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Background US is clinically established for breast imaging, but its diagnostic performance depends on operator experience. Computer-assisted (real-time) image analysis may help in overcoming this limitation. Purpose To develop precise real-time-capable US-based breast tumor categorization by combining classic radiomics and autoencoder-based features from automatically localized lesions. Materials and Methods A total of 1619 B-mode US images of breast tumors were retrospectively analyzed between April 2018 and January 2024. nnU-Net was trained for lesion segmentation. Features were extracted from tumor segments, bounding boxes, and whole images using either classic radiomics, autoencoder, or both. Feature selection was performed to generate radiomics signatures, which were used to train machine learning algorithms for tumor categorization. Models were evaluated using the area under the receiver operating characteristic curve (AUC), sensitivity, and specificity and were statistically compared with histopathologically or follow-up-confirmed diagnosis. Results The model was developed on 1191 (mean age, 61 years ± 14 [SD]) female patients and externally validated on 50 (mean age, 55 years ± 15]). The development data set was divided into two parts: testing and training lesion segmentation (419 and 179 examinations) and lesion categorization (503 and 90 examinations). nnU-Net demonstrated precision and reproducibility in lesion segmentation in test set of data set 1 (median Dice score [DS]: 0.90 [IQR, 0.84-0.93]; P = .01) and data set 2 (median DS: 0.89 [IQR, 0.80-0.92]; P = .001). The best model, trained with 23 mixed features from tumor bounding boxes, achieved an AUC of 0.90 (95% CI: 0.83, 0.97), sensitivity of 81% (46 of 57; 95% CI: 70, 91), and specificity of 87% (39 of 45; 95% CI: 77, 87). No evidence of difference was found between model and human readers (AUC = 0.90 [95% CI: 0.83, 0.97] vs 0.83 [95% CI: 0.76, 0.90]; P = .55 and 0.90 vs 0.82 [95% CI: 0.75, 0.90]; P = .45) in tumor classification or between model and histopathologically or follow-up-confirmed diagnosis (AUC = 0.90 [95% CI: 0.83, 0.97] vs 1.00 [95% CI: 1.00,1.00]; P = .10). Conclusion Precise real-time US-based breast tumor categorization was developed by mixing classic radiomics and autoencoder-based features from tumor bounding boxes. ClinicalTrials.gov identifier: NCT04976257 Published under a CC BY 4.0 license. Supplemental material is available for this article. See also the editorial by Bahl in this issue.

Objective: Preeclampsia and intrauterine growth restriction (IUGR) are related conditions. We aimed to characterise common lipid changes., Methods: Triglyceride and cholesterol levels of patients 24-42 weeks of gestation with IUGR (n = 52), hypertensive IUGR (HIUGR, n = 28), and preeclampsia without IUGR (PE, n = 56) were compared to a control group (CTRL, n = 167). In addition, 60 sera (n = 10 of each pathology IUGR, HIUGR, PE (without IUGR) compared to n = 30 matched CTRL) of severe early onset cases <34 weeks of gestation were chosen and further analysed by ultracentrifugation lipid subfractionation including VLDL, IDL, LDL, and HDL composition., Results: In the full cohort we found low cholesterol in IUGR (p = 0.0405), while triglyceride levels were high in PE (p < 0.0001). Lipid concentrations in HIUGR did not differ significantly from CTRL. In the 60 patients analysed by lipid subfractionation, triglyceride levels were increased in the VLDL subfraction in PE (p < 0.01), however, LDL-bound ApoB and cholesterol levels were lower in IUGR and HIUGR (p < 0.0001 for total cholesterol and p < 0.001 for ApoB in both groups), but not in PE when compared to CTRL., Conclusion: Low cholesterol, especially LDL cholesterol levels are a feature of IUGR while high triglyceride levels are a feature of preeclampsia. Increased VLDL-triglycerides suggest a disturbed conversion to LDL in preeclampsia. Of note, the presence of IUGR in hypertensive disorders further alters lipid profiles, which may explain heterogeneous data on lipid values for preeclampsia in the literature. Study groups have to be selected carefully to avoid misinterpretation., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The oxysterol 27-hydroxycholesterol (27-OHC) plays an important role in the regulation of cholesterol homeostasis. Pregnancy pathologies like preeclampsia (PE), HELLP-syndrome (HELLP), intrauterine growth restriction (IUGR) and intrahepatic cholestasis in pregnancy (ICP) are linked to disturbances in lipid metabolism. In the present study, we hypothesized a specific gestational regulation of 27-OHC and compromised 27-OHC levels due to placental and hepatic diseases in pregnancy resulting in a dysregulation of lipid metabolism., Methods: The 27-OHC was measured by gas-chromatography-mass spectrometry (GC-MS) and related to cholesterol concentrations. In the longitudinal cohort, a complete set of samples of healthy patients (n = 33) obtained at three different time points throughout gestation and once post-partum was analyzed. In the cross sectional cohort, patients with pregnancy pathologies (IUGR n = 14, PE n = 14, HELLP n = 7, ICP n = 7) were matched to a control group (CTRL) of equal gestational ages., Results: The 27-OHC levels already increased in the first trimester despite lower TC concentrations (p < 0.05). During the course of pregnancy, a subtle rise in 27-OHC concentrations results in an overall decrease of 27-OHC/TC ratio in between the first (p < 0.05) and second trimester. The ratio remains stable thereafter including the post-partum period. No significant differences have been observed in pregnancy pathologies as compared to the CTRL group., Conclusion: In conclusion, 27-OHC may have a compensatory role in cholesterol metabolism early in pregnancy. The conserved 27-OHC/TC ratio in pregnancy pathologies suggest that neither the placenta nor the liver is majorly involved in the regulation of 27-OHC metabolism.

The prognosis of lymphoid neoplasms has improved considerably during the last decades. However, treatment response for some lymphoid neoplasms is still poor, indicating the need for new therapeutic approaches. One promising new strategy is the inhibition of kinases regulating key signal transduction pathways, which are of central importance in tumorigenesis. Kinases of the CK1 family may represent an attractive drug target since CK1 expression and/or activity are associated with the pathogenesis of malignant diseases. Over the last years efforts were taken to develop highly potent and selective CK1-specific inhibitor compounds and their therapeutic potential has now to be proved in pre-clinical trials. Therefore, we analyzed expression and mutational status of CK1δ in several cell lines representing established lymphoma entities, and also measured the mRNA expression level in primary lymphoma tissue as well as in non-neoplastic blood cells. For a selection of lymphoma cell lines we furthermore determined CK1δ kinase activity and demonstrated therapeutic potential of CK1-specific inhibitors as a putative therapeutic option in the treatment of lymphoid neoplasms.

Purpose: To provide quantitative information on glucose utilization in cone-dominant ground squirrel retinas., Methods: Ground squirrel eyecups were incubated in medium containing (14)C-glucose, and the production of (14)CO(2) was measured. Measurements were also made of lactic acid production (glycolysis). Nuclear magnetic resonance (NMR) was used to track metabolites generated from (13)C-1 glucose., Results: Ground squirrel eyecups produced lactate at a high rate and exhibited normal histology. Light-adaptation reduced glycolysis by 20%. Ouabain decreased glycolysis by 25% and decreased (14)CO(2) production by 60%. Blockade of glutamate receptors had little effect on the glycolysis and (14)CO(2) produced. When metabolic responses were restricted to photoreceptors, light caused a 33% decrease in (14)CO(2) production. The rate of (14)CO(2) production was less than 10% of lactate production. Lactate was the major product formed from (13)C-glucose. Other (13)C-labeled compounds included glutamate, aspartate, glutamine, alanine, taurine, and GABA. Lactate was the only product detected in the medium bathing the ground squirrel retinas. The rod-dominant rat retina exhibited a similar pattern of metabolites formed from glucose., Conclusions: Lactate, not CO(2), is the major product of glucose metabolism in both ground squirrel and rat retinas. Active Na(+) transport, however, depends more on ATP produced by mitochondria than by glycolysis. A relatively high fraction of ATP production from glycolysis and glucose oxidation continues in the absence of active Na(+) pumping and glutamatergic transmission. Major neurotransmitters are synthesized from the aerobic metabolism of glucose; anoxia-induced impairment in retinal synaptic transmission may be due to depletion of neurotransmitters.

It is undisputed that glutathione (GSH) is an important cellular antioxidant. Although it is commonly believed that GSH is present in all retinal cells, several publications show that GSH is not immunologically detectable in outer segments of rod and cone photoreceptor cells, but is present in inner retinal cells and the pigment epithelium. Using these intriguing and surprising findings as a starting point, an hypothesis is proposed that the renewal of outer segments serves as a surrogate antioxidant for GSH and that the exceptional vulnerability of photoreceptor cells to certain toxic chemicals is linked to the deficiency in GSH in outer segments as a reductant, a detoxicant, and as an enzymatic cofactor. It is suggested that this deficiency of GSH is not damaging to outer segments under normal conditions, because renewal serves to replace any damaged molecules before they increase to detrimental levels. However, when photoreceptors are stressed, the renewal of outer segments alone is not capable of overcoming the higher rates of oxidizing and detrimental chemical reactions, and the health of the entire photoreceptor cell is at risk. The hypothesis is supported by a consideration of the essential role of the NADPH-dependent retinol reductase, by the different localization within photoreceptor cells of two key metabolic enzymes that are sensitive to oxidation, glyceraldehyde-3-phosphate dehydrogenase and the sodium-potassium ATPase, and by a consideration of the effects of toxic chemicals that selectively damage photoreceptor cells.

