bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Acetylcholine Muscarinic Receptor Subtypes in Human Brain Microcirculation: Identification and Cellular Localization
Authors: Elhusseiny Ahmed , Cohen Zvi , Olivier André , Stanimirović Danica B. , Hamel Edith
Published: November 2003

Acetylcholine is an important regulator of local cerebral blood flow. There is, however, limited information available on the possible sites of action of this neurotransmitter on brain intraparenchymal microvessels. In this study, a combination of molecular and functional approaches was used to identify which of the five muscarinic acetylcholine receptors (mAChR) are present in human brain microvessels and their intimately associated astroglial cells. Microvessel and capillary fractions isolated from human cerebral cortex were found by reverse transcriptase-polymerase chain reaction to express m2, m3, and, occasionally, m1 and m5 receptor subtypes. To localize these receptors to a specific cellular compartment of the vessel wall, cultures of human brain microvascular endothelial and smooth muscle cells were used, together with cultured human brain astrocytes. Endothelial cells invariably expressed m2 and m5 receptors, and occasionally the m1 receptor; smooth muscle cells exhibited messages for all except the m4 mAChR subtypes, whereas messages for all five muscarinic receptors were identified in astrocytes. In all three cell types studied, acetylcholine induced a pirenzepine-sensitive increase (62% to 176%, P < 0.05 to 0.01) in inositol trisphosphate, suggesting functional coupling of m1, m3, or m5 mAChR to a phospholipase C signaling cascade. Similarly, coupling of m2 or m4 mAChR to adenylate cyclase inhibition in endothelial cells and astrocytes, but not in smooth muscle cells, was demonstrated by the ability of carbachol to significantly reduce (44% to 50%, P < 0.05 to 0.01) the forskolin-stimulated increase in cAMP levels. This effect was reversed by the mAChR antagonist AF-DX 384. The results indicate that microvessels are able to respond to neurally released acetylcholine and that mAChR, distributed in different vascular and astroglial compartments, could regulate cortical perfusion and, possibly, blood–brain barrier permeability, functions that could become jeopardized in neurodegenerative disorders such as Alzheimer's disease.
Measurement of Changes in Opioid Receptor Binding in Vivo During Trigeminal Neuralgic Pain Using [11C]Diprenorphine and Positron Emission Tomography
Authors: Jones Anthony K. P. , Kitchen Niel D. , Watabe Hiroshi , Cunningham Vincent J. , Jones Terry , Luthra Savinda K. , Thomas David G. T.
Published: November 2003

The binding of [11C]diprenorphine to µ, κ, and Δ subsites in cortical and subcortical structures was measured by positron emission tomography in vivo in six patients before and after surgical relief of trigeminal neuralgia pain. The volume of distribution of [11C]diprenorphine binding was significantly increased after thermocoagulation of the relevant trigeminal division in the following areas: prefrontal, insular, perigenual, mid-cingulate and inferior parietal cortices, basal ganglia, and thalamus bilaterally. In addition to the pain relief associated with the surgical procedure, there also was an improvement in anxiety and depression scores. In the context of other studies, these changes in binding most likely resulted from the change in the pain state. The results suggest an increased occupancy by endogenous opioid peptides during trigeminal pain but cannot exclude coexistent down-regulation of binding sites.
Quantitative Magnetic Resonance Imaging in Experimental Hypercapnia: Improvement in the Relation between Changes in Brain R2* and the Oxygen Saturation of Venous Blood after Correction for Changes in Cerebral Blood Volume
Authors: Lin Weili , Celik Azim , Paczynski Richard P. , Hsu Chung Y. , Powers William J.
Published: November 2003

Acute hypercapnia simultaneously induces increases in regional cerebral blood volume (rCBV) and the oxygen saturation of cerebral venous blood (Yv). Changes in both physiologic parameters may influence the changes in R2* (ΔR2*) that can be measured in the brain with gradient echo magnetic resonance imaging. The authors examined the effect of incorporating independent measurements of the change in rCBV (ΔrCBV) on the fidelity of the relation between ΔR2* and ΔYv in the setting of experimental hypercapnia. A two-dimensional T2*-weighted gradient echo sequence was used to measure ΔR2* in the brain parenchyma of anesthetized rats in response to hypercapnia with respect to the control state. In parallel, estimates of rCBV were obtained using a three-dimensional steady-state approach in conjunction with a paramagnetic contrast agent during both control and hypercapnic states so that a ΔrCBV could be calculated. Regional CBV values of 2.96 ± 0.82% and 5.74 ± 1.21% were obtained during the control and hypercapnic states, respectively, and linear relations between rCBV and CO2 tension in both arterial ( r = 0.80) and jugular venous ( r = 0.76) blood samples were obtained. When correlating ΔR2* directly with ΔYv, no clear relation was apparent, but a strong linear relation ( r = 0.76) was observed when correction for ΔrCBV was incorporated into the data analysis. These results are consistent with the current understanding of the mechanisms of blood oxygen level–dependent (BOLD) contrast and underscore the potential importance of taking into account ΔrCBV when quantitative estimates of ΔYv from the “BOLD effect” are intended.
Increased Sarcolemmal Permeability in the Cerebral Artery during Chronic Spasm: An Assessment Using DNA-Binding Dyes and Detection of Apoptosis
Authors: Kim Phyo , Yoshimoto Yuhei , Nakaguchi Hiroshi , Mori Takashi , Asai Akio , Sasaki Tomio , Kirino Takaaki , Nonomura Yoshiaki
Published: November 2003

