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Biomedical subjects

C Cintron

Publications and source records attributed to C Cintron.

At least 19 recordsLinked to original sources
PubMed2000

CxGELSIX: a novel preparation of type VI collagen with possible use as a biomaterial.

PURPOSE: This study was initiated to evaluate tissue acceptance and stability of a novel type VI collagen preparation (CxGelsix) as a biomaterial in the rabbit corneal stroma. We hypothesized that CxGelsix, embedded intrastromally, does not have any adverse affect on surrounding corneal tissues, and remains intact in the presence of an acute inflammatory reaction during corneal wound healing. METHODS: Type VI collagen was extracted and purified from rabbit corneal stroma under nondenaturing conditions. This preparation, Gelsix, was concentrated and cross-linked with polyethylene glycol to produce a transparent film (CxGelsix). Discs of CxGelsix, 4.0-mm diameter, 9- to 35-microm thick were implanted intrastromally and clinically examined periodically for 4 months. In another experiment, implantation of CxGelsix, 2.0-mm-diameter, was followed by corneal wounding adjacent to the implant and examined clinically for 30 weeks. At the end of these periods, the tissues from these experiments were processed for light and transmission electron microscopy. RESULTS: An intralamellar 4.0-mm-diameter disc of CxGelsix does not alter the structure of corneal epithelium above the implant, suggesting normal transport of nutrients through CxGelsix. Moreover, no structural abnormalities were seen in the rest of the cornea, and the cornea remains transparent. Although the cornea accepts the presence of CxGelsix disc as judged by clinical criteria, gradual degradation of the implant is seen ultrastructurally. CxGelsix is remarkably stable despite its exposure to endogenous enzymes during inflammation and wound healing. Partial degradation of the implant occurs only after many months, and it is gradually replaced with bundles of fine collagen fibrils reminiscent of normal cornea. CONCLUSION: The results of this study suggest that CxGelsix is potentially useful as a biomaterial.

Animals↗
PubMed1998

mRNA levels of alpha1(VI) collagen, alpha1(XII) collagen, and beta ig in rabbit cornea during normal development and healing.

PURPOSE: Type VI and XII collagens and beta ig, major components of the interfibrillar matrix, may maintain proper spacing among collagen fibrils, necessary for corneal transparency. During normal corneal stroma development and healing, changes in mRNA levels of these proteins were measured to determine whether differences in steady state levels are indicative of the unique structure produced by each corneal tissue. METHODS: A full-thickness excision wound was made in each cornea of six adult rabbits and allowed to heal for 1, 2, or 4 weeks. Scar tissue from two rabbits (four scars) were used from each time period and processed for RNA extraction. Total RNA from 23-day-old fetal rabbit corneas (equivalent to approximately 1 week of stromal development) and 8-day-old neonate corneas (equivalent to approximately 3.5 weeks of stromal development) was also extracted. Relative quantities of alpha1(VI) collagen, alpha1(XII) collagen, beta ig, and beta-actin mRNAs were determined by competitive reverse transcriptase-polymerase chain reaction. Glyceraldehyde-3-phosphate dehydrogenase was used as a housekeeping gene. RESULTS: Increased mRNA levels for alpha1(VI) and alpha1(XII) collagens, beta ig, and beta-actin were seen during the first 2 weeks of healing and were followed by a decrease in 4-week-old scars. Similar increases were seen in fetal corneas with a further increase in the neonate. Differences in the beta ig mRNA levels relative to that of alpha1(XII) collagen in fetal stroma and in comparison with 1-week-old wounds suggest a higher production of beta ig in early healing tissue. CONCLUSIONS: Alterations of mRNA levels during healing and development are consistent with the cellular events and deposition of extracellular matrices in these corneal tissues. Assuming that extracellular matrix protein production is regulated at the transcriptional level, relative changes in beta ig and collagen mRNA levels reflect differences in protein deposition in early fetal and healing tissues. This is consistent with differences in the organization of the interfibrillar matrices of these tissues and their transparency.

