Profiling IOP-responsive genes in anterior and posterior ocular tissues in the rat CEI glaucoma model.

Purpose: The rat Controlled Elevation of Intraocular pressure (CEI) model allows study of in vivo responses to defined intraocular pressures (IOP). In this study, we use Nanostring technology to investigate in vivo IOP-related gene responses in the trabecular meshwork (TM) and optic nerve head (ONH) simultaneously from the same animals. Methods: Male and female rats (N=35) were subject to CEI for 8-hours at pressures simulating mean, daytime normotensive rat IOP (CEI-20), or 2.5x IOP (CEI-50). Naïve animals, receiving no anesthesia or surgical interventions, served as controls. Immediately after CEI, TM and ONH tissues were dissected, RNA isolated, and samples were analyzed with a Nanostring panel containing 770 genes. Post-processing, raw count data were uploaded to Rosalind® for differential gene expression analyses. Results: For the TM, 45 IOP-related genes were significant in the "CEI-50 vs. CEI-20" and "CEI-50 vs. naïve" comparisons, with 15 genes common to both comparisons. Bioinformatics analysis identified Notch and TGFß pathways to be the most up- and down-regulated KEGG pathways, respectively. For ONH, 22 significantly regulated genes were identified in the "CEI-50 vs. naïve" comparison. Pathway analysis identified 'defense response' and 'immune response' as two significantly upregulated biological process pathways. Conclusions: This study demonstrates the ability to assay IOP-responsive genes in both TM and ONH tissues simultaneously. In the TM, downregulation of TGFß pathway genes suggest that TM responses may prevent TGFß-induced extracellular matrix synthesis. For ONH, the initial response to elevated IOP may be protective, with astrocytes playing a key role in these gene responses.

A method describing the microdissection of trabecular meshwork tissue from Brown Norway rat eyes.

Glaucoma is often associated with elevated intraocular pressure (IOP), generally due to obstruction of aqueous humor outflow within the trabecular meshwork (TM). Despite many decades of research, the molecular cause of this obstruction remains elusive. To study IOP regulation, several in vitro models, such as perfusion of anterior segments or mechanical stretching of TM cells, have identified several IOP-responsive genes and proteins. While these studies have proved informative, they do not fully recapitulate the in vivo environment where IOP is subject to additional factors, such as circadian rhythms. Thus, rodent animal models are now commonly used to study IOP-responsive genes in vivo. Several single-cell RNAseq studies have been performed where angle tissue, containing cornea, iris, ciliary body tissue in addition to TM, is dissected. However, it is advantageous to physically separate TM from other tissues because the ratio of TM cells is relatively low compared to the other cell types. In this report, we describe a new technique for rat TM microdissection. Evaluating tissue post-dissection by histology and immunostaining clearly shows successful removal of the TM. In addition, TaqMan PCR primers targeting biomarkers of trabecular meshwork (Myoc, Mgp, Chi3l1) or ciliary body (Myh11, Des) genes showed little contamination of TM tissue by the ciliary body. Finally, pitfalls encountered during TM microdissection are discussed to enable others to successfully perform this microsurgical technique in the rat eye.

Effect of algorithms and covariates in glaucoma diagnosis with optical coherence tomography angiography.

PURPOSE: To assess the effects of algorithms and covariates in glaucoma diagnosis with optical coherence tomography angiography (OCTA). METHODS: In this prospective cross-sectional study, one eye each of 36 normal controls and 64 patients with glaucoma underwent 4.5 mm disc-centred and 6 mm macula-centred OCTA scans. The peripapillary nerve fibre layer plexus capillary density (NFLP-CD) and macular superficial vascular complex vessel density (SVC-VD) were measured using both a commercial algorithm (AngioAnalytics) and a custom algorithm (Center for Ophthalmic Optics & Lasers Angiography Reading Toolkit (COOL-ART)). The nerve fibre layer and ganglion cell complex thicknesses were measured on structural OCT. RESULTS: The overall peripapillary NFLP-CD and macular SVC-VD measured with the two algorithms were highly correlated but poorly agreed. Among the normal controls, the perfusion measurements made by both algorithms were significantly correlated with age. AngioAnalytics measurements were also correlated with signal strength index, while COOL-ART measurements were not. These covariates were adjusted. The diagnostic accuracy, measured as the area under the receiver operating characteristic curve for glaucoma detection, was not significantly different between algorithms, between structural and perfusion parameters and between the peripapillary and macular regions (All p>0.05). The macular SVC-VD in the 6 mm square had a significantly higher diagnostic accuracy than that of the central 3 mm square area (p=0.005). CONCLUSIONS: AngioAnalytics and COOL-ART vessel density measurements are not interchangeable but potentially interconvertible. Age and signal strength are significant covariates that need to be considered. Both algorithms and both peripapillary and macular perfusion parameters have similarly good diagnostic accuracy comparable to structural OCT. A larger macular analytic area provides higher diagnostic accuracy.

Focal Loss Analysis of Nerve Fiber Layer Reflectance for Glaucoma Diagnosis.

