Paul Sternberg

Oxidative damage and protection of the RPE

This review provides a model for the role of oxidative stress in the etiology of age-related macular degeneration (AMD). Epidemiological studies of diet, environmental and behavioral risk factors suggest that oxidative stress is a contributing factor of AMD. Pathological studies indicate that damage to the retinal pigment epithelium (RPE) is an early event in AMD. In vitro studies show that oxidant treated RPE cells undergo apoptosis, a possible mechanism by which RPE cells are lost during early phase of AMD. The main target of oxidative injury seems to be mitochondria, an organelle known to accumulate genomic damages in other postmitotic tissues during aging. The thiol antioxidant GSH and its amino acid precursors protect RPE cells from oxidant-induced apoptosis. Similar protection occurs with dietary enzyme inducers which increase GSH synthesis. These results indicate that therapeutic or nutritional intervention to enhance the GSH antioxidant capacity of RPE may provide an effective way to prevent or treat AMD.

Redox state of glutathione in human plasma

Thiol and disulfide forms of glutathione (GSH) and cysteine (Cys) were measured in plasma from 24 healthy individuals aged 25-35 and redox potential values (E(h)) for thiol/disulfide couples were calculated using the Nernst equation. Although the concentration of GSH (2.8 +/- 0.9 microM) was much greater than that of GSSG (0.14 +/- 0.04 microM), the redox potential of the GSSG/2GSH pool (-137 +/- 9 mV) was considerably more oxidized than values for tissues and cultured cells (-185 to -258 mV). This indicates that a rapid oxidation of GSH occurs upon release into plasma. The difference in values between individuals was remarkably small, suggesting that the rates of reduction and oxidation in the plasma are closely balanced to maintain this redox potential. The redox potential for the Cys and cystine (CySS) pool (-80 +/- 9 mV) was 57 mV more oxidized, showing that the GSSG/2GSH and the CySS/2Cys pools are not in redox equilibrium in the plasma. Potentials for thiol/disulfide couples involving CysGly were intermediate between the values for these couples. Regression analyses showed that the redox potentials for the different thiol/disulfide couples within individuals were correlated, with the E(h) for CySS-mono-Gly/(Cys. CysGly) providing the best correlation with other low molecular weight pools as well as protein disulfides of GSH, CysGly and Cys. These results suggest that E(h) values for GSSG/2GSH and CySS-mono-Gly/(Cys. CysGly) may provide useful means to quantitatively express the oxidant/antioxidant balance in clinical and epidemiologic studies.

Glutathione measurement in human plasma. Evaluation of sample collection, storage and derivatization conditions for analysis of dansyl derivatives by HPLC.

Literature values for human plasma GSH vary over 10-fold despite the use of apparently valid analytical procedures for GSH measurement. The purpose of this study was to develop a procedure to minimize error in sample collection, processing and storage that could contribute to such differences. HPLC with fluorescence detection of dansyl derivatives was used for quantification. The results show that collection of blood with a butterfly needle and syringe reduces overestimation due to limited hemolysis and that use of a preservation solution designed to inhibit autooxidation and enzymatic degradation allows quantitative recovery of both GSH and GSSG. Stability tests showed that non-derivatized samples were stable for at least 2 months at - 80 degrees while dansyl derivatives were stable in the dark at 0-4 degrees for 12 months. Results from 59 healthy individuals (20-43 years) provided a mean (+/-1 SD) GSH value of 2.09+/-1.14 micromolar.

