Journal of Cerebral Blood Flow & Metabolism

Regional cerebral blood flow (rCBF) was studied in type III nitric oxide (NO) synthase (endothelial, eNOS) mutant and wild type mice during mechanical whisker stimulation before and after nitro-L-arginine (L-NA) superfusion using the closed cranial window technique. rCBF increased equally in cortical barrel fields in both strains during stimulation, as measured by laser Doppler-flowmetry, and was inhibited by L-NA superfusion (1 mM) in both groups. Hence, coupling of blood flow and metabolism appears neuronal NOS- (nNOS) but not eNOS-dependent in cortical barrel fields of the mouse.

Basic fibroblast growth factor (bFGF) is a potent neurotrophic agent that promotes neuronal survival and outgrowth. Previous studies have shown that bFGF, administered intraventricularly or intravenously before or within hours after ischemia, reduces infarct size and neurological deficits in models of focal cerebral ischemia in rats. In the current study, we tested the hypothesis that bFGF, administered at later time points after ischemia, might improve behavioral recovery without affecting infarct size. Mature Sprague–Dawley rats received bFGF (1 μg/injection) or vehicle by biweekly intracisternal injection for 4 weeks, starting at 1 day following permanent proximal middle cerebral artery (MCA) occlusion. Animals were examined every other day using four different behavioral tests to assess sensorimotor and reflex function. At 4 weeks after ischemia, there was no difference in infarct volume between bFGF- and vehicle-treated animals. There was, however, an enhancement in the rate and degree of behavioral recovery among bFGF-treated animals, as measured by all four tests. There were no apparent side effects of bFGF treatment, except that bFGF-treated animals tended to recover body weight more slowly than did vehicle-treated animals following stroke. The mechanisms of enhancement of behavioral recovery by bFGF require further study, but may include protection against retrograde neuronal death and/or stimulation of neuronal sprouting.

The mechanisms of ischemic cell damage are still not fully understood. It has been shown that alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA)/kainate receptor antagonists, such as 6-nitro-7-sulphamoyl-benzo-(f)-quinoxaline-2,3-dione (NBQX), are neuroprotective in models of transient forebrain ischemia, even when applied during recovery, indicating that nonNMDA receptors may play a pivotal role in ischemic cell damage. In the present series of experiments, we studied whether transient cerebral ischemia causes changes in the extent of mRNA editing of AMPA/kainate receptor subunits, a reaction critical for the control of calcium flux through nonNMDA receptor ion channels. Transient cerebral ischemia was produced in rats using the four-vessel occlusion (4-VO) model. After 30 min of ischemia, brains were recirculated for 4, 8, or 24 h. Total RNA was extracted from the cortex, striatum, and hippocampus in order to analyze the extent of mRNA editing of the glutamate receptor subunits GluR2, GluR5, and GluR6. RNA was converted by reverse transcription into cDNA, which was used as a template for subunit-specific polymerase chain reaction (PCR) to amplify a product across the edited base A (A edited to I in the second transmembrane-spanning regions of GluR2, GluR5, and GluR6). PCR products were analyzed with the restriction enzyme Bbv 1, which recognizes the cDNA sequence GC AGC originating from unedited but not that originating from edited GluR2, GluR5, or GluR6 mRNA (GCGGC, the base I is read as G). Restriction digests were electrophoresed, and the bands visualized with ethidium bromide and then photographed. The extent of mRNA editing of the different subunits was quantified using image analysis and appropriate standards. In all control brains studied, GluR2 mRNA was completely edited and remained so after reversible cerebral ischemia. The extent of GluR5 mRNA editing was significantly upregulated in the striatum (from 39 ± 6% in controls to 57 ± 9 and 56 ± 7 after 4 and 8 h of recovery, respectively, p < 0.05 versus control) but not in the cortex and hippocampus. The extent of GluR6 mRNA editing was significantly reduced after 24 h of recovery: in the cortex, from 92 ± 1 to 78 ± 6% (p < 0.01); in the striatum, from 91 ± 2 to 79 ± 1% (p < 0.001); and in the hippocampus, from 90 ± 3 to 80 ± 2% (p < 0.05). A significant reduction was already apparent in the striatum after 4 h of recovery (p < 0.05). Results indicate that mRNA editing is regulated differently in each of the glutamate receptor subunits GluR2, GluR5, and GluR6 after transient cerebral ischemia. The ischemia-induced upregulation of GluR5 mRNA editing observed in the striatum may be indicative of a higher sensitivity to transient ischemia of neurons that exhibit a large fraction of unedited GluR5 mRNA. This assumption is corroborated by the observation (Mackler and Eberwine, 1993) that GluR5 mRNA is completely unedited in neurons of the hippocampal CA1-subfield, a region most vulnerable to transient cerebral ischemia. Whether the decrease in GluR6 mRNA editing observed in all brain structures after ischemia results from a disturbance of the editing reaction or from glial proliferation will have to be established in further experiments. Studying ischemia-induced changes in mRNA editing of glutamate receptor subunits GluR5 and GluR6 may help to elucidate the molecular mechanisms of ischemic cell damage.

