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In mice, burrowing is considered a species-typical parameter for assessing well-being, while this is less clear in rats. This exploratory study evaluated burrowing behaviour in three rat strains during training and in the direct postoperative phase after complex intracranial surgery in different neuroscience rat models established at Hannover Medical School or Aachen University Hospital. Male Crl:CD (SD; n = 18), BDIX/UlmHanZtm (BDIX; n = 8) and RjHan:WI (Wistar; n = 35) rats were individually trained to burrow gravel out of a tube on four consecutive days. Thereafter, BDIX rats were subjected to intracranial injection of BT4Ca cells and tumour resection (rat glioma model), SD rats to injection of 6-hydroxydopamine (6-OHDA) or vehicle (rat Parkinson’s disease model) and Wistar rats to endovascular perforation or sham surgery (rat subarachnoid haemorrhage (SAH) model). Burrowing was retested on the day after surgery. During training, BDIX rats burrowed large amounts (mean of 2370 g on the fourth day), while SD and Wistar rats burrowed less gravel (means of 846 and 520 g, respectively). Burrowing increased significantly during training only in Wistar rats. Complex surgery, that is, tumour resection (BDIX), 6-OHDA injection (SD) and endovascular perforation or sham surgery for SAH (Wistar) significantly reduced burrowing and body weight, while simple stereotactic injection of tumour cells or vehicle did not affect burrowing. Despite the training, burrowing differed between the strains. In the direct postoperative phase, burrowing was reduced after complex surgery, indicating reduced well-being. Reduced burrowing was accompanied with postoperative weight loss, a validated and recognised quantitative measure for severity assessment.

Introduction: Animal models for preclinical research of subarachnoid hemorrhage (SAH) are widely used as much of the pathophysiology remains unknown. However, the burden of these models inflicted on the animals is not well characterized. The European directive requires severity assessment-based allocation to categories. Up to now, the classification into predefined categories is rather subjective and often without underlying scientific knowledge. We therefore aimed at assessing the burden of rats after SAH or the corresponding sham surgery to provide a scientific assessment. Methods: We performed a multimodal approach, using different behavior tests, clinical and neurological scoring, and biochemical markers using the common model for SAH of intracranial endovascular filament perforation in male Wistar rats. Up to 7 days after surgery, animals with SAH were compared to sham surgery and to a group receiving only anesthesia and analgesia. Results: Sham surgery (n = 15) and SAH (n = 16) animals showed an increase in the clinical score the first days after surgery, indicating clinical deterioration, while animals receiving only anesthesia without surgery (n = 5) remained unaffected. Body weight loss occurred in all groups but was more pronounced and statistically significant only after surgery. The analysis of burrowing, open field (total distance, erections), balance beam, and neuroscore showed primarily an effect of the surgery itself in sham surgery and SAH animals. Only concerning balance beam and neuroscore, a difference was visible between sham surgery and SAH. The outcome of the analysis of systemic and local inflammatory parameters and of corticosterone in blood and its metabolites in feces was only robust in animals suffering from larger bleedings. Application of principal component analysis resulted in a clear separation of sham surgery and SAH animals from their respective baseline as well as from the anesthesia-only group at days 1 and 3, with the difference between sham surgery and SAH being not significant. Discussion/Conclusion: To our knowledge, we are the first to publish detailed clinical score sheet data combined with advanced behavioral assessment in the endovascular perforation model for SAH in rats. The tests chosen here clearly depict an impairment of the animals within the first days after surgery and are consequently well suited for assessment of the animals’ suffering in the model. A definitive classification into one of the severity categories named by the EU directive is yet pending and has to be performed in the future by including the assessment data from different neurological and nonneurological disease models.

BACKGROUND: Argon has shown neuroprotective effects after traumatic brain injury (TBI) and cerebral ischemia in vitro and in focal cerebral ischemia in vivo. The purpose of this study is to show if Argon beneficially impacts brain contusion volume (BCV) as the primary outcome parameter as well as secondary outcome parameters such as brain edema, intracranial pressure (ICP), neurological outcome, and cerebral blood flow (CBF) in an in vivo model.METHODS: Subjects were randomly assigned to either argon treatment or room air. After applying controlled cortical impact (CCI) onto the dura with 8 m/s (displacement 1 mm, impact duration 150 ms), treatment was administered by a recovery chamber with 25%, 50%, or 75% argon and the rest being oxygen for 4 h after trauma. Two control groups received room air for 15 min and 24 h, respectively. Neurological testing and ICP measurements were performed 24 h after trauma, and brains were removed to measure secondary brain damage. RESULTS: The primary outcome parameter BCV and the secondary outcome parameter brain edema were not significantly reduced by argon treatment at any concentration, respectively. There was a highly significant decrease in ICP at 50% argon (p=0.001), and significant neurological improvement (beamwalk missteps) at 25% and 50% argon (p=0.01; p=0.049 respectively) compared to control.CONCLUSIONS: Similar to prior in vitro studies argon exerts its best neuroprotective effects with regard to neurological outcome and ICP at a concentration of 50%. Furthermore, a significant improvement in neurological outcome was observed at an argon concentration of 25%. There was no significant reduction of BCV as the primary outcome parameter.

Effective pharmacological neuroprotection is one of the most desired aims in modern medicine. We postulated that a combination of two clinically used drugs&mdash, nimodipine (L-Type voltage-gated calcium channel blocker) and amiloride (acid-sensing ion channel inhibitor)&mdash, might act synergistically in an experimental model of ischaemia, targeting the intracellular rise in calcium as a pathway in neuronal cell death. We used organotypic hippocampal slices of mice pups and a well-established regimen of oxygen-glucose deprivation (OGD) to assess a possible neuroprotective effect. Neither nimodipine (at 10 or 20 &micro, M) alone or in combination with amiloride (at 100 &micro, M) showed any amelioration. Dissolved at 2.0 Vol.% dimethyl-sulfoxide (DMSO), the combination of both components even increased cell damage (p = 0.0001), an effect not observed with amiloride alone. We conclude that neither amiloride nor nimodipine do offer neuroprotection in an in vitro ischaemia model. On a technical note, the use of DMSO should be carefully evaluated in neuroprotective experiments, since it possibly alters cell damage.

