Corneal Cross-Linking With Verteporfin and Nonthermal Laser Therapy.

PURPOSE: To test whether verteporfin with a nonthermal laser increases corneal mechanical stiffness and resistance to enzymatic degradation ex vivo. METHODS: Thirty human corneas (n = 5 per group) were treated with verteporfin alone (V), irradiated with nonthermal laser therapy (689 nm) alone (NTL), or received combined treatment of verteporfin with nonthermal laser therapy for 1 sequence (V+NTL1) or 6 sequences (V+NTL6) of 1 minute of NTL exposure. Positive controls were pretreated with 0.1% riboflavin/20% dextran every 3 to 5 minutes for 30 minutes and irradiated with ultraviolet light type A (λ = 370 nm, irradiance = 3 mW/cm) for 30 minutes using the Dresden protocol (R+UVA). Untreated corneas were used as negative controls. The corneal biomechanical properties were measured with enzymatic digestion, compression, creep, and tensile strength testing. RESULTS: V+NTL6- and R+UVA-treated corneas acquired higher rigidity and more pronounced curvature than untreated corneas. The stress-strain tests showed that V+NTL6 and R+UVA corneas became significantly stiffer than controls (P < 0.005). The V+NTL6 group seemed to be slightly stiffer than the R+UVA group, although the differences were not statistically significant. V+NTL6 corneas were found to have a significantly lower absolute creep rate (-1.87 vs. -3.46, P < 0.05) and significantly higher maximum stress values (7.67 vs. 3.02 P < 0.05) compared with untreated corneas. CONCLUSIONS: Verteporfin-NTL (V+NTL6) increases corneal mechanical stiffness and resistance to enzymatic collagenase degradation. Although a clinical study is needed, our results suggest that V+NTL6 induces corneal cross-linking and corneal biomechanical changes that are similar to those induced by standard corneal collagen cross-linking.

Ex Vivo Study of Transepithelial Corneal Cross-linking.

PURPOSE: To perform in vitro assessment of different techniques of transepithelial corneal cross-linking (CXL) and to compare the results to deepithelialized CXL. METHODS: Transepithelial CXL was performed after pre-treatment with or without penetration enhancers (gum cellulose, 0.44% sodium chloride, and 0.01% benzalkonium chloride) for 15 or 60 minutes. Deepithelialized corneas underwent CXL after pretreatment with riboflavin for 15 minutes, according to the Dresden protocol. All corneas were incubated in 0.3% collagenase A solution and the time to total dissolution was measured. Corneas were also imaged with confocal microscopy to evaluate the corneal epithelium, subbasal nerve plexus, and depth of stromal keratocyte nuclei as a means of measuring the depth of collagen CXL. RESULTS: Deepithelialized CXL corneas with 15 minutes of pretreatment dissolved after 15.4 ± 3.1 hours, significantly longer (P = .001) than deepithelialized untreated corneas (8.5 ± 0.6 hours). Transepithelial CXL corneas with 15 minutes of pretreatment with or without penetration enhancers dissolved after 8.3 ± 2.1 and 7.4 ± 1.6 hours, respectively. A longer pretreatment of 60 minutes with penetration enhancers resulted in greater resistance to degradation of the transepithelial CXL corneas (14.6 ± 2.2 hours), which was similar to deepithelialized CXL corneas. The results of the biological assay correlated well with the imaging results obtained by confocal microscopy. CONCLUSIONS: Corneas treated by transepithelial CXL with an extended pretreatment time of 60 minutes and penetration enhancers exhibited similar characteristics as corneas treated by the deepithelialized CXL approach. By confocal imaging, the transepithelial approach with extended pretreatment time demonstrated evidence of epithelial damage, which may have improved the treatment effect of this group. [J Refract Surg. 2017;33(3):171-177.].

Elevated Neutrophil Elastase in Tears of Ocular Graft-Versus-Host Disease Patients.

