Tumor necrosis factor reduces the amplitude of rat cortical spreading depression in vivo.

OBJECTIVE: Brain damage and ischemia often trigger cortical spreading depression (CSD), which aggravates brain damage. The proinflammatory cytokine tumor necrosis factor (TNF) is significantly upregulated during brain damage, but it is unknown whether TNF influences spreading depression in cerebral cortex in vivo. This question is important because TNF not only furthers inflammatory reactions but might also be neuroprotective. Here we tested the hypothesis that TNF affects CSD, and we explored the direction in which CSD is modified by TNF. METHODS: CSD, elicited by pressure microinjection of KCl, was recorded in anesthetized rats and mice. TNF was administered locally into a trough, providing local TNF treatment of a cortical area. For further analysis, antibodies to TNF receptor (TNFR) 1 or 2 were applied, or CSD was monitored in TNFR1 and TNFR2 knockout mice. γ-Aminobutyric acid (GABA)A receptors were blocked by bicuculline. Immunohistochemistry localized the cortical expression of TNFR1 and TNFR2. RESULTS: Local application of TNF to the cortex reduced dose-dependently the amplitude of CSD. This effect was prevented by blockade or knockout of TNFR2 but not by blockade or knockout of TNFR1. TNFR2 was localized at cortical neurons including parvalbumin-positive inhibitory interneurons, and blockade of GABAA receptors by bicuculline prevented the reduction of CSD amplitudes by TNF. INTERPRETATION: We identified a functional link between TNF and CSD. TNF activates TNFR2 in cortical inhibitory interneurons. The resulting release of GABA reduces CSD amplitudes. In this manner, TNF might be neuroprotective in pathological conditions.

Cortical spreading depolarization is a potential target for rat brain excitability modulation by Galanin.

The inhibitory neuropeptide Galanin (Gal) has been shown to mediate anticonvulsion and neuroprotection. Here we investigated whether Gal affects cortical spreading depolarization (CSD). CSD is considered the pathophysiological neuronal mechanism of migraine aura, and a neuronal mechanism aggravating brain damage upon afflictions of the brain. Immunohistochemistry localized Gal and the Gal receptors 1-3 (GalR1-3) in native rat cortex and evaluated microglial morphology after exposure to Gal. In anesthetized rats, Gal was applied alone and together with the GalR antagonists M40, M871, or SNAP 37889 locally to the exposed cortex. The spontaneous electrocorticogram and CSDs evoked by remote KCl pressure microinjection were measured. In rat cortex, Gal was present in all neurons of all cortical layers, but not in astrocytes, microglia and vessels. GalR2 and GalR3 were expressed throughout all neurons, whereas GalR1 was preponderantly located at neurons in layers IV and V, but only in about half of the neurons. In susceptible rats, topical application of Gal on cortex decreased CSD amplitude, slowed CSD propagation velocity, and increased the threshold for KCl to ignite CSD. In some rats, washout of previously applied Gal induced periods of epileptiform patterns in the electrocorticogram. Blockade of GalR2 by M871 robustly prevented all Gal effects on CSD, whereas blockade of GalR1 or GalR3 was less effective. Although microglia did not express GalRs, topical application of Gal changed microglial morphology indicating microglial activation. This effect of Gal on microglia was prevented by blocking neuronal GalR2. In conclusion, Gal has the potential to ameliorate CSD thus reducing pathophysiological neuronal events caused by or associated with CSD.

From spreading depolarization to epilepsy with neuroinflammation: The role of CGRP in cortex.

