Trabecular Meshworks Research Articles

Evaluation of ciliary cleft changes after phacoemulsification using ultrasound biomicroscopy in dogs with cataracts.

Glaucoma is one of the most serious complications that causes irreversible blindness after phacoemulsification in dogs; however, a clear mechanism has not been elucidated. This study aimed to analyse the possible anatomical factors associated with glaucoma after phacoemulsification using parameters that reflect the anatomical characteristics of dogs. A total of 69 eyes of 48 dogs were included in this study. The patients were divided into three groups: normal eye (n = 18), cataract (n = 39), and post-phacoemulsification for at least 2 months after surgery (post-phaco, n = 12). For further analysis, the dogs were subdivided into two groups according to cataract stage: phacoemulsification non-candidate and candidate groups. Non-cataracts and incipient cataracts were categorized into the non-candidate group, whereas immature and mature cataracts were categorized into the candidate group. Measurements of the ciliary cleft parameters, including the area of the ciliary cleft (CCA), length of the ciliary cleft (CCL), width of the ciliary cleft (CCW), iridocorneal angle, and angle opening distance, were obtained using ultrasound biomicroscopy. CCA, CCL, and CCW were significantly higher in the candidate group than in the non-candidate group. CCA, CCL, and CCW were significantly reduced in the post-phaco group compared to those in the cataract group. Based on these results, we found that the ciliary cleft expanded in cataract-affected eyes and narrowed after phacoemulsification. This may indicate that the space between the trabecular meshworks became narrower, potentially leading to an increase in the resistance of the aqueous humor. A narrowed ciliary cleft after phacoemulsification may be an anatomical factor associated with glaucoma.

Low-Intensity Pulsed Ultrasound Accelerates Traumatic Vertebral Fracture Healing by Coupling Proliferation of Type H Microvessels.

Patients with traumatic vertebral fractures often have major associated postoperative morbidities such as healing failure and kyphosis. Low-intensity pulsed ultrasound (US) has been found to promote bone fracture healing. The objectives of our study were to determine whether low-intensity pulsed US could promote traumatic vertebral fracture healing and to explore its inner mechanisms. A rat model of traumatic vertebral fracture was created and treated with low-intensity pulsed US after surgery. At 4 weeks after surgery, radiographic, micro-computed tomography, and 3-dimensional reconstruction were used to assess the radiologic healing status; a histologic analysis was performed to evaluate the pathologic process and relationship between osteogenesis and type H microvessels. Well-remodeled trabecular meshworks were found in the low-intensity pulsed US treatment group compared to the control group. Micro-computed tomography and 3-dimensional reconstruction revealed more and thicker trabeculae after low-intensity pulsed US treatment. Abundant chondrocytes, a newly formed bone marrow cavity, trabeculae, and microvessels were formed at the fracture sites. More osterix-positive osteoblasts were circling the newly formed bone meshwork and were situated at the interface of chondrocytes in the low-intensity pulsed US treatment group. Type H microvessels were spreading around the newly formed trabecula, bone marrow cavity, osteoblasts, and interface of chondrocytes, with a larger mean vascular density in the low-intensity pulsed US group. Low-intensity pulsed US could accelerate traumatic vertebral fracture healing by temporally and spatially increasing chondrogenesis and osteoblast-induced osteogenesis coupled with angiogenesis of type H microvessels in a rat model of traumatic vertebral fracture.

Morphological Differences between the Trabecular Meshworks of Zebrafish and Mammals