PURPOSE: To investigate the levels of neutrophil elastase (NE), matrix metalloproteinases (MMPs), and myeloperoxidase (MPO) in tear washes of patients with ocular graft-vs-host disease (oGVHD). DESIGN: Case-control study. METHODS: Based on established criteria, oGVHD patients (n = 14; 28 eyes) and age-/sex-matched healthy controls (n = 14; 28 eyes) were enrolled. Tear washes were collected and analyzed for NE using a single-analyte enzyme-linked immunosorbent assay (ELISA). MMPs (1, 2, 3, 7, 8, 9, 12), MPO, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were analyzed using multianalyte bead-based ELISA assays. Total MMP activity was measured using a fluorimetric assay. Correlation studies were performed between NE, MMP-8, MMP-9, and MPO within study groups. RESULTS: NE, MMP-8, MMP-9, and MPO levels were elevated in oGVHD tears when compared with controls (P < .0001). NE was the most elevated analyte. MMP activity was higher and TIMP-1 levels were lower in oGVHD than in control (P < .0001). In oGVHD, NE significantly correlated with MMP-8 (r = 0.92), MMP-9 (r = 0.90), and MPO (r = 0.79) (P < .0001). MMP-8 correlated with MMP-9 (r = 0.96, P < .0001), and MPO (r = 0.60, P = .001). MMP-9 correlated with MPO (r = 0.55, P = .002). In controls, NE, MMP-9, and MPO significantly correlated with each other (P < .0001). CONCLUSIONS: The marked increase in NE in oGVHD tears that correlated strongly with elevated MMP-8, MMP-9, and MPO suggests a common neutrophilic source and provides evidence of neutrophil activity on the ocular surface of oGVHD patients.

Corneal Resistance to Keratolysis After Collagen Crosslinking With Rose Bengal and Green Light.

Purpose: The purpose of this study was to evaluate the resistance to degradation by collagenase A of corneas that have been crosslinked with Rose Bengal and green light (RGX). Methods: The ex vivo crosslinking procedure was performed on enucleated rabbit corneas. Corneas were deepithelialized after applying 30% alcohol. Corneas were stained with Rose Bengal (RB, 0.1%) for 2 minutes and then exposed to green light (532 nm) at 0.25 W/cm2 for times to deliver doses of 50, 100, 150, or 200 J/cm2 (n = 5 per group). Five corneas were pretreated with riboflavin solution (0.1% riboflavin) for 15 minutes and irradiated with ultraviolet A (UVA) light (370 nm, 3 mW/cm2) for 30 minutes. Five corneas underwent only de-epithelialization and were otherwise untreated. Five corneas were stained with RB without light exposure. The central corneas of each group was removed with a 8.5-mm trephine and incubated at 37°C in 0.3% collagenase A solution. Time to dissolution of each cornea was compared across treatments. Results: Corneas treated with RGX were treated with light fluences of 50, 100, 150, and 200 J/cm2; these corneas dissolved completely at 8.3 ± 1.2, 11.1 ± 1.4, 12.4 ± 1.7, and 15.7 ± 1.8 hours, respectively. Corneas treated by riboflavin and UVA light dissolved at 15.7 ± 1.7 hours, and nontreated corneas dissolved at 6.1 ± 1.3 hours. Corneas treated with only RB (no green light) dissolved at 9.3 ± 1.7 hours. Compared with the untreated corneas, all of the RB groups and the riboflavin-UVA-treated group of corneas degraded statistically significantly slower than untreated corneas (P < 0.05). Conclusions: Crosslinking with RGX increased corneal resistance to digestion by collagenase comparable to that produced by riboflavin and UVA treatment.

Tear Matrix Metalloproteinases and Myeloperoxidase Levels in Patients With Boston Keratoprosthesis Type I.

