Brain Advance Access originally published online on May 18, 2005
Brain 2005 128(9):2042-2051; doi:10.1093/brain/awh545
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Ion changes in spreading ischaemia induce rat middle cerebral artery constriction in the absence of NO
Departments of 1 Neurology and Experimental Neurology and 2 Physiology, Charité University Medicine, Berlin, Germany
Correspondence to: Jens P. Dreier, Department of Neurology, Campus Charité Mitte, Charité University Medicine, Schumannstr. 20/21, 10117 Berlin, Germany E-mail: jens.dreier{at}charite.de
 |
Summary |
|---|
 Top Summary IntroductionMaterial and methodsResultsDiscussionReferences |
|---|
In rats, cortical spreading hyperaemia is coupled to a spreading neuroglial depolarization wave (spreading depression) under physiological conditions, whereas cortical spreading ischaemia is coupled to it if red blood cell products are present in the subarachnoid space. Spreading ischaemia has been proposed as the pathophysiological correlate of the widespread cortical infarcts abundantly found in autopsy studies of patients with subarachnoid haemorrhage. The purpose of the present study was to investigate whether the extracellular ion changes associated with the depolarization wave may cause the vasoconstriction underlying spreading ischaemia. We induced spreading ischaemia in vivo with the nitric oxide (NO) scavenger oxyhaemoglobin and an elevated K+ concentration in the subarachnoid space while slow potential, pH, extracellular volume and concentrations of K+, Na+, Ca2+ and Cl– were measured in the cortex with microelectrodes. We then extraluminally applied an ionic cocktail (cocktailSI) to the isolated middle cerebral artery in vitro, matching the ionic composition of the extracellular space as measured during spreading ischaemia in vivo. Extraluminal application of cocktailSI caused middle cerebral artery dilatation in the absence and constriction in the presence of NO synthase inhibition in vitro, corresponding with the occurrence of spreading hyperaemia in the presence and spreading ischaemia in the absence of NO in vivo. The L-type Ca2+ inhibitor nimodipine caused the cocktailSI-induced vasoconstriction to revert to vasodilatation in the absence of NO in vitro similar to the reversal of spreading ischaemia to spreading hyperaemia in response to nimodipine in vivo. We found that K+ was the predominant vasoconstrictor contained in cocktailSI. Its vasoconstrictor action was augmented by NO synthase inhibition. Our results suggest that, under elevated baseline K+ as a hallmark of any condition of energy deficiency, the extracellular ion changes represent the essential mediator of the vascular response to spreading neuroglial depolarization. In the presence of NO they mediate vasodilatation and in its absence they mediate constriction.
Key Words: subarachnoid haemorrhage; spreading depression; ion measurements; coupling; cerebral blood flow
Abbreviations: ACSF = artificial cerebrospinal fluid; ANOVA = analysis of variance; [Ca2+]o = extracellular calcium concentration; cGMP = cyclic guanosine monophosphate; [Cl–]o = extracellular chloride concentration; cocktailSI = ionic cocktail applied to the isolated middle cerebral artery in vitro matching the ionic composition of the extracellular space as measured during spreading ischaemia in vivo (cocktailSI,K3 = cocktailSI, containing 3 mM potassium; cocktailSI,K50 = cocktailSI, containing 50 mM potassium); ECS = extracellular space; [K+]ACSF = potassium concentration of the artificial cerebrospinal fluid; [K+]e = extraluminal potassium concentration at the middle cerebral artery; [K+]o = extracellular potassium concentration; L-NNA = NG-nitro-L-arginine; [Mg2+]o = extracellular magnesium concentration; MOPS = 3-(N-morpholino)propanesulphonic acid; [Na+]ACSF = sodium concentration of the artificial cerebrospinal fluid; [Na+]o = extracellular sodium concentration; NOS = nitric oxide synthase; TPA = tetrapropylammonium
Received December 23, 2004. Revised April 18, 2005. Accepted April 23, 2005.
|
Introduction |
|---|
|
TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Spreading depression is a neuroglial depolarization wave, which propagates at a rate of approximately 3 mm/min in the cerebral cortex (Lauritzen, 1994
; Leão, 1944). During spreading depression, the neuronal metabolism is activated; regional cerebral blood flow is coupled to the activated metabolism and increases. The hyperaemic flow change propagates together with the neuroglial depolarization wave in the cerebral cortex (cortical spreading hyperaemia). The parenchyma recovers from spreading depression without neuronal damage (Nedergaard and Hansen, 1988).
In contrast to spreading depression, spreading depression-like depolarizations occur under energy compromise in the ischaemic penumbra (Strong et al., 2000). They propagate in a similar way to spreading depression (Strong et al., 2000) but the increased neuronal metabolism is not sufficiently matched by an increase in cerebral blood flow, as evidenced by a delay in the energy-dependent repolarization. As a consequence, spreading depression-like depolarizations apparently aggravate neuronal damage (Busch et al., 1996).
A third spreading depression variant has been discovered more recently. Here, the depolarization wave, similar to a regular spreading depression, may start while cerebral blood flow is normal but the coupling between the activated metabolism and cerebral blood flow is inverse. Thus, the neuroglial depolarization wave induces severe vasoconstriction of cerebral arteries and arterioles resulting in an ischaemic blood flow change (Dreier et al., 1998). This ischaemic flow change propagates together with the neuroglial depolarization wave in the cerebral cortex (cortical spreading ischaemia). Spreading ischaemia occurs locally where haemoglobin and an elevated K+ concentration are present in the subarachnoid space. The property of haemoglobin responsible for spreading ischaemia is its nitric oxide (NO)-scavenging function since the NO synthase (NOS) inhibitor NG-nitro-L-arginine (L-NNA) can replace haemoglobin in the protocol producing spreading ischaemia (Dreier et al., 1998). It has been proposed that the elevated subarachnoid K+ concentration increases the extracellular baseline K+ concentration ([K+]o) which may, in turn, down-regulate the Na, K-ATPase activity (Dreier et al., 2001).
