Search

To identify racial differences of oxidative damage and stress and mitochondrial function in human trabecular meshwork (TM).Experimental study.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.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.Oxidative damage in AH, ATP production, complex I activity, mitochondrial quantification, and intracellular ROS in cultured TM cells stratified by racial background.Aqueous humor samples (75 Black, 98 White) displayed significantly higher 8-OHdG levels (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.

Background/aimsMeibomian 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.MethodsHistological 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).ResultsThe 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) (pConclusionOur 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.

PURPOSE: To investigate correlations of oxygen (pO(2)) 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 presence of primary open angle glaucoma for statistical analyses. Intraocular pO(2) measurements with a fiberoptic probe were made in patients at the beginning of planned glaucoma and/or cataract surgery. Aqueous humor specimens were obtained for antioxidant analysis (AsA, TRAP). RESULTS: Following prior pars plana vitrectomy, pO(2) was significantly higher compared to the reference group (cataract; CAT) in the anterior chamber (AC) angle (16.2 ± 5.0 vs. 13.0 ± 3.9 mmHg, P=.0171) and in the posterior chamber (7.6 ± 3.1 vs. 3.9 ± 2.7 mmHg, P

The hypoxic environment around the lens is important for maintaining lens transparency. Lens epithelial cells (LECs) play a key role in lens metabolism. We measured oxygen consumption to assess the role of human LECs in maintaining hypoxia around the lens, as well as the impact of systemic and ocular diagnosis on these cells.Baseline cellular respiration was measured in rabbit LECs (NN1003A), canine kidney epithelial cells (MDCK), trabecular meshwork cells (TM-5), and bovine corneal endothelial cells (CCEE) using a XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA), which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro. Following informed written consent, lens capsule epithelial cells were obtained from patients during cataract surgery and were divided into small explants in 96-well plates. Capsules were removed when LECs became confluent. OCR was normalized to the number of cells per well using rabbit LECs as a standard. The effect of patient age, sex, race, and presence of diabetes or glaucoma on oxygen consumption was assessed by using the Mann-Whitney U test and multivariate regression analysis.Primary LECs were obtained from 69 patients. The OCR from donors aged 70 and over was lower than that of those under 70 years (2.21±1.037 vs. 2.86±1.383 fmol/min/cell; p0.05). Diabetic patients had lower OCR than non-diabetic patients (2.02±0.911 vs. 2.79±1.332fmol/min/cell; p0.05), and glaucoma patients had lower OCR than non-glaucoma patients (2.27±1.19 vs. 2.83±1.286 fmol/min/cell; p0.05). Multivariate regression analysis confirmed that donors aged 70 and over (p0.05), diabetic patients (p0.01), and glaucoma patients (p0.05) had significantly lower OCR, independent of other variables. Gender and race had no significant effect on OCR.The lower oxygen consumption rate of human LECs in older donors and patients with diabetes or glaucoma could contribute to cataract development. Diabetes and glaucoma are particularly important factors associated with decreased OCR, independent of age. Ongoing studies are examining pO2 at the anterior surface of the lens in vivo and oxygen consumption in the patient's LECs.

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.

PurposeVitreous 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.MethodsChondroitin 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.ResultsProteoglycan 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.ConclusionsProteoglycan 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.

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.

The lens is composed of a thin metabolically active outer layer, consisting of epithelial and superficial fibre cells. Lying within this outer shell are terminally differentiated, metabolically inactive fibre cells, which are divided into an outer cortex and central nucleus. Mature fibre cells contain a very high protein concentration, which is important for the transparency and refractive power of the lens. These proteins are protected from oxidation by reducing substances, like glutathione, and by the low-oxygen environment around the lens. Glutathione reaches the mature fibre cells by diffusing from the metabolically active cells at the lens surface. With age, the cytoplasm of the nucleus becomes stiffer, reducing the rate of diffusion and making nuclear proteins more susceptible to oxidation. Low pO2is maintained at the posterior surface of the lens by the physical and physiological properties of the vitreous body, the gel filling the space between the lens and the retina. Destruction or degeneration of the vitreous body increases exposure of the lens to oxygen from the retina. Oxygen reaches the lens nucleus, increasing protein oxidation and aggregation and leading to nuclear cataract. We suggest that maintaining low pO2around the lens should prevent the formation of nuclear cataracts.

A network of capillaries branches from the hyaloid vascular system and surrounds the mammalian lens throughout much of its embryonic development. These vessels are presumed to be important for the growth and maturation of the lens, although the lenses of non-mammalian vertebrates have no comparable vessels. Over expression of VEGF-A in the lens increases the extent of these capillaries, but it is not known whether VEGF-A from the lens is necessary for their formation or survival. To address this question, we deleted Vegfa in the lens. This prevented the formation of the capillary networks adjacent to the lens capsule, but did not alter nearby hyaloid vessels at the surface of the retina. Postnatal lenses lacking Vegfa were smaller than wild type and, by 1 month of age, many had mild nuclear opacities. These opacities regressed with age. The lens is hypoxic throughout most of life and VEGF-A expression is often regulated by the transcription factor, hypoxia inducible factor-1. Lenses lacking Hif1a were of apparently normal size, had markedly reduced levels of mRNA for VEGF-A and glyceraldehyde-3-phosphate dehydrogenase, but had normal-appearing capillaries covering their surface. We conclude that VEGF-A from the lens is necessary for the formation of the normal hyaloid vascular system and that lack of these capillaries was the most likely cause of growth retardation during fetal and early postnatal lens development. In the absence of HIF-1 function, sufficient VEGF-A is produced by the lens to promote capillary formation. Further study is needed to explain the formation of the mild opacities seen in some lenses lacking Vegfa and their regression later in life.

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 Aβ42. 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. Key Words: Lens opacity, cataract, Scheimpflug imaging, Alzheimer disease biomarkers, preclinical Alzheimer disease

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.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.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 (P0.03 and P0.01, respectively).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.

