Posterior Embryotoxon Revisited: An Immunohistologic Study.

PURPOSE: This series describes the immunopathologic features of posterior embryotoxon (PE) and demonstrates that it is not an anterior displaced Schwalbe's line as commonly described, but a peripheral corneal stromal nub variable in location with abnormal extracellular matrix. DESIGN: Case series. PARTICIPANTS: Archived specimens from patients with PE. METHODS: Sections from archived formalin-fixed, paraffin-embedded specimens (n = 9; 7 autopsy and 2 trabeculectomy specimens) were examined by light microscopy. Immunohistochemistry was performed on 5 specimens to characterize the extracellular matrix composition of PE. RESULTS: Posterior embryotoxon appeared as nubs of whorled collagen extending from the corneal stroma, lined in some instances, by Descemet membrane. These nubs were located anterior to Schwalbe's line (n = 4), posteriorly (n = 1), partially embedded in the trabecular meshwork (n = 1), or at Schwalbe's line (n = 2). Qualitatively, collagen I labeling of the PE stroma was similar or weaker than the corneal stroma, whereas collagen III staining was focal and slightly more intense compared with the corneal stroma. Lumican and keratan sulfate staining was similar or less intense in PE compared with the corneal stroma. MAIN OUTCOME MEASURES: Identify location of PE and its immunohistochemical features. CONCLUSIONS: In contrast to the widely accepted definition of PE as a prominent, anteriorly displaced Schwalbe line, histologic evidence suggests that it is a direct extension of the corneal stroma with variable locations that may displace the attenuated Descemet membrane when located anterior to or at Schwalbe's line. Immunohistochemical examination revealed that the composition of PE's extracellular matrix was similar to corneal stroma but with some variability in staining intensity.

Absence of Cytochrome P450-1b1 Increases Susceptibility of Pressure-Induced Axonopathy in the Murine Retinal Projection.

Mutations in the cytochrome P450-1B1 (Cyp1b1) gene is a common genetic predisposition associated with various human glaucomas, most prominently in primary congenital glaucoma (PCG). The role of Cyp1b1 in the eye is largely unknown, however, its absence appears to drive the maldevelopment of anterior eye structures responsible for aqueous fluid drainage in murine models. Nevertheless, vision loss in glaucoma ultimately results from the structural and functional loss of retinal ganglion cells (RGCs). Cyp1b1's influence in the development and support of retinal ganglion cell structure and function under normal conditions or during stress, such as elevated ocular pressure; the most common risk factor in glaucoma, remains grossly unknown. Thus, to determine the role of Cyp1b1 in normal retinal projection development we first assessed the strucutrual integrity of RGCs in the retina, optic nerve, and superior colliculus in un-manipulated (naïve) Cyp1b1-knockout (Cyp1b1-/-) mice. In addition, in a separate cohort of Cyp1b1-/- and wildtype mice, we elevated and maintained intraocular pressure (IOP) at glaucomatous levels for 5-weeks, after which we compared RGC density, node of Ranvier morphology, and axonal transport between the genotypes. Our results demonstrate that naïve Cyp1b1-/- mice develop an anatomically intact retinal projection absent of overt glaucomatous pathology. Following pressure elevation, Cyp1b1-/- accelerated degradation of axonal transport from the retina to the superior colliculus and altered morphology of the nodes of Ranvier and adjacent paranodes in the optic nerves. Together this data suggests the absence Cyp1b1 expression alone is insufficient to drive murine glaucomatous pathology, however, may increase the vulnerability of retinal axons to disease relevant elevations in IOP.

Prevalence and Clinical Characteristics of Childhood Glaucoma at a Tertiary Care Children's Hospital.

