Glaucoma is a blinding disease largely caused by increased resistance to drainage of fluid from the eye’s anterior chamber, resulting in elevated intraocular pressure (IOP). A major site of fluid outflow regulation and pathology is the trabecular meshwork (TM) at the entrance of the eye’s drainage system. We aimed to characterize the structural and functional properties of a newly developed tissue-engineered anterior segment eye culture model. We hypothesized that repopulation of a decellularized TM with non-native TM cells could restore aspects of normal TM. The decellularization protocol removed all cells and debris while preserving the ECM. Seeded cells localized to the TM region and progressively infiltrated the meshwork ECM. Cells reached a distribution comparable to control TM after four days of perfusion culture. After a perfusion rate increase challenge, tissue-engineered cultures reestablished normal IOPs (reseeded = 13.7±0.4 mmHg, decellularized = 35.2±2.2 mmHg, p < 0.0001). eGFP expressing CrFK control cells caused a high and unstable IOP (27.0±6.2 mmHg). In conclusion, we describe a readily available, storable, and biocompatible scaffold for anterior segment perfusion culture of non-native cells. Tissue-engineered organs demonstrated similarities to native tissues and may reduce the need for scarce donor globes in outflow research.

A 31-year-old male noticed blurred vision in his right eye for five days with no obvious predisposing causes, accompanied by mild dizziness. No obvious nodular lesions were found in the body. The patient’s binocular visual acuity was 20/20. Fundus photography showed the optic nerve swelling and radial superficial retinal hemorrhage of both eyes. Blood panel, urine routine, liver, and kidney function were all normal. Total cholesterol, triglycerides, high-density lipoprotein, and low-density lipoprotein were all in the normal limits. Head MRI showed a mass in the right temporal lobe, clear boundary, and multiple separations, which thinned and disappeared closer to the skull. The right temporal lobe and lateral ventricle were all compressed, with the midline structure shifted to the left. The patient was then transferred to Neurosurgery. During the operation, we observed the tumor had invaded the skull. The actual size of the tumor was 5.6 cm × 7.5 cm × 10.1 cm. Histology revealed foam cell accumulation in the mucous connective tissue of the right temporal lobe. The immunohistochemistry showed: CD34 (+), CD99 (+), EMA (−), GFAP (−), IDH-1 (−), Ki-67 (+) index about 10%, Oliga-2 (−), PR (−), S-100 (−), Vim (+), β-Catenin (+), CD1a (−), CD68 (+). Three months after the removal of the tumor, the visual acuity of both eyes was 20/20; The visual fields were normal, the optic disc edema and retinal hemorrhages had disappeared. MRI indicated the midline structure was back to normal.

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.

Purpose: This study investigated the hypotensive effect of RKI-1447, a Rho kinase inhibitor, in a porcine ex vivo pigmentary glaucoma model. Methods: Twenty-eight porcine anterior chambers were perfused with medium supplemented with 1.67 × 10⁷ pigment particles/mL for 48 hours before treatment with RKI-1447 (n = 16) or vehicle control (n = 12). Intraocular pressure (IOP) was recorded and outflow facility was calculated. Primary trabecular meshwork cells were exposed to RKI-1447 or vehicle control; effects on the cytoskeleton, motility, and phagocytosis were evaluated. Result: Compared to baseline, the perfusion of pigment caused a significant increase in IOP in the RKI-1447 group (P = 0.003) at 48 hours. Subsequent treatment with RKI-1447 significantly reduced IOP from 20.14 ± 2.59 mmHg to 13.38 ± 0.91 mmHg (P = 0.02). Pigment perfusion reduced the outflow facility from 0.27 ± 0.03 at baseline to 0.18 ± 0.02 at 48 hours (P < 0.001). This was partially reversed with RKI-1447. RKI-1447 caused no apparent histological changes in the micro- or macroscopic TM appearance. RKI-1447-treated primary TM cells showed significant disruption of the actin cytoskeleton both in the presence and absence of pigment (P < 0.001) but no effect on TM migration was observed. Pigment-treated TM cells exhibited a reduction in TM phagocytosis, which RKI-1447 reversed. Conclusion: RKI-1447 significantly reduces IOP by disrupting TM stress fibers and increasing TM phagocytosis. These features may make it useful for the treatment of secondary glaucomas with an increased phagocytic load.