This study evaluated in vivo imaging capabilities and safety of qualitative monitoring of oxygen saturation of hemoglobin (sO_2) 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 sO_2 measurements were obtained under the following conditions: when systemic sO_2 as measured by pulse oximetry was between 100% and 99% (level 1); systemic sO_2 as measured by pulse oximetry was between 98% and 90% (level 2); and systemic sO_2 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, sO_2 values (%) obtained with AR PAT were lower than sO_2 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 sO_2 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 sO_2 in the anterior segment is important to studies evaluating the role of oxidative damage, hypoxia and ischemia in pathogenesis of ocular diseases.

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.

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.

Purpose To measure oxygen (pO 2 ) 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: pO 2 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 pO 2 in the anterior chamber angle ( P = .048). pO 2 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 pO 2 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.

Purpose: To conduct an epidemiological survey of cataracts and examine the characteristics of lens opacities in Chinese Singaporeans. The results are then compared with those from two similar surveys previously done in Japan in Noto Area, Honshu, and Amami, Kyushu, respectively.Subjects and Methods: 468 subjects of*50 years of age, who responded to the invitation to participate, were examined. Examination principally included photo-documentation of the anterior and posterior segments of both eyes. Evaluation and grading of lens opacities were done using graphical analysis of Scheimpflug and retro-illumination images. Inter-group comparisons were based on statistical analysis of cataract prevalence and distribution.Results: The prevalence of clear lenses decreased with aging with no significant difference between males and females - a finding common to Singapore and the two Japanese study groups. The prevalence of cataracts (or lens opacities of Grade II and above) in 60-79 year-old Singapore subjects was significantly higher than Noto and Amami subjects in the same age group. Further, cortical opacity was the main type in Singapore subjects in their 50s and which was significantly higher than Noto subjects of the same age group. In all age groups, the distribution and prevalence of both nuclear and subcapsular types in the Singapore group were higher than the two Japanese study groups.Conclusions: Cataracts in Chinese Singaporeans are characterized by a high prevalence of nuclear opacities which was generally seen in tropics and sub-tropics. Our study also suggested the involvement of solar-UV in cortical cataracts as well as that of additional risk factors, such as environmental temperature and race, in nuclear and subcapsular cataract formation.J Epidemiol, 2001 ; 11 : 16-23

UV irradiation has the potential to induce the development of lens opacities. This has been demonstrated since long with animal experiments. Unfortunately these animal cataracts did not explain or elucidate the epidemiological observation that the frequency of human cataracts--such as the so called senile cataract--is remarkably higher in regions with increased cosmic UV irradiation or in the population being in close professional contact with UV-irradiation. The main problem was that the type of UV induced animal cataracts differs remarkably with respect to onset, localization of the opacity, size and its timely progression from the cataract classes observed in human. The research of the last 10 years comes to the conclusion that beside the direct (acute) damage--as seen in animal studies due to high UV dosages--we have to realize a syn- or co-cataractogenic potential of UV irradiation even below the threshold dose which is able to accumulate in the lens and to initiate together with other risk factors (chronic damage) the opacification of the lens. The mechanism for the animal cataract and the human cataract (with an UV risk participation) are different. The epidemiological research about cataract frequency in different regions of the world have to take into account that UV irradiation--even below a threshold dose--is a possible risk among the multifactorial pathogenesis of human cataract.

Cataract epidemiological surveys applying objective judgement through lens images in the climatically different places of Noto and Amami, Japan, Singapore and Reykjavik, Iceland yielded several significant results about the influence of solar UV. 1) The percentage of transparent and of lens opacification was significantly higher in the Reykjavik subjects than in the Singaporeans. 2) The percentages including early changes were higher in Amami and Singapore than in Noto and Reykjavik. 3) Progressed lens opacification was highest in Singapore. While the main type of lens opacification was cortical in Noto and Reykjavik, that of Singapore was nuclear. 4) A significant correlation between cortical opacification and the history of time spent outdoors was noticed. The UV risk for formation and/or progression of cortical opacification should be acceptable from the epidemiological standpoint. J Epidemiol, 1999; 9 : S33-S38.

Purpose Previous studies that measured liquefaction and oxygen content in human vitreous suggested that exposure of the lens to excess oxygen causes nuclear cataracts. Here, we developed a computational model that reproduced available experimental oxygen distributions for intact and degraded human vitreous in physiologic and environmentally perturbed conditions. After validation, the model was used to estimate how age-related changes in vitreous physiology and structure alter oxygen levels at the lens. Methods A finite-element model for oxygen transport and consumption in the human vitreous was created. Major inputs included ascorbate-mediated oxygen consumption in the vitreous, consumption at the posterior lens surface, and inflow from the retinal vasculature. Concentration-dependent relations were determined from experimental human data or estimated from animal studies, with the impact of all assumptions explored via parameter studies. Results The model reproduced experimental data in humans, including oxygen partial pressure (Po2) gradients (≈15 mm Hg) across the anterior-posterior extent of the vitreous body, higher oxygen levels at the pars plana relative to the vitreous core, increases in Po2 near the lens after cataract surgery, and equilibration in the vitreous chamber following vitrectomy. Loss of the antioxidative capacity of ascorbate increases oxygen levels 3-fold at the lens surface. Homogeneous vitreous degeneration (liquefaction), but not partial posterior vitreous detachment, greatly increases oxygen exposure to the lens. Conclusions Ascorbate content and the structure of the vitreous gel are critical determinants of lens oxygen exposure. Minimally invasive surgery and restoration of vitreous structure warrant further attention as strategies for preventing nuclear cataracts.

The aim of the study was to investigate the protective effects of intact vitreous gel on the lens after pharmacologic vitreolysis and hyperoxia exposure in rats in vivo.Eyes of Sprague-Dawley rats were induced to posterior vitreous detachment (PVD) by pharmacologic vitreolysis, and the rats with and without PVD were treated with hyperoxia 3 h per day for 5 months. Lens transparency was monitored by a slit-lamp biomicroscope. A series of biochemical measurements were made in extracts of the lens cortex and nucleus. Ascorbate levels were measured in the aqueous and vitreous humors.No significant differences in lens transparency or morphology were observed in all groups, and no significant biochemical changes were observed in the cortex or nucleus of lenses of the PVD group. In the lens nucleus, the values of water-soluble protein concentration in PVD + hyperoxia group were lower than that of the PVD group. The levels of water-soluble proteins, glutathione (GSH) and ascorbate decreased in the hyperoxia group with an intact vitreous body. Vitreolysis enhanced the effect of hyperoxia, decreasing soluble protein, GSH and ascorbate below the levels seen in eyes with vitreolysis alone. The levels of antioxidants and soluble proteins were lower in the lens nucleus, and the effects of vitreolysis plus hyperoxia were more significant in the nucleus. Hyperoxia and hyperoxia plus vitreolysis reduced catalase activity and increased oxidized GSH to a greater extent in the lens cortex, although these treatments increased protein-GSH mixed disulfides in both regions. Long-term hyperoxia also lowered ascorbate levels in the vitreous and aqueous humors, an effect that was enhanced by vitreolysis.Exposure to excess molecular oxygen produces significant oxidative damage to the lens, especially the lens nucleus. These effects were enhanced by pharmacologic vitreolysis, indicating that intact vitreous gel protects the lens from oxidative damage.

