Gerd Rosner

Ischemic flow threshold for extracellular glutamate increase in cat cortex.

Extracellular glutamate (Glu), cerebral blood flow (CBF), and auditory-evoked potentials (AEPs) were measured concurrently using microdialysis and hydrogen clearance in the auditory cortex of anesthetized cats during global ischemia of various severities. A threshold-type relationship was observed between extracellular Glu and CBF: Glu increased at CBF levels below about 20 ml/100 g/min. The Glu increase was related to the impairment of AEPs. The results suggest that Glu neurotoxicity is an important factor for ischemic neuronal injury even in penumbra.

Ischemia‐Induced Accumulation of Extracellular Amino Acids in Cerebral Cortex, White Matter, and Cerebrospinal Fluid

Abstract: In a global model of brain ischemia, accumulation of amino acids was studied in the extracellular space of the auditory cortex and the internal capsule using microdialysis, and in CSF of halothane anesthetized cats. In both brain regions, blood flow determined by hydrogen clearance decreased below 10 ml/100 g/min after extracranial multiple‐vessel occlusion, and extracellular potassium activity (Ke) measured in the dialysate increased significantly. A delayed rise in Ke was observed in CSF. In contrast, ischemic amino acid accumulation differed markedly between the two brain regions investigated. In cortex, transmitter amino acids glutamate, aspartate, and γ‐aminobutyric acid (GABA) rose almost immediately after onset of ischemia, and increased 30‐, 25‐, and 250‐fold, respectively, after 2 h of ischemia. The nontransmitter amino acids taurine, alanine, and serine increased 10‐, seven‐, and fourfold, respectively, whereas glutamine and essential amino acids (valine, phenylalanine, isoleucine, and leucine) increased only 1.5‐fold. In the internal capsule, increases in amino acids, if any, were delayed and much smaller than in cortex. The largest alteration was a fivefold elevation of GABA. In CSF, changes in amino acids were small and comparable to those in the internal capsule. Our results demonstrate that ischemia‐induced extracellular amino acid accumulation is a well localized phenomenon restricted to gray matter structures that possess release and reuptake systems for these substances. We assume that amino acids diffuse slowly into adjacent white matter structures, and into CSF.

Reduction of infarct volume by halothane : Effect on cerebral blood flow or perifocal spreading depression-like depolarizations

Halothane is a strong inhibitor of potassium evoked spreading depression (SD) in cats. In the current study, we investigate halothane effects on induction of perifocal SD-like depolarizations, CBF, and infarct evolution in focal ischemia. Calomel and platinum electrodes measured cortical direct current potential and CBF in ectosylvian, suprasylvian, and marginal gyri. Left middle cerebral artery occlusion (MCAO) induced permanent focal ischemia for 16 hours in artificially ventilated cats (30% oxygen, 70% nitrous oxide) under halothane (0.75%, n = 8) or α-chloralose anesthesia (60 mg/kg intravenously, n = 7). Under α-chloralose, MCAO induced severe ischemia in ectosylvian and suprasylvian gyri(mean CBF <10 mL/100 g/min), and direct current potentials turned immediately into terminal depolarization. In marginal gyri, CBF reduction was mild (more than 20 mL/100 g/min), and in six of seven animals, frequent SD-like depolarizations turned into terminal depolarization at a later stage of the experiments. Under halothane, MCAO induced severe ischemia (less than 10 mL/100 g/min) and immediate terminal depolarization only in ectosylvian gyrus. In suprasylvian gyrus, residual CBF remained significantly higher (more than 10 mL/100 g/min) than under α-chloralose, whereas in marginal gyri, CBF did not differ between groups. Compared with chloralose, the number of transient depolarizations was significantly reduced in marginal gyrus, and in suprasylvian gyrus transient but significantly longer depolarizations than in marginal gyrus were recorded. Except for one animal, transient depolarizations did not turn into terminal depolarization under halothane, and infarct volume reduction was particularly seen in suprasylvian gyrus. We conclude that halothane, the most commonly used anesthetic in studies of experimental brain ischemia, has protective properties, which may depend on both cerebrovascular and electrophysiologic influences.

