Journal of Cerebral Blood Flow & Metabolism

Positron emission tomography (PET) functional imaging is based on changes in regional cerebral blood flow (rCBF). Functional magnetic resonance imaging (fMRI) is based on a variety of physiological parameters as well as rCBF. This study is aimed at the cross validation of three-dimensional (3D) fMRI, which is sensitive to changes in blood oxygenation, with oxygen-15-labeled water (H₂¹⁵O) PET. Nine normal subjects repeatedly performed a simple finger opposition task during fMRI scans and during PET scans. Within-subject statistical analysis revealed significant (“activated”) signal changes (p < 0.05, Bonferroni corrected for number of voxels) in contralateral primary sensorimotor cortex (PSM) in all subjects with fMRI and with PET. With both methods, 78% of all activated voxels were located in the PSM. Overlap of activated regions occurred in all subjects (mean 43%, SD 26%). The size of the activated regions in PSM with both methods was highly correlated (rho = 0.87, p < 0.01). The mean distance between centers of mass of the activated regions in the PSM for fMRI versus PET was 6.7 mm (SD 3.0 mm). Average magnitude of signal change in activated voxels in this region, expressed as z-values adapted to timeseries, z_t, was similar (fMRI 5.5, PET 5.3). Results indicate that positive blood oxygen level-dependent (BOLD) signal changes obtained with 3D principles of echo shifting with a train of observations (PRESTO) fMRI are correlated with rCBF, and that sensitivity of fMRI can equal that of H₂¹⁵O PET.

When used to measure blood flow in the brain, water leaves a residue in the vascular bed that influences the estimation of blood flow by current methods. To assess the magnitude of this influence, we developed a two-compartment model of blood flow with separate parameters for transport and vascular distribution of brain water. Maps of the water clearance, K₁ into brain tissue, separated from the circulation by a measurably resistant blood–brain barrier (BBB), were generated by time-weighted integration. Depending on the validity of the assumptions underlying the two-compartment model presented here, the maps revealed a significant overestimation of the clearance of water when the vascular residue was ignored. Maps of V_o the estimate of the apparent vascular distribution volume of tracer H₂¹⁵O, clearly revealed major cerebral arteries. Thus, we claim that the accumulation of radioactive water in brain tissue also reflects the volume of the arterial vascular bed of the brain.

Recently, two methods have been proposed for regional cerebral blood flow (rCBF) quantitation using [¹²³I]iodoamphetamine (IMP) and single-photon emission computed tomography (SPECT). The table look-up (TLU) method has been shown to provide both rCBF and volume of distribution, V_d, images from two SPECT scans, while a single-scan autoradiographic (ARG) technique provided rCBF using a fixed and assumed V_d. In both methods, a single blood sample was referred to calibrate the previously determined standard input function The present multicenter project was designed to evaluate the accuracy of both methods for use as clinical investigative tools. Ten independent institutions performed [¹²³I]IMP-SPECT studies according to both methods in 76 subjects (10 normal volunteers, 32 patients with cerebrovascular disease, and 34 patients with other diseases). Calculated rCBF values were compared with those obtained by the following reference methods available in the participating institutions; [¹⁵O] H₂O positron emission tomography (PET) (five institutions), [¹³³Xe]SPECT (four institutions), and the [¹²³I]IMP microsphere method (three institutions). Both ARG and TLU methods provided rCBF values that were significantly correlated with those measured by the [¹⁵O] H₂O PET technique (p < 0.001 for all subjects; overall regression equation, y = 15.14 + 0.54×) and those measured by the [¹²³I]IMP-microsphere method (p < 0.001 for all subjects; y = 2.0 + 0.80×). Significant correlation (p < 0.05) was observed in 18 of 24 subjects studied with the [¹³³Xe] SPECT reference technique (overall regression equation, y = 15.0 + 0.55×). Mean cortical gray matter rCBF in a group of normal subject was 43.9 ± 3.3 and 43.4 ± 2.0 ml/min/100 g for the ARG and TLU methods, respectively. Regional V_d of [¹²³I]IMP estimated by the TLU method was 45 ml/ml ± 20% in the normal cortical region. Close agreement between ARG and TLU rCBF values was observed (y = −3.21 + 1.07×, r = 0.97), confirming the validity of assuming a fixed V_d in the ARG method. Results of this study demonstrate that both the ARG and TLU methods accurately and reliably estimate rCBF in a variety of clinical settings.

Using repeated positron emission tomography (PET) measures of regional cerebral counts, we investigated the regional cortical activations induced in eight normal subjects performing eight different frequencies of fingertapping (0.5–4 Hz) with the right index finger. The task was auditorially cued and the performance recorded during the scanning procedure. Performance evaluation showed increased error rates, during fingertapping, of high and low frequencies, and the best tapping performance was measured in the midrange of frequencies. Significantly activated areas (p < 0.05) of normalized cerebral counts were located in the left sensorimotor cortex (M1S1), right motor cortex, left thalamus, right insula, supplementary motor area (SMA), and bilaterally in the primary auditory cortex and the cerebellum. Statistical evaluation showed a significant (p < 0.01) and positive dependence of cerebral activation upon movement rate in the contralateral M1S1. There was no significant rate dependence of cerebral activation in other activated motor areas. The SMA and the right cerebellar hemisphere showed a more uniform activation throughout the tapping frequency range. Furthermore, we found a stimulus rate dependence of cerebral activation in the primary auditory cortex. We believe that the present data provide useful information for the preparation and interpretation of future motor activation studies of normal human subjects and may serve as reference points for studies of pathological conditions.

