Javier de Cristóbal

Neuroprotective effect of aspirin by inhibition of glutamate release after permanent focal cerebral ischaemia in rats.

Aspirin reduces the size of infarcts after ischaemic stroke. Although this fact has been attributed to its anti‐platelet actions, direct neuroprotective effects have also been reported. We have recently demonstrated that aspirin is neuroprotective by inhibiting glutamate release in ‘in vitro’ models of brain ischaemia, via an increase in ATP production. The present study was designed to determine whether the inhibition of glutamate release induced by aspirin might be protective in a whole‐animal model of permanent focal brain ischaemia. Focal brain ischaemia was produced in male adult Fischer rats by occluding both the common carotid and middle cerebral arteries. Central and serum glutamate levels were determined at fixed intervals after occlusion. The animals were then killed and infarct volume was measured. Aspirin (30 mg/kg i.p. administered 2 h before the occlusion) produced a significant reduction in infarct volume, an effect that correlated with the inhibition caused by aspirin on ischaemia‐induced increase in brain and serum glutamate concentrations after the onset of the ischaemia. Aspirin also inhibited ischaemia‐induced decrease in brain ATP levels. Our present findings show a novel mechanism for the neuroprotective effects of aspirin, which takes place at concentrations in the anti‐aggregant–analgesic range, useful in the management of patients with risk of ischaemic events.

Tau-knockout mice show reduced GSK3-induced hippocampal degeneration and learning deficits

It has been proposed that deregulation of neuronal glycogen synthase kinase 3 (GSK3) activity may be a key feature in Alzheimer disease pathogenesis. We have previously generated transgenic mice that overexpress GSK3beta in forebrain regions including dentate gyrus (DG), a region involved in learning and memory acquisition. We have found that GSK3 overexpression results in DG degeneration. To test whether tau protein modified by GSK3 plays a role in that neurodegeneration, we have brought GSK3 overexpressing mice to a tau knockout background. Our results indicate that the toxic effect of GSK3 overexpression is milder and slower in the absence of tau. Thus, we suggest that the hyperphosphorylated tau mediates, at least in part, the pathology observed in the brain of GSK3 overexpressing mice.

Inhibition of glutamate release by delaying ATP fall accounts for neuroprotective effects of antioxidants in experimental stroke

Excitotoxic neuronal injury related to excessive glutamate release is believed to play a key role in the pathogenesis of focal cerebral ischemia. Reversal of neuronal glutamate transporters caused by ATP fall and subsequent imbalance of membrane ionic gradients accounts for most glutamate release after cerebral ischemia. ATP synthesis from oxidative phosphorylation derives from the coupled functioning of the mitochondrial respiratory chain (MRC) and the ATP synthase; interestingly, the MRC is one of the main sites of cellular reactive oxygen species (ROS) generation even in physiological circumstances. Hence, we have studied the effect of the antioxidants glutathione, superoxide dismutase, and α‐tocopherol on infarct outcome, brain ATP, and glutamate levels after permanent middle cerebral artery occlusion (MCAO) in Fischer rats; we have also characterized the actions of antioxidants on MRC complexes. Our results show that intraperitoneal administration of antioxidants 2 h before MCAO enhances ATP synthesis and causes a neuroprotective effect concomitant to inhibition of ischemia‐induced increase in brain glutamate. Antioxidants also increased mitochondrial ATP and MRC complex I–III activity and respiration, suggesting that these actions are due to removal of the inhibition caused by endogenous ROS on MRC. These findings may possess important therapeutic repercussions in the management of ischemic stroke.

