The molecular basis of corneal transparency

The cornea consists primarily of three layers: an outer layer containing an epithelium, a middle stromal layer consisting of a collagen-rich extracellular matrix (ECM) interspersed with keratocytes and an inner layer of endothelial cells. The stroma consists of dense, regularly packed collagen fibrils arranged as orthogonal layers or lamellae. The corneal stroma is unique in having a homogeneous distribution of small diameter 25-30 nm fibrils that are regularly packed within lamellae and this arrangement minimizes light scattering permitting transparency. The ECM of the corneal stroma consists primarily of collagen type I with lesser amounts of collagen type V and four proteoglycans: three with keratan sufate chains; lumican, keratocan, osteoglycin and one with a chondroitin sulfate chain; decorin. It is the core proteins of these proteoglycans and collagen type V that regulate the growth of collagen fibrils. The overall size of the proteoglycans are small enough to fit in the spaces between the collagen fibrils and regulate their spacing. The stroma is formed during development by neural crest cells that migrate into the space between the corneal epithelium and corneal endothelium and become keratoblasts. The keratoblasts proliferate and synthesize high levels of hyaluronan to form an embryonic corneal stroma ECM. The keratoblasts differentiate into keratocytes which synthesize high levels of collagens and keratan sulfate proteoglycans that replace the hyaluronan/water-rich ECM with the densely packed collagen fibril-type ECM seen in transparent adult corneas. When an incisional wound through the epithelium into stroma occurs the keratocytes become hypercellular myofibroblasts. These can later become wound fibroblasts, which provides continued transparency or become myofibroblasts that produce a disorganized ECM resulting in corneal opacity. The growth factors IGF-I/II are likely responsible for the formation of the well organized ECM associated with transparency produced by keratocytes during development and by the wound fibroblast during repair. In contrast, TGF-β would cause the formation of the myofibroblast that produces corneal scaring. Thus, the growth factor mediated synthesis of several different collagen types and the core proteins of several different leucine-rich type proteoglycans as well as posttranslational modifications of the collagens and the proteoglycans are required to produce collagen fibrils with the size and spacing needed for corneal stromal transparency.     

Morphological characteristics and intercellular connections of corneal keratocytes

PURPOSE: To investigate the morphological characteristics of keratocytes and the interconnection of keratocytes with adjacent keratocytes using the flat preparation method and scanning electron microscopy with a frontal section of the human corneal stroma. METHODS: The thin, corneal collagen lamellae were carefully dissected from the cornea (n=7), which had been stained by the flat preparation method. The remaining tissue was fixed in 3% glutaraldehyde and observed by transmission electron microscopy following the frontal section. RESULTS: The flat preparation revealed the corneal fibroblasts between the lamellae of the collagen fibers and showed that the ramifying cellular processes of the keratocytes were in contact with the cytoplasmic processes or cell bodies of neighboring fibroblasts. Two types of discrete subpopulations of keratocytes were identified: a smaller, cellular type of keratocyte with spindle-shaped nucleus with heterochromatin, and a larger, cellular type with a large indented nucleus with relatively scanty cytoplasm. Collagen fibers ran parallel to each other toward the fenestration of the cytoplasmic wall of the keratocyte. CONCLUSIONS: These flat preparation method results showed that the keratocytes within the corneal stroma are interconnected with the adjacent keratocytes, which indicates the presence of a functional communicating network through the keratocyte circuits within the stroma. A smaller, cellular type of keratocyte with spindle-shaped nucleus was morphologically differentiated from a larger, cellular type with a large, indented nucleus by flat preparation and transmission electron microscopy.     

