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.

Estradiol biosynthesis in canine lens epithelial cells.

PURPOSE: To confirm that lens epithelial cells (LEC) synthesize 17ß-estradiol, active estrogen, and to identify the pathway(s) by which normal and cataractous LEC synthesize 17ß-estradiol. METHODS: ELISA was used to measure estradiol in aqueous humor; immunohistochemical staining was used to localize estradiol, testosterone and sulfatase; tritiated water release assay was used to measure aromatase activity; and qRT-PCR was used to quantify expression of aromatase and sulfatase in normal and cataractous canine and human LEC. RESULTS: Canine eyes with and without cataracts had no differences in aqueous humor estradiol levels; however, cataractous LEC had more intense immunoreactivity for estradiol than normal LEC. There were little to no differences in canine sulfatase protein and mRNA expression when normal and cataractous LEC were compared. qRT-PCR demonstrated that canine cataractous LEC had significantly higher expression of aromatase; this was confirmed with the tritiated water release assay. Similar to dogs, human cataracts had both sulfatase and aromatase mRNA expression. CONCLUSIONS: Normal and cataractous LEC can synthesize estradiol by the sulfatase pathway; however, cataractous LEC appear to use the aromatase pathway as well. Because no differences in aqueous humor estradiol levels were detected, we suspect that estradiol synthesized by the sulfatase pathway is secreted into the aqueous humor; whereas, estradiol synthesized by the aromatase pathway is used for, as yet unknown, intracrine purposes.

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.

Sustained-release celecoxib from incubated acrylic intraocular lenses suppresses lens epithelial cell growth in an ex vivo model of posterior capsule opacity.

PURPOSE: To determine whether celecoxib (CXB) can be released from incubated intraocular lenses (IOLs) sufficiently to inhibit lens epithelial cell (LEC) growth in an ex vivo model of posterior capsule opacification (PCO). MATERIALS: LEC growth was evaluated for 14 days in canine lens capsules (LCs) that had been exposed to media containing 20 µM CXB for 1-5 days. After the incubation of hydrophilic and hydrophobic IOLs in CXB solution, the determination of the in vitro release of CXB from the IOLs was performed for up to 28 days. The incubated and nonincubated IOLs were evaluated in the ex vivo model of PCO, and the rate of LEC growth was evaluated over 28 days. RESULTS: The treatment of LCs with 20 µM CXB for 4 and 5 days completely inhibited LEC growth. LEC repopulation did not occur after the removal of CXB. IOLs incubated in CXB for 24 h resulted in a sustained release of CXB in vitro at levels theoretically sufficient to inhibit PCO. LCs in the ex vivo model of PCO treated with acrylic IOLs incubated in CXB had significantly suppressed LEC ingrowth compared with untreated and IOL-only LCs. CONCLUSIONS: A 4-day treatment of LCs with a concentration of 20 µM CXB may effectively prevent PCO. IOLs incubated in CXB for 24 h resulted in a sustained release of CXB in vitro at levels sufficient to inhibit LEC growth in the ex vivo model of PCO. Further studies are needed to determine whether CXB-incubated IOLs can effectively prevent the development of PCO in vivo.

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.

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.

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.

Isolation and characterization of primary canine lens epithelial cells.

OBJECTIVE: The purpose of this study was to characterize the proliferation and differentiation of primary canine lens epithelial cells (LEC) under standard culture conditions. PROCEDURE: Canine LEC were isolated by mechanical dissection of the canine globe and enzymatic digestion of the lens capsule from fresh lenses. Isolated capsules and cell suspensions were seeded in laminin-coated culture flasks. Canine LEC proliferated and formed monolayers, which could be passaged and maintained for approximately 2 weeks. Cells were characterized morphologically and cell lysates examined for expression of protein markers of epithelial origin and differentiation. RESULTS: Canine LEC exhibit morphologic characteristics of epithelial cells when cultured on laminin/lysine coated flasks. Expression of epithelial cell marker, cytokeratin 5, was highest at passage 1 and diminished with increasing passage number. Expression of gamma-crystallin, a protein found only in differentiated lens fiber cells, increased at passage 6. A laminin/lysine-coated surface supported optimal proliferation of canine LEC. Both an initial seeding density of 1 x 10(5) cells/cm(2) and culture in Dulbecco's modified essential media (DMEM) supplemented with 10% FBS supported a doubling time of less than 48 h in canine LEC. CONCLUSION: This study demonstrates that primary canine LEC retain the characteristics of lens epithelial cells prior to passage 6 under the described culture conditions and represent a suitable in vitro model for investigating lens physiology and cataractogenesis.