Clinical presentation and neurological outcome in subarachnoid hemorrhage (SAH) is highly variable. Aneurysmal SAH (aSAH) is hallmarked by sudden increase of intracranial pressure (ICP) and acute hypoperfusion contributing to early brain injury (EBI) and worse outcome, while milder or non-aneurysmal SAH with comparable amount of blood are associated with better neurological outcome, possibly due to less dramatic changes in ICP. Acute pressure dynamics may therefore be an important pathophysiological aspect determining neurological complications and outcome. We investigated the influence of ICP variability on acute changes after SAH by modulating injection velocity and composition in an experimental model of SAH. Five hundred microliters of arterial blood (AB) or normal saline (NS) were injected intracisternally over 1 (AB1, NS1), 10 (AB10, NS10), or 30 min (AB30) with monitoring for 6 h (n = 68). Rapid blood injection resulted in highest ICP peaks (AB1 median 142.7 mmHg [1.Q 116.7–3.Q 230.6], AB30 33.42 mmHg [18.8–38.3], p

INTRODUCTION: The relationship between blood metabolites and hemoglobin degradation products (BMHDPs) formed in the cerebrospinal fluid and the development of vasospasm and delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH) has been the focus of several previous studies, but their molecular and cellular targets remain to be elucidated. METHODS: Because BMHDP‐induced changes in Ca(v)2.3 channel function are thought to contribute to DCI after aSAH, we studied their modulation by unconjugated bilirubin (UCB) in an organotypical neuronal network from wild‐type (WT) and Ca(v)2.3‐deficient animals (KO). Murine retinae were isolated from WT and KO and superfused with nutrient solution. Electroretinograms were recorded before, during, and after superfusion with UCB. Transretinal signaling was analyzed as b‐wave, implicit time, and area under the curve (AUC). RESULTS: Superfusion of UCB significantly attenuated the b‐wave amplitude in the isolated retina from wild‐type mice by 14.9% (P

Aneurysmal subarachnoid hemorrhage (aSAH) is a severe medical condition associated with a significant cause of mortality throughout the world. Cisterna magna injection model is accepted widely to mimic clinical aSAH and is performed on small animal models to study aSAH during neurosurgery. Coherent light scattered from the surface of the rat brain is used to infer information about the variations in blood flow during this condition. We obtained speckle images from the exposed cortex during the entire experiment using an external tissue imaging system. Contrast and fractal analyses are carried out for the recorded speckle pattern time series. Correlation analysis based on Hurst exponent for these images is found to be a more sensitive tool in studying aSAH as compared to routinely used laser speckle contrast analysis for assessing the changes in blood flow velocity. Additionally, our studies provide improved blood flow detection sensitivity with image Hurst exponent in combination with computed fractal dimension, during an event of aSAH.

Many details of the pathophysiology of subarachnoid haemorrhage (SAH) still remain unknown, making animal experiments an indispensable tool for assessment of diagnostics and therapy. For animal protection and project authorization, one needs objective measures to evaluate the severity and burden in each model. Corticosterone is described as a sensitive stress parameter reflecting the acute burden, and inflammatory markers can be used for assessment of the extent of the brain lesion. However, the brain lesion itself may activate the hypothalamic-pituitary-adrenal-axis early after SAH, as shown for ischemic stroke, probably interfering with early inflammatory processes, thus complicating the assessment of severity and burden on the basis of corticosterone and inflammation. To assess the suitability of these markers in SAH, we evaluated the courses of corticosterone, IL-6 and TNF-α up to 6h in an acute model simulating SAH in continuously anaesthetized rats, lacking the pain and stress induced impact on these parameters. Animals were randomly allocated to sham or SAH. SAH was induced by cisterna magna blood-injection, and intracranial pressure and cerebral blood flow were measured under continuous isoflurane/fentanyl anaesthesia. Withdrawn at predetermined time points, blood was analysed by commercial ELISA kits. After 6h the brain was removed for western blot analysis of IL-6 and TNF-α. Serum corticosterone levels were low with no significant difference between sham and SAH. No activation of the HPA-axis was detectable, rendering corticosterone a potentially useful parameter for stress assessment in future chronic studies. Blood IL-6 and TNF-α increased in both groups over time, with IL-6 increasing significantly more in SAH compared to sham towards the end of the observation period. In the basal cortex, IL-6 and TNF-α increased only in SAH. The pro-inflammatory response seems to start locally in the brain, reflected by an increase in peripheral blood. An additional surgery-induced systemic inflammatory response should be considered.

Journal of biomedical optics 24(7), 070601 (2019). doi:10.1117/1.JBO.24.7.070601, Published by SPIE, Bellingham, Wash.

In rats, spreading depolarization induces vasodilation/hyperemia in naïve tissue but the inverse response when artificial cerebrospinal fluid is topically applied to the brain containing (a) a nitric oxide–lowering agent and (b) elevated K+. The inverse response is characterized by severe vasoconstriction/ischemia. The perfusion deficit runs together with the depolarization in the tissue (=spreading ischemia). Here, we found in male Wistar rats that pre-treatment with artificial cerebrospinal fluid containing elevated K+in vivo led to a selective decline in α2/α3Na+/K+-ATPase activity, determined spectrophotometrically ex vivo. Moreover, spreading ischemia, recorded with laser-Doppler flowmetry and electrocorticography, resulted from artificial cerebrospinal fluid containing a nitric oxide–lowering agent in combination with the Na+/K+-ATPase inhibitor ouabain at a concentration selectively inhibiting α2/α3activity. Decline in α2/α3activity results in increased Ca2+uptake by internal stores of astrocytes, vascular myocytes, and pericytes since Ca2+outflux via plasmalemmal Na+/Ca2+-exchanger declines. Augmented Ca2+mobilization from internal stores during spreading depolarization might enhance vasoconstriction, thus, contributing to spreading ischemia. Accordingly, spreading ischemia was significantly shortened when intracellular Ca2+stores were emptied by pre-treatment with thapsigargin, an inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). These findings might have relevance for clinical conditions, in which spreading ischemia occurs such as delayed cerebral ischemia after subarachnoid hemorrhage.

