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
Authors: Miller EJ, Gemensky-Metzler AJ, Wilkie DA, Wynne RM, Curto EM, Chandler HL
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.
PMID: 29943637 [PubMed - in process]
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 01;251(11):1324-1330
Authors: Wehrman RF, Gemensky-Metzler AJ, Zibura AE, Nyhart AB, Chandler HL
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.
PMID: 29154707 [PubMed - in process]
Subconjunctival antimicrobial poloxamer gel for treatment of corneal ulceration in stranded California sea lions (Zalophus californianus).
Vet Ophthalmol. 2017 Sep;20(5):441-449
Authors: Simeone CA, Colitz CMH, Colegrove KM, Field CL, Rios C, Chandler HL, Johnson SP
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.
PMID: 27905668 [PubMed - in process]
Oxidative Stress Measures of Lipid and DNA Damage in Human Tears.
Invest Ophthalmol Vis Sci. 2017 May 01;58(6):BIO151-BIO157
Authors: Haworth KM, Chandler HL
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.
PMID: 28662237 [PubMed - indexed for MEDLINE]
Seasonal Effect on Ocular Sun Exposure and Conjunctival UV Autofluorescence.
Optom Vis Sci. 2017 Feb;94(2):219-228
Authors: Haworth KM, Chandler HL
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.
PMID: 27820717 [PubMed - indexed for MEDLINE]
Cyclosporine A prevents ex vivo PCO formation through induction of autophagy-mediated cell death.
Exp Eye Res. 2015 May;134:63-72
Authors: Chandler HL, Gervais KJ, Lutz EA, Curto EM, Matusow RB, Wilkie DA, Gemensky-Metzler AJ
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.
PMID: 25839646 [PubMed - indexed for MEDLINE]
Estradiol biosynthesis in canine lens epithelial cells.
Curr Eye Res. 2015 May;40(5):541-8
Authors: Colitz CM, Lu P, Sugimoto Y, Barden CA, Chandler HL
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.
PMID: 25260172 [PubMed - indexed for MEDLINE]
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
Authors: Lutz EA, Gemensky-Metzler AJ, Wilkie DA, Chandler HL
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.
PMID: 24447772 [PubMed - indexed for MEDLINE]
Aloe vera: an in vitro study of effects on corneal wound closure and collagenase activity.
Vet Ophthalmol. 2014 Nov;17(6):403-10
Authors: Curto EM, Labelle A, Chandler HL
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.
PMID: 24666433 [PubMed - indexed for MEDLINE]
Equine glaucoma: a histopathologic retrospective study (1999-2012).
Vet Ophthalmol. 2014 Sep;17(5):334-42
Authors: Curto EM, Gemensky-Metzler AJ, Chandler HL, Wilkie DA
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.
PMID: 23859597 [PubMed - indexed for MEDLINE]
Analysis of the transport of and cytotoxic effects for nalbuphine solution in corneal cells.
Am J Vet Res. 2012 Dec;73(12):1987-95
Authors: Spatola RA, Thangavelu M, Upadhyayula V, Lee S, Phelps MA, Chandler HL
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.
PMID: 23176428 [PubMed - indexed for MEDLINE]
Induction of posterior capsule opacification by hyaluronic acid in an ex vivo model.
Invest Ophthalmol Vis Sci. 2012 Apr 06;53(4):1835-45
Authors: Chandler HL, Haeussler DJ, Gemensky-Metzler AJ, Wilkie DA, Lutz EA
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.
PMID: 22408013 [PubMed - indexed for MEDLINE]
Evaluation of extractants and precipitants in tear film proteomic analyses.
Optom Vis Sci. 2010 Nov;87(11):854-60
Authors: Powell DR, Thangavelu M, Chandler HL, Nichols KK, Nichols JJ
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.
PMID: 20852451 [PubMed - indexed for MEDLINE]
The effect of phosphorylated Akt inhibition on posterior capsule opacification in an ex vivo canine model.
Mol Vis. 2010 Oct 29;16:2202-14
Authors: Chandler HL, Webb TR, Barden CA, Thangavelu M, Kulp SK, Chen CS, Colitz CM
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.
PMID: 21139685 [PubMed - indexed for MEDLINE]
Tear proteomics in keratoconus.
Mol Vis. 2010 Oct 02;16:1949-57
Authors: Pannebaker C, Chandler HL, Nichols JJ
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.
PMID: 21031023 [PubMed - indexed for MEDLINE]
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
Authors: Long AC, Bomser JA, Grzybowski DM, Chandler HL
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.
PMID: 20673043 [PubMed - indexed for MEDLINE]
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
Authors: Chandler HL, Gemensky-Metzler AJ, Bras ID, Robbin-Webb TE, Saville WJ, Colitz CM
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.
PMID: 20707747 [PubMed - indexed for MEDLINE]
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
Authors: Newkirk KM, Haines DK, Calvarese ST, Esson DW, Chandler HL
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.
PMID: 20447024 [PubMed - indexed for MEDLINE]
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
Authors: Chandler HL, Reuter KS, Sinnott LT, Nichols JJ
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.
PMID: 19710408 [PubMed - indexed for MEDLINE]