Ischemia induces immediate-early genes (IEGs) in brain. Since prolonged expression of some IEGs may precede neuronal death, some researchers have suggested that these IEGs mediate neuronal death. We therefore examined the effect of 5 and 10 min of global ischemia on the expression of the IEGs NGFI-A, NGFI-B, NGFI-C, egr-2, egr-3, and Nurr1 in gerbil brain. All of the IEGs were induced after 30 min of reperfusion in the hippocampus. Most of them were induced in several other regions as well, including cortex, hypothalamus, thalamus, and amygdala. The acute IEG induction decreased in most brain areas by 2–6 h. However, at 24 h following 5 min of ischemia NGFI-A continued to be expressed in the CA1 region and dentate gyrus. In the dentate gyrus, NGFI-C continued to be expressed for 24 h and egr-3 for as long as 72 h. In other brain areas, all of the IEGs returned to control levels by 72 h except in CA1, where most messenger RNA (mRNA) levels were decreased; this decrease correlated with marked neuronal loss. The persistent expression of NGFI-A in CA1 neurons destined to die and the persistent expression of NGFI-A, NGFI-C, and egr-3 genes in dentate granule cell neurons that survive may indicate that some transcription factors modulate cell death whereas others support cell survival when expressed for prolonged periods. The protein products of several transcription factors, including c-fos, are known to downregulate their own expression. The persistent expression of NGFI-A in the CA1 neurons destined to die could therefore be due to ischemia-induced transcriptional activation caused by, e.g., increased intracellular calcium levels plus a lack of negative feedback caused by the blockade of the translation of NGFI-A mRNA into protein.

Stress proteins are induced after a variety of neuronal injuries. The inducible 72-kDa heat shock protein (hsp70) is a stress protein that protects neurons from glutamate toxicity in vitro. Hsp70 has also been proposed to underlie the phenomenon of ischemic tolerance whereby brief sublethal intervals of global ischemia protect the hippocampus from subsequent lethal prolonged ischemia. To determine if the phenomenon of tolerance occurs in cortex after focal ischemia, the rat middle cerebral artery (MCA) was occluded by the suture method. Three 10-min intervals of transient ischemia (3 × 10-isc) separated by 45-min periods of reperfusion made up the most effective paradigm of preconditioning ischemia studied, and substantially reduced the volume of infarction 72 h after subsequent 100-min MCA occlusion. This approach induced protection if the interval between the 3 × 10-isc and the 100-min ischemia was 2, 3, or 5 days but not 1 or 7 days. Three 10-min intervals of transient ischemia alone produced minimal histological changes in the cortex at 72 h. Moreover, there were no significant changes in regional cerebral blood flow in the tolerant regions at 72 h after 3 × 10-isc before or during MCA occlusion. To explore the role of stress proteins in the induction of tolerance, expression of hsp70 and the glucose-regulated proteins grp75 and grp78 were studied. Samples from tolerant regions of the brain that had undergone preconditioning ischemia were evaluated at 1, 2, 3, 5, 7, and 14 days after 3 × 10-isc by Western blot analysis. The time course of hsp70 expression most closely correlated with tolerance. Hsp70 protein expression increased during times when tolerance was present (at 2–5 days) but did not increase thereafter (at 7 and 14 days). However, hsp70 was also increased before tolerance was present (at 1 day). Immunocytochemistry showed that hsp70 protein was expressed in neurons in the tolerant regions 24 h after 3 × 10-isc and was expressed in both neurons and glia after 72 h. Although immunocytochemistry suggested that there was increased neuronal expression of grp75 and grp78, no significant differences were found in protein expression as determined by Western blot before (at 1 day), during (at 2–5 days), and after (at 7 days and thereafter) tolerance. Thus, the time course of grp75 and grp78 expression did not correlate with that of tolerance. This model of ischemic tolerance is a useful method by which mechanisms of endogenous neuroprotection may be explored.

We tested the hypothesis that treatment of transient focal cerebral ischemia in rat with antibodies directed against adhesion molecules reduces apoptosis. Rats (n = 31) were subjected to 2 h of middle cerebral artery (MCA) occlusion induced by intraluminal insertion of a nylon monofilament into the internal carotid artery. Upon reperfusion, animals were treated with monoclonal antibodies directed against intercellular adhesion molecule (ICAM)-1) (n = 8) or integrin CD11b/CD18 (n = 10), or administered IgG1 as a control (n = 13). At 48 h after ischemia, animals were killed and the brains analyzed for ischemic cell damage, using hematoxylin and eosin (H/E); apoptosis, using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) method; and inflammatory cells, using immunohistochemistry with an anti-myeloperoxidase (MPO) antibody. Data revealed a significant reduction in the volume of infarction (p < 0.01) and a decline in the absolute (p < 0.001), and normalized (to the ischemic area, p < 0.05) numbers of apoptotic cells in both animals treated with anti-ICAM-1 and anti-CD11b antibodies compared to control animals. The numbers of immunoreactive MPO cells were also reduced in the treatment groups compared to those in the control group (p < 0.05). These data suggest that treatment with anti-adhesion molecule antibodies selectively reduce apoptosis, and that a contributing factor to the beneficial effect of antibody treatment for reducing ischemic cell damage may be a reduction in numbers of apoptotic cells.

