SDPR expression in human trabecular meshwork and its potential role in racial disparities of glaucoma.

In order to identify how differential gene expression in the trabecular meshwork (TM) contributes to racial disparities of caveolar protein expression, TM dysfunction and development of primary open angle glaucoma (POAG), RNA sequencing was performed to compare TM tissue obtained from White and Black POAG surgical (trabeculectomy) specimens. Healthy donor TM tissue from White and Black donors was analyzed by PCR, qPCR, immunohistochemistry staining, and Western blot to evaluate SDPR (serum deprivation protein response; Cavin 2) and CAV1/CAV2 (Caveolin 1/Caveolin 2). Standard transmission electron microscopy (TEM) and immunogold labeled studies were performed. RNA sequencing demonstrated reduced SDPR expression in TM from Black vs White POAG patients' surgical specimens, with no significant expression differences in other caveolae-associated genes, confirmed by qPCR analysis. No racial differences in SDPR gene expression were noted in healthy donor tissue by PCR analysis, but there was greater expression as compared to specimens from patients with glaucoma. Analysis of SDPR protein expression confirmed specific expression in the TM regions, but not in adjacent tissues. TEM studies of TM specimens from healthy donors did not demonstrate any racial differences in caveolar morphology, but a significant reduction of caveolae with normal morphology and immuno-gold staining of SDPR were noted in glaucomatous TM as compared to TM from healthy donors. Linkage of SDPR expression levels in TM, POAG development, and caveolar ultrastructural morphology may provide the basis for a novel pathway of exploration of the pathologic mechanisms of glaucoma. Differential gene expression of SDPR in TM from Black vs White subjects with glaucoma may further our understanding of the important public health implications of the racial disparities of this blinding disease.

Trabecular Meshwork Mitochondrial Function and Oxidative Stress: Clues to Racial Disparities of Glaucoma.

Purpose: To identify racial differences of oxidative damage and stress and mitochondrial function in human trabecular meshwork (TM). Design: Experimental study. Participants: One hundred seventy-three eyes of 173 patients undergoing intraocular surgery provided aqueous humor (AH) for analysis. Trabecular meshwork tissues from eye bank donors were used as healthy controls for primary cell culture. Methods: Enzyme-linked immunosorbent assay methods were used to measure 8-hydroxy-2-deoxyguanosine (8-OHdG), an oxidative damage marker, in AH comparing Black and White Americans. Human TM primary cultured cells from Black and White donors were used for adenosine triphosphate (ATP) measurement under high and low oxygen culture conditions. Complex I activity was measured in mitochondrial fractions isolated from cultured TM cells. Mitochondrial quantification was performed by translocase of outer mitochondrial membrane 20 (TOMM20) Western blot. Intracellular reactive oxygen species (ROS) production was measured in live TM cells. Main Outcome Measures: Oxidative damage in AH, ATP production, complex I activity, mitochondrial quantification, and intracellular ROS in cultured TM cells stratified by racial background. Results: Aqueous humor samples (75 Black, 98 White) displayed significantly higher 8-OHdG levels (P = 0.024) in Black compared with White patients with severe stage glaucoma. Using cultured healthy donor TM cells, ATP production was higher in Black than White TM cells (P = 0.002) in low oxygen culture conditions. Complex I activity was not statistically different in Black compared with White TM cells, but TOMM20 expression was higher in Black versus White cells (P = 0.001). In response to hydrogen peroxide challenge, ROS production was significantly higher in Black compared to White TM cells (P = 0.004). Conclusions: Significantly higher 8-OHdG levels in AH of Black compared with White patients with severe glaucoma indicated that oxidative damage may be a risk factor in glaucoma pathogenesis or the result of distinct pathologic features in the Black population. To identify potential origins or causes of this damage, our data showed that healthy Black cultured TM cells have higher ATP and ROS levels, with increased quantity of mitochondria, compared with White TM cells. These findings indicate that mitochondrial alterations and increased oxidative stress may influence racial disparities of glaucoma.

