Effect of Myopic LASIK on Corneal and Morphology of Subbasal Nerves Tuuli U. Linna, 1 Minna H. Vesaluoma, 1 Juan J. Pérez Santonja, 2 W. Matthew Petroll, 3 Jorge L. Alió, 2 and Timo M. T. Tervo 1 PURPOSE. To investigate whether the morphology of the subbasal nerves corresponds to corneal sensitivity after laser in situ keratomileusis (LASIK). METHODS. In a case series study, 59 patients were examined at 2 to 4 hours, 3 days, 1 to 2 weeks, 1 to 2 months, 3 months, or 6 or more months after undergoing LASIK for myopia, by using a Cochet Bonnet esthesiometer and an in vivo confocal microscope, and were compared with control subjects. Corneal sensitivity and confocal images of subbasal nerves were obtained centrally and 2 mm nasally and temporally. Subbasal nerve fiber bundles (NFBs) were grouped as follows: corneas with no nerve images; corneas with short ( 200 m), unconnected NFBs; corneas with long ( 200 m) NFBs without interconnections; and corneas with long NFBs with interconnections. RESULTS. Corneal sensitivity was at its lowest at 1 to 2 weeks after LASIK. of the hinge area was higher than temporal or central areas at every time point. At 6 or more months the sensitivity values were comparable with the values observed in control subjects. The central area showed mainly short, unconnected subbasal NFBs, even at 6 months. In general, the temporal area presented with long NFBs from 3 months onward, whereas the nasal area showed long NFBs at every time point. CONCLUSIONS. The results suggest that the corneal areas with no nerve images or short, unconnected NFBs are associated with lower sensitivities than corneal areas with long NFBs with or without interconnections. In vivo confocal microscopy reveals LASIK-induced alterations of subbasal nerve morphology and thus enables a direct comparison of corneal sensory innervation and sensitivity. (Invest Ophthalmol Vis Sci. 2000;41:393 397) Because keratorefractive surgery always disrupts the integrity of the corneal sensory innervation, the sensitivity is initially reduced. In laser in situ keratomileusis (LASIK), which is widely used for correcting myopia, the automated microkeratome cuts the subbasal nerve fiber bundles (NFBs) and the superficial stromal nerves in the flap margin. The nerves of the stromal bed are subsequently exposed to an excimer laser photoablation. Corneal reinnervation after LASIK has been studied histochemically. 1,2 In rabbit corneas, regenerating nerve fibers have been shown to emerge from the cut stromal nerves, penetrate the uppermost acellular stromal layer, and contribute to the formation of new subbasal NFBs and intraepithelial nerve endings. 2 From the 1 Department of Ophthalmology, University of Helsinki, Finland; the 2 Refractive Surgery and Cornea Unit, Alicante Institute of Ophthalmology, University of Alicante, School of Medicine, Spain; and the 3 Department of Ophthalmology, University of Texas, Southwestern Medical Center at Dallas. Supported by The Finnish Medical Council, Finnish Medical Foundation, Leiras Medical Manufacturers, The Instrumentarium Scientific Foundation, Alicante Institute of Ophthalmology, The Eye Foundation of Finland, the Ella and Georg Ehrnrooth Foundation, and The Finnish Eye and Tissue Bank Foundation. Submitted for publication June 22 1999; accepted August 17, 1999. Commercial relationships policy: N. Corresponding author: Tuuli U. Linna, Department of Ophthalmology, University of Helsinki, PL 220, 00029 HUCH, Finland. tuuli.linna@huch.fi Owing to its dense sensory innervation, the cornea is extremely sensitive to external stimuli. Normal sensation is important for tear secretion and normal physiology of the cornea. 3 Impaired innervation resulting in decreased sensitivity may compromise the epithelial properties affecting also the healing response of the cornea. 4 The sensory thresholds, which are initially reduced after LASIK, have been reported to return near to normal within 6 to 12 months. 5 7 Also, the depth of the corneal ablation has been shown to affect the extent of corneal sensitivity loss and recovery after LASIK. 8 The sensory subbasal nerve plexus is readily visualized with in vivo confocal microscopy 9,10 and has been used to evaluate in vivo human corneal alterations and reinnervation after LASIK. 11,12 However, a correlation between confocal microscopic images and corneal sensitivity after LASIK has not been established. In this study, we investigated for the first time whether the morphology of the subbasal NFBs corresponds with corneal sensitivity that occurs after LASIK. METHODS All patients were treated in accordance with the tenets of the Declaration of Helsinki, and informed consent was obtained from the patients after explanation of the nature of the study. Patients Fifty-six corneas of 56 patients (29 men and 36 women; age range, 19 53 years; mean, 34.6 8.8 years; preoperative mean Investigative Ophthalmology & Visual Science, February 2000, Vol. 41, No. 2 Copyright Association for Research in Vision and Ophthalmology 393
