ARTICLE
Recovery of corneal sensitivity after collagen crosslinking with and without epithelial debridement in eyes with keratoconus Leopoldo Spadea, MD, Serena Salvatore, MD, Maria Pia Paroli, MD, Enzo Maria Vingolo, MD
PURPOSE: To evaluate the changes in corneal sensitivity after corneal collagen crosslinking (CXL) with the epithelium off (epi-off) and with the epithelium on (epi-on) in eyes with keratoconus. DESIGN: Prospective interventional case series. SETTING: Eye Clinic, A. Fiorini Hospital, Terracina (Latina), Rome, Italy. METHODS: On the basis of the corneal pachymetry at the area of topographic steepening, eyes treated with CXL were divided in 2 groups: those with a corneal thickness of 400 mm or more (epi-off) and those with a corneal thickness of less than 400 mm (epi-on). Corneal sensitivity was measured using a contact esthesiometer (Cochet-Bonnet) before treatment and 1 and 7 days and 1, 3, 6, 9, and 12 months after treatment. RESULTS: The study evaluated 50 CXL-treated eyes in 50 patients (25 epi-on and 25 epi-off) from January 7, 2012, to December 15, 2012. In the epi-off group, corneal sensitivity was statistically significantly reduced for up to 3 months after CXL and gradually returned to normal levels. In the epi-on group, corneal sensitivity was statistically significantly reduced for up to 7 days but was not statistically significantly different from preoperative values at other measurement times. At 7 days, the corneal sensitivity was statistically significantly lower in the epi-off eyes than in the epi-on eyes. CONCLUSIONS: Epi-off and epi-on CXL both caused hypoesthesia, but corneal sensitivity subsequently recovered completely. The corneal hypoesthesia was more pronounced in eyes in which the epi-off technique was used, and the recovery time was shorter for eyes treated using epi-on CXL. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned. J Cataract Refract Surg 2015; 41:527–532 Q 2015 ASCRS and ESCRS
The aim of corneal collagen crosslinking (CXL) with riboflavin and ultraviolet-A (UVA) is to arrest the progression of keratoconus and secondary ectasia.1 This surgical technique is used to improve the mechanical stability of the cornea by increasing the formation of covalent intrafibrillar and interfibrillar bonds using photosensitized oxidation.1 It also might decrease corneal steepening and improve the refractive error by minimizing irregular astigmatism induced by biomechanical instability of the cornea.2,3 Under the CXL protocol recommended by Wollensak et al.,4 the corneal epithelium is debrided before the Q 2015 ASCRS and ESCRS Published by Elsevier Inc.
procedure to allow riboflavin to penetrate the stroma (hence, “epithelium off” [epi-off]). However, the epithelium debridement can increase the risk for ulcers, infections, haze, scarring, infiltrates, longer recovery time, patient discomfort, and postoperative pain.5,6 Partial modifications to the epi-off procedure were introduced to overcome the potential disadvantages of epithelium debridement. The application of multiple topical anesthetic eyedrops was proposed to loosen the tight junctions of the corneal epithelial cells, facilitating riboflavin penetration through an intact epithelium (“epithelium on” [epi-on]).7 Furthermore, http://dx.doi.org/10.1016/j.jcrs.2014.06.030 0886-3350
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epi-on CXL can also be safely used in keratoconic eyes with very thin corneas. In fact, the epi-off CXL technique is used only in eyes with a corneal thickness of at least 400 mm because of concerns about the cytotoxic effect on the corneal endothelium, crystalline lens, and other intraocular tissues.8 Riboflavin and UVA irradiation can cause photochemical damage, such as keratocyte apoptosis, by forming oxygen-free radicals.9 Although the efficacy of the epi-on technique is still under debate,10 some clinical studies have shown encouraging results.2,8,11,12 Filippello et al.11 reported a substantial arrest of keratoconus progression after epi-off CXL, with a statistically significant improvement in visual acuity and topographic parameters. Corneal sensitivity recently has gained attention given how important corneal innervation is to maintaining normal corneal architecture and functionality. The impairment of corneal innervation might diminish the cornea's ability to bear surgical challenges and could increase the risk for complications. Evaluating nerve integrity is essential for assessing the efficacy and long-term effects of corneal treatments. In vivo, ex vivo, and in vitro investigations have found significant alterations in the morphology and histology of corneal tissue, mainly at the anterior 300 mm after CXL.13 Despite several clinical and laboratory studies of the effect of CXL on the corneal microstructure,1 little is known about corneal sensitivity and the immediate and long-term postoperative effect of CXL on corneal innervation. Two recent studies reported transient hypoesthesia in keratoconic corneas 6 to 9 months after epi-off CXL.14,15 To our knowledge, to date there have been no published studies of corneal sensitivity after epi-on CXL. This study evaluated corneal sensitivity in keratoconic eyes following epion and epi-off CXL. PATIENTS AND METHODS This prospective interventional study included eyes with progressive keratoconus of patients with no other systemic or ocular disease. Keratoconus was diagnosed using computerized videokeratography (Keratograph, Oculus Optikger€ate GmbH) and ultrasound (US) pachymetry (Corneo Gage Plus, Sonogage, Inc.). Progression was defined as an increase in maximum keratometry (K) of 1.00 diopter
Submitted: January 5, 2014. Final revision submitted: June 4, 2014. Accepted: June 8, 2014.
