The effects of negative periocular pressure on intraocular pressure.

Glaucoma is a major cause of blindness, and IOP reduction remains the only clinically-validated therapy. In this study, we analyze a novel IOP-lowering strategy that uses a modest negative pressure (vacuum) applied locally to the periorbital region by a pair of goggles with each lens individually connected to a programmable pump. Motivated by clinical data showing an IOP reduction, we used an existing validated lumped-parameter model of the eye to understand the putative mechanism of this treatment. The model considers aqueous humor dynamics, episcleral venous pressure, and changes in ocular blood volume to describe how IOP changes with time in response to an external perturbation. We find that clinical data are qualitatively and quantitatively consistent with model predictions if we include two primary mechanisms in the model: first, negative pressure application causes a relatively rapid increase in globe volume accompanied by increased blood volume in the eye. Second, negative pressure application reduces episcleral venous pressure, causing a slower adjustment of IOP due to altered aqueous humor dynamics. These results provide testable hypotheses that hopefully will lead to a fuller experimentally-driven understanding of how negative periocular pressure influences IOP. Evaluating the long-term effects of such treatments on glaucoma patients requires further clinical study.

Imaging Distal Aqueous Outflow Pathways in a Spontaneous Model of Congenital Glaucoma.

PURPOSE: To validate the use of aqueous angiography (AA) in characterizing distal aqueous outflow pathways in normal and glaucomatous cats. METHODS: Ex vivo AA and optical coherence tomography (OCT) were performed in nine adult cat eyes (5 feline congenital glaucoma [FCG] and 4 normal), following intracameral infusion of 2.5% fluorescein and/or 0.4% indocyanine green (ICG) at physiologic intraocular pressure (IOP). Scleral OCT line scans were acquired in areas of high- and low-angiographic signal. Tissues dissected in regions of high- and low-AA signal, were sectioned and hematoxylin and eosin (H&E)-stained or immunolabeled (IF) for vascular endothelial and perivascular cell markers. Outflow vessel numbers and locations were compared between groups by Student's t-test. RESULTS: AA yielded circumferential, high-quality images of distal aqueous outflow pathways in normal and FCG eyes. No AA signal or scleral lumens were appreciated in one buphthalmic FCG eye, though collapsed vascular profiles were identified on IF. The remaining eight of nine eyes all showed segmental AA signal, distinguished by differences in time of signal onset. AA signal always corresponded with lumens seen on OCT. Numbers of intrascleral vessels were not significantly different between groups, but scleral vessels were significantly more posteriorly located relative to the limbus in FCG. CONCLUSIONS: A capacity for distal aqueous humor outflow was confirmed by AA in FCG eyes ex vivo but with significant posterior displacement of intrascleral vessels relative to the limbus in FCG compared with normal eyes. TRANSLATIONAL RELEVANCE: This report provides histopathologic correlates of advanced diagnostic imaging findings in a spontaneous model of congenital glaucoma.

Anterior-Segment Optical Coherence Tomography for Predicting Postoperative Outcomes After Trabeculectomy.

PURPOSE: To determine whether anterior-segment optical coherence tomography (AS-OCT) can be used to predict post-trabeculectomy bleb outcomes. MATERIALS AND METHODS: We divided 58 eyes of 47 trabeculectomy patients into success or failure groups based on their status at 12 months after surgery. We then compared various AS-OCT measurement parameters between the two groups at 1 and 2 weeks and 1, 3, 6, and 12 months. We also analyzed the early post-trabeculectomy bleb parameters with multiple logistic regression, stepwise multiple regression, and the receiver operating characteristic (ROC) curve, to evaluate the power of these parameters to predict long-term outcomes. RESULTS: Intraocular pressure 3 or more months after trabeculectomy was significantly lower in the success group than the failure group (all: P < 0.0016). Cleft volume was significantly higher 6 or more months after trabeculectomy in the success group than the failure group (P = 0.0027 and <0.0016). Reflectivity of the bleb wall was significantly higher in the failure group than the success group at 2 weeks and all later time points (all: P < 0.0016). Reflectivity of the bleb wall at 2 weeks after trabeculectomy was a risk factor for failure, with an odds ratio (OR) for failure of 2.48 (95% confidence interval, 1.31-4.68, increasing per 10 AU). The area under the ROC curve for reflectivity of the bleb wall at 2 weeks after trabeculectomy was 0.775 when the cutoff value was set at 122.8, with sensitivity, specificity, and OR of 78.3%, 80.0%, and 14.4, respectively. A stepwise multiple regression analysis showed that reflectivity of the bleb wall at 2 weeks was an independent factor indicating postoperative bleb survival period (ß = -0.39, P = 0.007). CONCLUSIONS: Reflectivity of the bleb wall, measured by AS-OCT, may be an early post-trabeculectomy predictor of bleb outcome.

