Contaminaçäo bacteriana em olhos doadores: estudo comparativo entre pacientes em coma "depassée" e cadáveres / Bacterial contamination in donor eyes: a comparative study between coma \"depassée\" e cadáveres

Estudaram-se a contaminaçäo bacteriana e antibiograma de prováveis doadores de córnea em 2 grupos: pacientes em coma "depassée" e cadáveres. Encontraram-se maior contaminaçäo e predominância de bactérias Gram negativas nos cadáveres. A resistência aos antibióticos de expectro mais amplo foram os aminoglicoideos e o perfloxacin

Resistência da córnea após ceratomia radial: estudo experimental em coelhos / Cornea resistance after radial keratomy: experimental study in rabitts

Estudo experimental prospectivo a resistência corneal após a ceratomia radial em coelhos. Foram utilizados 13 animais da raça cinzento europeu. Empregamos, em todos os casos, a mesma técnica e instrumental cirúrgico. As alteraçöes na resistência, sofridas pela córnea foram estudadas e registradas por intermédio de um diagrama tensäo deformaçäo. A análise estatística envolveu o test "t" para amostras pareadas, baseado na distribuiçäo t de Student. Esta análise envolveu a diferença entre os valores da Tensäo de Ruptura no olho operado e no olho näo operado. O teste "t", para amostras pareadas, mostrou ser significante a reduçäo da resistência da córnea após esta cirurgia refrativa

Córnea / Cornea

A córnea, rica em sinais e sintomas (relacionados alta sensibilidade e perdas de transparência), fundamental na óptica ocular ("in situ"), seu poder dióptrico mais do que o dobro do cristalino, ainda que, curiosamente, quando isolada, no ar, represente quase que apenas uma lâmina de faces paralelas) e estrutura em que as patologias säo detectadas e diferenciadas por minucias de observaçäo merece dos oftalmologistas uma atençäo especial. Progressos recentes nos conhecimentos sobre a fisiologia, etiopatogenia, diagnóstico e terapêutica das afaces da córnea traduzem a necessidade de suas atualizaçöes. Embora necessariamente sintética, a revisäo traz elementos variados sobre tais informaçöes e fornece uma extensa bibliografia com a qual os aprofundamentos, de cada tópico apresentado seräo possíveis

Immunohistochemical detection of bFGF and TNF-alpha in the course of inflammatory angiogenesis in the mouse cornea.

Tumor necrosis factor alpha (TNF-alpha) and basic fibroblast growth factor (bFGF) have been assigned a major role in the induction of angiogenesis. By performing immunohistochemical studies of cauterized mouse corneas, the in situ distribution of bFGF and TNF-alpha in the course of inflammatory neovascularization was investigated. bFGF was found throughout the corneal epithelium of untreated and cauterized eyes. However it was only rarely detected in the endothelium or in infiltrating cells before the onset of neovascularization. TNF-alpha was not expressed by any cell before the ingrowth of new blood vessels, which took place within 36 hours after cauterization. It is concluded that the release of these cytokines by infiltrating cells is of minor or no importance for the induction of sterile inflammatory angiogenesis.

A comparative biochemical and ultrastructural study of proteoglycan-collagen interactions in corneal stroma. Functional and metabolic implications.