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.

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.

Objective To compare the PO 2 distribution in different regions in the eyes of patients undergoing intraocular surgery. Methods Before initiation of intraocular cataract and/or glaucoma surgery, an optical oxygen sensor was introduced into the anterior chamber via a peripheral corneal paracentesis. The tip of the flexible fiberoptic probe was positioned by the surgeon for 3 measurements in all patients: (1) near the central corneal endothelium, (2) in the mid–anterior chamber, and (3) in the anterior chamber angle. In patients scheduled to undergo cataract extraction, PO 2 was also measured (4) at the anterior lens surface and (5) in the posterior chamber just behind the iris. Oxygen measurements at the 5 locations were compared using a 2-tailed unpaired t test and multivariate regression. Results The PO 2 value was significantly higher in African American patients at all 5 locations compared with Caucasian patients. Adjusting for age increased the significance of this association. Adjusting for race revealed that age was associated with increased PO 2 beneath the central cornea. Conclusions Racial differences in oxygen levels in the human eye reflect an important difference in oxidative metabolism in the cornea and lens and may reflect differences in systemic physiologic function. Increased oxygen or oxygen metabolites may increase oxidative stress, cell damage, intraocular pressure, and the risk of developing glaucoma. Oxygen use by the cornea decreases with age.

Introduction Alterations in intraocular oxygen levels are important contributors to, or indications of, ocular disease. Polarographic electrodes and fibre-optic sensors (optodes) have been used to measure oxygen and to map the distribution of oxygen in animal models and in human eyes. A recent study reported the use of a commercial electrode to compare oxygen distribution in the vitreous of patients undergoing vitrectomy related to central retinal vein occlusion, macular hole or preretinal membrane. The results of this study were at variance with previous measures of oxygen distribution in the human vitreous using polarographic or optical sensors. To resolve this discrepancy, the present study compared measurements made in vitro or in animal eyes, using the electrode employed in the previous study or a fibre-optic sensor of a different design. Study design Comparative in vitro and in vivo measurements. Results In vitro, the two devices reported similar levels of oxygen, although the electrode consistently detected levels above the calculated values. In rabbit eyes, the electrode had a slow response time and was unable to detect oxygen gradients that were readily measured by the smaller optode. When the electrode was inserted into an eye of similar size to the human eye, the reference thermistor measured the temperature outside the eye, not in the vitreous. Conclusions The design of the electrode used in the previous study makes it unsuitable for measurements of oxygen distribution in the eye.

Objective To investigate the rate and mechanism of oxygen consumption by the vitreous. Methods Oxygen consumption was measured with a microrespirometer. Vitreous ascorbate was measured spectrophotometrically and by gas chromatography–mass spectrometry. Vitreous degeneration was related to the rate of oxygen consumption and ascorbate concentration in samples obtained during vitrectomy. Results Prolonged exposure to oxygen or treatment with ascorbate oxidase eliminated oxygen consumption by the vitreous. Adding ascorbate restored oxygen consumption. Oxygen consumption persisted after boiling or treating the vitreous with the chelating agents EDTA and deferoxamine. In patients undergoing retinal surgery, liquefaction of the vitreous and previous vitrectomy were associated with decreased ascorbate concentration and lower oxygen consumption. Conclusions Ascorbate in the vitreous decreases exposure of the lens to oxygen. The catalyst for this reaction is not known, although free iron may contribute. The gel state of the vitreous preserves ascorbate levels, thereby sustaining oxygen consumption. Vitrectomy or advanced vitreous degeneration may increase exposure of the lens to oxygen, promoting the progression of nuclear cataracts. Clinical Relevance Determining how the eye is protected from nuclear cataracts should suggest treatments to reduce their incidence.

Cataract and glaucoma are two of the most common ocular morbidities. Previous studies by our group and others have suggested that exposure of the crystalline lens to excessive oxygen may have a critical role in the pathogenesis of age-related nuclear cataracts.1–9 Specifically, the oxygen level is relatively low in the vitreous gel and nuclear cataracts are prone to develop after vitrectomy by increasing lens exposure to oxygen from the posterior segment altering the normally hypoxic environment around the lens. Furthermore, Chang10 reported an increased risk of open angle glaucoma developing after vitrectomy and subsequent cataract surgery. The presence of the natural lens at the time of vitrectomy was associated with a 28-month delay in the onset of glaucoma.10,11 We hypothesized that exposure of the anterior segment to oxygen may increase the risk of glaucoma. A large body of evidence indicates that increased oxidative stress or the accumulation of oxidative damage contributes to the pathogenesis of open angle glaucoma.12–22 Recent studies by our group on oxygen distribution in the human eyes clearly demonstrated that the oxygen level was significantly elevated in aqueous humor near the trabecular meshwork (TM) after combined vitrectomy and cataract surgery. Therefore, it further supports the notion that the elevated oxygen near the TM may contribute to the development of open-angle glaucoma.19–22 Our most recent study also has revealed a steep oxygen gradient in the anterior segment of human eyes with an inverse correlation between central corneal thickness (CCT) and oxygen partial pressure (pO2) exclusively in the anterior chamber (AC) angle.22 As the corneal endothelial pumps require active oxygen consumption, we speculated that corneal endothelial dysfunctions could impact the oxygen tension in the anterior segment of human eyes. This study further explored the oxygen consumption by corneal endothelium and its implications for oxidative stress in the anterior segment. Measurements of pO2 were performed in the anterior segment of patients undergoing either cataract surgery (normal control) or Descemet's stripping automated endothelial keratoplasty (DSAEK) due to Fuchs' endothelial corneal dystrophy (FECD) or pseudophakic bullous keratopathy (PBK).

