bluuebunny/crossref_metadata_2025 huggingface dataset:
Manganese Superoxide Dismutase Affects Cytochrome c Release and Caspase-9 Activation after Transient Focal Cerebral Ischemia in Mice
Authors: Noshita Nobuo , Sugawara Taku , Fujimura Miki , Morita-Fujimura Yuiko , Chan Pak H.
Published: November 2003

Release of cytochrome c from mitochondria to cytosol is a critical step in the mitochondrial-dependent signaling pathways of apoptosis. The authors have reported that manganese superoxide dismutase (Mn-SOD) attenuated cytochrome c release and apoptotic cell death after focal cerebral ischemia (FCI). To investigate downstream to the cytochrome c-dependent pathway, the authors examined caspase-9 activation after transient FCI by immunohistochemistry and Western blotting in both wild-type and Sod2 −/+ mice. Mice were subjected to 60 minutes of middle cerebral artery occlusion followed by 1, 2, 4, or 24 hours of reperfusion. Two hours after reperfusion, cytochrome c and caspase-9 were observed in the cytosol and significantly increased in Sod2 −/+ mutants compared with wild-type mice as shown by Western blotting. Immunofluorescent double labeling for cytochrome c and caspase-9 showed cytosolic cytochrome c 1 hour after transient FCI. Cleaved caspase-9 first appeared in the cytosol at 2 hours and colocalized with cytochrome c. Terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick and labeling (TUNEL) showed significant increase of positive cells in Sod2 −/+ mice compared with the wild-type in the cortex, but not in the caudate putamen. The current study revealed Mn-SOD might affect cytochrome c translocation and downstream caspase activation in the mitochondrial-dependent cell death pathway after transient FCI.
Hyperglycemia Enhances DNA Fragmentation after Transient Cerebral Ischemia
Authors: Li Ping-An , Rasquinha Ingrid , He Qing Ping , Siesjö Bo K. , Csiszár Katalin , Boyd Charles D. , MacManus John P.
Published: November 2003

Previous histopathologic results have suggested that one mechanism whereby hyperglycemia (HG) leads to exaggerated ischemic damage involves fragmentation of DNA. DNA fragmentation in normoglycemia (NG) and HG rats subjected to 30 minutes of forebrain ischemia was studied by terminal deoxynucleotidyl transferase mediated DNA nick-labeling (TUNEL) staining, by pulse-field gel electrophoresis (PFGE), and by ligation-mediated polymerase chain reaction (LM-PCR). High molecular weight DNA fragments were detected by PFGE, whereas low molecular weight DNA fragments were detected using LM-PCR techniques. The LM-PCR procedure was performed on DNA from test samples with blunt (without Klenow polymerase) and 3′-recessed ends (with Klenow polymerase). In addition, cytochrome c release and caspase-3 activation were studied by immunocytochemistry. Results show that HG causes cytochrome c release, activates caspase-3, and exacerbates DNA fragments induced by ischemia. Thus, in HG rats, but not in control or NGs, TUNEL-stained cells were found in the cingulate cortex, neocortex, thalamus, and dorsolateral crest of the striatum, where neuronal death was observed by conventional histopathology, and where both cytosolic cytochrome c and active caspase-3 were detected by confocal microscopy. In the neocortex, both blunt-ended and stagger-ended fragments were detected in HG, but not in NG rats. Electron microscopy (EM) analysis was performed in the cingulate cortex, where numerous TUNEL-positive neurons were observed. Although DNA fragmentation was detected by TUNEL staining and electrophoresis techniques, EM analysis failed to indicate apoptotic cell death. It is concluded that HG triggers a cell death pathway and exacerbates DNA fragmentation induced by ischemia.
Absolute Quantification by Positron Emission Tomography of the Endogenous Ligand
Authors: Delforge Jacques , Bottlaender Michel , Pappata Sabina , Loc'h Christian , Syrota André
Published: November 2003

The results of several recent papers have shown a significant influence of the endogenous neurotransmitters on the exogenous ligand kinetics measured by positron emission tomography. For example, several groups found that the percentage of D2 receptor sites occupied by the endogenous dopamine ranged from 25% to 40% at basal level. An obvious consequence of this significant occupancy is that the ligand-receptor model parameters, usually estimated by a model that does not take into account the endogenous ligand (EL) kinetics, can be significantly biased. In the current work, the authors studied the biases obtained by using the multiinjection approach. The results showed that in the classical ligand-receptor model, the receptor concentration is correctly estimated and that only the apparent affinity is biased by not taking the EL into account. At present, all absolute quantifications of the EL have been obtained through pharmacologic manipulation of the endogenous transmitter concentration, which is often too invasive a method to be used in patients. A theoretical reasoning showed that a noninvasive approach is necessarily based on both the apparent affinity measurement and on a multiregion approach. The correlation between the receptor concentration and the apparent affinity, previously observed with some ligands, verifies these two conditions; thus, the authors suggest that this correlation could be the result of the EL effect. To test this assumption experimentally, the effect of reserpine-induced dopamine depletion on the interactions between the D2 receptor sites and the FLB 457 is studied. With untreated baboons, the apparent FLB 457 affinity was smaller in the receptor-rich regions (striatum) than in the receptor-poor regions. This discrepancy disappeared after dopamine depletion, strongly suggesting that this affinity difference was related to the EL effect. Therefore, the purpose of the current study was to test the ability to quantify the EL based on the observed correlation between the receptor concentration and the apparent affinity. This approach offers a method for estimating the percentage of receptor sites occupied by the EL and, if its affinity is known, the free EL concentration. From the data obtained using FLB 457 with baboons, the authors found that approximately 53% of the D2 receptor sites are occupied by dopamine in the striatum and that the free dopamine concentration is approximately 120 nmol/L at basal level. This approach is transferable to patients, because the experimental data are obtained without pharmacologically induced modification of the EL.
Stroke Genomics: Approaches to Identify, Validate, and Understand Ischemic Stroke Gene Expression
Authors: Read Simon J. , Parsons Andrew A. , Harrison David C. , Philpott Karen , Kabnick Karen , O'Brien Shawn , Clark Steven , Brawner Mary , Bates Stewart , Gloger Israel , Legos Jeffrey J. , Barone Frank C.
Published: November 2003