Increasing evidence indicates that glucocorticoids (GCs), produced in response to physical/emotional stressors, can exacerbate brain damage resulting from cerebral ischemia and severe seizure activity. However, much of the supporting evidence has come from studies employing nonphysiological paradigms in which adrenalectomized rats were compared with those exposed to constant GC concentrations in the upper physiological range. Cerebral ischemia and seizures can induce considerable GC secretion. We now present data from experiments using metyrapone (an 11-β-hydroxylase inhibitor of GC production), which demonstrate that the GC stress-response worsens subsequent brain damage induced by ischemia and seizures in rats. Three different paradigms of brain injury were employed: middle cerebral artery occlusion (MCAO) model of focal cerebral ischemia; four-vessel occlusion (4VO) model of transient global forebrain ischemia; and kainic acid (KA)-induced (seizure-mediated) excitotoxic damage to hippocampal CA3 and CA1 neurons. Metyrapone (200 mg/kg body wt) was administered systemically in a single i.p. bolus 30 min prior to each insult. In the MCAO model, metyrapone treatment significantly reduced infarct volume and also preserved cells within the infarct. In the 4VO model, neuronal loss in region CA1 of the hippocampus was significantly reduced in rats administered metyrapone. Seizure-induced damage to hippocampal pyramidal neurons (assessed by cell counts and immunochemical analyses of cytoskeletal alterations) was significantly reduced in rats administered metyrapone. Measurement of plasma levels of corticosterone (the species-typical GC of rats) after each insult showed that metyrapone significantly suppressed the injury-induced rise in levels of circulating corticosterone. These findings indicate that endogenous corticosterone contributes to the basal level of brain injury resulting from cerebral ischemia and excitotoxic seizure activity and suggest that drugs that suppress glucocorticoid production may be effective in reducing brain damage in stroke and epilepsy patients.

We tested the effects of administration of a selective neuronal nitric oxide synthase (nNOS) inhibitor, ARL 17477, on ischemic cell damage and regional cerebral blood flow (rCBF), in rats subjected to transient (2 h) middle cerebral artery (MCA) occlusion and 166 h of reperfusion (n = 48) and in rats without MCA occlusion (n = 25), respectively. Animals were administered ARL 17477 (i.v.): 10 mg/kg; 3 mg/kg; 1 mg/kg; N-nitro-L-arginine (L-NA) 10 mg/kg L-NA 1 mg/kg; and Vehicle. Administration of ARL 17477 1 mg/kg, 3 mg/kg and 10 mg/kg reduced ischemic infarct volume by 53 (p < 0.05), 23, and 6.5%, respectively. L-NA 1 mg/kg and 10 mg/kg increased infarct volume by 2 and 15%, respectively (p > 0.05). Administration of ARL 17477 (10 mg/kg) significantly (p < 0.05) decreased rCBF by 27 ± 5.3 and 24 ± 14.08% and cortical NOS activity by 86 ± 14.9 and 91 ± 8.9% at 10 min or 3 h, respectively, and did not alter mean arterial blood pressure (MABP). L-NA (10 mg/kg) significantly reduced rCBF by 23 ± 9.8% and NOS activity by 81 ± 7% and significantly (p < 0.05) increased MABP. Treatment with 3 mg/kg and 1 mg/kg ARL 17477 reduced rCBF by only 2.4 ± 4.5 and 0%, respectively, even when NOS activity was reduced by 63 ± 13.4 and 45 ± 15.7% at 3 h, respectively, (p < 0.05). The data demonstrate that ARL 17477 inhibits nNOS in the rat brain and causes a dose-dependent reduction in infarct volume after transient MCA occlusion.

Infarct volume and edema were assessed after transient focal ischemia in mice lacking neuronal nitric oxide synthase (NOS) gene expression. With use of an 8–0 coated monofilament, the middle cerebral artery (MCA) of mutant (n = 32) and wild-type mice [SV-129 (n = 31), C57Black/6 (n = 18)] were occluded for 3 h and reperfused for up to 24 h. Regional CBF (rCBF), neurological deficits, water content, and infarct volume were examined in all three strains. rCBF, blood pressure, and heart rate did not differ between groups when measured for 1 h after reperfusion. Neurological deficits were less severe in mutant mice after MCA occlusion. Brain water content at 3 h after reperfusion and infarct volume at 24 h after reperfusion were greater in wild-type mice. These data indicate that genetic deletion of neuronal NOS confers resistance to focal ischemic injury in a reperfusion model. The findings agree with previous studies showing that tissue injury is less extensive after both permanent MCA occlusion and global ischemia in mice lacking neuronal NOS gene expression. Hence, NO may play a pivotal role in the pathogenesis of ischemic brain damage.