Determining the appropriate sample size is a crucial component of positron emission tomography (PET) studies. Power calculations, the traditional method for determining sample size, were developed for hypothesis-testing approaches to data analysis. This method for determining sample size is challenged by the complexities of PET data analysis: use of exploratory analysis strategies, search for multiple correlated nodes on interlinked networks, and analysis of large numbers of pixels that may have correlated values due to both anatomical and functional dependence. We examine the effects of variable sample size in a study of human memory, comparing large (n = 33), medium (n = 16,17), small (n = 11, 11, 11), and very small (n = 6, 6, 7, 7, 7) samples. Results from the large sample are assumed to be the “gold standard.” The primary criterion for assessing sample size is replicability. This is evaluated using a hierarchically ordered group of parameters: pattern of peaks, location of peaks, number of peaks, size (volume) of peaks, and intensity of the associated t (or z) statistic. As sample size decreases, false negatives begin to appear, with some loss of pattern and peak detection; there is no corresponding increase in false positives. The results suggest that good replicability occurs with a sample size of 10–20 subjects in studies of human cognition that use paired subtraction comparisons of single experimental/baseline conditions with blood flow differences ranging from 4 to 13%.

Changes in cerebral blood oxygenation due to functional activation of the primary sensorimotor cortex during a unilateral finger opposition task were simultaneously mapped by deoxyhemoglobin-sensitive magnetic resonance imaging (MRI) and monitored by near-infrared spectroscopy (NIRS). Activation foci along the contralateral central sulcus displayed task-associated increases in MRI signal intensity, indicating a concomitant decrease of the focal concentration of deoxyhemoglobin. This interpretation was confirmed by simultaneous reductions in deoxyhemoglobin measured optically. Since observation of the latter effect required exact spatial matching of the MRI-detected activation foci and position of the fiber optic bundles (“optodes”) used for transmitting and receiving light, it may be concluded that optical recordings of changes in deoxyhemoglobin during functional challenge probe only a restricted brain tissue region. While deoxyhemoglobin responses seen by NIRS were smaller for ipsi- than for contralateral finger movements, task-related increases in oxyhemoglobin were rather similar between both conditions and, thus, seem to be less specific. Furthermore, no consistent changes were obtained for total hemoglobin during task performance, possibly due to the short timing of the repetitive protocol. In general, results underline, in humans, the hitherto assumed signal physiology for functional brain mapping by oxygenation-sensitive MRI and allow assessment of both constraints and practicability of functional studies by NIRS.

In a prospective study of the natural development of total cerebral blood flow volume (CBFV), the common, external and internal carotid and vertebral arteries were examined in 94 healthy children and adolescents between 3 and 18 years of age (sex and age evenly distributed) using a 7.0-MHz transducer of a computed sonography system. Intravascular flow volumes were calculated with the product of angle-corrected time-averaged flow velocity and the cross-sectional area of the vessel. CBFV was determined as the sum of flow volumes in the internal carotid and vertebral arteries of both sides. CBFV increased significantly between 3 and 6.5 years of age (from 687 ± 85 to 896 ± 110 ml/min; age correlation, p ≤ 0.01) and declined thereafter (p ≤ 0.001) to a constant level of ∼700 ml/min at 15 years of age. There was no difference in CBFV between sexes. The proportion of bilateral vertebral artery flow volume in total CBFV decreased significantly between the ages of 3 and 18 years (p ≤ 0.001). As the flow volumes of the external carotid arteries increased markedly from childhood to adulthood, flow volumes of the common carotid arteries were not representative of CBFV. Intrasession test-retest correlation of CBFV was high (r = 0.89, p ≤ 0.0001). Reference data for the childhood years presented here and previously described results from healthy adults allow us to outline the natural evolution of CBFV in humans. The reliability of the method has already been demonstrated. Thus, it may now be introduced into clinical application.

The distribution volume ratio (DVR), which is a linear function of receptor availability, is widely used as a model parameter in imaging studies. The DVR corresponds to the ratio of the DV of a receptor-containing region to a nonreceptor region and generally requires the measurement of an arterial input function. Here we propose a graphical method for determining the DVR that does not require blood sampling. This method uses data from a nonreceptor region with an average tissue-to-plasma efflux constant k₂ to approximate the plasma integral. Data from positron emission tomography studies with [¹⁵C]raclopride (n = 20) and [¹¹C]d-threo-methylphenidate ([¹¹C]dMP) (n = 8) in which plasma data were taken and used to compare results from two graphical methods, one that uses plasma data and one that does not. k₂ was 0.163 and 0.051 min⁻¹ for [¹¹C]raclopride and [¹¹C]dMP, respectively. Results from both methods were very similar, and the average percentage difference between the methods was −0.11% for [¹¹C]raclopride and 0.46% for [¹¹C]dMP for DVR of basal ganglia (BG) to cerebellum (CB). Good agreement between the two methods was also achieved for DVR images created by both methods. This technique provides an alternative method of analysis not requiring blood sampling that gives equivalent results for the two ligands studied. It requires initial studies with blood sampling to determine the average kinetic constant and to test applicability. In some cases, it may be possible to neglect the b̅₂ term if the BG/CB ratio becomes reasonably constant for a sufficiently long period of time over the course of the experiment.

