Biomedical subjects

C Cintron

Publications and source records attributed to C Cintron.

At least 37 records · Page 2Linked to original sources

PubMed1988

Developmental changes in proteoglycans of rabbit corneal stroma.

Proteoglycans have been extracted from rabbit corneal stromas at developmental stages from fetal to adult. Ion exchange fractionation and gel chromatography show that proteodermatan sulfates decrease in sulfation and relative amount and proteokeratan sulfates increase in sulfation and relative amount during development. There are small increases in size of all of the proteoglycans up to 2 weeks after birth, and the final adult composition is achieved by 8 weeks.

Animals↗

PubMed1986

Development of monoclonal antibodies recognizing collagenase from rabbit PMN; the presence of this enzyme in ulcerating corneas.

Rabbit uterine collagenase was purified from the medium of involuting uterus (1-2 days postpartum) in culture using ammonium sulfate fractionation, DEAE-cellulose, heparin-affinity, and high performance liquid chromatography. The enzyme was purified more than 1600 fold. Hybridoma cell-lines producing monoclonal antibodies were prepared by fusing the spleen cells of mice immunized with the purified enzyme with mouse myeloma cells (Sp2/O-Ag14). The hybridoma cells were selected with HAT medium, cloned, and screened by ELISA. Antibody-producing ascites were prepared by injecting hybridoma cell-lines into the peritoneal cavities of mice. Western-blot analysis indicated that the antibodies recognized a polypeptide having a molecular weight of 52,000. The IgG isolated from the ascites inhibited the enzyme. Indirect immunofluorescent staining demonstrated that polymorphonuclear leukocytes (PMNs) in the superficial layer of alkali-burned corneas contained collagenase, whereas stromal cells and PMNs within the stroma were not stained by the antibodies. Our results suggest that collagenases produced by rabbit PMNs are different from those produced by fibroblasts from cornea. We hypothesize that PMNs in alkali-burned corneas secrete all or most of their collagenases by degranulation at the anterior surface of the cornea, and then continue to migrate into the deeper portion of the stroma.

Animals↗

PubMed1985

Disparate effects of calmodulin inhibitors on corneal epithelial migration in rabbit and rat.

We investigated the effects of two calmodulin inhibitors, trifluoperazine and N-6-aminohexyl-5-chloro-1-naphthalenesulfonamide, on corneal epithelial wound closure in rabbits and rats. Measured, round epithelial defects were made on corneas by gentle scraping. After 20 h in organ culture with or without inhibitor, the remaining wound area was measured and samples were fixed for microscopy. In the rabbit, these inhibitors had little or no effect on the rate of wound coverage. In the rat, however, both trifluoperazine (3-40 microM) and N-6-aminohexyl-5-chloro-1-naphthalenesulfonamide (10-100 microM) inhibited wound closure in a dose-dependent manner. Thus we believe that calmodulin plays a crucial role in epithelial migration in the rat. In the rabbit, there seems to exist a mechanism that is not critically dependent upon an intact calmodulin pathway.

Animals↗

PubMed1985

Different corneal epithelial healing mechanisms in rat and rabbit: role of actin and calmodulin.

The authors investigated the effects of calmodulin inhibitors, trifluoperazine (10-20 microM) and W-7 (25-50 microM), and of cytochalasin B (5 micrograms/ml) on the F-actin distribution, surface morphology, and migration of rat and rabbit corneal epithelial cells in tissue culture. In the rat, actively migrating cells have abundant F-actin-containing stress fibers and numerous cytoplasmic extensions of the plasmalemma. These features, and ultimately cell migration, are inhibited by calmodulin inhibitors and cytochalasin B. In the rabbit, migrating cells are devoid of stress fibers and cytoplasmic extensions. Cell migration is not inhibited by calmodulin inhibitors but is arrested by cytochalasin B. The cell-to-substrate adhesion is reduced by calmodulin inhibitors in both rat and rabbit. These findings corroborate our earlier observations in organ culture studies and support the view that corneal epithelial cell migration is calmodulin-dependent in the rat, while it is not in the rabbit. The complete blockage of migration in both species by cytochalasin B suggests that actin polymerization is critical for corneal epithelial locomotion in both species.