Influence of cell and collagen concentration on the cell-matrix mechanical relationship in a corneal stroma wound healing model

The effect of different collagen and cell concentrations on the mechanical and remodeling behaviors of corneal stroma wound healing models consisting of collagen hydrogels seeded with human corneal fibroblasts during a 25 day culture period were examined. Human corneal fibroblasts were seeded at 1 × 10⁵, 3 × 10⁵ or 5 × 10⁵ cells per hydrogel, and collagen concentrations of 2.5 mg/ml, 3.5 mg/ml or 4.5 mg/ml were examined. Two non-destructive techniques, spherical indentation and optical coherence tomography, were used to measure the elastic modulus and dimensional changes respectively at several time-points over the culture period. The elastic modulus of the hydrogels increased continuously over 25 days. Hydrogels with higher initial cell seeding densities and lower initial collagen concentrations were found to increase in elastic modulus faster and possessed a higher elastic modulus by the end of the culture period when compared to the other hydrogels. A mathematical equation was applied to accurately fit the change in elastic modulus over time. This study demonstrates a robust in vitro technique able to monitor the effect of different parameters on the cell-matrix mechanical relationship in a corneal stroma model during prolonged culture periods and enhances our understanding on corneal wound healing processes.     

Microtubule regulation of corneal fibroblast morphology and mechanical activity in 3-D culture

The purpose of this study was to investigate the role of microtubules in regulating corneal fibroblast structure and mechanical behavior using static (3-D) and dynamic (4-D) imaging of both cells and their surrounding matrix. Human corneal fibroblasts transfected to express GFP-zyxin (to label focal adhesions) or GFP-tubulin (to label microtubules) were plated at low density inside 100 microm thick type I collagen matrices. After 24h, the effects of nocodazole (to depolymerize microtubules), cytochalasin D (to disrupt f-actin), and/or Y-27632 (to block Rho-kinase) were evaluated using 3-D and 4-D imaging of both cells and ECM. After 24h of incubation, cells had well organized microtubules and prominent focal adhesions, and significant cell-induced matrix compaction was observed. Addition of nocodazole induced rapid microtubule disruption which resulted in Rho activation and additional cellular contraction. The matrix was pulled inward by retracting pseudopodial processes, and focal adhesions appeared to mediate this process. Following 24h exposure to nocodazole, there was an even greater increase in both the number of stress fibers and the amount of matrix compaction and alignment at the ends of cells. When Rho-kinase was inhibited, disruption of microtubules resulted in retraction of dendritic cell processes, and rapid formation and extension of lamellipodial processes at random locations along the cell body, eventually leading to a convoluted, disorganized cell shape. These data suggest that microtubules modulate both cellular contractility and local collagen matrix reorganization via regulation of Rho/Rho-kinase activity. In addition, microtubules appear to play a central role in dynamic regulation of cell spreading mechanics, morphology and polarity in 3-D culture.     

Increased stromal extracellular matrix synthesis and assembly by insulin activated bovine keratocytes cultured under agarose

Previously, pharmacological levels of insulin have been shown to stimulate the synthesis of normal corneal stromal collagen and proteoglycans by bovine keratocytes in culture. Here we compared insulin to physiological levels of IGF-I and found that IGF-I also stimulated the synthesis of these extracellular matrix components, but less than that of insulin. Keratocytes in monolayer culture secreted most of the collagen synthesized into the media in the form of procollagen, a precursor of collagen. We found that an overlay of 3% agarose on the keratocytes in culture enhanced the conversion of procollagen to collagen and increased the deposition of collagen and proteoglycans into the cell layer. The extracellular matrix associated with the keratocytes cultured under agarose exhibited a corneal stromal-like architecture. These results suggest that enhancing the conversion of procollagen to collagen is a key step in the formation of extracellular matrix by keratocytes in vitro. Agarose overlay of insulin activated keratocytes in culture is a useful model for studying corneal stromal extracellular matrix assembly in vitro.     

Increased stromal extracellular matrix synthesis and assembly by insulin activated bovine keratocytes cultured under agarose

Previously, pharmacological levels of insulin have been shown to stimulate the synthesis of normal corneal stromal collagen and proteoglycans by bovine keratocytes in culture. Here we compared insulin to physiological levels of IGF-I and found that IGF-I also stimulated the synthesis of these extracellular matrix components, but less than that of insulin. Keratocytes in monolayer culture secreted most of the collagen synthesized into the media in the form of procollagen, a precursor of collagen. We found that an overlay of 3% agarose on the keratocytes in culture enhanced the conversion of procollagen to collagen and increased the deposition of collagen and proteoglycans into the cell layer. The extracellular matrix associated with the keratocytes cultured under agarose exhibited a corneal stromal-like architecture. These results suggest that enhancing the conversion of procollagen to collagen is a key step in the formation of extracellular matrix by keratocytes in vitro. Agarose overlay of insulin activated keratocytes in culture is a useful model for studying corneal stromal extracellular matrix assembly in vitro.     

