Journal of Refractive Surgery

Translational Science Supplemental Data

The Impact of Photorefractive Keratectomy and Mitomycin C on Corneal Nerves and Their Regeneration

Carla S. Medeiros, MD; Gustavo K. Marino, MD; Luciana Lassance, PhD; Shanmugapriya Thangavadivel, PhD; Marcony R. Santhiago, MD, PhD; Steven E. Wilson, MD

Abstract

This article has been amended to include factual corrections. To read the erratum, click here. The online article and its erratum are considered the version of record.

PURPOSE:

To determine how photorefractive keratectomy (PRK) and mitomycin C (MMC) affect corneal nerves and their regeneration over time after surgery.

METHODS:

Twenty-eight New Zealand rabbits had corneal epithelial scraping with (n = 3) and without (n = 3) MMC 0.02% or −9.00 diopter PRK with (n = 6) and without (n = 16) MMC 0.02%. Corneas were removed after death and corneal nerve morphology was evaluated using acetylcholinesterase immunohistochemistry and beta-III tubulin staining after 1 day for all groups, after 1 month for PRK with and without MMC, and 2, 3, and 6 months after PRK without MMC. Image-Pro software (Media Cybernetics, Rockville, MD) was used to quantitate the area of nerve loss after the procedures and, consequently, regeneration of the nerves over time. Opposite eyes were used as controls.

RESULTS:

Epithelial scraping with MMC treatment did not show a statistically significant difference in nerve loss compared to epithelial scraping without MMC (P = .40). PRK with MMC was significantly different from PRK without MMC at 1 day after surgery (P = .0009) but not different at 1 month after surgery (P = .90). In the PRK without MMC group, nerves regenerated at 2 months (P < .0001) but did not return to the normal preoperative level of innervation until 3 months after surgery (P = .05). However, the morphology of the regenerating nerves was abnormal—with more tortuosity and aberrant innervation compared to the preoperative controls—even at 6 months after surgery.

CONCLUSIONS:

PRK negatively impacts the corneal nerves, but they are partially regenerated by 3 months after surgery in rabbits. Nerve loss after PRK extended peripherally to the excimer laser ablated zone, indicating that there was retrograde degeneration of nerves after PRK. MMC had a small additive toxic effect on the corneal nerves when combined with PRK that was only significant prior to 1 month after surgery.

[J Refract Surg. 2018;34(12):790–798.]

Abstract

This article has been amended to include factual corrections. To read the erratum, click here. The online article and its erratum are considered the version of record.

PURPOSE:

To determine how photorefractive keratectomy (PRK) and mitomycin C (MMC) affect corneal nerves and their regeneration over time after surgery.

METHODS:

Twenty-eight New Zealand rabbits had corneal epithelial scraping with (n = 3) and without (n = 3) MMC 0.02% or −9.00 diopter PRK with (n = 6) and without (n = 16) MMC 0.02%. Corneas were removed after death and corneal nerve morphology was evaluated using acetylcholinesterase immunohistochemistry and beta-III tubulin staining after 1 day for all groups, after 1 month for PRK with and without MMC, and 2, 3, and 6 months after PRK without MMC. Image-Pro software (Media Cybernetics, Rockville, MD) was used to quantitate the area of nerve loss after the procedures and, consequently, regeneration of the nerves over time. Opposite eyes were used as controls.

RESULTS:

Epithelial scraping with MMC treatment did not show a statistically significant difference in nerve loss compared to epithelial scraping without MMC (P = .40). PRK with MMC was significantly different from PRK without MMC at 1 day after surgery (P = .0009) but not different at 1 month after surgery (P = .90). In the PRK without MMC group, nerves regenerated at 2 months (P < .0001) but did not return to the normal preoperative level of innervation until 3 months after surgery (P = .05). However, the morphology of the regenerating nerves was abnormal—with more tortuosity and aberrant innervation compared to the preoperative controls—even at 6 months after surgery.

CONCLUSIONS:

PRK negatively impacts the corneal nerves, but they are partially regenerated by 3 months after surgery in rabbits. Nerve loss after PRK extended peripherally to the excimer laser ablated zone, indicating that there was retrograde degeneration of nerves after PRK. MMC had a small additive toxic effect on the corneal nerves when combined with PRK that was only significant prior to 1 month after surgery.

[J Refract Surg. 2018;34(12):790–798.]

The cornea is a densely innervated structure that is richly supplied by sensory and autonomic nerve fibers originating from the ophthalmic division of the trigeminal nerve.1 Many corneal nerve receptors are polymodal nociceptors,2 which are stimulated by different mechanical, thermal, and chemical stimuli that serve perceptions such as touch, pain, and temperature. Many of these fibers also modulate functions such as the blink reflex, tear production, and corneal wound healing response.1,3

Corneal nerves also have a critical role in maintaining a healthy ocular surface and traumatic or surgical wounds commonly alter nerve morphology and function.3 Surgery-related injury to corneal innervation results in transient or persistent clinical changes to corneal function and may cause postoperative complications such as dry eye syndrome, neurotrophic epitheliopathy, photophobia, hypoesthesia, and persistent epithelial defects.4–7 Mitomycin C (MMC) used with photorefractive keratectomy (PRK) also has the potential to alter nerve morphology and function, but few studies have investigated this possibility.8 This study aimed to better characterize corneal nerve injury and regeneration after PRK performed with and without MMC in a rabbit model.

Materials and Methods

Animals and Surgeries

All animals were treated in accordance with the tenets of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and the Animal Control Committee at the Cleveland Clinic Foundation approved these studies.