CGRP release plays a major role in migraine pain by activating the trigeminal pain pathways. Here we explored putative additional effects of CGRP on cortical circuits and investigated whether CGRP affects cortical excitability, cortical spreading depolarization (CSD), a phenomenon associated with migraine aura, blood-brain-barrier (BBB) and microglial morphology. We used immunohistochemistry to localize CGRP and the CGRP receptor (CGRP-R) in native cortex and evaluated morphology of microglia and integrity of the BBB after exposure to CGRP. In anesthetized rats we applied CGRP and the CGRP-R antagonist BIBN4096BS locally to the exposed cortex and monitored the spontaneous electrocorticogram and CSDs evoked by remote KCl pressure microinjection. In mouse brain slices CGRP effects on neuronal activity were explored by multielectrode array. CGRP immunoreactivity was detectable in intracortical vessels, and all cortical neurons showed CGRP-R immunoreactivity. In rat cortex in vivo, topical CGRP induced periods of epileptiform discharges, however, also dose-dependently reduced CSD amplitudes and propagation velocity. BIBN4096BS prevented these effects. CGRP evoked synchronized bursting activity in mouse cortical but not in cerebellar slices. Topical application of CGRP to rat cortex induced plasma extravasation and this was associated with reduced ramification of microglial cells. From these findings we conclude that CGRP induces a pathophysiological state in the cortex, consisting in neuronal hyperexcitability and neuroinflammation. Thus, CGRP may have a pronounced impact on brain functions during migraine episodes supporting the benefit of CGRP antagonists for clinical use. However, increased cortical CGRP may end the CSD-induced aura phase of migraine.

Spreading depression in the brainstem of the adult rat: electrophysiological parameters and influences on regional brainstem blood flow.

Cortical spreading depression is a pathophysiological excitation wave that occurs during pathophysiological brain conditions such as ischemic brain infarction, migraine aura, and others. Judged from experiments in rodents, the brainstem is thought to be comparatively resistant to the generation of spreading depression. However, because spreading depression can be elicited in the brainstem of rat pups after superfusing the brainstem with solutions enhancing excitability, we reinvestigated spreading depression in the brainstem of the adult rat. Based on theoretical predictions indicating a major role of extracellular potassium in susceptibility to spreading depression, we used conditioning solutions in which chloride ions were replaced by acetate and tetraethylammonium chloride and a small amount of KCl were added. Under these conditions, spreading depression was reproducibly elicited in the brainstem either by topical application of KCl crystals to the brainstem surface or by local microinjection of KCl into the brainstem. The direct current shifts so elicited were accompanied by typical elevation of extracellular potassium ions, propagated in the brainstem, and were prevented by MK-801, an N-methyl D-aspartate blocker. During spreading depression, the regional blood flow in the brainstem was transiently increased. In addition, systemic arterial blood pressure, but not the heart rate, was transiently enhanced. In the nonconditioned brainstem, KCl stimulation neither elicited spreading depression nor induced changes in regional blood flow and blood pressure. These data show that proper conditioning renders the brainstem susceptible to spreading depression, and that spreading depression at this site elicits changes in local circulation and systemic blood pressure.

The potential of substance P to initiate and perpetuate cortical spreading depression (CSD) in rat in vivo.

The tachykinin substance P (SP) increases neuronal excitability, participates in homeostatic control, but induces brain oedema after stroke or trauma. We asked whether SP is able to induce cortical spreading depression (CSD) which often aggravates stroke-induced pathology. In anesthetized rats we applied SP (10-5, 10-6, 10-7, or 10-8 mol/L) to a restricted cortical area and recorded CSDs there and in remote non-treated areas using microelectrodes. SP was either applied in artificial cerebrospinal fluid (ACSF), or in aqua to perform a preconditioning. Plasma extravasation in cortical grey matter was assessed with Evans Blue. Only SP dissolved in aqua induced self-regenerating CSDs. SP dissolved in ACSF did not ignite CSDs even when excitability was increased by acetate-preconditioning. Aqua alone elicited as few CSDs as the lowest concentration of SP. Local pretreatment with 250 nmol/L of a neurokinin 1 receptor antagonist prevented the SP-induced plasma extravasation, the initiation of CSDs by 10-5 mol/L SP diluted in aqua, and the initiation of CSDs by aqua alone, but did not suppress KCl-induced CSD. Thus neurokinin 1 receptor antagonists may be used to explore the involvement of SP in CSDs in clinical studies.

Effects of interleukin-1ß on cortical spreading depolarization and cerebral vasculature.