The goal of research with receptor ligands and PET is the characterization of an in vivo system that measures rates of association and dissociation of a ligand-receptor complex and the density of available binding sites. It has been suggested that multiple injection studies of radioactive ligand are more likely to identify model parameters than are single injection studies. Typically, at least one of the late injections is at a low specific activity (SA), so that part of the positron emission tomography (PET) curve reflects ligand dissociation. Low SA injections and the attendant reductions in receptor availability, however, may violate tracer kinetic assumptions, namely, tracer may no longer be in steady state with the total (labeled and unlabeled) ligand. Tissue response becomes critically dependent on the dose of total ligand, and an accurate description of the cold ligand in the tissue is needed to properly model the system. Two alternative models have been applied to the receptor modeling problem, which reduces to describing the time-varying number of available receptor sites. The first (Huang et al., 1989) contains only compartments for the hot ligand, ‘hot only’ (HO), but indirectly accounts for the action of cold ligand at receptor sites via SA. The second stipulates separate compartments for the hot and cold ligands, ‘hot and cold’ (HC), thus explicitly calculating available number of receptors. We examined these models and contrasted their abilities to predict PET activity, receptor availability, and SA in each tissue compartment. For multiple injection studies, the models consistently predicted different PET activities—especially following the third injection. Only for very high rate constants were the models identical for multiple injections. In one case, simulated PET curves were quite similar, but discrepancies appeared in predictions of receptor availability. The HO model predicted nonphysiological changes in the availability of receptor sites and introduced errors of 30–60% into estimates of B′_max for test data. We, therefore, strongly recommend the use of the HC model for all analyses of multiple injection PET studies.

Fluorodopa (FDOPA) is an analogue of L-dihydroxyphenylalanine (L-dopa) used to assess the nigrostriatal dopamine system in vivo with positron emission tomography (PET). However, FDOPA/PET quantitation is complicated by the presence of the 3-O-methyl-FDOPA (30MFD) fraction in brain and plasma. Pretreatment with entacapone (OR-611), a peripheral catechol O-methyltransferase (COMT) inhibitor, greatly reduces the plasma 30MFD fraction and provides an ideal situation to evaluate the contribution of the plasma 30MFD fraction in several kinetic models of FDOPA uptake. We performed FDOPA/PET with and without the OR-611 preadministration in six Parkinson's disease (PD) patients. We measured the time-course of the plasma FDOPA and 30MFD fractions using high-pressure liquid chromatography (HPLC). We calculated striato-occipital ratios (SOR), and estimated the striatal FDOPA uptake rate constant graphically using the plasma FDOPA and occipital tissue time activity curves (K_i^FD and K_i^OCC, respectively). We also estimated striatal dopa decarboxylase (DDC) activity (k₃^D) using a model incorporating independent measurements of 30MFD transport kinetic rate constants. With the preadministration of OR-611, the pharmacological efficiency in plasma was prolonged significantly (21.1–37.7%; p < 0.01). We also observed significant mean elevations in SOR and K_i^OCC by 21.8 and 53.5%, respectively (p < 0.05). K_i^FD and k₃^D did not show significant change. We conclude that OR-611 prolongs the circulation time of FDOPA in the plasma but does not alter rate constants for striatal FDOPA uptake or decarboxylation.

Brain slice glucose utilization (SGU) can be measured by methods analogous to those used for in vivo cerebral glucose utilization. In order to make this technique more accessible and applicable to a broad range of experimental conditions, we have derived a simplified operational rate equation and generated the table of apparent rate coefficients necessary to apply the equation under different experimental situations. Calculations of the apparent rate coefficients were based upon an eight-parameter kinetic model combined with Michaelis–Menten theory to account for changes in the rate constants as a function of buffer glucose concentration. The theory was tested with a series of experiments using rat brain slices, [¹⁴C]-2-deoxyglucose (2DG) and [¹⁴C]-3–O-methylglucose (3OMG). The errors involved in the simplified technique were estimated by a variety of techniques and found to be acceptable over a broad range of conditions. A detailed, practical protocol for the simplified method is presented.

Magnetic resonance water diffusion imaging can detect early ischemic changes in stroke. Using a middle cerebral artery occlusion model, we examined which range of values of the orientation-independent diffusion quantity is an early noninvasive indicator of reduced cerebral perfusion and focal brain injury. Cats underwent either a 30-min occlusion followed by 3.5 h reperfusion (n = 7) or a 60-min occlusion followed by 4-h reperfusion (n = 6). Repeated measurements of CBF were made with radiolabeled microspheres, and acute focal injury was measured with triphenyltetrazolium chloride (TTC) staining. During occlusion, the decrease in D_av correlated with CBF for caudate [30-min occlusion (n = 13): p < 0.0001; 60-min occlusion (n = 6): p < 0.02] and for cortex [30-min occlusion (n = 12): p < 0.0001; 60-min occlusion (n = 5): p < 0.04]. Variable caudate and hemispheric injury levels were found among cats in both groups. The area of tissue injury demarcated by TTC began to correlate with the area of reduced D_av by 30 min of occlusion (p < 0.02), and this correlation improved (p < 0.0001) at 1, 1.5, and 2.0 h after the onset of occlusion. The time necessary to reach a one-to-one correspondence between the percent of hemisphere injured and the percent of hemispheric area with D_av < 0.65 × 10⁻⁹ m²/s was 2 h after occlusion. Thus, the absolute value of D_av is a good indicator of the risk of tissue injury, whereas the combination of D_av and the length of time of D_av reduction is an excellent predictor of acute focal tissue injury demarcated by TTC staining.