Actins↗
PubMed1997

Beta-ig. Molecular cloning and in situ hybridization in corneal tissues.

PURPOSE: To identify a protein that copurifies with type VI collagen from rabbit cornea and to determine its cell source in rabbit corneal tissues by in situ hybridization. METHODS: Type VI collagen was extracted from cornea with urea and purified by ammonium sulfate precipitation and gel chromatography. The purity of the collagen was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On reduction with mercaptoethanol or dithiothreitol, the alpha chains of type VI collagen ran into the gel. In addition to the type VI collagen polypeptides, an extra 68-kDa protein band appeared, suggesting that this protein is present as a large molecular weight component before reduction. Amino acid sequencing indicated that protein was related to beta ig-h3 from humans. Western blot analysis was used to determine immunologic similarity to this human protein. A rabbit stromal cell cDNA library was screened with human beta ig-h3 cDNA probe. Positive clones were sequenced and analyzed for sequence homology. Oligonucleotide probes prepared from rabbit cDNA sequences were used for Northern blot analysis and in situ hybridization of corneal tissues. RESULTS: Electroblotting of the SDS-PAGE and amino acid sequence analysis of the first 10 N-terminal amino acids of the 68-kDa band gave 100% homology with a known protein produced by human adenocarcinoma cells, beta ig-h3. This 68-kDa protein was identical immunologically to beta ig-h3 by Western blot analysis. Sequence analysis of a rabbit cDNA clone contained the whole coding region and had high identity with both human beta ig-h3 and mouse beta ig-m3. The deduced amino acid sequence had 92% identity with these species. An oligonucleotide probe from the rabbit cDNA sequence detected a single band of mRNA from cultures of stromal cells consistent in size with human beta ig-h3 mRNA. The authors refer to the rabbit form of beta ig-h3 as beta ig because the protein was obtained from normal rabbit cornea and the mRNA comes from primary cultures of rabbit stromal cells and not from a cloned cell line. In situ hybridization of rabbit corneal tissue indicated that the beta ig mRNA is located primarily in the epithelium of normal adult cornea, in fetal stromal cells, and both endothelium- and stroma-derived cells in healing corneal wounds. Normal adult endothelium and stroma did not show beta ig mRNA label. CONCLUSIONS: The highly conserved amino acid sequence homology between the human, mouse, and rabbit proteins and the temporal expression of beta ig message during corneal healing and development suggest this protein plays an important role in the morphogenesis of corneal tissues.

Amino Acid Sequence↗
PubMed1997

Repair phenotype in corneal fibroblasts is controlled by an interleukin-1 alpha autocrine feedback loop.

PURPOSE: To explore the role of autocrine interleukin-1 alpha (IL-1 alpha) as a central regulator of the repair phenotype in corneal fibroblasts. METHODS: Disruption of the actin cytoskeleton with cytochalasin B (CB), which mimics changes in shape that occur in repair tissues, was used to stimulate repair gene expression in early-passage fibroblasts. Changes in expression of IL-1 alpha, IL-8, collagenase, and ENA-78 were determined by Northern blot analysis, radioimmunoassay, and an enzyme-amplified sensitivity immunoassay (EASIA). Expression of repair genes was also examined in repair fibroblasts, isolated from healing, penetrating keratectomy wounds in rabbits. RESULTS: Blocking IL-1 alpha activity prevented both constitutive and stimulated increases in synthesis of IL-8 and collagenase in early-passage cultures of corneal fibroblasts, demonstrating the role of IL-1 alpha as a necessary intermediate for expression of these genes. Evidence is also presented that the IL-1 alpha autocrine controls expression of an IL-8 related factor, ENA-78. Unlike early-passage fibroblasts, fibroblasts freshly isolated from the uninjured cornea did not express IL-1 alpha. However, fibroblasts freshly isolated from remodeling corneal repair tissue 3 weeks after injury were found to express substantial levels of IL-1 alpha, regulated through an autocrine feedback loop. Neutralization experiments demonstrated that the IL-1 alpha autocrine is largely responsible for controlling both collagenase and IL-8 synthesis in repair fibroblasts, as it is in early-passage fibroblasts. CONCLUSIONS: These findings provide evidence that activation of an autocrine IL-1 alpha feedback loop is an important mechanism by which fibroblasts adopt a repair phenotype during remodeling of the cornea.