1. Corneas of mouse, rat, guinea pig, rabbit, sheep, cat, dog, pig and cow were quantitatively analysed for water, hydroxyproline, nucleic acid, total sulphated polyanion, chondroitin sulphate/dermatan sulphate and keratan sulphate, several samples or pools of tissue from each species being used. Ferret cornea was similarly analysed for water and hydroxyproline on one pool of eight corneas. Pooled frog (38) and ferret (eight) corneas and a single sample of human cornea were qualitatively examined for keratan sulphate and chondroitin sulphate/dermatan sulphate by electrophoresis on cellulose acetate membranes. Nine species (mouse, frog, rat, guinea pig, rabbit, sheep, cat, pig and cow) were examined by light microscopy and six (mouse, frog, rat, guinea pig, rabbit and cow) by electron microscopy, with the use of Alcian Blue or Cupromeronic Blue in critical-electrolyte-concentration (CEC) methods to stain proteoglycans. 2. Water (% of wet weight), hydroxyproline (mg/g dry wt.) and chondroitin sulphate (mg/g of hydroxyproline) contents were approximately constant across the species, except for mouse. 3. Keratan sulphate contents (mg/g of hydroxyproline) increased with corneal thickness, whereas dermatan sulphate contents decreased. The oversulphated domain of keratan sulphate was absent from mouse and frog corneas, increasing as percentage of total keratan sulphate with increasing corneal thickness. Sulphation of dermatan sulphate was essentially complete (i.e. one sulphate group per disaccharide unit). 4. Chondroitin sulphate/dermatan sulphate proteoglycans were present at the d bands of the collagen fibrils of all species examined, orthogonally arrayed, with high frequency, and occasionally at the e bands. Keratan sulphate proteoglycans were present at the a and c bands of all species examined, but with far higher frequency in the thicker corneas, where keratan sulphate contents were high. 5. Alcian Blue CEC staining showed much higher sulphation of keratan sulphate in thick corneas, e.g. that of cow, than in thin corneas, e.g. that of mouse, in keeping with biochemical analyses. 6. It is suggested that the constancy of interfibrillar volumes is regulated via the swelling and osmotic pressure of the interfibrillar polyanions, by adjustment of the extent of sulphation in two independent proteoglycan populations, to achieve an 'average sulphation' of the total polyanion similar to that of fully sulphated chondroitin sulphate/dermatan sulphate. 7. The balance of synthesis of the two kinds of proteoglycans may be determined by the O2 supply to the avascular cornea. O2 supply may also determine the conversion of chondroitin sulphate into dermatan sulphate.

Calibration of measurements in vivo of fluorescein in the cornea.

Quenching of fluorescence of fluorescein is not observed with broad field fluorophotometers. Fluorophotometric equipment which measures the fluorescence in a tiny spot has, however, been reported to underestimate the molarity of fluorescein in the rabbit corneal stroma by as much as a factor of two. In this experiment, quenching was measured in the rabbit cornea with two scanning fluorophotometers. The quenching was measured by four different techniques: (1) by elution of fluorescein, (2) by elution of albumin, (3) by polarization of fluorescence, and (4) by spectrofluorophotometry. It was estimated by all four methods that quenching in the living rabbit cornea with these instruments is approximately 20%. Taken together, the four experiments suggest that the quenching of fluorescence of fluorescein can be explained entirely on the basis of the interaction of fluorescein and albumin in the stroma.

Stimulatory effects of basic fibroblast growth factor on DNA synthesis in the human embryonic cornea.

First trimester human embryonic eye globes were micro-dissected so that a passage was opened between the outer environment and the anterior chamber, which rendered free access of tissue culture medium to the endothelial cell monolayer. The dissected eye globes were maintained in organ culture for 24h in the continuous presence of tritiated thymidine. Sections were cut through the whole eye globes and were subject to autoradiographic analysis in order to estimate the mitogenic response of human corneal endothelial cells to externally supplied growth factors and hormones. It was found that the corneal endothelial cells could be stimulated to initiate DNA synthesis by exposure to basic fibroblast growth factor (bFGF). The thymidine labelling index nearly doubled after bFGF addition. Northern blot analysis revealed the presence of bFGF transcripts in the embryonic eye. In contrast we were unable to trace any bFGF transcripts in other first trimester human embryonic organs. In an attempt to determine the topographical distribution of bFGF mRNA within the eye, we found that transcript levels were higher in the posterior regions of the eye globe. Immunostaining with the appropriate antibody showed conclusively that bFGF protein was present in both the anterior and posterior human eye. These results suggest that local production of bFGF may stimulate cell proliferation in vivo.

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.

Expression of two molecular forms of the complement decay-accelerating factor in the eye and lacrimal gland.