Morphometry of the trabecular meshwork in vivo in a healthy population using fourier-domain optical coherence tomography.

PURPOSE: We measured the length, thickness, and area of the trabecular meshwork (TM) in vivo using Fourier domain optical coherence tomography (FD-OCT) in a Caucasian population of healthy subjects. METHODS: A cross-sectional study was done of 1006 healthy subjects. Left eyes were randomly selected. Age, sex, IOP, and spherical refractive error were noted. The depth and volume of the anterior chamber and the central corneal thickness were measured with Pentacam, while IOL Master was used to measure the axial length. The length, thickness, and area of the TM were measured through FD-OCT RTVue. A study was done to determine the correlation between TM size, and other demographic and ocular parameters. Finally, the reproducibility of the measurements was assessed for a subgroup of 50 eyes from 50 patients. RESULTS: We were able to measure the TM in 91.1% of the total eyes studied. The mean TM length was 496.99 ± 92.77 µm (range, 275-800), TM thickness was 174.16 ± 28.14 µm (range, 100-276), and TM area was 0.069 ± 0.031 mm(2) (range, 0.023-0.133). No differences were found in terms of length and area for sex, although the TM was slightly thicker in men (P = 0.046). No correlation was observed between the TM measurements and any of the studied demographic or ocular parameters (R < 0.09, P < 0.001). Intra- and interobserver reproducibility of the measurements were good (intraclass correlation coefficient > 0.750, P < 0.001). CONCLUSIONS: The FD-OCT is an effective and reproducible examination technique to measure the length, thickness, and area of the TM in vivo.

A comprehensive model for correcting RNFL readings of varying signal strengths in cirrus optical coherence tomography.

PURPOSE: To develop a model for the Cirrus HD-OCT that allows for the comparison of retinal nerve fiber layer (RNFL) thickness measurements with dissimilar signal strengths (SS) and accounts for test-retest variability. METHODS: Retinal nerve fiber layers were obtained in normals using the Cirrus optic disc cube 200 × 200 protocol during a single encounter. Five RNFL scans were obtained with a SS of 9 or 10. Diffusion lens filters were used to degrade SS to obtain five scans at each SS group of 7 or 8, 5 or 6, and 3 or 4. The relationship between average RNFL thickness and SS was established, and an equation was developed to allow for adjustment of an RNFL measurement had it been a SS of 7. Intravisit interclass correlation coefficient (ICC) and coefficient of variation (CV) parameter estimates for each SS group were calculated. Repeatability and upper tolerance limit were calculated as 1.96 × âˆš2 × within-subject standard deviation (Sw) and 1.645 × âˆš2 × Sw, respectively. RESULTS: There was a linear relationship between average RNFL and SS. RNFLadj = RNFL - 1.03*SS + 7.21 allows for the adjustment of RNFL readings to the same SS. Interclass correlation coefficients and CVs were good for all measurements down to SS of 3 or 4. Repeatability and upper tolerance limit were 5.24 and 4.40 µm, respectively. CONCLUSIONS: Our model adjusts RNFL readings based on SS and includes an upper tolerance limit of 5 µm. If validated, this model could improve the detection of real RNFL changes. Further study to validate this model should be performed before widespread use is adopted.

Relationship between iris surface features and angle width in Asian eyes.

PURPOSE: To examine the associations between iris surface features with anterior chamber angle width in Asian eyes. METHODS: In this prospective cross-sectional study, we recruited 600 subjects from a large population-based study, the Singapore Epidemiology of Eye Diseases (SEED) study. We obtained standardized digital slit-lamp iris photographs and graded the iris crypts (by number and size), furrows (by number and circumferential extent), and color (higher grade denoting darker iris). Vertical and horizontal cross-sections of anterior chamber were imaged using anterior segment optical coherence tomography. Angle opening distance (AOD), angle recess area (ARA), and trabecular-iris space area (TISA) were measured using customized software. Associations of the angle width with the iris surface features in the subject's right eyes were assessed using linear regression analysis. RESULTS: A total of 464 eyes of the 464 subjects (mean age: 57.5 ± 8.6 years) had complete and gradable data for crypts and color, and 423 eyes had gradable data for furrows. After adjustment for age, sex, ethnicity, pupil size, and corneal arcus, higher crypt grade was independently associated with wider AOD750 (ß [change in angle width per grade higher] = 0.018, P = 0.023), ARA750 (ß = 0.022, P = 0.049), and TISA750 (ß = 0.011, P = 0.019), and darker iris was associated narrower ARA750 (ß = -0.025, P = 0.044) and TISA750 (ß = -0.013, P = 0.011). CONCLUSIONS: Iris surface features, assessed and measured from slit-lamp photographs, correlated well with anterior chamber angle width; irises with more crypts and lighter color were associated with wider angle. These findings may provide another imaging modality to assess angle closure risk based on iris surface features.