Recent reports have linked neuronal cell death by necrosis to poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation. It is believed that under stress, the activity of this enzyme is up-regulated, resulting in extensive poly(ADP-ribosyl)ation of nuclear proteins, using NAD(+) as its substrate, which, in turn, leads to the depletion of NAD(+). In efforts to restore the level of NAD(+), depletion of ATP occurs, resulting in the shutdown of ATP-dependent ionic pumps. This results in cell swelling and eventual loss of membrane selectivity, hallmarks of necrosis. Reports from in vitro and in vivo studies in the brain have shown that NMDA receptor activation stimulates PARP activity and that blockade of the enzyme provides substantial neuroprotection. The present study was undertaken to determine whether PARP activity is regulated by NMDA in the rat retina, and whether blockade of PARP activity provides protection against toxic effects of NMDA. Rat retinas exposed to intravitreal injections containing NMDA, with or without the PARP inhibitor N-(6-oxo-5, 6-dihydrophenanthridin-2-yl)-(N,-dimethylamino) acetamide hydrochloride (PJ-34), were assessed for changes in PARP-1 activity as evidenced by poly(ADP-ribosyl)ation (PAR), loss of membrane integrity, morphological indicators of apoptosis and necrosis, and ganglion cell loss. Results showed that: NMDA increased PAR formation in a concentration-dependent manner and caused a decline in retinal ATP levels; PJ-34 blockade attenuated the NMDA-induced formation of PAR and decline in ATP; NMDA induced the loss of membrane selectivity to ethidium bromide (EtBr) in inner retinal neurons, but loss of membrane selectivity was not prevented by blocking PARP activity; cells stained with EtBr, or reacted for TUNEL-labeling, displayed features characteristic of both apoptosis and necrosis. In the presence of PJ-34, greater numbers of cells exhibited apoptotic features; PJ-34 provided partial neuroprotection against NMDA-induced ganglion cell loss. These findings suggest that although blockade of PARP activity fully attenuates NMDA-induced PAR formation and loss of retinal ATP content, and improves the survival of select populations of ganglion cells, this approach does not provide full neuroprotection. In contrast, blockade of PARP activity promotes apoptotic-like cell death in the majority of cells undergoing cell death. Furthermore, these studies show that the loss of membrane selectivity is not dependent upon PAR formation or the resulting decline of ATP, and suggests that an alternative pathway, other than PARP activation, exists to mediate this event.

Purpose: To test the hypothesis that diabetes alters retinal NAD+-to-NADH ratios early in the course of the disease (e.g., the hyperglycemic pseudohypoxia hypothesis)., Methods: In freshly excised age-matched control and diabetic rat retinas, measurements were made of the NAD+ and NADH content as well as a surrogate marker of NAD+-to-NADH ratios obtained from lactate and pyruvate levels. In addition, the effect of various hyperglycemic levels was assessed from measurements of retinal lactate and pyruvate concentrations and the rate of lactic acid production in vitro (isolated rat retinas, monolayer cultures of human retinal pigment epithelial cells, and rabbit lens epithelial cells)., Results: No significant differences (P>0.05) were found between control and diabetic tissues in their amount of total NAD+ and NADH/retina, and the ratio of NAD+ to NADH, or in their content of lactate, pyruvate, and adenosine triphosphate (ATP) or in the ratio of lactate to pyruvate. The content of lactate and pyruvate in retinas incubated for 2 hours in media containing 10 or 30 mM glucose was the same as found in fresh tissues, but the levels of these metabolites in retinas incubated in media containing 5 mM glucose declined in comparison to the fresh values. There were no significant differences in lactate content in cultured retinal and lens cells that were exposed to 5 or 30 mM glucose-containing media., Discussion: The present results do not support the hyperglycemic pseudohypoxia hypothesis of diabetic retinopathy.

Glucose has long been considered the substrate for energy metabolism in the retina. Recently, an alternative hypothesis (metabolic coupling) suggested that mitochondria in retinal neurons utilize preferentially the lactate produced specifically by Müller cells, the principal glial cell in the retina. These two views of retinal metabolism were examined using confluent cultures of photoreceptor cells, Müller cells, ganglion cells, and retinal pigment epithelial cells incubated in modified Dulbecco's minimal essential medium containing glucose or glucose and lactate. The photoreceptor and ganglion cells represented neural elements, and the Müller and pigment epithelial cells represented non-neural cells. The purpose of the present experiments was two-fold: (1) to determine whether lactate is a metabolic product or substrate in retinal cells, and (2) to examine the evidence that supports the two views of retinal energy metabolism. Measurements were made of lactic acid production, cellular ATP levels, and cellular morphology over 4 h. Results showed that all cell types incubated with 5 mM glucose produced lactate aerobically and anaerobically at linear rates, the anaerobic rate being 2-3-fold higher (Pasteur effect). Cells incubated with both 5 mM glucose and 10 mM lactate produced lactate aerobically and anaerobically at rates similar to those found when cells were incubated with glucose alone. Anaerobic ATP content in the cells was maintained at greater than 50% of the control, aerobic value, and cellular morphology was well preserved under all conditions. The results show that the cultured retinal cells produce lactate, even in the presence of a high starting ambient concentration of lactate. Thus, the net direction of the lactic dehydrogenase reaction is toward lactate formation rather than lactate utilization. It is concluded that retinal cells use glucose, and not glial derived lactate, as their major substrate.

We have investigated the dependence of the rate of lactic acid production on the rate of Na(+) entry in cultured transformed rat Müller cells and in normal and dystrophic (RCS) rat retinas that lack photoreceptors. To modulate the rate of Na(+) entry, two approaches were employed: (i) the addition of L-glutamate (D-aspartate) to stimulate coupled uptake of Na(+) and the amino acid; and (ii) the addition of monensin to enhance Na(+) exchange. Müller cells produced lactate aerobically and anaerobically at high rates. Incubation of the cells for 2-4 h with 0.1-1 mM L-glutamate or D-aspartate did not alter the rate of production of lactate. ATP content in the cells at the end of the incubation period was unchanged by addition of L-glutamate or D-aspartate to the incubation media. Na(+)-dependent L-glutamate uptake was observed in the Müller cells, but the rate of uptake was very low relative to the rate of lactic acid production. Ouabain (1 mM) decreased the rate of lactic acid production by 30-35% in Müller cells, indicating that energy demand is enhanced by the activity of the Na(+)-K(+) pump or depressed by its inhibition. Incubation of Müller cells with 0.01 mM monensin, a Na(+) ionophore, caused a twofold increase in aerobic lactic acid production, but monensin did not alter the rate of anaerobic lactic acid production. Aerobic ATP content in cells incubated with monensin was not different from that found in control cells, but anaerobic ATP content decreased by 40%. These results show that Na(+)-dependent L-glutamate/D-aspartate uptake by cultured retinal Müller cells causes negligible changes in lactic acid production, apparently because the rates of uptake are low relative to the basal rates of lactic acid production. In contrast, the marked stimulation of aerobic lactic acid production caused by monensin opening Na(+) channels shows that glycolysis is an effective source of ATP production for the Na(+)-K(+) ATPase. A previous report suggests that coupled Na(+)-L-glutamate transport stimulates glycolysis in freshly dissociated salamander Müller cells by activation of glutamine synthetase. The Müller cell line used in this study does not express glutamine synthetase; consequently these cells could only be used to examine the linkage between Na(+) entry and the Na(+) pump. As normal and RCS retinas express glutamine synthetase, the role of this enzyme was examined by coapplication of L-glutamate and NH(4) (+) in the presence and absence of methionine sulfoximine, an inhibitor of glutamine synthetase. In normal retinas, neither the addition of L-glutamate alone or together with NH(4) (+) caused a significant change in the glycolytic rate, an effect linked to the low rate of uptake of this amino acid relative to the basal rate of retinal glycolysis. However, incubation of the RCS retinas in media containing L-glutamate and NH(4)(+) did produce a small (15%) increase in the rate of glycolysis above the rate found with L-glutamate alone and controls. It is unlikely that this increase was the result of conversion of L-glutamate to L-glutamine, as it was not suppressed by inhibition of glutamine synthetase with 5 mm methionine sulfoximine. It appears that the magnitude of Müller cell glycolysis required to sustain the coupled transport of Na(+) and L-glutamate and synthesis of L-glutamine is small relative to the basal glycolytic activity in a rat retina.

