bluuebunny/crossref_metadata_2025 huggingface dataset:
Mild Hypothermia Attenuates Cytochrome C Release but Does Not Alter Bcl-2 Expression or Caspase Activation after Experimental Stroke
Authors: Yenari Midori A. , Iwayama Satoshi , Cheng Danye , Sun Guo Hua , Fujimura Miki , Morita-Fujimura Yuiko , Chan Pak H. , Steinberg Gary K.
Published: November 2003

Mild hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not well known. The authors previously found that hypothermia reduces the density of apoptotic cells, but it is not certain whether temperature alters associated biochemical events. Mitochondrial release of cytochrome c has recently been shown to be a key trigger in caspase activation and apoptosis via the intrinsic pathway. Using a model of transient focal cerebral ischemia, the authors determined whether mild hypothermia altered expression of Bcl-2 family proteins, mitochondrial release of cytochrome c, and caspase activation. Mild hypothermia significantly decreased the amount of cytochrome c release 5 hours after the onset of ischemia, but mitochondrial translocation of Bax was not observed until 24 hours. Mild hypothermia did not alter Bcl-2 and Bax expression, and caspase activation was not observed. The present study provides the first evidence that intraischemic mild hypothermia attenuates the release of cytochrome c in the brain, but does not appear to affect other biochemical aspects of the intrinsic apoptotic pathway. They conclude that necrotic processes may have been interrupted to prevent cytochrome c release, and that the ameliorative effect of mild hypothermia may be a result of maintaining mitochondrial integrity. Furthermore, the authors show it is unlikely that mild hypothermia alters the intrinsic apoptotic pathway.
Antioxidant Tempol Enhances Hypothermic Cerebral Preservation during Prolonged Cardiac Arrest in Dogs
Authors: Behringer Wilhelm , Safar Peter , Kentner Rainer , Wu Xianren , Kagan Valerian E. , Radovsky Ann , Clark Robert S. B. , Kochanek Patrick M. , Subramanian Murugan , Tyurin Vladimir A. , Tyurina Yulia Y. , Tisherman Samuel A.
Published: November 2003

The authors are systematically exploring pharmacologic preservation for temporarily unresuscitable exsanguination cardiac arrest in dogs. They hypothesized that the antioxidant Tempol improves cerebral outcome when added to aortic saline flush at the start of cardiac arrest. In study A, no drug (n = 8), Tempol 150 mg/kg (n = 4), or Tempol 300 mg/kg (n = 4) was added to 25 mL/kg saline flush at 24°C (achieving mild cerebral hypothermia) at the start of 20-minute cardiac arrest. In study B, no drug (n = 8) or Tempol 300 mg/kg (n = 7) was added to 50 mL/kg saline flush at 2°C (achieving moderate cerebral hypothermia) at the start of 40-minute cardiac arrest. Cardiac arrest was reversed with cardiopulmonary bypass. Mild hypothermia lasted for 12 hours, controlled ventilation was sustained to 24 hours, and intensive care was provided for up to 72 hours. In study A, overall performance category 1 or 2 (good outcome) was achieved in all eight dogs treated with Tempol compared with three of eight dogs in the control group ( P = 0.03). In study B, good outcome was achieved in all seven dogs treated with Tempol versus only two of 8 dogs in the control group ( P = 0.007). In both studies, neurologic deficit scores were significantly better in the Tempol group, but not total histologic damage scores. At 72 hours, electron paramagnetic resonance spectroscopy of Tempol revealed direct evidence for its presence in the brain. Single- and double-strand DNA damage, nitrotyrosine immunostaining, total antioxidant reserve, and ascorbate acid levels were similar between groups, and thiol levels were decreased after Tempol in study B. The authors conclude that when added to aortic saline flush at the start of prolonged cardiac arrest, the antioxidant Tempol can enhance mild or moderate hypothermic cerebral preservation in terms of improved functional outcome. The mechanisms involved in this beneficial effect need further clarification.
Molecular Pathways of Protein Synthesis Inhibition during Brain Reperfusion: Implications for Neuronal Survival or Death
Authors: DeGracia Donald J. , Kumar Rita , Owen Cheri R. , Krause Gary S. , White Blaine C.
Published: November 2003

Protein synthesis inhibition occurs in neurons immediately on reperfusion after ischemia and involves at least alterations in eukaryotic initiation factors 2 (eIF2) and 4 (eIF4). Phosphorylation of the α subunit of eIF2 [eIF2(αP)] by the endoplasmic reticulum transmembrane eIF2α kinase PERK occurs immediately on reperfusion and inhibits translation initiation. PERK activation, along with depletion of endoplasmic reticulum Ca2+ and inhibition of the endoplasmic reticulum Ca2+-ATPase, SERCA2b, indicate that an endoplasmic reticulum unfolded protein response occurs as a consequence of brain ischemia and reperfusion. In mammals, the upstream unfolded protein response components PERK, IRE1, and ATF6 activate prosurvivial mechanisms (e.g., transcription of GRP78, PDI, SERCA2b) and proapoptotic mechanisms (i.e., activation of Jun N-terminal kinases, caspase-12, and CHOP transcription). Sustained eIF2(αP) is proapoptotic by inducing the synthesis of ATF4, the CHOP transcription factor, through “bypass scanning” of 5‘ upstream open-reading frames in ATF4 messenger RNA; these upstream open-reading frames normally inhibit access to the ATF4 coding sequence. Brain ischemia and reperfusion also induce μ-calpain–mediated or caspase-3–mediated proteolysis of eIF4G, which shifts message selection to m7G-cap–independent translation initiation of messenger RNAs containing internal ribosome entry sites. This internal ribosome entry site–mediated translation initiation (i.e., for apoptosis-activating factor-1 and death-associated protein-5) can also promote apoptosis. Thus, alterations in eIF2 and eIF4 have major implications for which messenger RNAs are translated by residual protein synthesis in neurons during brain reperfusion, in turn constraining protein expression of changes in gene transcription induced by ischemia and reperfusion. Therefore, our current understanding shifts the focus from protein synthesis inhibition to the molecular pathways that underlie this inhibition, and the role that these pathways play in prosurvival and proapoptotic processes that may be differentially expressed in vulnerable and resistant regions of the reperfused brain.
Late Reversal of Cerebral Perfusion and Water Diffusion after Transient Focal Ischemia in Rats
Authors: Wang Lei , Yushmanov Victor E. , Liachenko Serguei M. , Tang Pei , Hamilton Ronald L. , Xu Yan
Published: November 2003