Complement is present in ocular fluids, but the molecular mechanism(s) restricting its activation to exogenous targets and not to autologous ocular cells are currently unknown. To clarify how this control is achieved, monoclonal antibody (mAb)-based techniques were used to examine the eye, the lacrimal gland, and ocular fluids for the decay-accelerating factor (DAF), a membrane regulatory protein which protects blood cells from autologous complement activation on their surfaces. Immunohistochemical staining of tissue sections revealed DAF antigen on corneal and conjunctival epithelia, corneal endothelium, trabecular meshwork, and retina, as well as on lacrimal gland acinar cells and in adjacent lumens. By flow cytometry, cultures of conjunctival epithelium exhibited the highest DAF levels and levels on corneal epithelium greater than corneal endothelium greater than conjunctival fibroblasts. Biosynthetic labeling of corneal endothelium yielded de novo DAF protein with an apparent molecular weight (Mr) of 75 kD, approximating that of blood cell DAF protein, and digestions of conjunctival epithelium with phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme which cleaves glycoinositolphospholipid membrane anchors, released approximately 70% of the ocular surface DAF protein similar to leukocyte surface DAF protein. Quantitations of DAF by radioimmunometric assay employing mAbs against two DAF epitopes revealed 325 ng/ml (n = 12), 4.8 ng/ml (n = 10), and 22.0 ng/ml (n = 8) of soluble DAF antigen in tears, aqueous humor, and vitreous humor, respectively. Western blot analyses of the tear DAF antigen revealed two DAF forms, one with an apparent Mr of 72 kD resembling membrane DAF forms in other sites, and a second with an apparent Mr of 100 kD, which is previously undescribed. Since DAF activity is essential physiologically in protecting blood cells from autologous complement attack, the identification of DAF on the ocular surface, intraocularly, in the lacrimal gland, and in tears suggests that DAF-mediated control of complement activation is also required in these locations.

Characteristics and in vivo occurrence of type VIII collagen.

Type VIII collagen was isolated from bovine Descemet's membranes by pepsin treatment and salt fractionation, as described by Kapoor et al. [(1986) Biochemistry 25, 3930-3937]. Contaminating type IV collagen was removed by ion-exchange chromatography. Purified type VIII collagen consisted of two different polypeptide chains and, compared to the fiber forming collagens, showed a higher thermal stability. Corresponding fractions isolated from pepsinized human Ewing's sarcoma and fetal calf aorta reacted immunologically with a protein of similar molecular mass. After extraction of Descemet's membranes with guanidine hydrochloride, a peptide of about 60 kDa was obtained. This seems to be the tissue form of type VIII collagen.

Alpha-1 proteinase inhibitor levels in keratoconus.

The levels of alpha 1-proteinase inhibitor (alpha 1-antitrypsin) in keratoconus, normal human, and other diseased corneas were examined. Using an immunoperoxidase technique, the presence of this inhibitor was demonstrated in the epithelium, stroma and endothelium of all corneal sections. Compared with normal human controls, the staining intensity in the epithelium and stromal lamellae of keratoconus corneas was markedly reduced. Such a reduction was not seen in either scarred or other diseased corneas. Extracts of keratoconus and normal human corneas were subsequently analyzed for alpha 1-proteinase inhibitor by a dot blot assay using a monoclonal antibody against the inhibitor and a 125I-labelled secondary antibody. In agreement with the immunohistochemical findings, the alpha 1-proteinase inhibitor level found in the epithelium of keratoconus corneas was approximately one-fourth of that found in normal human controls. In addition, the stromal extracts of keratoconus corneas contained about one-sixth the inhibitor level of that in normal human extracts. These results lend further support to the hypothesis that degradation processes may be aberrant in keratoconus.

Isoforms of corneal keratan sulfate proteoglycan.

Bovine corneal keratan sulfate proteoglycan was found to contain three major protein components. Two proteins (37 and 25 kDa) were released from the proteoglycan by endo-beta-galactosidase, N-glycanase, or chemical deglycosylation. A smaller protein (20 kDa), not covalently linked to keratan sulfate, co-purified with the proteoglycan by conventional and high performance ion exchange chromatography, by ethanol precipitation, and by affinity purification on columns of monoclonal antibody to keratan sulfate, but could be separated from the proteoglycan by gel filtration chromatography in dissociative agents. The three proteins produced different fragmentation patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after digestion with V8 protease, and each had unique two-dimensional tryptic peptide maps. The N-terminal amino acid sequence of the core proteins differed. In addition, the proteoglycans containing these proteins differed in molecular size, suggesting different levels of glycosylation of the two core proteins. Similarity of the core proteins was suggested by similar amino acid composition, similarities in tryptic maps, and antigenic cross-reactivity. Corneal keratan sulfate proteoglycan, therefore, seems to occur in two different, but related, forms whose core proteins may represent members of a homologous family.

Neutral glycosphingolipids isolated from porcine corneas.