PRECIS: Glaucoma suspect was the most prevalent category in this study followed by glaucoma associated with acquired ocular anomaly and juvenile open-angle glaucoma. Primary congenital glaucoma was diagnosed in only 3% of the population studied. PURPOSE: To describe the prevalence and clinical characteristics of childhood glaucoma diagnosed over a 10-year period among patients aged 18 years or below who were seen at a tertiary care children's hospital using the new Childhood Glaucoma Research Network classification system. METHODS: Medical records of all patients aged 18 years or below (n=108) who were diagnosed with glaucoma between January 1, 2008 through September 30, 2018 were reviewed. Data collected included demographics (age at diagnosis, sex, and family history of glaucoma), intraocular pressure, disc-to-cup ratio, retinal nerve fiber layer thickness, and refractive errors. Clinical characteristics of each patient were evaluated according to the criteria established by Childhood Glaucoma Research Network. Categorical distributional equivalence comparisons were performed using the Pearson χ test. A P-value <0.05 was defined as statistically significant. RESULTS: A total of 108 patients with a diagnosis of childhood glaucoma or glaucoma suspect were included in this study. Sixty-four percent of these patients were males (P<0.0001). The mean age at the time of diagnosis was 7.07±5.4 years. "Glaucoma suspect" was the most prevalent category (46%, P=0.0002), followed by glaucoma associated with the acquired ocular anomaly (20%) and juvenile open-angle glaucoma (16%). Primary congenital glaucoma represented 3% and all these patients were males. Sixty-nine percent of the patients had bilateral involvement (P=0.0073). The highest intraocular pressure recorded in the study was 57 mm Hg, the largest cup-to-disc ratio was 0.96, and the lowest retinal nerve fiber layer measurement was 39 µm. Ninety-two percent of the patients had refractive errors and 85% of them had astigmatism. CONCLUSIONS: Establishing a pattern and the associated clinical characteristics of childhood glaucoma at tertiary care children's hospitals will help in developing collaborative research efforts and effective treatment/management strategies for children with these rare groups of disorders.

Differential Expression of Vitreous Proteins in Young and Mature New Zealand White Rabbits.

Different anatomical regions have been defined in the vitreous humor including central vitreous, basal vitreous, vitreous cortex, vitreoretinal interface and zonule. In this study we sought to characterize changes in the proteome of vitreous humor (VH) related to compartments or age in New Zealand white rabbits (NZW). Vitreous humor was cryo-collected from young and mature New Zealand white rabbit eyes, and dissected into anterior and posterior compartments. All samples were divided into 4 groups: Young Anterior (YA), Young Posterior (YP), Mature Anterior (MA) and Mature Posterior (MP) vitreous. Tryptic digests of total proteins were analyzed by liquid chromatography followed by tandem mass spectrometry. Spectral count was used to determine the relative protein abundances and identify proteins with statistical differences between compartment and age groups. Western blotting was performed to validate some of the differentially expressed proteins. Our results showed that 231, 375, 273 and 353 proteins were identified in the YA, YP, MA and MP respectively. Fifteen proteins were significantly differentially expressed between YA and YP, and 11 between MA and MP. Carbonic anhydrase III, lambda crystallin, alpha crystallin A and B, beta crystallin B1 and B2 were more abundant in the anterior region, whereas vimentin was less abundant in the anterior region. For comparisons between age groups, 4 proteins were differentially expressed in both YA relative to MA and YP relative to MP. Western blotting confirmed the differential expression of carbonic anhydrase III, alpha crystallin B and beta crystallin B2. The protein profiles of the vitreous humor showed age- and compartment-related differences. This differential protein profile provides a baseline for understanding the vitreous compartmentalization in the rabbit and suggests that further studies profiling proteins in different compartments of the vitreous in other species may be warranted.

Lymphatic and Blood Vessel Density in Human Conjunctiva After Glaucoma Filtration Surgery.

PURPOSE: To investigate the lymphatic vascular microvessel density (LVD) and the blood vascular microvessel density (MVD) and their distribution in excised leaking blebs after mitomycin C trabeculectomy and normal conjunctiva specimens. MATERIALS AND METHODS: LVD and MVD in normal human conjunctiva (n=8) and excised blebs in the hypocellular stroma and peribleb tissue (conjunctiva adjacent to hypocellular bleb tissue) (n=8) were evaluated by immunohistochemistry using antibodies raised against Lymphatic Vessel Endothelial Receptor 1 (D2-40, lymphatic endothelium) and CD34 (vascular endothelium). LVD and MVD counts were performed by light microscopy in 5 fields at ×20 magnification by 3 observers. Differences were determined using Mann-Whitney U test (P<0.05 was considered significant). RESULTS: The leaking blebs showed typical epithelial-stromal domes with areas of acellular stroma covered by attenuated epithelium and surrounded by normal conjunctival epithelium and a dense scar-like matrix replacing the substantia propria. The LVD and MVD were significantly reduced to nil in the hypocellular conjunctival stroma of the excised blebs compared with normal conjunctiva (21.42 vs. 1.16, P<0.002 and 24.28 vs. 1, P<0.008, respectively). The LVD and MVD was also reduced (2- to 2.5-fold) in the peribleb stroma when compared with normal conjunctiva specimens. CONCLUSIONS: In this study we show reduced LCD and MVD in the hypocellular and peribleb stroma. These results may suggest a role of these vessels in an altered immune response in leaking blebs leading to an increased risk for blebitis.

Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury.

Candidate biomarkers, indicative of disease or injury, are beginning to overwhelm the process of validation through immunological means. Recombinant antibodies developed through phage-display offer an alternative means of generating monoclonal antibodies faster than traditional immunization of animals. Peptide segments of putative biomarkers of laser induced injury in the rabbit, discovered through mass spectrometry, were used as targets for a selection against a library of phage-displayed human single-chain variable fragment (scFv) antibodies. Highly specific antibodies were isolated to four of these unique peptide sequences. One antibody against the retinal protein, Guanine Nucleotide-Binding Protein Beta 5 (GBB5), had a dissociation constant ~300 nM and recognized the full-length endogenous protein in retinal homogenates of three different animal species by western blot. Alanine scanning of the peptide target identified three charged and one hydrophobic amino acid as the critical binding residues for two different scFvs. To enhance the utility of the reagent, one scFv was dimerized through a Fragment-crystallizable hinge region (i.e., Fc) and expressed in HEK-293 cells. This dimeric reagent yielded a 25-fold lower detection limit in western blots.

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components.

Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

MicroRNA in aqueous humor from patients with cataract.

MicroRNAs (miRNAs) are small non-coding RNA molecules with regulatory function and marked tissue specificity that can modulate multiple gene targets. They have been detected in body fluids and are associated with various physiologic and pathologic processes. We analyzed aqueous humor (AH) from human subjects undergoing cataract surgery to establish the presence and relative quantities of known miRNAs. AH was collected from patients without known ocular diseases other than cataract and a normal systemic history. Quantitative real-time PCR in an array platform was used to detect known miRNAs present in the AH. Among the 264 miRNAs tested, 110 were present in the AH. The top 5 abundant miRNAs identified were miR-202, miR-193b, miR-135a, miR-365, and miR-376a. The presence of miRNAs in AH suggests that they may have functional roles in regulating target genes in tissues lining the anterior chamber. Further analysis of the AH miRNA population may identify potential gene targets and provide insights regarding their roles in AH regulation, glaucoma and anterior segment disease processes.

Immunohistochemical evidence of specific iris involvement in Blau syndrome.

PURPOSE: To evaluate iris involvement in Blau syndrome using histology and immunohistochemistry. METHODS: Iridectomy specimen of a patient with treated Blau syndrome and a normal control were evaluated by light microscopy and immunohistochemistry using antibodies against CD4(+), CD8(+), HLA-DR, CD68(+), NF-κB and IL-17. RESULTS: Blau iris tissue demonstrated increased numbers of CD4(+) lymphocytes and CD68 negative, HLA-DR positive spindle shaped cells compared to normal iris tissue. Blau iris tissue also demonstrated elevated CD4(+)/CD8(+) ratio and IL-17 and NF-κB immunolabeling. No macrophages, epithelioid cells, or granulomas were noted in the Blau specimen. CONCLUSIONS: The persistent immunolocalization of inflammatory markers in an iris specimen from an aggresively treated patient with proven Blau syndrome suggests that further pathologic and immunohistochemical investigation of Blau ocular tissue is necessary to better understand the complexities of NOD2 activating mutations in the eye.

Animal models of glaucoma.

Glaucoma is a heterogeneous group of disorders that progressively lead to blindness due to loss of retinal ganglion cells and damage to the optic nerve. It is a leading cause of blindness and visual impairment worldwide. Although research in the field of glaucoma is substantial, the pathophysiologic mechanisms causing the disease are not completely understood. A wide variety of animal models have been used to study glaucoma. These include monkeys, dogs, cats, rodents, and several other species. Although these models have provided valuable information about the disease, there is still no ideal model for studying glaucoma due to its complexity. In this paper we present a summary of most of the animal models that have been developed and used for the study of the different types of glaucoma, the strengths and limitations associated with each species use, and some potential criteria to develop a suitable model.

Congenital ectropion uvea and mechanisms of glaucoma in neurofibromatosis type 1: new insights.

