Abnormal deposition of laminin and type IV collagen at corneal epithelial basement membrane during wound healing in diabetic rats

PURPOSE: To understand the pathophysiology of the corneal basement membrane in diabetes, we compared the localization of laminin and type IV collagen in the epithelial basement membrane during corneal epithelial wound healing in diabetic and nondiabetic rats. METHODS: Streptozotocin was used to induce diabetes in half the rats. Two weeks later, the whole corneal epithelium was debrided. Diabetic and healthy rats (3-5 per group) were sacrificed before debridement and 1, 3, and 7 days and 1 month afterwards. The localization of laminin and type IV collagen was observed in cryosections by epifluorescence microscopy. RESULTS: In unwounded corneas of both diabetic and normal rats, laminin and type IV collagen were localized in the corneal epithelial basement. The intensity of fluorescence, however, was clearly stronger in the diabetic rats. In normal rats, wounding initially removed laminin and type IV collagen, but during healing these two proteins reappeared beneath the resurfacing corneal epithelium. Although similar results were observed in diabetic rats, the expression of laminin and type IV collagen was delayed, and their deposition was fragmented and irregular. CONCLUSIONS: These results suggest that delayed corneal epithelial wound healing in diabetes might involve delayed reappearance and abnormal reformation of epithelial basement membrane proteins.     

Removal of the basement membrane enhances corneal wound healing

Recurrent corneal erosions are painful and put patients’ vision at risk. Treatment typically begins with debridement of the area around the erosion site followed by more aggressive treatments. An in vivo mouse model has been developed that reproducibly induces recurrent epithelial erosions in wild-type mice spontaneously within two weeks after a single 1.5 mm corneal debridement wound created using a dulled-blade. This study was conducted to determine whether 1) inhibiting MMP9 function during healing after dulled-blade wounding impacts erosion development and 2) wounds made with a rotating-burr heal without erosions. Oral or topical inhibition of MMPs after dulled-blade wounding does not improve healing. Wounds made by rotating-burr heal with significantly fewer erosions than dulled-blade wounds. The localization of MMP9, β4 integrin and basement membrane proteins (LN332 and type VII collagen), immune cell influx, and reinnervation of the corneal nerves were compared after both wound types. Rotating-burr wounds remove the anterior basement membrane centrally but not at the periphery near the wound margin, induce more apoptosis of corneal stromal cells, and damage more stromal nerve fibers. Despite the fact that rotating-burr wounds do more damage to the cornea, fewer immune cells are recruited and significantly more wounds resolve completely.     

Corneal neovascularization after excimer keratectomy wounds in matrilysin-deficient mice

PURPOSE: Matrilysin, matrix metalloproteinase (MMP)-7, is upregulated in the corneal epithelium during wound healing after excimer keratectomy wounds. The purpose of this study was to determine the role of matrilysin in maintaining corneal avascularity during wound healing. METHODS: Matrilysin-deficient mice (n = 17) and their age-matched wild-type littermates (n = 18) were treated with 193 nm argon-fluoride excimer keratectomy (experiment I). The percentage of corneal surface occupied by neovascularization was measured with a computer image-analysis program adjusted for parallax. In another experiment (experiment II), epithelial closure was monitored with slit lamp biomicroscopy and fluorescein staining, and corneal neovascularization was confirmed by india ink perfusion, electron microscopy, and immunolocalization of CD31 and type IV collagen. Corneal micropocket assays were performed to compare the area of corneal neovascularization in matrilysin-deficient mice and wild-type littermates (experiment III). To determine whether the differences in corneal neovascularization were related to differences in angiogenic factors, the levels of basic fibroblast growth factor (bFGF) were compared with those of vascular endothelial growth factor (VEGF) in matrilysin-deficient and wild-type mouse corneas (experiment IV). RESULTS: The percentages of the corneal surface occupied by neovascularization after excimer laser keratectomy in the matrilysin-deficient mice measured 21.3% +/- 5.2% and 18.7% +/- 5.8% at days 3 and 7, respectively, compared with 5.3% +/- 2.4% and 5.5% +/- 3.4% in the wild-type littermates at days 3 (P < 0.01) and 7, respectively (P < 0.05; experiment I). No significant differences in the rates of epithelial closure of corneal wounds were observed between matrilysin-deficient and wild-type mice after wounding. Corneal neovascularization in the matrilysin-deficient mice was confirmed by india ink present in the corneal stromal blood vessels (extending from the limbus to the wound), immunohistochemical staining, and electron microscopy. Gram, Giemsa, calcofluor white, and acridine orange stains and electron microscopy showed no evidence of corneal infection (experiment II). The area of corneal neovascularization in matrilysin-deficient mice was not significantly different from that of wild-type littermates after implantation of bFGF pellets (0.91 +/- 0.55 mm(2) and 0.77 +/- 0.34 mm(2), respectively; experiment III). The levels of bFGF and VEGF (VEGF, VEGF-B, and VEGF-C) in corneal epithelial cells were not elevated in matrilysin-deficient mice compared with the wild-type mice (experiment IV). CONCLUSIONS: Matrilysin may play an important role in maintaining corneal avascularity during wound healing. The differences in corneal neovascularization between matrilysin-deficient mice and wild-type littermates seem unrelated to the bFGF and VEGF levels in the corneal epithelium.     

