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PubMed1981

Ultrahistochemical studies on tangential sections of the trabecular meshwork in normal and glaucomatous eyes.

The composition of the extracellular material of the cribriform meshwork was compared in five normal and 13 glaucomatous eyes of the same age group (58 to 70 years). Enzymatic digestion and histochemical methods applicable to electron microscopy were used. In both groups of eyes the ground substance was sensitive to chondroitinase ABC, and also the other methods showed no qualitative differences between the groups. In contrast, the fibrillar components of the extracellular material, which partly could be studied only after enzymatic treatment, showed qualitative differences between normal and glaucomatous eyes. In addition to the normal fibrous components, the glaucoma tissue contained large amounts of very fine fibrils. In seven eyes with end-stage glaucoma these fibrils filled the whole cribriform region. In these cases collagen fibers also appeared. Whether these were formed from the fine fibrils is not clear.

Aged↗
PubMed2003

CNP-induced changes in pHi, cGMP/cAMP and mRNA expression of natriuretic peptide receptors in human trabecular meshwork cells.

It has been demonstrated that natriuretic peptides lower intraocular pressure, however, the underlying cellular mechanism(s) mediating this response remain(s) to be determined. The purpose of this study was to investigate the effects of C-type natriuretic peptide (CNP) on pH(i), cGMP/cAMP and expression of atrial natriuretic peptide receptor (NPR-A), brain natriuretic peptide receptor (NPR-B) and C-type natriuretic peptide receptor (NPR-C), in HTM cells. At concentrations of 10(-7) M, CNP caused an acidification of pH(i). In addition, CNP caused a dose-dependent increase in cGMP formation and inhibition of forskolin-stimulated cAMP accumulation. These changes were not significantly altered in the absence of 10(-3) M isobutylmethylxanthine (IBMX). Treatment with the NPR-A antagonist, anantin, produced no influence on basal cGMP/cAMP levels, the CNP-stimulated cGMP accumulation and CNP-induced inhibition of forskolin-stimulated cAMP accumulation. However, CNP-induced reduction of forskolin-stimulated cAMP accumulation was inhibited by pretreatment with pertussis toxin (PTX). Furthermore, NPRB receptors were predominantly expressed and pretreatment with CNP (10(-7) M, 24hr) enhanced all NPR mRNAs expression which was not altered by higher concentrations or longer incubation. Results demonstrate that NPR-A, NPR-B and NPR-C receptors' expression can be up-regulated by CNP treatment. CNP activates NPR-B receptors preferentially to increase cGMP accumulation and acts through the PTX-sensitive cAMP-signaling pathway leading to a decrease in pH(i).

Cell Culture Techniques↗
PubMed2006

Constitutive signalling pathway activity in trabecular meshwork cells from glaucomatous eyes.

We recently described an IL-1-regulated stress response specific to the eye's aqueous outflow pathways that is diagnostic of glaucomas of diverse etiology. The goal of this study was to further identify IL-1-regulated signalling pathways in normal TM cells and determine whether their activity is altered in glaucomatous TM cells. Activity of the MAPK, p38, and JNK signalling pathways, represented by protein kinases ERK1/2, p38, and JNK-1, was followed by western blotting using antibodies specific for the active phosphorylated forms, after treatment of normal (N=5) or glaucomatous (N=5) cell lines by IL-1. Active forms of each of these kinases could be detected in normal and glaucomatous cells prior to treatment. When normal cells were stimulated with exogenous IL-1, an increase in activity of each of the kinases was observed. In contrast, treatment of glaucomatous cells with IL-1 resulted in little or no change in kinase activity. This difference was shown to be statistically significant by use of the paired two-tailed Student's t-test. Interference with IL-1 autocrine signalling in glaucomatous cell lines by treatment with IL-1 receptor antagonist (IL-1ra) had no effect on constitutive p38 or JNK activity (ERK was not examined). The results suggest that the MAPK, p38 and JNK signal transduction pathways are relatively unresponsive in glaucomatous cells as compared to normal cells. These results provide new information about the behaviour of glaucomatous TM cells, which may be important for understanding the pathophysiology of high-tension glaucoma.

Cadaver↗
PubMed1997

The effect of cytochalasin D on outflow facility and the trabecular meshwork of the human eye in perfusion organ culture.

