bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental and Regional Differences in Nitric Oxide Synthase Activity and Blood Flow in the Sheep Brain
Authors: Northington Frances J. , Tobin Joseph R. , Harris Andrew P. , Traystman Richard J. , Koehler Raymond C.
Published: November 2003

Nitric oxide synthase (NOS) participates in the regulation of cerebral blood flow and neurotransmitter release and as a second messenger of glutamatergic and cholinergic systems. Developmental differences in NOS activity have been described in the rat, but not in a species with longer gestation and a larger, lobulated brain at birth. We assayed NOS activity by conversion of [14C]L-arginine to [14C]L-citrulline in 50-mg tissue samples from eight brain regions in sheep at 70, 92, 110, and 135 days gestation (term = 145 days); newborns (<7 days); and adults to test the hypothesis that NOS activity in the brain is developmentally regulated from midgestation through adulthood and matures along the neuroaxis in parallel with the known development of cerebral blood flow and neuronal activity. Three patterns of maturation of NOS activity were evident: increasing to or exceeding adult levels before 70 days gestation in the thalamus, cerebellum, and medulla; increasing to adult levels between 70 and 92 days in the hippocampus; and increasing to adult levels after 92 days in the cortex and caudate. Additionally, there were regional differences in cortical NOS activity: at 70 and 92 days of gestation, frontal cortex NOS activity was greater than parietal or occipital activity, and at 135 days gestation and in the newborn and adult, cortical and caudate activity exceeded that in most of the more caudal regions. The up to fourfold increase in regional cortical NOS activity between 92 and 135 days gestation was associated with twofold increases in cerebral blood flow and oxygen consumption during this period. Inhibition of NOS activity with administration of 60 mg/kg of NG-nitro-L-arginine methylester (L-NAME) resulted in 27% and 25% reductions in cerebral blood flow at 93 and 133 days gestation. While the associated increases in NOS activity with increases in CBF and CMRO2 do not appear causative, at various points in gestation the development of NOS activity may participate in the development of mature patterns of cerebral blood flow regulation in parallel with development of synaptic and electrical activity.
Characterization of Metabotropic Glutamate Receptor-Mediated Nitric Oxide Production in Vivo
Authors: Bhardwaj Anish , Northington Frances J. , Martin Lee J. , Hanley Daniel F. , Traystman Richard J. , Koehler Raymond C.
Published: November 2003

We tested the hypothesis that stimulation of metabotropic glutamate receptors (mGluRs) increases nitric oxide (NO) production in the hippocampus in vivo. Microdialysis probes were placed bilaterally into the CA3 region of the hippocampus of adult Sprague–Dawley rats under pentobarbital anesthesia. Probes were perfused for 5 h with artificial cerebrospinal fluid (CSF) containing 3 μM [14C]-L-arginine. Recovery of [14C]-L-citrulline in the effluent was used as a marker of NO production. In nine groups of rats, increases in [14C]-L-citrulline recovery were compared between right- and left-sided probes perfused with various combinations of the selective mGluR agonist, trans-(1 S,3 R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD); the mGluR antagonist, (±)- α-methyl-4-carboxyphenylglycine (MCPG); the NO synthase inhibitor, N-nitro-L-arginine (LNNA); the ryanodine sensitive calcium-release channel inhibitor dantrolene, the non- N-methyl-D-aspartate (NMDA); receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX); the NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a,d] cyclohepten-5,10-imine (MK-801); and the Na+ channel blocker, tetrodotoxin. Recovery of [14C]-L-citrulline during perfusion with artificial CSF progressively increased to 90 ± 21 fmol/min (± SD) over 5 h. Perfusion in the contralateral hippocampus with 1 m M ACPD augmented [14C]-L-citrulline recovery to 250 ± 81 fmol/min. Perfusion of 1 m M nitroarginine + ACPD inhibited [14C]-L-citrulline recovery compared to that with ACPD alone. Perfusion with 1 m M MCPG + ACPD attenuated ACPD enhanced [14C]-L-citrulline recovery. Perfusion of 1 m M dantrolene + ACPD inhibited the ACPD-evoked increase in [14C]-L-citrulline recovery. Perfusion of 1 m M MCPG or dantrolene without ACPD did not decrease [14C]-L-citrulline recovery as compared to CSF alone. ACPD-enhanced [14C]-L-citrulline recovery was not attenuated by CNQX, MK-801, or tetrodotoxin (TTX). Using an indirect method of assessing NO production in vivo, these data demonstrate that mGluR stimulation enhances NO production in rat hippocampus. Inhibition with dantrolene suggests that calcium-induced calcium release amplifies the inositol triphosphate-mediated calcium signal associated with mGluR stimulation, thereby resulting in augmented calcium-dependent NO production.
Distribution of Glut1 Glucose Transporters in Different Brain Structures Compared to Glucose Utilization and Capillary Density of Adult Rat Brains
Authors: Zeller Karin , Rahner-Welsch Sylvia , Kuschinsky Wolfgang
Published: November 2003

Glut1 is a specific transporter system that mediates glucose transfer across the blood–brain barrier (BBB). Although the main location of Glut1 is in the capillary endothelium of the brain, its local distribution in different brain regions is not as well defined. In the present investigation, the local pattern of Glut1 distribution was determined in 13 brain structures using an immunoautoradiographic method developed for this purpose. A polyclonal antibody directed against the C-terminal amino acid sequence of Glut1 was applied to cryosections of rat brains. A secondary antibody was added that had been coupled to [35S]. Results show a heterogeneous distribution of Glut1 in the brain with activities of [35S] ranging from 65% below to 15% above the mean. White matter activity was lower than gray matter activity. For comparison, capillary sections were counted in corresponding cryosections by indirect immunofluorescence using fibronectin antibodies. In addition, local cerebral glucose utilization (LCGU) was analyzed in identical brain structures of conscious rats by the quantitative autoradiographic 2-deoxyglucose method. Significant correlations were found between Glut1 density and either LCGU or capillary density. Results indicate a tight coupling of Glut1 transporter density and capillary density to the LCGU of different BBB structures in adult rats.
Interleukin-8 Released into the Cerebrospinal Fluid after Brain Injury is Associated with Blood–Brain Barrier Dysfunction and Nerve Growth Factor Production
Authors: Kossmann Thomas , Stahel Philip F. , Lenzlinger Philipp M. , Redl Heinz , Dubs Rolf W. , Trentz Otmar , Schlag Guenter , Morganti-Kossmann Maria C.
Published: November 2003