Spreading ischaemia is a malignant type of the spreading depression variants. Where it propagates, focal cortical necrosis arises (Dreier et al., 2000). Haemoglobin and K+ are the protein and ion with the highest concentrations in red blood cells and are released when red blood cells lyse. The induction of spreading ischaemia by haemoglobin and K+ in the subarachnoid space has led to the hypothesis (Dreier et al., 1998) that spreading ischaemia could be the pathophysiological correlate of the widespread focal cortical necroses that are observed in approximately 80% of autopsy cases after subarachnoid haemorrhage in humans (Birse and Tom, 1960; Stoltenburg-Didinger and Schwarz, 1987; Neil-Dwyer et al., 1994). Interestingly, prophylactic treatment with the L-type Ca2+ channel antagonist nimodipine has been shown to significantly reduce the risk of ischaemic stroke and bad outcome in patients after subarachnoid haemorrhage (Feigin et al., 1998). In rats, nimodipine caused spreading ischaemia to revert to spreading hyperaemia (Dreier et al., 1998, 2002), which supported a relation between the animal model and the clinical condition.
In the present study, our goal was to find out whether extracellular ion changes mediate the vasoconstriction underlying spreading ischaemia. For this purpose, we first measured the ion changes during spreading ischaemia with ion-sensitive microelectrodes in vivo. The vascular effect of the ion changes could not be tested in vivo independently of the spreading depolarization wave since intracortical application of [K+]o as measured during spreading ischaemia would induce a depolarization wave (Kraig and Nicholson, 1978). Therefore, we investigated the effect of an ionic cocktail of matching composition applied extraluminally to the isolated rat middle cerebral artery in vitro in the presence and absence of NO.
|
Material and methods |
|---|
|
TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Animal preparation, experimental protocol, cerebral blood flow measurement and electrophysiology in vivo
All animal experiments were approved by the Governmental Animal Care and Use Committee [Landesamt für Arbeitsschutz, Gesundheitsschutz und technische Sicherheit Berlin (LAGetSi), G 0346/98]. Male Wistar rats (n = 44; 250–350 g) were anaesthetized with 100 mg/kg body weight thiopental sodium intraperitoneally (Trapanal; BYK Pharmaceuticals, Konstanz, Germany), tracheotomized, and artificially ventilated (Effenberger Rodent Respirator; Effenberger Med.-Techn. Gerätebau, Pfaffing/Attel, Germany). The left femoral artery was cannulated, and continuous saline solution was infused (0.5 ml/h). Body temperature was maintained at 38.0 ± 0.5°C using a heating pad. Systemic arterial pressure (RFT Biomonitor, Zwönitz, Germany) and end-expiratory partial pressure of carbon dioxide (Heyer CO2 Monitor EGM I, Bad Ems, Germany) were monitored. Arterial partial pressure of oxygen (PaO2), arterial partial pressure of carbon dioxide (PaCO2) and pH were serially measured using a Compact 1 Blood Gas Analyser (AVL Medizintechnik GmbH, Bad Homburg, Germany). Since the rats were not paralysed, we could assess the adequacy of the anaesthesia level with testing motor responses to tail-pinch. In addition, changes in blood pressure in response to tail pinch were used to control anaesthesia. Further thiopental doses (25 mg/kg body weight intraperitoneally) were applied when necessary. The animals were killed with KCl intravenously at the end of the experiments.
Parietally, a craniotomy was performed using a saline-cooled drill, as previously reported (Dreier et al., 1998). The dura mater was removed. An inflow tube made it possible to superfuse the brain cortex with artificial cerebrospinal fluid (ACSF) at the open window (Fig. 1). The composition of the control ACSF in mM was: NaCl 127.5; KCl 3.0; CaCl2 1.5; MgSO4 1.2; NaHCO3 24.5; glucose 3.7; urea 6.7. Increasing the ACSF K+ concentration ([K+]ACSF) from 3 to 20, 35 and 45 mM, respectively, determined the decrease in [Na+]ACSF from 152 to 135, 120 and 110 mM to maintain iso-osmolarity. The ACSF was equilibrated with a gas mixture containing 6.6% O2, 5.9% CO2, and 87.5% N2. The measured ion concentrations in the control ACSF were in mM (ABL System 625; Radiometer, Copenhagen, Denmark): Na+ 152.4 ± 0.6, K+ 2.84 ± 0.05, Ca2+ 1.35 ± 0.07 and glucose 3.5 ± 0.1 at a pH of 7.42 ± 0.03, a partial pressure of oxygen (pO2) of 109.0 ± 7.5 mmHg and a partial pressure of carbon dioxide (pCO2) of 40.5 ± 2.3 mmHg (n = 5). The Mg2+ concentration was 1.16 ± 0.03 mM and the Cl– concentration was 132.2 ± 0.5 mM (n = 5; Modular Analytics SWA, F. Hoffmann-La Roche, Basel, Switzerland). Cerebral blood flow was continuously monitored with two laser-Doppler flow probes (Perimed, Järfälla, Sweden). The subarachnoid steady (direct current) potential was measured with a silver chloride wire inserted into the space between the cortex and the coverslip. The electrode was connected to a differential amplifier (Jens Meyer, Munich, Germany). Cerebral blood flow, subarachnoid direct current shift and the voltage signals from the microelectrodes (see below) were continuously recorded using a personal computer and a chart recorder (Dash IV; Astro-Med, West Warwick, RI, USA).