Induction of trabecular meshwork cells from induced pluripotent stem cells.

PURPOSE: Loss or dysfunction of trabecular meshwork (TM) cells has been associated with the development of pathologically elevated IOP, and it is conceivable that replacement of damaged TM cells could restore function to the TM. We propose that the use of TM-like cells derived from induced pluripotent stem cells (iPSCs) created from a patient's own dermal fibroblasts offers the best solution to this challenge. Here we demonstrate that mouse iPSCs can be induced to differentiate into TM-like cells suitable for autologous transplantation. METHODS: Directed induction of stem cell differentiation was achieved through coculture of mouse iPSCs with human TM cells for up to 21 days. The resultant TM-like cells (iPSC-TM) were characterized morphologically, immunohistochemically, and functionally. RESULTS: The iPSC-TM cells closely resembled cultured human TM cells morphologically and began to express many markers of TM cells while ceasing to express pluripotency markers such as Nanog, Oct4, and Sox2. Functionally, these cells developed the ability to phagocytose particles. Finally, exposure to dexamethasone or phorbol 12-myristate acetate caused a distinct increase in the production and secretion of myocilin and matrix metalloproteinase-3, respectively, behavior characteristic of TM cells. CONCLUSIONS: Our data demonstrate that iPSCs can be induced to assume a phenotype that resembles native TM cells in many important aspects. Not only do these cells represent a valuable research tool, but transplantation into glaucomatous eyes with elevated IOP may also restore function to the TM, resulting in re-establishment of IOP.

Differential effects of caveolin-1 and -2 knockdown on aqueous outflow and altered extracellular matrix turnover in caveolin-silenced trabecular meshwork cells.

PURPOSE: A single nucleotide polymorphism (SNP) identified between caveolin-1 (CAV1) and caveolin-2 (CAV2) on chromosome 7 is associated with glaucoma. One function of CAVs is endocytosis and recycling of extracellular matrix (ECM) components. Here, we generated CAV-silencing lentivirus to evaluate the effects on ECM turnover by trabecular meshwork (TM) cells and to measure the effect on outflow facility in anterior segment perfusion culture. METHODS: Short hairpin CAV1 and CAV2 silencing and control lentivirus were generated, characterized, and applied to anterior segments in perfusion culture. Colocalization of CAVs with various ECM molecules in TM cells was investigated using immunofluorescence and confocal microscopy. Western immunoblotting and fluorogenic-based enzyme activity assays were used to investigate ECM protein levels and degradation, respectively. RESULTS: Endogenous CAVs colocalized with cortactin at podosome- or invadopodia-like structures (PILS), which are areas of focal ECM degradation. In perfusion culture, outflow rates increased significantly in CAV1-silenced anterior segments, whereas outflow significantly decreased in CAV2-silenced anterior segments. Matrix metalloproteinase (MMP)2 and MMP14, and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS4) colocalized with both CAVs in TM cells. Protein levels and enzyme activities of MMP/ADAMTS4, fibronectin protein levels, actin stress fibers, and α-smooth muscle actin were all increased in CAV-silenced cells. CONCLUSIONS: Caveolin-mediated endocytosis is one mechanism by which TM cells can alter the physiological catabolism of ECM in order to change the composition of the outflow channels in the TM to regulate aqueous outflow resistance. Dysregulation of CAV function could contribute to the pathological changes in ECM that are observed in glaucoma.

Improvement in outflow facility by two novel microinvasive glaucoma surgery implants.