Region-specific cerebral blood flow (CBF) and the apparent diffusion coefficient (ADC) of tissue water in the rat brain were quantified by high-field magnetic resonance imaging at 9.4 T in the rat suture occlusion model. Cerebral blood flow and ADC were compared during the short- (4.5 hours) and long-term (up to 6 days) reperfusion after 80 minutes of transient middle cerebral artery occlusion, and correlated with the histology analysis. On occlusion, average CBF fell from ∼100 to less than 50 mL 100 g−1 min−1 in the cortex, and to less than 20 mL 100 g−1 min−1 in the caudate putamen (CP). Corresponding ADC values decreased from (6.98 ± 0.82) × 10−4 to (5.49 ± 0.54) × 10−4 mm2/s in the cortex, and from (7.16 ± 0.58) × 10−4 to (4.86 ± 0.62) × 10−4 mm2/s in the CP. On average, CBF recovered to ∼50% of baseline in the first 24 hours of reperfusion. After 2 to 4 days, a strong hyperperfusion in the ipsilateral cortex and CP, up to ∼300 mL 100 g−1 min−1, was observed. The ADC ratio in the ipsilateral and contralateral CP was also inverted in the late reperfusion period. Histology revealed more severe tissue damage at the late stage of reperfusion than at 4.5 hours. Significant reversal of CBF and ADC during the late reperfusion period may reflect the impairment of autoregulation in the ischemic regions. Vascular factors may play an important role in the infarct development after 80-minute focal ischemia.
Quantitative Assessment of the Balance between Oxygen Delivery and Consumption in the Rat Brain after Transient Ischemia with T2-BOLD Magnetic Resonance Imaging
Authors: Kettunen Mikko I. , Gröhn Olli H. J. , Silvennoinen M. Johanna , Penttonen Markku , Kauppinen Risto A.
Published: November 2003

The balance between oxygen consumption and delivery in the rat brain after exposure to transient ischemia was quantitatively studied with single-spin echo T2-BOLD (blood oxygenation level–dependent) magnetic resonance imaging at 4.7 T. The rats were exposed to graded common carotid artery occlusions using a modification of the four-vessel model of Pulsinelli. T2, diffusion, and cerebral blood volume were quantified with magnetic resonance imaging, and CBF was measured with the hydrogen clearance method. A transient common carotid artery occlusion below the CBF value of approximately 20 mL·100 g−1·min−1 was needed to yield a T2 increase of 4.6 ± 1.2 milliseconds (approximately 9% of cerebral T2) and 6.8 ± 1.7 milliseconds (approximately 13% of cerebral T2) after 7 and 15 minutes of ischemia, respectively. Increases in CBF of 103 ± 75% and in cerebral blood volume of 29 ± 20% were detected in the reperfusion phase. These hemodynamic changes alone could account for only approximately one third of the T2 increase in luxury perfusion, suggesting that a substantial increase in blood oxygen saturation (resulting from reduced oxygen extraction by the brain) is needed to explain the magnetic resonance imaging observation.
Striking Differences in Glucose and Lactate Levels between Brain Extracellular Fluid and Plasma in Conscious Human Subjects: Effects of Hyperglycemia and Hypoglycemia
Authors: Abi-Saab Walid M. , Maggs David G. , Jones Tim , Jacob Ralph , Srihari Vinod , Thompson James , Kerr David , Leone Paola , Krystal John H. , Spencer Dennis D. , During Matthew J. , Sherwin Robert S.
Published: November 2003

Brain levels of glucose and lactate in the extracellular fluid (ECF), which reflects the environment to which neurons are exposed, have never been studied in humans under conditions of varying glycemia. The authors used intracerebral microdialysis in conscious human subjects undergoing electro-physiologic evaluation for medically intractable epilepsy and measured ECF levels of glucose and lactate under basal conditions and during a hyperglycemia–hypoglycemia clamp study. Only measurements from nonepileptogenic areas were included. Under basal conditions, the authors found the metabolic milieu in the brain to be strikingly different from that in the circulation. In contrast to plasma, lactate levels in brain ECF were threefold higher than glucose. Results from complementary studies in rats were consistent with the human data. During the hyperglycemia–hypoglycemia clamp study the relationship between plasma and brain ECF levels of glucose remained similar, but changes in brain ECF glucose lagged approximately 30 minutes behind changes in plasma. The data demonstrate that the brain is exposed to substantially lower levels of glucose and higher levels of lactate than those in plasma; moreover, the brain appears to be a site of significant anaerobic glycolysis, raising the possibility that glucose-derived lactate is an important fuel for the brain.
Aquaporins in Brain: Distribution, Physiology, and Pathophysiology
Authors: Badaut Jérôme , Lasbennes François , Magistretti Pierre J. , Regli Luca
Published: November 2003

