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PubMed1999

Na-K-Cl cotransport in normal and glaucomatous human trabecular meshwork cells.

PURPOSE: Previous results from this laboratory showed that intracellular volume of trabecular meshwork (TM) cells is regulated by the Na-K-Cl cotransport system. Other studies suggest that TM cell volume, in turn, is a determinant of permeability across the TM. Given that a decrease in outflow facility across the TM is thought to be the primary cause of elevated intraocular pressure in primary open-angle glaucoma, the present study was conducted to investigate the possibility that Na-K-Cl cotransport function may be altered in glaucomatous TM cells compared with normal TM cells. METHODS: Normal and glaucomatous human TM cells were cultured from donor eyes and trabeculectomy specimens, respectively. Trabecular meshwork cell monolayers were evaluated for Na-K-Cl cotransport activity, assessed as ouabain-insensitive, bumetanide-sensitive K influx using 86Rb as a tracer for K. Cotransporter protein expression was determined by western blot analysis, and intracellular volume was determined radioisotopically using [14C]urea and [14C]sucrose as markers of total and extracellular water space, respectively. RESULTS: Na-K-Cl cotransport activity of glaucomatous TM cells was found to be reduced by 32% +/- 2% compared with that of normal TM cells, whereas western blot analyses showed that cotransporter protein expression in glaucomatous TM cells was reduced by 64% +/- 14% compared with expression in normal TM cells. Also, exposure of normal TM cells to 10 microM norepinephrine or 50 microM 8-bromo-3',5'-cyclic adenosine monophosphate was found to diminish Na-K-Cl cotransport activity, whereas these agents were without effect on glaucomatous TM cell cotransport. Finally, resting cell volume of glaucomatous TM cells was found to be increased compared with that of normal TM cells, whereas intracellular volume of both cell types was reduced after exposure to 10 microM benzmetanide or 10 microM bumetanide. CONCLUSIONS: These findings indicate that Na-K-Cl cotransport function and regulation are altered in glaucomatous TM cells compared with that of normal TM cells. However, the observation that cell volume of glaucomatous TM cells is greater than that of normal TM cells, despite reduced Na-K-Cl cotransport activity, suggests that other volume-regulatory ion flux pathways may be involved in the reduced outflow of glaucoma.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1997

Dexamethasone decreases phagocytosis by human trabecular meshwork cells in situ.

PURPOSE: To determine the effect of dexamethasone on the phagocytic capacity of the human trabecular meshwork. A decrease in meshwork phagocytosis has been proposed in the pathogenesis of steroid glaucoma. METHODS: The anterior segments of seven pairs of human eyes were placed in perfusion organ culture. One eye of each pair received dexamethasone, and the fellow eye served as control. After 21 days, latex microspheres labeled with fluorescein isothiocyanate and coated with antibodies were added to the culture medium. Twenty-four hours later, the eyes were fixed, the trabecular meshworks were treated with a rhodamine-labeled secondary antibody and sectioned, and the number of ingested beads was determined using a laser scanning confocal microscope. Nuclei were counted and used to calculate the phagocytic index of each anterior segment (number of ingested beads divided by number of nuclei). RESULTS: Ingested beads appeared green and could be differentiated from noningested beads, which appeared red, using appropriate wavelengths of the laser. Bead ingestion was confirmed with electron microscopy and the use of secondary antibody labeled with horseradish peroxidase. Dexamethasone decreased phagocytosis by 57%, as shown by the fact that trabecular cells in dexamethasone-treated meshworks ingested significantly fewer beads than cells in fellow control meshworks (1.5 +/- 0.6 beads/cell versus 3.5 +/- 1.4 beads/cell; P = 0.008). No evidence of significant migration or loss of trabecular cells was noted; the number of trabecular cells appeared similar in dexamethasone-treated and control meshworks (144 +/- 36 versus 141 +/- 46). CONCLUSIONS: Dexamethasone inhibits phagocytosis by human trabecular meshwork cells in perfusion organ culture.

Adult↗

PubMedDate not supplied

Trabecular meshwork alteration and intraocular pressure change following pulsed near-infrared laser trabeculoplasty in cats.