Alteration of sarcolemmal permeability was evaluated in the cerebral artery after subarachnoid hemorrhage. Significance of membrane dysfunction in the pathogenesis of chronic spasm and contribution of apoptosis were investigated in a canine model. Permeability of the smooth muscle cell (SMC) membrane was assessed by double staining with a hydrophilic (ethidium bromide [EB]) and a lipophilic (Hoechst 33342) DNA-binding dye. Quantitative observations were made with a ultraviolet-fluorescence microscope and a ultraviolet-laser confocal microscope. Occurrence of apoptosis was studied using electrophoresis and TUNEL method. In the normal arteries, nuclei of SMC were stained with Hoechst 33342 but not with EB. In the spastic arteries, SMC in the inner layer of the tunica media were stained with EB. The incidence of EB-positive cells reached maximum on day 7 (45 ± 19%) and decreased in 2 to 4 weeks (13 ± 5.2% and 5.0 ± 2.1%, respectively), in parallel with amelioration of spasm. Electron and light microscopic observations revealed increased density of SMC cytoplasm with widening of the extracellular space. Necrosis was not evident. Apoptosis was not detected by the two methods. These results demonstrate that an augmentation in sarcolemmal permeability takes place during the course of chronic vasospasm and suggest its close correlation to pathogenesis.
Separate Neuronal and Glial Na+,K+-ATPase Isoforms Regulate Glucose Utilization in Response to Membrane Depolarization and Elevated Extracellular Potassium
Authors: Honegger Paul , Pardo Beatriz
Published: November 2003

The role of cell type-specific Na+,K+-ATPase isozymes in function-related glucose metabolism was studied using differentiated rat brain cell aggregate cultures. In mixed neuron-glia cultures, glucose utilization, determined by measuring the rate of radiolabeled 2-deoxyglucose accumulation, was markedly stimulated by the voltage-dependent sodium channel agonist veratridine (0.75 μmol/L), as well as by glutamate (100 μmol/L) and the ionotropic glutamate receptor agonist N-methyl-d-aspartate (NMDA) (10 μmol/L). Significant stimulation also was elicited by elevated extracellular potassium (12 mmol/L KCl), which was even more pronounced at 30 mmol/L KCl. In neuron-enriched cultures, a similar stimulation of glucose utilization was obtained with veratridine, specific ionotropic glutamate receptor agonists, and 30 mmol/L but not 12 mmol/L KCl. The effects of veratridine, glutamate, and NMDA were blocked by specific antagonists (tetrodotoxin, CNQX, or MK801, respectively). Low concentrations of ouabain (10−6 mol/L) prevented stimulation by the depolarizing agents but reduced only partially the response to 12 mmol/L KCl. Together with previous data showing cell type–specific expression of Na+,K+-ATPase subunit isoforms in these cultures, the current results support the view that distinct isoforms of Na+,K+-ATPase regulate glucose utilization in neurons in response to membrane depolarization, and in glial cells in response to elevated extracellular potassium.
Larger Anastomoses in Angiotensinogen-Knockout Mice Attenuate Early Metabolic Disturbances after Middle Cerebral Artery Occlusion
Authors: Maeda Keiichiro , Hata Ryuji , Bader Michael , Walther Thomas , Hossmann Konstantin-Alexander
Published: November 2003

Abnormalities in the homeostasis of the renin-angiotensin system have been implicated in the pathogenesis of vascular disorders, including stroke. The authors investigated whether angiotensinogen (AGN) knockout mice exhibit differences in brain susceptibility to focal ischemia, and whether such differences can be related to special features of the collateral circulation. Wild-type and AGN-knockout mice were submitted to permanent suture occlusion of the middle cerebral artery (MCA). The collateral vascular system was visualized by systemic latex infusion, and the ischemic lesions were identified by cresyl-violet staining. The core and penumbra of the evolving infarct were differentiated by bioluminescence and autoradiographic imaging of A TP and protein biosynthesis, respectively. In wild-type mice, mean arterial blood pressure was 95.0 ± 8.6 mm Hg, and the diameter of fully relaxed anastomotic vessels between the peripheral branches of the anterior and middle cerebral arteries 26.6 ± 4.0 μm In AGN knockouts, mean arterial blood pressure was significantly lower, 71.5 ± 8.5 mm Hg ( P <,01), and the anastomotic vessels were significantly larger, 29.4 ± 4.6 μm ( P < .01). One hour after MCA occlusion, AGN-knockout mice exhibited a smaller ischemic core (defined as the region of ATP depletion) but a larger penumbra (the area of disturbed protein synthesis with preserved ATP). At 24 hours after MCA occlusion, this difference disappeared, and histologically visible lesions were of similar size in both strains. The observations show that in AGN-knockout mice the more efficient collateral blood supply delays ischemic injury despite the lower blood pressure. Pharmacologic suppression of angiotensin formation may prolong the therapeutic window for treatment of infarcts.
Mice Deficient in Interleukin-1 Converting Enzyme are Resistant to Neonatal Hypoxic-Ischemic Brain Damage
Authors: Liu Xiao-Hong , Kwon Deborah , Schielke Gerald P. , Yang Guo-Yuan , Silverstein Faye S. , Barks John D. E.
Published: November 2003