Water homeostasis in the brain is of central physiologic and clinical importance. Neuronal activity and ion water homeostasis are inextricably coupled. For example, the clearance of K+ from areas of high neuronal activity is associated with a concomitant water flux. Furthermore, cerebral edema, a final common pathway of numerous neurologic diseases, including stroke, may rapidly become life threatening because of the rigid encasement of the brain. A water channel family, the aquaporins, facilitates water flux through the plasma membrane of many cell types. In rodent brain, several recent studies have demonstrated the presence of different types of aquaporins. Aquaporin 1 (AQP1) was detected on epithelial cells in the choroid plexus whereas AQP4, AQP5 and AQP9 were localized on astrocytes and ependymal cells. In rodent brain, AQP4 is present on astrocytic end-feet in contact with brain vessels, and AQP9 is found on astrocytic processes and cell bodies. In basal physiologic conditions, AQP4 and AQP9 appear to be implicated in brain homeostasis and in central plasma osmolarity regulation. Aquaporin 4 may also play a role in pathophysiologic conditions, as shown by the reduced edema formation observed after water intoxication and focal cerebral ischemia in AQP4-knockout mice. Furthermore, pathophysiologic conditions may modulate AQP4 and AQP9 expression. For example, AQP4 and AQP9 were shown to be upregulated after ischemia or after traumatic injuries. Taken together, these recent reports suggest that water homeostasis in the brain is maintained by regulatory processes that, by control of aquaporin expression and distribution, induce and organize water movements. Facilitation of these movements may contribute to the development of edema formation after acute cerebral insults such as ischemia or traumatic injury.
Neuroprotection by a Bile Acid in an Acute Stroke Model in the Rat
Authors: Rodrigues Cecilia M. P. , Spellman Stephen R. , Solá Susana , Grande Andrew W. , Linehan-Stieers Cheryle , Low Walter C. , Steer Clifford J.
Published: November 2003

Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, is a strong modulator of apoptosis in both hepatic and nonhepatic cells, and appears to function by inhibiting mitochondrial membrane perturbation. Excitotoxicity, metabolic compromise, and oxidative stress are major determinants of cell death after brain ischemia-reperfusion injury. However, some neurons undergo delayed cell death that is characteristic of apoptosis. Therefore, the authors examined whether TUDCA could reduce the injury associated with acute stroke in a well-characterized model of transient focal cerebral ischemia. Their model of middle cerebral artery occlusion resulted in marked cell death with prominent terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling (TUNEL) within the ischemic penumbra, mitochondrial swelling, and caspase activation. Tauroursodeoxycholic acid administered 1 hour after ischemia resulted in significantly increased bile acid levels in the brain, improved neurologic function, and an approximately 50% reduction in infarct size 2 and 7 days after reperfusion. In addition, TUDCA significantly reduced the number of TUNEL-positive brain cells, mitochondrial swelling, and partially inhibited caspase-3 processing and substrate cleavage. These findings suggest that the mechanism for in vivo neuroprotection by TUDCA is, in part, mediated by inhibition of mitochondrial perturbation and subsequent caspase activation leading to apoptotic cell death. Thus, TUDCA, a clinically safe molecule, may be useful in the treatment of stroke and possibly other apoptosis-associated acute and chronic injuries to the brain.
Calcium Compartments in Brain
Authors: Newman George C. , Hospod Frank E. , Patlak Clifford S. , Trowbridge Sean D. , Wilke Richard J. , Fuhrmann Mark , Jones Keith W.
Published: November 2003

Excellent progress has been made toward understanding the physiology and pharmacology of specific calcium-related cellular processes of the brain, but few studies have provided an integrated view of brain calcium kinetics. To further the knowledge of the size and binding properties of brain calcium compartments, the authors have conducted a series of experiments in hippocampal brain slices exposed to high and low extracellular calcium. Slices were incubated in buffers containing 0.001 to 4.5 mmol/L calcium for up to 75 minutes. Slice calcium content was analyzed by three methods: exchange equilibrium with 45Ca, synchrotron-radiation–induced x-ray emission, and inductively coupled plasma. Data were analyzed using a model based on a Langmuir isotherm for two independent sites, with additional extracellular and bound compartments. In parallel experiments, altered low calcium had no effect on slice histology and only mild effects on slice adenylates. When combined with prior 45Ca and fluorescent probe binding experiments, these results suggest that there are at least five kinetically distinct calcium compartments: (1) free extracellular (∼10%); (2) loosely associated extracellular plasma membrane (∼55%); (3) intracellular compartment with moderate avidity (∼17%); (4) tightly bound, nonexchangeable intracellular compartment (∼15%); and (5) free cytoplasmic (<0.01%). If only the third compartment is considered a potential calcium buffer, then the buffering ratio is calculated to be ∼2,700:1, but if the second compartment is also included, then the buffering ratio would be ∼13,000:1. This may explain the wide range of estimates observed by fluorescent probe studies.
Quantitative Analysis for Estimating Binding Potential of the Brain Serotonin Transporter with [11C]McN5652
Authors: Ikoma Yoko , Suhara Tetsuya , Toyama Hinako , Ichimiya Tetsuya , Takano Akihiro , Sudo Yasuhiko , Inoue Makoto , Yasuno Fumihiko , Suzuki Kazutoshi
Published: November 2003

