Jorge A. Alvarado

Trabecular Meshwork Cellularity in Primary Open-angle Glaucoma and Nonglaucomatous Normals

The trabecular meshwork cellularity (cells/unit tissue area) was compared in patients with primary open-angle glaucoma (POAG) with that of nonglaucomatous (NG) individuals. The NG specimens (n = 69) include specimens from the prenatal period (n = 14) as well as the postnatal period to age 98 years (n = 55). The glaucoma specimens (n = 49) covered a wide-range of ages (23-80 years) and were obtained at trabeculectomy (n = 31) or at autopsy (n = 18). Our results show that the trabecular cellularity in NG specimens decreases most rapidly and in a nonlinear manner in the late fetal period and for the first few years of postnatal life. This rapid decline in cellularity then slows down to proceed in a nearly linear manner for the remainder of the 98 years of life studied. The meshworks from patients with POAG have a lower cellularity than normals over the wide range of ages examined, but both types of specimens undergo similar declines in cellularity with age. Thus, the age-cellularity curves for both the NG and POAG specimens are parallel to each other. The loss of cells occurs in a gradient-like manner with the inner tissues being most affected and the outermost tissues least affected. A variety of statistical tests show that these changes in cellularity are highly significant and specific. These findings are compared to the loss of endothelial cells in the cornea and they are discussed in relation to the important clinical characteristics of POAG.

Trabecular Meshwork Cell Culture in Glaucoma Research:: Evaluation of Biological Activity and Structural Properties of Human Trabecular Cells In Vitro

The propagation of human trabecular cells in culture allows the study of the structural and functional properties of this distinct cell type under reproducible experimental conditions. Human trabecular cells can be effectively grown from dissected explants of trabecullar tissue, and the cultured cells can maintain the distinctive ultrastructural features of uncultured trabecular cells through at least five passages in vitro. The trabecular cell possesses a wide range of biochemical and structural properties that may be important for the maintenance of the aqueous outflow pathway. These properties include the growth of trabecular cells as an endothelial monolayer with a nonthrombogenic cell surface, the production of plasminogen activator, avid phagocytosis, and the ability to synthesize glycosaminoglycans, collagen, fibronectin, and other connective tissue elements. The presence of hyaluronidase and other lysosomal enzymes emphasizes that human trabecular cells are capable of metabolizing hyaluronic acid and other extracellular materials. Potential mechanisms of trabecular cell damage in vitro are examined by evaluating the effects of extended passage, peroxide exposure, and laser treatment on cellular morphology.

Endoscopic photocoagulation of the ciliary body for treatment of refractory glaucomas.

PURPOSE To evaluate the safety and efficacy of endoscopic cyclophotocoagulation in the treatment of refractory glaucomas. METHODS The preoperative and postoperative courses of 68 eyes of 68 patients who underwent endoscopic cyclophotocoagulation at our institution were retrospectively reviewed. Study patients had diverse forms of glaucoma, and most had failed maximal medical therapy as well as failed filtration or transscleral cyclodestructive procedures, or both. Endoscopic cyclophotocoagulation treatment encompassed 180 to 360 degrees of the ciliary body circumference and was performed through a limbal incision (56 eyes, 12 of which underwent concurrent cataract extraction) or pars plana incision (12 eyes). A second laser treatment was required in five eyes (7%). RESULTS During the mean follow-up period of 12.9 months, mean +/- SD intraocular pressure decreased from 27.7 +/- 10.3 mm Hg preoperatively to 17.0 +/- 6.7 mm Hg at the final postoperative visit (P < .0001), for a mean reduction of 10.7 mm Hg and a mean percent decrease of 34%. Sixty-one eyes (90%) achieved an intraocular pressure < or = 21 mm Hg. Using this definition of success, Kaplan-Meier analysis predicted a successful outcome in 94% of patients after 1 year and 82% after 2 years. The mean number of glaucoma medications used by each patient was reduced from 3.0 +/- 1.3 preoperatively to 2.0 +/- 1.3 postoperatively (P < .0001). Best-corrected visual acuity was stable or improved in 64 eyes (94%), with four (6%) losing 2 or more lines of Snellen acuity. No case of hypotony (intraocular pressure < 5 mm Hg) or phthisis was observed. CONCLUSION These early results suggest that endoscopic cyclophotocoagulation is a safe and effective therapeutic modality for refractory glaucomas.