Sequencing of the human genome is nearing completion and biologists, molecular biologists, and bioinformatics specialists have teamed up to develop global genomic technologies to help decipher the complex nature of pathophysiologic gene function. This review will focus on differential gene expression in ischemic stroke. It will discuss inheritance in the broader stroke population, how experimental models of spontaneous stroke might be applied to humans to identify chromosomal loci of increased risk and ischemic sensitivity, and also how the gene expression induced by stroke is related to the poststroke processes of brain injury, repair, and recovery. In addition, we discuss and summarise the literature of experimental stroke genomics and compare several approaches of differential gene expression analyzes. These include a comparison of representational difference analysis we have provided using an experimental stroke model that is representative of stroke evolution observed most often in man, and a summary of available data on stroke differential gene expression. Issues regarding validation of potential genes as stroke targets, the verification of message translation to protein products, the relevance of the expression of neuroprotective and neurodestructive genes and their specific timings, and the emerging problems of handling novel genes that may be discovered during differential gene expression analyses will also be addressed.
Hypoperfusion without Ischemia Surrounding Acute Intracerebral Hemorrhage
Authors: Zazulia Allyson R. , Diringer Michael N. , Videen Tom O. , Adams Robert E. , Yundt Kent , Aiyagari Venkatesh , Grubb Robert L. , Powers William J.
Published: November 2003

A zone of hypoperfusion surrounding acute intracerebral hemorrhage (ICH) has been interpreted as regional ischemia. To determine if ischemia is present in the periclot area, the authors measured cerebral blood flow (CBF), cerebral metabolic rate of oxygen (CMRO2), and oxygen extraction fraction (OEF) with positron emission tomography (PET) in 19 patients 5 to 22 hours after hemorrhage onset. Periclot CBF, CMRO2, and OEF were determined in a 1-cm-wide area around the clot. In the 16 patients without midline shift, periclot data were compared with mirror contralateral regions. All PET images were masked to exclude noncerebral structures, and all PET measurements were corrected for partial volume effect due to clot and ventricles. Both periclot CBF and CMRO2 were significantly reduced compared with contralateral values (CBF: 20.9 ± 7.6 vs. 37.0 ± 13.9 mL 100 g−1 min−1, P = 0.0004; CMRO2: 1.4 ± 0.5 vs. 2.9 ± 0.9 mL 100 g−1 min−1, P = 0.00001). Periclot OEF was less than both hemispheric OEF (0.42 ± 0.15 vs. 0.47 ± 0.13, P = 0.05; n = 19) and contralateral regional OEF (0.44 ± 0.16 vs. 0.51 ± 0.13, P = 0.05; n = 16). In conclusion, CMRO2 was reduced to a greater degree than CBF in the periclot region in acute ICH, resulting in reduced OEF rather than the increased OEF that occurs in ischemia. Thus, the authors found no evidence for ischemia in the periclot zone of hypoperfusion in acute ICH patients studied 5 to 22 hours after hemorrhage onset.
Protein Kinase C and Cerebral Vasospasm
Authors: Laher Ismail , Zhang John H.
Published: November 2003

Twenty-five years after the discovery of protein kinase C (PKC), the physiologic function of PKC, and especially its role in pathologic conditions, remains a subject of great interest with 30,000 studies published on these aspects. In the cerebral circulation, PKC plays a role in the regulation of myogenic tone by sensitization of myofilaments to calcium. Protein kinase C phosphorylates various ion channels including augmenting voltage-dependent Ca2+ channels and inhibiting K+ channels, which both lead to vessel contraction. These actions of PKC amplify vascular reactivity to different agonists and may be critical in the regulation of cerebral artery tone during vasospasm. Evidence accumulated during at least the last decade suggest that activation of PKC in cerebral vasospasm results in a delayed but prolonged contraction of major arteries after subarachnoid hemorrhage. Most of the experimental results in vitro or in animal models support the view that PKC is involved in cerebral vasospasm. Implication of PKC in cerebral vasospasm helps explain increased arterial narrowing at the signal transduction level and alters current perceptions that the pathophysiology is caused by a combination of multiple receptor activation, hemoglobin toxicity, and damaged neurogenic control. Activation of protein kinase C also interacts with other signaling pathways such as myosin light chain kinase, nitric oxide, intracellular Ca2+, protein tyrosine kinase, and its substrates such as mitogen-activated protein kinase. Even though identifying PKC revolutionized the understanding of cerebral vasospasm, clinical advances are hampered by the lack of clinical trials using selective PKC inhibitors.
The Key Role of Caveolin-1 in Estrogen-Mediated Regulation of Endothelial Nitric Oxide Synthase Function in Cerebral Arterioles In vivo
Authors: Xu Hao-Liang , Galea Elena , Santizo Roberto A. , Baughman Verna L. , Pelligrino Dale A.
Published: November 2003

