Protection by Pyruvate of Rat Retinal Cells against Zinc Toxicity In Vitro, and Pressure-Induced Ischemia In Vivo

Abstract

purpose. To examine whether zinc accumulation occurs during retinal neuronal death after pressure-induced ischemia in rats and whether pyruvate protects against such death.

methods. To induce transient retinal ischemia, intraocular pressure was increased above systolic pressure for 65 minutes. Pyruvate was administered through the tail vein for 12 hours after ischemia to determine its effect on degeneration of retinal neurons. Retinas were removed and sectioned, and zinc accumulation was visualized with N-(6-methoxy-8-quinolyul)-p-carboxybenzoyl-sylphonamide (TFL-Zn) fluorescence microscopy, and neuronal death was determined with acid fuchsin staining. For in vitro studies, retinal cell cultures were prepared from newborn rat pups and used for experiments at days in vitro (DIV) 7 to 10.

results. After retinal ischemia, staining revealed that most zinc-accumulating neurons were injured neurons, suggesting that endogenous zinc may contribute to ischemic neuronal death in the retina. In vitro studies showed that 15 minutes of exposure to 300 to 500 μM zinc resulted in the death of a substantial number of retinal cells in culture, and that this death was preceded by poly(ADP-ribose) polymerase (PARP)-mediated depletion of nicotinamide-adenine dinucleotide (NAD⁺) and adenosine triphosphate (ATP). Pyruvate, but not lactate, protected against this zinc-induced cell death in vitro. Consistent with this finding, in vivo studies showed that compared with control rats, pyruvate-treated rats had a substantial reduction in the number of cells showing signs of cell death.

conclusions. The present results suggest endogenous zinc contributes to retinal cell death after ischemia. Pyruvate potently protected against zinc toxicity in cultured rat retinal cells and reduced ischemia-induced cell death in rat retinas.

The transition metal zinc is involved in a diverse range of essential cellular functions in all eukaryotic cells. ¹ For example, metalloenzymes such as carbonic anhydrase and metalloproteases exhibit zinc-dependent catalytic activity. In addition, zinc provides structural motifs for a wide range of proteins including zinc-finger transcription factors. ²  

Because of high-affinity binding, protein-bound zinc cannot be easily visualized with conventional histochemical methods, such as modified Timm staining ^{3 4} or zinc-specific fluorophore staining. ^{5 6} In addition to the pool of tightly bound zinc in the CNS, a substantial amount of zinc is present within glutamatergic synaptic vesicles in a free or loosely bound state. ^{4 6} Zinc within the synaptic vesicle is more reactive and therefore more readily visualized histochemically. 

Neuronal activity leads to the release of zinc in synaptic vesicles into the extracellular space, ^{7 8 9 10 11} consistent with zinc’s important role in synaptic physiology. Indeed, zinc exerts diverse effects on both neurotransmitter receptors and ion channels. ^{12 13 14} However, acute neuron injury can result in the release of excessive amounts of zinc from terminals or zinc-binding proteins, and accumulation of such zinc may contribute to the death of cells in the cerebral cortex and hippocampus. ^{15 16 17}  

The intracellular mechanisms underlying zinc cytotoxicity are under intense investigation. ¹⁵ In cortical cultures, zinc cytotoxicity involves increased oxidative stress. ^{18 19 20 21} Zinc exposure results in mitochondrial injury ^{22 23} and increases intracellular levels of reactive oxygen species (ROS) and lipid peroxidation. ^{18 19 20 21} Furthermore, zinc toxicity may be attenuated by various antioxidative measures. ^{18 19} As well as causing mitochondrial damage, activation of PKC and induction of NADPH oxidase have been shown to participate in the generation of increased oxidative stress. ^{24 25} As a result of oxidative damage to DNA, poly(ADP-ribose) polymerase (PARP) may become activated and lead to the depletion of NAD⁺ and adenosine triphosphate (ATP), which, if severe, may cause cell necrosis. ²⁶  

Like other areas of the central nervous system (CNS), the retina contains a substantial amount of histochemically reactive zinc in various areas, including pigment epithelial cells and the outer and inner nuclear layers. ^{27 28 29} Labile zinc in pigment epithelial cells may serve functions in phagocytosis and antioxidant processes. ²⁸ In addition, zinc in photoreceptor cells may contribute to dark-light adaptation of the neural retina, because histochemically reactive zinc appears to exhibit dark-light status-dependent translocation between photoreceptor perikarya in the outer nuclear layer and the inner segments of photoreceptors. ³⁰ In addition, as in other parts of the CNS, labile zinc in the neural retina is released with neuronal activity and may modulate neurotransmission. ^{31 32 33 34} Such activity-dependent zinc release suggests that an endogenous zinc-based cytotoxic mechanism operates in the retina as in other areas of the CNS. 