Twenty-eight 12- to 15-week-old female New Zealand White rabbits weighing 2 to 3 kg each were included in this study. One eye of each rabbit was selected to have epithelial scraping with or without MMC 0.02% or photorefractive keratectomy (PRK) with or without MMC 0.02%. The opposite eye was used as a control. Each group had three animals at each time point, except four rabbits were included in the 6-month time point.

Epithelial scraping with MMC for 30 seconds (n = 3) and without MMC (n = 3) was studied 1 day after the procedure. PRK with MMC for 30 seconds (n = 3 at each time point) and PRK without MMC (n = 3 at each time point) were analyzed 1 day and 1 month after the surgery. In addition, PRK without MMC was studied at 2 (n = 3), 3 (n = 3), and 6 (n = 4) months after surgery.

General anesthesia for corneal scraping or PRK was performed by intramuscular injection of ketamine hydrochloride (30 mg/kg) and xylazine hydrochloride (5 mg/kg). Topical proparacaine hydrochloride 1% (Alcon Laboratories, Inc., Fort Worth, TX) was also applied to each treated eye and the control eye prior to surgery. The animals were killed by an intravenous injection of 100 mg/kg Beuthanasia (MERCK, Kenilworth, NJ) while the animal was under general anesthesia.

Epithelial Scrape Technique

With the animal under general and local anesthesia, a sterile eyelid speculum was positioned within interpalpebral fissure and an 8-mm diameter area of epithelium and epithelial basement membrane concentric with the pupil was removed by scraping with a #64 Beaver blade (Becton-Dickinson & Co., Franklin Lakes, NJ). Care was taken to insure all epithelial basement membrane was removed within the area of scraping.

PRK Technique

With the animal under general and local anesthesia, a sterile eyelid speculum was positioned within the interpalpebral fissure and an 8-mm diameter area of epithelium concentric with the pupil was removed by scraping with a #64 Beaver blade. A spherical PRK ablation of −9.00 diopters was performed with a 6-mm ablation zone centered over the entrance pupil using a VISX Star S4 IR excimer laser (Abbot Medical Optics, Irvine, CA).

MMC Application

MMC 0.02% (Intas Pharmaceutical LTD, Accord Health Care, Durham, NC) was applied after epithelial scraping or PRK using a round 8 × 1 mm thick surgical sponge that was wet with solution and placed on the central cornea for 30 seconds. The cornea was irrigated with 3 mL of balanced salt solution (BSS; Alcon Laboratories, Inc.) after removal of the sponge.

Postoperative Antibiotics

One drop of ciprofloxacin hydrochloride 0.3% (Ciloxan; Alcon Laboratories, Inc.) was applied after epithelial scraping, PRK, or to control eyes four times a day until the corneal epithelium was healed in the treated eye (4 to 6 days).

Corneal Nerve Staining Methods

Corneal nerves were stained using the Karnovsky-Roots acetylcholinesterase (AChE) technique.9–11 Briefly, after removing the corneoscleral rim, the cornea was rinsed in D-PBS (Dulbecco's Phosphate Buffered Saline, D8662; Sigma-Aldrich, St. Louis, MO) and the tissue was fixed in a 4% paraformaldehyde in 50 mM Na-K phosphate buffer (pH 7.2) and 8% sucrose solution for 30 minutes. The Descemet's membrane– endothelial complex was removed before fixation, otherwise the membrane becomes strongly adherent to the posterior stroma and this membrane has nonspecific AchE staining.10 The cornea was then rinsed overnight at 4°C in 0.1 M sodium phosphate buffer (pH 7.2). Preliminary experiments were performed to determine whether nerves were affected by removing the Descemet's membrane–endothelial complex prior to fixation and found no difference in nerve area per square millimeter in corneas whether the Descemet's membrane–endothelial complex was removed prior to (18.4 ± 0.9) or after (18.6 ± 0.5) paraformaldehyde fixation (n = 3 in each group, P = .40).

The following day, the corneas were preincubated for 7 hours at 4°C in 10 mL of 0.1 M sodium phosphate buffer (pH 5.5) solution containing 65 mM sodium acetate, 10 mM sodium citrate, 4 mM copper sulfate, and 0.5 mM potassium ferricyanide, and then reacted with acetylcholine iodide (1.0 mg/mL; Sigma-Aldrich) in incubation medium for 20 to 22 hours at 4°C. The acetylcholinesterase present in the corneal nerves reacted with acetylcholine iodide in the substrate to produce a brown staining of the nerves. Subsequently, the corneas were rinsed for 15 minutes in 0.1 mg/mL sodium sulfate. Finally, the corneoscleral rims were placed in dilute ammonium sulfide solution (Sigma-Aldrich) prepared by placing two drops of 0.1 mg/mL sodium sulphate in 10 mL of distilled water for 30 minutes and then rinsing twice for 5 minutes in distilled water. Four radial corneal incisions were made to flatten the cornea and the sample was mounted with the epithelium facing up in 1× PBS onto a microscope slide (Superfrost plus #1255015; Thermo Fisher Scientific, Pittsburgh, PA) and a cover slip was placed over the tissue.

Nerve morphology was also subsequently examined using three-dimensional analysis with beta-III tubulin staining.12 The corneas that had been fixed, as previously described, were cut using a 9-mm trephine (Beaver-Visitec, Waltham, MA). The corneal button was then washed with 0.1 M PBS containing 0.1% bovine serum albumin (PBS-BSA, #9048-46-8; Sigma-Aldrich) three times during a 24-hour interval. The button was incubated with 10% normal goat serum (#S-100; Vector Laboratories, Burlingame, CA) with 0.3% Triton X-100 solution (#9002-93-1; Sigma-Aldrich) in 0.1% BSA-PBS solution for 60 minutes at room temperature.