The marked impairment in cerebrovascular endothelial nitric oxide synthase (eNOS) function that develops after ovariectomy may relate to the observation that the abundance of cerebral vascular eNOS and its endogenous inhibitor, caveolin-1, vary in opposite directions with chronic changes in estrogen status. The authors endeavored, therefore, to establish a link between these correlative findings by independently manipulating, in ovariectomized female rats, eNOS and caveolin-1 expression, while monitoring agonist (acetylcholine)-stimulated eNOS functional activity. In the current study, the authors showed that individually neither the up-regulation of eNOS (through simvastatin treatment), nor the down-regulation of caveolin-1 (through antisense oligonucleotide administration) is capable of restoring eNOS function in pial arterioles in vivo in these estrogen-depleted rats. Only when eNOS up-regulation and caveolin-1 down-regulation are combined is activity normalized. These results establish a mechanistic link between the estrogen-associated divergent changes in the abundance of caveolin-1 and eNOS protein and eNOS functional activity in cerebral arterioles.
Oxidative Stress–Dependent Release of Mitochondrial Cytochrome c after Traumatic Brain Injury
Authors: Lewén Anders , Fujimura Miki , Sugawara Taku , Matz Paul , Copin Jean-Christophe , Chan Pak H.
Published: November 2003

Mitochondrial cytochrome c translocation to the cytosol initiates the mitochondrial-dependent apoptotic pathway. This event has not been previously reported in traumatic brain injury (TBI). The authors determined the expression of cytochrome c in cytosolic and mitochondrial fractions after severe TBI produced by the controlled cortical impact model in the mouse. One hour after trauma there was an increase in cytosolic cytochrome c immunoreactivity. The increases in cytosolic cytochrome c preceded DNA fragmentation, which started at 4 hours. Western blots of mitochondrial and cytosolic fractions confirmed that there was a translocation of cytochrome c from the mitochondria after TBI. Mice deficient in manganese superoxide dismutase (MnSOD) showed an increased loss of mitochondrial cytochrome c after trauma, but less apoptotic cell death 4 and 24 hours after injury compared with wild-type control mice. However, the overall cell death was increased in MnSOD mice, as illustrated by a larger cortical lesion in these animals. The results show that cytochrome c is released from the mitochondria after severe TBI partly by a free radical–dependent mechanism, and that massive mitochondrial cytochrome c release is a predictor of necrotic cell death rather than apoptosis.
Interactions between the Endothelium-Derived Relaxing Factor/Nitric Oxide System and the Endogenous Opiate System in the Modulation of Cerebral and Spinal Vascular CO2 Responsiveness
Authors: Komjáti Katalin , Greenberg Joel H. , Reivich Martin , Sándor Peter
Published: November 2003

The role of the L-arginine-nitric oxide (NO) system, the role of the endogenous morphine-like substances (endorphins), and the possible interaction between these two systems in the modulation of regional cerebral and spinal CO2 responsiveness was investigated in anesthetized, ventilated, normotensive, normoxic cats. Regional cerebral blood flow was measured with radiolabeled microspheres in hypocapnic, normocapnic, and hypercapnic conditions in nine individual cerebral and spinal cord regions. General opiate receptor blockade by 1 mg/kg naloxone intravenously alone or NO synthase blockade by 3 mg/kg Nω-nitro-L-arginine-methyl ester (L-NAME) intravenously alone caused no changes in regional CO2 responsiveness. Combined administration of these two blocking agents in the very same doses, however, resulted in a strong potentiation, with a statistically significant reduction of the CO2 responsiveness observed. Separation of the blood flow response to hypercapnia and hypocapnia indicates that this reduction occurs only during hypercapnia. Specific μ and δ opiate receptors were blocked by 0.5 mg kg−1 IV β-funaltrexamine and 0.4 mg kg−1 IV naltrindole, respectively. The role of specific μ and δ opiate receptors in the NO–opiate interaction was found to be negligible because neither μ nor δ receptor blockade along with simultaneous NO blockade were able to decrease CO2 responsiveness. The current findings suggest a previously unknown interaction between the endothelium-derived relaxing factor/nitric oxide (EDRF/NO) system and the endogenous opiate system in the cerebrovascular bed during hypercapnic stimulation, with the phenomenon not mediated by μ or δ opiate receptors.
Using 10CO2 for Single Subject Characterization of the Stimulus Frequency Dependence in Visual Cortex: A Novel Positron Emission Tomography Tracer for Human Brain Mapping
Authors: Law Ian , Jensen Mikael , Holm Søren , Nickles Robert J. , Paulson Olaf B.
Published: November 2003

Carbon-10–labeled carbon dioxide (10CO2) with a half-life of 19.3 seconds offers almost ideal characteristics as a positron emission tomography (PET) tracer for assessment of the regional cerebral blood flow (rCBF) distribution, enabling multiple independent measurements at short intervals. To appraise the feasibility of 10CO2 for localizing and characterizing human brain function in single subjects, the authors chose a well-characterized activation paradigm. In 6 healthy volunteers, 50 to 64 independent PET scans of the rCBF distribution were acquired while viewing an annular reversing checkerboard presented at 10 reversal frequencies between 0.03 and 30 Hz. Changes in regional cerebral activity as a function of reversal frequency were modeled in every subject using a set of polynomial basis functions, which, as predicted, showed highly significant second or third order relations located in the striatal cortex. Correlation coefficients (R2) ranged from 0.46 to 0.63. The average intersubject maximal response relative to the 0.03 Hz condition was 8.0% ± 1.7% SD occurring at stimulus contrast reversal frequencies between 6 and 15 Hz with an average of 11.8 ± 3.8 (SD) Hz. From the qualitative and quantitative replication of previous results it is concluded that 10CO2 PET is a feasible technique for human brain mapping studies and a great improvement compared with the existing oxygen-15–labeled water (H215 O) PET method, particularly for single subject studies and parametric design.
Hypoxic Preconditioning Induces Changes in HIF-1 Target Genes in Neonatal Rat Brain
Authors: Jones Nicole M. , Bergeron Marcelle
Published: November 2003