Six neutral glycosphingolipids were isolated from porcine corneas using silicic acid column chromatography and preparative thin-layer chromatography. Five of these glycolipids were partially identified by gas-liquid chromatography. Two were glucosylceramides, two were lactosylceramides and one was tetrahexosylceramide containing galactose, glucose and N-acetylgalactosamine in the molar ratio of 2:1:1. Glucosylceramides were found to be the predominating component, with lactosyl- and tetrahexosylceramides being the minor constituents. Sphingosine was the major long-chain base in all fractions. The fatty acids of the corneal neutral glycosphingolipids were variable in chain length. This represents the first investigation of neutral glycosphingolipids in corneas of any species.

Flow cytometric DNA analysis of corneal epithelium.

We have modified an existing technique in order to perform DNA analysis by flow cytometry (FCM) of corneal epithelium from the mouse, rat, chicken, rabbit, and human. This protocol permitted an investigation of human corneal scrapings from several categories: normal, aphakic bullous keratopathy (ABK), keratoconus (KC), Fuch's dystrophy, edema, epithelial dysplasia, and lipid degeneration. No abnormal characteristic cell-kinetic profile was detected when averaged DNA histograms were compared statistically between the normal and either ABK, KC, edema, or Fuch's dystrophy groups. Abnormal DNA histograms were recorded for cell samples that were taken 1) from three individuals who had epithelial dysplasia and 2) from one individual diagnosed with lipid degeneration. The former condition was characterized by histograms that had a subpopulation of cells with an aneuploid amount of DNA or had higher than normal percentages of cells in the S and G2 + M phases of the cell cycle. Corneal cells from the patient who had lipid degeneration had an abnormally high percentage of cells in the G2 + M phases of the cell cycle. The availability of accurate DNA flow cytometric analysis of corneal epithelium allows further studies on this issue from both experimental and clinical situations.

12(R)-hydroxyeicosatetraenoic acid, an endogenous corneal arachidonate metabolite, lowers intraocular pressure in rabbits.

12(R)-hydroxyeicosatetraenoic acid (12(R)HETE) is an endogenous corneal epithelial arachidonic acid metabolite formed by the cytochrome P450 system and a potent inhibitor of Na(+)-K(+)-ATPase activity. We studied the effect of topically applied 12(R)HETE, either derived endogenously from corneal epithelium or synthetically prepared, on the IOP of the rabbit eye and compared it to its stereoisomer 12(S)HETE. Topical application of 1 microgram of biologically derived 12(R)HETE to both eyes of rabbits resulted in a marked reduction in IOP: a reduction of 4-7 mmHg occurred within 30-120 min. The IOP reduction effect of a single application of 12(R)HETE was long-lasting (9 days), whereas no effect on IOP was found for the vehicle control. Using synthetic compound, we demonstrated that the effect of 12(R)HETE on IOP is dose-dependent. Single topical application of 1, 10, and 50 micrograms of 12(R)HETE caused a reduction in IOP of 4, 6, and 12 mmHg, respectively. The stereoisomer, 12(S)HETE, did not have any effect on IOP at doses up to 5 micrograms. The IOP reduction effect of 12(R)HETE was not associated with hyperemia, appearance of flare, miotic response, or increased protein concentration of the aqueous humor. This study was the first to demonstrate that an endogenous inhibitor of Na(+)-K(+)-ATPase generated by the corneal epithelium potently and specifically lowers IOP in rabbits. Further studies are needed to elucidate the mechanism by which 12(R)HETE lowers IOP.

Acid hematin pigmentation of the cornea in stillborn fetuses.

During the postmortem histopathologic evaluation of eyes from stillborn fetuses we noted the presence of a prominent undescribed corneal pigment in 18 of 55 stillborn fetuses. The corneal pigment was frequently associated with documented meconium-stained amniotic fluid, and in no instance was a stained cornea coupled with recorded clear amniotic fluid. Pigmented corneas came from stillborn fetuses with a longer duration of intrauterine death than nonstained corneas. The pigment stained black with the Fontana-Masson stain, was birefringent, and treatment of tissue sections with 5% potassium permanganate and 5% oxalic acid as well as with saturated alcoholic picric acid solution removed the pigment indicating that it is acid hematin. The most likely cause of the acid hematin-stained corneas was tissue acidity created in utero with prolonged intrauterine death.

Diamines and aminoalcohols: neutral solvents for native collagen.