Racial Disparities in Glaucoma: From Epidemiology to Pathophysiology.

Among individuals of African and Latinx descent compared to those of European background, there is a higher prevalence, earlier onset, more rapid progression of primary open angle glaucoma and greater incidence of blindness. Although some suggest that outreach, education and screening programs may expand earlier diagnosis, and attention to access, cost of treatment, and adherence will improve outcomes, there is increasing evidence of genetic and physiologic differences which may be associated with these disease disparities.

Bioinspired Thermosensitive Hydrogel as a Vitreous Substitute: Synthesis, Properties, and Progress of Animal Studies.

In many vitreal diseases, the surgeon removes the natural vitreous and replaces it with silicone oils, gases, or balanced salt solutions to fill the eyeball and hold the retina in position. However, these materials are often associated with complications and have properties that differ from natural vitreous. Herein, we report an extension of our previous work on the synthesis of a biomimetic hydrogel that is composed of thiolated gellan as an analogue of type II collagen and poly(methacrylamide-co-methacrylate-co-bis(methacryloyl)cystamine), a polyelectrolyte, as an analogue of hyaluronic acid. This thermosensitive hydrogel can be injected into the eye as a viscous solution at 45 °C. It then forms a physical gel in situ when it reaches body temperature, and later forms disulfide covalent crosslinks. In this article, we evaluated two different formulations of the biomimetic hydrogels for their physical, mechanical, and optical properties, and we determined their biocompatibility with several cell lines. Finally, we report on the progress of the four-month preclinical evaluation of our bio-inspired vitreous substitute in comparison to silicone oil or a balanced salt solution. We assessed the eyes with a slit-lamp examination, intraocular pressure measurements, electroretinography, and optical coherence tomography. Preliminary results are very encouraging for the continuing evaluation of our bio-inspired hydrogel in clinical trials.

Histopathology and selective biomarker expression in human meibomian glands.

BACKGROUND/AIMS: Meibomian gland dysfunction (MGD) is the most common form of evaporative dry eye disease, but its pathogenesis is poorly understood. This study examined the histopathological features of meibomian gland (MG) tissue from cadaver donors to identify potential pathogenic processes that underlie MGD in humans. METHODS: Histological analyses was performed on the MGs in the tarsal plates dissected from four cadaver donors, two young and two old adults, including a 36-year-old female (36F) and three males aged 30, 63 and 64 years (30M, 63M and 64M). RESULTS: The MGs of 36F displayed normal anatomy and structure, whereas the MGs of 30M showed severe ductal obstruction with mild distortion. The obstruction was caused by increased cytokeratin levels in association with hyperproliferation, but not hyperkeratinisation. In two older males, moderate to severe MG atrophy was noted. Cell proliferation was significantly reduced in the MG acini of the two older donors as measured by Ki67 labelling index (6.0%±3.4% and 7.9%±2.8% in 63M and 64M, respectively) when compared with that of the two younger donors (23.2%±5.5% and 16.9%±4.8% in 30M and 36F, respectively) (p<0.001). The expression patterns of meibocyte differentiation biomarkers were similar in the older and younger donors. CONCLUSION: Our histopathological study, based on a small sample size, suggests potentially distinct pathogenic mechanisms in MGD. In the young male adult, hyperproliferation and aberrant differentiation of the central ductal epithelia may lead to the obstruction by overproduced cytokeratins. In contrast, in older adults, decreased cell proliferation in acinar basal epithelia could be a contributing factor leading to MG glandular atrophy.

Intraocular Oxygen and Antioxidant Status: New Insights on the Effect of Vitrectomy and Glaucoma Pathogenesis.

