(a) Illustration of photodisruption process in corneal stroma. (b) Scanning electron microscopic micrograph showing the cavitation bubbles generated during the photodisruption process
2.2 Wound Healing After Refractive Lenticule Extraction (ReLEx®)
Corneal wound healing has an important effect on the safety, efficacy, and stability of laser vision correction [9, 10]. Biological differences in wound-healing responses are thought to be a major factor affecting the predictability of refractive surgery in some patients (overcorrection, undercorrection, regression, and irregular astigmatism) . Corneal wound healing has also been reported to be associated with corneal haze, myopia regression, and epithelial ingrowth after femtosecond laser in situ keratomileusis (Fs-LASIK) [11–13].
Corneal wound healing involves a complex cascade of pathways. On the molecular and cellular level, cytokines and growth factors, such as interleukin (IL)-1, tumor necrosis factor (TNF)-α, epidermal growth factor, and platelet-derived growth factor, are released from the injured epithelium [14–16]. This injury can take the form of an incision, femtosecond laser exposure, or other insults . The released cytokines and growth factors mediate stromal keratocytes apoptosis , followed by proliferation and migration of remaining stromal keratocytes within a few hours to restore stromal cellularity [18, 19]. Within 24 h, inflammatory cells migrate to phagocytize the apoptotic cells and enhance the transformation of keratocytes to fibroblasts . Transforming growth factor-β (TGF-β) and other cytokines then induce the differentiation of fibroblasts to myofibroblasts , and the appearance of myofibroblasts is the primary biological event associated with the development of corneal surface irregularity and corneal haze [20, 21] (Fig. 2.2). During the corneal wound-healing response, the balance between myofibroblast precursor apoptosis and myofibroblast development is a critical determinate of whether corneal haze develops . Moreover, corneal avascularity, or the maintenance of a corneal “angiogenic privilege” state, is important for corneal transparency [23, 24]. The maintenance of corneal avascularity depends on a fine balance between the production of angiogenic and anti-angiogeneic factors . One of the first molecules thought to have a major role in maintaining corneal avascularity was pigment epithelium-derived factor (PEDF) .
Illustration of corneal wound-healing cascade
2.2.1 Corneal Wound Healing and Inflammatory Response After ReLEx®: Animal Study
There are only a few studies investigating the corneal wound healing and inflammatory response after ReLEx in the literature. Our group has investigated and compared the early corneal wound repair and inflammatory responses after FLEx and Fs-LASIK 1 day after surgery . We demonstrated that (1) the expression of fibronectin, which is produced by activated keratocytes and plays an important role in cell adhesion, growth, migration, and differentiation during the corneal wound-healing process , showed a less abundant expression around the incision line in the corneas that underwent FLEx than Fs-LASIK. The differences became more marked as the power of correction was increased. This was because the higher energy of the excimer laser in Fs-LASIK caused an elevated intensity of fibronectin staining. Also in Fs-LASIK, a wider treatment zone including a 1.0-mm blend zone is performed (a conventional blend zone is not needed in ReLEx®). A similar trend was also seen in the number of CD11b-positive cells (a marker of monocytes) that play a role in inflammatory infiltration after injury. The results suggested that excimer laser treatment in Fs-LASIK stimulated a higher degree of inflammation. Furthermore, we found that there were no CD11b-positive cells seen along the laser vertical or lamellar cutting plane in the post-FLEx corneas when the lenticule was not removed. This indicated that the inflammatory response following ReLEx mainly comes from the surgical dissection, rather than the laser. (2) TUNEL-positive cells (indicating apoptotic cells) were detected in the corneal center and periphery of flap, suggesting femtosecond laser energy still induced keratocyte cell death in the absence of injured epithelium. There was no significant disparity in the number of apoptotic cells between Fs-LASIK group and ReLEx, although more apoptotic cells were observed in Fs-LASIK group. (3) Ki-67, a cell proliferation marker, was primarily present in the epithelial cells of the flap margin, rather than the epithelium of cornea center, for both groups. This indicated the proliferation activity was mainly seen in the areas where epithelium was damaged or displaced. No significant difference in the Ki-67 expression around the flap margin was observed between two groups.
Dong et al.  evaluated and compared the early corneal wound healing and inflammatory response after SMILE versus Fs-LASIK using a rabbit model. The authors reported that (1) TUNEL-positive cells were detected at the lamellar interface after SMILE and Fs-LASIK procedures at postoperative 4 and 24 h. A statistically significantly fewer TUNEL-positive stromal cells were observed in the SMILE group than in the Fs-LASIK group at postoperative 4 and 24 h. (2) There were statistically significantly fewer Ki67-positive cells in the stroma in the SMILE group as compared to the Fs-LASIK group, at day 3 and week 1 postoperatively, indicating that SMILE stimulated less stromal keratocyte proliferation. (3) The CD11b-positive cells were significantly less in the SMILE group at day 1, day 3, and week 1 postoperatively. The authors postulated this could be due to the following reasons: a small incision for SMILE produced fewer cytokines to attract inflammatory cells in the injury, the intrastromal dissection by a femtosecond laser contributed to less extent of tissue injury compared with the stromal ablation by an excimer laser, and there was less necrotic debris in the interface after SMILE.