BACKGROUND AND OBJECTIVE: To comparatively assess the safety and variation in intraocular pressure (IOP) of two pulsed near-infrared lasers (titanium:sapphire and alexandrite) for laser trabeculoplasty versus conventional blue-green argon laser trabeculoplasty in an animal model. MATERIALS AND METHODS: The left eyes of 15 healthy cats received a 180 degree laser trabeculoplasty treatment: 5 with a titanium:sapphire laser, 5 with an alexandrite laser, and 5 with an argon laser. Preoperatively and postoperatively, all animals underwent tonometry, gonioscopy, and slit-lamp examination. The cats were observed up to 12 weeks. Scanning electron microscopy and histologic examination were performed to evaluate potential alterations in the trabecular meshwork structure. RESULTS: IOP at 1 hour, 1 day, and 1 week following treatment was remarkably lower, irrespective of the laser source used. Following treatment with both near-infrared lasers, gonioscopy showed depigmentation underneath the area of the treated trabecular meshwork and histologic evaluation showed a decrease in pigment density. On scanning electron microscopy, damage to the trabecular meshwork structure could not be detected after treatment with near-infrared lasers. CONCLUSIONS: Near-infrared laser trabeculoplasty was found to be effective to temporarily lower IOP in cats. The lasers selectively altered pigment-containing cells, avoiding structural damage of the trabecular meshwork anatomy.

Animals↗

PubMed1989

The effect of epinephrine and benzalkonium chloride on cultured corneal endothelial and trabecular meshwork cells.

We evaluated the effect of dipivefrin hydrochloride, epinephrine hydrochloride, epinephrine borate and their respective vehicles with and without the preservative benzalkonium chloride, on the in vitro growth characteristics of human corneal keratocytes, endothelial cells and trabecular meshwork. Epinephrine hydrochloride and borate at low concentrations (0.0002%) significantly inhibited growth of both trabecular meshwork and corneal endothelial cells. Higher concentrations (0.02%) of these same drugs induced the same effect on the growth of keratocytes in vitro. Similar observations were made on the effect of dipivefrin hydrochloride on human corneal cells in vitro. Benzalkonium chloride alone was demonstrated to be responsible for the growth inhibitory effects on trabecular cells. The susceptibility of trabecular meshwork cells in culture to the commonly used ophthalmic preservative benzalkonium chloride is demonstrated.

Benzalkonium Compounds↗

PubMed1996

Human trabecular meshwork cell survival is dependent on perfusion rate.

PURPOSE: To determine whether suppression of flow may be detrimental to trabecular cell survival and to the morphologic characteristics of the trabecular meshwork. METHODS: The anterior segments of normal human eye bank eyes were placed in perfusion organ culture. The effect of various perfusion rates of culture medium, and of the constant flow and constant pressure methods of delivery of culture medium, were studied. Trabecular cell survival was determined by quantitation of cell nuclei in histologic sections and by morphologic observation. RESULTS: Trabecular meshworks with perfusion rates of 1 microliter/minute and higher had significantly more trabecular cells than meshworks with lower perfusion rates. A significant loss of trabecular cells was found in meshworks cultured with the constant pressure technique when compared with fellow eyes cultured with the constant flow of medium. Those constant pressure cultured meshworks with surviving cells had higher flow rates than those with necrotic cells. CONCLUSIONS: A minimum perfusion rate of 1 microliter/minute is required for trabecular cell survival in perfusion organ culture. Constant pressure perfusion of medium is unsuccessful in maintaining trabecular cells in long-term culture if low perfusion rates occur. Constant flow appears to mimic the in vivo situation more closely.

Adult↗

PubMed1997

Ultrastructural changes in the trabecular meshwork of human eyes treated with corticosteroids.

