Corneal Wound Repair After Rose Bengal and Green Light Crosslinking: Clinical and Histologic Study
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Cornea Corneal Wound Repair After Rose Bengal and Green Light Crosslinking: Clinical and Histologic Study Patricia Gallego-Munoz,˜ 1 Luc´ıa Ibares-Fr´ıas,1 Elvira Lorenzo,1 Susana Marcos,2 Pablo Perez-Merino,´ 2 Nandor Bekesi,2 Irene E. Kochevar,3 and M. Carmen Mart´ınez-Garc´ıa1 1Departamento de Biolog´ıa Celular, Histolog´ıa y Farmacolog´ıa, GIR de Tecnicas´ Opticas´ para el Diagnostico,´ Universidad de Valladolid, Valladolid, Spain 2Instituto de Optica,´ Consejo Superior de Investigaciones Cient´ıficas, Madrid, Spain 3Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States Correspondence: M. Carmen PURPOSE. To evaluate corneal wound healing after treatment with a new collagen crosslinking Mart´ınez-Garc´ıa, Departamento de protocol using rose bengal dye and green light (RGX). Biolog´ıa Celular, Histolog´ıa y Farm- acolog´ıa, Facultad de Medicina, Uni- METHODS. One cornea of 20 New Zealand rabbits was de-epithelialized (DE) in an 8-mm versidad de Valladolid, C/Ramon´ y diameter circle and, in another group (n ¼ 25), the DE corneas were then stained with 0.1% Cajal 7, 47005 Valladolid, Spain; rose bengal for 2 minutes and exposed to green light (532 nm) for 7 minutes (RGX). The mariacarmen.martinez.garcia@ contralateral eyes without treatment acted as controls. The animals were clinically followed uva.es. including fluorescein staining and pachymetry. Healing events were analyzed after euthanasia 0 Submitted: December 23, 2016 at 2, 30, and 60 days. Cell death (TUNEL assay), cell proliferation (5-bromo-2 -deoxyuridine Accepted: June 1, 2017 incorporation), and cell differentiation to myofibroblasts (a-SMA labeling) were carried out. In Citation: Gallego-Munoz˜ P, Ibares-Fr´ıas addition, loss of keratocytes and subsequent repopulation of the corneal stroma were L, Lorenzo E, et al. Corneal wound quantified on hematoxylin-eosin–stained sections. repair after rose bengal and green RESULTS. Wound closure was slower after RGX (4.4 days) then after DE (3.3 days). Cell death light crosslinking: clinical and histo- was restricted to the anterior central stroma, and the cellular decrease did not differ logic study. Invest Ophthalmol Vis Sci. significantly between RGX and DE corneas. Cell proliferation in the epithelium and stroma 2017;58:3471–3480. DOI:10.1167/ iovs.16-21365 appeared at 2 days. In both DE and RGX corneas, recovery of the epithelium was complete at day 30, although cell repopulation of the stroma was not complete at 60 days. CONCLUSIONS. The healing response in corneas after RGX is very similar to that observed after DE alone, suggesting that, along with its short treatment time and limited effect on keratocytes, RGX displays good potential for clinical cornea stiffening. Keywords: wound repair, crosslinking, keratoconus treatment, rose bengal, cornea orneal crosslinking (CXL) is currently used to stiffen the Alternatives, such as the use of hypo-osmolar riboflavin,12 C stroma, and thus inhibit the progressive cornea protrusion transepithelial corneal CXL,13,14 iontophoresis transcorneal characteristic of keratoconus and other ectatic corneal diseas- delivery,15 and accelerated CXL16,17 have been proposed. es.1–3 This simple and noninvasive technique is based on light- However, the safety and efficacy of these procedures is yet to initiated photochemical reactions that induce formation of co- be well established.18 valent crosslinks between collagens and between collagen and For these reasons, new crosslinking techniques are being proteoglycans.4 A photosensitizer, typically riboflavin (vitamin developed. One new method uses rose bengal (RB) as the B2), is applied to the de-epithelialized (DE) cornea, which is photosensitizer together with green light (532 nm) and only then exposed to long wavelength ultraviolet light (370-nm UVA) requires approximately 10 minutes for treatment. Rose bengal for up to 30 minutes using the conventional procedure. The is well known as a diagnostic agent for ocular surface damage. It photochemical reaction produces reactive oxygen species and has already been used with green light to seal corneal wounds radicals that induce formation of covalent bonds.5 As a result of without sutures,19 to attach amniotic membrane to the ocular these reactions, protein crosslinking stabilizes corneal biome- surface20 to repair nerves and blood vessels in other tissues and chanics and increases corneal stiffness.6–8 for a clinical study of skin wound sealing.21 Thin corneas may not contain enough riboflavin to block These studies have shown that RB–green light crosslinking, the UVA from the endothelium, thus making the procedure termed RGX, creates bonds in stromal collagen fibrils similar to unsuitable for these corneas. For this reason, treating corneas the mechanism involved in conventional CXL. Previous ex vivo thinner than 400 lm after epithelial removal is not recom- studies using rabbit eyes have demonstrated that