|
Ion-sensitive microelectrodes
To measure changes in the intracortical direct current potential and extracellular ion concentrations, we used ion-sensitive microelectrodes. They were prepared and tested as described previously from double-barrelled thetaglass capillaries (Kugelstätter, Garching, Germany) (Lux and Neher, 1973
). The tip diameter was 3 µm. The reference barrel for the measurement of the direct current potential was back-filled with 154 mM NaCl. The ion-selective barrel was first silanized with methyltrichlorosilane in dichloromethane (Fluka/Sigma-Aldrich, Deisenhofen, Germany) and then tip-filled with ion-exchanger. Ion-exchangers (Fluka/Sigma-Aldrich), filling and calibrating solutions are given in Table 1. Selectivity coefficients for interfering mono- or bivalent ions were less than 10–3 for H+, K+, Ca2+ and Cl– exchangers. The selectivity coefficient of the Na+ exchanger was only 0.6 for Ca2+. Given a measured change in the extracellular Ca2+ concentration ([Ca2+]o) from 1.19 to 0.09 during spreading ischaemia, the extracellular Na+ concentration ([Na+]o) would fall to 61.3 ± 19.3 mM without and to 61.4 ± 19.4 mM with correction for Ca2+ following the Nicolsky–Eisenman equation. A differential amplifier (Jens Meyer, Munich, Germany) was used to subtract the reference signal from the signal of the ion-selective barrel to obtain the voltage shift related to the ion concentration change. The change in ion concentration was then calculated using the Nernst equation. The measuring depth in the cerebral cortex was 100 µm. At the caudal part of the window, we used two microelectrodes glued together, whereas a single microelectrode was positioned rostrally (Fig. 1). Usually, one of the glued electrodes was K+-sensitive while the other varied. Gluing was microscopically controlled. One electrode tip was slightly bent so that the two electrode tips could be glued together in a parallel manner with a tip distance ranging between 5 and 25 µm (Lux and Neher, 1973; Heinemann and Lux, 1975).
|
Changes in the size of the extracellular space (ECS) were estimated with tetrapropylammonium (TPA)-sensitive electrodes containing the ion-exchanger Corning 474317. The cortex was superfused with ACSF containing TPA at a concentration of 2 mM for this purpose. The electrodes are blinded for K+ under this condition. The percentage change in the ECS was calculated from the change in TPA concentration with the following formula:
 |
(Dietzel et al., 1980).
It was not possible to obtain valid measurements of the extracellular Mg2+ concentration ([Mg2+]o) with currently available ion exchangers because of a high selectivity coefficient for Ca2+. Under physiological conditions with [Ca2+]o around 1.4 mM, Mg2+ electrodes were blind to Mg2+; with decline in [Ca2+]o during spreading ischaemia, they became more sensitive to Mg2+ but remained within the non-linear portion of the Nicolsky response curve. The magnitudes of all measured changes in [Na+]o, [K+]o, [Ca2+]o, [Cl–]o, pH and extracellular volume during spreading ischaemia were within the range of those previously reported for both anoxic depolarization and spreading depression (Hansen and Olsen, 1980; Hansen and Zeuthen, 1981; Mutch and Hansen, 1984; Vo
í
ek and Syková, 1997). Therefore, for the experiments in the isolated middle cerebral artery, we estimated the change in [Mg2+]o during spreading ischaemia based on measurements with microdialysis and graphite furnace atomic absorption spectroscopy in a gerbil middle cerebral artery occlusion model (Lee et al., 2002).
Haemoglobin preparation
Haemoglobin was freshly prepared from citrate blood of Wistar rats. The blood was centrifuged (3000 G, 5 min at 4°C) and the plasma discarded. The cells were washed twice with three to four volumes of cold 0.9% NaCl. The buffy coat was removed. The red blood cells were lysed by sonication. The suspension of lysed red blood cells was centrifuged (15 000 G, 10 min at 4°C) and the pellet was removed. The haemoglobin-containing supernatant was transferred by gel chromatography (Bio-Gel P-6; Bio Rad, Richmond, VA, USA) to the ACSF. Concentration and composition of haemoglobin and the electrolytes in the ACSF, which resulted in spreading ischaemia, were measured using a radiometer (ABL System 625; Radiometer): total haemoglobin 3.2 ± 1.0 mM; oxyhaemoglobin 90.9 ± 9.6%; CO haemoglobin 4.0 ± 1.3%; methaemoglobin 2.2 ± 0.9%; deoxyhaemoglobin 5.7 ± 12.3%; Na+ 117.5 ± 6.9 mM; K+ 32.4 ± 5.3 mM; Ca2+ 1.3 ± 0.1 mM; glucose 3.4 ± 0.3 mM with a pH of 7.32 ± 0.12 (n = 44; Cl– and Mg2+ not measured). For comparison, respective concentrations of fresh haemolysate in EDTA (ethylenediamine tetraacetate) tubes were: total haemoglobin 10.3 ± 0.8 mM; oxyhaemoglobin 96.7 ± 1.5%; CO haemoglobin 1.1 ± 0.1%; methaemoglobin 0 ± 0%; deoxyhaemoglobin 4.0 ± 0.7%; Na+ 81.6 ± 2.1 mM; K+ 71.6 ± 2.2 mM; glucose 2.9 ± 0.3 mM with a pH of 7.31 ± 0.03 (n = 7).
Isolation and cannulation of the rat middle cerebral artery
Male Wistar rats (n = 25; 250–350 g) were anaesthetized with isoflurane and decapitated. All experiments were approved by the Governmental Animal Care and Use Committee (LAGetSi, T 0032/99). The brain was rapidly removed from the skull and put in cold (4°C) 3-(N-morpholino)propanesulphonic acid (MOPS)-buffered saline solution with 1% dialysed bovine serum albumin containing in mM: NaCl 144.0; KCl 3.0; CaCl2 2.5; MgSO4 1.5; NaH2PO4 1.21; EDTA 0.02; pyruvate 2.0; MOPS 2.0; glucose 5.0 at pH 7.40 (Lindauer et al., 2001). For a detailed description of middle cerebral artery isolation/cannulation, see Lindauer et al. (2001). Briefly, approximately 1 cm middle cerebral artery was carefully dissected from the brain and cannulated on glass micropipettes. The vessel was continuously perfused with MOPS-buffered saline solution at a transmural pressure of 80 mmHg at a temperature of 37°C. The extraluminal bath contained MOPS-buffered saline solution at a temperature of 37°C without bovine serum albumin and was continuously exchanged at a rate of 20 ml/min. The measured osmolality of the extraluminal bath was 301 ± 1 mosmol/kg (n = 3, Osmomat 030; Gonotec, Gesellschaft für Mess- und Regeltechnik, Berlin, Germany). The vessel chamber was placed on an inverted microscope equipped with a video camera. A monitor was used for online analysis of the luminal diameter. After preparation, the artery was allowed to equilibrate for 1 h. During the entire experiment, temperature, perfusion inflow pressure and flow rate were kept constant. All pharmacologically active substances were added to the extraluminal bath.
