Corneal molecular and cellular biology update for the refractive surgeon

Abstract

Purpose: To review clinically relevant progress in understanding cellular and molecular interactions in the cornea that relate to refractive surgical outcomes in patients.

Methods: Recent published literature focused on femtosecond LASIK and surface ablation procedures, such as photorefractive keratectomy, was reviewed and correlated with clinical results of surgery.

Results: The femtosecond laser has a direct necrotic effect on stromal keratocytes, resulting in the release of cellular components that are chemotactic to bone marrow-derived inflammatory cells. Developments of the femtosecond laser led to lower energy delivery to the stroma and altered laser ablation profiles that decrease epithelial damage during the side-cut, and have markedly improved femtosecond LASIK to the point that the overall early postoperative healing response is indistinguishable from microkeratome LASIK. New studies have directly demonstrated the importance of surface irregularity and resulting structural and functional defects in the epithelial basement membrane, in the generation and persistence of anterior stromal myofibroblasts and haze following surface ablation procedures. These defects augment penetration of epithelium-derived TGF-beta, which is a critical modulator of myofibroblast development in the stroma. Studies on the mechanism of action of mitomycin C treatment to prevent haze have confirmed that the most powerful effect is on stromal cell proliferation and, therefore, decreased population of the anterior stroma with myofibroblast progenitor cells. An undesirable long-term effect of mitomycin C is diminished anterior stromal keratocyte density due to diminished keratocyte re-population. This raises concerns regarding future corneal anomalies in treated corneas.

Conclusions: Basic research studies of refractive procedures provide important insights into the effects of wound healing on surgical outcomes.

Figure 1

Death of stromal cells detected with the TUNEL assay (red cells [arrows]) at 24 hours after LASIK flap formation with the A) Hansatome microkeratome, B) 15-kHz femtosecond laser, and C) 30-kHz femtosecond laser. Blue 4′,6-diamidino-2-phenylindole (DAPI) stains all intact cell nuclei. In A and B, DAPI staining of stromal nuclei anterior and posterior to the region of TUNEL-stained cells is less distinct due to tissue edema, but keratocytes are present in these zones outside the region of cell death caused by the microkeratome or femtosecond laser. (Original magnification ×200.) Reprinted with permission from SLACK Incorporated.

Figure 2

CD-11b-positive (green stain [arrows]) monocyte cell infiltration into the cornea at 24 hours after LASIK flap formation with the A) Hansatome microkeratome, B) 15-kHz femtosecond laser, and C) 30-kHz femtosecond laser. Blue 4′,6-diamidino-2-phenylindole (DAPI) stains all intact cell nuclei. (Original magnification ×200.) Reprinted with permission from SLACK Incorporated.

Figure 3

Mitotic cells (red cells [arrows]) detected by immunohistochemical staining for mitosis-specific antigen Ki-67 24 hours after LASIK flap formation with A) the Hansatome microkeratome, B) the 15-kHz femtosecond laser, and C) the 30-kHz femtosecond laser. (Original magnification ×400.) Note the larger diameter gap (arrowheads) in the epithelium and stroma at the epithelio-stromal junction in eyes treated with the femtosecond lasers (B) and (C) compared with the small gap in eyes treated with a microkeratome (A). Blue 4′,6-diamidino-2-phenylindole (DAPI) stains all intact cell nuclei. Reprinted with permission from SLACK Incorporated.

Figure 4

Slit-lamp photograph of haze occurring in an eye that underwent treatment with 0.002% mitomycin C for 15 seconds after photorefractive keratectomy for -9.00 diopters of myopia. (Original magnification ×20.)

Figure 5

Basement membrane defects and myofibroblast cells after photorefractive keratectomy (PRK). A) Note the uniform basement membrane (green cells [arrows]) regeneration in a cornea without haze at 1 month after -4.50 diopter (D) PRK. B) At 1 month after -9.00-D PRK, the basement membrane has obvious disruptions (green cells [arrows]), and beneath this area, red-stained alpha-smooth muscle actin-positive myofibroblast cells (arrowheads) are noted. The basement membrane is highlighted by immunohistochemical staining for integrin beta4. Blue 4′,6-diamidino-2-phenylindole (DAPI) stains all intact cell nuclei. (Original magnification ×400.) Reprinted with permission from Elsevier.

Figure 6

Myofibroblast apoptosis in a rabbit cornea with severe haze after -9.00 diopter photorefractive keratectomy. DAPI staining of cell nuclei appears blue in all panels. A) When the section is stained for the myofibroblast marker alpha-smooth muscle actin, myofibroblasts (arrows) are detected as green in the anterior stroma beneath the epithelium. B) When the section is imaged with a red TUNEL assay for apoptosis, TUNEL-positive cells (arrows) are detected in the same location as myofibroblasts in A. C) When both the red and green are imaged, some myofibroblast cells in the anterior stroma are noted to be undergoing apoptosis (arrows) and therefore appear yellow. (Original magnification ×500). Reprinted with permission from Elsevier.

Figure 7

Mitomycin C blocks proliferation of cells in the anterior stroma after photorefractive keratectomy (PRK). A) At 24 hours after PRK and treatment with 0.02% mitomycin C for 2 minutes, there are very few cells undergoing mitosis (arrowheads) in the anterior stroma detected by immunohistochemical staining for the mitosis marker Ki-67. B) In contrast, at 24 hours after PRK and treatment with balanced salt solution vehicle for 2 minutes, numerous cells are proliferating (arrowheads) in the anterior stroma. 4′,6-diamidino-2-phenylindole (DAPI) staining of cell nuclei appears blue; green stain is Ki-67. (Original magnification ×400.) Reprinted with permission from SLACK Incorporated.