Live outflow imaging in porcine eyes

Purpose: Although outflow of aqueous humor can be estimated using fluorophotometry and tonography, no method exists to assess segmental outflow directly. This has hindered investigations into outflow resistance elements that are downstream of the trabecular meshwork and into causes of failure of canal-based minimally invasive glaucoma surgeries (MIGS). Here, we describe a method to observe and quantify conventional outflow directly in ex vivo whole eyes and an organotypic culture model using standard fluorescence visualization equipment. Results: Aqueous outflow paths could be distinguished from venous and arterial vessels. Intricate vascular tree filling patterns were observed down to estimated 50 micrometer diameter. Whole eyes demonstrated significantly earlier filling of the nasal (SN, IN) than of the temporal quadrants (ST, IT). The fastest, superonasal quadrant filled 1.6 times faster than the slowest, superotemporal quadrant (p≤0.05). The inferotemporal quadrant also filled faster than the superotemporal quadrant. In contrast, perfused anterior segment cultures had lost these characteristic filling time differences (p>0.05). One eye showed no outflow after 20 minutes of pressurization. After three days of culture, the same quadrant that was the fastest in whole eyes (SN), was 2.2 times as fast as the slowest (ST) but this did not reach statistical significance with the number of eyes tested. The eye without flow at 20 minutes did not recuperate during 3 days of culture. Reverse filling was occasionally seen in d3 anterior segments (bottom row, red arrowhead). Whole eyes could not be reliably cultured for 3 days. Discussion: Canalograms using a green fluorescent chromophore can be obtained with standard visualization equipment to estimate the local outflow function. Regionally different outflow patterns and filling times can be observed that match channel size equivalents in human eyes. Outflow is impaired in fresh anterior segment cultures but then normalizes to the pattern seen in whole eyes. The nasally increased flow may have implications for placement and study of MIGS

Ocular hypotension, actin stress fiber disruption and phagocytosis increase by RKI-1447, a Rho-kinase inhibitor

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

Stratification of phaco-trabectome surgery results using a glaucoma severity index

The outcomes of phacoemulsification combined with trabectome surgery was analyzed using a glaucoma severity index based on preoperative intraocular pressure (IOP), number of preoperative medications, and visual field damage. Despite a less absolute indication to lower IOP, a substantial pressure reduction was seen in patients with more advanced glaucoma

Impact of Pigment Dispersion on Trabecular Meshwork Cells

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 pigment glaucoma

Combined analysis of trabectome and phaco-trabectome outcomes by glaucoma severity.

Prior glaucoma severity staging systems were mostly concerned with visual field function and retinal nerve fiber layer, but did not include intraocular pressure or medications to capture resistance to treatment. We recently introduced a simple index that combines pressure, medications, and visual field damage and applied it to stratify outcomes of trabectome surgery. In the analysis presented here, we combined data of trabectome alone and trabectome with same session cataract surgery to increase testing power and chances of effect discovery. This microincisional glaucoma surgery removes the primary resistance to outflow in glaucoma, the trabecular meshwork, and has been mostly used in mild glaucoma. Traditional glaucoma surgeries have a relatively high complication rate and have been reserved for more advanced disease stages. In the analysis presented here we include our data of trabectome combined with cataract surgery. This is a common practice pattern as both occur in the same age group with increasing frequency. For patients in higher glaucoma index (GI) groups, the intraocular pressure (IOP) reduction was 2.34+/-0.19 mmHg more than those in a GI group one level lower while holding everything else constant. Those who had undergone trabectome combined with phacoemulsification had an IOP reduction that was 1.29+/-0.39 mmHg less compared to those with trabectome alone. No statistically significant difference was found between genders and age groups while holding everything else constant. Hispanics had a 3.81+/-1.08 mmHg greater IOP reduction. Pseudoexfoliation and steroid glaucoma patients had an IOP reduction that was greater by 2.91+/-0.56 and 3.86+/-0.81 mmHg, respectively, than those with primary open angle glaucoma. These results suggest a role for trabectome-mediated ab interno trabeculectomy beyond mild forms of glaucoma. Additionally, the multifactorial glaucoma index demonstrates a role in staging patients when comparing glaucoma surgical modalities

Stratification of phaco-trabectome surgery results using a glaucoma severity index

Purpose: Nonsurgical glaucoma therapy reduces intraocular pressure (IOP) by a percentage that is similar for most patients. Recently introduced microincisional glaucoma surgeries are different because they remove the trabecular meshwork that constitutes the primary resistance to outflow. We hypothesized that because of this, the resulting postoperative IOP, rather than the relative reduction, had to be independent of glaucoma severity. We applied a glaucoma index (GI) to analyze outcomes of trabectome surgery combined with phacoemulsification (PT) by glaucoma severity. Methods: Only PT with 12 month follow up and no other surgeries were included. GI incorporated preoperative IOP, medications (meds) and visual field (VF). Baseline IOP was divided into 4 groups

Trabecular Meshwork Engineering and Live Tracking in Perfused Porcine Anterior Segments

Purpose: To establish a trabecular meshwork ™ engineering model using porcine anterior segments of consistently high quality in a physiological, fixed perfusion system.\ud \ud Discussion: Compared to previously used human donor eyes, this inexpensive porcine anterior segment perfusion model is of sufficient, repeatable high quality to develop strategies to modify genetically, ablate and repopulate the TM. Despite significant anatomic differences, effects of transduction and ablation in the porcine model presented here replicate key aspects of previously explored human, feline and rodent models