(D) or more in the previous 12 months and patientreported deterioration of visual acuity (after excluding that from noncorneal-related causes). If progression was confirmed, CXL was proposed as a treatment to halt the keratoconus progression. Patients were thoroughly counseled about the current clinical experience, possible outcomes and complications of the procedure, and the research of the study team. On the basis of the corneal pachymetry (epithelium included) at the area of topographic steepening, the study eyes were divided into 2 groups: those with corneal thickness of 400 mm or more, in which epi-off CXL would be performed, and those with corneal thickness of less than 400 mm, in which epi-on CXL would be performed. The study followed the tenets of the Declaration of Helsinki. All patients gave informed written consent. The inclusion criteria for both groups were topographic progression of keratoconus, a steepest cone apex of less than 65.0 D, a corrected distance visual acuity (CDVA) with spectacles of 20/60 or better, and an endothelial density of at least 2000 cells/mm2. Exclusion criteria for both groups were corneal opacity, previous herpetic keratitis, previous ocular surgery, active ocular infection, autoimmune disease, diabetes, pregnancy, lactation, and smoking of tobacco (because of the presumed negative association between smoking and progression of keratoconus and corneal sensitivity).16 In each patient, the procedure was performed in the eye that showed more progression of the disease. When the disease progression was similar in both eyes, the eye with the worse CDVA was treated. The preoperative and postoperative evaluations included corneal topography, uncorrected distance visual acuity (UDVA) and CDVA with manifest refraction, corneal sensitivity, slitlamp biomicroscopy, applanation tonometry, US pachymetry, and noncontact endothelial specular microscopy. Follow-up evaluations were scheduled for 1 and 7 days and 1, 3, 6, 9, and 12 months. Patients were instructed to discontinue use of contact lenses at least 4 weeks before each visit.17 Before the instillation of eyedrops, corneal sensitivity was measured using a contact esthesiometer (CochetBonnet, Luneau Ophtalmologie). All eyes were measured by the same ophthalmologist (S.S.), who was masked to the nature of the treatment. A 0.12 mm diameter, 6.0 cm long nylon filament was gently placed on the center of the cornea perpendicular to the surface. Patients were asked to look straight ahead and indicate when they felt the stimulus. If there was no response to the first contact, the filament was shortened by 5.0 mm to increase its rigidity and the stimulus was reapplied. This process was repeated until the patient felt the stimulus. The mean filament length, derived from a minimum of 3 stimulus applications that produced a response from the eye, was considered to be the corneal touch threshold. All corneal sensitivity measurements were analyzed at the same time of day (between 9:00 AM and 11:00 AM).
Surgical Technique
From the Department of Biotechnology and Medical-Surgical Sciences, Latina (Spadea, Salvatore, Vingolo), and the Department of Ophthalmology, Rome (Paroli), Sapienza University of Rome, Italy. Corresponding author: Leopoldo Spadea, MD, Via Benozzo Gozzoli 34, 00142 Rome, Italy. E-mail: [email protected].
All procedures were performed by the same surgeon (L.S.). Epi-off CXL was performed under sterile conditions as follows: One drop of pilocarpine 1.0% was instilled 30 minutes preoperatively. Preoperatively, oxybuprocaine 0.4% eyedrops preserved with p-hydroxybenzoate (Benoxinato Cloridrato) were instilled initially and 3 times at 5-minute intervals as a topical anesthetic. The epithelium was
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removed using a blunt metal spatula to mechanically scrape a 9.0 mm diameter epithelial area of the cornea. Before irradiation, the cornea was soaked for 15 minutes in riboflavin solution (Ricrolin, Sooft Italia S.p.A.). Then the cornea was exposed to a solid-state UVA illuminator (CBM, X-linker, Costruzione Strumenti Oftalmici) for 30 minutes during which the riboflavin solution was applied every 2.5 minutes. The UVA illuminator was calibrated to 3.0 mW/cm2 of surface irradiance (5.4 J/cm2 surface dose) using a UV light meter at the specified working distance. The aiming beam was focused on the central cornea while the patient fixated on a pulsating green light. Epi-on CXL was performed under sterile conditions as follows: Riboflavin 0.1% solution in 15% dextran T500 with sodium ethylenediaminetetraacetic acid 0.01% and trometamol (tris[hydroxymethyl]amino methane) (Ricrolin TE, Sooft Italia S.p.A.) was instilled every 10 minutes for 2 hours. Preoperatively, oxybuprocaine 0.4% eyedrops preserved with p-hydroxybenzoate (Benoxinato Cloridrato) were instilled initially and 3 times at 5-minute intervals as a topical anesthetic. One drop of pilocarpine 1.0% was instilled 30 minutes preoperatively; then an eyelid speculum was inserted and riboflavin was instilled over the cornea every 3 minutes for 15 minutes. Using a CBM Vega X-Linker device (3.0 mW/cm2), UVA irradiation was delivered for 30 minutes during which the riboflavin solution was instilled every 5 minutes.8 At the end of both procedures, the eye was rinsed with a balanced salt solution and a bandage soft contact lens was applied. An antibiotic regimen of ofloxacin and flurbiprofen drops was administered 4 times a day for 1 week. Topical corticosteroid drops (clobetasone butyrate 0.1%) were then administered for 1 month and subsequently tapered.