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.

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.

Prevention of posterior capsular opacification through cyclooxygenase-2 inhibition.

PURPOSE: To determine if cyclooxygenase-2 (COX-2) is upregulated when lens epithelial cells (LEC) in clinical samples of cataracts and posterior capsule opacification (PCO) undergo epithelial-mesenchymal transition (EMT)-like changes. We also wanted to learn if inhibition of the enzymatic activity of COX-2 could prevent PCO formation. METHODS: To ensure that EMT-like changes were occurring in LEC, real-time RT-PCR was used to examine expression of EMT markers. Clinical samples of canine cataracts and PCO were examined for COX-2 expression using immunohistochemistry, western blot analysis, and real-time RT-PCR. The COX-2 inhibitors, rofecoxib and celecoxib, were used in an ex vivo model of PCO formation, and the effects on cellular migration, proliferation, and apoptosis were analyzed using immunohistochemistry and western blots. Prostaglandin E2 (PGE2) expression was examined with ELISA. RESULTS: Markers of EMT, such as lumican, Snail, Slug, and COX-2 were expressed in LEC. In clinical samples of cataracts and PCO, there was overexpression of COX-2 protein and mRNA. Both rofecoxib and celecoxib were effective at inhibiting PCO formation in our ex vivo model. Prevention of PCO with the COX-2 inhibitors appeared to work through decreased migration and proliferation, and increased apoptosis. Neither of the drugs had a toxic effect on confluent LEC and appeared to inhibit PCO through their pharmacologic action. Synthesis of PGE2 was inhibiting in the capsules treated with the COX-2 inhibiting drugs. CONCLUSIONS: Extracapsular phacoemulsification cataract surgery is the most common surgical procedure performed in human and veterinary ophthalmology. The most frequent postoperative complication is PCO. The LEC that remain adhered to the lens capsule undergo EMT-like changes, proliferate, and migrate across the posterior lens capsule causing opacities. We have shown that COX-2, a protein associated with EMT, is upregulated in canine cataracts and PCO. Inhibiting the enzymatic activity effectively prevented EMT of LEC in our ex vivo model of PCO through pharmacologic action, and not acute toxicity. These findings indicate that using COX-2 inhibitors in vivo may be an effective technique in preventing PCO.

Regulation of gap junction intercellular communication in primary canine lens epithelial cells: role of protein kinase C.

PURPOSE: Gap junction intercellular communication (GJIC) is important in maintaining lens epithelial cell homeostasis and reductions in GJIC may be associated with the development of cataract. Protein kinase C (PKC) activation can disrupt gap junction communication via phosphorylation of connexin 43 (C x 43) proteins that compose gap junction channels. This study examined the role of PKC activation in modulating GJIC in a primary canine lens epithelial cell (LEC) line. METHODS: TPA (12-O-tetradecanoyl-phorbol-acetate), a potent PKC activator and inhibitor of GJIC, was utilized in the present study. Primary cultures of canine LEC were treated with TPA (0-1000 ng/ml) for 0.5 hr and GJIC was assessed by scrape loading/dye transfer (SL/DT), and immunoblotting to detect phosphorylation of C x 43 protein. Inhibition of general and calcium-dependent PKC activity was achieved by pretreatment of cells with GF109203X and Gö6976, respectively. RESULTS: Treatment with TPA (1-1000 ng/ml) significantly decreased GJIC in canine LEC as assessed by SL/DT. Pretreatment with 10 and 100 ng/ml TPA decreased GJIC by 80% as compared to controls and increased Cx43 phosphorylation as assessed by immunoblotting. Pretreatment of cells with GF109203X and Gö6976, partially restored TPA-inhibited GJIC by 40% and 60%, respectively, and reduced C x 43 phosphorylation. Expression of calcium dependent PKC isoforms was detected in canine whole lens and LEC. CONCLUSIONS: Treatment with TPA significantly reduces GJIC in canine LEC. These effects are mediated, in part, by activation of calcium-dependent PKC isoforms. Primary canine LEC are a useful model in the study of the molecular mechanisms involved in GJIC and cataractogenesis.

