Journal of Cerebral Blood Flow & Metabolism

In vitro and in vivo parameters of flumazenil (FMZ) binding were measured in spiking and nonspiking neocortex identified by intraoperative elcctrocorticography in epileptic patients who underwent cortical resection for seizure control. In vitro measures of receptor affinity (K_D), number (B_max) and laminar distribution for [³H]-FMZ binding in the epileptic focus (n = 38) were compared to nonspiking cortex from a subgroup of the patients (n = 12) and to tissue obtained from trauma patients (n = 5). The in vitro binding parameters were compared to in vivo [¹¹C]-FMZ binding measured with positron emission tomography (PET) (n = 19). The B_max was higher in the 38 spiking tissues as compared to the 12 nonspiking tissues (P = .012). Paired comparison of spiking versus nonspiking binding in the 12 patients from whom nonspiking tissue was available showed increases in both K_D (P = .037) and B_max (P = .0047) in spiking cortex. A positive correlation was found between K_D and B_max values for 38 patients (r = 0.55, P < .0001), the magnitude of the K_D increase being twice that of the B_max increase. In addition, there was a significant correlation between the asymmetry indices of the in vivo FMZ binding on PET and in vitro K_D of spiking cortex (n = 19, r = 0.52, P = .02). The laminar distribution of [³H]-FMZ showed increased FMZ binding in cortical layers V-VI in spiking cortex compared to nonspiking and control cortex. The increased receptor number in spiking cortical layers V-VI may be a compensatory mechanism to decreased GABAergic input. The increased B_max in spiking cortex was accompanied by a larger decrease in the affinity of FMZ for the receptor suggesting that decreased FMZ binding in the epileptic focus measured with PET is due to a decrease in the affinity of the tracer for the receptor.

The calculated fraction of receptor ligands available for blood–brain barrier passage in vivo (f_avail) may differ from in vitro (f_eq) measurements. This study evaluates the protein–ligand interaction for iomazenil and flumazenil in rats by comparing f_eq and f_avail. Repeated measurements of blood–brain barrier permeability for two benzodiazepine antagonists were performed in 44 rats by the double-indicator technique. Cerebral blood flow was measured by intracarotid Xe-injection. The apparent permeability–surface product (PS_app) was measured while CBF or bolus composition was changed. Comparison of PS_app obtained in the absence and presence of 5% albumin in the injectate yielded f_avail, whereas f_eq was measured by equilibrium dialysis. Iomazenil and flumazenil f_avail was 62% and 82%, respectively, whereas f_eq was significantly lower, 42% and 61%. The PS_app for iomazenil and flumazenil increased significantly by 89% and 161% after relative CBF increases of 259% and 201%, respectively. The results demonstrate that application of f_eq in neuroreceptor studies underestimates the plasma input function to the brain. Model simulations render possible that the differences between f_avail and f_eq as well as the effect of CBF on PS_app can be caused by capillary heterogeneity.

The regional binding of N1′ -([¹¹C]methyl)naltrindole (MeNTI), a selective δ-opioid antagonist, was studied in healthy human subjects with positron emission tomography (PET). After the bolus intravenous administration of high specific activity [¹¹C]MeNTI, PET was performed over 90 minutes. Arterial plasma samples were obtained during the scanning period and assayed for the presence of radiolabeled metabolites. The data were analyzed with various kinetic (two-and three-compartment models, Patlak graphical analysis) and nonkinetic (apparent volume of distribution and activity at a late scanning time) approaches. This tracer showed irreversible binding characteristics during the scanning period used. The results of the analyses also were compared with the density and distribution of δ-opioid receptors in the human brain in vitro. Additionally, computer simulations were performed to assess the effects of changes in receptor binding and tracer transport changes on the perceived binding parameters obtained with the models. A constrained three-compartment kinetic model was demonstrated to be superior to other quantification models for the description of MeNTI kinetics and quantification of δ receptor binding in the human brain with ¹¹C-labeled MeNTI.

