Regional glycosaminoglycans composition of the human sclera.

Human sclera from different parts of the normal adult eye was analyzed for content of uronic acid and the distribution of specific glycosaminoglycan types. Uronic acid is widely determined as representative of glycosaminoglycans in biological substances and variations of about 2-fold were found. Differences of up to 50% were detected in the relative abundance of hyaluronic acid and of dermatan sulphate, depending on the site on the globe from which the sample was taken. Sclera from around the papilla was found to be rich in content of dermatan sulphate. Sclera from the posterior eyeball showed a higher percentage of chondroitin sulphate as compared with sclera from equator and limbus. Equatorial sclera was richer in hyaluronic acid than posterior sclera. Sclera from the posterior eyeball showed a higher content of uronic acid as compared with sclera from equator and limbus. This work is an addition to the understanding of the scleral biochemistry and this finding may have some relevance in connection with ocular disease, as the proteoglycans and their content of glycosaminoglycans play a key role in the regulation of collagen fibril assembly and in the biomechanical strength of collagen fibrils.

[Age-related changes in fibronectin of the eye outflow drainage system]. / Vozrastnaia dinamika fibronektina drenazhnogo apparata glaza.

The content of the fibronectin, an extracellular glycoprotein in the drainage outflow system of human eyes was determined by the indirect immunoperoxidase staining technique. The degree of fibronectin accumulation in ocular tissues was evaluated by quantitative morphometric analysis. It was shown that the fibronectin level was elevated in ageing. Increased deposit of fibronectin in trabecular tissues, mainly, in the inner wall of Schlemm's canal and juxta-canalicular zone was demonstrated along with ageing. Comparison of fibronectin accumulation in glaucoma and ageing support the idea that ageing is a risk factor of glaucoma.

The epithelial basement membrane zone of the limbus.

The basement membrane zone of the limbal epithelium adjacent to the cornea was examined by ultrastructural and immunohistochemical techniques to determine whether differences exist between this region and central cornea. In human limbus, the percentage of basal cell membrane occupied by hemidesmosomes was significantly less (14.9 +/- 3.5) than that in central cornea 27.9 +/- 9.2), whereas the area of basement membrane/100 microns of cell membrane did not differ significantly. In rabbits, both percentage of membrane occupied by hemidesmosomes and area of basement membrane were less in the limbal region. Comparison of laminin and type VII collagen (anchoring fibril collagen) localisation in limbus and in central cornea demonstrated that both matrix proteins had a more convoluted pattern of localisation in the limbus. In addition, short segments of basement membrane with associated anchoring fibrils were present in the zone between the basal cells' basement membrane and blood vessels. These areas of duplicated basement membrane with anchoring fibrils were separated from the epithelium by layers of extracellular matrix that included collagen fibrils. Scanning electron microscopy of the surface topography of human limbal and central corneal basement membrane, prepared by removal of the epithelium with EDTA, demonstrated that in the limbal zone between the Palisades of Vogt and cornea, a very rough undulating surface was present with papillae or 'pegs' of stroma extending upward, and that central cornea lacked such papillae. Rabbit limbal basement membrane surface showed no such papillae, only occasional indentations into the stroma.(ABSTRACT TRUNCATED AT 250 WORDS)

The autofluorescent products of lipid peroxidation may not be lipofuscin-like.

The fluorescent molecules of cellular age pigment granules (lipofuscin) are commonly thought to be end products of membrane lipid autoxidation. Lipofuscin fluorophores of the retinal pigment epithelium (RPE) appear to be derived from photoreceptor outer segment membranes. Experiments were therefore conducted to determine whether the in vitro oxidation of retinal homogenates would generate fluorophores similar to the naturally occurring lipofuscin fluorophores of the RPE. Neural retina and RPE-choroid homogenates from young (2-3 month old) albino rats were subjected to an iron-ascorbate-air pro-oxidant reaction medium, and compared to unoxidized control samples from young age-matched animals as well as senescent (24 month old) rats. In addition, neural retina and RPE-choroid homogenates from 3 month old albino rats were subjected to a 100% oxygen atmosphere to test whether the fluorescent products of autoxidation differ substantially from those generated in the pro-oxidant medium. The chloroform-soluble fluorophores of chloroform-methanol sample extracts were analyzed by corrected fluorescence spectroscopy and thin-layer chromatography (TLC). In vitro pro-oxidation of both the neural retina and the RPE from young rats produced blue-emitting fluorophores which differed from the orange- and yellow-emitting fluorophores extracted from the RPE of senescent rats. Corrected fluorescence spectroscopy of aged tissue extracts revealed vitamin A-related fluorescence (330 nm excitation maximum; 515 nm emission maximum) and a spectrally resolvable age-related fluorescence (420 nm excitation maximum; 600 nm emission maximum). Only the vitamin A-related fluorescence could be measured in the control of young samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localisation of tenascin during the development of scleral papillae and scleral ossicles in the embryonic chick.