394 Linna et al. IOVS, February 2000, Vol. 41, No. 2 refraction, 7.05 3.23 D), who had undergone LASIK 3 days, 1 to 2 weeks, 1 to 2 months, 3 months, or 6 or more months earlier, were examined in this case series study. Patients with dry eyes, or inability to cooperate during esthesiometry or confocal microscopic examination were excluded. Six healthy corneas that had not undergone surgery were examined for control purposes. In addition, three corneas were examined 2 to 3 hours after LASIK under the confocal microscope. Esthesiometry was not performed on these three corneas because of the recent instillation of a topical anesthetic before LASIK. LASIK Procedure LASIK procedures were performed with an automated corneal shaper microkeratome (ALK-E, Chiron Vision, Irvine, CA) to create the flap and an excimer laser (either model 217 C-Lasik Chiron Technolas, Dornach, Germany, equipped with the Plano Scan program ver 2.998, n 26; or a model 20/20, Visx, Santa Clara, CA, equipped with the multizone ablation algorithm ver.4.02c, n 33) for photoablation. The procedure was performed with patients under topical anesthesia with 0.4% oxybuprocaine. The diameter of the flap was 8.5 mm and the intended thickness 160 m. The Plano Scan algorithm (Chiron) is based on a 2.0-mm flying spot with pseudorandom positioning, with an energy fluence of 120 mj/cm 2 and a repetition rate of 50 Hz (a 5.5- or 6-mm single-zone ablation), whereas the Visx 20/20 with software version 4.02c is a wide-field beam laser using a multizone algorithm and an energy fluence of 160 mj/cm 2 and a frequency of 6 Hz (the first 6.0 D are corrected at 6-mm zone size, from 6.0 to 10.0 D at 5.5 mm zone size, and those diopters more than 10 D at 5.0-mm zone size). Eyes were not occluded after surgery. Antibiotic (tobramycin 0.3%; Tobrex; Alcon Berhis, Madrid, Spain) and corticosteroid (fluorometholone 0.1%; Allergan, Madrid, Spain) eye drops were instilled four times a day for the first 10 days. Measurement Cochet Bonnet esthesiometry 3 was performed on each cornea (59 patients and 6 control subjects) centrally and approximately 2 mm nasally and 2 mm temporally. The diameter of the nylon filament was 0.12 mm, and its length could be varied from 0 to 62 mm. The pressure applied to the cornea thus ranged from 11 to 200 mg/0.0113 mm 2. Each corneal area was tested three times with each filament length, which was sequentially reduced in 5-mm steps starting from 60 mm. Two positive responses in three attempts at each filament length were regarded as a positive result. The longest filament length resulting in a positive response was considered the corneal sensitivity threshold. All the measurements were performed under slit lamp by the same observer (TL). In Vivo Confocal Microscopy A tandem scanning confocal microscope (model 165A, Tandem Scanning, Reston, VA) equipped with a 24, 0.6 numeric aperture immersion-objective lens was used in the present study. The setup and operation of the confocal microscope has been described previously. 13 15 Briefly, the illumination was supplied by a 100-W mercury lamp, and it was designed for full-thickness examination of the cornea. The internal lenses of the objective were moved with a motorized focusing device (18011 Encoder Mike TM Controller; Oriel, Stratford, CT) interfaced with a Pentium (Intel, Mountain View, CA) computer system (Gateway 2000, N. Sioux City, SD), to vary the focal plane relative to the objective tip. Real-time images were captured using a low-light-level video camera (VE-1000 Sit System; Dage MTI, Michigan City, IN), and the images were recorded on an S-VHS videotape (Fuji Magnetics, Kleve, Germany) using a video cassette recorder (AG-7355; Panasonic, Tokyo, Japan), and printed in color (Stylus 800; Seiko Epson Corporation, Nagano, Japan). With this objective and camera the field of view was 450 360 m, and the optical slice thickness (z-axis resolution) was 9 m. Before the examination, 1 drop topical anesthetic (benoxinate hydrochloride, Oftan Obucain; Santen, Tampere, Finland) and 1 drop 2.5% hydroxymethylcellulose gel (Goniosol, Iolab Pharmaceuticals, Claremont, CA) were applied on the cornea. The patient fixated with the contralateral eye on a bright object to minimize eye movements during examination. The objective lens of the microscope was adjusted to provide an en face view of the central part of the cornea to confirm the proper alignment, after which special attention was paid to viewing the subbasal nerves. The nasal and temporal areas, including the wound edge, were additionally examined if the patient was cooperative. Accordingly, the patient