Journal of Cerebral Blood Flow & Metabolism

Cerebral infarction initiates a cascade of molecular events, leading to proteolytic cell death. Matrix-degrading metalloproteinases (MMPs) are neutral proteases involved in extracellular matrix damage. Type IV collagenase is an MMP that increases cerebral capillary permeability after intracerebral injection and may be important along with plasminogen activators (PA) in secondary brain edema in stroke. Therefore, we measured MMPs and PAs in spontaneously hypertensive (SHR) or Wistar-Kyoto (WKY) rats with permanent middle cerebral artery occlusion (MCAO). Brain tissue was assayed for MMPs and PAs at 1, 3, 12, and 24 h and 5 days after occlusion, using substrate gel Polyacrylamide electrophoresis (zymography). SHR showed an increase in 92-kDa type IV collagenase (gelatinase B) in the infarcted hemisphere compared with the opposite side at 12 and 24 h (p < 0.05). Gelatinase A remained the same in both infarcted and normal tissue until 5 days after injury, when it increased significantly (p < 0.05). Urokinase-type PA was increased significantly at 12 and 24 h and 5 days, while tissue-type PA was decreased significantly at 1, 12, and 24 h in the ischemic compared with the nonischemic hemisphere. Gelatinase B was markedly increased in SHR at 12 and 24 h compared with WKY (p < 0.05). Secondary vasogenic edema is maximal 1–2 days after a stroke, which is the time that gelatinase B was elevated. The time of appearance of gelatinase B suggests a role in secondary tissue damage and vasogenic edema, while gelatinase A may be involved in tissue repair.

A method for the detection and tracking of propagated fluorescence transients as indicators of depolarizations in focal cerebral ischemia is described, together with initial results indicating the potential of the method. The cortex of the right cerebral hemisphere was exposed for nonrecovery experiments in five cats anesthetized with chloralose and subjected to permanent middle cerebral artery (MCA) occlusion. Fluorescence with 370-nm excitation (attributed to the degree of reduction of the NAD/H couple) was imaged with an intensified charge-coupled device camera and digitized. Sequences of images representing changes in gray level from a baseline image were examined, together with the time courses of mean gray levels in specified regions of interest. Spontaneous increases in fluorescence occurred, starting most commonly at the edge of areas of core ischemia; they propagated usually throughout the periinfarct zone and resolved to varying degrees and at varying rates, depending on proximity of the locus to the MCA input. When a fluorescence transient reached the anterior cerebral artery territory, its initial polarity reversed from an increase to a decrease in fluorescence. An initial increase in fluorescence in response to the arrival of a transient may characterize cortex that will become infarcted, if pathophysiological changes in the periinfarct zone are allowed to evolve naturally.

We recently demonstrated that closed head injury (CHI) in the rat triggers the production of tumor necrosis factor alpha (TNFα) in the contused hemisphere. Other investigations have shown that this cytokine plays a role in the inflammatory response following trauma. The present study was designed to determine whether inhibition of TNFα production or activity affects the development of cerebral edema as well as neurological dysfunction and hippocampal cell loss after CHI. To this end, we used two pharmacological agents, each acting via a different mechanism: pentoxifylline (PTX), which attenuates the production of TNFα, and tumor necrosis factor binding protein (TBP), a physiological inhibitor of TNFα activity. Both agents significantly lessened peak edema formation at 24 h and facilitated the recovery of motor function for ≤14 days postinjury. In addition, TBP attenuated disruption of the blood-brain barrier and protected hippocampal cells. PTX significantly lowered the brain TNFα level (by ∼80%), and TBP completely abolished the activity of recombinant human TNF when they were added at the same time in the in vitro bioassay. We suggest, therefore, that a decrease in TNFα level or the inhibition of its activity is accompanied by reduced brain damage.