A new insight into the cellular regulation of aqueous outflow: how trabecular meshwork endothelial cells drive a mechanism that regulates the permeability of Schlemm’s canal endothelial cells

Aim: To test the hypothesis that trabecular meshwork endothelial cells (TMEs) increase the permeability of Schlemm’s canal endothelial cells (SCEs) by actively releasing ligands that modulate the barrier properties of SCEs. Methods: The TMEs were first irradiated with a laser light and allowed to condition the medium, which is then added to SCEs. The treatment response is determined by both measuring SCE permeability (flow meters) and the differential expression of genes (Affymetrix chips and quantitative polymerase chain reaction (PCR)). The cytokines secreted by the treated cells were identified using ELISA and the ability of these cytokines to increase permeability is tested directly after their addition to SCEs in perfusion experiments. Results: SCEs exposed to medium conditioned by the light activated TMEs (TME-cm) respond by undergoing a differential expression (DE) of 1120 genes relative to controls. This response is intense relative to a DE of only 12 genes in lasered SCEs. The TME-cm treatment of SCEs increased the SCE permeability fourfold. The role of cytokines in these responses is supported by two findings: adding specific cytokines established to be secreted by lasered TMEs to SCEs increases permeability; and inactivating the TME-cm by boiling or diluting, abrogates these conditioned media permeability effects. Conclusion: These experiments show that TMEs can regulate SCE permeability and that it is likely that TMEs have a major role in the regulation of aqueous outflow. This novel TME driven cellular mechanism has important implications for the pathogenesis of glaucoma and the mechanism of action of laser trabeculoplasty. Ligands identified as regulating SCE permeability have potential use for glaucoma therapy.

Localization of Extracellular Proteins of the Human Trabecular Meshwork by Indirect Immunofluorescence

We used monospecific antibodies on semithin frozen sections to identify and localize the major tissue constituents of the nonglaucomatous human trabecular meshwork. The trabecular beams (sheets and cords) consist of a basement membrane (subendothelial extracellular matrix) surrounding an interstitial central core of connective tissue (substantia propria). The basement membrane contains collagen types III, IV, and V, the glycoproteins laminin and fibronectin, and the basement membrane-associated heparan sulfate proteoglycan. The trabecular basement membrane is unlike most subendothelial basement membranes because it contains collagen type III and a relatively disorganized structure. The central core contains collagen types I and III, and elastin. The closely linked juxtacanalicular meshwork contains collagen type III, but no collagen type I or elastin. The connective tissue composition of the trabecular meshwork appears similar to other highly compliant and resilient tissues, such as lung, blood vessels, and conjunctiva.

Studies on human trabecular cells propagated in vitro

Abstract The propagation of human trabecular cells in culture allows the study of the structural and functional properties of this distinct cell type under reproducible experimental conditions. Human trabecular cells can be effectively grown from dissected expiants of trabecular tissue, using 250 ng/ml fibroblast growth factor (FGF) and either 10% human or fetal calf serum with DME medium. The cultured cells show few ultrastructural alterations through at least four passages, although cultured cells appear slightly more spread out in vitro when compared to cells from intact tissue. We have previously demonstrated that human trabecular cells are capable of producing a complex connective tissue matrix, show active phagocytosis, and have functional glucocorticoid receptors. We report here that use of cultured human trabecular cells readily permits the detection of direct glucocorticoid effects and the measurement of beta adrenergic receptors. Future studies on cultured human trabecular cells will examine the role of hormones and other regulatory substances on the biological function of this important cell type.