numbers differed in each (nasal, central, temporal) group. Grouping of NFBs Based on the confocal images, the subbasal NFBs were grouped into four different categories based on their morphology: no nerve images, only short ( 200 m) unconnected NFBs, long ( 200 m) NFBs without interconnections, and long NFBs with interconnections. Short, unconnected NFBs were sometimes observed among the long NFBs. Statistical Analyses Statistical analyses were performed by computer (SPSS for Windows, ver. 7.0; SPSS, Chicago, IL). Normality was tested using the Kolmogorov Smirnov test. Differences between groups were tested using parametric analysis of variance (ANOVA) or the nonparametric Kruskal Wallis H test. Data are expressed as means SD, and the differences were considered statistically significant at P 0.05. RESULTS Corneal Corneal sensitivity of the nasal hinge area was higher than in the temporal or central areas at every time point. The sensitivity of each area was at its lowest 1 to 2 weeks after LASIK and at 6 or more months was comparable to the values observed in control corneas (Fig. 1). The sensitivity values of each area differed significantly from each other at different time points (P 0.001, Kruskal Wallis). Morphology of Subbasal Nerves The morphology of the subbasal NFBs in each area and at each time point is indicated in Table 1. Figure 2 shows an example of each different subbasal NFB morphology group: long NFBs with interconnections (Fig. 2A, control cornea), long NFBs without interconnections (Fig. 2B), short, unconnected NFBs (Fig. 2C), and no NFB images (Fig. 2D).
IOVS, February 2000, Vol. 41, No. 2 and Subbasal Nerves after LASIK 395 DISCUSSION FIGURE 1. Mean corneal sensitivity ( SD) after LASIK shown as millimeters of Cochet Bonnet filament length at each time point. N, number of patients at each time point. In general, better subbasal morphology was associated with better sensitivity in each area, although statistically significant results were obtained only from central and nasal areas (Kruskal Wallis, P 0.008 and ANOVA, P 0.008, respectively; Table 2). In the temporal area, the result was not statistically significant (Kruskal Wallis, P 0.171), although the mean sensitivity values were greater with better NFB morphology. The ophthalmic and maxillary branches of the trigeminal nerve provide the sensory innervation of the cornea. 16,17 When penetrating the limbus in the anterior third of the stroma, the nerve bundles lose their myelin sheaths, divide dichotomously or trichotomously, bend at right angles, lose their Schwann cell sheet and penetrate the Bowman s layer to enter the epithelium. 18 20 Then, they bend again to form the subbasal nerve plexus between Bowman s layer and the basal epithelial cell layer. Fibers of the subbasal nerve plexus bend both horizontally and vertically, forming the nerve terminals between the epithelial cells. 21 LASIK, which was introduced in 1990 by Pallikaris and Siganos, 22 has gained worldwide and increasing popularity in correcting myopia. In LASIK, a hinged flap is created by an automated microkeratome, after which the stromal bed is photoablated with an excimer laser. The flap is subsequently repositioned without sutures. Because most of the corneal stromal nerves lie within the anterior two thirds of the cornea, only the deepest stromal nerves avoid the microkeratome cut at the flap margin. The regeneration of stromal and epithelial innervation after LASIK has been studied using acetylcholinesterase histochemistry. 1,2 Initially, in a rabbit study, only the hinge area was shown to preserve some of its stromal and epithelial innervation. 2 In addition, occasional deep stromal NFBs were observed to survive the microkeratome cut under the flap. The cut stromal NFBs were found to send thin regenerating nerve fibers sometimes anastomosing with the neighboring stromal NFBs. These regenerating NFBs sometimes penetrated the TABLE 1. Morphology Status of the Subbasal NFBs at Each Time Point in the Corneal Areas Studied No NFB Images Short, Unconnected NFBs Long NFBs without Interconnections Long NFBs with Interconnections Central area Before surgery 6 2 4 hours after LASIK 1 2 3 days after LASIK 2 8 2 1 1 2 weeks after LASIK 6 8 1 1 2 months after LASIK 5 6 1 3 months after LASIK 7 1 6 or more months after LASIK 2 2 4 Total n in each NFB group 15 31 10 9 Nasal area Before surgery 2 4 hours after LASIK 3 days after LASIK 3 2 1 2 weeks after LASIK 1 1 1 2 months after LASIK 1 2 2 3 months after LASIK 1 1 6 or more months after LASIK 3 2 Total n in each NFB group 1 4 9 5 Temporal area Before surgery 2 4 h after LASIK 1 3 days after LASIK 5 1 2 1 2 weeks after LASIK 5 4 1 1 2 months after LASIK 2 6 1 1 3 months after LASIK 3 6 or more months after LASIK 1 3 Total n in each NFB group 12 12 11 1 Data are number of patients.