A substantial fraction of interstitial collagens (types I, III, V and VI) can be dissolved from finely divided, connective tissues by homogenization in 0.5-1 M ethylene diamine and certain other organic amines neutralized by hydrochloric or other acids to pH 7-8.5. The amount of collagen solubilized from the tendons and other tissues of young animals is similar to that extracted by dilute acetic acid. In ethylene diamine hydrochloride solutions, native type I collagen denatures at 38 degrees and it has a sedimentation coefficient similar to that measured in acidic citrate solutions. From these amine hydrochloride solutions native collagen fibrils can be regenerated simply by tenfold dilution with water or by dialysis. The form of these precipitates is determined by temperature, pH, enzyme pretreatment of the collagen, and the dialysate salt concentrations and composition. Weak gels can be formed with less than 0.1 mg collagen per ml. Regenerated matrices containing types V and VI collagens, and with different fibril sizes can be used to support cell growth or to form sheets or hydrated gels possibly suitable for prosthetic uses.

The comparative chemical morphology of the mammalian cornea.

A simple model of mammalian corneal stroma has been tested against biochemical and ultrastructural measurements performed on a number of species. Contents of water, collagen and total sulphated polyanion were constant, as predicted from the model. Alcian blue CEC results showed great variability between species, with a rise in CEC as corneal size and thickness increased. These variations were due to changes in keratan sulphate content, and particularly to its oversulphated terminal domain, which is absent from mouse cornea. The increase in keratan sulphate content with corneal thickness was balanced by an increase in dermatan sulphate in thin corneas, thus maintaining total sulphated GAG levels at a constant "average", in all the mammals investigated. This balance is probably decided by oxygen tension, which will vary with corneal thickness.

Beta adrenergic binding sites in the human eye: an autoradiographic study.

Beta adrenergic binding sites were localized and characterized in the human eye by means of "in vitro" autoradiography, using [125I] (-) iodocyanopindolol (125ICYP) as radioligand. Binding sites were visualized by apposition of isotope sensitive film to slide mounted eye sections. Receptor sites were present in the extraocular muscles, in the conjunctiva, in the epithelium and endothelium of the cornea, in the trabeculum and in the ciliary muscle. They were also present in the lens epithelium and in the retina. The pigmented ocular structures were heavily labelled but the binding was nonspecific. Characterization of these binding sites was achieved by testing the ability of selective adrenergic compounds to displace 125ICYP binding. These studies suggested that the majority of adrenergic binding sites in nonpigmented structures of human eye were of a beta2 type.

Binding of Pseudomonas aeruginosa to neutral glycosphingolipids of rabbit corneal epithelium.

35S-labeled Pseudomonas aeruginosa isolates were shown to bind to neutral glycosphingolipids (NGSLs) of rabbit corneal epithelia in culture by a thin-layer chromatogram overlay procedure. The lipids of the corneal epithelial cells grown in culture were extracted and partitioned into a chloroform-rich lower phase containing NGSLs and an aqueous upper phase containing gangliosides. By using a dot-blot assay, at least six times more radiolabeled P. aeruginosa isolates were shown to bind to the lipids in the lower phase compared with those in the upper phase. Thin-layer chromatography of the lower-phase lipids followed by staining with an orcinol spray revealed at least 10 NGSL components and several fast-migrating, nonglycosylated neutral lipid components (including cholesterol). 35S-labeled P. aeruginosa was shown to bind to NGSL components 1, 2, 5, 6, and 9. P. aeruginosa-reactive NGSL components 6 and 9 migrated with chromatographic mobilities similar to those of the standards ceramide trihexoside (CT) and ceramide monohexoside, respectively. Components 1 and 2 migrated slightly ahead of asialo GM1, and component 5 migrated faster than globoside but slower than CT. Among the various standards tested, P. aeruginosa bound to asialo GM1 and, to a lesser extent, to ceramide dihexoside and CT but not to GM1, GD1A, GM3, or ceramide monohexoside. It remains to be determined whether any of the five P. aeruginosa-reactive NGSL components of corneal epithelium identified in this study plays a role in the development of corneal infection. However, we have previously shown that component 9, one of the five P. aeruginosa-reactive NGSL components identified in this study, is present in significantly greater amounts in migrating epithelia than it is in nonmigrating epithelia (N. Panjwani, G. Michalopoulos, J. Song, G. Yogeeswaran, and J. Baum, Invest. Ophthalmol. Vis. Sci., in press). This may prove to be of biological significance because it is generally believed that traumatized (migrating) epithelia are more susceptible to infection than normal (nonmigrating) epithelia are.