bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolution of Microcirculatory Disturbances after Permanent Middle Cerebral Artery Occlusion in Rats
Authors: Vogel Johannes , Hermes Alexander , Kuschinsky Wolfgang
Published: November 2003

Nonischemic brain capillaries show a continuous and heterogeneous plasma perfusion. In the current study, plasma perfusion was investigated in rats during 2 to 168 hours of permanent middle cerebral artery occlusion. Perfused capillaries were detected in brain cryosections by fluorescein isothyocanate (FITC) dextran after 10 minutes of circulation time. Heterogeneity of capillary perfusion was identified by Evans blue (EB), which circulated for 3 seconds. In this setting, the heterogeneity of intracapillary EB concentrations reflects heterogeneities in capillary flow velocities. The CBF was quantified by simultaneous iodo[14C]antipyrine autoradiography. When moving from normal flow to low-flow areas in the ischemic hemisphere, three states of capillary filling could be distinguished: state 1—fast perfusion, filling by FITC dextran and EB (CBF 0.33 mL • g–1 • min–1); state 2—delayed perfusion, only FITC dextran filling (CBF 0.104 mL • g–1 • min–1); state 3— minimal perfusion, no dye filling (CBF 0.056 mL • g–1 • min−1). In tissue of state 1 at the borderline to ischemic tissue, a higher heterogeneity of intracapillary EB concentration (85.7%) was found than in the contralateral nonischemic hemisphere (76.4%) ( P < 0.05), indicating a compromised microcirculation. The adjacent ischemic areas were filled by FITC dextran (state 2) 2 to 4 hours after middle cerebral artery occlusion, indicating a maintained, although slow, perfusion at this time. Later, minimal perfused areas (state 3) progressively replaced the delayed perfused areas (state 2). This study shows, for the first time, the evolution of microvascular disturbances in relation to CBF. In the low-flow areas, an early residual plasma perfusion is later followed by a lack of perfusion or minimal perfusion. In areas of higher, although reduced flow at the border between normal and ischemic tissue, an extreme capillary perfusion heterogeneity indicates permanent microcirculatory abnormalities.
Transplantation of Fetal Kidney Tissue Reduces Cerebral Infarction Induced by Middle Cerebral Artery Ligation
Authors: Chiang Yung-Hsiao , Lin Shinn-Zong , Borlongan Cesario V. , Hoffer Barry J. , Morales Marisela , Wang Yun
Published: November 2003

The authors, and others, have recently reported that intracerebral administration of glial cell line-derived neurotrophic factor (GDNF) or osteogenic protein-1 protects against ischemia-induced injury in the cerebral cortex of adult rats. Because these trophic factors are highly expressed in the fetal, but not adult, kidney cortex, the possibility that transplantation of fetal kidney tissue could serve as a cellular reservoir for such molecules and protect against ischemic injury in cerebral cortex was examined. Fetal kidneys obtained from rat embryos at gestational day 16, and adult kidney cortex, were dissected and cut into small pieces. Adult male Sprague-Dawley rats were anesthetized with chloral hydrate and placed in a stereotactic apparatus. Kidney tissues were transplanted into three cortical areas adjacent to the right middle cerebral artery (MCA). Thirty minutes after grafting, the right MCA was transiently ligated for 90 minutes. Twenty-four hours after the onset of reperfusion, animals were evaluated behaviorally. It was found that the stroke animals that received adult kidney transplantation developed motor imbalance. However, animals that received fetal kidney grafts showed significant behavioral improvement. Animals were later sacrificed and brains were removed for triphenyltetrazolium chloride staining, Pax-2 immunostaining, and GDNF mRNA expression. It was noted that transplantation of fetal kidney but not adult kidney tissue greatly reduced the volume of infarction in the cerebral cortex. Fetal kidney grafts showed Pax-2 immunoreactivity and GDNF mRNA in the host cerebral cortex. In contrast, GDNF mRNA expression was not found in the adult kidney grafts. Taken together, our data indicate that fetal kidney transplantation reduces ischemia/reperfusion-induced cortical infarction and behavioral deficits in adult rats, and that such tissue grafts could serve as an unique cellular reservoir for trophic factor application to the brain.
Adenovirus-Mediated Gene Transfer of Glial Cell Line-Derived Neurotrophic Factor Prevents Ischemic Brain Injury after Transient Middle Cerebral Artery Occlusion in Rats
Authors: Kitagawa Hisashi , Sasaki Chihoko , Sakai Kenichi , Mori Atsushi , Mitsumoto Yasuhide , Mori Toyoki , Fukuchi Yoshimosuke , Setoguchi Yasuhiro , Abe Koji
Published: November 2003