Background Impairment of neurovascular coupling (NVC) was recently reported in the context of subarachnoid hemorrhage and may correlate with disease severity and outcome. However, previous techniques to evaluate NVC required invasive procedures. Retinal vessels may represent an alternative option for non-invasive assessment of NVC. Methods A prototype of an adapted retinal vessel analyzer was used to assess retinal vessel diameter in mice. Dynamic vessel analysis (DVA) included an application of monochromatic flicker light impulses in predefined frequencies for evaluating NVC. All retinae were harvested after DVA and electroretinograms were performed. Results A total of 104 retinal scans were conducted in 21 male mice (90 scans). Quantitative arterial recordings were feasible only in a minority of animals, showing an emphasized reaction to flicker light impulses (8 mice; 14 scans). A characteristic venous response to flicker light, however, could observed in the majority of animals. Repeated measurements resulted in a significant decrease of baseline venous diameter (7 mice; 7 scans, p < 0.05). Ex-vivo electroretinograms, performed after in-vivo DVA, demonstrated a significant reduction of transretinal signaling in animals with repeated DVA (n = 6, p < 0.001). Conclusions To the best of our knowledge, this is the first non-invasive study assessing murine retinal vessel response to flicker light with characteristic changes in NVC. The imaging system can be used for basic research and enables the investigation of retinal vessel dimension and function in control mice and genetically modified animals.

Objective: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common inherited small-vessel disease, is associated with vascular aggregation of mutant Notch3 protein, dysfunction of cerebral vessels, and dementia. Pericytes, perivascular cells involved in microvascular function, express Notch3. Therefore, we hypothesize that these cells may play a role in the pathogenesis of CADASIL.Methods: Two-, 7-, and 12-month-old CADASIL mutant mice (TgNotch3(R169C)) and wild-type controls were examined regarding Notch3 aggregation in pericytes, the coverage of cerebral vessels by pericytes, pericyte numbers, capillary density, blood-brain barrier (BBB) integrity, astrocytic end-feet, and the expression of astrocytic gap junction and endothelial adherens junction protein using immunostaining and Western blot analysis. In addition, we examined cerebrovascular CO2 reactivity using laser Doppler fluxmetry and in vivo microscopy.Results: With increasing age, mutated Notch3 aggregated around pericytes and smooth muscle cells. Notch3 aggregation caused significant reduction of pericyte number and coverage of capillaries by pericyte processes (p < 0.01). These changes were associated with detachment of astrocytic end-feet from cerebral microvessels, leakage of plasma proteins, reduction in expression of endothelial adherens junction protein, and reduced microvascular reactivity to CO2. Smooth muscle cells were not affected by Notch3 accumulation.Interpretation: Our results show that pericytes are the first cells affected by Notch3 aggregation in CADASIL mice. Pericyte pathology causes opening of the BBB and microvascular dysfunction. Therefore, protecting pericytes may represent a novel therapeutic strategy for vascular dementia.

The vasculature in the brain is an actively regulated organ which, on the one hand, is influenced by global changes of systemic parameters (systemic blood pressure, blood gases, and blood pH) and, on the other hand, is capable of locally directing the blood to the regions of demand with high spatial and temporal resolution. Together with pericytes and astrocytes, as part of the neurovascular unit, cerebral vascular endothelium forms the tight blood-brain barrier and has an important immuno-regulatory function. Within this book chapter the main principles of the vascular architecture and the cellular components at the neurovascular unit forming the blood-brain barrier will be described before summarizing the main mechanisms of global blood flow regulation to changes in systemic parameters as well as giving an overview on the local regulation according to the cellular demand during neuronal activation.

The mammalian brain depends upon glucose as its main source of energy, and tight regulation of glucose metabolism is critical for brain physiology. Consistent with its critical role for physiological brain function, disruption of normal glucose metabolism as well as its interdependence with cell death pathways forms the pathophysiological basis for many brain disorders. Here, we review recent advances in understanding how glucose metabolism sustains basic brain physiology. We aim at synthesizing these findings to form a comprehensive picture of the cooperation required between different systems and cell types, and the specific breakdowns in this cooperation which lead to disease.

The most important acute neurological diseases seen at neurosurgery departments are traumatic brain injuries (TBI) and subarachnoid hemorrhages (SAH). In both diseases the pathophysiological sequela are complex and have not been fully understood up to now, and rodent models using rats and mice are most suitable for the investigation of the pathophysiological details. In both models, surgery is performed under anesthesia, followed by assessment of their functional outcome and behavioral testing before brain tissue analysis after euthanasia. Postoperative analgesia is mandatory, and supplementary care is highly recommended for refinement purposes. Pain and stress assessment is mainly based on clinical and behavioral signs, and further research is needed to improve the evaluation of severity in these models.

Cerebral blood flow (CBF) quantification is a valuable tool in stroke research. Mice are of special interest because of the potential of genetic engineering. Magnetic resonance imaging (MRI) provides repetitive, noninvasive CBF quantification. Many MRI techniques require the knowledge of the brain–blood partition coefficient ( BBPC) for water. Adopting an MRI protocol described by Roberts et al (1996) in humans, we determined the BBPC for water in 129S6/SvEv mice from proton density measurements of brain and blood, calibrated with deuterium oxide/water phantoms. The average BBPC for water was 0.89±0.03 mL/g, with little regional variation within the mouse brain.

Recently, a universal, simple, and fail-safe mechanism has been proposed by which cerebral blood flow (CBF) might be coupled to oxygen metabolism during neuronal activation without the need for any tissue-based mechanism. According to this concept, vasodilation occurs by local erythrocytic release of nitric oxide or ATP wherever and whenever hemoglobin is deoxygenated, directly matching oxygen demand and supply in every tissue. For neurovascular coupling in the brain, we present experimental evidence challenging this view by applying an experimental regime operating without deoxy-hemoglobin. Hyperbaric hyperoxygenation (HBO) allowed us to prevent hemoglobin deoxygenation, as the oxygen that was physically dissolved in the tissue was sufficient to support oxidative metabolism. Regional CBF and regional cerebral blood oxygenation were measured using a cranial window preparation in anesthetized rats. Hemodynamic and neuronal responses to electrical forepaw stimulation or cortical spreading depression (CSD) were analyzed under normobaric normoxia and during HBO up to 4 ATA (standard atmospheres absolute). Inconsistent with the proposed mechanism, during HBO, CBF responses to functional activation or CSD were unchanged. Our results show that activation-induced CBF regulation in the brain does not operate through the release of vasoactive mediators on hemoglobin deoxygenation or through a tissue-based oxygen-sensing mechanism.