Unlike in other areas of the CNS, a pathologic role for zinc in the retina is yet to be identified. Because the retina contains a substantial amount of histochemically reactive zinc, zinc-related toxicity may play an important role in pathologic cell death in the retina. 

Materials and Methods

Materials

ZnCl₂, pyruvate, oxaloacetate, ionomycin, hydrogen peroxide, disodium calcium EDTA (CaEDTA), disodium zinc EDTA (ZnEDTA), and lactate were purchased from Sigma-Aldrich (St. Louis, MO). Nicotinamide was obtained from Alexis (San Diego, CA), trolox from Aldrich (Milwaukee, WI), and 2,7-dichlorofluorescein diacetate (DCF) from Molecular Probes (Eugene, OR). 

Pressure-Induced Retinal Ischemia in Rats

All animal experiments were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Male Sprague-Dawley rats weighing 300 to 350 g were anesthetized by an intraperitoneal injection of ketamine HCl (90 mg/kg) and xylazine HCl (10 mg/kg). The anterior chamber of the right eye was cannulated with a 26-gauge needle attached to an infusion line for normal saline. The intraocular pressure was increased over systolic arterial pressure by elevating a saline bag to halt retinal arterial circulation for 65 minutes. The needle was then withdrawn, and the intraocular pressure was allowed to normalize. Levofloxacin ophthalmic solution was applied topically before and after cannulation. Sham-treated rats underwent similar procedures, but without elevation of the saline bag. To investigate the cytoprotective effect of pyruvate, within 1 hour of the 65-minute ischemia, osmolarity-matched saline (control) or sodium pyruvate (4.5 g/kg) dissolved in water was administered through the tail vein for 6 to 12 hours. 

Tissue Preparation, Zinc-Specific Fluorescence, Acid Fuchsin, and Hematoxylin and Eosin Staining

Eyes were enucleated at either 24 hours or 7 days after the 65-minute period of ischemia. Transretinal eyeball sections perpendicular to the retinal surface were prepared with a cryostat and mounted on prechilled glass slides coated with poly-l-lysine. For samples collected 24 hours after the 65-minute ischemia, unfixed eyeball sections were stained for 90 seconds with the zinc-specific fluorescent dye N-(6-methoxy-8-quinolyul)-p-carboxybenzoyl-sylphonamide (TFL-Zn) dissolved in Tris buffer. TFL-Zn-stained sections were examined under a fluorescence microscope and photographed with a digital camera. To identify dead neurons, eyeball sections were subjected to acid fuchsin staining. Zinc-accumulating neurons and acidophilic neurons in the inner nuclear layer and ganglion cell layer were counted in four adjacent areas within 1 mm of the optic nerve in a condition-blinded fashion. For samples collected 7 days after 65 minutes of ischemia, unfixed eyeball sections were stained with hematoxylin and eosin. Measurements of the thickness of retinal layers were performed from the outer limiting membrane to the inner limiting membrane in sections through the equator. Measurements were taken in three adjacent areas within 1 mm of the optic nerve in a condition-blinded fashion and are presented as mean values. 

Retinal Cell Culture

Primary cell cultures that included neurons, astrocytes, and photoreceptor cells were generated from the retinas of newborn (postnatal day 1 or 2) Sprague-Dawley rats. ³⁵ Briefly, retinas were isolated, placed in Hanks’ balanced Ca²⁺- and Mg²⁺-free salt solution (HBSS) and mechanically dissociated into single cells by trituration with fire-polished Pasteur pipettes. Dissociated cells were plated on poly-l-lysine-coated, 24-well plates (three retinas per plate). The plating medium was based on Eagle’s minimum essential medium (MEM; Invitrogen-Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (HIFBS) and 25 mM KCl. Retinal cultures were maintained at 37°C in a humidified 5% CO₂ incubator, and used in experiments after 10 days of culturing in vitro. 