To examine a possible protective effect of exogenous glial cell line-derived neurotrophic factor (GDNF) gene expression against ischemic brain injury, a replication-defective adenoviral vector containing GDNF gene (Ad-GDNF) was directly injected into the cerebral cortex at 1 day before 90 minutes of transient middle cerebral artery occlusion (MCAO) in rats. 2,3,5-Triphenyltetrazolium chloride staining showed that infarct volume of the Ad-GDNF-injected group at 24 hours after the transient MCAO was significantly smaller than that of vehicle- or Ad-LacZ-treated group. Enzyme-linked immunosorbent assay (ELISA) for immunoreactive GDNF demonstrated that GDNF gene products in the Ad-GDNF-injected group were higher than those of vehicle-treated group at 24 hours after transient MCAO. Immunoreactive GDNF staining was obviously detected in the cortex around the needle track just before or 24 hours after MCAO in the Ad-GDNF group, whereas no or slight GDNF staining was detected in the vehicle group. The numbers of TUNEL, immunoreactive caspase-3, and cytochrome c-positive neurons induced in the ipsilateral cerebral cortex at 24 hours after transient MCAO were markedly reduced by the Ad-GDNF group. These results suggest that the successful exogenous GDNF gene transfer ameliorates ischemic brain injury after transient MCAO in association with the reduction of apoptotic signals.
Secondary Reduction in the Apparent Diffusion Coefficient of Water, Increase in Cerebral Blood Volume, and Delayed Neuronal Death after Middle Cerebral Artery Occlusion and Early Reperfusion in the Rat
Authors: Campagne Menno van Lookeren , Thomas G. Roger , Thibodeaux Harold , Palmer James T. , Williams Simon P. , Lowe David G. , van Bruggen Nicholas
Published: November 2003

It has been reported recently that very delayed damage can occur as a result of focal cerebral ischemia induced by vascular occlusion of short duration. With use of diffusion-, T2-, and contrast-enhanced dynamic magnetic resonance imaging (MRI) techniques, the occlusion time dependence together with the temporal profile for this delayed response in a rat model of transient focal cortical ischemia have been established. The distal branch of the middle cerebral artery was occluded for 20, 30, 45, or 90 minutes. Twenty minutes of vascular occlusion with reperfusion exhibited no significant mean change in either the apparent diffusion coefficient of water (ADC) or the T2 relaxation time at 6, 24, 48, or 72 hours after reperfusion ( P = 0.97 and 0.70, respectively). Ninety minutes of ischemia caused dramatic tissue injury at 6 hours, as indicated by an increase in T2 relaxation times to 135% of the contralateral values ( P < 0.01). However, at intermediate periods of ischemia (30 to 45 minutes), complete reversal of the ADC was seen at 6 hours after reperfusion but was followed by a secondary decline over time, such that a 25% reduction in tissue ADC was seen at 24 as compared with 6 hours ( P < 0.02). This secondary response was accompanied by an increase in cerebral blood volume (CBV), as shown by contrast-enhanced dynamic MRI (120% of contralateral values; P < 0.001), an increase in T2 relaxation time (132%; P < 0.01), together with clear morphological signs of cell death. By day 18, the mean volume of missing cortical tissue measured with high-resolution MRI in animals occluded for 30 and 45 minutes was 50% smaller than that in 90-minute occluded animals ( P < 0.005). These data show that ultimate infarct size is reduced after early reperfusion and is occlusion time dependent. The early tissue recovery that is seen with intermediate occlusion times can be followed by cell death, which has a delayed onset and is accompanied by an increase in CBV.
Assessment of Extrastriatal Vesicular Monoamine Transporter Binding Site Density Using Stereoisomers of [11C]Dihydrotetrabenazine
Authors: Koeppe Robert A. , Frey Kirk A. , Kuhl David E. , Kilbourn Michael R.
Published: November 2003

Previous studies have demonstrated the utility of [nC]dihydrotetrabenazine ([11C]DTBZ) as a ligand for in vivo imaging of the vesicular monoamine transporter system. The (+)-isomer has a high affinity (approximately 1 nmol/L) for the vesicular monoamine transporter (VMAT2) binding site, whereas the (–)-isomer has an extremely low affinity (approximately 2 μmol/L). Efforts to model dynamic (+)-[11C]DTBZ data demonstrate the difficulty in separating the specific binding component from the free plus nonspecific component of the total positron emission tomography (PET) measure. The authors' previous*** PET work, as well as in vitro studies, indicate that there is little specific VMAT2 binding in neocortical regions. However, precise determination of in vivo binding levels have not been made, leaving important questions unanswered. At one extreme, is there sufficient specific binding in cortex or other extrastriate regions to be estimated reliably with PET? At the other extreme, is there sufficiently little binding in cortex so that it can be used as a reference region representing nonsaturable tracer uptake? The authors address these questions using paired studies with both active (+) and inactive (–) stereoisomers of [11C]DTBZ. Six normal control subjects were scanned twice, 2 hours apart, after injections of 16 mCi of (+)- and (–)-[11C]DTBZ (order counter-balanced). Three-dimensional PET acquisition consisted of 15 frames over 60 minutes for each scan. Arterial samples were acquired throughout, plasma counted, and corrected for radiolabeled metabolites. Analysis of specific binding was assessed by comparison of total distribution volume measures from the (+)- and (–)-[11C]DTBZ scans. The authors' findings indicate that only approximately 5% of the cortical signal in (+)-[11C]DTBZ scans results from binding to VMAT2 sites. The strongest extrastriatal signal comes from the midbrain regions where approximately 30% of the PET measure results from specific binding. The authors conclude that (1) the density of VMAT2 binding sites in cortical regions is not high enough to be quantified reliably with DTBZ PET, and (2) binding does appear to be low enough so that cortex can be used as a free plus nonspecific reference region for striatum.
Quantitative Measurement of Local Cerebral Blood Flow in the Anesthetized Mouse Using Intraperitoneal [14C]Iodoantipyrine Injection and Final Arterial Heart Blood Sampling
Authors: Maeda Keiichiro , Mies Günter , Oláh László , Hossmann Konstantin-Alexander
Published: November 2003

