Spreading depolarization remarkably exacerbates ischemia-induced tissue acidosis in the young and aged rat brain.

Spreading depolarizations (SDs) occur spontaneously in the cerebral cortex of subarachnoid hemorrhage, stroke or traumatic brain injury patients. Accumulating evidence prove that SDs exacerbate focal ischemic injury by converting zones of the viable but non-functional ischemic penumbra to the core region beyond rescue. Yet the SD-related mechanisms to mediate neurodegeneration remain poorly understood. Here we show in the cerebral cortex of isoflurane-anesthetized, young and old laboratory rats, that SDs propagating under ischemic penumbra-like conditions decrease intra and- extracellular tissue pH transiently to levels, which have been recognized to cause tissue damage. Further, tissue pH after the passage of each spontaneous SD event remains acidic for over 10 minutes. Finally, the recovery from SD-related tissue acidosis is hampered further by age. We propose that accumulating acid load is an effective mechanism for SD to cause delayed cell death in the ischemic nervous tissue, particularly in the aged brain.

Imaging reveals the focal area of spreading depolarizations and a variety of hemodynamic responses in a rat microembolic stroke model.

Spreading depolarizations (SDs) occur in stroke, but the spatial association between SDs and the corresponding hemodynamic changes is incompletely understood. We applied multimodal imaging to visualize the focal area of selected SDs, and hemodynamic responses with SDs propagating over the ischemic cortex. The intracarotid infusion of polyethylene microspheres (d=45 to 53 µm) produced multifocal ischemia in anesthetized rats (n=7). Synchronous image sequences captured through a cranial window above the frontoparietal cortex revealed: Changes in membrane potential (voltage-sensitive (VS) dye method); cerebral blood flow (CBF; laser speckle contrast (LSC) imaging); and hemoglobin (Hb) deoxygenation (red intrinsic optical signal (IOS) at 620 to 640 nm). A total of 31 SD events were identified. The foci of five SDs were seen in the cranial window, originating where CBF was the lowest (56.9±9%), but without evident signs of infarcts. The hyperemic CBF responses to propagating SDs were coupled with three types of Hb saturation kinetics. More accentuated Hb desaturation was related to a larger decrease in CBF shortly after ischemia induction. Microsphere-induced embolization triggers SDs in the rat brain, relevant for small embolic infarcts in patients. The SD occurrence during the early phase of ischemia is not tightly associated with immediate infarct evolution. Various kinetics of Hb saturation may determine the metabolic consequences of individual SDs.

Effects of NMDA receptor antagonists with different subtype selectivities on retinal spreading depression.

BACKGROUND AND PURPOSE: Spreading depression (SD) is a local, temporary disruption of cellular ionic homeostasis that propagates slowly across the cerebral cortex and other neural tissues such as the retina. Spreading depolarization associated with SD occurs in different types of stroke, and this phenomenon correlates also with the initiation of classical migraine aura. The aim of this study was to investigate how NMDA receptor antagonists with different subtype selectivity alter SD. EXPERIMENTAL APPROACH: Immunoblotting was applied to the chick retina for NMDA receptor subunit protein analysis, and an efficient in vitro chick retinal model used with SD imaging for NMDA receptor pharmacology. KEY RESULTS: The prominent NMDA receptor subtypes GluN1, GluN2A and GluN2B were found highly expressed in the chick retina. Nanomolar concentrations of NVP-AAM077 (GluN2A-preferring receptor antagonist) markedly suppressed high K(+) -induced SD; that is, ∼30 times more effectively than MK801. At sub-micromolar concentrations, Ro 25-6981 (GluN2B-preferring receptor antagonist) produced a moderate SD inhibition, whereas CP-101,606 (also GluN2B-preferring receptor antagonist) and UBP141 (GluN2C/2D-preferring receptor antagonist) had no effect. CONCLUSIONS AND IMPLICATIONS: The expression of major NMDA receptor subtypes, GluN1, GluN2A and GluN2B in the chick retina makes them pertinent targets for pharmacological inhibition of SD. The high efficacy of NVP-AAM077 on SD inhibition suggests a critical role of GluN2A-containing receptors in SD genesis. Such high anti-SD potency suggests that NVP-AAM077, and other GluN2A-selective drug-like candidates, could be potential anti-migraine agents.