PURPOSE: The purpose of this study was to investigate correlations of partial pressure of oxygen (pO2) in the ocular anterior segment of human eyes and aqueous humor antioxidant levels of ascorbate (AsA) and total reactive antioxidant potential (TRAP) with glaucoma and vitreous status. METHODS: This prospective, cross-sectional study stratified patients (n = 288 eyes) by lens and vitreous status and the presence of primary open-angle glaucoma for statistical analyses. Intraocular pO2 concentrations were measured using a fiberoptic probe in patients at the beginning of planned glaucoma and/or cataract surgery. Aqueous humor specimens were obtained for antioxidant analysis of AsA and TRAP. RESULTS: Following prior pars plana vitrectomy, pO2 levels were significantly higher than in the reference group of cataract surgery in the anterior chamber angle (16.2 ± 5.0 vs. 13.0 ± 3.9 mm Hg; P = .0171) and in the posterior chamber (7.6 ± 3.1 vs. 3.9 ± 2.7 mm Hg; P < .0001). AsA and TRAP levels were significantly lower (1.1 ± 0.4 vs. 1.4 ± 0.5 mM, respectively; 403.3 ±116.5 vs. 479.0 ± 146.7 Trolox units, respectively; P = .004 and P = .024, respectively) in patients after vitrectomy. In patients with an intact vitreous, neither pO2 nor antioxidant status correlated with lens status or glaucoma. CONCLUSIONS: Increased pO2 and antioxidant depletion following vitrectomy suggests an alteration of the intraocular oxidant-antioxidant balance. Our study links physiologic factors such as increased pO2 in the anterior chamber angle and the posterior chamber to decreased antioxidant levels in aqueous humor following vitrectomy. Oxidative stress/damage to the trabecular meshwork in such post-vitrectomy cases may contribute to intraocular pressure elevation and increased risk of glaucoma. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Bioinspired Fibrillary Hydrogel with Controlled Swelling Behavior: Applicability as an Artificial Vitreous.

The vitreous humor of the eye is mainly composed of fibrillary collagen and semiflexible hyaluronic acid (HA). To mimic this macromolecular composition of the vitreous, we previously developed an injectable two-component hydrogel composed of a fibrillary gellan and a semiflexible polyelectrolyte, poly[methacrylamide-co-(methacrylic acid)], both endowed with thiol cross-linkers. We optimized the hydrogel formulations for optical, physical, mechanical, and transport properties approximating those of the vitreous. Here, we studied 11 hydrogel formulations with varying concentrations of each component, and, as expected, we found that they all swelled in physiological solution. The two formulations that most closely matched the vitreous properties were investigated further. Judged against nonsurgical control and silicone oil, a clinically accepted vitreous replacement, both hydrogel formulations were biocompatible in rabbits for 30 days. Both hydrogels maintained optical clarity, physiological intraocular pressure, and intact retinal layers that displayed normal electroretinography. The swelling behavior of the gel led us to postulate that the native vitreous may also exhibit controlled swelling, where ionic HA's swelling capacity is restricted by fibrillary collagen. In conclusion, the two hydrogels merit further in vivo evaluation as an artificial vitreous for an extended duration and additionally in mini-pigs for their similarity to human eyes in size.

Pupillary light reaction in preclinical Alzheimer's disease subjects compared with normal ageing controls.

BACKGROUND/AIMS: We wished to determine whether the pupillary light reaction can differentiate preclinical Alzheimer's disease (AD) subjects from normal ageing controls. We performed a prospective study evaluating the pupillary light reaction in a cohort of well-characterised subjects with preclinical AD versus normal ageing controls. METHODS: We recruited 57 subjects from our institution's Memory and Aging Project, part of our Alzheimer's Disease Research Center. All subjects completed PET-PiB imaging, cerebrospinal fluid analysis and at least 1 neuropsychiatric assessment after their baseline assessment. All participants were assigned a clinical dementia rating and underwent a complete neuro-ophthalmic examination. Participants were divided into a dementia biomarker+ (preclinical AD) and biomarker- (normal ageing) group based on preclinical risk for Alzheimer's dementia. Pupillometry measurements were performed by using the NeurOptics PLR-200 Pupillometer. RESULTS: A total of 57 subjects were recruited with 24 dementia biomarker+ and 33 dementia biomarker- individuals. A variety of pupil flash response (PLR) parameters were assessed. Comparisons between groups were analysed using generalised estimating equations. None of the pupillary parameters showed a significant difference between groups. CONCLUSIONS: We found no significant differences in PLR between preclinical AD subjects and normal ageing controls. This suggests that the disease effect on the PLR may be small and difficult to detect at the earliest stages of the disease. Future studies could include larger sample size and chromatic pupillometry.

