Evaluation of a commercial NGS service for detection of bacterial and fungal pathogens in infectious ulcerative keratitis
Vet Ophthalmol. 2023 Nov;26(6):500-513. doi: 10.1111/vop.13069. Epub 2023 Mar 21.
OBJECTIVES: To compare results from a commercial next-generation sequencing (NGS) service to corneal cytology and culture for identification of causative organisms in veterinary patients presenting for infectious ulcerative keratitis (IUK).
PROCEDURE: Swabs for corneal aerobic and fungal cultures and DNA swabs for NGS were submitted for canine and equine normal controls (n = 11 and n = 4, respectively) and IUK patients (n = 22 and n = 8, respectively) for which microbrush cytology specimens confirmed the presence of infectious organisms. The sensitivity of the NGS results was compared with bacterial and fungal culture results. Concordance between the NGS and culture results was determined.
RESULTS: The NGS results were positive for bacterial and fungal organisms in 5 and 1 normal and 18 and 1 IUK cases, respectively. Bacterial and fungal cultures were positive for 7 and 2 normal and 20 and 5 IUK cases, respectively. Sensitivity of NGS was 82.14% (95% confidence interval (CI), 63.11% to 93.94%) and specificity was 76.47% (95% CI, 50.10% to 93.19%). Concordance (complete and partial) between identified bacterial and fungal organisms was found in 79% and 100% of cases, respectively. NGS identified organisms in 3 culture-negative IUK samples.
CONCLUSION: A commercial NGS service may be useful in the identification of causative agents in IUK cases with a sensitivity greater than the sensitivity previously reported for aerobic culture. Further testing is needed to determine the clinical significance of additional organisms isolated by NGS from infected cases, as well as organisms isolated from normal corneas.
Mouse Corneal Transplantation
Methods Mol Biol. 2023;2597:19-24. doi: 10.1007/978-1-0716-2835-5_3.
Corneal transplantation is the most common form of organ transplantation worldwide. Transplant survival depends on various factors, many of which are not fully understood. Due to the existence of many genetically defined strains, mouse models of corneal transplantation are most commonly used. Here, we describe a method for a mouse corneal transplantation.
Sustained release of heme-albumin as a potential novel therapeutic approach for age-related macular degeneration
Biomater Sci. 2022 Dec 6;10(24):7004-7014. doi: 10.1039/d2bm00905f.
Globally, age-related macular degeneration (AMD) is the third most common visual impairment. Most often attributed to cellular fatigue with aging, over expression of reactive oxygen species (ROS) causes ROS accumulation in the retina, leading to chronic inflammatory immune signaling, cellular and tissue damage, and eventual blindness. If left uncontrolled, the disease will progress from the dry form of AMD to more severe forms such as geographic atrophy or wet AMD, hallmarked by choroidal neovascularization. There is no cure for AMD and treatment options are limited. Treatment options for wet AMD require invasive ocular injections or implants, yet fail to address the disease progressing factors. To provide more complete treatment of AMD, the application of a novel anti-inflammatory heme-bound human serum albumin (heme-albumin) protein complex delivered by antioxidant ROS scavenging polydopamine (PDA) nanoparticles (NPs) for sustained treatment of AMD was investigated. Through the induction of heme oxygenase-1 (HO-1) by heme-albumin in retinal pigment epithelial (RPE) cells, anti-inflammatory protection may be provided through the generation of carbon monoxide (CO) and biliverdin during heme catabolism. Our results show that the novel protein complex has negligible cytotoxicity towards RPE cells (ARPE-19), reduces oxidative stress in both inflammatory and ROS in vitro models, and induces a statistically significant increase in HO-1 protein expression. When incorporated into PDA NPs, heme-albumin was sustainably released for up to 6 months, showing faster release at higher oxidative stress levels. Through its ability to react with ROS, heme-albumin loaded PDA NPs showed further reduction of oxidative stress with minimal cytotoxicity. Altogether, we demonstrate that heme-albumin loaded PDA NPs reduce oxidative stress in vitro and can provide sustained therapeutic delivery for AMD treatment.
Amphiphilic silicones to mitigate lens epithelial cell growth on intraocular lenses
J Mater Chem B. 2022 Apr 20;10(16):3064-3072. doi: 10.1039/d2tb00213b.
Silicone intraocular lenses (IOLs) that resist lens epithelial cell (LEC) growth would greatly improve patient outcomes. Herein, amphiphilic surface modifying additives (SMAs) were incorporated into an IOL-type diphenyl silicone to reduce LEC growth without compromising opto-mechanical properties. The SMAs were poly(ethylene oxide)-silane amphiphiles (PEO-SAs) [H-Si-ODMSm-block-PEO8-OCH3], comprised of a PEO segment and siloxane tether of varying lengths (m = 0, 13, and 30). These three SMAs were each blended into the addition cure diphenyl silicone at varying concentrations (5, 10, 15, 20, and 25 μmol g-1) wherein the wt% of PEO was maintained for all SMAs at a given molar concentration. The chemical crosslinking and subsequent retention of SMAs in modified silicones was confirmed. Key material properties were assessed following equilibration in both air and aqueous environments. Silicones modified with SMAs having longer tethers (m = 13 and 30) underwent rapid and substantial water-driven restructuring of PEO to the surface to form highly hydrophilic surfaces, especially as SMA concentration increased. The % transmittance was also maintained for silicones modified with these particular SMAs. The moduli of the modified silicones were largely unchanged by the SMA and remained in the typical range for silicone IOLs. When the three SMAs were introduced at the highest concentration, modified silicones remained non-cytotoxic and LEC count and associated alpha-smooth muscle actin (α-SMA) expression decreased with increasing tether length. These results demonstrate the potential of silicones modified with PEO-SA SMAs to produce LEC-resistant IOLs.