[11C](+)McN5652 is a selective serotonin reuptake inhibitor with subnanomolar potency for the serotonin transporter, and is currently being used for positron emission tomography studies. However, quantification of the regional [11C](+)McN5652 binding potential in vivo is a controversial issue because of its complex characteristics. The authors examined the regional differences in nonspecific binding and proposed simple methods for estimating the binding potential of [11C](+)McN5652. The regional difference in nonspecific binding was evaluated by the activity ratio of the thalamus compared with the cerebellum for inactive-isomer [11C](−)McN5652 and [11C](+)McN5652 saturation studies. The distribution volume of the thalamus was approximately 1.16 times larger than that of the cerebellum. The thalamus-to-cerebellum distribution volume ratio was estimated by nonlinear least square and graphical methods, with and without arterial input function. The graphical method with k2′ without blood sampling was practical and most applicable for estimation of the distribution volume ratio because this method is more stable than the nonlinear least square method in the simulation study. Binding potential estimated with the distribution volume ratio of [11C](+)McN5652 and the correction with distribution volume ratio of [11C](−)McN5652 represent the most reliable parameters for the assessment of serotonin transporter binding.
Nitric Oxide does not Inhibit Cerebral Cytochrome Oxidase In Vivo or in the Reactive Hyperemic Phase after Brief Anoxia in the Adult Rat
Authors: De Visscher Geofrey , Springett Roger , Delpy David T. , Van Reempts Jos , Borgers Marcel , van Rossem Koen
Published: November 2003

In this study, near-infrared spectroscopy was applied to examine whether cytochrome oxidase in the rat brain is inhibited by nitric oxide in vivo. During normoxia, intravenous NG-nitro-l-arginine methyl ester (l-NAME) administration significantly decreased the cerebral saturation of hemoglobin with oxygen but did not alter the cytochrome oxidase redox state. Anoxia significantly reduced the cytochrome oxidase. The time course of the recovery of the redox state during reoxygenation was not altered by l-NAME. The results suggest that in adult rats, cytochrome oxidase is not inhibited by nitric oxide, either in physiologic conditions or during reoxygenation after a brief anoxic period.
Cloning and Characterization of Rat Caspase-9: Implications for a Role in Mediating Caspase-3 Activation and Hippocampal Cell Death after Transient Cerebral Ischemia
Authors: Cao Guodong , Luo Yumin , Nagayama Tetsuya , Pei Wei , Stetler R. Anne , Graham Steven H. , Chen Jun
Published: November 2003

Delayed hippocampal neurodegeneration after transient global ischemia is mediated, at least in part, through the activation of terminal caspases, particularly caspase-3, and the subsequent proteolytic degradation of critical cellular proteins. Caspase-3 may be activated by the membrane receptor-initiated caspase-8–dependent extrinsic pathway and the mitochondria-initiated caspase-9–dependent intrinsic pathway; however, the precise role of these deduced apoptosis-signaling pathways in activating caspase-3 in ischemic neurons remains elusive. The authors cloned the caspase-9 gene from the rat brain and investigated its potential role in mediating ischemic neuronal death in a rat model of transient global ischemia. Caspase-9 gene expression and protease activity were extremely low in the adult brain, whereas they were developmentally upregulated in newborn rats, especially at postnatal 12 weeks, a finding consistent with the theory of an essential role for caspase-9 in neuronal apoptosis during brain development. After 15-minute transient global ischemia, caspase-9 was overexpressed and proteolytically activated in the hippocampal CA1 neurons at 8 to 72 hours of reperfusion. The temporal profile of caspase-9 activation coincided with that of cytochrome c release and caspase-3 activation, but preceded CA1 neuronal death. Immunoprecipitation experiments revealed that there was enhanced formation of Apaf-1/caspase-9 complex in the hippocampus 8 and 24 hours after ischemia. Furthermore, intracerebral ventricular infusion of the relatively specific caspase-9 inhibitor N-benzyloxycarbonyl-Leu-Glu-His-Asp-fluoro-methylketone before ischemia attenuated caspase-3–like activity and significantly enhanced neuronal survival in the CA1 sector. In contrast, inhibition of caspase-8 activity had no significant effect on caspase-3 activation or neuronal survival. These results suggest that the caspase-9–dependent intrinsic pathway may be the primary mechanism responsible for the activation of caspase-3 in ischemic hippocampal neurons.
Specific Binding of [11C]Raclopride and N-[3H]Propyl-Norapomorphine to Dopamine Receptors in Living Mouse Striatum: Occupancy by Endogenous Dopamine and Guanosine Triphosphate–Free G Protein
Authors: Cumming Paul , Wong Dean F. , Gillings Nicholas , Hilton John , Scheffel Ursula , Gjedde Albert
Published: November 2003

According to the ternary complex model of G-protein linkage to receptors, agonists increase the affinity of the receptors for the G protein. The model predicts that an endogenous agonist's constant of inhibition toward an agonist radioligand is lower than that toward an antagonistic radioligand. The authors hypothesized that competition from endogenous dopamine in striatum of living mice should have a greater effect on the binding of the D2,3 partial agonist N-[3H]propylnorapomorphine than on the binding of the D2,3 antagonist [11C]raclopride. The baseline binding potential ( pB(0)), defined as the ratio of bound-to-unbound ligand in the absence of competition from endogenous dopamine, was simultaneously measured in mouse striatum for [11C]raclopride ( pB(0) = 8.5) and N-[3H]propylnorapomorphine ( p′B(0) = 5.3). The baseline was established by treatment with α-methyl- p-tyrosine and reserpine. Relative to these baseline values in saline-treated mice, the pB of N-[3H]propylnorapomorphine decreased 52% whereas the pB of [11C]raclopride decreased only 30%, indicating greater sensitivity of the former compound to inhibition by synaptic dopamine. Furthermore, amphetamine decreased the pB of N-[3H]propylnorapomorphine to a greater extent (73%) than that of [11C]raclopride (43%) relative to the reserpine condition. For both radioligands, the occupancy of the dopamine receptors by endogenous agonist obeyed Michaelis-Menten kinetics over a wide range of agonist concentrations established by the pharmacologic treatments. The apparent inhibition constant of endogenous dopamine depended on the dopamine occupancy and decreased to a value 1.66 times greater for N-[3H]propylnorapomorphine than for [11C]raclopride at its highest occupancies. The results are consistent with the hypothesis that agonist binding is more sensitive than antagonist binding to competition from endogenous dopamine. Therefore, dopamine agonist ligands may be superior to benzamide antagonist ligands for the estimation of dopamine receptor occupancy by endogenous synaptic dopamine. The analysis of the effect of dopamine occupancy on the inhibition of N-[3H]propylnorapomorphine binding indicated a limited supply of G protein with a maximum ternary complex fraction of 40% of maximum agonist binding capacity.
Moment Analysis of Microflow Histogram in Focal Ischemic Lesion to Evaluate Microvascular Derangement after Small Pial Arterial Occlusion in Rats
Authors: Tomita Yutaka , Tomita Minoru , Schiszler Istvan , Amano Takahiro , Tanahashi Norio , Kobari Masahiro , Takeda Hidetaka , Ohtomo Manabu , Fukuuchi Yasuo
Published: November 2003