Hypoxic preconditioning induces tolerance to hypoxic-ischemic injury in neonatal rat brain and is associated with changes in gene expression. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that is strongly induced by hypoxia or the hypoxia-mimetic compound cobalt chloride (CoCl2). Hypoxia-inducible factor-1 modulates the expression of several target genes including the glycolytic enzymes, glucose transporter-1 (GLUT-1), and erythropoietin. Recently, HIF-1 expression was shown to increase after hypoxic and CoCl2 preconditioning in newborn rat brain. To study the involvement of HIF-1 target genes in neonatal hypoxia-induced ischemic tolerance, the authors examined the brains of newborn rats after exposure to hypoxia (8% O2 for 3 hours) or injection of CoCl2 (60 mg/kg). Preconditioning with hypoxia or CoCl2 24 hours before hypoxia-ischemia afforded a 96% and 76% brain protection, respectively, compared with littermate control animals. Hypoxic preconditioning increased the expression of GLUT-1 mRNA and protein, and of aldolase, phosphofructokinase, and lactate dehydrogenase proteins but not mRNA. This suggests that the modulation of glucose transport and glycolysis by hypoxia may contribute to the development of hypoxia-induced tolerance. In contrast, preconditioning with CoCl2 did not produce any change in HIF-1 target gene expression suggesting that different molecular mechanisms may be involved in the induction of tolerance by hypoxia and CoCl2 in newborn brain.
A Review and Rationale for the Use of Genetically Engineered Animals in the Study of Traumatic Brain Injury
Authors: Longhi Luca , Saatman Kathryn E. , Raghupathi Ramesh , Laurer Helmut L. , Lenzlinger Philipp M. , Riess Peter , Neugebauer Edmund , Trojanowski John Q. , Lee Virginia M.-Y. , Grady M. Sean , Graham David I. , McIntosh Tracy K.
Published: November 2003

The mechanisms underlying secondary cell death after traumatic brain injury (TBI) are poorly understood. Animal models of TBI recapitulate many clinical and pathologic aspects of human head injury, and the development of genetically engineered animals has offered the opportunity to investigate the specific molecular and cellular mechanisms associated with cell dysfunction and death after TBI, allowing for the evaluation of specific cause-effect relations and mechanistic hypotheses. This article represents a compendium of the current literature using genetically engineered mice in studies designed to better understand the posttraumatic inflammatory response, the mechanisms underlying DNA damage, repair, and cell death, and the link between TBI and neurodegenerative diseases.
Neuroprotective Effect of Sodium Orthovanadate on Delayed Neuronal Death after Transient Forebrain Ischemia in Gerbil Hippocampus
Authors: Kawano Takayuki , Fukunaga Kohji , Takeuchi Yusuke , Morioka Motohiro , Yano Shigetoshi , Hamada Jun-Ichiro , Ushio Yukitaka , Miyamoto Eishichi
Published: November 2003

In transient forebrain ischemia, sodium orthovanadate as well as insulinlike growth factor-1 (IGF-1) rescued cells from delayed neuronal death in the hippocampal CA1 region. Adult Mongolian gerbils were subjected to 5-minute forebrain ischemia. Immunoblotting analysis with anti–phospho-Akt/PKB (Akt) antibody showed that phosphorylation of Akt at serine-473 (Akt-Ser-473) in the CA1 region decreased immediately after reperfusion, and in turn transiently increased 6 hours after reperfusion. The decreased phosphorylation of Akt-Ser-473 was not observed in the CA3 region. The authors then tested effects of intraventricular injection of orthovanadate and IGF-1, which are known to activate Akt. Treatment with orthovanadate or IGF-1 30 minutes before ischemia blocked delayed neuronal death in the CA1 region. The neuroprotective effects of orthovanadate and IGF-1 were associated with preventing decreased Akt-Ser-473 phosphorylation in the CA1 region observed immediately after reperfusion. Immunohistochemical studies with the anti–phospho-Akt-Ser-473 antibody also demonstrated that Akt was predominantly in the nucleus and was moderately activated in the cell bodies and dendrites of pyramidal neurons after orthovanadate treatment. The orthovanadate treatment also prevented the decrease in phosphorylation of mitogen-activated protein kinase (MAPK). Pretreatment with combined blockade of phosphatidylinositol 3-kinase and MAPK pathways totally abolished the orthovanadate-induced neuroprotective effect. These results suggest that the activation of both Akt and MAPK activities underlie the neuroprotective effects of orthovanadate on the delayed neuronal death in the CA1 region after transient forebrain ischemia.
Positron Emission Tomography Quantification of [11C]-DASB Binding to the Human Serotonin Transporter: Modeling Strategies
Authors: Ginovart Nathalie , Wilson Alan A. , Meyer Jeffrey H. , Hussey Doug , Houle Sylvain
Published: November 2003