Cultured Cell Death Experiments

Cultured retinal cells were thoroughly washed by multiple rinsing with MEM and then left in MEM without HIFBS. Brief (15 minutes) exposure to zinc or other drugs was performed under sterile conditions at room temperature, after which cells were thoroughly washed and returned to a CO₂ incubator. After a morphologic assessment, cell injury was quantified by measurement of lactate dehydrogenase (LDH) released by injured cells 18 hours after zinc exposure ¹⁸ unless otherwise specified. LDH release is expressed relative to the value of 100, which represented the maximum neuronal LDH release that occurred after exposure of a parallel cell culture to 100 μM zinc for 24 hours, which induced near complete neuronal and glial damage. Where possible, data obtained from “sister” cultures were used for statistical comparisons. All experiments were performed at least three times using cell cultures derived from different platings. 

The LDH assay was not performed in experiments involving addition of pyruvate after zinc exposure, because pyruvate is a component of the LDH assay. In these cases, cell death was determined by counting dead cells. Briefly, retinal cells were stained with 0.4% trypan blue for 5 minutes, after which stained and unstained cells were counted in five randomly chosen fields using bright-field optics at 200× magnification. The percentage of cell death was calculated by dividing the mean number of dead cells by the mean number of total cells. 

Measurement of NAD⁺ and ATP

NAD⁺ levels were measured with an enzymatic cycling assay. ³⁶ After 15 minutes of exposure to zinc, cultures were returned to a CO₂ incubator for the indicated times. Cortical cultures were harvested and treated with 0.5 M perchloric acid (Merck, Germany) for 15 minutes at 4°C. The resultant extracts were neutralized with 0.5 M KOH for 1 hour on ice. Cell debris and KClO₄ were removed by centrifugation at 2000g for 15 minutes at 4°C. Fifty microliters of supernatant or 0 to 20 pmol standard NAD⁺ solution (Sigma-Aldrich) was reacted with 100 μL of freshly prepared reagent mixture (2 mM phenazine ethosulfate, 0.5 mM MTT (3- [4,5-dimethylthiazol-2-yo]-2,5-diphenyltetrazolium bromide), 600 mM ethanol (Merck, Darmstadt, Germany), 5 mM EDTA, 0.83 mg/mL BSA, and 4 U alcohol dehydrogenase (Sigma-Aldrich) in 120 mM sodium/bicine buffer [pH 7.8]) in the dark at 37°C for 1 hour. The absorbance at 570 nm was measured and corrected for the protein content determined by the bicinchoninic acid protein assay (BCA; Pierce, Rockford, IL). Data are expressed as percentages of the mean control values. 

ATP levels were measured by the luciferin-luciferase method using an ATP determination kit (Molecular Probes). Cell cultures were extracted with perchloric acid, neutralized with KOH and cell debris removed. Ten microliters of supernatant was mixed with reconstituted buffer (0.5 mM luciferin, 1.25 μg/mL luciferase, 25 mM tricine buffer [pH 7.8], 5 mM MgSO₄, 100 μM EDTA, and 1 mM dithiothreitol [DTT]), and chemiluminescence was measured with a luminometer (L_max; Molecular Devices, Sunnyvale, CA). The results were corrected for protein content and are presented as percentages of the mean of control values. 

Statistical Analysis

A two-tailed t-test with Bonferroni correction for appropriate multiple comparisons was performed. In all figures, one asterisk denotes P < 0.05 and two denote P < 0.01. 

Results

Accumulation of Zinc in Degenerating Neurons after Retinal Ischemia in Rats

Raising the intraocular pressure over systolic pressure in rats resulted in complete cessation of blood flow to the retina, as visualized by ophthalmoscope. At specific times after 65 minutes of ischemia, retinas were removed, stained with acid fuchsin and the zinc-specific fluorescent dye TFL-Zn, and examined by microscope. We found that up to 2 hours after ischemia, little TFL-Zn fluorescence was present in cell bodies, although tissues appeared markedly swollen. By 6 hours, while tissue swelling was still evident, scattered cell bodies in the inner nuclear layer (INL) and outer nuclear layer (ONL) began to exhibit faint fluorescence. The number of such fluorescent neurons increased over 12 to 24 hours 1 . In addition, some ganglion neurons started to show TFL-Zn fluorescence at these later time points. At the later time points, there was a close correlation between TFL-Zn fluorescence and acidophilic changes, suggesting that zinc accumulation may play a role in neuronal death in retinas. Seven days after ischemia, retinal thickness was markedly reduced. Although acid fuchsin or TFL-Zn staining of fiber layers appeared denser than in the control, perhaps due to tissue atrophy, there were no signs of acidophilic or TFL-Zn⁺ neurons at this late time point. 