Impairment of the blood-brain barrier (BBB) after cerebral ischemia leads to extravasation of plasma constituents into the brain parenchyma. We describe a novel method using non-invasive near-infrared fluorescence (NIRF) imaging and bovine serum albumin labeled with a NIRF dye (NIRF-BSA) to detect BBB impairment after middle cerebral artery occlusion (MCAO) in mice. We first explored the time course of BBB impairment after transient MCAO using Evans blue (EB), which binds to plasma albumin in vivo. An initial BBB impairment was observed at 4-8h and a second impairment at 12-16h after reperfusion. No EB extravasation was detected at 8-12h. Non-invasive NIRF imaging with NIRF-BSA confirmed biphasic BBB impairment. Upon co-injection of NIRF-BSA with EB we found a strong correlation between the detected NIRF signal and the amount of extravasated EB (r=0.857, P=0.00178). When MCAO mice received NIRF-BSA together with gadolinium-diethylene triamine penta-acetic acid (Gd-DTPA), T1-weighted images showed Gd-DTPA enhancement at all times while NIRF imaging showed biphasic BBB impairment. In conclusion, NIRF-BSA is a suitable marker of plasma albumin extravasation in the mouse brain. Non-invasive NIRF imaging with NIRF-BSA is a useful tool to study BBB integrity in preclinical models of central nervous system pathology.

Background and Purpose— Statins exert rapid cholesterol-independent vasoprotective effects. Here, we tested whether postevent treatment with intravenously (i.v.) administered rosuvastatin improves acute stroke outcome in mice. Methods— 129/SV wild-type mice were subjected to 1-hour filamentous middle cerebral artery occlusion (MCAo), followed by reperfusion, and were postevent treated with i.v. or intraperitoneal (i.p.) rosuvastatin given up to 6 hours after MCAo (dose range 0.02 to 20 mg kg −1 body weight). Results— Rosuvastatin, when administered i.v., significantly reduced lesion size when given up to 4 hours after MCAo and in doses as low as 0.2 mg kg −1 . In contrast, i.p. administration provided protection only when given directly on reperfusion at a dose of 20 mg kg −1 but not at lower doses or later time points. Lesion protection was evident as late as 5 days after brain ischemia and was associated with functional improvements in the pole-test and wire-hanging test (2.0 mg kg −1 dose). Neuroprotection with i.v. rosuvastatin was achieved with peak plasma concentrations −1 (ie, with 0.2 mg kg −1 ) and was associated with increased levels of phosphorylated Akt kinase and endothelial nitric oxide synthase in the vasculature. Conclusions— Rosuvastatin, given intravenously at pharmacologically relevant concentrations, protects from focal brain ischemia up to 4 hours after an event. In our opinion, the development of an intravenous statin formulation is warranted for acute stroke trials with statins in humans.

Fluorescence molecular brain imaging is a new modality allowing the detection of specific contrast agents down to very low concentration ranges (picomolar) in disease models. Here we demonstrate a first noninvasive application of fluorescence imaging in the human brain, where concentrations down to about 100 nM of a nonspecific dye were detected. We argue that due to its high sensitivity, optical molecular imaging of the brain is feasible, which – together with its bedside applicability – makes it a promising technique for use in patients.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common inherited small-vessel disease, is associated with vascular aggregation of mutant Notch3 protein, dysfunction of cerebral vessels, and dementia. Pericytes, perivascular cells involved in microvascular function, express Notch3. Therefore, we hypothesize that these cells may play a role in the pathogenesis of CADASIL.Two-, 7-, and 12-month-old CADASIL mutant mice (TgNotch3(R169C) ) and wild-type controls were examined regarding Notch3 aggregation in pericytes, the coverage of cerebral vessels by pericytes, pericyte numbers, capillary density, blood-brain barrier (BBB) integrity, astrocytic end-feet, and the expression of astrocytic gap junction and endothelial adherens junction protein using immunostaining and Western blot analysis. In addition, we examined cerebrovascular CO2 reactivity using laser Doppler fluxmetry and in vivo microscopy.With increasing age, mutated Notch3 aggregated around pericytes and smooth muscle cells. Notch3 aggregation caused significant reduction of pericyte number and coverage of capillaries by pericyte processes (p0.01). These changes were associated with detachment of astrocytic end-feet from cerebral microvessels, leakage of plasma proteins, reduction in expression of endothelial adherens junction protein, and reduced microvascular reactivity to CO2 . Smooth muscle cells were not affected by Notch3 accumulation.Our results show that pericytes are the first cells affected by Notch3 aggregation in CADASIL mice. Pericyte pathology causes opening of the BBB and microvascular dysfunction. Therefore, protecting pericytes may represent a novel therapeutic strategy for vascular dementia.

Laser Speckle Contrast Analysis (LASCA), a novel, high-resolution blood flow imaging method, was performed on rat somatosensory cortex during functional activation. In the same animals, cerebral blood flow (CBF) was measured with Laser Doppler Flowmetry. To obtain a quantitative estimate of the underlying neuronal activity, somatosensory evoked potentials were recorded simultaneously with an epidural EEG. Our results show that: 1. CBF changes measured by LASCA or LDF are nonlinearly dependent on the magnitude of electrical neural activity revealed by somatosensory evoked potentials. 2. The magnitude of relative CBF changes measured by LASCA and LDF shows a strong correlation. 3. LASCA imaging localizes the highest relative changes of CBF in microcirculatory areas, with a smaller contribution by larger vessels. This study demonstrates that LASCA is a reliable method that provides 2D-imaging of CBF changes that are comparable to LDF measurements. It further suggests that functional neuroimaging methods based on CBF enhance areas of microcirculation and thus might prove more accurate in localizing neural activity than oxygenation related methods like BOLD-fMRI.