Normal aging is associated with the degeneration of specific neural systems. We used [¹⁸F]fluorode-oxyglucose (FDG)/positron emission tomography (PET) and a statistical model of regional covariation to explore the metabolic topography of this process. We calculated global and regional metabolic rates for glucose (GMR and rCMR_glc) in two groups of normal subjects studied independently on different tomographs: Group 1—130 normal subjects (62 men and 68 women; range 21–90 years); Group 2—20 normal subjects (10 men and 10 women; range 24–78 years). In each of the two groups, the Scaled Subprofile Model (SSM) was applied to rCMR_glc data to identify specific age-related profiles. The validity of these profiles as aging markers was assessed by correlating the associated subject scores with chronological age in both normal populations. SSM analysis disclosed two significant topographic profiles associated with aging. The first topographic profile, extracted in an analysis of group 1 normals, was characterized by relative frontal hypometabolism associated with covariate metabolic increases in the parietooccipital association areas, basal ganglia, mid-brain, and cerebellum. Subject scores for this profile correlated significantly with age in both normal groups (R² = 0.48 and 0.33, p < 0.0001 for groups 1 and 2, respectively). Because of clinical similarities between normal motoric aging and parkinsonism, we explored the possibility of shared elements in the metabolic topography of both processes. We performed a combined group SSM analysis of the 20 group 2 normals and 22 age-matched Parkinson's disease patients, and identified another aging-related topographic profile. This profile was characterized by relative basal ganglia hypermetabolism associated with covariate decreases in frontal premotor cortex. Subject scores for this profile also correlated significantly with age in both normal groups (group 1: R² = 0.30, p < 0.00001; group 2: R² = 0.59, p < 0.01). Healthy aging is associated with reproducible topographic covariation profiles associated with specific neural systems. FDG/PET may provide a useful metabolic marker of the normal agingprocess.

There is a characteristic decrease in glucose metabolism in associative frontal and temporo-parietal cortices of patients suffering from Alzheimer's disease (AD). The decrease in metabolism might result from local neuronal loss or from a decrease of synaptic activity. We measured in vivo [¹¹C]methionine accumulation into proteins with positron emission tomography (PET) to assess cortical tissue loss in AD. Both global regional activity and compartmental analysis were used to express [¹¹C]methionine accumulation into brain tissue. Glucose metabolism was measured with [¹⁸F]fluorodeoxyglucose and autoradiographic method. Combined studies were performed in 10 patients with probable AD, compared to age-matched healthy volunteers. There was a significant 45% decrease of temporo-parietal glucose metabolism in patients with AD, and frontal metabolism was lowered in most patients. Temporo-parietal metabolism correlated to dementia severity. [¹¹C]methionine incorporation into temporo-parietal and frontal cortices was not significantly decreased in AD. There was no correlation with clinical symptoms. Data suggest that regional tissue loss, assessed by the decrease of [¹¹C]methionine accumulation, is not sufficient to explain cortical glucose hypometabolism, which reflects, rather, reduced synaptic connectivity.

Summation analysis strategies are recognized throughout diverse scientific fields as powerful means of differentially enhancing experimental signals over random fluctuations (noise). Such techniques, applied to emission tomographic cerebral blood flow scans, reveal subtle alterations in neuronal activity during specific behavioral states. In the present work, we extend the principles of intersubject image summation analysis to the evaluation of emission tomographic ligand-binding studies. A general methodology is presented that may be applied to a wide variety of binding site determinations. The procedure consists of anatomic standardization of individual brains to a common stereotaxic orientation, followed by statistical analyses of group versus group or individual versus group differences. We develop and evaluate performance of our technique with the use of positron emission tomographic [¹¹C]flumazenil scans from normal volunteers, depicting the regional cerebral distribution of benzodiazepine binding sites.

Serotonergic system abnormalities have been implicated in major depression, suicide, violence, alcoholism, and other psychopathologies. The prolactin response to fenfluramine has been widely used as a neuroendocrine probe to study brain serotonin responsivity. We have extended this methodology by using the positron emission tomography (PET) ¹⁸F-fluorodeoxyglucose (¹⁸FDG) method to examine the fenfluramine-induced changes in regional cerebral glucose metabolism (rCMR_glu), an indicator of changes in regional neuronal activity. We report results on 16 healthy controls, each of whom underwent two PET studies. One group of six subjects had a placebo on day 1 and a single 60 mg oral dose of fenfluramine on day 2. The second group, of 10 subjects, was tested on two consecutive occasions without drug or placebo. Data were analyzed for significant rCMR_glu changes on day 2 vs day 1 using the statistical parametric mapping method (p < 0.01). Subjects who did not receive drugs showed no statistically significant areas of rCMR_glu increase or decrease on day 2 versus day 1. In contrast, the group that received fenfluramine showed significant fenfluramine-induced responses. Areas of rCMR_glu increases involved mainly the left prefrontal and left temperoparietal cortex. Within the prefrontal cortex, two major areas of rCMR_glu increase included, first, an area centered on the anterior cingulate and, second, an area in the lateral prefrontal cortex involving principally the inferior, middle, and superior frontal gyri. Some decreases in rCMR_glu were observed, principally in the right hemisphere. This PET-fenfluramine paradigm is a potentially useful method for studying abnormalities of serotonin function in the prefrontal cortex.