PURPOSE. Excessive exposure to oxygen has been proposed to be a risk factor for nuclear cataracts. For a better understanding of the metabolism of oxygen in the eye, oxygen distribution was mapped in the intraocular fluids, and the rate of oxygen consumption by the lens in rabbits breathing different levels of oxygen was calculated. METHODS. Young albino rabbits were anesthetized, intubated, and exposed to normoxic, hypoxic, or hyperoxic conditions. The hemoglobin saturation of the blood was monitored with a pulse oximeter, and arterial oxygen levels were measured with a blood gas analyzer. A fiberoptic optical oxygen sensor (optode) was used to determine oxygen levels in different regions of the eye. Oxygen flux across the posterior of the lens was calculated from the measured oxygen gradients in the vitreous chamber. RESULTS. Oxygen levels in the ocular fluids changed markedly when rabbits breathed air made hypoxic or hyperoxic. Oxygen levels were highest near the retinal vasculature, the iris vasculature, and the inner surface of the central cornea. Compared with nearby regions, oxygen levels were decreased in the aqueous humor closest to the pars plicata of the ciliary body and near the anterior chamber angle. Oxygen levels were generally lower closer to the lens. From the oxygen gradients in the vitreous body, oxygen consumption by the posterior half of the lens was calculated to be 0.2 to 0.4 L/h under normoxic conditions. Oxygen consumption by the posterior of the lens increased in proportion to the amount of oxygen supplied. CONCLUSIONS. Intraocular oxygen is mostly derived from the retinal and iris vasculature and by diffusion across the cornea. Freshly secreted aqueous humor and the aqueous humor in the anterior chamber angle are relatively depleted of oxygen. The marked increase in oxygen consumption that occurs when the lens is exposed to increased oxygen is likely to result in the production of higher levels of reactive oxygen species and may provide a link between elevated oxygen levels and the risk of nuclear cataracts. (Invest Ophthalmol Vis Sci. 2006;47: 1571‐1580) DOI:10.1167/iovs.05-1475 A

In the mammalian lens, the expression of the beta- and gamma-crystallin families is thought to be limited to fiber cells. However, several studies detected these proteins or their mRNAs in human lens epithelial cells. To resolve this apparent discrepancy, 14 crystallin mRNAs were examined and the expression and subcellular distribution of selected crystallin proteins in lens epithelial cells determined.Transcript levels were analyzed by quantitative real-time PCR using mRNA from P3 rat lens epithelia cultured for 0 or 20 hours or 4 or 7 days in basal medium or with added FGF2. Antibodies to betaB1-, gammaS-, alphaA-, and alphaB-crystallins were used for Western blot analysis of proteins extracted from adult mouse, human, bovine, rabbit, and rat lens epithelial and fiber cells. Rat lenses or lens epithelia were rapidly fixed in situ, 30 minutes after death, or after dissection from the lens, and the intracellular distributions of crystallins were examined by immunostaining and confocal microscopy.Four patterns of crystallin gene expression were detected in cultured lens epithelia. Transcripts encoding most beta- and gamma-crystallins were detectable and, in some cases, abundant at the time of explantation. Changes in crystallin protein levels in P3 epithelia cultured in basal or FGF-supplemented medium generally reflected the changes in their mRNAs. betaB1- and gammaS-crystallins were abundant in adult human, mouse, rat, rabbit, and bovine lens epithelial cells. The alpha-, beta- and gamma-crystallins were found in distinct subcellular locations in adult lens epithelial cells. These proteins dramatically relocalized during fiber cell differentiation and after death and/or dissection of the lens epithelium.BetaB1- and gammaS-crystallins are normally abundant in adult mammalian lens epithelial cells. Complex programs of transcription and degradation regulate the accumulation of crystallin mRNAs in lens epithelial cells after stress, at different ages, and during cell differentiation. Because crystallins selectively localize in distinct subcellular compartments during differentiation or stress, they may function to protect lens cells from injury. After stress, most alphaA- and alphaB-crystallin subunits are not in the same macromolecular complexes.

Purpose To compare the characteristics of early cortical cataract localization in three groups in cataract epidemiologic surveys performed in Reykjavik, Melbourne, and Singapore. Methods Individuals who had right eyes with an area of cortical opacity less than 20% of the pupil when dilated 7 mm or more were selected as subjects. This included 197 subjects from the Reykjavik Eye Study, 231 from the Vitamin E, Cataract, and Age-Related Maculopathy (VECAT) study in Melbourne, and 92 from the Singapore-Japan Cooperative Cataract Study, all showing early-stage cataract in pupils dilated to 7 mm or more. Scheimpflug and retroilluminated photographs were used to locate opacities. Localization of cortical cataract was determined by dividing the retroillumination image into seven concentric circles with diameters of 1 through 7 mm, and eight sections of 45 degrees radial octants. The positive rate of opacification was then calculated for each quadrant. Results The highest positive rate of opacification was observed in the lower nasal quadrant in all groups. The relative risk of the prevalence of cortical opacity in the lower nasal oblique hemisphere to the upper temporal oblique hemisphere was the highest in the Singaporean subjects followed by those of Melbourne and then of Reykjavik. Conclusions The prevalence of cortical cataract was higher in the lower nasal quadrant than in the other quadrants for all subjects of diverse race in three climatically different locations. This higher prevalence was most pronounced in subjects living at low latitude. These results support the view that solar UV exposure is a possible risk factor for development of human cortical cataract.