Glutathione in Human Plasma: Decline in Association with Aging, Age-Related Macular Degeneration, and Diabetes

Blood samples were analyzed for GSH and GSH redox state in 40 age-related macular degeneration (ARMD) patients (> 60 y), 33 non-ARMD diabetic patients (> 60 years), 27 similarly aged non-ARMD and nondiabetic individuals (> 60 years), and 19 younger individuals (< 60 years) without ARMD or diabetes. Results showed a significantly lower plasma GSH in older individuals (ARMD, diabetes, and controls) than in younger individuals (p < .01). Total GSH (GSHt) obtained following treatment with dithiothreitol was significantly lower only in diabetic cases (p < .05) but also approached significance for ARMD cases (p = .089). Estimation of redox potential indicated that the plasma GSH pool is considerably more oxidized in all of the older groups. Analyses of whole blood GSH showed that GSH was significantly lower in diabetic cases compared to the other groups, but did not reveal any difference associated with age or ARMD. In contrast, GSSG in whole blood was significantly higher in the older groups compared to the younger controls. The results suggest that in studies of age-related pathologies, oxidation of GSH may be a more important parameter than a decline in pool size, while in specific pathologies such as diabetes, both oxidation and a decline in pool size may be important.

Retinal pigment epithelial changes after macular hole surgery with indocyanine green-assisted internal limiting membrane peeling

PURPOSE To report the results of macular hole surgery using indocyanine green to improve visualization and facilitate peeling of the internal limiting membrane. METHODS A retrospective noncomparative review of a consecutive series of 22 patients (22 eyes) who underwent macular hole repair using indocyanine green to facilitate visualization of the internal limiting membrane was performed. One patient was excluded because of a history of a rhegmatogenous retinal detachment. All patients underwent a three-port pars plana vitrectomy with internal limiting membrane peeling. Indocyanine green (0.1% solution) was used to assist in the visualization of the internal limiting membrane. The main outcome measures were postoperative visual acuity, macular hole status, and postoperative retinal pigment epithelial changes. RESULTS In 21 eyes, the median preoperative best-corrected visual acuity was 20/200 (range, 20/60 to counting fingers at 5 feet). The median postoperative visual acuity was 20/400 (range, 20/60-1/200) with an average follow-up of 13 weeks. The macular hole was closed in 18 eyes (86%) at the most recent follow-up. Ten eyes were found to have atrophic retinal pigment epithelium changes in the area of the previous macular hole. CONCLUSIONS Indocyanine green assists in visualization of the internal limiting membrane in macular hole surgery. In our series, 10 eyes had unusual atrophic changes in the retinal pigment epithelium at the site of the previous macular hole, or in the area where the indocyanine green solution would have had direct access to the bare retinal pigment epithelium cells. Although the use of indocyanine green improves visualization and assists with peeling of the internal limiting membrane, the safety and potential toxicity of indocyanine green to the retinal pigment epithelium require further investigation.

Redox analysis of human plasma allows separation of pro-oxidant events of aging from decline in antioxidant defenses

Oxidative stress is a component of diseases and degenerative processes associated with aging. However, no means are available to assess causative oxidative events separately from decline in function of protective antioxidant systems. Previous studies show that ongoing oxidative processes maintain plasma cysteine/cystine redox at a value that is more oxidized than the antioxidant glutathione/glutathione disulfide (GSH/GSSG) system, suggesting that redox analysis of these plasma thiols could allow separate evaluation of an increase in oxidative events from a decline in antioxidant function. The present study uses measurement of cysteine/cystine and GSH/GSSG redox in plasma of 122 healthy individuals aged 19-85 years to determine whether thiol-disulfide redox changes occur with age. The results show a linear oxidation of cysteine/cystine redox state with age at a rate of 0.16 mV/year over the entire age span. In contrast, GSH/GSSG redox was not oxidized prior to 45 years and subsequently was oxidized at a nearly linear rate of 0.7 mV/year. These data suggest that there is a continuous, linear increase in oxidative events throughout adult life but that the capacity of the GSH antioxidant system is maintained until 45 years and then declines rapidly. The data further suggest that redox states of cysteine/cystine and GSH/GSSG provide an approach to clinically distinguish between increased causative oxidative events and decreased GSH antioxidant function. In principle, such analyses can be used to assess efficacy of intervention strategies against oxidative stress prior to or early after onset of clinical symptoms in aging and age-related disease.