Disruption of the basement membrane after corneal débridement

PURPOSE: To determine whether the native basement membrane left behind after manual débridement 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.     

Comparison of wound healing of linear incisions in the rabbit cornea produced by the newly developed excimer laser, a metal blade, and a diamond blade]

We developed a new delivery system for corneal ablation with the 193 nm argon-fluoride excimer laser. The laser was used to make linear incisions in the rabbit cornea, and wound healing of the incisions was compared with the healing of incisions made with metal and diamond blades. The morphology of incisions made with excimer laser radiation (193 nm) was compared with the morphology of incisions produced by diamond and metal blades, and the corneal wound was examined by light and electron microscopy. The surface of the cornea at the site of the laser wound was smooth; the laser wound was wider than the blade wounds immediately after surgery. Two weeks after surgery, all wounds had healed equally. One month after surgery, there were fewer fibroblastic keratocytes in the laser wound, and the epithelial layer at the wound site was thinner than in the case of the blade wounds. These results suggest that wound healing of laser incisions is more rapid than healing of wounds created with metal or diamond blades.     

Reassembly of the anchoring structures of the corneal epithelium during wound repair in the rabbit

Reappearance of the structures involved in adhesion of the corneal epithelium to the stroma was studied in healing 7 mm keratectomy wounds in rabbit corneas. Corneas were taken at 48 and 66 hr, 1, 2, 3, 4, 6 and 8 weeks, and 4, 6 and 12 months post-wounding. Immunolocalization of bullous pemphigoid antigen (BPA), laminin and type VII collagen was used to determine time and sequence of appearance of hemidesmosomes, basement membrane and anchoring fibrils, respectively. Electron micrographs from three regions in the wound were used to correlate the immunohistochemical data and to quantitate the increase in basal cell membrane occupied by hemidesmosomes and the increase in basement membrane over healing time. Evidence of resynthesis of the adhesion structures was present at the wound margin before epithelial wound closure (48 hr). BPA, laminin and type VII collagen co-localized, indicating that hemidesmosomes, basement membrane and anchoring fibrils reappeared synchronously. Reappearance of the structures proceeded from wound margin to the center, and by 1 week BPA, laminin, and type VII collagen were present in discontinuous segments across the wound. From 2 weeks to 6 months, the segments became more continuous, and anchoring fibril networks were discerned at 4 weeks. Strata of type VII collagen and laminin were present within the newly synthesized stromal matrix at wound margin at 1 week, continuous across the wound bed by 2-4 weeks, and still present at 6 months; however, at 12 months, only a few strata of type VII collagen were present below the basement membrane at wound center.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biomechanical and wound healing characteristics of corneas after excimer laser keratorefractive surgery: is there a difference between advanced surface ablation and sub-Bowman's keratomileusis?