To investigate the relationship between vitamin E deficiency and prednisolone-induced cataract formation, long-term examination of lens changes was performed in rats under the condition of vitamin E deficiency or supplementation and administration of prednisolone.Rats were divided into six groups: normal chow (N), vitamin E-deficient chow (ED), normal chow with prednisolone instillation (NP), vitamin E-deficient chow with prednisolone instillation (EDP), NP treatment with vitamin E supplementation (NP+VE), and EDP treatment with vitamin E supplementation (EDP+VE). Prednisolone (1 mg/kg small middle dot d) and vitamin E (5%; 10 microL per administration per eye, 1 mg/kg small middle dot d) were applied in the cul-de-sac. Lens changes were documented and analyzed. Vitamin E status was confirmed by measuring peroxide-induced hemolysis.After 15 months, 91.7% of the eyes in the EDP group showed development of anterior and posterior cortical cataracts. Supplementation with vitamin E significantly reduced cataract formation (to 38.9% of eyes). Neither a vitamin E-deficient diet nor prednisolone treatment alone significantly increased cataract formation. Hemolysis-susceptibility tests confirmed the expected vitamin E status of rats fed vitamin E-deficient chow and rats supplemented with eye drops containing vitamin E.Vitamin E deficiency and long-term prednisolone treatment together cause cataracts. Singly, however, both conditions are subliminal cataractogenic risk factors.

PURPOSE Vitreous degeneration contributes to several age-related eye diseases, including retinal detachment, macular hole, macular traction syndrome, and nuclear cataracts. Remarkably little is understood about the molecular interactions responsible for maintaining vitreous structure. The purpose of this study was to measure the structural properties of the vitreous body after enzymatic degradation of selected macromolecules. METHODS Mechanical properties of plugs of bovine and porcine vitreous were analyzed using a rheometer. Oscillatory and extensional tests measured vitreous stiffness and adhesivity, respectively. Major structural components of the vitreous were degraded by incubation overnight in collagenase, trypsin, or hyaluronidase, singly or in combination. Vitreous bodies were also incubated in hyper- or hypotonic saline. Effects of these treatments on the mechanical properties of the vitreous were measured by rheometry. RESULTS Enzymatic digestion of each class of macromolecules decreased the stiffness of bovine vitreous by approximately half (P < 0.05). Differential effects were observed on the damping capacity of the vitreous (P < 0.05), which was shown to correlate with material behavior in extension (P < 0.01). Digestion of hyaluronan significantly decreased the damping capacity of the vitreous and increased adhesivity. Collagen degradation resulted in the opposite effect, whereas digestion of proteins and proteoglycans with trypsin did not alter behavior relative to controls. Osmotic perturbations and double-enzyme treatments further implicated hyaluronan and hyaluronan-associated water as a primary regulator of adhesivity and material behavior in extension. CONCLUSIONS Collagen, hyaluronan, and proteoglycans act synergistically to maintain vitreous stiffness. Hyaluronan is a key mediator of vitreous adhesivity, and mechanical damping is an important factor influencing dynamic vitreous behavior.

Editor, We read with interest the recent editorial on the physiological effects of vitreous surgery, which highlighted the importance of the viscosity of the vitreous fluid on the rate of diffusion of small molecules of physiological importance (Stefansson & Loftsson 2006). From the Stokes–Einstein equation and previous measurements of the viscosity of the human vitreous body (Lee et al. 1992), it was suggested that the rate of diffusion should be 300–2000 times slower before vitrectomy than after. We agree that the rate at which small molecules are distributed in the eye is an important aspect of ocular physiology. However, the published measures of the viscosity of the vitreous were obtained from vitreous gel, while the Stokes–Einstein equation describes the effect of the viscosity on the rate of diffusion in a liquid. David Maurice measured the rate of loss of radioactive protein from the centre of the vitreous body of living rabbit eyes. Using these measures and physical modelling, he concluded that the rate of diffusion of molecules in the intact vitreous body was experimentally indistinguishable from water (Maurice 1959). Dr Maurice’s measurements have since been used to model the distribution of many kinds of molecules in the eye. To make more direct observations of diffusion in the vitreous body at the microscopic scale, we used fluorescence correlation spectroscopy (FCS) (Elson 2004). FCS measures the time spent by single fluorescent molecules in a tiny volume at the focal point of a laser beam. When their molecular weight is known, FCS provides a measure of the viscosity of the medium in which the molecules are moving. We collected data from the diffusion of two fluorescent molecules of different molecular weight, Alexa 488 (~ 0.5 kDa) and recombinant green fluorescent protein [GFP (~ 29 kDa)], in physiological salt solutions, homogenized porcine vitreous or intact porcine vitreous gel. Correlation spectra were collected using a Zeiss Confocor™ (Thornwood, NY, USA) attachment on a LSM510 confocal microscope (Zeiss Confocor™) at a point 100 μm into the solution or gel. The correlation spectra obtained are shown in Fig. 1; mean diffusion times and calculated diffusion coefficients for Alexa 488 and GFP are shown in Tables 1 and ​and2,2, respectively. Fig. 1 Correlation spectra for Alexa 488 (A) and recombinant green fluorescent protein [GFP (B)] in different media. BSS, balanced salt production; PBS, phosphate-buffered saline. Table 1 Mean diffusion times and calculated diffusion coefficients for Alexa 488. Table 2 Mean diffusion times and calculated diffusion coefficients for recombinant green fluorescent protein (GFP). As expected from its greater molecular weight, GFP had a longer diffusion time and lower diffusion coefficient than Alexa 488 under all conditions. However, the diffusion times and calculated diffusion coefficients of each fluorescent molecule measured in saline solutions, homogenized vitreous or vitreous gel were similar. According to the Stokes–Einstein equation, if the microscopic viscosity of the vitreous fluid or the vitreous gel were greater than saline, the calculated diffusion coefficients in the vitreous fluids would have been proportionally smaller in vitreous (gel or liquid) than in saline. Since these values were similar, the microscopic viscosity of liquid or gel vitreous is similar to saline. These measurements and the work of Dr Maurice suggest that the rate of diffusion of a molecule in the vitreous chamber is expected to be similar before and after vitrectomy and in eyes with an intact vitreous body or with extensive vitreous liquefaction. It has been argued that the loss of the gel structure of the vitreous body, as a consequence of vitrectomy or age-related liquefaction, has important effects on the distribution of small molecules in the eye (Stefansson et al. 1990; Harocopos et al. 2004; Stefansson 2006). The available evidence suggests that the increased rate at which molecules are redistributed in the vitreous compartment in the absence of the vitreous gel or after vitreous degeneration is more likely to be because of an increase in fluid circulation than a difference in the rate of diffusion.