Each corneal button was subsequently incubated with shaking in 1:3,000 monoclonal antibody specific for neuronal class beta-III tubulin (Tuj1, MMS-435P; Covance Antibody Services Inc., Berkeley, CA) in 0.1 M PBS containing 1.5% normal goat serum with 0.1% Triton X-100 for 72 hours at 4°C. Then, the corneal buttons were washed with PBS-BSA for 24 hours and incubated with gentle shaking in the secondary antibody Alexa Fluor 568 goat anti-mouse IgG (Cat #A11004; ThermoFisher Scientific, Rockford, IL) at a dilution of 1:1500 in 0.1 M PBS containing 1.5% normal goat serum with 0.1% Triton X-100 for 24 hours at 4°C. Finally, the corneal buttons were washed in PBSBSA solution for 24 hours and mounted with Vectashield (Vector Laboratories Inc., Burlingame, CA) onto microscope slides (Superfrost plus #1255015; Thermo Fisher Scientific) with the epithelium facing up and covered with a coverslip.

Microscopy

Corneas that had acetylcholinesterase staining were imaged using a Retiga SRV Cooled charge-coupled device camera with liquid crystal tunable RGB filter (QImaging, Surrey, British Columbia, Canada) on a Leica MZ-16FA stereomicroscope (Leica Microsystems, GmbH, Wetzlar, Germany) using Image-Pro Plus and Scope-Pro software (Media Cybernetics, Rockville, MD).

Confocal images of corneal buttons that were stained with beta-III tubulin were obtained using a Leica SP8 confocal microscope (Leica Microsystems, GMbH, Wetzlar, Germany). Confocal images were montaged using the Leica LAS-X capture and processing software.

Computerized Quantification of Nerve Area

The nerves located in the intended area of laser ablation (6-mm diameter, total area 28.2 mm2) and in the surrounding cornea out to a 9.4-mm diameter circle were analyzed in control corneas using Image-Pro software. To always measure the same area in all corneas, a 9.4-mm diameter circle with a total area of 69.3 mm2 was drawn on each image and saved for future analyses. Subsequently, in the area selected, a filter was applied to convert the stained nerve images to black and white (nerves were white) to increase the contrast and facilitate the measurements. Image-Pro software automatically measured the area of the nerves in square millimeters inside the area of interest. Identical measurements were performed to calculate the nerve damage area after epithelial scraping or PRK at the different time points.

Statistical Analysis

Data were analyzed using SAS statistical software (version 9.4; SAS Inc., Cary, NC). Variations were expressed as standard deviation of the mean. Statistical comparisons between the groups were performed using analysis of variance and, for comparisons between two groups, a t test assuming unequal variances (Satterthwaite). All statistical tests were conducted at an alpha level at 0.01 due to the large number of statistical comparisons. Normality of the data is assumed for statistical analyses.

Results

Acetylcholinesterase Staining of Corneal Nerves in the Central Cornea

Corneas that had epithelial scraping had lower nerve density compared to control corneas at 1 day after scraping (Figure A [available in the online version of this article], Tables 12), indicating there was mild damage to the superficial nerves produced by scraping. PRK had a major impact on corneal nerve density at 1 day after surgery (Figure A, Tables 12). There was progressive recovery of corneal nerve density within the ablated zone from 1 to 6 months after PRK (Figures AB [available in the online version of this article], Tables 12) compared to control corneas. At 3 and 6 months, the difference in nerve density in the PRK ablated zone compared to controls was not statistically significant (P < .01), although there was a trend toward a persistent decrease in nerve density at these time points (P = .05). There was no difference between scraping with and without MMC at 1 day after injury (Figure A, Tables 12), indicating MMC itself had no effect on corneal nerve density after a scrape injury. However, in the context of PRK injury, there was a small effect of MMC on corneal nerve density within the ablated zone at 1 day after surgery (P = .0009, Figure A, Tables 12), but this effect of MMC was no longer present at 1 month after PRK and, therefore, was not examined at later time points.

Acetylcholinesterase histochemistry staining in corneas at 1 day after different injuries. The scrape–MMC group had 8-mm epithelial scrape without the use of MMC. The scrape+MMC group had MMC 0.02% applied for 30 seconds after 8-mm epithelial scrape. The PRK–MMC group had −9.00 D PRK performed without the use of MMC. The PRK+MMC group had −9.00 D PRK with MMC treatment. Three corneas were analyzed per group. MMC = mitomycin C 0.02%; PRK = photorefractive keratectomy; D = diopters

Figure A.

Acetylcholinesterase histochemistry staining in corneas at 1 day after different injuries. The scrape–MMC group had 8-mm epithelial scrape without the use of MMC. The scrape+MMC group had MMC 0.02% applied for 30 seconds after 8-mm epithelial scrape. The PRK–MMC group had −9.00 D PRK performed without the use of MMC. The PRK+MMC group had −9.00 D PRK with MMC treatment. Three corneas were analyzed per group. MMC = mitomycin C 0.02%; PRK = photorefractive keratectomy; D = diopters

Nerve Area per mm3 Over Time

Table 1:

Nerve Area per mm3 Over Time

ANOVA P Values per Group/per Time-point

Table 2:

ANOVA P Values per Group/per Time-point

Acetylcholinesterase histochemistry staining in corneas at different time points after −9.00 D PRK with and without MMC. The PRK+MMC-1m group had a −9.00 D PRK with 0.02% MMC for 30 seconds and analyzed after 1 month after surgery. The PRK–MMC-1m, −2m, −3m and −6m groups had −9.00 D PRK without the use of MMC and were analyzed at 1, 2, 3, and 6 months, respectively, after surgery. Three corneas were analyzed per group, per time-point, except the sixth month time point had four corneas. MMC = mitomycin C 0.02%; PRK = photorefractive keratectomy; D = diopters

Figure B.

