keratan-sulfate and Corneal-Opacity

To examine the immunohistochemical alterations in the corneal stroma in Peters anomaly (PA) and congenital glaucoma (CG) compared with age-matched normals and acquired adult corneal scarring (AACS).. The clinical features of PA and CG patients who underwent penetrating keratoplasty were recorded. Immunohistochemistry of cornea and control tissue (normal and acquired corneal scars) was performed with antibodies against collagen types I, III, keratan sulfate, lumican, decorin, and smooth muscle actin followed by semiquantitative analysis of immunolabeling.. Clinical features in 2 groups were consistent with PA and CG. Microscopy showed thickened stromal collagen bundles in PA (n = 15), CG (n = 11), and AACS (n = 20) compared with normals (n = 18). PA and CG had distinct immunophenotypes compared with controls. Type I collagen labeling was more intense in CG compared with PA (intensity grading (IG) 2.73 vs. 2.07; P < 0.001). Decorin, lumican, and keratan sulfate labeling was significantly less intense in PA versus AACS (IG; 1.91, 0.38, 1.75 in PA and 2.7, 1.11, 2.61 in AACS. respectively; P = 0.002, P = 0.001 and P = 0.004) and normals (IG 1.92, 1.06, 2.59 respectively; P < 0.001, P < 0.001 and P = 0.005). Collagen I labeling was less intense in CG versus AACS (IG 2.73 vs. 3.09) (P = 0.007). Collagen III labeling was more intense in PA/CG than in normals (IG 0.9, 0.64, 0.62 retrospectively) (P < 0.001 in both).. The immunophenotype of the corneal scar in PA and CG differs from normal corneas and so does PA from AACS. The similarities between CG and AACS suggest that CG scarring has an acquired component.

Topics: Adolescent; Anterior Eye Segment; Child; Child, Preschool; Chondroitin Sulfate Proteoglycans; Collagen Type I; Collagen Type III; Cornea; Corneal Opacity; Decorin; Eye Abnormalities; Female; Humans; Hydrophthalmos; Immunohistochemistry; Infant; Keratan Sulfate; Keratoplasty, Penetrating; Lumican; Male; Proteoglycans; Retrospective Studies

The corneal stroma is enriched in small leucine-rich proteoglycans (SLRPs), including both class I (decorin and biglycan) and class II (lumican, keratocan and fibromodulin). Transparency is dependent on the assembly and maintenance of a hierarchical stromal organization and SLRPs are critical regulatory molecules. We hypothesize that cooperative interclass SLRP interactions are involved in the regulation of stromal matrix assembly. We test this hypothesis using a compound Bgn(-/0)/Lum(-/-) mouse model and single Lum(-/-) or Bgn(-/0) mouse models and wild type controls. SLRP expression was investigated using immuno-localization and immuno-blots. Structural relationships were defined using ultrastructural and morphometric approaches while transparency was analyzed using in vivo confocal microscopy. The compound Bgn(-/0)/Lum(-/-) corneas demonstrated gross opacity that was not seen in the Bgn(-/0) or wild type corneas and greater than that in the Lum(-/-) mice. The Bgn(-/0)/Lum(-/-) corneas exhibited significantly increased opacity throughout the stroma compared to posterior opacity in the Lum(-/-) and no opacity in Bgn(-/0) or wild type corneas. In the Bgn(-/0)/Lum(-/-) corneas there were abnormal lamellar and fibril structures consistent with the functional deficit in transparency. Lamellar structure was disrupted across the stroma with disorganized fibrils, and altered fibril packing. In addition, fibrils had larger and more heterogeneous diameters with an abnormal structure consistent with abnormal fibril growth. This was not observed in the Bgn(-/0) or wild type corneas and was restricted to the posterior stroma in Lum(-/-) mice. The data demonstrate synergistic interclass regulatory interactions between lumican and biglycan. These interactions are involved in regulating both lamellar structure as well as collagen fibrillogenesis and therefore, corneal transparency.

Topics: Animals; Biglycan; Chondroitin Sulfate Proteoglycans; Collagen; Corneal Opacity; Corneal Stroma; Immunoblotting; Keratan Sulfate; Leucine-Rich Repeat Proteins; Lumican; Mice; Mice, Knockout; Microscopy, Confocal; Proteins; Pseudopodia

To describe the histopathology of the cornea in microphthalmia with linear streaks (MLS) syndrome.. Two patients with MLS syndrome underwent penetrating keratoplasty. This study describes the histopathology and investigates immunophenotype of the corneal extracellular matrix by using keratan sulfate and collagen type III antibodies.. Clinical examination revealed bilateral sclerocornea and characteristic skin changes. By light microscopy, central corneal stroma in both patients showed vascularization and irregular thick collagen lamellae typical of sclerocornea. In addition, corneal thinning, anterior synechiae, and the absence of the Descemet membrane were noted, which was suggestive of Peters anomaly. Diffuse and intense anti-keratan sulfate staining and minimal anti-collagen type III stromal staining were seen in both corneal buttons.. The cornea in MLS may clinically resemble sclerocornea. Histologic features resemble those previously described in sclerocornea and also seen in anterior segment dysgeneses. Keratan sulfate and collagen type III labeling suggests that the corneal extracellular matrix resembled cornea and not sclera.