It has long been known that mammalian retinas metabolize glucose aerobically to lactic acid and carbon dioxide. The classical view holds that glucose is the primary substrate for energy metabolism in all retinal cells, and that photoreceptor cells have the highest rates of glycolysis and respiration. A different and more recent view is that the Müller cells are the principal, if not sole aerobic producers of lactate, which then serves as the primary fuel for the mitochondria in photoreceptor cells and other retinal neurons. In this paper, we have examined these two competing hypotheses in rat and guinea pig retinas by identifying the cellular sites of glucose uptake and phosphorylation via hexokinase by means of autoradiographic studies with 3H-2-deoxyglucose (3H-2DG). The rat retina serves as a vascular model and the guinea pig retina serves as an avascular model. Rat and guinea pig eyecups were incubated in oxygenated, bicarbonate-buffered media containing glucose in the presence of labeled and unlabeled 2DG. Biochemical measurements of lactate production and ATP content were made on rat retinas incubated with different concentrations of glucose and 2DG in order to establish the optimal condition for conducting the autoradiographic studies with 3H-2DG. The optimal substrate concentrations were 1mM glucose and 0.25 mM 2DG. Results showed that following incubation of dark-adapted rat eyecups for 1 hr in media containing 1mM glucose/0.25 mM 2DG and supplemented with 3H-2DG, the label was distributed throughout all the layers of the retina, from the ganglion cell layer to the retinal pigment epithelium, with denser label associated with the outer retina (photoreceptors) relative to the density of label in the inner retina, as evaluated by counts of silver grains in individual retinal layers. Exposure of rat eyecups to light did not alter the relative distribution of label, but did increase total grain counts by 70%. However, uptake of labeled 2DG, as measured by scintillation counting of radioactivity in trichloroacetic acid extracts, was not significantly different between light- and dark-adapted rat retinas. In guinea pig eyecups, labeled 2DG was distributed throughout all the retinal layers. Addition of 10mM lactate or pyruvate to the glucose/2DG media produced no measurable change in the density or distribution of label in the eyecups. Measurements of the activity of hexokinase in rat retinas revealed that this enzyme was present in both the mitochondrial and cytosolic fractions. The present results suggest that as long as the availability of ambient glucose is adequate, retinal neurons use glucose, rather than glial-derived lactate, as the major substrate for the production of high energy phosphates.

The purpose of the present experiments was to enhance understanding of the factors that are critical for the survival of retinal cells exposed to mitochondrial inhibition. Confluent cultures of Müller cells (rMC-1) and human retinal pigment epithelial cells (hRPE) were incubated in Dulbecco's minimal essential medium in the presence and absence of 1x10(-5)M Antimycin A, an inhibitor of mitochondrial electron transport. To modulate the rates of aerobic and anaerobic glycolysis, cells were incubated in media containing varying concentrations of glucose and 1-100 micro M of iodoacetic acid (IAA), an inhibitor of glyceraldehdye-3-phosphate dehydrogenase (G3PDH). Measurements were made of G3PDH, lactic acid production, and cellular ATP levels, along with an examination of cellular morphology, the latter providing an index of cellular viability. Control rMC-1 and hRPE produced lactate aerobically, respectively, at 0.48 and 1.50 micro molhr(-1)/10(6) cells. Anaerobically, lactate production increased 2-fold in rMC-1 and 3-fold in hRPE. Anaerobic ATP levels in both types of cells were maintained at control levels over 8hr. Experimental conditions were sought that would modulate only the capacity of rMC-1 and hRPE to increase glycolysis following mitochondrial inhibition, i.e. alter their Pasteur effect. We used low concentrations of IAA to partially inhibit G3PDH. Incubation of rMC-1 with IAA for 6hr caused a graded inhibition of G3PDH: 70% inhibition with 1 micro M, 90% with 5 micro M, 97% with 10 micro M, and 100% with 100 micro M. While the aerobic and anaerobic rates of lactic acid production were not altered by 1 micro M IAA, both were suppressed completely by 100 micro M IAA. However, incubation of rMC-1 with 5 micro M IAA caused a decrease of 30% in the rate of anaerobic lactic acid production but no change in the rate of aerobic glycolysis. Moreover, with 5 micro M IAA, rMC-1 incubated aerobically maintained ATP levels, but anaerobic ATP content decreased to a low level and cell morphology and viability were compromised. Essentially similar results were observed with hRPE. Both rMC-1 and hRPE are remarkably resistant to mitochondrial inhibition. This resistance is linked directly to the magnitude of the increase in the Pasteur effect. When the capacity of rMC-1 and hRPE to generate a Pasteur effect is selectively curtailed, these cells no longer are resistant to mitochondrial inhibition. It is suggested that in an intact tissue the ability of a cell to withstand a metabolic challenge will depend very much on the adequacy of the supply of glucose. Even a small limitation in the availability of this utilizable substrate and in the rate of the compensatory increase in the rate of anaerobic glycolysis could put the cell at greater risk during the challenge.

Purpose: To measure selected parameters of energy metabolism and adenosine triphosphate (ATP) production in passaged monolayer cultures of human retinal glial (Müller) cells to assess the effects of varying substrate and oxygen availability on the biochemistry and histologic integrity of these cells., Methods: Confluent Müller cell cultures were incubated for up to 4 hours at 37 degrees C in a modified minimal essential medium (no serum) under aerobic or mitochondrial-inhibited conditions in the presence and absence of 5 mM glucose or in the presence of lactate, pyruvate, glutamate, or glutamine. Cellular ATP levels, lactic acid production, and (14)CO(2) production from labeled glucose or glutamate were measured along with an examination of cellular morphology. Immunohistochemistry with antibodies to glial cell-specific proteins was also performed. Cells were positive for vimentin, but negative for glial fibrillary acidic protein and glutamine synthetase., Results: Human Müller cells maintained ATP content aerobically at the same level for 4 hours in the presence and absence of glucose. ATP content was also maintained anaerobically at a value equal to that found aerobically, but only in the presence of glucose. ATP content in human Müller cells declined to a very low level when glycolysis was blocked by iodoacetate, and inclusion of lactate, pyruvate, glutamate, or glutamine did not restore the level of ATP. Aerobically, lactic acid production accounted for 99% of the total glucose used, whereas the oxidation of glucose by the mitochondria accounted for only 1%. When mitochondria were inhibited with antimycin A, there was only a modest (1.3-fold) increase in the rate of lactic acid production. No significant differences were found in the histologic appearance of the cells after mitochondrial blockade, but there was massive death of cells after inhibition of glycolysis with iodoacetate., Conclusions: These results suggest that, in the presence of glucose and oxygen, cultured Müller cells obtain their ATP principally from glycolysis and have a low rate of oxygen consumption. This metabolic pattern may spare oxygen for retinal neurons, particularly in the inner nuclear and ganglion cell layers under normal physiological conditions. Furthermore, retinal Müller cells in culture are resistant to anoxia or absence of glucose, which provides a basis for understanding why Müller cells are less susceptible than neurons to ischemia or hypoglycemia.

This article provides current information on the potential role of oxidation in relation to age-related macular degeneration (AMD). The emphasis is placed on the generation of oxidants and free radicals and the protective effects of antioxidants in the outer retina, with specific emphasis on the photoreceptor cells, the retinal pigment epithelium and the choriocapillaris. The starting points include a discussion and a definition of what radicals are, their endogenous sources, how they react, and what damage they may cause. The photoreceptor/pigment epithelium complex is exposed to sunlight, is bathed in a near-arterial level of oxygen, and membranes in this complex contain high concentrations of polyunsaturated fatty acids, all considered to be potential factors leading to oxidative damage. Actions of antioxidants such as glutathione, vitamin C, superoxide dismutase, catalase, vitamin E and the carotenoids are discussed in terms of their mechanisms of preventing oxidative damage. The phototoxicity of lipofuscin, a group of complex autofluorescent lipid/protein aggregates that accumulate in the retinal pigment epithelium, is described and evidence is presented suggesting that intracellular lipofuscin is toxic to these cells, thus supporting a role for lipofuscin in aging and AMD. The theory that AMD is primarily due to a photosensitizing injury to the choriocapillaris is evaluated. Results are presented showing that when protoporphyric mice are exposed to blue light there is an induction in the synthesis of Type IV collagen synthesis by the choriocapillary endothelium, which leads to a thickened Bruch's membrane and to the appearance of sub-retinal pigment epithelial fibrillogranular deposits, which are similar to basal laminar deposits. The hypothesis that AMD may result from oxidative injury to the retinal pigment epithelium is further evaluated in experiments designed to test the protective effects of glutathione in preventing damage to cultured human pigment epithelial cells exposed to an oxidant. Experiments designed to increase the concentration of glutathione in pigment epithelial cells using dimethylfumarate, a monofunctional inducer, are described in relation to the ability of these cells to survive an oxidative challenge. While all these models provide undisputed evidence of oxidative damage to the retinal pigment epithelium and the choriocapillaris that is both light- and oxygen-dependent, it nevertheless is still unclear at this time what the precise linkage is between oxidation-induced events and the onset and progression of AMD.

