Prior studies have demonstrated that rabbit corneas with high −9D PRK corrections develop dense slit-lamp haze and high density of subepithelial myofibroblasts, similar to some human corneas that generate haze after PRK for high myopia when mitomycin C prophylaxis is not used,
13 whereas most rabbit and human corneas with lower PRK corrections for myopia (−4.5D PRK in rabbits) do not.
7,13 In a study of haze generation after irregular PTK in mice,
5 which is the method used to generate haze in this normally haze-resistant species, disruptions in epithelial basement membrane were noted in areas where myofibroblasts were present. In the current study, a near absence of basement membrane regeneration was noted in all rabbit corneas at 1 month after −9D PRK (
Fig. 5). Conversely, in rabbit corneas that had moderate −4.5D PRK, all corneas had basement membrane regenerated with morphology similar to that in unwounded corneas, except in one cornea where localized areas of haze lacked normal epithelial basement membrane and had associated myofibroblasts (
Fig. 4D).
Abnormalities in the epithelial basement membrane have been noted previously in monkey or rabbit models at 4 weeks after PRK.
10,14,15 In another study, an irregular basement membrane was associated with late corneal epithelial healing defects and altered corneal wound healing.
11
Basement membranes are specialized extracellular matrices that underlie cells and separate them from, and adhere them to, connective tissues. They have been shown to be important for cell adhesion, migration, differentiation, and signal transduction.
16–18 A wide array of human disorders result from, or are associated with, defects in basement membrane assembly or composition, such as Alport syndrome, epidermolysis bullosa, and Fraser syndrome.
19 Moreover, basement membranes are involved in embryonic development,
20,21 remodeling of tissues,
22 and wound healing.
23 At the light microscopic level, matrix molecules have been localized in a linear staining pattern noted beneath epithelial cells in the basement membrane zone. Basement membrane components such as laminins, nidogens, collagen type IV, and perlecan have been localized to basement membranes in immunohistologic and electron microscopic ultrastructural studies.
24 Epithelial basement membranes range from approximately 50 to 100 nm in thickness
25 and typically display three layers at the EM level—lamina lucida, lamina densa, and lamina fibroreticulares.
26
The importance of the integrity of the regenerating epithelial basement membrane as a factor in the development of haze has been suggested by prior studies.
5,6 Late haze that occurs after PRK is localized to the anterior subepithelial stroma on slit-lamp examination.
7 This proximity of the haze and the associated myofibroblasts to the epithelium suggested that epithelial–stromal interactions are involved in myofibroblast development.
3 Other work suggested that normally functioning epithelial basement membrane modulates myofibroblast development through barrier function limiting penetration of epithelium-derived TGF beta and platelet-derived growth factor (PDGF) into the stroma at sufficient levels to drive myofibroblast development and maintain myofibroblast viability once the mature cells are generated.
4 Corneal myofibroblasts may be generated from either keratocyte-derived or bone marrow–derived precursor cells.
27
Many corneas that develop haze return to transparency over a period of months to years. This disappearance of haze is associated with apoptosis of myofibroblasts, movement of keratocytes back into the subepithelial stroma, and reabsorption of abnormal extracellar matrix produced by the myofibroblasts.
4 Studies have shown that interleukin-1 (IL-1) produced by the myofibroblasts (autocrine) or surrounding stromal cells (paracrine) triggers apoptosis of the myofibroblasts when TGF beta levels decline in the stroma.
9,28 The return of normal epithelial basement membrane structure and function is thought to lead to a drop in the penetration of epithelium-derived TGF beta in the anterior stroma. The abnormalities of the basement membrane noted at 1 month after −9D PRK in the present study are consistent with this hypothesis. Conversely, in corneas showing a rapid restoration of basement membrane structure, for example as noted at 1 month after −4.5D PRK in the present study, apoptosis of myofibroblast precursors and mature myofibroblasts likely outstrips myofibroblast generation from precursor cells since epithelial cell–derived TGF beta penetration into the anterior cornea stroma is rapidly reduced after the injury.
Epithelial and other basement membranes regenerate after injury primarily through self-assembly on cell surfaces
25 —in the case of corneal epithelial basement membrane, this would occur on the basal epithelial surface. In vivo and culture studies have suggested that laminins are principally responsible for initial organizing of basement membrane assembly since they are uniquely able to self-assemble into sheet-like structures on cell surfaces without the contribution of other components required for the assembly of a fully functional basement membrane, such as type IV collagens bound to nidogen-1 and nidogen-2, heparan sulfate proteoglycans agrin and perlecan, and many other components.
29 Collagen type IV can also self-assemble but is usually not present until later in the basement membrane regeneration process.
30 Although corneal epithelial cells can produce most of these basement membrane components, some may be contributed by normal stromal cells (keratocytes in the cornea), and not by other stromal cell types such as stromal fibroblasts and myofibroblasts.
31 Also, components such as enzymes and signaling molecules provided by stromal cells likely contribute to formation of the basement membrane and associated structures such as the anchoring fibrils. For example, bone morphogenic protein (BMP) 1 produced by keratocytes is a procollagen protease that converts procollagen VII to produce mature anchoring fibril collagen.
32
It has long been known that higher-correction PRK produces a greater early wave of keratocyte apoptosis/necrosis, and greater and more prolonged acellularity of the anterior stroma, than lower-correction PRK.
7 Our working hypothesis, based on the results of the current study, is that the prolonged recovery of the basement membrane is attributable to a deficiency of components and/or signaling from keratocytes, and that becomes especially limiting in the higher PRK corrections. Surface irregularity can also contribute to difficulty in regenerating the basement membrane.
5 The delay in restoration of a fully functional basement membrane then allows prolonged penetration of high levels of TGF beta and possibly other factors into the stroma from the regenerated epithelium to drive myofibroblast generation from precursor cells.
27 Presumably myofibroblasts do not contribute the necessary components to complete assembly of the basement membrane; and their presence, plus the large amount of random extracellular matrix they produce, further hinders keratocytes from reoccupying a position in the anterior stroma. Therefore, in a cornea with persistent haze, a vicious cycle of sorts is set up whereby myofibroblasts are maintained by the epithelium in the absence of a functional basement membrane; and myofibroblasts themselves, along with the disorganized matrix they produce, provide a barrier against keratocytes that are present deeper in the stroma moving into a subepithelial position to contribute to basement membrane regeneration. In some corneas, over a period of many months to years, this cycle is somehow interrupted and the basement membrane is regenerated; myofibroblasts deprived of TGF beta undergo apoptosis; abnormal extracellular matrix is reabsorbed; keratocytes reoccupy the anterior stroma; and the cornea is returned to transparency. Further work is needed to delineate the specific mechanisms involved in these processes leading to corneal scarring and recovery after scarring is produced.