The difference between ¹H nuclear magnetic resonance (NMR) spectra obtained from the human brain during euglycemia and during hyperglycemia is depicted as well-resolved glucose peaks. The time course of these brain glucose changes during a rapid increase in plasma glucose was measured in four healthy subjects, aged 18–22 years, in five studies. Results demonstrated a significant lag in the rise of glucose with respect to plasma glucose. The fit of the integrated symmetric Michaelis–Menten model to the time course of relative glucose signals yielded an estimated plasma glucose concentration for half maximal transport, K_t, of 4.8 ± 2.4 mM (mean ± SD), a maximal transport rate, T_max, of 0.80 ± 0.45 μmol g^–1 min^–1, and a cerebral metabolic glucose consumption rate (CMR)_glc of 0.32 ± 0.16 μmol g“¹ min”¹. Assuming cerebral glucose concentration to be 1.0 μmol/g at euglycemia as measured by ¹³C NMR, the fit of the same model to the time course of brain glucose concentrations resulted in K_t = 3.9 ± 0.82 mM, T_max = 1.16 ± 0.29 μmol g^–1 min^–1, and CMR_glc = 0.35 ± 0.10 μmol g^–1 min^–1. In both cases, the resulting time course equaled that predicted from the determination of the steady-state glucose concentration by ¹³C NMR spectroscopy within the experimental scatter. The agreement between the two methods of determining transport kinetics suggests that glucose is distributed throughout the entire aqueous phase of the human brain, implying substantial intracellular concentration.

The equilibrium brain/plasma distribution ratio for 3-O-methyl-D-glucose (methylglucose) varies with plasma and tissue glucose contents and can be used to determine local glucose levels in brain. This ratio was previously found to rise as brain glucose concentration fell in response to lowered plasma glucose content. The ratios, however, differed with the same tissue glucose levels in conscious and pentobarbital-sedated rats, suggesting that changes in metabolic demand might alter the quantitative relationship between the methylglucose distribution ratio and brain glucose concentration. To examine this possibility, metabolic rate was varied by focal drug application, and hexose concentrations measured in treated and surrounding tissue. When tissue glucose levels were reduced by raised metabolic demand, methylglucose distribution ratios also fell. When brain glucose levels rose due to reduced consumption, the methylglucose distribution ratio also rose. Thus, in contrast to the inverse relationship between brain/plasma methylglucose ratio and brain glucose concentration when brain glucose content is altered secondarily to changes in plasma glucose level, changes in brain glucose content induced by altered glucose utilization cause the brain glucose level and methylglucose distribution ratio to rise and fall in a direct relationship. Determination of brain glucose content from methylglucose distribution ratios must take into account rates of glucose delivery and consumption.

Water-suppressed chemical shift magnetic resonance imaging was used to detect neurochemical alterations in vivo in neurotoxin-induced rat models of Huntington's and Parkinson's disease. The toxins were: N-methyl-4-phenylpyridinium (MPP⁺), aminooxyacetic acid (AOAA), 3-nitropropionic acid (3-NP), malonate, and azide. Local or systemic injection of these compounds caused secondary excitotoxic lesions by selective inhibition of mitochondrial respiration that gave rise to elevated lactate concentrations in the striatum. In addition, decreased N-acetylaspartate (NAA) concentrations were noted at the lesion site over time. Measurements of lactate washout kinetics demonstrated that t_1/2 followed the order: 3-NP ≈ MPP⁺ » AOAA ≈ malonate, which parallels the expected lifetimes of the neurotoxins based on their mechanisms of action. Further increases in lactate were also caused by intravenous infusion of glucose. At least part of the excitotoxicity is mediated through indirect glutamate pathways because lactate production and lesion size were diminished using unilateral decortectomies (blockade of glutamatergic input) or glutamate antagonists (MK-801). Lesion size and lactate were also diminished by energy repletion with ubiquinone and nicotinamide. Lactate measurements determined by magnetic resonance agreed with biochemical measurements made using freeze clamp techniques. Lesion size as measured with MR, although larger by 30%, agreed well with lesion size determined histologically. These experiments provide evidence for impairment of intracellular energy metabolism leading to indirect excitotoxicity for all the compounds mentioned before and demonstrate the feasibility of small-volume metabolite imaging for in vivo neurochemical analysis.