The purpose of the present experiments was to evaluate the contribution of the glutamate-glutamine cycle in retinal glial (Müller) cells to photoreceptor cell synaptic transmission. Dark-adapted isolated rat retinas were superfused with oxygenated bicarbonate-buffered media. Recordings were made of the b-wave of the electroretinogram as a measure of light-induced photoreceptor to ON-bipolar neuron transmission. L-methionine sulfoximine (1-10 mM) was added to superfusion media to inhibit glutamine synthetase, a Müller cell specific enzyme, by more than 99% within 5-10 min, thereby disrupting the conversion of glutamate to glutamine in the Müller cells. Threo-hydroxyaspartic acid and D-aspartate were used to block glutamate transporters. The amplitude of the b-wave was well maintained for 1-2 h provided 0.25 mM glutamate or 0.25 mM glutamine was included in the media. Without exogenous glutamate or glutamine the amplitude of the b-wave declined by about 70% within 1 h. Inhibition of glutamate transporters led to a rapid (2-5 min) reversible loss of the b-wave in the presence and absence of the amino acids. In contrast, inhibition of glutamine synthetase did not alter significantly either the amplitude of the b-wave in the presence of glutamate or glutamine or the rate of decline of the b-wave found in the absence of these amino acids. Excellent recovery of the b-wave was found when 0.25 mM glutamate was resupplied to L-methionine sulfoximine-treated retinas. The results suggest that in the isolated rat retina uptake of released glutamate into photoreceptors plays a more important role in transmitter recycling than does uptake of glutamate into Müller cells and its subsequent conversion to glutamine.

Purpose: To determine which processes or factors that regulate corneal hydration are responsible for the hydration-modulating effects of adenosine. Influx of fluid to the stroma and efflux to the aqueous humor are governed, respectively, by the imbibition pressure of the stromal matrix and the transendothelial ionic gradients determined by the permeability and active transport characteristics of this monolayer. The focus of this study was to assess the effects of adenosine on these endothelial parameters., Methods: Isolated corneas freshly dissected from rabbit eyes were used throughout. Active ion transport was assessed by measurement of 86Rb+ uptake by the endothelial cells of intact corneas incubated for 30 minutes in 25 mM HCO3(-)-Ringer with agents promoting corneal deturgescence or corneal swelling. Intracellular and extracellular fluid in the scraped endothelial cell mass was estimated from simultaneous counts of 3H-mannitol and 14C-urea, allowing calculation of tissue-to-medium (T-M) ratios of 86Rb+ in cell water. Permeability of the endothelium was determined by measuring the efflux into the superfusate of 5-carboxyfluorescein (CF) applied to the stroma of deepithelialized corneas superfused at the endothelial surface with the same media described for 86Rb+ uptake. Thickness of these corneas and of others fixed for scanning electron microscopy was monitored with a specular microscope., Results: In the control medium, 25 mM HCO3(-)-Ringer, 86Rb+ was accumulated to yield a T-M ratio of 6.21. Neither adenosine nor other agents that increase cyclic adenosine monophosphate (cAMP)--that is, forskolin and dibutyryl cAMP--changed this value to a significant extent. Bumetanide had no effect, but ouabain caused a decrease in T-M to 1.30, a 79% inhibition. Elimination of Na+ or HCO3- also caused marked decreases in uptake. Permeability to CF in control medium was 3.40 x 10(-4) cm/min. A decrease of more than 20% (P < 0.05) was seen in the presence of adenosine and cAMP promoters and also with the protein kinase inhibitor H-8, whereas phorbol myristate acetate caused an increase to 4.50 x 10(-4) cm/min (P < 0.01). Ouabain caused no change, but blocked the effects of adenosine. Reducing the Ca2+ concentration of the superfusing medium caused time-dependent increases in permeability to 4.57 at 15 to 45 minutes and 12.5 at 80 to 110 minutes. At the earlier time, this increase in permeability could be prevented by the addition of adenosine or H-8. Elimination of Na+ or HCO3- ions from the medium caused a small decrease in permeability and, like ouabain, blocked the effect of adenosine. Changes in thickness of corneas were consistent, in most cases, with the observed alterations in 86Rb+ uptake or permeability to CF. Scanning electron microscopy showed contraction and rounding of endothelial cells in low Ca2+ medium, with stretching of intercellular borders, features that were largely eliminated when adenosine was also present., Conclusions: Adenosine, through increasing cAMP, decreases permeability of the corneal endothelium. This effect, rather than a change in the active transport (fluid pump) mechanism, is responsible for the promotion of deturgescence and maintenance of lower steady state thickness of corneas exposed to adenosine. The mechanism may involve the phosphorylation state of cytoskeletal proteins and seems to be dependent on an undisturbed environment of monovalent ions.

The role of Na(+)-K(+)-2Cl- cotransport in ion and fluid transport of the corneal endothelium was examined by measuring changes in corneal hydration and uptake of 86Rb by the endothelial cell layer. Isolated, intact rabbit corneas maintain normal hydration when they are superfused at the endothelial surface with bicarbonate (HCO3-)-Ringer solutions as a result of equilibrium between active ion and fluid transport out of the stromal tissue and leak of fluid into stromal tissue from the aqueous humor. Furosemide and bumetanide did not alter this equilibrium when they were added to the superfusion medium. Uptake of 86Rb by the endothelium of the incubated cornea was increased 25% by bumetanide, but uptake in the presence of ouabain (70% less than that of controls) was not changed by bumetanide. In Na(+)-free medium, uptake of 86Rb was reduced by 58%, but it was unchanged in Cl(-)-free medium. Calyculin A, a protein phosphatase inhibitor and activator of Na(+)-K(+)-Cl- cotransport, was without effect on 86Rb uptake. Hypertonicity (345 mosmol/kg) increased uptake slightly, whereas hypotonicity (226 mosmol/kg) caused a 33% decrease. Neither of these changes was significantly different when bumetanide was present in the media. It is concluded that Na(+)-K(+)-2Cl- cotransporter activity is not exhibited by the in situ corneal endothelium and does not play a role in the ion and fluid transport of this cell layer. Its presence in cultured endothelial cells may reflect the reported importance of this protein in growth, proliferation, and differentiation.

Purpose: To report results of functional, biochemical and structural studies of photoreceptor mitochondria in isolated rat retinas under conditions of mitochondrial inhibition., Methods: Dark-adapted rat retinas were incubated in a modified Ringer's bicarbonate medium under aerobic and anaerobic conditions. Several different procedures were used to inhibit mitochondrial function; N2, 0.01 mM antimycin A, and 1 and 10 mM potassium cyanide (KCN). Measurements were made of lactic acid production, retinal adenosine triphosphate (ATP) content, and receptor potentials. Morphology of the inner segment mitochondria was examined by electron microscopy., Results: In the presence of N2, 0.01 mM antimycin, or 1 mM KCN, lactic acid production was linear throughout the 60- minute period; and the rate was similar for each condition. Retinal ATP content and the amplitude of the receptor potential were also maintained at high levels after short-term incubations with either N2, antimycin A, or 1 mM KCN. In contrast, use of 10 mM KCN produced an entirely different set of results. These effects were studied both at the alkaline pH (8.9) found when this concentration of KCN was simply added to bicarbonate-buffered media and at the normal pH (after readjustment) of 7.4. With 10 mM KCN (pH 8.9), retinal lactate production was severely depressed, retinal ATP content was nearly depleted within 5 to 10 minutes, and the amplitude of the receptor potential rapidly declined to a low level. The deleterious effects of 10 mM KCN on these parameters were lessened to varying degrees when pH was readjusted to 7.4. Electron microscopic observations of rat rod inner segments indicated generally excellent survival of these organelles after incubation with either N2, antimycin A, or 1 mM KCN in comparison with their appearance under oxygenated conditions. However, the inner segments were significantly disrupted after incubation of retinas with 10 mM KCN., Conclusions: Findings suggest that the loss of the receptor potential and depletion of ATP observed with minutes after exposing isolated rat retinas to media containing 10 mM KCN results from the inhibition of both respiration and glycolysis by this high concentration of KCN. In contrast, when conditions are chosen so that only respiration is impaired (as with N2, antimycin A, or 1 mM KCN) photoreceptor cells are resistant to short-term episodes of mitochondrial inhibition, principally because the upregulation of glycolysis generates sufficient ATP to compensate reasonably well for the loss in mitochondrially produced ATP.

The metabolic competence and histological integrity of the frog retina in vitro were evaluated as a function of the presence/absence of exogenous glucose and of oxygen tension. Dark- and light-adapted frog neural retinas were incubated for 1-8 hr at 23 degrees C in a modified Ringer's-bicarbonate medium under aerobic and anaerobic conditions, in the presence and absence of 10 mM glucose. Control retinas (+glucose, aerobic conditions) maintained ATP levels comparable to those of freshly excised tissue (ave. 17 nmol mg protein-1), produced minimal lactate (ave. 0.12 mumol mg protein-1 hr-1), and exhibited normal histology. In the absence of any exogenous carbon source, retinas incubated aerobically maintained ATP levels, produced lactate, incorporated [3H]acetate into nonsaponifiable lipids, and exhibited histology comparable to controls. In the presence of 1 mM iodoacetate, aerobic ATP levels declined markedly, with or without exogenous glucose. Under anaerobic conditions with glucose present, lactate production increased ca. 8.5-fold, while ATP levels were maintained at control levels, demonstrating a marked Pasteur effect; under these conditions, retinas exhibited only moderate histopathological changes. However, in the absence of both glucose and oxygen, ATP levels declined precipitously, with concomitant massive cytological deterioration. No major differences in the biochemical measurements or histological appearance were observed as a function of light adaptation. These results demonstrate the remarkable resilience of the frog retina to anoxia and hypoglycemic stress. Aerobically, with or without exogenous glucose, ATP production and de novo lipid synthesis are maintained, apparently by recruitment of an endogenous carbohydrate substrate (e.g., glycogen).