Although brain ischemia has been extensively studied using diffusion-weighted magnetic resonance imaging, most studies performed so far have not had adequate time resolution to follow the temporal changes in the water apparent diffusion coefficient (ADC) in hyperacute ischemia. Using diffusion echo planar imaging, we obtained ADC maps (calculated from measurements made with 8 b-values) with a time resolution of 43 s in a feline model of global brain ischemia and reperfusion. Different protocols were performed: 10-min hypoperfusion, 10- and 22-min ischemia followed by reperfusion, and cardiac arrest. ADC values were obtained from white matter of the internal capsule and from the thalamus. Cortical gray matter measurements were not deemed reliable due to the close proximity of CSF in the cortical sulci. Following occlusion, the ADC declined in the thalamus to <2 SD of its normal baseline value within 1.5–2.5 min. This decay was exponential with a time constant (τ ± SD) of 6.0 ± 2.6 min; no further decrease in the ADC was observed 10 min following ischemia. Following reperfusion, in animals that showed ADC recovery, the ADC began increasing immediately, returning to its preischemic value in ∼15 min. No significant ADC changes were observed during hypoperfusion. Following cardiac arrest, the decay of ADC was more rapid in the thalamus (τ = 2.6 ± 0.6 min) than in white matter (τ = 6.6 ± 1.8 min). We observed that the ADC at 40 min after cardiac arrest was similar to the ADC at 10 min after ischemia. Given that all animals subjected to 10-min ischemic episodes showed ADC recovery with reperfusion, doubt is cast on whether it is possible to define a threshold value of the ADC below which brain tissue is irreversibly damaged. Finally, despite variability in the time constants of the ADC decay induced by ischemia, the ADC values at 10 min were very similar in all the animals. This suggests that when blood flow is diminished sufficiently to induce an ADC reduction, differences in perfusion affect the rapidity of the decrease but not the final asymptotic value reached.

Changes of neuronal excitability and γ-aminobutyric acid (GABA_A)-receptor expression were studied in the surround of photothrombotic infarcts, which were produced in the sensorimotor cortex of the rat by using the rose bengal technique. In a first series of experiments, multiunit recordings were performed on anesthetized animals 2–3 mm lateral from the lesion. Mean discharge frequency was considerably higher in recordings from lesioned animals (>100 Hz in the first postlesional week) compared with control animals (mean, 15 Hz). These alterations were already present after 1 day but were most pronounced 3 to 7 days after lesion induction. Thereafter the hyperexcitability declined again, although it remained visible up to 4 months. In a second series of experiments, the GABA_A-receptor expression was studied autoradiographically. This revealed a reduction of GABA_A receptors in widespread brain areas ipsilateral to the lesion. The reduction was most pronounced in the first days after lesion induction and declined with longer intervals. It is concluded that cortical infarction due to photothrombosis leads to a long-lasting and widespread reduction of GABA_A-receptor expression in the surround of the lesion, which is associated with an increased neuronal excitability. Such alterations may be responsible for epileptic seizures that can be observed in some patients after stroke and may contribute to neurologic deficits after stroke.

Hippocampal CA1 neurons exposed to a nonlethal period (2 min) of ischemia, acquired tolerance to a subsequent lethal 5-min period of ischemia, which usually causes delayed-type neuronal death. Intracelluar Ca²⁺ movements before and after the 5 min of forebrain ischemia were evaluated in gerbil hippocampal CA1 pyramidal neurons, had acquired tolerance in comparison with nonischemia-tolerant CA1 neurons. Evaluation was performed by observing the ultrastructural intracellular Ca²⁺ distribution and the Ca²⁺ adenosine triphosphatase (Ca²⁺-ATPase) activity using electron microscopic cytochemistry. In comparison with nonischemia-tolerant CA1 neurons, mitochondria of ischemia-tolerant CA1 neurons sequestered more Ca²⁺ from the cytosomal fraction 15 min after the 5-min period of ischemia, and Ca²⁺ deposits in these mitochondria were rapidly decreased. Plasma membrane Ca²⁺-ATPase activities were already significantly elevated before the 5 min of ischemia, and remained at a higher level subsequently compared to nonischemia-tolerant CA1 neurons. Changes in the mitochondrial Ca²⁺ distribution and Ca²⁺-ATPase activities in ischemia-tolerant CA1 neurons after the 5-min period of ischemia showed a strong resemblance to those in CA3 neurons, which originally possess resistance to such periods of ischemia. These findings suggest that enhanced or maintained activities of mitochondrial Ca²⁺ sequestration and plasma membrane Ca²⁺-ATPase reduced Ca²⁺ toxicity following 5-min ischemia in terms of time, resulting in escape from delayed neuronal death.

This study ascertains whether high extracellular glutamate contributes to the initiation of spreading depression (SD) by K⁺. Two microdialysis probes, each incorporating an electrode to record the extracellular direct current (DC) potential at the elicitation site, were implanted symmetrically in the cortex of anesthetized rats. Recurrent SD was triggered by perfusion of 130 mM K⁺ through the microdialysis probe for 20 min. On one side, this medium was supplemented with increasing concentrations of glutamate (0.1–1 mM) or of the selective glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC; 1–10 mM). The effects of L-trans-PDC on extracellular glutamate and basal DC potential were studied in separate experiments. Application of K⁺ for 20 min consistently elicited five to seven waves of SD. Increasing the concentration of glutamate in the perfusion medium did not alter SD elicitation. Application of L-trans-PDC concentration dependently increased the dialysate levels of glutamate (by ∼ 19-fold with 10 mM L-trans-PDC) but, unexpectedly, reduced SD elicitation. These data do not support the hypothesis that SD is elicited because high extracellular glutamate resulting from exocytosis and/or reversal of glutamate uptake depolarizes adjacent neurons. As SD elicitation requires activation of N-methyl-D-aspartate (NMDA) receptors, these results also illustrate that sensitivity of a pathological or experimental event to NMDA receptor antagonists does not necessarily imply involvement of increased extracellular glutamate. This does not rule out a selective action of glutamate, transiently released from presynaptic vesicles, on immediately juxtaposed postsynaptic receptors.