After development of spontaneous tone (at least 20% reduction of resting diameter compared with diameters measured immediately after pressurizing), experiments started with an isolated increase in the extraluminal K+ concentration ([K+]e) to 20 mM (hypertonic solution) to test arterial smooth muscle function. Arteries were excluded if they did not show a K+-induced vasodilatation of at least 30%. Thereafter, we lowered [K+]e again to 3 mM. The lumen diameter after equilibration served as the baseline diameter. In group 1 (n = 5), we then applied MOPS-buffered saline solution with an ion composition matching the extracellular changes measured during spreading ischaemia (cocktailSI,K50) containing in mM: NaCl 60.0; KCl 50.0; CaCl2 0.1; MgSO4 0.7; NaH2PO4 1.21; EDTA 0.02; pyruvate 2.0; MOPS 2.0; glucose 5.0; pH 6.90; measured osmolality 224 ± 1 mosmol/kg (n = 3). Then cocktailSI,K50 was washed out again and the experiment was repeated once. Each solution was applied until the effect on the arterial diameter was stable (compare Figs 3–7 for detailed experimental paradigms). Experiments of group 2 (n = 5) started with the application of cocktailSI,K50 followed by wash-out similar to those of group 1. However, L-NNA (Sigma-Aldrich) at 10 µM was then washed in, and after equilibration cocktailSI,K50 was co-applied with L-NNA. In group 3 (n = 5) we co-applied cocktailSI,K50 with nimodipine (Bayer, Leverkusen, Germany) (10 nM) alone and, in group 4 (n = 5), with nimodipine and L-NNA (10 µM). In the experiments of group 5 (n = 5), cocktailSI did not contain [K+]e at 50 mM but [K+]e remained at 3 mM (cocktailSI,K3) and [Na+]e was increased accordingly to 107 mM to maintain iso-osmolarity, but otherwise group 5 was not different from group 2: cocktailSI,K3 was applied first in the absence and then in the presence of L-NNA.
Data analysis
The in vivo data were analysed by comparing relative changes in cerebral blood flow and absolute changes in direct current potential and ion concentrations. Cerebral blood flow changes were calculated in relation to baseline at onset (100%, a zero level was established at the end after global cerebral ischaemia). The subarachnoid direct current and cerebral blood flow parameters related to spreading ischaemia were the same as reported previously (Dreier et al., 2001). The absolute changes in arterial diameter in vitro were calculated by subtraction of the baseline diameter. All data in text and figures are given as mean ± SD. The statistical tests are given in the text. P < 0.05 was accepted as statistically significant.
|
Results |
|---|
|
TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Changes in pH, [K+]o, [Na+]o, [Ca2+]o, [Cl–]o and extracellular volume during spreading ischaemia in vivo
All systemic variables remained within normal limits throughout preparation and experiment (pH 7.41 ± 0.03; PaCO2 42.3 ± 3.0 mmHg; PaO2 117 ± 112 mmHg; mean arterial pressure 86 ± 19 mmHg; haematocrit 45.3 ± 3.1%; total haemoglobin 14.8 ± 1.0 g/dl). To measure the changes in [K+]o, [Na+]o, [Ca2+]o, [Cl–]o and pH, we generated spreading ischaemia by increasing [K+]ACSF in a stepwise manner (3, 25, 35, 45 mM) at 60-min intervals during continuous application of the NO scavenger oxyhaemoglobin. Spreading ischaemias occurred in a similar manner as in a closed window preparation (Dreier et al., 1998
). The first spreading ischaemia occurred at 25 mM [K+]ACSF in four of 36 animals, at 35 mM [K+]ACSF in 29 of 36 and at 45 mM [K+]ACSF in the remaining three animals. Table 2 gives the changes in cerebral blood flow and direct current potential parameters, while Table 3 gives the changes in the extracellular ions during spreading ischaemia. Importantly, similar to normal spreading depression but in contrast to anoxic depolarization (Kraig et al., 1983), spreading ischaemia did not start with a pronounced gradual decline in pH but with an alkaline shift from 7.35 to 7.68 ± 0.14 followed by a decline in pH to 6.85 ± 0.26 (Fig. 2). The delay between the onsets of the negative intracortical direct current shifts at the caudal and rostral recording sites was 46 ± 40 s and the delay between the onsets of the hypoperfusions was 39 ± 40 s, suggesting propagation of both depolarization wave and ischaemic flow change.
|
|
|
Eight experiments were performed to calculate the ECS shrinkage during spreading ischaemia. The first spreading ischaemia occurred at 25 mM [K+]ACSF in three of eight and at 35 mM [K+]ACSF in the remaining five animals under superfusion of TPA (2 mM). The changes in cerebral blood flow and the intracortical as well as subarachnoid direct current potential during spreading ischaemia were not significantly different from those in the absence of TPA. The calculated ECS shrinkage was 70.4 ± 16.9% during spreading ischaemia.
In vitro model for spreading ischaemia in the isolated middle cerebral artery
Here we tested whether the extracellular ion changes measured in vivo during the spreading neuroglial depolarization wave would cause middle cerebral artery dilatation in the presence of NO but constriction in its absence. Under control conditions, cocktailSI,K50 reproducibly caused the middle cerebral artery diameter to increase compared with baseline (group 1, Fig. 3). In group 2, cocktailSI,K50 also caused the arterial diameter to increase under control conditions, whereas it induced a decrease in the presence of L-NNA (P < 0.001, Fig. 4). The difference in arterial diameters between cocktailSI,K50-induced dilatation in the absence and constriction in the presence of NOS inhibition was 72 ± 4 µm (Fig. 4).
|
|
In group 3, nimodipine alone augmented cocktailSI,K50-induced middle cerebral artery dilatation compared with control (P < 0.001, Fig. 5).
|
In group 4, the combination of L-NNA (10 µM) and nimodipine (10 nM) had no significant effect on the baseline diameter (Fig. 6; compare measuring points 45 and 65 min after onset of the experiment). However, nimodipine caused cocktailSI,K50-induced middle cerebral artery constriction under NOS inhibition to revert to dilatation (compare Fig. 6 with Fig. 4, measuring point 80 min). The cocktailSI,K50-induced dilatation was significantly more pronounced under L-NNA and nimodipine than under control conditions (P < 0.001, Fig. 6).