OBJECTIVES: To study the ultrastructure of the trabecular meshwork in human eyes with corticosteroid-induced glaucoma and to determine whether the changes noted also occur in the eyes of patients with primary open-angle glaucoma (POAG) who have been treated with corticosteroids. METHODS: The trabecular meshwork from 5 patients in whom corticosteroid-induced glaucoma was diagnosed and from 6 patients with POAG who had been treated with systemic or topical corticosteroids for months to years was investigated with light and electron microscopy. None of the eyes with POAG were considered to have corticosteroid-induced elevation of the intraocular pressure. RESULTS: Eyes with corticosteroid-induced glaucoma had the accumulation of extracellular material distinct from the sheath-derived plaques typical of POAG. A finger-printlike arranged material resembling basement membranes (FBM material), considered characteristic of corticosteroid-induced glaucoma, was found in all eyes with corticosteroid-induced glaucoma. In addition, an abnormal accumulation of densely packed, fine fibrils immediately beneath the inner wall endothelium of Schlemm's canal was present. The findings were similar among patients receiving topical or systemic treatment and among patients of different ages. In the eyes from donors with POAG who had been treated with corticosteroids, the fine fibrillar material and FBM material were present in small amounts in 3 of 6 donors and were not found in the other 3 donors. CONCLUSIONS: The extracellular material that accumulates in eyes with corticosteroid-induced glaucoma differs from that seen in eyes with POAG. Eyes with POAG exposed to long-term corticosteroid treatment did not all respond with the formation of the abnormal extracellular materials characteristic of those found in eyes with corticosteroid-induced glaucoma.

Adolescent↗

PubMed1996

Contractile response of the isolated trabecular meshwork and ciliary muscle to cholinergic and adrenergic agents.

To characterize the contractile properties of isolated trabecular meshwork strips, we measured the effect of various cholinergic and adrenergic substances on the contractility of trabecular meshwork (TM) strips in comparison with the effects on ciliary muscle (CM) strips. Using an electromagnetic force length transducer we performed measurements of isometric tension on isolated bovine TM and CM strips. Isolated strips were contracted by cholinergic agonists, the relative potency being carbachol > pilocarpine > acetylcholine. The half-maximal effective concentration was 2 x 10(-7) mol l(-1) for carbachol and 2 x 10(-6) mol l(-1) for pilocarpine. To characterize muscarinic receptors, we tested selective antagonists for M1 (pirenzepine) and M3 (4-DAMP). Pharmacologically, the functional muscarinic receptors are of the M3 subtype in TM as well as CM. The M1 subtype seems to be less important. The alpha 1-agonist phenylephrine was more effective in inducing contractions in TM than in CM. The alpha 2-agonist brimonidine induced contractions only in TM. In precontracted tissues the beta-agonist isoproterenol induced a relaxation in both tissues. This relaxation could be inhibited by metipranolol. Epinephrine (or dipivefrin) induced small contractions in TM and CM, which became more prominent, especially in TM, when the beta-adrenoreceptors were inhibited by metipranolol. The data indicate the presence of functional muscarinic, alpha-adrenergic, and beta-adrenergic receptors in bovine TM and CM. The contractile properties of TM and CM are differently modulated by the various drugs. Cholinergic and alpha-adrenergic agonists induced contraction, whereas beta-agonists induced relaxation.

Adrenergic Agents↗

PubMed1999

Modulation of myocilin/TIGR expression in human trabecular meshwork.

PURPOSE: To study factors that modulate myocilin/trabecular meshwork inducible glucocorticoid response protein (TIGR) mRNA expression in human trabecular meshwork (TM). METHODS: mRNA from fresh TM of four human donors, from perfused anterior segment organ cultured TM of three donors, and from four primary TM cell lines of different donors was isolated. The full length cDNA of myocilin/TIGR was cloned from TM mRNA using a polymerase chain reaction approach and used as probe for northern blot analysis hybridization. Trabecular meshwork cell cultures were treated with transforming growth factor (TGF)-beta1 (1 ng/ml), dexamethasone (10(-7) M), and mechanical stretch (10%). RESULTS: mRNA for myocilin/TIGR could be readily detected by northern blot analysis hybridization in 2 to 3 microg of total RNA from all fresh and all organ-cultured TM samples. In contrast, no mRNA for myocilin/TIGR could be detected in 20 microg of total RNA isolated from three different primary TM cell lines. Only one TM cell line had a baseline expression of myocilin/TIGR, which was 35- to 55-fold lower than that of fresh or organ-cultured TM samples. Treatment of TM cell cultures with dexamethasone for 1 day markedly increased expression of myocilin/TIGR mRNA, an effect that was even more pronounced after 3 days of treatment. Treatment with TGF-beta1 for 24 hours had no effect; however, after 3 and 12 days of treatment a 3.8- and 4-fold increase in myocilin/TIGR mRNA expression was observed. Expression of myocilin/TIGR mRNA was also increased after 10% mechanical stretch; however, in contrast to the effects of TGF-beta-1, this effect was observed much earlier (8-24 hours) after treatment. CONCLUSIONS: Dynamic mechanical stimuli maintain myocilin/TIGR expression in TM in situ and lack of these stimuli in monolayer cell cultures might be involved in downregulation of myocilin/TIGR expression.

