bluuebunny/crossref_metadata_2025 huggingface dataset:
Performance of a Randomization Test for Single-Subject 15O-Water PET Activation Studies
Authors: Halber Marco , Herholz Karl , Wienhard Klaus , Pawlik Gunter , Heiss Wolf-Dieter
Published: November 2003

We adapted and implemented a permutation test ( Holmes 1994 ) to single-subject positron emission tomography (PET) activation studies with multiple replications of conditions. That test determines the experimentwise α error as well as location and extent of focal activations in each individual. Its performance was assessed in five normal volunteers, using 15O-H2O-PET data acquired on a high-resolution scanner, with septa retracted (3D mode), during functional activation by repeating words versus resting (four replications each). Calculated α errors decreased and the size of activated tissue volumes (voxels with P ≤ 0.05) increased with increasing filter kernel size applied to the difference images. At a filter kernel of 12 mm Gaussian full width at half maximum, significant focal activations were seen bilaterally in superior temporal cortex, including Brodmann's areas 41 and 42, in all five subjects. Additional foci were detected in the precentral gyrus, left inferior frontal gyrus, supplementary motor area, and cerebellum of several subjects. The average CBF increase in activated voxels ranged from 17.6% to 28.7%. Activated volumes were smaller than those detected with a standard parametric test procedure. We conclude that the permutation test is a less sensitive procedure, having the advantage of not depending on unproven distributional assumptions, that detects strong activation foci in individual subjects with high reproducibility.
In Vivo Uptake of [3H]Nimodipine in Focal Cerebral Ischemia: Modulation by Hyperglycemia
Authors: Osuga Sachiko , Hogan Matthew J.
Published: November 2003

Cell membrane depolarization and tissue acidosis occur rapidly in severely ischemic brain. Preischemic hyperglycemia is recognized to increase ischemic tissue acidosis and the present studies were undertaken to correlate depolarization and tissue acidosis during acute focal cerebral ischemia and hyperglycemia. We used a dual-label autoradiography method to simultaneously measure the in vivo distribution of [3H]nimodipine and [14C]DMO (5,5-dimethyl-2,4-oxazolidinedione) in brain to identify regions of ischemic depolarization and measure regional net tissue pH. Regional cerebral blood flow (CBF) was measured in separate studies. Measurements were made 30 minutes after combined middle cerebral artery and ipsilateral common carotid artery occlusion in normoglycemic and hyperglycemic rats. Tissue pH in the ischemic cortex was depressed to 6.76 ± 0.11 in normoglycemic rats (n = 12) and 6.57 ± 0.13 in hyperglycemic rats (n = 12), with significantly greater acidosis in the hyperglycemic group ( P < 0.001). In contrast the ratio of [3H]nimodipine uptake in the ischemic cortex relative to the contralateral nonischemic cortex was significantly greater in normoglycemic (1.83 ± 0.45) than hyperglycemic (1.40 ± 0.50) rats ( P < 0.05). Within this region of ischemic cortex CBF was 31 ± 22 mL/100 g in normoglycemic rats (n = 8) and 33 ± 22 mL/100 g/min in hyperglycemic rats (n = 9). Cerebral blood flow did not differ between these two groups in any region. Thus hyperglycemia reduced the extent of ischemic depolarization within the cortex during the first 30 minutes of focal cerebral ischemia. This effect may be related to the increased tissue acidosis or to other factors that may lessen calcium influx and preserve cellular energy stores in the ischemic cortex of the hyperglycemic rats.
Regional Cerebral Blood Flow during and after 2 Hours of Middle Cerebral Artery Occlusion in the Rat
Authors: Tsuchidate Ryoichi , He Qing-Ping , Smith Maj-Lis , Siesjö Bo K.
Published: November 2003

In this study we explored if the secondary bioenergetic failure, which occurs a few hours after recirculation, following transient middle cerebral artery occlusion (MCAO) in rats, is caused by a compromised reflow. We induced 2 hours of MCAO and measured CBF at the end of the ischemia, as well as 15 minutes, 1, 2, and 4 hours after the start of recirculation, using autoradiographic or tissue sampling 14C-iodoantipyrine techniques. After 2 hours of MCAO, the autoradiographically measured CBF in the ischemic core areas was reduced to 3 to 5% of contralateral values. The reduction in CBF was less in neighboring, penumbral areas. After recirculation, flow already normalized in core tissues after 15 minutes, and remained close to normal for the 4 hours recirculation period studied. However, in penumbral tissues, recovery CBF values were usually below normal. The results show that tissues that are heavily compromised by the 2-hour period of ischemia and are destined to incur infarction, show a “relative hyperemia” during recirculation. In fact, some areas of the previously densely ischemic tissue showed overt hyperperfusion. This finding raises the question whether the relative or absolute hyperemia reflects events that are pathogenetically important. Because drugs that clearly ameliorate the final damage incurred fail to alter the relative hyperperfusion of previously ischemic tissues, it is concluded that vascular events in the reperfusion period do not play a major role in causing the final damage.
l-Arginine-Induced Regional Cerebral Blood Flow Increase is Abolished after Transient Focal Cerebral Ischemia in the Rat
Authors: Sporer Bernd , Martens K. Helge , Koedel Uwe , Haberl Roman L.
Published: November 2003