Interleukin-1 (IL-1) converting enzyme (ICE) is a cysteine protease that cleaves inactive pro-IL-1β to active IL-1β. The pro-inflammatory cytokine IL-1β is implicated as a mediator of hypoxic-ischemic (HI) brain injury, both in experimental models and in humans. ICE is a member of a family of ICE-like proteases (caspases) that mediate apoptotic cell death in diverse tissues. The authors hypothesized that in neonatal mice with a homozygous deletion of ICE (ICE-KO) the severity of brain injury elicited by a focal cerebral HI insult would be reduced, relativefto wild-type mice. Paired litters of 9- to 10-day-old ICE-KO and wild-type mice underwent right carotid ligation, followed by 70 or 120 minutes of exposure to 10% O2, In this neonatal model of transient focal cerebral ischemia followed by reperfusion, the duration of hypoxia exposure determines the duration of cerebral ischemia and the severity of tissue damage. Outcome was evaluated 5 or 21 days after lesioning; severity of injury was quantified by morphometric estimation of bilateral cortical; striatal, and dorsal hippocampal volumes. In animals that underwent the moderate HI insult (70-minute hypoxia), damage was attenuated in ICE-KO mice, when evaluated at 5 or 21 days post-lesioning. In contrast, in mice that underwent the more severe HI insult (120-minute hypoxia), injury severity was the same in both groups. Reductions in intra-HI CBF, measured by laser Doppler flowmetry, and intra- and post-HI temperatures did not differ between groups. These results show that ICE activity contributes to the progression of neonatal HI brain injury in this model. Whether these deleterious effects are mediated by proinflammatory actions of IL-lβ and/or by pro-apoptotic mechanisms is an important question for future studies.
Interleukin-1 Receptor Antagonist Attenuates Regional Neuronal Cell Death and Cognitive Dysfunction after Experimental Brain Injury
Authors: Sanderson Kristin L. , Raghupathi Ramesh , Saatman Kathryn E. , Martin David , Miller Gerald , McIntosh Tracy K.
Published: November 2003

The effect of systemic administration of human recombinant interleukin-1 receptor antagonist (rhIL-1ra) on behavioral outcome and histopathologic damage after lateral fluid-percussion brain injury of moderate severity was evaluated. In study 1, brain-injured Sprague Dawley rats received timed subcutaneous injections beginning 15 minutes after injury of either 100 mg/kg rhIL-1ra (high dose, total dose = 1900 mg/kg), 10 mg/kg rhIL-1ra (low dose, total dose = 190 mg/kg), or vehicle over 7 days. No effect of low-dose rhIL-1ra was observed in study 1. High-dose rhIL-1ra significantly attenuated posttraumatic neuronal loss in the injured hippocampal CA3 region ( P < 0.05), dentate hilus ( P < 0.05), and cortex ( P < 0.05) but impaired recovery of motor function at 7 days after trauma ( P < 0.05). In study 2, rats were pretrained to learn a visuospatial task in a Morris water maze, subjected to fluid-percussion brain injury or sham treatment, and randomly assigned to receive multiple subcutaneous injections at timed intervals of 100 mg/kg rhIL-1ra (total dose = 900 mg/kg) or vehicle over 42 hours, followed by continuous infusion of a lower concentration of rhIL-1ra (20 mg/kg/day, total dose = 100 mg/kg), or vehicle for 5 days using subcutaneously implanted osmotic minipumps. Postinjury administration of rhIL-1ra significantly attenuated cognitive deficits compared with vehicle-treated animals at 42 hours ( P < 0.05) but did not affect motor function at 48 hours, 1 week, and 2 weeks. These results suggest that inhibitors of cytokine pathways may be therapeutically useful for the treatment of brain trauma.
A Comparison of 11C-Labeled l-DOPA and l-Fluorodopa as Positron Emission Tomography Tracers for the Presynaptic Dopaminergic System
Authors: Torstenson Richard , Tedroff Joakim , Hartvig Per , Fasth Karl-Johan , Lågström Bengt
Published: November 2003

11C-labeled 3,4-Dihydroxy-phenyl-l-alanine (L-DOPA) and l-fluorodopa were used as tracers for the functional state of the presynaptic dopamine system in anesthetized monkeys with positron emission tomography, The radiotracer disposition in brain tissue and plasma were studied and effects induced by pharmacologic challenges were evaluated, 6 R-l-erythro-5,6,7,8-tetrahydrobiopterin (6 R-BH4) increased the striatal influx rate constant, e.g., striatal Ki for l-[β-11C]DOPA, but it induced no effect on the Ki-value using l-[β-11C]-6-fluorodopa. Studies of radiolabeled tracer and metabolites in plasma showed substantial differences between the two tracers. At baseline conditions, 60% unchanged l-[β-11C]DOPA was detected in plasma 50 minutes after tracer injection and the 3- O-methylated fraction accounted for 25% of total radioactivity. For l-[β-11C]-6-fluorodopa, the relation was inverse; about 25% unchanged tracer and 60% 3- O-methyl metabolite were present in plasma after 50 minutes. A site-specific 11C-labeling in the carboxylic position in the molecules revealed a significant specific retention of radioactivity in striatum with l-[carboxy-11C]-6-fluorodopa but not with l-[carboxy-11C]DOPA. The 3- O-methyl metabolite of l-DOPA is known to pass the blood-brain barrier and may interfere with the calculation of the Ki value using a brain reference region. Thus, extensive 3- O-methylation in circulation of the fluorinated analog could obscure the detectability of potential functional change in striatal Ki of the tracer when using a reference tissue model for calculation.
Respiratory Chain Inhibition Induces Tolerance to Focal Cerebral Ischemia
Authors: Wiegand F. , Liao W. , Busch C. , Castell S. , Knapp F. , Lindauer U. , Megow D. , Meisel A. , Redetzky A. , Ruscher K. , Trendelenburg G. , Victorov I. , Riepe M. , Diener H. C. , Dirnagl V.
Published: November 2003