PURPOSE: To investigate the tear levels of matrix metalloproteinases (MMPs), myeloperoxidase (MPO), and tissue inhibitor of metalloproteinase-1 in eyes after Boston keratoprosthesis type I (B-KPro) implantation and to correlate these markers with the established B-KPro prognostic categories. METHODS: Tear washes were collected from 40 patients (7 with autoimmune disease, 2 with chemical burn, and 31 with other noncicatrizing diagnoses). Tear levels of MMPs, MPO, and tissue inhibitor of metalloproteinase-1 were quantified using multianalyte bead-based enzyme-linked immunosorbent assays. The total MMP activity was determined using a fluorimetric assay. The analytes were compared to the underlying diagnosis and other clinical factors. RESULTS: The MMP-8, MMP-9, and MPO levels were markedly elevated in the eyes with B-KPro (80 ± 31, 291 ± 77, and 244 ± 33 pg/µg, respectively). Chemical burn was associated with significantly higher tear MMP-8 (474 ± 376 pg/µg) and MMP-9 levels (1300 ± 635 pg/µg) compared with noncicatrizing diseases (MMP-8: 41 ± 15 pg/µg, P = 0.02 and MMP-9: 196 ± 57 pg/µg, P = 0.02) and higher MMP-9 levels compared with autoimmune diseases (MMP-8: 96 ± 65 pg/µg, P = 0.21 and MMP-9: 306 ± 196 pg/µg, P = 0.04). Similar analyte levels were observed in the B-KPro eye and the contralateral non-B-KPro eye of patients with bilateral diseases. MMP-8, MMP-9, and total MMP activities correlated strongly with each other. CONCLUSIONS: In the eyes with B-KPro, tear MMP-8 and MMP-9 levels seem to be related to the underlying ocular surface pathology and not significantly influenced by the presence of the prosthesis.

Collagen cross-linking of the Boston keratoprosthesis donor carrier to prevent corneal melting in high-risk patients.

OBJECTIVE: To examine the clinical relevance and pathophysiology of Boston keratoprosthesis (B-KPro)-related corneal keratolysis (cornea melt) and to describe a novel method of preventing corneal melt using ex vivo crosslinked cornea tissue carrier. METHODS: A review of B-KPro literature was performed to highlight cases of corneal melt. Studies examining the effect of corneal collagen cross-linking (CXL) on the biomechanical properties of corneal tissue are summarized. The use of crosslinked corneal tissue as a carrier to the B-KPro is illustrated with a case. RESULTS: Corneal melting after B-KPro is a relatively rare event, occurring in 3% of eyes during the first 3 years of postoperative follow-up. The risk of post-KPro corneal melting is heightened in eyes with chronic ocular surface inflammation such as eyes with Stevens-Johnson syndrome and mucous membrane pemphigoid. This chronic inflammation results in high tear levels of matrix metalloproteinases, the enzymes responsible for collagenolysis and corneal melt. Crosslinked corneal tissue has been shown to have stiffer biomechanical properties and to be more resistant to degradation by collagenolytic enzymes. We have previously optimized the technique for ex vivo corneal CXL and are currently studying its impact on the prevention of corneal melting after B-KPro surgery in high-risk eyes. Crosslinked carrier tissue was used in a 52-year-old man with familial aniridia and severe post-KPro corneal melt. The patient maintained his visual acuity and showed no evidence of corneal thinning or melt in the first postoperative year. CONCLUSION: Collagen crosslinking was previously shown to halt the enzymatic degradation of corneal buttons ex vivo. This study demonstrates the safety and potential benefit of using crosslinked corneal grafts as carriers for the B-KPro, especially in eyes at higher risk of postoperative melt.

UV cross-linking of donor corneas confers resistance to keratolysis.

PURPOSE: The aim of this study was to develop a modified ex vivo corneal cross-linking method that increases stromal resistance to enzymatic degradation for use as a carrier for the Boston keratoprosthesis. METHODS: Ex vivo cross-linking of human corneas was performed using Barron artificial anterior chambers. The corneas were deepithelialized, pretreated with riboflavin solution (0.1% riboflavin/20% dextran), and irradiated with ultraviolet A (UV-A) light (λ = 370 nm, irradiance = 3 mW/cm) for various durations. The combined effect of UV-A and gamma (γ) irradiation was also assessed using the commercially available γ-irradiated corneal donors. The corneas were then trephined and incubated at 37°C with 0.3% collagenase A solution. The time to dissolution of each cornea was compared across treatments. RESULTS: Deepithelialized corneas (no UV light, no riboflavin) dissolved in 5.8 ± 0.6 hours. Cross-linked corneas demonstrated increased resistance to dissolution, with a time to dissolution of 17.8 ± 2.6 hours (P < 0.0001). The corneal tissues' resistance to collagenase increased with longer UV-A exposure, reaching a plateau at 30 minutes. Cross-linking both the anterior and posterior corneas did not provide added resistance when compared with cross-linking the anterior corneas only (P > 0.05). γ-irradiated corneas dissolved as readily as deepithelialized controls regardless of whether they were further cross-linked (5.6 ± 1.2 hours) or not (6.1 ± 0.6 hours) (P = 0.43). CONCLUSIONS: Collagen cross-linking of the deepithelialized anterior corneal surface for 30 minutes conferred optimal resistance to in vitro keratolysis by collagenase A.