[11C]-DASB, namely [11C]-3-amino-4-(2-dimethylaminomethyl-phenylsulfanyl)-benzonitrile, is a new highly selective radioligand for the in vivo visualization of the serotonin transporter (SERT) using positron emission tomography (PET). The current study evaluates different kinetic modeling strategies for quantification of [11C]-DASB binding in five healthy humans. Kinetic analyses of tissue data were performed with a one-tissue (1CM) and a two-tissue (2CM) compartment model. Time-activity curves were well described by a 1CM for all regions. A 2CM model with four parameters failed to converge reliably. Reliable fits of the data were obtained only if no more than three parameters were allowed to vary. However, even then, the rate constants k3 and k4 were estimated with poor precision. Only the ratio k3/k4 was stable. Goodness of fit was not improved by using a 2CM as compared with a 1CM. The minimal study duration required to obtain stable k3/k4 estimates was 80 minutes. For routine use of [11C]-DASB, several simplified methods using the cerebellum as a reference region to estimate nonspecific binding were also evaluated. The transient equilibrium, the linear graphical analysis, the ratio of target to reference region, and the simplified reference tissue methods all gave binding potential values consistent with those obtained with the 2CM. The suitability of [11C]-DASB for research on the SERT using PET is thus supported by the observations that tissue data can be described using a kinetic analysis and that simplified quantitative methods, using the cerebellum as reference, provide reliable estimates of SERT binding parameters.
Relationship of Spikes, Synaptic Activity, and Local Changes of Cerebral Blood Flow
Authors: Lauritzen Martin
Published: November 2003

The coupling of electrical activity in the brain to changes in cerebral blood flow (CBF) is of interest because hemodynamic changes are used to track brain function. Recent studies, especially those investigating the cerebellar cortex, have shown that the spike rate in the principal target cell of a brain region (i.e. the efferent cell) does not affect vascular response amplitude. Subthreshold integrative synaptic processes trigger changes in the local microcirculation and local glucose consumption. The spatial specificity of the vascular response on the brain surface is limited because of the functional anatomy of the pial vessels. Within the cortex there is a characteristic laminar flow distribution, the largest changes of which are observed at the depth of maximal synaptic activity (i.e. layer IV) for an afferent input system. Under most conditions, increases in CBF are explained by activity in postsynaptic neurons, but presynaptic elements can contribute. Neurotransmitters do not mediate increases in CBF that are triggered by the concerted action of several second messenger molecules. It is important to distinguish between effective synaptic inhibition and deactivation that increase and decrease CBF and glucose consumption, respectively. In summary, hemodynamic changes evoked by neuronal activity depend on the afferent input function (i.e. all aspects of presynaptic and postsynaptic processing), but are totally independent of the efferent function (i.e., the spike rate of the same region). Thus, it is not possible to conclude whether the output level of activity of a region is increased based on brain maps that use blood-flow changes as markers.
Matrix Metalloproteinase Inhibition Prevents Oxidative Stress-Associated Blood–Brain Barrier Disruption after Transient Focal Cerebral Ischemia
Authors: Gasche Yvan , Copin Jean-Christophe , Sugawara Taku , Fujimura Miki , Chan Pak H.
Published: November 2003

Oxidative stress generated during stroke is a critical event leading to blood–brain barrier (BBB) disruption with secondary vasogenic edema and hemorrhagic transformation of infarcted brain tissue, restricting the benefit of thrombolytic reperfusion. In this study, the authors demonstrate that ischemia-reperfusion–induced BBB disruption in mice deficient in copper/zinc-superoxide dismutase (SOD1) was reduced by 88% ( P < 0.0001) and 73% ( P < 0.01), respectively, after 3 and 7 hours of reperfusion occurring after 1 hour of ischemia by the inhibition of matrix metalloproteinases. Accordingly, the authors show that local metalloproteinase-generated proteolytic imbalance is more intense in ischemic regions of SOD1 mice than in wild-type litter mates. Moreover, active in situ proteolysis is, for the first time, demonstrated in ischemic leaking capillaries that produce reactive oxygen species. By showing that oxidative stress mediates BBB disruption through metalloproteinase activation in experimental ischemic stroke, this study provides a new target for future therapeutic strategies to prevent BBB disruption and potentially reperfusion-triggered intracerebral hemorrhage.
Increased Susceptibility to Ischemic Brain Injury in Cyclooxygenase-1–Deficient Mice
Authors: Iadecola Costantino , Sugimoto Koreaki , Niwa Kiyoshi , Kazama Ken , Ross M. Elizabeth
Published: November 2003

Cyclooxygenase-1 (COX-1), a rate-limiting enzyme in the synthesis of prostanoids, is involved in selected vasodilatatory responses of the cerebral circulation. Cyclooxygenase-1–null mice were used to determine whether COX-1 influences cerebral ischemic damage. The middle cerebral artery was occluded in COX-1 −/− and +/+ mice (n = 9/group), and lesion volume was determined in thionin-stained sections 24 or 96 hours later. Middle cerebral artery occlusion produced larger infarcts in COX-1 −/− mice, both at 24 (35 ± 17%; P < 0.05) and 96 hours (41 ± 16%; P < 0.05) after ischemia. The enlargement was not due to increased susceptibility to glutamate excitotoxicity, because microinjection of N-methl- d -asparatate or kainate in the parietal cortex produced comparable lesions in COX-1 +/+ and −/− mice ( P > 0.05; n = 8/group). To examine the contribution of hemodynamic factors to the enlargement of the infarct, cerebral blood flow was monitored by laser-Doppler flowmetry in the ischemic territory (n = 6/group). Although the reduction in cerebral blood flow was comparable in the ischemic core ( P > 0.05), at the periphery of the ischemic territory the reduction was greater in COX-1 −/− mice (−58 ± 4%) than in COX-1 +/+ mice (−34 ± 5%; P < 0.05). It is concluded that mice lacking COX-1 are more susceptible to focal cerebral ischemia, an effect that can be attributed to a more severe cerebral blood flow reduction in vulnerable regions at the periphery of the ischemic territory. Thus, the vascular effects of COX-1 may contribute to maintain cerebral blood flow in the postischemic brain and, as such, play a protective role in ischemic brain injury.
Evidence of Phosphorylation of Akt and Neuronal Survival after Transient Focal Cerebral Ischemia in Mice
Authors: Noshita Nobuo , Lewén Anders , Sugawara Taku , Chan Pak H.
Published: November 2003

