Choi HJ, Sun D, Jakobs TC. Isolation of intact astrocytes from the optic nerve head of adult mice. Exp Eye Res 2015;137:103-10.Abstract

The astrocytes of the optic nerve head are a specialized subtype of white matter astrocytes that form the direct cellular environment of the unmyelinated ganglion cell axons. Due to their potential involvement in glaucoma, these astrocytes have become a target of research. Due to the heterogeneity of the optic nerve tissue, which also contains other cell types, in some cases it may be desirable to conduct gene expression studies on small numbers of well-characterized astrocytes or even individual cells. Here, we describe a simple method to isolate individual astrocytes. This method permits obtaining astrocytes with intact morphology from the adult mouse optic nerve and reduces contamination of the isolated astrocytes by other cell types. Individual astrocytes can be recognized by their morphology and collected under microscopic control. The whole procedure can be completed in 2-3 h. We also discuss downstream applications like multiplex single-cell PCR and quantitative PCR (qPCR).

Houston KE, Woods RL, Goldstein RB, Peli E, Luo G, Bowers AR. Asymmetry in the Collision Judgments of People With Homonymous Field Defects and Left Hemispatial Neglect. Invest Ophthalmol Vis Sci 2015;56(6):4135-42.Abstract

PURPOSE: Although the impact of homonymous visual field defects (HFDs) on mobility has been investigated previously, the emphasis has been on obstacle detection. Relatively little is known about HFD patients' ability to judge collisions once an obstacle is detected. We investigated this using a walking simulator. METHODS: Patients with HFDs (n = 29) and subjects with normal vision (NV; n = 21) were seated in front of a large screen on which a visual simulation of walking was displayed. They made collision judgments for a human figure that appeared for 1 second at lateral offsets from the virtual walking path. A perceived-collision threshold was calculated for right and left sides. RESULTS: Symmetrical collision thresholds (same on left and right sides) were measured for participants with NV (n = 21), and right (n = 9) and left (n = 7) HFD without hemispatial neglect. Participants with left neglect (n = 10) showed significant asymmetry with thresholds smaller (compared to the NV group and other HFD groups) on the blind (P < 0.001) and larger on the seeing (P = 0.05) sides. Despite the asymmetry, the overall width of the zone of perceived collision risk was not different, suggesting a relatively uniform rightward deviation in judgments of the left neglect group. CONCLUSIONS: Left neglect was associated with rightward asymmetry in collision judgments, which may cause collisions on the left side even when an obstacle is detected. These behaviors may represent the spatial misperceptions in body midline described previously in patients with left neglect.

Quiroz YT, Schultz AP, Chen K, Protas HD, Brickhouse M, Fleisher AS, Langbaum JB, Thiyyagura P, Fagan AM, Shah AR, Muniz M, Arboleda-Velasquez JF, Munoz C, Garcia G, Acosta-Baena N, Giraldo M, Tirado V, Ramírez DL, Tariot PN, Dickerson BC, Sperling RA, Lopera F, Reiman EM. Brain Imaging and Blood Biomarker Abnormalities in Children With Autosomal Dominant Alzheimer Disease: A Cross-Sectional Study. JAMA Neurol 2015;72(8):912-9.Abstract

