCO) in an ex vivo canine model and evaluate the mechanism of CsA-induced cell death. Canine lens epithelial cells (LEC) were treated with CsA and changes in cell migration, proliferation, and density were monitored over time. CsA-treated LEC underwent transmission electron microscopy (TEM), immunofluorescence, and immunoblotting in the presence or absence of autophagy inhibitors to evaluate the mechanism of cell death. Lens capsules were harvested from canine cadaver eyes for an ex vivo model of PCO. Lens capsules were treated with CsA for 1, 2, 3, 4, 5, 6, or 7 days, and subsequently maintained in culture for a total of 28 days in the absence of drug. CsA reduced LEC viability in a dose dependent manner. Morphologically, CsA-treated LEC were swollen, had intact nuclei, lacked peripheral chromatin condensation, and demonstrated prominent vacuolization; TEM revealed autophagosomes. LC3-II protein expression and acridine orange fluorescence increased in CsA-treated cells. A small non-significant induction of cleaved caspase-3 was observed in CsA-treated LEC. Lens capsules treated with 5, 6, or 7 days of 10 µg/mL CsA showed a significant decrease in ex vivo PCO formation; 6 days of drug delivery prevented PCO. This study finds that morphologic changes, formation of acidic vesicles, and increased expression of LC3-II supports the hypothesis that CsA mediates LEC death via autophagy; this is a novel finding in the lens. Induction of CsA-induced apoptosis was minimal. Six days of intracapsular CsA drug delivery prevented ex vivo PCO formation.

Retrospective study of the prevalence of keratoconjunctivitis sicca in diabetic and nondiabetic dogs after phacoemulsification.

OBJECTIVE: To evaluate the prevalence of keratoconjunctivitis sicca (KCS) in diabetic and nondiabetic dogs following phacoemulsification. PROCEDURES: Medical records were reviewed for signalment, body weight, diabetes status, and STT at the preoperative and all postoperative examinations. A clinical diagnosis of KCS was defined as a STT <15 mm/min with compatible clinical signs. Schirmer tear test values and the prevalence of KCS based on STT <15 mm/min were evaluated at 2-4, 5-8, 9-14, 15-20, and 41-52 weeks postoperatively. Size of dog (large, >10 kg; small, ≤10 kg) and STT ranges (<15 mm/min, 15-22 mm/min and >22 mm/min) were categorized for analysis of effect of body weight and STT on prevalence of KCS. RESULTS: One hundred and seventeen nondiabetic (198 eyes) and 118 diabetic dogs (228 eyes) were evaluated. KCS was diagnosed in nearly twice as many diabetics as nondiabetics (27.4% vs. 15.4%) within 2 weeks postoperatively (P = 0.0088). Percentage of eyes with KCS among all dogs decreased over time and did not differ significantly after the first postoperative visit. Diabetic small dogs were 1.7 times more likely to have KCS than nondiabetic small dogs (P = 0.0052). Preoperative STT was significantly associated with likelihood of postoperative KCS among large (P < 0.0001), but not small dogs (P = 0.0781). Among large dogs, eyes with STTs between 15 and 22 mm/min were approximately 3 times more likely to be diagnosed with KCS after surgery than those with STT >22 mm/min. CONCLUSION: The greatest risk for KCS for all dogs is during the first 2 weeks postoperatively. Populations at greatest risk for postoperative KCS are small dogs, small diabetic dogs, and large dogs with preoperative STT ≤22.

Effects of pulsed fluid lens capsule washing following phacoemulsification on lens epithelial cells and posterior capsule opacification formation ex vivo.