The authors show that the inhibitor of the succinate dehydrogenase, 3-nitroproprionic acid (3-NPA), which in high doses and with chronic administration is a neurotoxin, can induce profound tolerance to focal cerebral ischemia in the rat when administered in a single dose (20 mg/kg) 3 days before ischemia. Infarcts were approximately 70% and 35% smaller in the 3-NPA preconditioned groups of permanent and transient focal cerebral ischemia, respectively. This regimen of 3-NPA preconditioning neither induced necrosis, apoptosis, or any other histologically detectable damage to the brain, nor did it affect behavior of the animals. 3-NPA led to an immediate (1-hour) and long-lasting (3-day) decrease in succinate dehydrogenase activity (30% reduction) throughout the brain, whereas only a short metabolic impairment occurred (ATP decrease of 35% within 30 minutes, recovery within 2 hours). The authors found that 3-NPA induces a burst of reactive oxygen species and the free radical scavenger dimethylthiourea, when administered shortly before the 3-NPA stimulus, completely blocked preconditioning. Inhibition of protein synthesis with cycloheximide given at the time of 3-NPA administration completely inhibited preconditioning. The authors were unsuccessful in showing upregulation of mRNA for the manganese superoxide dismutase, and did not detect increased activities of the copper-zinc and manganese superoxide dismutases, prototypical oxygen free radicals scavenging enzymes, after 3-NPA preconditioning. The authors conclude that it is possible to pharmacologically precondition the brain against focal cerebral ischemia, a strategy that may in principal have clinical relevance. The data show the relevance of protein synthesis for tolerance, and suggests that oxygen free radicals may be critical signals in preconditioning.
Functional Calcitonin Gene-Related Peptide Type 1 and Adrenomedullin Receptors in Human Trigeminal Ganglia, Brain Vessels, and Cerebromicrovascular or Astroglial Cells in Culture
Authors: Moreno Maria Jesús , Cohen Zvi , Stanimirovic Danica B. , Hamel Edith
Published: November 2003

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) are potent dilators of human brain arteries, and they have been implicated in the neurogenic inflammation underlying migraine headache and in the evolution of stroke, respectively. However, little is known about the presynaptic and postsynaptic distribution of their respective receptors in the human cerebrovascular bed and trigeminovascular system. In the current study, the expression of mRNA for ADM and the two cloned human CGRP1 receptors (identified here as A-CGRP1 receptors [ Aiyar et al., 1996 ] and K-CGRP1 receptors) [ Kapas and Clark, 1995 ] were evaluated in human brain vessels and trigeminal ganglia. Further, the ability of CGRP and ADM to activate adenylate cyclase in cerebromicrovascular and astroglial cell cultures was determined, and the receptors involved were characterized pharmacologically. Isolated human pial vessels, intracortical microvessels, and capillaries, as well as cultures of brain endothelial (EC), smooth muscle (SMC), and astroglial (AST) cells, all expressed mRNA for the two cloned CGRP1 receptors; however, message for the K-CGRP1 receptor was barely detectable in microvascular tissues and cells. In contrast, only isolated capillaries and cultured AST exhibited message for the ADM receptor. In human trigeminal ganglia, mRNA for ADM and the two CGRP1 receptors was systematically present The CGRP dose-dependently increased (up to 50-fold) cAMP formation in cell cultures, an effect significantly blocked by 0.1 to 10 μmol/L of the CGRP1 receptor antagonist CGRP8–37. The ADM receptor agonist, ADM13–52 (1 μmol/L), similarly increased cAMP production in all cell types, and this response was virtually abolished by 1 μmol/L CGRP8–37. Low concentrations (1 to 10 μmol/L) of the ADM receptor antagonist ADM22–52 blocked the ADM13–52-induced cAMP formation in AST (26% at 10 μmol/L, P < 0.05), whereas they potentiated this response in brain EC and SMC (40% and 100%, P < 0.001, respectively). Even at a higher dose (50 μmol/L), ADM22–52 inhibited the ADM13–52 effect in vascular cells (45%) much less effectively than in AST (95%). These results indicate that both CGRP and ADM can affect human brain vessels through a CGRP1 receptor, and they further suggest the presence of functional ADM receptors in human astroglial cells.
Mutually Protective Actions of Kainic Acid Epileptic Preconditioning and Sublethal Global Ischemia on Hippocampal Neuronal Death: Involvement of Adenosine A1 Receptors and KATP Channels
Authors: Plamondon Hélène , Blondeau Nicolas , Heurteaux Catherine , Lazdunski Michel
Published: November 2003

Preconditioning with sublethal ischemia attenuates the detrimental effects of subsequent prolonged ischemic insults. This research elucidates potential in vivo cross-tolerance between different neuronal death-generating treatments such as kainate administration, which induces seizures and global ischemia. This study also investigates the effects of a mild epileptic insult on neuronal death in rat hippocampus after a subsequent, lethal epileptic stress using kainic acid (KA) as a model of epilepsy. Three preconditioning groups were as follows: group 1 was injected with 5 mg/kg KA before a 6-minute global ischemia; group 2 received a 3-minute global ischemia before 7.5 mg/kg KA; and group 3 was injected with a 5-mg/kg dose of KA before a 7.5-mg/kg KA injection. The interval between treatments was 3 days. Neuronal degeneration, revealed by the silver impregnation method and analysis of cresyl violet staining, was markedly reduced in rats preconditioned with a sublethal ischemia or a 5-mg/kg KA treatment. Labeling with terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′triphosphate-biotin nick-end labeling and DNA laddering confirmed the component of DNA fragmentation in the death of ischemic and epileptic neurons and its reduction in all preconditioned animals. The current study supports the existence of bidirectional cross-tolerance between KA excitotoxicity and global ischemia and suggests the involvement of adenosine A1 receptors and sulfonylurea- and ATP-sensitive K+ channels in this protective phenomenon.
Relationship between Inward Rectifier Potassium Current Impairment and Brain Injury after Cerebral Ischemia/Reperfusion
Authors: Bastide Michèle , Bordet Régis , Pu Qian , Robin Emmanuel , Puisieux François , Dupuis Bernard
Published: November 2003