The serine-threonine kinase, Akt, prevents apoptosis by phosphorylation at serine-473 in several cell systems. After phosphorylation, activated Akt inactivates other apoptogenic factors, such as Bad or caspase-9, thereby inhibiting cell death. The present study examined phosphorylation of Akt at serine-473 and DNA fragmentation after transient focal cerebral ischemia in mice subjected to 60 minutes of focal cerebral ischemia by intraluminal blockade of the middle cerebral artery. Phospho-Akt was analyzed by immunohistochemistry and Western blot analysis. The DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end-labeling (TUNEL). Immunohistochemistry showed the expression of phospho-Akt was markedly increased in the middle cerebral artery territory cortex at 4 hours of reperfusion compared with the control, whereas it was decreased by 24 hours. Western blot analysis showed a significant increase of phospho-Akt 4 hours after focal cerebral ischemia in the cortex, whereas phospho-Akt was decreased in the ischemic core. Double staining with phospho-Akt and TUNEL showed different cellular distributions of phospho-Akt and TUNEL-positive staining. Phosphorylation of Akt was prevented after focal cerebral ischemia by LY294002, a phosphatidylinositol 3-kinase inhibitor, which facilitated subsequent DNA fragmentation. These results suggest that phosphorylation of Akt may be involved in determining cell survival or cell death after transient focal cerebral ischemia.
Differences in Neurotransmitter Synthesis and Intermediary Metabolism between Glutamatergic and GABAergic Neurons during 4 Hours of Middle Cerebral Artery Occlusion in the Rat: The Role of Astrocytes in Neuronal Survival
Authors: Håberg Asta , Qu Hong , Sæther Oddbjørn , Unsgård Geirmund , Haraldseth Olav , Sonnewald Ursula
Published: November 2003

Astrocytes are intimately involved in both glutamate and γ-aminobutric acid (GABA) synthesis, and ischemia-induced disruption of normal neuroastrocytic interactions may have important implications for neuronal survival. The effects of middle cerebral artery occlusion (MCAO) on neuronal and astrocytic intermediary metabolism were studied in rats 30, 60, 120, and 240 minutes after MCAO using in vivo injection of [1-13C]glucose and [1,2-13C]acetate combined with ex vivo13C magnetic resonance spectroscopy and high-performance liquid chromatography analysis of the ischemic core (lateral caudoputamen and lower parietal cortex) and penumbra (upper frontoparietal cortex). In the ischemic core, both neuronal and astrocytic metabolism were impaired from 30 minutes MCAO. There was a continuous loss of glutamate from glutamatergic neurons that was not replaced as neuronal glucose metabolism and use of astrocytic precursors gradually declined. In GABAergic neurons astrocytic precursors were not used in GABA synthesis at any time after MCAO, and neuronal glucose metabolism and GABA-shunt activity declined with time. No flux through the tricarboxylic acid cycle was found in GABAergic neurons at 240 minutes MCAO, indicating neuronal death. In the penumbra, the neurotransmitter pool of glutamate coming from astrocytic glutamine was preserved while neuronal metabolism progressively declined, implying that glutamine contributed significantly to glutamate excitotoxicity. In GABAergic neurons, astrocytic precursors were used to a limited extent during the initial 120 minutes, and tricarboxylic acid cycle activity was continued for 240 minutes. The present study showed the paradoxical role that astrocytes play in neuronal survival in ischemia, and changes in the use of astrocytic precursors appeared to contribute significantly to neuronal death, albeit through different mechanisms in glutamatergic and GABAergic neurons.
Parametric Images of the Extrastriatal D2 Receptor Density Obtained Using a High-Affinity Ligand (FLB 457) and a Double-Saturation Method
Authors: Delforge Jacques , Bottlaender Michel , Loc'h Christian , Dolle Frédéric , Syrota André
Published: November 2003

The potential of positron emission tomography for the quantitative estimation of receptor concentration in extrastriatal regions has been limited in the past because of the low density of the D2 receptor sites in these regions and the insufficient affinity of the most widely used radioligands for dopamine receptors. The new method described in this paper permits the estimate of the D2 receptor concentration in the extrastriatal regions using a two-injection protocol and FLB 457, a ligand with a high affinity (20 pmol/L in vitro) with D2 dopamine receptors. This approach is not valid for the striatal regions because some hypotheses cannot be verified (because of the high receptor concentration in these regions). The experimental protocol includes two injections with ligand doses designed to significantly occupy the extrastriatal receptor sites (≈ 90%), while leaving less than 60% of the receptor sites occupied by the ligand in the striatal regions. The results obtained using this double-saturation method are in line with the concentration estimates previously obtained using the multiinjection approach. The receptor concentration is 2.9 ± 0.5 pmol/mL in the thalamus, 1.0 ± 0.2 pmol/mL in the temporal cortex, and 0.35 ± 0.13 pmol/mL in the occipital cortex. This study provides new arguments supporting the presence of a small receptor-site concentration in the cerebellum, estimated at 0.35 ± 0.16 pmol/mL The simplicity of the calculation used to estimate the receptor concentration lends itself easily to parametric imaging. The receptor concentration is estimated pixel by pixel, without filtering. This method permits estimation of the extrastriatal D2 receptor concentration using an experimental protocol that can easily be used in patient studies (i.e., single experiment, no blood sampling, short experiment duration).
Induction of Caspase-Activated Deoxyribonuclease Activity after Focal Cerebral Ischemia and Reperfusion
Authors: Luo Yumin , Cao Guodong , Pei Wei , O'Horo Cristine , Graham Steven H. , Chen Jun
Published: November 2003