The potential of nitric oxide (NO) to influence positively or negatively the outcome of mechanically induced focal cerebral ischemia is still controversial. Recent evidence suggests that NO of vascular origin, whether synthesized from exogenously administered L-arginine (L-Arg) or from NO donor compounds, is beneficial but that of neuronal origin is not. However, the therapeutic potential of NO to ameliorate stroke induced by arterial thrombosis has not been reported. We assessed the therapeutic effect of L-Arg administration in spontaneously hypertensive rats (SHR) subjected to permanent photothrombotic occlusion of the distal middle cerebral artery (dMCA). The ipsilateral carotid artery was left unligated to enhance L-Arg delivery into the putative penumbral region. Local CBF (LCBF) was assessed at 30 min by the [¹⁴C]iodoantipyrine technique (n = 9), while histological infarct volumes and index of peripheral ischemic cell change were determined at 3 days (n = 7). Rats (n = 9) given 300 mg/kg L-Arg at 18 and 3 h before photothrombotic dMCA occlusion and at 5 min afterward displayed no significant differences in LCBF compared with animals (n = 8) injected with water (the carrier vehicle) and similarly irradiated. Infarct volumes were also similar, being 37.0 ± 9.7 mm³ (SD) in the vehicle-treated and 49.1 ± 17.2 mm³ (SD) in the L-Arg-treated groups (both n = 7), as were assessments of ischemic neuronal density in the penumbra. In contrast, L-Arg administered intravenously in a dose of 300 mg/kg to nonischemic SHR (n = 5) increased cortical CBF by ∼75% during a 70-min observation period. We conclude that thrombotic processes superimposed upon cerebral ischemia may facilitate tissue reactions that offset the potentially beneficial effect of L-Arg, and this caveat must be considered when proposing L-Arg for clinical treatment of focal thrombotic stroke.

The present study was designed to investigate the influence of the endothelium and that of the L-arginine pathway on the contractile responses of isolated human cerebral arteries to electrical field stimulation (EFS) and norepinephrine. Rings of human middle cerebral artery were obtained during autopsy of 19 patients who had died 3–8 h before. EFS (1–8 Hz) induced frequency-dependent contractions that were abolished by tetrodotoxin, prazosin, and guanethidine (all at 10^-6 M). The increases in tension were of greater magnitude in arteries denuded of endothelium. N^G-monomethyl L-arginine (L-NMMA 10^-4 M) potentiated the contractile response to EFS in artery rings with endothelium but did not influence responses of endothelium-denuded arteries. L-arginine (10^-4 M) reversed the potentiating effects of L-NMMA on EFS-induced contractions. Norepinephrine induced concentration-dependent contractions, which were similar in arteries with and without endothelium or in arteries treated with L-NMMA. Indomethacin (3 × 10⁻⁶ M) had no significant effect on the contractile response to EFS or on the inhibition by L-NMMA of acetylcholine-induced relaxation. These results suggest that the contractile response of human cerebral arteries to EFS is modulated by nitric oxide mainly derived from endothelial cells; although adrenergic nerves appear to be responsible for the contraction, the transmitter involved in the release of nitric oxide does not appear to be norepinephrine. The effects of L-NMMA in this preparation appear to be due to inhibition of nitric oxide formation rather than caused by cyclooxygenase activation.

Ischemic cell death occurs when extracellular glutamate levels increase, causing tissue depolarization and an excessive rise in intracellular calcium concentrations. The relative occurrence of the depolarization events and the changes in glutamate concentration in ischemia have not been studied. In a model of focal cerebral ischemia in the rat, three measurements were made simultaneously in vivo: cerebral blood flow (CBF) by the H₂-clearance method, extracellular glutamate concentration by microdialysis, and activation of the voltage-sensitive calcium channel (VSCC) by its binding to [³H]nimodipine. Effects of probe implantation on these measurements were accounted for. The CBF to control ratio obtained during the experiments spanned the range of 1.08 to 0.07. Binding to [³H]nimodipine became significantly activated when CBF fell to ∼0.49 of its control value while extracellular glutamate concentrations increased significantly only at a CBF ratio of <0.33. Activation of the VSCC at this high CBF ratio may be due to ischemic depolarization, which has been shown to activate the binding to [³H]nimodipine. It may be useful to define a CBF threshold of 50% of normal in focal ischemia for opening of the VSCC. The same threshold has been linked to an overall depression of protein synthesis and to activation of a number of molecular responses.

Intracerebral microdialysis (MD) was applied in patients with subarachnoid hemorrhage. The regional CBF, the CMRO₂, and oxygen extraction ratio (OER) were measured with simultaneous positron emission tomography (PET). The aim was to directly correlate alterations in dialysate levels of energy-related metabolites (lactate, lactate/pyruvate ratio, hypoxanthine) and excitatory amino acids (EAAs) (glutamate and aspartate) to the energy state in the MD probe region as determined by PET. Regional ischemia was defined according to Heiss et al. and Lassen (Heiss et al., 1992; Lassen, 1966). Whole-brain ischemia was considered present when the OER for the whole brain exceeded the mean whole-brain OER + 2 SD of six reference patients. In general, the presence of whole-brain ischemia and/or regional ischemia within the region of the MD probe was associated with increased levels of energy-related metabolites and EAAs retrieved by MD. Increased levels of energy-related metabolites and EAAs were only occasionally seen when PET did not show any signs of ischemia or when signs of regional ischemia were found remote from the MD probe region. Thus, the energy-related metabolites and EAAs may be used as extracellular “markers” of ischemia. PET may be of use in defining critical ischemic regions (tissue at risk) where the MD probe can be inserted for- chemical monitoring.