PURPOSE: To describe the biomechanical and wound healing characteristics of corneas after excimer laser keratorefractive surgery. METHODS: Histologic, ultrastructural, and cohesive tensile strength evaluations were performed on 25 normal human corneal specimens, 206 uncomplicated LASIK specimens, 17 uncomplicated sub-Bowman's keratomileusis (SBK) specimens, 4 uncomplicated photorefractive keratectomy (PRK) specimens, 2 uncomplicated advanced surface ablation (ASA) specimens, 5 keratoconus specimens, 12 postoperative LASIK ectasia specimens, and 1 postoperative PRK ectasia specimen and compared to previously published studies. RESULTS: Histologic and ultrastructural studies of normal corneas showed significant differences in the direction of collagen fibrils and/or the degree of lamellar interweaving in Bowman's layer, the anterior third of the corneal stroma, the posterior two-thirds of the corneal stroma, and Descemet's membrane. Cohesive tensile strength testing directly supported these morphologic findings as the stronger, more rigid regions of the cornea were located anteriorly and peripherally. This suggests that PRK and ASA, and secondarily SBK, should be biomechanically safer than conventional LASIK with regard to risk for causing keratectasia after surgery. Because adult human corneal stromal wounds heal slowly and incompletely, all excimer laser keratorefractive surgical techniques still have some distinct disadvantages due to inadequate reparative wound healing. Despite reducing some of the risk for corneal haze compared to conventional PRK, ASA cases still can develop corneal haze or breakthrough haze from the hypercellular fibrotic stromal scarring. In contrast, similar to conventional LASIK, SBK still has the short- and long-term potential for interface wound complications from the hypocellular primitive stromal scar. CONCLUSIONS: Ophthalmic pathology and basic science research show that SBK and ASA are improvements in excimer laser keratorefractive surgery compared to conventional LASIK or PRK, particularly with regard to maintaining corneal biomechanics and perhaps moderately reducing the risk of corneal haze. However, most of the disadvantages caused by wound healing issues remain.     

Anterior stromal micropuncture electron microscopic changes in the rabbit cornea

Anterior stromal micropuncture has become an effective treatment for recurrent erosion. The healing process in rabbit corneas was investigated. Following micropuncture of the corneal surface with a 27-gauge needle knife, electron microscopy was carried out at regular intervals from time 0 through 5 months. The corneal incisions began to fill with epithelium by day 1. Activated keratocytes were adjacent to the basement membrane defect by 7 days. The basement membrane appeared to be healed at 2 and 4 weeks. Epithelial projections into the stromal incisions with underlying mature basement membrane persisted at 5 months postsurgery. Basement membrane reproduction occurred much more rapidly following needle puncture than after microdiathermy. This was thought to occur because the corneal epithelial cell was immediately exposed to type I collagen, whereas following microdiathermy, new type I collagen must be secreted on the necrotic collagen before the corneal epithelium will secrete basement membrane.     

A mouse model for the study of recurrent corneal epithelial erosions: alpha9beta1 integrin implicated in progression of the disease