|
In group 5, cocktailSI did not contain [K+]e at 50 but only at 3 mM (cocktailSI,K3), whereas the other ion changes remained the same. Thus, by comparing groups 2 and 5, we were able to isolate the effect of [K+]e from the effects of the other ion changes. We found that K+ was a potent vasoconstrictor contained in cocktailSI,K50: in the absence of L-NNA, the middle cerebral artery was 110 µm less dilated by cocktailSI,K50 than by cocktailSI,K3 (Fig. 7). In the presence of L-NNA, the difference between the cocktails was 147 µm (P < 0.01, Fig. 7). This suggested that NOS inhibition significantly augmented the vasoconstrictor effect of K+ by about 37 µm, which is half of the total shift from cocktailSI,K50-induced dilatation to constriction by NOS inhibition (37 of 72 µm; compare Figs 4 and 7).
|
The direct effect of the reduced basal NO concentration ([NO]) on the vascular smooth muscle was responsible for about 17 of 72 µm (difference between group 1 [control] and groups 2/5 [L-NNA alone] at measuring point 65 min, P < 0.05, one-way ANOVA with Bonferroni post hoc test). The remaining quarter of augmented vasoconstriction by NOS inhibition [18 µm = 72 – (37 + 17) µm] remained unexplained. In Table 4, groups 2–5 are compared at each measuring point with the control group using one-way ANOVAs and post hoc Bonferroni tests.
|
|
Discussion |
|---|
|
TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Extracellular ion changes during spreading ischaemia
We measured the extracellular ion changes during spreading ischaemia in the rat. The microelectrodes were positioned close to the cortical surface, where the spreading, massive constriction of pial arteries and arterioles is directly seen during spreading ischaemia. It has been previously reported that the ion changes during spreading depression and anoxic depolarization are very similar in different species (Kraig and Nicholson, 1978
; Hansen and Olsen, 1980; Hansen and Zeuthen, 1981; Mutch and Hansen, 1984; Lauritzen 1994; Jing et al., 1994; Voíek and Syková, 1997; Mazel et al., 2002; Hrab
tová et al., 2003). Therefore, it was not surprising that the magnitudes of the ion changes, extracellular shrinkage and decline in apparent extracellular osmolarity during spreading ischaemia were also within the same range. The measured change in [Cl–]o was somewhat smaller than that previously reported for the rat during spreading depression and anoxic depolarization (Hansen and Zeuthen, 1981). Approximately 9 mM of this 20 mM difference could be explained by the superior selectivity of chloride ionophore I - cocktail A over interfering bicarbonate compared with Corning 477315 exchanger, used in previous studies (Kraig and Nicholson, 1978; Baumgarten, 1981; Kondo et al., 1989).
In vitro model of spreading ischaemia in the isolated middle cerebral artery
We extraluminally applied a saline solution to the isolated middle cerebral artery matching the ionic composition of the extracellular space as measured during spreading ischaemia. We found that application of this ion cocktail caused significant middle cerebral artery dilatation in the presence of NO and constriction in the absence of NO, similar to the conversion of spreading hyperaemia to spreading ischaemia if NO was not available in vivo (Dreier et al., 1998). Nimodipine caused the ion cocktail-induced middle cerebral artery constriction to revert to dilatation in the continued absence of NO, similar to the reversal of spreading ischaemia to spreading hyperaemia by nimodipine in vivo (Dreier et al., 1998, 2002). These analogies of the in vitro to the in vivo model suggested that we successfully translated important features of the coupling between the spreading neuroglial depolarization wave and cerebral blood flow into the in vitro model. A limitation was that we used neither pial nor cortical arteries and arterioles but the proximal segment of the middle cerebral artery. There are distinct functional differences between proximal and distal middle cerebral artery segments (Edwards et al., 1988). Distal segments were found to have a lower membrane potential and to hyperpolarize as well as dilate more when the inward rectifier K+ channel was activated by moderate increases in [K+]e. However, in the higher range of [K+]e, as studied here, the effect on the membrane potential became similar between proximal and distal segments. Another limitation was that ion changes occurred rapidly during spreading ischaemia in vivo, whereas wash-in of the ion cocktail in vitro took several minutes. Furthermore, the anion gap in vivo (
16 mM), which may consist of lactate, bicarbonate and negatively charged amino acids (Kraig and Nicholson, 1978; Taylor et al., 1996), was filled with Cl– ions. Since a reduction in [Cl–]e was shown to contract rat posterior cerebral arteries, this possibly led to a reduced vasoconstrictor effect of the ion cocktail (Nelson et al., 1997).
Modulation of L-type Ca2+ channels may underlie the permissive effect of NO on K+-induced vasoconstriction
If [K+]e remained at 3 mM instead of increasing to 50 mM, middle cerebral artery dilatation in response to the ion cocktail in the presence of NO was significantly augmented, and constriction in the absence of NO was caused to revert to dilatation. This result supported the previous hypothesis that K+ was critically involved in the vasoconstriction underlying spreading ischaemia (Dreier et al., 1998). To our knowledge, Nishiye et al. (1989) demonstrated in guinea-pig basilar arteries for the first time that K+-induced vasoconstriction was augmented when [NO] was reduced by the NO scavenger oxyhaemoglobin. Saline solution containing elevated [K+]e was prepared by iso-osmotic replacement of NaCl with KCl in this study. Later, Minato et al. (1995) confirmed this finding in dog basilar arteries with a NOS inhibitor instead of oxyhaemoglobin. Golding et al. (2000) found that K+-induced middle cerebral artery constriction was stronger if the solution was isotonic compared with a hypertonic solution; K+-induced vasoconstriction was only significantly augmented by NOS inhibition if the solution was hypertonic. In our study, NOS inhibition significantly augmented K+-induced vasoconstriction of a hypotonic solution. Golding et al. (2001) and Schuh-Hofer et al. (2001) demonstrated a permissive role of NO countering K+-induced vasoconstriction in vitro. Analogously, Dreier et al. (2001) showed a permissive role of NO for the coupling between spreading neuroglial depolarization and cerebral blood flow in vivo. Iadecola and Zhang (1996) had originally coined the concept of the permissive role of NO. Following this concept, NO is not the active vasodilator but a basal NO concentration is required in order for dilatation to occur. Thus, the basal release of NO from the endothelium and perivascular nerves possibly acts to maintain a more dilated state at high extraluminal K+ concentrations.