IMPORTANCE: Brain imaging and fluid biomarkers are characterized in children at risk for autosomal dominant Alzheimer disease (ADAD). OBJECTIVE: To characterize and compare structural magnetic resonance imaging (MRI), resting-state and task-dependent functional MRI, and plasma amyloid-β (Aβ) measurements in presenilin 1 (PSEN1) E280A mutation-carrying and noncarrying children with ADAD. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional measures of structural and functional MRI and plasma Aβ assays were assessed in 18 PSEN1 E280A carriers and 19 noncarriers aged 9 to 17 years from a Colombian kindred with ADAD. Recruitment and data collection for this study were conducted at the University of Antioquia and the Hospital Pablo Tobon Uribe in Medellín, Colombia, between August 2011 and June 2012. MAIN OUTCOMES AND MEASURES: All participants had blood sampling, structural MRI, and functional MRI during associative memory encoding and resting-state and cognitive assessments. Outcome measures included plasma Aβ1-42 concentrations and Aβ1-42:Aβ1-40 ratios, memory encoding-dependent activation changes, resting-state connectivity, and regional gray matter volumes. Structural and functional MRI data were compared using automated brain mapping algorithms and search regions related to AD. RESULTS: Similar to findings in adult mutation carriers, in the later preclinical and clinical stages of ADAD, mutation-carrying children were distinguished from control individuals by significantly higher plasma Aβ1-42 levels (mean [SD]: carriers, 18.8 [5.1] pg/mL and noncarriers, 13.1 [3.2] pg/mL; P < .001) and Aβ1-42:Aβ1-40 ratios (mean [SD]: carriers, 0.32 [0.06] and noncarriers, 0.21 [0.03]; P < .001), as well as less memory encoding task-related deactivation in parietal regions (eg, mean [SD] parameter estimates for the right precuneus were -0.590 [0.50] for noncarriers and -0.087 [0.38] for carriers; P < .005 uncorrected). Unlike carriers in the later stages, mutation-carrying children demonstrated increased functional connectivity of the posterior cingulate cortex with medial temporal lobe regions (mean [SD] parameter estimates were 0.038 [0.070] for noncarriers and 0.190 [0.057] for carriers), as well as greater gray matter volumes in temporal regions (eg, left parahippocampus; P < . 049, corrected for multiple comparisons). CONCLUSIONS AND RELEVANCE: Children at genetic risk for ADAD have functional and structural brain changes and abnormal levels of plasma Aβ1-42. The extent to which the underlying brain changes are either neurodegenerative or developmental remains to be determined. This study provides additional information about the earliest known biomarker changes associated with ADAD.

Patel AV, Rashid A, Jakobiec FA, Lefebvre DR, Yoon MK. Orbital Branch of the Infraorbital Artery: Further Characterization of an Important Surgical Landmark. Orbit 2015;34(4):212-5.Abstract

The orbital branch of the infraorbital artery, a key vascular structure that is not universally noted in orbital textbooks and atlases, is clinically significant, since injury to it can result in perioperative hemorrhage. We conducted a cadaver dissection to document its presence, measure its location, and evaluate it histopathologically. It was present in 8 of 9 orbits and was a mean distance of 16.6 mm (range 10-23) from the inferior orbital rim. In half of the specimens, there were 2 separate structures seen. Histopathology confirmed these structures to be neurovascular bundles.

Shen LQ, Kloek CE, Turalba AV. Assessing the Effect of a Glaucoma Surgical Curriculum in Resident Physicians. JAMA Ophthalmol 2015;133(9):1077-80.Abstract

IMPORTANCE: Subspecialty surgical training is an important part of resident education. We changed the glaucoma rotation in which postgraduate year 4 residents worked with multiple attending physicians with varying teaching styles to a structured surgical curriculum led by 2 dedicated preceptors, and we evaluated the effect on residents' surgical performance prospectively. OBSERVATIONS: A curriculum consisting of preoperative training, intraoperative teaching, postoperative feedback, and repetition was implemented for postgraduate year 4 residents between July 2, 2012, and June 30, 2014. In a class of 8 residents per year, the mean (SD) glaucoma surgical volume increased from 8.9 (0.8) cases in the prior year to 13.6 (2.5) in 2013 (mean difference, 4.8 cases; 95% CI, 2.4-7.1; P = .001) and 14.8 (4.2) in 2014 (mean difference, 5.9 cases; 95% CI, 2.1-9.6; P = .007). A self-assessment survey showed improvement in suturing (scores for each section range from 1 [worst] to 5 [best]; mean rating, 3.9 in the prior year vs 4.4 in 2013 [P = .04] and 4.5 in 2014 [P = .02]). A validated survey assessing overall surgical competency revealed improvement in handling adverse events (mean rating, 4.1 in the prior year vs 5.0 for both 2013 and 2014; both P < .001). CONCLUSIONS AND RELEVANCE: Despite the small sample size and nonrandomized study design, these data suggest that a structured surgical curriculum has advantages in teaching subspecialty surgery and might be considered by other ophthalmology training programs.