Effects of pterygium surgery on front and back corneal surfaces and anterior segment parameters

The purpose of this study was to evaluate the effects of pterygium surgery on front and back corneal surfaces and anterior segment parameters. This prospective study included 96 eyes with primary pterygium that underwent surgery. Preoperatively and at 1, 3, and 6 months postoperatively, Pentacam was used to evaluate front and back corneal surfaces, anterior chamber depth (ACD), anterior chamber angle (ACA) and anterior chamber volume (ACV). Mean simulated keratometry at the front corneal surface increased from 42.73 ± 2.21 D preoperatively to 44.45 ± 2.05 D at 1 month (P < 0.001); it then decreased to 44.32 ± 2.07 D at 3 months (P < 0.001) and 44.19 ± 2.10 D at 6 months (P = 0.01). There was no statistically significant change in mean simulated keratometry at the back corneal surface. Radius of the front corneal best-fit sphere (BFS) decreased from 7.99 ± 0.29 mm preoperatively to 7.77 ± 0.25 mm at 1 month postoperatively (P < 0.001), without further change up to 6 months. Radius of the back corneal BFS increased from 6.42 ± 0.24 mm preoperatively to 6.50 ± 0.24 mm at 1 month (P < 0.001), without further change. Postoperative changes in mean simulated keratometry and radii of BFS had statistically significant positive correlations with pterygium extension onto the cornea and grade of pterygium morphology but, not with the surgical technique. There were no significant changes in ACD, ACA, and ACV values after pterygium surgery. Furthermore, the spherical equivalent of manifest refraction changed from +0.75 ± 1.06 D preoperatively to −0.72 ± 1.33 D at 1 month postoperatively (P = 0.001), with no further significant change. In conclusion, after pterygium surgery there were significant changes in front mean keratometry and front and back corneal radii of BFS. These were correlated with preoperative pterygium size and morphology grade. No significant changes in anterior segment parameters were noted postoperatively.     

Capillaries in the epithelium of pterygium

AIM—To present new morphological observations of intraepithelial capillaries in pterygium and to provide some explanations for this phenomenon.
METHODS—The ultrastructural features of pterygia from 26 patients were examined. Surgically excised tissue was processed for conventional light and transmission electron microscopy.
RESULTS—Individual capillaries within the epithelium of the anterior half towards the head of pterygia were identified in 11 specimens out of 26 pterygia examined (42.3%). The perivascular connective tissue of the intraepithelial capillaries contained fibroblasts, collagen fibrils, and elastin-like material. Epithelial cells surrounding these capillaries showed defects in the basal lamina in contrast with the continuous basal lamina of the endothelium. In the intercellular space of the epithelium an amorphous substance, occasional fibroblast processes, and collagen fibrils were frequently observed.
CONCLUSION—Capillaries in the epithelium of pterygia are rare, but not exceptional. The ingrowth of these vessels from the stroma into the epithelium can be interpreted as a reaction to hypoxia or deficiency of any other substance transported via the bloodstream. Apparently, the perivascular connective tissue can be used by ingrowing fibroblasts as a migration pathway. The migrating fibroblasts appear to use the defects of the epithelial basal lamina (whether partially or complete) in order to reach the intercellular space. It is possible that collagen fibrils in the epithelial intercellular space have been laid down by fibroblasts which contribute to the pathological dedifferentiation of the conjunctival epithelium.

Keywords: pterygium; ultrastructure; epithelium; blood vessels     

Long-term storage of endocytosed latex beads in keratocytes in vivo.