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 (cocktail(SI)) 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 cocktail(SI) 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 cocktail(SI)-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 cocktail(SI). 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.

Albeit controversely discussed, it has been suggested by several authors that nitric oxide (NO) serves as a permissive factor in the cerebral blood flow response to systemic hypercapnia. Potassium channels are important regulators of cerebrovascular tone and may be modulated by a basal perivascular NO level. To elucidate the functional targets of the proposed NO modulation during hypercapnia-induced vasodilation, the authors performed experiments in isolated, cannulated, and pressurized rat middle cerebral arteries (MCA). Extracellular pH was reduced from 7.4 to 7.0 in the extraluminal bath to induce NO dependent vasdilation. Acidosis increased vessel diameter by 35 ± 10%. In separate experiments, ATP-sensitive potassium channels (KATP) were blocked by extraluminal application of glibenclamide (Glib), Ca2+-activated potassium channels (KCa) by tetraethylammonium (TEA), voltage-gated potassium channels (Kv) by 4-aminopyridine, and inward rectifier potassium channels (KIR) by BaCl2. Na+-K+-ATP-ase was inhibited by ouabain. Application of TEA slightly constricted the arteries at pH 7.4 and slightly but significantly attenuated the vasodilation to acidosis. Inhibition of the other potassium channels or Na+-K+-ATP-ase had no effect. Combined blockade of KATP and KCa channels further reduced resting diameter, and abolished acidosis induced vasodilation. The authors conclude that mainly KCa channels are active under resting conditions. KATP and KCa channels are responsible for vasodilation to acidosis. Activity of one of these potassium channel families is sufficient for vasodilation to acidosis, and only combined inhibition completely abolishes vasodilation. During NO synthase inhibition, dilation to the KATP channel opener pinacidil or the KCa channel opener NS1619 was attenuated or abolished, respectively. The authors suggest that a basal perivascular NO level is necessary for physiologic KATP and KCa channel function in rat MCA. Future studies have to elucidate whether this NO dependent effect on KATP and KCa channel function is a principle mechanism of NO induced modulation of cerebrovascular reactivity and whether the variability of findings in the literature concerning a modulatory role of NO can be explained by different levels of vascular NO/cGMP concentrations within the cerebrovascular tree.

We describe a nonradioactive, fluorescence-based method to assess plasma protein extravasation (PPE) in rat dura mater using confocal laser scanning microscopy (CLSM). Unilateral PPE can be induced by electrical stimulation of the ipsilateral trigeminal ganglion (TG) and is widely used as an experimental migraine model. The gold standard to determine PPE in the meninges is based on the detection of radiolabeled albumin ([125]I-BSA). The aim of this study was to develop a nonradioactive, histological method to quantify PPE in the meninges. The fluorescent dye Evans Blue (50 mg/kg) was injected intravenously to the rat 7 min prior to TG stimulation. PPE in dura mater was detected by a CLSM. The amount of extravasated Evans Blue in the dura mater was measured at six to eight regions of interest (ROIs) in the vicinity of large meningeal vessels. The ratio of the average fluorescence intensity within dura mater of the "stimulus side", compared to the contralateral "control side", was calculated for each animal. By using this method, The PPE ratio was 1.67+/-0.12 (n=5). Intravenous injection of three different dosages of the 5HT(1B/1D)-receptor agonist sumatriptan (25, 50, and 100 microg/kg) 15 min prior to stimulation attenuated PPE by 42+/-12%, 49+/-9%, and 86+/-15%, respectively (p0.01). The approximated ED(50) value was 48 microg/kg. Our results are in accordance with previous reports in the literature using the radioactive approach. We conclude that CLSM is a safe, sensitive, and reliable method to assess PPE in rat meninges in an experimental migaine model.

Regular physical activity is associated with a decrease of cerebrovascular and cardiovascular events, which may relate to enhanced endothelium-dependent vasodilation. Here, we provide evidence that physical activity protects against ischemic stroke via mechanisms related to the upregulation of endothelial nitric oxide synthase (eNOS) in the vasculature. Voluntary training on running wheels or exercise on a treadmill apparatus for 3 weeks, respectively, reduced cerebral infarct size and functional deficits, improved endothelium-dependent vasorelaxation, and augmented cerebral blood flow in wild-type mice. The neuroprotective effects of physical training were completely absent in eNOS-deficient mice, indicating that the enhanced eNOS activity by physical training was the predominant mechanism by which this modality protects against cerebral injury. Our results suggest that physical activity not only decreases stroke risk, but also provides a prophylactic treatment strategy for increasing blood flow and reducing brain injury during cerebral ischemia.

To investigate the effect of resting blood oxygen concentration on the hemodynamic response to functional brain activation, we compared activation-induced changes in hemoglobin oxygenation during normoxia with systemic hyperoxia or mild hypoxia. Hemoglobin oxygenation changes were measured by microfiber optical spectroscopy (500–590 nm) in response to physiological whisker barrel cortex activation by whole whisker pad deflection (4 s, 4 Hz) in α-chloralose/urethane anesthetised male Wistar rats. During systemic hyperoxia (n=6), the stimulation-induced hyperoxygenation response was decreased and prolonged, whereas during mild systemic hypoxia (n=7) the peak response was significantly increased followed by a faster return to baseline. During normoxia, a poststimulation under- (oxy-hemoglobin) and overshoot (deoxy-hemoglobin) was observed, which disappeared during systemic hyperoxia and was pronounced during systemic hypoxia. Although averaging out below statistical significance when combining all animals, during mild systemic hypoxia a very small early increase of deoxy-Hb at the beginning of the stimulation period was conspicuous more often than during normoxic or even hyperoxic conditions. This small early increase of deoxy-Hb never preceded the onset of the oxy-Hb response, and was not accompanied by a concomitant decrease in oxy-Hb. Hyperoxia or hypoxia did not affect the induced neuronal responses. Our findings support the concept that the hemodynamic response is regulated according to the metabolic demand of oxygen within the activated tissue.