Effects of early aging and cerebral hypoperfusion on spreading depression in rats.

Cortical spreading depression (CSD) is a feature of stroke pathophysiology. As stroke incidence increases with age, we have examined the effects of early aging and chronic cerebral hypoperfusion on CSD in rats. Three groups were studied: Young, 2-month-old animals; Middle-aged-2VO, subjected to 8 months of bilateral carotid occlusion from 2-month-of-age; and Middle-aged-SHAM, sham-operated. At 2- and 10-month-of-age for the Young and Middle-aged groups, recurrent CSD were induced under halothane anesthesia, by sustained application of 1 M KCl to the cortex for 2 h. Propagating CSD (i.e., cortical EEG, direct current potential) and associated laser Doppler blood flow changes were recorded anteriorly. Susceptibility to CSD and event duration were both decreased by early aging (frequency: 21±0.5 and 6±0.5 CSD/h; duration: 139±7 and 63±8 s; in Young and Middle-aged-SHAM, respectively). There was also a tendency for CSD-associated hyperemia to be reduced in the Middle-aged-2VO group (8.9±2.1 vs. 32.8±12.6% × min in Young). These data suggest reduced sensitivity of the cortex to CSD elicitation with early aging, and a less responsive cerebrovascular system with chronic hypoperfusion.

Multi-modal imaging of anoxic depolarization and hemodynamic changes induced by cardiac arrest in the rat cerebral cortex.

We have reported previously that, in otherwise physiological conditions, spreading depression (SD) can be visualized directly by using a fluorescent, voltage-sensitive (VS) dye. However, in stroke models, where depolarizations occur spontaneously near the ischemic core, marked hemodynamic changes interfere significantly with VS dye imaging. This study provides the scientific basis necessary for accurate interpretation of VS dye images captured from ischemic brains. Using two cameras and carefully selected illuminations, multiple image sequences of the cortex were captured through a cranial window during cardiac arrest and subsequent anoxic depolarization (AD). This multi-modal strategy, used in anesthetized rats, allowed the study of synchronous changes in the following variables: (i) membrane potential (VS dye method); (ii) cerebral blood volume (CBV) with green (540-550 nm) illumination; (iii) hemoglobin (Hb) deoxygenation with red (620-640 nm) illumination, and cerebral blood flow (CBF) by laser speckle contrast imaging. Careful analysis of the data and their relationship revealed two important points: (i) as long as hemoglobin deoxygenation is not too pronounced, vascular changes interfere little with VS dye signals; (ii) in contrast, when the local, blood oxygen carrying capacity is close to exhaustion, higher absorption of both red light excitation and VS dye emission by deoxy-Hb, results in marked decreases of VS dye signals. Multiple, synchronous imaging of cellular depolarization, CBF, CBV and Hb deoxygenation is required for reliable data interpretation - but this combination is a powerful tool to examine the coupling between membrane potential and hemodynamic changes, with high spatial and temporal resolution.

Simultaneous, live imaging of cortical spreading depression and associated cerebral blood flow changes, by combining voltage-sensitive dye and laser speckle contrast methods.

Cortical spreading depression (i.e. waves of cellular depolarization, CSD) causes the aura symptoms in classical migraine, and may contribute to delayed cellular damage after an ischemic or traumatic insult to the brain. In the latter cases, secondary neuronal injury may be worsened by some of the cerebral blood flow (CBF) changes that are associated with CSD. Here, we describe a new method for the simultaneous, live imaging of local cellular depolarization and CBF changes (i.e. two variables with well-defined and important biological significance), through a closed cranial window prepared in anesthetized rats. This novel experimental strategy was validated by imaging the changes associated with CSD elicited by application of high K(+) medium on the cortical surface. CSD was visualized directly by using a fluorescent voltage-sensitive (VS) dye, whereas laser speckle contrast (LSC) imaging allowed the visualization of the corresponding CBF changes. In addition to the high temporal and spatial resolution of VS dye and LSC imaging, their novel combination allows to determine how CBF changes relate to a heterogeneous and evolving pattern of cellular depolarization, in any area of interest of the cortical region under study. This methodological development is especially pertinent and timely for investigations into the peri-lesion depolarizations that occur in models of focal brain injury, situations where their site of spontaneous elicitation and propagation pattern cannot be predicted. It should also help advance our knowledge in epilepsy, CBF pharmacology, and neurovascular coupling under normal and pathophysiological conditions.