Purpose: The zebrafish has been used as an animal model to study ocular development and diseases, including glaucoma. However, there are still many concerns about the morphological differences between zebrafish and mammals. Before using the zebrafish for glaucoma studies, we should understand the morphological differences in the trabecular meshworks (TMs) of zebrafish and other animal models. This study investigated and compared the histological morphologies and compositions of the extracellular matrices of the TMs of the zebrafish and some commonly used animal models, including the mouse, rat, rabbit, and cow. Methods: Sections of the angular portions from the studied species (mouse, rat, rabbit, cow, zebrafish, and human) were prepared for immunohistochemical and electron microscopic analyses. Antibodies directed against cytoskeletal and extracellular matrix components (AE1/AE3, vimentin, α-smooth muscle actin, keratocan, and lumican) were used for immunolocalization. Reverse transcription polymerase chain reaction (RT-PCR) for keratocan and lumican was also performed. Results: The TMs of the mouse, rat, and human consist of extracellular matrix organized into a network of beams covered in trabecular endothelial cells. However, no lamellate meshwork exists in the TMs of the rabbit, cow, or zebrafish. Instead, a reticular meshwork (rabbit and cow) and an annular ligament (zebrafish) develop. Immunohistological analysis revealed that vimentin is expressed in the TMs of the rat, rabbit, and human, and α-smooth muscle actin is expressed in the TMs of the mouse, rat, rabbit, and human. Only the annular ligament of the zebrafish stained positively with anti-AE1/AE3 antibody. The annular ligament of the zebrafish also expresses keratocan and lumican. The human TM showed weakly positive staining of lumican. A prominent distribution of mitochondria and intracellular vacuoles is observed in the trabecular cells of the mouse, rat, rabbit, and cow, but not the zebrafish. The analysis of RT-PCR shows the keratocan and lumican mRNAs are expressed in the annular ligament of zebrafish, but not in mouse, rat, rabbit, and cow. Conclusions: We conclude that the zebrafish expresses different extracellular matrix proteins and has a distinctive ultrastructure in the TM. Therefore, zebrafish should be used with caution for glaucoma studies.

Presence of an Established Calcification Marker in Trabecular Meshwork Tissue of Glaucoma Donors

To determine the presence of calcification markers in the trabecular meshwork tissue from glaucoma donors and in trabecular meshwork cells insulted by dexamethasone (DEX) and transforming growth factor beta2 (TGFbeta2), factors associated with glaucoma. To investigate as well the effect of silencing the inhibitor of calcification matrix Gla (MGP) in the trabecular meshwork cells. Trabecular meshwork tissue was obtained from perfused postmortem anterior segments of glaucomatous and normal eyes. Primary trabecular meshwork cells were obtained from residual corneal rims after surgical corneal transplantation. Calcification marker alkaline phosphatase (ALP) enzyme activity was assayed by fluorescence produced after substrate cleavage. DNA quantification was evaluated by fluorescence produced after binding to the Hoechst dye. Transfection of siRNA to primary cells was accomplished by nucleofector electroporation with trabecular meshwork-optimized conditions. cDNA quantification was performed with the use of TaqMan real-time PCR. Human trabecular meshworks from glaucoma donors exhibited significantly higher levels of ALP activity than their matched counterparts with normal eyes. The normalized ALP of the control specimens was 7.3 +/- 1.6 ng ALP/microg DNA (n = 4), whereas that of the glaucomatous tissue was 37.0 +/- 10.7 ng ALP/microg genomic DNA (n = 5; P </= 0.04). DEX and TGFbeta2 significantly induced the upregulation of ALP activity in two trabecular meshwork primary cell lines. Expression of the gene encoding MGP was reduced in the glaucomatous tissue by -4.4 +/- 1.7-fold (n = 9; P </= 0.006). Silencing MGP by siRNA resulted in ALP activity that was increased by 197% +/- 8.4% (P </= 0.0003). The increased activity of the calcification marker, ALP, in glaucomatous trabecular meshworks might be indicative of an undergoing mineralization process during development of the disease. Inhibition of the calcification mechanism represented by the presence of active MGP appears to be compromised in glaucomatous tissue.

The identification of myocilin-associated proteins in the human trabecular meshwork

Myocilin forms high molecular weight complexes in vivo presumably due to interaction with itself and other myocilin binding proteins. To identify myocilin interacting proteins, yeast 2-hybrid analysis was performed on >1×10 6 human trabecular meshwork cDNA clones. Coimmunoprecipitation and Far Western analysis were also performed on cell lysates obtained from fresh human trabecular meshworks or cultured human monolayer trabecular cell lines. Among the different methods, 46 candidate myocilin-associated proteins were identified, including molecules associated with the extracellular matrix, cytoskeleton, signaling, and metabolism. The most consistent interaction was myocilin–myocilin binding. Yeast-2 hybrid and Far Western analysis also found an association between myocilin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). None of the other candidate myocilin interacting proteins were identified in more than one method. Characterization of these potential interacting proteins may help to better understand the function of myocilin in the trabecular meshwork and aqueous outflow pathway.