The authors' high-spatial-resolution optical method was used to examine microvascular derangement in a focal cerebral cortex lesion in 12 Sprague-Dawley rats anesthetized with α-chloralose-urethane. A pial artery (approximately 40- to 50-μm diameter) was occluded by laser-beam cauterization (n = 6). Diluted carbon black suspension was injected into the internal carotid artery, and images in a 2-mm x 2-mm region of interest during tissue dye-dilution were recorded. Sequential frames were analyzed with Matlab software to evaluate blood distribution and mean transit time, affording a two-dimensional microflow map and histogram with first, second, third, and fourth moments. In the early phase of ischemia, blood distribution and average flow decreased (both P < 0.01), and the second moment (microflow heterogeneity) and third moment (skew to the left owing to increase in low-flow components) increased ( P < 0.05 and P < 0.01, respectively). At approximately 2 hours, blood distribution decreased further in 3 cases, apparently because capillary stasis prevented carbon black filling. However, average microflow unexpectedly increased in 4 of 5 rats, presumably due to exclusion of unper-fused (low flow at the earlier stage) channels from the calculation. The authors conclude that flow in ischemic tissue is quite heterogeneous and that an averaged flow value tends to smear important information about ischemic microvascular derangement.
Nitric Oxide Formation during Cortical Spreading Depression is Critical for Rapid Subsequent Recovery of Ionic Homeostasis
Authors: Obrenovitch Tihomir P. , Urenjak Jutta , Wang Minyan
Published: November 2003

Cortical spreading depression (CSD) is a temporary disruption of local ionic homeostasis that propagates slowly across the cerebral cortex. Cortical spreading depression promotes lesion progression in experimental stroke, and may contribute to the initiation of migraine attacks. The purpose of this study was to investigate the roles of the marked increase of nitric oxide (NO) formation that occurs with CSD. Microdialysis electrodes were implanted in the cortex of anesthetized rats to perform the following operations within the same region: (1) elicitation of CSD by perfusion of high K+ medium; (2) recording of CSD elicitation; (3) application of the NO synthase inhibitor, NG-nitro-l-arginine methyl ester (l-NAME); and (4) recording of dialysate pH changes. The primary effect of l-NAME (0.3 to 3.0 mmol/L in the perfusion medium) was a marked widening of individual CSD wave, resulting essentially from a delayed initiation of the repolarization phase. This change was due to NO synthase inhibition because it was not observed with the inactive isomer d-NAME, and was reversed by l-arginine. This effect did not appear to be linked to the suppression of a sustained, NO-mediated vascular change associated with the superposition of NO synthase inhibition on high levels of extracellular K+. The delayed initiation of repolarization with local NO synthase inhibition may reflect the suppression of NO-mediated negative feedback mechanisms acting on neuronal or glial processes involved in CSD genesis. However, the possible abrogation of a very brief, NO-mediated vascular change associated with the early phase of CSD cannot be ruled out.
Effect of Simple Motor Performance on Regional Dopamine Release in the Striatum in Parkinson Disease Patients and Healthy Subjects: A Positron Emission Tomography Study
Authors: Ouchi Yasuomi , Yoshikawa Etsuji , Futatsubashi Masami , Okada Hiroyuki , Torizuka Tatsuo , Sakamoto Masanobu
Published: November 2003

To investigate changes in dopamine release in the striatum during motor exercise in human subjects with and without striatal dopamine denervation, eight healthy subjects and eight patients with Parkinson disease (PD) were measured during unilateral foot extension/flexion movement using positron emission tomography with [11C]raclopride. Five subjects in each group were later scanned in the resting condition. Estimation of binding potential (k3/k4) of [11C]raclopride was based on Logan plot method. Significant reductions in [11C]raclopride k3/k4 were found in the dorsal putamen contralateral to the exercise side in the healthy group and ipsilaterally in the PD group. Spearman rank correlation analysis showed that [11C]raclopride k3/k4 correlated inversely with the decrease in performance (velocity and motion range) in the dorsal putamen contralaterally in the healthy group and ipsilaterally in the PD group. These results suggest that simple but laborious motor exercise (motor stimulation) generates significant dopamine release in the dorsal striatum contralateral to the motor execution in humans. Lack of the crossed pattern and ipsilateral increase in dopamine release in the dorsal striatum during the unilateral limb movement may reflect the pathophysiology for hypokinetic and insufficient coordinating movement in PD.
TAK-779, A Nonpeptide CC Chemokine Receptor Antagonist, Protects the Brain against Focal Cerebral Ischemia in Mice
Authors: Takami Shinya , Minami Masabumi , Katayama Takahiro , Nagata Izumi , Namura Shobu , Satoh Masamichi
Published: November 2003

