In Vivo Confocal Microscopy Analyses of Corneal Microstructural Changes in a Prospective Study of Collagen Cross-linking in Keratoconus Charlotte Jordan, PhD, Dipika V. Patel, PhD, MRCOphth, Nandoun Abeysekera, BE (Hons), Charles N.J. McGhee, PhD, FRCOphth Purpose: To use in vivo confocal microscopy (IVCM) to quantitatively analyze microstructural changes over time, after corneal collagen cross-linking for keratoconus. Design: Prospective cohort study. Participants: A total of 38 eyes of 38 patients undergoing collagen cross-linking for keratoconus. Methods: Prospective, clinical cohort study of corneal collagen cross-linking in progressive keratoconus. Laser scanning IVCM performed preoperatively and at 1, 3, 6, and 12 months postoperatively. Main Outcome Measures: Density of corneal sub-basal nerves, anterior and posterior keratocytes, and corneal endothelium. Results: Compared with baseline values, the mean sub-basal nerve density decreased significantly at 1, 3, and 6 months postoperatively (P < 0.01); however, this returned to preoperative values at 12 months (P ¼ 0.57). One month postoperatively, there was complete absence of keratocyte nuclei in 86% of corneas. Anterior stromal edema with hyper-reflective cytoplasm and extracellular lacunae in a honeycomb-like appearance was observed and persisted at 3 months postoperatively. Scattered, presumed fragmented keratocyte nuclei, were observed at 1 and 3 months, but by 6 months, keratocyte repopulation of the anterior stroma was apparent. Quantitative analysis confirmed a significant decrease in the mean anterior keratocyte density 1, 3, and 6 months postoperatively (P 0.01) with return to baseline values at 12 months postoperatively (P ¼ 0.57). The demarcation between treated and untreated corneal stroma appeared as a region where normal keratocytes transitioned into elongated, hyper-reflective, needle-like structures and then into large hyper-reflective stromal bands. There was no significant change in posterior keratocyte density or endothelial density at any postoperative time point. Conclusions: This prospective IVCM study revealed complete loss of the sub-basal nerve plexus and loss of anterior stromal keratocytes in the early postoperative period, with complete regeneration of the sub-basal nerve plexus and keratocyte repopulation by 12 months postoperatively. The posterior stroma and corneal endothelium were unaffected. Ophthalmology 2014;121:469-474 ª 2014 by the American Academy of Ophthalmology. Keratoconus is a noninflammatory, progressive ectasia of the cornea that usually presents in early puberty and progresses until the third to fourth decade of life when the disease typically tends to stabilize, although progression can be variable throughout a patient’s life.1 Our knowledge of keratoconus has increased exponentially since its earliest description; however, the precise cause of the disease remains enigmatic but seems to have both genetic and environmental elements.1e3 The Collaborative Longitudinal Evaluation of Keratoconus study noted that over 8 years, the mean rate of progression of the flattest keratometric value was 0.20.8 diopters (D) per year (mean progression of 1.60 D over 8 years), whereas the flattest corneal curvature increased by 3.0 D or more in at least 1 eye in 24% of subjects.4 Corneal collagen cross-linking is a relatively recently introduced treatment for progressive keratoconus that aims to “stiffen” the cornea by stimulating collagen cross-linking in the corneal stroma using ultraviolet A (UVA) and riboflavin, 2014 by the American Academy of Ophthalmology Published by Elsevier Inc.