During brain damage and ischemia, the cytokine interleukin-1ß is rapidly upregulated due to activation of inflammasomes. We studied whether interleukin-1ß influences cortical spreading depolarization, and whether lipopolysaccharide, often used for microglial stimulation, influences cortical spreading depolarizations. In anaesthetized rats, cortical spreading depolarizations were elicited by microinjection of KCl. Interleukin-1ß, the IL-1 receptor 1 antagonist, the GABAA receptor blocker bicuculline, and lipopolysaccharide were administered either alone or combined (interleukin-1ß + IL-1 receptor 1 antagonist; interleukin-1ß + bicuculline; lipopolysaccharide + IL-1 receptor 1 antagonist) into a local cortical treatment area. Using microelectrodes, cortical spreading depolarizations were recorded in a non-treatment and in the treatment area. Plasma extravasation in cortical grey matter was assessed with Evans blue. Local application of interleukin-1ß reduced cortical spreading depolarization amplitudes in the treatment area, but not at a high dose. This reduction was prevented by IL-1 receptor 1 antagonist and by bicuculline. However, interleukin-1ß induced pronounced plasma extravasation independently on cortical spreading depolarizations. Application of lipopolysaccharide reduced cortical spreading depolarization amplitudes but prolonged their duration; EEG activity was still present. These effects were also blocked by IL-1 receptor 1 antagonist. Interleukin-1ß evokes changes of neuronal activity and of vascular functions. Thus, although the reduction of cortical spreading depolarization amplitudes at lower doses of interleukin-1ß may reduce deleterious effects of cortical spreading depolarizations, the sum of interleukin-1ß effects on excitability and on the vasculature rather promote brain damaging mechanisms.

Voltage-gated calcium channels are not involved in generation and propagation of spreading depression (SD) in the brainstem of immature rats.

Spreading depression (SD) can be elicited in the brainstem of rats younger than 13 days when excitability is enhanced by acetate superfusion [F. Richter, S. Rupprecht, A. Lehmenkühler, H.-G. Schaible, Spreading depression can be elicited in brain stem in immature but not adult rats, J. Neurophysiol. 90 (2003) 2163--2170]. To investigate whether voltage-gated calcium channels (VGCCs) modify initiation and propagation of SD in this type of tissue, we applied specific blockers to L-, T-, P/Q-, and N-type VGCCs locally or systemically. SD-related d.c. potentials and concomitant increases in extracellular potassium concentration ([K(+)](e)) were unaffected by the L- and T-type VGCC blocker flunarizine that was applied either systemically (up to 2mg/kg body weight) or by superfusion onto the brainstem (40 microM). In addition, local application of the P/Q-type VGCC blocker omega-agatoxin (1 microM) or of the N-type VGCC blocker omega-conotoxin (1 microM) to the brainstem surface did not influence SD. The results indicate that VGCCs do not modify the generation or propagation of SDs in the brainstem of the immature rat. Blockade of N-type VGCCs disturbed the normal breathing rhythm. Application of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) (250-1000 microM) that elicited SD in the immature cortex, failed to elicit SD in the immature brainstem. In summary, it is likely that K(+) initiates and propagates brainstem SDs.

Scalp-recorded contingent negative variation (CNV) increases during experimentally induced sustained ischemic pain in humans.

Contingent negative variations (CNV) after acoustic stimuli (S1) followed by optical ones (S2) were recorded using electroencephalography in 22 healthy students both under control conditions and during ischemic pain to study the effects of sustained pain on CNV. Mean negative CNV-amplitudes and integrated areas below CNV were significantly larger during periods of ischemic pain than under control conditions (16.53 versus 13.11 microV, respectively (P=0.0028) and 8.318 versus 6.357 microV*s, respectively (P=0.00071)). We conclude that deep somatic pain augments CNV. Reduced CNV amplitudes occurring during migraine attacks, however, reflect other mechanisms which may mask the effects of migraine headache on CNV.

Enhanced neuronal excitability in adult rat brainstem causes widespread repetitive brainstem depolarizations with cardiovascular consequences.

The brainstem of the adult rat is relatively resistant to spreading depolarization (SD) but after enhancement of excitability SD can be evoked by local application of KCl. In the present experiments, we observed that the enhanced excitability even triggers prolonged periods of repetitive depolarizations (RDs), which elicit significant cardiovascular changes. In contrast to KCl-evoked SDs with amplitudes of ∼24 mV and spreading velocity of 4 mm/min, spontaneous RDs had amplitudes of 7 to 12 mV, propagated up to 30 times faster than KCl-evoked SDs, and depolarized larger brainstem areas including the contralateral side. Similarly as SD, RDs depended on glutamatergic neurotransmission and were blocked by MK-801 or by the calcium channel blocker agatoxin. They depended on sodium channels and were blocked by tetrodotoxin. Functionally, the invasion of RDs into the spinal trigeminal and other nuclei evoked bursts of action potentials, indicating that specific neuronal systems are affected. In fact, during episodes of RDs the blood pressure and the local blood flow at the surface of the brainstem and the cortex increased substantially. Brainstem RDs did not propagate into the cerebral cortex. We propose to consider brainstem RPs as a pathophysiological mechanism whose significance for brainstem disease states should be further explored.