Autoradiographic measurement of local cerebral blood flow (CBF) with [14C]iodoantipyrine (IAP) is limited in mice by the difficulty in cannulating vessels and the blood loss for repeated blood sampling. The authors modified and validated the method to measure local CBF with [14C]IAP in mice by combining intraperitoneal tracer application with a single blood sampling from the heart at the end of the experiment. Experiments were carried out in male SV129 mice under halothane anesthesia. After intraperitoneal administration of 15 μCi [14C]IAP, arterial blood samples were collected repeatedly and anesthetized animals were immersed in liquid nitrogen. In addition, frozen blood from the heart was sampled to obtain the final blood [14C]radioactivity. Correlation analysis between the sampling time and [14C] radioactivity of the arterial blood revealed a highly significant linear relationship ( P < 0.001, r = 0.978) and a lag time of the [14C]tracer in arterial blood of 3.3 ± 0.6 seconds. [14C]radioactivity of the final arterial blood sample (444 ± 264 nCi/mL) was almost equal to that of the heart blood (454 ± 242 nCi/mL), and the absolute difference in each animal was 3.3 ± 4.2% (mean ± SD). The convolution integrals for the CBF calculation were determined either by integrating the radioactivity of individual arterial blood samples or by assuming a linear rise from [14C]tracer lag time after intraperitoneal [14C]IAP injection to the value measured in the blood sample from the frozen heart. Regional flow values calculated by the two methods differed by less than 11 % (not significant). This method allows the quantitative measurement of local CBF in anesthetized mice without any vessel catheterization and will make mutant mice a more powerful tool to elucidate the molecular mechanisms of brain injuries by combining flow studies with molecular-biological methods.
Detection of Tumor Necrosis Factor-α mRNA Induction in Ischemic Brain Tolerance by Means of Real-Time Polymerase Chain Reaction
Authors: Wang Xinkang , Li Xiang , Erhardt Joseph A. , Barone Frank C. , Feuerstein Giora Z.
Published: November 2003

A short duration of ischemia (i.e., ischemic preconditioning) results in significant brain protection to subsequent severe ischemic insult. Because previous studies suggest that tumor necrosis factor-α (TNF-α) plays a role in both promoting ischemic damage and neuroprotection, the present work aimed to evaluate the expression of TNF-α mRNA in an established model of ischemic preconditioning using a transient 10-minute occlusion of the middle cerebral artery. Because the level of TNF-α mRNA expression in the brain was too low to be consistently detected by Northern technique, a real-time polymerase chain reaction method was applied to quantitate the absolute copy number of TNF-α transcript in rat brain after the preconditioning procedure. TNF-α mRNA was induced in the ipsilateral cortex as early as 1 hour (27 ± 1 copies of mRNA per microgram of tissue compared to 11 ± 3 copies in sham-operated samples) after preconditioning, reached a peak level at 6 hours (49 ± 10 copies of transcript, n = 4, P < 0.01), and persisted up to 2 days. These data not only demonstrate the utility of real-time polymerase chain reaction for sensitive and accurate measurement of mRNA expression in normal and injured tissues but also suggest a potential role of TNF-α in the phenomenon of ischemic preconditioning.
Magnetic Resonance Imaging Indexes of Therapeutic Efficacy of Recombinant Tissue Plasminogen Activator Treatment of Rat at 1 and 4 Hours after Embolic Stroke
Authors: Jiang Quan , Zhang Rui Lan , Zhang Zheng Gang , Ewing James R. , Jiang Ping , Divine George W. , Knight Robert A. , Chopp Michael
Published: November 2003

With use of magnetic resonance imaging (MRI), the effects of early and delayed treatment of embolic stroke in rat with recombinant tissue plasminogen activator (rt-PA) were investigated. Rats with embolic stroke were treated with rt-PA at 1 (n = 9) or 4 (n = 7) hours after stroke onset or were untreated (n = 15). Diffusion-weighted imaging, perfusion-weighted imaging, and T2-weighted imaging were performed before and after embolization from 1 hour to 7 days. No significant differences were detected in the relative areas with low cerebral blood flow (CBF), apparent diffusion coefficient of water (ADCw), and T2 between the 4-hour treated group and the untreated group. Significant decreases in the average relative areas with low CBF were detected in the 1-hour treated group from 4 to 48 hours after embolization as compared with the untreated group. The increase in T2 in the 1-hour treated group was significantly lower than in the untreated and 4-hour treated groups. A significant increase in ADCw was detected in the 1-hour treated group at 3 and 24 hours after embolization as compared with the untreated and 4-hour treated groups. Secondary embolization was detected by both MRI and laser scanning confocal microscopy. The data suggest that MRI can detect the efficacy of rt-PA treatment and secondary ischemic damage.
Periinfarct and Remote Excitability Changes after Transient Middle Cerebral Artery Occlusion
Authors: Neumann-Haefelin Tobias , Witte Otto W.
Published: November 2003