BACKGROUND: The aim of the study was to evaluate ex vivo the effects of using a custom tip to direct a pulsed stream of fluid to remove residual lens epithelial cells (LECs) and reduce posterior capsule opacification (PCO) formation following phacoemulsification. METHODS: Twenty-four canine cadaver eyes were assigned to one of three treatment groups. Six eyes (Control Group) had standard phacoemulsification only, nine eyes (Group 1) had standard phacoemulsification followed by capsular washing using intermediate settings (power = 40%, pulses per second [PPS] = 50, 30 s washing per capsule hemisphere), and nine eyes (Group 2) had standard phacoemulsification followed by aggressive capsular washing (power = 60%, PPS = 50, 60 s washing per capsule hemisphere). RESULTS: Control lens capsules had diffuse LECs remaining following standard phacoemulsification and complete ex vivo PCO formation (confluent LECs on the posterior capsule) within 4 ± 2 days (range 2-8 days). Group 1 lens capsules had focal, equatorial LEC clusters remaining following treatment, and complete PCO formation within 9 ± 2 days (range 5-11 days). Group 2 lens capsules had little to no LECs observed following treatment; 5 of 9 capsules had complete PCO formation within 13 ± 2 days (range 9-14 days), and 4 of 9 capsules had no PCO formation by 24 days post-treatment. CONCLUSIONS: Pulsed fluid lens capsule washing is capable of removing LECs and delaying PCO formation in canine eyes following phacoemulsification ex vivo. Use of more aggressive capsular washing settings resulted in more effective LEC removal and subsequent delay of ex vivo PCO.

Induction of posterior capsule opacification by hyaluronic acid in an ex vivo model.

PURPOSE: Because hyaluronic acid (HA) is found in many surgical viscoelastic agents, this study aimed to determine (1) if HA receptors are present in the canine lens, (2) if the rate of lens epithelial cell (LEC) migration is altered following treatment with HA, and (3) if introduction of exogenous HA into the lens capsule promotes lenticular migration, thus contributing to posterior capsule opacification (PCO). METHODS: Normal and cataractous canine LECs were evaluated for expression of the HA receptor CD44 and the receptor for HA mediated motility (RHAMM) using immunohistochemistry, immunoblotting, and real-time PCR. Canine LEC were treated with various concentrations of HA, and induction of migration was monitored over time. Commercially available surgical viscoelastics were utilized ex vivo, and rates of PCO formation were analyzed. RESULTS: Basal protein and mRNA expression of both CD44 and RHAMM was noted. Cataractous canine LEC demonstrated significantly (P < 0.01) higher expression of CD44 but not RHAMM. Treatment with higher concentrations of HA resulted in a significant (P < 0.01) increase in CD44 mRNA and increased LEC migration in vitro. Use of CD44-neutralizing antibodies confirmed the role of CD44 in HA-induced lenticular migration. Viscoelastic material containing higher concentrations of HA led to increased rates of ex vivo PCO. CONCLUSIONS: Exogenous HA can induce lenticular migration and CD44 expression. Use of surgical viscoelastics that contained HA resulted in increased rates of ex vivo PCO suggesting that judicious selection and use of viscoelastic material during cataract surgery is warranted.

Semiconductor diode laser transscleral cyclophotocoagulation for the treatment of glaucoma in horses: a retrospective study of 42 eyes.

OBJECTIVE: To evaluate the outcome of diode laser transscleral cyclophotocoagulation (TSCP) for the treatment of glaucoma in horses. PROCEDURE: Medical records at The Ohio State University were reviewed. All horses that underwent diode laser TSCP between the years of 1995 and 2007 were included. Preoperative, procedural and clinical follow-up data were collected, and telephone follow-up was performed. RESULTS: Forty-two eyes of 36 horses were included. Twenty-four hours prior to surgery mean intraocular pressure (IOP) was 37.17 + or - 13.48 mmHg (42 eyes). Forty-one of 42 eyes (98%) were sighted and 39 of 39 (100%) of eyes were receiving topical glaucoma medication. At 3-5 weeks postoperatively the average IOP was 19.36 + or - 12.04 mmHg (22 eyes). IOP remained significantly lower than pretreatment values at all periods of clinical follow-up (P < 0.05). There was no significant difference in vision outcome, or the requirement for topical glaucoma medication relative to pretreatment values at any follow-up period. Hyphema in 5 of 42 eyes was the only complication noted. Of the 27 eyes seen for clinical follow-up, 2 were enucleated because of refractory elevation of IOP. Mean telephone follow-up was 49 months. Twenty-one of 22 owners contacted (95%) reported that the treatment had been of value, 14 of 22 eyes (64%) were receiving topical glaucoma medication, and 13 of 22 eyes (59%) were considered sighted. CONCLUSIONS: Diode laser TSCP aided in the control of IOP and maintenance of vision but did not eliminate the need for topical glaucoma medication during the period of clinical follow-up.

Exophthalmus secondary to a sinonasal cyst in a horse.

