Increasing prevalence and cost of glaucoma have increased the demand for surgeons well trained in newer, microincisional surgery. These procedures occur in a highly confined space, making them difficult to learn by observation or assistance alone as is currently done. We hypothesized that our ex vivo outflow model is sensitive enough to allow computing individual learning curves to quantify progress and refine techniques. Seven trainees performed nine trabectome-mediated ab interno trabeculectomies in pig eyes (n = 63). An expert surgeon rated the procedure using an Operating Room Score (ORS). The extent of outflow beds accessed was measured with canalograms. Data was fitted using mixed effect models. ORS reached a half-maximum on an asymptote after only 2.5 eyes. Surgical time decreased by 1.4 minutes per eye in a linear fashion. The ablation arc followed an asymptotic function with a half-maximum inflection point after 5.3 eyes. Canalograms revealed that this progress did not correlate well with improvement in outflow, suggesting instead that about 30 eyes are needed for true mastery. This inexpensive pig eye model provides a safe and effective microsurgical training model and allows objective quantification of outcomes for the first time.

Purpose: To correlate outflow function and outflow tract vessel diameter changes induced by nitric oxide (NO). Methods: In a porcine anterior segment perfusion model, the effects of a nitric oxide donor (100 μM DETA-NO) on outflow facility were compared with controls (n = 8 per group) with trabecular meshwork (TM) and after circumferential ab interno trabeculectomy (AIT). Outflow structures were assessed with spectral-domain optical coherence tomography (SD-OCT) before and after NO, or an NO synthase inhibitor (100 μM L-NAME) and the vasoconstrictor, endothelin-1 (100 pg/mL ET-1). Scans were processed with a custom macroscript and aligned for automated reslicing and quantification of cross-sectional outflow tract areas (CSA). Results: The facility increased after DETA-NO (Δ of 0.189 ± 0.081 μL/min·mm Hg, P = 0.034) and AIT (Δ of 0.251 ± 0.094 μL/min·mm Hg, P = 0.009), respectively. Even after AIT, DETA-NO increased the facility by 61.5% (Δ of 0.190 ± 0.074 μL/min·mm Hg, P = 0.023) and CSA by 13.9% (P < 0.001). L-NAME + ET-1 decreased CSA by −8.6% (P < 0.001). NO increased the diameter of focal constrictions 5.0 ± 3.8-fold. Conclusions: NO can dilate vessels of the distal outflow tract and increase outflow facility in a TM-independent fashion. There are short, focally constricting vessel sections that display large diameter changes and may have a substantial impact on outflow.

Objective: The trabecular meshwork (TM) is the primary substrate of outflow resistance in glaucomatous eyes. Repopulating diseased TM with fresh, functional TM cells might be a viable therapeutic approach. Decellularized TM scaffolds have previously been produced by ablating cells with suicide gene therapy or saponin, which risks incomplete cell removal or dissolution of the extracellular matrix, respectively. We hypothesized that improved trabecular meshwork cell ablation would result from freeze-thaw cycles compared to chemical treatment. Materials and Methods: We obtained 24 porcine eyes from a local abattoir, dissected and mounted them in an anterior segment perfusion within two hours of sacrifice. Intraocular pressure (IOP) was recorded continuously by a pressure transducer system. After 72 h of IOP stabilization, eight eyes were assigned to freeze-thaw (F) ablation (−80 °C × 2), to 0.02% saponin (S) treatment, or the control group (C), respectively. The TM was transduced with an eGFP expressing feline immunodeficiency viral (FIV) vector and tracked via fluorescent microscopy to confirm ablation. Following treatment, the eyes were perfused with standard tissue culture media for 180 h. TM histology was assessed by hematoxylin and eosin staining. TM viability was evaluated by a calcein AM/propidium iodide (PI) assay. The TM extracellular matrix was stained with Picro Sirius Red. We measured IOP and modeled it with a linear mixed effects model using a B-spline function of time with five degrees of freedom. Results: F and S experienced a similar IOP reduction of 30% from baseline (P = 0.64). IOP reduction of about 30% occurred in F within 24 h and in S within 48 h. Live visualization of eGFP demonstrated that F conferred a complete ablation of all TM cells and only a partial ablation in S. Histological analysis and Picro Sirius staining confirmed that no TM cells survived in F while the extracellular matrix remained. The viability assay showed very low PI and no calcein staining in F in contrast to many PI-labeled, dead TM cells and calcein-labeled viable TM cells in S. Conclusion: We developed a rapid TM ablation method that uses cyclic freezing that is free of biological or chemical agents and able to produce a decellularized TM scaffold with preserved TM extracellular matrix in an organotypic perfusion culture.

Purpose: The rate of conventional aqueous humor outflow is the highest nasally. We hypothesized that this is reflected in regionally different outflow structures and analyzed the entire limbus by high-resolution, full-thickness ribbon-scanning confocal microscopy (RSCM). Methods: We perfused pig eyes by anterior chamber cannulation with eight lectin-fluorophore conjugates, followed by optical clearance with benzyl alcohol benzyl benzoate (BABB). RSCM and advanced analysis software (Imaris) were used to reconstruct a three-dimensional (3D), whole-specimen rendering of the perilimbal outflow structures. We performed morphometric analyses of the outflow tract from the level of the trabecular meshwork (TM) to the scleral vascular plexus (SVP). Results: Except for pigmented structures, BABB cleared the entire eye. Rhodamine-conjugated Glycine max agglutinin (soybean [SBA]) labeled the outflow tract evenly and retained fluorescence for months. RSCM produced terabyte-sized files allowing for in silico dissection of outflow tract vessels at a high resolution and in 3D. Networks of interconnected lumens were traced from the TM to downstream drainage structures. The collector channel (CC) volumes were 10 times smaller than the receiving SVP vessels, the largest of which were in the inferior limbus. Proximal CC diameters were up to four times the size of distal diameters and more elliptical at their proximal ends. The largest CCs were found in the superonasal and inferonasal quadrants where the highest outflow occurs. Conclusion: RSCM of cleared eyes enabled high-resolution, volumetric analysis of the outflow tract. The proximal structures had greater diameters nasally, whereas the SVP was larger in the inferior limbus.

Pigment dispersion can lead to pigmentary glaucoma, a poorly understood condition of younger myopic eyes with fluctuating high intraocular pressure. It has been difficult to investigate its pathogenesis without a model similar to human eyes in size and behavior. Here we present a porcine ex vivo model that recreates several features of pigmentary glaucoma, including intraocular hypertension, accumulation of pigment in the trabecular meshwork, and declining phagocytosis. We found that trabecular meshwork cells regulate outflow, form actin stress fibers, and have a decreased phagocytic activity. Gene expression microarrays and a pathway analysis of TM monolayers as well as ex vivo anterior segment perfusion cultures indicated that RhoA plays a central role in regulating the cytoskeleton, motility, and phagocytosis in the trabecular meshwork, providing new insights and targets to investigate in pigmentary glaucoma.