Effects of Vitrectomy and Lensectomy on Older Rhesus Macaques: Oxygen Distribution, Antioxidant Status, and Aqueous Humor Dynamics.

Purpose: The purpose of this study is to evaluate effects of vitrectomy (PPV) and lens extraction with intraocular lens implantation (PE/IOL) on molecular oxygen (pO2) distribution, aqueous humor antioxidant-oxidant balance, aqueous humor dynamics, and histopathologic changes in the trabecular meshwork (TM) in the older macaque monkey. Methods: Six rhesus monkeys underwent PPV followed by PE/IOL. pO2, outflow facility, and intraocular pressure (IOP) were measured. Aqueous and vitreous humor specimens were analyzed for antioxidant status and 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative damage. TM specimens were obtained for immunohistochemical and quantitative PCR analysis. Results: pO2 at baseline revealed steep gradients in the anterior chamber and low levels in the posterior chamber (PC) and around the lens. Following PPV and PE/IOL, pO2 significantly increased in the PC, around the IOL, and angle. IOP increased following both surgical interventions, with no change in outflow facility. Histopathologic analysis did not show changes in TM cell quantification, but there was an increase in 8-OHdG. Quantitative PCR did not reveal significant differences in glaucoma-related gene expression. Aqueous and vitreous humor analysis revealed decreased ascorbate and total reactive antioxidant potential and increased 8-OHdG in the aqueous humor only in the surgical eyes. Conclusions: Oxygen distribution in the older rhesus monkey is similar to humans at baseline and following surgical interventions. Our findings of histopathologic changes of TM oxidative damage and alterations in the oxidant-antioxidant balance suggest a potential correlation of increased oxygen exposure with oxidative stress/damage and the development of open angle glaucoma.

GDF15 is elevated in mice following retinal ganglion cell death and in glaucoma patients.

Glaucoma is the second leading cause of blindness worldwide. Physicians often use surrogate endpoints to monitor the progression of glaucomatous neurodegeneration. These approaches are limited in their ability to quantify disease severity and progression due to inherent subjectivity, unreliability, and limitations of normative databases. Therefore, there is a critical need to identify specific molecular markers that predict or measure glaucomatous neurodegeneration. Here, we demonstrate that growth differentiation factor 15 (GDF15) is associated with retinal ganglion cell death. Gdf15 expression in the retina is specifically increased after acute injury to retinal ganglion cell axons and in a murine chronic glaucoma model. We also demonstrate that the ganglion cell layer may be one of the sources of secreted GDF15 and that GDF15 diffuses to and can be detected in aqueous humor (AH). In validating these findings in human patients with glaucoma, we find not only that GDF15 is increased in AH of patients with primary open angle glaucoma (POAG), but also that elevated GDF15 levels are significantly associated with worse functional outcomes in glaucoma patients, as measured by visual field testing. Thus, GDF15 maybe a reliable metric of glaucomatous neurodegeneration, although further prospective validation studies will be necessary to determine if GDF15 can be used in clinical practice.

Expression Profiling of Ascorbic Acid-Related Transporters in Human and Mouse Eyes.