Purpose: To measure glucose-dependent metabolic activities and selected parameters of the polyol pathway in retinas isolated from normal rats to test the hypothesis recently proposed by Van den Enden et al that incubation of whole retinas for 2 hours with elevated concentrations of glucose results in activation of the polyol pathway, which is the cause of a redox imbalance, as measured by an increase in the retinal cytosolic lactate-pyruvate ratio and a diabetic-like state., Methods: Retinas obtained from nondiabetic rats and separated from other ocular tissues were incubated for several hours in incubation medium containing glucose at concentrations ranging from 5 to 30 mM. Measurements were made under aerobic and anaerobic conditions of lactic acid production, retinal adenosine triphosphate (ATP), lactic acid content, the hexose monophosphate shunt pathway, aldose reductase activity, and levels of sorbitol and galactitol. Morphology was examined by light microscopy at the end of the incubations., Results: Incubation of isolated rat retinas with 20 mM glucose increased lactic acid production by approximately 25% in comparison to the rate observed in 5mM glucose under aerobic and anaerobic conditions. The content of ATP and lactate in the retinas after a 2-hour incubation in the presence of oxygen and 20 mM glucose was equal to the amounts found in fresh tissues, whereas these metabolites declined, respectively, by 25% and 45% when 5 mM glucose was used. The activity of the hexose monophosphate shunt pathway in isolated rat retinas was not increased increased significantly when the concentration of glucose was raised from 5 to 30 mM. Aldose reductase activity and polyols were below our limits of detection, 0.5 nmol/minute.mg protein and 3.5 nmol/retina, respectively, under all conditions tested. The morphologic appearance of the retina was similar in the presence of normal and high concentrations of glucose., Conclusions: These results show that incubation of isolated rat retinas, obtained from nondiabetic rats, with elevated concentrations of glucose for 2 hours leads to increases in glycolysis and a higher tissue content of lactic acid and ATP in comparison to values obtained with 5 mM glucose. However, the magnitude of the glucose-dependent increase in the retinal level of lactate in the current study and in that of Van den Enden et al is six to seven times greater than the calculated flux of glucose through the polyol pathway. These results, therefore, do not support the hypothesis of Van den Enden et al. Rather, it is suggested that supranormal concentrations of glucose yield more lactate and ATP in a whole retina because they optimize the supply of this essential nutrient to cells throughout the tissue by overcoming diffusional limitations that result when the retina is separated from its normal choroidal and intraretinal blood supplies.

Purpose: To investigate the cellular mechanisms whereby adenosine increases net transendothelial fluid transport by the endothelial cells of the cornea., Methods: Rabbit corneas were isolated and the endothelial surface was superfused while thickness was measured with the specular microscope. Cyclic adenosine monophosphate (cAMP) was measured in endothelia from fresh and incubated corneas, and adenylyl cyclase and phosphodiesterase activities were measured in homogenates or the particulate fraction of endothelia from bovine or rabbit. Adenosine, adenosine-receptor agonists, dibutyryl cAMP, forskolin, and phosphodiesterase inhibitors were used to modulate physiological and biochemical parameters., Results: Adenosine, N-ethyl(carboxamido)adenosine, dibutyryl cAMP, forskolin, and phosphodiesterase inhibitors all promoted deturgescence of swollen corneas and maintained fresh corneas at lower steady state thicknesses than in controls. These effects were abolished in the presence of ouabain or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or after complete removal of HCO3- from the media. Intracellular cAMP was significantly increased by forskolin and phosphodiesterase inhibitors and, to a lesser extent, by agonists. Increases in cAMP concentration declined rapidly with time. Cyclase activity in the bovine tissue was enhanced by agonists and by G-protein activators. Dose-response curves of corneal swelling indicated a greater sensitivity to N-ethyl(carboxamido)adenosine than to the A2 alpha specific agonist CGS 21680., Conclusions: Adenosine increases net endothelial fluid transport through an increase in cAMP. The effects are mediated by stimulation of adenylyl cyclase through a G-protein coupled to an adenosine receptor, which is most probably of the A2 beta subtype. Results suggest that the regulation of corneal hydration by adenosine is more probably through stimulation of active transport than through a change in permeability, involving either transmembrane fluxes of Na+ or HCO3- or another step tightly coupled to these primary events in fluid movement.

Purpose: In view of the antioxidant role of ascorbic acid and the glutathione redox cycle in the lens, the authors have studied the relationship of the cycle to reduction of the oxidized product of ascorbic acid, dehydroascorbic acid (DHA), in lens epithelium., Methods: Cultured dog lens epithelial cells and intact rabbit lenses were exposed to various concentrations of DHA in experiments performed at 20 degrees C to minimize hydrolysis of the compound (t1/2 of 5 minutes at 37 degrees C). Levels of glutathione (GSH) and oxidized glutathione (GSSG) were measured in lens cells and whole lens epithelial by electrochemical detection., Results: Treatment of lens cells with 1 mM DHA for 0.5 to 3 hours in the absence of glucose (glucose is required for the reduction of GSSG through the glutathione redox cycle) produced from 60% to complete oxidation of GSH (controls contained negligible GSSG) and distinct morphologic changes (cell contraction and blebbing), as shown by scanning electron microscopy. Glucose prevented these effects and allowed nearly immediate recovery of GSH after DHA exposure in the absence of glucose. A dose-dependent response was observed for the formation of GSSG in cultured cells from 0.05 to 0.5 mM DHA in the absence of glucose. The results of experiments performed with DHA plus an inhibitor of glutathione reductase mimicked those obtained using DHA minus glucose. DHA produced a 3- to 10-fold stimulation of hexose monophosphate shunt activity in cultured lens cells and whole lenses, which was prevented by the inhibition of glutathione reductase. Treatment of whole lenses with DHA minus glucose also produced oxidation of epithelial GSH and was accompanied by the loss of lens transparency. No evidence was found for dehydroascorbate reductase activity in the lens epithelium., Conclusions: The exposure of lenses and lens epithelial cells to DHA under conditions in which the glutathione redox cycle was compromised resulted in the disappearance of GSH in the tissues and the appearance of GSSG. The reduction of DHA was shown to be linked to the glutathione redox cycle by a nonenzymatic interaction between GSH and DHA. Reduction of DHA in the lens is important because of the potential toxicity of this oxidant and/or its degradation products.

Purpose: To determine whether maintenance of corneal hydration is dependent on bicarbonate ions and whether these ions can be derived from metabolic or exogenous CO2, and to investigate the relationship of transendothelial fluid movement to control of hydration., Methods: The thickness of intact or deepithelialized rabbit corneas was measured while superfused on the endothelial surface with either 33 mM HGO3-/5% CO2 buffered media or 10 mM HPO4- buffered media in the presence and absence of inhibitors of ion transport and respiration. The corneal surface was covered with either silicone oil ("normal" corneas) or with the same media used for superfusion ("swollen" corneas). ATP and Na+,K(+)-ATPase activity were measured in endothelia scraped from the tissues after superfusion., Results: Intact and deepithelialized corneas covered with oil swelled at a negligible rate (4 to 8 microns/hour) in 33 mM HCO3- medium but at 45 to 60 microns/hour in HPO4- medium. Antimycin A altered neither of these swelling rates, but ethoxzolamide (0.1 mM) caused swelling in HCO3-/CO2 (approximately 12 microns/hour above controls) with no change of rate in HPO4-. Ouabain (0.1 mM) increased swelling to 45 to 50 microns/hour in HCO3-/CO2 but had no effect in HPO4-. Saturating the oil on deepithelialized corneas with 5% CO2, or putting HCO3-/CO2 medium on the epithelial surface of intact corneas, did not alter the swelling rates seen with HPO4- superfusion. The equilibrium thickness of deepithelialized corneas swollen with HCO3-/CO2 on both surfaces was 35 microns less than that of corneas swollen in HPO4-. The difference was abolished by ouabain, which caused corneas in HCO3-/CO2 to swell an additional 30 microns but did not alter the equilibrium thickness of corneas swollen in HPO4-. Ethoxzolamide and DIDS (0.2 mM) increased the thickness in HCO3-/CO2 but not in HPO4-. Na+,K(+)-ATPase activities of endothelia were similar after HCO3-/CO2 and HPO4- superfusions, but the concentration of ATP in the HPO4(-)-superfused tissues was increased 35%., Conclusions: Normal corneal thickness can be maintained in vitro only in media that contain HCO3- at concentrations of more than 20 mM. Neither metabolic CO2 nor CO2 present in air-equilibrated, nominally HCO3(-)-free media can supply this requirement for HCO3-, even though these sources support the presumably related processes of transendothelial fluid movement and intracellular pH regulation.