Recombinant human interleukin-1 receptor antagonist (rhIL-1ra) markedly protects against focal cerebral ischaemia in the rat, implicating endogenous IL-1 in the events leading to cerebral infarction. The present experiments investigated the effect of intracerebroventricular (i.c.v.) administration of IL-1β or rhIL-1ra on ischaemia damage and physiological parameters after permanent middle cerebral artery occlusion in the rat. IL-1β (5 ng, i.c.v.) markedly (92%) enhanced infarct volume and caused a significant rise in body temperature, but rhIL-1ra (10 μg, i.c.v.) significantly reduced infarct volume and did not significantly affect heart rate, blood pressure, or body temperature. rhIL-1ra administered 30 min before, or at the time of ischaemia significantly reduced infarct volume in cortex (55 and 60%, respectively) and striatum (52 and 41%, respectively). rhIL-1ra administered 30 min after ischaemia significantly reduced total and cortical infarct volume (26 and 29%, respectively), but did not significantly protect striatal tissue. The effects of rhIL-1ra were still evident in both cortex and striatum 7 days after ischaemia. These results support the role of IL-1 in ischaemic brain damage, revealing potent, sustained, neuroprotective effects of rhIL-1ra in the cortex and striatum, which cannot be attributed directly to changes in physiological parameters.

Our previous study on the ischemia-induced expression of platelet-derived growth factor (PDGF)-B chain in the rat brain prompted us to examine expression of PDGF β-receptor in the ischemic brain. Focal ischemia was induced by permanent tandem occlusion of middle cerebral and common carotid arteries in spontaneously hypertensive rats. Northern analysis revealed that ischemia significantly increased expression of the receptor in the ischemic neocortex at 4 and 7 days (328 ± 109%; 323 ± 119%, respectively, over control: n = 4, p < 0.05 versus sham). Neurons in infarct transiently snowed increased immunostaining for the receptor at 1 day, whereas neurons in periinfarct area showed sustained and increased immunoreactivity from 1 to 14 days postischemia. Reactive glial cells in the external capsule and in molecular layer of the neocortex adjacent to infarct possessed enhanced immunoreactivity from 1 to 21 days. Furthermore, marked immunoreactivity was observed on brain macrophages in infarct and on the abluminal side of capillaries surrounding infarct from 4 to 7 days. These results demonstrated that ischemic insult increases expression of the PDGF β-receptor at both the mRNA and protein level in the brain, suggesting its important role in cellular cascade of the ischemic brain.

These investigations characterised the cerebrovascular effects of an endothelin ET_A-receptor antagonist PD156707 in normal and ischaemic cat brain. A dose of PD156707 that inhibited the effects of exogenous endothelin-1 was established in nonischaemic cerebral resistance arterioles. Perivascular microapplication of the endothelin–receptor antagonist PD156707 (0.03–3 μM) had a minimal effect on nonischaemic pial resistance arterioles. The perivascular coapplication of PD156707 and ET-1 (10 nM) effected a dose-dependent attenuation of the ET-1 vasoconstrictive response (IC₅₀ = 0.1 μM). Intravenous administration of PD156707 (3 μmol/kg bolus + 5 μmol/kg/h infusion) attenuated the vasoconstriction elicited by perivascular ET-1 (10 nM) in normal pial arterioles (ET-1 vasoconstriction: −37 ± 13% from preinjection baseline; after intravenous PD156707: 6 ± 10% from preinjection baseline). In the focal ischaemia studies, cerebral perfusion was measured in the suprasylvian and ectosylvian gyri (by laser Doppler flowmetry). Occlusion of the middle cerebral artery reduced cerebral perfusion in the suprasylvian and ectosylvian gyri by ∼50%. Intravenous administration of PD156707 (3 μmol/kg bolus + 5 μmol/kg/h infusion), initiated 30 min after middle cerebral artery occlusion, effected a progressive increase in cerebral perfusion up to preocclusion baseline levels, whereas cerebral perfusion in vehicle-treated animals did not vary from its postocclusion level. In these animals, the intravenous administration of PD156707 reduced the hemispheric volume of ischaemic damage by 45% (vehicle: 2,376 ± 1,107 mm³; PD156707: 1,307 ± 548 mm³; p < 0.05). Our investigations indicate that endothelin receptor antagonism may be a new therapeutic strategy for the amelioration of focal ischaemic damage.

We studied the effect of a novel α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate antagonist, YM90K [6-(1H-imidazol-1-yl)-7-nitro-2, 3(1H, 4H)-quinoxalinedione monohydrochloride], in a focal cerebral ischemia model using anesthetized cats. Cats were subjected to permanent occlusion of the middle cerebral artery (MCA) for 6 h, then killed and examined histologically. The amount of ischemic damage was assessed in 12 stereotaxic coronal sections. Treatment with YM90K (i.v. infusion of 0.5 mg/5 ml/kg/h) starting 10 min after MCA occlusion markedly reduced the volume of ischemic damage (from 2823 ± 164 mm³ of the cerebral hemisphere in saline-treated cats to 1737 ± 305 mm³ in YM90K-treated cats). No essential differences were observed between YM90K- and saline-treated cats concerning physiological variables or brain temperature. These results further support the notion that the AMPA/kainate receptor plays an important role in the pathogenesis of focal cerebral ischemia. This evidence for the neuroprotective efficacy of YM90K in a gyrencephalic species suggests its therapeutic potential in the treatment of human stroke.