Interleukin (IL) 8 was measured in CSF of 14 patients with severe traumatic brain injury. IL-8 levels were significantly higher in CSF (up to 8,000 pg/ml) than serum (up to 2,400 pg/ml) (p < 0.05), suggesting intrathecal production. Maximal IL-8 values in CSF correlated with a severe dysfunction of the blood–brain barrier. Nerve growth factor (NGF) was detected in CSF of 7 of 14 patients (range of maximal NGF: 62–12,130 pg/ml). IL-8 concentrations were significantly higher in these patients than in those without NGF (p < 0.01). CSF containing high IL-8 (3,800–7,900 pg/ml) induced greater NGF production in cultured astrocytes (202–434 pg/ml) than samples with low IL-8 (600–1,000 pg/ml), which showed a smaller NGF increase (0–165 pg/ml). Anti-IL-8 antibodies strongly reduced (52–100%) the release of NGF in the group of high IL-8, whereas in the group with low IL-8, this effect was lower (0–52%). The inability of anti-IL-8 antibodies to inhibit the synthesis of NGF completely may depend on cytokines like tumor necrosis factor α and IL-6 found in these CSF samples, which may act in association with IL-8. Thus, IL-8 may represent a pivotal cytokine in the pathology of brain injury.
Expression of Ionotropic Glutamate Receptor Subunits in Glial Cells of the Hippocampal CA1 Area following Transient Forebrain Ischemia
Authors: Gottlieb Miroslav , Matute Carlos
Published: November 2003

We examined by immunohistochemistry the expression of ionotropic glutamate receptor subunits (GluRs) in glial cells of the rat dorsal hippocampus 3 to 28 days after transient forebrain ischemia. In general, the expression of GluRs at all time points studied underwent a drastic reduction that was primarily restricted to the CA1 region. In addition to the disappearance of GluRs as a result of neuronal cell death, we observed their expression in reactive glial cells, The time course of expression and the subunits involved were different for astrocytes and microglia. Reactive astrocytes exhibited kainate, GluR5–7, and N-methyl-D-aspartate (NMDA), NR2A/B, receptor subunits, both of which were maximally expressed approximately 4 weeks after ischemia. In contrast, reactive microglia expressed GluR4 and NRI subunits, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and NMDA receptor subtypes, respectively, with maximal expression observed between 3 and 7 days after ischemia. These results demonstrate that specific types of GluRs are expressed in reactive glial cells after ischemia and that, overall, their expression levels peak around or after the periods of maximal astrogliosis and microgliosis. Thus, modulation of GluR expression may be one of the molecular components accompanying the gliotic process.
Quantification of Neuroreceptors in the Living Human Brain: IV. Effect of Aging and Elevations of D2-Like Receptors in Schizophrenia and Bipolar Illness
Authors: Wong Dean F. , Pearlson Godfrey D. , Tune Larry E. , Young L. Trevor , Meltzer Carolyn Cidis , Dannals Robert F. , Ravert Hayden T. , Reith Jakob , Kuhar Michael J. , Gjedde Albert
Published: November 2003

In a previous study of 10 drug-naive schizophrenic patients, the density of D2 dopamine receptors was found to be elevated in the caudate nucleus. The study raised questions about the influence of the age of the patients, the relationship of receptor density to psychosis, and the accuracy of the method used to obtain this evidence. Using positron emission tomography and constrained analysis of the brain uptake of the radioligand N-[11C] methylspiperone ([11C]NMSP), we tested four questions: Were the assumptions underlying the quantitation valid? Is there an age decline of the density of D2-like dopamine receptors in drug-naive schizophrenia and bipolar illness? If so, is it different from that observed in normal aging? Are D2-like dopamine receptors elevated at any age in either drug-naive schizophrenic or psychotic bipolar illness patients? NMSP and haloperidol partition volumes and plasma protein fractions were not significantly different among patient groups and normal volunteers. The model-derived assay of radioligand metabolites in plasma was confirmed by high-performance liquid chromatography in the patient groups. D2-like dopamine receptors declined with age, and the slope did not differ significantly between the schizophrenic patients, bipolar affective illness patients, and normal controls. Taking the effect of age into account, increases in D2 dopamine receptor density were found in seven psychotic patients with bipolar affective illness compared with seven nonpsychotic patients and 24 control subjects as well as in 22 drug-naive schizophrenic patients compared with the 24 control subjects.
Up-Regulation of the Nedd2 Gene Encoding an ICE/Ced-3-Like Cysteine Protease in the Gerbil Brain after Transient Global Ischemia
Authors: Kinoshita Makoto , Tomimoto Hidekazu , Kinoshita Ayae , Kumar Sharad , Noda Makoto
Published: November 2003

We assessed the expression of several genes encoding pro-apoptotic cysteine proteases similar to interleukin-1β converting enzyme (ICE) and nematode Ced-3 in association with delayed neuronal death (DND) after transient forebrain ischemia in Mongolian gerbil. The levels of the two species of Nedd2 mRNA concomitantly increased about twofold in the whole forebrain at 3–6 h after 10-min ischemia and declined to the basal level by 24 h. In situ hybridization revealed that the Nedd2 gene was up-regulated in some neuronal populations in CA1 and CA3 regions of the hippocampus. In contrast, expression of ICE, CPP32/Yama/Apopain, and TX/ICErelII did not change within 48 h. These observations raise the possibility that up-regulation of Nedd2 in the vulnerable neurons may contribute to the proteolytic processes preceding the manifestation of apoptosis and/or necrosis after ischemic insult.
Delayed Neuronal Death after Global Incomplete Ischemia in Dogs is Accompanied by Changes in Phospholipase C Protein Expression
Authors: Sieber Frederick E. , Traystman Richard J. , Martin Lee J.
Published: November 2003