Endocytosis by keratocytes (corneal fibroblasts) is an important part of the host defense system. To investigate the long-term fate of endocytosed materials, we injected polystyrene latex beads into the corneal stroma of four rabbits. The corneal stroma was observed under a transmission electron microscope 4 and 800 days after the injection. After 4 days, the beads were found not only between the collagen fibers of the stroma, but also in some keratocytes. After 800 days, no extracellular beads were seen, but endocytosed beads remained, surrounded by limiting membranes, in the cytoplasm of keratocytes. These observations demonstrate that keratocytes endocytose latex beads and store them for a long time, isolating these foreign materials from the corneal stroma. These observations suggest that keratocytes, like some other fibroblasts perform a noninflammatory and nonimmunological defense function.     

Morphologic characterization of organized extracellular matrix deposition by ascorbic acid-stimulated human corneal fibroblasts

PURPOSE: To characterize the structure and morphology of extracellular matrix (ECM) synthesized by untransformed, cultured human corneal fibroblasts in long-term cultures. METHODS: Human corneal stromal keratocytes were expanded in transwell culture in the presence of fetal bovine serum and a stable derivative of vitamin C. The cells were allowed to synthesize a fibrillar ECM for up to 5 weeks. Constructs were assessed by light (phase-contrast and differential interference-contrast) and transmission (standard and quick freeze/deep etch) microscopy. RESULTS: Electron micrographs revealed stratified constructs with multiple parallel layers of cells and an extracellular matrix comprising parallel arrays of small, polydisperse fibrils (27-51 nm) that often alternate in direction. Differential interference contrast images demonstrated oriented ECM fibril arrays parallel to the plane of the construct, whereas quick-freeze, deep-etch micrographs showed the details of the matrix interaction with fibroblasts through arrays of membrane surface structures. CONCLUSIONS: Human keratocytes, cultured in a stable vitamin C derivative, are capable of assembling extracellular matrix, which comprises parallel arrays of ECM fibrils. The resultant constructs, which are highly cellular, are morphologically similar to the developing mammalian stroma, where organized matrix is derived. The appearance of arrays of structures on the cell membranes suggests a role in the local organization of synthesized ECM. This model could provide critical insight into the fundamental processes that govern the genesis of organized connective tissues such as the cornea and may provide a scaffolding suitable for tissue engineering a biomimetic stroma.     

Corneal nerve fiber loss in diabetes with chronic kidney disease

AimsPatients with chronic kidney disease (CKD) in type 2 diabetes typically manifest with severe peripheral neuropathy. Corneal confocal microscopy is a novel technique that may serve as a marker of nerve injury in peripheral neuropathy. This study examines the changes that occur in corneal nerve morphology as a result of peripheral neuropathy due to renal dysfunction in people with type 2 diabetes.MethodsSixty-two participants (mean age, 62 ± 12 years) with type 2 diabetes and 25 age-matched healthy controls underwent a comprehensive assessment of neuropathy using the total neuropathy score (TNS). The corneal sub-basal nerve plexus was imaged using corneal confocal microscopy. Corneal nerve fiber length, fiber density, branch density, total branch density, nerve fractal dimension, inferior whorl length and inferior whorl nerve fractal dimension were quantified. Based on the eGFR, participants were classified into those with diabetic CKD (eGFR < 60; n = 22) and those without CKD (eGFR ≥ 60; n = 40).ResultsParticipants with diabetic CKD had significantly lower corneal nerve fiber density (P = 0.037), length (P = 0.036) and nerve fractal dimension (P = 0.036) compared to those without CKD. Multiple linear regression analysis revealed that reduced corneal nerve fiber density (ß coefficient = 0.098, P = 0.017), length (ß coefficient = 0.006, P = 0.008) and nerve fractal dimension (ß coefficient = 0.001, P = 0.007) was associated with low eGFR levels when adjusted for age, duration of diabetes and severity of neuropathy.ConclusionCorneal confocal microscopy detects corneal nerve loss in patients with diabetic CKD and reduction in corneal nerve parameters is associated with the decline of kidney function.     