Kruh JN, Kruh-Garcia NA, Foster SC. Evaluation of the Effect of N-Acetylcysteine on Protein Deposition on Contact Lenses in Patients with the Boston Keratoprosthesis Type I. J Ocul Pharmacol Ther 2015;31(6):314-22.Abstract

PURPOSE: To establish the efficacy of topical N-acetylcysteine (NAC) as a treatment to reduce protein deposition on the contact lens surface. METHODS: In this prospective, nonrandomized clinical trial, a total of 10 eyes (9 patients) were enrolled from a single center. All patients had a prior ocular history of either a Boston Keratoprosthesis type I or trichiasis from Stevens-Johnson syndrome, which necessitated full-time contact lens wear. Four visits were required to complete the study. During visit 1, a new contact lens was inserted and a baseline examination was performed. Visit 2 served as the control month, whereas visits 3 and 4 were month 1 and 2 on treatment with 20% NAC. At the end of each visit the contact lens was replaced. The lenses from visit 2 (control month-without NAC) and from visit 3 (treatment month-with NAC) were collected for proteomic analysis. The main outcome measures were to quantify protein deposition, as well as to assess the visual acuity and ocular surface symptoms before and after treatment. RESULTS: Topical NAC resulted in a 20% decrease in protein deposition. This correlated with a trend for improvement in visual acuity and increased subjective improvement in vision at month 1 (P=0.0153) and 2 (P=0.0016). CONCLUSIONS: NAC reduced protein deposition, decreased ocular surface symptoms, and improved contact lens transparency, thereby providing increased optical clarity.

Silva PS, Cavallerano JD, Tolson AM, Rodriguez J, Rodriguez S, Ajlan R, Tolls D, Patel B, Sehizadeh M, Thakore K, Sun JK, Aiello LP. Real-Time Ultrawide Field Image Evaluation of Retinopathy in a Diabetes Telemedicine Program. Diabetes Care 2015;38(9):1643-9.Abstract

OBJECTIVE: To evaluate the ability of trained nonphysician retinal imagers to perform diabetic retinopathy (DR) evaluation at the time of ultrawide field retinal (UWF) imaging in a teleophthalmology program. RESEARCH DESIGN AND METHODS: Clinic patients with diabetes received Joslin Vision Network protocol retinal imaging as part of their standard medical care. Retinal imagers evaluated UWF images for referable DR at the time of image capture. Training of the imagers included 4 h of standardized didactic lectures and 12 h of guided image review. Real-time evaluations were compared with standard masked gradings performed at a centralized reading center. RESULTS: A total of 3,978 eyes of 1,989 consecutive patients were imaged and evaluated. By reading center evaluation, 3,769 eyes (94.7%) were gradable for DR, 1,376 (36.5%) had DR, and 580 (15.3%) had referable DR. Compared with the reading center, real-time image evaluation had a sensitivity and specificity for identifying more than minimal DR of 0.95 (95% CI 0.94-0.97) and 0.84 (0.82-0.85), respectively, and 0.99 (0.97-1.00) and 0.76 (0.75-0.78), respectively, for detecting referable DR. Only three patients with referable DR were not identified by imager evaluation. CONCLUSIONS: Point-of-care evaluation of UWF images by nonphysician imagers following standardized acquisition and evaluation protocols within an established teleophthalmology program had good sensitivity and specificity for detection of DR and for identification of referable retinal disease. With immediate image evaluation, <0.1% of patients with referable DR would be missed, reading center image grading burden would be reduced by 60%, and patient feedback would be expedited.

