January 2008
Volume 49, Issue 1
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Physiology and Pharmacology  |   January 2008
The Role of Nitric Oxide and cGMP in Somatostatin’s Protection against Retinal Ischemia
Author Affiliations
  • Niki Mastrodimou
    From the Laboratory of Pharmacology, Department of Basic Sciences, Faculty of Medicine, University of Crete, Heraklion, Crete, Greece.
  • Foteini Kiagiadaki
    From the Laboratory of Pharmacology, Department of Basic Sciences, Faculty of Medicine, University of Crete, Heraklion, Crete, Greece.
  • Kyriaki Thermos
    From the Laboratory of Pharmacology, Department of Basic Sciences, Faculty of Medicine, University of Crete, Heraklion, Crete, Greece.
Investigative Ophthalmology & Visual Science January 2008, Vol.49, 342-349. doi:10.1167/iovs.07-0341
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      Niki Mastrodimou, Foteini Kiagiadaki, Kyriaki Thermos; The Role of Nitric Oxide and cGMP in Somatostatin’s Protection against Retinal Ischemia. Invest. Ophthalmol. Vis. Sci. 2008;49(1):342-349. doi: 10.1167/iovs.07-0341.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To investigate whether nitric oxide (NO) and/or cGMP protects the retina from chemical ischemia and underlie somatostatin’s neuroprotective effects.

methods. Eyecups of female Sprague-Dawley rats were incubated with PBS or the chemical ischemia mixture [iodoacetic acid (5 mM)/sodium cyanate (25 mM)] in the absence or presence of (1) arginine (0.05–2.0 mM), the substrate of nitric oxide synthase (NOS); (2) the NO donors sodium nitroprusside (SNP; 0.25–4.0 mM), 3-morpholinosydnonimine (SIN-1; 0.1, 0.3, 1.0 mM), SIN-1 (0.1 mM)/l-cysteine (5 mM, peroxynitrite scavenger), and NONOate (1, 5, 10 μΜ, slow NO releaser); (3) 8-Br-cGMP (0.1, 0.5, 1.0 mM); (4) BIM23014 (sst2 receptor agonist; 1 μM), alone or in the presence of (5) the NOS inhibitor N γ-monomethyl-l-arginine (NMMA; 0.5 mM); or (6) the guanylyl cyclase inhibitors 1H-[1,2,4]oxadiazolol [4,3-a]quinoxalin-1-one (ODQ;100 μM) and NS2028 (50 μM) for 60 minutes, at 5%CO2/air in 37°C. The effect of SIN-1 (0.1, 0.3, 1.0, or 3.0 mM) on the retina was also examined. Subsequently, the eyecups were fixed and sectioned for choline acetyltransferase (ChAT) immunoreactivity and TUNEL staining.

results. Arginine and SNP had no effect on the chemical ischemia–induced toxicity. SIN-1, NONOate, and 8-Br-cGMP produced a concentration-dependent protective effect, as shown by ChAT immunoreactivity. TUNEL staining also confirmed the neuroprotective effect of these agents. l-Cysteine partially reduced the SIN-1–induced protective effect. SIN-1 alone was toxic only at the highest concentration used (3 mM). NMMA, ODQ, and NS2028 reversed the protective effect of BIM23014.

conclusions. The results suggest that a NO/peroxynitrite/cGMP mechanism may be important in the protection of the retina from ischemic insult. Furthermore, the NO/sGC/cGMP pathway is involved in the neuroprotective effects of sst2 ligands against retinal ischemia.