Actins↗

PubMed1984

Fibronectin in developing rabbit cornea.

Fibronectin is believed to be important in tissue morphogenesis. We examined the distribution of fibronectin in developing rabbit cornea by immunohistofluorescence. Cryostat sections of cornea from 13, 15, and 20-day-old fetuses, 3-day neonates, and adults were incubated with affinity-purified fluoresceinated guinea pig anti-rabbit fibronectin antiserum (aFN). aFN bound to components within the presumptive stromal region and along the basal surfaces of corneal and lens epithelia during early stages of mesenchymal invasion. At 15 days of gestation, fluorescence was associated with the stromal extracellular matrix of the cornea, the subepithelial zone, and the lens capsule. In the 20-day fetus an intense aFN fluorescence was present along the inner corneal stromal border coincident with the formation of Descemet's membrane. Fluorescence within the corneal stroma appeared as fine lines, restricted to the collagen lamellae, remaining through birth and disappearing in the adult. Although stromal fluorescence disappeared in the adult, Descemet's membrane continued to fluoresce, albeit to a lesser extent. The results of our studies indicate the presence of fibronectin in developing rabbit cornea. Because fibronectin is important to cell adhesion in vitro, and because intercellular and cell-extracellular matrix interactions, including adhesion, are necessary for tissue morphogenesis, our observation suggests that fibronectin plays an important role in corneal morphogenesis.

Animals↗

PubMed1984

Beta-adrenergic and serotonergic stimulation of rabbit corneal tissues and cultured cells.

The adult rabbit cornea synthesizes cyclic AMP in response to both serotonin and isoproterenol. The authors have examined the postnatal development of these pathways and attempted to localize the responsive cell type(s) by dissection, cell culture, and surgical denervation. Full thickness corneas of neonatal rabbits have beta-adrenergic responses similar to the adult but fail to respond to serotonin until the animals are 9-12 weeks old. When adult corneas are separated into epithelia, stromal, and endothelial layers, only the stromal layer synthesizes cyclic AMP in response to serotonin, whereas all layers respond to isoproterenol. When grown in tissue culture, keratocytes, epithelial, and endothelial cells are unresponsive to serotonin but respond to isoproterenol. Neither adrenergic nor sensory denervation abolishes the corneal adrenergic or serotonergic response pathways. These results indicate that the epithelial cells do not contain the serotonin stimulated, cyclic AMP-mediated pathway as originally postulated. The cell population that does contain this pathway is within the stroma and may be the Schwann cells.

Animals↗

PubMed1983

A microassay for UDP-glucose dehydrogenase.

An assay for UDP-glucuronic acid [J. Singh, L. R. Schwarz, and F. J. Wiebel, Biochem. J. 189, 369-372 (1980)] has been utilized for determining UDP-glucose dehydrogenase activity. The assay for UDP-glucuronic acid, a product of UDP-glucose dehydrogenase, is based on the fluorometric determination of D-glucuronosyl benzo(a)pyrene. This compound is formed from UDP-glucuronic acid and 3-hydroxybenzo(a)pyrene in a reaction catalyzed by the glycuronosyl transferase of guinea pig microsomes. Unreacted 3-hydroxybenzo(a)pyrene is removed by extraction with chloroform-methanol, and the amount of gluconosylbenzo(a)pyrene formed is determined fluorometrically. Because this assay for UDP-glucose dehydrogenase is about 500 times more sensitive than spectrophotometric assays, it can be used to measure the amount of enzyme extractable from milligram quantities of connective tissue. Some kinetic properties of UDP-glucose dehydrogenase extracted from rabbit tissue have been determined. No evidence of different forms of the enzyme in rabbit liver, cartilage, or corneal stroma was found.

Animals↗

PubMed1983

Proteoglycan changes during restoration of transparency in corneal scars.