Purpose: To evaluate nerve fiber layer (NFL) reflectance for glaucoma diagnosis. Methods: Participants were imaged with 4.5 × 4.5 mm volumetric disc scans using spectral-domain optical coherence tomography. The normalized NFL reflectance map was processed by an azimuthal filter to reduce directional reflectance bias caused by variation of beam incidence angle. The peripapillary area of the map was divided into 160 superpixels. Average reflectance was the mean of superpixel reflectance. Low-reflectance superpixels were identified as those with NFL reflectance below the fifth percentile normative cutoff. Focal reflectance loss was measured by summing loss in low-reflectance superpixels. Results: Thirty-five normal, 30 preperimetric, and 35 perimetric glaucoma participants were enrolled. Azimuthal filtering improved the repeatability of the normalized NFL reflectance, as measured by the pooled superpixel standard deviation (SD), from 0.73 to 0.57 dB (P < 0.001, paired t-test) and reduced the population SD from 2.14 to 1.78 dB (P < 0.001, t-test). Most glaucomatous reflectance maps showed characteristic patterns of contiguous wedge or diffuse defects. Focal NFL reflectance loss had significantly higher diagnostic sensitivity than the best NFL thickness parameter (from map or profile): 77% versus 55% (P < 0.001) in glaucoma eyes with the specificity fixed at 99%. Conclusions: Azimuthal filtering reduces the variability of NFL reflectance measurements. Focal NFL reflectance loss has excellent glaucoma diagnostic accuracy compared to the standard NFL thickness parameters. The reflectance map may be useful for localizing NFL defects. Translational Relevance: The high diagnostic accuracy of NFL reflectance may make population-based screening feasible.

Utilizing a Professionalism Mentor to Address Sexual Harassment in Academic Ophthalmology.

Objective This study assesses a new departmental role-a professionalism mentor-who receives sexual harassment reporting, liaisons with campus resources, and organizes educational sessions. Study Design Multicenter randomized controlled survey study. Methods Academic ophthalmology departments in the United States were randomized to a professionalism mentor group ( n = 9) and a control group ( n = 7). Among both pre- and postsurveys, 605 faculty and trainee responses were received and 546 were complete. The intervention group was assigned a professionalism mentor with educational session for a 6- to 10-month period. Sexual harassment and reporting rate change over time were compared between the two groups. Results Among 546 anonymous responses, 16% experienced workplace sexual harassment during the prior 10 months. Location in the South or Midwest was a risk factor ( p < 0.001). Victims were mostly women (76%), including residents/fellows (46%) and academic attendings (49%); perpetrators included patients (35%) and academic attendings (35%). Departments with and without a professionalism mentor had stable harassment from pre- to postsurvey ( p = 0.95 comparing change). The professionalism mentor group had an increase in reporting to an authority from pre- to postsurvey (7-23%), whereas the control group had a decrease (27-12%; p = 0.07 comparing change). Most faculty and trainees in the interventional arm of this study recommended instituting a professionalism mentor with educational session (66% presurvey and 68% postsurvey), compared with educational session alone (25% presurvey and 23% postsurvey), or neither (9% presurvey and 9% postsurvey). Residency program directors in the professionalism mentor group even more strongly supported instituting both a professionalism mentor and educational program (100% presurvey and 100% postsurvey) as opposed to educational program alone (0% presurvey and 0% postsurvey) or neither (0% presurvey and 0% postsurvey). Conclusion This study identified a high sexual harassment rate in academic ophthalmology departments over a brief period. The presence of a professionalism mentor was viewed favorably and may lead to increased reporting.

Case Series of Stickler Syndrome Presenting With Acute Angle Closure.

PURPOSE: Stickler syndrome is associated with open-angle glaucoma, but no reports exist of adults presenting with acute angle closure. Here, we report the clinical associations and short-term outcomes in 2 patients with Stickler syndrome who presented with acute angle closure at a single tertiary referral center. OBSERVATIONS: A 36-year-old female with Stickler syndrome presented with acute onset of red and painful left eye with cloudy vision. Initial intraocular pressure (IOP) was measured to be 54 mm Hg. Gonioscopy revealed no angle structures or peripheral anterior synechiae in the left eye. Two laser peripheral iridotomies were created 5 days apart without adequate IOP lowering. Left eye cataract extraction with intraocular lens implantation and Ahmed glaucoma drainage device implantation were performed with resolution of ocular hypertension.A 35-year-old male with Stickler syndrome presented with intermittent right eye pain and blurry vision. IOP was 42 mm Hg and gonioscopy revealed 270 degrees of angle closure in the right eye. The patient underwent laser peripheral iridotomy in the right eye with resolution of ocular hypertension by the next day. CONCLUSIONS: Recognizing angle closure as a potential feature of Stickler syndrome can help providers adequately screen and manage patients with Stickler syndrome presenting with acutely elevated IOP.

Sectorwise Visual Field Simulation Using Optical Coherence Tomographic Angiography Nerve Fiber Layer Plexus Measurements in Glaucoma.