Several lines of evidence suggest that increased oxidative stress or the accumulation of oxidative damage contributes to the pathogenesis of glaucoma.1–7 Antioxidant protective mechanisms are decreased, and enzymes induced by oxidative stress are increased in the aqueous humor of glaucoma patients, compared with those undergoing cataract surgery.3,8 Oxidative damage to DNA increases in the trabecular meshwork cells of glaucoma patients, and these cells are more susceptible to oxidative DNA damage than are other cells in the anterior segment.9 In one study, the lymphocytes of glaucoma patients had pathogenic mutations in their mitochondrial DNA that were not present in control subjects.10 These alterations are consistent with oxidative damage. The mitochondria of glaucoma patients also had significantly lower oxidative activity than in control subjects.10 Despite the abundant evidence linking glaucoma's pathogenesis to oxidative damage, the source of the oxidative stress in these patients is not known. It is also not clear whether the increased oxidative damage in glaucoma patients is the result of increased exposure to oxidants, decreased antioxidative protection, or a combination of these factors. Ocular surgery can increase the risk of developing glaucoma. Elevated intraocular pressure and glaucoma have long been associated with corneal transplantation (penetrating keratoplasty) or the implantation of an artificial cornea.11–19 Recently, two studies reported that vitrectomy is also associated with increased risk of elevated intraocular pressure and glaucoma. In both studies, the presence of the natural lens delayed the onset of glaucoma.20,21 In most older patients who undergo vitrectomy, nuclear sclerotic cataracts develop within 2 years.22–26 Nuclear cataract is associated with increased oxidative damage to the lens, and vitrectomy leads to increased exposure of the lens to oxygen.23,27,28 These observations led Chang20 to propose that, after vitrectomy, metabolites of oxygen, like hydrogen peroxide, may damage the tissues of the outflow pathway, contributing to the increased risk of glaucoma. Since the presence of the natural lens reduces the risk of glaucoma, he proposed that the lens protects the anterior segment from oxygen or oxygen metabolites. The present study was designed to test Chang's hypothesis that oxygen is relatively low in the anterior segment of the non–surgically altered eye and that it increases after vitrectomy and cataract surgery. The distribution of oxygen in the anterior segment of the eye was measured in a reference group of patients who were undergoing surgery for glaucoma or cataract. Oxygen distribution in these patients was compared with that in patients who had had vitrectomy, cataract surgery, or both procedures. The results of these measurements supported the predictions of the Chang hypothesis, which suggest that increased exposure to oxygen or its metabolites causes open-angle glaucoma. These studies also revealed an unexpected physiologic mechanism that regulates the distribution of oxygen in the anterior chamber angle of the human eye.

To determine whether diabetes mellitus is protective for nuclear sclerotic cataract at baseline and 6 and 12 months after vitrectomy surgery.Prospective, interventional cohort study.Phakic diabetic and nondiabetic patients undergoing vitrectomy surgery for a variety of retinal conditions underwent Scheimpflug lens photography in the operated and fellow eye at baseline and at 6 and 12 months after vitrectomy surgery.Of 52 eyes included in the analysis, 23 eyes were from diabetic patients, 14 of which had surgery for ischemic retinopathy. At baseline, eyes with ischemic diabetic retinopathy had less nuclear sclerotic cataract than nonischemic diabetic and nondiabetic eyes. This was true for eyes undergoing vitrectomy surgery (P = .0001) and for fellow eyes (P = .003). Nuclear sclerotic cataract developed after vitrectomy surgery in nonischemic diabetic eyes and nondiabetic eyes at the same rate. Diabetic eyes with ischemic retinopathy showed no significant progression of nuclear opacification, and therefore had significantly less postvitrectomy nuclear cataract at 6 months (P1 × 10(-6)) and at 12 months (P.001) than nondiabetic or nonischemic diabetic eyes. Normalizing to baseline opacity and adjusting for age and other comorbidities did not alter this result.Ischemic diabetic retinopathy, not just systemic diabetes mellitus, protected against nuclear sclerotic cataract at baseline and after vitrectomy surgery. These findings are consistent with the hypothesis that increased exposure to oxygen is responsible for nuclear cataract formation.

Purpose—The lens grows throughout life, and lens size is a major risk factor for nuclear and cortical cataracts. A previous study showed that the hypoxic environment around the lens suppressed lens growth in older rats. The present study was conducted to investigate the mechanism responsible for the age-dependent decline in lens cell proliferation. Methods—Transgenic mice expressing Cre recombinase in the lens were bred to mice containing floxed Hif1a alleles. Transgenic mice expressing oxygen insensitive forms of HIF-1α in lens epithelial cells were exposed to room air or 60% oxygen. Proliferation was measured by BrdU labeling and cell death by using the TUNEL assay. Morphology was assessed in histologic sections. HIF-1α and p27 KIP1 levels were determined by Western blot. The expression of HIF-regulated genes was assessed on microarrays. Results—Lenses lacking Hif1a degenerated, precluding study in older animals. Breathing 60% oxygen reduced HIF-1α levels and HIF-1-regulated transcripts in lens epithelial cells from young and older lenses. Overexpression of oxygen-insensitive HIF-1α had no effect on lens size, but suppressed increased proliferation in response to oxygen. Systemic injection of the iron chelator, 1,10-phenanthroline prevented the degradation of HIF-1α and reduced oxygen-induced proliferation. Increasing oxygen decreased levels of p27 KIP1 in the epithelial cells of older mice, which was prevented by expressing oxygen-insensitive forms of HIF-1α. Conclusions—HIF-1α is present and HIF-1 is transcriptionally active throughout life, but suppresses growth only in older lenses. Maintaining elevated levels of p27 KIP1 in older lenses requires HIF-1. p27 KIP1 and other growth regulators may selectively suppress the proliferation of older lens epithelial cells. The human lens grows continuously throughout life. 1–3 Epidemiologic studies found that older subjects with small lenses are at increased risk of cortical cataracts and those with larger lenses are more likely to have nuclear cataracts. 4–6 A recent study showed that increasing the level of oxygen in the eyes of older rats increases the rate of lens cell proliferation and lens growth. 7 Based on this observation, it has been suggested that the hypoxic environment around the lens normally inhibits the rate of lens growth. It was not clear from these studies whether hypoxia directly inhibits proliferation by altering intracellular factors that control lens cell proliferation, by decreasing the growth