thereby halting, or at least slowing, the progression of the disease.5,6 Contemporary in vivo confocal microscopy (IVCM) is a noninvasive method of examining the living human cornea at the cellular and microstructural level in healthy and pathologic states, making it a powerful clinical and research tool7 that has been applied to analysis of the pathologic changes of keratoconus.8 Indeed, IVCM has demonstrated alterations to all layers of the cornea in keratoconus with a particular reduction in density of the sub-basal nerve plexus and stromal keratocytes.8e10 As collagen crosslinking has become more widely adopted, IVCM has been used to identify the microstructural changes in the keratoconic cornea associated with this treatment.11 The aim of this prospective study was to use IVCM to quantitatively analyze the microstructural corneal changes occurring over time, after collagen cross-linking for keratoconus. ISSN 0161-6420/14/$ - see front matter http://dx.doi.org/10.1016/j.ophtha.2013.09.014
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Ophthalmology Volume 121, Number 2, February 2014
Methods Subjects All patients were recruited as part of a prospective clinical study of corneal collagen cross-linking in patients with progressive keratoconus. Inclusion criteria included patients aged 14 to 30 years with progressive keratoconus, maximal keratometry of 60 D, minimal corneal thickness of 400 mm, best spectacle-corrected visual acuity of 20/80, clear cornea with no scarring, and rigid gas permeable contact lens intolerance. Exclusion criteria included previous ocular trauma or surgery, ocular disease (other than keratoconus) or systemic disease that may affect the cornea, and stable keratoconus. Progression was defined as an increase in maximal keratometry of 0.75 D in the preceding 3 months, a change in refractive astigmatism of 0.75 D in the preceding 12 months, a change of 0.2 mm in base curve of contact lens fit in the preceding 12 months, or a decrease in corneal thickness by 30 mm in the preceding 6 months. All patients were recruited from Ophthalmology and Optometry practices across New Zealand and treated and examined at the Department of Ophthalmology, Greenlane Clinical Centre, Auckland District Health Board, Auckland, New Zealand. One eye was randomized to be treated by corneal collagen cross-linking. Informed consent was obtained from all participants, and the study design adhered to the tenets of the Declaration of Helsinki. The study protocol was approved by the Northern X regional ethics committee.
Corneal Collagen Cross-linking Protocol The central 7 to 8 mm of the corneal epithelium was mechanically removed using a bevel-up crescent blade. For corneas with a postepithelial debridement corneal thickness of at least 400 mm, 0.1% solution of isotonic riboflavin ophthalmic solution was instilled (10 mg riboflavin-5-phosphate in 10 ml dextran-T- 500 solution) (Opto Ribolink, Opto Global Pty Ltd, Adelaide, Australia). For corneas with a postepithelial debridement corneal thickness of less than 400 mm, hypotonic riboflavin 0.1% ophthalmic solution was instilled (Opto Ribolink, Opto Global Pty Ltd). After epithelial debridement, riboflavin drops were instilled every 5 minutes for 30 minutes. Light irradiation of the cornea was then commenced using a UVA double diode 370 nm light source located 10 to 12 mm in front of the corneal apex. This produced a radiant energy of 3 mW/cm2 or 5.4 J/cm2 (monitored via a potentiometer/ultraviolet power meter). Irradiation was performed for 30 minutes with further instillation of riboflavin drops every 5 minutes. On completion of the procedure, the eye was flushed with balanced salt solution and a soft bandage contact lens was inserted for the immediate 24-hour postoperative period. Postoperative treatment included preservative-free chloramphenicol eye drops 0.5% (Chauvin Pharmaceuticals, Surrey, UK) 4 times daily for 1 week and analgesia for 4 days (oral tramadol [50 mg every 4e6 hours] and oral diazepam [4 mg at night]).
Examinations Laser scanning IVCM was performed on all treated eyes of all subjects using the Heidelberg Retina Tomograph II Rostock Corneal Module (Heidelberg Engineering GmBH, Heidelberg, Germany). A 60 objective water immersion lens with a numeric aperture of 0.9 (Olympus, Tokyo, Japan) and a working distance, relative to the applanating cap, of 0.0 to 3.0 mm was used. All eyes were anesthetized using a drop of 0.4% benoxinate hydrochloride (Chauvin Pharmaceuticals, Kingston-upon-Thames, England).
470
Viscotears (Carbomer 980, 0.2%, Novartis, North Ryde, Australia) was used as a coupling agent between the applanating lens cap and the cornea. During the examination, all subjects were asked to fixate on a distance target aligned to enable examination of the central cornea. The full thickness of the central cornea was scanned using the device’s “section mode.” The section mode enables instantaneous imaging of a single area of the cornea at a desired depth. The overall examination took approximately 10 minutes to perform for each subject, and none of the subjects experienced any corneal complications as a result of examination. The IVCM images of the corneal endothelium were obtained using the Confoscan 4 (Nidek Technologies, Gamagori, Japan). This system was chosen for endothelial imaging and analysis because good intraobserver and interobserver reproducibility of endothelial density measurement using the automatic endothelial analysis have been reported.12 In contrast, corneal endothelial cell density has been shown to be significantly overestimated with the Rostock Corneal Module laser scanning system.13 All participants were examined preoperatively and at 1, 3, 6, and 12 months postoperatively.
