Johannes W. Rohen

Quantitative analysis of ‘plaque material’ in the inner- and outer wall of Schlemm's canal in normal- and glaucomatous eyes

The amount of plaque material derived from the sheaths of the elastic-like fibers in the cribriform layer of the trabecular meshwork (sheath-derived or SD plaques) was quantitatively evaluated in normal eyes and in trabeculectomy specimens of chronic simple, intermittent angle closure, and pseudoexfoliation glaucoma. The amount of SD plaque material increases with age but no correlation between age and SD plaques was found in any of the glaucomas evaluated. In comparison to normal eyes, the amount of SD plaque material was significantly greater in cases of chronic simple glaucoma and in cases of intermittent narrow angle glaucoma, but not in pseudoexfoliation glaucoma. In the latter, number and distribution of SD plaques resembled those of normal eyes. SD plaques were also found in the outer wall of Schlemms canal. In normal eyes there was a correlation between outer-wall plaques and age as well as between inner- and outer-wall plaques. These correlations were not found in the different types of glaucoma. Our findings indicate that in addition to the age-related increase in plaque material, there is in glaucomatous eyes some plaque material which is distributed unevenly in the inner- and outer-wall of Schlemms canal. This additional material was not found in pseudoexfoliation glaucoma, indicating that elevated intraocular pressure alone can not be responsible for plaque formation.

Why is Intraocular Pressure Elevated in Chronic Simple Glaucoma?: Anatomical Considerations

Ultrastructural analysis of 400 trabeculectomy specimens of glaucomatous eye revealed three types of extracellular deposits within the cribriform layer of the trabecular meshwork. One of these derives from the sheath of the subendothelial elastic-like fibres. Tissue culture and ultrahistochemical studies led to the assumption that these deposits contain glycoproteins, probably secreted by the cribriform layer cells.

Contractile cells in the human scleral spur

The scleral spur in 37 human (age 17-87 years) and six cynomolgus monkey eyes (2-4 years) was investigated. Serial meridional and tangential sections were studied with ultrastructural and immunocytochemical methods. The bundles of the ciliary muscle do not enter the scleral spur, but their tendons, which consist of elastic fibres join the elastic fibres in the scleral spur. Within the scleral spur a population of circularly oriented and spindle-shaped cells is found. In contrast to the ciliary muscle cells, the scleral spur cells form no bundles, but are loosely aggregated. They have long cytoplasmic processes and are connected to each other by adherens-type and gap junctions. They stain intensely for alpha-smooth muscle actin, myosin and vimentin. In contrast to the ciliary muscle cells, they do not stain for desmin. Ultrastructurally, the scleral spur cells contain abundant thin (actin) filaments, but do not otherwise show the typical ultrastructural features of ciliary muscle cells. The scleral spur cells do not express a complete basal lamina. They form individual tendinous connections with the elastic fibres in the scleral spur, which are continuous with the elastic fibres of the trabecular meshwork. The scleral spur cells are in close contact with nerve terminals containing small agranular (30-60 nm) and large granular (65-110 nm) vesicles but also with terminals containing small granular (30-60 nm) vesicles which are regarded as typical for adrenergic terminals. We conclude that the scleral spur cells are contractile myofibroblasts. Their contraction might influence the rate of the aqueous outflow.

Production of glycosaminoglycans by cell cultures of the trabecular meshwork of the primate eye