The neuropeptide somatostatin 1 is found in the retina, 2 where it activates the somatostatin receptors 3 (ssts) found in retinal neurons and the retinal pigment epithelium (RPE) (see review by Thermos 4 ). Specifically, sst2A receptors are localized in rod bipolar, amacrine, and photoreceptor neurons in the retina, 5 6 7 8 9 whereas sst2B receptors are localized in photoreceptors and the RPE. 8 10 In addition, sst1 and sst4 receptors were found to be present in amacrine 11 and ganglion cells, 12 13 respectively. 
Retinal ischemia is the underlying cause of many ocular diseases and leads to neuronal damage and blindness. The importance of somatostatin ligands in the inhibition of ischemia-induced neovascularization, one of the major causes of retinal diseases that results in visual loss has been investigated. Somatostatin and its sst2 agonists inhibited the ischemia-induced neovascularization in a mouse model of oxygen-induced retinopathy. 14 15  
In a recent study, an in vitro model 16 of chemical ischemia was used in the retina. 17 Chemical ischemia involves the blockade of oxidative phosphorylation and glycolysis and is believed to be useful in the understanding of the early events underlying the pathophysiology of ischemia. In this model, somatostatin analogues selective for the sst2 subtype protect the retina from ischemic insult. 17 The mechanisms by which the somatostatin analogues prevent the damage produced by chemical ischemia or other neurotoxic insults are not known. However, somatostatin’s ability to inhibit voltage-gated Ca+2 channels may be responsible for the lowering of the intracellular calcium ion concentrations, 18 responsible for the toxic effects. The rise in intracellular calcium levels resulting from ischemia-induced activation of voltage-gated calcium channels and ionotropic glutamate receptors is believed to be the underlying cause of retinal cell death. 19  
A study by Mastrodimou et al. 17 suggested that protein kinase C (PKC) and tyrosine hydroxylase (TH)-sst2–containing neurons 5 6 7 8 13 20 21 were protected from ischemic insult, possibly by sst2 involvement in the attenuation of calcium levels. However, the neuroprotection afforded to the ChAT- and bNOS-containing neurons, which lack sst2 receptors, could not be explained. 
Somatostatin and the novel neuropeptide cortistatin, 22 which resembles somatostatin structurally and binds to somatostatin receptors, 23 have been shown to have neuroprotective effects against different paradigms of neurotoxicity in the central nervous system, such as NMDA, and kainate-induced neurotoxicity 24 25 and middle cerebral artery. 26 The somatostatin reduction of NMDA-induced neuronal death in cortical neurons was mediated by a cGMP-dependent mechanism. 24  
Somatostatin-induced inhibition of neuronal Ca2+ currents has been suggested to be mediated via a cGMP-dependent protein kinase. 27 Recent studies in our laboratory have shown that somatostatin increases NO 12 and cGMP levels in rat retinal explants 28 via an sst2 mechanism. Therefore, one can conjecture that NO triggers the synthesis of cGMP in neighboring cells that do not contain somatostatin receptors and provides neuroprotection. 
The purpose of the present study was to investigate whether NO and/or cGMP protects the retina from chemical ischemia insult and whether this effect represents a putative mechanism for somatostatin’s neuroprotection of the retina. 
Materials and Methods
Animals
Female Sprague-Dawley rats (250–300 g) were housed two to three animals per cage with free access to food and water. A 12-hour light-dark cycle was maintained. Euthanasia was performed with ether inhalations. All procedures that were performed on the animals were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and in compliance with Greek National Laws (Animal Act, P.D. 160/91). 
Effect of Arginine, Nitroprusside, NONOate, SIN-1, or 8-Br-cGMP on Chemical Ischemia
After dissecting the eyes, the anterior poles were cut away, and the eyecups were immersed in 0.1 M phosphate-buffered saline (PBS). To produce chemical ischemia and study the possible protection by the different agents, we used the protocol previously used by Mastrodimou et al. 17 Eyecups were incubated with (1) PBS, (2) a chemical ischemia mixture (5 mM IAA/25 mM NaCN), and (3) arginine, the NOS substrate (0.05, 0.125, 0.25, 0.5, 1.0, or 2.0 mM), or the NO donors sodium nitroprusside (SNP; 0.25, 0.50, 1.0, 2.0, or 4.0 mM), spermine NONOate (slow-release NO donor; 1, 5, or 10 μM), SIN-1 (0.1, 0.3, or 1 mM) or 8-Br-cGMP (0.1, 0.5, or 1 mM), together with the chemical ischemia mixture, for 60 minutes (two times for 30 minutes), followed by incubation two times for 30 minutes in PBS (control and ischemia groups) or arginine/nitroprusside/NONOate/SIN-1/8-Br-cGMP, respectively, in PBS (neuroprotection groups) at the concentrations used earlier. All incubations were performed at 37°C and 5% CO2/95% air. These experiments were performed three times. 
Effect of SIN-1/l-Cysteine on Chemical Ischemia
Eyecups were incubated in (1) PBS or in the presence of (2) chemical ischemia mixture, and (3) SIN-1 (0.1 mM)/l-Cysteine, a peroxynitrite scavenger (5 mM), 29 together with the chemical ischemia mixture for 60 minutes (two times for 30 minutes each), followed by incubation two times for 30 minutes each in PBS (control and ischemia groups) or SIN-1/l-cysteine in PBS (neuroprotection groups) at the concentrations used earlier. All incubations were performed at 37°C and 5% CO2/95% air. These experiments were performed three times. 
Effect of SIN-1 Alone
The effect of SIN-1 alone on the retina was also examined. The eyecups were incubated either in PBS or in PBS containing SIN-1 (0.1, 0.3, 1, or 3 mM) for 120 minutes (four times for 30 minutes each). The experiments were performed twice. 
Effect of the NO Synthase Inhibitor NMMA on the BIM23014-Induced Neuroprotective Effect