Morphology and Time Course of Zinc Toxicity in Retinal Cell Cultures

We examined morphologic changes in cultured retinal cells after 15 minutes of exposure to 400 μM zinc. Little change was observed up to 4 hours after zinc exposure 2 , whereas some cells began to exhibit marked cell body swelling at approximately 6 hours (2 , arrows), and a substantial fraction of cells were trypan blue positive at this time, indicating loss of plasma membrane integrity. Further cell degeneration occurred later, with almost all cells being trypan blue positive by 8 hours 2 . This temporal loss of plasma membrane integrity was confirmed using LDH release assay which showed LDH levels began to rise around 5 hours after a 15-minute exposure to 400 μM zinc, and reached maximum levels by 8 hours 2 . 

Zinc Concentration-Toxicity Relationship in Retinal Cell Cultures

The relationship between the concentration of zinc and retinal cell death was examined. Cell death was estimated by measuring LDH release from dead cells, 24 hours after a 15-minute exposure to zinc 3 or immediately after 24 hours of continual exposure to zinc 3 . Very little cell death resulted from 15 minutes of exposure to 100 and 200 μM zinc, whereas almost complete cell death occurred at 400 and 500 μM. Exposure to 300 μM zinc for 15 minutes resulted in the death of between 40% and 80% of cells. For cells exposed to zinc for 24 hours, 20 to 40 μM zinc induced little cell death, whereas 50 μM zinc induced 30% to 40% cell death, and 60 to 80 μM zinc resulted in almost 100% death of cells in culture. 

Depletion of NAD⁺ and ATP by Zinc: Contribution by PARP

It appears that PARP activation is an important common final pathway after various types of oxidative cell injury. ²⁶ Injury to DNA strands by ROS triggers PARP activation that transfers the ADP-ribose moiety from NAD⁺ to target proteins. As a result, NAD⁺ and ATP levels become severely depleted, resulting in cell death. Consistent with such a process, we found that levels of NAD⁺ decreased rapidly after 15 minutes of exposure to 400 μM zinc 4 . The level of NAD⁺ dropped to less than 50% of the control level at 3 hours, and less than 10% at 5 hours after zinc exposure. ATP levels were also drastically reduced in these cells, with ATP depletion after a similar time course 4 . Addition of the PARP inhibitor nicotinamide (10 mM) to cell cultures significantly attenuated NAD⁺ and ATP depletion 5 hours after zinc exposure, which is consistent with an important role for PARP in depletion of NAD⁺ and ATP (4 4 , respectively). Furthermore, nicotinamide reduced retinal cell death as indicated by less LDH release measured 24 hours after 15 minutes of zinc exposure 4 , indicating that PARP contributes to zinc-induced cell death. 

Protection against Zinc-Induced Retinal Cell Death in Culture by Pyruvate

We examined the effect of pyruvate on zinc-induced death in cultured retinal cells. We found that addition of 5 mM pyruvate markedly attenuated cell death measured by the counting of dead cells, 24 hours after 15 minutes of exposure to 400 μM zinc 5 5 5 . In contrast, 6 mM lactate had no protective effect 5 5 . In addition, pyruvate attenuated the decrease in NAD⁺ and ATP levels, whereas lactate had no effect 5 5 . 

Protection against Ischemic Retinal Cell Death in Rats by Pyruvate

Because pyruvate markedly protected against zinc toxicity in retinal cell cultures, we examined its effect on pressure-induced retinal ischemia in rats. After 65 minutes of ischemia, pyruvate (4.5 g/kg) or osmolarity-matched saline (control) was infused through the tail vein for 12 hours. We found that pyruvate-treated rats 6 had fewer TFL-Zn⁺ neurons than saline-treated rats 6 . Moreover, the number of acidophilic neurons was reduced to a similar extent in the pyruvate-treated group 6 compared with the saline-treated group 6 . Quantitative data showing the number of TFL-Zn⁺ and acidophilic neurons are presented in 6 . 

This protective effect of pyruvate persisted up to 7 days after ischemia 7 . At this time, retinal thickness was markedly atrophied 7 days after ischemia 7 compared with the normal control 7 . Although all the layers were thinned, the inner plexiform layer exhibited the most drastic reduction in thickness. Pyruvate treatment substantially prevented tissue atrophy 7 . Quantitative data are presented in 7 . 