Functional alterations of barium-sensitive potassium inward rectifier (Kir) current, which is involved in the vasodilation of middle cerebral arteries (MCA) in rat brain, have been described during brain ischemiaireperfusion (I/R). The authors investigate the effects of I/R on Kir current recorded in isolated myocytes from MCA of control rats and from contralateral and ipsilateral MCA of ischemic rats by the whole-cell patch-clamp technique, and the relationship between its alteration and The severity of brain injury. The vascular smooth muscle cells exhibited similar morphologic features in all conditions, and the Kir was present in the three groups of myocytes, exhibiting a characteristic inward rectification and a normal external potassium dependence. The Kir density was significantly reduced in cell of MCA ipsilateral to occlusion with a maximum at −135 mV, whereas there was no difference between control and contralateral cells. This alteration in Kir density in occluded MCA was significantly correlated with severity of brain injury and brain edema. These results suggest that the alteration of Kir density in MCA myocytes after I/R and the consecutive impaired dilation of MCA may contribute to aggravation of the brain injury.
The Relationship between the Apparent Diffusion Coefficient Measured by Magnetic Resonance Imaging, Anoxic Depolarization, and Glutamate Efflux during Experimental Cerebral Ischemia
Authors: Harris Neil G. , Zilkha TElias , Houseman John , Symms Mark R. , Obrenovitch Tihomir P. , Williams Stephen R.
Published: November 2003

A reduction in the apparent diffusion coefficient (ADC) of water measured by magnetic resonance imaging (MRI) has been shown to occur early after cerebrovascular occlusion. This change may be a useful indicator of brain tissue adversely affected by inadequate blood supply. The objective of this study was to test the hypothesis that loss of membrane ion homeostasis and depolarization can occur simultaneously with the drop in ADC. Also investigated was whether elevation of extracellular glutamate ([GLU]e) would occur before ADC changes. High-speed MRI of the trace of the diffusion tensor (15-second time resolution) was combined with simultaneous recording of the extracellular direct current (DC) potential and on-line [GLU]e from the striatum of the anesthetized rat. After a control period, data were acquired during remote middle cerebral artery occlusion for 60 minutes, followed by 30 minutes of reperfusion, and cardiac arrest-induced global ischemia. After either focal or global ischemia, the ADC was reduced by 10 to 25% before anoxic depolarization occurred. After either insult, the time for half the maximum change in ADC was significantly shorter than the corresponding DC potential parameter ( P < 0.05). The [GLU]e remained at low levels during the entire period of varying ADC and DC potential and did not peak until much later after either ischemic insult. This study demonstrates that ADC changes can occur before membrane depolarization and that high [GLU]e has no involvement in the early rapid ADC decrease.
Microglia and Macrophages Are the Major Source of Tumor Necrosis Factor in Permanent Middle Cerebral Artery Occlusion in Mice
Authors: Gregersen Rikke , Lambertsen Kate , Finsen Bente
Published: November 2003

The proinflammatory cytokine tumor necrosis factor (TNF) is known to be expressed in brain ischemia; however, its cellular and temporal appearance is not fully settled. In this study, nonradioactive in situ hybridization for murine TNF mRNA was performed on brain sections from adult C57×129 mice at 6 hours, 12 hours, 24 hours, 2 days, 5 days, or 10 days (six to eight mice per group) after induction of permanent focal cerebral ischemia. Cortical infarct volumes were estimated, and TNF mRNA-expressing cells were counted within the infarct and infarct border using Cast-Grid analysis. At 12 hours, a peak of 19.2 ± 5.1 TNF mRNA-expressing cells/mm2 was counted, contrasting two to three times lower values at 6 and 24 hours (6.4 ± 4.6 and 9.2 ± 3.4 cells/mm2, respectively) and <2 cells/ mm2 at 48 hours and later stages. The TNF mRNA-expressing cells were distributed along the entire rostrocaudal axis of the cortical infarcts and occasionally within the caudate putamen. At all time points, TNF mRNA colocalized with Mac-1-positive microglia/macrophages but not with Ly-6G (Gr-1)-positive polymorphonuclear leukocytes. Similarly, combined in situ hybridization for TNF mRNA and immunohistochemistry for glial fibrillary acidic protein at 12 and 24 hours revealed no TNF mRNA-expressing astrocytes at these time points. Translation of TNF mRNA into bioactive protein was demonstrated in the neocortex of C57Bl/6 mice subjected to permanent middle cerebral artery occlusion. In summary, this study points to a time-restricted microglial/macrophage production of TNF in focal cerebral ischemia in mice.
Carrier-Mediated Delivery of Metabotropic Glutamate Receptor Ligands to the Central Nervous System: Structural Tolerance and Potential of the l-system Amino Acid Transporter at the Blood-Brain Barrier
Authors: Reichel Andreas , Begley David J. , Abbott N. Joan
Published: November 2003