Recombinant Human MG53 Protein Protects Against Alkaline-Induced Corneal Injuries in Mice
Mil Med. 2021 Jan 25;186(Suppl 1):486-490. doi: 10.1093/milmed/usaa357.
INTRODUCTION: The current study was designed to test the potential role of recombinant human MG53 (rhMG53) protein on protecting against alkaline-induced corneal injury in mice.
MATERIALS AND METHODS: A round filter paper with 2-mm diameter was soaked in 1 mol/L of NaOH solution. The mouse alkaline injury was generated by placing the filter paper directly on the cornea for 30 seconds and washed with 30-mL saline; 10 µL of rhMG53 solution (20 µg/mL) or saline control was topically administrated on the mouse corneas (twice per day for 10 days). Re-epithelialization was measured by fluorescein staining and imaged by a slit lamp equipped with a digital camera. Clinical neovascularization and opacity scores were measured every day after injury. Ten days after injury, mice were sacrificed and corneas were dissected out for flat mount staining of CD31 for neovascularization.
RESULTS: MG53 was present in both dog aqueous humor and human tears. mg53-/- corneas were more susceptible to alkaline-induced corneal injury. Topical treatment of rhMG53 improved re-epithelialization, suppressed neovascularization, and fibrosis induced by alkaline injury.
CONCLUSIONS: rhMG53 may be an effective means to treat corneal wounding.
Using minimum inhibitory concentration values of common topical antibiotics to investigate emerging antibiotic resistance: A retrospective study of 134 dogs and 20 horses with ulcerative keratitis
Vet Ophthalmol. 2020 Sep;23(5):806-813. doi: 10.1111/vop.12801. Epub 2020 Jul 1.
OBJECTIVES: To identify the minimum inhibitory concentration (MIC) distribution for commonly used topical antibiotics from isolates of dogs and horses with ulcerative bacterial keratitis, and to investigate changes in MIC values over time and following treatment with topical fluoroquinolones.
ANIMALS STUDIED: One hundred thirty-four client-owned dogs and 20 client-owned horses with bacterial ulcerative keratitis.
PROCEDURE: Minimum inhibitory concentration values for 14 topical antibiotics were reported for canine and equine cases of bacterial ulcerative keratitis between 2013 and 2018. Changes in MIC values over time and after treatment with topical fluoroquinolones were reported.
RESULTS: The three most common bacterial genera isolated were Staphylococcus, Streptococcus, and Pseudomonas. Together, these represented 79.4% of canine cases and 77.4% of equine cases. Overall, isolates from horses tended to have lower MIC values, as did Pseudomonas isolates from both dogs and horses, compared to other bacterial genera, especially Staphylococcus spp. The MIC values of erythromycin and trimethoprim sulfa for Staphylococcus spp., and the MIC value of moxifloxacin for Pseudomonas significantly increased over time. Previous topical fluoroquinolone use was associated with a significant increase in the MIC value of ofloxacin in canine and equine Staphylococcus isolates and current topical fluoroquinolone use was associated with significant increases in the MIC values of ciprofloxacin, moxifloxacin, and ofloxacin in canine Staphylococcus isolates.
CONCLUSION: Patients previously or currently treated with topical fluoroquinolones, particularly in Staphylococcus infections, may require alternative antibiotics or additional antibiotic classes other than fluoroquinolones. Bacterial culture with MIC susceptibility testing should be highly recommended when a Staphylococcal infection is suspected.
A Hydrogel Vitreous Substitute that Releases Antioxidant
Macromol Biosci. 2020 Feb;20(2):e1900305. doi: 10.1002/mabi.201900305. Epub 2019 Dec 17.
Current experimental vitreous substitutes only replace the physical functions of the natural vitreous humor. Removal of the native vitreous disrupts oxygen homeostasis in the eye, causing oxidative damage to the lens that likely results in cataract formation. Neither current clinical treatments nor other experimental vitreous substitutes consider the problem of oxidative stress after vitrectomy. To address this problem, biomimetic hydrogels are prepared by free radical polymerization of poly(ethylene glycol) methacrylate and poly(ethylene glycol) diacrylate. These hydrogels have similar mechanical and optical properties to the vitreous. The hydrogels are injectable through small-gauge needles and demonstrate in vitro biocompatibility with human retinal and lens epithelial cells. The hydrogels and added vitamin C, an antioxidant, show a synergistic effect in protecting ocular cells against reactive oxygen species, which fulfills a chemical function of the natural vitreous. These hydrogels have the potential to prevent post-vitrectomy cataract formation and reduce the cost of additional surgeries.
Lens Stretching Modulates Lens Epithelial Cell Proliferation via YAP Regulation
Invest Ophthalmol Vis Sci. 2019 Sep 3;60(12):3920-3929. doi: 10.1167/iovs.19-26893.
PURPOSE: The continuous growth of the lens throughout life may contribute to the onset of age-related conditions in the lens (i.e., presbyopia and cataract). Volumetric growth is the result of continuous proliferation of lens epithelial cells (LECs). The driving factors controlling LEC proliferation are not well understood. This study tested the hypothesis that mechanical stretching modulates LEC proliferation.