PURPOSE: To simulate 24-2 visual field (VF) using optical coherence tomographic angiography (OCTA) for glaucoma evaluation. DESIGN: Cross-sectional study. METHODS: One eye each of 39 glaucoma and 31 age-matched normal participants was scanned using 4.5-mm OCTA scans centered on the disc. The peripapillary retinal nerve fiber layer plexus capillary density (NFLP-CD, %area) was measured. The NFLP-CD and 24-2 VF maps were divided into 8 corresponding sectors using an extension of Garway-Heath scheme. RESULTS: Sector NFLP-CD was transformed to a logarithmic dB scale and converted to sector simulated VF deviation maps. Comparing simulated and actual 24-2 VF maps, the worst sector was in the same or adjacent location in the same hemisphere 97% of the time. VF mean deviation (VF-MD) was simulated by NFLP mean deviation (NFLP-MD). The differences between NFLP-MD and VF-MD in early, moderate, and severe glaucoma stages were -0.9 ± 2.0, 0.9 ± 2.9, and 5.8 ± 3.2 dB. NFLP-MD had better (P = .015) between-visit reproducibility (0.63 dB pooled standard deviation) than VF-MD (1.03 dB). NFLP-MD had a significantly higher sensitivity than VF-MD (P < .001) and overall NFL thickness (P = .031). CONCLUSIONS: OCTA-based simulated VF agreed well with actual 24-2 VF in terms of both the location and severity of glaucoma damage, with the exception of severe glaucoma in which the simulation tended to underestimate severity. The NFLP-MD had better reproducibility than actual VF-MD and holds promise for improving glaucoma monitoring. The NFLP-MD had better diagnostic accuracy than both VF-MD and overall NFL thickness and may be useful for early glaucoma diagnosis.

Cupping in the Monkey Optic Nerve Transection Model Consists of Prelaminar Tissue Thinning in the Absence of Posterior Laminar Deformation.

PURPOSE: To use optical coherence tomography (OCT) to test the hypothesis that optic nerve head (ONH) "cupping" in the monkey optic nerve transection (ONT) model does not include posterior laminar deformation. METHODS: Five monkeys (aged 5.5-7.8 years) underwent ONH and retinal nerve fiber layer (RNFL) OCT imaging five times at baseline and biweekly following unilateral ONT until euthanization at ∼40% RNFL loss. Retinal nerve fiber layer thickness (RNFLT) and minimum rim width (MRW) were calculated from each pre- and post-ONT imaging session. The anterior lamina cribrosa surface (ALCS) was delineated within baseline and pre-euthanasia data sets. Significant ONT versus control eye pre-euthanasia change in prelaminar tissue thickness (PLTT), MRW, RNFLT, and ALCS depth (ALCSD) was determined using a linear mixed-effects model. Eye-specific change in each parameter exceeded the 95% confidence interval constructed from baseline measurements. RESULTS: Animals were euthanized 49 to 51 days post ONT. Overall ONT eye change from baseline was significant for MRW (-26.2%, P = 0.0011), RNFLT (-43.8%, P < 0.0001), PLTT (-23.8%, P = 0.0013), and ALCSD (-20.8%, P = 0.033). All five ONT eyes demonstrated significant eye-specific decreases in MRW (-23.7% to -31.8%) and RNFLT (-39.6% to -49.7%). Four ONT eyes showed significant PLTT thinning (-23.0% to -28.2%). The ALCS was anteriorly displaced in three of the ONT eyes (-25.7% to -39.2%). No ONT eye demonstrated posterior laminar displacement. CONCLUSIONS: Seven weeks following surgical ONT in the monkey eye, ONH cupping involves prelaminar and rim tissue thinning without posterior deformation of the lamina cribrosa.

Maternal hemoglobin concentration and its association with birth weight in newborns of mothers with preeclampsia.

OBJECTIVE: Maternal hemoglobin concentration is inversely related to newborn size presumably through plasma volume constriction. We sought to determine whether birth weight would show an inverse relationship to hemoglobin concentration in a group of infants whose mothers had preeclampsia, where plasma volume constriction is common. METHODS: Electronic and paper chart review identified 142 nulliparous women with preeclampsia (excluding hemolysis, elevated liver enzymes, low platelets syndrome). Birth weight percentile was determined based on cross-sectional hybrid growth curves. Maximal third trimester maternal hemoglobin concentrations were obtained and standardised to z-scores based on gestational age matched normative data. Birth weight percentile was examined as a function of hemoglobin z-score using appropriate statistics. RESULTS: Average gestational age at delivery was 35.9 +/- 1.9 weeks. Mean birth weight percentile for infants of preeclamptic mothers was 34 +/- 32. Mean hemoglobin z-score for mothers with preeclampsia was 0.3 +/- 1.5, significantly higher than a control population (p = 0.04). Maternal hemoglobin z-score was inversely associated with birth weight percentile (r = -0.18, p = 0.03). CONCLUSION: Maternal hemoglobin concentrations are significantly elevated prior to delivery in women with preeclampsia. There is a statistically significant inverse correlation of maternal hemoglobin concentration to birth weight percentile.