Treatments that postpone hypoxic spreading depression (SD)-like depolarization (also called anoxic depolarization) facilitate recovery of function after transient cerebral hypoxia. Hypertonia reduces cerebral excitability, and we tested whether it also offers protection against SD-like depolarization and hypoxia. Oxygen was withdrawn from hippocampal slices bathed in normal artificial cerebrospinal fluid (ACSF) and, simultaneously, from slices cut from the same hippocampus but bathed in strongly hypertonic ACSF. Extracellular osmolarity (π_o) was increased by adding 100 mM mannitol or fructose to ACSF. Slices in normal π_o underwent SD-like negative extracellular voltage shift (ΔV_o). The hypertonic slices usually showed no SD-like ΔV_o but only a small, gradual negative voltage shift. Hypertonia also prevented the precipitate drop of interstitial calcium level ([Ca²⁺]_o). When oxygenation and normal osmolarity were restored, synaptic transmission in the previously hypertonic slices recovered completely, but 3 h after reoxygenation orthodromically transmitted population spikes of the control slices recovered only 25.1% of the initial control amplitude. We conclude that hypertonic treatment during hypoxia improves subsequent recovery of synaptic function. The protection is probably due to the prevention of calcium uptake by blocking the SD-like depolarization, with the prevention of hypoxic cell swelling playing a lesser role.

The flow threshold for alterations of the in vitro [³H]cyclic AMP (cAMP) binding, an indicator of the total amount of particulate cAMP-dependent protein kinase, was evaluated in the gerbil brain after 30 min, 2 h, and 6 h of unilateral common carotid artery occlusion, respectively. The autoradiographic method developed in our laboratory enabled us to measure the [³H]cAMP binding and local CBF in each region of the same brain. The ischemic flow thresholds for reduction of the cAMP binding in the hippocampus CA₁ were 18, 34, and 49 ml 100 g^–1 min^–1 after 30-min, 2-h, and 6-h ischemia, respectively. These values were higher than those in other regions such as the hippocampus CA₃ and temporal cerebral cortex in each duration of ischemia. These findings indicate that (a) the ischemic flow threshold for perturbation of the cAMP system may be higher in the hippocampus CA₁ than in other brain regions, suggesting that the hippocampus CA₁ could be especially vulnerable to acute ischemic stress; and (b) the level of the aforementioned threshold may increase progressively during the time course of ischemia in particular regions such as the hippocampus CA₁ and CA₃, suggesting that the duration of ischemia exerts a definite influence on the viability of the ischemic neuronal cells in these regions.

Stroke trials are initiated after demonstrated pharmacological protection in animal models. NBQX protects CA₁ neurons against global ischemia; however, this glutamate antagonist induces a period of subnormal temperature (e.g., a decrease of only 1.0–1.5°C) lasting several days. In this study, NBQX (3 × 30 mg/kg, i.p.) was administered starting 60 min after reperfusion, and brain temperature had declined significantly below vehicle-treated animals by 2 h after reperfusion. When the postischemic brain temperature of NBQX-treated gerbils was regulated, no neuronal protection was found. Mimicking an NBQX-induced temperature profile for 28 h postischemia yielded histological protection 4 days later comparable to that of NBQX. However, both the NBQX and temperature simulation groups showed decreased protection after 10-day survival. Our data suggest that a protracted period of subnormal temperature during the postischemic period can obscure the interpretation of preclinical drug studies.