Neutrophil collagenase, gelatinase, and myeloperoxidase in tears of patients with stevens-johnson syndrome and ocular cicatricial pemphigoid.

OBJECTIVE: To investigate the levels of matrix metalloproteinases (MMPs), myeloperoxidase (MPO), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in tears of patients with Stevens-Johnson syndrome (SJS) and ocular cicatricial pemphigoid (OCP). DESIGN: Prospective, noninterventional cohort study. PARTICIPANTS: Four SJS patients (7 eyes), 19 OCP patients (37 eyes), and 20 healthy controls who underwent phacoemulsification (40 eyes). METHODS: Tear washes were collected from all patients and were analyzed for levels of MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MPO, and TIMP-1 using multianalyte bead-based enzyme-linked immunosorbent assays. Total MMP activity was determined using a fluorometric assay. Correlation studies were performed between the various analytes within study groups. MAIN OUTCOME MEASURES: Levels of MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MPO, and TIMP-1 (in nanograms per microgram of protein) and total MMP activity (in relative fluorescent units per minute per microgram of protein) in tears; MMP-8-to-TIMP-1 ratio; MMP-9-to-TIMP-1 ratio; and the correlations between MMP-8 and MMP-9 and both MMP and MPO. RESULTS: MMP-8, MMP-9, and MPO levels were elevated significantly in SJS and OCP tears (SJS>OCP) when compared with controls. The MMP activity was highest in SJS patients, whereas OCP patients and controls showed lower and similar activities. The TIMP-1 levels were decreased in SJS and OCP patients when compared with those in controls, with levels in OCP patients reaching significance. The MMP-8-to-TIMP-1 and MMP-9-to-TIMP-1 ratios were markedly elevated in SJS and OCP tears (SJS>OCP) when compared with those of controls. Across all study groups, MMP-9 levels correlated strongly with MMP-8 and MPO levels, and MMP-8 correlated with MPO, but it did not reach significance in SJS patients. There was no relationship between MMP-7 and MPO. CONCLUSIONS: Because MMP-8 and MPO are produced by inflammatory cells, particularly neutrophils, the correlation data indicate that they may be the common source of elevated enzymes, including MMP-9, in SJS and OCP tears. Elevated MMP-to-TIMP ratios and MMP activity suggest an imbalance in tear MMP regulation that may explain the predisposition of these patients to demonstrate corneal melting and chronic complications associated with persistent inflammation. Myeloperoxidase in tears may be a sensitive and specific marker for the quantification of ocular inflammation.

Ranibizumab monotherapy versus single-session verteporfin photodynamic therapy combined with as-needed ranibizumab treatment for the management of neovascular age-related macular degeneration.

PURPOSE: To compare verteporfin photodynamic therapy combined with intravitreal ranibizumab (combination therapy) versus ranibizumab monotherapy for management of neovascular age-related macular degeneration. METHODS: Thirty patients (40 eyes) with neovascular age-related macular degeneration were prospectively allocated to combination therapy or monotherapy. In monotherapy, the induction phase consisted of 3 consecutive monthly ranibizumab injections (0.5 mg), while the combination therapy had a single session of photodynamic therapy with intravitreal ranibizumab. Follow-up treatment for either group consisted only of additional as-needed ranibizumab injections. The main outcome measure was that a proportion of eyes losing <15 letters of visual acuity after 12 months. RESULTS: Except for 1 eye in combination therapy, all eyes in both groups lost <15 letters of visual acuity. At 12 months, there was a mean gain of +12 letters and +3.2 letters for monotherapy and combination therapy, respectively (relative percent change of 32% vs. 7%, P = 0.03). Anatomical improvement was similar in both groups. After induction, the time until ranibizumab retreatment was longer for combination therapy (P = 0.002) while ranibizumab injections were required more frequently with monotherapy (P = 0.015). CONCLUSION: Ranibizumab monotherapy showed greater improvement in visual acuity versus combination therapy. However, combination therapy required fewer ranibizumab injections. Larger trials need to confirm the findings of this pilot study.