Acetylcholinesterase histochemistry staining in corneas at different time points after −9.00 D PRK with and without MMC. The PRK+MMC-1m group had a −9.00 D PRK with 0.02% MMC for 30 seconds and analyzed after 1 month after surgery. The PRK–MMC-1m, −2m, −3m and −6m groups had −9.00 D PRK without the use of MMC and were analyzed at 1, 2, 3, and 6 months, respectively, after surgery. Three corneas were analyzed per group, per time-point, except the sixth month time point had four corneas. MMC = mitomycin C 0.02%; PRK = photorefractive keratectomy; D = diopters

Corneal Nerve Morphology Using Beta-III Tubulin Staining After PRK without MMC

Extensive damage to the corneal nerves was noted at 1 day after PRK at the level of the subbasal nerve plexus (Figure 1), in the anterior stroma at a depth of approximately 125 μm (Figure 2), and in the mid-stroma at a depth of approximately 250 μm (Figure 3). It can be noted that nerve damage extended beyond the 6-mm excimer laser ablated zone at 1 day after PRK (Figures 1B, 2B, and 3B), especially in the superficial zone of the subbasal nerve plexus (Figure 1B).

Beta-III tubulin histochemistry staining the corneal subbasal plexus over time after −9.00 D PRK without MMC. This composite shows the early damage peripheral to the (B) ablated zone and (C and D) subsequent regeneration following the margin of the wound in a perpendicular pattern. An oblique orientation of the regenerated fibers at the edge of the wound was observed (E and F). However, the original central ablated zone is not fully regenerated. The 6-mm white circle delineates the estimated excimer laser ablation zone. Note that nerve density is decreased beyond the ablated zone. MMC = mitomycin C 0.02%; PRK = photorefractive keratectomy; D = diopters

Figure 1.

Beta-III tubulin histochemistry staining the corneal subbasal plexus over time after −9.00 D PRK without MMC. This composite shows the early damage peripheral to the (B) ablated zone and (C and D) subsequent regeneration following the margin of the wound in a perpendicular pattern. An oblique orientation of the regenerated fibers at the edge of the wound was observed (E and F). However, the original central ablated zone is not fully regenerated. The 6-mm white circle delineates the estimated excimer laser ablation zone. Note that nerve density is decreased beyond the ablated zone. MMC = mitomycin C 0.02%; PRK = photorefractive keratectomy; D = diopters

Beta-III tubulin histochemistry staining the corneal anterior stroma at a depth of approximately 125 μm over time after −9.00 D PRK without MMC. This composite shows that the anterior corneal stroma contributed substantially to the neuro-remodeling process. (C) Abnormal morphology and a very tortuous pattern are observed 1 month after the initial injury. (D) The small and tortuous neurites were replaced by longer fibers that repopulate the treated area but with dichotomous or trichotomous branching pattern. (E) A high density of nerves, but with thinner fibers, is observed in the central cornea. (F) A more organized pattern is noted at 6 months after surgery than 3 months after surgery. The 6-mm white circle delineates the estimated excimer laser ablation zone. MMC = mitomycin C 0.02%; PRK = photorefractive keratectomy; D = diopters

Figure 2.

Beta-III tubulin histochemistry staining the corneal anterior stroma at a depth of approximately 125 μm over time after −9.00 D PRK without MMC. This composite shows that the anterior corneal stroma contributed substantially to the neuro-remodeling process. (C) Abnormal morphology and a very tortuous pattern are observed 1 month after the initial injury. (D) The small and tortuous neurites were replaced by longer fibers that repopulate the treated area but with dichotomous or trichotomous branching pattern. (E) A high density of nerves, but with thinner fibers, is observed in the central cornea. (F) A more organized pattern is noted at 6 months after surgery than 3 months after surgery. The 6-mm white circle delineates the estimated excimer laser ablation zone. MMC = mitomycin C 0.02%; PRK = photorefractive keratectomy; D = diopters

Beta-III tubulin histochemistry staining the corneal mid-stroma at a depth of approximately 250 μm overtime after −9.00 D PRK without MMC. This composite shows that the nerve loss extends to deeper cornea layers in the mid-stroma. There is (B) retrograde axonal death and then (C) neuron recovery. (D) The first attempt of regeneration was also observed on the deeper layers. (E) At 3 months after PRK, some long fibers are present at the ablated zone. (F) This population was increased after 6 months, but not fully recovered compared to unwounded controls. The 6-mm white circle delineates the estimated excimer laser ablation zone. MMC = mitomycin C 0.02%; PRK = photorefractive keratectomy; D = diopters

Figure 3.

Beta-III tubulin histochemistry staining the corneal mid-stroma at a depth of approximately 250 μm overtime after −9.00 D PRK without MMC. This composite shows that the nerve loss extends to deeper cornea layers in the mid-stroma. There is (B) retrograde axonal death and then (C) neuron recovery. (D) The first attempt of regeneration was also observed on the deeper layers. (E) At 3 months after PRK, some long fibers are present at the ablated zone. (F) This population was increased after 6 months, but not fully recovered compared to unwounded controls. The 6-mm white circle delineates the estimated excimer laser ablation zone. MMC = mitomycin C 0.02%; PRK = photorefractive keratectomy; D = diopters

One month following PRK, signs of nerve regeneration were noted in the subbasal plexus (Figure 1C) and anterior stroma (Figure 2C). Collateral sprouts arose from the edge of the wound on the subepithelial plexus and migrated perpendicularly to reach the subbasal plexus and epithelium (Figure 1). In addition, stromal nerve changes were also present in the deeper ablated zone, where there were limited larger fibers even at 6 months after surgery (Figure 3). Small nerves that exhibited aberrant morphology with localized twisting, looping, and multiple branches were also observed and tended to be concentrated at the margin of the excimer laser ablation (Figure 3C).