Transient middle cerebral artery (MCA) occlusion results in substantially smaller cortical infarcts than permanent MCA occlusion if reperfusion is initiated within the first few hours. Only little information is available on the long-term functional outcome of the cortical regions “salvaged” by early reperfusion. To address this issue we examined basic electrophysiologic parameters in vitro using standard extracellular recording techniques at 7 and 28 days after transient MCA occlusion (1- and 2-hour ischemia) in rats. Both neocortical areas ipsi- and contralateral to MCA occlusion were systematically mapped to delineate the extent of periinfarct and remote alterations. In the periinfarct region we found a significant reduction of field potential amplitudes up to 3 mm when measuring from the infarct border at 7 days and up to 7 mm at 28 days. Paired-pulse inhibition, an indicator of GABAergic transmission, was only moderately impaired in this region at 7 days and not significantly different from control at 28 days. Remote effects were observed both ipsi- and contralaterally. Ipsilaterally they were restricted to a region close to the midline (presumably motor cortex) and were most likely attributable to the degeneration of corticostriatal connections. The extent of the contralateral excitability changes was clearly related to the size of the neocortical infarcts with large infarcts resulting in the widespread reduction of field potential amplitudes and an impairment of paired-pulse inhibition. The results show that there is a relatively large periinfarct region with decreased overall excitability after transient MCA occlusion which is likely to have a profound effect on perilesional processes involved in functional recovery. Remote excitability changes may contribute to the functional deficit and are probably related to deafferentation.
Thromboembolic Events Lead to Cortical Spreading Depression and Expression of c-fos, Brain-Derived Neurotrophic Factor, Glial Fibrillary Acidic Protein, and Heat Shock Protein 70 mRNA in Rats
Authors: Dietrich W. Dalton , Truettner Jessie , Prado Ricardo , Stagliano Nancy E. , Zhao Weizhao , Busto Raul , Ginsberg Myron D. , Watson Brant D.
Published: November 2003

The hypotheses that cerebral embolic events lead to repetitive episodes of cortical spreading depression (CSD) and that these propagating waves trigger the expression of c-fos, brain-derived neurotrophic factor (BDNF), glial fibrillary acidic protein (GFAP), and heat shock protein 70 (HSP70) mRNA were tested. Wistar rats underwent photochemically induced right common carotid artery thrombosis (CCAT) (n = 18) or sham (n = 8) procedures. In a subgroup of rats (n = 5), laser-Doppler flowmetry probes were placed overlying the right parietal cortex to record CSD-like changes in cortical blood flow during the initial 2-hour postinjury period. Rats were killed by decapitation at 2 or 24 hours after CCAT, and brains were processed for in situ localization of the gene expression. Two to five intermittent transient hyperemic episodes lasting 1 to 2 minutes were recorded ipsilaterally after CCAT. At 2 hours after CCAT, the widespread expression of c-fos and BDNF mRNAs was observed throughout the ipsilateral cerebral cortex. Pretreatment with the N-methyl-d-aspartate receptor blocker MK-801 (2 mg/kg) 1 hour before CCAT reduced the expression of BDNF mRNA expression at 2 hours. At 24 hours after CCAT, increased expression of GFAP mRNA was present in cortical and subcortical regions. In contrast, multifocal regions of HSP70 expression scattered throughout the thrombosed hemisphere were apparent at both 2 and 24 hours after injury. These data indicate that thromboembolic events lead to episodes of CSD and time-dependent alterations in gene expression. The ability of embolic processes to induce widespread molecular responses in neurons and glia may be important in the pathogenesis of transient ischemic attacks and may influence the susceptibility of the postembolic brain to subsequent insults including stroke.
Excitotoxicity is Required for Induction of Oxidative Stress and Apoptosis in Mouse Striatum by the Mitochondrial Toxin, 3-Nitropropionic Acid
Authors: Kim Gyung W. , Copin Jean-Christophe , Kawase Makoto , Chen Sylvia F. , Sato Shuzo , Gobbel Glenn T. , Chan Pak H.
Published: November 2003