Discussion

Although intracellular accumulation of zinc has been shown to contribute to neuronal death in various models of acute brain injury, it is unknown whether the same mechanism operates in the retina. The present study demonstrates that zinc accumulates in degenerating neurons after retinal ischemia, indicating that a similar phenomenon occurs in the retina as occurs in other brain areas. 

Histochemically reactive zinc is enriched in glutamatergic terminals in the CNS and is released under both physiological and pathologic conditions. Released zinc may enter the cytoplasm through various calcium-permeable channels, such as N-methyl-d-aspartate (NMDA) receptors, calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors and voltage-gated calcium channels. ¹⁵ High cytoplasmic zinc levels may trigger a variety of intracellular events that lead to cell death through apoptosis and oxidative stress. ³⁷  

Although it is rarely mentioned, retinas also contain zinc in synaptic vesicles, especially in the inner nuclear layers. ^{27 29} In addition, zinc release has been demonstrated in the retina. ^{30 34} No information is currently available regarding the possible elevation of extracellular zinc concentrations in the retina after acute injury. The concentration of zinc in the hippocampal synaptic cleft can reach several tens to hundreds of micromoles per liter with intense stimuli. ⁸ Because zinc content in the retina is as high as that in forebrain, it is reasonable to speculate that after severe injury, extracellular zinc concentrations in the retina could rise to cytotoxic levels, which is several tens of micromolar in retinal cell culture, as shown in the present study. 

The role of endogenous zinc in pathologic neuronal death is highlighted by the neuroprotective effect of zinc chelators such as CaEDTA. ¹⁶ However, intraocular administration of CaEDTA results in severe retinal detachment, making it difficult to test the protective effects of CaEDTA in ischemic retinal cell death. Because degenerating cells in both the retina and other brain areas accumulate zinc, it appears that endogenous zinc also contributes to ischemic cell death in the retina. 

The characteristics of zinc toxicity in retinal cell cultures were very similar to those in cortical cell cultures. First, morphologic changes such as cell-body swelling and membrane disruption suggest zinc-induced cell death occurs mainly by necrosis. Second, the concentration-dependence of zinc toxicity was similar for both cell types. ¹⁸ A 15-minute exposure to 300 μM zinc induced partial (40%–80%) retinal cell death, whereas a 24-hour exposure to only 60 μM zinc induced nearly complete death. Third, NAD⁺ and ATP depletion, probably mediated by PARP activation, ²⁶ was common for zinc toxicity in both retinal and cortical cell cultures. ^{38 39} Finally, as in cortical cell cultures, ^{38 40} pyruvate markedly attenuated zinc-induced cell death in retinal cultures. Therefore, it is highly likely that intracellular events mediating zinc toxicity are largely shared by retinal and cortical cells. 

Pyruvate is a natural intermediate of glucose energy metabolism and has no serious side effects. Our cell culture data suggest that pyruvate may be useful as a neuroprotective agent against ischemic retinal cell death. Indeed, we found that pyruvate substantially reduced zinc accumulation and cell death after pressure-induced retinal ischemia in rats. We previously reported that pyruvate had a remarkable neuroprotective effect in a rat model of transient cerebral ischemia, ⁴⁰ where the role of endogenous zinc has been well established. ¹⁶ Taken together, the zinc accumulation in degenerating neurons and the protection by pyruvate suggest a significant contribution by zinc to cell death after retinal ischemia. 

Retinal ischemia has been implicated as a mechanism contributing to cell death in various conditions, such as arterial and venous occlusion and glaucoma. The present results suggest the interesting and novel possibility that endogenous zinc plays a significant role as a trigger of cell death in retinal ischemia. Furthermore, the data indicate that pyruvate can provide in vivo protection against the apparent cytotoxic effects of zinc in the retina. Further studies seem warranted to determine the precise roles of endogenous zinc in the various conditions associated with retinal cell death. 

² Contributed equally to the work and therefore should be considered equivalent senior authors.

 Supported by Health and Medical Technology Development Program Grant 02-PJ1-PG1-CH02-0003 from the Korean Ministry of Health and Welfare (YHY).

 Submitted for publication December 5, 2003; revised January 15, 2004; accepted January 15, 2004.