Adult↗

PubMed1976

[Electron-microscopic delineation of acid mucopolysaccharides in the trabecular meshwork (author's transl)].

The presence of acid mucopolysaccharides in the trabecular meshwork was repeatedly investigated histochemically. The electron microscopical demonstration of glycosaminoglycanes with Ruthenium red was to be emphasized. In the material gained by trabeculectomy in 13 patients, a Ruthenium-positive, up to 30 nm thick film of varying degree could be demonstrated on the endothelium of the trabecular meshwork.

Connective Tissue↗

PubMed1990

[An ultrahistochemical study of the trabecular meshwork in normal and open-angle glaucomatous eyes].

The acid mucopolysaccharides (AMPs) in the human trabecular meshwork were studied ultrahistochemically with hyaluronidase and chondroitinase ABC digestion in 15 normal eyes and 27 cases of primary open-angle glaucoma (POAG). It was found that in normal eyes, hyaluronidase-sensitive AMPs existed in the connective tissue of cribriform meshwork and trabeculae. They could play an important role in regulating the aqueous outflow resistance. In POAG, the amount of AMPs in the trabecular meshwork was increased, leading to increased aqueous outflow resistance through the combination of hyaluronic acid with water, forming electron-dense "plaque" materials in a matrix of chondroitin sulfate.

Adolescent↗

PubMed1990

Characterization of lysosomal enzymes from cultured cynomolgus monkey trabecular meshwork cells.

The current study characterizes selected properties of lysosomal enzymes associated with cynomolgus monkey trabecular meshwork (MTM) cells. These proteins may participate in the turnover of macromolecules involved in regulating the aqueous outflow. Intracellular levels of lysosomal enzymes in MTM cells were similar to those found in cultured human fibroblasts. The presence of ammonium chloride increased the secretion rate of certain lysosomal enzymes from 47 to 122% of normal. Column chromatography of the secreted enzymes on the galactose-specific lectin Ricinus communis I demonstrated an increase in the number of accessible galactose residues on lysosomal enzymes secreted in the presence of ammonium chloride. The presence of mannose-6-phosphate receptors on the trabecular meshwork cells was demonstrated by the specific uptake of purified 125I-beta-D-glucosidase. This uptake represented 20% of that observed with cultured human fibroblasts and was inhibited only 50% by the presence of mannose-6-phosphate.

Ammonium Chloride↗

PubMed1994

Segmental variability of the trabecular meshwork in normal and glaucomatous eyes.

PURPOSE: Although numerous studies have examined morphologic changes in the trabecular meshwork of glaucomatous eyes, few have systematically looked for segmental variations around the circumference of the meshwork. If segmental variations occur, studies based on random sections or on single trabeculectomy specimens may not give a complete picture of the histologic changes in glaucoma. The purpose of this study was to determine the extent of segmental variability in human trabecular meshwork. METHODS: Eyes from five normal donors and eyes from five donors with primary open-angle glaucoma were obtained at autopsy. Histologic sections of each quadrant from nine of the normal eyes and eight of the glaucomatous eyes were analyzed. Light microscopy was used to measure the length of Schlemm's canal, the thickness of the trabecular lamellae, and the area of empty space in the juxtacanalicular (JCT) region. Transmission electron microscopy was used to examine the thickness of the JCT and the ultrastructural components of the JCT, which were quantitated with a computerized image analysis system. Ultrastructural analysis included JCT area, area of empty space, area of empty space touching Schlemm's canal, area of solid tissue, and area of sheath and tendon material. RESULTS: Differences among quadrants were qualitative rather than quantitative, because significant variations in structure were not found within single eyes. The variability among quadrants was similar for normal and glaucomatous eyes, with a coefficient of variation of approximately 20% for most tissue components. A greater variability was found for the thickness of the JCT. The largest variability was found for the area of empty space touching Schlemm's canal: 81.4% for normal eyes and 86.5% for glaucomatous eyes. No quadrant had consistently higher or lower values of any parameter. The length of Schlemm's canal did not differ significantly between normal and glaucomatous eyes (normal: 256.6 +/- 66.0 microns, glaucomatous: 298.8 +/- 57.7 microns), nor did the mean thickness of the trabecular lamellae (normal: 4.7 +/- 0.8 microns, glaucomatous: 4.8 +/- 0.5 microns). Light microscopy underestimated the total amount of empty space in the JCT by 20% compared with electron microscopy. CONCLUSIONS: Significant segmental differences were not found within single eyes in either normal or glaucomatous eyes. The magnitude of the variability of most tissue components was similar for normal and glaucomatous eyes. The most variable component was the area of empty space touching Schlemm's canal, which may represent the effective pathway for aqueous flow to enter Schlemm's canal. Future quantitative studies of the JCT should include samples from at least three quadrants per eye. Quantitative analysis of the JCT from a single quadrant, as occurs in the study of trabeculectomy specimens, may overestimate or underestimate the amount of solid tissue or empty space by 200%.