We investigated the l-arginine-induced, regional cerebral blood flow (rCBF) enhancement after different durations of transient focal cerebral ischemia in the rat to determine if l-arginine increases rCBF after transient focal cerebral ischemia. Focal ischemia (5 minutes and 20 minutes) followed by 90 minutes of reperfusion was induced in a normotensive rat suture-model. Regional cerebral blood flow in both hemispheres was measured by laser-Doppler-flowmetry. Reactivity of rCBF to l-arginine (300 mg/kg) was measured 45 minutes after reperfusion, and hypercapnia 90 minutes after reperfusion. The effect of d-arginine and pretreatment with the nitric oxide (NO) synthase inhibitor Nω-nitro-l-arginine (l-NA) (10 mg/kg) was examined in additional groups. Hypercapnia and l-arginine increased rCBF in sham operated controls and on the nonischemic hemispheres. d-arginine did not. Twenty-minute long ischemia significantly reduced the response to l-arginine (control side: 115 ± 5.9%; ischemic side: 107 ± 6.1%, n = 7) and hypercapnia, 5 minutes of ischemia did not. Nω-nitro-l-arginine pretreatment partly restored the l-arginine-induced rCBF increase. Thus, rCBF increase caused by l-arginine in the reperfusion period was unaffected by 5 minutes of ischemia, but reduced by 20 minutes of ischemia. The restoration after pretreatment with l-NA may be caused by attenuated production of cytotoxic substances, e.g., NO and related compounds.
Platelet-Derived Growth Factor-BB, but Not -AA, Prevents Delayed Neuronal Death after Forebrain Ischemia in Rats
Authors: Iihara Koji , Hashimoto Nobuo , Tsukahara Tetsuya , Sakata Masahiro , Yanamoto Hiroji , Taniguchi Takashi
Published: November 2003

Our previous studies demonstrated coordinate expression of platelet-derived growth factor (PDGF) -B chain and β-receptor in neurons at risk in the rat brain with focal ischemia. To clarify a role of the -B chain in the brain further, we examined whether PDGF-A or -B chain protects CA1 pyramidal neurons from delayed neuronal death after forebrain ischemia in rats. Pretreatment with PDGF-BB, but not -AA, at 120 ng/d for 2 days until forebrain ischemia was performed markedly ameliorated delayed neuronal death in CA1 pyramidal neurons on day 7 after ischemia. This neuroprotective effect of PDGF-BB was dose-dependent, and pretreatment with PDGF-BB at 240 ng/d showed almost complete inhibition of delayed neuronal death. In contrast, posttreatment with PDGF-BB at 120 ng/d starting 20 minutes after ischemia demonstrated no significant neuroprotective effect. The current study established marked neuroprotective actions of PDGF-BB in ischemic neuronal damage.
Calcium-Activated K+ Channels in Cerebral Arterioles in Piglets are Resistant to Ischemia
Authors: Bari Ferenc , Louis Thomas M. , Busija David W.
Published: November 2003

Our previous studies indicate that function of ATP-dependent K+ channels (KATP) in cerebral arterioles is suppressed after ischemia. In the current study, we examined pial arteriolar responses to forskolin, dibutyryl-cAMP, NS-1619, and methionine (met)-enkephalin, activators of calcium-dependent K+ channels (KCa) before and 1 hour after 10 minutes of total, global ischemia in anesthetized piglets. Arteriolar diameters were measured using a closed cranial window and intravital microscopy. All pharmacologic agents were given topically. Baseline diameters were approximately 100 μm, and diameters had returned to normal by 1 hour after ischemia. Forskolin dilated arterioles by 9 ± 3%, 18 ± 4%, and 31 ± 12% at 5 × 10−8, 5 × 10−7, and 10−6 mol/L, respectively ( P < 0.05, n = 10). In addition, dibutyryl-cAMP dilated arterioles by 8 ± 2% at 10−4 mol/L and 14 ± 2% at 3 × 10−4 mol/L ( P < 0.05, n = 6). Also, NS-1619 increased diameter of arterioles by 9 ± 2% at 10−7 mol/L and 17 ± 9% at 10−5 mol/L ( P < 0.05, n = 5). Finally, met-enkephalin dilated arterioles by 9 ± 2% at 10−8 mol/L and 16 ± 3% at 10−6 mol/L ( P < 0.05, n = 5). At 1 hour after ischemia, arteriolar dilator effects to forskolin, dibutyryl-cAMP and NS-1619, and met-enkephalin were intact. Thus, in contrast to KATP, KCa in cerebral arterioles are resistant to ischemic stress.
The Effect of Nitric Oxide Synthase Inhibition on Acute Platelet Accumulation and Hemodynamic Depression in a Rat Model of Thromboembolic Stroke
Authors: Stagliano Nancy E. , Zhao Weizhao , Prado Ricardo , Dewanjee Mrinal K. , Ginsberg Myron D. , Dietrich W. Dalton
Published: November 2003