In a recent article in the November 2006 issue of the Archives, Kubo et al1 report that an increase in axial length or myopia is associated with a higher grade of nuclear cataract. In the “Comment” section, they state, “The mechanism of the apparent relationship between axial length and nuclear cataract at the time of cataract surgery is unclear.” They go on to suggest that “changes in vitreous circumstances caused by longer axial length may induce nuclear or other types of cataract.” We would like to propose a mechanism to explain their observations. In 2004, we published an article titled “Importance of Vitreous Liquefaction in Age-Related Cataract.”2 We found that the extent of vitreous liquefaction was positively correlated with the extent of nuclear opacification, even after adjusting for age. In myopic eyes, vitreous syneresis occurs earlier, is more extensive, and increases with the degree of myopia.3 Therefore, myopia increases the extent of vitreous liquefaction, which is associated with an increase in the risk of nuclear cataract. Kubo and colleagues cite research by us and others to support the oxidative theory of nuclear cataract formation. The lens normally exists in a low-oxygen environment.4 Increased exposure of the lens to oxygen causes nuclear cataract.5 A potential source of oxidative damage to the lens is increased exposure to molecular oxygen from the retina. The intact vitreous gel ensures that oxygen from the retina can only reach the lens by diffusion, a relatively inefficient process over macroscopic distances. Replacement of the vitreous gel with liquid, as occurs after vitreous syneresis or vitrectomy, will increase fluid circulation within the vitreous chamber. Increased circulation of the vitreous fluid readily distributes oxygen from the retinal surface throughout the eye. We suggest that an intact vitreous gel helps to maintain the low level of oxygen around the lens by preventing bulk flow of the vitreous fluid. We are pleased that the observations by Kubo and colleagues are consistent with one aspect of a hypothesis central to our research: that liquefaction of the vitreous is important in the pathogenesis of nuclear cataract.

It has been difficult to discover the aspects of aging that are responsible for the exponential increase in cataract risk after age 50. Environmental risk factors for cataract are often difficult to modify (smoking), or they account for only a small fraction of the total cataract burden (sunlight exposure).1 Family history is a major contributor to cataract risk.2–6 However, the genes that contribute to increased risk for age-related cataract have not been identified, and the pathways in which these genes act to increase or decrease cataract risk are unknown. Therefore, understanding the physiology of the normal lens is important for identifying the aspects of lens biology that are altered with age. The lens is an epithelial tissue with cells that exist in three states of growth (Fig. 1A). The anterior surface of the lens is covered by a simple cuboidal epithelium. In adults, epithelial cells in the central region of the epithelium are mitotically quiescent. However, cells at the periphery of the epithelium, in the germinative zone, proliferate throughout life. When germinative zone cells divide, their daughter cells move posteriorly, withdraw from the cell cycle, and differentiate into fiber cells. Fiber cells, which make up the bulk of the lens, are greatly elongated and accumulate high levels of crystallins, which account for its transparency and refractive power. As the lens grows, fiber cells that were originally near the surface are buried deeper within the lens by the differentiation of more superficial fiber cells. At the completion of their elongation and maturation, fiber cells lose all their membrane-bound organelles.7 However, the remaining cellular components persist for the life of the lens. By these processes, the lens grows in cell number, mass, and size throughout life.8 Figure 1 (A) Diagram of the adult lens. Inset: details of the equatorial region, including the location of the germinative zone at the periphery of the lens epithelium. Red asterisk: nucleus at the lowest point of the “lens bow.” In one experiment, ... Lens growth is rapid during fetal and early postnatal life but slows thereafter. In humans, lens mass increases logarithmically until soon after birth, then transitions to linear growth for the remainder of life.9–11 Unlike the remainder of the mammalian eye, the lens continuously increases in cell number and size. The consequences of this sustained growth in an eye that is not growing are thought to contribute to presbyopia, the decreased ability to focus on near objects that develops in middle age. Several growth factors promote the proliferation of lens epithelial cells in culture. These include platelet-derived growth factor,12–15 fibroblast growth factor,16–20 insulin and insulin-like growth factors,21,22 epidermal growth factor,23–28 and hepatocyte growth factor.29,30 These mitogens are present in ocular tissues near the lens or in the aqueous humor that bathes the lens epithelium. However, none has been demonstrated to regulate the normal growth of the lens in vivo. It has been assumed that the decline in the rate of lens growth that occurs during postnatal life results from decreased levels of lens mitogens or decreased responses of lens cells to these agents. The avascular lens exists in a hypoxic environment.31–35 Increased exposure to oxygen has been shown to be a risk factor for the most common type of age-related cataract, nuclear cataract.36,37 During aging, degeneration of the vitreous body, the gel between the lens and the retina, may expose the lens to increased oxygen.38 In the present study, examination of the effects of oxygen on the lens in vivo revealed that the normal level of intraocular hypoxia is required to suppress lens cell proliferation and to maintain a smaller lens. Given that increased lens thickness is a risk factor for nuclear cataract formation,39,40 these observations provide a potential link between oxygen exposure and cataract formation.