Calcium Ion Transients in Peri-Infarct Depolarizations May Deteriorate Ion Homeostasis and Expand Infarction in Focal Cerebral Ischemia in Cats

Background and Purpose — Harmful effects of peri-infarct depolarizations (PIDs) may depend on recurrent Ca2+ influx. Thus far, few studies have documented the relevance of PIDs in gyrencephalic animals, and the progressive nature of this process has not been investigated over extended periods. We therefore studied in prolonged focal ischemia in cats spatial and temporal profiles of extracellular calcium ([Ca2+]o) shifts in relation to direct current (DC) potential, nitric oxide (NO) concentration and regional cerebral blood flow alterations, and final pathological outcome. Methods — In halothane-anesthetized cats receiving either vehicle (n=12) or MK-801 treatment (5 mg/kg IV; n=10), the left middle cerebral artery was permanently occluded. Laser-Doppler probes, ion-selective microelectrodes, and NO electrodes measured simultaneously regional cerebral blood flow, DC potential, electrocorticogram, [Ca2+]o, and NO concentrations in ectosylvian and suprasylvian gyri of the left cerebral cortex. Results — Persistent depolarization immediately after middle cerebral artery occlusion occurred in 10 ectosylvian and 4 suprasylvian gyri of vehicle-treated animals and in 9 ectosylvian and 3 suprasylvian gyri of MK-801–treated animals. PIDs associated with transient decreases of [Ca2+]o were detected in suprasylvian gyri of only 4 vehicle-treated animals, of which 3 developed recurrent PIDs. Electrocorticogram was suppressed during PIDs, and electrocorticogram recovery worsened in a stepwise manner with consecutive depolarizations. PID duration increased slightly with ongoing ischemia and evolved to persistent depolarization at a final stage. NO transients were not detected during PID, and regional cerebral blood flow transients were not pronounced. Infarction was larger with initial persistent depolarization than with PID and was smallest in MK-801–treated animals. Conclusions — PID is not a common finding in peri-infarct zones in cats, and it is suppressed by the N-methyl-d-aspartate antagonist MK-801. However, if repeated PIDs are generated, they result in a stepwise, progressive breakdown of neuronal function and ion homeostasis, probably contributing to the growth of infarction in focal cerebral ischemia. Recurrent Ca2+ influx is a mechanism that presumably contributes to this process.

Halothane, but not α-chloralose, blocks potassium-evoked cortical spreading depression in cats

The effects of two anesthetics, halothane and alpha-chloralose, on induction of spreading depression and on extracellular glutamate elevation after intracortical potassium administration were investigated in artificially ventilated (30% oxygen/70% nitrous oxide) cats. High potassium concentrations were achieved using either direct KCl injections (7 microliters, 150 mM via a micropipette) or microdialysis by supplementing 100, 300 or 500 mM KCl, respectively, for 10 min to the perfusion solution (Ringers). Changes of the cortical DC potential were recorded adjacent (1-2 mm: electrode DC1) and distant (6-7 mm: electrode DC2) to the injection site. Either under halothane (0.75% in the respiratory gas mixture) or under alpha-chloralose (60 mg/kg i.v.) anesthesia, prolonged negative shifts of the DC potential reflecting the elevated potassium levels after KCl injection were measured near the injection site (electrode DC1). In contrast, spreading depressions (transient short DC deflections) were almost exclusively observed under alpha-chloralose. Spreading depressions recorded with electrode DC1 were superimposed on the prolonged negative DC shifts and they propagated frequently to the more distant site (DC2). Upon KCl administration, dose dependent elevations of extracellular glutamate were measured. These elevations were not significantly altered by the type of anesthesia. Our results suggest that in cats, spreading depression induction is affected by anesthesia, i.e., spreading depression induction is inhibited by halothane as compared to alpha-chloralose. Furthermore, factors other than glutamate or high potassium seem to contribute to spreading depression induction.