PURPOSE: To describe an in vivo mouse model for the study of recurrent corneal erosion syndrome (RCES) in mice and to characterize the changes in alpha9 integrin expression during wound healing. METHODS: Corneal epithelial debridement wounds of two sizes (1.5 and 2.5 mm) were made on the ocular surface of BALB/c mice and were evaluated at various times after wounding. Corneas were processed either as whole mounts and stained with propidium iodide and an antibody against alpha9 integrin or for bromodeoxyuridine analyses of cell proliferation. A separate study involved analyses of corneal wound healing over time in individual mice with large and small debridement wounds. Mice were anesthetized once per week and their corneas stained with fluorescein to assess the quality of the corneal epithelium. After 6 weeks, mice were killed and eyes processed for study by immunofluorescence in either whole mounts or frozen sections. RESULTS: Whole mount confocal microscopy showed open wounds on the ocular surface of mice at 1 and 2 weeks after large wounds were created, but not after small wounds. In addition, alpha9 integrin was upregulated during healing, and changes were observed in alpha9 integrin localization at the limbus with large wounds but not with small wounds. Although only 1 of 16 corneas with small wounds had erosions at 1 and 2 weeks, 11 of 16 with large wounds had erosions. However, by 6 weeks, 13 of 16 eyes showed signs of erosion whether wounds were small or large. With large wounds, RCES corneas frequently showed numerous goblet cells adjacent to a limbus lacking alpha9 integrin. Corneas from mice with documented RCES showed both retention of alpha9 integrin and tenascin-C expression at the anterior stromal-epithelial interface as well as impaired relocalization of alpha3beta1 integrin to the basement membrane zone. CONCLUSIONS: These data show that spontaneous recurrent corneal erosions occur in a mouse model after manual creation of a single wound by debridement. Differences between the healing of small (1.5 mm) and large (2.5 mm) wounds were observed. Large wounds often resulted in the presence of goblet cells on the central cornea and a loss of alpha9 integrin at the limbus. Small wounds never showed differences in the localization of alpha9 integrin at the limbus, and no goblet cells were observed in the central cornea. More studies are needed to understand the causes of erosions in these mice.     

Expression of fibronectin isoforms bearing the alternatively spliced EIIIA, EIIIB, and V segments in corneal alkali burn and keratectomy wound models in the rat

PURPOSE: To better understand the healing process in the wounded cornea, fibronectin (FN) isoforms bearing the alternatively spliced EIIIA, EIIIB, and V segments (EIIIA+, EIIIB+, and V+ FNs) were evaluated in alkali burn and keratectomy wound models in the rat. METHODS: Alkali burn or keratectomy wounds (both 2 mm) were created, and corneas were harvested at various time points and analyzed by indirect immunofluorescence using antibodies specific for the EIIIA, EIIIB, and V segments as well as for the total pool of FN (total FN). RESULTS: There was minimal staining for any variety of FN in the epithelium or basement membrane zone (BMZ) in normal cornea, but each antibody produced granular staining in the stroma. Bright staining for V+ and total FNs was evident at the denuded stromal surface 1 day following keratectomy. In contrast, staining for EIIIA+ and EIIIB+ FNs was negligible at 24 hours but appeared on the wound surface under the migrating unstained epithelium by the second day. BMZ staining for FN then gradually subsided, such that there was little or no staining by 6 weeks. In contrast, alkali burn wounds exhibited very little BMZ staining throughout the time course. Although there was preferential staining of the anterior aspect of Descemet membrane by anti-EIIIA and anti-EIIIB antibodies under normal conditions, the staining intensity of the anterior and posterior aspects became similar following corneal wounding. CONCLUSION: Deposition of EIIIA+ and EIIIB+ FNs in the BMZ of the keratectomy wound occurs more slowly than deposition of V+ and total FNs. EIIIA+ FN is expressed in a distribution that overlaps with that previously described for the alpha 9 integrin subunit following corneal debridement, suggesting that EIIIA-alpha 9 interactions could occur during corneal wound healing. In contrast, the relative lack of FN deposition in alkali burn wounds suggests that proteolytic degradation of FN may occur; and this, along with impairment of new FN synthesis because of cellular damage, could play a role in the high prevalence of recurrent epithelial erosions in alkali-wounded corneas.     