The impulse response function of a radioligand is the most fundamental way to describe its pharmacokinetics and to assess its tissue uptake and retention pattern. This study investigates the impulse response function of [¹¹C](+)-McN5652, a radioligand used for positron emission tomography (PET) imaging of the serotonin transporter (SERT) in the brain. Dynamic PET studies were performed in eight healthy volunteers injected with [¹¹C](+)McN5652 and subsequently with its pharmacologically inactive enantiomer [¹¹C](–)-McN5652. The impulse response function was calculated by deconvolution analysis of regional time–activity curves, and its peak value (f_max), its retention value at 75 minutes (f_T), and its normalized retention (f_rel = f_T/f_max) were obtained. Alternatively, compartmental models were applied to calculate the apparent total distribution volume (DV_T) and its specific binding component (DV_s). Both the noncompartmental (f_T, f_rel) and the compartmental parameters (DV) were investigated with and without correction for nonspecific binding by simple subtraction of the corresponding value obtained with [¹¹C](–)-McN5652. The impulse response function obtained by deconvolution analysis demonstrated high tracer extraction followed by a slow decline in the form of a monoexponential function. Statistical analysis revealed that the best compartmental model in terms of analysis of variance F and condition number of the parameter variance–covariance matrix was the one that was based on a single tissue compartment with parameters k₁ and k₂ and that also included the parameter of regional cerebral blood volume (BV). The parameter f_rel demonstrated low between-subject variance (coefficient of variation [CV] = 19%), a midbrain to cerebellum ratio of 1.85, and high correlation with the known density of SERT (r = 0.787 where r is the coefficient of linear correlation between the parameter and the known density of SERT). After correction for nonspecific binding, f_rel demonstrated further improvement in correlation (r = 0.814) and midbrain to cerebellum ratio (3.09). The variance of the distribution volumes was acceptable when the logarithmic transform lnDV was used instead of DV (17% for the three-parameter model), but correlation of this compartmental parameter was slightly less (r = 0.652 for the three-parameter model) than the correlation of the noncompartmental f_rel with the known density of SERT, and the midbrain to cerebellum ratio was only 1.5 (uncorrected) and 1.8 (corrected). At the expense of increasing variance, the correlation was increased after correction for nonspecific binding using the inactive enantiomer (r = 0.694; CV = 22%). These results indicate that the kinetics of [¹¹C](+)McN5652 can best be described by a one-tissue compartment model with three parameters (k₁, k₂, and BV), and that both the noncompartmental parameter f_rel and the compartmental distribution volumes have the potential for quantitative estimation of the density of SERT. Further validation of the radioligand in experimental and clinical situations is warranted.

[¹⁵O]Butanol has been shown to be superior to [¹⁵O]water for measuring cerebral blood flow with positron emission tomography. This work demonstrates that it is also superior for performing activation studies. Data were collected under three conditions: a visual confrontation animal-naming task, nonsense figure size discrimination, and a nonvisual darkroom control task. Time–activity curves (TAC) were obtained for regions known to be activated by the confrontation naming task to compare absolute uptake and the different kinetics of the two tracers. Also, t statistic maps were calculated from the data of 10 subjects for both tracers and compared for magnitude of change and size of activated regions. Peak uptake in the whole-brain TAC were similar for the two tracers. For all regions and conditions, the washout rate of [¹⁵O]butanol was 41% greater than that of [¹⁵O]water. At a threshold of 0, the [¹⁵O]water and [¹⁵O]butanol percent difference (nonnormalized) and t statistic (global normalization) images are nearly identical, indicating that the same property is being measured with both tracers. The [¹⁵O]butanol parametric images displayed at a threshold of |t| = 5 look similar to the [¹⁵O]water parametric maps displayed at a threshold of |t| = 4, which is consistent with the observation that t statistic values in [¹⁵O]butanol images are generally greater. The t statistic values were equal when the [¹⁵O]butanol parametric map was created from any subset of 6 subjects and the [¹⁵O]water parametric map was created from all 10 subjects. Fewer subjects need to be studied with [¹⁵O]butanol to reach the same statistical power as an [¹⁵O]water-based study.