PURPOSE: To determine the effect of cytochalasin D on outflow facility in the human anterior segment, and the histologic changes that accompany the effect. METHODS: Human anterior segments were studied in perfusion organ culture. The anterior segment from one eye received cytochalasin D, and that from the fellow control eye received vehicle; doses ranged from 0.06 mg/ml to 27.7 mg/ml. The duration of action and the effect of repeated doses were studied, and the accompanying histologic changes were assessed in 12 pairs of anterior segments. RESULTS: Cytochalasin D in concentrations of 0.6 mg/ml and 1.1 mg/ml caused increases in outflow facility of 42% and 37%, respectively (P < 0.05), with a peak effect 2 to 6 hours after infusion and a duration of action of approximately 14 hours. Anterior segments were not responsive to repeated doses (24 hours apart). Compared with the effect of vehicle in control anterior segments, cytochalasin D caused scattered breaks in the inner wall endothelial lining of Schlemm's canal (4.6 +/- 2.5% versus 0.7 +/- 0.6%; P = 0.02; anterior segments fixed during maximum drug effect). No increase in the amount of optically empty space within the juxtacanalicular tissue was seen. Inner wall breaks persisted, even in eyes in which the outflow facility had returned to baseline; the basement membrane and subendothelial matrix of the inner wall remained intact. Final intraocular pressure was inversely correlated with the length of optically empty space immediately adjacent to the inner wall. CONCLUSIONS: Cytochalasin D can increase outflow facility in the anterior segment of the human eye and causes ruptures of the inner wall of Schlemm's canal. These breaks persist, even when interocular pressure returns to baseline; the basement membrane and subendothelial matrix of the inner wall appear to remain intact. The final intraocular pressure was inversely correlated with the length of optically empty space immediately adjacent to the inner wall.

Aged↗
PubMed1997

Keratan sulphate in the trabecular meshwork and cornea.

PURPOSE: A study was made of the distribution of keratan sulphate in the human anterior chamber. METHODS: The monoclonal antibody, 5-D-4, was used in immuno-electron microscopy to visualise keratan sulphate distribution in the anterior chamber of 16 normal eyes, 7 Fuchs' dystrophy corneas, and a macular dystrophy cornea. RESULTS: Keratan sulphate was detected in normal human aqueous humour and also on the surface of trabecular cells in the uveal meshwork. Normal corneal stroma showed an increase in keratan sulphate labelling from anterior to posterior, with marked labelling in the posterior region of Descemet's membrane. The apical surface of the corneal endothelium labelled positively, but showed considerable variation in the level of labelling from cell to cell. The macular dystrophy cornea had the classic histopathological features of a type I case, including a highly abnormal Descemet's membrane. No keratan sulphate was detected in the macular dystrophy patient's corneal stroma or serum. The Fuchs' endothelial dystrophy corneas showed a normal distribution of keratan sulphate labelling in the stroma. The Fuchs' endothelial cells labelled for keratan sulphate but were highly abnormal in appearance, often exhibiting long filopodia and appearing to be actively migrating. CONCLUSIONS: This work has shown that keratan sulphate has a much wider distribution than was previously believed. The detection of keratan sulphate on the trabecular and endothelial cell surfaces also suggests a possible role for this molecule in cell adhesion and/or migration.

Antibodies, Monoclonal↗
PubMed2005

Characterization of free-floating spheres from human trabecular meshwork (HTM) cell culture in vitro.