PURPOSE: Ascorbic acid (AsA) is an important antioxidant in the eye. Ascorbic acid is usually transported by sodium-dependent AsA transporters (SVCTs), and dehydroascorbic acid (DHA) by glucose transporters (GLUTs). This study investigates these AsA-related transporters in human compared with mouse eyes. METHODS: Five pairs of human donor eyes and 15 pairs of mouse eyes were collected. Immunofluorescence and in situ hybridization were performed to detect SVCTs and GLUTs expression in the ciliary epithelium, retina, and lens epithelial cells (LECs). These tissues were isolated with laser microdissection followed by extraction of total RNA. Quantitative PCR (qPCR) was performed to examine the mRNA level of SVCTs and GLUTs in human and mouse ocular tissues. RESULTS: Immunofluorescence and in situ hybridization showed SVCT2 and GLUT1 expression in human ciliary epithelium with varied distributions. Sodium-dependent AsA transporter 2 is expressed only in the pigmented epithelium (PE), and GLUT1 is predominately expressed in the nonpigmented epithelium (NPE). However, SVCT2 was not identified in mouse ciliary epithelium, whereas GLUT1 expressed in both PE and NPE. Laser microdissection and qPCR revealed high levels of SVCT2 mRNA in human RPE cells and murine neural retina. Sodium-dependent AsA transporter 1 mRNA could be detected only in human and murine LECs. Glucose transporter 3 and GLUT4 mRNA could not be detected in either the human or mouse ciliary processes or in the lens epithelium. CONCLUSIONS: These fundamental findings indicate AsA transporter expression in eyes of humans is significantly different compared with mice. This may explain why human aqueous and vitreous humors contain higher AsA levels compared with other animals.

O2 -sensitive MRI distinguishes brain tumor versus radiation necrosis in murine models.

PURPOSE: The goal of this study was to quantify the relationship between the (1) H longitudinal relaxation rate constant, R1 , and oxygen (O2 ) concentration (relaxivity, r1 ) in tissue and to quantify O2 -driven changes in R1 (ΔR1 ) during a breathing gas challenge in normal brain, radiation-induced lesions, and tumor lesions. METHODS: R1 data were collected in control-state mice (n = 4) during three different breathing gas (and thus tissue O2 ) conditions. In parallel experiments, pO2 was measured in the thalamus of control-state mice (n = 4) under the same breathing gas conditions using an O2 -sensitive microprobe. The relaxivity of tissue O2 was calculated using the R1 and pO2 data. R1 data were collected in control-state (n = 4) mice, a glioma model (n = 7), and a radiation necrosis model (n = 6) during two breathing gas (thus tissue O2 ) conditions. R1 and ΔR1 were calculated for each cohort. RESULTS: O2 r1 in the brain was 9 × 10(-4) ± 3 × 10(-4) mm Hg(-1) · s(-1) at 4.7T. R1 and ΔR1 measurements distinguished radiation necrosis from tumor (P< 0.03 and P< 0.01, respectively). CONCLUSION: The relaxivity of O2 in the brain is determined. R1 and ΔR1 measurements differentiate tumor lesions from radiation necrosis lesions in the mouse models. These pathologies are difficult to distinguish by traditional imaging techniques; O2 -driven changes in R1 holds promise in this regard. Magn Reson Med 75:2442-2447, 2016. © 2015 Wiley Periodicals, Inc.

Impact of Corneal Endothelial Dysfunctions on Intraocular Oxygen Levels in Human Eyes.