Activation of phospholipase C (PLC) increases intracellular Ca++ and may play a role in delayed neuronal death after ischemia. Because changes in intracellular Ca++ are believed to participate in ischemic neuronal injury, we tested the hypothesis that PLCβ protein levels are temporally altered in brain regions that undergo neurodegeneration after global incomplete ischemia. Dogs (n = 12) were subjected to 20 minutes of global incomplete ischemia followed by recovery of either 1 (n = 5) or 7 days (n = 7). Six sham-operated animals were used as nonischemic controls. In hematoxylin and eosin-stained brain sections, neuronal density at 1 day after ischemia was unchanged relative to nonischemic controls in hippocampus CA1, caudate, and cerebellar cortex (anterior lobule). However, at 7 days after ischemia, neuronal densities were decreased to 56 ± 15% (mean ± SD) and 75 ± 17% of control in CA1 and caudate, respectively. At 1 and 7 days after ischemia, the percentage of neurons showing ischemic injury increased from 13 ± 10 to 40 ± 35% in CA1, 24 ± 25 to 59 ± 16% in cerebellum, and 4 ± 2 to 18 ± 12% in caudate. Densitometric analysis of immunocytochemically stained brain sections from controls (n = 3), 1 day after ischemia (n = 3), and 7 days after ischemia (n = 5) revealed that PLCβ immunoreactivity was increased in cerebellum at 1 day (0.274 ± 0.013 v 0.295 ± 0.005 optical density units [OD] in control and 1 day, respectively) and 7 days (0.108 ± 0.009 v 0.116 ± 0.005 O.D. in control and 7 days, respectively). PLCβ immunoreactivity was unchanged after ischemia in caudate and hippocampus. Western blot analysis of PLCβ immunoreactivity in the cerebellar cortex and hippocampus in the control (n = 3), 1 day (n = 2), and 7 days after ischemia (n = 2) groups showed that PLCβ levels were increased after ischemia in cerebellum 266% and 227% above control at 1 and 7 days, respectively, However, in hippocampus, PLC expression after ischemia was unchanged at 97% and 84% of control at 1 and 7 days, respectively. These results show that delayed neuronal degeneration after global incomplete ischemia is accompanied by regional abnormalities in PLC levels. Elevated PLC levels early may represent an aberrant signal transduction mechanism resulting in delayed cell damage, whereas decreased PLC levels later after ischemia may reflect ongoing neurodegeneration.
In Vivo Uptake of [3H]Nimodipine into Brain during Cortical Spreading Depression
Authors: Osuga Sachiko , Hakim Antoine M. , Osuga Hitoshi , Hogan Matthew J.
Published: November 2003

We report autoradiographic measurements of the in vivo uptake of [3H]nimodipine during the nonischemic depolarization of cortical spreading depression (CSD) in rat brain. [3H]Nimodipine uptake in brain was determined regionally in rats undergoing CSD (n = 8) and was significantly increased in cortex (14 ± 7%) and hippocampus (10 ± 6%) on the stimulated side relative to the contralateral hemisphere when compared with the same measurements in a control group (n = 8). A similar measurement using the physiologically inert radiotracer [14C]iodoantipyrine to control for potential effects of CSD on radioligand distribution showed a minimal increase (2.4 ± 0.7%) of radiotracer uptake in cortex after CSD. This increase was significantly less than that observed in the [3H]nimodipine uptake studies. We hypothesize that increased in vivo [3H]nimodipine uptake in CSD identifies regions of depolarization and thus infers activation of the L-type voltage sensitive calcium channels.
Hypoxia-Ischemia, but Not Hypoxia Alone, Induces the Expression of Heme Oxygenase-1 (HSP32) in Newborn Rat Brain
Authors: Bergeron Marcelle , Ferriero Donna M. , Vreman Hendrik J. , Stevenson David K. , Sharp Frank R.
Published: November 2003

Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme to produce bile pigments and carbon monoxide. The HO-1 isozyme is induced by a variety of agents such as heat, heme, and hydrogen peroxide. Evidence suggests that the bile pigments serve as antioxidants in cells with compromised defense mechanisms. Because hypoxia-ischemia (HI) increases the level of oxygen free radicals, the induction of HO-1 expression in the brain during ischemia could modulate the response to oxidative stress. To study the possible involvement of HO-1 in neonatal hypoxia-induced ischemic tolerance, we examined the brains of newborn rat pups exposed to 8% O2 (for 2.5 to 3 hours), and the brain of chronically hypoxic rat pups with congenital cardiac defects (Wistar Kyoto; WKY/ NCr). Heme oxygenase-1 immunostaining did not change after either acute or chronic hypoxia, suggesting that HO-1 is not a good candidate for explaining hypoxia preconditioning in newborn rat brain. To study the role of HO-1 in neonatal HI, 1-week-old rats were subjected to right carotid coagulation and exposure to 8% O2/92% N2 for 2.5 hours. Whereas HO enzymatic activity was unchanged in ipsilateral cortex and subcortical regions compared with the contralateral hemisphere or control brains, immunocytochemistry and Western blot analysis showed increased HO-1 staining in ipsilateral cortex, hippocampus, and striatum at 12 to 24 hours up to 7 days after HI. Double fluorescence immunostaining showed that HO-1 was expressed mostly in ED-1 positive macrophages. Because activated brain macrophages have been associated with the release of several cytotoxic molecules, the presence of HO-1 positive brain macrophages may determine the tissue vulnerability after HI injury.
Noninvasive near Infrared Spectroscopy Monitoring of Regional Cerebral Blood Oxygenation Changes during Peri-Infarct Depolarizations in Focal Cerebral Ischemia in the Rat
Authors: Wolf Tilo , Lindauer Ute , Reuter Uwe , Back Tobias , Villringer Arno , Einhäupl Karl , Dirnagl Ulrich
Published: November 2003