The brain endothelial large neutral amino acid carrier (l-system) is well suited for facilitated drug transport to the brain because of its high transport capacity and relatively broad structural substrate tolerance. The authors have examined the potential of this transporter for central nervous system (CNS) delivery of a new family of compounds derived from the large neutral amino acid phenylglycine. These compounds are highly selective for specific isoforms of metabotropic glutamate receptors (mGluRs) but will only become effective therapeutics for CNS diseases such as ischemic disorders, stroke, and epilepsy if they can effectively cross the blood-brain barrier. Using the immortalized rat brain endothelial cell line RBE4 as in vitro blood-brain barrier model, the authors have studied the interaction of phenylglycine and selected derivatives with the l-system-mediated transport of l-[3H]-histidine. The transport of l-histidine was characteristic of the l-system in vivo with the following kinetic parameters: Km 135 ± 18 μmol/L, Vmax 15.3 ± 1.13 nmol/min/mg protein, and KD 2.38 ± 0.84 μL/min/mg protein. The affinities of the l-system for phenylglycine and the derivatives investigated increased in the order S-4-carboxy-phenylglycine (Ki = 16 mmol/L) < R-phenylglycine (2.2 mmol/L) < S-3-hydroxy-phenylglycine (48 μmol/L) < S-phenylglycine (34 μmol/L), suggesting that a negative charge at the side chain or R-configuration is detrimental for carrier recognition, whereas neutral side chain substituents are well tolerated. The authors have further shown (1) that the mode of interaction with the l-system of S-phenylglycine and S-3-hydroxy-phenylglycine is competitive, and (2) that the transporter carries these two agents into the cell as shown by high-performance liquid chromatography (HPLC) analysis of the RBE4 cell contents. The study provides the first evidence for the potential of S-phenylglycine derivatives for carrier-mediated delivery to the CNS and outlines the substrate specificity of the l-system at the blood-brain barrier for this class of mGluR ligands. As the affinities of S-phenylglycine and S-3-hydroxy-phenylglycine for the l-system carrier are even higher than those of some natural substrates, these agents should efficiently enter CNS via this route. Possible strategies for a synergistic optimization of phenylglycine-derived therapeutics with respect to desired activity at the CNS target combined with carrier-mediated delivery to overcome the blood-brain barrier are discussed.
Mapping the Visual Recognition Memory Network with PET in the Behaving Baboon
Authors: Blaizot Xavier , Landeau Brigitte , Baron Jean-Claude , Chavoix Chantal
Published: November 2003

By means of a novel 18F-fluoro-deoxyglucose PET method designed for cognitive activation imaging in the baboon, the large-scale neural network involved in visual recognition memory in the nonhuman primate was mapped for the first time. In this method, the tracer is injected in the awake, unanesthetized, and unrestrained baboon performing the memory task, and brain imaging is performed later under light anesthesia. Brain maps obtained during a computerized trial-unique delayed matching-to-sample task (lists of meaningless geometrical patterns and delay > 9 seconds) were statistically compared pixel-by-pixel to maps obtained during a specially designed visuomotor control task. When displayed onto the baboon's own anatomic magnetic resonance images, foci of significant activation were distributed along the ventral occipitotemporal pathway, the inferomedial temporal lobe (especially the perirhinal cortex and posterior hippocampal region), and the orbitofrontal cortex, consistent with lesion, single-unit, and autoradiographic studies in monkeys, as well as with activation studies in healthy humans. Additional activated regions included the nucleus basalis of Meynert, the globus pallidus and the putamen. The results also document an unexpected left-sided advantage, suggesting hemispheric functional specialization for recognition of figural material in nonhuman primates.
The Suitability of [11C]-α-Methyl-L-tryptophan as a Tracer for Serotonin Synthesis: Studies With Dual Administration of [11C] and [14C] Labeled Tracer
Authors: Shoaf Susan E. , Carson Richard E. , Hommer Daniel , Williams Wendol A. , Higley J. Dee , Schmall Bernard , Herscovitch Peter , Eckelman William C. , Linnoila Markku
Published: November 2003

The tracer [11C]-α-methyl-L-tryptophan (αMTP) has been used to measure brain serotonin synthesis rates with positron emission tomography (PET). To address questions about the accuracy of the kinetic model, [14C]αMTP was used to directly measure conversion to [14C]-α-methyl-serotonin (αM5HT) in monkeys that had been previously studied with PET and [11C]αMTP. Four male, fasted, isoflurane-anesthetized rhesus monkeys were studied with [11C]αMTP and PET. Immediately after the initial 3-hour scan, a second dose of [11C]αMTP was coinjected with 1 mCi of [14C]αMTP, and additional PET data were collected. Approximately 90 minutes after the second αMTP administration, the animals were killed with an overdose of phenobarbital, and brain samples from 21 regions were taken and analyzed by HPLC. Minimal conversion of αMTP to αM5HT occurred; HPLC analysis of 14C radioactivity showed that greater than 96% of the total counts were in fractions corresponding to the αMTP peak. Brain concentrations of serotonin, tryptophan, 5-hydroxyindole-3-acetic acid, and αMTP also were determined fluorometrically using external quantification. Patlak plots generated from PET images acquired over 3 hours showed no time period of linear increase, and final slopes were not significantly different from zero, consistent with the finding of minimal conversion to [14C]αM5HT. These data indicate that in the 3-hour period after injection, [11C]αMTP is acting predominantly as a tracer of tryptophan uptake, not serotonin synthesis.
Determinants of Cerebral Fractional Oxygen Extraction Using Near Infrared Spectroscopy in Preterm Neonates
Authors: Wardle Stephen P. , Yoxall C. William , Weindling A. Michael
Published: November 2003