Animals↗
PubMed1996

Beta ig-h3 is synthesized by corneal epithelium and perhaps endotheliumin Fuchs' dystrophic corneas.

PURPOSE: Deposition of abnormal sub-epithelial matrix and posterior collagenous layer by epithelium and endothelium, respectively, in Fuchs' dystrophy gives us the opportunity to determine if these tissues synthesize beta ig-h3. METHODS: Immunohisto-/immunocytochemistry of corneas were conducted with rabbit anti-human beta ig-h3 and monoclonal anti-human type VI collagen. Labeled sense and anti-sense beta ig-h3 oligonucleotide probes were used for in situ hybridization. RESULTS: beta ig-h3-specific fluorescence was found just beneath detached epithelium in the sub-epithelial matrix, abnormal Descemet's membrane and posterior collagenous layer. Type VI collagen co-localized with beta ig-h3 within abnormal sub-epithelial matrix and corneal stroma adjacent to Descemet's membrane. beta ig-h3 mRNA was detected in corneal epithelium of dystrophic corneas. CONCLUSIONS: Expression of beta ig-h3 in sub-epithelial matrix and posterior collagenous layer of Fuchs' dystrophy is consistent with the synthesis of new extracellular matrices by epithelial and endothelial tissues. beta ig-h3 mRNA in corneal epithelium further supports an epithelial source of this protein. Endothelial synthesis of beta ig-h3 is based on circumstantial evidence due to cell loss during surgical and histological procedures. Co-localization of beta ig-h3 with type VI collagen in abnormal sub-epithelial matrix and at the stromal/Descemet's membrane interface suggest this collagen in association with beta ig-h3 interacts with these tissues and anchors them to the adjacent stroma.

Animals↗
PubMed1996

Transplanted corneal stromal cells in vitreous reproduce extracellular matrix of healing corneal stroma.

PURPOSE: To characterize the extracellular matrix (ECM) formed by corneal stromal cells after injection into the vitreous. This will provide a basis for future studies on the function of corneal ECM macromolecules. METHODS: Cell line from rabbit dermal fibroblasts (RAB9) and primary cultures of rabbit corneal stroma fibroblasts (NRCF) were grown to confluence. For each cell type, approximately 1 x 10(6) cells suspended in basal medium were injected into the vitreous of normal rabbits and observed periodically with a slit lamp. After 1,2, and 4 weeks, eyes were processed for transmission electron microscopy (TEM), immunohistochemistry, immunocytochemistry, and in situ hybridization. RESULTS: All cells showed gradual growth within the vitreous along the needle track. Occurrence of retinal detachment and inflammation was variable. Transmission electron microscopy of NRCF confirmed the deposition of ECM reminiscent of the organization of normal fetal corneal stroma. Similar matrices were produced by RAB9. NRCF deposited collagen fibrils similar in diameter to those seen in normal developing and healing corneal stroma. RAB9 produced collagen fibrils with larger diameters. NRCF-transplanted cells synthesized proteoglycans and collagen immunologically identical to decorin proteins and type VI collagen, indicating that the expression of specific ECM is maintained after transplantation. In addition, in situ hybridization showed that type XII collagen mRNA is synthesized by transplanted NRCF similar to healing corneas. CONCLUSIONS: Corneal stromal cells transplanted into vitreous produce a matrix morphologically and biochemically similar to that in healing corneal stroma.

Animals↗
PubMed1995

Localization of type XII collagen in normal and healing rabbit cornea by in situ hybridization.