Up to 6 months after PRK (Figures 13), the corneal nerves continued to regenerate and the morphology became more normal, but even at 6 months after surgery had not returned fully to the control density at any of the three levels in the cornea (Figures 1 and 3).

Nerve damage at all layers of the cornea compared to the control (Video 1, available in the online version of this article) can be best noted in videos of beta-III tubulin staining at 1 day (Video 2, available in the online version of this article), 1 month (Video 3, available in the online version of this article), 2 months (Video 4, available in the online version of this article), 3 months (Video 5, available in the online version of this article), and 6 months (Video 6, available in the online version of this article) after PRK.

Discussion

Important observations have been made to provide a detailed understanding of the distribution of innervation in the cornea.13,14 The nerves penetrate the cornea from the limbus in the mid-stroma and terminate as free nerve endings in the epithelial layer of the cornea.1 Most of the nerve fibers are located in the anterior third of the stroma, but the thick stromal nerve trunks are located beneath the anterior third of the stroma. The path of the stromal nerves makes a 90° turn in the superficial cornea and they divide into several branches that parallel the basal epithelium and give rise to the subbasal plexus.1

The delicate nerve network within the epithelium and the subbasal plexus are damaged during epithelial removal as a first step of PRK surgery. Subsequently, the excimer laser injures deeper stromal nerves during the ablation process and the amount of damage is related to the level of correction and, therefore, the amount of stromal ablation, as well as whether the treatment is for myopia or hyperopia. The greater the intended correction, the deeper the excimer laser ablation extends into the cornea and the more nerves that are negatively impacted.

Several different methods can be used to study corneal nerve architecture—including probing tissue sections with dyes or immunohistochemistry for nerve components, as in the current study or in vivo confocal microscopy (IVCM) or electron microscopy.1 Most studies of corneal nerves used IVCM. This approach has provided important insights into corneal nerve morphology and function, but it has disadvantages when studying the impact of PRK on corneal nerves. A major disadvantage is a decrease in the contrast of the images attributable to backscattered light that originates from “activated” keratocytes (in cell biology referred to as corneal fibroblasts) and altered extracellular matrix that occurs in the months after PRK and is referred to as normal haze. These factors make the use of IVCM to evaluate corneal nerves more difficult, especially when examining small fine bundles.15,16

The acetylcholinesterase staining method with a stereomicroscope was used in the current study as the first method to analyze whole rabbit corneas. The stereomicroscope allows the examination of thicker specimens by providing a full-thickness visualization of the sample. The acetylcholinesterase staining was used to quantify nerve damage after the different procedures because it allows the visualization of every type of fiber present in corneal tissue. Thus, the combination of the acetylcholinesterase staining method with the stereomicroscope analysis of the whole cornea provided high-contrast images of the total nerve network for quantitative analysis.

The second nerve staining method used in this study was immunofluorescent detection of the tubulin beta-III subunit of tubulin III, a protein that is primarily expressed in neurons and is considered to play a critical role in proper axon guidance and maintenance.17 The use of the in vitro confocal microscope for this study enabled imaging at different depths within the stroma to provide a more complete analysis of nerve damage after PRK at different levels in the cornea and, therefore, provided a better understanding of nerve regeneration after surgery.

As expected, the corneal nerves were damaged at 1 day in all groups, including after manual epithelium layer scraping alone. Epithelial debridement damaged the free nerve endings in the epithelium and the complex network of the fibers in the subbasal plexus. The excimer laser PRK ablation for high myopia used in this study caused nerve loss that extended peripherally to the zone treated with excimer laser (Figures 3B and 4B). This indicates that the ablation of nerves caused a retrograde nerve degeneration beyond the 6-mm central ablated area.18–21 Thus, PRK does not merely ablate the nerve endings, as has been suggested by clinicians, but rather the damage to the nerves extends well beyond the excimer laser ablation and, therefore, regeneration of the nerves requires months to approach the preoperative density in the center of the ablated zone.

Different from previous reports that used subepithelial plexus to study the corneal nerves,22–24 this confocal three-dimensional analysis of the different corneal depths throughout the whole corneal thickness showed extensive nerve damage at all corneal depths from the epithelium to the mid-stroma. However, the degree of nerve loss would be variable and likely less when PRK ablations were made to correct lower levels of myopia.25,26

Neurotoxicity has been described with the use of MMC in other organs. For example, the topical application of MMC produced a substantial sensory-neural hearing loss—demonstrating toxicity to acoustic nerves.27 When used for advanced breast cancer treatment, it also produced peripheral neurotoxicity.28 A decrease of the thickness of the myelin sheath was observed after local use of MMC at concentrations greater than 0.7 mg/mL for preventing post-laminectomy epidural scar formation, indicating an adverse effect on the peripheral nerves.29 The neurotoxicity effect of MMC was also noted.30,31 The current study revealed only mild MMC nerve toxicity in the early postoperative period that did not persist at 1 month after PRK with MMC 0.02% and was not noted at all after simple epithelial debridement with MMC treatment. This finding agrees with a study of the long-term safety of MMC in the cornea.32 Other authors reported a dose-dependent neurotoxicity of MMC in the eye and other tissues.29–31 Thus, this study did not find any long-term effects of MMC treatment on corneal nerves when used in conjunction with PRK for the correction of high myopia.

After the nerve damage produced by PRK, there is gradual recovery and reorganization of the nerve fibers extending over a period of at least 6 months. In this study, the area of regenerated nerve fibers in the central cornea reached a value comparable with un-operated control eyes at 3 months after surgery, which agrees with other studies.22,33 However, other studies differ considerably in relation to the functional recovery time of the corneal nerves, varying between 5 to 8 months,24,34–36 1 year,37 2 years,38–40 3 years,16 and 5 years23 depending on the methods used for the nerve study.