The relative importance of hemodynamic factors in the pathogenesis of thrombotic or embolic stroke is unclear. Of particular therapeutic interest are those substances that facilitate vasodilation and the clearance of platelet aggregates in the compromised microvasculature. A likely contributor to these functions is nitric oxide because it is known to inhibit platelet aggregability and promote vascular relaxation. To investigate the involvement of nitric oxide in the hemodynamic changes after experimental ischemia, photochemically induced nonocclusive common carotid artery thrombosis (CCAT) was studied. CCAT is a rat model of unilateral carotid artery stenosis and platelet embolization to the brain. This study characterized the acute hemodynamic consequences of CCAT and the resultant pattern of platelet deposits with and without nitric oxide synthase inhibition by nitro-l-arginine methyl ester (l-NAME). In addition, the subacute local cerebral blood flow changes were studied at 24 hours. Right CCAT was produced in 30 male Wistar rats injected with 111In-labeled platelets. Between 5 and 15 minutes after thrombosis, rats were treated with either 15 mg/kg of l-NAME (intravenously) or saline vehicle. Hemodynamic changes were studied 30 to 45 minutes after thrombosis using [14C]iodoantipyrine autoradiography. Eight coronal levels were analyzed, and cortical and subcortical regions of interest were defined. Significant increases were observed in total platelets in the ipsilateral hemisphere after l-NAME treatment, and in the distribution of platelets in the anterior frontal and occipital cortices with nitric oxide synthase inhibition, encompassing the anterior and posterior border zone areas of the ipsilateral cortex. Otherwise, foci of labeled platelets were detected throughout the ipsilateral and contralateral hemispheres. Mean local cerebral blood flow images (n = 5) revealed a moderate bilateral global reduction in flow acutely, which normalized in the untreated thrombosed group by 24 hours. In contrast, the l-NAME–treated groups (sham and experimental) had lasting, widespread reductions in flow of approximately 25%. Pairwise comparisons between groups showed that CCAT/l-NAME was significantly different from shams in the corpus callosum and different from L-NAME shams in the internal capsule ( P < 0.05) These hemodynamic and platelet accumulation changes may partially account for the aggravation of cognitive and sensorimotor deficits previously reported in this model of thromboembolic stroke.
Heparin Inhibits Leukocyte Rolling in Pial Vessels and Attenuates Inflammatory Changes in a Rat Model of Experimental Bacterial Meningitis
Authors: Weber Joerg R. , Angstwurm Klemens , Rosenkranz Thomas , Lindauer Ute , Freyer Dorette , Bürger Wolf , Busch Christina , Einhäupl Karl M. , Dirnagl Ulrich
Published: November 2003

Heparin is a natural proteoglycan that was first described in 1916. In addition to its well characterized effect on blood coagulation, it is becoming clear that heparin also modulates inflammatory processes on several levels, including the interference with leukocyte–endothelium interaction. Anecdotal observations suggest a better clinical outcome of heparin-treated patients with bacterial meningitis. The authors demonstrate that heparin, a glycosaminoglycan, inhibits significantly in the early phase of experimental pneumococcal meningitis the increase of 1) regional cerebral blood flow (125 ± 18 versus 247 ± 42%), 2) intracranial pressure (4.5 ± 2.0 versus 12.1 ± 2.2 mm Hg), 3) brain edema (brain water content: 78.23 ± 0.33 versus 79.49 ± 0.46%), and 4) influx of leukocytes (571 ± 397 versus 2400 ± 875 cells/μL) to the cerebrospinal fluid compared with untreated rats. To elucidate the possible mechanism of this observation, the authors investigated for the first time leukocyte rolling in an inflammatory model in brain venules by confocal laser scanning microscopy in vivo. Heparin significantly attenuates leukocyte rolling at 2, 3, and 4 hours (2.8 ± 1.3 versus 7.9 ± 3.2/100 μm/min), as well as leukocyte sticking at 4 hours (2.1 ± 0.4 versus 3.5 ± 1.0/100 μm/min) after meningitis induction compared with untreated animals. The authors conclude that heparin can modulate acute central nervous system inflammation and, in particular, leukocyte–endothelium interaction, a key process in the cascade of injury in bacterial meningitis. They propose to evaluate further the potential of heparin in central nervous system inflammation in basic and clinical studies.
Transient Middle Cerebral Artery Occlusion by Intraluminal Suture: II. Neurological Deficits, and Pixel-Based Correlation of Histopathology with Local Blood Flow and Glucose Utilization
Authors: Zhao Weizhao , Belayev Ludmila , Ginsberg Myron D.
Published: November 2003

We conducted a pixel-based analysis of the acute hemodynamic and metabolic determinants of infarctive histopathology in a reproducible model of temporary (2-hour) middle cerebral artery occlusion (MCAO) produced in rats by an intraluminal suture. Three-dimensional averaged image data sets of local cerebral blood flow (LCBF) and glucose utilization (LCMRglc) acquired in the companion study ( Belayev et al., 1997 ) either at the end of a 2-hour period of MCAO or after 1 hour of recirculation were comapped (using digitized atlas-templates) with data sets depicting the frequency of histological infarction in a matched animal group (n = 8) in which 2 hours of MCAO was followed by 3-day survival, sequential neurobehavioral examinations, and perfusion-fixation and paraffin-embedding of brains for light-microscopic analysis. All rats developed marked postural-reflex and forelimb-placing deficits at 60 minutes of MCAO, signifying high-grade ischemia. Tactile placing deficits persisted during the 72-hour observation period while visual placing and postural-reflex abnormalities variably improved. Comapping of LCBF and histopathology showed that in those pixels destined to undergo infarction, LCBF measured at 2 hours of MCAO showed a sharp distributional peak centered at 0.14 mL/g/min. In 70% of pixels destined to infarct, LCBF at 2 hours of MCAO was 0.24 mL/g/min or below, and in 89% LCBF was below 0.47 mL/g/min (the upper limits of the ischemic core and penumbra, respectively, as defined in the companion study [ Belayev et al., 1997 ]). Local cerebral glucose utilization measured at ~1 hour after 2 hours of MCAO was distributed bimodally in the previously ischemic hemisphere. The major peak, at 22 μmol/100 g/min, coincided exactly with the distribution peak of pixels destined to undergo infarction, while in pixels with a zero probability of infarction, LCMRglc was higher by 12 to 13 μmol/100 g/min. These results indicate that local blood flow at 2 hours of MCAO is a robust predictor of eventual infarction. Pixels with ischemic-core levels of LCBF (0% to 20% of control) have a 96% probability of infarction, while the fate of the penumbra is more heterogeneous: below LCBF of 0.35 mL/g/min, the probability of infarction is 92%, while approximately 20% pixels in the upper-penumbral LCBF range (30% to 40% of control) escape infarction. Our data strongly support the view that the likelihood of infarction within the ischemic penumbra is highly influenced by very subtle differences in early perfusion.
Blockade of Cerebral Blood Flow Response to Insulin-Induced Hypoglycemia by Caffeine and Glibenclamide in Conscious Rats
Authors: Horinaka Naoaki , Kuang Tang-Yong , Pak Hazel , Wang Robert , Jehle Jane , Kennedy Charles , Sokoloff Louis
Published: November 2003