Statistical Analysis Statistical analysis was performed using the Statistical Package for the Social Sciences for Windows software (version 19.0). Comparisons between preoperative and postoperative values at each timepoint were performed for the epi-on and epi-off groups. The differences between preoperative and postoperative values were assessed using the Wilcoxon signed-rank test. Comparisons of corneal sensitivity values between the epi-off and epi-on groups at each follow-up time were made using repeated-measures analysis of variance. The Bonferroni post hoc test was used for post hoc analysis. Statistical differences in corneal sensitivity at baseline between the 2 groups were evaluated using a Student t test, and P values less than 0.05 were considered statistically significant.
RESULTS Fifty eyes of 50 patients had CXL. Of these, 25 (9 women and 16 men; mean age 29.2 years G 11.0 [SD]; mean corneal thickness 421.6 G 21.4 mm) were assigned to the epi-off CXL group and 25 (12 women and 13 men; mean age 31.5 G 9.9 years; mean corneal thickness 379.6 G 19.2 mm) were placed in the epi-on CXL group. In the epi-off group, the epithelium in all eyes recovered within 4 G 1 days after deepithelialization, whereas in the epi-on group, it healed completely in all eyes within 1 day. Figure 1 plots the mechanical sensitivity in the center of the cornea before and after
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Figure 1. The mean values for preoperative and postoperative corneal sensitivity for the epi-off and epi-on CXL groups. The vertical bars denote 95% confidence intervals. The asterisks indicate a statistically significant reduction in corneal sensitivity over baseline in both groups. For the epi-off group, the postoperative P values were ! .001 at 1 day, 7 days, and 1 month; ! .049 at 3 months; ! .28 at 6 months; ! .82 at 9 months, and ! .9 at 12 months. For the epi-on group, the postoperative P values were ! .001 at 1 day, ! .02 at 7 days, ! .061 at 1 month, ! .061 at 3 months, ! .78 at 6 months, ! .91 at 9 months, and ! .9 at 12 months.
treatment with epi-on CXL and epi-off CXL There was no statistically significant difference in preoperative corneal sensitivity between the 2 groups (P Z .9). In the epi-off CXL group, central corneal sensitivity was statistically significantly lower for the first 7 days than preoperatively (P ! .001). The corneal sensitivity recovered in 3 months but remained statistically significantly lower (P ! .05) than it was preoperatively until 6 months after treatment, when it regained preoperative levels. In the epi-on CXL group, corneal sensitivity was statistically significantly lower 1 and 7 days after surgery (P ! .05) than it was preoperatively but was not statistically significantly different at subsequent measurements (Figure 1). Corneal sensitivity 1 and 7 days after epi-off CXL was statistically significantly lower than after epi-on CXL (P ! .001). DISCUSSION The corneal epithelium is the most densely innervated and sensitive surface of the body. Epithelial nerve density in the cornea is 300 to 600 times higher than that of skin.18 Corneal sensory nerves protect the cornea from external insults by initiating nerve reflex mechanisms.18,19 Corneal nerves are crucial to the maintenance of epithelial integrity, the modulation of cell proliferation, the stimulation of ion transport, and the
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regulation of wound healing after corneal injury. In addition to these functions, corneal nerves exert various trophic effects on the cornea by liberating neuropeptides, neurotrophins, and growth factors that promote corneal epithelial homeostasis and by activating brainstem circuits that stimulate reflex tear production and blinking.20 Damage to corneal nerves from surgery, trauma, or disease leads to diminished corneal sensitivity and possible transient or longterm alterations to the functional integrity of the ocular surface.21 A notable number of authors have analyzed corneal sensitivity in keratoconic eyes.22 Dogru et al.23 found that 48% of keratoconic eyes had reduced corneal sensitivity. They also found that corneal sensitivity was statistically significantly decreased in eyes with severe keratoconus than in eyes with mild or moderate disease. Several studies proposed that hypoesthesia in eyes with severe keratoconus implied the presence of corneal epithelial/stromal disease.23,24 Other studies noted that keratoconic eyes of patients who wore contact lenses had statistically significantly less corneal sensitivity than normal eyes (controls) of patients who wore contact lenses.25 Cho et al.26 analyzed corneal sensitivity in patients with asymmetric keratoconus and found less corneal sensitivity in all eyes, in clinical and subclinical keratoconus, than in normal eyes. They suggested that the hypoesthesia was associated with tear deficiency, abnormal impression cytological results, and thinning of the cornea. Our results, similar to those in other studies,14,15,27 showed an initial reduction in corneal sensitivity after epi-off CXL and a recovery of sensitivity during the 6month follow-up. Although corneal sensitivity during the 7 days after epi-off CXL was significantly lower than after epi-on CXL, after the epi-on treatment the cornea was significantly less sensitive than it was preoperatively. This shows that epi-on CXL might affect corneal sensitivity, but to a lesser degree than epi-off CXL. It also suggests that the reduction in corneal sensitivity after the epi-off CXL was probably induced by the intraepithelial nerve damage caused when the epithelium was mechanically removed and by the deeper corneal nerve damage induced by the CXL treatment. A study of eyes of New Zealand white rabbits27 found that corneal sensitivity decreased after deepithelialization alone, without UVA irradiation. This result seems to confirm that the sensory innervation of the corneal epithelium is crucial to the sensory functions of the cornea. Furthermore, the study found a significant difference between the recovery time of corneal sensitivity after epi-off CXL and after deepithelialization alone. This might be indirect evidence
