Clinical management of Candida albicans keratomycosis in a bottlenose dolphin (Tursiops truncatus).

OBJECTIVE: Corneal ulceration secondary to trauma commonly affects marine mammals, often with opportunistic secondary bacterial or fungal infections. This report characterizes the combined use of auriculopalpebral and ophthalmic nerve blocks, adipose-derived stem cells, and subconjunctival injections for successful treatment of corneal trauma and infection in dolphins. ANIMAL STUDIED: An 11-year-old, female bottlenose dolphin (Tursiops truncatus) presented with bilateral diffuse corneal opacities, which progressed to keratomycosis caused by Candida albicans. PROCEDURE: Aggressive medical management was employed, including the use of subconjunctival injections of adipose-derived stem cells, plasma, topical and oral antifungals and antibiotics, and anti-inflammatory and pain medications. Anesthetic block of the auriculopalpebral and ophthalmic nerves was employed to evaluate the corneas. CONCLUSION: Subconjunctival injections were employed over 52 days, followed by topical drops for 5 months. At last evaluation, there was no evidence of blepharospasm bilaterally. Only a faint superficial gray corneal opacity remained OS. A temporal paraxial corneal opacity was present OD, with receding inactive vascularization and a small amount of melanosis temporally.

Subconjunctival antimicrobial poloxamer gel for treatment of corneal ulceration in stranded California sea lions (Zalophus californianus).

OBJECTIVE: Corneal ulcers are commonly encountered in pinnipeds. Prolonged oral antibiotics and topical ophthalmic solutions may not be practical to administer, and novel treatment techniques are desired. Thermodynamic gels are a potential solution because they hold antimicrobials at the site of injection, slowly releasing drug. This study investigated the clinical efficacy of antibiotic-impregnated poloxamer gel in management of corneal ulceration. ANIMAL STUDIED: Twenty-six California sea lions undergoing rehabilitation at The Marine Mammal Center. PROCEDURES: A poloxamer gel mixed with 2% enrofloxacin was subconjunctivally injected in the treatment group. Control animals received oral doxycycline. Systemic anti-inflammatories and analgesics were administered as needed. Corneal examinations under general anesthesia were repeated weekly, and included sampling for bacterial culture and corneal cytology, collection of high-quality corneal images, and treatment administration until the ulcers were healed. RESULTS: There was no gross or histologic evidence of a localized tissue reaction to the gel administration in the conjunctiva, and no evidence of systemic reaction to therapy in animals that died due to unrelated causes during the study period (n = 17). In animals that experienced a superficial corneal ulcer involving only epithelium or superficial stroma (n = 12), all lesions resolved completely, in both treatment and control groups. Of those animals with deeper or more complex ulcers involving keratomalacia or descemetoceles (n = 15), four demonstrated complete lesion resolution (all four received gel treatment). CONCLUSIONS: This study demonstrates that subconjunctival antibiotic poloxamer gel administration is a safe and effective alternative therapeutic option to traditional treatments for superficial corneal ulceration in pinnipeds.

CHARACTERIZATION OF ANTERIOR SEGMENT OPHTHALMOLOGIC LESIONS IDENTIFIED IN FREE-RANGING DOLPHINS AND THOSE UNDER HUMAN CARE.