The possibility that adenosine and ATP-sensitive potassium channels (KATP) might be involved in the mechanisms of the increases in cerebral blood flow (CBF) that occur in insulin-induced hypoglycemia was examined. Cerebral blood flow was measured by the [14C]iodoantipyrine method in conscious rats during insulin-induced, moderate hypoglycemia (2 to 3 mmol/L glucose in arterial plasma) after intravenous injections of 10 to 20 mg/kg of caffeine, an adenosine receptor antagonist, or intracisternal infusion of 1 to 2 μmol/L glibenclamide, a KATP channel inhibitor. Cerebral blood flow was also measured in corresponding normoglycemic and drug-free control groups. Cerebral blood flow was 51% higher in untreated hypoglycemic than in untreated normoglycemic rats ( P < 0.01). Caffeine had a small, statistically insignificant effect on CBF in normoglycemic rats, but reduced the CBF response to hypoglycemia in a dose-dependent manner, i.e., 27% increase with 10 mg/kg and complete elimination with 20 mg/kg. Chemical determinations by HPLC in extracts of freeze-blown brains showed significant increases in the levels of adenosine and its degradation products, inosine and hypoxanthine, during hypoglycemia ( P < 0.05). Intracisternal glibenclamide had little effect on CBF in normoglycemia, but, like caffeine, produced dose-dependent reductions in the magnitude of the increases in CBF during hypoglycemia, i.e., +66% with glibenclamide-free artificial CSF administration, +25% with 1 μmol/L glibenclamide, and almost complete blockade (+5%) with 2 μmol/L glibenclamide. These results suggest that adenosine and KATP channels may play a role in the increases in CBF during hypoglycemia.
Laminar Analysis of Cerebral Blood Flow in Cortex of Rats by Laser-Doppler Flowmetry: A Pilot Study
Authors: Fabricius Martin , Akgören Nuran , Dirnagl Ulrich , Lauritzen Martin
Published: November 2003

Laser-Doppler flowmetry (LDF) is a reliable method for estimation of relative changes of CBF. The measurement depth depends on wavelength of the laser light and the separation distance of transmitting and recording optical fibers. We designed an LDF probe using two wavelengths of laser light (543 nm and 780 nm), and three separation distances of optical fibers to measure CBF in four layers of the cerebral cortex at the same time. In vitro comparison with electromagnetic flow measurements showed linear relationship between LDF and blood flow velocity at four depths within the range relevant to physiologic measurements. Using artificial brain tissue slices we showed that the signal for each channel decreased in a theoretically predictable fashion as a function of slice thickness. Application of adenosine at various depths in neocortex of halothane-anesthetized rats showed a predominant CBF increase at the level of application. Electrical stimulation at the surface of the cerebellar cortex demonstrated superficial predominance of increased CBF as predicted from the distribution of neuronal activity. In the cerebellum, hypercapnia increased CBF in a heterogeneous fashion, the major increase being at apparent depths of approximately 300 and 600 μm, whereas in the cerebral cortex, hypercapnia induced a uniform increase. In contrast, the CBF response to cortical spreading depression in the cerebral cortex was markedly heterogeneous. Thus, real-time laminar analysis of CBF with spatial resolution of 200 to 300 μm may be achieved by LDF. The real-time in depth resolution may give insight into the functional organization of the cortical microcirculation and adaptive features of CBF regulation in response to physiologic and pathophysiologic stimuli.
Vasodilator Effects on Canine Basilar Artery Induced by Intracisternal Interleukin-1β
Authors: Osuka Koji , Suzuki Yoshio , Watanabe Yasuo , Dogan Aclan , Takayasu Masakazu , Shibuya Masato , Yoshida Jun
Published: November 2003

The effect of interleukin-1β (IL-1β) on a cerebral artery was investigated in anesthetized dogs. Intracisternal administration of IL-1β (0.03 and 0.3 μg) dilated the canine basilar artery in a dose-dependent manner, without affecting systemic blood pressure or heart rate. The increase in diameter induced by 0.3 μg of IL-1β was 28.4% ± 13.4% of control at 2 hours and was inhibited by 30 μg of the IL-1β receptor antagonist, zinc protoporphyrin (4.5% ± 13.5%, P < 0.05). Interleukin-1β did not affect the concentration of nitric oxide metabolites in CSF. However, there was an increase in the concentration of eicosanoids in CSF, and the elevation of 6-keto-PGF1α paralleled the vasodilation. Pretreatment with 30 μg of the selective inducible cyclooxygenase (COX-2) inhibitor NS-398 also inhibited the IL-1β-induced vasodilation significantly (5.9% ± 9.4% at 2 hours, P < 0.01). Western blot analysis revealed the expression of a 68-kD COX-2-like protein in basilar artery extracts. These findings suggest that the IL-1β-induced vasodilator effect is linked to the prostaglandin cascade, predominantly to prostaglandin I2, by induction of COX-2, but not to the stimulation of nitric oxide metabolism.
Anoxic and Ischemic Injury of Myelinated Axons in CNS White Matter: From Mechanistic Concepts to Therapeutics
Authors: Stys Peter K.
Published: November 2003