Objective: The Rho GTPase/Rho kinase pathway is an important target in glaucoma treatment. This study investigated the hypotensive effect of RKI-1447, a Rho kinase inhibitor developed for cancer treatment, in a porcine ex vivo pigmentary glaucoma model. Materials and Methods: Twenty-eight fresh porcine anterior chambers were perfused with pigment medium (1.67 × 10⁷ pigment particles/mL) for 48 hours before being subjected to the RKI-1447 (n = 16) or the vehicle control (n = 12). Another twelve eyes with normal medium perfusion served as the control. The intraocular pressure (IOP) was recorded at two-minute intervals and the outflow facility was calculated. To investigate the intracellular mechanism of the IOP reduction, primary trabecular meshwork cells were exposed to RKI-1447 or the vehicle control and then analyzed for changes in cytoskeleton, motility, and phagocytosis. Results: Compared to the baseline, the perfusion of pigment caused a significant increase in IOP in the RKI-1447 group (P = 0.003) at 48 hours. Subsequent treatment with RKI-1447 significantly reduced IOP from 20.14 ± 2.59 mmHg to 13.38 ± 0.91 mmHg (P = 0.02). Pigment perfusion reduced the outflow facility from 0.27 ± 0.03 at baseline to 0.18 ± 0.02 at 48 hours (P < 0.001). This was partially reversed with RKI-1447. RKI-1447 exhibited no apparent changes in the micro- or macroscopic appearance, including histology. Primary TM cells exposed to RKI-1447 showed a significant disruption of the actin cytoskeleton both in the presence and absence of pigment exposure (P < 0.001) but no effect on TM migration was observed. Pigment-treated TM cells exhibited a reduction in TM phagocytosis, which RKI reversed. Conclusions: RKI-1447 is a novel ROCK inhibitor that significantly reduces IOP by disrupting TM stress fibers and increasing TM phagocytosis. These features may make it especially useful for the treatment of secondary glaucomas with an increased phagocytosis load but also for other open angle glaucomas.

Purpose: To investigate the effect of pigment dispersion on trabecular meshwork (TM) cells. Methods: Porcine TM cells from ab interno trabeculectomy specimens were exposed to pigment dispersion, then analyzed for changes in morphology, immunostaining, and ultrastructure. Their abilities to phagocytose, migrate, and contract were quantified. An expression microarray, using 23,937 probes, and a pathway analysis were performed. Results: TM cells readily phagocytosed pigment granules. Pigment induced stress fiber formation (pigment (P): 60.1 ± 0.3%, n = 10, control (C): 38.4 ± 2.5%, n = 11, P < 0.001) and contraction at 24 hours onward (P < 0.01). Phagocytosis declined (P: 68.7 ± 1.3%, C: 37.0 ± 1.1%, n = 3, P < 0.001) and migration was reduced after 6 hours (P: 28.0.1 ± 2.3, n = 12, C: 40.6 ± 3.3, n = 13, P < 0.01). Microarray analysis revealed that Rho, IGF-1, and TGFβ signaling cascades were central to these responses. Conclusions: TM cell exposure to pigment dispersion resulted in reduced phagocytosis and migration, as well as increased stress fiber formation and cell contraction. The Rho signaling pathway played a central and early role, suggesting that its inhibitors could be used as a specific intervention in treatment of pigmentary glaucoma.

