Nina Vartiainen

Astrocytes protect neurons from nitric oxide toxicity by a glutathione-dependent mechanism

Nitric oxide (NO) contributes to neuronal death in cerebral ischemia and other conditions. Astrocytes are anatomically well positioned to shield neurons from NO because astrocyte processes surround most neurons. In this study, the capacity of astrocytes to limit NO neurotoxicity was examined using a cortical co‐culture system. Astrocyte‐coated dialysis membranes were placed directly on top of neuronal cultures to provide a removable astrocyte layer between the neurons and the culture medium. The utility of this system was tested by comparing neuronal death produced by glutamate, which is rapidly cleared by astrocytes, and N‐methyl‐d‐aspartate (NMDA), which is not. The presence of an astrocyte layer increased the LD50 for glutamate by approximately four‐fold, but had no effect on NMDA toxicity. Astrocyte effects on neuronal death produced by the NO donors S‐nitroso‐N‐acetyl penicillamine and spermine NONOate were examined by placing these compounds into the medium of co‐cultures containing either a control astrocyte layer or an astrocyte layer depleted of glutathione by prior exposure to buthionine sulfoximine. Neurons in culture with the glutathione‐depleted astrocytes exhibited a two‐fold increase in cell death over a range of NO donor concentrations. These findings suggest that astrocytes protect neurons from NO toxicity by a glutathione‐dependent mechanism.

Long-term Induction of Haem Oxygenase-1 (HSP-32) in Astrocytes and Microglia Following Transient Focal Brain Ischaemia in the Rat

Haem oxygenase‐1 (HO‐1) is a stress protein and a rate‐limiting enzyme in haem degradation, generating ferrous iron, carbon monoxide and bile pigments. HO‐1 has been suggested to be protective against oxidative stress. In the normal rodent brain the enzyme is localized in selected neuron populations, but heat shock, glutathione depletion in vivo and oxidative stress in vitro induce HO‐1 predominantly in glial cells. We studied HO‐1 expression in the brain following transient occlusion of the middle cerebral artery, and found increased mRNA levels in the ischaemic region from 4 h to 7 days after 90 min of ischaemia. The mRNA levels peaked at 12 h, and were localized perifocally. HO‐1‐immunoreactive astrocytes and microglial cells were seen in the perifocal area, in the ipsilateral and occasionally in the contralateral hippocampus. Some perifocal neurons were also HO‐14mmunoreactive. In the infarcted area HO‐1‐positive microglia/macrophages were detected in double‐labelling experiments. A microassay measuring the conversion of [14C]haem to [14C]bilirubin showed a two‐fold increase in haem oxygenase activity in the infarcted core. These observations show a long‐term induction of HO‐1 protein and its activity following ischaemia‐reperfusion brain injury, and indicate increased capacity for haem degradation and the generation of biologically active bile products, carbon monoxide and iron in astrocytes and some microglia/macrophages during focal brain ischaemia.

Pyrrolidine dithiocarbamate inhibits translocation of nuclear factor kappa-B in neurons and protects against brain ischaemia with a wide therapeutic time window

Pyrrolidine dithiocarbamate (PDTC) is an antioxidant and inhibitor of transcription factor nuclear factor kappa‐B (NF‐κB). Because the role of NF‐κB in brain injury is controversial and another NF‐κB inhibiting thiocarbamate, DDTC, was recently shown to increase ischaemic brain damage, we investigated the effect of PDTC on transient brain ischaemia. Ischaemia was induced by occlusion of the middle cerebral artery (MCAO). In Wistar rats, the PDTC treatment started even 6 h after MCAO reduced the infarction volume by 48%. PDTC protected against MCAO also in spontaneously hypertensive rats and against forebrain ischaemia in Mongolian gerbils. PDTC prevented NF‐κB activation in the ischaemic brain as determined by reduced DNA binding and nuclear translocation of NF‐κB in neurons. PDTC had anti‐inflammatory effect by preventing induction of NF‐κB‐regulated pro‐inflammatory genes. In ischaemic rats, NF‐κB was localized in cyclo‐oxygenase‐2‐immunoreactive neurons. Blood cytokine levels were not altered by ischaemia or PDTC. When cultured neurons were exposed to an excitotoxin, no production of reactive oxygen species was detected, but PDTC provided protection and prevented nuclear translocation of NF‐κB. The clinically approved PDTC and its analogues may act as anti‐inflammatories and may be safe therapies in stroke with a wide time window.