Abstract Monolayer cultures derived from the isolated trabecular meshwork tissue of the eyes of three monkeys have been established as primary cultures (designated as TS and TI) and as regularly subcultivated cultures (designated as TI5) for time-periods ranging from 6 months to 3 years. These cells produce glycosaminoglycans which are secreted into the media. Sodium [ 35 S]-sulfate or [ 14 C]glucosamine were used as radioactive precursors for the study of glycosaminoglycan synthesis. As revealed by electrophoretic separation of the labeled glycosaminoglycans on cellulose acetate strips and by enzymatic characterization, TS- and TI-cultures incorporated the labeled glucosamine mainly (between 60 and 85%) in hyaluronic acid and chondroitin-4-sulfate (between 11 and 34%), while TI5-cultures incorporated glucosamine predominantly into hyaluronic acid (63%) and dermatan sulfate (31%). [ 35 S]sulfate was mainly incorporated by TS- and TI-cells into chondroitin-4-sulfate and by TI5-cells into dermatan sulfate. Under the assumption that these glycosaminoglycans are also synthesized by the trabecular meshwork in situ, it is suggested that the gel-like properties of hyaluronic acid and the ion-binding capacity of the sulfated glycosaminoglycans might have a function in the filtering mechanism of the aqueous humor outflow.

Outflow facility studies in the perfused human ocular anterior segment.

We have recently developed a tissue model of the human aqueous outflow pathway involving placement of the eviscerated anterior corneoscleral shell, [with lens and uveal tissue removed but trabecular meshwork (TM) attached] onto a specialized perfusion apparatus. The TM and associated outflow tissues are perfused with culture medium at a physiologically-relevant perfusion pressure in a 5% CO2 environment at 37 degrees C. Under these conditions, the perfused outflow tissues are similar for several days, to the human and/or subhuman primate outflow system in vivo with regard to morphology as well as several functional parameters. Measured facility of outflow (0.271 +/- 0.018 microliters min-1 mmHg-1, n = 79) is similar to facility values obtained by tonography in living human beings. Moreover, outflow facility decreases in a linear fashion with increased perfusion pressure by 1.4% mmHg-1. Finally the removal of the TM results in a 41% decrease in measured outflow resistance. The ability to study viable human outflow tissue for at least several days and the opportunity to establish a model which serves as an alternative to animal testing, point to the potential importance of this technique in investigating the biology of the aqueous outflow system.

Outflow facility studies in the perfused bovine aqueous outflow pathways

We have recently developed a technique for constant pressure perfusion of the aqueous outflow pathway of the eye. Our preliminary studies, conducted in the calf eye, show surprisingly that the manipulations necessary for preparing the outflow pathways and attached corneoscleral shell for perfusion do not greatly disrupt normal aqueous outflow physiology and anatomy according to the following criteria: 1. facility of outflow is similar before and during outflow pathway perfusion 2. as in the intact eye, facility of outflow decreases with increased IOP 3. removal of outflow resistance tissue greatly increases facility of outflow 4. morphology of outflow tissues remains normal Use of the perfused outflow pathway model may enable the creation of valuable in vitro preparations which may provide much needed information about the pathogenesis of primary open-angle glaucoma.

Synthesis and composition of glycosaminoglycans by cultured human trabecular meshwork cells

A human trabecular meshwork cell line with a limited number of population doublings was established in monolayer culture. All cultures produced hyaluronic acid, heparan sulfate, chondroitin sulfate, and dermatan sulfate. Following [14C]-glucosamine incorporation into proliferating (phase II) cultures, 70%–80% of the medium glycosaminoglycan label was found in hyaluronic acid and 8%–14% in heparan sulfate. About 60% of the cell-bound activity was associated with hyaluronic acid and 30% with heparan sulfate.Long-term cultivation under nondividing (“senescent”) conditions resulted in a change of the pattern of synthesized and excreted (medium) [14C]-glucosamine-labeled glycosaminoglycans with a relative decrease of hyaluronic acid and a relative increase of heparan sulfate.