We have used a polyclonal antibody against tenascin (a 240 kDa extracellular matrix glycoprotein) and indirect immunofluorescence to investigate the distribution of tenascin in cryostat sections of the chick sclera during the six stages of scleral papilla development (Murray, 1943) and formation of membrane bone anlagen (mesenchymal condensations between 6-12 days of incubation - HH Stage 29-37). At Stages 1 and 2, when the papilla was a slight thickening in the conjunctival epithelium and mesenchymal condensation formation was initiated, tenascin was sparse in the sclera. At Stage 3, when the papilla had an epithelial mass intruding into the mesenchyme, there was an accumulation of tenascin fibrils along the subsurface of the papilla with fibrils extending from this region into the mesenchymal condensation. Interestingly, tenascin was sparse between papillae and between mesenchymal condensations. The Stage 4 papilla had a similar localisation of tenascin fibrils and in addition fine arborizing fibrils of tenascin were observed within the basal epithelia of the papilla adjacent to the epithelial-mesenchymal interface (which suggested that the fibrils infiltrated through the basement membrane region). The Stage 5 and 6 papillae had a column of vertical tenascin fibrils extending from the subsurface of the papilla to the interior mesenchyme corresponding exactly to the location of the mesenchymal condensation which was then forming the anlage of the ossicle in a flat bed about 100 microns from the conjunctival surface. The column of tenascin disappeared as the osteoid appeared in the ossicular bed on the 12th day of incubation but a dense accumulation of tenascin remained along the subsurface of the papilla. With the exception of tenascin fluorescence in the basal region of the Stage 4 papilla, tenascin fibrils were not observed in the other stages of papilla development in the epithelium covering areas between the mesenchymal condensation. This restricted distribution of tenascin may be important in the morphogenesis of scleral papillae and scleral ossicles.

Age-related changes in protein profiles of the normal human trabecular meshwork.

The protein profile of the trabecular meshwork from 44 normal human eyes of various ages, ranging from 5 months to 87 years, was investigated by using sodium dodecyl sulfate-polyacrylamide-gel (SDS-PAGE) electrophoresis and a highly sensitive silver-stain technique. Samples from each eye were analysed individually to avoid anomalies that may occur with tissue pooling. More than 20 protein bands were consistently visible at all ages and varied in molecular weight from 20,000 to 290,000 MW. The shifts which occurred in band prominence with increasing age included a consistent decrease in the intensity of bands at MWs 42,000 (tentatively identified as G-actin) and 58,000; a noticeable increase in the intensity of the bands at MWs 140,000 and 160,000 that correspond to alpha I and II components of type IV collagen; and a gradual disappearance of a faint band at MW 68,000 in eyes older than 31 years. Our study shows that molecular aging does occur in the trabecular meshwork. The data presented also provides a basis for comparison of the polypeptide alterations that may occur in primary open-angle glaucoma and in other disease processes.

Ultrastructural localization of proteoglycans in tissue using cuprolinic blue according to the critical electrolyte concentration method: comparison with biochemical data from the literature.

Several connective tissues were stained for proteoglycans using the cationic dye Cuprolinic Blue according to the critical electrolyte concentration method. With this method, proteoglycans are visualized as electron-dense filaments. In most tissues, two types of proteoglycan filaments are present: a small (maximum length 60 nm), thin, collagen fibril-associated filament, and a thick, heavily-staining filament which is predominantly localized between bundles of collagen fibrils. Cartilage contains very large (about 300 nm) proteoglycan filaments while in cornea they are very small. Comparison with biochemical data from the literature suggests that the appearance of the proteoglycan filaments may be indicative for the glycosaminoglycan-protein ratio and for the molecular weight of the part of the protein core to which glycosaminoglycans are attached. The data thus obtained on the localization and structure of a proteoglycan may be useful when planning a strategy for its isolation.

Dermatan sulphate proteoglycans from sclera examined by rotary shadowing and electron microscopy.

Two dermatan sulphate-containing proteoglycans from bovine sclera were examined by rotary shadowing and electron microscopy, and the results were compared with previous biochemical findings. Both the large iduronate-poor proteoglycan (PGI) and the small iduronate-rich proteoglycan (PGII) possessed a globular proteinaceous region. Whereas PGI had a branched extension from the globular region, with five to eight side chains attached to it, PGII had only a single tail, which was of glycosaminoglycuronan. PGII aggregated via globular-region interactions, which were much diminished by reduction and alkylation. PGI aggregated via side chains and globular-region interactions. Although a few PGI aggregates were large, and similar to the hyaluronan-cartilage proteoglycan aggregates [Weidemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333], hyaluronan did not cause enhanced aggregation. PGII is very similar in shape to the small cartilage chondroitin sulphate proteoglycan, whereas PGI somewhat resembles the large cartilage chondroitin sulphate proteoglycan, although with many fewer glycosaminoglycan side chains, and probably only one globular region as opposed to two in the cartilage proteoglycan.