White matter of the brain and spinal cord is susceptible to anoxia and ischemia. Irreversible injury to this tissue can have serious consequences for the overall function of the CNS through disruption of signal transmission. Myelinated axons of the CNS are critically dependent on a continuous supply of energy largely generated through oxidative phosphorylation. Anoxia and ischemia cause rapid energy depletion, failure of the Na+−K+-ATPase, and accumulation of axoplasmic Na+ through noninactivating Na+ channels, with concentrations approaching 100 mmol/L after 60 minutes of anoxia. Coupled with severe K+ depletion that results in large membrane depolarization, high [Na+]i stimulates reverse Na+–Ca2+ exchange and axonal Ca2+ overload. A component of Ca2+ entry occurs directly through Na+ channels. The excessive accumulation of Ca2+ in turn activates various Ca2+-dependent enzymes, such as calpain, phospholipases, and protein kinase C, resulting in irreversible injury. The latter enzyme may be involved in “autoprotection,” triggered by release of endogenous γ-aminobutyric acid and adenosine, by modulation of certain elements responsible for deregulation of ion homeostasis. Glycolytic block, in contrast to anoxia alone, appears to preferentially mobilize internal Ca2+ stores; as control of internal Ca2+ pools is lost, excessive release from this compartment may itself contribute to axonal damage. Reoxygenation paradoxically accelerates injury in many axons, possibly as a result of severe mitochondrial Ca2+ overload leading to a secondary failure of respiration. Although glia are relatively resistant to anoxia, oligodendrocytes and the myelin sheath may be damaged by glutamate released by reverse Na+–glutamate transport. Use-dependent Na+ channel blockers, particularly charged compounds such as QX-314, are highly neuroprotective in vitro, but only agents that exist partially in a neutral form, such as mexiletine and tocainide, are effective after systemic administration, because charged species cannot penetrate the blood–brain barrier easily.

These concepts may also apply to other white matter disorders, such as spinal cord injury or diffuse axonal injury in brain trauma. Moreover, whereas many events are unique to white matter injury, a number of steps are common to both gray and white matter anoxia and ischemia. Optimal protection of the CNS as a whole will therefore require combination therapy aimed at unique steps in gray and white matter regions, or intervention at common points in the injury cascades.
Interictal Seizure Resections Show Two Configurations of Endothelial Glut1 Glucose Transporter in the Human Blood–Brain Barrier
Authors: Cornford Eain M. , Hyman Shigeyo , Cornford Marcia E. , Landaw Elliot M. , Delgado-Escueta Antonio V.
Published: November 2003

Immunogold electron microscopy was used to analyze and quantify the Glut1 glucose transporter in brain tissue from five patients undergoing surgery for treatment of seizures. Samples were prepared from two different regions of each resection: (1) the most actively spiking epileptogenic site, and (2) the least actively spiking region, as indicated by intraoperative EEG monitoring. Two configurations of endothelial cell Glut1 were observed. About one half of the capillary profiles examined displayed abundant Glut1 immunoreactivity on both luminal and abluminal endothelial membranes. In the remainder of the profiles, reduced Glut1 labeling was seen, but adjacent erythrocyte membranes remained highly Glut1 immunoreactive, suggesting that reduced endothelial Glut1 reactivity was not attributable to method artifacts. Immunogold studies using antisera to human glial fibrillary acidic protein and human serum albumin demonstrated increased quantities of these two epitopes in the extravascular regions in which more EEG spiking activity had been demonstrated. These observations were consistent with the hypotheses that capillary integrity was more compromised, and gliosis was quantitatively increased, in the more actively spiking region of the resection. Altered glucose transporter activity in the blood–brain barrier was characterized by a bimodal Glut1 distribution in which the smaller (type B) endothelial cells displayed low Glut1 immunoreactivity, whereas adjacent (and even contiguous) larger (type A) endothelial cells showed 5- to 10-fold greater expression of membrane Glut1 transporter protein. Because this transporter facilitates glucose entry to the brain, small pericapillary volumes of brain tissue may have quite different concentrations of glucose. We hypothesize that in complex partial seizures and other forms of brain insult, an alteration of blood–brain barrier Glut1 glucose transporter activity is indicated by the appearance of these two subpopulations of endothelial cells. In comparison with previous studies of human brain capillaries in hemangioblastoma and brain injury, endothelial Glut1 density was apparently reduced (interictally) in affected temporal lobes of patients with complex partial seizures.
The Intraischemic and Early Reperfusion Changes of Protein Synthesis in the Rat Brain. eIF-2α Kinase Activity and Role of Initiation Factors eIF-2α and eIF-4E
Authors: Burda Jozef , Martín M. Elena , Gottlieb Miroslav , Chavko Mikulas , Marsala Jozef , Alcázar Alberto , Pavón Miguel , Fando Juan L. , Salinas Matilde
Published: November 2003

Rats were subjected to the standard four-vessel occlusion model of transient cerebral ischemia (vertebral and carotid arteries). The effects of normothermic ischemia (37°C) followed or not by 30-minute reperfusion, as well as 30-minute postdecapitative ischemia, on translational rates were examined. Protein synthesis rate, as measured in a cell-free system, was significantly inhibited in ischemic rats, and the extent of inhibition strongly depended on duration and temperature, and less on the model of ischemia used. The ability of reinitiation in vitro (by using aurintricarboxylic acid) decreased after ischemia, suggesting a failure in the synthetic machinery at the initiation level. Eukaryotic initiation factor 2 (eIF-2) presented almost basal activity and levels after 30-minute normothermic ischemia, and the amount of phosphorylated eIF-2α in these samples, as well as in sham-control samples, was undetectable. The decrease in the levels of phosphorylated initiation factor 4E (eIF-4E) after 30-minute ischemia (from 32% to 16%) could explain, at least partially, the impairment of initiation during transient cerebral ischemia. After reperfusion, eIF-4E phosphorylation was almost completely restored to basal levels (29%), whereas the level of phosphorylated eIF-2α was higher (13%) than in controls and ischemic samples (both less than 2%). eIF-2α kinase activity in vitro as measured by phosphorylation of endogenous eIF-2 in the presence of ATP/Mg2+, was higher in ischemic samples (8%) than in controls (4%). It seems probable that the failure of the kinase in phosphorylating eIF-2 in vivo during ischemia is due to the depletion of ATP stores. The levels of the double-stranded activated eIF-2α kinase were slightly higher in ischemic animals than in controls. Our results suggest that the modulation of eIF-4E phosphorylation could be implicated in the regulation of translation during ischemia. On the contrary, phosphorylation of eIF-2α, by an eIF-2α kinase already activated during ischemia, represents a plausible mechanism for explaining the inhibition of translation during reperfusion
Endothelin B Receptor–Mediated Increase of Cerebral Blood Flow in Experimental Pneumococcal Meningitis
Authors: Koedel Uwe , Lorenzl Stefan , Gorriz Corinna , Arendt Rainer M. , Pfister Hans-Walter
Published: November 2003