Deep spontaneous vasodilatatory events are frequently recorded in various cerebral diseases, causing dramatic increases (A-waves) in intracranial pressure (ICP) and subsequently provoking ischemic brain insults, The relationship between fluctuations in CBF, ICP, and arterial blood pressure (ABP) is influenced by properties of cerebrovascular control mechanisms and the cerebrospinal pressure-volume compensation, The goal of this study was to construct a mathematical model of this relationship and to assess its ability to predict the occurrence and time course of A-waves, A group of 17 severely head-injured patients were included in the study, In our model ICP was derived from the ABP waveform using a linear signal transformation. The transformation was modified during the simulation by a relationship between ABP and flow velocity, i.e., by the characterization of the cerebrovascular bed. In this way the ICP could be calculated from the ABP waveform. This model was verified by comparison of simulated and directly measured ICP during A-waves recorded in seven of the patients. In all simulations, plateau elevations of ICP were well replicated. The mean absolute error between real and simulated ICP was 8.3 ± 5.4 mm Hg at the baseline and 7.9 ± 4.3 mm Hg at the top of plateau waves. The correlation coefficient between real and simulated increase in ICP was R = 0.98; P < .001. Similarly, correlation between real and simulated increase in pulse amplitude of ICP was highly significant (R = 0.94; P < .001). The mathematical model of the relationship between ABP, flow velocity, and ICP is of potential clinical use for the noninvasive detection of A-waves in patients in whom invasive ICP assessment is not conducted.

Early overuse of a lesioned forelimb, induced by immediate immobilization of the intact forelimb after a cortical lesion, has been reported to increase tissue damage and delay functional recovery. To investigate if early training without immobilization of the intact forelimb could increase tissue loss and reduce recovery, the middle cerebral artery was ligated distal to the striatal branches in 25 male spontaneously hypertensive rats. Control rats were housed in standard cages, training rats were transferred to larger cages allowing various activities and received additional special training 1 hour a day starting either 24 hours or 7 days after the ligation. The rats were tested on a rotating pole, in a leg placement test, and in a water maze and they were killed 6 weeks after the ligation. Delayed training resulted in the best overall performance; however, both training groups performed better than standard rats on the rotating pole. The cortical infarct volume was larger in the early training group than in the other two groups (P < .005), possibly related to increased glutamate release and peri-infarct cortical hyperexcitability.

Cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α can play pathogenetic or protective roles in stroke. They are increased in the brain after experimental ischemia and in the CSF of patients with stroke. However, their presence in the periphery is still controversial. To determine the source and time-course of cytokines in blood of stroke patients, IL-6 and TNF-α release from blood cells and serum levels were determined in 40 patients on days 1 through 2, 4, 10, 30, and 90 after stroke. Twenty healthy age-matched volunteers were used as controls. IL-6 and TNF-α release from stimulated blood cells was increased in stroke patients, compared to controls. A peak response (+224%) was observed at day 4 for IL-6, while TNF-α release was largely and significantly increased (about three-fold compared to controls) from day 1 to 2 until day 90 after stroke. The increase in IL-6 release was significantly higher in ischemic, compared to hemorragic strokes, at days 1 and 4. Circulating IL-6 was increased at each time point. The ischemic processes in the CNS induces a long-lasting activation of IL-6 and TNF-α production in peripheral blood cells, which are a major source of serum cytokines after stroke.