Cerebrovascular damage leading to subsequent reductions in local cerebral blood flow (lCBF) may represent an important secondary injury mechanism following traumatic brain injury (TBI). We determined whether patterns of ¹¹¹indium-labeled platelet accumulation were spatially related to alterations in lCBF determined autoradiographically 30 min after TBI. Sprague–Dawley rats (n = 8), anesthetized with halothane and maintained on a 70:30 (vol/vol) mixture of nitrous oxide/oxygen and 0.5% halothane, underwent parasagittal fluid percussion brain injury (1.7–2.2 atm). ¹¹¹Indiumtropolone–labeled platelets were injected 30 min prior to TBI while [^l4C]-iodoantipyrine was infused 30 min after trauma. Sham-operated animals (n = 7) underwent similar surgical procedures but were not injured. In autoradiographic images of the indium-labeled platelets, focal sites of platelet accumulation within the traumatized hemisphere were restricted to the pial surface (five of eight rats), the external capsule underlying the lateral parietal cortex (five of eight rats), and within cerebrospinal fluid (CSF) compartments (six of eight rats). In contrast, mild-to-moderate reductions in lCBF, not restricted to sites of platelet accumulation, were seen throughout the traumatized hemisphere. Flow reductions were most severe in coronal sections underlying the impact site. For example, within the lateral parietal cortex and hippocampus, lCBF was significantly reduced [p < 0.01; analysis of variance (ANOVA)] from 1.71 ± 0.34 (mean ± SD) and 0.78 ± 0.12 ml/g/min, respectively, versus 0.72 ± 0.17 and 0.41 ± 0.06 ml/g/min within the traumatized hemisphere. Significant flow reductions were also seen in remote cortical and subcortical areas, including the right frontal cortex and striatum. These results indicate that focal platelet accumulation and widespread hemodynamic depression are both early consequences of TBI. Therapeutic strategies directed at these early microvascular consequences of TBI may be neuroprotective by attenuating secondary ischemic processes.

The role of endogenous endothelins in mediating postischaemic hypoperfusion after transient global ischaemia was investigated in halothane-anaesthetised rats. Pretreatment with the broad-spectrum (ET_A and ET_B) endothelin antagonist, Bosentan (17μmol/kg) had minimal effect on postischaemic hypoperfusion, measured by hydrogen clearance, in the caudate nucleus and the parietal cortex in the 3 h after bilateral common carotid artery occlusion with concomitant haemorrhagic hypotension (transient global ischaemia). In a separate series of rats with CBF measured by [¹⁴C]iodoantipyrine autoradiography at 90 min after carotid occlusion with concomitant haemorrhagic hypotension, Bosentan treatment failed to significantly alter CBF in any of the 35 brain regions examined. No significant alterations in CBF, measured by hydrogen clearance, were observed after transient bilateral common carotid artery occlusion. [¹⁴C]Iodoantipyrine autoradiography at 90 min after occlusion failed to demonstrate any significant increases in CBF after transient bilateral common carotid artery occlusion in any of the 35 brain regions examined in anaesthetised rats. The failure of the broad-spectrum endothelin antagonist Bosentan, at concentrations known to inhibit the cerebrovascular effects of exogenous ET-1, provide no support for the view that endothelins have a major role in mediating acute postischaemic hypoperfusion.

The goal of this study was to test the hypothesis that administration of L-arginine, a substrate for the synthesis of nitric oxide, restores endothelium-dependent dilatation of the basilar artery during diabetes mellitus. We measured the diameter of the basilar artery in vivo in nondiabetic and diabetic (streptozotocin; 50–60 mg/kg i.p.) rats in response to endothelium-dependent agonists (acetylcholine and bradykinin) and an endothelium-independent agonist (nitroglycerin) before and during application of L-arginine. Topical application of acetylcholine (1.0 and 10 μM) and bradykinin (1.0 and 10 μM) produced dilatation in nondiabetic rats of the basilar artery which was impaired in diabetic rats. Topical application of nitroglycerin (0.1 and 1.0 μM) produced similar dilatation of the basilar artery in nondiabetic and diabetic rats. Topical application of L-arginine (0.1 and 3 mM) did not enhance dilatation of the basilar artery in response to acetylcholine and bradykinin in diabetic rats. Thus, impairment of dilatation of the basilar artery in diabetic rats in response to acetylcholine and bradykinin appears to be related to a mechanism unrelated to the availability of L-arginine for nitric oxide synthase.