Study investigates the role of endothelin (ET) receptors in mediating early changes in cerebral blood flow—as measured by laser Doppler flowmetry (CBFLDF)—during experimental pneumococcal meningitis. Meningitis was induced with heat-killed pneumococci and confirmed by a significant increase in CBFLDF (baseline 100%; 225.3 ± 21.8% after 6 hours; mean ± SD), intracranial pressure (ICP), brain water content, and white blood cell count in the CSF. Intravenous administration of the selective endothelin B (ETB) receptor antagonist BQ-788 immediately before pneumococcal challenge (but not 4 hours afterward) significantly attenuated these pathophysiologic alterations (e.g., CBFLDF 6 hours after pneumococcal challenge: 116.7 ± 17.4%). Pretreatment with BQ-123, a selective endothelin A receptor antagonist, had no significant effect on ICP and brain water content, but augmented the increase in CBFLDF and CSF white blood cell count. Since ET is known to trigger the release of nitric oxide (NO) by ETB receptor activation, we examined specific ET–NO interactions in primary rat cerebromicrovascular endothelial cells after stimulation with heat-killed pneumococci. Pneumococci induced a significant increase in both ET and NO concentrations in endothelial cell culture medium. Treatment with phosphoramidon, an inhibitor of the endothelin-converting enzyme, prevented the production of endothelin and markedly reduced NO generation. Our data provide evidence that ET is involved as a mediator in early pneumococcal meningitis in the rat and contributes to the increase in CBFLDF, ICP, brain water content, and CSF pleocytosis, presumably through ETB receptor–mediated NO production.
Cerebral Ischemia/Reperfusion Increases Endothelial Nitric Oxide Synthase Levels by An Indomethacin-Sensitive Mechanism
Authors: Beasley Tracy C. , Bari Ferenc , Thore Clara , Thrikawala Nishadi , Louis Thomas , Busija David
Published: November 2003

In anesthetized piglets, endothelial and neuronal nitric oxide synthase (eNOS and nNOS, respectively) levels were investigated after global cerebral ischemia. Increased intracranial pressure was used to produce 5 or 10 minutes of global ischemia, which was verified visually by observing pial arteriolar blood flow and by a microsphere technique. At 4 to 6 hours of reperfusion, parietal cortex, hippocampus, and cerebellum were collected for immunohistochemical or immunoblot analysis. Immunohistochemical examination localized eNOS only to blood vessels and nNOS only to nonvascular cells, which were primarily neurons in all regions examined. Analysis of immunoblot data revealed significant increases in eNOS levels from 47 ± 22 pixels/μg protein for time controls to 77 ± 36 pixels/μg protein (75% increase) for ischemia in parietal cortex (n = 9 to 10) and 22 ± 10 for control to 40 ± 16 pixels/μg protein (40% increase) for ischemia in hippocampus (n = 7 to 8). Levels of eNOS in cerebellum also tended to be higher but were variable and not significant (n = 5 to 6). In contrast, changes in nNOS levels were not detected at 4 or 6 hours. The increase in eNOS levels detected on immunoblots also was apparent on tissue sections as an increase in intensity of staining. Cyclooxygenase-dependent mechanisms were investigated with respect to the ischemia-induced increase in eNOS levels. Pretreatment with the cyclooxygenase inhibitor indomethacin (5 mg/kg intravenously) abolished the ischemia-induced eNOS increase in parietal cortex and hippocampus (n = 7). Thus, we conclude that the eNOS response is rapid, specific to vessels, and involves an indomethacin-sensitive mechanism.
Metabolism of Lactate in Cultured GABAergic Neurons Studied by 13C Nuclear Magnetic Resonance Spectroscopy
Authors: Waagepetersen Helle S. , Bakken Inger J. , Larsson Orla M. , Sonnewald Ursala , Schousboe Arne
Published: November 2003