METHODS: Biomechanical regulation of LEC proliferation was investigated by culturing whole porcine lenses and connective tissues ex vivo under varying physiologically relevant stretching conditions using a bespoke lens stretching device. Additionally, some lenses were treated with a YAP function inhibitor to determine the Hippo signaling pathway's role in regulating lens growth. Resulting changes in LEC labeling index were analyzed using EdU incorporation and flow cytometry for each lens.
RESULTS: LEC proliferation was found to be modulated by mechanical strain. Increasing both the magnitude of static stretching and the stretching frequency in cyclic stretching resulted in a proportional increase in the labeling indices of the LECs. Additionally, treatment with the YAP function inhibitor effectively eliminated this relationship.
CONCLUSIONS: These data demonstrate that LEC proliferation is regulated in part, by the mechanotransduction of stresses induced in the lens capsule and that YAP plays an important role in mechanosensing. These results have important implications for understanding lens growth and morphogenesis. The model may also be used to identify and evaluate targets for modulating lens growth.
Determination of trypan blue efficacy in the mitigation of ex vivo canine PCO formation
Vet Ophthalmol. 2019 Nov;22(6):902-909. doi: 10.1111/vop.12669. Epub 2019 Apr 3.
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
Commun Biol. 2019 Feb 20;2:71. doi: 10.1038/s42003-019-0316-7. eCollection 2019.
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-β-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
Am J Vet Res. 2018 Jul;79(7):770-778. doi: 10.2460/ajvr.79.7.770.
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.
Objective evaluation of the systemic effects of topical application of 1% atropine sulfate ophthalmic solution in healthy horses
J Am Vet Med Assoc. 2017 Dec 1;251(11):1324-1330. doi: 10.2460/javma.251.11.1324.
OBJECTIVE To determine the safety of topical administration of 1% atropine ophthalmic solution in healthy horses by objectively measuring gastrointestinal transit time. DESIGN Randomized, masked, controlled crossover study. ANIMALS 6 adult geldings. PROCEDURES Horses were randomly assigned (3/group) to first receive topical treatment of the left eye with 1% atropine or artificial tears solution; the right eye was left untreated. After 24 hours of treatment every 6 hours, 200 nontoxic beads were administered to each horse via nasogastric intubation and treatment frequency was decreased to every 12 hours for 4 more days. Pupillary light reflexes (PLRs), mydriasis, heart rate, fecal bead passage, abdominal girth measurements, auscultable gut sounds, fecal weight, and clinical signs of abdominal pain were monitored. Following a 4-week washout period, horses received the opposite treatment in the left eye and measurements were repeated. Serum atropine concentration (reflecting systemic absorption) was measured with an ELISA at various points after initial atropine administration. RESULTS No horse had subjective or objective evidence of colic or ileus at any monitoring point. Complete mydriasis of the left eye with absence of the PLR was identified in 5 horses within 6 hours and in all 6 horses within 12 hours after initial atropine administration. One horse had mydriasis with an absent PLR in the untreated eye by day 5 of atropine treatment. At no point was atropine detected in serum samples of any horse. CONCLUSIONS AND CLINICAL RELEVANCE Topical atropine application at clinically appropriate doses induced no evidence of ileus in healthy horses.
Oxidative Stress Measures of Lipid and DNA Damage in Human Tears
Invest Ophthalmol Vis Sci. 2017 May 1;58(6):BIO151-BIO157. doi: 10.1167/iovs.17-21436.
PURPOSE: We evaluate feasibility and repeatability of measures for lipid peroxidation and DNA oxidation in human tears, as well as relationships between outcome variables, and compared our findings to previously reported methods of evaluation for ocular sun exposure.
METHODS: A total of 50 volunteers were seen for 2 visits 14 ± 2 days apart. Tear samples were collected from the inferior tear meniscus using a glass microcapillary tube. Oxidative stress biomarkers were quantified using enzyme-linked immunosorbent assay (ELISA): lipid peroxidation by measurement of hexanoyl-lysine (HEL) expression; DNA oxidation by measurement of 8-oxo-2'-deoxyguinosone (8OHdG) expression. Descriptive statistics were generated. Repeatability estimates were made using Bland-Altman plots with mean differences and 95% limits of agreement were calculated. Linear regression was conducted to evaluate relationships between measures.
RESULTS: Mean (±SD) values for tear HEL and 8OHdG expression were 17368.02 (±9878.42) nmol/L and 66.13 (±19.99) ng/mL, respectively. Repeatability was found to be acceptable for both HEL and 8OHdG expression. Univariate linear regression supported tear 8OHdG expression and spring season of collection to be predictors of higher tear HEL expression; tear HEL expression was confirmed as a predictor of higher tear 8OHdG expression.
CONCLUSIONS: We demonstrate feasibility and repeatability of estimating previously unreported tear 8OHdG expression. Seasonal temperature variation and other factors may influence tear lipid peroxidation. Support is demonstrated to suggest lipid damage and DNA damage occur concurrently on the human ocular surface.
Subconjunctival antimicrobial poloxamer gel for treatment of corneal ulceration in stranded California sea lions (Zalophus californianus)
Vet Ophthalmol. 2017 Sep;20(5):441-449. doi: 10.1111/vop.12447. Epub 2016 Dec 1.
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.
Seasonal Effect on Ocular Sun Exposure and Conjunctival UV Autofluorescence
Optom Vis Sci. 2017 Feb;94(2):219-228. doi: 10.1097/OPX.0000000000001014.
PURPOSE: To evaluate feasibility and repeatability of measures for ocular sun exposure and conjunctival ultraviolet autofluorescence (UVAF), and to test for relationships between the outcomes.