Selective Laser Trabeculoplasty (SLT) Complicated by Intraocular Pressure Elevation in Eyes With Heavily Pigmented Trabecular Meshworks

To report and assess the complication of intraocular pressure (IOP) elevations after selective laser trabeculoplasty (SLT) in patients with heavily pigmented trabecular meshworks. Noncomparative, observational case series. Retrospective analysis of the medical files of four glaucoma patients with heavily pigmented trabecular meshwork, who presented with IOP elevations after SLT. All four glaucoma patients presented with post-SLT IOP elevations. Three had features of pigmentary dispersion syndrome, and the fourth had a heavily pigmented trabecular meshwork. Two patients had previous argon laser trabeculoplasty (ALT) in the same eye in which SLT was performed, and one had a previous ocular trauma. Eventually, three of the patients required surgical trabeculectomy. This case series suggests that post-SLT IOP elevations can be a serious adverse event in some glaucomatous patients. It is recommended by the authors that patients with a deeply pigmented trabecular meshwork, taking multiple topical medications and having previous ALT treatment, should be considered at higher risk for this complication.

Carbohydrate binding proteins galectin-1 and galectin-3 in human trabecular meshwork

Purpose. Galectins are a family of carbohydrate binding proteins involved in a variety of biological processes including cell-cell and cell-matrix interactions. We examined galectin-1 and galectin-3 to determine if galectins are expressed in the human trabecular meshwork and Schlemm's canal. Methods. Human trabecular meshworks were dissected from donor eyes within 12 hr of death. Galectin-1 and galectin-3 expression was examined by RT-PCR and Western blot analysis. Immunohistochemistry of galectin-1 and galectin-3 was analyzed in normal and glaucomatous tissue. Results. Expression of mRNA and protein of Galectin-1 (14 kDa) and galectin-3 (31 kDa) was found in the outflow pathway. Immunostaining revealed galectin-1 and galectin-3 throughout the meshwork, cells lining Schlemm's canal, and extracellular spaces in the inner and outer walls of the canal. Comparison of normal, POAG and PEX samples revealed no difference in location or intensity for either galectin-1 and galectin-3. Conclusion. Galectin-1 and galectin-3 are present in human trabecular meshwork of normal and glaucomatous eyes.

The myocilin (MYOC) gene expression in the human trabecular meshwork

Purpose. We previously reported a novel cytoskeletal protein with a myosin-like domain which is localized in the ciliary rootlet and basal body of connecting cilium of photoreceptor and hence we named it ‘myocilin’. It was soon realized that myocilin is identical to a protein called TIGR (trabecular meshwork inducible glucocorticoid response protein) which was found to be responsible for the pathogenesis of juvenile open angle glaucoma. In this study, we employed in situ RNA hybridization to examine the myocilin (MYOC)/ TIGR gene expression in the trabecular meshworks of glaucomatous and nonglaucomatous eyes. Methods. The glaucomatous specimens were obtained by trabeculectomy from the patients with primary open angle glaucoma (POAG), chronic angle closure glaucoma (CACG) and steroid glaucoma, respectively, and the nonglaucomatous specimens were obtained from a victim of traffic accident at autopsy and from a patient with maxillary sinus carcinoma at enucleation for the operation. The in situ RNA hybridization was carried out with digoxigenin-labeled sense and antisense RNA probes. Results. In all cases, hybridization signals were detected primarily in the trabecular meshwork cells and secondarily in the fibroblast-like cells of corneoscleral wall. Conclusions. Myocilin gene is expressed clearly in the trabecular meshwork cells of both glaucomatous and nonglaucomatous eyes.

Immunohistochemical localization of MYOC/TIGR protein in the trabecular tissue of normal and glaucomatous eyes

Purpose. To examine immunohistochemically the localization of myocilin/trabecular meshwork inducible glucocorticoid response (MYOC/TIGR) protein in the glaucomatous and normal trabecular meshworks. Methods. Trabecular tissues were used from one eye with late-onset goniodysgenetic glaucoma, three with primary open angle glaucoma (one of which had the MYOC/TIGR gene mutation), two with exfoliation glaucoma and one without glaucoma. For light microscopic immunohistochemistry, frozen sections were stained by the avidin-biotin complex method using anti-MYOC/TIGR polyclonal antibody. For electron microscopic immunohistochemistry, the pre-embedding method using the same antibody was performed. Double immunostaining using both anti-MYOC/TIGR and anti-type VI collagen antibodies was done by the immunofluorescence method. Results. With light microscopy, immunoreactivity was seen in the whole trabecular meshwork of each of the specimens. No notable differences were detected in staining among the types of glaucoma, or between the eyes with and those without the gene mutation. Under electron microscopy, immunoreaction products were observed not only in the cytoplasm of the trabecular cells but also in the extracellular matrix, where staining was associated with the long-spacing collagen, fine granular materials and possibly microfibrils. With double immunohistochemistry, MYOC/TIGR was colocalized with type VI collagen in the trabecular meshwork. Conclusions. In glaucomatous and normal trabecular meshworks, the MYOC/TIGR protein is distributed in the extracellular matrix colocalizing with type VI collagen.