To identify the cell types responsible for type XII collagen synthesis in normal and healing rabbit cornea, a partial cDNA sequence of rabbit type XII collagen, obtained from an adult rabbit cornea cDNA library, was used to develop highly specific oligonucleotide probes for Northern blot analysis and in situ hybridization. Approximately 2000 bases of a type XII collagen 2.2 kb cDNA clone were sequenced. Comparative sequence analysis of the bases showed a 74% identity with chick alpha 1 (XII) chain of type XII collagen. The deduced amino acid sequence indicated a 72% identity with chick type XII collagen. Northern blot analysis showed that cultures of cornea stromal and endothelial cells each contain two RNA species, greater than 10 kb, that hybridize to rabbit type XII collagen oligonucleotide probes. Although normal stromal cells failed to show type XII collagen mRNA, normal endothelial cells contain mRNA for this collagen. These results indicate that endothelium of normal rabbit cornea has a potential to synthesize type XII collagen. During corneal wound healing, both endothelium-derived and stroma-derived cells in the developing scar tissue contained type XII mRNA. In view of the known presence of type XII collagen in corneal stromas from chick and mouse, the distribution of mRNA in normal cornea is puzzling.

Amino Acid Sequence↗
PubMed1995

Stratified squamous epithelia produce mucin-like glycoproteins.

The stratified squamous epithelia of the ocular surface, larynx, and vagina are mucus-coated epithelia, apices of which are subject to abrasive pressure from epithelia-epithelia interactions from eyelid, vocal cords, or vaginal folds, respectively. Mucus coats on these epithelia have generally been considered to be derived from the specialized mucin-producing cells embedded either in the epithelia or in adjacent tissues. Here we report the isolation, partial characterization, and cellular localization of a mucin-like glycoprotein produced by these stratified epithelia. In all three epithelia, the mucin-like molecule is present on cytoplasmic vesicles in subapical cells. As cells differentiate to their apical-most position adjacent to their mucus coat, the mucin-like molecule moves to the cell membrane where it is particularly prominent on microplicae folds. Lectin affinity chromatography was used to isolate the molecule from rat vaginal and corneal epithelium. Isolated material was approximately 60% carbohydrate and 40% protein. The major monosaccharide was N-acetylgalactosamine with lesser amounts of N-acetylglucosamine, galactose, mannose, xylose and fucose. Amino acid analysis demonstrated the predominant amino acids to be glycine, serine, threonine and proline. These data plus PAS and Alcian blue binding to the isolate indicate a mucin-like glycoprotein.

Animals↗
PubMed1995

Cloning and in situ hybridization of rabbit decorin in corneal tissues.

PURPOSE: To develop molecular probes and to identify the cell types responsible for decorin synthesis in healing cornea. METHODS: Adult rabbit cornea and rabbit corneal stromal cell (keratocyte) culture cDNA libraries were constructed. The libraries were screened with commercially available human cDNA and oligonucleotide probes. Positive clones were sequenced to obtain a full length rabbit decorin cDNA. Synthetic oligonucleotides for rabbit decorin were chosen as probes for Northern blot analysis and in situ hybridization of healing rabbit corneas. RESULTS: The cDNA sequences of the positive clones from the two libraries were identical in areas of overlap. The combined cDNA sequence indicated a 1.5-kb length with a complete open reading frame for decorin. The cDNA and deduced amino acid sequences are 90% and 88% identical, respectively, to previously reported human fibroblast and bovine bone decorin sequences. A hypervariable region near the N-terminal has little homology to decorins of other species or other rabbit protein. Northern blot analysis detected a 2.0-kb and a 2.3-kb band in mRNA from rabbit keratocyte cultures. Decorin mRNA was detected in keratocytes of normal and healing rabbit corneas by in situ hybridization. Label in the healing tissue was markedly increased above normal. Normal endothelium and epithelium in normal and healing cornea failed to show label. CONCLUSIONS: Decorin mRNA from normal adult rabbit cornea is identical to decorin mRNA from keratocytes in culture and is highly homologous to decorin from other animal species. In situ hybridization indicated an upregulation of decorin message in cells adjacent to and within the healing tissue. Both stroma-derived and endothelium-derived cells in the wound synthesize message for decorin.