Image Analysis For each time point, 2 of the clearest images from each layer were selected by an experienced observer (C.J.). The sub-basal nerve plexus images were defined as the first clear images of the nerves at the level of Bowman’s layer. The anterior stromal images were defined as the first clear images immediately posterior to Bowman’s layer, whereas the posterior stromal images were defined as the first clear images immediately anterior to the endothelium. All selected images were de-identified and randomized by an independent examiner (N.A.). Quantitative analysis was subsequently performed by a single masked examiner (C.J.) using Image J software (US National Institutes of Health, Bethesda, MD) for keratocyte density and analySIS software (analySIS 3.1 Soft Imaging System, Münster, Germany) for sub-basal nerve density. The full 400400-mm frame of each image of sub-basal nerve plexus and stromal image was used for analysis. Only keratocyte nuclei that were in focus, with all edges within the image frame, were analyzed and counted. Sub-basal nerve density was calculated by measuring the total length of the nerves per image. Endothelial analysis was performed using the automated endothelial analysis tool on the NAVIS application on the Confoscan 4 (Nidek Technologies, Gamagori, Japan).
Statistical Analysis Statistical analysis was undertaken using the Statistical Analysis System version 9.1 (SAS Inc, Cary, NY). Repeated-measures analysis of variance was performed to compare pre- and postoperative values for all parameters. P values
thereby halting, or at least slowing, the progression of the disease.5,6 Contemporary in vivo confocal microscopy (IVCM) is a noninvasive method of examining the living human cornea at the cellular and microstructural level in healthy and pathologic states, making it a powerful clinical and research tool7 that has been applied to analysis of the pathologic changes of keratoconus.8 Indeed, IVCM has demonstrated alterations to all layers of the cornea in keratoconus with a particular reduction in density of the sub-basal nerve plexus and stromal keratocytes.8e10 As collagen crosslinking has become more widely adopted, IVCM has been used to identify the microstructural changes in the keratoconic cornea associated with this treatment.11 The aim of this prospective study was to use IVCM to quantitatively analyze the microstructural corneal changes occurring over time, after collagen cross-linking for keratoconus. ISSN 0161-6420/14/$ - see front matter http://dx.doi.org/10.1016/j.ophtha.2013.09.014
469
Ophthalmology Volume 121, Number 2, February 2014
Methods Subjects All patients were recruited as part of a prospective clinical study of corneal collagen cross-linking in patients with progressive keratoconus. Inclusion criteria included patients aged 14 to 30 years with progressive keratoconus, maximal keratometry of 60 D, minimal corneal thickness of 400 mm, best spectacle-corrected visual acuity of 20/80, clear cornea with no scarring, and rigid gas permeable contact lens intolerance. Exclusion criteria included previous ocular trauma or surgery, ocular disease (other than keratoconus) or systemic disease that may affect the cornea, and stable keratoconus. Progression was defined as an increase in maximal keratometry of 0.75 D in the preceding 3 months, a change in refractive astigmatism of 0.75 D in the preceding 12 months, a change of 0.2 mm in base curve of contact lens fit in the preceding 12 months, or a decrease in corneal thickness by 30 mm in the preceding 6 months. All patients were recruited from Ophthalmology and Optometry practices across New Zealand and treated and examined at the Department of Ophthalmology, Greenlane Clinical Centre, Auckland District Health Board, Auckland, New Zealand. One eye was randomized to be treated by corneal collagen cross-linking. Informed consent was obtained from all participants, and the study design adhered to the tenets of the Declaration of Helsinki. The study protocol was approved by the Northern X regional ethics committee.