Topics: Collagen Type III; Cornea; Corneal Opacity; Extracellular Matrix; Female; Fluorescent Antibody Technique, Indirect; Humans; Immunophenotyping; Infant; Keratan Sulfate; Keratoplasty, Penetrating; Male; Microphthalmos; Sclera; Skin Abnormalities; Syndrome

A retrospective clinical and a genetic study was carried out of severe subepithelial corneal haze occurring after photorefractive keratectomy (PRK). Since this clinical condition resembles the lumican-null mouse phenotype, mutation analysis of lumican and keratocan was carried out to investigate whether germline genetic alterations have an effect on development of severe corneal haze in humans. Corneal thickness, photoablation depth, and severity of persistent corneal haze were also analyzed. In vivo confocal microscopy examination was also performed to study corneal structure and endothelial cells.. Severity of corneal haze was evaluated by slit-lamp biomicroscopy according to Hanna's scale. Corneal structure and endothelial cell shapes and density were viewed with a scanning confocal microscope. PCR-based mutational analysis was performed using temperature gradient gel electrophoresis (TGGE) and direct sequencing.. Preoperative corneal thickness was normal (539+/-23.13 microm, mean+/-SD), and the photoablation depth was 88.94+/-18.64 microm (mean+/-SD). The most severe corneal haze was grade 2.0 on Hanna's scale one year after PRK. In vivo confocal microscopy also showed normal endothelial cell density and morphology. Aside from an intronic polymorphism in a control, no genetic alterations were found in the lumican and keratocan genes.. There was no evidence that endothelial dysfunction and germline mutation of lumican and keratocan genes participate in the etiology of subepithelial corneal haze. Our findings suggest that the mechanisms of the development of severe corneal opacity are different in humans after PRK compared to the lumican deficient knockout mouse model.

Topics: Adult; Case-Control Studies; Cell Count; Chondroitin Sulfate Proteoglycans; Cornea; Corneal Opacity; Endothelium, Corneal; Female; Humans; Keratan Sulfate; Lasers, Excimer; Lumican; Male; Microscopy, Confocal; Photorefractive Keratectomy; Proteoglycans; Retrospective Studies; Severity of Illness Index

The purpose of this study was to characterize temporally stromal growth and transparency in lumican-deficient and normal neonatal mice.. Lumican-deficient mice and CD1 wild-type mice were evaluated by in vivo confocal microscopy through-focusing (CMTF) to quantify stromal and epithelial thickness and corneal light-scattering and by laser scanning CM to determine density of keratocytes from 1 day to 12 weeks after birth.. CD1 corneas showed a rapid loss of light-scattering, decreasing by 50% from day 1 to day 12, that paralleled a 60% decrease in density of keratocytes. By contrast, the stroma demonstrated a marked swelling from day 8 to day 12, followed by thinning at day 14. Compared to corneas from CD1 mice, lumican-deficient corneas showed significantly increased (P < 0.05) light-scattering beginning at week 3 that remained elevated above wild-type levels for the duration of the study. Stromal development was also markedly altered, with thinning detected at week 3, followed by no detectable stromal growth for the duration of the study. Density of keratocytes was significantly increased, but the total cell number was similar compared with that in the wild-type cornea, suggesting no effect on keratocyte differentiation.. Development of normal neonatal corneal transparency appears related to changes in density of keratocytes. The stroma, however, undergoes a marked swelling and thinning at the time of eyelid opening (days 8-14). In the lumican-deficient mouse, stromal swelling is abolished, indicating that this critical phase in stromal development is lumican dependent and essential for normal stromal growth and maintenance of stromal transparency.

Topics: Animals; Animals, Newborn; Cell Count; Chondroitin Sulfate Proteoglycans; Corneal Opacity; Corneal Stroma; Epithelium, Corneal; Female; Fibroblasts; Keratan Sulfate; Light; Lumican; Mice; Mice, Knockout; Microscopy, Confocal; Scattering, Radiation