Deoxyribonucleic acid fragmentation at nucleosomal junctions is a hallmark of neuronal apoptosis in ischemic brain injury, for which the mechanism is not fully understood. Using the in vitro cell-free apoptosis assay, the authors found that caspase-3–dependent deoxyribonuclease activity caused internucleosomal DNA fragmentation in brain-cell extracts in a rat model of transient focal ischemia. This in vitro deoxyribonuclease activity was completely inhibited by purified inhibitor of caspase-activated deoxyribonuclease protein, the specific endogenous inhibitor of caspase-activated deoxyribonuclease, or by caspase-activated deoxyribonuclease immunodepletion. The induction of the deoxyribonuclease activity was correlated with caspase-3 activation and caspase-3–mediated degradation of inhibitor of caspase-activated deoxyribonuclease. Furthermore, inhibiting caspase-3–like protease activity prevented the endogenous induction of internucleosomal DNA fragmentation in the ischemic brain. These results suggest that caspase-3–dependent caspase-activated deoxyribonuclease activity plays an important role in mediating DNA fragmentation after focal ischemia.
Water Compartmentalization and Spread of Ischemic Injury in Thick-Slice Ischemia Model
Authors: Hrabětová Sabina , Chen Kevin C. , Masri Daniel , Nicholson Charles
Published: November 2003

Water compartmentalization was studied in a thick-slice (1000 μm) model of ischemia by combining water-content measurements with extracellular diffusion analysis. Thick slices bathed in artificial cerebrospinal fluid continually gained water. Total tissue water content was increased by 67% after 6 hours of the incubation. Diffusion measurements using the tetramethylammonium method showed that the extracellular space, typically occupying 20% of brain tissue in vivo, was decreased to 10% at 30 minutes and 15% at 6 hours in both deep and superficial layers of thick slices. Quantification of water compartmentalization revealed that water moved initially from the extracellular space into the cells. Later, however, both compartments gained water. The initial cell swelling was accompanied by dramatic shifts in potassium. An initial rise of extracellular potassium to about 50 mmol/L was measured with a potassium-selective microelectrode positioned in the center of the thick slice; the concentration decreased slowly afterwards. Potassium content analysis revealed a 63% loss of tissue potassium within two hours of the incubation. In thick slices, ionic shifts, water redistribution, and a loss of synaptic transmission occur in both deep and superficial layers, indicating the spread of ischemic conditions even to areas with an unrestricted supply of nutrients.
Rapid Tolerance to Focal Cerebral Ischemia in Rats is Attenuated by Adenosine A1 Receptor Antagonist
Authors: Nakamura Michiko , Nakakimura Kazuhiko , Matsumoto Mishiya , Sakabe Takefumi
Published: November 2003

Two types of ischemic tolerance in the brain, rapid and delayed, have been reported in terms of the interval between the conditioning and test insults. Although many reports showed that delayed-phase neuroprotection evoked by preconditioning is evident after 1 week or longer, there have been a few investigations about rapidly induced tolerance, and the reported neuroprotective effects become ambiguous 7 days after the insults. The authors examined whether this rapid ischemic tolerance exists after 7 days of reperfusion in a rat focal ischemic model, and investigated modulating effects of the adenosine A1 receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine). Preconditioning with 30 minutes of middle cerebral artery occlusion reduced infarct volume 7 days after 180 minutes of subsequent focal ischemia given after 1-hour reperfusion. The rapid preconditioning also improved neurologic outcome. These beneficial effects were attenuated by pretreatment of 0.1 mg/kg DPCPX, which did not influence the infarct volume after conditioning (30 minutes) or test (180 minutes) ischemia when given alone. The results show that preconditioning with a brief focal ischemia induces rapid tolerance to a subsequent severe ischemic insult, the effect of which is still present after 7 days of reperfusion, and that the rapid ischemic tolerance is possibly mediated through an adenosine A1 receptor–related mechanism.
Imaging Brain Amyloid of Alzheimer Disease in Vivo in Transgenic Mice with an Aβ Peptide Radiopharmaceutical
Authors: Lee Hwa Jeong , Zhang Yun , Zhu Chunni , Duff Karen , Pardridge William M.
Published: November 2003

