Disruption of the Basement Membrane after Corneal Débridement Animal Model

PURPOSE. To determine whether the native basement membrane left behind after manual débride-ment wounding is retained throughout healing in the Balb/c mouse. METHODS. Mouse corneas were subjected to either 1.5 mm (small) or limbus-to-limbus (large) epithelial débridement wounds and allowed to heal for times ranging from 12 hours to 3 days. For the larger wounds, care was taken to leave an approximately 0.5-mm zone of epithelial cells near the limbal border. Unwounded corneas served as control specimens. At each time point, confocal immunofluorescence microscopy was used to localize several proteins found in the basement membrane including laminin-5, entactin, and perlecan. In addition, ultrastructural studies were performed using transmission electron microscopy (TEM) to assess the basement membrane zone (BMZ) of the corneas at various times after injury. RESULTS. The smaller (1.5-mm) wounds healed within 24 hours, and the larger wounds healed at approximately 48 hours. Both wound sizes healed with little scarring or neovascularization. At all time points after 1.5-mm wounding, immunofluorescence confocal microscopy and TEM showed that both basement membrane proteins and the lamina densa were retained at the BMZ throughout healing. For the larger wounds, at time points after 24 hours, confocal microscopy showed patches along the denuded corneal stroma where there was a partial or complete loss of basement membrane markers at the BMZ. TEM confirmed that the lamina densa was partly or completely absent along the anterior surface of the exposed cornea at time points of more than 24 hours after the larger wounds. CONCLUSIONS. The denuded epithelial basement membrane was shown to be partially disassembled in response to manual débridement wounds when re-epithelialization took more than 24 hours. Regulated disassembly of the epithelial basement membrane probably plays a role in the healing of large-diameter débridement wounds. (Invest Ophthalmol Vis Sci. 2000;41:1045-1053 S tudies of corneal wound healing after manual débride-ment were first conducted more than 30 years ago and have been shown by numerous groups to leave the lamina densa of the basement membrane intact and in its native state after wounding. 1-5 Based on previous studies, 6,7 we know that the mouse cornea re-epithelializes after manual débridement between 20 to 24 hours in the 8-week-old Balb/c mouse after wounds removing no more than 40% of the epithelial surface (1.5 mm; small wounds). Re-epithelialization of small wounds is accompanied by increased expression of ␣6␤4 integrin, 8 a structural component of the hemidesmosomes and a signaling molecule known to regulate epithelial cell proliferation. 9 -11 Extracellular ligands for ␣6␤4 integrin are members of the laminin family of adhesive glycoproteins, primarily laminin-1 and -5. Laminin-5 is a component of the anchoring filaments of hemidesmosome adhesion complexes. It also has been shown recently to be secreted by migrating epithelial cells in the skin in response to blisters and deeper, more penetrating wounds 12 and by corneal epithelial cells in response to manual keratectomy wounds. 13 Laminin-1 is also a basement membrane component, 7 Unlike ␣6␤4, the functions of ␣9␤1 in epithelial cells are unclear. To determine whether alterations in the nature of the substrate, the basement membrane zone (BMZ), are associated with the increased expression of either ␣6␤4 or ␣9␤1 observed in response to corneal débridement wounding, we performed both small and large débridement wounds and evaluated tissues by immunohistochemistry for the localization of several different basement membrane proteins at a variety of different time points after wounding. We also evaluated the morphology of the corneal epithelial basement membrane directly by transmission electron microscopy (TEM)

Characteristics of Progenitor Cells Derived from Adult Ciliary Body in Mouse, Rat, and Human Eyes

PURPOSE. To isolate and characterize progenitor cells derived from adult mammalian ciliary body. METHODS. The authors isolated progenitor cells from the ciliary body of adult mice, rats, and human cadaver eyes and determined quantitative growth characteristics of groups of progenitor cells called neurosphere (NS) cells, including individual cell diameter, NS diameter, percentage of NS-forming cells, and cell number per eye in mouse, rat, and human eyes. The immunolabeling and ultrastructure of NS cells were investigated by confocal and transmission electron microscopy. RESULTS. Average diameters of individual cells and neurospheres after 1 week in culture were similar in mice, rats, and humans (cell diameters: 22 Ϯ 1.1, 21 Ϯ 0.3, 25 Ϯ 0.4 m; NS diameters: 139 Ϯ 22, 137 Ϯ 9, 141 Ϯ 11 m, respectively). Mean numbers of cells per NS were estimated to be 1183 in mice, 5360 in rats, and 685 in humans. Molecules that were identified by immunolabeling in NS cells included nestin, Chx-10, vimentin, GFAP, and Pax-6. Thy-1 was expressed in some NS cells. Ultrastructurally, NS cells displayed abundant rough endoplasmic reticulum and many cellular processes but no characteristics of mature retinal neurons or glia. CONCLUSIONS. Progenitor cells from adult mammalian ciliary body have significant, but limited, proliferation potential and express markers characteristic of other progenitor cells and seen during early retinal development. The ciliary body could be a source of cells for transplantation in experimental rodent eyes and for autotransplantation in human eyes. (Invest Ophthalmol Vis Sci