KERATOCYTE DENSITY IN VIVO AFTER PHOTOREFRACTIVE KERATECTOMY IN HUMANS* By Jay C. Erie, MD (BY INV'ITATION), Sanjay V. Patel, BMBS (BY INNVITATION), Jay W. McLaren, PhD (BY INV7ITATION), Leo J. Maguire, MD (BY INVITATION), Manuel Ramirez, MD (BY INVITATION), AND William M. Bourne, MD ABSTRACT Purpose: To determine changes in keratocyte density in central huiman corneas in vivo after photorefractive keratectomy (PRK). Methods: Fifteen patients (25 eyes) received excimer PRK (VISX Star) with epithelial removal by laser-scrape (43 iim ablation followed by man- ual scrape) to correct myopia between -1.5D and -7.25D. Corneas were examined by using confocal microscopy in vivo before PRK and at 1 day, 5-days, 1 month, and 3 months after PRK. A cuistom automated image-pro- cessing algorithlm identified bright objects (keratocytes) against a dark background and estimated keratocyte density by using the number and size of the objects. Cell density was quantified in anteroposterior stromal regionis after PRK and compared to cell density in corresponding pre-PRK regions. Resuilts: One day after PRK, keratocyte density inereased 9% in the ante- rior third of the stroma (1l=.003), was unchanged in the middle third of the stroma (Hl=.481), and decreased 6% in the posterior third of the stro- ma (H=.038). Keratocyte density remained elevated in the anterior stroma to 3 months after PRK; at this time, there was a 13% increase in kerato- cyte density throughout the full-thickness stroma (HI<.001). Conclusions: Keratocyte density was increased in the anterior stroma immediately after PRK in humans. Three months later, keratocyte density was increased in all anteroposterior stromal regions, suggesting that PRK affects keratocytes throughout the entire central cornea. °Fromii the Departmiieiot of Ophthalmology, Mayo Clinic, Rochester, Minniiesota. Suipported io part byl grant EY02037 fromii the National Inistituites of Health; Research to Prevent Blindniess, Imic, New7, York; and the Mavo Founiidationi, Rochester, Minniiesota. T1o. ANl. OPIIrrI. SOC. V'OL. XCV7II 1999 222 Erze INTRODUCTION Excimer laser photorefractive keratectomy (PRK) is a widespread sLirgical procedure used to correct myopia, astigmatism, and hyperopia. Although clinical results are good, variable predictability of refractive and visual out- comes, regression of initially achieved refractive effect, and subepithelial haze remiain clinical conicerns. 2 These uindesirable side effects have been attributed to qualitative or quiantitative variations in corneal stromal kera- tocytes and their subsequient effect on corneal wound healing. Previouis histologic studies of corneal changes after PRK in humans7-' have shown that keratocyte density increases in the anterior corneal stro- ma after PRK. Human specimens, however, are rarely available for histol- ogy, requiiring most histologic data to come from rabbits or primates. Since the corneas of these animlals differ in their structure and the nature of their wound healing, comparison to humans should be made cautiously. In addition, histologic data from rabbits and monkeys after PRK have been conflicting. Keratocyte loss in the anterior stroma has been noted in rab- bits after PRK,"' " whereas in monkeys, keratocyte density increased in the anterior stroma after PRK.'2'- Histologic studies also have other limitations when studying corneal wound healing after PRK. Analysis of fixed tissue can only provide infor- mation abouit the appearanice of a cornea at a single time in an individuial subject. Therefore, serial investigations of anJ ongoing process, such as wound healinig, must rely on specimens fromi several subjects at different times. In addition, histologic preparation of specimens alters the tissue being studied from its state in vivo. Thus, alternative methods of studying wound healing in human corneas in vivo after PRK are needed. The development of confocal microscopes enables human corineas to be examained in vivo while avoiding the problems encountered in histolog- ic stuidies.' Conf'ocal microscopy has already proved to be a valuable tool f'or examining corneal wound healing,','9 but so far, few reports of kerato- cyte density after PRK in huiman corneas halve been published.35-2' An accurate quantification of clhanges in keratocyte density after PRK may enhance our understanding of the corneal wound healing process. The aim of our present prospective studcly was to quantify anteroposterior regional keratocyte density in the central human corneal stroma before and after PRK by uising confocal microscopy in vivo and a custom automated cell- counting algorithm. We have previously shown this method to be repeat- able and valid for estimating keratocyte (lensity in vivo in rabbits2' and humians.