Cetaceans in the wild and under human care develop a variety of ocular lesions. Although they have echolocation, cetacean species have good sight, making ocular health an important part of overall health care. The cornea is the primary site of abnormalities in both populations. Typical lesions of cetaceans under human care are characterized in this retrospective review of cases. One hundred eighty animals (n = 360 eyes) were chosen from the author's ophthalmologic examination reports from different geographic areas; they included Atlantic bottlenose dolphins (Tursiops truncatus), Pacific bottle nose dolphins (Tursiopstruncatus gilli), Indopacific bottlenose dolphins (Steno bredanensis), Indopacific humpback dolphins (Sousa chinensis), and roughtooth dolphins (Steno bredanensis). These animals were examined at least once, although most were examined numerous times over many years; lesions were categorized and are described. Seventy-seven eyes from 47 animals were normal. Medial keratopathy was the most common lesion and identified in 180 eyes from 97 animals, with 83 affected bilaterally. Horizontal keratopathy was identified in 69 eyes from 41 animals, with 28 affected bilaterally. Axial keratopathy and nonspecific axial opacities were identified in 67 eyes from 44 animals, with 21 affected bilaterally. Seventy-eight eyes from 50 animals, with 28 affected bilaterally, had more than one type of corneal lesion. Cataracts were identified in 32 eyes from 19 animals, with 13 affected bilaterally. Traumatic injuries were also common and involved eyelids and cornea. Sixteen eyes from 11 animals were blind; five dolphins were blind bilaterally due to phthisis bulbi secondary to corneal perforation or severe trauma. None of the diseases had a sex predisposition; however, medial keratopathy was significantly more common as a bilateral presentation than as a unilateral presentation. Cetaceans under human care with impaired sight can use echolocation; however, ocular health should definitely be a priority in their overall health plan.

Safety study of capsular tension ring use in canine phacoemulsification and IOL implantation.

OBJECTIVE: To evaluate and compare surgical outcome and complications in canine eyes with stable, cataractous lenses undergoing routine phacoemulsification and intraocular lens (IOL) implantation with or without implantation of a capsular tension ring device (CTR). PROCEDURES: Dogs undergoing routine bilateral phacoemulsification and IOL implantation had one eye randomly chosen to have a CTR (47 eyes) inserted while the contralateral eye served as a control (47 eyes). The CTR was placed in the capsular bag following phacoemulsification and immediately prior to IOL implantation. All eyes received acrylic foldable IOLs. Patients were evaluated intra- and postoperatively, and complications were compared between eyes with and without a CTR. RESULTS: There were no intra-operative complications associated with implantation of the CTR. Postoperatively, the overall incidences of glaucoma (6%), intraocular hemorrhage (4%), and retinal detachment (2%) were equal between control and CTR eyes at the time of last follow-up. There was no significant difference in overall incidence of immediate postoperative uveitis (35% vs. 38%) or fibrin (19% vs. 15%) between control and CTR eyes. Follow-up time ranged from 2 weeks to 21 months. CONCLUSION: In canine eyes with stable cataractous lenses undergoing routine phacoemulsification and IOL implantation placement of a CTR does not increase the risk of intra- or postoperative complications.

Bilateral ocular anomalies in a South African fur seal (Arctocephalus pusillus pusillus).

A female South African fur seal (Arctocephalus pusillus pusillus) began having obvious clinical ophthalmologic problems by 8 weeks of age. The initial clinical sign was diffuse corneal edema, which progressed to bullae formation and ulcers; the underlying cause of corneal edema and bullous keratopathy was not identified antemortem.An ophthalmological evaluation was performed when the fur seal was approximately 6 months of age, but due to the diffuse corneal edema, intraocular structures could not be easily evaluated. An underlying infectious etiology was suspected; therefore,appropriate diagnostics were pursued, but did not identify a cause. Initial improvement was noted, but the fur seal then became blind and eventually became very painful.Due to decreased quality of life and aggressive behavior, the fur seal was euthanized.Histopathological diagnoses were persistent tunica vasculosa lentis and persistent hyperplastic primary vitreous with bilateral hypermature resorbed cataracts and retinal detachments with rosette formation.

A retrospective survey of the ocular histopathology of the pinniped eye with emphasis on corneal disease.