Induction of Thymosin β4 mrna Following Focal Brain Ischemia

THYMOSIN β4 is a protein expressed in most rodent and human tissues, including the brain, and is thought to participate in neurite outgrowth during development by sequestration of G-actin necessary for growth cone extension. Under normal conditions in the adult rat brain, the gene has been suggested

Aspirin Inhibits p44/42 Mitogen-Activated Protein Kinase and Is Protective Against Hypoxia/Reoxygenation Neuronal Damage

Background and Purpose— Acetylsalicylic acid (ASA) is preventive against stroke and protects against focal brain ischemia in rats. We studied the mechanisms of the manner in which ASA provides neuroprotection against hypoxia/reoxygenation (H/R) injury. Methods— Spinal cord cultures exposed to 20 hours of hypoxia followed by reoxygenation were treated with a vehicle, ASA or inhibitors of inducible nitric oxide synthase (iNOS), mitogen-activated protein kinases p38 MAPK and ERK1/2, or an N-methyl-d-aspartic acid (NMDA) receptor antagonist. Cell viability was assessed by LDH release measurement and cell counts. Prostaglandin production was measured by enzyme immunoassay, MAPK signaling by immunoblotting, and DNA binding of nuclear factor-&kgr;B (NF-&kgr;B) and activating protein-1 (AP-1) by electrophoretic mobility shift assay. Results— One to 3 mmol/L ASA inhibited H/R-induced neuronal death when present during H/R but not when administered only for the reoxygenation period. Prostaglandin E2 production was very low and was not altered by ASA. The AP-1 and NF-&kgr;B DNA binding activities increased after H/R. ASA increased the H/R-induced AP-1 binding but had no effect on NF-&kgr;B binding. H/R induced a sustained ERK1/2 activation followed by neuronal death, whereas no changes in p38 or c-Jun N-terminal kinase were detected. ASA strongly inhibited this ERK1/2 activation. PD98059, an ERK1/2 inhibitor, was also neuroprotective, prevented H/R-induced ERK1/2 activation, and had no effect on NF-&kgr;B binding activity. Inhibition of NMDA receptors, iNOS, or p38 MAPK did not provide neuroprotection. Conclusions— Inhibition of the sustained activation of ERK1/2 may partially contribute to neuroprotection achieved by ASA against H/R injury.

Aspirin provides cyclin-dependent kinase 5-dependent protection against subsequent hypoxia/reoxygenation damage in culture

Aspirin [acetylsalicylic acid (ASA)] is an anti‐inflammatory drug that protects against cellular injury by inhibiting cyclooxygenases (COX), inducible nitric oxide synthase (iNOS) and p44/42 mitogen‐activated protein kinase (p44/42 MAPK), or by preventing translocation of nuclear factor κB (NF‐κB). We studied the effect of ASA pre‐treatment on neuronal survival after hypoxia/reoxygenation damage in rat spinal cord (SC) cultures. In this injury model, COX, iNOS and NF‐κB played no role in the early neuronal death. A 20‐h treatment with 3 mm ASA prior to hypoxia/reoxygenation blocked the hypoxia/reoxygenation‐induced lactate dehydrogenase (LDH) release from neurons. This neuroprotection was associated with increased phosphorylation of neurofilaments, which are substrates of p44/42 MAPK and cyclin‐dependent kinase 5 (Cdk5). PD90859, a p44/42 MAPK inhibitor, had no effect on ASA‐induced tolerance, but olomoucine and roscovitine, Cdk5 inhibitors, reduced ASA neuroprotection. Hypoxia/reoxygenation alone reduced both the protein amount and activity of Cdk5, and this reduction was inhibited by pre‐treatment with ASA. Moreover, the protein amount of a neuronal Cdk5 activator, p35, recovered after reoxygenation only in ASA‐treated samples. The prevention of the loss in Cdk5 activity during reoxygenation was crucial for ASA‐induced protection, because co‐administration of Cdk5 inhibitors at the onset ofreoxygenation abolished the protection. In conclusion, pre‐treatment with ASA induces tolerance against hypoxia/reoxygenation damage in spinal cord cultures by restoring Cdk5 and p35 protein expression.