Effect of wound type on Smad 2 and 4 translocation

PURPOSE: In a prior study, it was reported that both TGF-beta receptors type-I and -II are upregulated after wounding, suggesting that TGF-beta signaling may play a role in corneal epithelial repair. The Smad proteins, which translocate into the nucleus after activation of the TGF-beta receptors, are key factors in the major TGF-beta signaling pathway. The present study was undertaken to examine whether Smads 2 and 4 translocate into the nucleus during wound repair and whether the wound type affects the extent of translocation. METHODS: Either a 3-mm superficial keratectomy or epithelial debridement was performed on adult Sprague-Dawley rats. The eyes were allowed to heal from 4 hours to 2 weeks. Indirect immunofluorescence was performed with anti-Smads 2 and 4, anti-laminin, a marker of basement membrane, and anti-alphavbeta6 integrin, which has been implicated in TGF-beta activation. In addition, the effect of the p38MAPK inhibitor SB202190 on healing rates of debridement and keratectomy wounds was determined in organ culture. RESULTS: In unwounded tissue, Smad 2 was cytoplasmic. By 4 hours after keratectomy, nuclear localization was visible in a few epithelial basal cells at the leading edge of the wound. The number of basal cells expressing nuclear Smad 2 in the wound area increased with time, peaking at 48 hours (95%). However, in the debridement model, Smad 2 localization remained primarily cytoplasmic. Smad 4 showed similar localization. In both wound models, p38MAPK inhibitor slowed epithelial migration, and alphavbeta6 integrin appeared to be upregulated with localization primarily observed in the basal cells migrating over the wound area. CONCLUSIONS: The presence of the basement membrane appears to have an effect on the extent and duration of translocation of the Smad 2 and 4 proteins during corneal epithelial wound repair. The Smad pathway does not appear to be essential for migration; rather, it may play a role in resynthesis of the basement membrane.     

Role of fibronectin in the healing of superficial keratectomies in vitro

A fibronectin (Fn)-fibrinogen (Fg) surface matrix is not essential for epithelial cell migration in a corneal epithelial scrape wound model, in which the basement membrane is preserved. We have therefore tested whether such a provisional scaffolding becomes more critical in a superficial keratectomy model, when the basement membrane is surgically removed. Exogeneous Fn at 0.3 mg/ml was added to the medium of organ cultures of rabbit superficial keratectomies. At 48 hr after wounding, the healing rate was 1.12 +/- 0.03 mm2/hr in control corneas and 1.11 +/- 0.03 mm2/hr in those cultured with Fn. At 64 hr after wounding, the healing rates were also not significantly different (P greater than 0.5). Immunofluorescence studies showed that Fn was not detectable on the surface of control corneas but could be depicted under the migrating epithelium. In corneas cultured with Fn, it diffused throughout the entire stroma but did not deposit as a surface matrix. We therefore attempted to obtain formation of a provisional Fn-Fg surface matrix before establishment of the in vitro organ culture by leaving the superficial keratectomies in vivo for 8 hr, 24 hr or 64 hr. At 64 hr after wounding, their healing rate was 0.80 +/- 0.04 mm2/hr, 0.86 +/- 0.04 mm2/hr and 0.85 +/- 0.06 mm2/hr, respectively, which were not significantly different from that of contralateral ex vivo-wounded cultured corneas (0.83 +/- 0.04 mm2/hr, P greater than 0.5). Immunofluorescence studies revealed a Fn matrix on the bare surface of in vivo-wounded specimen, which was not detectable on ex vivo-wounded cultured corneas; there was also no diffuse stromal Fn.(ABSTRACT TRUNCATED AT 250 WORDS)     

The contribution of blood flow by the anterior ciliary arteries to the anterior segment in the primate eye