OBJECTIVE: A retrospective review of globes from 70 pinnipeds submitted to the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW) describing the type and frequency of ocular disease. ANIMALS STUDIED: The study included 50 California sea lions, four animals listed only as 'sea lion', nine Northern elephant seals, five harbor seals, 1 Northern fur seal, and 1 Hooded seal. PROCEDURES: Globes were classified by microscopic findings. Categories were not mutually exclusive. RESULTS: The largest category was corneal disease (63 globes from 40 pinnipeds). The second largest was cataractous changes (35 globes from 23 pinnipeds). Additional ocular diseases included traumatic ocular injuries (nine globes from eight animals), phthisis bulbi (nine globes from eight pinnipeds), neoplasia (nine globes from six adult California sea lions), amyloid deposition in the corneal stroma, ciliary body, or both locations (five globes from four pinnipeds), and fungal disease (three globes from two pinnipeds). Pinnipeds with corneal disease were further categorized: stromal pathology (39 globes from 27 pinnipeds); epithelial pathology (37 globes from 27 pinnipeds); Descemet's pathology (11 globes from eight pinnipeds); endothelial attenuation or absence (33 globes from 22 pinnipeds); presence of retrocorneal membranes (15 globes from 10 pinnipeds); anterior synechia (eight globes from six animals), and keratitis (seven globes from five pinnipeds). CONCLUSIONS: This is the first report of ocular amyloid in pinniped eyes. All cases of neoplasia were in a pattern suggesting metastatic disease. In this study, there was a higher prevalence of ocular disease in captive pinnipeds, particularly in the posterior cornea.

Characterization of ocular gland morphology and tear composition of pinnipeds.

OBJECTIVE: The importance of tear film integrity to ocular health in terrestrial mammals is well established, however, in marine mammals, the role of the tear film in protection of the ocular surface is not known. In an effort to better understand the function of tears in maintaining health of the marine mammal eye surface, we examined ocular glands of the California sea lion and began to characterize the biochemical nature of the tear film of pinnipeds. PROCEDURES: Glands dissected from California sea lion eyelids and adnexa were examined for gross morphology, sectioned for microscopic analysis, and stained with hematoxylin and eosin. The tear film was examined using interferometry. Tears were collected from humans and pinnipeds for the analysis of protein and carbohydrate content. RESULTS: The sea lion has sebaceous glands in the lid, but these glands are different in size and orientation compared with typical meibomian glands of terrestrial mammals. Two other accessory ocular glands located dorsotemporally and medially appeared to be identical in morphology, with tubulo-acinar morphology. An outer lipid layer on the ocular surface of the sea lion was not detected using interferometry, consistent with the absence of typical meibomian glands. Similar to human tears, the tears of pinnipeds contain several proteins but the ratio of carbohydrate to protein was greater than that in human tears. CONCLUSIONS: Our findings indicate that the ocular gland architecture and biochemical nature of the tear film of pinnipeds have evolved to adapt to the challenges of an aquatic environment.

Absorption and ocular deposition of dietary lutein in marine mammals.

Cataracts and ocular disease are common lesions of marine mammals in zoological collections. Lutein, an oxygenated carotenoid, may have therapeutic or prophylactic effects on ocular disorder. Therefore, this study examined the ability of marine mammals to absorb dietary lutein. Two preliminary trials examined lutein in two forms (beadlet or ester) in a small sample size of marine mammals representing pinnipeds and cetaceans. Lutein was fed daily in tablets providing 0.89-3.6 mg lutein/kg body weight(0.75) per day for 15 days to 2 years. A third study was conducted using lutein beadlet fed at 3.6 mg lutein/kg body weight(0.75) per day for 15-21 days. Blood was analyzed for lutein pre- and postsupplementation. In the preliminary trials, lutein beadlet was observed to result in greater blood lutein levels than lutein esters, and cetaceans had more noticeable responses than pinnipeds. In Study 3, serum lutein and zeaxanthin increased postsupplementation in beluga whales (P < 0.05), and serum lutein tended to increase postsupplementation in dolphins (P < 0.10), but little change was seen in serum lutein in pinnipeds or manatee. Opportunistic retinal samples demonstrated some detectable lutein in the retina of a dolphin and several harp seals. The lutein levels in dolphins after supplementation are similar to those reported in free-ranging animals. Ocular lutein in harp seals demonstrates that ocular deposition occurs despite low circulating lutein levels.