Brain reperfusion causes prompt, severe, and prolonged protein synthesis suppression and increased phosphorylation of eukaryotic initiation factor 2α [eIF2α(P)] in hippocampal CA1 and hilar neurons, The authors hypothesized that eIF2α(P) dephosphorylation would lead to recovery of protein synthesis. Here the effects of insulin, which activates phosphatases, were examined by immunostaining for eIF2α(P) and autoradiography of in vivo ³⁵S amino acid incorporation. Rats resuscitated from a 10-minute cardiac arrest were given 0, 2, 10 or 20U/kg of intravenous insulin, underwent reperfusion for 90 minutes, and were perfusion fixed. Thirty minutes before perfusion fixation, control and resuscitated animals received 500 μCi/kg of ³⁵S methionine/cysteine. Alternate 30-μm brain sections were autoradiographed or immunostained for eIF2α(P). Controls had abundant protein synthesis and no eIF2α(P) in hippocampal neurons. Untreated reperfused neurons in the CA1, hilus, and dentate gyrus had intense staining for eIF2α(P) and reduced protein synthesis; there was little improvement with treatment with 2 or 10 U/kg of insulin. However, with 20 U/kg of insulin, these neurons recovered protein synthesis and were free of eIF2α(P). These results show that the suppression of protein synthesis in the reperfused brain is reversible; they support a causal association between eIF2α(P) and inhibition of protein synthesis, and suggest a mechanism for the neuroprotective effects of insulin.

During cerebral ischemia blood–brain barrier (BBB) disruption is a critical event leading to vasogenic edema and secondary brain injury. Gelatinases A and B are matrix metalloproteinases (MMP) able to open the BBB. The current study analyzes by zymography the early gelatinases expression and activation during permanent ischemia in mice (n = 15). ProMMP-9 expression was significantly (P < 0.001) increased in ischemic regions compared with corresponding contralateral regions after 2 hours of ischemia (mean 694.7 arbitrary units [AU], SD ± 238.4 versus mean 107.6 AU, SD ± 15.6) and remained elevated until 24 hours (mean 745,7 AU, SD ± 157.4). Moreover, activated MMP-9 was observed 4 hours after the initiation of ischemia. At the same time as the appearance of activated MMP-9, we detected by the Evan's blue extravasation method a clear increase of BBB permeability, Tissue inhibitor of metalloproteinase-1 was not modified during permanent ischemia at any time. The ProMMP-2 was significantly (P < 0.05) increased only after 24 hours of permanent ischemia (mean 213.2 AU, SD ± 60.6 versus mean 94.6 AU, SD ± 13.3), and no activated form was observed. The appearance of activated MMP-9 after 4 hours of ischemia in correlation with BBB permeability alterations suggests that MMP-9 may play an active role in early vasogenic edema development after stroke.

The present study was designed to determine the effect of recombinant endothelial nitric oxide synthase (eNOS) gene expression on reactivity of canine basilar arteries to endothelin-1 (ET-1). Experiments were performed ex vivo. The arteries were exposed (30 minutes at 37°C) to adenoviral vectors encoding eNOS gene (AdCMVeNOS) or β-galactosidase reporter gene (AdCMVβ-Gal). Twenty-four hours after transduction, transgene expression was evident mainly in the vascular adventitia. Rings of control (nontransduced), AdCMVβ-Gal- and AdCMVeNOS-transduced arteries with and without endothelium were suspended for isometric tension recording. Levels of guanosine 3′,5′ -cyclic monophosphate (cGMP) were measured by radioimmunoassay. During contractions to uridine 5′-triphosphate, ET-1 (10⁻¹⁰ to 3×10⁻9 mol/L) caused further increase in tension in control and AdCMVβ-Gal-transduced arteries. In contrast, ET-1 caused concentration-dependent relaxations of AdCMVeNOS-transduced arteries. The relaxations to ET-1 in AdCMVeNOS-transduced arteries were endothelium-independent They were abolished by N^G-nitro-L-argininemethyl ester or by chemical treatment of adventitia with paraformaldehyde before gene transfer. ET-1 (10⁻⁹ mol/L) significantly increased intracellular cGMP levels in AdCMVeNOS-transduced arteries without endothelium. In arteries transduced with AdCMVeNOS, higher concentrations (10⁻⁹ to 3×10⁻⁸ mol/L) of ET-2 also caused relaxations, whereas ET-3 and sarafotoxin, a selective ET_B receptor agonist, did not produce any relaxations. The relaxations to ET-1 in AdCMVeNOS-transduced arteries were strongly reduced by BQ-123 (10⁻⁷ mol/L), an ET_A receptor antagonist, but were not affected by BQ-788 (3×10⁻⁷ mol/L), an ET_B receptor antagonist These results suggest that genetically modified adventitia can produce nitric oxide and cause relaxations in response to ET-1 via activation of ET_A receptors. Our findings support a novel concept that successful transfer and expression of recombinant eNOS gene can lead to a qualitative change in responsiveness to vasoconstrictor substances.