We describe the implementation and validation of a combined dynamic–autoradiographic approach for measuring the regional cerebral blood flow (rCBF) with ¹⁵O-butanol. From arterial blood data sampled at a rate of 1 s and list mode data of the cerebral radioactivity accumulated over 100 s, the time shift between blood and tissue curves, the dispersion constant DC, the partition coefficient p, and the CBF were estimated by least squares fitting. Using the fit results, a pixel-by-pixel parametrization of rCBF was computed for a single 40-s (autoradiographic) ¹⁵O-butanol uptake image. The mean global CBF found in 27 healthy subjects was 49 ± 8 ml 100 g⁻¹ min⁻¹. Gray and white matter rCBF were 83 ± 20 and 16 ± 3 ml 100 g⁻¹ min⁻¹, respectively, with a corresponding partition coefficient p of 0.77 ± 0.18 and 0.77 ± 0.29 ml/g in both compartments. The quantitative images resulted in a significantly higher gray matter rCBF than the autoradiographic images.

Partial volume and mixed tissue sampling errors can cause significant inaccuracy in quantitative positron emission tomographic (PET) measurements. We previously described a method of correcting PET data for the effects of partial volume averaging on gray matter (GM) quantitation; however, this method may incompletely correct GM structures when local tissue concentrations are highly heterogeneous. We have extended this three-compartment algorithm to include a fourth compartment: a GM volume of interest (VOI) that can be delineated on magnetic resonance (MR) imaging. Computer simulations of PET images created from human MR data demonstrated errors of up to 120% in assigned activity values in small brain structures in uncorrected data. Four-compartment correction achieved full recovery of a wide range of coded activity in GM VOIs such as the amygdala, caudate, and thalamus. Further validation was performed in an agarose brain phantom in actual PET acquisitions. Implementation of this partial volume correction approach in [¹⁸F]fluorodeoxyglucose and [¹¹C]-carfentanil PET data acquired in a healthy elderly human subject was also performed. This newly developed MR-based partial volume correction algorithm permits the accurate determination of the true radioactivity concentration in specific structures that can be defined by MR by accounting for the influence of heterogeneity of GM radioactivity.

The deoxyglucose method for calculation of regional cerebral glucose metabolism by PET using ¹⁸F-2-fluoro-2-deoxy-d-glucose (FDG) requires knowledge of the lumped constant, which corrects for differences in the blood–brain barrier (BBB) transport and phosphorylation of FDG and glucose. The BBB transport rates of FDG and glucose have not previously been determined in humans. In the present study these transport rates were measured with the intravenous double-indicator method in 24 healthy subjects during normoglycemia (5.2 ± 0.7 mM). Nine subjects were restudied during moderate hypoglycemia (3.4 ± 0.4 mM) and five subjects were studied once during hyperglycemia (15.0 ± 0.7 mM). The global ratio between the unidirectional clearances of FDG and glucose (K₁^*/K₁) was similar in normoglycemia (1.48 ± 0.22), moderate hypoglycemia (1.41 ± 0.23), and hyperglycemia (1.44 ± 0.20). This ratio is comparable to what has been obtained in rats. We argue that the global ratio is constant throughout the brain and may be applied for the regional determination of LC. We also determined the transport parameters of the two hexoses from brain back to blood and, assuming symmetrical transport across the BBB, we found evidence of a larger initial distribution volume of FDG in brain (0.329 ± 0.236) as compared with that of glucose (0.162 ± 0.098, p < 0.005). The difference can be explained by the very short experimental time, in which FDG may distribute both intra- and extracellularly, whereas glucose remains in a volume comparable to the interstitial fluid of the brain.

In this work, we introduce 6-[¹⁸F]fluoro-L-m-tyrosine (6-FMT) and compare its in-vivo kinetic and biochemical behaviors in monkeys and rodents with those of 4-FMT and 6-[¹⁸F]fluoro-L-3,4-dihydroxyphenylalanine (DOPA) (FDOPA). These radiofluorinated m-tyrosine presynaptic dopaminergic probes, resistant to peripheral 3-O-methylation, offer a nonpharmacological alternative to the use of catechol-O-methyltransferase inhibitors. Like FDOPA, 4-FMT and 6-FMT are analogs that essentially follow the L-DOPA pathway of central metabolism. After i.v. administration in nonhuman primates and rodents, these new radiofluorinated m-tyrosine analogs accumulate selectively in striatal structures and allow for the detection of additional innervation sites (e.g., brain stem) rich in aromatic amino acid decarboxylase. Biochemical analyses in rodents and monkeys revealed the specificity of their central and peripheral metabolism. Molecular and enzymatic mechanisms involved in their retention in central brain structures are consistent with involvement of dopaminergic neurons. The high signal-to-noise ratios observed make these radiofluorinated m-tyrosine analogs outstanding candidates for probing the integrity of central dopaminergic mechanisms in humans.

