Cellular and extracellular matrix modulation of corneal stromal opacity

Abstract

Stromal transparency is a critical factor contributing to normal function of the visual system. Corneal injury, surgery, disease and infection elicit complex wound healing responses that serve to protect against insults and maintain the integrity of the cornea, and subsequently to restore corneal structure and transparency. However, in some cases these processes result in prolonged loss of corneal transparency and resulting diminished vision. Corneal opacity is mediated by the complex actions of many cytokines, growth factors, and chemokines produced by the epithelial cells, stromal cells, bone marrow-derived cells, lacrimal tissues, and nerves. Myofibroblasts, and the disorganized extracellular matrix produced by these cells, are critical determinants of the level and persistence of stromal opacity after corneal injury. Decreases in corneal crystallins in myofibroblasts and corneal fibroblasts contribute to cellular opacity in the stroma. Regeneration of a fully functional epithelial basement membrane (BM) appears to have a critical role in the maintenance of corneal stromal transparency after mild injuries and recovery of transparency when opacity is generated after severe injuries. The epithelial BM likely has a regulatory function whereby it modulates epithelium-derived growth factors such as transforming growth factor (TGF) β and platelet-derived growth factor (PDGF) that drive the development and persistence of myofibroblasts from precursor cells. The purpose of this article is to review the factors involved in the maintenance of corneal transparency and to highlight the mechanisms involved in the appearance, persistency and regression of corneal opacity after stromal injury.

Keywords: Bone marrow-derived cells; Cornea; Epithelial basement membrane; Fibrocytes; Myofibroblasts; Stroma Haze; Wound healing.

Fig. 1

Keratocyte apoptosis (red, arrows) detected with the TUNEL assay 4 hours after −9D PRK. DAPI stains intact nuclei in deeper keratocytes. 400x

Fig. 2

Corneal haze at 4 months after −7D PRK in a human cornea that was not treated with mitomycin C. 15X.

Fig. 3

Bone marrow-derived cells (green, arrowheads) infiltrate a mouse cornea at 24 hours after epithelial injury in a chimeric mouse that had a total body irradiation and a bone marrow transplant from a mouse expressing green fluorescent protein in all of its cells several months earlier. (see Wilson et al., 2004) Magnification 10X

Fig. 4

α-SMA+ cells (green, arrows) in the anterior stroma at one month after −9D PRK in a rabbit. * indicates artifact separation of the epithelium from the stroma that occurs during cutting of the section with a cryolathe. Blue is DAPI staining for cell nuclei. e is epithelium and s is the stroma. 400X.

Fig. 5

Vimentin+ cells (arrows) in the anterior stroma at one week after −9D PRK in a rabbit. e indicates epithelium. This is a rabbit cornea one week after −9D PRK that underwent double immunohistochemistry for vimentin (orange) and α-SMA (green). At this time point after surgery, none of the cells in the anterior stroma express α-SMA. However, many of these vimentin+ cells are likely myofibroblasts in early development that will begin to express α-SMA with further maturation at about two weeks after surgery. Note keratocytes (arrowheads) that have been shown in prior studies to also express vimentin, but at much lower levels, and this expression was not detected with the concentration of primary antibody for vimentin used in this staining (see Chaurasia et al., 2009). Thus, these early myofibroblasts that are vimentin+SMA-express vimentin at high levels. 400X

Fig. 6

α-SMA+ Desmin+ myofibroblasts in a rabbit cornea at 1 month after −9D PRK. At this point after surgery, corneal myofibroblasts express vimentin (not shown), α-SMA (left panel, green), and desmin (center panel, red). In this double-stained section there is nearly 100% concurrence of α-SMA and desmin expression (right panel, overlay). Blue is DAPI staining of cell nuclei. 400X. After Chaurasia et al., 2009.

Fig. 7

Defective epithelial basement membrane regeneration in corneas with stromal haze after PRK. (A) In a rabbit cornea at one month after −4.5D PRK that did not develop significant stromal haze there is normal regeneration of the epithelial basement membrane (arrows) with the lamina lucida and lamina densa clearly visible with transmission electron microscopy, as it is in the normal unwounded corneas (not shown). The black arrowhead indicates a keratocyte in the stroma (s). (B) At one month after PRK for high myopia (−9D), in a rabbit cornea that developed severe stromal haze, there is defective regeneration of the epithelial basement membrane with no visible lamina lucida or lamina densa. White arrowheads indicate layers of myofibroblasts with large amounts of rough endoplasmic reticulum and the Xs indicate disordered extracellular matrix these cells secrete in the anterior stroma (s). e indicates epithelium in both panels. Magnification 23,000X

Fig. 8

Apoptosis of myofibroblasts at one month after −9D PRK. TUNEL assay was used to detect apoptosis and immunohistochemistry to detect α-SMA in myofibroblasts (arrowheads) revealed one myofibroblast undergoing apoptosis (arrow). E indicates epithelium. Blue stain is DAPI for cell nuclei. 400X. The balance between myofibroblast generation and myofibroblast apoptosis in a particular cornea after injury determines whether haze is increasing, persisting, or disappearing over time. After Wilson, Chaurasia, and Medeiros, F.W. 2007 with permission.