Amino Acid Sequence↗
PubMed1994

Structure of corneal scar tissue: an X-ray diffraction study.

Full-thickness corneal wounds (2 mm diameter) were produced in rabbits at the Schepens Eye Research Institute, Boston. These wounds were allowed to heal for periods ranging from 3 weeks to 21 months. The scar tissue was examined using low- and wide-angle x-ray diffraction from which average values were calculated for 1) the center-to-center collagen fibril spacing, 2) the fibril diameter, 3) the collagen axial periodicity D, and 4) the intermolecular spacing within the collagen fibrils. Selected samples were processed for transmission electron microscopy. The results showed that the average spacing between collagen fibrils within the healing tissue remained slightly elevated after 21 months and there was a small increase in the fibril diameter. The collagen D-periodicity was unchanged. There was a significant drop in the intermolecular spacing in the scar tissues up to 6 weeks, but thereafter the spacing returned to normal. The first-order equatorial reflection in the low-angle pattern was visible after 3 weeks and became sharper and more intense with time, suggesting that, as healing progressed, the number of nearest neighbor fibrils increased and the distribution of nearest neighbor spacings reduced. This corresponded to the fibrils becoming more ordered although, even after 21 months, normal packing was not achieved. Ultrastructural changes in collagen fibril density measured from electron micrographs were consistent with the increased order of fibril packing measured by x-ray diffraction. The results suggest that collagen molecules have a normal axial and lateral arrangement within the fibrils of scar tissue. The gradual reduction in the spread of interfibrillar spacings may be related to the progressive decrease in the light scattered from the tissue as the wound heals.

Animals↗
PubMed1993

Keratan sulfate and dermatan sulfate proteoglycans associate with type VI collagen in fetal rabbit cornea.

Keratan sulfate proteoglycan (KSPG) and dermatan sulfate proteoglycan (DSPG) are associated with collagen fibrils in adult rabbit cornea. Because certain cytochemical data suggested that proteoglycans are associated with type VI collagen in the fetal rabbit cornea, we developed polyclonal antibodies specific to the core proteins of rabbit corneal KSPG (lumican and/or fibromodulin) and DSPG (decorin and/or biglycan) and used the antibodies as immunocytochemical probes to determine proteoglycan ultrastructural location. Immunogold particles were associated with Type VI collagen filaments but not with collagen fibrils in fetal and neonate rabbit cornea. Association of corneal KSPG and DSPG with Type VI collagen was immunocytochemically confirmed with monoclonal antibodies to low-sulfated keratan sulfate glycosaminoglycan (GAG) and chondroitin-4-sulfate GAG of DSPG. The monospecificity of the polyclonal and monoclonal antibodies and exclusive binding of antibodies to Type VI collagen filaments, together with previous chemical identification of low-sulfated KSPG and DSPG in developing rabbit cornea, support our contention that corneal proteoglycans in developing rabbit cornea are associated with Type VI collagen.

Animals↗
PubMed1993

Stromal fibroblasts synthesize collagenase and stromelysin during long-term tissue remodeling.