Proteoglycan expression by human trabecular meshworks

PURPOSE. Proteoglycans may serve important roles in trabecular meshwork structure or function. Detailed molecular characterization and identification of specific trabecular proteoglycan core proteins has been limited. METHODS. Radiolabeled proteoglycans were extracted from cultured human trabecular meshworks and subjected to ion exchange and molecular sieve chromatography. Peaks were subjected to glycosaminoglycan content analysis. Reverse transcription with polymerase chain reaction was used to identify trabecular mRNAs of several common proteoglycan core proteins. Western immunoblots of trabecular extracts were also utilized to identify these proteoglycan core proteins. RESULTS. The proteoglycans elute from ion exchange columns at high salt and molecular sieve column profiles, and they exhibit broad peaks typical of the proteoglycan microheterogeneity seen in other tissues. The four common glycosaminoglycan side-chains were identified on these proteoglycans. Trabecular cells in organ or cell culture contain mRNAs coding for decorin, biglycan, versican, perlecan and a basement membrane glycoprotein, SPARC. Syndecan-1 transcripts were present at very low levels, while aggrecan transcripts were not detectable. Decorin, biglycan, versican and perlecan core proteins were also identified by immunoblots of trabecular cell extracts. CONCLUSIONS. Several common proteoglycans are expressed by trabecular cells in organ explant or cell culture. Their general characteristics are not unlike those found in other tissues. These proteoglycans may serve important functions in the trabecular outflow pathway.

Reduced trabecular meshwork height in juvenile primary open-angle glaucoma.

To compare trabecular meshwork height in a series of patients with juvenile primary open-angle glaucoma (JPOAG) with that in normal control patients. Ultrasound biomicroscopy and A-scan biometry were performed on 16 eyes with JPOAG and 24 normal eyes. A radial, perpendicular image in the horizontal temporal meridian detailing the line of Schwalbe, scleral spur, and angle anatomy was obtained for each eye by a single examiner. Trabecular meshwork height was defined as the distance from the scleral spur to the Schwalbe line. Mean patient age (P = .85, t test), refractive error (P = .68), sex distribution (P = .26, Fisher exact test) and axial length (P = .39) were similar between the groups. Mean +/- SE trabecular meshwork heights were 0.36 +/- 0.03 mm (range, 0.19-0.53 mm) for JPOAG and 0.58 +/- 0.02 mm (range, 0.40-0.80 mm) for controls (P < .001). Eyes with greater axial length tended to have larger trabecular meshworks in both groups (P = .012, multivariate regression). A trabecular meshwork height-axial length ratio of 0.021 or less was associated with a significantly increased risk for JPOAG being present (odds ratio, 57; 95% confidence interval, 6.0-541). The trabecular meshwork is smaller in eyes with JPOAG compared with that in normal eyes. This finding suggests a structural abnormality that may underlie the reduced outflow.

Selective targeting of trabecular meshwork cells: In vitro studies of pulsed and CW laser interactions

The purpose of the present study was to selectively target pigmented trabecular meshwork cells without producing collateral damage to adjacent non-pigmented cells or structures. The ability to selectively target trabecular meshwork cells without coagulation, while preserving the structural integrity of the meshwork, could be a useful approach to study whether the biological response of non-coagulative damage to the trabecular meshwork and trabecular meshwork cells is similar to that seen with coagulative damage to the trabecular meshwork which occurs with argon laser trabeculoplasty. This approach also may be useful to non-invasively deplete trabecular meshwork cells while preserving the structural integrity of the trabecular meshwork in an animal model. A mixed cell culture of pigmented and non-pigmented trabecular meshwork cells were irradiated with Q-switched Nd-YAG and frequency-doubled Nd-YAG lasers, microsound pulsed dye-lasers, and an argon ion laser in order to define a regime where laser absorption would be confined to pigmented trabecular meshwork cells, thereby permitting selective targeting of these cells without producing collateral thermal damage to adjacent non-pigmented cells. Pulse durations ranged from 10 nsec to 0.1 sec. A fluorescent viability/cytotoxicity assay was used to evaluate laser effects and threshold energies, and cells were examined morphologically by light and TEM. Selective targeting of pigmented trabecular meshwork cells was achieved with pulse durations between 10 nsec and 1 microsec and 1 microsec without producing collateral thermal or structural damage to adjacent non-pigmented trabecular meshworks cells when examined by light and transmission electron microscopy. Pulse durations greater than 1 microsec resulted in non-selective killing of non-pigmented trabecular meshwork cells. Threshold radiant exposures were as low as 18 mJ cm-2, and increased at longer wavelengths, longer pulse durations and lower melanin contents within the cells. It is concluded that selective targeting of pigmented trabecular meshwork cells can be achieved using pulsed lasers with low threshold radiant exposures avoiding collateral thermal damage to adjacent non-pigmented trabecular meshwork cells. This approach can be readily applied in vivo.

