Cell-specific caspase expression by different neuronal phenotypes in transient retinal ischemia

Emerging evidence supports an important role for caspases in neuronal death following ischemia-reperfusion injury. This study assessed whether cell specific caspases participate in neuronal degeneration and whether caspase inhibition provides neuroprotection following transient retinal ischemia. We utilized a model of transient global retinal ischemia. The spatial and temporal pattern of the active forms of caspase 1, 2 and 3 expression was determined in retinal neurons following ischemic injury. Double-labeling with cell-specific markers identified which cells were expressing different caspases. In separate experiments, animals received various caspase inhibitors before the induction of ischemia. Sixty minutes of ischemia resulted in a delayed, selective neuronal death of the inner retinal layers at 7 days. Expression of caspase 1 was not detected at any time point. Maximal expression of caspase 2 was found at 24 h primarily in the inner nuclear and ganglion cell layers of the retina and localized to ganglion and amacrine neurons. Caspase 3 also peaked at 24 h in both the inner nuclear and outer nuclear layers and was predominantly expressed in photoreceptor cells and to a lesser extent in amacrine neurons. The pan caspase inhibitor, Boc-aspartyl fmk, or an antisense oligonucleotide inhibitor of caspase 2 led to significant histopathologic and functional improvement (electroretinogram) at 7 days. No protection was found with the caspase 1 selective inhibitor, Y-vad fmk. These observations suggest that ischemia-reperfusion injury activates different caspases depending on the neuronal phenotype in the retina and caspase inhibition leads to both histologic preservation and functional improvement. Caspases 2 and 3 may act in parallel in amacrine neurons following ischemia-reperfusion. These results in the retina may shed light on differential caspase specificity in global cerebral ischemia.     

Transplantation of photoreceptor and total neural retina preserves cone function in P23H rhodopsin transgenic rat

Background

Transplantation as a therapeutic strategy for inherited retinal degeneration has been historically viewed to restore vision as a method by replacing the lost retinal cells and attempting to reconstruct the neural circuitry with stem cells, progenitor cells and mature neural retinal cells.

Methods and Findings

We present evidence for an alternative strategy aimed at preventing the secondary loss of cones, the most crucial photoreceptors for vision, by transplanting normal photoreceptors cells into the eye of the P23H rat, a model of dominant retinitis pigmentosa. We carried out transplantation of photoreceptors or total neural retina in 3-month-old P23H rats and evaluated the function and cell counts 6 months after surgery. In both groups, cone loss was significantly reduced (10%) in the transplanted eyes where the cone outer segments were found to be considerably longer. This morphological effect correlated with maintenance of the visual function of cones as scored by photopic ERG recording, but more precisely with an increase in the photopic b-wave amplitudes by 100% and 78% for photoreceptor transplantation and whole retinal transplantation respectively.

Conclusions

We demonstrate here that the transplanted tissue prevents the loss of cone function, which is further translated into cone survival.     

Activation of autophagy in a rat model of retinal ischemia following high intraocular pressure

Acute primary open angle glaucoma is an optic neuropathy characterized by the elevation of intraocular pressure, which causes retinal ischemia and neuronal death. Rat ischemia/reperfusion enhances endocytosis of both horseradish peroxidase (HRP) or fluorescent dextran into ganglion cell layer (GCL) neurons 24 h after the insult. We investigated the activation of autophagy in GCL-neurons following ischemia/reperfusion, using acid phosphatase (AP) histochemistry and immunofluorescence against LC3 and LAMP1. Retinal I/R lead to the appearance of AP-positive granules and LAMP1-positive vesicles 12 and 24 h after the insult, and LC3 labelling at 24 h, and induced a consistent retinal neuron death. At 48 h the retina was negative for autophagic markers. In addition, Western Blot analysis revealed an increase of LC3 levels after damage: the increase in the conjugated, LC3-II isoform is suggestive of autophagic activity. Inhibition of autophagy by 3-methyladenine partially prevented death of neurons and reduces apoptotic markers, 24 h post-lesion. The number of neurons in the GCL decreased significantly following I/R (I/R 12.21±1.13 vs controls 19.23±1.12 cells/500 μm); this decrease was partially prevented by 3-methyladenine (17.08±1.42 cells/500 μm), which potently inhibits maturation of autophagosomes. Treatment also prevented the increase in glial fibrillary acid protein immunoreactivity elicited by I/R. Therefore, targeting autophagy could represent a novel and promising treatment for glaucoma and retinal ischemia.     