-- Keratocyte Density in Vivo 223 METHODS PATIENT SELECTION Fifteen patients were studied. Inclusion criteria were normal results on anterior segment and dilated fuindus examination, normal intraocular pressure (<22 mm Hg), any eye color, and any race. All subjects were older than 21 years, and either or both eyes were studied. Subjects were exclud- ed if they wore contact lenses within 6 months of the study; had diabetes mellitus, glaucomna or ocular hypertension (>21 mm Hg), anterior segment pathology or such previous history, or a family history of corneal patholo- gy; or currently used any topical ocular medication. Our Institutional Review Board approved this study, and informed consent was obtained from all subjects after explanation of the nature and possible conse- quences of the study. SURGICAL PROCEDURE All eyes underwent PRK for myopia or myopic astigmatism using the VISX Star excimer laser (VISX, Santa Ana, Calif with wavelength of 193 nm, fixed pulse rate of 6 Hz, and radiaint exposure of 160 mJ/cm.2 Patient self- fixation was used. The epithelium was removed by using the laser-scrape technique (43 jtim epithelial ablation followed by manual scrape of the remaining epithelial cells with a blunt spatula). The profile of the ablation was determined by computer-controlled expansion of the aperture in an iris diaphragm. Immediately upon completion of the ablation, the cornea was cooled for 30 seconds by irrigation of cold balanced salt solution. POSTOPERATIVE REGIMEN AND EXAMINATIONS A bandage soft contact lens (SofLens 66, Bausch & Lomb Inc, Rochester, NY) was worn until the cornea was re-epithelialized (2 to 5 days). Topical medications consisted of preservative-free ketorolac tromethamine 0.5% (Acular PF, Allergan Inc, Irvine, Calif) for 4 doses over 2 days, ofloxacin 0.3% (Ocuflox, Allergan Inc, Irvine, CalifI 4 times daily until re-epithelial- ization was complete, and fluorometholone 0.1% (FML, Allergan Inc, Irvine, Calif) 4 times daily with taper over 3 to 4 months. Slit-lamp biomi- croscopy and confocal microscopy were carried out preoperatively and at 1 day, 5 days, 1 month, and 3 months after PRK. CONFOCAL MICROSCOPY IN VIVO A Tandem Scanning Confocal Microscope (Tandem Scanning Corporation, Reston, Va) was use(d to examine corneas in vivo by using a technique described previously.2-22 The microscope had a 24X, 0.6 NA 9224 Erle objective lens with a concave surface and a working distance of 0 to 1.5 mm. The position of the focal plane (optical section) couldl be advanced or retracted at a predetermined rate under computer control (Silicon Graphics INDY, Silicon Graphics Inc, Mountain View, Calif via an encoder mike controller (Oriel 18011, Oriel Instruments, Stratford, Conn). The position of' the focal plane was adjusted by an internal lens without changing the position of the front surface of the objective. Digital images were recorded by a low-light camera (DAGE-MTI VE-1000 SIT, DAGE-MTI Inc, Michigan City, Ind) directly into compuiter memory. The microscope had been calibrated as outlined in a previous study.-2 Proparacaine hydrochloride 0.5% (Bausch & Lomb Pharmaceuticals, Inc, Tampa, Fla) was instilled into the eye to be examined. Patients wore a bandage soft contact lens during the examination at 1 and 5 days and in some instances at 1 and 3 months. The objective lens was disinfected by using 70% isopropvl alcohol wipes before and after each examlination. A drop of 2.5% hydroxypropyl methylcellulose (CIBANVision Ophthallmics, Atlanta, Ga) optical coupling mediuimi was placed on the tip of the objec- tive lens, and the lens Wcas manually advanced until the mediuml contacted the central cornea (or contact lens). A series of confiocal images was recorded as the focal plane was advaniced at 78 iim/sec from anterior to the epitheliumi (or contact lens) to posterior to the endotlhelium. Digital images were directly acquired by the comiputer workstation and stored in its memory at 30 frames/sec. Each image represented a coronal section approximately 475 jm x 350 mm (horizontal x vertical) and was separated from adjacent images by 2.6 mm.-- Imnages were acquiired from the central corniea by usinlg 2 camera modes: a fixed mode in which the camera was set at a constant gain, voltage, and black level, and an automatic mode in whiclh the levels of these parameters were automatically adjuisted by the camera throughout acquisition. KERATOCYTE DENSITY MEASURENIENT Keratocyte density measuremnent required numnerous digital image-piro- cessing steps that were automatically performed by a custom program2-22 written using the comprehensive AVW library of image processing and visuialization functions (Mayo Biomedical Imagiing Resource and Mayo Medical Ventures, Rochester, Minn).2324 The images uised to estimate ker- atocyte