RGX mended.9 In addition, the conventional and most widely used significantly increases corneal stiffness3 and causes no toxicity procedure is lengthy, and a tendency toward progression of to keratocytes 24 hours post treatment.22 In a recent study of keratometry values 24 months after CXL is sometimes reported, RGX in vivo in rabbit eyes, the green light level that increased particularly among the pediatric population.10,11 corneal stiffness was shown to be safe for the retina, as Copyright 2017 The Authors iovs.arvojournals.org j ISSN: 1552-5783 3471 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 10/01/2021 Healing Response After RGX IOVS j July 2017 j Vol. 58 j No. 9 j 3472 evidenced by the lack of damage to the blood–retinal barrier, Germany) before and after treatments and at different time the retinal photoreceptors, the retinal pigment epithelium, and points (1, 2, 3, 4, 15, 30, and 60 days, immediately prior to the choriocapillaris.23 euthanasia). In the clinical follow-up, the epithelial wound was In order to further evaluate the safety and effectiveness of stained by sodium fluorescein (Fluotest; Alcon, Cusi, Barcelo- the RGX technique a greater understanding of the post- na) every day until epithelial closure. Conjunctival hyperemia treatment healing process is required; thus, necessitating the was quantified from 0 to 4 according to the Efron Scale for use of an experimental animal model. We selected the rabbit hyperemia.25 Corneal pachymetry was carried out with an because it has been used as a model of corneal wound healing ultrasonic pachymeter (Corneo-Gage Plus; Sonogage, Inc., after different surgeries, including conventional crosslink- Cleveland, OH, USA). ing24–27 and RGX.23,24 The goal of the present study was to characterize the Tissue Processing and Light Microscopy wound healing process after RGX. We compared clinical and histologic outcomes in RGX and DE rabbit eyes in vivo. Eyes Animals were euthanized on days 2, 30, and 60 after treatments were followed for 60 days in order to monitor the healing by intracardiac injection of sodium pentobarbital (Dolethal process. 0737-ESP; Vetoquinol, Madrid, Spain) under general anesthesia. Eyes were enucleated and divided into three groups: group 1, DE eyes (DE); group 2, RGX-treated eyes (RGX); and group 3, MATERIALS AND METHODS contralateral untreated eyes (control). They were then fixed with 4% buffered paraformaldehyde and embedded in paraffin. The Animal Ethics Committee at the University of Valladolid Sections, 5-lm thick were stained with hematoxylin-eosin (H- approved the animal studies described in this research. E). Sections were examined under an Olympus BX41 Animals were cared for following the guidelines of the ARVO microscope (Olympus Life Science, Hamburg, Germany) and Statement for the Use of Animals in Ophthalmic and Vision photomicrographs were obtained with an Olympus DP20 Research. Digital Camera. Quantitative measurements of the photographs Female adult albino New Zealand rabbits, weighing 2.5 to 3 were taken using the program Cell A (Olympus Soft Imaging kg, were supplied by a center listed in the official register as a Solutions GmbH, Munster,¨ Germany). provider of lab animals (Granja San Francisco, Navarra, Spain). Treatment Procedure Cell Counting Forty-five New Zealand white rabbits were divided into two In each cornea, three measures of corneal full thickness were groups.Bothwereanesthetizedwithanintramuscular taken at 340 magnification. All the H-E stained cells were then injection of ketamine hydrochloride (37.5 mg/kg, Ketolar; counted at 3100 magnification using the Touch Count function Parke Davis SA, Barcelona, Spain) and xylazine hydrochloride from Cell A software (Olympus Soft Imaging Solutions GmbH) 2 (5 mg/kg, Rompun; Bayer, Leverkusen, Germany) in the thigh, in three columns of 90,000 lm (from epithelium to followed by topical application of 0.5% tetracaine hydrochlo- endothelium) in the center of the cornea (wound in DE and ride and 1 mg of oxybuprocaine (Colircusi Anestesico´ Doble; RGX) and limbus. Each column was divided into anterior, 2 Alconcus´ı SA, Barcelona, Spain). medial, and posterior layers, each approximately 30,000 lm . In one group (20 rabbits), the central corneal epithelium Layers were 146-lm thick in the control and RGX, and 176-lm was manually removed by gentle scraping with a sharp scraper thick in DE because of the greater thickness of the DE corneas. in the area of an 8-mm diameter circle demarcated by a All H-E-stained sections were prepared identically to facilitate trephine (DE group). Contralateral eyes were not DE and were comparison. used as controls. The second group (25 rabbits) was treated with RGX to Cell Death: TUNEL Assay crosslink corneal proteins. After demarcating a central area with an 8-mm trephine, the epithelial layer was removed. In order to detect DNA fragmentation associated with Sterile RB (0.1% in PBS, 0.5 mL) was then dropped onto the apoptosis, TUNEL assays were performed in deparaffinized cornea and the cornea was exposed to green light.