The role of the slug transcription factor in cell migration during corneal re-epithelialization in the dog.

Epithelial cell migration during corneal wound re-epithelialization shares features with the developmental process of epithelial-mesenchymal transition (EMT) modulated by Snail family transcription factors, including Slug. Our studies demonstrated that Slug expression was enhanced at sites of epithelial cell migration at the margins of normally healing corneal wounds in dogs, but significantly decreased at the margins of non-healing canine corneal erosions. Increased Slug expression was associated with internalization of E-cadherin and beta-catenin from the cell membrane and with enhanced expression of smooth-muscle-specific alpha-actin, tropomyosin, and matrix metalloproteinases at wound margins. Enhanced Slug expression in corneal explants due to an adenoviral expression construct or to oxytetracycline treatment resulted in significantly higher rates of corneal epithelial cell migration. Oxytetracycline appeared to act by stimulating transforming growth factor-beta activity, thus increasing Slug expression and enhancing corneal epithelial migration. These findings highlight the similarities between epithelial migration during EMT and during successful corneal wound healing, support an important role for the Snail family in the process, and indicate that modulating Slug expression may be clinically useful in treating non-healing corneal wounds.

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.

Ultraviolet irradiation up-regulates telomerase transcription and activity in lens epithelial cells.

PURPOSE: Ultraviolet irradiation (UVR) increases telomerase activity in various cell types including skin, a sun-exposed organ. The lens is also continually exposed to UVR and we hypothesized that lenses exposed to UVR would have increased telomerase activity, with up-regulated TERT and TR, the two main components of the telomerase holoenzyme. To evaluate whether the cornea would protect lenses from such changes, whole globes, as well as isolated lenses, were exposed to UVR, and lenses were evaluated for changes in telomerase activity. METHODS: There were three parts to this project. The first part of this experiment evaluated freshly harvested normal adult canine lenses exposed to 0, 300, 600, or 1200 J/m(2) UVR, and then allowed to recover for 1, 2, 3 and 4 h. Since only 600 J/m(2) UVR increased telomerase activity, four more postexposure recovery time-points for this UVR dose were evaluated: 10 min, 30 min, 8 h and 24 h. The second part of this experiment used freshly enucleated whole canine globes exposed to 0, 50, 100, 150, 300, 600 or 1200 J/m(2) and incubated overnight; lens epithelial cells (LEC) were evaluated for telomerase activity. The third part evaluated lenses that were exposed to 0 or 600 J/m(2) UVR, and then allowed to recover for 8 and 24 h, before TERT and TR mRNA levels were measured. RESULTS: Isolated lenses exposed to 600 J/m(2) UVR had significantly higher telomerase activity than unexposed controls and other UVR doses, at all time-points except 24 h postexposure. Lenses from whole globes exposed to UVR showed a dose-dependent increase in telomerase activity except at 50 J/m(2) and 1200 J/m(2). Isolated lenses exposed to 600 J/m(2) UVR and then allowed to recover for 8 and 24 h significantly up-regulated TERT and TR mRNAs compared to unexposed control lenses. CONCLUSIONS: Telomerase activity is regulated at both the transcriptional and post-translational levels in canine LEC. Previous work in our laboratory showed dose, time, and age-dependent changes in telomerase activity in the lens. The present study showed that TERT and TR mRNA transcription was increased for up to 24 h following an acute dose of UVR. Both telomerase activity and TERT and TR mRNA levels were elevated until 24 h post-UVR exposure, TERT in combination with TR functions in proliferation-related telomerase activity, but TERT alone has an anti-apoptotic function and its up-regulation may protect LEC from the acute effects of UVR. We are continuing to evaluate the mechanisms by which telomerase is regulated in normal and cataractous LEC.

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.