The effect of a nonpeptide CC chemokine receptor antagonist, TAK-779, on ischemic brain injury resulting from 1-hour middle cerebral artery occlusion followed by 48-hour reperfusion was examined in ddY mice. On intracerebroventricular injection of vehicle or TAK-779, infarct volume in the vehicle-treated group was 44.2 ± 13.2% of the contralateral hemispheric volume, and TAK-779 (25 and 250 ng/mouse) dose-dependently reduced the infarct volume to 35.0 ± 12.2% and 31.1 ± 12.9%, respectively. On intravenous injection, infarct volume in the vehicle-treated group was 32.0 ± 16.1%, and TAK-779 (5 μg per 20 g body weight) significantly reduced this to 22.0 ± 10.5%. The results showed for the first time that a nonpeptide chemokine receptor antagonist is protective against ischemic brain injury.
Microglial—Macrophage Synthesis of Tumor Necrosis Factor after Focal Cerebral Ischemia in Mice is Strain Dependent
Authors: Lambertsen Kate Lykke , Gregersen Rikke , Finsen Bente
Published: November 2003

Commonly used inbred mouse strains display substantial differences in sensitivity to focal cerebral ischemia. Such differences can often be ascribed to differences in vascular anatomy. The authors investigated whether a contributing factor could be strain-related differences in cellular synthesis of the pleiotropic and potential neurotoxic cytokine tumor necrosis factor (TNF) in the border zone of and within the focal cerebral infarct. In all mouse strains investigated they found that TNF was synthesized by infarct and periinfarct infiltrating Mac-1 immunopositive microglia—macrophages. BALB/c mice, which developed the largest infarcts, contained significantly fewer TNF-producing microglia—macrophages compared with SJL and C57BL/6 mice at both 12 and 24 hours after permanent occlusion of the distal part of the middle cerebral artery. SJL mice developed larger infarcts than C57BL/6 mice, whereas the number of TNF-producing microglia—macrophages per infarct volume unit was comparable. Western blotting data confirmed the increased TNF levels in SJL mice compared with BALB/c and C57BL/6 mice. Furthermore, mice with 12-hour postischemic survival consistently contained two-to threefold more TNF-producing microglia—macrophages than mice with 24-hour survival. The data show that the magnitude of the cellular TNF response to cerebral ischemia is strain dependent, while the time-profile and the cellular sources of TNF are similar irrespective of genetic background. Furthermore, the lack of correlation between infarct size and cellular TNF response suggests that the functionally important TNF is produced in the very early phase (minutes to a few hours) after induction of ischemia, just as it raises the possibility that different mouse strains display different sensitivities to TNF.
Involvement of Superoxide in Excitotoxicity and DNA Fragmentation in Striatal Vulnerability in Mice after Treatment with the Mitochondrial Toxin, 3-Nitropropionic Acid
Authors: Kim Gyung W. , Chan Pak H.
Published: November 2003

Oxidative stress and excitotoxicity have been implicated in selective striatal vulnerability caused by the mitochondrial toxin, 3-nitropropionic acid (3-NP), which may simulate Huntington's disease in animals and humans. The detailed mechanism of the role of superoxide in striatal vulnerability induced by 3-NP is still unknown. The authors investigated oxidative cellular injury and DNA fragmentation after systemic 3-NP injection in wild-type (Wt) mice and mutant mice with a deficiency in manganese superoxide dismutase (MnSOD; Sod2 −/+). Furthermore, they investigated the effects of decortication after 3-NP treatment in Sod2 −/+ mice, and copper/zinc SOD (CuZnSOD) treatment in recently developed Sod2 −/+ mice that overexpress CuZnSOD (SOD1 +/− / Sod2 −/+ mice). Oxidized hydroethidine, 8-hydroxyguanosine immunoreactivity, and nitrotyrosine immunoreactivity were increased in the Sod2 −/+ mice compared with the Wt mice after 3-NP treatment ( P < 0.001). Decortication completely abolished oxidative striatal damage after 3-NP treatment in the Sod2 −/+ mice. Increased CuZnSOD attenuated DNA fragmentation and striatal lesion volume after 3-NP treatment in the Sod2 −/+ mice ( P < 0.001). These data suggest that production of superoxide may be a critical step to excitotoxicity and subsequent DNA fragmentation in selective striatal vulnerability after 3-NP treatment.
Temporal Profiles of the Subcellular Localization of Bim, a BH3-Only Protein, during Middle Cerebral Artery Occlusion in Mice
Authors: Shibata Mamoru , Hattori Hidenori , Sasaki Takahiro , Gotoh Jun , Hamada Junichi , Fukuuchi Yasuo
Published: November 2003

BH3-only proteins are a subfamily of proapoptotic Bcl-2 proteins that act upstream of the mitochondrially mediated cell death pathway, and their association with the pathogenesis of brain ischemia remains largely unknown. The authors explored the temporal profiles of the expression levels and subcellular localization of BH3-only proteins in permanent middle cerebral artery occlusion (MCAO) by Western blot analysis. They observed an increased mitochondrial distribution of Bim at 3 to 6 hours of MCAO that appeared unrelated to transcriptional upregulation, as assessed by semiquantitative reverse transcription—polymerase chain reaction. At 3 to 6 hours of MCAO, Bim immunoreactivity was enhanced in neurons and oligodendrocytes in the ischemic regions. The increased mitochondrial localization of Bim coincided with a marked cytochrome c release and preceded the peak of caspase-9 activation. The authors observed an association of Bim with the dynein intermediate chain, a major component of the dynein motor complex, in the brain using a coimmunoprecipitation assay. Cerebral ischemia induced a time-dependent significant decrease in dynein expression, which started at 3 hours of MCAO. The authors deduced that the liberation of Bim from the dynein motor complex is a likely mechanism for the increased mitochondrial localization of Bim. During MCAO, Bad did not show any change in phosphorylation state or subcellular localization.
A Potent Protective Role of Lysophospholipids against Global Cerebral Ischemia and Glutamate Excitotoxicity in Neuronal Cultures
Authors: Blondeau Nicolas , Lauritzen Inger , Widmann Catherine , Lazdunski Michel , Heurteaux Catherine
Published: November 2003