Nicotinamide attenuates retinal ischemia and light insults to neurones

The aim of the present studies was to determine whether nicotinamide is effective in blunting the negative influence of ischemia/reperfusion to the rat retina in situ and of light to transformed retinal ganglion cells (RGC-5 cells) in culture. Ischemia was delivered to the retina of one eye of rats by raising the intraocular pressure. Nicotinamide was administered intraperitoneally just before ischemia and into the vitreous immediately after the insult. Electroretinograms (ERGs) of both eyes were recorded before and 5 days after ischemia. Seven days after ischemia, retinas were analysed for the localization of various antigens. Retinal and optic nerve extracts were also prepared for analysis of specific proteins and mRNAs. Also, RGC-5 cells in culture were given a light insult (1000 lux, 48 and 96 h) and evidence for reduced viability and apoptosis determined by a variety of procedures. Nicotinamide was added to some cultures to see whether it reversed the negative effect of light. Ischemia/reperfusion to the retina affected the localization of Thy-1, neuronal nitric oxide synthase (NOS) and choline acetyltransferase (ChAT), the a- and b-wave amplitudes of the ERG, the content of various retinal and optic nerve proteins and mRNAs. Significantly, nicotinamide statistically blunted many of the effects induced by ischemia/reperfusion which included the activation of poly-ADP-ribose polymerase (PARP). Light-induced apoptosis of RGC-5 cells in culture was attenuated by nicotinamide and the PARP inhibitor NU1025. The presented data show that nicotinamide attenuates injury to the retina and RGC-5 cells in culture caused by ischemia/reperfusion and by light, respectively. Evidence is provided to suggest that nicotinamide acts as a PARP inhibitor and possibly an antioxidant.     

Global and Ocular Hypothermic Preconditioning Protect the Rat Retina from Ischemic Damage

Retinal ischemia could provoke blindness. At present, there is no effective treatment against retinal ischemic damage. Strong evidence supports that glutamate is implicated in retinal ischemic damage. We investigated whether a brief period of global or ocular hypothermia applied 24 h before ischemia (i.e. hypothermic preconditioning, HPC) protects the retina from ischemia/reperfusion damage, and the involvement of glutamate in the retinal protection induced by HPC. For this purpose, ischemia was induced by increasing intraocular pressure to 120 mm Hg for 40 min. One day before ischemia, animals were submitted to global or ocular hypothermia (33°C and 32°C for 20 min, respectively) and fourteen days after ischemia, animals were subjected to electroretinography and histological analysis. Global or ocular HPC afforded significant functional (electroretinographic) protection in eyes exposed to ischemia/reperfusion injury. A marked alteration of the retinal structure and a decrease in retinal ganglion cell number were observed in ischemic retinas, whereas global or ocular HPC significantly preserved retinal structure and ganglion cell count. Three days after ischemia, a significant decrease in retinal glutamate uptake and glutamine synthetase activity was observed, whereas ocular HPC prevented the effect of ischemia on these parameters. The intravitreal injection of supraphysiological levels of glutamate induced alterations in retinal function and histology which were significantly prevented by ocular HPC. These results support that global or ocular HPC significantly protected retinal function and histology from ischemia/reperfusion injury, probably through a glutamate-dependent mechanism.     

Taurine deficiency damages retinal neurones: cone photoreceptors and retinal ganglion cells

In 1970s, taurine deficiency was reported to induce photoreceptor degeneration in cats and rats. Recently, we found that taurine deficiency contributes to the retinal toxicity of vigabatrin, an antiepileptic drug. However, in this toxicity, retinal ganglion cells were degenerating in parallel to cone photoreceptors. The aim of this study was to re-assess a classic mouse model of taurine deficiency following a treatment with guanidoethane sulfonate (GES), a taurine transporter inhibitor to determine whether retinal ganglion cells are also affected. GES treatment induced a significant reduction in the taurine plasma levels and a lower weight increase. At the functional level, photopic electroretinograms were reduced indicating a dysfunction in the cone pathway. A change in the autofluorescence appearance of the eye fundus was explained on histological sections by an increased autofluorescence of the retinal pigment epithelium. Although the general morphology of the retina was not affected, cell damages were indicated by the general increase in glial fibrillary acidic protein expression. When cell quantification was achieved on retinal sections, the number of outer/inner segments of cone photoreceptors was reduced (20?%) as the number of retinal ganglion cells (19?%). An abnormal synaptic plasticity of rod bipolar cell dendrites was also observed in GES-treated mice. These results indicate that taurine deficiency can not only lead to photoreceptor degeneration but also to retinal ganglion cell loss. Cone photoreceptors and retinal ganglion cells appear as the most sensitive cells to taurine deficiency. These results may explain the recent therapeutic interest of taurine in retinal degenerative pathologies.     