Background and Purpose— Statins (3-hydroxy-3-methylglutaryl–coenzyme A [HMG-CoA] reductase inhibitors) reduce stroke damage independent of lipid lowering by upregulation of endothelial nitric oxide synthase (eNOS). Acute withdrawal of statin treatment may suppress endothelial NO production and impair vascular function. Methods— To test this hypothesis, we treated 129/SV mice with atorvastatin (10 mg/kg) for 14 days and then withdrew treatment. Results— Treatment with atorvastatin conferred stroke protection by 40% after filamentous occlusion of the middle cerebral artery followed by reperfusion. Withdrawal of statin treatment, however, resulted in the loss of stroke protection after 2 and 4 days. In mouse aortas and brain vasculature, statins upregulated eNOS message 2.3- and 1.7-fold, respectively, as measured by reverse transcription–polymerase chain reaction. Withdrawal of statins resulted in 5- and 2.7-fold downregulation of eNOS in aorta and brain, respectively, after 2 days. Statin treatment decreased RhoA GTPase membrane expression to 48%, while withdrawal of statins resulted in 4-fold increase of RhoA in the membrane. Moreover, platelet factor 4 and β-thromboglobulin in plasma were significantly downregulated by statin treatment, but withdrawal of statins resulted in a 2.9- and 3.1-fold upregulation after 2 days, respectively. Thrombus formation induced by ligature of the inferior vena cava was significantly reduced by statin treatment. When statin treatment was withdrawn, however, protection was lost between 2 and 4 days. Conclusions— Acute termination of statin treatment results in a rapid loss of protection in mouse models of cerebral ischemia and thrombus formation independent of lipid lowering. In patients with acute or impending stroke, withdrawal of statins may impair outcome.

A tight temporal and spatial relationship exists between neuronal activity, metabolism, and blood flow in the brain, and different temporal and spatial kinetics of oxygen consumption and blood flow lead to complex changes in regional cerebral haemoglobin oxygenation. Until now, it remains unclear whether neuronal activation leads to an early deoxygenation termed ‘initial dip’ preceding the rCBF response accompanied by hyperoxygenation. Although several studies have reported the ‘initial dip’, other studies did not confirm these findings, regardless of the species used. In an extensive series of experiments in anesthetized rats, we did not robustly find the dip using various modes of cortical O 2 -measurements. Our findings for the first time demonstrate that the appearance of an initial dip and its amplitude may be correlated to the kinetics of the rCBF response. In addition, we show that the mode of analysis of spectroscopic data critically affects results. In our experiments, the finding of an initial dip was sensitive to the mode of spectroscopic analysis. Advanced models of spectroscopic analysis, including differential pathlength correction, should become standard in optical imaging spectroscopy studies. We conclude that in the rodent the initial dip is not a robust phenomenon, the occurrence of which may depend on the level of baseline CBF and thus mean transit time.

According to the 'neuronal' theory, cortical spreading depression (CSD) is the pathophysiological correlate of migrainous aura. However, the 'vascular' theory has implicated altered vascular function in the induction of aura symptoms. The possibility of a vascular origin of aura symptoms is supported, e.g. by the clinical observation that cerebral angiography frequently provokes migrainous aura. This suggests that endothelial irritation may somehow initiate one of the pathways resulting in migrainous aura. Up to now, an endothelium-derived factor has never been shown to trigger CSD. Here, for the first time, we demonstrate and characterize the ability of the vasoconstrictor and astroglial/neuronal modulator endothelin-1 to trigger Leao's 'spreading depression of activity' in vivo in rats. At a concentration range between 10 nM and 1 microM, endothelin-1 induced changes characteristic of CSD with regard to the rate of propagation, steady (direct current) potential and extracellular K(+)-concentration. A spreading hyperaemia followed by oligaemia was observed similar to those in K(+)-induced CSD. Endothelin-1 did not provoke changes characteristic of a terminal depolarization. The mechanism by which endothelin-1 generated CSD involved the N-methyl-D-asparate receptor. Cerebral blood flow decreased slightly, but significantly, before endothelin-1 generated CSD. A vasodilator (NO*-donor) shifted the threshold for CSD induction to higher concentrations of endothelin-1. Endothelin-1, in contrast to K(+), did not induce CSD in rat brain slices suggesting indirectly that endothelin-1 may require intact perfusion to exert its effects. In conclusion, endothelin-1 was found in the experiment to be the most potent inducer of CSD currently known. We propose endothelin-1 as a possible candidate for the yet enigmatic link between endothelial irritation and migrainous aura.

Characterizing disease progression and identifying possible therapeutic interventions in stroke is greatly aided by the use of longitudinal function imaging studies. In this study, we investigate the applicability of real-time multispectral optoacoustic tomography (MSOT) as a tool for non-invasive monitoring of the progression of stroke in the whole brain. The middle cerebral artery occlusion (MCAO) method was used to induce stroke. Mice were imaged under isoflurane anesthesia preoperatively and at several time points during and after the 60-minute occlusion. The animals were sacrificed after 24 hours and their excised brains frozen at -80°C for sectioning. The cryosection were stained using H&E staining to identify the ischemic lesion. Major vessels are readily identifiable in the whole mouse head in the in vivo optoacoustic scans. During ischemia, a reduction in cerebral blood volume is detectable in the cortex. Post ischemia, spectral unmixing of the optoacoustic signals shows an asymmetry of the deoxygenated hemoglobin in the hemisphere affected by MCAO. This hypoxic area was mainly located around the boundary of the ischemic lesion and was therefore identified as the ischemic penumbra. Non-invasive functional MSOT imaging is able to visualize the hypoxic penumbra in brains affected by stroke. Stopping the spread of the infarct area and revitalizing the penumbra is central in stroke research, this new imaging technique may therefore prove to be a valuable tool in the monitoring and developing new treatments.