The process of connective tissue remodeling is an important mechanism contributing to tissue morphogenesis in development and homeostasis. Although it has long been known that remodeling tissues actively mediate collagenolysis, little is understood about the molecular mechanisms controlling this cell-regulated process. In this study, we examined the biosynthesis of collagenase and the related metalloproteinase, stromelysin, during remodeling of repair tissue deposited after mechanical injury to the rabbit cornea. Neither enzyme was synthesized by uninjured corneas; however, synthesis and secretion was detectable within one day after injury. Collagenase accumulated in its latent form while stromelysin appeared to be partially activated. Enzymes were synthesized by cells having a fibroblast phenotype. These cells were found within the stroma. New synthesis was correlated with accumulation of enzyme-specific mRNA. Highest levels of enzyme synthesis were observed in the repair tissue. However, stromal cells outside of the repairing area also synthesized both enzymes. The level of synthesis decreased in a gradient radiating from the repair tissue. Total synthetic levels in a given area of cornea were dependent on both the number of cells expressing enzyme and the rate of enzyme synthesis. Synthesis of collagenase was detected in repair tissue as long as nine months after injury. Our findings provide direct support for the hypothesis that new collagenase synthesis by cells in repair tissue is the first step in collagen degradation during long-term tissue remodeling.

Animals↗
PubMed1991

Immunohistochemistry of proteoglycans in human lamina cribrosa.

Proteoglycans are macromolecular components of connective tissue, which are believed to have an important role in the organization of other extracellular matrix components and in the hydration and rigidity of tissue. Using antibodies to the heparan sulfate-, dermatan sulfate-, and keratan sulfate-proteoglycans core proteins, we used indirect immunofluorescence to determine the presence of these core proteins and to characterized their distribution in the lamina cribrosa of healthy human eyes of various ages. Findings with heparan sulfate-proteoglycan core proteins support the multilayered organization of basement membranes in the lamina cribrosa as proposed in previous reports. Dermatan sulfate-proteoglycan core protein results indicate colocalization with fibrillar collagen. Application of monoclonal antibodies to the glycosaminoglycan side chain disclosed an increasing predominance of chondroitin-4-sulfate over chondroitin-6-sulfate with age. This may indicate a decreasing water-holding capacity of the cribriform plates and suggest a decreasing ability to withstand pressure. This study attempts to identify the core proteins of proteoglycans in human lamina cribrosa and serves as a basis for study of glaucomatous eyes.

Adolescent↗
PubMed1991

Differential roles for two gelatinolytic enzymes of the matrix metalloproteinase family in the remodelling cornea.

We have documented changes in collagenolytic/gelatinolytic enzymes of the matrix metalloproteinase family (MMP) in remodelling rabbit cornea. MMP-2 (65 kDa gelatinase) in the proenzyme form is synthesized by the cells of the normal corneal stroma. After keratectomy the level of MMP-2 is increased in the stroma and enzyme appears in both pro- and activated forms. In addition, corneal cells synthesize MMP-9 (92 kDa gelatinase) in the proenzyme form after keratectomy; expression occurs in both the epithelial as well as stromal corneal layers. Changes in expression of both enzymes are precisely localized to the repairing portion of cornea, but demonstrate important differences in timing that correlate with the timing of specific events of matrix remodelling. Our data suggest that each of the gelatinases plays a different role in tissue remodelling after injury. We hypothesize that MMP-2 performs a surveillance function in normal cornea, catalyzing degradation of collagen molecules that occasionally become damaged. After wounding, this enzyme appears to participate in the prolonged process of collagen remodelling in the corneal stroma that eventually results in functional regeneration of the tissue. MMP-9 expression does not correlate with stromal remodelling, but we suggest that the enzyme might play a part in controlling resynthesis of the epithelial basement membrane.

Animals↗
PubMed1990

Proteoglycan distribution in developing rabbit cornea.

We used a staining procedure specific for sulfated glycosaminoglycans, cuprolinic blue dye (CBD), and immunohistochemical techniques to determine the histological distribution and ultrastructural organization of proteoglycans in developing rabbit cornea. We found several types of CBD-stained structures located throughout the corneal stroma, indicative of the distribution and perhaps the chemical heterogeneity of proteoglycans in this tissue. Keratan sulfate-specific immunohistochemical evidence supports our cytochemical findings. Our results suggest that low-sulfated keratan sulfate proteoglycans are found throughout most of the developing stroma, with the exception of the posterior margin of this tissue. Highly sulfated keratan sulfate proteoglycans in young fetal corneas, initially restricted to the subepithelial stroma, progressively extend to deeper portions of the stroma with development. Dermatan sulfate proteoglycans are located throughout the stroma, including the posterior margin. Invoking a recently published "oxygen-lack hypothesis" and correlating the tissue location of proteoglycans with the source of oxygen, we hypothesize that the distribution of proteoglycans in the developing rabbit cornea is related to the selective synthesis of keratan sulfate glycosaminoglycans under hypoxic conditions.