Gene targeted lumican-null mutants (lum(tm1sc)/lum(tm1sc)) have cloudy corneas with abnormally thick collagen fibrils. The purpose of the present study was to analyze the loss of transparency quantitatively and to define the associated corneal collagen fibril and stromal defects.. Backscattering of light, a function of corneal haze and opacification, was determined regionally using in vivo confocal microscopy in lumican-deficient and wild-type control mice. Fibril organization and structure were analyzed using transmission electron microscopy. Biochemical approaches were used to quantify glycosaminoglycan contents. Lumican distribution in the cornea was elucidated immunohistochemically. RESULTS; Compared with control stromas, lumican-deficient stromas displayed a threefold increase in backscattered light with maximal increase confined to the posterior stroma. Confocal microscopy through-focusing (CMTF) measurement profiles also indicated a 40% reduction in stromal thickness in the lumican-null mice. Transmission electron microscopy indicated significant collagen fibril abnormalities in the posterior stroma, with the anterior stroma remaining relatively unremarkable. The lumican-deficient posterior stroma displayed a pronounced increase in fibril diameter, large fibril aggregates, altered fibril packing, and poor lamellar organization. Immunostaining of wild-type corneas demonstrated high concentrations of lumican in the posterior stroma. Biochemical assessment of keratan sulfate (KS) content of whole eyes revealed a 25% reduction in KS content in the lumican-deficient mice.. The structural defects and maximum backscattering of light clearly localized to the posterior stroma of lumican-deficient mice. In normal mice, an enrichment of lumican was observed in the posterior stroma compared with that in the anterior stroma. Taken together, these observations indicate a key role for lumican in the posterior stroma in maintaining normal fibril architecture, most likely by regulating fibril assembly and maintaining optimal KS content required for transparency.

Topics: Animals; Chondroitin Sulfate Proteoglycans; Collagen; Corneal Opacity; Corneal Stroma; Gene Targeting; Keratan Sulfate; Lumican; Mice; Mice, Knockout; Microscopy, Confocal; Microscopy, Fluorescence

Lumican, a prototypic leucine-rich proteoglycan with keratan sulfate side chains, is a major component of the cornea, dermal, and muscle connective tissues. Mice homozygous for a null mutation in lumican display skin laxity and fragility resembling certain types of Ehlers-Danlos syndrome. In addition, the mutant mice develop bilateral corneal opacification. The underlying connective tissue defect in the homozygous mutants is deregulated growth of collagen fibrils with a significant proportion of abnormally thick collagen fibrils in the skin and cornea as indicated by transmission electron microscopy. A highly organized and regularly spaced collagen fibril matrix typical of the normal cornea is also missing in these mutant mice. This study establishes a crucial role for lumican in the regulation of collagen assembly into fibrils in various connective tissues. Most importantly, these results provide a definitive link between a necessity for lumican in the development of a highly organized collagenous matrix and corneal transparency.

Topics: Animals; Chondroitin Sulfate Proteoglycans; Collagen; Corneal Opacity; Gene Expression; Gene Targeting; In Situ Hybridization; Keratan Sulfate; Lumican; Mice; Skin Diseases, Metabolic; Tensile Strength

Corneal opacification associated with glycosaminoglycan (GAG) deposition occurs in canine mucopolysaccharidosis I (MPS I), a deficiency of the lysosomal enzyme alpha-L-iduronidase. In affected dogs corneal lesions appear similar to those in children with the same disease. Transplantation of bone marrow from unaffected littermates was performed in 5 MPS I affected dogs at 5 months of age. In three recipients that became long-term survivors corneal clouding was largely alleviated compared to affected control dogs. In no case, however, did the corneas remain totally clear throughout the course of the study (594, 628 and 1425 days). Light and electron microscopic findings correlated with the clinical impression of partial improvement. Glycosaminoglycan analysis of corneal tissue from two transplant recipients, one normal littermate, and one MPS I-affected, untransplanted dog showed quantitative and qualitative changes in stored GAG following bone marrow transplantation.

Topics: Animals; Bone Marrow Transplantation; Corneal Opacity; Dermatan Sulfate; Dogs; Electrophoresis; Glycosaminoglycans; Keratan Sulfate; Mucopolysaccharidosis I; Time Factors

Macular corneal dystrophy is an autosomal recessive disorder in which abnormal deposits in the corneal stroma have been identified. We examined the corneal buttons of 12 patients, who had clinical features of macular dystrophy, by histochemical staining, transmission electron microscopy, and immunohistochemical techniques. All corneas exhibited positive staining with Muller Mowry's colloidal iron. Using monoclonal antibodies 1/20/5-D-4, J-10, J-19, and J-36 that recognize specific sites on the sulfated keratan sulfate molecule, we stained corneal sections by an avidin-biotin-peroxidase complex method and identified two groups of macular corneal dystrophy. One group consisting of four corneas reacted positively with all four antibodies, and the other group consisting of eight corneas did not react with any of the antibodies used. These results confirmed those recently presented by Yang et al that there may be subgroups of macular dystrophy that can be identified by immunohistochemical methods. Also, serum levels of sulfated keratan sulfate were determined in seven patients. One patient who displayed a normal level of serum keratan sulfate had positive corneal immunoreactivity. Of the six patients who lacked serum keratan sulfate, four showed negative and two had positive corneal immunostaining, suggesting at least three subgroups in the disease. An attempt was made to correlate the clinical features, histochemical-staining characteristics, and ultrastructural morphology with the immunoreactivity to keratan sulfate antibodies, but no correlations could be made.

Topics: Adult; Aged; Antibodies, Monoclonal; Corneal Dystrophies, Hereditary; Corneal Opacity; Female; Humans; Immunoenzyme Techniques; Keratan Sulfate; Male; Microscopy, Electron; Middle Aged