A prior study in rabbits also made important contributions regarding nerve regeneration after corneal lesions.25 One important observation was that new fibers grew in an arrangement perpendicular to the edge of the wound,25 which was also observed in the current study at 1 month after PRK (Figure 1C). These wound-oriented neurites were the primary nerves to repopulate the cornea and migrated from the subbasal plexus into the epithelium, but they were also detected in the anterior stroma (Figure 2C), albeit with abnormal morphology.

Nerve regeneration in the mid-stroma also started at 1 month after PRK (Figure 3C), but with a different pattern than was noted in unwounded control corneas. A dense, multiple-branched, and tortuous morphology was noted specifically at the margin of the ablation zone in rabbit corneas that had −9.00 diopter PRK in the current study, and was similar to findings previously reported in the literature as an early, and probably immature or aborted, attempt to regenerate into the ablated zone that was described as the first phase of regeneration.25,41

Rósza et al.25,41 described a subsequent event that consisted of degeneration of the neurites from the first phase and recovery of the nerve terminals. This process is known as the second phase of nerve regeneration and refers to the previously described vestiges of wound-oriented neurites being replaced by growing axon terminals, with higher density and shorter length.25,42 In the current study, this neuron behavior was observed at the third month and was not completed until the sixth month (Figures 2E–2F). The oblique arrangement of the nerve fibers noted at 3 months is a characteristic sign of this neuroregeneration phase.

When acetylcholinesterase images at 3 months after −9.00 diopter PRK were observed alone, there was no statistical difference in the area of the regenerated nerves compared to the unwounded control corneas in the central cornea (Figure B). However, in the beta-III tubulin confocal images, where the nerves were analyzed not only by area but also by depth, it could be observed that the nerve regeneration of the PRK-treated area occurred mainly in the anterior stroma at this point (Figure 2). The second phase of nerve growth is considered the first sign of neurogenesis.25 This phase was initiated in the corneal stroma 3 months after −9.00 diopter PRK in rabbits, but a substantial intraepithelial nerve population did not reach the central cornea even after 6 months (Figure 1F). Also, a persistent abnormal architecture and orientation of the nerve fibers was still observed at 6 months after PRK. Thus, the neural remodeling is not completed until after 6 months following high-correction PRK, and abnormalities could persist for months or years in some corneas. The slow neural recovery present at the subepithelial and subbasal plexus could be a consequence of the dense and complex network of the nerve fibers that compose these structures, which likely need a longer time to recover fully, although longer term studies would be needed to study this. What can be concluded from this study is that nerve defects persist for a minimum of 6 months following PRK surgery for high myopia.