Intermittent peri-infarct depolarizations (PID), which spread from the vicinity of the infarction over the cortex, have been reported in focal ischemia. These depolarizations resemble cortical spreading depression except that they damage the cortex and enlarge the infarct volume possibly because of compromised oxygen delivery. The main purpose of this study was to evaluate the noninvasive technique of near-infrared spectroscopy (NIRS) for the identification of PID and to evaluate its capability for further pathophysiological studies. We used male barbiturate-anesthetized Wistar rats (n = 10) in which middle cerebral artery occlusion had been performed with a surgical thread. Middle cerebral artery occlusion resulted in a drop in parietally measured regional cerebral blood flow (laser Doppler flowmetry) to 31 ± 8% of baseline flow. Six ± 4 minutes after the induction of focal ischemia, 5 ± 2 direct current deflections were recorded during a one-hour measurement period which may be regarded as PID. Measuring regional cerebral blood oxygenation changes with a NIRO 500 revealed dynamic concentration changes in the three chromophores oxyhemoglobin [HbO2], deoxyhemoglobin [Hb], and the oxidized form of cytochrome aa3 [CytO] during PID. Typically, an initial slight decrease of [HbO2] (−6.1 ± 1.7 arbitrary units [AU]) and an increase of [Hb] (+1 1.5 ± 7.7 AU) were followed by an increase of [HbO2] (+10.8 ± 4.7 AU) and a decrease of [Hb] (−4.7 ± 5.5 AU); [CytO] decreased during the depolarizations (−2.0 ± 1.2 AU). We conclude that NIRS can detect typical PID-associated changes in blood oxygenation. We hypothesize that during the course of PID, unlike “normal” spreading depression, hypoxygenation precedes hyperoxygenation of the microcirculation in a given cortex volume as the depolarization wave propagates through hemodynamically compromised to intact tissue. This would accord with the known damaging effect of PID. The NIRS “fingerprint” of PID encourages the search for PID during early stroke in patients.
Transient Ischemia Induces an Early Decrease of Synaptic Transmission in CA1 Neurons of Rat Hippocampus: Electrophysiologic Study in Brain Slices
Authors: Shinno Kiyohito , Zhang Liang , Eubanks James H. , Carlen Peter L. , Wallace M. Christopher
Published: November 2003

We examined the functionality of hippocampal CA1 neurons at early times after transient global ischemia, by electrophysiologic recordings in brain slices. Transient ischemia was conducted on rats using the method of 15-minute four-vessel occlusion, and brain slices were obtained from these animals at different times after ischemia. Within 24 hours after insult, CA1 neurons showed no substantial damage as identified by morphologic means, but exhibited dramatic decreases in synaptic activities by 12 hours after insult, which became further decreased at more extended times after recovery. Blocking γ-aminobutyric acid A (GABAA) receptors with bicuculline produced a reversible augmentation of the diminished synaptic responses in slices prepared from 12-hour postinsult animals, but failed to do so in slices obtained from rats 24 hours after insult. Recorded in whole-cell mode, the minimum depolarizing current required to elicit an action potential was about twofold larger in the ischemic CA1 neurons than in sham controls, suggesting that an elevated spiking threshold exists in these neurons. We suggest that decreases in electrophysiologic activities precede the morphologic deterioration in postischemic CA1 neurons. The early decrease in CA1 synaptic activities may be associated with an imbalance between glutamate-mediated synaptic excitation and GABAA-mediated synaptic inhibition, whereas substantial impairments in synaptic transmission likely take place after prolonged postischemic recovery.
DNA Fragmentation Follows Delayed Neuronal Death in CA1 Neurons Exposed to Transient Global Ischemia in the Rat
Authors: Petito Carol K. , Torres-Munoz Jorge , Roberts Brenda , Olarte John-Paul , Nowak Thaddeus S. , Pulsinelli William A.
Published: November 2003

Apoptosis is an active, gene-directed process of cell death in which early fragmentation of nuclear DNA precedes morphological changes in the nucleus and, later, in the cytoplasm. In ischemia, biochemical studies have detected oligonucleosomes of apoptosis whereas sequential morphological studies show changes consistent with necrosis rather than apoptosis. To resolve this apparent discrepancy, we subjected rats to 10 minutes of transient forebrain ischemia followed by 1 to 14 days of reperfusion. Parameters evaluated in the CA1 region of the hippocampus included morphology, in situ end labeling (ISEL) of fragmented DNA, and expression of p53. Neurons were indistinguishable from controls at postischemic day 1 but displayed cytoplasmic basophilia or focal condensations at day 2; some neurons were slightly swollen and a few appeared normal. In situ end labeling was absent. At days 3 and 5, approximately 40 to 60% of CA1 neurons had shrunken eosinophilic cytoplasm and pyknotic nuclei, but only half of these were ISEL. By day 14, many of the necrotic neurons had been removed by phagocytes; those remaining retained mild ISEL. Neither p53 protein nor mRNA were identified in control or postischemic brain by in situ hybridization with riboprobes or by northern blot analysis. These results show that DNA fragmentation occurs after the development of delayed neuronal death in CA1 neurons subjected to 10 minutes of global ischemia. They suggest that mechanisms other than apoptosis may mediate the irreversible changes in the CA1 neurons in this model.
Oxidative Glucose Metabolism in Rat Brain during Single Forepaw Stimulation: A Spatially Localized 1H[13C] Nuclear Magnetic Resonance Study
Authors: Hyder Fahmeed , Rothman Douglas L. , Mason Graeme F. , Rangarajan Anand , Behar Kevin L. , Shulman Robert G.
Published: November 2003