CASE DESCRIPTION: A 13-year-old female Miniature Horse was evaluated for progressive unilateral exophthalmia of the left globe of 3 weeks' duration. CLINICAL FINDINGS: Results of a physical examination were unremarkable. Ophthalmic examination identified exophthalmus of the left globe with complete resistance to retropulsion and mild blepharoconjunctivitis. Computed tomography revealed a large, space-occupying mass within the left caudal maxillary and left conchofrontal sinuses. The mass extended into the left retrobulbar space and contacted the cribriform plate. Trephination yielded copious amounts of turbid yellow fluid. The diagnosis was a sinonasal cyst. TREATMENT AND OUTCOME: Subtotal surgical excision of the cyst via a frontonasal osteoplastic flap was curative, with complete resolution of the exophthalmus. Histologic examination confirmed diagnosis of a sinonasal cyst. There was no evidence of cyst recurrence by 4 months after surgery. CLINICAL RELEVANCE: Sinonasal cyst should be a differential diagnosis for retrobulbar disease in horses. Exophthalmia may be the only clinical finding in horses with a sinonasal cyst.

Evaluation of advanced glycation end-products in diabetic and inherited canine cataracts.

BACKGROUND: The receptor for advanced glycation end-products (RAGE) increases in the human cataract and should correlate with increased DNA damage and proliferation of lens epithelial cells (LECs). The purpose of this study was to measure and immunolocalize RAGE in normal and cataractous canine LECs, and to determine whether there was a correlation between RAGE and DNA damage (gadd45), cell-cycle regulation (p21), and LEC proliferation (proliferating cell nuclear antigen, PCNA). METHODS: Thirty-two anterior lens capsules from 22 dogs that underwent cataract surgery and 10 lenses from dogs with normal eyes were evaluated. Eleven of the cataractous lenses were from diabetic patients (n=16), and eleven were from patients with inherited cataracts (n=16). Standard immunohistochemical staining was performed using antibodies against RAGE, gadd45, p21, PCNA, alpha-smooth muscle actin, and TGF-beta. Immunostaining intensity for each antibody was given a score of 0-4+. Standard Western blot analysis on normal and cataractous lens capsules was performed using the same antibodies as in the immunohistochemical staining. Comparisons were also made based on age and sex. Real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed for RAGE. RESULTS: There was an increase in RAGE expression with age in normal LECs, but no significant difference was seen when normal adult LECs were compared to cataractous LECs. The stage of the cataract and the presence of LIU were not associated with a significant increase in RAGE expression. There was no age-dependent difference in the normal lenses for gadd45, p21, or PCNA. Significant up-regulation of p21 (P < 0.05) and PCNA (P < 0.05) was seen in diabetic cataracts compared to inherited cataracts. CONCLUSION: RAGE and PCNA expression did not increase with cataractogenesis, possibly due to overexpression associated with normal aging and constant exposure to oxidative stress from sunlight-related ultraviolet irradiation, respectively. However, p21 and PCNA increased in diabetic cataractogenesis suggesting cell cycle and proliferation dysregulation. This may be related to the rapid onset in this type of cataract compared with the more chronic and slower-to-develop inherited cataracts.

Posterior capsular opacification in diabetic and nondiabetic canine patients following cataract surgery.

Posterior capsular opacification (PCO) is the most common postoperative complication of contemporary cataract surgery. Limited information is available regarding PCO formation and factors that influence PCO development in the dog. Two hundred sixty-five eyes (144 from diabetic dogs and 121 from dogs with breed-related cataracts) were prospectively evaluated for PCO formation for up to 12 months postoperatively. The mean age of all dogs in the study was 7.77 years and diabetic dogs were significantly older than dogs with breed-related cataracts. There were 73 males (61 neutered, 12 intact) and 74 females (70 neutered, 4 intact) in the study. Statistical analysis was performed based on age, breed/size, gender, stage of cataract at the time of surgery, PCO score at each time point, breed-related vs. diabetic cataract, right eyes compared to left eyes, and presence/absence of uveitis. Age and gender did not significantly influence PCO formation. Small and medium-sized breeds developed significantly more PCO in comparison to the large/giant breeds at 2 weeks and 2-4 months postoperatively, but the differences were not significant at later time points. There was an overall significant increase in PCO formation in eyes with early immature cataracts when compared to other stages of cataract up to 4 months postoperatively but not at later time points. There were no statistical differences in PCO score at 6 months or at 1 year postoperatively in eyes with breed-related and diabetic cataracts. Right eyes did not differ from left eyes in PCO score. PCO score significantly increased over time in breed-related and diabetic groups and in the overall population. No difference was found in the degree of PCO formation in eyes with inflammation prior to or after surgery compared with those without inflammation. In summary, age, gender, presence of inflammation, and cause of cataract (breed-related vs. diabetes mellitus) do not influence the development of PCO in canine cataract dogs. Small and medium-sized breeds develop significant PCO earlier than larger breeds. It is important to note that all eyes from all dogs in this study developed PCO in a time dependent manner.