that UVA exposure causes additional nerve damage. These data also suggest that the corneal epithelium might protect the deeper corneal nerves from the damaging effects of UVA exposure. In fact, the theoretical basis for epi-on CXL presents 2 major challenges: the role of the corneal epithelium as a barrier to UVA penetration and the difficulty of penetration by a hydrophilic macromolecule such as riboflavin through the corneal epithelium. The epithelium and Bowman layer have high absorption coefficients in the UV spectrum. The UV-filtering ability of the epithelium might be the result of its molecular composition, which gives it a high absorption coefficient. Furthermore an appropriate distribution of riboflavin in the cornea is critical because inadequate stromal absorption of riboflavin is likely to reduce the effectiveness of the CXL process.28 In vivo corneal confocal microscopy analysis of the postoperative impact of CXL on the cornea revealed clear differences between epi-off CXL and epi-on CXL. One study showed that the subbasal nerve plexus was essentially destroyed immediately after epi-off CXL and that there was virtually no nerve fiber regeneration by 6 months, whereas the cornea appeared mostly unaltered following epi-on CXL.29 A corneal optical coherence tomography analysis and histological study after epi-on CXL treatments showed a line of demarcation approximately 100 mm from the corneal epithelial layer, slightly beneath Bowman membrane.11,12 In contrast, after epi-off CXL the demarcation line was deeper, 320 to 340 mm from the corneal epithelial layer.28 Thus, less of the cornea is involved and therefore fewer nerve fibers are altered with epi-on CXL than with epi-off CXL. These data could explain why less corneal hypoesthesia occurred in the epi-on group than in the epi-off group. Contact lens use also affects corneal sensitivity.17 To minimize the effect of contact lens use on measurements, patients were asked to discontinue their use at least 4 weeks before each examination. A limitation of this study is that we used only the contact esthesiometer to monitor corneal sensitivity. This instrument uses mechanical stimuli to test the response of the cornea's mechanonociceptors. Consequently, the study could not evaluate the corneal sensitivity's recovery to chemical and temperature stimuli. Other aesthometers, eg, the air-jet esthesiometer (CRCERT-Belmonte), can test the response of all 3 types of neuroreceptors on the ocular surface.30 In addition, the contact esthesiometer cannot detect subtle changes in sensitivity and hypersensitivity because it has a lower threshold for detection.30 In our study, the measurement of corneal sensitivity recovery at 12 months was truncated at 6.0 cm only in
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16% of eyes. Furthermore, because most studies evaluating the effect of surgical procedures on corneal sensitivity used the contact esthesiometer, their results were somewhat comparable to ours. Since the completion of our study, flurbiprofen has been found to cause a slight reduction in the corneal sensitivity to mechanical and chemical stimulation,31 which might have affected corneal sensitivity in our study. However, in our study, flurbiprofen was used in both groups in the same way, which should have limited any confounding effect on our analysis. In conclusion, the results in our study indicate that epi-off CXL and epi-on CXL in keratoconic eyes only transiently affects corneal innervation and sensitivity and that there is a complete recovery over the follow-up period. WHAT WAS KNOWN There are few published studies investigating the immediate and long-term postoperative effects of epi-off CXL on corneal innervation. Corneal sensitivity after epi-on CXL has not been evaluated in the literature.
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Epi-off CXL and epi-on CXL in keratoconic eyes only transiently affected corneal innervation and sensitivity, and the corneal sensitivity recovered completely.
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REFERENCES 1. Spoerl E, Huhle M, Seiler T. Induction of cross-links in corneal tissue. Exp Eye Res 1998; 66:97–103 2. Spadea L, Mencucci R. Corneal cross-linking [letter]. Ophthalmology 2011; 118:2520.e1–2520.e4 3. Spadea L. Corneal collagen cross-linking with riboflavin and UVA irradiation in pellucid marginal degeneration. J Refract Surg 2010; 26:375–377 4. Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-A– induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol 2003; 135:620–627. Available at: http:// grmc.ca/assets/files/collagen_crosslinking_2003_wollensak. pdf. Accessed November 11, 2014 5. Kymionis GD, Portaliou DM, Bouzoukis DI, Suh LH, Pallikaris AI, Markomanolakis M, Yoo SH. Herpetic keratitis with iritis after corneal crosslinking with riboflavin and ultraviolet A for keratoconus. J Cataract Refract Surg 2007; 33:1982–1984 6. Pollhammer M, Cursiefen C. Bacterial keratitis early after corneal crosslinking with riboflavin and ultraviolet-A. J Cataract Refract Surg 2009; 35:588–589 7. Leccisotti A, Islam T. Transepithelial corneal collagen crosslinking in keratoconus. J Refract Surg 2010; 26:942–948 8. Spadea L, Mencucci R. Transepithelial corneal collagen cross-linking in ultrathin keratoconic corneas. Clin Ophthalmol 2012; 6:1785–1792. Available at: http://www.ncbi.nlm.nih.