This article provides a comprehensive analysis of the redox reaction between glutathione/glutathione disulfide and ascorbic acid/dehydroascorbic acid. It includes an historical perspective of the progression of the experiments, first begun more than 60 years ago and continuing today with heightened importance. Indeed, the antioxidant capacity of glutathione and ascorbic acid, whether singly or in combination, linked via the redox couple, is a subject of intense interest for studies by bench scientists and clinicians, particularly because a growing body of evidence suggests that free radicals may be involved in a variety of diseases. The authors begin with a detailed summary of "test tube" experiments (the chemical perspective) that have revealed the conditions that regulate the rate of the redox coupling between glutathione and dehydroascorbic acid and that promote or inhibit the decomposition of dehydroascorbic acid in ordinary, buffered aqueous media; results obtained in the authors' laboratory are used for illustration purposes and uniformity of presentation. The authors then proceed to a critical examination of the extent to which the redox couple between glutathione and ascorbic acid operates in a cell, using the often published antioxidant cascade (See Fig. 1) as the model for the analysis (the physiological perspective). The evidence for and the evidence against the presence of the enzyme dehydroascorbate reductase in animal cells is outlined in a balanced way in an attempt to make sense of this continuing controversy. Next, the authors carefully document the many studies showing that exogenous dehydroascorbic acid is transported into cells where it is reduced to ascorbic acid by glutathione. Finally, they probe the functional significance and efficiency of the redox couple in monolayer cultures of human retinal pigment epithelial (RPE) cells, as a prototypical cellular model. The authors include the results of new experiments showing that incubation of RPE cells with a nitroxide, TEMPOL, leads to the selective oxidation of intracellular ascorbic acid. This approach is desirable because it dissects the cascade at a specific site and permits measurements of the levels of ascorbic acid and glutathione in the cells before, during, and after oxidation. The results show that only partial regeneration of ascorbic acid is obtained when control conditions are restored. However, if either ascorbic acid or dehydroascorbic acid is added to the media during the recovery period following treatment of cells with TEMPOL, then full recovery of ascorbic acid is observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Purpose: To examine the relationship between the activity of the sodium pump of the corneal endothelium and corneal thickness. It was postulated that because inhibition pressure of the stroma decreases as thickness increases, a partially inhibited sodium pump would result in a new steady-state thickness of the cornea when reduced rates of fluid influx and efflux were equal. Measurements of physiologic behavior and biochemical activity were to be made in the same tissue and thus establish the relationship directly., Methods: Rabbit corneas were superfused with a bicarbonate Ringer solution containing different concentrations of ouabain. Exposure to ouabain was either continuous for 4 hours or for an initial 10 minutes followed by ouabain-free superfusion. Thickness was measured, and, after superfusion, endothelium was removed from the corneas, sonicated, and assayed for Na(+)-K+ adenosine triphosphatase (ATPase) activity without further addition of ouabain to the assay medium. Thickness was also measured during superfusion with suboptimal concentrations of Na+ or HCO3- and with brefeldin A, an inhibitor of protein trafficking., Results: Continuous exposure to ouabain caused corneas to swell, but no new steady-state thickness was reached. At low concentrations, swelling rates increased with time, as did the extent of inhibition of the Na(+)-K+ ATPase. With only a 10-minute exposure to ouabain, swelling rates with 10(-4) M to 10(-5) M decreased with the duration of ouabain-free superfusion. Similar swelling curves were obtained by reductions in Na+ or HCO3- concentrations in the superfusion medium, indicating that partial inhibition of the endothelial fluid transport processes, whether via the Na(+)-K+ ATPase or by suboptimal ionic conditions, led toward a new equilibrium thickness of the cornea. However, when superfusion was continued for more than 4 hours, the corneas exposed for 10 minutes to 3 x 10(-5) M or lower-concentration ouabain showed increasing Na(+)-K+ ATPase activity and began to thin, indicating a recovery of fluid transport capability. This recovery was blocked by addition of brefeldin A during the ouabain-free superfusion., Conclusions: Inhibition of Na(+)-K+ ATPase by low concentrations of ouabain increases with time. Temporary exposure to ouabain causes swelling at rates that decline with time as ouabain dissociates from enzyme sites. This dissociation, together with the turnover of Na(+)-K+ ATPase in the plasma membrane, can lead to recovery of normal thickness in ouabain-exposed corneas. Twenty percent of Na(+)-K+ ATPase in the endothelium is estimated to be intracellular, and about 20% of the activity can be inhibited without inducing swelling.

Objective: To assay the proteolytic activity of plasmin on the vitreoretinal junction and to assess a potential facilitating effect on posterior vitreous detachment., Methods: We injected 1 U of plasmin into the vitreous of rabbits. Some eyes underwent vitrectomy after plasmin injection. Electroretinography and electron microscopy were performed., Results: In plasmin-treated eyes, electroretinography displayed a transient (3 days) decreased b-wave amplitude. Histologic examination demonstrated posterior vitreous detachment in eyes that received intravitreal plasmin followed by vitrectomy., Conclusion: Plasmin may prove to be a useful biochemical adjunct to mechanical vitrectomy.

Experiments were performed to evaluate the nonenzymatic reaction between glutathione (GSH) and dehydroascorbic acid (DHA). Though both ascorbic acid and glutathione disulfide (GSSG) are formed from this reaction, previous work has focused almost exclusively on measurements of ascorbic acid. In contrast, there is very little information about the formation of GSSG under the same conditions as those used to produce ascorbic acid. The emphasis on ascorbic acid stems from the fact that a spectrophotometric technique is available for its measurement, whereas 1H-NMR or an amino acid analyzer has been used to measure GSSG. The present experiments use a simple, rapid method for accurately and precisely measuring the concentrations of GSSG in a solution. The spectrophotometric (340 nm) procedure uses NADPH and glutathione reductase; analysis time is very short, many replicate samples can be tested and as little as 0.05-0.1 mM GSSG can be detected. Using this method, it is shown that there is an equimolar production of GSSG and ascorbic acid from GSH and DHA and that the decrease in GSH is stoichiometrically related to the increase in the concentration of GSSG. The present findings provide additional insight into the interaction between the GSH/GSSG redox couple and the ascorbic acid/DHA redox couple.

Experiments were undertaken to measure the pH of solutions of commonly used intravitreal antibiotics in various irrigating solutions to determine whether a change in pH might be a factor in antibiotic-induced retinal toxicity. Such retinal toxicity has been particularly damaging when solutions of gentamicin have been used. The pHs of the following solutions were measured: gentamicin, amikacin, methicillin, tobramycin, and vancomycin, combined with balanced salt solution (BSS) PLUS (bicarbonate buffer), BSS (citrate/acetate buffer), and lactated Ringer's solution (lactate buffer). Each of these antibiotics induced a concentration-dependent decrease in pH of the solutions; gentamicin, amikacin and tobramycin produced the largest shifts. The results also demonstrated that BSS PLUS acts as the strongest buffer and lactated Ringer's as the weakest. We conclude that it is important to determine the pH of intraocular antibiotic irrigating solutions, not just the pH of the antibiotics.

The role of chloride in fluid transport of the rabbit corneal endothelium was examined by measuring changes in corneal thickness following ion substitutions or addition of ion transport inhibitors in media superfusing the isolated tissue. Normal fluid transport is indicated by maintenance of constant thickness in a fresh cornea or thinning (deturgescence) of a preswollen deepithelialized cornea to its initial thickness at approximately 40 microns/h. These patterns are seen when tissues are superfused with HCO(3-)-Ringer containing 114 mM Cl-. When Cl- was substituted with gluconate, glucuronate, or SO4(2-) fresh and preswollen corneas immediately thinned at greater than 150 microns/h to a value less than 300 microns and then began to swell at 30 microns/h to above their original thickness. Substitution of Cl- with NO3- or Br- had a negligible immediate thinning effect, but fresh corneas subsequently swelled and preswollen corneas failed to deturgesce fully. The rapid thinning (called a "downtransient") observed with gluconate, glucuronate, and SO4(2-) also occurred in these media when ion and fluid transport were completely inhibited with ouabain or stilbenes or by absence of HCO3-, indicating that the thinning results from osmotic gradients induced by ionic reflection coefficients different from that of Cl-. When the downstransient was avoided in deepithelialized corneas by preswelling with the same Cl(-)-free media on both sides of the cornea, corneas maintained a constant but swollen thickness in gluconate and in NO3- or Br- deturgesced slowly and incompletely; ouabain or stilbenes caused further swelling in all media. We conclude that absence of Cl- partially impairs fluid transport, most probably via its role in a Cl(-)-HCO3- exchanger which has been proposed in a recent model of endothelial fluid transport.