The actions of Bosentan and PD155080, non-peptide endothelin receptor antagonists, were examined in feline pial arterioles in situ following middle cerebral artery (MCA) occlusion to gain insight into the cerebrovascular influence of endogenous endothelins in focal cerebral ischaemia. Immediately following permanent MCA occlusion, all pial arterioles overlying the suprasylvian and ectosylvian gyri displayed marked dilatations, which were maintained in a population of vessels but differentiated into sustained constrictions in others. Perivascular subarachnoid microinjections of Bosentan (30 μM), PD155080 (30 μM), and artificial CSF (pH 7.2) were performed between 30 and 210 min following MCA occlusion. The perivascular microapplication of Bosentan (30 μM) and PD155080 (30 μM) around pial vessels overlying the suprasylvian and ectosylvian gyri, which are within the territory of the occluded MCA, elicited an increase in the calibre of postocclusion dilated and constricted pial arterioles. The perivascular microapplication of PD155080 (30 μ/M) around postocclusion constricted arterioles overlying the ectosylvian and suprasylvian gyri elicited an increase in the calibre of arterioles (69 ± 49% from preinjection baseline; n = 8). The perivascular microapplication of Bosentan (30 μM) around postocclusion constricted arterioles overlying the ectosylvian and suprasylvian gyri also elicited an increase in the calibre of arterioles (68 ± 60% from preinjection baseline; n = 13). In contrast, the microapplication of CSF (pH 7.2) elicited small reductions in pial arteriolar calibre of postocclusion constricted arterioles (—8 ± 13% from preinjection baseline; n = 8). The perivascular microapplication of PD155080 (30 μM) around postocclusion dilated pial arterioles overlying the ectosylvian and suprasylvian gyri elicited an increase in the calibre of arterioles (11 ± 10% from preinjection baseline; n = 38). The perivascular microapplication of Bosentan (30 μM) around postocclusion dilated arterioles elicited an increase in the calibre of arterioles (16 ± 15% from preinjection baseline; n = 36). In contrast, the microapplication of CSF (pH 7.2) elicited small reductions in pial arteriolar calibre of postocclusion dilated arterioles (—9 ± 6% from preinjection baseline; n = 44). Perivascular microapplication of Bosentan or PD155080 had minimal effect on the calibre of pial arterioles on the parasagittal gyrus (anterior cerebral artery territory), although these arterioles had also displayed sustained dilatation following MCA occlusion. These results indicate that contractile factors (whose effects can be reversed with endothelin receptor antagonists) constrict or impair dilatation of cortical resistance arterioles in an acute cerebral ischaemic episode.

These studies tested the hypothesis that the cerebral vasospasm that follows subarachnoid hemorrhage (SAH) is due to alterations in endothelin (ET) and ET receptor expression. Eight monkeys underwent cerebral angiography and induction of SAH. Angiography was repeated 7 days later to confirm the presence of cerebral vasospasm, and animals were killed. RNA was isolated from right (vasospastic) and left (control) side middle cerebral arteries and surrounding cerebral cortex. The levels of prepro (PP) ET-1 (ppET-1) and ppET-3 and ET_A and ET_B receptor mRNAs were determined using a quantitative reverse transcriptase polymerase chain reaction-based assay. ET-1 peptide was also measured in CSF at baseline and after 7 days. Specific agonist binding to ET_A and ET_B receptors in both middle cerebral arteries and in surrounding brain cortex was measured in three animals by autoradiographic binding assays. Levels of ET_B receptor mRNA were 3.4 ± 2.2-fold higher in the right than in the left cerebral arteries (p < 0.01). There were no significant differences in the levels of ppET-1, ppET-3, or ET_A receptor mRNA in cerebral arteries. ET-1 peptide was not elevated in CSF. Levels of ET_A and ET_B receptor mRNAs were 2.6 ± 1.1- and 2.1 ± 1.3-fold higher, respectively, in the right than in the left cerebral cortex, while the level of ppET-3 mRNA was 2.1 ± 1.0-fold lower. There were no differences in ppET-1 mRNA levels between right and left cerebral cortex. Binding to ET_A and ET_B receptors in cerebral arteries and cortex did not differ significantly between right and left sides. These results do not support the hypothesis that overexpression of ET-1 is the principal cause of vasospasm, but rather they suggest that SAH causes complex changes in the ET system that together are responsible for the cellular response to SAH.