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29. Touboul D, Efron N, Smadja D, Praud D, Malet F, Colin J. Corneal confocal microscopy following conventional, transepithelial, and accelerated corneal collagen cross-linking procedures for keratoconus. J Refract Surg 2012; 28:769–776 30. Golebiowski B, Papas E, Stapleton F. Assessing the sensory function of the ocular surface: implications of use of a noncontact air jet aesthesiometer versus the Cochet–Bonnet aesthesiometer. Exp Eye Res 2011; 92:408–413 lvez A, Perea31. Acosta MC, Berenguer-Ruiz L, Garcıa-Ga Tortosa D, Gallar J, Belmonte C. Changes in mechanical, chemical, and thermal sensitivity of the cornea after topical application of nonsteroidal anti-inflammatory drugs. Invest
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First author: Leopoldo Spadea, MD Department of Biotechnology and Medical-Surgical Sciences, Latina, Sapienza, University of Rome, Rome, Italy
Recovery of corneal sensitivity after collagen crosslinking with and without epithelial debridement in eyes with keratoconus Leopoldo Spadea, MD, Serena Salvatore, MD, Maria Pia Paroli, MD, Enzo Maria Vingolo, MD
PURPOSE: To evaluate the changes in corneal sensitivity after corneal collagen crosslinking (CXL) with the epithelium off (epi-off) and with the epithelium on (epi-on) in eyes with keratoconus. DESIGN: Prospective interventional case series. SETTING: Eye Clinic, A. Fiorini Hospital, Terracina (Latina), Rome, Italy. METHODS: On the basis of the corneal pachymetry at the area of topographic steepening, eyes treated with CXL were divided in 2 groups: those with a corneal thickness of 400 mm or more (epi-off) and those with a corneal thickness of less than 400 mm (epi-on). Corneal sensitivity was measured using a contact esthesiometer (Cochet-Bonnet) before treatment and 1 and 7 days and 1, 3, 6, 9, and 12 months after treatment. RESULTS: The study evaluated 50 CXL-treated eyes in 50 patients (25 epi-on and 25 epi-off) from January 7, 2012, to December 15, 2012. In the epi-off group, corneal sensitivity was statistically significantly reduced for up to 3 months after CXL and gradually returned to normal levels. In the epi-on group, corneal sensitivity was statistically significantly reduced for up to 7 days but was not statistically significantly different from preoperative values at other measurement times. At 7 days, the corneal sensitivity was statistically significantly lower in the epi-off eyes than in the epi-on eyes. CONCLUSIONS: Epi-off and epi-on CXL both caused hypoesthesia, but corneal sensitivity subsequently recovered completely. The corneal hypoesthesia was more pronounced in eyes in which the epi-off technique was used, and the recovery time was shorter for eyes treated using epi-on CXL. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned. J Cataract Refract Surg 2015; 41:527–532 Q 2015 ASCRS and ESCRS
The aim of corneal collagen crosslinking (CXL) with riboflavin and ultraviolet-A (UVA) is to arrest the progression of keratoconus and secondary ectasia.1 This surgical technique is used to improve the mechanical stability of the cornea by increasing the formation of covalent intrafibrillar and interfibrillar bonds using photosensitized oxidation.1 It also might decrease corneal steepening and improve the refractive error by minimizing irregular astigmatism induced by biomechanical instability of the cornea.2,3 Under the CXL protocol recommended by Wollensak et al.,4 the corneal epithelium is debrided before the Q 2015 ASCRS and ESCRS Published by Elsevier Inc.
procedure to allow riboflavin to penetrate the stroma (hence, “epithelium off” [epi-off]). However, the epithelium debridement can increase the risk for ulcers, infections, haze, scarring, infiltrates, longer recovery time, patient discomfort, and postoperative pain.5,6 Partial modifications to the epi-off procedure were introduced to overcome the potential disadvantages of epithelium debridement. The application of multiple topical anesthetic eyedrops was proposed to loosen the tight junctions of the corneal epithelial cells, facilitating riboflavin penetration through an intact epithelium (“epithelium on” [epi-on]).7 Furthermore, http://dx.doi.org/10.1016/j.jcrs.2014.06.030 0886-3350
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epi-on CXL can also be safely used in keratoconic eyes with very thin corneas. In fact, the epi-off CXL technique is used only in eyes with a corneal thickness of at least 400 mm because of concerns about the cytotoxic effect on the corneal endothelium, crystalline lens, and other intraocular tissues.8 Riboflavin and UVA irradiation can cause photochemical damage, such as keratocyte apoptosis, by forming oxygen-free radicals.9 Although the efficacy of the epi-on technique is still under debate,10 some clinical studies have shown encouraging results.2,8,11,12 Filippello et al.11 reported a substantial arrest of keratoconus progression after epi-off CXL, with a statistically significant improvement in visual acuity and topographic parameters. Corneal sensitivity recently has gained attention given how important corneal innervation is to maintaining normal corneal architecture and functionality. The impairment of corneal innervation might diminish the cornea's ability to bear surgical challenges and could increase the risk for complications. Evaluating nerve integrity is essential for assessing the efficacy and long-term effects of corneal treatments. In vivo, ex vivo, and in vitro investigations have found significant alterations in the morphology and histology of corneal tissue, mainly at the anterior 300 mm after CXL.13 Despite several clinical and laboratory studies of the effect of CXL on the corneal microstructure,1 little is known about corneal sensitivity and the immediate and long-term postoperative effect of CXL on corneal innervation. Two recent studies reported transient hypoesthesia in keratoconic corneas 6 to 9 months after epi-off CXL.14,15 To our knowledge, to date there have been no published studies of corneal sensitivity after epi-on CXL. This study evaluated corneal sensitivity in keratoconic eyes following epion and epi-off CXL. PATIENTS AND METHODS This prospective interventional study included eyes with progressive keratoconus of patients with no other systemic or ocular disease. Keratoconus was diagnosed using computerized videokeratography (Keratograph, Oculus Optikger€ate GmbH) and ultrasound (US) pachymetry (Corneo Gage Plus, Sonogage, Inc.). Progression was defined as an increase in maximum keratometry (K) of 1.00 diopter
Submitted: January 5, 2014. Final revision submitted: June 4, 2014. Accepted: June 8, 2014.