Localization of sialic acid in the human trabecular meshwork

The localization of sialic acid in the trabecular meshworks of the normal human primary open-angle (POAG), congenital and juvenile glaucomatous eyes was investigated using both an electron microscope and lectins specifically binding with sialic acid. Sialic acid was always localized in fine fibrils lying underneath the canal wall and around collagen fibrils of the whole trabecular tissue. In both congenital and juvenile glaucoma, sialic acid was detected not only in the fine fibrils but also in the basement membrane-like materials in compact tissue present 3 μm underneath the canal wall. Sialated glycoprotein appears to play an important role in the pathogenesis of both diseases.

Effect of hyaluronidase on aqueous outflow resistance in normotensive and glaucomatous eyes of dogs

Summary The anterior chambers in 16 dogs with normotensive eyes and 3 Beagles with glaucomatous eyes were treated with 0, 25, 50, or 100 IU of bovine testicular hyaluronidase. Aqueous outflow resistance was then determined by constant-pressure perfusion of 0.9% NaCl solution for 30 or 60 minutes. In normotensive eyes, 25, 50, or 100 IU of hyaluronidase significantly (P &lt; 0.02) increased the rate of constant-pressure perfusion compared with that of untreated eyes during 30- or 60-minute perfusions. Treatment of glaucomatous eyes with 25, 50, or 100 IU of hyaluronidase did not significantly increase the rate of constant-pressure perfusion over controls during a 30-minute perfusion. Bovine testicular hyaluronidase at all doses removed the staining of colloidal iron from the trabecular meshwork in normotensive eyes. In Beagles with glaucoma, the trabecular meshworks remained stained with colloidal iron when treated with the hyaluronidase, which suggested that some glycosaminoglycans were resistant to this enzyme's action.

Clinical and pathological features of chronic glaucoma in north-east Ghana

Of 34 consecutive patients with chronic glaucoma seen in north-east Ghana, 22 (65%) were male and seven (21%) were aged under 40 years. Only 17% of eyes had a visual acuity better than 6/18 at presentation. Sixteen of 23 patients who underwent gonioscopy had PAS of which 13 had positive skin snips for onchocerciasis, compared with two out of seven patients with positive skin snips who had open angle glaucoma (p = 0.003). Of 22 trabecular meshworks examined by light microscopy ten (45%) showed marked melanin pigmentation which was more common in younger patients but did not correlate with onchocerciasis infection.

Argon Laser Trabeculoplasty: Studies of Mechanism of Action

The effects of argon laser trabeculoplasty (LTP) on outflow facility, Schlemm's canal morphology, trabecular cellularity, and trabecular glycosaminoglycan composition were determined in 33 pairs of eye-bank human eyes. At two levels of intraocular pressure, measured outflow facility did not immediately drop in response to LTP. Schlemm's canal distention was observed only at 40 mmHg intraocular pressure, where the canal normally tends to collapse. Trabecular cell density was reduced by about 40% in the eyes receiving laser treatment. The organ cultured trabecular meshworks in response to LTP altered their incorporation of 35S-sulfate, compared to controls, suggesting a change in the synthesis or turnover of the extracellular matrix of the trabeculum after trabeculoplasty.At least three potential mechanisms were identified in response to laser trabeculoplasty, including some mechanical distortion of the trabeculum at high intraocular pressures. We also hypothesize that laser trabeculoplasty dislodges some trabecular cells and may stimulate the remaining cells to renew more active synthesis and/or turnover of the trabecular extracellular matrix.