Bilateral ocular abnormalities in a wild stranded harp seal (Phoca groenlandica) suggestive of anterior segment dysgenesis and persistent hyperplastic primary vitreous.

A male yearling harp seal (Phoca groenlandica) stranded and was brought to Mystic Aquarium & Institute for Exploration's Seal Rescue and Rehabilitation Center. The seal presented with a bilateral pendular vertical nystagmus, negative menace response, and a positive palpebral response. Ophthalmological examination by slit lamp biomicroscopy revealed perilimbal corneal edema, excessive iridal surface structures, pupils that appeared to be shaped improperly (dyscoria), and suspected cataracts. Attempts to dilate the pupils with both dark-lighted conditions and repeated dosages of 10% phenylephrine and 1% atropine ophthalmic solution in each eye (OU) were unsuccessful. Ocular ultrasonography findings suggested bilateral cataracts with flattened anterior-posterior (A-P) diameter and possible persistent hyperplastic primary vitreous. It is possible that these structural congenital abnormalities could produce further ocular complications for this seal including uveitis, secondary glaucoma, retinal detachment, and/or vitreal hemorrhage in the future. This case demonstrates the importance of a thorough ophthalmological examination in stranded wild animals, especially if their symptoms appear neurological.

The effect of phosphorylated Akt inhibition on posterior capsule opacification in an ex vivo canine model.

PURPOSE: To evaluate whether inhibition of phosphorylated Akt (pAkt) would reduce or prevent posterior capsule opacification (PCO) in an ex vivo canine lens capsule model. METHODS: Normal and cataractous lenses (n=6) were evaluated for pAkt via immunohistochemistry and immunoblotting. Primary cultures of lens epithelial cells (LEC) were exposed to ultraviolet light (UV) to induce pAkt. Cultures were then incubated in 0, 2.5, 5, or 10 µM (n=6) of a novel Akt inhibitor (AR-12) for either 8 or 24 h. Cultures were harvested and pAkt expression and telomerase activity examined by immunoblotting and telomeric repeat amplification protocol (TRAP)-enzyme linked immunosorbent assay (ELISA), respectively. Lens capsules were harvested post-sham cataract surgery and exposed to 0, 2.5, 5, 7.5, or 10 µM (n=8) of AR-12 for a total of 14 days treatment. Additional lens capsules (n=6) were exposed to 10 µM of AR-12 for 1 week followed by media alone for 1 week; or exposed to media alone for 1 week followed by 10 µM of AR-12 for 1 week. Histopathology and immunohistochemical staining were performed to evaluate PCO formation. Analysis of telomerase activity on the lens capsules was performed by TRAP-ELISA. RESULTS: pAkt protein expression was increased in clinical samples of canine cataracts compared to normal lenses. Following exposure to UV, cultures of LEC significantly (p<0.05) increased expression of pAkt and telomerase activity. Treatment with AR-12 for both 8 and 24 h following UV irradiation significantly (p<0.01) decreased pAkt expression. When UV-exposed LEC were allowed to recover in the presence of either 5.0 or 10.0 µM AR-12, there was a significant (p<0.05) decrease in telomerase activity. In the ex vivo model of PCO, within the region of the capsulorhexis, PCO inhibition was maximally achieved with 10 µM of AR-12. A significant decrease in LEC was noted on the posterior capsules containing 5.0, 7.5, and 10 µM AR-12 compared to the control capsules (p<0.01). Telomerase activity decreased in a dose-dependent manner. One week of treatment with 10 µM AR-12, immediately following capsule excision, was sufficient to inhibit PCO formation, while a delay in exposure to AR-12 after 1 week of media incubation alone did not prevent PCO formation. CONCLUSIONS: pAkt is known to have roles in cell survival, proliferation, and migration, and this study suggests its inhibition immediately following cataract surgery may be a useful approach to prevent PCO.