Eyecups were incubated in the presence of the chemical ischemia mixture alone or in the presence of the somatostatin sst2 analogue BIM23014 (1 μM) and BIM23014 (1 μM) plus N γ-monomethyl-l-arginine (NMMA; 0.5 mM) for 60 minutes (two times for 30 minutes each), followed by incubation two times for 30 minutes each in BIM23014 (1 μM) in the absence or presence of NMMA (0.5 mM), respectively. Control samples received PBS. These experiments were performed three times. 
Effect of the Guanylate Cyclase Inhibitors ODQ and NS2028 on the BIM23014-Induced Neuroprotective Effect
Eyecups were incubated in the presence of the chemical ischemia mixture, alone or in the presence of (1) the somatostatin sst2 analogue BIM23014 (1 μM) and (2) BIM23014 (1 μM) and ODQ (100 μM) or NS2028 (50 μM) for 60 minutes (two times for 30 minutes each), followed by incubation two times for 30 minutes each in BIM23014 (1 μM), in the absence or presence of ODQ (100 μM) or NS2028 (50 μM) respectively. ODQ is the most widely used soluble guanylyl cyclase (sGC) inhibitor, yet we also examined the effect of NS2028, chosen for its better potency and specificity. 30 Control samples received PBS. These experiments were performed three times. 
Immunohistochemical Studies
Tissue Preparation.
After completion of the chemical ischemia protocol, the eyecups were fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 1 hour at 4°C. After fixation, eyecups were rinsed in PB and incubated in 30% sucrose overnight, at 4°C for cryoprotection. Tissues were frozen in isopentane at −45°C for 1 minute and kept at −80°C until further use. Eyecups were sectioned vertically at 10 μm thickness using a cryostat, thaw mounted on slides (Superfrost; Fisher Scientific, Pittsburgh, PA) and stored at −20°C. Slices were cut near the optic nerve, every 100 μm. Nine slices were put on every slide. 
ChAT Immunoreactivity.
A mouse monoclonal antibody raised against ChAT (1:100; Biotrend, Cologne, Germany), was used as a marker for acetylcholine amacrine cells. Cryostat sections were incubated in 0.1 M Tris-HCl buffer; TBS (pH 7.4), containing 3.3% normal goat serum for 30 minutes, washed in 0.1 M TBS and incubated with the primary antibodies in 0.1 M TBS containing 0.3% Triton X-100 and 0.5% normal goat serum overnight at room temperature. The sections were washed again and incubated for 2 hours with the secondary antibody, Alexa Fluor488 goat anti-mouse IgG(H+L) (1:300; Invitrogen-Molecular Probes, Eugene, OR) Finally, the sections were washed and coverslipped with mounting medium (Vector Laboratories, Burlingame, CA). 
TUNEL Staining.
To determine cell loss, enzymatic in situ labeling of apoptosis-induced DNA strand breaks was performed using the terminal deoxynucleotidyl transferase (TDT)–mediated TMR-dUTP nick-end labeling (TUNEL) assay (Roche, Mannheim, Germany). 
Microscopy
Light microscopy images were taken with a microscope equipped with a ×20/0.50 or ×40/0.75 lens (Axioskop with a Plan-Neofluar lens; Carl Zeiss Meditec, Oberkochen, Germany). Confocal images were taken with a laser-scanning microscope (model DM RE with a He/Ne Laser; Leica, Wetzlar, Germany; with HP Plan APO; Hewlett-Packard, Palo Alto, CA). Optic sections were taken with a z-axis resolution of 1.1 μm through the immunolabeled cells. Light and contrast adjustment of images were processed with image analysis software (Photoshop, ver. 7.0; Adobe Systems, San Jose, CA). 
Results
The chemical ischemia model was successful in producing cholinergic cell loss (Figs. 1A 1B) , as previously shown. 17 To examine whether NO could provide neuroprotection against chemical ischemia in the retina, arginine, and NO donors were used in different concentrations. Neither arginine nor SNP protected the retina from the ischemic insult when these agents were co-incubated with the chemical ischemia mixture. In Figure 1 , the effect of a low and a high concentration of arginine (0.05, 2.0 mM; Figs. 1C 1D , respectively) and SNP (0.5, 4 mM; Fig. 1E 1F , respectively) is shown, while similar results were obtained with the other concentrations used (data not shown). 
The slow NO releaser NONOate afforded protection from chemical ischemia in a concentration-dependent manner (Figs. 2C 2D 2E) . The concentration of 5 μM (Fig. 2D)gave the best protection, yet it did not reach control levels. TUNEL staining (Figs. 2F 2G 2H 2I)was in agreement with the ChAT immunoreactivity data depicting an attenuation of apoptotic cells in the presence of NONOate (5 μM). 
Similar results were also obtained in the presence of SIN-1 (Fig. 3) . ChAT immunoreactivity was brought to control levels when SIN-1 (0.1 mM) was co-incubated with the chemical ischemia mixture. The protection was attenuated at higher concentrations of SIN-1 (0.3 and 1.0 mM) (Figs. 3C 3D 3E) . To assess whether this effect is due to peroxynitrite, we incubated SIN-1 in the presence of the chemical ischemia mixture and the peroxynitrite scavenger l-cysteine. As shown in Figure 3F , l-cysteine partially reduced the protective effect of SIN-1. The immunohistochemistry data were substantiated by TUNEL staining (Figs. 3G 3H 3I 3J)
To examine whether SIN-1 alone is toxic to the retina, eyecups were incubated with SIN-1 at the concentrations of 0.1, 0.3, 1.0, and 3 mM, and for the total time used in the neuroprotection protocol (120 min). As shown in Figure 4 , ChAT immunoreactivity was not affected at the concentrations used in the protection experiment (0.1, 0.3, and 1.0 mM). However, a decrease in cholinergic neurons, but not in the processes, became evident at the higher concentration of 3 mM. TUNEL immunostain also showed an increase in apoptotic cells in retinas treated with SIN-1 (3 mM; Fig. 4 ). 
While arginine/nitroprusside at the concentrations used do not support a role for NO in the neuroprotection from chemical ischemia insults, the NONOate and SIN-1 data support NO/cGMP-mediated neuroprotection. To study the direct involvement of cGMP in the neuroprotection, the cell-permeable analogue of cGMP, 8-Br-cGMP was co-incubated with the chemical ischemia mixture. 8-Br-cGMP produced a concentration dependent protective effect. The concentration of 0.5 mM gave maximum protection, as shown by ChAT immunoreactivity (Fig. 5) . The neuroprotective effect of 8-Br-cGMP was also observed with TUNEL staining (Fig. 5) , substantiating the concentration-dependent protection. 