Cerebral fractional oxygen extraction (FOE) represents the balance between cerebral oxygen delivery and consumption. This study aimed to determine cerebral FOE in preterm infants during hypotension, during moderate anemia, and with changes in the PaCO2. Three groups of neonates were studied: stable control neonates (n = 43), anemic neonates (n = 46), and hypotensive neonates (n = 19). Cerebral FOE was calculated from the arterial oxygen saturation measured by pulse oximetry, and cerebral venous oxygen saturation was measured using near infrared spectroscopy with partial jugular venous occlusion. Mean ± SD cerebral FOE was similar in control (0.292 ± 0.06), anemic (0.310 ± 0.08; P = 0.26), and hypotensive (0.278 ± 0.06; P = 0.41) neonates. After anemic neonates were transfused, mean ± SD cerebral FOE decreased to 0.274 ± 0.05 ( P = 0.02). There was a weak negative correlation with the hemoglobin concentration (n = 89, r = −0.24, P = 0.04) but not with the hemoglobin F fraction (n = 56, r = 0.24, P = 0.09). In the hypotensive neonates, there was no relationship between cerebral FOE and blood pressure (n = 19, r = 0.34, P = 0.15). There was a significant negative correlation between cerebral FOE and PaCO2 within individuals (n = 14, r = −0.63, P = 0.01), but there was no relationship between individuals (n = 14, r = 0, P = 1). Cerebral FOE was not significantly altered in neonates with either mild anemia or hypotension. There were, however, changes in cerebral FOE when physiological changes occurred over a relatively short period; Cerebral FOE decreased after blood transfusion and increased with decreasing PaCO2. As no change in cerebral FOE was seen during hypotension, it was speculated that cerebral oxygen delivery may have been maintained by cerebral blood flow autoregulation.
Oxygen Dependency of Cerebral Oxidative Phosphorylation in Newborn Piglets
Authors: Springett Roger , Wylezinska Marzena , Cady Ernest B. , Cope Mark , Delpy David T.
Published: November 2003

Changes in hemoglobin oxygenation and oxidation state of the CuA centre of cytochrome oxidase were measured with full spectral near infrared spectroscopy simultaneously with phosphorus metabolites using nuclear magnetic resonance 31P spectroscopy at high time resolution (10 seconds) during transient anoxia (FiO2 = 0.0 for 105 seconds) in the newborn piglet brain. During the onset of anoxia, there was no change in either phosphocreatine (PCr) concentration or the oxidation state of the CuA centre of cytochrome oxidase until there was a substantial fall in cerebral hemoglobin oxygenation, at which point the CuA centre reduced simultaneously with the decline in PCr. At a later time during the anoxia, intracellular pH decreased rapidly, consistent with a fall in cerebral metabolic rate for O2 and reduced flux through the tricarboxylic acid cycle. The simultaneous reduction of CuA and decline in PCr can be explained in terms of the effects of the falling mitochondrial electrochemical potential. From these observations, it is concluded that, at normoxia, oxidative phosphorylation and the oxidation state of the components of the electron transport chain are independent of cerebral oxygenation and that the reduction in the CuA signal occurs when oxygen tension limits the capacity of oxidative phosphorylation to maintain the phosphorylation potential.
Reduction in Water and Metabolite Apparent Diffusion Coefficients During Energy Failure Involves Cation-Dependent Mechanisms. A Proton Nuclear Magnetic Resonance Study of Rat Cortical Brain Slices
Authors: Hakumäki Juhana M. , Pirttilä Tiina R. M. , Kauppinen Risto A.
Published: November 2003

Proton (1H) nuclear magnetic resonance (NMR) diffusion spectroscopy was used to assess apparent diffusion coefficients (ADCs) in rat brain slices. Aglycemic hypoxia caused reductions in the ADC of N-acetylaspartate (NAA) (0.15 to 0.09 × 10−3 mm2/s) and “slow” diffusion coefficient ( D2) of tissue water (0.51 to 0.37 × 10−3 mm2/s), together with a 32 ± 11% increase in tissue water volume, attributable to tissue swelling. The ADC and D2 reductions were diminished, however, by removing external Ca2+, and under 10 mmol/L Mg2+, normoxic diffusion coefficients persisted until 40 minutes of hypoxia. The data suggest that the shift of water into the intracellular space alone cannot satisfactorily explain the reduced cerebral diffusion upon energy failure and that external Mg2+ and Ca2+ play crucial modulatory roles.
Noninvasive Functional Imaging of Human Brain Using Light
Authors: Benaron David A. , Hintz Susan R. , Villringer Arno , Boas David , Kleinschmidt Andreas , Frahm Jens , Hirth Christina , Obrig Hellmuth , van Houten John C. , Kermit Eben L. , Cheong Wai-Fung , Stevenson David K.
Published: November 2003

Analysis of photon transit time for low-power light passing into the head, and through both skull and brain, of human subjects allowed for tomographic imaging of cerebral hemoglobin oxygenation based on photon diffusion theory. In healthy adults, imaging of changes in hemoglobin saturation during hand movement revealed focal, contralateral increases in motor cortex oxygenation with spatial agreement to activation maps determined by functional magnetic resonance imaging; in ill neonates, imaging of hemoglobin saturation revealed focal regions of low oxygenation after acute stroke, with spatial overlap to injury location determined by computed tomography scan. Because such slow optical changes occur over seconds and co-localize with magnetic resonance imaging vascular signals whereas fast activation-related optical changes occur over milliseconds and co-localize with EEG electrical signals, optical methods offer a single modality for exploring the spatio-temporal relationship between electrical and vascular responses in the brain in vivo, as well as for mapping cortical activation and oxygenation at the bedside in real-time for clinical monitoring.
The Window of Opportunity for Neuronal Rescue with Insulin-Like Growth Factor-1 after Hypoxia—Ischemia in Rats is Critically Modulated by Cerebral Temperature during Recovery
Authors: Guan Jian , Gunn Alistair J. , Sirimanne Ernest S. , Tuffin Julie , Gunning Mark I. , Clark Ross , Gluckman Peter D.
Published: November 2003