In the α-chloralose-anesthetized rat during single forepaw stimulation, a spatially localized 1H[13C] nuclear magnetic resonance spectroscopic method was used to measure the rate of cerebral [C4]-glutamate isotopic turnover from infused [1,6-13C]glucose. The glutamate turnover data were analyzed using a mathematical model of cerebral glucose metabolism to evaluate the tricarboxylic acid (TCA) cycle flux (VTCA). During stimulation the value of VTCA in the sensorimotor region increased from 0.47 ± 0.06 (at rest) to 1.44 ± 0.41 μmol·g−1 min−1 ( P < 0.01) in the contralateral hemispheric compartment (24 mm3) and to 0.65 ± 0.10 μmol·g−1min−1 ( P < 0.03) in the ipsilateral side. Each VTCA value was converted to the cerebral metabolic rates of glucose oxidation ( oxidative-CMRglC) and oxygen consumption (CMRO2). These rates were corrected for partial-volume based on activation maps obtained by blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI). The percent increase and the absolute value of oxidative-CMRglc in the activated regions are similar to values reported previously for total-CMRglc using the same activation paradigm. This indicates that the large majority of energy required for brain activation, in going from the resting to an activated state, is supplied by glucose oxidation. The level of activity during stimulation is relevant to awake animals because the oxidative-CMRglc (1.05 ± 0.28 μmol·g−1·min−1; current study) is in the range of total-CMRglc previously reported for awake rats undergoing physiologic activation (0.7–1.4 μmol·g−1 min−1). It is concluded that oxidative glycolysis is the main source of energy for increased brain activity and a positive BOLD fMRI signal-change occurs in conjunction with a large increase in CMRO2.
Delayed Triphenyltetrazolium Chloride Staining Remains Useful for Evaluating Cerebral Infarct Volume in a Rat Stroke Model
Authors: Li Fuhai , Irie Katsumi , Anwer M. Sawkat , Fisher Marc
Published: November 2003

Sixteen of 24 Sprague-Dawley rats with permanent middle cerebral artery occlusion for 24 hours were subjected to immediate or 8-hour delayed 2,3,5-triphenyltetrazolium chloride (TTC) staining (n = 8 at each time point); the other 8 animals were subjected to immediate or 8-hour delayed measurement of succinate dehydrogenase activity (n = 4 at each time point). The TTC staining was of good quality good in all animals, and the infarcted region could be distinguished easily from normal tissue. There was no significant difference in corrected infarct volume between the two groups (263.8 ± 43.1 versus 264.4 ± 54.8 mm3 [mean ± standard deviation]). The activity of succinate dehydrogenase was not significantly different when normal or infarcted tissue was measured immediately after death or with an 8 hour delay, although less activity was detected at both time points in the infarcted tissue. These results demonstrate that an 8-hour delay of TTC staining is reliable for evaluating brain infarct volume in a rat stroke model and this probably is attributable to the slow deterioration of mitochondrial enzyme activity in nonischemic brain over this time period.
In Vivo Regulation of DOPA Decarboxylase by Dopamine Receptors in Rat Brain
Authors: Cumming Paul , Ase Ariel , Laliberté Christine , Kuwabara Hiroto , Gjedde Albert
Published: November 2003

To test the hypothesis that dopamine (DA) receptors influence cerebral DOPA-decarboxylase (DDC) activity in vivo, we used HPLC to measure the kinetics of the cerebral uptake and metabolism of [3H]DOPA in carbidopa-treated rats, and in rats also treated acutely with a DA receptor antagonist (flupenthixol, 2 mg/kg, intraperitoneally) or a DA receptor agonist (apomorphine, 200 μg/g, subcutaneously). The unidirectional blood-brain clearance of [3H]DOPA (K1DOPA, 0.030 mL g−1 min−1) increased by 50% after flupenthixol. The magnitudes of the relative DDC activity (k3DOPA) in striatum (0.20 min−1), olfactory tubercle (0.11 min−1), and hypothalamus (0.15 min−1) of carbidopa-treated rats were doubled with flupenthixol, but cortical DDC activity was unaffected (0.02 min−1). Apomorphine reduced the magnitude of k3DOPA in striatum by 20%. The rate constant for catabolism of [3H]DA formed in brain (k7′, monoamine oxidase [MAO] activity), which ranged from 0.025 min−1 in striatum to 0.08 min−1 in hypothalamus of carbidopa-treated rats, globally increased 2- to 4-fold after flupenthixol, and decreased to 0.003 min−1 in striatum after apomorphine. These in vivo results confirm the claim that acute blockade of DA receptors with flupenthixol stimulates the synthesis of [3H]DA from [3H]DOPA, and that this [3H]DA is subject to accelerated catabolism. Conversely, activation of the DA receptors with apomorphine inhibits DDC activity and DA catabolism.
Cerebral Blood Flow during Hypoxemia and Hemodilution in Rabbits: Different Roles for Nitric Oxide?
Authors: Todd Michael M. , Farrell Stella , Wu Bo
Published: November 2003