Glutamate (Glu) neurotoxicity is an important element of a number of neurological disorders including central nervous system (CNS) ischemia. We evaluated the effects of the novel AMPA Glu antagonist LY293558 on functional neurological outcome in two rabbit stroke models. In the reversible spinal cord ischemia model, ischemia of the caudal lumbar spinal cord was produced by temporary occlusion of the abdominal aorta. LY293558 was administered 5 min after recirculation as a 16 mg/kg i.v. bolus followed by 2.2 mg/kg infused over 1 h. Control animals received saline. LY293558 significantly increased the duration of ischemia required to produce paraplegia, from 30.5 ± 15.8 min (mean ± SD) controls to 50.1 ± 11.5 in treated animals (p < 0.01). In an irreversible model of cerebral ischemia, 50 μm plastic microspheres were injected into the carotid artery and lodged in the cerebral microvasculature. LY293558 did not significantly reduce neurological damage in this model. These data suggest that LY293558 may have therapeutic benefit following some types of ischemic injury.

We investigated the impairment of retention of spatial memory in rats with chronic focal cerebral ischemia, and examined the correlation between this impairment and pathological outcomes. A preoperative acquisition trial of the Morris water-maze task was performed twice a day for 14 successive days, and then the middle cerebral artery (MCA) was occluded. A retention trial was performed 8 weeks after MCA occlusion. Escape latency and swimming path length to the platform were significantly increased in MCA-occluded rats compared with those of sham-operated rats, and these deficits significantly correlated with massive shrinkage of the brain. Retention latency of a passive avoidance task, which was trained preoperatively, was also significantly shortened in MCA-occluded rats compared with that in sham-operated rats. These results suggest that chronic focal cerebral ischemia causes prolonged spatial memory disturbance in rats and is associated with pathological changes, and that this rat model may be useful for assessing not only anterograde but also retrograde amnesia caused by focal cerebral ischemia.

Infarct size and vascular hemodynamics were measured 24 h after middle cerebral artery (MCA) occlusion in mice genetically deficient in the endothelial nitric oxide synthase (eNOS) isoform. eNOS mutant mice developed larger infarcts (21%) than the wild-type strain when assessed 24 h after intraluminal filament occlusion. Moreover, regional CBF values recorded in the MCA territory by laser-Doppler flowmetry were more severely reduced after occlusion and were disproportionately reduced during controlled hemorrhagic hypotension in autoregulation experiments. Unlike the situation in wild-type mice, nitro-L-arginine superfusion (1 mM) dilated pial arterioles of eNOS knockout mice in a closed cranial window preparation. As noted previously, eNOS mutant mice were hypertensive. However, infarct size remained increased despite lowering blood pressure to normotensive levels by hydralazine treatment. Systemic administration of nitro-L-arginine decreased infarct size in eNOS mutant mice (24%) but not in the wild-type strain. This finding complements published data showing that nitro-L-arginine increases infarct size in knockout mice expressing the eNOS but not the neuronal NOS isoform (i.e., neuronal NOS knockout mice). We conclude that NO production within endothelium may protect brain tissue, perhaps by hemodynamic mechanisms, whereas neuronal NO overproduction may lead to neurotoxicity.

This experiment examined the effects of nitric oxide (NO) synthase inhibition on brain intracellular pH, regional cortical blood flow, and NADH fluorescence before and during 3 h of focal cerebral ischemia using in vivo fluorescence imaging. Thirty fasted rabbits under 1% halothane were divided into four treatment groups receiving N^ω-nitro-L-arginine methyl ester (L-NAME) intravenously at 20 min prior to ischemia (0.1, I, and 10 mg/kg and 1 mg/kg + 5 mg/kg L-arginine) and two control groups (nonischemic and ischemic). In ischemic controls, brain pH_i declined to 6.73 ± 0.03 at 30 min and remained acidotic through the remainder of the ischemic period. In the 0.1 mg/kg group, brain pH_i fell after 30 min of ischemia to 6.76 ± 0.05 (p < 0.05), but then improved progressively despite occlusion. In the 1 mg/kg group, brain pH_i remained normal despite middle cerebral artery (MCA) occlusion. In the 10 mg/kg group and in the combined L-NAME + L-arginine group, pH_i fell after 30 min of ischemia to 6.81 ± 0.03 (p < 0.05) and remained acidotic. During occlusion, regional cortical blood flow dropped in a dose-dependent manner. After 3 h of ischemia, regional cortical blood flow was 33.9 ± 10.9 and 25.1 ± 8.9 ml/100 g/min at doses of 0.1 and 10.0 mg/kg, respectively. L-NAME treatment did not significantly alter the increased NADH fluorescence that accompanied occlusion. This study shows that L-NAME can prevent intracellular brain acidosis during focal cerebral ischemia independent from regional cortical blood flow changes. This experiment suggests that NO is involved in pH_i regulation during focal cerebral ischemia.

Intraspinal microinjection of the nonspecific nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME) was used to determine if inhibition of NOS results in morphological changes in the rat spinal cord. Following spinal injections of 100–750 mM L-NAME (pH 7.0), 1.0–500 mM L-NAME (pH 2.5–5.4), or L-NAME + L-arginine, quantitative analysis of morphological changes revealed a positive dose-response relationship between L-NAME and neuronal loss. This effect was blocked by L-arginine and was inversely related to spinal levels of NOS enzyme activity. Results of this study have shown the importance of basal NOS activity in maintaining the structural integrity of spinal neurons. It is proposed that the effects of L-NAME on nitric oxide (NO) production leads to decreased blood flow, secondary to vasoconstriction, and a hypoxic–ischemic reaction in spinal tissue. The results suggest that a potential contributing factor to neuronal damage in pathological conditions such as spinal cord injury may be the decreased production of nitric oxide.