McAlvin JB, Zhan C, Dohlman JC, Kolovou PE, Salvador-Culla B, Kohane DS. Corneal Anesthesia With Site 1 Sodium Channel Blockers and Dexmedetomidine. Invest Ophthalmol Vis Sci 2015;56(6):3820-6.Abstract

PURPOSE: Amino-amide or amino-ester local anesthetics, which are currently used for topical ocular anesthesia, are short acting and may delay corneal healing with long-term use. In contrast, site 1 sodium channel blockers (S1SCBs) are potent local anesthetics with minimal adverse tissue reaction. In this study, we examined topical local anesthesia with two S1SCBs, tetrodotoxin (TTX) or saxitoxin (STX) individually or in combination with α2-adrenergic receptor agonists (dexmedetomidine or clonidine), and compared them with the amino-ester ocular anesthetic proparacaine. The effect of test solutions on corneal healing was also studied. METHODS: Solutions of TTX ± dexmedetomidine, TTX ± clonidine, STX ± dexmedetomidine, dexmedetomidine, or proparacaine were applied to the rat cornea. Tactile sensitivity was measured by recording the blink response to probing of the cornea with a Cochet-Bonnet esthesiometer. The duration of corneal anesthesia was calculated. Cytotoxicity from anesthetic solutions was measured in vitro. The effect on corneal healing was measured in vivo after corneal debridement followed by repeated drug administration. RESULTS: Addition of dexmedetomidine to TTX or STX significantly prolonged corneal anesthesia beyond that of either drug alone, whereas clonidine did not. Tetrodotoxin or STX coadministered with dexmedetomidine resulted in two to three times longer corneal anesthesia than did proparacaine. S1SCB-dexmedetomidine formulations were not cytotoxic. Corneal healing was not delayed significantly by any of the test solutions. CONCLUSIONS: Coadministration of S1SCBs with dexmedetomidine provided prolonged corneal anesthesia without delaying corneal wound healing. Such formulations may be useful for the management of acute surgical and nonsurgical corneal pain.

Tandon A, Chen CJ, Penman A, Hancock H, James M, Husain D, Andreoli C, Li X, Kuo JZ, Idowu O, Riche D, Papavasilieou E, Brauner S, Smith SO, Hoadley S, Richardson C, Kieser T, Vazquez V, Chi C, Fernandez M, Harden M, Cotch MF, Siscovick D, Taylor HA, Wilson JG, Reich D, Wong TY, Klein R, Klein BEK, Rotter JI, Patterson N, Sobrin L. African Ancestry Analysis and Admixture Genetic Mapping for Proliferative Diabetic Retinopathy in African Americans. Invest Ophthalmol Vis Sci 2015;56(6):3999-4005.Abstract

PURPOSE: To examine the relationship between proportion of African ancestry (PAA) and proliferative diabetic retinopathy (PDR) and to identify genetic loci associated with PDR using admixture mapping in African Americans with type 2 diabetes (T2D). METHODS: Between 1993 and 2013, 1440 participants enrolled in four different studies had fundus photographs graded using the Early Treatment Diabetic Retinopathy Study scale. Cases (n = 305) had PDR while controls (n = 1135) had nonproliferative diabetic retinopathy (DR) or no DR. Covariates included diabetes duration, hemoglobin A1C, systolic blood pressure, income, and education. Genotyping was performed on the Affymetrix platform. The association between PAA and PDR was evaluated using logistic regression. Genome-wide admixture scanning was performed using ANCESTRYMAP software. RESULTS: In the univariate analysis, PDR was associated with increased PAA (odds ratio [OR] = 1.36, 95% confidence interval [CI] = 1.16-1.59, P = 0.0002). In multivariate regression adjusting for traditional DR risk factors, income and education, the association between PAA and PDR was attenuated and no longer significant (OR = 1.21, 95% CI = 0.59-2.47, P = 0.61). For the admixture analyses, the maximum genome-wide score was 1.44 on chromosome 1. CONCLUSIONS: In this largest study of PDR in African Americans with T2D to date, an association between PAA and PDR is not present after adjustment for clinical, demographic, and socioeconomic factors. No genome-wide significant locus (defined as having a locus-genome statistic > 5) was identified with admixture analysis. Further analyses with even larger sample sizes are needed to definitively assess if any admixture signal for DR is present.