The L-type Ca2+ channel antagonist nimodipine apparently blocked the vasoconstricting effect of high extraluminal K+ consistent with previous studies (Towart, 1981; Nosko et al., 1986). This effect was independent of NOS inhibition. Since the L-type Ca2+ channel has such a salient importance for K+-induced vasoconstriction, it is obvious that modulation of this channel may underlie the permissive role of NO countering K+-induced vasoconstriction. In support of this idea, Mukundan and Kanagy (2001) showed an increased sensitivity and maximal contraction in response to the L-type Ca2+ channel agonist BAY K 8644 in aortic rings from L-NNA-treated rats compared with controls. The same group also showed that, as in cerebral arteries, K+-induced vasoconstriction was augmented by NOS inhibition in aortic rings and mesenteric arteries (Kanagy, 1997). Modulation of vascular, including cerebrovascular, L-type Ca2+ channels by NO has been ascribed to both voltage-dependent and -independent mechanisms. Voltage-dependent inhibition resulted from cyclic guanosine monophosphate (cGMP)-dependent protein kinase, which caused an increase in the open probability of Ca2+-activated K+ (KCa) channels (Robertson et al., 1993; Archer et al., 1994; Gerzanich et al., 2001). Activation of Kca channels deactivated L-type Ca2+ channels by polarizing the cellular membrane. Voltage-independent inhibition by NO resulted from cGMP-dependent protein kinase phosphorylating the L-type Ca2+ channel itself, or, more likely, a closely related regulatory phosphoprotein (Ishikawa et al., 1993; Tewari and Simard, 1997). Interestingly, nicotine was shown to block this pathway in vascular smooth muscle from lenticulostriate cerebral arteries (Gerzanich et al., 2001). However, NOS inhibition may also augment the Ca2+ sensitivity of the vascular smooth muscle contractile apparatus via a reduction in cGMP/myosin light chain phosphatase activity (Bolz et al., 2003). This effect would also be antagonized by nimodipine, although indirectly, since Ca2+ influx is reduced.
Other vasoeffectors in the ion cocktail
We found that a quarter of the augmented vasoconstrictor effect of the ion cocktail by NOS inhibition was not related to the direct effect of reduced basal [NO] on vascular smooth muscle and did not reflect augmented K+-induced vasoconstriction. This part was probably related to the loss of the permissive NO effect for other vasodilators, particularly H+. Under normal conditions, a decline in pH from 7.4 to 6.9 would potently dilate cerebral arteries (Kontos et al., 1977). However, this effect is significantly inhibited when [NO] is reduced (Niwa et al., 1993; Iadecola et al., 1994; Iadecola and Zhang, 1996). This permissive function of NO for the vascular effect of H+ has been attributed to increased availability of both KATP and KCa channels (Lindauer et al., 2003). Another dilator component of the ion cocktail was the reduction in [Ca2+]e (Harder, 1985). This could be diminished by NOS inhibition if L-type Ca2+ channels are modulated. Reductions in [Cl–]e and [Mg2+]e, as in the ion cocktail, were shown to constrict cerebral arteries (Murakawa et al., 1990; Farago et al., 1991; Nelson et al., 1997). A permissive role of NO for Cl– or Mg2+ has not been yet investigated to our knowledge.
|
Acknowledgements |
|---|
This study was supported by grants DFG-SFB 507 A1 (Dreier, Einhäupl) and A6 (Lindauer). Support of the Wilhelm Sander foundation (2002.028.1) (Dreier) and the Hermann and Lilly Schilling Foundation (Dirnagl) is gratefully acknowledged.
|
References |
|---|
|
TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Archer SL, Huang JM, Hampl V, Nelson DP, Shultz PJ, Weir EK. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc Natl Acad Sci USA 1994; 91: 7583–87.
Baumgarten CM. An improved liquid ion exchanger for chloride ion-selective microelectrodes. Am J Physiol 1981; 241: C258–63.[Web of Science][Medline]
Birse SH, Tom MI. Incidence of cerebral infarction associated with ruptured intracranial aneurysms. A study of 8 unoperated cases of anterior cerebral aneurysm. Neurology 1960; 10: 101–6.[Medline]
Bolz SS, Vogel L, Sollinger D, Derwand R, de Wit C, Loirand G, et al. Nitric oxide-induced decrease in calcium sensitivity of resistance arteries is attributable to activation of the myosin light chain phosphatase and antagonized by the RhoA/Rho kinase pathway. Circulation 2003; 107: 3081–7.
Busch E, Gyngell ML, Eis M, Hoehn-Berlage M, Hossmann KA. Potassium-induced cortical spreading depressions during focal cerebral ischemia in rats: contribution to lesion growth assessed by diffusion-weighted NMR and biochemical imaging. J Cereb Blood Flow Metab 1996; 16: 1090–9.[CrossRef][Web of Science][Medline]
Dietzel I, Heinemann U, Hofmeier G, Lux HD. Transient changes in the size of the extracellular space in the sensorimotor cortex of cats in relation to stimulus-induced changes in potassium concentration. Exp Brain Res 1980; 40: 432–9.[Web of Science][Medline]
Dreier JP, Körner K, Ebert N, Görner A, Rubin I, Back T, et al. Nitric oxide scavenging by haemoglobin or nitric oxide synthase inhibition by N-nitro-L-arginine induces cortical spreading ischemia when K+ is increased in the subarachnoid space. J Cereb Blood Flow Metab 1998; 18: 978–90.[CrossRef][Web of Science][Medline]
Dreier JP, Ebert N, Priller J, Megow D, Lindauer U, Klee R, et al. Products of hemolysis in the subarachnoid space induce cortical spreading ischemia and focal necrosis in rats, a model for delayed ischemic neurological deficits after subarachnoid haemorrhage? J Neurosurg 2000; 93: 668–76.