Post-treatment with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 reduces hypoxic–ischemic brain injury in immature animals. To elucidate possible mechanisms, cerebral glucose utilization (CMR_glc) and cerebral blood flow (CBF) were measured 1–5 h after hypoxia–ischemia and administration of MK-801 in 7-day-old rats. After 100 min of unilateral hypoxia–ischemia, half of the pups were injected with MK-801. CMR_glc was assessed by the [¹⁴C]deoxyglucose (2-DG) method. The brains were analyzed either by autoradiography or for energy metabolites and chromatographic separation of 2-DG-6-phosphate and 2-DG. CBF was measured by the autoradiographic [¹⁴C]iodoantipyrine method. Mean CMR_glc in the cerebral cortex was increased ipsilaterally after hypoxia–ischemia to 15 ± 3.3 μmol 100 g⁻¹ min⁻¹ (p < 0.01) and areas with CMR_glc >20 μmol 100 g⁻¹ min⁻¹ amounted to 8.0 ± 7.7 mm² in the ipsilateral hemisphere compared with 1.2 ± 1.6 mm² contralateral (p < 0.001). Treatment with MK-801 decreased CMR_glc bilaterally (p < 0.05) and reduced ipsilateral areas with increased CMR_glc by 64% (p < 0.01). CBF was unaltered after hypoxia–ischemia and by MK-801 treatment. In conclusion, regional glucose hyper-utilization in the parietal cortex after hypoxia–ischemia was attenuated by MK-801; this may have relevance to the neuroprotective effect of NMDA-receptor antagonists in this model.

The effects of the centrally acting anticholinesterase metrifonate (MFT) and its metabolite dichlorvos (2,2-dichlorovinyl dimethyl phosphate; DDVP) on local cerebral glucose utilization (LCGU) have been studied in 3- and 27-month-old rats, using the autoradiographic [¹⁴C]deoxyglucose technique. In 3-month-old rats, MFT (80 mg/kg i.p.) increased LCGU significantly in 17 of the 54 regions studied, including insular, cingulate, and temporal cortices, ventral hippocampus, thalamus, lateral habenula, substantia nigra, and superior colliculus. In these regions, the average MFT-induced increase in LCGU was 23% above control. The average hemispheric LCGU increased by 10% (p < 0.01). DDVP (5 mg/kg) increased LCGU in 19 regions (average increase 26%). The average hemispheric LCGU increased by 9% (p < 0.01). Regional distributions of MFT- and DDVP-induced increases in LCGU were similar and overlapped the distribution of the acetylcholinesterase activity. In 27-month-old rats, MFT was active in 18 regions (average increase 25%). The whole-brain mean LCGU increased by 10% (p < 0.01). MFT compensated for the age-related hypometabolism in some brain areas including insular, temporal, and retrosplenial cortices, substantia nigra, and superior colliculus. The effects of MFT on LCGU were preserved in old rats, at variance with other anticholinesterases (tacrine, physostigmine), which are less active in the aged rat brain.

Unlike adults, hyperglycemia with circulating glucose concentrations of 25–35 mM/L protects the immature brain from hypoxic–ischemic damage. To ascertain the effect of hyperglycemia on cerebral oxidative metabolism during the course of hypoxia–ischemia, 7-day postnatal rats underwent unilateral common carotid artery ligation followed by exposure to 8% O₂ for 2 h at 37°C. Experimental animals received 0.2 cc s.c. 50% glucose at the onset of hypoxia–ischemia, and 0.15 cc 25% glucose 1 h later to maintain blood glucose concentrations at 20–25 mML for 2 h. Control rat pups received equivalent concentrations or volumes of either mannitol or 1 N saline at the same intervals. The cerebral metabolic rate for glucose (CMR_glc.) increased from 7.1 (control) to 20.2 μmol 100 g⁻¹ min⁻¹ in hyperglycemic rats during the first hour of hypoxia–ischemia, 79 and 35% greater than the rates for saline- and mannitol-injected animals at the same interval, respectively (p < 0.01). Brain intracellular glucose concentrations were 5.2 and 3.0 mM/kg in the hyperglycemic rat pups at 1 and 2 h of hypoxia–ischemia, respectively; glucose levels were near negligible in mannitol- and saline-treated animals at the same intervals. Brain intracellular lactate concentrations averaged 13.4 and 23.3 mM/kg in hyperglycemic animals at 1 and 2 h of hypoxia–ischemia, respectively, more than twice the concentrations estimated for the saline- and mannitol-treated littermates. Phosphocreatine (PCr) and ATP decreased in all three experimental groups, but were preserved to the greatest extent in hyperglycemic animals. Results indicate that anaerobic glycolytic flux is increased to a greater extent in hyperglycemic immature rats than in normoglycemic littermates subjected to cerebral hypoxia–ischemia, and that the enhanced glycolysis leads to greater intracellular lactate accumulation. Despite cerebral lactosis, energy reserves were better preserved in hyperglycemic animals than in saline-treated controls, thus accounting for the greater resistance of hyperglycemic animals to hypoxic–ischemic brain damage.