Profiles of Cortical Tissue Depolarization in Cat Focal Cerebral Ischemia in Relation to Calcium Ion Homeostasis and Nitric Oxide Production

Cortical depolarization was investigated in a topographic gradient of ischemic density after 1-hour transient middle cerebral artery occlusion in halothane-anesthetized cats. A laser Doppler flow probe, an ion-selective microelectrode, and a nitric oxide (NO) electrode measured regional CBF (rCBF), direct current (DC) potential, extracellular Ca2+ concentration ([Ca2+]o), and NO concentration in ectosylvian and suprasylvian gyri of nine animals. Recordings revealed 12 of 18 sites with persistent negative shifts of the DC potential, severe rCBF reduction, and a drop of [Ca2+]o characteristic for core regions of focal ischemia. Among these sites, two types were distinguished by further analysis. In Type 1 (n = 5), rapid, negative DC shifts resembled anoxic depolarization as described for complete global ischemia. In this type, ischemia was most severe (8.9 ± 2.5% of control rCBF), [Ca2+]o dropped fast and deepest (0.48 ± 0.20 mmol/L), and NO concentration increased transiently (36.1 ± 24.0 nmol/L at 2.5 minutes), and decreased thereafter. In Type 2 (n = 7), the DC potential fell gradually over the first half of the ischemic episode, rCBF and [Ca2+]o reductions were smaller than in Type 1 (16.2 ± 8.2%; 0.77 ± 0.41 mmol/L), and NO increased continuously during ischemia (53.1 ± 60.4 nmol/L at 60 minutes) suggesting that in this type NO most likely exerts its diverse actions on ischemia-threatened tissue. In the remaining six recording sites, a third type (Type 3) attributable to the ischemic periphery was characterized by minimal DC shifts, mild ischemia (37.2 ± 13.3%), nonsignificant alterations of [Ca2+]o, but decreased NO concentrations during middle cerebral artery occlusion. Reperfusion returned the various parameters to baseline levels within 1 hour, the recovery of [Ca2+]o and NO concentration being delayed in Type 1. An NO synthase inhibitor (NG-nitro-l-arginine, 50 mg/kg intravenously; four animals) abolished NO elevation during ischemia. In conclusion, even in the core of focal cerebral ischemia and reperfusion, different ischemic densities produce different types of cortical tissue manifesting distinctive chronological profiles of depolarization, Ca2+ influx, and NO synthesis.

Cortical Deafferentation in Cat Focal Ischemia: Disturbance and Recovery of Sensory Functions in Cortical Areas with Different Degrees of Cerebral Blood Flow Reduction

During and after 15-min occlusion of the middle cerebral artery (MCA) in cats, local CBF and neuronal activity were measured in cortical areas varying in the degree of CBF reduction. In an area within the ischemic center (primary auditory cortex, middle ectosylvian gyrus), CBF was severely suppressed. Click-induced auditory evoked potentials and evoked as well as spontaneous single-unit activity ceased within 1 min after occlusion. Recirculation resulted in a recovery of the different neurophysiological parameters with a time delay ranging from several minutes to 2 h. In two areas surrounding the ischemic focus (a visual area in the marginal gyrus and the forelimb representation area in the primary somatosensory cortex), CBF was reduced but remained above 30 ml/100 g/min during MCA occlusion. Visual flash-induced evoked potentials and somatosensory evoked potentials induced by median nerve electrical stimulation ceased in the corresponding areas with a somewhat slower time course as compared to the auditory responses and they recovered faster after recirculation. In another somatosensory area (hindlimb projection area in the primary somatosensory cortex), CBF stayed nearly at control levels during occlusion. Evoked potentials and single-unit activity induced by tibial nerve electrical stimulation decreased ∼5 min after occlusion and were abolished ∼5 min later. At that time, single-unit activity had changed to a nonresponsive pattern but persisted. However, potentials evoked transcallosally by electrical stimulation of the contralateral hemisphere were still recorded. After reopening the MCA, the recovery of neuronal functions was usually complete and occurred within ∼5 min. We conclude that attention has to be focused on those areas surrounding an acute ischemic focus that show either no or only slight CBF alterations. The functional impairment found in such areas is caused by the injury of subcortical structures leading to a cortical deafferentation. Considering the apparent lack of a CBF disturbance, such a condition should be distinguished from the so-called cortical ischemic penumbra.