(D) or more in the previous 12 months and patientreported deterioration of visual acuity (after excluding that from noncorneal-related causes). If progression was confirmed, CXL was proposed as a treatment to halt the keratoconus progression. Patients were thoroughly counseled about the current clinical experience, possible outcomes and complications of the procedure, and the research of the study team. On the basis of the corneal pachymetry (epithelium included) at the area of topographic steepening, the study eyes were divided into 2 groups: those with corneal thickness of 400 mm or more, in which epi-off CXL would be performed, and those with corneal thickness of less than 400 mm, in which epi-on CXL would be performed. The study followed the tenets of the Declaration of Helsinki. All patients gave informed written consent. The inclusion criteria for both groups were topographic progression of keratoconus, a steepest cone apex of less than 65.0 D, a corrected distance visual acuity (CDVA) with spectacles of 20/60 or better, and an endothelial density of at least 2000 cells/mm2. Exclusion criteria for both groups were corneal opacity, previous herpetic keratitis, previous ocular surgery, active ocular infection, autoimmune disease, diabetes, pregnancy, lactation, and smoking of tobacco (because of the presumed negative association between smoking and progression of keratoconus and corneal sensitivity).16 In each patient, the procedure was performed in the eye that showed more progression of the disease. When the disease progression was similar in both eyes, the eye with the worse CDVA was treated. The preoperative and postoperative evaluations included corneal topography, uncorrected distance visual acuity (UDVA) and CDVA with manifest refraction, corneal sensitivity, slitlamp biomicroscopy, applanation tonometry, US pachymetry, and noncontact endothelial specular microscopy. Follow-up evaluations were scheduled for 1 and 7 days and 1, 3, 6, 9, and 12 months. Patients were instructed to discontinue use of contact lenses at least 4 weeks before each visit.17 Before the instillation of eyedrops, corneal sensitivity was measured using a contact esthesiometer (CochetBonnet, Luneau Ophtalmologie). All eyes were measured by the same ophthalmologist (S.S.), who was masked to the nature of the treatment. A 0.12 mm diameter, 6.0 cm long nylon filament was gently placed on the center of the cornea perpendicular to the surface. Patients were asked to look straight ahead and indicate when they felt the stimulus. If there was no response to the first contact, the filament was shortened by 5.0 mm to increase its rigidity and the stimulus was reapplied. This process was repeated until the patient felt the stimulus. The mean filament length, derived from a minimum of 3 stimulus applications that produced a response from the eye, was considered to be the corneal touch threshold. All corneal sensitivity measurements were analyzed at the same time of day (between 9:00 AM and 11:00 AM).
Surgical Technique
From the Department of Biotechnology and Medical-Surgical Sciences, Latina (Spadea, Salvatore, Vingolo), and the Department of Ophthalmology, Rome (Paroli), Sapienza University of Rome, Italy. Corresponding author: Leopoldo Spadea, MD, Via Benozzo Gozzoli 34, 00142 Rome, Italy. E-mail: [email protected].
All procedures were performed by the same surgeon (L.S.). Epi-off CXL was performed under sterile conditions as follows: One drop of pilocarpine 1.0% was instilled 30 minutes preoperatively. Preoperatively, oxybuprocaine 0.4% eyedrops preserved with p-hydroxybenzoate (Benoxinato Cloridrato) were instilled initially and 3 times at 5-minute intervals as a topical anesthetic. The epithelium was
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removed using a blunt metal spatula to mechanically scrape a 9.0 mm diameter epithelial area of the cornea. Before irradiation, the cornea was soaked for 15 minutes in riboflavin solution (Ricrolin, Sooft Italia S.p.A.). Then the cornea was exposed to a solid-state UVA illuminator (CBM, X-linker, Costruzione Strumenti Oftalmici) for 30 minutes during which the riboflavin solution was applied every 2.5 minutes. The UVA illuminator was calibrated to 3.0 mW/cm2 of surface irradiance (5.4 J/cm2 surface dose) using a UV light meter at the specified working distance. The aiming beam was focused on the central cornea while the patient fixated on a pulsating green light. Epi-on CXL was performed under sterile conditions as follows: Riboflavin 0.1% solution in 15% dextran T500 with sodium ethylenediaminetetraacetic acid 0.01% and trometamol (tris[hydroxymethyl]amino methane) (Ricrolin TE, Sooft Italia S.p.A.) was instilled every 10 minutes for 2 hours. Preoperatively, oxybuprocaine 0.4% eyedrops preserved with p-hydroxybenzoate (Benoxinato Cloridrato) were instilled initially and 3 times at 5-minute intervals as a topical anesthetic. One drop of pilocarpine 1.0% was instilled 30 minutes preoperatively; then an eyelid speculum was inserted and riboflavin was instilled over the cornea every 3 minutes for 15 minutes. Using a CBM Vega X-Linker device (3.0 mW/cm2), UVA irradiation was delivered for 30 minutes during which the riboflavin solution was instilled every 5 minutes.8 At the end of both procedures, the eye was rinsed with a balanced salt solution and a bandage soft contact lens was applied. An antibiotic regimen of ofloxacin and flurbiprofen drops was administered 4 times a day for 1 week. Topical corticosteroid drops (clobetasone butyrate 0.1%) were then administered for 1 month and subsequently tapered.