In the isolated rat middle cerebral artery (MCA) we investigated the role of nitric oxide (NO)/cGMP in the vasodilatory response to extraluminal acidosis. Acidosis increased vessel diameter from 140 +/- 27 microm (pH 7.4) to 187 +/- 30 microm (pH 7.0, P0.01). NO synthase (NOS) inhibition by N(omega)-nitro-L-arginine (L-NNA, 10 microM) reduced baseline diameter (103 +/- 20 microm, P0.01) and attenuated response to acidosis (9 +/- 8 microm). Application of the NO-donors 3-morpholinosydnonimine (1 microM) or S-nitroso-N-acetylpenicillamine (1 microM), or of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 100 microM) reestablished pre-L-NNA diameter at pH 7.4 and reversed L-NNA-induced attenuation of the vessel response to acidosis. Restoration of pre-L-NNA diameter (pH 7.4) by papaverine (20 microM) or nimodipine (30 nM) had no effect on the attenuated response to acidosis. Guanylyl cyclase inhibition with 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (5 microM) or NOS-inhibition with 7-nitroindazole (7-NI, 100 microM) reduced baseline vessel diameter (109 +/- 8 or 127 +/- 11 microm, respectively) and vasodilation to acidosis, and restoration of baseline diameter with 8-BrcGMP (30 microM) completely restored dilation to pH 7.0. Chronic denervation of NOS-containing perivascular nerves in vivo 14 days before artery isolation significantly reduced pH-dependent reactivity in vitro (diameter increase sham: 48 +/- 14 microm, denervated: 14 +/- 8 microm), and 8-BrcGMP (30 microM) restored dilation to pH 7.0 (denervated: 49 +/- 31 microm). Removal of the endothelium did not change vasodilation to acidosis. We conclude that NO, produced by neuronal NOS of perivascular nerves, is a modulator in the pH-dependent vasoreactivity.

Using optical methods through a closed cranial window over the rat primary sensory cortex in chloralose/urethane-anesthetized rats we evaluated the time course of oxygen delivery and consumption in response to a physiological stimulus (whisker deflection). Independent methodological approaches (optical imaging spectroscopy, single fiber spectroscopy, oxygen-dependent phosphorescence quenching) were applied to different modes of whisker deflection (single whisker, full whisker pad). Spectroscopic data were evaluated using different algorithms (constant pathlength, differential pathlength correction). We found that whisker deflection is accompanied by a significant increase of oxygenated hemoglobin (oxy-Hb), followed by an undershoot. An early increase in deoxygenated hemoglobin (deoxy-Hb) proceeded hyperoxygenation when spectroscopic data were analyzed by constant pathlength analysis. However, correcting for the wavelength dependence of photon pathlength in brain tissue (differential pathlength correction) completely eliminated the increase in deoxy-Hb. Oxygen-dependent phosphorescence quenching did not reproducibly detect early deoxygenation. Together with recent fMRI data, our results argue against significant early deoxygenation as a universal phenomenon in functionally activated mammalian brain. Interpreted with a diffusion-limited model of oxygen delivery to brain tissue our results are compatible with coupling between neuronal activity and cerebral blood flow throughout stimulation, as postulated 110 years ago by C. Roy and C. Sherrington (1890, J. Physiol. 11:85–108).

It has been previously shown that spreading neuronal activation can generate a cortical spreading ischaemia (CSI) in rats. The purpose of the present study was to investigate whether vasodilators cause CSI to revert to a normal cortical spreading depression (CSD). A KCl-induced CSD travelled from an open cranial window to a closed window where the cortex was superfused with physiological artificial cerebrospinal fluid (ACSF). At the closed window, recordings revealed a short-lasting negative slow potential shift accompanied by a variable, small and short initial hypoperfusion followed by hyperaemia and then oligaemia. In contrast, spreading neuronal activation locally induced CSI at the closed window when ACSF contained a NO. synthase (NOS) inhibitor, NG-nitro-l-arginine, and an increased K+ concentration ([K+]ACSF). CSI was characterised by a sharp and prolonged initial cerebral blood flow decrease to 29 ± 11 % of the baseline and a prolonged negative potential shift. Co-application of a NO. donor, S-nitroso-N-acetylpenicillamine, and NOS inhibitor with high [K+]ACSF re-established a short-lasting negative potential shift and spreading hyperaemia typical of CSD. Similarly, the NO.-independent vasodilator papaverine caused CSI to revert to a pattern characteristic of CSD. In acute rat brain slices, NOS inhibition and high [K+]ACSF did not prolong the negative slow potential shift compared to that induced by high [K+]ACSF alone. The data indicate that the delayed recovery of the slow potential was caused by vasoconstriction during application of high [K+]ACSF and a NOS inhibitor in vivo. This supports the possibility of a vicious circle: spreading neuronal activation induces vasoconstriction, and vasoconstriction prevents repolarisation during CSI. Speculatively, this pathogenetic process could be involved in migraine-induced stroke. The coupling between neuronal activity and cerebral blood flow (CBF) is a fundamental process which underpins all cerebral functions (for review see Villringer & Dirnagl, 1995). The underlying mechanisms and how they might become disrupted in cerebrovascular disease are still far from being completely understood. Cortical spreading depression (CSD) represents a specific phenomenon associated with neuronal activation and secondary changes of CBF (for review see Lauritzen, 1994). CSD is characterised as a depolarisation wave in the cerebral cortex propagating at a rate of approximately 3 mm min−1. The wave is induced by factors interfering with neuronal and astroglial function, such as glutamate and K+. Large changes in the ionic homoeostasis and release of excitatory amino acids are associated with CSD. Inter- and extracellular communication between neurones and astrocytes are likely to be involved in the propagation of CSD under physiological conditions (Nedergaard et al. 1995; Basarsky et al. 1998; Kunkler & Kraig, 1998). Interactions between neurones and astrocytes during neuronal activation are also assumed at the level of neuronal nutrition (neurometabolic coupling) (Pellerin & Magistretti, 1994). Hence, both neurones and astrocytes probably collaborate in the process of matching the increased metabolic demand with CBF during spreading depression. In the brains of rodents, CBF may slightly decrease during the onset of CSD but this initial vasoconstriction is variable and short. CSD then induces a brief cortical spreading hyperaemia, followed by a longer-lasting cortical spreading oligaemia (for review see Lauritzen, 1994). Adequate tissue oxygenation during CSD is reflected by the lack of histopathological changes (Nedergaard & Hansen, 1988), except for microglial activation (Caggiano & Kraig, 1996). The physiological mechanism of the CBF response to CSD is not well understood. Much attention has been focused on the role of the potent vasodilator NO. (Goadsby et al. 1992; Duckrow, 1993; Zhang et al. 1994; Wahl et al. 1994; Fabricius et al. 1995; Wolf et al. 1996; Colonna et al. 1997; Dreier et al. 1998). A conceivable mechanism linking CSD to NO. is the coupling of NO. production to changes of intracellular [Ca2+]. Intracellular [Ca2+] rises during CSD in neurones (Kunkler & Kraig, 1998). This stimulates the neuronal NO. synthase (NOS) (Moncada et al. 1991). Endothelial NOS could be indirectly activated by acetylcholine or monoamines released during CSD. Indeed, an increase of NO. was directly measured at the initial phase of CSD (Read & Parsons, 2000). Hence, the vasodilator NO. may mask an initial vasoconstriction. Accordingly, Duckrow (1993) found that NOS inhibition augmented the initial CBF decrease which may depend on the rise of the extracellular K+ concentration ([K+]o), a strong vasoconstrictive stimulus (Hansen et al. 1980; McCulloch et al. 1982). The findings by Duckrow (1993) were confirmed in some (Fabricius et al. 1995; Dreier et al. 1998) but not all (Zhang et al. 1994; Wolf et al. 1996) studies in rats. The reasons for these discrepancies remain unclear. However, a substantial augmentation and prolongation of the initial vasoconstriction was achieved by applying either a NOS inhibitor or the NO. scavenger oxyhaemoglobin in the presence of increased subarachnoid K+ yielding a cortical spreading ischaemia (CSI) (Dreier et al. 1998). CSI was similarly obtained by combined administration of oxyhaemoglobin, physiological K+ and low glucose in the subarachnoid space (Dreier et al. 2000). In these experiments, histopathological analysis revealed that CSIs produce widespread cortical infarction (Dreier et al. 2000). Thus, experimental evidence was provided that spreading neuronal activation can generate significant ischaemia if the coupling between neuronal metabolism and blood flow is inverted (Dreier et al. 1998, 2000). In the present study, CSI was induced by topical superfusion of ACSF containing a NOS inhibitor and increased [K+]ACSF in rats. We tested whether the NO. donor S-nitroso-N-acetylpenicillamine (SNAP) or the NO.-independent vasodilator papaverine re-established a short-lasting CSD with hyperaemia. We also investigated the effects of NOS inhibition and high [K+]ACSF on CSD in brain slices in which vascular effects are excluded.