Corneal Collagen Cross-linking Protocol The central 7 to 8 mm of the corneal epithelium was mechanically removed using a bevel-up crescent blade. For corneas with a postepithelial debridement corneal thickness of at least 400 mm, 0.1% solution of isotonic riboflavin ophthalmic solution was instilled (10 mg riboflavin-5-phosphate in 10 ml dextran-T- 500 solution) (Opto Ribolink, Opto Global Pty Ltd, Adelaide, Australia). For corneas with a postepithelial debridement corneal thickness of less than 400 mm, hypotonic riboflavin 0.1% ophthalmic solution was instilled (Opto Ribolink, Opto Global Pty Ltd). After epithelial debridement, riboflavin drops were instilled every 5 minutes for 30 minutes. Light irradiation of the cornea was then commenced using a UVA double diode 370 nm light source located 10 to 12 mm in front of the corneal apex. This produced a radiant energy of 3 mW/cm2 or 5.4 J/cm2 (monitored via a potentiometer/ultraviolet power meter). Irradiation was performed for 30 minutes with further instillation of riboflavin drops every 5 minutes. On completion of the procedure, the eye was flushed with balanced salt solution and a soft bandage contact lens was inserted for the immediate 24-hour postoperative period. Postoperative treatment included preservative-free chloramphenicol eye drops 0.5% (Chauvin Pharmaceuticals, Surrey, UK) 4 times daily for 1 week and analgesia for 4 days (oral tramadol [50 mg every 4e6 hours] and oral diazepam [4 mg at night]).
Examinations Laser scanning IVCM was performed on all treated eyes of all subjects using the Heidelberg Retina Tomograph II Rostock Corneal Module (Heidelberg Engineering GmBH, Heidelberg, Germany). A 60 objective water immersion lens with a numeric aperture of 0.9 (Olympus, Tokyo, Japan) and a working distance, relative to the applanating cap, of 0.0 to 3.0 mm was used. All eyes were anesthetized using a drop of 0.4% benoxinate hydrochloride (Chauvin Pharmaceuticals, Kingston-upon-Thames, England).
470
Viscotears (Carbomer 980, 0.2%, Novartis, North Ryde, Australia) was used as a coupling agent between the applanating lens cap and the cornea. During the examination, all subjects were asked to fixate on a distance target aligned to enable examination of the central cornea. The full thickness of the central cornea was scanned using the device’s “section mode.” The section mode enables instantaneous imaging of a single area of the cornea at a desired depth. The overall examination took approximately 10 minutes to perform for each subject, and none of the subjects experienced any corneal complications as a result of examination. The IVCM images of the corneal endothelium were obtained using the Confoscan 4 (Nidek Technologies, Gamagori, Japan). This system was chosen for endothelial imaging and analysis because good intraobserver and interobserver reproducibility of endothelial density measurement using the automatic endothelial analysis have been reported.12 In contrast, corneal endothelial cell density has been shown to be significantly overestimated with the Rostock Corneal Module laser scanning system.13 All participants were examined preoperatively and at 1, 3, 6, and 12 months postoperatively.
Image Analysis For each time point, 2 of the clearest images from each layer were selected by an experienced observer (C.J.). The sub-basal nerve plexus images were defined as the first clear images of the nerves at the level of Bowman’s layer. The anterior stromal images were defined as the first clear images immediately posterior to Bowman’s layer, whereas the posterior stromal images were defined as the first clear images immediately anterior to the endothelium. All selected images were de-identified and randomized by an independent examiner (N.A.). Quantitative analysis was subsequently performed by a single masked examiner (C.J.) using Image J software (US National Institutes of Health, Bethesda, MD) for keratocyte density and analySIS software (analySIS 3.1 Soft Imaging System, Münster, Germany) for sub-basal nerve density. The full 400400-mm frame of each image of sub-basal nerve plexus and stromal image was used for analysis. Only keratocyte nuclei that were in focus, with all edges within the image frame, were analyzed and counted. Sub-basal nerve density was calculated by measuring the total length of the nerves per image. Endothelial analysis was performed using the automated endothelial analysis tool on the NAVIS application on the Confoscan 4 (Nidek Technologies, Gamagori, Japan).
Statistical Analysis Statistical analysis was undertaken using the Statistical Analysis System version 9.1 (SAS Inc, Cary, NY). Repeated-measures analysis of variance was performed to compare pre- and postoperative values for all parameters. P values