The activities of Mg(2+)-dependent and Na(+)-K(+)-stimulated ATPase in homogenates of rat retina were measured in the presence of increasing concentrations of oxidized glutathione (GSSG). The Mg(2+)-ATPase was not inhibited by GSSG at any of the concentrations tested. The Na(+)-K(+)-stimulated ATPase was not inhibited by 1 mM GSSG, but its activity was decreased by 20 and 35%, respectively, in the presence of 5 and 10 mM GSSG. Other enzymatic measurements using supernatant fractions of rat retina showed that 1-10 mM GSSG did not inhibit the activities of hexokinase, glucose-6-phosphate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase. These results suggest that GSSG is not likely to exert significant deleterious changes on cellular processes, at least in cells and tissues in which normal glutathione (GSH) concentration is 2 mM or lower.

The adenosine triphosphate (ATP) content was measured independently in separated capsule-epithelium and fibers from whole rabbit lenses, both fresh and after incubation under various combinations of glucose and oxygen deprivation. Lactate production was also measured during aerobic and anaerobic incubations of whole lenses and of monolayers of cultured epithelial cells. The fresh capsule-epithelium contained 3.3 nmoles ATP, whereas the decapsulated lens contained 410 nmoles ATP, a value that was indistinguishable from that of the whole, intact lens. In the presence of glucose, the fibers and epithelium each maintained their respective ATP content under aerobic and anaerobic conditions. In the absence of glucose, the ATP content in each fraction declined with time, but only in the epithelium was the rate of decline of ATP significantly faster in nitrogen than in oxygen. In whole lens, the rates of anaerobic and aerobic lactate production were similar, whereas in the cultured epithelial monolayers, the anaerobic rate was two-fold greater than in oxygen. From this it is concluded that approximately 50% of the ATP of the epithelial cells is derived from oxidative metabolism. A Pasteur response shown here for the first time with the cultured epithelium allows these cells to compensate for the loss of ATP production when mitochondrial oxidation is curtailed. The epithelium does not contribute to the ATP content of the lens fibers under aerobic or anaerobic conditions.

1. The hydration and transparency of the mammalian cornea are maintained by a metabolically dependent fluid transport system located in the endothelial cell layer. The purpose of the study was to determine whether this pump activity is dependent upon aerobic or anaerobic metabolism. 2. The ability of the cornea, superfused in vitro with a bicarbonate-Ringer solution containing glucose and adenosine, to maintain normal hydration (thickness) when respiration is inhibited has been examined in intact and de-epithelialized preparations and correlated with glycolytic activity and cellular concentrations of ATP. 3. In respiring intact and de-epithelialized corneas thickness was maintained for periods up to 5 h during superfusion with the control Ringer solution. 4. KCN (10(-3) M) or antimycin A (10(-6) M) caused the intact cornea to swell at 15 +/- 3 microns h-1, whereas the de-epithelialized tissue maintained normal thickness under these conditions. This swelling of the intact tissue appears to be due to the osmotic effect of increased epithelial lactate production under anaerobic conditions. 5. Pre-swollen de-epithelialized corneas deturgesced fully to original thickness at a rate of 43 +/- 7 microns h-1 under aerobic conditions, but with KCN or antimycin they deturgesced at only 65% of that rate and generally failed to return to their original thickness. 6. Ouabain (10(-4) M) caused de-epithelialized corneas to swell in the presence of KCN or antimycin, as it did under aerobic conditions, showing that maintenance of hydration or deturgescence are pump-dependent processes under both conditions. 7. Lactate production was markedly stimulated by KCN or antimycin in intact and de-epithelialized preparations, but not in the stroma alone. The magnitude of the Pasteur effect was calculated to be 5-fold in the endothelium and 2.5-fold in the epithelium. Ouabain inhibited anaerobic lactate production in the endothelium by 50%. 8. ATP content of the epithelium following 5 h superfusion was 22.0 nmol cm-2 in control (aerobic) corneas, but fell to 1.9 nmol cm-2 in the presence of 10(-3) M-KCN, whereas the endothelial value fell only from 1.1 to 0.7 nmol cm-2 under these conditions. 9. Omission of glucose from the medium containing KCN or antimycin caused immediate swelling of tissues and a rapid decline of ATP content to less than 1% of that in control conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Measurements of lactate production and ATP concentration in superfused rat retinas were compared with extracellular photoreceptor potentials (Fast PIII). The effect of glucose concentration, oxygen tension, metabolic inhibition, and light were studied. Optimal conditions were achieved with 5-20 mM glucose and oxygen. The isolated retina had a high rate of lactate production and maintained the ATP content of a freshly excised retina, and Fast PIII potentials were similar to in vivo recordings. Small (less than 10%) decreases in aerobic and anaerobic lactate production were observed after illumination of dark-adapted retinas. There were no significant differences in ATP content in dark- and light-adapted retinas. In glucose-free medium, lactate production ceased, and the amplitude of Fast PIII and the level of ATP declined, but the rates of decline were slower in oxygen than in nitrogen. ATP levels were reduced and the amplitude of Fast PIII decreased when respiration was inhibited, and these changes were dependent on glucose concentration. Neither glycolysis alone nor Krebs cycle activity alone maintained the superfused rat retina at an optimal level. Retinal lactate production and utilization of ATP were inhibited by ouabain. Mannose but not galactose or fructose produced lactate and maintained ATP content and Fast PIII. Iodoacetate blocked lactate production and Fast PIII and depleted the retina of ATP. Pyruvate, lactate, and glutamine maintained ATP content and Fast PIII reasonably well (greater than 50%) in the absence of glucose, even in the presence of iodoacetate. addition of glucose, mannose, or 2-deoxyglucose to medium containing pyruvate and iodoacetate abolished Fast PIII and depleted the retina of its ATP. It is suggested that the deleterious effects of these three sugars depend upon their cellular uptake and phosphorylation during the blockade of glycolysis by iodoacetate.

This study investigates the possible role of glutathione (GSH) in defending the retina against oxidative damage. Freshly excised rat retina was found to contain 1.2 mumol/g wet wt GSH and an undetectable level of oxidized glutathione (GSSG). Whole retinas were either incubated or superfused with various concentrations of the GSH-oxidant diamide in order to study the effects of oxidation of GSH on the activity of the hexose monophosphate shunt (HMS) and on the receptor potential of the retina. It was found that exposure of the retina to diamide produced a stimulation of HMS activity up to 26-times that of the control. Significant changes in GSH content and receptor potential were observed at concentrations of diamide that produced more than a 5.4-fold stimulation of HMS activity. The diamide-induced electrical alterations included an increase in latency and peak time of the receptor potential, a delay in the onset of the off response and an increase in the time required for the potential to return to the baseline. It was found that nearly 80% of GHS could be regenerated and that most of the electrical effects of diamide could be reversed by superfusion with normal medium. The results indicate that the retina possesses an active system for maintaining GSH in the reduced state and that this may be essential for the normal function of this tissue.

The activity of Mg2+-ATPase and Na+-K+-ATPase was measured in media of calcium concentrations ranging from 10(-9)M to 10(-3)M, with retinal homogenates from rat and rabbit. In both species calcium stimulated the Mg2+-ATPase and inhibited Na+-K+-ATPase. In the rat, activity of Na+-K+-ATPase fell by 75% as calcium was increased from 10(-8)M to 10(-7)M and reached 90% inhibition only at 10(-3)M. By contrast, the activity in the rabbit fell gradually to a 90% inhibited state, over the range 10(-8)M to 10(-3)M. Calcium activated the Mg2+-ATPase by a maximum of 50% in each species, at a concentration of 10(-8)M in the rat and over a broader concentration range between 10(-5)M and 10(-4)M in the rabbit. It is postulated that maintenance of intracellular calcium at low levels by the Ca2+-activated Mg2+-ATPase or other cellular mechanisms is essential for the activity of the membrane-bound Na+-K+-ATPase of the retina.

The interaction of glutathione (GSH) with ascorbic acid and dehydroascorbic acid was examined in in-vitro experiments in order to examine the role of GSH in protecting against the autoxidation of ascorbic acid and in regenerating ascorbic acid by reaction with dehydroascorbic acid. If a buffered solution (pH 7.4) containing 1.0 mM ascorbic acid was incubated at 37 degrees C, there was a rapid loss of ascorbic acid in the presence of oxygen. When GSH was added to this solution, ascorbic acid did not disappear. Maximum protection against ascorbic acid autoxidation was achieved with as little as 0.1 mM GSH. Cupric ions (0.01 mM) greatly accelerated the rate of autoxidation of ascorbic acid, an effect that was inhibited by 0.1 mM GSH. Other experiments showed that GSH complexes with cupric ions, resulting in in a lowering of the amount of GSH in solution as measured in GSH standard curves. These results suggest that the inhibition of ascorbic acid autoxidation by GSH involves complexation with cupric ions that catalyze the reaction. When ascorbic acid was allowed to autoxidize at 37 degrees C the subsequent addition of GSH (up to 10 mM) did not lead to the regeneration of ascorbic acid. This failure to detect a direct reaction between GSH and the dehydroascorbic acid formed by oxidation of ascorbic acid under this condition was presumably due to the rapid hydrolysis of dehydroascorbic acid. When conditions were chosen, i.e., low temperature, that promote stability of dehydroascorbic acid, the direct reaction between GSH and dehydroascorbic acid to form ascorbic acid was readily detected. The marked instability of dehydroascorbic acid at 37 degrees C raises questions regarding the efficiency of the redox couple between GSH and dehydroascorbic acid in maintaining the concentration of ascorbic acid in mammalian cells exposed to an oxidative challenge.