Aβ1–40 is a potential peptide radiopharmaceutical that could be used to image the brain Aβ amyloid of Alzheimer disease in vivo, should this peptide be made transportable through the blood–brain barrier in vivo. The blood–brain barrier transport of [125I]-Aβ1–40 in a transgenic mouse model was enabled by conjugation to the rat 8D3 monoclonal antibody to the mouse transferrin receptor. The Aβ1–40–8D3 conjugate is a bifunctional molecule that binds the blood–brain barrier TfR and undergoes transport into brain and binds the Aβ amyloid plaques of Alzheimer disease. App SW/ Psen1 double-transgenic and littermate control mice were administered either unconjugated Aβ1–40 or the Aβ1–40–8D3 conjugate intravenously, and brain scans were obtained 6 hours later. Immunocytochemical analysis showed abundant Aβ immunoreactive plaques in the brains of the App SW/ Psen1 transgenic mice and there was a selective retention of radioactivity in the brains of these mice at 6 hours after intravenous administration of the conjugate. In contrast, there was no selective sequestration either of the conjugate in control littermate mouse brain or of unconjugated Aβ1–40 in transgenic mouse brain. In conclusion, the results show that it is possible to image the Aβ amyloid burden in the brain in vivo with an amyloid imaging agent, provided the molecule is conjugated to a blood–brain barrier drug-targeting system.
Vascular Genomics of the Human Brain
Authors: Shusta Eric V. , Boado Ruben J. , Mathern Gary W. , Pardridge William M.
Published: November 2003

The microvasculature of the human brain plays an important role in the development and maintenance of the central nervous system and in the pathogenesis of brain diseases, and is the site of differential gene expression within the brain. However, human brain microvascular-specific genes may not be detected in whole-brain gene microarray because the volume of the brain microvascular endothelium is relatively small (0.1%) compared with the whole brain. Therefore, the differential gene expression within the human brain microvasculature was evaluated using suppression subtractive hybridization with RNA isolated from human brain microvessels. Gene identification was restricted to the first 71 clones that were differentially expressed at the brain microvasculature. Twenty of these were genes encoding proteins with known function that were involved in angiogenesis, neurogenesis, molecular transport, and maintenance of endothelial tight junctions or the cytoskeleton. Eighteen genes coding for proteins of an unknown function were identified, including five genes containing satellite DNA sequences. The results provide the initial outline of the genomics of the human brain microvasculature, and have implications for the identification of both targets for brain-specific drug transport and changes in microvascular gene expression in brain diseases.
Differential Activation of Mitogen-Activated Protein Kinase Pathways after Traumatic Brain Injury in the Rat Hippocampus
Authors: Otani Naoki , Nawashiro Hiroshi , Fukui Shinji , Nomura Namiko , Yano Akiko , Miyazawa Takahito , Shima Katsuji
Published: November 2003

Mitogen-activated protein kinases, which play a crucial role in signal transduction, are activated by phosphorylation in response to a variety of mitogenic signals. In the present study, the authors used Western blot analysis and immunohistochemistry to show that phosphorylated extracellular signal-regulated protein kinase (p-ERK) and c-Jun NH(2)-terminal kinase (p-JNK), but not p38 mitogen-activated protein kinase, significantly increased in both the neurons and astrocytes after traumatic brain injury in the rat hippocampus. Different immunoreactivities of p-ERK and p-JNK were observed in the pyramidal cell layers and dentate hilar cells immediately after traumatic brain injury. Immunoreactivity for p-JNK was uniformly induced but was only transiently induced throughout all pyramidal cell layers. However, strong immunoreactivity for p-ERK was observed in the dentate hilar cells and the damaged CA3 neurons, along with the appearance of pyknotic morphologic changes. In addition, immunoreactivity for p-ERK was seen in astrocytes surrounding dentate and CA3 pyramidal neurons 6 hours after traumatic brain injury. These findings suggest that ERK and JNK but not p38 cascades may be closely involved in signal transduction in the rat hippocampus after traumatic brain injury.
Correlation of VEGF and Angiopoietin Expression with Disruption of Blood–Brain Barrier and Angiogenesis after Focal Cerebral Ischemia
Authors: Zhang Zheng Gang , Zhang Li , Tsang Wayne , Soltanian-Zadeh Hamid , Morris Daniel , Zhang Ruilan , Goussev Anton , Powers Cecylia , Yeich Thomas , Chopp Michael
Published: November 2003

In an effort to elucidate the molecular mechanisms underlying cerebral vascular alteration after stroke, the authors measured the spatial and temporal profiles of blood–brain barrier (BBB) leakage, angiogenesis, vascular endothelial growth factor (VEGF), associated receptors, and angiopoietins and receptors after embolic stroke in the rat. Two to four hours after onset of ischemia, VEGF mRNA increased, whereas angiopoietin 1 (Ang 1) mRNA decreased. Three-dimensional immunofluorescent analysis revealed spatial coincidence between increases of VEGF immunoreactivity and BBB leakage in the ischemic core. Two to 28 days after the onset of stroke, increased expression of VEGF/VEGF receptors and Ang/Tie2 was detected at the boundary of the ischemic lesion. Concurrently, enlarged and thin-walled vessels were detected at the boundary of the ischemic lesion, and these vessels developed into smaller vessels via sprouting and intussusception. Three-dimensional quantitative analysis of cerebral vessels at the boundary zone 14 days after ischemia revealed a significant ( P < 0.05) increase in numbers of vessels (n = 365) compared with numbers (n = 66) in the homologous tissue of the contralateral hemisphere. Furthermore, capillaries in the penumbra had a significantly smaller diameter (4.8 ± 2.0 μm) than capillaries (5.4 ± 1.5 μm) in the homologous regions of the contralateral hemisphere. Together, these data suggest that acute alteration of VEGF and Ang 1 in the ischemic core may mediate BBB leakage, whereas upregulation of VEGF/VEGF receptors and Ang/Tie2 at the boundary zone may regulate neovascularization in ischemic brain.