PURPOSE: We studied the implications of corneal endothelial dysfunctions on oxidative stress in the anterior segment via in vivo measurements of oxygen partial pressure (pO2) in the anterior chamber (AC) of human eyes. METHODS: We recruited 51 patients undergoing cataract surgery and/or endothelial keratoplasty (EK). Endothelial cell density (ECD; n = 33) and central corneal thickness (CCT; n = 41) were measured on patients with relatively clear corneas. Before surgery, an oxygen sensor was introduced into the AC via a peripheral corneal paracentesis. In all patients, seven measurements of pO2 were obtained by positioning the flexible tip near the endothelium at the central cornea, at four cardinal subendothelial locations near the midperipheral cornea, and in the mid-AC and AC angle. In patients with pseudophakia or eyes undergoing cataract surgery, pO2 also was measured near the lens surface and in the posterior chamber. RESULTS: Consistent with our previous reports, a steep oxygen gradient was noted in the anterior segment of normal controls (n = 24). In patients with endothelial dysfunctions (n = 27), there was a significant increase of pO2 at all five subendothelial locations without a significant increase of pO2 in the AC angle. By regression analyses, subendothelial pO2 correlated inversely with ECD and positively with CCT in patients with endothelial dysfunctions. CONCLUSIONS: This study demonstrates an even steeper intraocular oxygen gradient in eyes with corneal endothelial dysfunctions. It suggests that the reduced oxygen consumption in corneal endothelial cells may increase oxidative stress in the AC and the existence of an alternative aqueous inflow pathway that maintains a relatively low and constant pO2 at the AC angle.

Photoacoustic tomography imaging and estimation of oxygen saturation of hemoglobin in ocular tissue of rabbits.

This study evaluated in vivo imaging capabilities and safety of qualitative monitoring of oxygen saturation of hemoglobin (sO2) of rabbit ciliary body tissues obtained with acoustic resolution (AR) photoacoustic tomography (PAT). AR PAT was used to collect trans-scleral images from ciliary body vasculature of seven New Zealand White rabbits. The PAT sO2 measurements were obtained under the following conditions: when systemic sO2 as measured by pulse oximetry was between 100% and 99% (level 1); systemic sO2 as measured by pulse oximetry was between 98% and 90% (level 2); and systemic sO2 as measured by pulse oximetry was less than 90% (level 3). Following imaging, histological analysis of ocular tissue was conducted to evaluate for possible structural damage caused by the AR PAT imaging. AR PAT was able to resolve anatomical structures of the anterior segment of the eye, viewed through the cornea or anterior sclera. Histological studies revealed no ocular damage. On average, sO2 values (%) obtained with AR PAT were lower than sO2 values obtained with pulse oximetry (all p < 0.001): 86.28 ± 4.16 versus 99.25 ± 0.28, 84.09 ± 1.81 vs. 95.3 ± 2.6, and 64.49 ± 7.27 vs. 71.15 ± 10.21 for levels 1, 2 and 3 respectively. AR PAT imaging modality is capable of qualitative monitoring for deep tissue sO2 in rabbits. Further studies are needed to validate and modify the AR PAT modality specifically for use in human eyes. Having a safe, non-invasive method of in vivo imaging of sO2 in the anterior segment is important to studies evaluating the role of oxidative damage, hypoxia and ischemia in pathogenesis of ocular diseases.

A test of lens opacity as an indicator of preclinical Alzheimer Disease.

Previous studies reported that characteristic lens opacities were present in Alzheimer Disease (AD) patients postmortem. We therefore determined whether cataract grade or lens opacity is related to the risk of Alzheimer dementia in participants who have biomarkers that predict a high risk of developing the disease. AD biomarker status was determined by positron emission tomography-Pittsburgh compound B (PET-PiB) imaging and cerebrospinal fluid (CSF) levels of Aß42. Cognitively normal participants with a clinical dementia rating of zero (CDR = 0; N = 40) or with slight evidence of dementia (CDR = 0.5; N = 2) were recruited from longitudinal studies of memory and aging at the Washington University Knight Alzheimer's Disease Research Center. The age, sex, race, cataract type and cataract grade of all participants were recorded and an objective measure of lens light scattering was obtained for each eye using a Scheimpflug camera. Twenty-seven participants had no biomarkers of Alzheimer dementia and were CDR = 0. Fifteen participants had biomarkers indicating increased risk of AD, two of which were CDR = 0.5. Participants who were biomarker positive were older than those who were biomarker negative. Biomarker positive participants had more advanced cataracts and increased cortical light scattering, none of which reached statistical significance after adjustment for age. We conclude that cataract grade or lens opacity is unlikely to provide a non-invasive measure of the risk of developing Alzheimer dementia.