Insulin-like growth factor (IGF-1) is induced in damaged brain tissue after hypoxia—ischemia, and exogenous administration of IGF-1 shortly after injury has been shown to be neuroprotective. However, it is unknown whether treatment with IGF-1 delayed by more than a few hours after injury may be protective. Hypothermia after brain injury has been reported to delay the development of ischemic neuronal death. The authors therefore hypothesize that a reduction in the environmental temperature during recovery from hypoxia—ischemia could prolong the window of opportunity for IGF-1 treatment. Unilateral brain damage was induced in adult rats using a modified Levine model of right carotid artery ligation followed by brief hypoxia (6% O2 for 10 minutes). The rats were maintained in either a warm (31°C) or cool (23°C) environment for the first 2 hours after hypoxia. All rats were subsequently transferred to the 23°C environment until the end of the experiment. A single dose of IGF-1 (50 μg) or its vehicle was given intracerebroventricularly at either 2 or 6 hours after hypoxia. Histologic outcome in the lateral cortex was quantified 5 days after hypoxia. Finally, cortical temperature was recorded from 1 hour before and 2 hours after hypoxia in separate groups of rats exposed to the “warm” and “cool” protocols. In rats exposed to the warm recovery environment, IGF-1 reduced cortical damage ( P < 0.05) when given 2 hours but not 6 hours after insult. In contrast, with early recovery in the cool environment, a significant protective effect of IGF-1 in the lateral cortex ( P < 0.05) was found with administration 6 hours after insult. In conclusion, a reduction in cerebral temperature during the early recovery phase after severe hypoxia—ischemia did not significantly reduce the severity of injury after 5 days' recovery; however, it markedly shifted and extended the window of opportunity for delayed treatment with IGF-1.
Hypothermia during Reperfusion after Asphyxial Cardiac Arrest Improves Functional Recovery and Selectively Alters Stress-Induced Protein Expression
Authors: Hicks Shawn D. , DeFranco Donald B. , Callaway Clifton W.
Published: November 2003

This study examined whether prolonged hypothermia induced 1 hour after resuscitation from asphyxial cardiac arrest would improve neurologic outcome and alter levels of stress-related proteins in rats. Rats were resuscitated from 8 minutes of asphyxia resulting in cardiac arrest. Brain temperature was regulated after resuscitation in three groups: normothermia (36.8°C × 24 hours), immediate hypothermia (33°C × 24 hours, beginning immediately after resuscitation), and delayed hypothermia (33°C × 24 hours, beginning 60 minutes after resuscitation). Mortality and neurobehavioral deficits were improved in immediate and delayed hypothermia rats relative to normothermia rats. Furthermore, both immediate and delayed hypothermia improved neuronal survival in the CA1 region of the hippocampus assessed at 14 days. In normothermia rats, the 70-kDa heat shock protein (Hsp70) and 40-kDa heat shock protein (Hsp40) were increased within 12 hours after resuscitation in the hippocampus. Delayed hypothermia attenuated the increase in Hsp70 levels in the hippocampus but did not affect Hsp70 induction in the cerebellum. Hippocampal expression of Hsp40 was not affected by hypothermia. These data indicate that prolonged hypothermia during later reperfusion improves neurologic outcome after experimental global ischemia and is associated with selective changes in the pattern of stress-induced protein expression.
Extracellular–Intracellular Distribution of Glucose and Lactate in the Rat Brain Assessed Noninvasively by Diffusion-Weighted 1H Nuclear Magnetic Resonance Spectroscopy In Vivo
Authors: Pfeuffer Josef , Tkáč Ivan , Gruetter Rolf
Published: November 2003

To determine the distribution of cerebral glucose and lactate between the intracellular and the extracellular space of the rat brain in vivo, the diffusion characteristic of glucose and lactate was compared with that of metabolites known to be mainly intracellular ( N-acetylaspartate, choline, creatine, glutamate, myo-inositol, and taurine) using a pulsed-field-gradient 1H nuclear magnetic resonance technique. The detection of a glucose signal at large diffusion weighting provided direct experimental evidence of intracellular glucose in the rat brain. At large diffusion weighting, the apparent diffusion coefficient (ADC) of glucose and lactate was similar to that of the intracellular metabolites such as N-acetylaspartate, creatine, and glutamate. At small diffusion weighting, the ADC of glucose and lactate was increased, which was explained by a decreased relative contribution of intracellular glucose to the total signal. The calculated extracellular volume fraction of glucose (0.19 ± 0.05) and lactate (0.17 ± 0.06) was consistent with a substantial fraction of glucose and lactate signals being intracellular. The findings were direct in vivo evidence that the largest concentration gradient of glucose is at the blood-brain barrier and that glucose is evenly distributed in the brain in vivo between the intracellular and extracellular space.
Modulation by Nitric Oxide of Cerebral Neutrophil Accumulation after Transient Focal Ischemia in Rats
Authors: Batteur-Parmentier Sophie , Margaill Isabelle , Plotkine Michel
Published: November 2003

A beneficial role of nitric oxide (NO) after cerebral ischemia has been previously attributed to its vascular effects. Recent data indicate a regulatory role for NO in initial leukocyte-endothelial interactions in the cerebral microcirculation under basal and ischemic conditions. In this study, the authors tested the hypothesis that endogenous NO production during and/or after transient focal cerebral ischemia can also be neuroprotective by limiting the process of neutrophil infiltration and its deleterious consequences. Male Sprague-Dawley rats were subjected to 2 hours occlusion of the left middle cerebral artery and the left common carotid artery. The effect of NG-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg, intraperitoneally), an NO synthase inhibitor, was examined at 48 hours after ischemia on both infarct size and myeloperoxidase activity, an index of neutrophil infiltration. L-NAME given 5 minutes after the onset of ischemia increased the cortical infarct volume by 34% and increased cortical myeloperoxidase activity by 60%, whereas administration of L-NAME at 1, 7, and 22 hours of reperfusion had no effect. Such exacerbations of infarction and myeloperoxidase activity produced when L-NAME was given 5 minutes after the onset of ischemia were not observed in rats rendered neutropenic by vinblastine. These results suggest that after transient focal ischemia, early NO production exerts a neuroprotective effect by modulating neutrophil infiltration.