Excitotoxicity is implicated in the pathogenesis of several neurologic diseases, such as chronic neurodegenerative diseases and stroke. Recently, it was reported that excitotoxicity has a relationship to apoptotic neuronal death, and that the mitochondrial toxin, 3-nitropropionic acid (3-NP), could induce apoptosis in the striatum. Although striatal lesions produced by 3-NP could develop through an excitotoxic mechanism, the exact relationship between apoptosis induction and excitotoxicity after 3-NP treatment is still not clear. The authors investigated the role of excitotoxicity and oxidative stress on apoptosis induction within the striatum after intraperitoneal injection of 3-NP. The authors demonstrated that removal of the corticostriatal glutamate pathway reduced superoxide production and apoptosis induction in the denervated striatum of decorticated mice after 3-NP treatment. Also, the N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801, prevented apoptosis in the striatum after 3-NP treatment for 5 days, whereas the non-NMDA receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline, was ineffective. The authors also evaluated the initial type of neuronal death by 3-NP treatment for different durations from 1 to 5 days. In early striatal damage, apoptotic neuronal death initially occurred after 3-NP treatment. Our data show that excitotoxicity related to oxidative stress initially induces apoptotic neuronal death in mouse striatum after treatment with 3-NP.
Apoptosis and Necrosis Occur in Separate Neuronal Populations in Hippocampus and Cerebellum after Ischemia and Are Associated with Differential Alterations in Metabotropic Glutamate Receptor Signaling Pathways
Authors: Martin Lee J. , Sieber Frederick E. , Traystman Richard J.
Published: November 2003

It was evaluated whether postischemic neurodegeneration is apoptosis and occurs with alterations in phosphoinositide-linked metabotropic glutamate receptors (mGluRs) and their associated signaling pathways. A dog model of transient global incomplete cerebral ischemia was used. The CA1 pyramidal cells and cerebellar Purkinje cells underwent progressive delayed degeneration. By in situ end-labeling of DNA, death of CA1 and Purkinje cells was greater at 7 days than 1 day after ischemia, whereas death of granule neurons in dentate gyrus and cerebellar cortex was greater at 1 than at 7 days. Ultrastructurally, degenerating CA1 pyramidal neurons and cerebellar Purkinje cells were necrotic; in contrast, degenerating granule neurons were apoptotic. In agarose gels of regional DNA extracts, random DNA fragmentation coexisted with internucleosomal fragmentation. By immunoblotting of regional homogenates, mGluR1α, mGluR5, phospholipase Cβ (PLCβ), and Gαq/11 protein levels in hippocampus at 1 and 7 days after ischemia were similar to control levels, but in cerebellar cortex, mGluR1α and mGluR5 were decreased but PLCβ was increased. By immunocytochemistry, mGluR and PLCβ immunoreactivity dissipated in CA1 and cerebellar Purkinje cell/ molecular layers, whereas immunoreactivities for these proteins were enhanced in granule neurons. It was concluded that neuronal death after global ischemia exists as two distinct, temporally overlapping forms in hippocampus and cerebellum: necrosis of pyramidal neurons and Purkinje cells and apoptosis of granule neurons. Neuronal necrosis is associated with a loss of phosphoinositide-linked mGluR transduction proteins, whereas neuronal apoptosis occurs with increased mGluR signaling.
N-Type Ca2+ Channels in Cultured Rat Sphenopalatine Ganglion Neurons: An Immunohistochemical and Electrophysiological Study
Authors: Liu Jie , Evans M. Steven , Brewer Gregory J. , Lee Tony J. F.
Published: November 2003

Results from pharmacological studies have suggested that presynaptic N-type Ca2+ channels play an important role in regulating neuronal Ca2+ influx and transmitter nitric oxide (NO) release in isolated cerebral arteries. However, the presence of N-type Ca2+ channels in cerebral perivascular nerves has not been directly demonstrated. As a major source of cerebral perivascular NOergic innervation is the sphenopalatine ganglion (SPG), adult rat SPGs were cultured and examined by whole-cell patch-clamp technique. One week after growing in the culture medium, significant neurite outgrowth from the SPG neuronal cells was observed. Both soma and neurites of these cells were immunoreactive for N-type Ca2+ channels, transmitter-synthesizing enzymes (choline acetyltransferase and NO synthase), and several neuropeptides (vasoactive intestinal peptide, neuropeptide Y, calcitonin gene-related peptide, substance P, and pituitary adenylate cyclase-activating peptide-38) that had been found in cerebral perivascular nerves in whole-mount vascular preparations. In current-clamp recordings, injection of a small depolarizing current caused action potential firing. In voltage-clamp recordings, the fast inward currents were blocked by tetrodotoxin and outward currents by tetraethylammonium, which is typical for neurons. Most Ca2+ currents isolated by blockade of sodium and potassium currents were blocked by ω-conotoxin, indicating that N-type Ca2+ channels are the dominant voltage-dependent Ca2+ channels regulating Ca2+ influx during membrane depolarization of SPG neurons. The ability to culture postganglionic SPG neurons provides an opportunity to directly study the electrophysiological and pharmacological properties of these neurons.
Measurement of Cerebral Blood Flow Volume by Extracranial Sonography
Authors: Dörfler Petra , Puls Imke , Schließer Mira , Mäurer Mathias , Becker Georg
Published: November 2003