Sampson JF, Hasegawa E, Mulki L, Suryawanshi A, Jiang S, Chen W-S, Rabinovich GA, Connor KM, Panjwani N. Galectin-8 Ameliorates Murine Autoimmune Ocular Pathology and Promotes a Regulatory T Cell Response. PLoS One 2015;10(6):e0130772.Abstract

Galectins have emerged as potent immunoregulatory agents that control chronic inflammation through distinct mechanisms. Here, we report that treatment with Galectin-8 (Gal-8), a tandem-repeat member of the galectin family, reduces retinal pathology and prevents photoreceptor cell damage in a murine model of experimental autoimmune uveitis. Gal-8 treatment increased the number of regulatory T cells (Treg) in both the draining lymph node (dLN) and the inflamed retina. Moreover, a greater percentage of Treg cells in the dLN and retina of Gal-8 treated animals expressed the inhibitory coreceptor cytotoxic T lymphocyte antigen (CTLA)-4, the immunosuppressive cytokine IL-10, and the tissue-homing integrin CD103. Treg cells in the retina of Gal-8-treated mice were primarily inducible Treg cells that lack the expression of neuropilin-1. In addition, Gal-8 treatment blunted production of inflammatory cytokines by retinal T helper type (TH) 1 and TH17 cells. The effect of Gal-8 on T cell differentiation and/or function was specific for tissues undergoing an active immune response, as Gal-8 treatment had no effect on T cell populations in the spleen. Given the need for rational therapies for managing human uveitis, Gal-8 emerges as an attractive therapeutic candidate not only for treating retinal autoimmune diseases, but also for other TH1- and TH17-mediated inflammatory disorders.

Milman T, Kao AA, Chu D, Gorski M, Steiner A, Simon CZ, Shih C, Aldave AJ, Eagle RC, Jakobiec FA, Udell I. Paraproteinemic Keratopathy: The Expanding Diversity of Clinical and Pathologic Manifestations. Ophthalmology 2015;122(9):1748-56.Abstract

PURPOSE: To describe 7 patients with paraproteinemic keratopathy and to highlight the clinical and pathologic diversity of this rare entity and the importance of timely, systemic evaluation. DESIGN: Retrospective, multicenter collaborative case series. PARTICIPANTS: Seven patients with paraproteinemic keratopathy. METHODS: Clinical and pathologic records were reviewed to identify patients with well-documented corneal immunoglobulin deposits. Detailed ophthalmologic and medical histories were assembled. In 6 patients, corneal tissue was evaluated histochemically and immunohistochemically; in selected cases, corneal tissue was evaluated by in situ hybridization and ultrastructurally. MAIN OUTCOME MEASURES: Visual acuity and anterior segment examination at presentation and follow-up; local therapy; systemic diagnosis and management; and histopathologic, immunohistochemical, in situ hybridization, and ultrastructural findings. RESULTS: Seven patients were identified with corneal immunoglobulin deposition. In addition to previously reported crystalline, nummular, patch-like, and lattice-like corneal opacities, prominent corneal vascularization was present in 2 patients mimicking interstitial keratitis and limbal stem cell deficiency. All patients had evidence of paraproteinemia in a setting of monoclonal gammopathy of undetermined significance, smoldering plasma cell myeloma, or Waldenström macroglobulinemia. Corneal findings were the first manifestation of systemic disease in 4 patients, and the diagnosis was not suspected in 3 of these patients. Pathologic evaluation of biopsied corneal and conjunctival tissues demonstrated immunoglobulin deposits. Previously unreported ultrastructural patterns in the cornea were noted: large scroll-like immunotactoid deposits, immune complex-like deposits, and randomly arranged fibrils morphologically intermediate between amyloid and immunotactoid deposits. Surgical intervention to improve vision was performed in 4 patients, with recurrence of deposits in 3 patients. Three patients underwent systemic therapy with diminution of the deposits and improvement in vision in 1 patient. CONCLUSIONS: The clinical and pathologic expressions of corneal immunoglobulin deposits are protean and present a diagnostic challenge. Early recognition of this rare entity is important to address the potentially serious associated systemic disease.