Direct, live imaging of cortical spreading depression and anoxic depolarisation using a fluorescent, voltage-sensitive dye.

Perilesion depolarisations, whether transient anoxic depolarisation (AD) or spreading depression (SD), occur in stroke models and in patients with acute brain ischaemia, but their contribution to lesion progression remains unclear. As these phenomena correspond to waves of cellular depolarisation, we have developed a technique for their live imaging with a fluorescent voltage-sensitive (VS) dye (RH-1838). Method development and validation were performed in two different preparations: chicken retina, to avoid any vascular interference; and cranial window exposing the cortical surface of anaesthetised rats. Spreading depression was produced by high-K medium, and AD by complete terminal ischaemia in rats. After dye loading, the preparation was illuminated at its excitation wavelength and fluorescence changes were recorded sequentially with a charge-coupled device camera. No light was recorded when the VS dye was omitted, ruling out the contribution of any endogenous fluorophore. With both preparations, the changes in VS dye fluorescence with SD were analogous to those of the DC (direct current) potential recorded with glass electrodes. Although some blood quenching of the emitted light was identified, the VS dye signatures of SD had a good signal-to-noise ratio and were reproducible. The changes in VS dye fluorescence associated with AD were more complex because of additional interferents, especially transient brain swelling with subsequent shrinkage. However, the kinetics of the AD-associated changes in VS dye fluorescence was also analogous to that of the DC potential. In conclusion, this method provides the imaging equivalent of electrical extracellular DC potential recording, with the SD and AD negative shifts translating directly to fluorescence increase.

Is anoxic depolarisation associated with an ADC threshold? A Markov chain Monte Carlo analysis.

A Bayesian nonlinear hierarchical random coefficients model was used in a reanalysis of a previously published longitudinal study of the extracellular direct current (DC)-potential and apparent diffusion coefficient (ADC) responses to focal ischaemia. The main purpose was to examine the data for evidence of an ADC threshold for anoxic depolarisation. A Markov chain Monte Carlo simulation approach was adopted. The Metropolis algorithm was used to generate three parallel Markov chains and thus obtain a sampled posterior probability distribution for each of the DC-potential and ADC model parameters, together with a number of derived parameters. The latter were used in a subsequent threshold analysis. The analysis provided no evidence indicating a consistent and reproducible ADC threshold for anoxic depolarisation.

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.

Temporal relation between the ADC and DC potential responses to transient focal ischemia in the rat: a Markov chain Monte Carlo simulation analysis.

Markov chain Monte Carlo simulation was used in a reanalysis of the longitudinal data obtained by Harris et al. (J Cereb Blood Flow Metab 20:28-36) in a study of the direct current (DC) potential and apparent diffusion coefficient (ADC) responses to focal ischemia. The main purpose was to provide a formal analysis of the temporal relationship between the ADC and DC responses, to explore the possible involvement of a common latent (driving) process. A Bayesian nonlinear hierarchical random coefficients model was adopted. DC and ADC transition parameter posterior probability distributions were generated using three parallel Markov chains created using the Metropolis algorithm. Particular attention was paid to the within-subject differences between the DC and ADC time course characteristics. The results show that the DC response is biphasic, whereas the ADC exhibits monophasic behavior, and that the two DC components are each distinguishable from the ADC response in their time dependencies. The DC and ADC changes are not, therefore, driven by a common latent process. This work demonstrates a general analytical approach to the multivariate, longitudinal data-processing problem that commonly arises in stroke and other biomedical research.

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.