References

  1. Müller LJ, Marfurt CF, Kruse F, Tervo TM. Corneal nerves: structure, contents and function. Exp Eye Res. 2003;76:521–542. doi:10.1016/S0014-4835(03)00050-2 [CrossRef]
  2. Belmonte C, Acosta MC, Gallar J. Neural basis of sensation in intact and injured corneas. Exp Eye Res. 2004;78:513–525. doi:10.1016/j.exer.2003.09.023 [CrossRef]
  3. Shaheen BS, Bakir M, Jain S. Corneal nerves in health and disease. Surv Ophthalmol. 2014;59:263–285. doi:10.1016/j.survophthal.2013.09.002 [CrossRef]
  4. Kohlhaas M, Draeger J, Böhm A, et al. Aesthesiometry of the cornea after refractive corneal surgery [article in German]. Klin Monbl Augenheilkd. 1992;201:221–223. doi:10.1055/s-2008-1045898 [CrossRef]
  5. Wilson SE. Laser in situ keratomileusis-induced (presumed) neurotrophic epitheliopathy. Ophthalmology. 2001;108:1082–1087. doi:10.1016/S0161-6420(01)00587-5 [CrossRef]
  6. Ambrósio R Jr, Tervo T, Wilson SE. LASIK-associated dry eye and neurotrophic epitheliopathy: pathophysiology and strategies for prevention and treatment. J Refract Surg. 2008;24:396–407.
  7. Wilson SE, Medeiros CS, Santhiago MR. Pathophysiology of corneal scarring in persistent epithelial defects after PRK and other corneal injuries. J Refract Surg. 2018;34:59–64. doi:10.3928/1081597X-20171128-01 [CrossRef]
  8. Santhiago MR, Netto MV, Wilson SE. Mitomycin C: biological effects and use in refractive surgery. Cornea. 2012;31:311–321. doi:10.1097/ICO.0b013e31821e429d [CrossRef]
  9. Koelle GB, Friedenwald JS. The histochemical localization of cholinesterase in ocular tissues. Am J Ophthalmol. 1950;33:253–256. doi:10.1016/0002-9394(50)90845-2 [CrossRef]
  10. Tervo T. Histochemical demonstration of cholinesterase activity in the cornea of the rat and the effect of various denervations on the corneal nerves. Histochemistry. 1976;47:133–143. doi:10.1007/BF00492561 [CrossRef]
  11. Xia Y, Chai X, Zhou C, Ren Q. Corneal nerve morphology and sensitivity changes after ultraviolet A/riboflavin treatment. Exp Eye Res. 2011;93:541–547. doi:10.1016/j.exer.2011.06.021 [CrossRef]
  12. He J, Bazan NG, Bazan HEP. Mapping the entire human corneal nerve architecture. Exp Eye Res. 2010;91:513–523. doi:10.1016/j.exer.2010.07.007 [CrossRef]
  13. Zander E, Weddell G. Observations on the innervation of the cornea. J Anat. 1951;85:68–99.
  14. Marfurt CF, Cox J, Deek S, Dvorscak L. Anatomy of the human corneal innervation. Exp Eye Res. 2010;90:478–492. doi:10.1016/j.exer.2009.12.010 [CrossRef]
  15. Tervo T, Moilanen J. In vivo confocal microscopy for evaluation of wound healing following corneal refractive surgery. Prog Retin Eye Res. 2003;22:339–358. doi:10.1016/S1350-9462(02)00064-2 [CrossRef]
  16. Linna T, Tervo T. Real-time confocal microscopic observations on human corneal nerves and wound healing after excimer laser photorefractive keratectomy. Curr Eye Res. 1997;16:640–649. doi:10.1076/ceyr.16.7.640.5058 [CrossRef]
  17. Moskowitz PF, Smith R, Pickett J, Frankfurter A, Oblinger MM. Expression of the class III beta-tubulin gene during axonal regeneration of rat dorsal root ganglion neurons. J Neurosci Res. 1993;34:129–134. doi:10.1002/jnr.490340113 [CrossRef]
  18. Catapano J, Zhang J, Scholl D, Chiang C, Gordon T, Borschel GH. N-Acetylcysteine prevents retrograde motor neuron death after neonatal peripheral nerve injury. Plast Reconstr Surg. 2017;139:1105e–1115e. doi:10.1097/PRS.0000000000003257 [CrossRef]
  19. Al-Louzi O, Button J, Newsome SD, Calabresi PA, Saidha S. Retrograde trans-synaptic visual pathway degeneration in multiple sclerosis: a case series. Mult Scler. 2017;23:1035–1039. doi:10.1177/1352458516679035 [CrossRef]
  20. Handley SE, Panteli VS, Liasis A. Trans-synaptic retrograde degeneration following hemispherectomy in childhood. Neuroophthalmology. 2017;41:103–107. doi:10.1080/01658107.2016.1276935 [CrossRef]
  21. Uchihara T. An order in Lewy body disorders: retrograde degeneration in hyperbranching axons as a fundamental structural template accounting for focal/multifocal Lewy body disease. Neuropathology. 2017;37:129–149. doi:10.1111/neup.12348 [CrossRef]
  22. Moller-Pedersen T, Cavanagh HD, Petroll WM, Jester JV. Stromal wound healing explains refractive instability and haze development after photorefractive keratectomy: a 1-year confocal microscopic study. Ophthalmology. 2000;107:1235–1245. doi:10.1016/S0161-6420(00)00142-1 [CrossRef]
  23. Moilanen JA, Vesaluoma MH, Müller LJ, Tervo TM. Long-term corneal morphology after PRK by in vivo confocal microscopy. Invest Ophthalmol Vis Sci. 2003;44:1064–1069. doi:10.1167/iovs.02-0247 [CrossRef]
  24. Kauffmann T, Bodanowitz S, Hesse L, Kroll P. Corneal reinnervation after photorefractive keratectomy and laser in situ keratomileusis: an in vivo study with a confocal videomicroscope. Ger J Ophthalmol. 1996;5:508–512.
  25. Rózsa AJ, Guss RB, Beuerman RW. Neural remodeling following experimental surgery of the rabbit cornea. Invest Ophthalmol Vis Sci. 1983;24:1033–1051.
  26. De Felipe C, Belmonte C. c-Jun expression after axotomy of corneal trigeminal ganglion neurons is dependent on the site of injury. Eur J Neurosci. 1999;11:899–906. doi:10.1046/j.1460-9568.1999.00498.x [CrossRef]
  27. Moody MW, Lang H, Spiess AC, Smythe N, Lambert PR, Schmiedt RA. Topical application of mitomycin C to the middle ear is ototoxic in the gerbil. Otol Neurotol. 2006;27:1186–1192. doi:10.1097/01.mao.0000226306.43951.c8 [CrossRef]
  28. Kornek GV, Haider K, Kwasny W, et al. Effective treatment of advanced breast cancer with vinorelbine, 5-fluorouracil and lleucovorin plus human granulocyte colony-stimulating factor. Br J Cancer. 1998;78:673–678. doi:10.1038/bjc.1998.558 [CrossRef]
  29. Sui T, Zhang J, Du S, Su C, Que J, Cao X. Potential risk of mitomycin C at high concentrations on peripheral nerve structure. Neural Regen Res. 2014;9:821–827. doi:10.4103/1673-5374.131598 [CrossRef]
  30. Mietz H, Addicks K, Diestelhorst M, Krieglstein GK. Intraocular toxicity to ciliary nerves after extraocular application of mitomycin C in rabbits. Int Ophthalmol. 1995;19:89–93. doi:10.1007/BF00133178 [CrossRef]
  31. Mietz H, Prager TC, Schweitzer C, Patrinely J, Valenzuela JR, Font RL. Effect of mitomycin C on the optic nerve in rabbits. Br J Ophthalmol. 1997;81:584–589. doi:10.1136/bjo.81.7.584 [CrossRef]
  32. Gambato C, Miotto S, Cortese M, Ghirlando A, Lazzarini D, Midena E. Mitomycin C- assisted photorefractive keratectomy in high myopia: a long-term safety study. Cornea. 2011;30:641–645. doi:10.1097/ICO.0b013e31820123c8 [CrossRef]
  33. Tervo K, Latvala TM, Tervo TM. Recovery of corneal innervation following photorefractive keratoablation. Arch Ophthalmol. 1994;112:1466–1470. doi:10.1001/archopht.1994.01090230080025 [CrossRef]
  34. Heinz P, Bodanowitz S, Wiegand W, Kroll P. In vivo observation of corneal nerve regeneration after photorefractive keratectomy with a confocal videomicroscope. Ger J Ophthalmol. 1996;5:373–377.
  35. Nejima R, Miyata K, Tanabe T, et al. Corneal barrier function, tear film stability, and corneal sensation after photorefractive keratectomy and laser in situ keratomileusis. Am J Ophthalmol. 2005;139:64–71. doi:10.1016/j.ajo.2004.08.039 [CrossRef]
  36. Alio JL, Javaloy J. Corneal inflammation following corneal photoablative refractive surgery with excimer laser. Surv Ophthalmol. 2013;58:11–25. doi:10.1016/j.survophthal.2012.04.005 [CrossRef]
  37. Erie JC, Patel SV, Bourne WM. Aberrant corneal nerve regeneration after PRK. Cornea. 2003;22:684–686. doi:10.1097/00003226-200310000-00014 [CrossRef]
  38. Frueh BE, Cadez R, Bohnke M. In vivo confocal microscopy after photorefractive keratectomy in humans: a prospective, long-term study. Arch Ophthalmol. 1998;116:1425–1431. doi:10.1001/archopht.116.11.1425 [CrossRef]
  39. Erie JC, McLaren JW, Hodge DO, Bourne WM. Recovery of corneal subbasal nerve density after PRK and LASIK. Am J Ophthalmol. 2005;140:1059–1064. doi:10.1016/j.ajo.2005.07.027 [CrossRef]
  40. Neira-Zalentein W, Moilanen JA, Tiusku IS, Holopainem JM, Tervo TM. Photorefractive keratectomy retreatment after LASIK. J Refract Surg. 2008;24:710–712.
  41. Rózsa AJ, Beuerman RW. Density and organization of free nerve endings in the corneal epithelium of the rabbit. Pain. 1982;14:105–120. doi:10.1016/0304-3959(82)90092-6 [CrossRef]
  42. Beuerman RW, Schimmelpfennig B. Sensory denervation of the rabbit cornea affects epithelial properties. Exp Neurol. 1980;69:196–201. doi:10.1016/0014-4886(80)90154-5 [CrossRef]