von Alpen D, Tran HV, Guex N, Venturini G, Munier FL, Schorderet DF, Haider NB, Escher P. Differential Dimerization of Variants Linked to Enhanced S-Cone Sensitivity Syndrome (ESCS) Located in the NR2E3 Ligand-Binding Domain. Hum Mutat 2015;36(6):599-610.Abstract

NR2E3 encodes the photoreceptor-specific nuclear hormone receptor that acts as a repressor of cone-specific gene expression in rod photoreceptors, and as an activator of several rod-specific genes. Recessive variants located in the ligand-binding domain (LBD) of NR2E3 cause enhanced short wavelength sensitive- (S-) cone syndrome (ESCS), a retinal degeneration characterized by an excess of S-cones and non-functional rods. We analyzed the dimerization properties of NR2E3 and the effect of disease-causing LBD missense variants by bioluminescence resonance energy transfer (BRET(2) ) protein interaction assays. Homodimerization was not affected in presence of p.A256V, p.R039G, p.R311Q, and p.R334G variants, but abolished in presence of p.L263P, p.L336P, p.L353V, p.R385P, and p.M407K variants. Homology modeling predicted structural changes induced by NR2E3 LBD variants. NR2E3 LBD variants did not affect interaction with CRX, but with NRL and rev-erbα/NR1D1. CRX and NRL heterodimerized more efficiently together, than did either with NR2E3. NR2E3 did not heterodimerize with TLX/NR2E1 and RXRα/NR2C1. The identification of a new compound heterozygous patient with detectable rod function, who expressed solely the p.A256V variant protein, suggests a correlation between LBD variants able to form functional NR2E3 dimers and atypical mild forms of ESCS with residual rod function.

Hwang J, Hwang TJ, Ciolino JB. Pivotal clinical trials of novel ophthalmic drugs and medical devices: retrospective observational study, 2002-2012. BMJ Open 2015;5(6):e007987.Abstract

OBJECTIVES: Novel therapeutics are an important part of ophthalmologists' armamentarium, and the risks and benefits of these therapies must be carefully evaluated. We sought to quantify the characteristics of the pivotal clinical trials supporting the regulatory approval of new ophthalmic drugs and medical devices. DESIGN: Retrospective observational study. SETTING AND DATA SOURCE: Medical review dossiers for new ophthalmic drug and high-risk device approvals released publicly by the US Food and Drug Administration (FDA). MAIN OUTCOME MEASURES: Proportion of pivotal trials with randomisation, masking, active or placebo controls and subgroup analyses; total and median number of trial enrollees; and the number of drugs and devices approved with required postapproval studies. RESULTS: From 2002 to 2012, the FDA approved 11 ophthalmic drugs and 25 devices. The pivotal trials underlying the approvals of ophthalmic drugs in our study cohort enrolled a median of 809 patients. Virtually all drug trials were randomised and masked (91%), of which 7 (70%) used a placebo control. Pivotal trials for ophthalmic devices enrolled 324 patients on average, and significantly fewer trials for ophthalmic devices versus drugs were randomised (16% vs 91%; p<0.001) or masked (12% vs 91%; p<0.001). 8 (32%) ophthalmic devices and 6 (55%) ophthalmic drugs were approved with required postapproval studies. CONCLUSIONS: Ophthalmic therapeutics were approved based on varying levels of evidence. Postapproval studies could be used to confirm or refute early indications of safety and effectiveness of these therapeutics, with the study results accessible to patients and clinicians who need to make informed treatment decisions.