The changes in cerebral blood flow that accompany spreading depression are well-described, as are parallel changes in cellular activity, with a wave of hyperemia followed by a prolonged oligemic phase. In this study, a cat model of the CBF changes associated with spreading depression and in vitro pharmacology were used to determine if there is a role for the powerful peptide vasoconstrictor endothelin in this response. For the pharmacological studies, the middle cerebral artery was harvested from cats postmortem. For the physiological studies, cats were anesthetized with halothane induction and α-chloralose (60 mg/kg, intraperitoneal loading; 20 mg/kg i.v. 2-h maintenance). CBF was monitored continuously in the parietal cortex using laser Doppler flowmetry (CBF_LDF) after exposure of the dura mater. The in vitro work demonstrated that endothelin-1 (ET-1) mediates a strong and potent contraction of cerebral vessels. Both the selective ET_A receptor antagonist FR139317 and the combined ET_A and ET_B receptor antagonist Bosentan caused a rightward shift of the concentration-response curve without attenuation of the maximum effect. The calculated pA₂ values were 6.28 and 6.90, respectively. The slope did not differ from unity, suggesting that the ET-1-mediated contraction is evoked by a single population of ET_A receptors, which were effectively antagonized by these compounds. Spreading depression was induced with a needle stick injury to the cortex. Local administration of the endothelin antagonists FR139317 (10 μM) and Bosentan (10 μM) did not affect resting blood flow in the cortex. Induction of spreading depression following local administration of FR139317 and Bosentan resulted in responses no different from those in control cortex. These data demonstrate that endothelin does not play a significant role in the vasoconstrictor portion of the CBF change seen in spreading depression, nor does it affect resting flow. Since it is widely held that spreading depression, or a very similar mechanism, underlies the aura phase of migraine, it may be suggested from these studies that endothelin antagonists are unlikely to be useful in migraine.

We tested the hypothesis that increasing durations of focal ischemia that have been shown to result in enlargement of cortical infarct will be associated with progression of behavioral dysfunction that can be measured by a battery of tests sufficiently sensitive and reproducible to detect a positive effect of pharmacotherapy. Untreated or N-methyl-D-aspartate receptor antagonist (CNS-1102)-treated spontaneously hypertensive rats underwent 45, 60, 90, or 120 min of tandem middle cerebral and common carotid artery occlusion followed by reperfusion. We then evaluated the extent of damage and its recovery for up to 21 days using nine behavioral tests aimed at analyzing strength, coordination, and bilateral asymmetry. Also using a graded bioassay that employs a curve-fitting computer program (ALLFIT) to correlate duration of ischemia with degree of behavioral dysfunction, we calculated the average maximal behavioral dysfunction and duration of ischemia required to produce half-maximal behavioral dysfunction and compared these values in untreated controls with analogous values obtained from animals treated with CNS-1102. Three behavioral tests, forearm flex, tape (somatosensory neutralization), and foot-fault placing, were each separately and combined able to distinguish between the degrees of damage produced by increasing durations of ischemia. The behavioral abnormalities assessed using the tape test were reversible within a week, whereas those using forearm flex or foot-fault tests persisted for at least 21 days. CNS-1102 significantly reduced behavioral dysfunction measured by all three tests. This analysis of behavioral dysfunction represents a useful experimental model to grade efficacy of therapies aimed at protecting the brain from damage produced by acute stroke and might also be used to assess recovery from preexisting ischemic damage.

There is evidence of an intrinsic renin–angiotensin system in the brain. The goal of the study was to determine whether stimulation of endogenous angiotensin production by applying renin to the brain surface has an effect on pial arteriolar caliber and CBF. Pial vessel diameters were measured through a closed cranial window in anesthetized rabbits. Percent changes of blood flow in the cortical area under the cranial window were simultaneously measured by laser-Doppler flowmetry. Topical application of 0.01–0.1 U/ml renin induced maximum dilation of 18.9 ± 4% (mean ± SD) of pial arterioles within 2 min. Arteriolar calibers thereafter decreased slowly. Flow gradually increased to peak at 38 ± 15% 50 min after renin application. Angiotensin I levels in jugular blood, as measured by radioimmunoassay, increased to a peak 40 min after topical renin application. Angiotensin II levels in jugular blood and both angiotensin I and II levels in blood samples from the femoral artery did not change. Diameter and flow changes were inhibited by intravenous pretreatment with the converting enzyme blocker captopril (10 mg/kg body wt i.v.). Captopril did not affect the vasodilation and flow increase in response to hypercapnia. Topically applied Captopril (10⁻⁵ M) blocked renin-induced arteriolar dilation. We conclude that renin increases pial arteriolar diameters and cortical blood flow in the rabbit brain. Stimulation of angiotensin production is likely to be a mediator of this response.

The symptoms following sinus and vein occlusion observed in patients and experimental animals display a considerable variability that so far remains largely unexplained. In a rat cortical vein occlusion model using a photochemical thrombotic technique, we examined changes in the cerebral venous flow pattern by fluorescence angiography and regional cerebral blood flow (rCBF) and cerebral blood volume fraction (CBVF) by a modern laser Doppler “scanning” technique. Brain damage was assessed histologically. Fluorescence angiographic findings fell into two groups: group A, rats with an altered venous flow pattern after occlusion (n = 12), and group B, rats with interruption of blood flow and/or a growing venous thrombus (n = 5). In addition, sham-operated animals made up group C (n = 5). Extravasation of fluorescein, a massive decrease in rCBF, a short-lasting increase in CBVF, and regional brain damage were typical for group B. In addition, cortical CBF mapping revealed a transient hyperperfusion zone with hyperemia surrounding a hypoperfused ischemic core in group B. A circulation perturbation following venous occlusion appeared near those occluded cerebral veins without sufficient collateral flow. Furthermore, the venous thrombus continued to grow, accompanied by local critical ischemia and severe brain damage. Conversely, 71% of the animals (12 of 17) tolerated occlusion of a solitary vein without major flow disturbances or histological evidence of damage to the CNS (group A).