Penetrating Ocular Injuries: Types of Injuries and Visual Results

The authors studied the hospital records of 453 patients who underwent primary surgical repair of penetrating ocular injuries at the Wilmer Institute from January 1970 through December 1981. The injury was due to blunt force in 22%, a sharp laceration in 37%, and a missile in 41%. The following factors correlated with the final visual outcome: (1) initial visual acuity after the injury, (2) presence of an afferent pupillary defect, (3) type of injury, (4) location and extent of the penetrating wound, (5) type of lens damage, (6) presence and severity of vitreous hemorrhage, and (7) type of intraocular foreign body. This study emphasizes that the prognosis after a penetrating injury is strongly influenced by the nature of the injury and the extent of initial damage.

Indocyanine green effect on cultured human Retinal pigment epithelial cells : Implication for macular hole surgery

PURPOSE To evaluate potential toxic effects of indocyanine green dye on cultured human retinal pigment epithelial cells. METHODS Controlled laboratory experiment. Cultured human retinal pigment epithelial cells were exposed to balanced saline solution, balanced saline solution with endoillumination, indocyanine green or indocyanine green with endoillumination. Cells were evaluated by light microscopy, electron microscopy, and a mitochondrial dehydrogenase assay. RESULTS Retinal pigment epithelial cells exposed to indocyanine green showed no histologic or ultrastructural changes. Those exposed to indocyanine green alone or indocyanine green plus light demonstrated a significant decrease in mitochondrial enzyme activity (P = 0.0002 and 0.005, respectively). CONCLUSION Brief exposure of cultured human retinal pigment epithelial cells to indocyanine green results in decreased mitochondrial enzyme activity but does not appear to influence cellular morphology or ultrastructure.

Pathologic Features of Surgically Excised Subretinal Neovascular Membranes in Age-related Macular Degeneration

The histopathologic features of ten consecutive surgically excised subfoveal neovascular membranes from patients with age-related macular degeneration were examined. Ultrastructural features included the following in decreasing order of frequency: endothelium-lined vascular channels, new collagen, fibrocytes, retinal pigment epithelium, erythrocytes, and myofibroblasts. Chronic inflammatory cells were frequently evident and included macrophages, lymphocytes, and plasma cells. Basal laminar deposit or diffuse drusen were observed in six of the membranes. Photoreceptors and Bruchs membrane were each observed in three of the specimens, but were not associated with decreased postoperative visual acuity. Fibrin was observed in eight membranes, either within the stroma of the membrane or in association with subretinal hemorrhage.

Age-Related Retinopathy in NRF2-Deficient Mice

Background Cumulative oxidative damage is implicated in the pathogenesis of age-related macular degeneration (AMD). Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that plays key roles in retinal antioxidant and detoxification responses. The purposes of this study were to determine whether NRF2-deficient mice would develop AMD-like retinal pathology with aging and to explore the underlying mechanisms. Methods and Findings Eyes of both wild type and Nrf2−/− mice were examined in vivo by fundus photography and electroretinography (ERG). Structural changes of the outer retina in aged animals were examined by light and electron microscopy, and immunofluorescence labeling. Our results showed that Nrf2−/− mice developed age-dependent degenerative pathology in the retinal pigment epithelium (RPE). Drusen-like deposits, accumulation of lipofuscin, spontaneous choroidal neovascularization (CNV) and sub-RPE deposition of inflammatory proteins were present in Nrf2−/− mice after 12 months. Accumulation of autophagy-related vacuoles and multivesicular bodies was identified by electron microcopy both within the RPE and in Bruchs membrane of aged Nrf2−/− mice. Conclusions Our data suggest that disruption of Nfe2l2 gene increased the vulnerability of outer retina to age-related degeneration. NRF2-deficient mice developed ocular pathology similar to cardinal features of human AMD and deregulated autophagy is likely a mechanistic link between oxidative injury and inflammation. The Nrf2−/− mice can provide a novel model for mechanistic and translational research on AMD.