Statistical Analysis Statistical analysis was performed using the Statistical Package for the Social Sciences for Windows software (version 19.0). Comparisons between preoperative and postoperative values at each timepoint were performed for the epi-on and epi-off groups. The differences between preoperative and postoperative values were assessed using the Wilcoxon signed-rank test. Comparisons of corneal sensitivity values between the epi-off and epi-on groups at each follow-up time were made using repeated-measures analysis of variance. The Bonferroni post hoc test was used for post hoc analysis. Statistical differences in corneal sensitivity at baseline between the 2 groups were evaluated using a Student t test, and P values less than 0.05 were considered statistically significant.
RESULTS Fifty eyes of 50 patients had CXL. Of these, 25 (9 women and 16 men; mean age 29.2 years G 11.0 [SD]; mean corneal thickness 421.6 G 21.4 mm) were assigned to the epi-off CXL group and 25 (12 women and 13 men; mean age 31.5 G 9.9 years; mean corneal thickness 379.6 G 19.2 mm) were placed in the epi-on CXL group. In the epi-off group, the epithelium in all eyes recovered within 4 G 1 days after deepithelialization, whereas in the epi-on group, it healed completely in all eyes within 1 day. Figure 1 plots the mechanical sensitivity in the center of the cornea before and after
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Figure 1. The mean values for preoperative and postoperative corneal sensitivity for the epi-off and epi-on CXL groups. The vertical bars denote 95% confidence intervals. The asterisks indicate a statistically significant reduction in corneal sensitivity over baseline in both groups. For the epi-off group, the postoperative P values were ! .001 at 1 day, 7 days, and 1 month; ! .049 at 3 months; ! .28 at 6 months; ! .82 at 9 months, and ! .9 at 12 months. For the epi-on group, the postoperative P values were ! .001 at 1 day, ! .02 at 7 days, ! .061 at 1 month, ! .061 at 3 months, ! .78 at 6 months, ! .91 at 9 months, and ! .9 at 12 months.
treatment with epi-on CXL and epi-off CXL There was no statistically significant difference in preoperative corneal sensitivity between the 2 groups (P Z .9). In the epi-off CXL group, central corneal sensitivity was statistically significantly lower for the first 7 days than preoperatively (P ! .001). The corneal sensitivity recovered in 3 months but remained statistically significantly lower (P ! .05) than it was preoperatively until 6 months after treatment, when it regained preoperative levels. In the epi-on CXL group, corneal sensitivity was statistically significantly lower 1 and 7 days after surgery (P ! .05) than it was preoperatively but was not statistically significantly different at subsequent measurements (Figure 1). Corneal sensitivity 1 and 7 days after epi-off CXL was statistically significantly lower than after epi-on CXL (P ! .001). DISCUSSION The corneal epithelium is the most densely innervated and sensitive surface of the body. Epithelial nerve density in the cornea is 300 to 600 times higher than that of skin.18 Corneal sensory nerves protect the cornea from external insults by initiating nerve reflex mechanisms.18,19 Corneal nerves are crucial to the maintenance of epithelial integrity, the modulation of cell proliferation, the stimulation of ion transport, and the
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regulation of wound healing after corneal injury. In addition to these functions, corneal nerves exert various trophic effects on the cornea by liberating neuropeptides, neurotrophins, and growth factors that promote corneal epithelial homeostasis and by activating brainstem circuits that stimulate reflex tear production and blinking.20 Damage to corneal nerves from surgery, trauma, or disease leads to diminished corneal sensitivity and possible transient or longterm alterations to the functional integrity of the ocular surface.21 A notable number of authors have analyzed corneal sensitivity in keratoconic eyes.22 Dogru et al.23 found that 48% of keratoconic eyes had reduced corneal sensitivity. They also found that corneal sensitivity was statistically significantly decreased in eyes with severe keratoconus than in eyes with mild or moderate disease. Several studies proposed that hypoesthesia in eyes with severe keratoconus implied the presence of corneal epithelial/stromal disease.23,24 Other studies noted that keratoconic eyes of patients who wore contact lenses had statistically significantly less corneal sensitivity than normal eyes (controls) of patients who wore contact lenses.25 Cho et al.26 analyzed corneal sensitivity in patients with asymmetric keratoconus and found less corneal sensitivity in all eyes, in clinical and subclinical keratoconus, than in normal eyes. They suggested that the hypoesthesia was associated with tear deficiency, abnormal impression cytological results, and thinning of the cornea. Our results, similar to those in other studies,14,15,27 showed an initial reduction in corneal sensitivity after epi-off CXL and a recovery of sensitivity during the 6month follow-up. Although corneal sensitivity during the 7 days after epi-off CXL was significantly lower than after epi-on CXL, after the epi-on treatment the cornea was significantly less sensitive than it was preoperatively. This shows that epi-on CXL might affect corneal sensitivity, but to a lesser degree than epi-off CXL. It also suggests that the reduction in corneal sensitivity after the epi-off CXL was probably induced by the intraepithelial nerve damage caused when the epithelium was mechanically removed and by the deeper corneal nerve damage induced by the CXL treatment. A study of eyes of New Zealand white rabbits27 found that corneal sensitivity decreased after deepithelialization alone, without UVA irradiation. This result seems to confirm that the sensory innervation of the corneal epithelium is crucial to the sensory functions of the cornea. Furthermore, the study found a significant difference between the recovery time of corneal sensitivity after epi-off CXL and after deepithelialization alone. This might be indirect evidence
