Normal wound healing after epithelial debridement involves three distinct but continuous phases.
18 19 In the first phase, hemidesmosomes are lost and provisional attachment complexes, called focal contacts, form.
20 21 22 The epithelial cells flatten, migrate, and slide as a single-layered intact sheet to cover the denuded surface.
23 During the second phase, cells distal to the original wound proliferate to repopulate the wound area, and cell stratification and differentiation occur.
19 20 In the third phase, hemidesmosomes reform, and extracellular matrix is synthesized and reassembled.
18 19
After epi-LASIK, healing of the corneal epithelium may differ from normal epithelial healing. The replaced epithelial flap, whether dead or alive, stays in front of the leading edge, and a denuded surface is lacking. In addition, preservation of the basement membrane under the epithelial flap may exert specific effects.
9 10 Moreover, laser treatment of the stromal bed may induce an epithelial–stromal interaction and interfere with wound healing.
24 25 26 Finally, pharmacologic agents used during surgery may contribute to wound healing.
We examined specific epithelial wound healing after epi-LASIK by using in vivo confocal microscopy. This method has been used to observe corneal epithelial healing in various conditions. Cho et al.
27 found that healing superficial cells in rabbit corneas after epithelial debridement were initially elliptical and large. By the end of the second week, the cells were almost the same size and shape as normal cells. In limbal stem cell–deficient corneas, healing epithelial cells were smaller and more variable in size than were normal corneal cells.
27 In post-PRK rabbit corneas, small superficial corneal epithelial cells with high N/C ratios were found at 1 week after surgery. The corneal epithelium recovered normal cellular morphology at 2 weeks.
28 Although these studies provided important information about epithelial wound healing, the authors did not describe observations in different layers of corneal epithelial cells.
Discrepant results from different studies on the survival of epikeratome-created epithelial flaps have been reported. Pallikaris et al.
10 29 initially concluded that the epikeratome produces epithelial flaps with a sharply separated basement membrane and intact basal cells, although occasional focal disruption of the basal lamina was also observed. Their more recent study showed that most of the epithelial cells were morphologically normal with only minor cell degeneration.
9 Tanioka et al.
16 demonstrated that most of the basal cells in epithelial flaps created with different epikeratome devices were PI-positive dead cells. Because of the difficulty in obtaining human corneal epithelial sheets for evaluation, these studies observed only flap survival in the first few days after epi-LASIK.
9 10 16 Our long-term results are more similar to those of Tanioko et al.
16 In the group without MMC treatment, 31.8% of the eyes had basal cells with normal morphology
(Fig. 1A)on postoperative day 1; the incidence decreased dramatically on days 3 and 7. Those cells with morphology as shown in
Figures 1B 1C and 1Dwere thought to have undergone severe pathologic changes—most likely, cell death. The healing basal epithelial cells during the first few days after surgery showed high N/C ratios without visible cellular borders
(Fig. 1E) . Visible cellular borders usually reappeared at 1 to 2 weeks after surgery
(Fig. 1F) . At that time, basal cellular nuclei were usually seen, but the N/C ratios markedly decreased. After 3 weeks, 72.7% of the eyes had no visible nuclei. To our knowledge, we are the first to report the appearance and subsequent disappearance of basal cellular nuclei, combined with the disappearance and subsequent reappearance of basal cellular borders during wound healing, evaluated by in vivo confocal microscopy. Although not proved, a large and visible nucleus may represent new, actively growing cells that are metabolically active and unstable. The reestablishment of cellular borders may represent reconstruction of the cellular junction, which implies a return to normal and stable conditions.
The recovery of normal morphology took longer in apical surface cells than in basal cells. In both the MMC and non-MMC treatment groups, most eyes had abnormal apical surface epithelial cells during the first few weeks. Abnormalities included elongated, amorphous, multidirectional, or unidentifiable morphology. Abnormal cells were thought to have undergone severe pathologic changes—most likely, cell death. In the intermediate stage of healing between 3 weeks and 3 months, most eyes had small and compact apical surface cells, with high N/C ratios but no squamous appearance
(Fig. 2C) . These cells were thought to be in higher metabolic, earlier differentiation stages than normal squamous cells.
