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
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
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 1–3), 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.
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.