Nerve Area per mm3 Over Time

Group Mean ± SD
Control 18.4 ± 0.9
Scrape–MMC 1 day 16.9 ± 0.3
Scrape+MMC 1 day 17.3 ± 0.4
PRK–MMC 1 day 11.2 ± 0.1
PRK+MMC 1 day 9.5 ± 0.6
PRK–MMC 1 month 14.1 ± 0.4
PRK+MMC 1 month 14.2 ± 0.3
PRK–MMC 2 months 15.3 ± 0.1
PRK+MMC 2 months 17.5 ± 0.4
PRK+MMC 3 months 17.6 ± 0.3

ANOVA P Values per Group/per Time-point

Group Control Scrape –MMC 1 Day Scrape +MMC 1 Day PRK–MMC 1 Day PRK+MMC 1 Day PRK–MMC 1 Month PRK+MMC 1 Month PRK–MMC 2 Months PRK–MMC 3 Months PRK–MMC 6 Months
Control .002 .02 < .0001 < .0001 < .0001 < .0001 < .0001 .05 .05
Scrape–MMC 1 day .40 < .0001 < .0001 < .0001 < .0001 .002 .10 .10
Scrape+MMC 1 day < .0001 < .0001 < .0001 < .0001 .0002 .60 .50
PRK–MMC 1 day .0009 < .0001 < .0001 < .0001 < .0001 < .0001
PRK+MMC 1 day < .0001 < .0001 < .0001 < .0001 < .0001
PRK–MMC 1 month .90 .01 < .0001 < .0001
PRK+MMC 1 month .01 < .0001 < .0001
PRK–MMC 2 months < .0001 < .0001
PRK+MMC 2 months .90
PRK+MMC 3 months

Authors

From The Cole Eye Institute, The Cleveland Clinic, Cleveland, Ohio (CSM, GKM, LL, TS, SEW); the Department of Ophthalmology at University of São Paulo, São Paulo, Brazil (CSM, GKM, MRS); and the Department of Ophthalmology at Federal University of Rio de Janeiro, Rio de Janeiro, Brazil (MRS).

Supported in part by U.S. Public Health Service grants RO1EY10056 (SEW) and P30-EY025585 from the National Eye Institute, National Institutes of Health, Bethesda, MD, and Research to Prevent Blindness, Inc., New York, NY. Dr. Lassance is supported by NEI training grant T32 EY007157. Dr. Medeiros is supported by CAPES training grant PDSE2016 Brasília, Brazil. Research reported in this publication was supported by the Office of the Director, National Institutes of Health under award number 1S10OD019972-01 for light and confocal microscopy imaging.

The authors have no financial or proprietary interest in the materials presented herein.

The authors thank James F. Bena, MS, biostatistician in the Quantitative Health Sciences Department, Cleveland Clinic, for help with statistical analyses, and Paramananda Saikia, PhD, and Rodrigo Carlos de Oliveira, MD, Cleveland Clinic, for help with animal work during the last time-point and data analysis.

AUTHOR CONTRIBUTIONS

Study concept and design (CSM, GKM, MRS, SEW); data collection (CSM, LL, TS); analysis and interpretation of data (CSM, SEW); writing the manuscript (CSM, SEW); critical revision of the manuscript (CSM, GKM, LL, TS, MRS, SEW); statistical expertise (CSM); administrative, technical, or material support (GKM); supervision (CSM, GKM, SEW)

Correspondence: Steven E. Wilson, MD, Cole Eye Institute, i-32, The Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail: wilsons4@ccf.org

Received: August 24, 2018
Accepted: November 12, 2018

10.3928/1081597X-20181112-01

Sign up to receive

Journal E-contents