OBJECTIVE: To describe the clinicopathologic features of congenital ectropion uvea associated with glaucoma in neurofibromatosis-1 (NF-1). DESIGN: Retrospective case series. PARTICIPANTS AND CONTROLS: Five cases of NF-1 associated with glaucoma, from which enucleated eyes were available, and 2 eye bank eyes used as controls. METHODS: The clinical features and courses of these patients were reviewed. Formalin-fixed, paraffin-embedded eyes were examined by light and electron microscopy. Immunohistochemistry using antineurofibromin, anti-glial fibrillary acidic protein, and antivimentin was performed in 3 patients. Gene expression of the mitogen-activated protein kinase (MAPK) signaling pathway was examined in corneal endothelial cells in 1 patient. MAIN OUTCOME MEASURES: Cause of glaucoma in patients with ectropion uvea and NF-1. RESULTS: The age of patients at the time of glaucoma diagnosis ranged from birth to 13 years. Four of the 5 patients had megalocornea and buphthalmos at presentation. Ectropion uvea was noted clinically in 2 patients, but was demonstrated histopathologically in all 5 patients. On histopathologic examination, all patients had varying degrees of angle closure secondary to endothelialization of the anterior chamber angle. Uveal neurofibromas were noted in all patients; anteriorly displaced ciliary processes were noted in 4 of 5 patients who demonstrated ciliary body involvement with neurofibromas. Absence of Schlemm's canal was observed. The endothelial cells lining the closed angle demonstrated positive stain results with the vimentin antibody. Positive antineurofibromin immunolabeling was detected in normal control corneal endothelium, but was absent in corneal endothelium in patients with endothelialization of the angle. Upregulation of genes from the MAPK signaling pathway was demonstrated in the corneal endothelial cells isolated from the NF-1 eyes. CONCLUSIONS: Ectropion uvea in NF-1 glaucoma is secondary to endothelialization of the anterior chamber angle and is associated commonly with severe pediatric glaucoma in NF-1 patients. The endothelial cell proliferation may be related to overexpression of the Ras (Rat sarcoma)-MAPK genes in these eyes.

Anti-retinal antibodies in serum of laser-treated rabbits.

PURPOSE: Retinal injuries that affect the photoreceptors and/or the retinal pigment epithelium (RPE) may result in the leakage of retinal proteins into the systemic circulation. This study was designed to determine whether an immune response is elicited after an acute retinal injury resulting in circulating anti-retinal antibodies in the serum. METHODS: Fifty laser burns of different grades (minimally visible lesion [MVL], grade II [GII], or grade III [GIII] lesions) were created in the retinas of Dutch Belted rabbits. The degree of laser burns was confirmed by fundus imaging and histology. Serum samples were collected from the animals 3 months after the retinal injury. Candidate autoantigens were identified by two-dimensional (2-D) Western blots of rabbit retinal lysate probed with sera from either control or laser-treated animals. Candidate autoantigens were further characterized by immunostaining to confirm their retinal localization. RESULTS: Seven and 11 protein spots were selected from the MVL and GII laser-treated samples, respectively, for autoantigen identification. No protein spots were detected in the GIII laser-treated samples. Four candidate autoantigens were common to both MVL and GII lesions: dihydropyrimidinase-related protein 2, fructose-bisphosphate aldolase C, chaperonin-containing T-complex polypeptide 1 subunit zeta, and pyruvate kinase isozyme. CONCLUSIONS: Laser-induced retinal injuries resulted in circulating anti-retinal antibodies that were detectable 3 months after the injury. The response appeared to vary with the severity of the laser retinal damage. The identification of the candidate antigens in this study suggest that this approach may permit future development of new diagnostic methods for retinal injuries.

Identification of differentially expressed proteins in the aqueous humor of primary congenital glaucoma.

Primary Congenital Glaucoma (PCG) is an autosomal recessive disease caused by an abnormal development of the anterior chamber angle. Although, PCG has been linked to several genetic loci, the role that the genes at these loci or their encoded proteins play in the pathophysiology of PCG and development of the anterior chamber is not known. To identify proteins that may be altered in PCG and that may help in understanding the underlying pathophysiology of the disease, we took a global proteomics approach. Tryptic digests of the complex mixtures of proteins in aqueous humor were analyzed using Liquid Chromatography/Mass Spectrometry (LC-MS/MS). Proteins were identified by searching the data against the human subset of the UniProt database. The proteomes of aqueous humor in PCG (n = 7) and patients undergoing cataract surgery as control (n = 4) were compared based on the scan counts of comparable proteins. Using stringent filtering criteria, Apolipoprotein A-IV (APOA-IV), Albumin and Antithrombin 3 (ANT3) were detected at significantly higher levels in PCG AH compared to control, whereas Transthyretin (TTR), Prostaglandin-H2 D-isomerase (PTGDS), Opticin (OPT) and Interphotoreceptor Retinoid Binding Protein (IRBP) were detected at significantly lower levels. Many of these proteins play a role in retinoic acid (RA) binding/transport and have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's (AD). It is possible that similar to AD, the pathologic changes in PCG during development could be influenced by the availability of RA in the anterior chamber.