Animals↗
PubMed1990

Morphologic analyses of proteoglycans in rabbit corneal scars.

Ultrastructural localization of proteoglycans (PGs) in 1-week- to 2-year-old scar was determined by staining with cuprolinic blue dye (CBD) after specific enzymatic digestion of keratan sulfate (KS) glycosaminoglycans (GAGs) or chondroitin sulfate glycosaminoglycans (CSs). High critical electrolyte conditions were maintained for CBD-staining, specific for high-sulfated GAGs. Although KS was detected in the 1-week-old wound, no CBD-stained KS was seen in the anterior stroma adjacent to the wound. The CS was present throughout the 1-week-old wound and adjacent stroma, and PGs were biosynthetically 35SO4-labeled in normal stroma. Subsequently, radioactivity from labeled PGs in normal stroma adjacent to the wound moved into scar tissue during healing. Marked sensitivity of PGs to Chondroitinase ABC indicated an abundance of CS in 2-week-old scars. Punctate CBD-staining and immunohistochemical evidence suggested chemically altered KS is present in the 2-week-old anterior scar. The pattern of CBD-staining in 1- and 2-week scars, after chondroitinase treatment, suggested KS in the younger scar is similar to adult high-sulfated GAG, whereas KS in the 2-week scar contains primarily newly synthesized low-sulfated KS. The latter is consistent with previous immunochemical and biochemical analyses. Cytochemical and immunohistochemical evidence indicated that KS is not present in the 2-week-old posterior scar. By the week 8 of healing, CBD-stained KS was present throughout most of the scar, except along the posterior margin, consistent with earlier stages of healing. The CBD-stained structures in the first 8 weeks of healing were reminiscent of stained GAGs in normal developing cornea. This fetal-like CBD-staining pattern seen in scar, however, changed to that of the normal adult by the 2nd year of healing. The significance of these observations relate to our contention that healing adult cornea recapitulates some ontogenetic events of the normal cornea, and that the nonuniform distribution and chemical properties of GAGs in scar tissue are a function of the movement of existing proteoglycans and de novo synthesis of altered macromolecules.

Animals↗
PubMed1990

Immunolocalization of type VI collagen in developing and healing rabbit cornea.

We have localized type VI collagen in normal developing and corneal scar tissue. Indirect immunofluorescence showed that type VI collagen was distributed throughout the normal stroma and most of the scar. No fluorescence was detected along the posterior margin of the scar and in a retrocorneal membrane continuous with the scar. Since the corneal endothelium in rabbits contributes to the formation of scar tissue and retrocorneal membrane, our observations suggest that the endothelium does not synthesize type VI collagen. Indirect immunoelectron microscopy showed that type VI collagen was located abundantly between collagen fibrils as fine filamentous structures containing beads with a periodicity of 100 nm, consistent with published observations of other tissues. Because these filaments are more prominent when stained with ruthenium red, and predigestion of tissue with Chondroitinase ABC enhances binding of monoclonal antibody to type VI collagen, proteoglycans probably are associated with this collagen in the cornea. Ultrastructural observations supported by previous biochemical analyses show that the proportion of type VI collagen to fibrillar collagen is smaller in scar tissue compared with fetal cornea. The abundance of type VI collagen and its distribution and association with proteoglycans in rabbit corneal tissues suggest that this macromolecule plays a role in the tensile strength and transparency of the stroma.

Animals↗