To examine whether NOS and sGC are involved in somatostatin’s sst2-mediated neuroprotection of the retina, eyecups were incubated with the chemical ischemia mixture and the sst2-selective analogue BIM23014, in the absence and presence of the NOS inhibitor NMMA and the soluble guanylate cyclase inhibitors ODQ and NS2028. If NO and cGMP are critical in neuroprotection, inhibition of the NOS and guanylate cyclase activity would reverse the protective effect of BIM23014. As shown in Figure 6 , NMMA and the sGC inhibitors reversed the BIM23014-induced neuroprotection against chemical ischemia. The involvement of cGMP in the mediation of BIM23014’s protective effects in the chemical ischemia model was also suggested by TUNEL staining. Retinas that were incubated with BIM23014 in the presence of ODQ were characterized by an increase in apoptotic cells, mimicking the retinas that received the chemical ischemia insult. 
Discussion
The major findings of this study are the following: (1) the slow NO donor NONOate, the NO/peroxynitrite donor SIN-1, and 8-Br-cGMP protected the retina from chemical ischemia and (2) a NO/sGC/cGMP pathway was involved in the neuroprotective effects of the sst2 analogue BIM23014 in the same model. 
Ischemia is the underlying cause of retinal neovascularization, the major cause of many ocular diseases that lead to blindness. Ischemia induces the activation of voltage-gated calcium channels and ionotropic glutamate receptors, which results in an increase in intracellular calcium levels and the subsequent formation of NO. These events are believed to be the underlying cause of cell death. 19 The neuropeptide somatostatin and its sst2-specific analogues inhibited ischemia-induced neovascularization in a mouse model of oxygen-induced retinopathy. 14 15 Also, somatostatin depicted neuroprotective actions in different paradigms of neurotoxicity in the brain. 24 25 26 In a recent study, a chemical model of ischemia, initially used in hippocampal slices, 16 was used in the retina 17 and found to be a good model for examining putative neuroprotective agents. 
Incubation of the rat eyecup with the chemical ischemia mixture (IAA/NaCN) for 60 minutes affected several retinal cell populations, including cholinergic, rod bipolar and TH- and NOS-positive amacrine cells. However, the viability of photoreceptors and ganglion cells remained intact. 17 These data are in agreement with other studies showing that incubation of specific retinal cell types to chemical (KCN) and environmental hypoxia has no effect on photoreceptors. 31 Also, in a model of simulated ischemia, the removal of oxygen and N2 replacement of 95% O2 resulted in the degeneration of retinal neurons in the INL. 32  
Somatostatin analogues specific for the sst2 subtype were successful in reversing retinal cell death in this ischemia model. 17 The mechanisms by which somatostatinergic ligands act as neuroprotectants is still under investigation. The ability of somatostatin and analogues to inhibit the release of growth factors such as GH and IGF have implicated somatostatin as an antiangiogenic agent. 14 15 Somatostatin inhibits IGF-1-mediated induction of VEGF in hRPE cells, 33 and octreotide has been shown to prevent growth factor–induced proliferation of bovine retinal endothelial cells under hypoxia. 34  
Somatostatin is also known to inhibit voltage-gated calcium channels, 18 and neuronal calcium currents, the latter via a mechanism involving a cGMP-dependent protein kinase. 27 cGMP was also important in somatostatin’s protective actions against NMDA-induced neuronal death in cortical cultures. 24 The second-messenger cGMP is the product of the catalysis of GTP by the cytosolic enzyme sGC. 35 36 sGC is the physiological target of NO and NO donors such as SIN-1. 36 37 NO binds with high affinity to the heme iron of sGC which leads to its stimulation. 36  
NO has been found to promote but also antagonize ocular neovascularization. Pharmacologic inhibition of NOS reduced choroidal neovascularization and VEGF-induced neovascularization but did not reduce ischemia-induced retinal neovascularization. These studies, complemented with a genetic approach—namely, the employment of mice lacking individual or all three NOS isoforms—suggest that iNOS and/or nNOS in cells adjacent to endothelial cells in the presence of retinal ischemia has an antiangiogenic effect. 38 Also, in the developing rat retina, it has been shown that arginine and the NO donor SNAP block cell death induced by the protein synthesis inhibitor anisomycin. The antiapoptotic effect is partially mediated by cGMP. 39  
In the present study, a wide range of concentrations of arginine and the NO donor SNP were used to induce NO release that would be beneficial against chemical ischemia, but not toxic to the tissue. However, no protection was observed at any of the concentrations used. It may be that higher concentrations than the 2 mM arginine and 4 mM SNP, used in this study, are needed. Arginine at concentrations of 1, 3, and 10 mM and the NO donor SNAP (10 mM) protect the developing retina from anisomycin-induced cell death, suggesting a paracrine neuroprotective effect of nitric oxide. 39  
NaCN, an inhibitor of oxidative phosphorylation, is used for its ability to produce hypoxia. One must take into consideration the known effects of hypoxia on the arginine transporter. 40 41 It has been shown that hypoxia inhibits l-arginine uptake, an effect that would influence NO production. The lack of effect of SNP may be due to abrupt and rapid release of NO, which would enhance NaCN′s toxic effects. Alternatively, the formation of NO by SNP, which is accompanied by cyanide (CN) formation, may be inhibited by the exogenous cyanide. 37 42  