Nitric oxide generation from sodium nitroprusside and hydroxylamine in brain

WE aimed to demonstrate different modes of in situ nitric oxide (NO) generation from two NO donors in cerebral cortex of halothane anesthetized cats. NO donors were delivered by in vivo microdialysis into the cortical tissue where an NO electrode measured real-time changes in extracellular NO concentration. In vitro testing in Ringers solution revealed that sodium nitroprusside (SNP) liberates NO spontaneously in the presence of light, whereas hydroxylamine does not generate any NO under these conditions. Supplementation with 200 mM of the NO donors SNP and hydroxylamine evoked dose-related increases in NO concentration (52.0 ± 6.7 nM, n = 5, and 14.0 ± 3.8 nM, n = 5, respectively). The increase in NO concentration was significantly greater during SNP than during hydroxylamine delivery (p < 0.001). In contrast, CBF increased similarly under the influence of both NO donors. Electrocorticogram and cortical direct current potential remained unaffected. In conclusion, hydroxylamine is degraded into NO intracellularly and efficiently dilates cerebral blood vessels. SNP, in contrast, generates NO mainly in the extracellular space.

Adenosine in Relation to Calcium Homeostasis: Comparison between Gray and White Matter Ischemia

In vitro studies suggest that adenosine may attenuate anoxic white matter damage as an intrinsic protective substance. The authors investigated ischemic alterations of purines in relation to tissue depolarization and extracellular calcium and amino acid concentrations in vivo using microdialysis and ion-selective electrodes in cortical gray and subcortical white matter of 10 cats during 120 minutes of global brain ischemia. Immediately on induction of ischemia, regional cerebral blood flow ceased in all cats in both gray and white matter. The direct current potential rapidly decreased, the decline being slower and shallower in white matter. Extracellular calcium levels decreased in gray matter. In contrast, they first increased in white matter and started to decrease below control levels only after approximately 30 minutes. Adenosine levels transiently increased in both tissue compartments; the peak was delayed by 30 minutes in white matter. Thereafter, levels declined faster in gray than in white matter and remained elevated in the latter tissue compartment. Inosine and hypoxanthine elevations were progressive in both regions but smaller in white matter. Levels of gamma-aminobutyric acid, another putatively protective agent, steadily increased, starting immediately in gray matter and delayed by almost 1 hour in white matter. The delayed and prolonged accumulation of adenosine correlates with a slower adenosine triphosphate breakdown in white matter ischemia and may result in protection of white matter by suspending cellular calcium influx.

Early nitric oxide increase in depolarized tissue of cat focal cerebral ischaemia

TISSUE nitric oxide (NO) concentration was investigated in relation to ion-homeostasis disturbance in the cat model of focal cerebral ischaemia. An NO electrode, a Ca2+ microelectrode and a laser Doppler probe were applied to the cerebral cortex in the core and periphery of the middle cerebral artery. NO concentration increased by 25.1 ± 6.3 nM at 5 min in severely ischaemic regions exhibiting anoxic depolarization (n = 5, p < 0.0005). This occurred with no reduction in extracellular Ca2+ concentration and before a massive Ca2+ influx into cells started several minutes later. The NO increase was abolished by NG-nitro-L-arginine treatment (n = 6, p < 0.05) and was absent in regions with no depolarization (n = 5, p < 0.0005). We conclude that the early increase in NO associated with depolarization is achieved by activation of constitutive NO synthase, possibly triggered by intra- cellular Ca2+ release.