The dermatan sulfate proteoglycans of bovine sclera and their relationship to those of articular cartilage. An immunological and biochemical study.

Dermatan sulfate proteoglycans were isolated from adult bovine sclera and adult bovine articular cartilage. Their immunological relationships were studied by enzyme-linked immunosorbent assays using polyclonal antibodies raised against the large and small dermatan sulfate proteoglycans from sclera and a polyclonal and monoclonal antibody directed against the small dermatan sulfate proteoglycans from cartilage. The small dermatan sulfate proteoglycans from sclera and cartilage displayed immunological cross-reactivity while there was no convincing evidence of shared epitope(s) with the larger dermatan sulfate proteoglycans, nor did these larger proteoglycans share any common epitopes with each other. A hyaluronic acid binding region was detected immunologically on the larger scleral dermatan sulfate proteoglycan but was absent from the larger dermatan sulfate proteoglycan of cartilage and both the small dermatan sulfate proteoglycans. These antibodies were used in immunofluorescence microscopy to localize the scleral proteoglycans and molecules containing these epitopes in the eye. The large scleral dermatan sulfate proteoglycan was restricted to sclera while molecules related to the small scleral and cartilage proteoglycans were found in the sclera, anterior uveal tract, iris, and cornea. Amino acid sequencing of the amino-terminal regions of the core proteins of the small dermatan sulfate proteoglycans from sclera and articular cartilage showed that all the first 14 amino acids analyzed were identical and the same as reported earlier for the small bovine skin and tendon dermatan sulfate proteoglycans. These studies demonstrate that the larger dermatan sulfate proteoglycans of sclera and cartilage are chemically unrelated to each other and to the smaller dermatan sulfate proteoglycans isolated from these tissues. The latter have closely related core proteins and probably represent a molecule with a widespread distribution in which the degree of epimerization of glucuronic acid and iduronic acid varies between tissues.

Lipid pattern of plasma in the presence of episcleral lipid deposits.

Examination of 40 subjects aged under 70 with episcleral lipid deposits revealed an abnormal serum lipid pattern in 48% of all. No correlation was noticed between the number of lipid deposits and the raised plasma lipid concentration or the distribution of fatty acids (saturated, unsaturated).

Changes in the content and composition of collagen in the glaucomatous eye--basis for a new hypothesis for the genesis of chronic open angle glaucoma--a preliminary report.

The pressure theory is still predominant in explaining the pathophysiology of the chronic open angle glaucoma. An insufficient drainage system resulting in an increased intraocular pressure is the basis for this theory. The pressure will exert an effect upon the optic disc which either directly on the nerve fibres or indirectly via the vascular system will result in a characteristic optic atrophy. The collagen fibres, both in the trabecular meshwork of the anterior chamber and in the lamina cribrosa of the optic disc, form a mesh through which the aqueous humour and the nerve fibres, respectively, pass through the wall of the eye. A hypothesis explaining the pathophysiology of this disease, and based on the assumption that there is a primary change in the collagen molecules, resulting in a weaker structure than normal both in the trabeculae and in the laminae, is forwarded. The structures analysed for the content of hydroxyproline, hydroxylysine and proline were the trabecular meshwork, the sclera and the lamina cribrosa. Three categories of autopsy eyes were studied, i.e. normal eyes, glaucomatous eyes, and eyes under a suspicion of glaucoma. In the normal eyes, the collagen composition in the trabecular meshwork was different from that in the sclera and the lamina cribrosa. There is also a difference in the composition between the sclera and the lamina cribrosa. In glaucoma, the content and/or the composition of the collagen molecules in the lamina were significantly changed. In the eyes under suspicion of glaucoma the same changes as in the glaucomatous eyes could be demonstrated. However, 5 of the 7 eyes in this category had no demonstrable nerve atrophy. The findings suggest that the change in collagen pattern is primary. This study has not demonstrated which types of collagen are present or the physical properties of this collagen. Further tests to demonstrate the different types of collagen and their rigidity are planned.

The collagens of the developing bovine cornea.

