Effects of phosphodiesterase inhibition on cortical spreading depression and associated changes in extracellular cyclic GMP.

Cortical spreading depression (CSD) is a temporary disruption of local ionic homeostasis that propagates slowly across the cerebral cortex, and may contribute to the pathophysiology of stroke and migraine. Previous studies demonstrated that nitric oxide (NO) formation promotes the repolarisation phase of CSD, and this effect may be cyclic GMP (cGMP)-mediated. Here, we have examined how phosphodiesterase (PDE) inhibition, either alone or superimposed on NO synthase (NOS) inhibition, alters CSD and the associated changes in extracellular cGMP. Microdialysis probes incorporating an electrode were implanted into the frontoparietal cortex of anaesthetised rats for quantitative recording of CSD, pharmacological manipulations, and dialysate sampling for cGMP measurements. CSD was induced by cathodal electrical stimulation in the region under study by microdialysis. Extracellular cGMP increased, but only slightly, during CSD. Perfusion of either zaprinast or sildenafil through the microdialysis probe, at concentrations that inhibited both PDE5 and PDE9 (and possibly other PDE), increased significantly extracellular cGMP. Unexpectedly, these levels remained high when NOS was subsequently inhibited with N(omega)-nitro-l-arginine methyl ester hydrochloride (l-NAME, 1mM). The most interesting pharmacological effect on CSD was obtained with sildenafil. This drug altered neither CSD nor the subsequent characteristic effect of NOS inhibition, i.e. a marked widening of CSD. The fact that NOS inhibition still widened CSD in the presence of the high extracellular levels of cGMP associated with PDE inhibition, suggests that NO may promote CSD recovery, independently of cGMP formation.

Oxygen-induced DNA damage in freshly isolated brain cells compared with cultured astrocytes in the Comet assay.

Brain cells are continuously exposed to reactive oxygen species generated by oxidative metabolism, and in certain pathological conditions defence mechanisms against oxygen radicals may be weakened and/or overwhelmed. DNA is a potential target for oxidative damage, and genomic damage can contribute to neuropathogenesis. It is important, therefore, to identify tools for the quantitative analysis of DNA damage in models of neurological disorders. The aim of this study was to compare the susceptibility of DNA to oxidative stress in cells freshly dissociated from the mouse brain, to that in cultured brain cells. Both primary cultures and a continuous cell line of astrocytes were considered. All cells were treated by xanthine/xanthine oxidase, a superoxide generator or hydrogen peroxide, applied alone or in the presence of the oxygen radical scavengers, superoxide dismutase, catalase, or ascorbic acid. DNA damage, quantified with the Comet assay, was consistent in all the different cell preparations exposed to oxidative stress, and was attenuated in similar ways by superoxide dismutase and catalase, scavengers of superoxide anion and hydrogen peroxide, respectively. The results with ascorbic acid were more variable, presumably because this compound may switch from anti- to pro-oxidant status depending on its concentration and other experimental conditions. Overall, similar responses were found in freshly dissociated and cultured brain cells. These results suggest that the Comet assay can be directly applied to cells freshly dissociated from the brain of rodents, including models of neurological disorders, such as stroke models and animals with targeted mutations that mimic human diseases.

Accumulation of quinolinic acid with neuroinflammation: does it mean excitotoxicity?

The quinolinic acid (QUIN) accumulation that is associated with neuroinflammation is often considered capable of promoting excitotoxic neuronal damage, but QUIN is a relatively weak agonist of N-methyl-D-aspartate (NMDA) receptors. Our study aimed to determine, in vivo, which extracellular concentrations of QUIN must be reached to initiate electrophysiological changes indicative of excitotoxic stress in the cerebral cortex of rats, under normal conditions and when superimposed to a challenge involving NMDA-receptor activation, i.e. repeated cortical spreading depression (CSD). Our experimental strategy relied on microdialysis probes incorporating an electrode, implanted in the brain of halothane-anaesthetised rats. These devices were used to apply QUIN or NMDA locally to the cortical area under study (with or without co-perfusion of high K+ for repetitive induction of CSD), and to record the associated changes in the extracellular DC potential (for information on the membrane polarisation of the cellular population surrounding the probe) and lactate (for the detection of increased local energy demand). The extracellular EC50 for induction of local depolarisation in the normal cortex was around 30 times higher than the extracellular QUIN levels measured in the immunoactivated brain of gerbils. Within the range of concentrations 0.03 to 0.3 mM in the perfusion medium, QUIN suppressed concentration-dependently the elicitation of CSD by K+, presumably because of NMDA-receptor desensitisation. Finally, on-line monitoring of changes in extracellular lactate with local application of QUIN indicated that extracellular concentration of QUIN in the low micromolar range are well tolerated by the brain parenchyma, at least in cortical regions. All these data do not support the notion that QUIN accumulation adds an excitotoxic component to neuroinflammation.

Nitric oxide formation during cortical spreading depression is critical for rapid subsequent recovery of ionic homeostasis.

Cortical spreading depression (CSD) is a temporary disruption of local ionic homeostasis that propagates slowly across the cerebral cortex. Cortical spreading depression promotes lesion progression in experimental stroke, and may contribute to the initiation of migraine attacks. The purpose of this study was to investigate the roles of the marked increase of nitric oxide (NO) formation that occurs with CSD. Microdialysis electrodes were implanted in the cortex of anesthetized rats to perform the following operations within the same region: (1) elicitation of CSD by perfusion of high K+ medium; (2) recording of CSD elicitation; (3) application of the NO synthase inhibitor, NG-nitro-l-arginine methyl ester (l-NAME); and (4) recording of dialysate pH changes. The primary effect of l-NAME (0.3 to 3.0 mmol/L in the perfusion medium) was a marked widening of individual CSD wave, resulting essentially from a delayed initiation of the repolarization phase. This change was due to NO synthase inhibition because it was not observed with the inactive isomer d-NAME, and was reversed by l-arginine. This effect did not appear to be linked to the suppression of a sustained, NO-mediated vascular change associated with the superposition of NO synthase inhibition on high levels of extracellular K+. The delayed initiation of repolarization with local NO synthase inhibition may reflect the suppression of NO-mediated negative feedback mechanisms acting on neuronal or glial processes involved in CSD genesis. However, the possible abrogation of a very brief, NO-mediated vascular change associated with the early phase of CSD cannot be ruled out.