The exact process of epithelial healing after epi-LASIK is still not clearly understood despite the information provided by in vivo confocal microscopy. Gradual sloughing of the damaged epithelial sheets may have occurred, followed by replacement with multilayered, highly active, new and growing cells. Because no denuded surface area was seen ahead of the leading edge (as noted in corneal debridement or PRK), we can easily explain why we saw no single layer of migrated cells, as is observed in phase I of normal epithelial healing. Features indicating the highly metabolic and undifferentiated status of the new growing cells were the increased N/C ratios of the basal and apical surface cells, the disappearance of cellular borders in basal cells, and the disappearance of the large squamous cells on the apical surface. However, we cannot rule out the possibility that new growing cells from the periphery of the cornea may have mixed with surviving cells on the flap and that together they composed the final cellular sheet.
Several of our findings need further clarification. First, our observational period and methods differed from those of previous studies. In published reports about epi-LASIK, healthy corneal epithelial sheets were observed only in the first 24 hours, and immunohistochemistry and electron microscopy were used for the examination.
9 10 16 Our cross-sectional views of the epithelial sheets, long observation times, and in vivo observations may add important information. Second, use of different epikeratomes could lead to different cutting levels and affect the survival of the cellular sheet, although Tanioka et al.
16 reported dead cells in epithelial flaps prepared with different epikeratomes. Third, because no similar study has been published, we have justified the grading scheme of corneal epithelial wound healing on the basis of the presumed correlation with severity just by morphologic interpretation. Further histologic studies are needed to confirm our hypothesis. Fourth, during postoperative week 1, most examinations were performed with therapeutic bandage contact lenses in place. To rule out the possibility that contact lens fitting might change the in vivo microscopic appearance of corneal epithelial cells, we did a preliminary test in rabbit eyes and found that placement of a contact lens did not change the appearance of the healing basal and apical surface epithelial cells (data not shown). We also compared the appearance of the basal and apical cells in the patients before and after removal of the contact lenses, and found no difference in apical cellular appearance (data not shown). Fifth, the stromal reaction based on the reflectivity tied to the light intensity in this study should be interpreted carefully. Potentially strong confounding factors may exist and make the results difficult to analyze. Sixth, to inhibit potential haze, we used MMC and a chilled physiologic saline solution in patients with high degrees of myopia. MMC has been widely accepted as an adjunctive therapy for corneal haze after higher myopic corrections by PRK and LASEK.
30 31 32 33 34 It has been hypothesized that MMC inhibits fibroblast proliferation and differentiation, consequently blocking myofibroblast formation through its various potent effects.
35 36 Although epi-LASIK is intended to create a cellular sheet with an intact basement membrane, the morphologically unhealthy epithelial cells we found may still be able to induce an epithelial–stromal reaction as in PRK or LASIK. There are only limited studies reported on the use of MMC in epi-LASIK. In this study, we found that higher myopic groups (> −6.0 D) treated with MMC may have similar stromal reactions compared with the low myopic group (< −6.0 D) without MMC treatment. Our results support the widely accepted view that MMC can prevent corneal haze formation in surface ablation for higher myopic corrections. We also found that the group with MMC treatment had a higher incidence of early corneal basal epithelial damage compared with the group without MMC treatment. In addition, the only two eyes that had morphologically intact basal epithelial morphology during the entire observational period were without MMC treatment. Although we tried to avoid direct contact between the MMC solution and the epithelial sheets and we protected the epithelial sheet during vigorous irrigation of the stromal bed with the chilled saline solution, mechanical or pharmacologic cellular damage caused by MMC or irrigation itself may still have occurred during surgery. Because almost all eyes in the groups with and without MMC treatment have severe cellular damage of the apical surface epithelial cells within 2 weeks after surgery, it is difficult to evaluate the effect of MMC on these cells. We found no differences in corneal epithelial thickness between the two groups at 1, 3, and 6 months after surgery. The healing patterns of basal and apical cells were also similar at 3 and 6 months after surgery. The effect of MMC on corneal epithelial wound healing was thus thought to occur only in the early postoperative period.
In conclusion, our study demonstrated several important new findings. First, we believe this is the first human study in which in vivo confocal microscopy was used to study early and late wound healing after surface ablation, specifically epi-LASIK. Second, the corneal epithelial flaps after epi-LASIK were not as healthy as previously thought, as cells underwent severe pathologic changes within or after 24 hours. Third, at least several weeks were required for the epithelial cells to return to their normal morphology, and apical surface cells needed more time than basal cells. Finally, the stromal reaction was the same in the eyes with higher myopia treated with MMC as in the eyes with lesser myopia without MMC treatment. Because cellular conditions cannot be properly assessed by morphologic analysis alone, sequential evaluations using apoptosis assays, immunohistochemical staining, and/or electron microscopy are needed to provide further information about the corneal wound-healing process after epi-LASIK.