A model of anuran retina relating interneurons to ganglion cell responses

A model is presented which accounts for many characteristic response properties used to classify anuran ganglion cell types while being consistent with data concerning interneurons. In the model color is ignored and input stimuli are assumed to be only black and white at high contrast. We show that accurate ganglion cell responses are obtained even with simplified receptors and horizontal cells: Receptors are modeled as responding with a step change, while horizontal cells respond only to global changes in intensity brought about by full field illumination changes. A hyperpolarizing and depolarizing bipolar cell are generated y subtracting local receptor and horizontal potentials. Two transient amacrine cells (On and Off) are generated using a high-pass filter like mechanism with a thresholded output which responds to positive going changes in the corresponding bipolar cell potentials. The model shows how a selective combination of bipolar and amacrine channels can account for many of the response properties used to classify the anuran ganglion cell types (class-0 through 4) and makes several experimental predictions.     

Intraoperative Electroretinograms before and after Core Vitrectomy

Purpose

To evaluate retinal function by intraoperative electroretinograms (ERGs) before and after core vitrectomy.

Design

Retrospective consecutive case series.

Method

Full-field photopic ERGs were recorded prior to the beginning and just after core vitrectomy using a sterilized contact lens electrode in 20 eyes that underwent non-complicated vitreous surgery. A light-emitted diode was embedded into the contact lens, and a stimulus of 150 ms on and 350 ms off at 2 Hz was delivered. The amplitudes and latencies of the a-, b-, and d-waves, photopic negative response (PhNR), and oscillatory potentials (OPs) were analyzed. The intraocular temperature at the mid-vitreous was measured at the beginning and just after the surgery with a thermoprobe.

Results

The intraocular temperature was 33.2 ± 1.3°C before and 29.4 ± 1.7°C after the vitrectomy. The amplitudes of the PhNR and OPs were significantly smaller after surgery, and the latencies of all components were prolonged after the surgery. These changes were not significantly correlated with the changes of the temperature.

Conclusion

Retinal function is reduced just after core vitrectomy in conjunction with significant temperature reduction. The differences in the degree of alterations of each ERG component suggests different sensitivity of each type of retinal neuron.     

Retinal cell type‐specific prevention of ischemia‐induced damages by LPS‐TLR4 signaling through microglia

Reprogramming of toll‐like receptor 4 (TLR4) by brief ischemia or lipopolysacharide (LPS) contributes to superintending tolerance against destructive ischemia in brain. However, beneficial roles of TLR4 signaling in ischemic retina are not well known. This study demonstrated that preconditioning with LPS 48 h prior to the retinal ischemia prevents the cellular damage in morphology with hematoxylin and eosin (H&E) staining and functions of retina with electroretinogram (ERG), while post‐ischemia treatment deteriorated it. The preventive effects of LPS preconditioning showed the cell type‐specificity of retinal cells. There was complete rescue of ganglion cells, partial rescue of bipolar and photoreceptor cells or no rescue of amacrine cells, respectively. LPS treatment caused the proliferation and migration of retinal microglia and its preconditioning prevented the ischemia‐induced microglial activation. Preventive actions from cell damages following LPS preconditioning prior to retinal ischemia were abolished in TLR4 knock‐out mice, and by pre‐treatments with anti‐TLR4 antibody or minocycline, a microglia inhibitor, which themselves had no effects on the retinal ischemia‐induced damages or microglia activation. Thus, this study revealed that TLR4 mediates the LPS preconditioning‐induced preventive effects through microglial activation in the retinal ischemia model.     