Immunohistochemical study of localization of extracellular matrix after holmium YAG laser irradiation in rat cornea

PURPOSE: To better understand the corneal responses to holmium YAG (Ho:YAG) laser irradiation, we used immunofluorescent microscopy to examine changes in the localization of the extracellular matrix components, which play important roles in the maintenance of corneal morphology and functions. METHODS: Rats were irradiated with a Ho:YAG laser. On days 1, 3, and 7 after irradiation, the eyes were enucleated and frozen. Cryosections were made with a cryostat and were stained with antibodies against type I collagen, fibronectin, type IV collagen, or laminin for immunohistochemical study. RESULTS: One day after Ho:YAG laser irradiation, contraction of the stromal collagen fibrils was observed. Keratocytes could not be observed at the irradiated stromal region on day 1 after irradiation. One week later, however, keratocytes returned to the irradiated area. Although the stromal collagen fibrils had contracted, they were stained by an antibody against type I collagen. Dense fluorescence for fibronectin was observed at the margin of the stromal acellular zone. Both laminin and type IV collagen were observed at the basement membrane under the corneal epithelium, regardless of whether or not the corneas had been irradiated. CONCLUSION: These results suggest that Ho:YAG laser irradiation might be useful for the collagen contraction of stroma, without causing serious damage to the corneal epithelium and the basement membrane.     

Neutrophil interactions with keratocytes during corneal epithelial wound healing: a role for CD18 integrins

PURPOSE: To determine the role of keratocytes and leukocyte beta(2) (CD18) integrins in neutrophil (PMN) migration through the corneal stroma after epithelial scrape injury. METHODS: Using C57BL/6 wild-type and CD18(-/-) mice, corneas were excised at 6 hours (wild-type) or 24 hours (CD18(-/-)) after central corneal epithelial abrasion, time points determined previously to have similar levels of emigrated PMNs. Corneas were prepared for ultrastructural morphometric analysis of PMNs, keratocyte networks, and collagen. RESULTS: Transmission electron microscopy revealed intact keratocyte networks within the paralimbus that were morphometrically similar, regardless of epithelial injury or mouse genotype. Secondary to epithelial abrasion, extravasated PMNs within the paralimbus developed close contacts with keratocytes and collagen. In wild-type mice, 40% of the PMN surface was in contact with the keratocyte surface, and this value decreased to 10% in CD18(-/-) mice. PMN contact with collagen was similar in wild-type and CD18(-/-) mice, with approximately 50% of the PMN surface contacting the collagen fibrils. Since corneal edema resulting from scrape injury was similar, regardless of genotype and did not involve structural changes in collagen fibrils, these data favor a direct role for CD18 in mediating PMN contact with keratocytes. CONCLUSIONS: The data show that in response to epithelial scrape injury, PMN migration in the corneal stroma involves close contact between keratocytes and collagen. Although PMN-keratocyte contacts require CD18 integrins, contact with collagen is CD18 independent. Fundamentally, PMN migration along keratocyte networks constitutes the beginning of a new experimental concept for understanding leukocyte migration within the wounded cornea.     

Protein expression pattern of collagen type XV in mouse cornea

Purpose To examine the alteration of protein expression pattern of collagen type XV in cornea during embryonic development and adult tissue repair. Collagen type XV is a basement membrane collagen of a subfamily of multiplexins (multiple triple helix domains and interruptions). Its COOH-terminal peptide has an anti-angiogenic effect and its distribution in avascular tissue of cornea is of interest.Methods Eyes of mouse embryos [day (E) 10.5–18.5] and healing adult mouse corneas following either débridement injury or incision were embedded in paraffin. Deparaffinized sections were processed for immunofluorescent staining with anti-collagen XV antibody.Results At E14.5 embryonic corneal epithelium, as well as fibroblasts in eyelids, began to express this collagen type very faintly, and at E18.5, besides corneal epithelial expression, epidermis, palpebral conjunctiva, and keratocytes started to express collagen type XV. In adult mouse cornea, collagen type XV was observed in basal and suprabasal epithelial cells and stroma, but not in the subepithelial basement membrane. Healing epithelial cells following débridement or incision injury down-regulated its protein expression.Conclusions Mouse embryonic corneal epithelium and keratocytes begin to express collagen type XV before birth. Healing murine corneal epithelium down-regulates collagen XV expression. The presence of collagen XV in corneal stroma may play a part in avascularity.     