We used Monte Carlo simulations and the diffusion approximation to estimate correction terms for the analysis of reflectance spectra of cortical intrinsic optical signals. These corrections depend on scattering and absorption properties, i.e. they are dependent on assumptions on the tissue blood content and oxygen saturation. The analysis was applied to reflectance spectra acquired during whisker barrel stimulation in the rat where attenuation spectra were converted to changes in oxygenated and deoxygenated haemoglobin concentration. The description of the experimental data as judged by the residual and sensitivity to variations of wavelength was considerably improved when the correction terms were included. Inclusion of the correction does have a considerable impact on the time course of deoxyhaemoglobin concentration changes. In contrast to the calculation without correction terms, there is no indication for an early increase in deoxyhaemoglobin ('early dip'). This finding might further current interpretation of the coupling between neuronal activation and oxygen extraction and supply.

We investigated the combined effect of increased brain topical K+ concentration and reduction of the nitric oxide(NO.) level caused by nitric oxide scavenging or nitric oxide synthase (NOS) inhibition on regional cerebral blood flow and subarachnoid direct current (DC) potential. Using thiopental-anesthetized male Wistar rats with a closed cranial window preparation, brain topical superfusion of a combination of the NO. scavenger hemoglobin(Hb; 2 mmol/L) and increased K+ concentration in the artificial cerebrospinal fluid ([K+]ACSF) at 35 mmol/L led to sudden spontaneous transient ischemic events with a decrease of CBF to 14 ± 7% (n = 4) compared with the baseline (100%). The ischemic events lasted for 53 ± 17 minutes and were associated with a negative subarachnoid DC shift of −7.3 ± 0.6 mV of 49 ± 12 minutes' duration. The combination of the NOS inhibitor N-nitro-L-arginine(L-NA, 1 mmol/L) with [K+]ACSF at 35 mmol/L caused similar spontaneous transient ischemic events in 13 rats. When cortical spreading depression was induced by KCl at a 5-mm distance, a typical cortical spreading hyperemia (CSH) and negative DC shift were measured at the closed cranial window during brain topical superfusion with either physiologic artificial CSF (n = 5), or artificial CSF containing increased [K+]ACSF at 20 mmol/L (n = 4), [K+]ACSF at 3 mmol/L combined with L-NA (n = 10), [K+]ACSF at 10 mmol/L combined with L-NA (five of six animals) or [K+]ACSF at 3 mmol/L combined with Hb (three of four animals). Cortical spreading depression induced long-lasting transient ischemia instead of CSH, when brain was superfused with either[K+]ACSF at 20 mmol/L combined with Hb (CBF decrease to 20± 20% duration 25 ± 21 minutes, n = 4), or [K+]ACSF at 20 mmol/L combined with L-NA (n = 19). Transient ischemia induced by NOS inhibition and [K+]ACSF at 20 mmol/L propagated at a speed of 3.4 ± 0.6 mm/min, indicating cortical spreading ischemia (CSI). Although CSH did not change oxygen free radical production, as measured on-line by in vivo lucigenin-enhanced chemiluminescence, CSI resulted in the typical radical production pattern of ischemia and reperfusion suggestive of brain damage (n = 4). Nimodipine (2 μg/kg body weight/min intravenously) transformed CSI back to CSH (n = 4). Vehicle had no effect on CSI(n = 4). Our data suggest that the combination of decreased NO. levels and increased subarachnoid K+ levels induces spreading depression with acute ischemic CBF response. Thus, a disturbed coupling of metabolism and CBF can cause ischemia. We speculate that CSI may be related to delayed ischemic deficits after subarachnoid hemorrhage, a clinical condition in which the release of Hb and K+ from erythrocytes creates a microenvironment similar to the one investigated here.