Qazi Y, Kheirkhah A, Blackie C, Cruzat A, Trinidad M, Williams C, Korb DR, Hamrah P. In vivo detection of clinically non-apparent ocular surface inflammation in patients with meibomian gland dysfunction-associated refractory dry eye symptoms: a pilot study. Eye (Lond) 2015;29(8):1099-110.Abstract

PurposeThe utility of in vivo confocal microscopy (IVCM) in the investigation of palpebral conjunctival and corneal inflammation in patients with meibomian gland dysfunction (MGD)-associated refractory dry eye symptoms following gland expression, despite objective clinical improvement.MethodsA retrospective, observational pilot study was conducted evaluating five patients with MGD-associated refractory dry eye symptoms and three control groups: symptomatic untreated MGD patients (n=3), treatment-responsive MGD patients with improved symptoms (n=3) and asymptomatic healthy normals (n=11). Ocular surface disease index (OSDI) scores, tear break-up time (TBUT), the number of meibomian glands yielding liquid secretion (MGYLS), palpebral conjunctival epithelial and substantia propria immune cell (EIC, SIC), and corneal dendritic cell (DC) densities were measured.ResultsDespite clinical improvement (TBUT: 6.4±1.2 s to 10.1±2.1 s, P=0.03; MGYLS: 3.5±0.8 glands to 7.0±1.1 glands, P=0.13) and a normal clinical examination post treatment, MGD patients remained symptomatic. IVCM revealed increased immune cells in the palpebral conjunctiva (refractory MGD EIC=592.6±110.1 cells/mm(2); untreated MGD EIC=522.6±104.7 cells/mm(2), P=0.69; responsive MGD EIC=194.9±119.4 cells/mm(2), P<0.01; normals EIC=123.7±19.2 cells/mm(2), P< 0.001), but not the cornea (refractory MGD DC=60.9±28.3 cells/mm(2); normals DC=25.9±6.3 cells/mm(2); P=0.43). EIC did not correlate with TBUT (Rs=-0.26, P=0.33). OSDI scores correlated with both EIC (Rs=0.76, P<0.001) and TBUT (Rs=-0.69, P<0.01) but not SIC. Intraglandular immune cells were also seen.ConclusionMGD-associated refractory symptoms and the symptom-sign disparity may be explained by clinically non-apparent, active inflammation of the palpebral conjunctiva as detected by IVCM. These patients may benefit from anti-inflammatory therapy.

Kim LA, Wong LL, Amarnani DS, Bigger-Allen AA, Hu Y, Marko CK, Eliott D, Shah VA, McGuone D, Stemmer-Rachamimov AO, Gai X, D'Amore PA, Arboleda-Velasquez JF. Characterization of cells from patient-derived fibrovascular membranes in proliferative diabetic retinopathy. Mol Vis 2015;21:673-87.Abstract

PURPOSE: Epiretinal fibrovascular membranes (FVMs) are a hallmark of proliferative diabetic retinopathy (PDR). Surgical removal of FVMs is often indicated to treat tractional retinal detachment. This potentially informative pathological tissue is usually disposed of after surgery without further examination. We developed a method for isolating and characterizing cells derived from FVMs and correlated their expression of specific markers in culture with that in tissue. METHODS: FVMs were obtained from 11 patients with PDR during diabetic vitrectomy surgery and were analyzed with electron microscopy (EM), comparative genomic hybridization (CGH), immunohistochemistry, and/or digested with collagenase II for cell isolation and culture. Antibody arrays and enzyme-linked immunosorbent assay (ELISA) were used to profile secreted angiogenesis-related proteins in cell culture supernatants. RESULTS: EM analysis of the FVMs showed abnormal vessels composed of endothelial cells with large nuclei and plasma membrane infoldings, loosely attached perivascular cells, and stromal cells. The cellular constituents of the FVMs lacked major chromosomal aberrations as shown with CGH. Cells derived from FVMs (C-FVMs) could be isolated and maintained in culture. The C-FVMs retained the expression of markers of cell identity in primary culture, which define specific cell populations including CD31-positive, alpha-smooth muscle actin-positive (SMA), and glial fibrillary acidic protein-positive (GFAP) cells. In primary culture, secretion of angiopoietin-1 and thrombospondin-1 was significantly decreased in culture conditions that resemble a diabetic environment in SMA-positive C-FVMs compared to human retinal pericytes derived from a non-diabetic donor. CONCLUSIONS: C-FVMs obtained from individuals with PDR can be isolated, cultured, and profiled in vitro and may constitute a unique resource for the discovery of cell signaling mechanisms underlying PDR that extends beyond current animal and cell culture models.