Central corneal thickness correlates with oxygen levels in the human anterior chamber angle.

PURPOSE: To measure oxygen (pO2) in eyes of patients undergoing intraocular surgery and identify correlations with central corneal thickness (CCT). DESIGN: Prospective, cross-sectional study. METHODS: setting: Institutional. patient population: 124 patients undergoing cataract and/or glaucoma surgery. observation procedure: Prior to surgery, an oxygen sensor was introduced into the anterior chamber (AC) via peripheral corneal paracentesis. The tip of the flexible fiberoptic probe was positioned for 3 measurements in all patients: (1) near central corneal endothelium; (2) in mid-AC; and (3) in AC angle. In patients undergoing cataract extraction, additional measurements were taken (4) at the anterior lens surface and (5) in the posterior chamber. main outcome measures: pO2 measurements at 5 locations within the eye were compared to central corneal thickness measurements by multivariate regression analyses. RESULTS: There was a statistically significant inverse correlation between CCT and pO2 in the anterior chamber angle (P = .048). pO2 was not significantly related to CCT at any other location, including beneath the central cornea. Regression analysis relating CCT to age, race, and oxygen levels in all 5 locations in the anterior segment revealed an association of a thinner cornea with increasing age (P = .007). CONCLUSIONS: Physiologic correlations with central corneal thickness may provide clues to understanding why a thinner cornea increases the risk of open glaucoma. Associations between glaucoma risk, CCT, and pO2 in the AC angle suggest that exposure of the outflow system to increased oxygen or oxygen metabolites may increase oxidative damage to the trabecular meshwork cells, resulting in elevation of intraocular pressure.

Quantitative proteomics analysis by iTRAQ in human nuclear cataracts of different ages and normal lens nuclei.

PURPOSE: The goal of this study was to quantitatively identify the differentially expressed proteins in nuclear cataracts of different ages and normal lens nuclei in humans. EXPERIMENTAL DESIGN: Forty-eight human lens nucleus samples with hardness grades III, IV were obtained during cataract surgery by extracapsular cataract extraction. Seven normal transparent human lens nuclei were obtained from fresh normal cadaver eyes during corneal transplantation surgery. Lens nuclei were divided into seven groups according to age and optic axis: Group A (average age 80.8 ± 1.2 years), Group B (average age 57.0 ± 4.0 years), Group C average age 80.3 ± 4.5 years), Group D (average age 56.9 ± 4.2 years), Group E (average age 78.1 ± 2.5 years), Group F (average age 57.6 ± 3.3 years) and Group G (seven normal transparent human lenses from normal cadaver eyes, average age 34.7 ± 4.2 years). Water-soluble, water-insoluble, and water-insoluble-urea-soluble protein fractions were extracted from samples. The three-part protein fractions from the individual lenses were combined to form the total proteins of each sample. The proteomic profiles of each group were further analyzed using 8-plex iTRAQ labeling combined with 2D-LC-MS/MS. The data were analyzed with the ProteinPilot software for peptide matching, protein identification, and quantification. Differentially expressed proteins were validated by Western blotting. RESULTS: We employed biological and technical replicates and selected the intersection of the two results, which included 80 proteins. Nine proteins were differentially expressed among the 80 proteins identified using proteomic techniques. In age-related nuclear cataracts (ARNC), the expression levels of fatty acid-binding protein and pterin-4-alpha-carbinolamine dehydratase were upregulated, whereas the levels of alpha-crystallin B chain (CRYAB), GSH synthetase, phakinin, gamma-crystallin C, phosphoglycerate kinase 1, betaine-homocysteine S-methyltransferase 1 (BHMT1), and spectrin beta chain were downregulated. These proteins may be associated with abnormal protein aggregation and oxidative stress. GSH synthetase and CRYAB expression levels in the nuclear cataract decreased with age. The mass spectrometric analysis results were consistent with the Western blot validation. CONCLUSION AND CLINICAL RELEVANCE: The results indicate that CRYAB and GSH synthetase may be involved in ARNC pathogenesis. iTRAQ combined with 2D-LC-MS/MS provides new methods for future studies of pathological mechanisms and protective drug development for ARNC.