The slow NO donor NONOate protected the retina from chemical ischemia, but it did not offer full protection under the experimental paradigm. SIN-1 protected the retina from chemical ischemia in a concentration-dependent manner and offered maximum retinal protection at a concentration of 0.1 mM. No protection was observed at the higher concentration of 1.0 mM. SIN-1, the vasoactive metabolite of molsidomine, 37 43 is metabolized in two steps to SIN-1A and subsequently to NO and nitrite, nitrate, superoxide anions, and peroxynitrite. 43 44  
To examine whether the SIN-1 neuroprotective effect is due to peroxynitrite, 29 we examined whether the peroxynitrite scavenger l-cysteine would reverse its actions. The data show that l-cysteine only partially decreased the neuroprotective effects, suggesting that an NO and a peroxynitrite mechanism may be involved in SIN′s actions. The putative toxic effect of SIN-1 on the retina was also examined. Eyes cups were treated with PBS in the absence and presence of different concentrations of SIN-1. SIN-1 did not influence ChAT immunoreactivity or TUNEL staining in the retina when used at the concentrations of 0.1, 0.3, and 1 mM, concentrations that were used for the protection study. However, at the higher concentration of 3 mM a reduction of cholinergic and an increase in apoptotic cells were evident. 
It is obvious from these studies that SIN-1 at low concentrations (0.1 mM) provides protection against chemical ischemia, whereas at high concentrations (3 mM), it leads to cell death (Fig. 3) . It is impossible to make any suggestions as to the resultant concentrations of NO that may play a role in the actions of SIN-1. In a recent study in which an NO electrode and in vivo microdialysis were used, SIN-1 (1 mM) did not increase NO levels in the striatum. 45 There are reports suggesting that SIN-1 has a direct stimulant effect on the soluble guanylate cyclase. 44 46 47 The present data suggest that at low concentrations, SIN-1 may promote neuroprotection via a cGMP mechanism, whereas at higher concentrations, it can be toxic. Whether the toxic effects are mediated by its metabolite peroxynitrite, known to induce toxicity in different paradigms, 48 49 could not be ascertained by the present experiments. Instead, a peroxynitrite mechanism may be partially involved in the protective actions of SIN-1, as suggested by the experiments with the peroxynitrite scavenger l-cysteine. Both NO and peroxynitrite have the ability to activate a soluble guanylyl cyclase and increase cGMP levels that may be neuroprotective. 44 50  
The involvement of cGMP in the protection of the retina from chemical ischemia was substantiated by the direct use of 8-Br-cGMP. This cell-permeable analogue of cGMP protected the retina from chemical ischemia in a concentration-dependent manner with maximum protection at 0.5 mM and partial protection at 1 mM. 8-Br-cGMP (1 mM) was also shown to have a partial protective effect against anisomycin-induced cell death in the developing retina. 39  
It is evident from the present study that a cGMP mechanism is involved in the protection of the retina from chemical ischemia. As stated earlier, somatostatin has been shown to increase NO and cGMP levels in the retina by activating the sst2 receptor subtype, 12 28 whereas sst2 selective ligands were found to protect the retina from chemical ischemia, 17 as SIN-1 and 8-Br-cGMP did in the present study. In paradigms of neurotoxicity in the brain, somatostatin was shown to have neuroprotective effects by a cGMP dependent mechanism. 24 To examine whether a NO/cGMP mechanism mediates the neuroprotection offered by the sst2 receptor activation to the retina, 17 we examined whether the inhibition of NOS and the soluble guanylyl cyclase was able to reverse the protective effect of the sst2 agonist. NMMA and the two sGC inhibitors ODQ and NS 2028 reversed the protective effect of BIM23014, thus implicating NO/sGC and cGMP in the neuroprotection. To further ascertain the importance of cGMP, assays were performed to assess cGMP levels in retinas treated with PBS or chemical ischemia alone or in the presence of BIM23014 and BIM23014 plus NS 2028. However, no statistically significant differences in cGMP levels were observed, perhaps for technical reasons or because of the abundance of sGC in the vasculature. Under the experimental conditions used, the isolation of the latter may have yielded differences in cGMP levels. 
The subsequent signaling by which cGMP offers protection may involve the regulation of calcium channels 27 and the reduction of the toxic high levels of intracellular calcium induced by ischemia. 19 Although there are no relevant reports in retinal circuitry to support this conjecture, it has been shown that NO/cGMP mediates the inhibition of calcium channels in retinal pericytes and reduces calcium influx. 51  
In addition, it has been shown that peroxynitrite activates voltage-dependent calcium channels (VDCCs) 52 and influences neurotransmitter release. 53 Recent studies have indicated that NO, cGMP and SIN-1 can stimulate the release of GABA. 54 Inhibitory neurotransmitters such as GABA could counteract the toxic influence of glutamate on retinal neurons during retinal ischemia and would be expected to provide protection. 19 Actually, GABA has been suggested as a neuroprotective agent in brain acute ischemic stroke. 55 Therefore, one cannot exclude the possibility that NO, cGMP, and SIN-1 increase GABA levels in rat retina and assist in the neuroprotection in the present paradigm. However, this conjecture must be substantiated. 
In conclusion, the present study reports for the first time that NO/peroxynitrite and cGMP are important mediators in the protection of rat retina from chemical ischemia. Furthermore, the data support the involvement of the NO/sGC/cGMP signaling pathway in the neuroprotective effects bestowed on the retina by the sst2 somatostatin ligands in the same model. 
 