METHODS: Fifty volunteers were seen for two visits 14 ± 2 days apart. Ocular sun exposure was estimated over a 2-week time period using questionnaires that quantified time outdoors and ocular protection habits. Conjunctival UVAF was imaged using a Nikon D7000 camera system equipped with appropriate flash and filter system; image analysis was done using ImageJ software. Repeatability estimates were made using Bland-Altman plots with mean differences and 95% limits of agreement calculated. Non-normally distributed data was transformed by either log10 or square root methods. Linear regression was conducted to evaluate relationships between measures.
RESULTS: Mean (±SD) values for ocular sun exposure and conjunctival UVAF were 8.86 (±11.97) hours and 9.15 (±9.47) mm, respectively. Repeatability was found to be acceptable for both ocular sun exposure and conjunctival UVAF. Univariate linear regression showed outdoor occupation to be a predictor of higher ocular sun exposure; outdoor occupation and winter season of collection both predicted higher total UVAF. Furthermore, increased portion of day spent outdoors while working was associated with increased total conjunctival UVAF.
CONCLUSIONS: We demonstrate feasibility and repeatability of estimating ocular sun exposure using a previously unreported method and for conjunctival UVAF in a group of subjects residing in Ohio. Seasonal temperature variation may have influenced time outdoors and ultimately calculation of ocular sun exposure. As winter season of collection and outdoor occupation both predicted higher total UVAF, our data suggests that ocular sun exposure is associated with conjunctival UVAF and, possibly, that UVAF remains for at least several months after sun exposure.
Heat-shock protein expression in canine corneal wound healing
Vet Ophthalmol. 2016 May;19(3):262-6. doi: 10.1111/vop.12302. Epub 2015 Aug 24.
OBJECTIVE: Heat-shock proteins, particularly the 70-kDa member (Hsp70), have been implicated in facilitating wound healing in multiple tissues. Expression and localization of three HSPs were assessed in normal and wounded canine corneas to elucidate a role in epithelial healing.
METHODS: Paraffin-embedded normal corneas, acute and repeatedly abraded corneas, and keratectomies of spontaneous chronic corneal epithelial defects (SCCEDs) were subjected to routine immunohistochemistry for Hsp27, 47, and 70 expression. Ex vivo corneal defects were created and treated with anti-HSPs or IgG controls, and wound healing was monitored. Primary cultures of canine corneal stromal fibroblasts and corneal epithelial cells were treated with exogenous Hsp70, and an artificial wound was created in vitro to monitor restoration of the monolayer.
RESULTS: Normal canine corneas exhibited constitutive expression of all HSPs evaluated. Inducible expression was demonstrated in acutely wounded tissues, and expression in the chronically abraded corneas was relocalized. All HSP expression was below the limits of detection in the epithelium of SCCED samples. Inhibition of HSPs in culture resulted in delayed wound healing when compared to controls. Hsp70-treated fibroblasts demonstrated significantly (P < 0.001) increased migration and proliferation compared to the vehicle control; however, there was no significant effect of exogenous Hsp70 on corneal epithelial cells.
CONCLUSIONS: These findings suggest that HSPs are induced in the normal canine cornea during re-epithelialization. Hsp70 expression is likely important for inducing the cytoarchitectural remodeling, migration, and proliferation necessary early in the canine corneal healing response, and suppressed expression may contribute to the pathophysiology of nonhealing defects.
Cyclosporine A prevents ex vivo PCO formation through induction of autophagy-mediated cell death
Exp Eye Res. 2015 May;134:63-72. doi: 10.1016/j.exer.2015.03.020. Epub 2015 Mar 31.
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.
Estradiol biosynthesis in canine lens epithelial cells
Curr Eye Res. 2015 May;40(5):541-8. doi: 10.3109/02713683.2014.935446. Epub 2014 Sep 26.
PURPOSE: To confirm that lens epithelial cells (LEC) synthesize 17β-estradiol, active estrogen, and to identify the pathway(s) by which normal and cataractous LEC synthesize 17β-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.
Aloe vera: an in vitro study of effects on corneal wound closure and collagenase activity
Vet Ophthalmol. 2014 Nov;17(6):403-10. doi: 10.1111/vop.12163. Epub 2014 Mar 25.
PURPOSE: To evaluate the in vitro effects of an aloe vera solution on (i) the viability and wound healing response of corneal cells and (ii) the ability to alter collagenase and gelatinase activities.
METHODS: Primary cultures of corneal epithelial cells and fibroblasts were prepared from grossly normal enucleated canine globes and treated with an aloe solution (doses ranging from 0.0-2 mg/mL). Cellular viability was evaluated using a colorimetric assay. A corneal wound healing model was used to quantify cellular ingrowth across a defect made on the confluent surface. Anticollagenase and antigelatinase activities were evaluated by incubating a bacterial collagenase/gelatinase with aloe solution (doses ranging from 0.0-500 μg/mL) and comparing outcome measures to a general metalloproteinase inhibitor, 1, 10-phenanthroline, and canine serum (doses ranging from 0.0-100%).
RESULTS: None of the concentrations of aloe solution tested significantly affected the viability of corneal epithelial cells or fibroblasts. Concentrations ≤175 μg/mL slightly accelerated corneal epithelial cell wound closure; this change was not significant. Concentrations ≥175 μg/mL significantly (P ≤ 0.001) slowed the rate of corneal fibroblast wound closure, while aloe concentrations <175 μg/mL did not significantly alter fibroblast wound closure. Aloe solution did not alter the ability for collagenase to degrade gelatin or collagen Type I but increased the ability for collagenase to degrade Type IV collagen.