Lysophospholipids (LPLs) are important intermediates in the synthesis and degradation of membrane phospholipids. Here we show that certain LPLs, particularly lysophosphatidylcholine and lysophosphatidylinositol, prevent neuronal death both in an in vivo model of transient global ischemia and in an in vitro model of excitotoxicity using primary cultures of cerebellar granule cells exposed to high extracellular concentrations of glutamate (20–40 μmol/L). The intravenous injection of lysophosphatidylcholine or lysophosphatidylinositol at a concentration of 200 nmol/kg induced a survival of CA1 pyramidal neurons as high as approximately 95%, even when the treatment was started 30 minutes after 15-minute global ischemia. In contrast, lysophosphatidic acid induced no protection. This work also provides evidence that a pretreatment with lysophosphatidylcholine or lysophosphatidylinositol (200 nmol/kg) injected as long as 3 days before a severe 6-minute ischemia provided a potent tolerance against neurodegeneration. Neuroprotection was also observed in in vitro experiments with LPLs. Taken together, in vivo and in vitro data suggest a potential therapeutic use of LPLs as antiischemic compounds. The potential role of 2P-domain K+ channels as targets of LPLs in this potent neuroprotective effect is discussed.
Unique Profile of Spreading Depression in a Primate Model
Authors: Yokota Chiaki , Kuge Yuji , Hasegawa Yasuhiro , Tagaya Masafumi , Abumiya Takeo , Ejima Norimasa , Tamaki Nagara , Yamaguchi Takenori , Minematsu Kazuo
Published: November 2003

Spreading depression (SD) is considered to play a role in pathologic conditions of humans such as in the evolution of ischemic brain injury and migraine aura. Because many studies have demonstrated spreading hypoperfusion in patients with migraine and persistent hypoperfusion in nonprimate animal models of SD, these changes in cerebral blood flow (CBF) were regarded as an epiphenomenon of SD. However, there is no direct evidence of the occurrence of SD in primates. The authors attempted to elicit SD by applying 3.3 mol/L potassium chloride to the cerebral cortex of nine male cynomolgus monkeys. The CBF was monitored by positron emission tomography in five animals. Propagated direct-current shifts were found by the two neighboring microelectrodes only in one animal. The direct-current wave propagated at a speed of 4 mm/min and its amplitude was 20 mV, being consistent with the SD findings. Except in one animal with 6 SD episodes, SD waves were recorded infrequently at the rostral site (none in three animals, once in three, and twice in two). Focal hyperemia accompanied SD. Neither spreading hypoperfusion nor persistent hypoperfusion was found. These unique features of SD in primates raise a doubt as to whether the role of SD in nonprimate animals is the same as that in stroke and migraine in humans.
Impairment of Autoregulatory Vasodilation by NAD(P)H Oxidase—Dependent Superoxide Generation during Acute Stage of Subarachnoid Hemorrhage in Rat Pial Artery
Authors: Shin Hwa Kyoung , Lee Jeong Hyun , Kim Ki Young , Kim Chi Dae , Lee Won Suk , Rhim Byung Yong , Hong Ki Whan
Published: November 2003

This study assessed the mechanism(s) by which the autoregulatory vasodilation of rat pial artery in response to acute hypotension during the acute phase of subarachnoid hemorrhage (SAH) was markedly blunted. Increased superoxide production from the cerebral vessels in response to NAD(P)H at 24 hours after SAH + NG-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg) was inhibited by intracisternal administration of a tyrosine kinase inhibitor genistein (10 μmol/L) and Rac inhibitor Clostridium difficile toxin B (1 ng/mL) and a flavoenzyme inhibitor diphenyleneiodonium (10 μmol/L). The expression of gp91phox was enhanced by SAH + L-NAME from 12 to 24 hours, which was inhibited by genistein and toxin B, but not the p22phox. Increased membrane translocation of Rac after SAH + L-NAME was attenuated by both genistein and toxin B, whereas increased tyrosine kinase activity was blocked by genistein, but not by toxin B. The blunted autoregulatory vasodilation to acute hypotension was effectively recovered by genistein and C. difficile toxin B as well as by diphenyleneiodonium. In conclusion, SAH during acute stage causes an increase in NAD(P)H oxidase—dependent superoxide formation in cerebral vessels, which is due to activation of tyrosine phosphorylation-dependent increased expression of gp91phox mRNA and translocation of Rac protein, thereby resulting in a significant reduction of autoregulatory vasodilation.
Decreased Akt Activity is Associated with Activation of Forkhead Transcription Factor after Transient Forebrain Ischemia in Gerbil Hippocampus
Authors: Kawano Takayuki , Morioka Motohiro , Yano Shigetoshi , Hamada Jun-ichiro , Ushio Yukitaka , Miyamoto Eishichi , Fukunaga Kohji
Published: November 2003