Anatomy of the California sea lion globe.

OBJECTIVE: This study analyzed the morphology of the California sea lion globe to determine what features may contribute to their characteristic visual abilities. PROCEDURE: Globes from the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW) collection were examined from gross photographs and microscopic sections stained with hematoxylin and eosin, trichrome, smooth muscle actin, and alcian blue periodic acid-Schiff (PAS). Transmission electron microscopy of the cornea and iris was also performed. CLINICAL RESULTS: There was a round, flattened area ventromedial to the axial cornea. The pupil was tear-drop shaped. Pectinate ligaments were visible without magnification. The retina was holangiotic, containing numerous spoke-like venules and arterioles. The tapetum was green encompassing the entire fundus. The optic nerve was unmyelinated. HISTOLOGICAL RESULTS: The sclera was thinnest equatorially and thickest at the limbus and posterior pole. Bowman's layer was difficult to see by light microscopy but clear with transmission electron microscopy. The cornea had a thick epithelium, thin endothelium and Descemet's membrane, and the stroma thinned axially. The dilator muscle was absent near the pupil, but enlarged and mingled with the sphincter muscle near the iris base. A large, wide ciliary cleft with prominent trabeculae and a single continuous pectinate ligament was present. The corneoscleral trabecular meshwork was discontinuous. A round lens attached to the ciliary body via direct attachment to ciliary processes and delicate zonular ligaments. There was a circumferential muscle at the base of the ciliary processes. A thick tapetum covered the entire fundus except peripherally. The retina was characterized by sparse, large ganglion cells.

Characterization of progressive keratitis in otariids.

OBJECTIVE: To characterize a form of progressive keratitis that occurs commonly in otariids. MATERIALS AND METHODS: One hundred and thirteen captive otariids were evaluated by ophthalmologic examination and digital photography. RESULTS: Forty-six females and 67 males were in the reference population, average age of 14 years. California sea lions predominated (n = 100); there were also six Steller sea lions, five brown fur seals, one Guadalupe fur seal and one northern fur seal. Three stages of progressive keratitis are described. Overall, 64.6% animals and 142 eyes from 113 animals (62.8%) were affected with one of three stages. The mildest form, Stage 1 keratitis, occurred in 78 of 226 eyes (34.5%); the intermediate Stage 2 keratitis occurred in 30 of 226 eyes (13.3%); and the most severe, Stage 3 keratitis, occurred in 34 of 226 eyes (15%). All but six animals had bilateral disease. Animals with Stage 1 keratitis were significantly younger than those in more advanced stages. DISCUSSION: 'Otariid Keratitis' occurs in all populations of eared seals evaluated. A large-scale epidemiological study is ongoing to identify the risk factors that contribute to this disease. Exposure to chronic sunlight appears to be an important risk factor as shade diminishes clinical signs; animals kept out of sunlight the majority of the time have less severe clinical signs. Age may be important since exposure accumulates with aging. Progression of the disease is also associated with secondary opportunistic bacterial and fungal infections. The surface immune system may be imbalanced contributing to these infections and progression.

In vivo effects of adjunctive tetracycline treatment on refractory corneal ulcers in dogs.