Hypoxemia and anemia are associated with increased CBF, but the mechanisms that link the changes in Pao2 or arterial O2 content (Cao2) with CBF are unclear. These experiments were intended to examine the contribution of nitric oxide. Cao2 in pentobarbital-anesthetized rabbits was reduced to approximately 6.5 mL O2/dL by hypoxemia (Pao2 approximately 24 to 26 mm Hg) or hemodilution with hetastarch (hematocrit approximately 14% to 15%). Animals with normal Cao2 (approximately 17.5 to 18 mL O2/dL) served as controls. In part I, each animal was given 3, 10, and 30 mg/kg Nω-nitro-l-arginine methyl ester (l-NAME) intravenously (total 43 mg/kg) to inhibit production of nitric oxide. Forebrain CBF was measured with radioactive microspheres approximately 15 to 20 minutes after each dose. Baseline CBF was greater in hypoxemic rabbits (111 ± 31 mL·100 g−1·min−1, mean ± SD) than in hemodiluted (70 ± 22 mL·100 g−1·min−1) or control animals (39 ± 12 mL·100 g−1·min−1). l-NAME (which reduced brain tissue nitric oxide synthase activity by approximately 65%) reduced CBF in hypoxemic animals to 80 ± 23 mL·100 g−1·min−1 ( P < 0.0001), but had no significant effect on CBF in either anemic or control animals. In four additional rabbits, further hemodilution to a Cao2 of approximately 3.5 mL O2/dL increased baseline CBF to 126 ± 21 mL·100 g−1·min−1, but again there was no effect of l-NAME. In part II, animals were anesthetized as above, and a closed cranial window was prepared. The cyclic GMP (cGMP) content of the artificial CSF superfusate was measured under baseline conditions, and then after the reduction of Cao2 to approximately 6.5 mL O2/dL by either hypoxemia or hemodilution. Concentrations of cGMP did not change during either control conditions or after hemodilution. However, cGMP increased significantly with the induction of hypoxemia. The cGMP increase in hypoxemic animals could be blocked with l-NAME. These results suggest that nitric oxide plays some role in hypoxemic vasodilation, but not during hemodilution.
Neuronal Nitric Oxide Synthase Expression is Induced in Neocortical Astrocytes after Spreading Depression
Authors: Caggiano Anthony O. , Kraig Richard P.
Published: November 2003

Spreading depression (SD) confers either increased susceptibility to ischemic injury or a delayed protection. Because nitric oxide modulates ischemic injury, we investigated if altered expression of nitric oxide synthase (NOS) by SD could account for the effect of SD on ischemia. Furthermore, the identity of cells expressing NOS after SD is important, since SD results in heterogeneous, cell type–specific changes in intracellular environment, which can control NOS activity. Immunohistochemical, computer-based image analyses and Western blotting show that the number of neuronal NOS (nNOS)–positive cells in the somatosensory cortex was significantly increased at 6 hours and 3 days after SD ( P < 0.05 and 0.01, respectively), whereas inducible NOS expression remained unchanged. Double-labeling of nNOS and glial fibrillary acidic protein identified these nNOS-positive cells as astrocytes. The effect of altered NO production on induced nNOS expression was examined by treating rats with sodium nitroprusside or NA-nitro-L-arginine methyl ester (LNAM) during SD. Increased nNOS expression was prevented by sodium nitroprusside and phenylephrine or phenylephrine alone, but not LNAM. Because SD increased astrocytic nNOS expression at time points correlating with both ischemic hypersensitivity and ischemic tolerance, the ability of SD to modulate ischemic injury must be complex, perhaps involving NOS but other factors as well.
Regional Differences in Cerebral Blood Flow and Glucose Utilization in Diabetic Man: The Effect of Insulin
Authors: Cranston Iain , Marsden Paul , Matyka Krystyna , Evans Mark , Lomas Jill , Sonksen Peter , Maisey Michael , Amiel Stephanie A.
Published: November 2003

To determine the effect of insulin on regional cerebral blood flow (rCBF) and glucose metabolism (CMRglu), we performed quantitative dynamic PET scanning of labeled water (H215O) and deoxyglucose (18FDG) using two protocols in 10 diabetic men. In protocol A, to test reproducibility of the technique, insulin was infused at 1.5 mU·kg−1·min−1 twice (n = 5). In protocol B, low (0.3 mU·kg−1·min−1) and high (3 mU·kg−1·min−1) dose insulin was given on separate occasions (n = 5). Euglycemia (5 mmol/L) was maintained by glucose infusion. In protocol A, CMRglu was 6% higher during the first infusion, and catecholamines were also increased, indicating stress. Blood flow was not different. Changing free insulin levels from 20.5 ± 4.8 to 191 ± 44.5 mU/L ( P < 0.001, low versus high dose, protocol B) did not alter total or regional CMRglu (whole brain 36.6 ± 4.0 versus 32.8 ± 6.2 μmol·100 g−1·min−1, P = 0.32) or CBF (41.7 ± 5.1 and 45.6 ± 9.7 mL·100 g−1·min−1, P = 0.4) or rCBF. In cerebellum, CMRglu was lower than in cortex and the ratio between rate constants for glucose uptake and phosphorylation ( K1 and k3) was reversed. There are regional differences in cerebral metabolic capacity that may explain why cerebral cortex is more sensitive to hypoglycemia than cerebellum. Brain glucose metabolism is not sensitive to insulin concentration within the physiologic range. This suggests that intracerebral insulin receptors have a different role from those in the periphery.
Calculation of the FDG Lumped Constant by Simultaneous Measurements of Global Glucose and FDG Metabolism in Humans
Authors: Hasselbalch Steen G. , Madsen Peter L. , Knudsen Gitte M. , Holm Søren , Paulson Olaf B.
Published: November 2003

The lumped constant defined as the conversion factor between the net uptake of fluoro-2-deoxy-D-glucose (FDG) and glucose was calculated from global CMRglc and from positron emission tomography (PET) using FDG as tracer (CMRFDG). Fifteen healthy, normal volunteers (mean age 24 ± 4 years) were studied. Global CBF and CMRglc were measured with the Kety-Schmidt technique using 133Xe as tracer, and values were corrected for errors from incomplete diffusion equilibrium for inert gas tracer between brain tissue and cerebral venous blood. Measurements of CMRFDG were obtained with PET using the dynamic and single-scan methods and the K1–k3 model. Measurements with the Kety-Schmidt technique and PET-FDG were performed simultaneously. Global CBF was 47.1 ± 8.0 mL · 100 g−1 · min−1, and CMRglc was 22.8 ± 4.1 μmol · 100 g−1 · min−1. No difference in CMRFDG was found with the two methods (17.8 ± 1.6 and 18.2 ± 1.3 μmol · 100 g−1 · min−1, dynamic and single scan methods, respectively). Accordingly, the lumped constant ranged from 0.80 ± 0.16 to 0.82 ± 0.15, with a mean value of 0.81 ± 0.15. The mean ratio between phosphorylation of FDG and glucose (k3*/k3) was 0.39 ± 0.25. The discrepancy between the lumped constant determined in this study and previously obtained values can be explained partly by methodologic problems, and we conclude that most of the discrepancy results from previous overestimation of global CBF. Key Words: Positron emission tomography—18F-Fluro-deoxy-D-glucose—Lumped constant—Brain glucose metabolism.
Cerebral Interleukin-6 is Neuroprotective during Permanent Focal Cerebral Ischemia in the Rat
Authors: Loddick Sarah A. , Turnbull Andrew V. , Rothwell Nancy J.
Published: November 2003