The morphology of the developing bovine eye has been examined and the collagens in fetal bovine eyes from three months' gestation to maturity have been solubilized by pepsin treatment and analyzed to determine the ratios of the predominant types of collagen. The type I collagen decreased, while the type V collagen increased with age. Type III collagen comprised less than 1% of all the corneas, except for the three-month fetal calf. The anterior to posterior thickness of the paraffin-embedded fetal calf cornea increased from the third to the seventh month, decreased from the seventh month to birth, and then increased after birth. Descemet's membrane increased in thickness with age. Analysis of dissected regions of the calf cornea showed a uniform distribution of the collagen populations from the center to the limbus (89% type I, 10% type V and less than 1% type III collagen) and uniformity through the depth of the stroma, except that type III was concentrated around Bowman's layer, and type IV in Descement's membrane. The localization of the different collagens was consistent with the immunofluorescent staining studies with anticollagen antibodies, but the ratios of the intensities of the fluorescence did not correspond to the quantitative analyses. These results are concordant with other studies that have shown that antibody binding may be masked or diminished in certain tissues and therefore immunofluorescence cannot be used reliably for quantitative measurements.

Characterization of collagen from normal human sclera.

Human scleral tissue contains approximately 50% collagen by weight, consisting predominantly of type I collagen. There is little or no evidence for the presence of substantial quantities of type II, type III or other collagen types. There appears to be no difference in either collagen content or genetic type in sclera between adult and juvenile tissues or between anterior and posterior segments of the sclera, although, with increased age there is a marked increase both in the extent of glycosylation of the collagen and its resistance to solubilization by treatment with pepsin.

Regulation of type I collagen fibril assembly by link protein and proteoglycans.

Link protein, a glycoprotein, that is present both in cartilaginous and non-cartilaginous tissues, has previously been shown to bind to collagen and to proteoglycan. Here, we have examined the effects of link protein and proteoglycans, both alone and in combination, on the assembly of type I collagen fibrils in vitro. Link protein alone had no effect on the kinetics of fibril formation or on the size of the fibrils. Link protein, however, modulated the effects of various proteoglycans including those from bone, cartilage, cornea and sclera. Link protein had the most significant effect on fibril assembly in the presence of the low molecular weight bone proteoglycan. Although the bone proteoglycan alone had no effect on fibril formation, the fibrils were wider in the presence of link protein and proteoglycan. Cartilage proteoglycan alone increased the extent of fibril formation and the resultant fibrils were wider in diameter with a complement of incompletely assembled fibrils. In the presence of both link protein and cartilage proteoglycan, the fibrils were fully formed with the characteristic banding pattern. Further, corneal and scleral proteoglycans alone decreased the extent of fibril formation and the width of the fibrils was either unaltered or slightly decreased in the presence of the link protein. Our results indicate that both link protein and tissue-specific proteoglycans may regulate the organization of collagen fibrils in tissues.

Glucocorticoid target cells in human outflow pathway: autopsy and surgical specimens.

Target cells for glucocorticoids were found in trabeculectomy specimens obtained surgically from humans with glaucoma (primary and secondary) and from nonglaucomatous autopsy eyes using an autoradiographic technique. Specific nuclear localization of 3H-dexamethasone was found in cells of the trabecular meshwork, scleral fibroblasts, and in the endothelial lining of both Schlemm's canal and the outflow vessels. Glucocorticoids may alter the outflow facility by a direct effect on the metabolism of these cells. The autoradiographic method is suitable for studying competitors of glucocorticoid binding in small surgical specimens of human tissue involved in the outflow of aqueous humor.

Episcleral and subconjunctival lipid deposits. One year follow-up.

A series of 39 subjects, aged under 70, had the numbers of lipid globules counted in the slit lamp within 4 regions of the right eye and 4 regions of the left. The counting was repeated after an averaged period of 12 months (range 7-20 months). The investigation disclosed that the number of lipid globules rises. No instances were detected of a fall in their number, allowance being made for errors of measurement, assessed on the basis of blind double counting. The degeneration is not positively correlated to xanthelasma, diabetes mellitus, or presenile arcus.

Prevalence of episcleral and subconjunctival lipid deposits in Eskimos.

Slit lamp examinations of 340 Eskimos in Greenland revealed lipid deposits in 12%. The prevalence was significantly lower than in a Copenhagen series of Caucasians (45% out of 689 subjects) recorded for 10-year age groups. The numbers of lipid deposits seemed to be equal in lipid-positive subjects of the 2 ethnic groups, but the globules were more often localized temporally and more rarely superiorly in Eskimos than in Caucasians. The relatively rare occurrence of lipid deposits in Eskimos may possibly be due to the special composition of lipids on the blood and the rare occurrence of arteriosclerosis in Eskimos.

Phase transitions in ocular tissue - NMR and temperature measurements.

The human cornea, sclera and lens is studied by nuclear magnetic resonance (NMR) measurements and temperature curves. The bound water fractions of these tissues have been determined as 3% in the cornea, 20% in the sclera and 17% in the lens. In contrast to other investigations we found freezing intervals near -1 degree C.