PURPOSE: To determine improvement in outflow facility (C) in human anterior segments implanted with a novel Schlemm's canal scaffold or two trabecular micro-bypasses. METHODS: Human anterior segments were isolated from 12 pairs of eyes from donors with no history of ocular disease and then perfused at 50, 40, 30, 20, and 10 mm Hg pressures for 10 minutes each. Baseline C was calculated from perfusion pressures and flow rates. The scaffold was implanted into Schlemm's canal of one anterior segment, and two micro-bypasses were implanted three clock-hours apart in the contralateral anterior segment. Outflow facility and resistance were compared at various standardized perfusion pressures and between each device. RESULTS: Compared to baseline, C increased by 0.16 ± 0.12 µL/min/mm Hg (74%) with the scaffold, and 0.08 ± 0.12 µL/min/mm Hg (34%) with two micro-bypasses. The scaffold increased C at perfusion pressures of 50, 40, 30, and 20 mm Hg (P < 0.005). Two micro-bypasses increased C at a perfusion pressure of 40 mm Hg (P < 0.05). CONCLUSIONS: Both implants effectively increased C in human eyes ex vivo. The scaffold increased C by a greater percentage (73% vs. 34%) and at a greater range of perfusion pressures (20 to 50 mm Hg vs. 40 mm Hg) than the two micro-bypasses, suggesting that the 8-mm dilation of Schlemm's canal by the scaffold may have additional benefits in lowering the outflow resistance. The Hydrus Microstent scaffold may be an effective therapy for increasing outflow facility and thus reducing the IOP in patients with glaucoma.

Scleral intraocular pressure measurement in cadaver eyes pre- and postkeratoprosthesis implantation.

PURPOSE: We correlated scleral IOP to assigned IOP using pneumatonometry in cadaver eyes before and after Boston type I keratoprosthesis (KPro) implantation. METHODS: Corneal IOP and scleral IOP at the superonasal, superotemporal, inferotemporal, and inferonasal quadrants were measured using pneumatonometry in six cadaver eyes cannulated with an infusion line with assigned IOP held at 20, 30, 40, and 50 mm Hg. Measurements of scleral IOP at the same location were repeated after a KPro was implanted. Correlations between scleral IOP and assigned IOP were analyzed for the entire group of eyes, and for each individual eye before and after KPro. One eye was tested by another masked grader for interobserver variability. RESULTS: Scleral IOP measured higher than corneal IOP by a mean of 13.2 mm Hg. For group analysis, pre-KPro scleral IOP had a positive and linear correlation with assigned IOP in all quadrants (P < 0.00001), and this correlation was preserved after KPro implantation (P < 0.00001). There was strong interobserver agreement in all measurement sites (P < 0.001). In analyses of individual eyes, scleral IOP measured at the inferotemporal quadrant confirmed the strong linear association between scleral IOP and assigned IOP before and after KPro for all study eyes. A Bland-Altman plot showed that the difference in scleral IOP between pre-KPro and post-KPro eyes fell mostly within ± 5 mm Hg. CONCLUSIONS: Scleral IOP measured by pneumatonometry may be used to estimate IOP in cadaver eyes with and without keratoprosthesis. This may be a potential modality for assessing IOP for patients with corneal pathology or keratoprosthesis.

Pro-inflammatory cytokines in glaucomatous aqueous and encysted Molteno implant blebs and their relationship to pressure.

PURPOSE: To ascertain the presence of additional pro-inflammatory cytokines in glaucomatous aqueous, and their relationship with IOP. METHODS: To quantify the levels of 23 pro-inflammatory cytokines, and correlate levels with IOP, aqueous humor samples were analyzed from 23 eyes with open angle glaucoma (OAG) undergoing glaucoma filtration procedures, and from 24 Molteno blebs during the hypertensive phase. Control aqueous was derived from 13 eyes without glaucoma undergoing cataract removal. RESULTS: A significant difference (P<0.05) was noted between hypertensive bleb aqueous and controls in the amount TGF-ß2, interleukins IL-6, IL-10, and chemokine (C-X-C motif) ligand 1 (CXCL1; GROα). The levels of these cytokines were higher in the glaucomatous aqueous, but not significantly so. A significant difference was noted in levels of chemokine (C-C motif) ligand 2 (CCL2; MCP-1, monocyte chemotactic protein-1) in the glaucoma eye and bleb aqueous compared with controls. Of the 23 cytokines tested for, 19 were found in the bleb group, 14 in the glaucoma group, and 16 in the control group. Compared with controls, all cytokines levels were higher in the glaucoma group and highest in the bleb group. CONCLUSIONS: The study confirms the well documented presence of TGF-ß2 in glaucomatous aqueous. The presence of significant levels of CCL2 in glaucomatous aqueous is a new finding. The finding of higher levels of all the cytokines in the aqueous from the encysted blebs, in which the IOP was the highest, suggests that their levels increase with an increase in IOP, as well as the possibility that encysted blebs form cytokines.