The rate and mode of corneal wound healing in severely diabetic rats were studied by light microscopy and scanning electron microscopy. Diabetes mellitus was induced in 52 rats by alloxan injection, and 52 nondiabetic rats were used as controls. After 3 weeks, a nonpenetrating razor-blade wound was made in the central cornea of both eyes in 48 diabetic and 48 normal rats. The incision passed through the epithelium and into the stroma. The effects of diabetes on the unwounded cornea were observed by comparison with corneas from eight unwounded rats (four diabetic and four normal). Whole corneas from wounded diabetic and normal rats were studied at 0, 1, 3, 6, 12 and 24 hr and at 2–7 days after wounding. The rate and mode of healing were not found to differ between diabetics and normals. The surfaces of corneal wounds in both groups appeared to be completely healed and indistinguishable from the surrounding unwounded epithelium after 24 hr. The epithelial cells involved in the initial healing process were derived primarily from the layer of wing cells which progressed across the wound close to the connective-tissue base. Only in the final stages of healing, after the wound had been filled by the deeper epithelial cells, did superficial epithelial cells migrate. There appeared to be more exfoliating superficial epithelial cells over the entire cornea in diabetic rats than in normals. Because the healing of central corneal incisions occurs initially and primarily by sliding of the deeper epithelial cells, and because the diabetic condition appears to be associated with increased exfoliation of surface cells, the healing of central incisions may be less affected by diabetes than the healing of defects of the whole corneal surface, where the superficial epithelial cells have been reported to be the main migratory cells in the initial healing process and where healing in diabetics is delayed.     

角膜上皮细胞基底膜的研究进展

角膜上皮细胞基底膜是一层很薄的组织,它的结构与其他部位的基底膜既有相似之处,也有独特的地方,在角膜创伤修复中有着重要的作用.基底膜组织的破坏会导致一系列棘手的角膜病变,而近来对角膜上皮细胞基底膜的研究使我们在这些病变的治疗方面有了新的人手点.就近年来角膜上皮细胞基底膜的研究进展做一综述.     

Connective tissue growth factor expression and action in human corneal fibroblast cultures and rat corneas after photorefractive keratectomy

PURPOSE: Connective tissue growth factor (CTGF) has been linked to fibrosis in several tissues. In this study, the interactions between CTGF and transforming growth factor (TGF)-beta were assessed in human corneal fibroblasts, and the levels and location of CTGF protein and mRNA were measured during healing of excimer laser ablation wounds in rat corneas. METHODS: Human corneal fibroblasts were incubated with TGF-beta1, -beta2, and -beta3 isoforms, and CTGF mRNA and protein were measured. CTGF was immunolocalized in the cultured fibroblasts by using a specific antibody. Regulation of collagen synthesis by TGF-beta and CTGF was assessed in human corneal fibroblasts with a neutralizing antibody and an antisense oligonucleotide to CTGF. CTGF mRNA and protein were measured in rat corneas up to day 21 after excimer ablation of the cornea. CTGF protein was immunolocalized in rat corneas after photorefractive keratectomy (PRK), and the presence of CTGF mRNA and protein in ex vivo rat corneal scrapings was established. RESULTS: All three TGF-beta isoforms stimulated expression of CTGF in human corneal fibroblasts, and CTGF was immunolocalized in the cells. Both TGF-beta and CTGF increased collagen synthesis in corneal fibroblasts. Furthermore, CTGF antibody or antisense oligonucleotide blocked TGF-beta-stimulated collagen synthesis. CTGF protein and mRNA increased in rat corneas through day 21 after PRK. CTGF expression was also detected in ex vivo scrapings of rat corneas. CONCLUSIONS: These data demonstrate that CTGF is expressed by corneal cells after stimulation by TGF-beta, that CTGF expression increases significantly during corneal wound healing, and that CTGF mediates the effects of TGF-beta induction of collagen synthesis by corneal fibroblasts. These data support the hypothesis that CTGF promotes corneal scar formation and imply that regulating CTGF synthesis and action may be an important goal for reducing corneal scarring.     