Aged↗

PubMedDate not supplied

Optineurin gene expression level in human trabecular meshwork does not change in response to pressure elevation.

Mutations in the gene optineurin (OPTN) have been associated with primary-open angle glaucoma. Here we present a study on the level of OPTN gene expression in the human trabecular meshwork in response to increased perfusion pressure in the anterior chamber perfusion model of the human eye. Perfusion pressure was raised from 10 to 30 mm Hg for periods ranging between 1 and 24 h. OPTN transcript levels in the trabecular meshwork were determined using real-time quantitative polymerase chain reaction. The results show no statistically significant alteration of the OPTN transcript level after raising the pressure. Moreover, no changes were detected in the transcript levels of the 3 known OPTN isoforms. This result shows that enhanced pressure levels do not lead to rapid changes in gene expression levels of OPTN in human trabecular meshwork. This suggests that alterations in OPTN gene expression are not involved in the mechanisms regulating aqueous humor outflow after an increase in intraocular eye pressure.

Aged↗

PubMed1987

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

Specimens from 17 human eyes, ranging in age from 3 to 80 years, were subjected to morphometric studies using light microscope, transmission electron microscope and scanning electron microscope, in order to clarify the age-related changes in the normal trabecular tissue. Statistical analyses showed that the cellularity in the various regions of the trabecular meshwork significantly declined with age. The spaces corresponding to the aqueous outflow pathway in each region of the meshwork also significantly decreased with age. On the other hand, extracellular materials significantly increased in amount with age in all regions of the trabecular meshwork. However, the decrease in the cellularity and the outflow pathway spaces did not show any statistically significant regional difference. These results suggest that general narrowing of the outflow pathway spaces due to the accumulation of extracellular materials with age is the cause of the increase in aqueous outflow resistance with age, and that each region of the trabecular meshwork is equally responsible for the increased resistance.

Adolescent↗

PubMed1999

Development of characterization of a immortal and differentiated murine trabecular meshwork cell line.