OBJECTIVE: To evaluate effect of adjunctive treatment with tetracycline analogues on time to complete corneal reepithelialization in dogs with nonhealing (ie, refractory) corneal ulcers. DESIGN: Randomized controlled clinical trial. ANIMALS: 89 dogs with refractory corneal ulcers. PROCEDURES: Corneal ulcers were treated via debridement and grid keratotomy. Dogs were assigned to receive 1 of 3 treatment regimens for up to 6 weeks: doxycycline (5 mg/kg [2.27 mg/lb], PO, q 12 h) with topically applied ophthalmic ointment containing neomycin, polymyxin B, and bacitracin (ie, triple antibiotic ointment; q 8 h); cephalexin (22 mg/kg [10 mg/lb], PO, q 12 h) with topically applied oxytetracycline ophthalmic ointment (q 8 h); or a control treatment of cephalexin (22 mg/kg, PO, q 12 h) with topically applied triple antibiotic ointment (q 8 h). Healing was monitored via measurements of the wound with calipers and evaluation of photographs obtained every 2 weeks. Treatment effectiveness was evaluated by wound healing and decreased signs of pain. RESULTS: The Boxer breed was overrepresented in all groups. At the 2-week time point, wound healing was significantly more common in small-breed dogs, compared with large-breed dogs. Dogs treated with oxytetracycline ophthalmic ointment had a significantly shorter healing time than did dogs receiving the control treatment. Corneal ulcers in dogs that received doxycycline PO healed more rapidly than did ulcers in dogs in the control treatment group; however, this difference was not significant. CONCLUSIONS AND CLINICAL RELEVANCE: Topical tetracycline ophthalmic ointment was a safe, inexpensive, and effective adjunctive treatment for refractory corneal ulcers in dogs.

Risk factors associated with cataracts and lens luxations in captive pinnipeds in the United States and the Bahamas.

OBJECTIVE: To determine risk factors for lens luxation and cataracts in captive pinnipeds in the United States and the Bahamas. DESIGN: Cross-sectional study. ANIMALS: 111 pinnipeds (99 California sea lions [Zalophus californianus], 10 harbor seals [Phoca vitulina], and 2 walruses [Odobenus rosmarus]) from 9 facilities. PROCEDURES: Eyes of each pinniped were examined by a veterinary ophthalmologist for the presence of cataracts or lens luxations and photographed. Information detailing husbandry practices, history, and facilities was collected with a questionnaire, and descriptive statistical analyses were performed for continuous and categorical variables. Odds ratios and associated 95% confidence intervals were estimated from the final model. RESULTS: Risk factors for lens luxation, cataracts, or both included age >or= 15 years, history of fighting, history of ocular disease, and insufficient access to shade. CONCLUSIONS AND CLINICAL RELEVANCE: Diseases of the lens commonly affect captive pinnipeds. Access to UV-protective shade, early identification and medical management of ocular diseases, and prevention of fighting can limit the frequency or severity of lens-related disease in this population. An extended life span may result from captivity, but this also allows development of pathological changes associated with aging, including cataracts.

ERalpha increases expression and interacts with TERT in cataractous canine lens epithelial cells.

PURPOSE: Estrogen receptor alpha (ERalpha) expression has previously been evaluated in lens epithelial cells (LEC). However, its function in the lens has not been determined. One potential function may be its interaction with the catalytic subunit of telomerase (TERT), which is present in normal LEC and higher in LEC that have undergone epithelial to mesenchymal transition (EMT). ERalpha is known to play a role in EMT, a process that may also involve TERT. METHODS: A commercially available transcription factor array was used to evaluate potential interactions between TERT and other proteins in normal and cataractous LEC. Based on these findings, ERalpha protein and mRNA expressions were measured using western blot analysis, immunohistochemical staining, and quantitative reverse transcription polymerase chain reaction (RT-PCR). Co-immunoprecipitation assays were used to evaluate the interaction of TERT with ERalpha as well as their phosphorylation in normal and cataractous LEC. RESULTS: The transcription factor array suggested that TERT interacted with ERalpha via the estrogen response element (ERE) in cataractous LEC but not in normal LEC. Expression of ERalpha protein and mRNA increased in cataractous LEC compared with normal LEC. Co-immunoprecipitation assays confirmed the interaction of TERT with ERalpha in cataractous LEC while no interaction was found in normal LEC. LEC that have undergone EMT, e.g., cataracts, are rapidly proliferating and migrating along the posterior lens capsule. CONCLUSIONS: ERalpha is known to play a role in EMT, and our data suggests that TERT and phosphorylated protein kinase B (pAkt) may be involved in the regulation of this process in cataractous LEC.