CONCLUSIONS: Although additional experiments are required, lower concentrations of aloe solution may be beneficial in healing of superficial corneal wounds to help decrease fibrosis and speed epithelialization. An increase in collagenase activity with aloe vera warrants further testing before considering in vivo studies.
Effects of pulsed fluid lens capsule washing following phacoemulsification on lens epithelial cells and posterior capsule opacification formation ex vivo
Vet Ophthalmol. 2015 May;18(3):221-8. doi: 10.1111/vop.12143. Epub 2014 Jan 22.
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.
Equine glaucoma: a histopathologic retrospective study (1999-2012)
Vet Ophthalmol. 2014 Sep;17(5):334-42. doi: 10.1111/vop.12080. Epub 2013 Jul 16.
PURPOSE: To characterize and describe the histopathologic findings in equine globes enucleated due to glaucoma.
METHODS: Medical records at The Ohio State University from 1999 to 2012 were reviewed retrospectively. Signalment, history, and treatment data were collected, and histologic slides of enucleated globes were examined and lesions recorded. Twenty-three eyes from 23 horses were eligible for inclusion in this study.
RESULTS: The majority of affected horses were > 15 years of age (65%). The ages ranged from 5 to 35 years (mean = 17.4 years). The left eye was affected in 10 cases (43%) and the right eye in 13 cases (57%). There were 13 mares (56%) and 10 geldings (44%). Quarter Horses (30%), Appaloosas (26%), and Thoroughbreds (22%) were the most common breeds in the study population. The most common histopathologic changes included hypercellularity of the optic nerve (93%), retinal atrophy (89%), corneal vascularization (83%), descemetization of pectinate ligaments (83%), hypercellularity of the anterior corneal stroma (75%), posterior bowing of the iris base (74%), ciliary body atrophy (74%), corneal striae (70%), pars plana elongation (60%), cataract (53%), and collapsed ciliary cleft/trabecular meshwork (52%). Evidence of uveitis (cataract, lymphoplasmacytic infiltration of the uvea, and/or anterior or posterior synechiae) was present in 20/23 eyes (87%).
CONCLUSIONS: Equine glaucoma most commonly occurs secondary to uveitis with Appaloosas and older horses predisposed. Histologic changes are comparable to prior reports of chronic glaucoma; notable findings not previously described in the horse were posterior bowing of the iris base and relative sparing of the superior retina from atrophy associated with elevated IOP.
Analysis of the transport of and cytotoxic effects for nalbuphine solution in corneal cells
Am J Vet Res. 2012 Dec;73(12):1987-95. doi: 10.2460/ajvr.73.12.1987.
OBJECTIVE: To assess the in vitro effects of various nalbuphine concentrations on viability and wound healing ability of corneal cells and potential drug transport through the corneal epithelium.
SAMPLE: Cultured canine and human corneal epithelial cells (CECs) and cultured canine corneal stromal fibroblasts.
PROCEDURES: CECs and stromal fibroblasts were exposed to nalbuphine (concentration of solutions ranged from 0% to 1.2%) for up to 30 minutes, and viability was assessed with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A standard scratch test technique was used. Wound healing of CECs and stromal fibroblasts was evaluated following treatment with nalbuphine solutions < 0.1%. Liquid chromatography-mass spectrometry-mass spectrometry analysis was used to evaluate drug transport across a monolayer and a multilayer of human CECs.
RESULTS: A progressive decrease in viability was detected in canine CECs for all nalbuphine treatment groups, whereas treatment with only 0.5% or 1.2% nalbuphine significantly reduced corneal stromal fibroblast viability, compared with results for control cells. Within 24 hours, treatment with 0.1% nalbuphine solution significantly altered the healing rate of both canine CECs and stromal fibroblasts. Continuous increases in transport rates of nalbuphine were detected with time for both the monolayer and multilayer of human CECs.
CONCLUSIONS AND CLINICAL RELEVANCE: In vitro, nalbuphine potentially could penetrate through corneal tissue, but it may cause damage to the corneal epithelium and stromal fibroblasts. Therefore, nalbuphine potentially may impair corneal wound healing.
Induction of posterior capsule opacification by hyaluronic acid in an ex vivo model
Invest Ophthalmol Vis Sci. 2012 Apr 6;53(4):1835-45. doi: 10.1167/iovs.11-8735.
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.
The effect of phosphorylated Akt inhibition on posterior capsule opacification in an ex vivo canine model
Mol Vis. 2010 Oct 29;16:2202-14.
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.
Tear proteomics in keratoconus
Mol Vis. 2010 Oct 2;16:1949-57.
PURPOSE: The purpose of this work was to identify potential tear-film based proteins expressed in keratoconus.
METHODS: Recruited subjects were normal gas permeable (GP) contact lens wearers, keratoconus subjects wearing GP contact lenses, and keratoconus subjects without contact lenses. Subjects wearing soft lenses or having previous ocular surgeries were excluded from participating. Approximately 5 µl of tears were sampled from both eye of each subject using glass microcapillaries. Additional testing included a brief history, visual acuity, slit lamp examination, and topography. Proteomic analyses used to compare samples included Bradford assays, cytokine arrays, SDS-PAGE, and mass spectrometry.