Figure 1.
 
Effect of Arg and SNP on the chemical ischemia (CI) induced damage of ChAT containing neurons in the retina. (A) ChAT immunoreactivity was localized in amacrine cells in the inner nuclear layer (INL), in displaced amacrine cells in the ganglion cell layer (GCL), and in processes in the inner plexiform layer (IPL) in retinas treated with PBS (control). No immunoreactivity was observed in retinas treated with the CI mixture (B) or with the CI mixture and Arg (0.05 mM, C; 2 mM, D), and SNP (0.5 mM, Ε; 4 mM, F). Bars, 40 μm.
Figure 1.
 
Effect of Arg and SNP on the chemical ischemia (CI) induced damage of ChAT containing neurons in the retina. (A) ChAT immunoreactivity was localized in amacrine cells in the inner nuclear layer (INL), in displaced amacrine cells in the ganglion cell layer (GCL), and in processes in the inner plexiform layer (IPL) in retinas treated with PBS (control). No immunoreactivity was observed in retinas treated with the CI mixture (B) or with the CI mixture and Arg (0.05 mM, C; 2 mM, D), and SNP (0.5 mM, Ε; 4 mM, F). Bars, 40 μm.
Figure 2.
 
Effect of NONOate on the chemical ischemia (CI)-induced damage of ChAT-containing neurons and TUNEL staining in the retina. ChAT immunoreactivity was present or absent in (A) control and (Β) CI–treated retinas, respectively. In retinas treated with the CI mixture and NONOate (1 μM, C; 5 μM, D; 10 μM, E), a concentration-dependent recovery of ChAT immunoreactivity was observed. TUNEL staining depicted major cell loss in retinas treated with CI (G) compared with control retinas (F). NONOate (1 μM, H; 5 μM, I) reduced the apoptotic damage in a concentration-dependent manner compared with (G). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 50 μm.
Figure 2.
 
Effect of NONOate on the chemical ischemia (CI)-induced damage of ChAT-containing neurons and TUNEL staining in the retina. ChAT immunoreactivity was present or absent in (A) control and (Β) CI–treated retinas, respectively. In retinas treated with the CI mixture and NONOate (1 μM, C; 5 μM, D; 10 μM, E), a concentration-dependent recovery of ChAT immunoreactivity was observed. TUNEL staining depicted major cell loss in retinas treated with CI (G) compared with control retinas (F). NONOate (1 μM, H; 5 μM, I) reduced the apoptotic damage in a concentration-dependent manner compared with (G). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 50 μm.
Figure 3.
 
Effect of SIN-1 and SIN+l-cysteine on the chemical ischemia(CI)-induced damage of ChAT containing neurons and TUNEL staining in the retina. ChAT immunoreactivity was present and absent in control (A) and CI-treated (Β) retinas, respectively. In retinas treated with the CI mixture and SIN-1 (0.1 mM, C; 0.3 mM, D; 1 mM, E) a concentration-dependent recovery of ChAT immunoreactivity was observed. l-Cysteine (5 mM) partially reversed the protective effect of 0.1 mM SIN-1 (F). TUNEL staining in PBS (G), CI (H), CI/SIN (I), and CI/SIN/l-Cys (J). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 50 μm.
Figure 3.
 
Effect of SIN-1 and SIN+l-cysteine on the chemical ischemia(CI)-induced damage of ChAT containing neurons and TUNEL staining in the retina. ChAT immunoreactivity was present and absent in control (A) and CI-treated (Β) retinas, respectively. In retinas treated with the CI mixture and SIN-1 (0.1 mM, C; 0.3 mM, D; 1 mM, E) a concentration-dependent recovery of ChAT immunoreactivity was observed. l-Cysteine (5 mM) partially reversed the protective effect of 0.1 mM SIN-1 (F). TUNEL staining in PBS (G), CI (H), CI/SIN (I), and CI/SIN/l-Cys (J). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 50 μm.
Figure 4.
 
Effect of SIN-1 on ChAT immunoreactivity and TUNEL staining in the retina. Retinas were treated with (A) PBS, (B) 0.1 mM SIN-1, (C) 0.3 mM SIN-1, (D) 1 mM SIN-1, and (E) 3 mM SIN-1. Only at the highest concentration, SIN-1 (3 mM) alone produced loss of ChAT-stained cell bodies (E), and an increase in TUNEL staining (H). No TUNEL staining was observed in control retinas (F) and in retinas treated with the lower concentration of 0.1 mM SIN-1 (G). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 20 μm.
Figure 4.
 
Effect of SIN-1 on ChAT immunoreactivity and TUNEL staining in the retina. Retinas were treated with (A) PBS, (B) 0.1 mM SIN-1, (C) 0.3 mM SIN-1, (D) 1 mM SIN-1, and (E) 3 mM SIN-1. Only at the highest concentration, SIN-1 (3 mM) alone produced loss of ChAT-stained cell bodies (E), and an increase in TUNEL staining (H). No TUNEL staining was observed in control retinas (F) and in retinas treated with the lower concentration of 0.1 mM SIN-1 (G). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 20 μm.
Figure 5.
 