396 Linna et al. IOVS, February 2000, Vol. 41, No. 2 most anterior acellular stromal layer and sent subbasal NFBs forming the nerve terminals between the epithelial cells. By 2.5 months the anterior stromal, subbasal, and intraepithelial innervation was restored to near normal. The architecture of the deep stromal NFBs, however, remained abnormal even at 5 months. Confocal microscopy has made it possible to investigate human corneal micromorphology in vivo. This technique enables visualization of cells in all corneal layers, stromal and subbasal nerves, scars, and foreign material with a high degree of resolution and contrast. Subbasal nerve fiber bundles are readily visualized by this technique, but because of the narrow field of view, a false-negative result may be obtained. The alignment of the microscope tip must be adjusted parallel to the surface of the cornea, otherwise the images of the subbasal NFBs are oblique, and long NFBs may falsely be considered short. Microfolding of Bowman s layer (Fig. 2D) is a general finding after LASIK. This phenomenon, which generates undulation of the support and surface on which the subbasal NFBs grow, interferes with the visualization of these nerves and also contributes to the potential risk of considering long NFBs falsely as short. Although the confocal microscope is indispensable in studying subbasal NFBs in vivo, the intraepithelial nerve endings are not normally visualized. Stromal nerves are readily perceived, but the narrow field of view causes some problems in providing a comprehensive impression on the stromal innervation. Therefore, we studied solely subbasal NFBs, and gained only indirect information on the stromal nerves and intraepithelial nerve endings, both of which, however, are as important as subbasal NFBs in transmitting sensory stimuli. In the present study, three patients who were examined at 2 to 4 hours to 3 days after LASIK, still had long NFBs with interconnections at the central corneal area that were comparable to the subbasal NFBs examined in control patients. These NFBs were considered to be degenerating NFBs, because the corneas examined from 1 week to 6 months did not have such interconnecting long NFBs, but rather showed mainly short, FIGURE 2. Example of nerve morphology in each different subbasal NFB group. (A) Long parallel-running NFBs (arrows) with interconnections (arrowhead) in a control cornea. (B) Crossing of two long NFBs without interconnections (arrow). (C) Short, unconnected NFBs (arrows). (D) Image with no NFB-like structures. Note a superficial fold (arrow) at Bowman s layer in (D). Image size, 265 220 m. TABLE 2. Corneal unconnected NFBs or no nerve images at all. The short NFBs may, at the earliest time points, represent degenerating remnants of long NFBs. At later time points, they may represent regenerating growing NFBs. Only 3 of 38 corneas examined at 1 week to 6 months had long NFBs without interconnections at the central area. In general, not until 1 to 2 years after LASIK did the central corneal area show long NFBs. In the temporal area these were observed from 3 months onward, whereas in the nasal area they were perceived at every time point. The hinge of the flap thus contributes to preservation of the nasal subbasal innervation. Corneal sensation is essential for the maintenance of normal corneal physiology. The blinking reflex, and normal tear secretion, which are affected by corneal hypesthesia, are essential to the well-being of the corneal surface. 3,4 Various keratorefractive procedures (radial keratotomy, epikeratophakia, photorefractive keratectomy, LASIK) damage corneal sensory innervation and result in initially reduced sensory thresholds. 5 7,23 26 The present study, as well as the previous studies, was performed using the Cochet Bonnet esthesiometer. Three different corneal test points were selected for the sensitivity measurement because of the geometry of the hinged keratomileusis flap. A marked decrease in corneal sensitivity was observed in the central and temporal areas in patients who had undergone LASIK 1 to 2 weeks earlier. The sensitivity of the nasal hinge area was also at its lowest at 1 to 2 weeks, but the decrease was not as marked as in the central and temporal areas. All three test points showed near normal sensitivity thresholds in patients examined 6 or more months after LASIK. These results are in accordance with the results reported earlier in a prospective study. 7 A correlation between corneal sensitivity and morphology of the subbasal nerves after LASIK was found in this series of patients. According to our data, the corneal areas with no nerve images or short, unconnected subbasal NFBs were associated with lower sensitivity values than corneal areas showing long NFBs with or without interconnections. Confocal microscopy makes it possible to obtain in vivo information about human corneal innervation after keratorefractive surgery and enables a direct comparison of corneal sensory innervation and sensitivity. 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