The authors recently reported that sodium orthovanadate rescues cells from delayed neuronal death in gerbil hippocampus after transient forebrain ischemia through phosphatidylinositol 3-kinase-protein kinase B (Akt) pathway ( Kawano et al., 2001 ). In the current study, they demonstrated that the activation of FKHR, a Forkhead transcription factor and a substrate for Akt, preceded delayed neuronal death in CA1 regions after transient forebrain ischemia. Adult Mongolian gerbils were subjected to 5-minute forebrain ischemia. Immunoblotting analysis with anti—phospho-FKHR antibody showed that phosphorylation of FKHR at serine-256 in the CA1 region decreased immediately after and 0.5 and 1 hour after reperfusion. The dephosphorylation of FKHR was correlated with the decreased Akt activity. Intracerebroventricular injection of orthovanadate 30 minutes before ischemia inhibited dephosphorylation of FKHR after reperfusion, and blocked delayed neuronal death in the CA1 region. Gel mobility shift analysis using nuclear extracts from the CA1 region prepared immediately after reperfusion revealed increases in DNA binding activity for the FKHR-responsive element on the Fas ligand promoter. The orthovanadate injection administered before ischemia inhibited its binding activity. Two days after reperfusion, expression of Fas ligand increased in the CA1 region and the orthovanadate injection inhibited this increased expression. These results suggest that the inactivation of Akt results in the activation of FKHR and, in turn, relates to the expression of Fas ligand in the CA1 region after transient forebrain ischemia.
Immature and Mature Cortical Neurons Engage Different Apoptotic Mechanisms Involving Caspase-3 and the Mitogen-Activated Protein Kinase Pathway
Authors: Lesuisse Christian , Martin Lee J.
Published: November 2003

The authors used cultured mouse cortical neurons to study mechanisms of DNA damage-induced apoptosis in immature and mature neurons. Neurons were maintained viably for 60 days in vitro (DIV60). The increased levels of glutamate receptors, synaptic proteins, and glycolytic enzyme were used to track maturation. Exposure of neurons to the DNA-damaging agent camptothecin induced apoptosis in immature (DIV5) and mature (DIV25–30) neurons. Internucleosomal fragmentation of DNA emerged more rapidly in mature neurons than in immature neurons. Immunoblotting revealed that cleaved caspase-3 increased in apoptotic DIV5 neurons but not in DIV30 neurons, but immunolocalization showed accumulation of cleaved caspase-3 in DIV5 and DIV30 neurons. A reversible caspase-3 inhibitor blocked apoptosis in DIV5 neurons but not in DIV30 neurons. Phosphorylation of extracellular signal-regulated kinase/mitogen-activated protein kinase (Erk/MAP kinase)-42/44 occurred preapoptotically in mature but not immature neurons, while Erk54 nuclear translocation and MAP kinase kinase kinase-1 cleavage into putative caspase-3–generated proapoptotic fragments occurred in DIV5 but not DIV30 neurons. Inhibition of Erk activation with MAP kinase kinase inhibitor blocked apoptosis at both ages. The results show that immature and mature cortical neurons engage different signaling mechanisms in MAP kinase and caspase pathways during apoptosis; thus, neuron age influences the mechanisms and progression of apoptosis.
Elevated Intracranial IL-18 in Humans and Mice after Traumatic Brain Injury and Evidence of Neuroprotective Effects of IL-18—Binding Protein after Experimental Closed Head Injury
Authors: Yatsiv Ido , Morganti-Kossmann Maria C. , Perez Daniel , Dinarello Charles A. , Novick Daniela , Rubinstein Menachem , Otto Viviane I. , Rancan Mario , Kossmann Thomas , Redaelli Claudio A. , Trentz Otmar , Shohami Esther , Stahel Philip F.
Published: November 2003

Proinflammatory cytokines are important mediators of neuroinflammation after traumatic brain injury. The role of interleukin (IL)-18, a new member of the IL-1 family, in brain trauma has not been reported to date. The authors investigated the posttraumatic release of IL-18 in murine brains following experimental closed head injury (CHI) and in CSF of CHI patients. In the mouse model, intracerebral IL-18 was induced within 24 hours by ether anesthesia and sham operation. Significantly elevated levels of IL-18 were detected at 7 days after CHI and in human CSF up to 10 days after trauma. Published data imply that IL-18 may play a pathophysiological role in inflammatory CNS diseases; therefore its inhibition may ameliorate outcome after CHI. To evaluate the functional aspects of IL-18 in the injured brain, mice were injected systemically with IL-18–binding protein (IL-18BP), a specific inhibitor of IL-18, 1 hour after trauma. IL-18BP—treated mice showed a significantly improved neurological recovery by 7 days, accompanied by attenuated intracerebral IL-18 levels. This demonstrates that inhibition of IL-18 is associated with improved recovery. However, brain edema at 24 hours was not influenced by IL-18BP, suggesting that inflammatory mediators other than IL-18 induce the early detrimental effects of intracerebral inflammation.
Expression of 150-kd Oxygen-Regulated Protein in the Hippocampus Suppresses Delayed Neuronal Cell Death
Authors: Miyazaki Mayuki , Ozawa Kentaro , Hori Osamu , Kitao Yasuko , Matsushita Kohji , Ogawa Satoshi , Matsuyama Tomohiro
Published: November 2003

ORP150—150-kd oxygen-regulated protein—is a novel stress protein localized in the endoplasmic reticulum (ER). To investigate the role of ORP150 in delayed neuronal cell death, the authors examined its expression in the gerbil brain after an ischemic insult. The expression of ORP150 antigen, as well as its transcripts, was observed in the CA1 region after the occlusion of the common carotid artery, and the preconditioning enhanced this expression. In cultured neurons, exposure either to hypoxia or to glutamate induced the expression of ORP150, and this effect was also observed by treating the culture with breferdin A or thapsigargin, indicating that both glutamate and hypoxia can cause stress in the ER (ER stress). Neurons became more vulnerable to these stresses following treatment with cycloheximide or after infection with an adenovirus carrying the ORP150-antisense structure. In contrast, the overexpression of ORP150 by an adenovirus suppressed neuronal cell death, and this was accompanied by the suppression of Ca2+ elevation and proteolytic activity induced by glutamate. Further, overexpression of ORP150 in CA1 neurons by an adenovirus carrying the ORP150-sense structure suppressed delayed neuronal cell death after ischemia. These data suggest a possible function of ORP150 as an intracellular apparatus that participates in a protective response in ischemic tolerance.