Effect of 8-Br-cGMP on the chemical ischemia (CI)-induced damage of ChAT-containing neurons and TUNEL staining in the retina. ChAT immunoreactivity in control (A) and CI-treated (Β) retinas. A concentration-dependent recovery of ChAT immunoreactivity was observed in the presence of the CI mixture and 8-Br-cGMP (0.1 mM, C; 0.5 mM, D; 1 mM, E). No TUNEL staining was observed in control retinas (F), whereas a major cell loss was found in retinas treated with CI (G). Retinas treated with CI and 8-Br-cGMP (0.5 mM, H; or 0.1 mM, I) displayed no cell death or minimal cell death, respectively. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 50 μm.
Figure 5.
 
Effect of 8-Br-cGMP on the chemical ischemia (CI)-induced damage of ChAT-containing neurons and TUNEL staining in the retina. ChAT immunoreactivity in control (A) and CI-treated (Β) retinas. A concentration-dependent recovery of ChAT immunoreactivity was observed in the presence of the CI mixture and 8-Br-cGMP (0.1 mM, C; 0.5 mM, D; 1 mM, E). No TUNEL staining was observed in control retinas (F), whereas a major cell loss was found in retinas treated with CI (G). Retinas treated with CI and 8-Br-cGMP (0.5 mM, H; or 0.1 mM, I) displayed no cell death or minimal cell death, respectively. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 50 μm.
Figure 6.
 
Effect of ODQ, NS 2028, and NMMA on the neuroprotective actions of BIM23014. ChAT immunoreactivity is present in retinas treated with PBS (A) and BIM23014 in the presence of the CI mixture (C). It is absent in retinas treated with the CI mixture alone (Β) or with the CI mixture and BIM23014/ODQ (D), BIM23014/NS2028 (E), and BIM23014/NMMA (F). No TUNEL staining is observed in control retinas (G) and in retinas treated with BIM23014 (I), in contrast to retinas treated with CI mixture alone (H) or with BIM23014/ODQ (J). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 20 μm.
Figure 6.
 
Effect of ODQ, NS 2028, and NMMA on the neuroprotective actions of BIM23014. ChAT immunoreactivity is present in retinas treated with PBS (A) and BIM23014 in the presence of the CI mixture (C). It is absent in retinas treated with the CI mixture alone (Β) or with the CI mixture and BIM23014/ODQ (D), BIM23014/NS2028 (E), and BIM23014/NMMA (F). No TUNEL staining is observed in control retinas (G) and in retinas treated with BIM23014 (I), in contrast to retinas treated with CI mixture alone (H) or with BIM23014/ODQ (J). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 20 μm.
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Figure 1.
 
Effect of Arg and SNP on the chemical ischemia (CI) induced damage of ChAT containing neurons in the retina. (A) ChAT immunoreactivity was localized in amacrine cells in the inner nuclear layer (INL), in displaced amacrine cells in the ganglion cell layer (GCL), and in processes in the inner plexiform layer (IPL) in retinas treated with PBS (control). No immunoreactivity was observed in retinas treated with the CI mixture (B) or with the CI mixture and Arg (0.05 mM, C; 2 mM, D), and SNP (0.5 mM, Ε; 4 mM, F). Bars, 40 μm.
Figure 1.
 
Effect of Arg and SNP on the chemical ischemia (CI) induced damage of ChAT containing neurons in the retina. (A) ChAT immunoreactivity was localized in amacrine cells in the inner nuclear layer (INL), in displaced amacrine cells in the ganglion cell layer (GCL), and in processes in the inner plexiform layer (IPL) in retinas treated with PBS (control). No immunoreactivity was observed in retinas treated with the CI mixture (B) or with the CI mixture and Arg (0.05 mM, C; 2 mM, D), and SNP (0.5 mM, Ε; 4 mM, F). Bars, 40 μm.
Figure 2.
 
Effect of NONOate on the chemical ischemia (CI)-induced damage of ChAT-containing neurons and TUNEL staining in the retina. ChAT immunoreactivity was present or absent in (A) control and (Β) CI–treated retinas, respectively. In retinas treated with the CI mixture and NONOate (1 μM, C; 5 μM, D; 10 μM, E), a concentration-dependent recovery of ChAT immunoreactivity was observed. TUNEL staining depicted major cell loss in retinas treated with CI (G) compared with control retinas (F). NONOate (1 μM, H; 5 μM, I) reduced the apoptotic damage in a concentration-dependent manner compared with (G). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 50 μm.
Figure 2.
 
Effect of NONOate on the chemical ischemia (CI)-induced damage of ChAT-containing neurons and TUNEL staining in the retina. ChAT immunoreactivity was present or absent in (A) control and (Β) CI–treated retinas, respectively. In retinas treated with the CI mixture and NONOate (1 μM, C; 5 μM, D; 10 μM, E), a concentration-dependent recovery of ChAT immunoreactivity was observed. TUNEL staining depicted major cell loss in retinas treated with CI (G) compared with control retinas (F). NONOate (1 μM, H; 5 μM, I) reduced the apoptotic damage in a concentration-dependent manner compared with (G). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 50 μm.
Figure 3.
 
