Normal laser-evoked cortical responses in patients with chronic hemibody pain.

BACKGROUND: Patients with widespread unilateral chronic pain associated with recurrent herpes simplex virus (HSV) infections show functional and/or structural changes in the insula, anterior cingulate cortex, frontal and prefrontal cortices, as well as the thalamus, suggesting central dysfunction of the pain system in these patients. Central pain has been associated with attenuated laser-evoked cortical responses. We aimed to clarify whether the observed deficient activation of these areas to acute nociceptive stimuli is due to a lesion at a lower level of pain processing pathways. METHODS: We explored the functional integrity of the ascending nociceptive pathways by recording the cortical-evoked responses to noxious laser stimulation using magnetoencephalography and electroencephalography in eight patients (age 41-51 years, mean 46) with recurrent HSV infections and a history of chronic, spontaneous, widespread unilateral pain, and in nine age-matched healthy control subjects. RESULTS: The cortical-evoked fields of the HSV patients originating from the secondary somatosensory and posterior parietal cortices, as well as the evoked potentials recorded from the midline, did not differ from those of the control subjects, indicating functionally intact ascending nociceptive pathways. CONCLUSIONS: The present results show that our patients with chronic hemibody pain do not show signs of spinothalamic tract lesion. This indicates normal processing of sensory aspects of painful stimuli, while higher pain processing areas show altered activation. We conclude that normal laser-evoked magnetic fields (LEF) or laser-evoked potentials (LEP) may not exclude central pain condition.

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 LD(50) 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.

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 microM) and NS-398 (30 microM). 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-kappaB) 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-kappaB 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.

Molecular cloning and characterization of the rat inducible nitric oxide synthase (iNOS) gene.

We have cloned and characterized the rat inducible nitric oxide synthase (iNOS) gene. It spans approx. 36kb and is divided into 27 exons and 26 introns. The distribution and length of exons are similar to those in the human iNOS gene. In the 5' flanking regulatory region of the rat iNOS gene, there are a number of putative transcription factor binding sites (>20), many of them probably indispensable for the gene's nuclear factor kappaB (NFkappaB)-dependent induction, but also many which may have a role in its NFkappaB-independent induction pathway. These include cyclic adenosine 3', 5'-monophosphate (cAMP) response elements (CRE), hypoxia responsive element (HRE) and GATA-core elements. Rat models are powerful tools in studies of neurological diseases. Because iNOS is most likely responsible for the harmful consequences of nitric oxide (NO) in general, the cloned rat iNOS gene will further reveal the mechanisms of iNOS inducibility in different cell types during development and disease, including brain diseases, and to promote studies of pharmacological intervention in cases where extensive NO production plays a critical role.

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-1-immunoreactive. 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.

Induction of thymosin beta(4) mRNA following focal brain ischemia.

Thymosin beta 4 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 to be expressed in microglia and CA1-CA2 hippocampal pyramidal cells. Here we show by using in situ hybridization that t beta 4 is dramatically up-regulated in the infarcted brain area after focal ischaemia. In addition, moderate t beta 4 induction is detected in the perifocal zone, substantia nigra and CA1-CA3 hippocampal pyramidal neurones. Macrophages are probably responsible for t beta 4 induction in the infarcted region, and activated non-neuronal cells probably contribute to the induction seen in the thalamus, substantia nigra and perifocal zone. However, the pyramidal neurones in the hippocampus show t beta 4, upregulation which may be related to restoration of neurite circuits after focal ischemic damage.