Vinpocetine regulates cation channel permeability of inner retinal neurons in the ischaemic retina

Vinpocetine is a natural drug which exerts neuroprotective effects in ischaemia of the brain through actions on cation channels, glutamate receptors and other pathways. This study investigated the effect of vinpocetine on cation channel permeability of inner retinal neurons after acute retinal metabolic insult. We focused on amacrine and ganglion cells immunoreactive for calretinin or parvalbumin due to their previously documented susceptibility to ischaemia. Using the probe, 1-amino-4-guanidobutane (AGB), we observed increased cation channel permeability across amacrine and ganglion cells under ischaemia and hypoglycaemia but not anoxia. Calretinin and parvalbumin immunoreactivity was also reduced during ischaemia and hypoglyacemia but not anoxia. Vinpocetine decreased AGB entry into ischaemic and hypoglycaemic ganglion cells indicating that the drug can modulate unregulated cation entry. In addition, vinpocetine prevented the loss of calretinin and parvalbumin immunoreactivity following ischaemia suggesting it may indirectly regulate intracellular calcium. Vinpocetine also reduced AGB permeability in selected amacrine and ganglion cell populations following N-methyl-D-aspartate (NMDA) but not kainate activation suggesting that vinpocetine’s regulation of cation channel permeability may partly involve NMDA sensitive glutamate receptors.     

Neuroprotective Effect of Tauroursodeoxycholic Acid on N-Methyl-D-Aspartate-Induced Retinal Ganglion Cell Degeneration

Retinal ganglion cell degeneration underlies the pathophysiology of diseases affecting the retina and optic nerve. Several studies have previously evidenced the anti-apoptotic properties of the bile constituent, tauroursodeoxycholic acid, in diverse models of photoreceptor degeneration. The aim of this study was to investigate the effects of systemic administration of tauroursodeoxycholic acid on N-methyl-D-aspartate (NMDA)-induced damage in the rat retina using a functional and morphological approach. Tauroursodeoxycholic acid was administered intraperitoneally before and after intravitreal injection of NMDA. Three days after insult, full-field electroretinograms showed reductions in the amplitudes of the positive and negative-scotopic threshold responses, scotopic a- and b-waves and oscillatory potentials. Quantitative morphological evaluation of whole-mount retinas demonstrated a reduction in the density of retinal ganglion cells. Systemic administration of tauroursodeoxycholic acid attenuated the functional impairment induced by NMDA, which correlated with a higher retinal ganglion cell density. Our findings sustain the efficacy of tauroursodeoxycholic acid administration in vivo, suggesting it would be a good candidate for the pharmacological treatment of degenerative diseases coursing with retinal ganglion cell loss.     

Modulation of pro-survival and death-associated pathways under retinal ischemia/reperfusion: effects of NMDA receptor blockade

Loss of retinal ganglion cells occurs in a variety of pathological conditions, including central retinal artery occlusion, diabetes and glaucoma. Using an experimental model of retinal ischemia induced by transiently raise the intraocular pressure (IOP), In this study, we report the original observation that ischemic retinal ganglion cells death is associated with the transient deactivation of the pro-survival kinase Akt and activation of GSK-3beta followed, during reperfusion, by a longer lasting, PI3K-dependent, activation of Akt and phosphorylation of GSK-3beta. Under these experimental conditions, retinal ischemia induced the expression of Bad, a pro-apoptotic protein, member of the Bcl-2 family. The detrimental effects yielded by the ischemic stimulus were minimized by intravitreal administration of the NMDA receptor antagonist, MK801, that reduced the expression of Bad and significantly increased Akt phosphorylation. In conclusion, our present results contribute to unravel the mechanisms underlying retinal damage by high IOP-induced transient ischemia in rat. In addition, these data implicate the pro-survival PI3K/Akt pathway and the observed reduced expression of Bad in the neuroprotection afforded by MK801.     

Gap junctions with amacrine cells provide a feedback pathway for ganglion cells within the retina.

In primates, one type of retinal ganglion cell, the parasol cell, makes gap junctions with amacrine cells, the inhibitory, local circuit neurons. To study the effects of these gap junctions, we developed a linear, mathematical model of the retinal circuitry providing input to parasol cells. Electrophysiological studies have indicated that gap junctions do not enlarge the receptive field centres of parasol cells, but our results suggest that they make other contributions to their light responses. According to our model, the coupled amacrine cells enhance the responses of parasol cells to luminance contrast by disinhibition. We also show how a mixed chemical and electrical synapse between two sets of amacrine cells presynaptic to the parasol cells might make the responses of parasol cells more transient and, therefore, more sensitive to motion. Finally, we show how coupling via amacrine cells can synchronize the firing of parasol cells. An action potential in a model parasol cell can excite neighbouring parasol cells, but only when the coupled amacrine cells also fire action potentials. Passive conduction was ineffective due to low-pass temporal filtering. Inhibition from the axons of the coupled amacrine cells also produced oscillations that might synchronize the firing of more distant ganglion cells.     