The identification and localization of fibronectin in cultured corneal endothelial cells: cell surface polarity and physiological implications.

Abstract Bovine corneal endothelial cells can be maintained for over 200 generations in tissue culture provided that fibroblast growth factor is present in the medium. The cultured cells exhibited at confluence the morphology of a highly contact inhibited cell monolayer composed of flattened and closely apposed cells. Transmission electron microscopy of the confluent endothelial cell monolayer demonstrated that the cellular organelles and cytoskeletal elements had a morphological appearance and distribution indistinguishable from those seen in the corneal endothelium in vivo. The confluent cells were also capable of synthesizing a basement membrane. In both sparse and subconfluent cultures fibronectin detected by indirect immunofluorescence was distributed on the apical cell surface and in the regions of cell-cell contacts. In contrast, when the cultures reached confluence, fibronectin could no longer be detected on the apical cell surface, but rather accumulated in the extracellular meshwork underlying the cell monolayer. Metabolic labeling with [ 35 S]-methionine demonstrated that at confluence fibronectin is deposited toward the extracellular matrix and secreted in large quantities into the tissue culture medium. Lactoperoxidase catalyzed iodination of confluent cultures showed that the fibronectin meshwork associated with the cell surface and the extracellular matrix is disulfide-bonded to form dimers and higher complexes. In contrast, actively growing cells that are not yet in contact showed little or no fibronectin on their cell surfaces and no production of an extracellular matrix. The apical surface of the confluent monolayer, which is not covered by fibronectin, is a non-thrombogenic surface, as indicated by the lack of platelet binding and release reaction. In contrast, the extracellular matrix, which is extensively coated with fibronectin, is thrombogenic. Our results therefore demonstrate that at confluence fibronectin becomes closely associated with the basel cell surface and can no longer be detected on top of the cells, thus maintaining the polarity of cell surfaces observed in vivo. It is suggested that the underlying meshwork of fibronectin may play a physiological role in maintaining the flattened and closely apposed morphology characteristic of differentiated corneal endothelial cells in vitro and in vivo and that the absence of fibronectin from the apical cell surface is related to the non-thrombogenic properties of these cells.

Ahmed Valve Implantation with Adjunctive Mitomycin C and 5-Fluorouracil: Long-term Outcomes

PURPOSE To evaluate long-term outcomes after Ahmed valve implantation in patients with glaucoma when using adjunctive intraoperative mitomycin C (MMC) and postoperative 5-fluorouracil (5-FU). DESIGN Retrospective, interventional, consecutive case series. METHODS A consecutive series of eyes undergoing Ahmed valve implantation, either alone (AHMED eyes) or in combination with cataract surgery (AHMED+PHACO), using both intraoperative MMC and postoperative 5-FU were evaluated. Failure was defined as the first occurrence of any of the following: 1) the first of three consecutive visits where intraocular pressure (IOP) was >18 mm Hg or <20% IOP reduction from baseline and the final number of topical medications was not reduced by at least two from baseline, 2) the need for additional surgery, or 3) the development of serious complications. RESULTS A total of 130 eyes underwent Ahmed valve implantation with intraoperative exposure to 0.5 mg/ml MMC (median time: eight minutes; range, four to 10) and postoperative subconjunctival injections of 5 mg of 5-FU (median: five injections; range, zero to nine). Kaplan-Meier estimates of the cumulative probability of valve success and confidence interval (CI) at the sixth follow-up year were 0.72 (95% CI, 0.59 to 0.82) for AHMED eyes (n = 88), 0.84 (95% CI, 0.65 to 0.93) for AHMED+PHACO eyes (n = 42). A median of two fewer medications were required relative to baseline for both AHMED and AHMED+PHACO eyes. CONCLUSIONS The adjunctive use of both intraoperative MMC and postoperative 5-FU with Ahmed valve implantation results in high success rates. IOP was well controlled in the majority of patients within the six-year postoperative period.