Interleukin-6 (IL-6) is a neurotrophic cytokine expressed in both neurons and glia. The present study shows that cerebral ischemia produced by permanent occlusion of the middle cerebral artery (MCAO) produces a dramatic increase in IL-6 bioactivity in the ischemic hemisphere within 2 hours of MCAO (167 ± 55 IU versus sham: 50 ± 35 IU), with further increases at 8 hours (3,456 ± 1,162 IU) and 24 hours (6,088 ± 1,772 IU). In a separate series of experiments, intracerebroventricular injection of recombinant IL-6 (3,100 or 31,000 IU) significantly reduced ischemic brain damage after MCAO (to 52% and 65% of controls, respectively). The large increase in endogenous IL-6 bioactivity in response to ischemia, together with the marked neuroprotection produced by exogenous IL-6 suggest that this cytokine is an important endogenous inhibitor of neuronal death during cerebral ischemia.
Subarachnoid Hemorrhage and the Role of Potassium Channels in Relaxations of Canine Basilar Artery to Nitrovasodilators
Authors: Onoue Hisashi , Katusic Zvonimir S.
Published: November 2003

This study was designed to determine the effect of subarachnoid hemorrhage (SAH) on potassium (K+) channels involved in relaxations of cerebral arteries to nitrovasodilators. The effects of K+ channel inhibitors on relaxations to 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP) were studied in rings of basilar arteries obtained from untreated dogs and dogs exposed to SAH. The levels of cyclic GMP were measured by radioimmunoassay. In rings without endothelium, concentration-dependent relaxations to SIN-1 (10−9 − 10−4 mol/L) and SNP (10−9 − 10−4 mol/L) were not affected by SAH, whereas increase in cyclic GMP production stimulated by SIN-1 (10−6 mol/L) was significantly suppressed after SAH. The relaxations to SIN-1 and SNP were reduced by charybdotoxin (CTX; 10−7 mol/L), a selective Ca2+-activated K+ channel inhibitor, in both normal and SAH arteries; however, the reduction of relaxations by CTX was significantly greater in SAH arteries. By contrast, the relaxations to these nitrovasodilators were not affected by glyburide (10−5 mol/L), an ATP-sensitive K+ channel inhibitor, in both normal and SAH arteries. These findings suggest that in cerebral arteries exposed to SAH, Ca2+-activated K+ channels may play a compensatory role in mediation of relaxations to nitric oxide. This may help to explain mechanisms of relaxations to nitrovasodilators in arteries with impaired production of cyclic GMP.
Heme Oxygenase-1 is Induced in Glia Throughout Brain by Subarachnoid Hemoglobin
Authors: Turner Christopher P. , Bergeron Marcelle , Matz Paul , Zegna Angelo , Noble Linda J. , Panter S. Scott , Sharp Frank R.
Published: November 2003

The heme oxygenase-1 gene, HO-1, induced by heme, ischemia, and heat shock, metabolizes heme to biliverdin, free iron, and carbon monoxide. Though the distribution of HO-1 has been described in normal rat brain, little is known about how extracellular heme proteins in the subarachnoid space distribute in brain. To address this issue, hemoglobin was injected into the cisterna magna of adult rats. Expression of HO-1 in these animals was compared with saline-injected, BSA-injected, and uninjected controls. Western blot analysis showed that 24 hours after injection oxyhemoglobin increased HO-1 levels approximately four- to fivefold in all brain regions studied compared with saline-injected and BSA-injected controls. In the brain, HO-1 immunoreactivity was evident at 4 hours and peaked at 24 hours after oxyhemoglobin injections, returning to control levels by 4 to 8 days. This HO-1 induction was detected mainly in cells with small, rounded somas bearing two to four truncated processes, a morphology consistent with that of microglia. These cells were double-stained with the microglial marker, OX42, in every brain region examined. It is proposed that subarachnoid hemoglobin may be taken up into microglia wherein heme induces HO-1.
Cerebroprotective Effect of the Nitric Oxide Synthase Inhibitors, 1-(2-Trifluoromethylphenyl) Imidazole and 7-Nitro Indazole, after Transient Focal Cerebral Ischemia in the Rat
Authors: Escott Katherine J. , Beech John S. , Haga Kristin K. , Williams Steve C. R. , Meldrum Brian S. , Bath Philip M. W.
Published: November 2003

The novel neuronal nitric oxide synthase inhibitors, 1-(2-trifluoromethylphenyl)imidazole (TRIM) and 7-nitro indazole (7-NI), were used to investigate the role of nitric oxide in a model of transient focal cerebral ischemia in vivo. In halothane-anesthetized rats, the middle cerebral artery (MCA) was occluded for 2 hours using an intravascular thread and then reperfused for 22 hours before histologic evaluation. TRIM (10, 20, or 50 mg/kg), 7-NI (60 mg/kg), TRIM (50 mg/kg) plus L-arginine (300 mg/kg), or L-arginine (300 mg/kg) alone was administered intraperitoneally, either at 5 or 90 minutes after MCA occlusion. Immediate administration (5 minutes after MCA occlusion) of TRIM produced a dose-related reduction in lesion size, which was reversed with L-arginine coadministration. Similarly, delayed administration of TRIM (90 minutes after MCA occlusion, 50 mg/kg) decreased total lesion volume by 48.4% ± 13.0% in comparison to a reduction of 39.3% ± 10.9% when TRIM (50 mg/kg) was administered immediately (5 minutes) after occlusion. 7-NI (60 mg/kg) reduced the total lesion volume by 38.5% ± 13.7% when administered immediately (5 minutes) after MCA occlusion, but had no effect when administration was delayed (90 minutes). Neither TRIM (50 mg/kg) nor 7-NI (60 mg/kg), administered 5 minutes after MCA occlusion, had any significant effect on mean arterial blood pressure throughout the ischemic period or for up to 10 minutes after reperfusion. These results indicate that immediate or delayed administration of the selective neuronal NOS inhibitor TRIM reduces the lesion volume after transient MCA occlusion. In contrast, only immediate administration of 7-NI reduces lesion volume.
Frequency Domain Analysis of Cerebral Blood Flow Velocity and its Correlation with Arterial Blood Pressure
Authors: Kuo Terry Bo-Jau , Chern Chang-Ming , Sheng Wen-Yung , Wong Wen-Jang , Hu Han-Hwa
Published: November 2003

