Antonio Cárdenas

In Vitro Ischemic Tolerance Involves Upregulation of Glutamate Transport Partly Mediated by the TACE/ADAM17-Tumor Necrosis Factor-α Pathway

A short ischemic event [ischemic preconditioning (IPC)] can result in a subsequent resistance to severe ischemic injury (ischemic tolerance). Although tumor necrosis factor-α (TNF-α) contributes to the brain damage found after cerebral ischemia, its expression and neuroprotective role in models of IPC have also been described. Regarding the role of TNF-α convertase (TACE/ADAM17), we have recently shown its upregulation in rat brain after IPC induced by transient middle cerebral artery occlusion and that subsequent TNF-α release accounts for at least part of the neuroprotection found in this model. We have now used an in vitro model of IPC using rat cortical cultures exposed to sublethal oxygen-glucose deprivation (OGD) to investigate TACE expression and activity after IPC and the subsequent mechanisms of ischemic tolerance. OGD-induced cell death was significantly reduced in cells exposed to IPC by sublethal OGD 24 hr before, an effect that was inhibited by the TACE inhibitor BB3103 (1 μm) and anti-TNF-α antibody (2 μg/ml) and that was mimicked by TNF-α (10 pg/ml) preincubation. Western blot analysis showed that TACE expression is increased after IPC. IPC caused TNF-α release, an effect that was blocked by the selective TACE inhibitor BB-3103. In addition, IPC diminished the increase in extracellular glutamate caused by OGD and increased cellular glutamate uptake and expression of EAAT2 and EAAT3 glutamate transporters; however, only EAAT3 upregulation was mediated by increased TNF-α. These data demonstrate that neuroprotection induced by IPC involves upregulation of glutamate uptake partly mediated by TACE overexpression.

Implication of Glutamate in the Expression of Inducible Nitric Oxide Synthase After Oxygen and Glucose Deprivation in Rat Forebrain Slices

Abstract: Nitric oxide synthesis by inducible nitric oxide synthase (iNOS) has been postulated to contribute to ischemia‐reperfusion neurotoxicity. The expression of this enzyme has been demonstrated in cells present in the postischemic brain. The mechanisms of iNOS expression after cerebral ischemia are a subject of current research. We therefore decided to investigate whether glutamate, which is released in ischemia and is implicated in neurotoxicity, might be involved in the mechanisms by which oxygen and glucose deprivation (OGD) leads to the expression of iNOS in rat forebrain slices. In this model, we have shown previously that 20 min of OGD causes the expression of iNOS. We have now found that the NMDA receptor antagonist MK‐801 blocks the expression of iNOS, suggesting that the activation of the NMDA subtype of glutamate receptor is implicated in the mechanisms that lead to the expression of this isoform. Moreover, we have found that glutamate alone could trigger the induction process, as shown by the appearance of a Ca2+‐independent NOS activity and by the detection of iNOS mRNA and protein in slices exposed to glutamate. Glutamate‐dependent iNOS expression was concentration‐dependent and was blocked by EGTA and by the inhibitors of nuclear factor κB (NF‐κB) activation pyrrolidine dithiocarbamate and MG132. In addition, glutamate induced NF‐κB translocation to the nucleus, an effect that was inhibited by MG132. Taken together, our data suggest that activation of NMDA receptors by glutamate released in ischemia is involved in the expression of iNOS in rat forebrain slices via a Ca2+‐dependent activation of the transcription factor NF‐κB. To our knowledge, this is the first report showing an implication of excitatory amino acids in the expression of iNOS caused by ischemia.