Measurement of global cerebral blood flow (CBF) using extracranial duplex sonography has been described, but normal values are still lacking. In 85 healthy adults (median age 43.4 years, range 20 to 80 years), CBF was determined by duplex sonographic examination of both internal carotid arteries and vertebral arteries. The mean global CBF was 630 ± 97 mL/min. Global CBF declined with age; sex did not influence the total CBF. When measurements were repeated, the intraindividual variability was low. This noninvasive sonographic measurement of CBF is reproducible, and values correspond closely to those obtained with positron emission tomography and magnetic resonance imaging.
Effects of Variations in Interstimulus Interval on Activation–Flow Coupling Response and Somatosensory Evoked Potentials with Forepaw Stimulation in the Rat
Authors: Ances Beau M. , Greenberg Joel H. , Detre John A.
Published: November 2003

In functional neuroimaging studies, the hemodynamic response to functional activation is used as a surrogate marker for neuronal activity, typically in response to task paradigms that use periodic stimuli. With use of a model system of electrical forepaw stimulation in rats (n = 14) with laser-Doppler (LD) monitoring of cerebral blood flow (CBF) changes in the somatosensory cortex, the effects of variations in the interstimulus interval (ISI) on the hemodynamic response to periodic stimuli were examined. A characteristic peak flow response was seen for 4-second stimuli and a peak and plateau response were seen for all 8-second stimuli regardless of ISI. However, both the amplitude of the LDCBF response and the integrated response were significantly reduced for shorter ISIs, whereas the baseline flow was not altered. Somatosensory evoked potential responses were also recorded in some rats (n = 8) and remained unchanged for the various ISIs for a particular stimulus duration. These results suggest that the decrease in the LDCBF responses observed with shorter ISIs likely represents a refractoriness of the hemodynamic response and not neuronal function. These results may have important implications for the optimization and interpretation of functional activation paradigms that use periodic stimuli.
Cerebral Metabolism of Lactate in Vivo: Evidence for Neuronal Pyruvate Carboxylation
Authors: Hassel Bjørnar , Bråthe Anders
Published: November 2003

The cerebral metabolism of lactate was investigated. Awake mice received [3-13C]lactate or [1-13C]glucose intravenously, and brain and blood extracts were analyzed by 13C nuclear magnetic resonance spectroscopy. The cerebral up-take and metabolism of [3-13C]lactate was 50% that of [1-13C]glucose. [3-13C]Lactate was almost exclusively metabolized by neurons and hardly at all by glia, as revealed by the 13C labeling of glutamate, γ-aminobutyric acid and glutamine. Injection of [3-13C]lactate led to extensive formation of [2-13C]lactate, which was not seen with [1-13C]glucose, nor has it been seen in previous studies with [2-13C]acetate. This formation probably reflected reversible carboxylation of [3-13C]pyruvate to malate and equilibration with fumarate, because inhibition of succinate dehydrogenase with nitropropionic acid did not block it. Of the [3-13C]lactate that reached the brain, 20% underwent this reaction, which probably involved neuronal mitochondrial malic enzyme. The activities of mitochondrial malic enzyme, fumarase, and lactate dehydrogenase were high enough to account for the formation of [2-13C]lactate in neurons. Neuronal pyruvate carboxylation was confirmed by the higher specific activity of glutamate than of glutamine after intrastriatal injection of [1-14C]pyruvate into anesthetized mice. This procedure also demonstrated equilibration of malate, formed through pyruvate carboxylation, with fumarate. The demonstration of neuronal pyruvate carboxylation demands reconsideration of the metabolic interrelationship between neurons and glia.
Plasminogen Activation in Focal Cerebral Ischemia and Reperfusion
Authors: Pfefferkorn T. , Staufer B. , Liebetrau M. , Bültemeier G. , Vosko M. R. , Zimmermann C. , Hamann G. F.
Published: November 2003

In focal cerebral ischemia the plasminogen-plasmin system plays a role in the fibrinolysis of vessel-occluding clots and also in the proteolysis of extracellular matrix components, which potentially contributes to brain edema and bleeding complications. The authors investigated the plasminogen activation after middle cerebral artery occlusion with and without reperfusion (reperfusion intervals 9 and 24 hours) in rats by histologic zymography and compared areas of increased plasminogen activation to areas of structural injury, which were detected immunohistochemically. After 3 hours of ischemia, increased plasminogen activation was observed in the ischemic hemisphere. The affected area measured 5.2% ± 8.5% and 19.4% ± 30.1% of the total basal ganglia and cortex area, respectively. Reperfusion for 9 hours after 3 hours of ischemia led to a significant expansion of plasminogen activation in the basal ganglia (68.8% ± 42.2%, P < 0.05) but not in the cortex (43.0% ± 34.6%, P = 0.394). In the basal ganglia, areas of increased plasminogen activation were related to areas of structural injury ( r = 0.873, P < 0.001). No such correlation was found in the cortex ( r = 0.299, P = 0.228). In this study, increased plasminogen activation was demonstrated early in focal cerebral ischemia. This activation may promote early secondary edema formation and also secondary hemorrhage after ischemic stroke.
Differing Effects of Copper, Zinc Superoxide Dismutase Overexpression on Neurotoxicity Elicited by Nitric Oxide, Reactive Oxygen Species, and Excitotoxins
Authors: Ying Weihai , Anderson Christopher M. , Chen Yongmei , Stein Becky A. , Fahlman Christian S. , Copin Jean-Christophe , Chan Pak H. , Swanson Raymond A.
Published: November 2003