Purpose In the mammalian lens, the expression of the beta- and gamma-crystallin families is thought to be limited to fiber cells. However, several studies detected these proteins or their mRNAs in human lens epithelial cells. To resolve this apparent discrepancy, 14 crystallin mRNAs were examined and the expression and subcellular distribution of selected crystallin proteins in lens epithelial cells determined. Methods Transcript levels were analyzed by quantitative real-time PCR using mRNA from P3 rat lens epithelia cultured for 0 or 20 hours or 4 or 7 days in basal medium or with added FGF2. Antibodies to betaB1-, gammaS-, alphaA-, and alphaB-crystallins were used for Western blot analysis of proteins extracted from adult mouse, human, bovine, rabbit, and rat lens epithelial and fiber cells. Rat lenses or lens epithelia were rapidly fixed in situ, 30 minutes after death, or after dissection from the lens, and the intracellular distributions of crystallins were examined by immunostaining and confocal microscopy. Results Four patterns of crystallin gene expression were detected in cultured lens epithelia. Transcripts encoding most beta- and gamma-crystallins were detectable and, in some cases, abundant at the time of explantation. Changes in crystallin protein levels in P3 epithelia cultured in basal or FGF-supplemented medium generally reflected the changes in their mRNAs. betaB1- and gammaS-crystallins were abundant in adult human, mouse, rat, rabbit, and bovine lens epithelial cells. The alpha-, beta- and gamma-crystallins were found in distinct subcellular locations in adult lens epithelial cells. These proteins dramatically relocalized during fiber cell differentiation and after death and/or dissection of the lens epithelium. Conclusions BetaB1- and gammaS-crystallins are normally abundant in adult mammalian lens epithelial cells. Complex programs of transcription and degradation regulate the accumulation of crystallin mRNAs in lens epithelial cells after stress, at different ages, and during cell differentiation. Because crystallins selectively localize in distinct subcellular compartments during differentiation or stress, they may function to protect lens cells from injury. After stress, most alphaA- and alphaB-crystallin subunits are not in the same macromolecular complexes.

PURPOSE. Vitrectomy is associated with the rapid progression of nuclear cataracts. This study was conducted to test the hypothesis that age-related liquefaction of the vitreous gel may also be associated with lens opacification. METHODS. Lenses from eye bank eyes were graded for nuclear, cortical, and posterior subcapsular opacities, and the amount of liquid vitreous was measured. RESULTS. Nuclear sclerosis (NS) grade, percent liquefaction, and age all correlated highly (P 0.0001). After adjustment was made for age, the correlation between NS and percent liquefaction was 0.37 (P 0.0001), and the correlation between age and NS after adjustment for liquefaction was 0.57 (P 0.0001). After the eyes were stratified into age groups, the correlation between NS and percent liquefaction was found to be highest in eyes from donors aged 51 to 70. Cortical and posterior subcapsular opacities increased with age, but scores for these cataract types did not significantly correlate with vitreous liquefaction. CONCLUSIONS. Loss of vitreous gel during aging increases the risk of nuclear cataracts. Preservation or replacement of the vitreous gel may protect patients from nuclear cataract. (Invest Ophthalmol Vis Sci. 2004;45:77‐ 85) DOI:10.1167/iovs.030820

Purpose Previous studies have identified sequences encoding vascular endothelial growth factor (VEGF)-A and one of the VEGF receptors (VEGFR2, Flk-1, KDR) in lens fiber cells. The current study was undertaken to determine the distribution of VEGF-A protein in the lens, whether signaling through VEGF receptors occurs in lens cells, the pattern of VEGF-A expression during lens development, and the effect of hypoxia on VEGF-A expression. Methods VEGF-A and VEGFR2 were localized using immunocytochemistry. VEGF-A and VEGFR2 protein were identified and quantified by Western blot analysis. Activated (tyrosine phosphorylated) VEGFR2 was detected by immunoprecipitation with an anti-phosphotyrosine antibody followed by Western blot analysis with antibody to VEGFR2. Levels of VEGF-A mRNA were measured by quantitative PCR. Suturing the lids of adult mouse or rabbit eyes for 3 days was used to induce lens hypoxia. Results VEGFR2 sequences were present in adult human lens epithelial cells, and VEGF-A transcripts were detected in chicken embryo, adult human, and mouse lens epithelial cells. VEGF-A protein localized to the ends of mouse embryo lens fiber cells at developmental stages when the fetal vasculature was forming. At later stages, VEGF-A was distributed uniformly throughout the cytoplasm of cortical fiber cells. VEGFR2 was present in mouse lens epithelial and fiber cells and was tyrosine phosphorylated at all stages examined. VEGF-A protein was barely detectable in lens epithelial cells during the first postnatal week, but increased as the capillaries of the anterior pupillary membrane regressed. VEGF-A levels were highest in adult lenses. Suturing the eyelid caused an increase in VEGF-A mRNA and protein in lens epithelial and fiber cells. Conclusions VEGF-A secreted by lens cells may stimulate the formation of the fetal vasculature, but regression of these vessels is not likely to be caused by a reduction in VEGF-A production by the lens. An active VEGF-A signaling system of unknown function appears to be active in the lens. It is likely that VEGF-A expression is regulated by tissue hypoxia at all stages of lens development.

PURPOSE. Previous studies have implicated members of the fibroblast growth factor (FGF) and insulin-like growth factor (IGF) families as stimulators of lens fiber cell differentiation in rodent and chicken embryo lenses, respectively. In the present study, the role of FGFs in fiber cell differentiation and epithelial cell proliferation in chicken embryos was examined. METHODS. Lenses were injected on embryonic day (E)3 with replication-defective retroviruses that express full-length or truncated FGF receptor (FGFR)-1 or a secreted form of FGF1. Lens epithelial explants were cultured in defined medium or medium supplemented with FGFs or vitreous humor, in the presence or absence of the FGF receptor antagonist SU5402. Explants were also cultured in vitreous humor that had been depleted of heparin-binding growth factors. Cell elongation was measured optically and protein accumulation by densitometry and Western blot analysis. RESULTS. Lens fiber cell differentiation was not inhibited in cells infected with virus expressing truncated FGFR1. Epithelial cells infected with virus encoding a secreted form of FGF1 did not differentiate into ectopic fiber cells. Viral transduction of FGFR1, truncated FGFR1, or FGF1 did not appreciably alter the proliferation of lens epithelial cells. Bovine vitreous humor stimulated chicken embryo lens epithelial cells to elongate and express markers of lens fiber cell differentiation. Bovine vitreous humor, but not FGF2, protected lens epithelial cells from apoptosis. Depleting vitreous humor of heparin-binding growth factors or treatment of lens cells with SU5402 did not inhibit the initial, rapid phase of lens cell elongation. Both treatments, used separately or together, reduced but did not prevent the expression of later markers of fiber cell differentiation. CONCLUSIONS. Fiber differentiation factors that are not members of the FGF family are present in chicken and mammalian vitreous humor. The factors that stimulate fiber cell differentiation in avian and mammalian eyes are similar. (Invest Ophthalmol Vis Sci. 2003;44:680 ‐ 690) DOI:10.1167/iovs.01-1177