 Disclosure: M.H. Yoo, None; J.-Y. Lee, None; S.E. Lee, None; J.-Y. Koh, None; Y.H. Yoon, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Figure 1.

 

Zinc accumulation in degenerating cells after retinal ischemia. (A) Fluorescence photomicrographs of rat retina stained with TFL-Zn at the indicated time after 65 minutes of pressure-induced retinal ischemia. (B) Photomicrographs of rat retina stained with acid fuchsin at the indicated time after 65 minutes of ischemia. 1, ganglion cell layer; 2, inner plexiform layer; 3, inner nuclear layer; 4, outer plexiform layer; 5, outer nuclear layer. Scale bar, 100 μm.

Figure 2.

 

Zinc-induced retinal cell death. (A–D) Photomicrographs of trypan blue-stained retinal cell cultures 1 (A), 4 (B), 6 (C), and 8 hours (D) after 15 minutes of exposure to 400 μM zinc. Arrows: cell body swelling in zinc-treated cells at 6 hours (C). Scale bar, 50 μm. (E) Relative LDH activity in cell culture medium (mean ± SEM, n = 6) at the indicated time points after 15 minutes exposure to 400 μM zinc.

Figure 3.

 

Relationship between zinc concentration and cell death. (A) LDH release (mean ± SEM, n = 9) from cells 24 hours after 15 minutes of exposure to the indicated concentrations of zinc. (B) LDH release (n = 9) after 24 hours of exposure to the indicated concentrations of zinc.

Figure 4.

 

Depletion of NAD⁺ and ATP and the effect of PARP inhibitors. Relative levels of (A) NAD⁺ or (B) ATP (each as a percentage of control, mean ± SEM, n = 6) in retinal cultures at the indicated time points after 15 minutes of exposure to 400 μM zinc. Levels of (C) NAD⁺ and (D) ATP in retinal cultures 5 hours after 15 minutes of exposure to 400 μM zinc, alone or in the presence of 10 mM nicotinamide, added both during and after exposure to zinc. (E) LDH release (mean ± SEM, n = 9) in retinal cultures 24 hours after 15 minutes of exposure to 400 μM zinc, alone or in the presence of 10 mM nicotinamide, added both during and after zinc exposure. *Significant difference from Zn (P < 0.05, two-tailed t-test).

Figure 5.

 

Cytoprotection by pyruvate. (A–C) Photomicrographs of retinal cultures 24 hours after 15 minutes of exposure to 400 μM zinc alone (A), zinc plus 5 mM pyruvate (B), or zinc plus 6 mM lactate (C). Scale bar, 50 μm. Levels of (D) NAD⁺ or (E) ATP (each as a percentage of control, n = 6) in retinal cultures 5 hours after 15 minutes of exposure to 400 μM zinc alone or in the presence of 5 mM pyruvate or 6 mM lactate, added both during and after zinc exposure. (F) Cell death in retinal cultures 24 hours after 15 minutes of exposure to 400 μM zinc alone or in the presence of 5 mM pyruvate or 6 mM lactate, which were added both during and after zinc exposure. *P < 0.05 and **P < 0.01: significant differences from Zn (two tailed t-test with Bonferroni correction for two comparisons).

Figure 6.

 

Pyruvate protected against zinc accumulation and neuronal death after retinal ischemia. TFL-Zn fluorescence in retinas from (A) saline- or (B) pyruvate-treated rats 24 hours after 65 minutes of pressure-induced ischemia. (C, D) Acid fuchsin-stained sections adjacent to (A) and (B), respectively. Numbered layers are as in 1 . (E) Number of TFL-Zn⁺ and acidophilic neurons (mean ± SEM, n = 10 each) in the respective retinal sections. *P < 0.05 and **P < 0.01: significant differences from Zn (two tailed t-test with Bonferroni correction for two comparisons).

Figure 7.

 

Long-lasting protective effect of pyruvate against retinal ischemia. (A–C) Hematoxylin and eosin-stained retinal sections. Normal control (A) or 7 days after 65 minutes of ischemia with saline (B) or pyruvate treatment (C). Scale bar, 50 μm. Numbered layers are as in 1 . (D) Retinal thickness (in millimeters, mean ± SEM, n = 11–13) in normal rats, ischemia- and saline-treated rats, or ischemia- and pyruvate-treated rats. **P < 0.01: significant difference (two tailed t-test with Bonferroni correction for two comparisons).