Glial cell-derived neurotrophic factor gene delivery enhances survival of human corneal epithelium in culture and the overexpression of GDNF in bioengineered constructs

This paper evaluates the effects of adenoviral vector-mediated glial cell-derived neurotrophic factor (GDNF) gene delivery on survival of primary human corneal epithelial cells (PHCEC) established from limbal explants in vitro and the overexpression of GDNF gene in bioengineered human corneal constructs on substrate of corneal stromal discs followed by autograft ex vivo. In vitro, the overexpression of GDNF in the supernatant of PHCEC peaked at day 4, but lasted for at least 4 weeks after the transduction mediated by adenoviral vector. At day 10, the cell viability was 2-fold greater (P < 0.001), the number of terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL)-positive cells was more than 50% lower (P < 0.01) in the GDNF transduction group than the non-transduction group. 5 weeks after the transduction, the living cell population was greater in the GDNF transduction group than the non-transduction group (P < 0.01). In the ex vivo autograft of the bioengineered human corneal constructs, outgrowth of enhanced green fluorescent protein (eGFP) positive cells on the recipient corneoscleral tissue was observed. Overexpression of GDNF in the supernatant peaked at day 2, but was observed for at least 4 weeks after transplantation. At day 5, immunofluorescent staining showed expression of GDNF by all layers of epithelial cells on the graft. Our findings revealed that GDNF is a survival growth factor for cultured human corneal epithelium. The use of bioengineered human corneal constructs containing GDNF-transduced epithelial cells represents a novel method for delivering of this gene to promote survival of transplanted corneal epithelium to treat various corneal surface diseases.     

Evaluation of corneal biomechanical properties in mustard gas keratopathy

Background

Degenerative biomechanical factors and immunologic processes with effect on collagen and corneal reparative process are known as the main cause of ocular surface dysfunction in mustard gas keratopathy (MGK) and may cause changes in the corneal biomechanical values. Therefore, we evaluate corneal biomechanical properties of these patients.

The fate of antigen-antibody complexes in the rabbit cornea

The mechanisms involved in the clearance of immune deposits in tissues are not yet clear. The cornea was chosen as a model to examine this question due to its avascularity and transparency. Bovine serum albumin (BSA) and rabbit anti BSA serum were injected at opposite sites into the corneal stroma of unsensitized rabbits. Within a day, a sharp opaque line was seen macroscopically between the two injection sites. Sections of the corneas were examined by light microscopy and electron microscopy; furthermore, immunohistochemical techniques were used. With the light microscope, a precipitation line was seen in the corneal stroma, which was identified as an antigen-antibody complex by immunofluorescence techniques. In the same area infiltrating polymorphonuclear cells and swollen keratocytes were observed. In the ultrathin sections precipitates were seen lying between the collagen fibrils without affecting the structure of the collagen. The swollen keratocytes had an activated rough endoplasmic reticulum. In certain cases the precipitates appeared to be intracellular, both in the polymorphonuclear cells, as well as in the keratocytes. These findings suggest that stromal keratocytes may play an important role in the degradation of corneal immune deposits.     