In the present study the topographical distribution of the intrinsic nerve plexuses of the basal cerebral arteries in humans was quantified and the relation between vessel diameter and nerve density was investigated. Whole-mount preparations of various segments of the basal cerebral arteries from middle-aged patients were stained for protein gene product (PGP) 9.5. The deep nerve plexuses, located at the adventitial–medial border, were quantified by image analysis. Confocal scanning laser microscopy was used to study nerve plexuses throughout the adventitia. Transverse cryostat sections were stained for PGP 9.5, tyrosine hydroxylase and neurofilament, and quantified. The results showed a three-layered configuration of the adventitial nerves. Measurements on whole-mounts demonstrated that nerve densities were highest in the posterior communicating artery (PCom), and next highest in the proximal parts of the posterior cerebral artery (PCA) and anterior choroidal artery. There appeared to be no clear relation between nerve density and vessel diameter. The measurements on sections confirmed the high nerve densities in the PCom and PCA. Tyrosine hydroxylase- and neurofilament-immunoreactivities appeared to demonstrate separate subpopulations of the overall nerve plexuses, representing sympathetic and, possibly, sensory fibers, respectively. Densities of both subgroups generally followed those of PGP 9.5-immunoreactive nerves. Transmission electron microscopy suggested motor function of the deep nerve plexuses. The results indicate a stronger neuronal influence on this part of the cerebral circulation than hitherto reported. It is concluded that human basal cerebral arteries display a topographical distribution of deep perivascular nerves, and that nerve density is determined by locality rather than by vascular diameter.

In the present study the intrinsic nerve plexuses of the basal cerebral arteries, derived from aged non-Alzheimer's and aged Alzheimer's disease patients were quantified and compared. A previous study described and quantified nerve density on similar arteries from healthy middle-aged patients. Whole-mount preparations of various segments of the basal cerebral arteries were stained for protein gene product 9.5. The deep nerve plexuses, located at the adventitial–medial border, were quantified by image analysis. Transverse cryostat sections were stained for various markers and quantified. Measurements on whole mounts demonstrated that nerve densities were highest in the posterior communicating artery and in the postcommunicating part of the posterior cerebral artery (PCA) for both aged and Alzheimer's groups. Statistical comparison showed a tendency toward decreased nerve density with aging, which was significant for the internal carotid artery, precommunicating part of the PCA, and the anterior choroidal artery in both non-Alzheimer's and Alzheimer's aged groups. In addition, in Alzheimer's patients nerve density was significantly lower in the precommunicating part of the anterior cerebral artery compared with the healthy aged group. Measurements on sections confirmed the tendency to decreased innervation with aging. It is concluded that densities of deep perivascular nerves of human basal cerebral arteries are subject to localized changes caused by aging and Alzheimer's disease.

Chronically implanted ultrasonic Doppler flowmeters were used to obtain simultaneously recorded flow velocity signals from internal carotid and vertebral arteries, and the sagittal sinus, in rabbits. All three signals increased to 144 ± 7–215 ± 35% of baseline during hypercapnia (arterial Pco₂ 55 mmHg) in both anesthetized and conscious animals. During the period of change in inspired CO₂, the relationship between simultaneously recorded mean internal carotid and mean sagittal sinus signals was linear, with the correlation ranging from 0.83 to 0.96. Since forebrain arterial inflow must approximate forebrain venous outflow, the high correlation between internal carotid and sagittal sinus signals indicates that these measures provide reliable and valid indices of cerebral blood flow (CBF). Vertebral and internal carotid angiography confirmed the location of Doppler probe. Chronically implanted ultrasonic Doppler flowmeters can, thus, provide continuous noninvasive measurements of cerebral arterial flow in both anesthetized and conscious rabbits.

The present study was designed to determine whether relaxations induced by hypercapnia depend upon nitric oxide (NO) derived from the endothelium, and whether NO-mediated relaxant response to electrical and chemical stimulation of vasodilator nerves is modulated by hypercapnia. In canine and monkey cerebral arterial strips contracted with K⁺, raising the level of CO₂ of the aerating gas in the bathing media from 5 to 10% produced a moderate relaxation, together with an increased Pco₂ (from 29.8 to 59.3 mm Hg) and a decreased pH (from 7.43 to 7.15). Relaxation was not influenced by endothelium denudation and treatment with N^G-nitro-_L-arginine. Contractions elicited by the NO synthase inhibitor were attenuated by the removal of the endothelium. Relaxations, caused by transmural electrical stimulation and nicotine, of canine cerebral arterial strips contracted with prostaglandin F_2α were potentiated only slightly by hypercapnia, but the potentiation of the response to exogenous NO (acidified NaNO₂) was clearly greater. It is concluded that as far as the arteries used are concerned, hypercapnia does not seem to liberate NO from the endothelium but does potentiate the effect of NO. The reason for lesser potentiation, by hypercapnia, of the response to nitroxidergic nerve stimulation than to NO action may be associated with an impairment by intracellular acidosis of NO synthase activation.

We investigated the role of carbon monoxide as a neural modulator of extracellular glutamate concentration in rat hippocampus CA₁ in transient forebrain ischemia by using metalloporphyrins, which block the production of carbon monoxide through the inhibition of heme oxygenase (HO) activity. Infusion of 10 and 100 μM zinc protoporphyrin IX, which inhibits nitric oxide synthase activity as well as HO activity, significantly increased glutamate concentration compared with that on the vehicle-treated side. However, infusion of 100 μM tin mesoporphyrin IX, which inhibits only HO activity, did not affect glutamate concentration in ischemia. Our results therefore do not support the hypothesis that carbon monoxide acts as a neural messenger through the modulation of extracellular glutamate concentration in ischemia.