Effect of SIN-1 and SIN+l-cysteine on the chemical ischemia(CI)-induced damage of ChAT containing neurons and TUNEL staining in the retina. ChAT immunoreactivity was present and absent in control (A) and CI-treated (Β) retinas, respectively. In retinas treated with the CI mixture and SIN-1 (0.1 mM, C; 0.3 mM, D; 1 mM, E) a concentration-dependent recovery of ChAT immunoreactivity was observed. l-Cysteine (5 mM) partially reversed the protective effect of 0.1 mM SIN-1 (F). TUNEL staining in PBS (G), CI (H), CI/SIN (I), and CI/SIN/l-Cys (J). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 50 μm.
Figure 3.
 
Effect of SIN-1 and SIN+l-cysteine on the chemical ischemia(CI)-induced damage of ChAT containing neurons and TUNEL staining in the retina. ChAT immunoreactivity was present and absent in control (A) and CI-treated (Β) retinas, respectively. In retinas treated with the CI mixture and SIN-1 (0.1 mM, C; 0.3 mM, D; 1 mM, E) a concentration-dependent recovery of ChAT immunoreactivity was observed. l-Cysteine (5 mM) partially reversed the protective effect of 0.1 mM SIN-1 (F). TUNEL staining in PBS (G), CI (H), CI/SIN (I), and CI/SIN/l-Cys (J). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 50 μm.
Figure 4.
 
Effect of SIN-1 on ChAT immunoreactivity and TUNEL staining in the retina. Retinas were treated with (A) PBS, (B) 0.1 mM SIN-1, (C) 0.3 mM SIN-1, (D) 1 mM SIN-1, and (E) 3 mM SIN-1. Only at the highest concentration, SIN-1 (3 mM) alone produced loss of ChAT-stained cell bodies (E), and an increase in TUNEL staining (H). No TUNEL staining was observed in control retinas (F) and in retinas treated with the lower concentration of 0.1 mM SIN-1 (G). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 20 μm.
Figure 4.
 
Effect of SIN-1 on ChAT immunoreactivity and TUNEL staining in the retina. Retinas were treated with (A) PBS, (B) 0.1 mM SIN-1, (C) 0.3 mM SIN-1, (D) 1 mM SIN-1, and (E) 3 mM SIN-1. Only at the highest concentration, SIN-1 (3 mM) alone produced loss of ChAT-stained cell bodies (E), and an increase in TUNEL staining (H). No TUNEL staining was observed in control retinas (F) and in retinas treated with the lower concentration of 0.1 mM SIN-1 (G). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 20 μm.
Figure 5.
 
Effect of 8-Br-cGMP on the chemical ischemia (CI)-induced damage of ChAT-containing neurons and TUNEL staining in the retina. ChAT immunoreactivity in control (A) and CI-treated (Β) retinas. A concentration-dependent recovery of ChAT immunoreactivity was observed in the presence of the CI mixture and 8-Br-cGMP (0.1 mM, C; 0.5 mM, D; 1 mM, E). No TUNEL staining was observed in control retinas (F), whereas a major cell loss was found in retinas treated with CI (G). Retinas treated with CI and 8-Br-cGMP (0.5 mM, H; or 0.1 mM, I) displayed no cell death or minimal cell death, respectively. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 50 μm.
Figure 5.
 
Effect of 8-Br-cGMP on the chemical ischemia (CI)-induced damage of ChAT-containing neurons and TUNEL staining in the retina. ChAT immunoreactivity in control (A) and CI-treated (Β) retinas. A concentration-dependent recovery of ChAT immunoreactivity was observed in the presence of the CI mixture and 8-Br-cGMP (0.1 mM, C; 0.5 mM, D; 1 mM, E). No TUNEL staining was observed in control retinas (F), whereas a major cell loss was found in retinas treated with CI (G). Retinas treated with CI and 8-Br-cGMP (0.5 mM, H; or 0.1 mM, I) displayed no cell death or minimal cell death, respectively. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 50 μm.
Figure 6.
 
Effect of ODQ, NS 2028, and NMMA on the neuroprotective actions of BIM23014. ChAT immunoreactivity is present in retinas treated with PBS (A) and BIM23014 in the presence of the CI mixture (C). It is absent in retinas treated with the CI mixture alone (Β) or with the CI mixture and BIM23014/ODQ (D), BIM23014/NS2028 (E), and BIM23014/NMMA (F). No TUNEL staining is observed in control retinas (G) and in retinas treated with BIM23014 (I), in contrast to retinas treated with CI mixture alone (H) or with BIM23014/ODQ (J). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 20 μm.
Figure 6.
 
Effect of ODQ, NS 2028, and NMMA on the neuroprotective actions of BIM23014. ChAT immunoreactivity is present in retinas treated with PBS (A) and BIM23014 in the presence of the CI mixture (C). It is absent in retinas treated with the CI mixture alone (Β) or with the CI mixture and BIM23014/ODQ (D), BIM23014/NS2028 (E), and BIM23014/NMMA (F). No TUNEL staining is observed in control retinas (G) and in retinas treated with BIM23014 (I), in contrast to retinas treated with CI mixture alone (H) or with BIM23014/ODQ (J). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 20 μm.
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