Localization of thrombospondin-1 and myofibroblasts during corneal wound repair

Thrombospondin-1 (TSP-1) is a multifunctional matrix protein that has recently been examined in various wound processes, primarily for its ability to activate the latent complex of transforming growth factor-beta (TGF-β). TGF-β has been shown to play a major role in stimulating mesenchymal cells to synthesize extracellular matrix. After injury, corneal keratocytes become activated and transform into fibroblasts and myofibroblasts. Our hypothesis is that TSP-1 regulates the transformation of keratocytes into myofibroblasts (MF) via TGF-β. In the current study, we examined the expression of TSP-1 and α-smooth muscle actin (SMA), a marker of MF, during rat corneal wound healing. Three-mm keratectomy or debridement wounds were made in the central rat cornea and allowed to heal from 8 hours to 8 weeks in vivo. Unwounded rat corneas served as controls. Expression of TSP-1, SMA and Ki67, a marker of proliferating cells, were examined by indirect-immunofluorescence microscopy. In unwounded corneas, TSP-1 expression was observed primarily in the endothelium. No expression was seen in the stroma, and only low levels were detected in the epithelium. Ki67 was localized in the epithelial basal cells and no SMA was present in the central cornea of unwounded eyes. After keratectomy wounds, TSP-1 expression was seen 24 h after wounding in the stroma immediately subjacent to the wound-healing epithelium. The expression of TSP-1 increased daily and peaked 7–8 days after wounding. SMA expression, however, was not observed until 3–4 days after wounding. Interestingly, SMA-positive cells were almost exclusively seen in the stromal zone expressing TSP-1. Peak expression of SMA-positive cells was observed 7–8 days after wounding. Ki67-expressing cells were seen both in the area expressing TSP-1 and the adjacent area. In the debridement wounds, no SMA expressing cells were observed at any time point. TSP-1 was localized in the basement membrane zone from 2 to 5 days after wounding, and the localization did not appear to penetrate into the stroma. These data are in agreement with our hypothesis that TSP-1 localization in the stromal matrix is involved in the transformation of keratocytes into myofibroblasts.     

Expression of HGF, KGF, EGF and receptor messenger RNAs following corneal epithelial wounding

Hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), epidermal growth factor (EGF), and their receptors have been associated with homeostasis and wound healing in the cornea. The purpose of this study was to examine the expression of the messenger RNAs for these growth factors and receptors in a wounded series of mouse corneas using in situ hybridization. In situ hybridization was performed with 3H-labeled riboprobes on unwounded corneas and corneas at 30 minutes, 4, 12, 24, 48 and 72 hr, and 7 days after epithelial scrape wounds in Balb/C mice. Qualitative and semi-quantitative analyses were performed. Expression of HGF, KGF and EGF mRNAs in keratocytes in the unwounded cornea was low. EGF mRNA was also expressed in unwounded corneal epithelium. Following wounding, however, these growth factor mRNAs were markedly upregulated in keratocytes. EGF mRNA expression in the epithelium appeared unaffected by wounding. At seven days after wounding and several days following closure of the epithelial defect, HGF mRNA and KGF mRNA were still expressed at higher levels in keratocytes compared with unwounded corneas. No difference in expression of HGF or KGF mRNAs between limbal, peripheral corneal, or central corneal keratocytes was noted in the unwounded cornea, KGF receptor mRNA was prominently expressed throughout the unwounded corneal epithelium. HGF receptor mRNA and EGF receptor mRNAs were expressed at low levels in unwounded cornea epithelium. Following scrape injury, expression of HGF receptor mRNA and KGF receptor mRNA were markedly upregulated in the corneal epithelium, while no significant increase in EGF receptor mRNA expression was noted. These studies suggest a prominent role for HGF and KGF in modulating corneal epithelial wound healing following injury. Less prominent changes in EGF mRNA and EGF receptor mRNA in the corneal epithelium following wounding may suggest that EGF has more of a role in homeostasis in the mouse corneal epithelium.     

Healing response of incised pig corneas in subcutaneously implanted diffusion chambers