Rosiglitazone and 15.deoxy-Δ12,14-prostaglandin J2 cause potent neuroprotection after experimental stroke through noncompletely overlapping mechanisms

Stroke triggers an inflammatory cascade which contributes to a delayed cerebral damage, thus implying that antiinflammmatory strategies might be useful in the treatment of acute ischaemic stroke. Since two unrelated peroxisome proliferator-activated receptor-γ (PPARγ) agonists, the thiazolidinedione rosiglitazone (RSG) and the cyclopentenone prostaglandin 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), have been shown to possess antiinflammatory properties, we have tested their neuroprotective effects in experimental stroke. Rosiglitazone or 15d-PGJ2 were administered to rats 10 mins or 2 h after permanent middle cerebral artery occlusion (MCAO). Stroke outcome was evaluated by determination of infarct volume and assesment of neurological scores. Brains were collected for protein expression, gene array analyses and gene shift assays. Our results show that both compounds decrease MCAO-induced infarct size and improve neurological scores. At late times, the two compounds converge in the inhibition of MCAO-induced brain expression of inducible NO synthase and the matrix metalloproteinase 9. Interestingly, at early times, complementary DNA microarrays and gene shift assays show that different mechanisms are recruited. Analysis of early nuclear p65 and late cytosolic IκBα protein levels shows that both compounds inhibit nuclear factor-κB signalling, although at different levels. All these results suggest both PPARγ-dependent and independent pathways, and might be useful to design both therapeutic strategies and prognostic markers for stroke.

TNFR1 upregulation mediates tolerance after brain ischemic preconditioning

A short ischemic event (ischemic preconditioning (IPC)) can result in subsequent resistance to severe ischemic injury (ischemic tolerance (IT)). The expression and neuroprotective role of tumor necrosis factor (TNF-α) have been described in models of IPC and we have showed the participation of its processing enzyme, the TNF-α convertase enzyme (TACE) in this process. We have now decided to explore the expression and localization of TNF receptors (TNFR) as well as other signalling mechanisms involved in IT. A period of 10 mins of temporary middle cerebral artery occlusion (tMCAO) was used for focal IPC. To evaluate the ability of IPC to produce IT, permanent MCAO was performed 48 hours after IPC. Ischemic preconditioning produced a reduction in infarct volume, as we showed previously. Ischemic preconditioning caused upregulation of neuronal TNFR1 that was reduced by the selective TACE inhibitor BB1101. Intracerebral administration of TNFR1 antisense oligodeoxynucleotide, which caused a reduction in TNFR1 expression, inhibited the IPC-induced protective effect, showing that TNFR1 upregulation is implicated in IT. Moreover, treatment with BB1101, TNFR1 antisense and lactacystin—a specific proteasome inhibitor—blocked IPC-induced NF-κB. Immunohistochemical studies showed the expression of TACE and TNFR1 in neurons. In summary, these data show that IPC produces neuronal upregulation of TACE and TNFR1, and that the pathway TACE/TNF-α/TNFR1/NF-κB is involved in IT.

The nonthiazolidinedione PPARγ agonist L-796,449 is neuroprotective in experimental stroke

Some agonists of the peroxisome proliferator-activated receptor γ (PPARγ) belonging to the thiazolidinedione (TZD) family, as well as the cyclopentenone prostaglandin 15-dPGJ2, have been shown to cause neuroprotection in animal models of stroke. We have tested whether the TZD-unrelated PPARγ agonist L-796,449 is neuroprotective after permanent middle cerebral artery occlusion (MCAO) in the rat brain. Our results show that L-796,449 decreases MCAO-induced infarct size and improves neurologic scores. This protection is concomitant to inhibition of MCAO-induced brain expression of inducible NO synthase (iNOS) and the matrix metalloproteinase MMP-9 and to upregulation of the cytoprotective stress protein heme oxygenase-1 (HO-1). Analysis of the NF-κB p65 monomer and the NF-κB inhibitor IκBα protein levels as well as gel mobility shift assays indicate that L-796,449 inhibits NF-κB signaling, and that it may be recruiting both PPARγ-dependent and independent pathways. In summary, our results provide new insights for stroke treatment.

Effect of subacute and chronic immobilisation stress on the outcome of permanent focal cerebral ischaemia in rats.