Overexpression of Cu,Zn superoxide dismutase (SOD1) reduces ischemic injury in some stroke models but exacerbates injury in a neonatal stroke model and in other settings. The current study used a SOD1 transgenic (SOD1-Tg) murine cortical culture system, derived from the same mouse strain previously used for the stroke models, to identify conditions that determine whether SOD1 overexpression in neurons is protective or detrimental. The nitric oxide (NO) donors S-nitroso- N-acetylpenicillamine, spermine-NONOate, and diethylamine-NONOate produced less death in SOD1-Tg neurons than in wild-type neurons ( p < 0.01). Also, NO produced markedly less 3-nitrotyosine in SOD1-Tg cells. In contrast, the superoxide generator menadione produced significantly greater death and nearly twice as much 2′7′-dichlorofluorescein fluorescence in SOD1-Tg neurons than in wild-type neurons, suggesting increased peroxide formation in the SOD1-Tg cells. No significant difference was observed in the vulnerability of the two cell types to H2O2, the product of the SOD reaction. Over-expression of SOD1 also had no effect on neuronal vulnerability to glutamate, N-methyl-D-aspartate, or kainate. These observations suggest that SOD1 overexpression can reduce neuronal death under conditions where peroxynitrite formation is a significant factor, but may exacerbate neuronal death under conditions of rapid intracellular superoxide formation or impaired H2O2 disposal.
Imaging Synaptic Neurotransmission with in Vivo Binding Competition Techniques: A Critical Review
Authors: Laruelle Marc
Published: November 2003

Several groups have provided evidence that positron emission tomography (PET) and single-photon emission computed tomography (SPECT) neuroreceptor imaging techniques might be applied to measure acute fluctuations in dopamine (DA) synaptic concentration in the living human brain. Competition between DA and radioligands for binding to D2 receptor is the principle underlying this approach. This new application of neuroreceptor imaging provides a dynamic measurement of neurotransmission that is likely to be informative to our understanding of neuropsychiatric conditions. This article reviews and discusses the body of data supporting the feasibility and potential of this imaging paradigm. Endogenous competition studies performed in rodents, nonhuman primates, and humans are first summarized. After this overview, the validity of the model underlying the interpretation of these imaging data is critically assessed. The current reference model is defined as the occupancy model, since changes in radiotracer binding potential (BP) are assumed to be directly caused by changes in occupancy of D2 receptors by DA. Experimental data supporting this model are presented. The evidence that manipulation of DA synaptic levels induces change in the BP of several D2 radiotracers (catecholamines and benzamides) is unequivocal. The fact that these changes in BP are mediated by changes in DA synaptic concentration is well documented. The relationship between the magnitude of BP changes measured with PET or SPECT and the magnitude of changes in DA concentration measured by microdialysis supports the use of these noninvasive techniques to measure changes in neurotransmission. On the other hand, several observations remain unexplained. First, the amphetamine-induced changes in the BP of D2 receptor antagonists [123I]IBZM and [11C]raclopride last longer than amphetamine-induced changes in DA extracellular concentration. Second, nonbenzamide D2 receptor antagonists, such as spiperone and pimozide, are not affected by changes in DA release, or are affected in a direction opposite to that predicted by the occupancy model. Similar observations are reported with D1 radiotracers. These results suggest that the changes in BP following changes in DA concentration might not be fully accounted by a simple occupancy model. Specifically, the data are reviewed supporting that agonist-mediated receptor internalization might play an important role in characterizing receptor-ligand interactions. Finally, it is proposed that a better understanding of the mechanism underlying the effects observed with benzamides is essential to develop this imaging technique to other receptor systems.
Reduction of Tissue Plasminogen Activator-Induced Hemorrhage and Brain Injury by Free Radical Spin Trapping after Embolic Focal Cerebral Ischemia in Rats
Authors: Asahi Minoru , Asahi Kazuko , Wang Xiaoying , Lo Eng H.
Published: November 2003