Primary cultures of mouse cerebral cortical neurons (GABAergic) were incubated for 4 hours in media without glucose containing 1.0 mmol/L [U-13C]lactate in the absence or presence of 0.5 mmol/L glutamine. Redissolved, lyophilized cell extracts were analyzed by 13C nuclear magnetic resonance spectroscopy to investigate neuronal metabolism of lactate and by HPLC for determination of the total amounts of glutamate (Glu), γ-aminobutyric acid (GABA), and aspartate (Asp). The 13C nuclear magnetic resonance spectra of cell extracts exhibited multiplets for Glu, GABA, and Asp, indicating pronounced recycling of labeled tricarboxylic acid cycle constituents. There was extensive incorporation of 13C label into amino acids in neurons incubated without glutamine, with the percent enrichments being approximately 60% for Glu and Asp, and 27% for GABA. When 0.5 mmol/L glutamine was added to the incubation medium, the enrichments for Asp, Glu, and GABA were 25%, 35%, and 25%, respectively. This strongly suggests that glutamine is readily converted to Glu and Asp but that conversion to GABA may be complex. The observation that enrichment in GABA was identical in the absence and presence of glutamine whereas cycling was decreased in the presence of glutamine indicates that only C-2 units derived from glutamine are used for GABA synthesis, that is, that metabolism through the tricarboxylic acid cycle is a prerequisite for GABA synthesis from glutamine. The current study gives further support to the hypothesis that cellular metabolism is compartmentalized and that lactate is an important fuel for neurons in terms of energy metabolism and extensively labels amino acids synthesized from tricarboxylic acid cycle intermediates (Asp and Glu) as well as the neurotransmitter in these neurons (GABA).
Cerebrovascular Adaptation after Unilateral Carotid Artery Ligation in the Rat: Preservation of Blood Flow and ATP during Forebrain Ischemia
Authors: Bronner Guri , Mitchell Kendall , Welsh Frank A.
Published: November 2003

To investigate long-term adaptations after unilateral carotid artery ligation, the effect of forebrain ischemia on cerebral blood flow and ATP levels was determined at various times after ligation. Unilateral carotid artery ligation was performed in male Wistar rats 0, 3, or 7 days before forebrain ischemia. Laser-Doppler blood flow was monitored bilaterally over the parietal cortex and ATP was measured in the subadjacent cortex of both hemispheres at the end of a 10-minute episode of ischemia. In the 0-day group, forebrain ischemia reduced cortical blood flow to 12% ± 8% (mean ± SD) of preischemic values and lowered cortical ATP to 26% ± 35% of control levels in the ipsilateral hemisphere. Delaying the onset of forebrain ischemia for 3 days after carotid artery ligation significantly improved cortical blood flow (29% ± 12%, P < 0.05) and ATP levels (92% ± 11%, P < 0.05) in the ipsilateral hemisphere. Delaying forebrain ischemia for 7 days also significantly improved ipsilateral blood flow (36% ± 11%, P < 0.05) and ATP levels (81% ± 29%, P < 0.05) compared with the 0-day group. In the contralateral hemisphere, the reduction in blood flow and ATP levels was not significantly altered by delaying the onset of forebrain ischemia for 3 or 7 days. These results show that unilateral carotid artery ligation induces long-term vascular adaptations that improve the collateral circulation and preserve ATP levels during a subsequent episode of ischemia.
Peroxynitrite Formation in Focal Cerebral Ischemia—Reperfusion in Rats Occurs Predominantly in the Peri-Infarct Region
Authors: Fukuyama Naoto , Takizawa Shunya , Ishida Hideyuki , Hoshiai Kiyotaka , Shinohara Yukito , Nakazawa Hiroe
Published: November 2003

Peroxynitrite (ONOO−) exhibits potent neurotoxicity and plays an important role in neu ronal death, but no evidence shows that it is formed in the brain during ischemia or subsequent reperfusion. To detect the formation of ONOO−, we used a hydrolysis/HPLC procedure to measure the formation of 3-nitro-l-tyrosine (NO2-Tyr), which is considered to reflect attack of ONOO− on l-tyrosine residues of cellular components in the brain. Focal ischemia was produced by occluding the right common carotid and right middle cerebral arteries for 2 hours, and the ischemic area was reperfused by reopening the middle cerebral artery. After 2 hours of ischemia, the values of the ratio of NO2-Tyr to l-tyrosine were 0% ± 0%, 0.42% ± 0.13% and 0.29% ± 0.10% in the noninfarct, periinfarct, and core-of-infarct regions, respectively. After 3 hours of reperfusion following 2 hours of ischemia, the ratio in the periinfarct region reached 0.89 ± 0.22%, which was significantly higher than that in the core-of-infarct region (0.35 ± 0.09%). The NO2-Tyr was not detected in 50 mg/kg of N-monomethyl-l-arginine–treated or sham-operated rats. Regional CBF in the periinfarct region decreased to 30.8 ± 15.9 mL/100 g/min during occlusion, but recovered more rapidly than did that in the core-of-infarct region.
Global Cerebral Blood Flow Decreases during Pain
Authors: Coghill Robert C. , Sang Christine N. , Berman Karen Faith , Bennett Gary J. , Iadarola Michael J.
Published: November 2003

Positron emission tomography studies have identified a common set of brain regions activated by pain. No studies, however, have quantitatively examined pain-induced CBF changes. To better characterize CBF during pain, 14 subjects received positron emission tomography scans during rest, during capsaicin-evoked pain (250 μg, intradermal injection), and during innocuous vibration. Using the H215O intravenous bolus method with arterial blood sampling, global CBF changes were assessed quantitatively. Painful stimulation produced a 22.8% decrease in global CBF from resting levels ( P < 0.0005). This decrease was not accounted for by arterial PCO2 or heart rate changes. Although the exact mechanism remains to be determined, this pain-induced global decrease represents a previously unidentified response of CBF.
Calcium Influx from the Extracellular Space Promotes NADH Hyperoxidation and Electrical Dysfunction after Anoxia in Hippocampal Slices
Authors: Pérez-Pinzón Miguel A. , Mumford Patricia L. , Carranza VeróAnica , Sick Thomas J.
Published: November 2003