The aim of this study was to determine the effect of mood disorders, including psychological distress and depression, on stroke outcome. Male Fischer rats were exposed to immobilisation stress, an animal paradigm of psychological stress, major depression and post-traumatic stress disorder. Either a subacute (1 h for 7 days) or a chronic (6 h for 21 days) exposure to stress was applied 24 h before permanent middle cerebral artery occlusion (MCAO). Stroke outcome was assessed by measurement of infarct size and behavioural characterisation. Serum glutamate and brain ATP levels as well as brain glutamate transporter function and expression were studied in the search for the molecular mechanisms involved. Subacute stress exposure increased infarct size and decreased behavioural scores after stroke. On the contrary, chronic stress exposure decreased infarct size. Peak serum glutamate levels correlated with infarct size after MCAO. Expression of glutamate transporters was decreased by subacute stress, whereas the expression of EAAT1, a glial glutamate carrier, was increased after the chronic stress protocol. Our results indicate that distinct patterns of stress determine different stroke outcomes, and that expressional changes of brain glutamate transporters, able to affect glutamate release after stroke, are involved.

Mechanisms of the neuroprotective effect of aspirin after oxygen and glucose deprivation in rat forebrain slices

Acetylsalicylic acid (ASA, Aspirin) is an anti-inflammatory drug with a wide spectrum of pharmacological activities and multiple sites of action. Apart from its preventive actions against stroke due to its antithrombotic properties, recent data in the literature suggest that high concentrations of ASA also exert direct neuroprotective effects. We have used an in vitro model of brain ischaemia using rat forebrain slices deprived of oxygen and glucose to test ASA neuroprotective properties. We have found that ASA inhibits neuronal damage at concentrations lower than those previously reported (0.1-0.5 mM), and that these effects correlate with the inhibition of excitatory amino acid release, of NF-kappaB translocation to the nucleus and iNOS expression caused by ASA. All of these three mechanisms may mediate the neuroprotective effects of this drug. Our results also show that the effects of ASA are independent of COX inhibition. Taken together, our present findings show that ASA is neuroprotective in an in vitro model of brain ischaemia at doses close to those recommended for its antithrombotic effects.

A chronic treatment with CDP-choline improves functional recovery and increases neuronal plasticity after experimental stroke

Chronic impairment of forelimb and digit movement is a common problem after stroke that is resistant to therapy. Although in the last years some studies have been performed to increase the efficacy of rehabilitative experience and training, the pharmacological approaches in this context remain poorly developed. We decided to study the effect of a chronic treatment with CDP-choline, a safe and well-tolerated drug that is known to stabilize membranes, on functional outcome and neuromorphological changes after stroke. To assess the functional recovery we have performed the staircase reaching test and the elevated body swing test (EBST), for studying sensorimotor integration and asymmetrical motor function respectively. The treatment with CDP-choline, initiated 24 h after the middle cerebral artery occlusion (MCAO) and maintained during 28 days, improved the functional outcome in both the staircase test (MCAO+CDP=87.0+/-6.6% pellets eaten vs. MCAO+SAL=40.0+/-4.5%; p<0.05) and the EBST (MCAO+CDP=70.0+/-6.8% vs. MCAO+SAL=88.0+/-5.4%; contralateral swing p<0.05). In addition, to study potential neuronal substrates of the improved function, we examined the dendritic morphology of layer V pyramidal cells in the undamaged motor cortex using a Golgi-Cox procedure. The animals treated with CDP-choline showed enhanced dendritic complexity and spine density compared with saline group. Our results suggest that a chronic treatment with CDP-choline initiated 24 h after the insult is able to increase the neuronal plasticity within noninjured and functionally connected brain regions as well as to promote functional recovery.