Immunohistochemical characterization of a malignant intraocular teratoid medulloepithelioma in a cockatiel.

A 3-year-old cockatiel was presented for evaluation of a buphthalmic right eye (OD). The history included a traumatic event 5 months prior to presentation, and the referring veterinarian diagnosed cataract and secondary glaucoma. Computed tomography of the bird's orbital region revealed a large right orbital soft tissue mass that extended lateral to the orbital rim with obliteration of the globe without bone involvement. There was also no evidence of metastatic disease within the lungs or any abnormalities within the celomic cavity, therefore enucleation was recommended to achieve a histopathologic diagnosis and prognosis, and to palliate the clinical signs. Final diagnosis was malignant intraocular teratoid medulloepithelioma.

The use of semiconductor diode laser for deflation and coagulation of anterior uveal cysts in dogs, cats and horses: a report of 20 cases.

OBJECTIVE: To describe semiconductor diode laser use for anterior uveal cyst deflation and coagulation in dogs, horses and cats. ANIMALS STUDIED: The presenting clinical signs, surgical technique and postoperative results for four dogs, nine horses and seven cats with anterior uveal cysts treated with diode laser are described. Treated cysts were of sufficient size and/or number to potentially impair vision, damage the corneal endothelium, or increase intraocular pressure (IOP). One dog with free-floating cysts exhibited 'fly biting' behavior. Cysts were suspected of causing shying on the affected side and/or head-shaking behavior in seven horses. Cysts were free floating within the anterior chamber in dogs, occurred in the corpora nigrum in horses and were attached to the posterior iris surface in cats. In cats, shallowing of the anterior chamber and dyscoria were observed. In all cats prior to cyst deflation, IOP increased after pharmacologic pupil dilation. Cats were more likely than dogs and horses to have bilateral and multiple cysts. PROCEDURE: Two dogs and all horses were treated without general anesthesia and two dogs and all cats were treated under general anesthesia. Diode laser was used to perforate, deflate and coagulate the cysts. RESULTS: Postoperatively, all eyes were free of discomfort or significant inflammation and minimal or no topical or systemic anti-inflammatory therapy was required. Abnormal behavior improved or resolved in all cases. In all cats, IOP 24 h after photocoagulation was lower than the postdilation IOP. Cysts did not recur, but new cysts were discovered in several cases. CONCLUSION: Semiconductor diode laser coagulation of anterior uveal cysts is safe, effective and noninvasive.

Agents for intraocular surgery.

No single viscoelastic material has all the properties required for every surgical procedure. Ophthalmic surgeons should be familiar with the advantages and disadvantages of several viscoelastics and realize the limitations encountered if the surgeon chooses to rely on a single viscoelastic. It is possible in the future that newer viscoelastics may become available offering aspects of cohesive and dispersive properties that make them easy to remove and not associated with postoperative hypertension. Investigation into other types of viscoelastic materials, such as poly-acrylamide (Orcolon) [33,51,52], human collagen [33,53], polytriethylene-glycol monomethacrylate, and polyglycerol monomethacrylate [48], may result in the availability of synthetic viscoelastic materials. Rethinking how we use viscoelastics may also be indicated. The approach of a single or repeat instillation of a viscoelastic only to have it aspirated during the procedure could be modified by using a continuous infusion ofa dilute viscoelastic [54]. One study diluted hyaluronic acid to a concentration of 0.06% to 0.12% and maintained continuous infusion of the viscofluid through the PE irrigation line [54]. It has been suggested that this may reduce turbulence and improve tissue protection [54].