Sphere formation from corneal keratocytes and phenotype specific markers

The keratocytes are specialized mesenchymal cells that produce and maintain the extracellular matrix of the corneal stroma. With a typical dendritic and flattened appearance, these cells can morph into fibroblasts and myofibroblasts upon injury, and produce abnormal or fibrotic extracellular matrices detrimental to corneal transparency. Insights into mechanisms that regulate these phenotypic switches and optimal culture conditions that preserve the keratocyte phenotype are important for tissue engineering of the corneal stroma. Like other cell types with self-renewing capacity, keratocytes can form spheres in culture. Here we investigated human and bovine keratocytes with respect to their sphere forming capabilities, and sought to identify potentially distinguishing markers for the keratocyte and fibroblast phenotypes. Keratocytes, isolated from bovine and human corneas, cultured in serum-free medium supplemented with insulin, selenium and transferrin, assumed typical keratocyte morphology, converted to fibroblasts in serum-containing medium and reverted to keratocytes after serum-deprivation. The bovine keratocytes produced spheres under adherent or low attachment conditions, while the human keratocytes produced spheres under low attachment conditions only. The primary keratocytes and fibroblasts expressed vimentin, confirming their mesenchymal origin. Keratocan, considered to be a marker for keratocytes, was also detected in early passage bovine fibroblasts. BMP3 was expressed in keratocytes and keratocyte-derived spheres, while cadherin 5 in keratocytes only, suggesting these as potential keratocyte markers.     

In vivo confocal microscopic study of corneal innervation in Sjögren's Syndrome with or without small fiber neuropathy

PurposeTo study changes in the subbasal nerve plexus by In vivo confocal microscopy (IVCM) in Sjögren's Syndrome (SS) with or without associated Small Fiber Neuropathy (SFN), in order to prevent diagnostic delay.MethodsSeventy-one patients with SS, including 19 with associated SFN, 20 healthy volunteers and 20 patients with Meibomian gland dysfunction (MGD) were included in this retrospective case-control study. IVCM was used to investigate subbasal nerve plexus density and morphology.ResultsCorneal sensitivity as evaluated with the Cochet-Bonnet aesthesiometer was significantly reduced in the SS group versus the control group (P = 0.026) and the MGD group (P = 0.037). The number of inflammatory cells was significantly increased in the SS group to 86.2 ± 82.1 cells/mm² compared to the control group (P < 0.001). The density of the subbasal nerve plexus was significantly reduced to 16.7 ± 6.5 mm/mm² in the SS group compared to the control group (P < 0.005) and the MGD group (P = 0.042). The tortuosity of the nerves in the SS group was significantly increased compared to the control group (P < 0.001) and the MGD group (P = 0.025). The average number of subbasal nerve plexus neuromas was significantly increased in the SS group compared to the control group (P = 0.001), with a significant increase in the average number of neuromas in SS patients with associated SFN compared to SS patients without SFN (P = 0.008).ConclusionIVCM can be useful to detect corneal nerve changes in SS patients and may allow earlier diagnosis of the disease and to consider new therapeutic approaches.     

Galardin inhibits collagen degradation by rabbit keratocytes by inhibiting the activation of pro-matrix metalloproteinases.

We investigated the inhibitory action of a synthetic peptidyl hydroxamate inhibitor of matrix metalloproteinase (MMP), Galardin (GM6001), on collagen degradation by rabbit corneal stromal fibroblasts (keratocytes) cultured three-dimensionally in the type I collagen gel with medium containing interleukin 1alpha (IL-1alpha) and/or plasminogen. Degradation of collagen fibrils during culture was measured by the release of hydroxyproline, and activation of MMPs was also analyzed by gelatin zymography and Western blotting. Plasmin activity was measured using a synthetic substrate. In the absence of plasminogen, treatment of the cells with IL-1alpha in collagen gel greatly enhanced the production of proMMP-1, -3 and -9, but no significant degradation of collagen was detected. In the presence of plasminogen, IL-1alpha stimulated collagen degradation by keratocytes in a dose-dependent manner. This resulted from the plasminogen activator-plasmin system-dependent activation of proMMP-1, -3 and -9. Galardin inhibited the collagen degradation in a dose-dependent fashion in the presence of plasminogen, whether IL-1alpha was present or not. Galardin inhibited the activation of proMMP-3, and also prevented the activation of proMMP-9 and the conversion of MMP-1 intermediates to the fully active MMP-1. Galardin did not affect plasmin activity. The present results suggest that Galardin inhibits IL-1alpha-stimulated collagen degradation in the presence of plasminogen, resulting from not only inhibiting active MMPs but also preventing the conversion of proMMPs to active MMPs.