that UVA exposure causes additional nerve damage. These data also suggest that the corneal epithelium might protect the deeper corneal nerves from the damaging effects of UVA exposure. In fact, the theoretical basis for epi-on CXL presents 2 major challenges: the role of the corneal epithelium as a barrier to UVA penetration and the difficulty of penetration by a hydrophilic macromolecule such as riboflavin through the corneal epithelium. The epithelium and Bowman layer have high absorption coefficients in the UV spectrum. The UV-filtering ability of the epithelium might be the result of its molecular composition, which gives it a high absorption coefficient. Furthermore an appropriate distribution of riboflavin in the cornea is critical because inadequate stromal absorption of riboflavin is likely to reduce the effectiveness of the CXL process.28 In vivo corneal confocal microscopy analysis of the postoperative impact of CXL on the cornea revealed clear differences between epi-off CXL and epi-on CXL. One study showed that the subbasal nerve plexus was essentially destroyed immediately after epi-off CXL and that there was virtually no nerve fiber regeneration by 6 months, whereas the cornea appeared mostly unaltered following epi-on CXL.29 A corneal optical coherence tomography analysis and histological study after epi-on CXL treatments showed a line of demarcation approximately 100 mm from the corneal epithelial layer, slightly beneath Bowman membrane.11,12 In contrast, after epi-off CXL the demarcation line was deeper, 320 to 340 mm from the corneal epithelial layer.28 Thus, less of the cornea is involved and therefore fewer nerve fibers are altered with epi-on CXL than with epi-off CXL. These data could explain why less corneal hypoesthesia occurred in the epi-on group than in the epi-off group. Contact lens use also affects corneal sensitivity.17 To minimize the effect of contact lens use on measurements, patients were asked to discontinue their use at least 4 weeks before each examination. A limitation of this study is that we used only the contact esthesiometer to monitor corneal sensitivity. This instrument uses mechanical stimuli to test the response of the cornea's mechanonociceptors. Consequently, the study could not evaluate the corneal sensitivity's recovery to chemical and temperature stimuli. Other aesthometers, eg, the air-jet esthesiometer (CRCERT-Belmonte), can test the response of all 3 types of neuroreceptors on the ocular surface.30 In addition, the contact esthesiometer cannot detect subtle changes in sensitivity and hypersensitivity because it has a lower threshold for detection.30 In our study, the measurement of corneal sensitivity recovery at 12 months was truncated at 6.0 cm only in
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16% of eyes. Furthermore, because most studies evaluating the effect of surgical procedures on corneal sensitivity used the contact esthesiometer, their results were somewhat comparable to ours. Since the completion of our study, flurbiprofen has been found to cause a slight reduction in the corneal sensitivity to mechanical and chemical stimulation,31 which might have affected corneal sensitivity in our study. However, in our study, flurbiprofen was used in both groups in the same way, which should have limited any confounding effect on our analysis. In conclusion, the results in our study indicate that epi-off CXL and epi-on CXL in keratoconic eyes only transiently affects corneal innervation and sensitivity and that there is a complete recovery over the follow-up period. WHAT WAS KNOWN There are few published studies investigating the immediate and long-term postoperative effects of epi-off CXL on corneal innervation. Corneal sensitivity after epi-on CXL has not been evaluated in the literature.
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WHAT THIS PAPER ADDS
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Epi-off CXL and epi-on CXL in keratoconic eyes only transiently affected corneal innervation and sensitivity, and the corneal sensitivity recovered completely.
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29. Touboul D, Efron N, Smadja D, Praud D, Malet F, Colin J. Corneal confocal microscopy following conventional, transepithelial, and accelerated corneal collagen cross-linking procedures for keratoconus. J Refract Surg 2012; 28:769–776 30. Golebiowski B, Papas E, Stapleton F. Assessing the sensory function of the ocular surface: implications of use of a noncontact air jet aesthesiometer versus the Cochet–Bonnet aesthesiometer. Exp Eye Res 2011; 92:408–413 lvez A, Perea31. Acosta MC, Berenguer-Ruiz L, Garcıa-Ga Tortosa D, Gallar J, Belmonte C. Changes in mechanical, chemical, and thermal sensitivity of the cornea after topical application of nonsteroidal anti-inflammatory drugs. Invest
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First author: Leopoldo Spadea, MD Department of Biotechnology and Medical-Surgical Sciences, Latina, Sapienza, University of Rome, Rome, Italy