RESULTS: Forty-four subjects were enrolled in the study including 20 normals (GP wearers), 18 with keratoconus and wearing GPs, and six with keratoconus (non-lens wearers). Across all proteomic approaches, several proteins were identified as possibly being unique to keratoconus. Increased expression of matrix metalloproteinase-1 (MMP-1) was found in keratoconus subjects with and without gas permeable contact lenses (p=0.02). Unique proteins more associated with keratoconus included several keratins, immunoglobulins alpha and kappa, precursors to prolactin, lysozyme C, and lipocalin.
CONCLUSIONS: Initial analyses indicate that keratoconus may be associated with the differential expression of several proteins. Further testing is needed to determine any causal relationship or correlation with the etiology of this condition.
Evaluation of extractants and precipitants in tear film proteomic analyses
Optom Vis Sci. 2010 Nov;87(11):854-60. doi: 10.1097/OPX.0b013e3181f6fb71.
PURPOSE: To determine the efficiency of several protein extraction or precipitation treatments used in proteomic analyses.
METHODS: Tear samples were taken from each eye of 40 normal subjects using glass microcapillaries. Tear volume was measured followed by storage at -86°C. Lotrafilcon B contact lenses were fitted and worn for 14 days, followed by removal and storage at -86°C. Tear samples from each eye within a subject were randomly assigned to either one of four chemical treatments (acetone, trichloroacetic acid, urea, and trifluoroacetic acid/acetonitrile [TFA/ACN]) or no chemical treatment in groups of 10. Contact lens samples were subjected to the same treatments as tear samples for each subject, with a second treatment preceding the first. Protein concentrations were quantified by Bradford assay.
RESULTS: For tear samples, a significant reduction in total protein was observed when subjected to any of the four treatments studied compared with those samples left untreated. A positive relationship was noted between protein concentration and tear volume for treated, untreated, and combined tear samples. For contact lens samples, there was a significant reduction in the amount of deposited protein removed when comparing acetone, trichloroacetic acid, and urea with TFA/ACN. A second extraction from contact lenses assigned to the urea and TFA/ACN groups yielded a significant amount of additional protein compared with the amount removed initially.
CONCLUSIONS: Tear samples subjected to any of the evaluated chemical treatments provided significantly less protein than untreated samples. For contact lenses, TFA/ACN extraction provided the highest yield of available protein out of the four treatments evaluated.
In vivo effects of adjunctive tetracycline treatment on refractory corneal ulcers in dogs
J Am Vet Med Assoc. 2010 Aug 15;237(4):378-86. doi: 10.2460/javma.237.4.378.
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.
All-trans retinoic Acid regulates cx43 expression, gap junction communication and differentiation in primary lens epithelial cells
Curr Eye Res. 2010 Aug;35(8):670-9. doi: 10.3109/02713681003770746.
PURPOSE: To examine the effect of all-trans retinoic acid (ATRA) treatment on connexin 43 (Cx43) expression, gap junction intercellular communication (GJIC), and cellular differentiation in primary canine lens epithelial cells (LEC).
METHODS AND MATERIALS: Dose and time-dependent effects of ATRA on Cx43 protein, mRNA and GJIC, were assessed by immunoblotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and scrape loading/dye transfer assays, respectively. Expression of beta crystallin was evaluated by immunoblotting.
RESULTS: Treatment with ATRA at non-cytotoxic concentrations significantly increased Cx43 protein, mRNA and GJIC in primary canine LEC. Treatment with ATRA for five and seven days increased levels of beta crystallin, a protein marker of LEC differentiation. Inhibition of GJIC via pre-treatment with a synthetic inhibitor, 18-alpha glycyrrethinic acid (AGA), reduced ATRA-induced increases in Cx43 and GJIC and partially blocked ATRA-induced beta crystallin protein.
CONCLUSIONS: Treatment with ATRA significantly increased Cx43 expression and GJIC in canine LEC, and these effects were associated with increased LEC differentiation. Results from this study suggest that functional gap junctions may play a role in the modulation of cellular differentiation in primary canine LEC.
Distribution and amount of pigment within the ciliary body and iris of dogs with blue and brown irides
Vet Ophthalmol. 2010 Mar;13(2):76-80. doi: 10.1111/j.1463-5224.2009.00756.x.
OBJECTIVE: Compare the amount and distribution of pigment in normal canine eyes with blue and brown irides.
ANIMAL STUDIED: Eighteen mixed-breed dogs euthanized for population control.
PROCEDURES: Intact globes were removed immediately following euthanasia. Iris color was noted, and globes were evaluated histologically to determine the distribution of pigment. High magnification (x400) digital images were taken of the dorsal and ventral ciliary processes (CP) of 13 eyes with blue irides and 13 eyes with brown irides. Low magnification (x20) images were taken of the dorsal and ventral portions of the ciliary body (CB) of 14 eyes with blue irides and 14 eyes with brown irides. The images were digitally analyzed to determine the percent of the CP and CB that were pigmented in the high and low magnification images, respectively.
RESULTS: Eyes with brown irides contained abundant melanin pigment around the CB musculature. This pigment was absent in eyes with blue irides, and the difference was statistically significant (P = 0.001) when digitally analyzed using the low magnification images. Iris color did not have a significant (P = 0.34) impact on the amount of melanin within the CP, as determined using the high magnification images.
CONCLUSIONS: Compared to eyes with brown irides, eyes with blue irides lack pigment around the CB musculature, but contain comparable amounts of pigment in the CP.
ERalpha increases expression and interacts with TERT in cataractous canine lens epithelial cells
Mol Vis. 2009 Nov 9;15:2259-67.
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.
Prevention of UV-induced damage to the anterior segment using class I UV-absorbing hydrogel contact lenses
Invest Ophthalmol Vis Sci. 2010 Jan;51(1):172-8. doi: 10.1167/iovs.09-3996. Epub 2009 Aug 26.