Aspirin inhibits stress-induced increase in plasma glutamate, brain oxidative damage and ATP fall in rats

The precise mechanisms by which stress induces brain damage are still being elucidated. The high-output, inducible isoform of nitric oxide (NO) synthase (iNOS) is expressed in rat brain after immobilisation stress and its inhibition protects against cell damage in this condition. We have hereby explored some mechanisms involved in iNOS expression and studied the effects of aspirin, a NSAID with neuroprotective actions, in this model. Acute (6 h) stress exposure in rats caused brain expression of iNOS, an increase in plasma glutamate and brain TNF-&agr;, induction of oxidative indicators in brain and a fall in brain ATP levels. Prior administration of aspirin (10 mg/kg i.p.) inhibited all these effects caused by stress, suggesting possible therapeutic implications of this drug in this condition.

Subacute administration of fluoxetine prevents short-term brain hypometabolism and reduces brain damage markers induced by the lithium-pilocarpine model of epilepsy in rats

The role of serotonin (5-hydroxytryptamine; 5-HT) in epileptogenesis still remains controversial. In this regard, it has been reported that serotonergic drugs can alter epileptogenesis in opposite ways. The main objective of this work was to investigate the effect of the selective 5-HT selective reuptake inhibitor (SSRI) fluoxetine administered subacutely (10mg/kg/day×7 days) on the eventual metabolic impairment induced by the lithium-pilocarpine model of epilepsy in rats. In vivo 2-deoxy-2-[(18)F]fluoro-d-glucose ([(18)F] FDG) positron emission tomography (PET) was performed to assess the brain glucose metabolic activity on days 3 and 30 after the insult. In addition, at the end of the experiment (day 33), several histochemical and neurochemical assessments were performed for checking the neuronal functioning and integrity. Three days after the insult, a marked reduction of [(18)F] FDG uptake (about 30% according to the brain region) was found in all brain areas studied. When evaluated on day 30, although a hypometabolism tendency was observed, no statistically significant reduction was present in any region analyzed. In addition, lithium-pilocarpine administration was associated with medium-term hippocampal and cortical damage, since it induced neurodegeneration, glial activation and augmented caspase-9 expression. Regarding the effect of fluoxetine, subacute treatment with this SSRI did not significantly reduce the mortality rate observed after pilocarpine-induced seizures. However, fluoxetine did prevent not only the short-term metabolic impairment, but also the aforementioned signs of neuronal damage in surviving animals to lithium-pilocarpine protocol. Finally, fluoxetine increased the density of GABAA receptor both at the level of the dentate gyrus and CA1-CA2 regions in pilocarpine-treated animals. Overall, our data suggest a protective role for fluoxetine against pilocarpine-induced brain damage. Moreover, this action may be associated with an increase of GABAA receptor expression in hippocampus.

A longitudinal FDG-PET study of transgenic mice overexpressing GSK- 3β in the brain.

Increased Glycogen synthase kinase-3 (GSK-3) activity is believed to contribute to the etiology of chronic disorders such as Alzheimers disease, one of the earliest diseases linked to GSK-3 dysfunction. Numerous mouse models with modified GSK-3 have been generated in order to study the physiology of GSK-3, its implication in diverse pathologies and the potential effect of GSK-3 inhibitors. In this study we have characterised and evaluated the brain metabolic changes induced by GSK-3β overexpression in transgenic mice throughout their lifespan. The conditional Tet/GSK-3β transgenic line used in this study has been previously extensively characterized at the pathological, biochemical and cognitive levels. Now we have investigated the effect GSK-3β overexpression on the (18)F-fluoro-deoxyglucose (FDG) uptake by positron emission tomography (PET), taking advantage from this non-invasive technique which has allowed us to track individually the same animals throughout their lives. The results obtained during the longitudinal analysis showed a reduction of metabolic activity in several brain regions, such as cortex, striatum and hippocampus, consistent with the areas where the transgene is being expressed. The reduction of the metabolic activity in these mice is observed from the first time point, performed at the age of 3 months, and maintained throughout the whole study, until the oldest age tested (19 months). This effect seems to be reverted in a satellite group of 3-month transgenic animals treated with the classical GSK-3 inhibitor lithium, as they show higher FDG uptake values compared with untreated age-matched transgenic animals.