To evaluate the 2-year efficacy and safety of ab interno trabeculectomy with the Trabectome in Chinese primary open-angle glaucoma (POAG) patients. This was a multicenter, retrospective study and included POAG patients with or without visually-significant cataracts. The primary outcome was intraocular pressure (IOP) reduction. Secondary outcomes included reduction of glaucoma medications, surgical complications, and success at 2-years. Success was defined as: 1) IOP < 21 mmHg and at least 20% IOP reduction from baseline after 3 months at any two consecutive visits, and 2) no additional glaucoma surgery required. A total of 42 patients were enrolled; thirteen had a history of failed glaucoma surgery. Twelve patients underwent Trabectome surgery combined with phacoemulsification while the remainder underwent Trabectome surgery alone. Overall, the mean preoperative IOP was 21.4±1.23 mmHg. The Trabectome lowered IOP to 18.5± 0.94 mmHg at 2 years (P<0.05). The number of glaucoma medications also decreased from a baseline of 2.0±0.9 to 1.2± 0.21 at 2 years (P=0.04). The overall success was 78%, with patients undergoing combined surgery having a higher success than those with Trabectome surgery alone (100% vs. 76%). No major complications were seen. The Trabectome appears to be an efficient and safe procedure in Chinese POAG patients in the long-term.

Purpose: To evaluate three different microincisional ab interno trabeculectomy procedures in a porcine eye perfusion model. Methods: In perfused porcine anterior segments, 90 degrees of trabecular meshwork (TM) were ablated using the Trabectome (T; n = 8), Goniotome (G; n = 8), or Kahook device (K; n = 8). After 24 hours, additional 90 degrees of TM were removed. Intraocular pressure (IOP) and outflow facility were measured at 5 µL/min and 10 µL/min perfusion to simulate an elevated IOP. Structure and function were assessed with canalograms and histology. Results: At 5 µL/min infusion rate, T resulted in a greater IOP reduction than G or K from baseline (76.12% decrease versus 48.19% and 47.96%, P = 0.013). IOP reduction between G and K was similar (P = 0.420). Removing another 90 degrees of TM caused an additional IOP reduction only in T and G but not in K. Similarly, T resulted in the largest increase in outflow facility at 5 µL/min compared with G and K (first ablation: 3.41 times increase versus 1.95 and 1.87; second ablation: 4.60 versus 2.50 and 1.74) with similar results at 10 µL/min (first ablation: 3.28 versus 2.29 and 1.90 (P = 0.001); second ablation: 4.10 versus 3.01 and 2.01 (P = 0.001)). Canalograms indicated circumferential flow beyond the ablation endpoints. Conclusions: T, G and K significantly increased the outflow facility. In this model, T had a larger effect than G and K.

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 to 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 macro script and aligned for automated reslicing and quantification of cross-sectional outflow tract areas (CSA). Results: The facility increased after DETA-NO (0.189±0.081 μL/min·mmHg, p=0.034) and AIT (0.251±0.094 μL/min·mmHg, p=0.009), respectively. Even after AIT, DETA-NO increased the facility by 61.5% (0.190±0.074 μL/min·mmHg, 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.

Purpose: To investigate whether microsurgical excision of trabecular meshwork (TM) in an ex vivo pigmentary glaucoma model can normalize the hypertensive phenotype. Methods: Eight eyes of a porcine pigmentary glaucoma model underwent 90° of microsurgical TM excision with an aspirating dual-blade (Goniotome (G)). 24 hours later, an additional 90° of TM were removed. Anterior segments with sham surgeries served as the control (C). Outflow facility and intraocular pressure (IOP) were analyzed. Histology with hematoxylin and eosin (H&E) was obtained. Results: After the first 90° TM excision, IOP was significantly lower in G (10.23±2.39 mmHg, n=7) than C (20.04±1.97mmHg, n=8, P<0.01). Outflow facility in G (0.38±0.07 µl/min/mmHg) was higher than C (0.16±0.02 µl/min/mmHg, P<0.01). After the second 90° TM excision, IOP in G (6.46±0.81 mmHg, n=7) was significantly lower than C (20.25±1.66 mmHg, n=8, P<0.001), while the outflow facility in G (0.50±0.05 µl/min/mmHg, n=7) was higher than C (0.16±0.01 µl/min/mmHg, n=8, P<0.001). Compared to the first excision, excision of an additional 90° did not change of IOP (P=0.20) or outflow facility (P=0.17) further. Conclusion: Excision of 90° of TM in a pigmentary glaucoma model using an aspirating dual-blade decreased IOP and increased outflow facility. Translational Relevance: Microsurgical TM excision over 90° can effectively restore outflow in pigmentary glaucoma.