Differential effects of Hsp90 inhibition on corneal cells in vitro and in vivo.

The transformation of quiescent keratocytes to activated fibroblasts and myofibroblasts (KFM transformation) largely depends on transforming growth factor beta (TGFß) signaling. Initiation of the TGFß signaling cascade results from binding of TGFß to the labile type I TGFß receptor (TGFßRI), which is stabilized by the 90 kDa heat shock protein (Hsp90). Since myofibroblast persistence within the corneal stroma can result in stromal haze and corneal fibrosis in patients undergoing keratorefractive therapy, modulation of TGFß signaling through Hsp90 inhibition would represent a novel approach to prevent myofibroblast persistence. In vitro, rabbit corneal fibroblasts (RCFs) or stratified immortalized human corneal epithelial cells (hTCEpi) were treated with a Hsp90 inhibitor (17AAG) in the presence/absence of TGFß1. RCFs were cultured either on tissue culture plastic, anisotropically patterned substrates, and hydrogels of varying stiffness. Cellular responses to both cytoactive and variable substrates were assessed by morphologic changes to the cells, and alterations in expression patterns of key keratocyte and myofibroblast proteins using PCR, Western blotting and immunocytochemistry. Transepithelial electrical resistance (TEER) measurements were performed to establish epithelial barrier integrity. In vivo, the corneas of New Zealand White rabbits were wounded by phototherapeutic keratectomy (PTK) and treated with 17AAG (3× or 6× daily) either immediately or 7 days after wounding for 28 days. Rabbits underwent clinical ophthalmic examinations, SPOTS scoring and advanced imaging on days 0, 1, 3, 7, 10, 14, 21 and 28. On day 28, rabbits were euthanized and histopathology/immunohistochemistry was performed. In vitro data demonstrated that 17AAG inhibited KFM transformation with the de-differentiation of spindle shaped myofibroblasts to dendritic keratocyte-like cells accompanied by significant upregulation of corneal crystallins and suppression of myofibroblast markers regardless of TGFß1 treatment. RCFs cultured on soft hydrogels or patterned substrates exhibited elevated expression of α-smooth muscle actin (αSMA) in the presence of 17AAG. Treatment of hTCEpi cells disrupted zonula occludens 1 (ZO-1) adherens junction formation. In vivo, there were no differences detected in nearly all clinical parameters assessed between treatment groups. However, rabbits treated with 17AAG developed greater stromal haze formation compared with controls, irrespective of frequency of administration. Lastly, there was increased αSMA positive myofibroblasts in the stroma of 17AAG treated animals when compared with controls. Hsp90 inhibition promoted reversion of the myofibroblast to keratocyte phenotype, although this only occurred on rigid substrates. By contrast, in vivo Hsp90 inhibition was detrimental to corneal wound healing likely due to impairment in corneal epithelial closure and barrier function restoration. Collectively, our data demonstrated a strong interplay in vitro between biophysical cues and soluble signaling molecules in determining corneal stromal cell phenotype.

Comparison of automated vs manual analysis of corneal endothelial cell density and morphology in normal and corneal endothelial dystrophy-affected dogs.

OBJECTIVE: To determine the efficacy of automated imaging software of the Nidek ConfoScan 4 confocal biomicroscope at analyzing canine corneal endothelial cell density and morphology in health and disease, by comparing to a manual analysis method. ANIMAL STUDIED: Nineteen eyes of 10 dogs were evaluated and include three Beagles, three Jack Russell Terriers, and four miscellaneous breeds. Twelve clinically normal and seven eyes affected with corneal endothelial dystrophy (CED) were scanned and analyzed. PROCEDURES: Endothelial cell density (ECD), mean and standard deviation (SD) of cell area, percent polymegathism, mean and SD of the number of cell sides, and percent pleomorphism were calculated using automated and manual methods for each scan. RESULTS: The automated analysis showed significantly greater ECD in comparison with the manual frame method due to misidentification of cell domains in CED-affected dogs. No significant differences in ECD were observed between normal and CED-affected dogs in automated analysis, while CED-affected dogs showed significantly lower ECD in manual frame method and planimetry. Using both automated and manual methods, CED-affected dogs showed greater variability of cell area or the number of cell sides than normal dogs. CONCLUSION: The automated imaging software is unable to accurately identify cell borders in CED-affected dogs resulting in inaccurate estimates of ECD. Thus, manual analysis is recommended for use in clinical trials assessing adverse events associated with novel medical treatments and/or surgical procedures.

Comprehensive Clinical, Diagnostic, and Advanced Imaging Characterization of the Ocular Surface in Spontaneous Aqueous Deficient Dry Eye Disease in Dogs.

