Carmen M. H. Colitz

Apoptotic and Necrotic Mechanisms of Stress-Induced Human Lens Epithelial Cell Death

Exposure to ultraviolet radiation (UVR) and reactive oxygen species (ROS) can damage the human lens and contribute to cataract formation. Recent evidence suggests that apoptosis in lens epithelial cells (LEC) is an initiating event in noncongenital cataract formation in humans and animals. The present study examines the cellular and molecular mechanisms by which environmental (ultraviolet B [UVB]) and chemical (hydrogen peroxide [H2O2], t-butyl hydroperoxide [TBHP]) stress induces cell death in an SV-40 immortalized human lens epithelial (HLE) cell line. Treatment of HLE cells with UVB, H2O2, and TBHP significantly decreased cell density with LD50 values of 350 J/m2, 500 μM, and 200 μM, respectively. Cellular morphology, DNA fragmentation, and annexin/propidium iodide staining consistent with apoptosis was observed only in UVB-treated cells, whereas lactate dehydrogenase (LDH) release was significantly higher in H2O2- and TBHP-treated cells. In addition, activation of apoptotic stress-signaling proteins, including c-Jun NH2-terminal kinase (JNK), caspase-3, and DNA fragmentation factor 45 (DFF45) was observed only in UVB-treated cells. Inhibition of JNK activity increased UVB-induced cell death, suggesting that this pathway may serve a prosurvival role in HLE cells. These findings suggest UVB predominantly induces apoptosis in HLE cells, whereas H2O2 and TBHP induce necrosis.

Role of bacteria in the pathogenesis of recurrent uveitis in horses from the southeastern United States

OBJECTIVE To determine the role of intraocular bacteria in the pathogenesis of equine recurrent uveitis (ERU) in horses from the southeastern United States by evaluating affected eyes of horses with ERU for bacterial DNA and intraocular production of antibodies against Leptospira spp. SAMPLE POPULATION Aqueous humor, vitreous humor, and serum samples of 24 clinically normal horses, 52 horses with ERU, and 17 horses with ocular inflammation not associated with ERU (ie, non-ERU inflammation). PROCEDURES Ribosomal RNA quantitative PCR (real-time PCR) assay was used to detect bacterial DNA in aqueous humor and vitreous humor from clinically normal horses (n = 12) and horses with chronic (> 3-month) ERU (28). Aqueous humor and serum were also evaluated for anti-Leptospira antibody titers from clinically normal horses (n = 12), horses with non-ERU inflammation (17), and horses with confirmed chronic ERU (24). RESULTS Bacterial DNA was not detected in aqueous humor or vitreous humor of horses with ERU or clinically normal horses. No significant difference was found in titers of anti-Leptospira antibodies in serum or aqueous humor among these 3 groups. Only 2 horses, 1 horse with ERU and 1 horse with non-ERU inflammation, had definitive intraocular production of antibodies against Leptospira organisms. CONCLUSIONS AND CLINICAL RELEVANCE In horses from the southeastern United States, Leptospira organisms may have helped initiate ERU in some, but the continued presence of the organisms did not play a direct role in the pathogenesis of this recurrent disease.

Ultraviolet Radiation-Induced Corneal Degeneration in 129 Mice

Ultraviolet radiation (UVR) is a risk factor for the development of ocular disease in humans, including acute photokeratitis, chronic corneal spheroidal degeneration, and cataract formation. This report describes the ocular lesions seen in 21 mice chronically exposed to UVR as part of a skin carcinogenicity study. All globes were affected to varying degrees. The primary lesion, not previously reported in UVR-exposed mice, was marked loss of keratocytes relative to age-matched controls. Secondary lesions included corneal stromal thinning, keratoconus, corneal vascularization and fibrosis, keratitis, globe rupture, and phthisis bulbi. In addition, more than 90% of UVR-exposed and unexposed lenses had evidence of cataract formation; this is the first report of the occurrence of spontaneous cataracts in 129 mice. In a subsequent study, apoptotic cells were identified histologically and by cleaved caspase 3 immunoreactivity in the corneal epithelium and, less commonly, in the corneal stroma after acute UVR exposure. Based on this finding, we propose that the loss of keratocytes observed in the chronic study was due to UVR-induced apoptosis.