Our group has also studied the early corneal wound-healing inflammatory response following SMILE in a rabbit model. The small vertical incision and lamellar dissection wounds were observed at day 1 (Fig. 2.3a), but both of them almost healed at week 1 postoperatively (Fig. 2.3b). The CD11b-positive cells were apparent at 1 day postoperatively, more abundant around the vertical incision site (Fig. 2.4a) than the lamellar incision plane (Fig. 2.4b). This is understandable since the vertical incision cuts through the epithelium and basement membrane. In healthy and intact corneas, the basement membrane can function to bind cytokines , suggesting that it may act as a physical barrier for signaling molecules that are produced by the epithelial cells or tear fluid . Thus, when the barrier is compromised, the underlying stroma is exposed to the signaling molecules, and the inflammatory cell infiltration is augmented. Moreover, the surgical manipulations around the incision, such as inserting instruments via the small incision to dissect the anterior and posterior surfaces of the lenticule and to extract the lenticule, might elicit cytokines to attract inflammatory cells because of more disturbances on the basement membrane. It might be also due to the some inadvertent minor epithelial abrasions or small tears at the incision site, which have been reported to be a common postoperative complication after SMILE . At 1 week postoperatively, the CD11b expression was significantly reduced (Figs. 2.4c and 2.4d). This observation was different from what was reported by Dong et al. . They showed an increase in CD11b-positive cells in the central cornea at 1 week compared to 1 day postoperatively. However, CD11b is an early inflammatory marker expressed on the surface of neutrophils, monoctyes, and macrophages, which have been reported to be attracted to the wound site within 24 h and be replaced by lymphocytes 3 days after the corneal insult . Since we had previously shown that the expression of CD11b was related to the surgical trauma of lenticule extraction, the difference in the results between the two studies may be explained by differences in surgical technique in lenticule extraction. The expression of fibronectin appeared around the incision as well as along the anterior and posterior extracted lenticule planes at day 1 postoperatively (Figs. 2.5a and 2.5b), and the staining intensity in these two sites increased at week 1 postoperatively (Figs. 2.5c and 2.5d). Studies on rabbit wound-healing models have shown that 1 day after an incision on the cornea, fibronectin appeared at the site of injury. During the following 1–2 weeks, the increased fibronectin provided a provisional matrix to support the migration of the remaining epithelial cells or keratocytes to cover the area of the defect [33, 34]. After 2 weeks, the wound-healing response was complete, and the expression of fibronectin began to decrease . This explains why the expression of fibronectin appeared more distinct around both the small vertical incision and extracted lenticule plane at week 1 after SMILE. Heat shock protein 47 (HSP47) is a stress protein and functions as a collagen-specific molecular chaperon. It is induced in response to stress applied to cells [35, 36]. Unlike the staining pattern of CD11b that mainly appeared in the vertical incision and resolved at 1 week following SMILE, the expression of HSP47 was observed throughout the whole cornea at day 1 postoperatively (Figs. 2.6a, b), suggesting that the laser-induced cell stress may affect the whole layer of cornea, as well as the small incision site. The HSP47-positive cells significantly reduced at 1 week postoperatively (Figs. 2.6c, d).
Light microscopic cross-sectional histologic specimen in rabbit corneas stained by hematoxylin-eosin (H & E) stain showing the small incision (arrowhead) and lamellar dissection wound (arrows) were observed at day 1 postoperatively (a) but almost healed at week 1 postoperatively (b). Original magnification: 100×, scale bar 100 μm
Immunohistochemical staining showing the expression of CD11b in rabbit corneas 1 day (a, b) and 1 week (c, d) at the incision (a, c; arrowheads) and extracted lenticule plane (b, d) after −4.0 D SMILE. The CD11b-positive cells were apparent at 1 day, more abundant around the incision (a) than around the lamellar cutting plane (b) and significantly reduced at 1 week postoperatively (c, d). Original magnification: 200×, scale bar 100 μm
Immunohistochemical staining showing the expression of fibronectin in rabbit corneas 1 day (a, b) and 1 week (c, d) at the incision (a, c; arrowheads) and extracted lenticule plane (b, d) after −4.0 D SMILE. The expression of fibronectin appeared around the incision as well as extracted lenticule plane at day 1 (a, b), and the staining intensity in these two sites became greater at week 1 postoperatively (c, d). Original magnification: 200×, scale bar 100 μm
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