Marianne Nielsen

Ischemia induced changes in expression of the astrocyte glutamate transporter GLT1 in hippocampus of the rat.

Changes in cellular uptake of glutamate following transient cerebral ischemia is of possible importance to ischemia induced cell death. In the present study, we employed in situ hybridization and immunohistochemistry to investigate the influence of cerebral ischemia on expression of mRNA and protein of the astrocyte glutamate transporter GLT1, and of glial fibrillary acidic protein. Different subfields of CA1 and CA3 of the rat hippocampus were studied at various time-points after ischemia (days 1, 2, 4, and 21). In CA1, GLT1-mRNA was decreased at all time-points after ischemia except from day 2, whereas in CA3, decreases were seen only on day 1. Expression of GLT1-protein in CA1 was unchanged during the initial days after ischemia, but decreased markedly from day 2 to 4. In CA3, GLT1-protein increased progressively throughout the observation period after ischemia. Following the degeneration of CA1 pyramidal cells, a positive correlation between the number of CA1 pyramidal cells and expression of either GLT1-mRNA or -protein was evident selectively in CA1. Increases in expression of mRNA and protein of glial fibrillary acidic protein were present from day 2, most notable in CA1. The present data provide evidence that expression of GLT1 in CA1 of the hippocampus is not decreased persistently before the degeneration of CA1 pyramidal cells, but is downregulated in response to loss of these neurons. Since the reduction in GLT1 expression evolved concomitantly with the degeneration of CA1 pyramidal cells, it may contribute to the severity of CA1 pyramidal cell loss. A progressive postischemic increase in GLT1 expression in CA3 may be linked to the resistance of CA3 neurons to ischemic cell damage.

Electroconvulsive stimulations normalizes stress-induced changes in the glucocorticoid receptor and behaviour.

Animal models of chronic stress, such as 21 days of 6h/daily restraint stress cause changes in neuronal morphology in the hippocampus and alter behaviour. These changes are partly mediated by the glucocorticoids. The objective of this study was threefold: (1) to study how this particular chronic stress paradigm influences expression of hippocampal glucocorticoid receptor mRNA, (2) to study the effect of previous repeated restraint stress on the behaviours executed in the forced swim test (FST) (e.g. a novel inescapable stress situation) and (3) to investigate the modulating effect of electroconvulsive stimulations (ECS) on the neural and behavioural effects of the stress paradigm. The study shows that restraint stress lowered glucocorticoid receptor mRNA levels in all hippocampal regions, including the CA3 region which is the site of the characteristic dendritic reorganization seen in this model. Furthermore, stressed rats displayed higher increases in immobility and decreased latency to immobility subjected to the novel stressor of the FST than non-stressed rats. ECS abolished both the neural and behavioural effects of the restraint stress and thus protected against the deleterious effects of the stress paradigm. The clinical relevance of these findings is discussed.

Neuroprotective effects of the AMPA antagonist PNQX in oxygen-glucose deprivation in mouse hippocampal slice cultures and global cerebral ischemia in gerbils

PNQX (9-methyl-amino-6-nitro-hexahydro-benzo(F)quinoxalinedione) is a selective AMPA antagonist with demonstrated neuroprotective effects in focal ischemia in rats. Here we report corresponding effects in mouse hippocampal slice cultures subjected to oxygen and glucose deprivation (OGD) and in transient global cerebral ischemia in gerbils. For in vitro studies, hippocampal slice cultures derived from 7-day-old mice and grown for 14 days, were submersed in oxygen-glucose deprived medium for 30 min and exposed to PNQX for 24 h, starting together with OGD, immediately after OGD, or 2 h after OGD. For comparison, other cultures were exposed to the NMDA antagonist MK-801 using the same protocol. Both PNQX and MK-801 displayed significant neuroprotective effects in all hippocampal subfields when present during and after OGD. When added just after OGD, only PNQX retained some neuroprotective effect. When added 2 h after OGD neither PNQX nor MK-801 had an effect. Transient global cerebral ischemia was induced in Mongolian gerbils by occlusion of both common carotid arteries for 4.5 min, with PNQX (10 mg/kg) being injected i.p. 30, 60 and 90 min after the insult. Subsequent analysis of brain sections stained for the neurodegeneration marker Fluoro-Jade B and immunostained for the astroglial marker glial fibrillary acidic protein revealed a significant PNQX-induced decrease in neuronal cell death and astroglial activation. We conclude that, PNQX provided neuroprotection against both global cerebral ischemia in gerbils in vivo and oxygen-glucose deprivation in mouse hippocampal slice cultures.