Dreier JP, Petzold G, Tille K, Lindauer U, Arnold G, Heinemann U, et al. Ischaemia triggered by spreading neuronal activation is inhibited by vasodilators in rats. J Physiol (Lond) 2001; 531: 515–26.
Dreier JP, Windmüller O, Petzold G, Lindauer U, Einhäupl KM, Dirnagl U. Ischemia triggered by red blood cell products in the subarachnoid space is inhibited by nimodipine administration or moderate volume expansion/hemodilution in rats. Neurosurgery 2002; 51: 1457–65.[CrossRef][Web of Science][Medline]
Edwards FR, Hirst GD, Silverberg GD. Inward rectification in rat cerebral arterioles; involvement of potassium ions in autoregulation. J Physiol (Lond) 1988; 404: 455–66.
Farago M, Szabo C, Dora E, Horvath I, Kovach AG. Contractile and endothelium-dependent dilatory responses of cerebral arteries at various extracellular magnesium concentrations. J Cereb Blood Flow Metab 1991; 11: 161–4.[Web of Science][Medline]
Feigin VL, Rinkel GJ, Algra A, Vermeulen M, van Gijn J. Calcium antagonists in patients with aneurysmal subarachnoid hemorrhage: a systematic review. Neurology 1998; 50: 876–83.
Gerzanich V, Zhang F, West GA, Simard JM. Chronic nicotine alters NO signaling of Ca2+ channels in cerebral arterioles. Circ Res 2001; 88: 359–65.
Golding EM, Steenberg ML, Johnson TD, Bryan RM Jr. The effects of potassium on the rat middle cerebral artery. Brain Res 2000; 880: 159–66.[CrossRef][Web of Science][Medline]
Golding EM, Steenberg ML, Johnson TD, Bryan RM Jr. Nitric oxide in the potassium-induced response of the rat middle cerebral artery: a possible permissive role. Brain Res 2001; 889: 98–104.[CrossRef][Web of Science][Medline]
Hansen AJ, Olsen CE. Brain extracellular space during spreading depression and ischemia. Acta Physiol Scand 1980; 108: 355–65.[Web of Science][Medline]
Hansen AJ, Zeuthen T. Extracellular ion concentrations during spreading depression and ischemia in the rat brain cortex. Acta Physiol Scand 1981; 113: 437–45.[Web of Science][Medline]
Harder DR. A cellular mechanism for myogenic regulation of cat cerebral arteries. Ann Biomed Eng 1985; 13: 335–9.[Web of Science][Medline]
Heinemann U, Lux HD. Undershoots following stimulus-induced rises of extracellular potassium concentration in cerebral cortex of cat. Brain Res 1975; 93: 63–76.[CrossRef][Web of Science][Medline]
Hrabtová S, Hrabe J, Nicholson C. Dead-space microdomains hinder extracellular diffusion in rat neocortex during ischemia. J Neurosci 2003; 23: 8351–9.
Iadecola C, Zhang F. Permissive and obligatory roles of NO in cerebrovascular responses to hypercapnia and acetylcholine. Am J Physiol 1996; 271: R990–1001.[Web of Science][Medline]
Iadecola C, Zhang F, Xu X. SIN-1 reverses attenuation of hypercapnic cerebrovasodilation by nitric oxide synthase inhibitors. Am J Physiol 1994; 267: R228–35.[Web of Science][Medline]
Ishikawa T, Hume JR, Keef KD. Regulation of Ca2+ channels by cAMP and cGMP in vascular smooth muscle cells. Circ Res 1993; 73: 1128–37.
Jing J, Aitken PG, Somjen GG. Interstitial volume changes during spreading depression (spreading depression) and spreading depression-like hypoxic depolarization in hippocampal tissue slices. J Neurophysiol 1994; 71: 2548–51.
Kanagy NL. Increased vascular responsiveness to alpha 2-adrenergic stimulation during NOS inhibition-induced hypertension. Am J Physiol 1997; 273: H2756–64.[Web of Science][Medline]
Kondo Y, Buhrer T, Seiler K, Fromter E, Simon W. A new double-barrelled, ionophore-based microelectrode for chloride ions. Pflügers Arch 1989; 414: 663–8.[CrossRef][Web of Science][Medline]
Kontos HA, Raper AJ, Patterson JL. Analysis of vasoactivity of local pH, PCO2 and bicarbonate on pial vessels. Stroke 1977; 8: 358–60.
Kraig RP, Nicholson C. Extracellular ionic variations during spreading depression. Neuroscience 1978; 3: 1045–59.[CrossRef][Web of Science][Medline]
Kraig RP, Ferreira-Filho CR, Nicholson C. Alkaline and acid transients in cerebellar microenvironment. J Neurophysiol 1983; 49: 831–50.
Lauritzen M. Pathophysiology of the migraine aura, the spreading depression theory. Brain 1994; 117: 199–210.
Leão AAP. Spreading depression of activity in the cerebral cortex. J Neurophysiol 1944; 7: 359–90.