Selenium functionalized intraocular lenses inhibit posterior capsule opacification in an ex vivo canine lens capsular bag assay.

The purpose of this study was to determine the inhibitory effect of selenocystamine coated intraocular lenses (IOLs) on the formation of posterior capsule opacification (PCO) in an ex vivo canine lens capsular bag assay. Selenocystamine was covalently bound to the surface of poly(2-hydroxyethyl methacrylate) (poly(HEMA)) discs. Three groups of canine lens capsules (6 coated IOLs (SeIOLs), 7 non-coated control IOLs and 8 empty capsules) were cultured for 10 days. During the culture period PCO was scored based on visual inspection of the capsules using phase contrast microscopy. On day 10 all the capsules were prepared for light microscopic examination and lens epithelial cells (LECs) were quantified. Proliferating cell nuclear antigen (PCNA), alpha-smooth muscle actin (alpha-SMA) and cleaved caspase-3 were examined by immunohistochemistry. Additionally, cell viability assays were performed on LECs cultured in tissue culture medium pre-incubated with either a SeIOL or control IOL. The viability assays demonstrated that no detectable cytotoxic leachables were associated with the functionalized IOLs. The central posterior capsule was free of cells underneath all SeIOLs, although large numbers of LECs populated the capsular periphery. Apoptotic cells were observed underneath the periphery of some SeIOLs. Both the PCO scores and LEC counts of SeIOL containing capsules were significantly lower than those of control group capsules (p < 0.01 and p = 0.0004, respectively). The use of selenium functionalized IOLs resulted in a significant reduction of PCO in this ex vivo model. Binding of selenocystamine to a foldable IOL may provide an effective method to prevent population of the central posterior capsule with LECs.

Immunohistochemical analysis of ocular hemangiomas and hemangiosarcomas in dogs.

PURPOSE: To determine if molecular markers typically associated with ultraviolet exposure could be detected in canine ocular hemangiomas (HA) and hemangiosarcomas (HSA). METHODS: Paraffin-embedded samples of canine ocular HA (n = 6) and HSA (n = 6) were examined for the presence of p53, p21, p16, cyclin D, PCNA, pAkt, telomerase, and estrogen receptor (ER)-alpha using immunohistochemistry. RESULTS: p53 and cyclin D protein were not detected in any of the canine HA or HSA samples. The majority of the HA and HSA were negative for both p21 and telomerase. pAkt immunoreactivity was absent in one HA, one HSA, but was present in five HA and five HSA. All of the HA or HSA samples were strongly positive for p16 and PCNA. ERalpha was expressed in all of the samples examined; there was more intense staining in the HSA samples compared to the HA samples. CONCLUSIONS: Results from this study describe the protein expression, via immunohistochemistry, that might be altered in UV exposure in HA and HAS formation. p53 may not play an important role in tumor development; rather, in the tumors examined, expression of cell cycle regulators independent of the p53 pathway appear central in HA and HSA formation and progression. In addition, this study finds that ERalpha may be involved in promoting the invasive behavior associated with HSA.

Factors associated with keratopathy in captive pinnipeds.