Trigger features and excitation in the retina

This review focuses on recent advances in our understanding of how neural divergence and convergence give rise to complex encoding properties of retinal ganglion cells. We describe the apparent mismatch between the number of cone bipolar cell types, and the diversity of excitatory input to retinal ganglion cells, and outline two possible solutions. One proposal is for diversity in the excitatory pathways to be generated within axon terminals of cone bipolar cells, and the second invokes narrow-field glycinergic amacrine cells that can apparently act like bipolar cells by providing excitatory drive to ganglion cells. Finally we highlight two advances in technique that promise to provide future insights; automation of electron microscope data collection and analysis, and the use of the ideal observer to quantitatively compare neural performance at all levels.     

Neuroprotective Effects of Bis(7)-tacrine in a Rat Model of Pressure-Induced Retinal Ischemia

The retinal ischemia–reperfusion model has been studied extensively and is an ideal animal model for studying clinical situations such as acute glaucoma and optic neuropathy. Our previous reports showed that bis(7)-tacrine had neuroprotective effects against glutamate-induced retinal ganglion cells damage through the drug’s anti-NMDA receptor effects. Here, we investigated whether bis(7)-tacrine protects the retina from ischemic injury in a rat model. Retinal ischemia was induced by raising the intraocular pressure to 120 mmHg for 90 min. Rats received intraperitoneal injections of 0.2 mg/kg bis(7)-tacrine or saline at 30 min before ischemia, and then twice a day after retinal ischemia. Morphometric evaluation showed that bis(7)-tacrine dramatically reduced the retinal damage compared with the control group. Moreover, bis(7)-tacrine suppressed ischemia-induced reductions in a- and b-wave amplitudes of electroretinography. Protein levels of p53, the tumor suppressor gene known to induce apoptosis, were increased after ischemic injury, and treatment with bis(7)-tacrine reduced the expression of the protein. Our results suggest that bis(7)-tacrine has a neuroprotective effect against ischemic injury in the rat retina, possibly through the drug’s anti-apoptotic effects. Bis(7)-tacrine may potentially be useful as a therapeutic drug in the management of ischemic retinal diseases.     

Axonal Synapses Utilize Multiple Synaptic Ribbons in the Mammalian Retina

In the mammalian retina, bipolar cells and ganglion cells which stratify in sublamina a of the inner plexiform layer (IPL) show OFF responses to light stimuli while those that stratify in sublamina b show ON responses. This functional relationship between anatomy and physiology is a key principle of retinal organization. However, there are at least three types of retinal neurons, including intrinsically photosensitive retinal ganglion cells (ipRGCs) and dopaminergic amacrine cells, which violate this principle. These cell types have light-driven ON responses, but their dendrites mainly stratify in sublamina a of the IPL, the OFF sublayer. Recent anatomical studies suggested that certain ON cone bipolar cells make axonal or ectopic synapses as they descend through sublamina a, thus providing ON input to cells which stratify in the OFF sublayer. Using immunoelectron microscopy with 3-dimensional reconstruction, we have identified axonal synapses of ON cone bipolar cells in the rabbit retina. Ten calbindin ON cone bipolar axons made en passant ribbon synapses onto amacrine or ganglion dendrites in sublamina a of the IPL. Compared to the ribbon synapses made by bipolar terminals, these axonal ribbon synapses were characterized by a broad postsynaptic element that appeared as a monad and by the presence of multiple short synaptic ribbons. These findings confirm that certain ON cone bipolar cells can provide ON input to amacrine and ganglion cells whose dendrites stratify in the OFF sublayer via axonal synapses. The monadic synapse with multiple ribbons may be a diagnostic feature of the ON cone bipolar axonal synapse in sublamina a. The presence of multiple ribbons and a broad postsynaptic density suggest these structures may be very efficient synapses. We also identified axonal inputs to ipRGCs with the architecture described above.     