PURPOSE: To determine whether class I ultraviolet (UV) light-blocking contact lenses prevent UV-induced pathologic changes in a rabbit model.
METHODS: Twelve rabbits were assigned to 1 of 3 treatment groups (n = 4), as follows: senofilcon A (class I UV blocking) contact lenses; lotrafilcon A contact lenses (no reported UV blocking); no contact lens. The contralateral eye was patched without a contact lens. Animals received UV-B (1.667 J/cm(2)) exposure daily for 5 days. Postmortem tissue was examined as follows: in the cornea, the expression of matrix-metalloproteinases (MMPs) was evaluated by zymography, and apoptosis was evaluated by TUNEL and caspase-3 ELISA; ascorbate in the aqueous humor was evaluated by nuclear magnetic resonance spectroscopy; crystalline lens apoptosis was evaluated by TUNEL and caspase-3 ELISA.
RESULTS: Exposed corneas showed a significant increase in MMP-2 and -9, TUNEL-positive cells, and caspase-3 activity in the lotrafilcon A group compared with the senofilcon A group (all P = 0.03). A significant decrease in aqueous humor ascorbate was observed in the exposed lotrafilcon A lens-wearing group compared with the exposed senofilcon A lens-wearing group (P = 0.03). Exposed crystalline lenses had significantly increased caspase-3 activity in the lotrafilcon A group compared with the senofilcon A group (P = 0.03). Increased numbers of TUNEL-positive cells were noted in both the lotrafilcon A and the non-contact lens groups.
CONCLUSIONS: The authors show that senofilcon A class I UV-blocking contact lenses are capable of protecting the cornea, aqueous humor, and crystalline lens of rabbits from UV-induced pathologic changes.
Cutaneous wound reepithelialization is compromised in mice lacking functional Slug (Snai2)
J Dermatol Sci. 2009 Oct;56(1):19-26. doi: 10.1016/j.jdermsci.2009.06.009. Epub 2009 Jul 29.
BACKGROUND: Keratinocytes at wound margins undergo partial epithelial to mesenchymal transition (EMT). Based on previous in vitro and ex vivo findings, Slug (Snai2), a transcriptional regulator of EMT in development, may play an important role in this process.
OBJECTIVES: This study was designed to validate an in vivo role for Slug in wound healing.
METHODS: Excisional wounds in Slug null and wild type mice were examined histologically at 6, 24, 48, and 72h after wounding; reepithelialization was measured and immunohistochemistry for keratins 8, 10, 14, and 6 and E-cadherin was performed. In 20 Slug null and 20 wild type mice exposed three times weekly to two minimal erythemal doses of UVR, the development of non-healing cutaneous ulcers was documented. Ulcers were examined histologically and by immunohistochemistry.
RESULTS: The reepithelialization component of excisional wound healing was reduced 1.7-fold and expression of the Slug target genes keratin 8 and E-cadherin was increased at wound margins in Slug null compared to wild type mice. In contrast, no differences in expression of keratins 10 or 14 or in markers of proliferation K6 and Ki-67 were observed. Forty per cent of Slug null mice but no wild type mice developed non-healing cutaneous ulcers in response to chronic UVR. Keratinocytes at ulcer margins expressed high levels of keratin 8 and retained E-cadherin expression, thus resembling excisional wounds.
CONCLUSION: Slug is an important modulator of successful wound repair in adult tissue and may be critical for maintaining epidermal integrity in response to chronic injury.
Selenium functionalized intraocular lenses inhibit posterior capsule opacification in an ex vivo canine lens capsular bag assay
Exp Eye Res. 2009 Nov;89(5):728-34. doi: 10.1016/j.exer.2009.06.016. Epub 2009 Jul 5.
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
Vet Ophthalmol. 2009 Mar-Apr;12(2):83-90. doi: 10.1111/j.1463-5224.2008.00684.x.
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.
Role of bacteria in the pathogenesis of recurrent uveitis in horses from the southeastern United States
Am J Vet Res. 2008 Oct;69(10):1329-35. doi: 10.2460/ajvr.69.10.1329.
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.
The acute cutaneous inflammatory response is attenuated in Slug-knockout mice
Lab Invest. 2008 Aug;88(8):831-41. doi: 10.1038/labinvest.2008.37. Epub 2008 May 5.
We previously reported ultraviolet radiation (UVR) induction of Slug, a Snail family zinc-finger transcription factor, in the epidermis of mice; we now report that Slug-knockout mice are, unexpectedly, more resistant to sunburn than wild-type mice. There was a marked difference between the cutaneous inflammatory response in the skin of Slug-knockout and wild-type mice from 12 h to 1 week following a single exposure to 3 minimal erythemal doses of UVR. Slug-knockout mice showed a much reduced immediate increase in skin thickness and neutrophil infiltration compared to wild-type mice. However, there were as many or more intraepidermal T cells, dermal mast cells, and dermal blood vessels in the UVR-exposed skin of Slug-knockout mice as in the skin of wild-type mice. Differences in cytokine and chemokine expression following UVR appeared to account for at least some differences between the genotypes in cutaneous inflammatory response. Despite the reported antiapoptotic and antiproliferative role for Slug in some cell types, we observed little difference between the genotypes in UVR-induced keratinocyte apoptosis or proliferation. Our findings indicate an unexpected but important role for Slug in the acute cutaneous inflammatory response to UVR.