Simple methods for the direct assembly, functionalization, and patterning of acid-terminated monolayers on Si111.

This article describes mild methods to directly assemble, functionalize, and pattern monolayers of undecylenic acid on hydrogen-terminated Si(111). These monolayers were assembled under very mild conditions from a neat solution of undecylenic acid containing 0.1 mol % 4-(decanoate)-2,2,6,6-tetramethylpiperidinooxy at room temperature without the need for UV light. Because of these mild conditions, monolayers exposing carboxylic acids could be assembled in one step without the need to protect the acid prior to its assembly. The monolayers were extensively characterized by horizontal attenuated total reflection infrared spectroscopy, X-ray photoelectron spectroscopy (XPS), and contact angle goniometry. The monolayers bonded to the silicon surface preferentially through the olefin with no detectable bonds between the carboxylic acids and silicon. The crystallinity of the monolayer was studied by infrared spectroscopy through the antisymmetric--v(a)(CH(2))--and symmetric--v(s)(CH(2))--stretches for methylene. Because it is important for future applications to assemble functional surfaces, methods to react the acid-terminated monolayers with trifluoroacetic anhydride and triethylamine to yield a symmetric anhydride on the monolayer were studied. These anhydrides were reacted with a variety of milligram-quantity amines to yield amide-terminated surfaces. This method was general, and a variety of amines could be bonded to the monolayer. The stabilities of these monolayers upon exposure to ambient conditions and under a variety of solvents were described. Because patterned monolayers have found wide applications, we have developed methods to pattern 1-octadecylamine and poly(ethylenimine) on the micrometer scale using soft lithography. In addition, polymer brushes of polynorbornene with thicknesses from 32 to 150 nm were grown from monolayers patterned with the Grubbs' catalyst. The patterned surfaces were imaged by scanning electron microscopy, scanning probe microscopy, and ellipsometry to determine the thicknesses of the patterns and the fidelity of the method.

Mild methods to assemble and pattern organic monolayers on hydrogen-terminated Si(111).

Mild methods to assemble well-ordered organic monolayers of olefins on Si(111) using 2,2,6,6-tetramethyl-1-piperidinyloxy and to pattern these monolayers on the micrometer-size scale using soft lithography are reported.

Detection of lameness and determination of the affected forelimb in horses by use of continuous wavelet transformation and neural network classification of kinematic data.

OBJECTIVE: To investigate continuous wavelet transformation and neural network classification of gait data for detecting forelimb lameness in horses. ANIMALS: 12 adult horses with mild forelimb lameness. PROCEDURE: Position of the head and right forelimb foot, metacarpophalangeal (ie, fetlock), carpal, and elbow joints was determined by use of kinematic analysis before and after palmar digital nerve blocks. We obtained 8 recordings from horses without lameness, 8 with right forelimb lameness, and 8 with left forelimb lameness. Vertical and horizontal position of the head and vertical position of the foot, fetlock, carpal, and elbow joints were processed by continuous wavelet transformation. Feature vectors were created from the transformed signals and a neural network trained with data from 6 horses, which was then tested on the remaining 2 horses for each category until each horse was used twice for training and testing. Correct classification percentage (CCP) was calculated for each combination of gait signals tested. RESULTS: Wavelet-transformed vertical position of the head and right forelimb foot had greater CCP (85%) than untransformed data (21%). Adding data from the fetlock, carpal, or elbow joints did not improve CCP over that for the head and foot alone. CONCLUSIONS AND CLINICAL RELEVANCE: Wavelet transformation of gait data extracts information that is important for the detection and differentiation of forelimb lameness of horses. All of the necessary information to detect lameness and differentiate the side of lameness can be obtained by observation of vertical head movement in concert with movement of the foot of 1 forelimb.