Measurements were made of the activities of the NADP+-dependent isocitrate dehydrogenase, malic enzyme and glucose-6-phosphate dehydrogenase in cytosolic supernatants of whole lens and capsule-epithelium. The activities of all three NADP+-dependent enzymes were concentrated in the capsule-epithelium relative to the activities measured in the whole lens. These results show for the first time that the rabbit lens epithelial cells contain substantial activities of the NADP+-dependent isocitrate dehydrogenase and malic enzyme. It is proposed that these enzymes together with glucose-6-phosphate dehydrogenase are involved in the regulation of the intracellular level of glutathione during oxidative stress by providing the NADPH necessary for the activity of glutathione reductase.

Using the incubated isolated rat retina, the effects of hyaluronidase on the electroretinogram (ERG) and metabolic activities were investigated. Initial experiments established the activity of hyaluronidase needed to liquefy, within 15 to 30 minutes, the vitreous of postmortem human eyes; this concentration was 1,000 units/mL. Rat retinas were superfused with a bicarbonate-buffered, oxygenated medium to which hyaluronidase was added in activities ranging from 100 to 5,000 units/mL. These concentrations of hyaluronidase did not significantly alter the amplitudes of the a waves and b waves of the ERG in comparison to their control amplitudes. Measurements were also made of lactic acid production, oxygen consumption, glutathione content, and adenosine triphosphatase activities in control and hyaluronidase-exposed retinas. In the presence of hyaluronidase, their respective values were similar to the controls for all biochemical factors studied. The present experiments demonstrate that addition of hyaluronidase to an "ocular irrigating" solution results in normal ERGs and normal retinal metabolic activity and suggests the possibility that hyaluronidase may be useful in enzyme-assisted vitrectomy.

The effects of several intraocular irrigating solutions on the rate of glycolysis and the levels of ATP and glutathione in the isolated rat retina were investigated. The media were HCO3-Ringer, balanced salt solution (BSS) (to which 5 mM glucose was added), and BSS PLUS. There were no significant differences in the metabolic activities and metabolite levels in retinas incubated with either HCO3-Ringer or BSS PLUS. Retinas incubated with BSS PLUS had higher rates of aerobic and anaerobic glycolysis and more ATP than did retinas incubated with BSS. There were no significant differences, however, in the glutathione content of retinas incubated with BSS PLUS and BSS. These results show that retinal energy metabolism is maintained better with BSS PLUS because this irrigating solution contains glucose and bicarbonate.

Glutathione (GSH), together with NADPH-producing pathways and glutathione reductase, provides a defense system against oxidants. Oxidation of GSH causes stimulation of the hexose monophosphate shunt and increased production of NADPH. We have asked if hexose monophosphate shunt activity is required for the recovery of GSH following exposure of the isolated rat retina to an oxidant. Hexose monophosphate shunt activity was decreased by depleting the retina of hexose stores, before exposing the tissue to diamide (0.04-1.0mM), an oxidant for GSH, for 30 min. After exposure, retinas were transferred to either glucose-containing or glucose-free recovery medium for an additional 30 min. Control retinas kept in glucose-free, oxygenated medium (no diamide) for 90-120 min maintained GSH at 90% of the value found in retinas incubated with glucose. After exposure of hexose-depleted retinas to 0.4 mM diamide, a nearly 90% decrease in GSH was observed. When the oxidant was removed, the level of GSH returned to more than 80% of the control value in the presence or absence of glucose. In contrast, no recovery of GSH was observed after diamide treatment if the retinas were transferred to ice-cold (1-5 degrees C) media with or without glucose or if the retinas were pre-treated with 2 mM 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to inhibit glutathione reductase. Measurements of two NADPH-producing cytosolic enzymes, namely NADP+-dependent malic enzyme and NADP+-dependent isocitrate dehydrogenase, revealed high activities. Optimum production of NADPH from malic enzyme was 0.90 nmol NADPH produced min-1 per retina, while with isocitrate dehydrogenase the average rate was 6.9 nmol NADPH produced min-1 per retina. We suggest that these enzymes together with a long-lived endogenous substrate (probably glutamate) are responsible for the recovery of GSH in hexose-depleted retinas. The present results suggest that more than one NADPH-producing system is capable of controlling the GSH concentration in retina. Studies that have focused on the hexose monophosphate shunt pathway as the sole source of NADPH for glutathione reductase in retina and other tissues may require re-evaluation depending on the overall metabolic capacity and substrate utilization of the particular tissue. Thus, the present findings are significant not only with respect to the retina but also for other tissues whose metabolic characteristics are similar to those found in the retina.

The concentrations of guanosine 3',5' monophosphate (cyclic GMP) and adenosine 3',5' monophosphate (cyclic AMP) were measured in rat retinas incubated under control conditions and in retinas incubated with diamide, a relatively specific glutathione oxidant. Retinas incubated with either glucose or pyruvate as the substrate for 30 min in the dark contained about 50 picomoles cyclic GMP/mg protein and 7 picomoles cyclic AMP/mg protein. Light-exposed control retinas contained about 70% less cyclic GMP (14.4 picomoles/mg protein) and 20% less cyclic AMP (5.4 picomoles/mg protein). Addition of diamide to the incubation medium at concentrations between 0.1 and 1.0 mM produced a concentration-dependent decrease in the dark level of cyclic GMP, but did not affect its concentration in the light or the concentration of cyclic AMP in dark-maintained and light-exposed retinas. The major effect of diamide was to reduce the normal dark-light difference in the concentration of cyclic GMP. Oxidizing conditions thus appear to alter selectively the light-sensitive compartment of retinal cyclic GMP.

The Mg2+-, the Na+-K+-, and the HCO3--stimulated ATPase activities have been measured in the retina and pigment epithelium of postmortem human donor eyes. Activities were measured between 24 and 72 hr after death, following storage of the eyes in moistened chambers kept in a refrigerator. There appeared to be no significant differences in the ATPase activities between the 24 and 72 hr tissues. The human retina demonstrated both Mg2+- and Na+-K+-stimulated ATPase activity, but no HCO3+-stimulated ATPase activity, but not HCO3+-stimulated ATPase activity. The Na+-K+ ATPase activity was 1.54 mumol Pi/hr/mg protein, which amounted to 37% of the total ATP hydrolysis of the retina. The pigment epithelium-choroid showed significant Mg2+-, Na+-K+-, and HCO3--stimulated ATPase activities, with the stimulation caused by HCO3- about 25% greater than the effect due to Na+-K+, 0.64 vs. 0.51 mumol Pi/hr/mg protein. These findings in human retina and pigment epithelium-choroid show that certain metabolic processes continue to operate for several days after death.

The relative inhibitory effects of ATP depletion, ouabain and calcium on the receptor potential of the isolated rat retina were investigated. The principal experimental test used to assess these effects was the rate and extent of recovery of the receptor potential after re-establishing the transmembrane sodium gradient. Retinas were incubated in 25 mM-sodium for 10-20 min and then the external concentration of sodium was increased to 155 mM in control retinas, in retinas exposed to 10(-4) M-ouabain, and in retinas depleted of ATP (glucose-free medium plus 10 mM 2-deoxyglucose). Measurements showed that by 10-20 min ouabain completely inhibited the activity of the sodium-potassium ATPase and 2-deoxyglucose caused almost total loss of retinal ATP. In control retinas, the increase in sodium led to a rapid, full recovery of the receptor potential. When the sodium-potassium pump was blocked by ouabain, a return to the normal level of sodium led within seconds to a small (30%) recovery of the potential which decayed over 5 min. In ATP depleted retinas no recovery of the receptor potential was observed. However, if glucose was added at the time of changing to 155 mM-sodium in depleted retinas then a substantial recovery of the receptor potential was observed, but the return of the potential was delayed relative to the instantaneous re-establishment of the sodium gradient. After ouabain caused the receptor potential to decay in the presence of 2 mM-calcium and 155 mM-sodium, the potential was restored within seconds upon a reduction of external calcium to 10(-7)M. At this low calcium concentration, but not at 2 mM-calcium, an inverted receptor potential was observed transiently when the membrane sodium gradient was reversed during ouabain treatment. No recovery of the receptor potential was seen if the concentrations of calcium and sodium were raised to their control levels after incubation of the retina in a low sodium, low calcium, ouabain-containing medium. These results show that the receptor potential is elicited by changing the sodium gradient when the sodium-potassium pump is blocked, but is not evoked when either retinal ATP content is low or the external level of calcium is increased. The similarity in the inhibitory effects of ATP depletion and calcium suggest that high energy phosphates play a role in maintaining a low intracellular concentration of calcium, possibly by influencing sodium-calcium exchange or calcium pumps.