Modulation of matrix metalloproteinases by ultraviolet radiation in the canine cornea
Vet Ophthalmol. 2008 May-Jun;11(3):135-44. doi: 10.1111/j.1463-5224.2008.00575.x.
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
Am J Vet Res. 2008 Jan;69(1):94-100. doi: 10.2460/ajvr.69.1.94.
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
Toxicol Pathol. 2007 Oct;35(6):819-26. doi: 10.1080/01926230701584197.
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.
Snai2 expression enhances ultraviolet radiation-induced skin carcinogenesis
Am J Pathol. 2007 Nov;171(5):1629-39. doi: 10.2353/ajpath.2007.070221. Epub 2007 Oct 4.
Snai2, encoded by the SNAI2 gene, has been shown to modulate epithelial-mesenchymal transformation (EMT), the conversion of sessile epithelial cells attached to adjacent cells and to the basement membrane into dissociated and motile fibroblastic cells. EMT occurs during development, wound healing, and carcinoma progression. Using Snai2-null mice (Snai2(lacZ)), we evaluated the role of Snai2 in UV radiation (UVR)-induced skin carcinogenesis. In chronically UVR-exposed nontumor-bearing skin from Snai2-null mice, inflammation and epidermal proliferation were decreased compared with wild-type (+/+) skin. Snai2-null mice had a consistently lower tumor burden than +/+ mice. In addition, null mice developed fewer aggressive spindle cell tumors, believed to arise from squamous cell carcinomas that have undergone EMT, than +/+ mice; however, the difference in tumor type distribution between the two genotypes was not statistically significant. No metastases were observed in either the +/+ or Snai2-null mice. Using quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry, we showed that the spindle cell tumors in the Snai2-null mice demonstrated impaired EMT, as shown by decreased vimentin and increased cadherin 1 expression. This study confirms a role for Snai2 in EMT, but demonstrates that Snai2 expression is not required for the development or progression of UVR-induced skin tumors.
Prevention of posterior capsular opacification through cyclooxygenase-2 inhibition
Mol Vis. 2007 Apr 30;13:677-91.
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.
The role of the slug transcription factor in cell migration during corneal re-epithelialization in the dog
Exp Eye Res. 2007 Mar;84(3):400-11. doi: 10.1016/j.exer.2006.10.010. Epub 2006 Dec 29.
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.
Expression and characterization of the catalytic subunit of telomerase in normal and cataractous canine lens epithelial cells
Mol Vis. 2006 Sep 13;12:1067-76.
PURPOSE: To determine whether the catalytic subunit of telomerase reverse transcriptase (TERT) is regionally distributed in canine lens epithelial cells (LEC), compare TERT and the RNA subunit of telomerase (TR) mRNA expression and TERT protein expression in normal and cataractous LEC, and to evaluate whether telomerase activity is present in the cytoplasm and nucleus from normal LEC. Finally, the expression of p23 and heat shock protein 90 (hsp90), coactivators of TERT in neoplastic cells, were evaluated in normal and cataractous LEC.
METHODS: TERT protein was detected by imunohistochemical staining and western immunoblotting in normal and cataractous LEC. Quantitative RT-PCR (qRT-PCR) was used to measure TERT and TR expression. Separated cytoplasmic and nuclear extracts from primary cultures of normal canine LEC were evaluated for TERT protein expression and telomerase activity. Western immunoblotting was performed on normal and cataractous LEC for p23 and hsp90, and coimmunoprecipitation was used to determine whether p23 and hsp90 were interacting with TERT in LEC.
RESULTS: TERT expression in normal lens capsule whole mounts varied by region in normal LEC. All cataractous LEC demonstrated more intense TERT immunostaining in both the nucleus and cytoplasm when compared to normal LEC. Normal LEC expressed less TERT protein and less TERT and TR mRNA than cataractous LEC. Normal LEC expressed hsp90 while cataractous LEC did not; p23 was not significantly expressed in either normal or cataractous LEC. Neither hsp90 nor p23 interacted with TERT.
CONCLUSIONS: The localization of TERT in normal LEC corresponded with the LEC's regional functions. There was more cytoplasmic TERT in the central region that corresponds with the need for inhibited apoptosis and for proliferative capabilities; there was more nuclear TERT in the germinal and equatorial regions corresponding with the need for proliferative capabilities. In addition, cataractous LEC demonstrated increased TERT protein and increased TERT and TR mRNA expression than normal LEC corresponding with their increased proliferative potential. However, the telomerase coactivators, p23 and hsp90, are not overexpressed and do not associate with TERT in cataractous LEC, suggesting that telomerase regulation in cataractous LEC, a somatic cell type, differs from that in neoplastic cells.
Molecular biology for the clinician: understanding current methods
J Am Anim Hosp Assoc. 2006 Sep-Oct;42(5):326-35. doi: 10.5326/0420326.
The basics of molecular biology involve the replication of deoxyribonucleic acid and its transcription and translation into proteins. Biochemical assays such as the Southern blot analysis, polymerase chain reaction (PCR), Northern blot analysis, reverse-transcriptase PCR, microarray technology, Western blot analysis, immunohistochemistry, enzyme-linked immunosorbent assay, and flow cytometry utilize various aspects of molecular biology. To understand these assays requires some basic understanding of the principles of molecular biology. This paper provides basic information on the methodology and techniques used in these assays.
Ultraviolet irradiation up-regulates telomerase transcription and activity in lens epithelial cells
Vet Ophthalmol. 2006 Sep-Oct;9(5):379-85. doi: 10.1111/j.1463-5224.2006.00499.x.
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.