Corneal scars generated in rabbits by penetrating wounds are initially opaque but become transparent within a year. Previous studies have shown that the corneal stroma consists of proteoglycans and collagen fibrils spaced at regular intervals and that the interfibrillar spaces, the presumed location of proteoglycans, are abnormally large in opaque scars. In the present study, the size and glycosaminoglycan composition of the corneal stromal proteoglycans were determined in corneal scars during the restoration of transparency. The results showed that initially opaque scars which contained the large interfibrillar spaces also contained unusually large chondroitin sulfate proteoglycans with glycosaminoglycan side chains of normal size. These opaque scars also lacked the keratan sulfate proteoglycan but did contain hyaluronic acid. In the 1-year-old scars there was a restoration of normal interfibrillar spacing, and a return to corneal stromal proteoglycans of normal size and composition. These correlations suggest that the corneal stromal proteoglycans may play a fundamental role in regulating corneal collagen fibril spacing.

Animals↗

PubMed1983

Effects of prednisolone and medroxyprogesterone on corneal wound healing, ulceration, and neovascularization.

Albino rabbits were treated four or six times daily with 1% prednisolone acetate, 1% medroxyprogesterone acetate, or a control vehicle, after one of three conditions. First, after 3-mm linear perforating stromal incisions; drugs were given for seven days, and wound bursting strength was determined. Prednisolone suppressed wound tensile strength by 20%; medroxyprogesterone suppressed it by 11%. Second, after trephination, drug administration for 14 days decreased collagen formation in the scar buttons by 43% in the prednisolone-treated group and 39% in the medroxy-progesterone-treated group. Third, after thermal burns; when drug application followed the burn immediately, deep ulceration or perforation developed in 85% of the controls, in none of the prednisolone-treated group, and in 17% of the medroxyprogesterone-treated group. When drug delivery was withheld until day 6, severe ulceration developed in 44% of both groups. In both experiments, stromal neovascularization was markedly suppressed by prednisolone, but only moderately decreased by medroxyprogesterone.

Animals↗

PubMed1983

Proteoglycans of rabbit cornea: labelling in organ culture and in vivo.

Rabbit corneas maintained with radioactively-labelled precursors in organ culture for up to 42 hr produced labelled proteoglycans of the same kind as those that exist normally or that are produced by labelling in vivo. Whole corneas, including a narrow strip of sclera, were kept in culture in the presence of [3H]-glucosamine and [35S]-sulfate. The rate of incorporation of sulfate into extractable proteoglycans was linear over the time investigated, as was the rate of incorporation of glucosamine after a short lag. Three labelled proteoglycans were isolated and found to behave in ion-exchange chromatography and gel chromatography in the same way as they did in previous studies by chemical analysis. Their labelled glycosaminoglycans were primarily dermatan sulfate and keratan sulfate, with traces of hyaluronic acid and heparan sulfate. When labelled precursors were injected directly into the anterior chamber of rabbit eyes, the resulting labelled proteoglycans were similar to those obtained in organ culture. Both in vivo and during organ culture, the specific activity of hexosamine in the keratan sulfate proteoglycans was about one-half that in dermatan sulfate, probably because of different synthetic rates or different specific activities of immediate precursors.

Animals↗

PubMed1983

Morphogenesis of rabbit corneal stroma.

Corneas of fetal and young albino rabbits were examined by light and transmission electron microscopy. In addition, DNA and hydroxyproline content were measured in developing stroma. The results were compared with similar data from healing corneas in adult rabbits and from developing corneas of other animal species. In the fetal rabbit, the prospective corneal stroma region contains an unorganized, sparse extracellular matrix until about the 13th day of gestation, when mesenchymal cells and capillaries from the hyaloid vessels move in to form the vascular pupillary membrane, endothelium, and stroma. Stromal growth is due to alteration in the density and morphology of the cell population early in development, along with a sequential thickening and thinning of the whole stroma. These events are similar to those reported in primates, but differ markedly from those reported in avian species. Normal developing cornea and healing adult cornea both involve migration of stomal fibroblasts and deposition of extracellular matrix. Stromal fibroblasts in the rabbit fetus are oriented with their long axis parallel to the corneal surface early in development compared with randomly oriented fibroblasts in the early healing wound of adult rabbit corneas. Although collagen and cell number progressively increase throughout the developmental periods studied, the ratio of cells to collagen is high initially but decreases with development. In contrast, the proportion of cells to collagen in the young scar tissue of adult cornea is low initially, indicating a marked deposition of collagen in comparison to that in the early normal developing stroma. The results suggest that the healing tissue differs from the normal fetal stroma in its coordination of cell population growth with collagen deposition and cellular organization.