Secretory phospholipase A2 induces delayed neuronal COX‐2 expression compared with glutamate

Agonists of the binding site for secretory phospholipase A2 (sPLA2) potentiate glutamate‐induced neuronal cell death in primary cell cultures and in vivo (Kolko et al. [ 1996 ] J. Biol. Chem. 271:32722; Kolko et al. [ 1999 ] Neurosci. Lett. 274:167]. Here, we tested the hypothesis that COX‐2 expression participates in the brain response to sPLA2. sPLA2‐OS2, a selective ligand of a neuronal sPLA2‐binding site, was injected into the rat striatum, and early‐response gene expression was monitored by in situ hybridization using 35S‐radiolabeled oligonucleotide probes and immunohistochemistry. An up‐regulation of COX‐2, c‐fos, and c‐jun, but not COX‐1, was observed around the lesion as well as in the neocortex 4 hr after the injection. Hippocampal up‐regulation of COX‐2 was seen in dentate gyrus 8 hr after injection. When glutamate was injected, up‐regulation of the early‐response genes peaked after 2 hr. Our studies showed 1) that sPLA2 selectively induced neuronal COX‐2; 2) that this induction was delayed (4 hr after injection of sPLA2) compared with that elicited by glutamate (2 hr after injection), suggesting different signaling; and 3) that c‐fos and c‐jun were induced around the infarct area as soon as 2 hr after injection, but in other aspects followed a time course similar to that of COX‐2. We conclude that sPLA2 may modulate neuronal COX‐2 expression through mechanisms that differ from those of glutamate‐induced COX‐2 expression.

Ischemic tolerance affects the adenylation state of GluR2 mRNA.

To analyse GluR2 regulations in the rat hippocampal CA1 region following global and tolerance-inducing ischemia in situ hybridization (ISH) and quantitative PCR (Q-PCR) was applied. In addition, cDNA was synthesised from two different primer combinations in order to elucidate possible differences in the adenylation state of GluR2 mRNA. Following global ischemia, ISH and Q-PCR both showed reductions to half of control levels of GluR2 mRNA in consent with previously published results. Following tolerance induction, ISH showed no changes, whereas PCR analysis showed up-regulation to 228% of control value for the general cDNA synthesis, and no change for the specific cDNA synthesis. This indicates that tolerance-inducing ischemia does not increase the amount of GluR2 mRNA; instead polyadenylation of the existing GluR2 mRNA pool takes place.

Extended Studies on the Effect of Glutamate Antagonists on Ischemic CA-1 Damage

Glutamate receptors are numerous on the ischemia vulnerable CA-1 pyramidal cells. Postischemic use of the AMPA antagonist NBQX has shown up to 80% protection against cell death. Three aspects of this were studied: In the first study, male Wistar rats were given NBQX (30 mg/kg x 3) either 20 hours or immediately (0 h) before 12 min of 4-vessel occlusion with hypotension. After six days of reperfusion comparison with an untreated group showed almost full protection in the 0 h group (4% cell loss, p < 0.001) but only slight protection in the 20 h group (62% cell loss, p < 0.05). After 12 min of ischemia in the present model, eosinophilic CA-1 cells are seen from day 2 on. Since there could be a late, deleterious calcium influx via NMDA receptors, one group of ischemic rats was given MK-801 (5 mg/kg i.p.) 24 hours after ischemia. However, quantitation 6 days later of remaining CA-1 cells showed no protection. In the third study referred here, two groups of ischemic rats were given NBQX (30 mg/kg x 3) immediately after ischemia. The groups survive for six and 21 days, respectively. Counting of CA-1 pyramidal cells showed an equal, significant protection in both groups (approx 20% cell loss).

Glutamate-Mediated Mechanisms in Delayed Neuronal Death After Cerebral Ischemia

The density of ionotropic glutamate receptors on the ischemia-vulnerable CA1 pyramidal cells is one of the highest in the CNS. Postischemic use of the AMPA antagonist NBQX has shown up to 80% protection against cell death. Three groups of rats given NBQX (30 mg/kg × 3) immediately after ischemia and with a survival period of 6 days, 3 weeks, and 8 weeks were compared to untreated groups of ischemic rats. Countings of CA1 pyramidal cells showed an equal, significant protection in all three groups (20%–40% cell loss). Thus, if glutamate antagonist treatment is instituted immediately after ischemia, the obtained protection is permanent. Others [11] have shown that a 6-h delay of treatment results in loss of protection. The very delayed loss in the latter situation could be a variant of the maturation phenomeon described by Ito and colleague (1975) [9].