Both vascular endothelial growth factor (VEGF) and integrin α_vβ₃ play roles in angiogenesis. In noncerebral vascular systems, VEGF can induce endothelial integrin α_vβ₃ expression. However, it is unknown whether VEGF, like integrin α_vβ₃, appears in the initial response of microvessels to focal brain ischemia. Their coordinate expression in microvessels of the basal ganglia after middle cerebral artery occlusion (MCAO) in the nonhuman primate model was examined quantitatively. Cells incorporating deoxyuridine triphosphate (dUTP⁺) by the polymerase I reaction at 1 hour (n = 3), 2 hours (n = 3), and 7 days (n = 4) after MCAO defined the ischemic core (Ic) and peripheral regions. Both VEGF and integrin α_vβ₃ were expressed by activated noncapillary (7.5- to 30.0-μm diameter) microvessels in the Ic region at 1 and 2 hours after MCAO. At 7 days after MCAO, the number of VEGF+, integrin α_vβ₃⁺, or proliferating cell nuclear antigen-positive microvessels had decreased within the Ic region. The expressions of VEGF, integrin α_vβ₃, and proliferating cell nuclear antigen were highly correlated on the same microvessels using hierarchical log-linear statistical models. Also, VEGF and subunit α_v messenger ribonucleic acids were coexpressed on selected microvessels. Here, noncapillary microvessels are activated specifically early during a focal cerebral ischemic insult and rapidly express VEGF and integrin α_vβ₃ together.

The role of cell type-specific Na⁺,K⁺-ATPase isozymes in function-related glucose metabolism was studied using differentiated rat brain cell aggregate cultures. In mixed neuron-glia cultures, glucose utilization, determined by measuring the rate of radiolabeled 2-deoxyglucose accumulation, was markedly stimulated by the voltage-dependent sodium channel agonist veratridine (0.75 μmol/L), as well as by glutamate (100 μmol/L) and the ionotropic glutamate receptor agonist N-methyl-d-aspartate (NMDA) (10 μmol/L). Significant stimulation also was elicited by elevated extracellular potassium (12 mmol/L KCl), which was even more pronounced at 30 mmol/L KCl. In neuron-enriched cultures, a similar stimulation of glucose utilization was obtained with veratridine, specific ionotropic glutamate receptor agonists, and 30 mmol/L but not 12 mmol/L KCl. The effects of veratridine, glutamate, and NMDA were blocked by specific antagonists (tetrodotoxin, CNQX, or MK801, respectively). Low concentrations of ouabain (10⁻⁶ mol/L) prevented stimulation by the depolarizing agents but reduced only partially the response to 12 mmol/L KCl. Together with previous data showing cell type–specific expression of Na⁺,K⁺-ATPase subunit isoforms in these cultures, the current results support the view that distinct isoforms of Na⁺,K⁺-ATPase regulate glucose utilization in neurons in response to membrane depolarization, and in glial cells in response to elevated extracellular potassium.