It has been observed in several tissues that direct isolation of cells in serum-free media and on nonadhesive substrates results in the formation of spherical clusters of cells known as free-floating spheres. Such free-floating spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Our goal was to isolate and characterize such free-floating spheres from HTM cell primary cultures. For this purpose, HTM cells were incubated in serum-free media and on a nonadhesive substrate. Individual free-floating spheres generated in these conditions were isolated in 96-well plates, and their proliferative capacity was evaluated by monitoring their size increase over time. The expression of the TM markers, MGP and CHI3L1, was examined using recombinant adenoviruses containing the respective promoters. Morphology of the free-floating spheres was analysed in semithin sections, and the gene expression profile was obtained using Human Genome U133 Plus 2.0 Affymetrix microarrays. HTM cells incubated in serum-free media and on nonadhesive substrate generated free-floating spheres that could be grown for more than 3 months. Addition of serum to the culture media promoted the attachment of the spheres to the substrate, migration of cells from the spheres, and differentiation into cells phenotypically similar to normal TM cells. Gene profiling analysis demonstrated strong similarities between the gene expression profiles of the spheres and HTM cell monolayers. Both infection with the recombinant adenoviruses and gene array analysis demonstrated the expression of CHI3L1 and MGP, indicating that free-floating spheres likely originate from HTM cells. Gene array analysis also showed expression of the marker for neural precursor cells nestin, as well as leukemia inhibitory factor, a gene involved in the maintenance of the undifferentiated state of progenitor cells. Analysis of semithin sections indicated that these TM free-floating spheres were highly dynamic structures demonstrating a distinct radial gradient of cell proliferation, survival, and apoptosis. Extensive up- and down-regulation of gene expression was associated with the processes of sphere attachment and cell migration after the addition of serum. These results suggest that HTM primary cultures might contain relatively undifferentiated or progenitor cells. The availability of TM progenitor cell cultures could constitute a useful tool to investigate cell therapy approaches targeting the TM in glaucoma.

Adipokines↗
PubMed1975

Demonstration of acid mucopolysaccharides in the trabecular meshwork of the Rhesus monkey.

The ability to demonstrate AMPS in the trabecular region in the normal eye of the Rhesus monkey was shown to be critically dependent upon technical variation. Staining the fixed specimen prior to dehydration and embedding permits the uniform demonstration of AMPS in the trabecular region of the Rhesus monkey and shows it to be hyaluronidase-sensitive. Electron microscopy using the modified technique shows the reaction products to be present within the trabecular band, the intertrabecular spaces, and the canal of Schlemm. More impressive distribution was seen in the basement membrane of trabecular endothelium intimately related to the cell wall and in the ground substance and basement membrane of the endothelium of the inner wall of the canal Schlemm. The technique is also successful in the human eye and suggests a greater abundance of trabecular AMPS in open-angle glaucoma.

Animals↗
PubMed1994

[Histopathological study of trabecular meshwork after trabeculotomy in monkeys].

Trabeculotomy is a commonly indicated surgical procedure for primary open angle glaucoma and congenital glaucoma. In order to clarify the mechanisms of lowering the intraocular pressure (IOP) following the intervention, we performed this procedure in monkey eyes, and did postoperative histopathological examinations using light microscopy, and scanning and transmission electronmicroscopy for trabecular mesh work specimens incised at various time intervals up to one year after operation. Immediately after the operation, a direct communication was seen between the Schlemm's canal and the anterior chamber. Subsequently, the repairing process of trabecular tissue occurred initially in the corneoscleral and endothelial meshwork and finally in the uveal meshwork. At one year after operation the chamber angle was almost completely repaired by newly-formed trabecular tissue which was identical to the normal trabecular tissue. There was no direct communication between the Schlemm's canal and anterior chamber at that time. These results suggest that the decreased resistance for the aqueous outflow might be caused by direct communication between the Schlemm's canal and anterior chamber at an early postoperative stage, and the repairing process of the endothelial meshwork increases the resistance of aqueous outflow.

Animals↗
PubMed2000

Histopathological Features of the Trabecular Meshwork in a Case of Presumed Bilateral Chandler Syndrome.

Background: We describe a patient who had no-table decrease in the number of corneal endothelial cells in both eyes and developed open angle glaucoma without evident iris atrophy and peripheral anterior synechia.Case: A 74-year-old woman. The trabeculectomized angle tissue and iris tissue of her left eye were observed under light and transmission electron microscopy. From Schwalbe's line to the anterior chamber angle, degenerated endothelium-like cells were observed and overgrowth of layers of collagen fiber with varied cycles and basement membrane-like material were noticed. In addition, the morphology of intra-trabecular space was atrophic and occlusive with marked degeneration and exfoliation of endothelial cells in the trabecular space. On the side of the anterior chamber, degenerated endothelium-like cells were observed, the morphology of which was considered to result from abnormal metabolic function in corneal endothelium. Overgrowth of basement membrane-like material onto iridic tissue was not observed.Conclusion: Although slit lamp examination revealed no obvious abnormality, the result of histological examination suggested that this was a case of Chandler's syndrome in a broad sense or was in the early stage of this disease. We also discussed differential diagnosis from other diseases.

Journal Article↗