OBJECTIVE To identify factors associated with keratopathy in captive pinnipeds and to provide guidance for preventive measures. ANIMALS 319 captive pinnipeds (229 otariids [sea lions and fur seals], 74 phocids [true seals], and 16 odobenids [walrus]) from 25 facilities. PROCEDURES Descriptive data collected from questionnaires completed by facilities and from medical records and physical examinations of pinnipeds were compiled and evaluated. Variables were assessed with χ2 tests of homogeneity to determine potential association with keratopathy, and variables with values of P ≤ 0.25 were inserted into the multivariable logistic regression model. RESULTS Results indicated that variables associated with significantly increased odds of keratopathy in captive pinnipeds included lighter or reflective pool color (OR, 2.11; 95% confidence interval [CI], 1.20 to 3.97), pool water salinity < 29 g/L (OR, 3.48; 95% CI, 1.89 to 6.56), and history of eye disease (OR, 3.30; 95% CI, 1.85 to 5.98), trauma (OR, 3.80; 95% CI, 1.72 to 8.89), and having been tested for leptospirosis (OR, 3.83; 95% CI, 1.54 to 10.26). However, odds of keratopathy decreased with UV index ≤ 6 (OR, 0.39; 95% CI, 0.2 to 0.72) and age < 20 years (OR, 0.32; 95% CI, 0.15 to 0.66). CONCLUSIONS AND CLINICAL RELEVANCE Findings indicated that odds of keratopathy in pinnipeds could be reduced by maintenance of pool water salinity ≥ 29 g/L and reduction of UV radiation exposure (eg, with adequate shade structures and use of darker, natural colors). Because UV radiation exposure is cumulative, even small attempts to reduce lifetime exposure to it could help control keratopathy in pinnipeds.

Modulation of matrix metalloproteinases by ultraviolet radiation in the canine cornea.

PURPOSE: To determine whether ultraviolet (UV) radiation can modulate expression and regulation of matrix metalloproteinases (MMP) in the canine cornea and to examine the expression of MMPs in canine chronic superficial keratitis (CSK). METHODS: Immunohistochemistry for MMP-2 and MMP-9 was performed on samples of CSK. In vitro, canine corneal epithelial cell (CEC) and stromal cell cultures were exposed to UV-irradiation. Following 2, 8 or 24 h, cells were harvested. MMP expression was examined by zymography, and RT-PCR was used to examine expression of Slug and Snail. CEC cultures treated with an EGFR inhibitor or a p38 inhibitor were UV-exposed and harvested 24 h later to examine expression of MMPs, Slug and Snail. RESULTS: Canine CSK had increased immunopositivity for both MMP-2 and MMP-9 compared to normal canine corneas. In vitro, CEC and stromal cell cultures exposed to UV showed generally increased expression of MMP-2, -9, Slug, and Snail; this response was dose and time dependent. Inhibition of the EGFR pathway did not prevent increased expression of MMP-2, -9, Slug or Snail in UV-exposed CEC; however, p38 inhibition did attenuate UV induction. CONCLUSIONS: We have found increased expression of MMPs in clinical samples of CSK compared to normal corneas. In addition, we have shown that there is a temporal association and dose dependency between UV exposure and production of MMPs, Slug, and Snail. These findings suggest that overexpression of MMPs due to UV-exposure may be linked to changes in the cornea that allow an influx of inflammatory cells and vascularization.

Effect of grape polyphenols on oxidative stress in canine lens epithelial cells.

OBJECTIVE: To evaluate whether the effects of oxidative stress could be attenuated in cultures of canine lens epithelial cells (LECs) by incubation with grape seed proanthocyanidin extract (GSE), resveratrol (RES), or a combination of both (GSE+RES). SAMPLE POPULATION: Primary cultures of canine LECs. PROCEDURES: LECs were exposed to 100MM tertiary butyl-hydroperoxide (TBHP) with or without GSE, RES, or GSE+RES. The dichlorofluorescein assay was used to detect production of reactive oxygen species (ROS), and immunoblot analysis was used to evaluate the expression of stress-induced cell-signaling markers (ie, the mitogen-activated protein kinase [MAPK] and phosphoinositide-3 kinase [PI3K] pathways). RESULTS: GSE and GSE+RES significantly reduced ROS production after a 30-minute exposure to TBHP. Only GSE significantly reduced ROS production after a 120-minute exposure to TBHP. Incubation with GSE reduced TBHP-induced activity of the MAPK and PI3K pathways. CONCLUSIONS AND CLINICAL RELEVANCE: GSE inhibited key components associated with cataractogenesis, ROS production, and stress-induced cell signaling. On the basis of the data reported here, there is strong evidence that GSE could potentially protect LECs from the damaging effects of oxidative stress.