Piroxicam and NS-398 rescue neurones from hypoxia/reoxygenation damage by a mechanism independent of cyclo-oxygenase inhibition

We studied whether NS‐398, a selective cyclo‐oxygenase‐2 (COX‐2) enzyme inhibitor, and piroxicam, an inhibitor of COX‐2 and the constitutively expressed COX‐1, protect neurones against hypoxia/reoxygenation injury. Rat spinal cord cultures were exposed to hypoxia for 20 h followed by reoxygenation. Hypoxia/reoxygenation increased lactate dehydrogenase (LDH) release, which was inhibited by piroxicam (180–270 µm) and NS‐398 (30 µm). Cell counts confirmed the neuroprotection. Western blotting revealed no COX‐1 or COX‐2 proteins even after hypoxia/reoxygenation. Production of prostaglandin E2 (PGE2), a marker of COX activity, was barely measurable and piroxicam and NS‐398 had no effect on the negligible PGE2 production. Hypoxia/reoxygenation increased nuclear factor‐kappa B (NF‐κB) binding activity, which was inhibited by piroxicam but not by NS‐398. AP‐1 binding activity after hypoxia/reoxygenation was inhibited by piroxicam but strongly enhanced by NS‐398. However, both COX inhibitors induced activation of extracellular signal‐regulated kinase (ERK) in neurones and phosphorylation of heavy molecular weight neurofilaments, cytoskeletal substrates of ERK. It is concluded that piroxicam and NS‐398 protect neurones against hypoxia/reperfusion. The protection is independent of COX activity and not solely explained by modulation of NF‐κB and AP‐1 binding activity. Instead, piroxicam and NS‐398‐induced phosphorylation through ERK pathway may contribute to the increased neuronal survival.

Glutamatergic receptors regulate expression, phosphorylation and accumulation of neurofilaments in spinal cord neurons

Glutamatergic regulation of neurofilament expression, phosphorylation and accumulation in cultured spinal cord neurons was studied. At seven days in culture, 0.15% of the neurons were immunoreactive for non-phosphorylated neurofilaments, but essentially no cells immunoreactive for phosphorylated neurofilaments were seen. The number and size of the immunoreactive cells in culture corresponded well to those of rat and human spinal cord neurons in vivo. In spinal cord cultures, sublethal, long-lasting stimulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate or metabotrophic receptors, but not N-methyl-D-aspartate receptors, dose-dependently increased the number of non-phosphorylated neurofilament-immunoreactive cells, which was blocked by nifedipine, an antagonist of voltage-sensitive Ca2+ channels. Stimulation of kainate or all non-N-methyl-D-aspartate receptors decreased the expression of medium-molecular-weight neurofilament messenger RNA. Blockade of AMPA/kainate receptors, but not of N-methyl-D-aspartate receptors, increased the amount of phosphorylated neurofilament protein and the number of phosphorylated neurofilament-immunoreactive cell bodies. The phosphorylated neurofilament-immunoreactive cell population was different from the non-phosphorylated neurofilament-immunoreactive neurons, which lost their axonal non-phosphorylated neurofilament immunoreactivity but showed intense cytoplasmic labeling in response to the blockade of AMPA/ kainate receptors. Immunoreactivity for phosphoserine did not change upon glutamate receptor stimulation and blockade. The results show that activation of AMPA/kainate receptors decreases the expression of neurofilament messenger RNA and neurofilament phosphorylation in spinal cord neurons by a mechanism involving active voltage-sensitive Ca2+ channels. Blockade of these receptors seems to disturb axonal neurofilament transport. Because AMPA/kainate receptors mediate chronic glutamatergic death of spinal motor neurons and these receptors have been suggested to be involved in the pathogenesis of amyotrophic lateral sclerosis, the observed alteration in neurofilament phosphorylation and distribution may contribute to the pathogenesis of chronic motor neuron diseases.