The relationship between sudden severe hypoxia and ischemia-associated spreading depolarization in adult rat brainstem in vivo.

Severe ischemia can induce spreading depolarization (SD) in the cerebral cortex, which is thought to contribute significantly to cerebral dysfunction. Whether the mature brainstem shows SD upon reduced oxygen supply has not been investigated although SDs may significantly influence brainstem functions. In anesthetized adult rats, we induced severe short-lasting hypoxia (SSH) by stopping artificial respiration for about 1 min or by ventilation with pure nitrogen for 1, 2 or 3 min, and milder hypoxia by ventilation with 6% O(2) in N(2) for 10 min. We measured DC potentials in the brainstem and cerebral cortex, systemic arterial blood pressure, heart rate and local blood flow at the brainstem or cerebral cortex surface. SSH lasting up to 1 min did not induce DC shifts in native brainstem but reduced heart rate, systemic blood pressure and blood flow in cortex and brainstem. Longer lasting SSH protocols both reduced systemic blood pressure and induced SD in the brainstem, but the magnitude of the cardiovascular response was not influenced by the simultaneous occurrence of SD. When neuronal excitability in the brainstem was artificially enhanced, SSH of 1 min evoked SD but again the magnitude of cardiovascular changes during SSH was not increased. SSH lasting 3 min evoked non-reversible sustained depolarization. SSH did not render the brainstem more excitable for classical SD evoked by local KCl application. Thus, sudden severe hypoxia/ischemia evokes SDs in the brainstem, but the occurrence of the so-elicited SD does not influence the immediate cardiovascular response to SSH.

Extracellular diffusion parameters in the rat somatosensory cortex during recovery from transient global ischemia/hypoxia.

Changes in the extracellular space diffusion parameters during ischemia are well known, but information about changes during the postischemic period is lacking. Extracellular volume fraction (alpha) and tortuosity (lambda) were determined in the rat somatosensory cortex using the real-time iontophoretic method; diffusion-weighted magnetic resonance imaging was used to determine the apparent diffusion coefficient of water. Transient ischemia was induced by bilateral common carotid artery clamping for 10 or 15 mins and concomitant ventilation with 6% O(2) in N(2). In both ischemia groups, a negative DC shift accompanied by increased potassium levels occurred after 1 to 2 mins of ischemia and recovered to preischemic values within 3 to 5 mins of reperfusion. During ischemia of 10 mins duration, alpha typically decreased to 0.07+/-0.01, whereas lambda increased to 1.80+/-0.02. In this group, normal values of alpha=0.20+/-0.01 and lambda=1.55+/-0.01 were registered within 5 to 10 mins of reperfusion. After 15 mins of ischemia, alpha increased within 40 to 50 mins of reperfusion to 0.29+/-0.03 and remained at this level. Tortuosity (lambda) increased to 1.81+/-0.02 during ischemia, recovered within 5 to 10 mins of reperfusion, and was increased to 1.62+/-0.01 at the end of the experiment. The observed changes can affect the diffusion of ions, neurotransmitters, metabolic substances, and drugs in the nervous system.

Spreading depression can be elicited in brain stem of immature but not adult rats.

Spreading depression (SD), a neuronal mechanism involved in brain pathophysiology, occurs in brain areas with high neuronal density such as the cerebral cortex. By contrast, the brain stem is thought to be resistant to SD. Here we show that DC shifts resembling cortical SD can be elicited in rat brain stem by topical application of KCl but not by pricking the brain stem. However, this was only possible until postnatal day 13, and, in addition, susceptibility for SD had to be enhanced. The latter was achieved by superfusion of the brain stem for 45 min with a solution containing acetate instead of chloride ions. Transient asphyxia or hypoxia by 2 min breathing 6% O2 in N2 had a similar effect. Negative brain stem DC deflections were paralleled by an increase of extracellular potassium concentration