In exercise, little is known about local cerebral glucose utilization (LCGU), which is an index of functional neurogenic activity. We measured LCGU in resting and running (≈85% of maximum O₂ uptake) rats (n = 7 in both groups) previously equipped with a tail artery catheter. LCGU was measured quantitatively from 2-deoxy-D-[1-¹⁴C]glucose autoradiographs. During exercise, total cerebral glucose utilization (TCGU) increased by 38% (p < 0.005). LCGU increased (p < 0.05) in areas involved in motor function (motor cortex 39%, cerebellum ≈110%, basal ganglia ≈30%, substantia nigra ≈37%, and in the following nuclei: subthalamic 47%, posterior hypothalamic 74%, red 61%, ambiguus 43%, pontine 61%), areas involved in sensory function (somatosensory 27%, auditory 32%, and visual cortex 42%, thalamus ≈75%, and in the following nuclei: Darkschewitsch 22%, cochlear 51%, vestibular 30%, superior olive 23%, cuneate 115%), areas involved in autonomic function (dorsal raphe nucleus 30%, and areas in the hypothalamus ≈35%, amygdala ≈35%, and hippocampus 29%), and in white matter of the corpus callosum (36%) and cerebellum (52%). LCGU did not change with exercise in prefrontal and frontal cortex, cingulum, inferior olive, nucleus of solitary tract and median raphe, lateral septal and interpenduncular nuclei, or in areas of the hippocampus, amygdala, and hypothalamus. Glucose utilization did not decrease during exercise in any of the studied cerebral regions. In summary, heavy dynamic exercise increases TCGU and evokes marked differential changes in LCGU. The findings provide clues to the cerebral areas that participate in the large motor, sensory, and autonomic adaptation occurring in exercise.

The purpose of this study was to identify the trigger mechanism activating the 5-lipoxygenase pathway during blood–brain cell contact and to estimate the contribution of blood and brain cells to the cysteinyl–leukotriene (LT) biosynthesis observed under these conditions. Incubation of dissociated rat brain cells in Krebs-Henseleit solution for up to 60 min did not stimulate any detectable cysteinyl–LT biosynthesis. Incubation of recalcified rat whole blood in vitro for up to 60 min led to release of only small amounts of cysteinyl–LT into the serum samples. However, coincubation of dissociated rat brain cells with physiologically recalcified autologous whole blood triggered a time-dependent release of large amounts of immunoreactive cysteinyl–LT into the serum samples. By reverse-phase HPLC, immunoreactive cysteinyl–LT was identified as a mixture of LTC₄, LTD₄, and LTE₄. The extent of the 5-lipoxygenase stimulation depended on the amount of autologous blood coincubated with the dissociated brain cells. Activation of the 5-lipoxygenase pathway also occurred with coincubation of dissociated rat brain cells with recalcified autologous plasma. Stimulation of cysteinyl–LT biosynthesis during blood–brain cell contact remained unaffected by aprotinin, but concentration-dependent inhibition by the structurally and functionally unrelated thrombin inhibitors D-Phe-Pro-Arg-CH₂Cl and recombinant hirudin was seen. Finally, when dissociated rat brain cells were incubated in Krebs-Henseleit solution in the presence of human α-thrombin, a concentration-dependent release of cysteinyl–LT into the buffer samples was observed. These data demonstrate that, in rats, during blood–brain cell contact, stimulation of the 5-lipoxygenase pathway in brain cells proceeds via α-thrombin as effector molecule.

Current smoking is a risk factor for stroke. The aim of this study was to evaluate the effect of smoking one cigarette on cerebral hemodynamics. Using transcranial Doppler ultrasound, we studied the changes of flow velocity after hypercapnia in the middle cerebral arteries (MCAs) of 24 healthy young smokers and 24 healthy controls matched for age and sex. We obtained hypercapnia with breath-holding and evaluated cerebrovascular reactivity with the breath-holding index. In smokers, the evaluation was performed during basal condition, immediately after smoking one cigarette, and at 10-, 20-, and 30-min intervals thereafter. In controls, the evaluation was performed at corresponding time intervals. Breath-holding index (BHI) values at rest were similar for both controls and smokers. In the former, breath-holding index values remained constant for each of the different evaluations. On the contrary, in smokers, breath-holding index values were significantly lower immediately (p < 0.0001), at 10 min (p < 0.001), and at 20 min (p < 0.0001) after smoking with respect to baseline values. Smoking also caused more short-lasting changes, in this case increases in mean flow velocity (MFV), heart rate (HR), and mean blood pressure (MBP). These results suggest that a failure of cerebrovascular regulation occurs after smoking. This phenomenon might contribute to the increased risk of cerebrovascular disease in current smokers.