Characteristics of glaucoma drainage implants during dynamic and steady-state flow conditions

OBJECTIVE This study aimed to ascertain whether the Optimed, Krupin, and Ahmed drainage devices function as valves that vary resistance depending on flow conditions to maintain pressure within a desired range. STUDY DESIGN Experimental study. INTERVENTION The three devices and a control cannula were submerged in fluid and perfused with balanced salt solution using a computer-driven apparatus that continuously monitors flow (Q) and pressure (P). In one set of experiments, the flow rates were maintained at 2, 5, 10, 25, or 50 microliters/min until steady-state pressures were achieved. In another set of experiments, the flow rate was increased linearly from 0 to 100 microliters/min over 15 to 20 minutes. MAIN OUTCOME MEASURES The resistance of each implant was calculated from the first set of experiments by dividing the change in pressure (P) by the change in flow (Q) between successive perfusion rates. Flow-pressure curves were plotted from the experiments in which perfusion rate was increased linearly. RESULTS Resistance remained relatively constant for the cannula (0.18-0.24 mmHg/microliter/min), the Krupin (0.09-0.25 mmHg/microliter/min), and the Optimed implants (0.04-0.08) throughout the tested flow rates. For the Ahmed device, conversely, resistance decreased proportionally (2.86-0.05 mmHg/microliter/min) to the increase in flow. When flow rate was increased linearly from 0 to 100 microliters/min, the Optimed and Krupin devices as well as the cannula generated a linear pressure response with a constant slope. The pressure in the two devices increased at a rate of 0.11 mmHg/microliter compared to 0.23 mmHg/microliter/min for the cannula. The flow-pressure curve for the Ahmed implant was distinct with a steep initial pressure rise and an essentially constant pressure of 12 mmHg thereafter. CONCLUSION The Optimed and Krupin devices displayed resistance and pressure responses to various flow conditions that were similar to those of a cannula or flow resistor. In these devices, resistance remained relatively stable and pressure increased linearly with flow. The Ahmed device, conversely, functioned as a valve that closely regulated pressure within a desired range by decreasing or increasing resistance as a function of flow.

Monocyte Modulation of Aqueous Outflow and Recruitment to the Trabecular Meshwork Following Selective Laser Trabeculoplasty

OBJECTIVES To determine whether selective laser trabeculoplasty (SLT) induces monocyte recruitment to the trabecular meshwork (TM) in human and monkey eyes and whether monocytes increase both aqueous outflow in vivo and the conductivity of human Schlemm canal endothelial cells (SCEs) in vitro. METHODS Monocyte recruitment was examined morphometrically in control human and monkey eyes and compared with that following SLT applied 1 to 3 days earlier. Outflow facility was measured for up to 4 days after the intracameral infusion of autologous macrophages in rabbits. Schlemm canal endothelial cell conductivity was measured using flow meters after exposing cultured SCEs to monocytes and monocyte-secreted factors for 24 hours. RESULTS Our estimates show that the TM in the human eye normally had an average of 15 003 monocytes, while in the monkey eye there were 3181 monocytes, and this number increased 4- to 5-fold following SLT. The intracameral infusion of autologous macrophages in rabbits increased outflow facility 2-fold in a rapid and sustained manner. Human monocytes and monocyte-secreted factors increased SCE conductivity 2-fold in vitro. CONCLUSIONS The number of monocytes/macrophages in the TM increases substantially after SLT and monocytes augment both outflow facility and SCE conductivity. Clinical Relevance These findings indicate that the innate immune system in general and monocytes in particular play an important role in aqueous outflow homeostasis. The recruitment of monocytes in increased numbers after SLT likely plays a role in lowering the intraocular pressure after this procedure. The intracameral introduction of autologous monocytes harvested from a vein could have therapeutic potential as a cell-based individualized treatment of glaucoma.