Quantitative imaging of enzymatic vitreolysis-induced fiber remodeling.

PURPOSE: Collagen fiber remodeling in the vitreous body has been implicated in cases of vitreomacular traction, macular hole, and retinal detachment, and also may occur during pharmacologic vitreolysis. The purpose of this study was to evaluate quantitative polarized light imaging (QPLI) as a tool for studying fiber organization in the vitreous and near the vitreoretinal interface in control and enzymatically perturbed conditions. METHODS: Fiber alignment was measured in anterior-posterior sections of bovine and porcine vitreous. Additional tests were performed on bovine lenses and nasal-temporal vitreous sections. Effects of proteoglycan degradation on collagen fiber alignment using trypsin and plasmin were assessed at the microstructural level using electron microscopy and at the global level using QPLI. RESULTS: Control vitreous showed fiber organization patterns consistent with the literature across multiple-length scales, including the global anterior-posterior coursing of vitreous fibers, as well as local fibers parallel to the equatorial vitreoretinal interface and transverse to the posterior interface. Proteoglycan digestion with trypsin or plasmin significantly increased fiber alignment throughout the vitreous (P < 0.01). The largest changes (3×) occurred in the posterior vitreous where fibers are aligned transverse to the posterior vitreoretinal interface (P < 0.01). CONCLUSIONS: Proteoglycan loss due to enzymatic vitreolysis differentially increases fiber alignment at locations where tractions are most common. We hypothesize that a similar mechanism leads to retinal complications during age-related vitreous degeneration. Structural changes to the entire vitreous body (as opposed to the vitreoretinal interface alone) should be evaluated during preclinical testing of pharmacological vitreolysis candidates.

Preservation of the structure of enzymatically-degraded bovine vitreous using synthetic proteoglycan mimics.

PURPOSE: Vitreous liquefaction and subsequent posterior vitreous detachment can lead to several sight-threatening diseases, including retinal detachment, macular hole and macular traction syndrome, nuclear cataracts, and possibly, open-angle glaucoma. In this study, we tested the ability of three novel synthetic chondroitin sulfate proteoglycan mimics to preserve the structure and physical properties of enzymatically-degraded bovine vitreous. METHODS: Chondroitin sulfate proteoglycan mimics, designed to bind to type II collagen, hyaluronic acid, or both, were applied to trypsin- or collagenase-treated bovine vitreous in situ and in vitro. Rheology and liquefaction tests were performed to determine the physical properties of the vitreous, while Western blots were used to detect the presence and degradation of soluble collagen II (α1). Deep-etch electron microscopy (DEEM) identified the ultrastructure of mimic-treated and untreated enzyme-degraded bovine vitreous. RESULTS: Proteoglycan mimics preserved the physical properties of trypsin-degraded bovine vitreous and protected against vitreous liquefaction. Although the collagen-binding mimic maintained the physical properties of collagenase-treated vitreous, liquefaction still occurred. Western blots indicated that the mimic provided only marginal protective ability against soluble collagen degradation. Deep-etch electron microscopy, however, showed increased density and isotropy of microstructural components in mimic-treated vitreous, supporting the initial result that vitreous structure was preserved. CONCLUSIONS: Proteoglycan mimics preserved bovine vitreous physical properties after enzymatic degradation. These compounds may be useful in delaying or preventing the pathological effects of age-related, or enzymatically-induced, degradation of the vitreous body.