PURPOSE: To perform a comprehensive clinical, diagnostic, and imaging characterization of the ocular surface in West Highland White Terriers (WHWTs) diagnosed with aqueous deficient dry eye (ADDE) disease. METHODS: Six ADDE-affected and 13 ADDE-unaffected WHWT dogs were enrolled and underwent clinical assessment and disease scoring, tear osmolarity, phenol red thread test, Schirmer tear test, tear film breakup time, fluorescein staining, Rose bengal and lissamine green vital dye staining, meibometry, corneal esthesiometry, ultrasound pachymetry, optical coherence tomography, in vivo confocal microscopy, and conjunctival biopsy. Subjective assessment of their condition was provided by owner-reported surveys. RESULTS: ADDE-affected WHWT dogs had higher median clinical disease (conjunctiva: 5.75 vs. 0.00; cornea: 14.00 vs. 5.00; total: 17.50 vs. 5.00), vital staining (Rose bengal: 2.25 vs. 1.50; lissamine green: 2.00 vs. 1.00), and histologic disease (conjunctiva: 2 vs. 0) scores when compared with the controls. In addition, ADDE-affected WHWTs had significantly lower phenol red thread test (5.0 vs. 17.5, mm/15 s), Schirmer tear test (3 vs. 20, mm/min), tear film breakup time (3.6 vs. 13.9, s) values and higher area under the curve values for meibometry (394 vs. 245, meibometry units [MU]). There were no significant differences in other tear film tests performed. Advanced imaging revealed decreased tear meniscus height (optical coherence tomography) and variable pigment deposition within corneal epithelial cells (in vivo confocal microscopy). CONCLUSIONS: This comprehensive assessment of ADDE-affected WHWTs depicts the ocular surface changes associated with quantitative lacrimal gland dysfunction. Importantly, ADDE-affected WHWTs may prove a valuable naturally occurring ADDE model for investigating underlying pathophysiological mechanisms and the development of novel therapeutics.

In Vivo Imaging of Corneal Endothelial Dystrophy in Boston Terriers: A Spontaneous, Canine Model for Fuchs' Endothelial Corneal Dystrophy.

PURPOSE: Boston Terriers (BTs) have a greater prevalence of corneal endothelial dystrophy (CED), in comparison to other canine breeds. Similar to Fuchs' endothelial corneal dystrophy (FECD), this condition is characterized by endothelial cell degeneration with secondary corneal edema. This study assessed corneal morphology using in vivo confocal microscopy (IVCM) and Fourier-domain optical coherence tomography (FD-OCT) in BTs with and without CED. METHODS: The corneas of 16 BTs with CED and 15 unaffected, age-matched BTs underwent clinical evaluation and were imaged using IVCM and FD-OCT. A two-sample t-test or Mann-Whitney rank sum test were used to statistically compare parameters between groups. Data are presented as mean ± SD or median (range). RESULTS: Mean age did not significantly differ between affected and unaffected dogs at 10.0 ± 2.0 and 10.6 ± 2.4 years, respectively (P = 0.437). Females (69%) were overrepresented among the CED-affected dogs. In CED patients, IVCM demonstrated endothelial polymegathism and pleomorphism. Corneal endothelial density was significantly less (P < 0.001) in dogs with CED (1026 ± 260 cells/mm2) versus age-matched controls (2297 ± 372 cells/mm2). Fourier-domain OCT demonstrated a significant increase (P < 0.01) in central corneal and endothelium-Descemet's complex thickness in dogs with CED versus age-matched controls at 1019 (485-1550) or 536 (464-650) µm and 32 (22-56) or 25 (15-34) µm, respectively. CONCLUSIONS: Corneal endothelial dystrophy in BTs is a bilateral, adult-onset condition that shares many similarities with FECD. Thus, CED could serve as a spontaneous disease model to study the pathogenesis of and develop novel treatments for FECD.

Pupil dilation using a standard cataract surgery regimen alone or with atropine 1.0% pretreatment: prospective comparative evaluation.

PURPOSE: To compare the amount of pupil dilation produced by a set of commonly used preoperative mydriatic agents for cataract surgery, with the same regimen preceded by topical administration of atropine 1.0%. SETTING: Department of Ophthalmology, Loma Linda University, Loma Linda, California, USA. METHODS: In this prospective unmasked study, the baseline pupil size in eyes of volunteers was measured. Pupil size was then measured 30 minutes after instillation of the institution's standard dilation regimen for cataract surgery, which included phenylephrine 2.5%, tropicamide 1.0%, and cyclopentolate 1.0%. Several days later, the subjects returned for repeat measurements after pretreating the study eye(s) with atropine 1.0% 3 times a day the day previously and once on the morning of repeat dilation and measurements. Pupil size was again measured after administration of the standard regimen. RESULTS: The study included 72 eyes of 54 patients. A paired t test showed a statistically significant difference in mean pupil dilation between the standard regimen alone and the standard regimen with atropine 1.0% pretreatment. The mean pupil dilation was 7.3 mm +/- 1.2 (SD) with the standard regimen alone and 6.9 +/- 1.2 mm with the standard regimen with atropine pretreatment; the difference was statistically significant (P<.001). CONCLUSION: The addition of atropine 1.0% 1 day before administration of a standard preoperative dilating regimen for cataract surgery resulted in a smaller dilated pupil diameter than administration of the standard set of preoperative mydriatic agents alone. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.