PURPOSE: To study mouse trabecular meshwork (TM) and to develop a murine TM cell line. METHODS: Mouse TM in situ was studied by light and electron microscopy (EM). In addition, TM was isolated from the H-2K(b)-tsA58 transgenic mouse strain in which promoter sequences of the major histocompatibility complex H-2Kb class 1 gene are fused to sequences of the SV40 mutant temperature-sensitive (ts) strain tsA58. The promoter is inducible by interferon (IFN)-gamma, and the tsA58 gene product is active at 33 degrees C (permissive conditions), but not at 37 degrees C (nonpermissive conditions). The TM explant was cultured in permissive conditions. Outgrowing cells were passaged through two rounds of single-cell cloning. One clonal cell line (MUTM-NEI/1) was characterized in nonpermissive conditions by EM, immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and northern blot hybridization. In addition, MUTM-NEI/1 cells were transfected with plasmid DNA. RESULTS: The mouse eye has a circumferentially oriented outflow vessel and a TM that is subdivided in an outer juxtacanalicular or cribriform part and an inner lamellated or trabecular part. From the TM of the H-2Kb-tsA58 mouse, a clonal cell line (MUTM-NEI/1) was established. In permissive conditions, MUTM-NEI/1 cells remained proliferative through at least 80 generations without change in phenotype. In nonpermissive conditions, proliferation was slower, and MUTM-NEI/1 cells differentiated and synthesized collagen types I, III, IV, and VI; laminin; and fibronectin. MUTM-NEI/1 cells were immunoreactive for vimentin, alphaB-crystallin, and neural cell adhesion molecule (NCAM), but not for desmin or cytokeratin. Less than 10% of MUTM-NEI/1 cells stained for alpha-smooth muscle actin, whereas after 3 days of treatment with transforming growth factor-beta1 almost all cells were positive. MUTM-NEI/1 cells expressed mRNA for NCAM, aquaporin 1, myocilin/trabecular meshwork glucocorticoid-inducible protein, and alphaB-crystallin, which was increased after oxidative stress. MUTM-NEI/1 cells could be successfully transfected with plasmid DNA. CONCLUSIONS: The architecture of the murine outflow system is comparable to that in primates. The MUTM-NEI/1 cell line is a clonal, immortal, and differentiated TM cell line that will be an important tool for study of the expression of TM genes.

Animals↗

PubMed1987

Age-related depletion of the cell population in the human trabecular meshwork.

The number of cells in the human trabecular meshwork was assessed for counting meshwork cell nuclei in semithin Araldite sections cut in the meridonal plane. The counts of nuclei decreased with increasing age and the decrease was associated with all regions of the meshwork. The appropriate regression equation and a mean length for meshwork nuclei was used to estimate cell numbers. At 20 years of age the estimated cell number for the whole meshwork was 763,000 which decreased to 403,000 cells by 80 years with a loss rate of 6,000 cells per year.

Adolescent↗

PubMed1984

Developmental immaturity of the trabecular meshwork in juvenile glaucoma.

We used light and electron microscopy to compare ten specimens of anterior chamber angle tissue obtained by trabeculectomy from seven patients with juvenile glaucoma (primary glaucoma with open iridocorneal angle in young humans with specimens of normal trabecular meshwork from humans of the same ages. In all cases of juvenile glaucoma, we observed a thick compact tissue consisting of cells with fine processes and extracellular substances at the anterior chamber side of Schlemm's canal and abnormal deposits of ground substances in the thick tissue. These observations strongly suggest that the thick compact tissue represents an immature development of the trabecular meshwork and may be one of the primary causes of increased intraocular pressure in juvenile glaucoma. Our studies indicate that both congenital and juvenile glaucoma occur because of developmental immaturity of the trabecular meshwork in anterior chamber angle tissue and that the more extensive the immaturity, the earlier the glaucoma will become manifest.

Adolescent↗

PubMed2005

Oxidative DNA damage in the human trabecular meshwork: clinical correlation in patients with primary open-angle glaucoma.

OBJECTIVE: To evaluate the intensity of oxidative molecular damage and its clinical correlations: visual field damage, intraocular pressure, age, and disease duration. METHODS: DNA was extracted from human trabecular meshwork specimens collected from 17 glaucoma-affected patients using standard filtration surgery. Twenty-one specimens from healthy eyes collected for cornea transplants serve as controls. Oxidative DNA damage was evaluated by determining 8-hydroxy-2'-deoxyguanosine levels. All patients underwent a Humphrey 30-2 visual field examination and diurnal tonometry before surgery. RESULTS: The mean +/- SD DNA oxidative damage was 8.51 +/- 5.44 and 1.75 +/- 1.80 8-hydroxy-2'-deoxyguanosine molecules/10(5) normal nucleotides in patients with glaucoma and controls, respectively. A statistically significant correlation was found among human trabecular meshwork DNA oxidative damage, visual field damage, and intraocular pressure. No other statistically significant correlations were found. CONCLUSIONS: Oxidative stress may represent an important pathogenetic step in primary open-angle glaucoma because it could induce human trabecular meshwork degeneration, favoring an intraocular pressure increase, thus priming the glaucoma pathogenetic cascade.

8-Hydroxy-2'-Deoxyguanosine↗