In cats anesthetized with α-chloralose, auto-regulation of blood flow (F) in the middle meningeal and common carotid arteries was assessed by bleeding and subsequently reinfusing the animals to achieve a 25% step reduction in mean arterial blood pressure (P), while maintaining the systolic blood pressure >80 mmHg. The integrity of autoregulation was assessed by calculating the gain factor G_f = 1 — [(ΔF/F)/(ΔP/P)]. Cats were examined intact, after hexamethonium (10 mg/kg), and after papaverine (6 mg/kg). Reduction of blood pressure of 25 to 60 mmHg produced equivalent drops in carotid blood flow (G_f = 0.041 ± 0.34; mean ± standard deviation, n = 12). There were only small changes in flow in the middle meningeal artery during this procedure (G_f = 0.91 ± 0.29). Hexamethonium did not block autoregulation in the middle meningeal artery (G_f = 0.92 ± 0.13, n = 4). However, papaverine almost completely abolished the ability of the artery to autoregulate (G_f = 0.10 ± 0.16, n = 7). The results suggest that the middle meningeal artery possesses an ability similar to that of the cortical circulation to autoregulate its blood flow through intrinsic, non-neuronal mechanisms. This will have important implications for the study of disturbances of dural arterial control in migraine and other headaches.

Two populations of scattered neurons containing nitric oxide synthase activity were detected in the wall of the third and lateral cerebral ventricles of rat brain, using histochemistry for NADPH-diaphorase activity. One type was multipolar and lay supraependymally, with dendrites oriented in the plane of the ependymal layer. The second type was bipolar and was situated subependymally, with dendrites extending in opposite directions, either into the surrounding brain tissue or to the ventricular surface. Moreover, multipolar neurons, situated in the corpus callosum and in the subcortical white matter, had long varicose dendrites extending toward the roof of the lateral ventricles. As a result, numerous NADPH-diaphorase neurites spread out on the free surface of the ependymal layer in contact with the CSF. These observations raise the possibility that periventricular nitrergic neurons play an essential role in registering the composition of the CSF and in modulating subcortical cerebral blood flow. A further possibility is that supraependymal nitrergic neuronal processes are effectors regulating activity of ependymal cells.

Color duplex flowmetry of internal carotid and vertebral arteries permits estimation of intravascular flow volumes and global cerebral blood flow volume (CBFV) by summing the flow volumes measured in each of the four extracranial vessels. Intravascular flow volumes were calculated as the product of angle-corrected time-averaged flow velocity and the cross-sectional area of the vessel. The reliability of this new method was tested in a prospective, intra- and interdiane, intra- and interobserver reproducibility study of 32 healthy subjects aged 7–57 years. In each subject, CBFV was tested by each observer twice on day 1 and once on day 2 in consecutive recordings. In each artery, both examiners found closely similar mean intravascular flow volumes. Intradiane interobserver reproducibility of CBFV was high on both days (correlation coefficient, CC, 0.90 and 0.85, p ≤ 0.0001; coefficient of variance, CV, 10.0 and 10.4%, respectively), as was the interdiane comparison (CC = 0.81, p ≤ 0.0001; CV ≤ 13.3%). Intraobserver reproducibility was even higher. On both days, there was a progressive decrease in CBFV from each subject's first to the last examination within a 1-h examination period (day 1: 717 ± 150 ml/min to 690 ± 120 ml/min; difference, p ≤ 0.05; day 2: 700 ± 120 ml/min to 665 ± 126 ml/min; difference, p ≤ 0.01). This habituation effect was more pronounced in subjects with high initial CBFV. Reproducibility of CBFV is comparable to that of mean CBF measurements with ¹³³Xe inhalation and H₂¹⁵O positron emission tomography techniques reported by other groups. This method makes serial bedside-monitoring of CBFV feasible without posing the risks of radiation exposure.

A method is described for the reliable production of controlled hypotension in experimental animals. Reduction in arterial blood pressure was obtained in rabbits by withdrawing arterial blood using a computer-driven pump operating within a feedback control system. Arterial blood pressure, blood flow velocity in the basilar artery (measured using transcranial Doppler), and anterior cortical microcirculation (measured using laser Doppler) were monitored. The aim of the experiments was to compare stability of hypotension produced using arterial blood pressure or basilar flow velocity as feedback control variables. Basilar artery flow velocity provided the most stable profound hypotension and during reinfusion when animals were not autoregulating. However, arterial blood pressure provided the most accurate stepwise control in autoregulating animals.