Age-related changes in the composition of proteins in the trabecular meshwork of the human eye

The composition of the trabecular meshwork proteins of human eyes ranging in age from 36 days to 84 years was examined by polyacrylamide gel electrophoresis and amino acid analysis. Proteins of different molecular weights could be extracted from the tissue with acetic acid. Although their electrophoretic patterns became less distinct with increasing age, proteins of molecular weights ranging from 50 000 to 69 000 always prevailed. The amino acid compositions of the acetic acid-insoluble trabecular meshwork residues revealed the prevalence of collagenous proteins. The peptide maps produced by treatment with cyanogen bromide indicate that most of the fragments solubilized from the trabecular meshwork of younger eyes are derived from type I collagen. Beyond 40 years of age, the trabecular meshwork was resistant to cyanogen bromide and pepsin digestion. A rough estimate of the distribution of collagen types in the trabecular meshwork was based on 3-hydroxyproline/4-hydroxyproline ratios, indicating an age-related increase of type I collagen from about 55 to 70 per cent, and of type IV collagen from about 2 to 5 per cent of the total protein present. During ageing, some of the protein-bound methionine is oxidized to methionine sulfoxide, reaching about 35 per cent of the total methionine content at the age of 20 years and, with a slower rate of oxidation, a mean value of 40 per cent at 80 years of age. Electron-microscopic analysis of specimens remaining undissolved after cyanogen bromide cleavage and pepsin treatment no longer revealed regular collagenous fibrils but rather elastic-like fibers surrounded by wide sheaths consisting of fine fibrils with a regular cross-banding periodicity of 40-50 nm. In addition, clusters of so-called curly (lattice) collagen were found. The amino acid composition of this insoluble material suggests that altered collagen-like molecules prevail among the proteins of the residues.

Quantitative analysis of ‘plaque material’ between ciliary muscle tips in normal- and glaucomatous eyes

Between the anterior ciliary muscle tips in normal and glaucomatous eyes, different forms of plaque material were found within the intermuscular connective tissue. These plaques derive from either the sheaths of the elastic-like fibers or the elastic tendons of the anterior ciliary muscle tips. In addition, there are plaques which have the characteristics of the hyalinized basement membranes of the trabecular beams. The quantitative evaluation of all three types of ciliary muscle (c.m.) plaques showed that in normal eyes the amount of c.m. plaques increases with age and correlates with the amount of plaques in the inner wall of Schlemms canal. In cases of chronic simple glaucoma the amount of c.m. plaques did not significantly correlate with age, but the absolute amount of this material was significantly higher than in normal eyes. No correlation between inner wall and c.m. plaques was found, indicating that in glaucoma additional factors besides age contribute to plaque formation.

Scanning electron microscopic study on the vasculature of the human anterior eye segment, especially with respect to the ciliary processes

The architecture of the vasculature of the human anterior eye segment was studied by scanning electron microscopy of vascular resin casts. Regarding the major vessels it was found that the perforating branches of the anterior ciliary arteries (ACA) form an anastomozing circle which lies in the posterior portion of the ciliary muscle (intramuscular circle). The ACAs supply the outer and posterior parts of the ciliary muscle, partly the iris, and the peripheral choroid by recurrent ACA branches. The major arterial circle of iris (MACI) which lies more anteriorly is formed mainly by the long posterior ciliary arteries and supplies the inner and anterior portion of the ciliary muscle, the iris and the ciliary processes. The ciliary process vasculature consists of three different vascular territories with discrete arterioles and venules. The first vascular territory which is located at the anterior end of the major processes, is drained posteriorly by venules which pass the ciliary body without greater connections to the venules of the major ciliary processes. The second and third territories comprise the vasculature of the major and minor ciliary processes drained posteriorly by venules which are located at the margin of the ciliary processes. In supravital experiments with human autopsy eyes, a characteristic segment of arterioles supplying the first and second territory was found to be constricted after immersion with epinephrine in a similar way as in cynomolgus monkeys in vivo. Although the general arrangement of the ciliary process vessels is similar to that of the cynomolgus monkey, there are characteristic differences in the size of the territories and in the pattern of the capillary networks. The division of the ciliary process vasculature into three vascular territories may reflect a functional differentiation in the process of aqueous humour production.