Animals↗

PubMed1982

Scanning electron microscopy of rabbit corneal scars.

Central full-thickness perforating excision wounds were made in rabbit corneas and were examined by light and scanning electron microscopy at various times after wounding to study the three-dimensional morphologic changes in the tissue during healing and remodeling. Formation of a fibrin clot soon after wounding seals the hole and functions as a substrate for the healing epithelium. Changes in the histologic appearance of the fibrin lot immediately below the new epithelium are followed by migration of adjacent stromal cells under the epithelium, parallel to the basal surface of this tissue. Further healing is characterized by the organization of stromal fibroblasts into several layers parallel to the corneal surface and the deposition of collagen as a matted meshwork of fibrils tangential to the cell surface. Although remodeling of the collagenous matrix of corneal scar is evident and the scar eventually appears less opaque, the lamellae of the scar are narrower and shorter than normal. Evidence from this and other studies suggests that the orientation of the fibroblasts in healing tissues is determined by the organization of the newly formed epithelium. Furthermore, our observations are consistent with the hypothesis that collagen fibrils are deposited parallel to the flat surface of the fibroblasts during scar formation. Subsequent reorganization of this collagenous matrix approaches the normal lamellar appearance, but the matrix fails to regenerate even after 2 years.

Animals↗

PubMed1982

The healing of linear nonperforating wounds in rabbit corneas of different ages.

Linear nonperforating incisions were made in the corneas of 2-week-old and 2-year-old rabbits. The resulting wounds were examined by light microscopy and transmission and scanning electron microscopy. A corneal incision of a 2-week-old rabbit produced a wide gaping wound caused by retraction of the cut stromal lamellae away from the incision. The wound became wider with time as the developing eye enlarged and the cut lamellae retracted further. Polymorphonuclear leukocytes, presumably from the tear film, penetrating into the wound area before it was covered over by the sliding epithelium. Most of the leukocytes disappeared by 3 days after wounding. Three to six layers of fibroblasts appeared beneath the epithelial plug. The tissue eventually rebuilt approximately one third of the corneal depth lost to the wound. The stroma of the wounded region did not return to its normal width, but the epithelium was thicker than that of the unwounded cornea. An incision in a 2-year-old rabbit cornea produced a narrow V-shaped wound that did not change shape with time. This wound was repaired by fibroblasts resulting in collagenous repair tissue being the same depth as the normal stroma. There appears to be no evidence for wide gaping wounds in humans in the literature, as was found in this study in rabbits.

Age Factors↗

PubMed1981

Quantitative analysis of collagen from normal developing corneas and corneal scars.

We measured the relative solubility of collagen in acetic acid after pepsin digestion and tentatively identified the types of collagen present in corneas of rabbits of various ages and in corneal scar tissue, using hydroxyproline assays and polyacrylamide gel electrophoretic analyses. More than 80% of the collagen in normal developing rabbit cornea was soluble after pepsin treatment; no more than 45% of that in two-week-old corneal scars was soluble. The predominant collagens in normal cornea and healing tissue were types I and AB. Type AB increased from 6% of the total collagen in fetal cornea to 11% in cornea from young adults. Collagen from two-week-old corneal wounds contained 16% type AB. Corneal type AB collagen was less soluble and more resistant to degradation by mammalian collagenase than was type I collagen. Unlike the normal cornea, in healing tissue the relative rate of synthesis of type I to type AB collagens did not correspond to their deposition. These results suggest a basic alteration in the molecular structure of the corneal scar, which may be instrumental in preventing the healing tissue from producing a normal, functioning organ.

Age Factors↗

PubMed1980