Inhibiting Matrix Metalloproteinase 3 Ameliorates Neuronal Loss in the Ganglion Cell Layer of Rats in Retinal Ischemia/Reperfusion

It has been demonstrated that matrix metalloproteinase 3 (MMP3) is integrally involved in the neuronal degeneration of the central nervous system by promoting glial activation, neuronal apoptosis and damage to the brain–blood barrier. However, whether MMP3 also contributes to the neuronal degeneration induced by retinal ischemia/reperfusion is still uncertain. In the present study, we detected the cellular localization of MMP3 in adult rat retinae and explored the relationship of its expression with neuronal loss in the ganglion cell layer (GCL) in retinal ischemia/reperfusion. We found that MMP3 was widely expressed in many cells throughout the layers of the rat retinae, including Vertebrate neuron-specific nuclear protein (NeuN)-, parvalbumin-, calbindin-, protein kinase C-α-, glial fibrillary acidic protein-, glutamine synthetase- and CD11b-positive cells. Furthermore, all rats were treated with high intraocular pressure (HIOP) for 1 h (h) and sacrificed at 6 h, 1 day (d), 3 d, and 7 d after HIOP. Compared to the normal control, the expression of both proenzyme MMP3 and active MMP3 were significantly up-regulated after HIOP treatment without alteration of the laminar distribution pattern. Moreover, inhibiting MMP3 ameliorated the loss of NeuN-positive cells in the GCL following HIOP. In summary, our data demonstrates that MMP3 is expressed in multiple types of neurons and glial cells in normal rat retinae. Simultaneously, the up-regulation of its expression and activity are closely involved in neuronal loss in the GCL in retinal ischemia/reperfusion.     

Two caspase-2 transcripts are expressed in rat hippocampus after global cerebral ischemia

Caspase family genes play a critical role in the initiation and execution of programmed cell death. Programmed cell death is an important contributor to neuronal loss following cerebral ischemia. We have performed a series of experiments to investigate the role of a specific caspase, caspase-2, in the development of delayed neuronal death following transient global ischemia in the rat. A rat ischemic brain cDNA library was screened, and two splice-variants of caspase-2 mRNA were identified, caspase-2S and caspase-2L, which were highly homologous with the sequences of human and mouse caspase-2S and caspase-2L genes, respectively. RT-PCR demonstrated an increase in expression of both caspase-2S and caspase-2L mRNA at 8, 24 and 72 h of reperfusion after global ischemia. The ratio of the two PCR fragments did not change significantly throughout the time course of reperfusion. Western blot with monoclonal antibody specific to the pro-apoptotic caspase-2L splice variant revealed an increase in procaspase-2 (51 kDa) protein from 4 to 72 h following ischemia compared with sham-operated controls. Furthermore, an approximately 30-kDa cleavage product appeared at 8 h and increased with increasing duration of reperfusion. Thus, caspase-2L is both translated and activated following transient global ischemia. Finally, intraventricular administration of the caspase-2-like inhibitor (VDVAD-FMK) 30 min before induction of ischemia decreased the number of CA1 neurons staining positively for DNA damage (Klenow-labeling assay) and increased the number of healthy-appearing CA1 neurons (cresyl violet) compared with vehicle-treated controls. Taken together, the data suggest that caspase-2 induction and activation are important mediators of delayed neuronal death following transient global ischemia.     

Assessment of the relative contribution of COX-1 and COX-2 isoforms to ischemia-induced oxidative damage and neurodegeneration following transient global cerebral ischemia

We investigated the relative contribution of COX-1 and/or COX-2 to oxidative damage, prostaglandin E2 (PGE2) production and hippocampal CA1 neuronal loss in a model of 5 min transient global cerebral ischemia in gerbils. Our results revealed a biphasic and significant increase in PGE2 levels after 2 and 24-48 h of reperfusion. The late increase in PGE2 levels (24 h) was more potently reduced by the highly selective COX-2 inhibitor rofecoxib (20 mg/kg) relative to the COX-1 inhibitor valeryl salicylate (20 mg/kg). The delayed rise in COX catalytic activity preceded the onset of histopathological changes in the CA1 subfield of the hippocampus. Post-ischemia treatment with rofecoxib (starting 6 h after restoration of blood flow) significantly reduced measures of oxidative damage (glutathione depletion and lipid peroxidation) seen at 48 h after the initial ischemic episode, indicating that the late increase in COX-2 activity is involved in the delayed occurrence of oxidative damage in the hippocampus after global ischemia. Interestingly, either selective inhibition of COX-2 with rofecoxib or inhibition of COX-1 with valeryl salicylate significantly increased the number of healthy neurons in the hippocampal CA1 sector even when the treatment began 6 h after ischemia. These results provide the first evidence that both COX isoforms are involved in the progression of neuronal damage following global cerebral ischemia, and have important implications for the potential therapeutic use of COX inhibitors in cerebral ischemia.