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.

Extra-adrenal paraganglioma of the equine orbit: six cases.

At The Ohio State University from 1994-2006 six of seven horses evaluated for primary orbital disease were diagnosed with extra-adrenal paraganglioma (EAPG). The horses ranged in age from 14 to 24 years, with a mean of 16.8 years. Duration of clinical signs was 1.5 years to 5 years, with a mean of 2.8 years. Clinical signs varied, but all six had non-painful exophthalmus of the right eye. Five horses had complete ocular exams reported; three of five had decreased to absent vision, two of five had pale optic nerves, and in three of five, difficulty of retropulsion of the globe was noted. Diagnostic tests performed included complete blood count, serum profile, radiography, ultrasound, computed tomography, true-cut biopsy, ocular examination, guttural pouch endoscopy, oral examination, and physical examination. Expulsive hemorrhage during orbital exenteration occurred in all horses. In five of six cases, tumor extension through the orbital foramen was apparent intra-operatively. Histopathologic appearance of all surgically removed tissues consisted of sheets of polygonal cells with abundant lightly granular cytoplasm, round nuclei with vesicular chromatin, and rare mitoses. Neoplastic cells were arranged into small groups separated by a fine fibrovascular stroma. All six cases were chromagranin positive on immunohistochemical staining. Follow-up ranged from six months to six years, with a mean of two years. Four of the five horses that recovered from surgery had no apparent tumor recurrence in 6-48 months.

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.

Tear production in normal juvenile dogs

OBJECTIVE To establish a baseline range or average for tear production in normal juvenile dogs and evaluate the effects of age, weight, and gender on Schirmer tear test (STT) in juvenile dogs. MATERIALS AND METHODS Healthy puppies of various breeds <6 months of age. STT1 and STT2 were performed in both eyes of each subject. Statistical analysis was performed using a backwards stepwise regression model with repeated measures. Using continuous variables of STT1 and STT2 as the dependent variables separately, the independent variables were age (days), eye (left or right), gender (male or female), ocular disease processes in eyes or not, and weight (kg). RESULTS Eighty-six eyes from 27 males and 16 females were included in this study. Ages ranged from 25 to 133 (mean ± SEM: 61.74 ± 24.15) days and weights ranged from 0.88 to 8.86 (3.27 ± 2.22) kg. STT1 results ranged from 0 to 26 (15.76 ± 5.79) mm/min. STT2 results ranged from 0 to 24 (8.79 ± 5.01) mm/min. Age, weight, and gender significantly affected STT1 results. Weight and gender significantly affected STT2 results. STT1 values increased by 0.15 mm/min for each 1 day increase in age and by 0.84 mm/min for each 1 kg increase in body weight. STT2 values increased by 0.57 mm/min for each 1 kg increase in body weight. CONCLUSIONS Age, weight, and gender significantly affect tear production in normal juvenile dogs. STT1 increases to adult values at approximately 9-10 weeks of age.

Feline Uveitis: Diagnosis and Treatment

Uveitis is the inflammation of any or all parts of the vascular tunic of the eye; the vascular tunic includes the iris, the ciliary body, and choroid. A good knowledge base, up-to-date reference materials, and good instruments will improve the diagnosis of uveitis. Feline uveitis can be caused by numerous infectious agents in addition to neoplasia and less likely trauma. The infectious causes most commonly associated with feline uveitis include feline leukemia virus, feline immunodeficiency virus, feline infectious peritonitis, systemic fungal infections, toxoplasmosis, and bartonellosis. Neoplastic causes of uveitis can be primary or secondary. Iris melanoma is the most common primary uveal neoplasia and trauma-associated sarcoma is the second most common primary uveal neoplasia. Treatment for the clinical signs of anterior uveitis include topical steroidal or non-steroidal anti-inflammatory agents, parasympatholytic agents for ciliary spasm, to keep the pupil dilated, and to prevent posterior synechia. Posterior uveitis should be treated with systemic medications that will address the underlying cause. Enucleation of blind, painful eyes not responsive to medications is a means to alleviate the animal’s discomfort and to further diagnose the underlying cause.

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. Bowmans layer was difficult to see by light microscopy but clear with transmission electron microscopy. The cornea had a thick epithelium, thin endothelium and Descemets 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.