A characteristic event during reperfusion after cerebral ischemia in vivo, and reoxygenation after anoxia in vitro, is hyperoxidation of the electron carriers of the mitochondrial respiratory chain. Current studies have tested the hypothesis that there is a relation among calcium molecules derived from extracellular sources, mitochondrial hyperoxidation, and electrical recovery after anoxia in hippocampal slices. Rat hippocampal slices were superfused with artificial cerebrospinal fluids (ACSF) containing calcium chloride (CaCl2) in concentrations of: 0.5, 1, 2, and 4 mmol/L. Slices were made anoxic and then allowed to recover for 60 minutes. Reduction–oxidation shifts of NADH were measured by rapid-scanning spectrofluorometry. Synaptic activity was indicated by population spike amplitudes in the CA1 pyramidal cell subfield of the hippocampus in response to stimulation of the Schaffer collaterals. Low calcium ACSF concentrations ameliorated NADH hyperoxidation and improved synaptic transmission recovery after anoxia. High calcium ACSF concentrations had opposite effects. These data suggest a link between mitochondrial hyperoxidation and electrical recovery after postanoxia reoxygenation and support the hypothesis that cytosolic calcium overload promotes mitochondrial hyperoxidation and limits electrical recovery.
Temporal Changes of Regional Glucose Use, Blood Flow, and Microtubule-Associated Protein 2 Immunostaining after Hypoxia—Ischemia in the Immature Rat Brain
Authors: Gilland Eric , Bona Elsa , Hagber Henrik
Published: November 2003

In a situation with normal CBF and without increased energy utilization, increased glucose utilization (CMRglc) can be a sign of impaired mitochondrial metabolism, which may be an early step in the injury cascade during reperfusion after hypoxia–ischemia (HI). Seven-day-old rats underwent unilateral carotid artery ligation and 70 minutes of HI. At 3, 6, 12, 24, and 48 or 72 hours after the insult, the CMRglc was measured by the 2-deoxyglucose method, and CBF by the iodoantipyrine method. These were compared with hematoxylin-eosin staining and microtubule-associated protein 2 (MAP 2) immunostaining in adjacent sections. In the ipsilateral hemisphere, there appeared regions with increased CMRglc compared with the contralateral hemisphere 3 to 12 hours after HI that also showed partial loss of MAP 2 immunostaining and early ischemic changes. These areas receded, leaving central glucose hypoutilizing areas with complete loss of MAP 2 immunostaining and histologic infarction, surrounded by only a rim of tissue with increased CMRglc. At 24 and 72 hours after the insult, no regions with increased CMRglc remained. Despite loss of MAP 2 immunostaining and histologic signs of infarction at 24 hours, cortical CBF was not reduced until 48 hours after HI, whereas the CBF in the caudate-putamen already was decreased compared with the contralateral side at 3 hours after HI. In conclusion, early reperfusion is characterized by glucose hyperutilizing areas in the cerebral cortex, followed by a secondary phase with low CMRglc and infarction.
Mitochondrial Heterogeneity in the Brain at the Cellular Level
Authors: Sonnewald Ursula , Hertz Leif , Schousboe Arne
Published: November 2003

Classically, compartmentation of glutamate metabolism in the brain is associated with the fact that neurons and glia exhibit distinct differences with regard to metabolism of this amino acid. The recent use of 13C-labeled compounds to study this metabolism in conjunction with the availability of cell type-specific tissue culture modes has led to the notion that such compartmentation may even be present in individual cell types, neurons as well as glia. To better understand and explain this, it is proposed that mitochondrial heterogeneity may exist resulting in tricarboxylic acid cycles with different properties regarding cycling rates and ratio as well as coupling to amino acid biosynthesis, primarily involving glutamate and aspartate. These hypotheses are evaluated in the light of current knowledge about mitochondrial structure and function.
Dexamethasone Aggravates Ischemia-Induced Neuronal Damage by Facilitating the Onset of Anoxic Depolarization and the Increase in the Intracellular Ca2+ Concentration in Gerbil Hippocampus
Authors: Adachi Naoto , Chen Junfeng , Liu Keyue , Tsubota Shinzo , Arai Tatsuru
Published: November 2003

The Ca2+ mobilization across the neuronal membrane is regarded as a crucial factor in the development of neuronal damage in ischemia. Because glucocorticoids have been reported to aggravate ischemic neuronal injury, the effects of dexamethasone on ischemia-induced membrane depolarization, histologic outcome, and changes in the intracellular Ca2+ concentration in the gerbil hippocampus were examined in vivo and in vitro. The effects of metyrapone, an inhibitor of glucocorticoid synthesis, were also evaluated. Changes in the direct-current potential shift in the hippocampal CA1 area produced by transient forebrain ischemia for 2.5 minutes were compared among animals pretreated with dexamethasone (3 μg, intracerebroventricularly), metyrapone (100 mg/kg, intraperitoneally), and saline. The histologic outcome was evaluated 7 days after ischemia by assessing the delayed neuronal death in the hippocampal CA1 pyramidal cells of these animals. A hypoxia-induced intracellular Ca2+ increase was evaluated by in vitro microfluorometry in gerbil hippocampal slices, and the effect of dexamethasone (120 μg/L in the medium) on the cytosolic Ca2+ accumulation was examined. The effect in a Ca2+-free ischemialike condition was also investigated. Preischemic administration of dexamethasone reduced the onset latency of ischemia-induced membrane depolarization by 22%, and aggravated neuronal damage in vivo. In contrast, pretreatment with metyrapone improved the histologic outcome. The onset time of the increase in the intracellular concentration of Ca2+ provoked by in vitro hypoxia was advanced in dexamethasone-treated slices. The Ca2+-free in vitro hypoxia reduced the elevation compared with that in the Ca2+-containing condition. Treatment with dexamethasone facilitated the increase on both the initiation and the extent in the Ca2+-free condition. Aggravation of ischemic neuronal injury by endogenous or exogenous glucocorticoids is thus thought to be caused by the advanced onset times of both the ischemia-induced direct-current potential shift and the increase in the intracellular Ca2+ concentration.