Incised corneas were implanted subcutaneously within diffusion chambers to determine the healing response of resident corneal cells to injury in the absence of a cellular contribution from the normally-occurring inflammatory reaction. Viable corneas were recovered up to 15 days after implantation. By day 2 the epithelium had migrated over the incisions, penetrating them where the wound edges were nonapposed, and over the cut outer edge on to the millipore membrane. The normal epithelial configuration was progressively lost, with regression from the upper regions of incisions, until it was reduced by day 10 to an extremely flattened squamosal type, one or two cells thick. A zone of stromal paraincisional a cellularity first observed on day 1 largely persisted although it became progressively repopulated with cells. The endothelial cells quickly died and there was no healing of Descemet's membrane. While many of the stromal cells appeared pyknotic from days 4–8, those corneas biopsied on days 10 and 15 were healthy. Fibroblast-like cells and mitotic figures were present adjacent to, and within, incisions; in some cases associated with argyrophilic fibres and apparent collagen fibre reunion. Fibroblastic outgrowths on to the millipore membrane contained new collagen fibres. This limited stromal response is considered in relation to previous accounts of in vitro cultured corneas and the possible contribution to in situ corneal healing by inflammatory cells.     

Proteoglycans of rabbit corneas with nonperforating wounds

Rabbit corneal proteoglycans were labeled by intrastromal injection of 3H-glucosamine and 35S-sulfate 1 and 2 weeks after partial-thickness radial scalpel incisions. Proteoglycans were extracted with guanidine-HCl and purified by ion exchange chromatography. Wounding caused a marked decrease in the total incorporation of labeled precursors into proteoglycans. The labeled proteoglycans were more readily extracted with guanidine-HCl after wounding. Labeled proteoglycans from wounded corneas had a larger molecular size on gel filtration chromatography than did proteoglycans from control corneas, a result of an increased amount of keratan sulfate in the large molecular size fractions. Analysis of labeled glycosaminoglycan (GAG) from guanidine-extracted proteoglycans and from the corneal tissue after guanidine-HCl extraction showed an increase in the relative amount of heparan sulfate and keratan sulfate after wounding, and a decrease in relative amount of dermatan sulfate. The 35S:3H ratio of heparan and dermatan sulfates increased after wounding, and that of keratan sulfate decreased, suggesting changes in sulfation. Degradation of labeled dermatan sulfate with hyaluronidase and with periodate revealed a 2-fold increase in iduronic acid content and 2-4-fold increase in hyaluronidase-resistant dermatan sulfate in the wounded corneas. Reduction in proteoglycan content, reduced sulfation of keratan sulfate, and accumulation of a high-sulfate, high-iduronic acid dermatan sulfate are previously reported properties of proteoglycan in scar tissue from perforating corneal wounds. Demonstration of these properties in proteoglycan after wounds similar to radial keratotomy incisions suggests that deposition of scar tissue can result from wounds which do not damage Descemet's membrane.     

Corneal wound-associated glycoconjugates analyzed by lectin histochemistry.

PURPOSE. To examine changes in corneal glycoconjugates during wound healing in a pig model of refractive surgery. METHODS. Pig corneas were wounded using a mechanized microkeratome. Lamellar keratectomy, without replacement of a corneal cap, or automated lamellar keratoplasty, with replacement of a corneal cap, was performed. Corneas were prepared for microscopy 1, 7, or 28 days after surgery. Cryosections of corneas were probed with lectins recognizing a variety of carbohydrate moieties. Glycoconjugates were characterized by glycosidase digestion and chemical extraction. RESULTS. Of the lectins used, only that from Dolichus biflorus (DBA), specific for terminal N-acetylgalactosamine, exhibited a dramatic change in staining pattern after wounding. In unwounded corneas, DBA labeled the superficial epithelium, Bowman's layer, and occasional keratocytes. After wounding, DBA intensely labeled exposed stromal surfaces and keratocytes near wound sites. RESULTS of glycosidase treatments indicated that the DBA-binding material contains alpha-linked N-acetylgalactosamine. After extraction with lipid solvents, stromal DBA labeling was unchanged, while that of keratocytes was greatly diminished. CONCLUSIONS. Corneal wounding results in the accumulation of specific glycoconjugates at surfaces and in keratocytes. After keratectomy, the exposed stromal surface is infiltrated with ocular surface components, including DBA-binding glycoproteins. The presence of these components may influence the course of wound healing. In addition, changes in keratocyte glycoconjugates may accompany their mobilization to the wound-repair phenotype.