We applied frequency domain analysis to detect and quantify spontaneous fluctuations in the blood flow velocity of the middle cerebral artery (MCAFV). Instantaneous MCAFV of normal volunteers was detected using transcranial Doppler sonography. Spectral and transfer function analyses of MCAFV and arterial blood pressure (ABP) were performed by fast Fourier transform. We found the fluctuations in MCAFV, like ABP, could be diffracted into three components at specific frequency ranges, designated as high-frequency (HF, 0.15 to 0.4 Hz), low-frequency (LF, 0.04 to 0.15 Hz), and very low-frequency (VLF, 0.016 to 0.04 Hz) components. The HF and LF components of MCAFV exhibited high coherence with those of ABP, indicating great similarity of MCAFV and ABP fluctuations within the two frequency ranges. However, it was not the case for the VLF component. Transfer function analysis revealed that the ABP-MCAFV phase angle was frequency-dependent in the LF range ( r = −0.79, P < 0.001) but not in the HF range. The time delay between LF fluctuations of ABP and those of MCAFV was evaluated as 2.1 seconds. We conclude that in addition to traditional B-wave equivalents, there are at least two different mechanisms for MCAFV fluctuations: the HF and LF fluctuations of MCAFV are basically secondary to those of ABP, and cerebral autoregulation may operate efficiently in LF rather than HF range. Frequency domain analysis offers an opportunity to explore the nature and underlying mechanism of dynamic regulation in cerebral circulation.
Monitoring of Cerebral Oxygen Metabolism in the Jugular Bulb: Reliability of Unilateral Measurements in Severe Head Injury
Authors: Metz Christoph , Holzschuh Matthias , Bein Thomas , Woertgen Chris , Rothoerl Ralf , Kallenbach Beatrix , Taeger Kai , Brawanski Alexander
Published: November 2003

To investigate the reliability of unilateral jugular venous monitoring and to determine the appropriate side, we performed bilateral jugular venous monitoring in 22 head-injured patients. Fiberoptic catheters were placed in both jugular bulbs. Arterial and bilateral jugular venous blood samples were obtained simultaneously for in vitro determination of jugular venous oxygen saturation (SJO2), arterial minus jugular venous lactate content difference (AJDL), and modified lactate-oxygen index (mLOI). Ischemia was assumed if one of the following pathologic values occurred at least unilaterally: SJO2 <54%, AJDL <−0.37 mmol/L, mLOI >0.08. The sensitivity of calculated unilateral monitoring in detecting ischemia was evaluated by comparing the incidence detected unilaterally with that disclosed bilaterally. The mean and maximum bilateral SJO2 differences varied between 1.4% and 21.0%, and 8.1% and 44.3%, respectively. The bias and limits of agreement (mean differences ± 2 SD) between paired samples were 0.4% ± 12.8%. There was no significant variation in bilateral SJO2 differences with time. Decreasing cerebral perfusion pressure (r = −0.559, P < 0.001) and arterial Pco2 (r = −0.342, P < 0.001) were associated with increasing bilateral SJO2 differences. Regarding AJDL, the maximum bilateral differences varied between 0.04 mmol/L and 1.52 mmol/L. The bias and limits of agreement were −0.01 ± 0.18 mmol/L. At best, 87% of ischemic events were disclosed by monitoring on the side of predominant lesion or, in diffuse injuries, on the side of the larger jugular foramen (computed tomographic [CT] approach). We conclude that in severe head injury, even calculated unilateral jugular venous monitoring has an unpredictable risk for misleading or missing data. Therefore, the reliability of unilateral jugular venous monitoring appears suspicious. For diagnosing ischemia the CT approach is recommended.
Metyrapone Reduces Rat Brain Damage and Seizures after Hypoxia—Ischemia: An Effect Independent of Modulation of Plasma Corticosterone Levels?
Authors: Krugers Harm J. , Kemper Richard H. A. , Korf Jakob , Horst Gert J. Ter , Knollema Siert
Published: November 2003

Hypoxia—ischemia is accompanied by abundant corticosterone secretion that could exacerbate brain damage after the insult. The authors demonstrate that the steroid synthesis inhibitor metyrapone (150 mg/kg subcutaneously) suppresses the hypoxia—ischemia-induced rise of plasma corticosterone levels (17.3 ± 3.6 μg/dL) when compared with corticosterone-treated animals (72.2 ± 4.8 μg/dL) immediately after hypoxia—ischemia. In parallel, metyrapone reduced brain damage ( P < 0.05). Moreover, none of the metyrapone-treated animals displayed seizures, whereas seven of eight corticosterone-treated animals had seizures after hypoxia—ischemia. Although corticosterone administration in metyrapone-treated animals elevated plasma corticosterone levels (39.0 ± 5.3 μg/dL), this did not result in a subsequent increase in brain damage and seizures when compared with metyrapone-treated animals. The authors conclude that metyrapone reduces brain damage and the incidence of seizures after hypoxia—ischemia but that this effect might partially be independent from its effect on modulating plasma corticosterone levels.