Lee MS, Wu YS, Yang DY, Lee JB, Cheng FC. Significantly decreased extracellular magnesium in brains of gerbils subjected to cerebral ischemia. Clin Chim Acta 2002; 318: 121–5.[CrossRef][Web of Science][Medline]
Lindauer U, Kunz A, Schuh-Hofer S, Vogt J, Dreier JP, Dirnagl U. Nitric oxide from perivascular nerves modulates cerebral arterial pH reactivity. Am J Physiol 2001; 281: H1353–63.[Web of Science]
Lindauer U, Vogt J, Schuh-Hofer S, Dreier JP, Dirnagl U. Cerebrovascular vasodilation to extraluminal acidosis occurs via combined activation of ATP-sensitive and Ca2+-activated potassium channels. J Cereb Blood Flow Metab 2003; 23: 1227–38.[CrossRef][Web of Science][Medline]
Lux HD, Neher E. The equilibration time course of [K+]o in cat cortex. Exp Brain Res 1973; 17: 190–205.[Web of Science][Medline]
Mazel T, Richter F, Vargová L, Syková E. Changes in extracellular space volume and geometry induced by cortical spreading depression in immature and adult rats. Physiol Res 2002; 51 Suppl 1: 85–93.[Web of Science][Medline]
Minato H, Hashizume M, Masuda Y, Hosoki K. Modulation of extraluminally induced vasoconstrictions by endothelium-derived nitric oxide in the canine basilar artery. Arzneimittelforschung 1995; 45: 675–8.[Medline]
Mutch WA, Hansen AJ. Extracellular pH changes during spreading depression and cerebral ischemia: mechanisms of brain pH regulation. J Cereb Blood Flow Metab 1984; 4: 17–27.[Web of Science][Medline]
Mukundan H, Kanagy NL. Ca2+ influx mediates enhanced 
2-adrenergic contraction in aortas from rats treated with NOS inhibitor. Am J Physiol 2001; 281: H2233–40.[Web of Science]
Murakawa T, Altura BT, Altura BM. Extracellular magnesium and potassium concentrations interact to modulate tone and reactivity of isolated canine cerebral vascular muscle. Magnes Trace Elem 1990; 9: 79–93.[Web of Science][Medline]
Nedergaard M, Hansen AJ. Spreading depression is not associated with neuronal injury in the normal brain. Brain Res 1988; 449: 395–8.[CrossRef][Web of Science][Medline]
Neil-Dwyer G, Lang DA, Doshi B, Gerber CJ, Smith PW. Delayed cerebral ischaemia: the pathological substrate. Acta Neurochir (Wien) 1994; 131: 137–45.[CrossRef][Medline]
Nelson MT, Conway MA, Knot HJ, Brayden JE. Chloride channel blockers inhibit myogenic tone in rat cerebral arteries. J Physiol (Lond) 1997; 502: 259–64.
Nishiye E, Nakao K, Itoh T, Kuriyama H. Factors inducing endothelium-dependent relaxation in the guinea-pig basilar artery as estimated from the actions of haemoglobin. Br J Pharmacol 1989; 96: 645–55.[Web of Science][Medline]
Niwa K, Lindauer U, Villringer A, Dirnagl U. Blockade of nitric oxide synthesis in rats strongly attenuates the CBF response to extracellular acidosis. J Cereb Blood Flow Metab 1993; 13: 535–9.[Web of Science][Medline]
Nosko M, Krueger CA, Weir BK, Cook DA. Effects of nimodipine on in vitro contractility of cerebral arteries of dog, monkey, and man. J Neurosurg 1986; 65: 376–81.[Web of Science][Medline]
Robertson BE, Schubert R, Hescheler J, Nelson MT. cGMP-dependent protein kinase activates Ca-activated K channels in cerebral artery smooth muscle cells. Am J Physiol 1993; 265: C299–303.[Web of Science][Medline]
Schuh-Hofer S, Lobsien E, Brodowsky R, Vogt J, Dreier JP, Klee R, et al. The cerebrovascular response to elevated potassium–role of nitric oxide in the in vitro model of isolated rat middle cerebral arteries. Neurosci Lett 2001; 306: 61–4.[CrossRef][Web of Science][Medline]
Stoltenburg-Didinger G, Schwarz K. Brain lesions secondary to subarachnoid hemorrhage due to ruptured aneurysms. In: Cervós-Navarro J, Ferszt R, editors. Stroke and microcirculation. New York: Raven Press; 1987. p. 471–80.
Strong AJ, Smith SE, Whittington DJ, Meldrum BS, Parsons AA, Krupinski J, et al. Factors influencing the frequency of fluorescence transients as markers of peri-infarct depolarizations in focal cerebral ischemia. Stroke 2000; 31: 214–22.
Taylor DL, Obrenovitch TP, Symon L. Changes in extracellular acid-base homeostasis in cerebral ischemia. Neurochem Res 1996; 21: 1013–21.[CrossRef][Web of Science][Medline]
Tewari K, Simard JM. Sodium nitroprusside and cGMP decrease Ca2+ channel availability in basilar artery smooth muscle cells. Pflügers Arch 1997; 433: 304–11.[CrossRef][Web of Science][Medline]
Towart R. The selective inhibition of serotonin-induced contractions of rabbit cerebral vascular smooth muscle by calcium-antagonistic dihydropyridines. Circ Res 1981; 48: 650–7.
Voríek I, Sykova E. Ischemia-induced changes in the extracellular space diffusion parameters, K+, and pH in the developing rat cortex and corpus callosum. J Cereb Blood Flow Metab 1997; 17: 191–203.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. P. Dreier, S. Major, A. Manning, J. Woitzik, C. Drenckhahn, J. Steinbrink, C. Tolias, A. I. Oliveira-Ferreira, M. Fabricius, J. A. Hartings, et al. Cortical spreading ischaemia is a novel process involved in ischaemic damage in patients with aneurysmal subarachnoid haemorrhage Brain, July 1, 2009; 132(7): 1866 - 1881. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. K. Shin, P. B. Jones, M. Garcia-Alloza, L. Borrelli, S. M. Greenberg, B. J. Bacskai, M. P. Frosch, B. T. Hyman, M. A. Moskowitz, and C. Ayata Age-dependent cerebrovascular dysfunction in a transgenic mouse model of cerebral amyloid angiopathy Brain, September 1, 2007; 130(9): 2310 - 2319. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Blondeau, O. Petrault, S. Manta, V. Giordanengo, P. Gounon, R. Bordet, M. Lazdunski, and C. Heurteaux Polyunsaturated Fatty Acids Are Cerebral Vasodilators via the TREK-1 Potassium Channel Circ. Res., July 20, 2007; 101(2): 176 - 184. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Dreier, J. Woitzik, M. Fabricius, R. Bhatia, S. Major, C. Drenckhahn, T.-N. Lehmann, A. Sarrafzadeh, L. Willumsen, J. A. Hartings, et al. Delayed ischaemic neurological deficits after subarachnoid haemorrhage are associated with clusters of spreading depolarizations Brain, December 1, 2006; 129(12): 3224 - 3237. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||