Up-regulation of TNF-α convertase (TACE/ADAM17) after oxygen–glucose deprivation in rat forebrain slices

Tumour necrosis factor-alpha (TNF-alpha) is a major immunomodulatory and proinflammatory cytokine which is shed in its soluble form by a membrane-anchored zinc protease, identified as a disintegrin and metalloproteinase (ADAM) called TNF-alpha convertase (TACE; ADAM17). The role of this protease in the adult nervous system remains poorly understood. During cerebral ischemia and subsequent reperfusion, expression and release of TNF-alpha have been shown. We have investigated the expression and activity of TACE in an in vitro model of brain ischemia consisting of rat forebrain slices exposed to oxygen-glucose deprivation (OGD). OGD caused the release of TNF-alpha, an effect which was inhibited by a hydroxamate-based metalloprotease inhibitor, BB-3103, with an IC(50) of 0.1 microM, suggesting that TNF-alpha release results selectively from TACE activity. Assay of TACE enzymatic activity on a fluorescein-labelled peptide spanning the cleavage site in pro-TNF-alpha, as well as Western blot and RT-PCR analyses showed that TACE is present in control forebrain and, more interestingly, that TACE expression is increased in OGD-exposed tissue. TACE enzymatic activity from OGD-exposed slices was significantly inhibited by cycloheximide, suggesting that de novo synthesis of TACE contributes to TNF-alpha release after ischaemia. Moreover, it was also inhibited by bisindolylmaleimide I, indicating that TACE activity is regulated by PKC. These findings posed the question of what was its function therein. Among other actions, TNF-alpha has been described to be involved in the expression of inducible nitric oxide synthase (iNOS), a high-output NOS isoform associated to cellular damage, but the link between TNF-alpha release after brain ischaemia and iNOS expression in this condition has not been shown. We have now found that iNOS expression in OGD-subjected brain slices is inhibited by BB-3103 at concentrations below 1 microM, indicating that shedding of TNF-alpha by TACE plays a necessary part in the induction of this NOS isoenzyme after OGD. Taken together, these data demonstrate that (1) TACE/ADAM17 activity accounts for the majority of TNF-alpha shedding after OGD in rat forebrain slices, (2) an increase in TACE expression contributes, at least in part, to the rise in TNF-alpha after OGD and (3) iNOS expression in OGD-subjected brain slices results from TACE activity and subsequent increase in TNF-alpha levels.

Upregulation of TACE/ADAM17 after ischemic preconditioning is involved in brain tolerance.

A short ischemic event (ischemic preconditioning [IPC]) can result in a subsequent resistance to severe ischemic injury (ischemic tolerance [IT]). Although tumor necrosis factor-α (TNF-α) contributes to the brain damage, its expression and neuroprotective role in models of IPC have also been described. However, the role of TNF-α convertase (TACE) in IPC and IT is not known. Using in vitro models, the authors previously demonstrated that TACE is upregulated after ischemic brain damage. In the present study, the authors used a rat model of transient middle cerebral artery occlusion as IPC to investigate TACE expression, its involvement in TNF-α release, and its role in IT. Western blot analysis showed that TACE expression is increased after IPC. Ischemic preconditioning caused TNF-α release, an effect that was blocked by the selective TACE inhibitor BB-1101 (10 mg · kg−1 · day−1; SHAM, 1,050 ± 180; IPC, 1,870 ± 290; IPC + BB, 1,320 ± 260 ng/mg; n = 4, P < 0.05). Finally, IPC produced a reduction in infarct volume, which was inhibited by treatment with BB-1101 and with anti–TNF-α (10 μg/5 doses; SHAM + permanent middle cerebral artery occlusion [pMCAO], 335 ± 20; IPC + pMCAO, 244 ± 14; IPC + BB + pMCAO, 300 ± 6; IPC + anti-TNF + pMCAO, 348 ± 22 mm3; n = 6–10, P < 0.05). Taken together, these data demonstrate that TACE is upregulated after IPC, plays a major role in TNF-α shedding in IPC, and has a neuroprotective role in IT.