Thrombolytic stroke therapy with tissue plasminogen activator (tPA) remains complicated by serious risks of cerebral hemorrhage and brain injury. In this study, a novel model of tPA-induced hemorrhage was used in spontaneously hypertensive rats to examine the correlates of hemorrhage, and test methods of reducing hemorrhage and brain injury. Homologous blood clot emboli were used to occlude the middle cerebral artery in spontaneously hypertensive rats, and delayed administration of tPA (6 hours postischemia) resulted in high rates of cerebral hemorrhage 24 hours later. Compared with untreated rats, tPA significantly increased hemorrhage volumes by almost 85%. Concomitantly, infarction and neurological deficits were worsened by tPA. A parallel experiment in normotensive Wistar-Kyoto rats showed markedly reduced rates of hemorrhage, and tPA did not significantly increase hemorrhage volumes. To examine whether tPA-induced hemorrhage was caused by the delayed onset of reperfusion per se, another group of spontaneously hypertensive rats was subjected to focal ischemia using a mechanical method of arterial occlusion. Delayed (6 hours) reperfusion via mechanical means did not induce hemorrhage. However, administration of tPA plus delayed mechanical reperfusion significantly increased hemorrhage volumes. Since reperfusion injury was implicated, a final experiment compared outcomes in spontaneously hypertensive rats treated with tPA plus the free radical spin trap α-phenyl tert butyl nitrone (α-PBN) versus tPA alone. tPA-induced hemorrhage volumes were reduced by 40% with α-PBN, and infarction and neurological deficits were also decreased. These results indicate that (1) blood pressure isanimportant correlate of tPA-induced hemorrhage, (2) tPA interacts negatively with reperfusion injury to promote hemorrhage, and (3) combination therapies with anti-free radical treatments may reduce the severity of tPA-induced hemorrhage and brain injury after cerebral ischemia.
Neural Substrate for the Effects of Passive Training on Sensorimotor Cortical Representation: A Study with Functional Magnetic Resonance Imaging in Healthy Subjects
Authors: Carel Christophe , Loubinoux Isabelle , Boulanouar Kader , Manelfe Claude , Rascol Olivier , Celsis Pierre , Chollet François
Published: November 2003

Repetitive passive movements are part of most rehabilitation procedures, especially in patients with stroke and motor deficit. However, little is known about the consequences of repeated proprioceptive stimulations on the intracerebral sensorimotor network in humans. Twelve healthy subjects were enrolled, and all underwent two functional magnetic resonance imaging (fMRI) sessions separated by a 1-month interval. Passive daily movement training was performed in six subjects during the time between the two fMRI sessions. The other six subjects had no training and were considered as the control group. The task used during fMRI was calibrated repetitive passive flexion-extension of the wrist similar to those performed during training. The control task was rest. The data were analyzed with SPM96 software. Images were realigned, smoothed, and put into Talairach's neuroanatomical space. The time effect from the repetition of the task was assessed in the control group by comparing activation versus rest in the second session with activation versus rest in the first session. This time effect then was used as null hypothesis to assess the training effect alone in our trained group. Passive movements compared with rest showed activation of most of the cortical areas involved in motor control (i.e., contralateral primary sensorimotor cortex, supplementary motor area [SMA], cingulum, Brodmann area 40, ipsilateral cerebellum). Time effect comparison showed a decreased activity of the primary sensorimotor cortex and SMA and an increased activity of ipsilateral cerebellar hemisphere, compatible with a habituation effect. Training brought about an increased activity of contralateral primary sensorimotor cortex and SMA. A redistribution of SMA activity was observed. The authors demonstrated that passive training with repeated proprioceptive stimulation induces a reorganization of sensorimotor representation in healthy subjects. These changes take place in cortical areas involved in motor preparation and motor execution and represent the neural basis of proprioceptive training, which might benefit patients undergoing rehabilitative procedures.
Dependence of Oxygen Delivery on Blood Flow in Rat Brain: A 7 Tesla Nuclear Magnetic Resonance Study
Authors: Hyder Fahmeed , Kennan Richard P. , Kida Ikuhiro , Mason Graeme F. , Behar Kevin L. , Rothman Douglas
Published: November 2003

Magnetic resonance imaging (MRI) and spectroscopy (MRS) were used at a magnetic field strength of 7 T to measure CBF and CMRO2 in the sensorimotor cortex of mature rats at different levels of cortical activity. In rats maintained on morphine anesthesia, transitions to lower activity and higher activity states were produced by administration of pentobarbital and nicotine, respectively. Under basal conditions of morphine sulfate anesthesia, CBF was 0.75 ± 0.09 mL · g−1 · min−1 and CMRO2 was 3.15 ± 0.18 μmol · g−1 · min−1. Administration of sodium pentobarbital reduced CBF and CMRO2 by 66% ± 16% and 61% ± 6%, respectively (i.e., “deactivation”). In contrast, administration of nicotine hydrogen tartrate increased CBF and CMRO2 by 41% ± 5% and 30% ± 3%, respectively (i.e., “activation”). The resting values of CBF and CMRO2 for α-chloralose anesthetized rats were 0.40 ± 0.09 mL · g−1 · min−1 and 1.51 ± 0.06 μmol · g−1 · min−1, respectively. Upon forepaw stimulation, CBF and CMRO2 were focally increased by 34% ± 10% and 26% ± 12%, respectively, above the resting nonanesthetized values (i.e., “activation”). Incremental changes in CBF and CMRO2, when expressed as a percentage change for “deactivation” and “activation” from the respective control conditions, were linear (R2 = 0.997) over the entire range examined with the global and local perturbations. This tight correlation for cerebral oxygen delivery in vivo is supported by a recent model where the consequence of a changing effective diffusivity of the capillary bed for oxygen, D, has been hypothetically shown to be linked to alterations in CMRO2 and CBF. This assumed functional characteristic of the capillary bed can be theoretically assessed by the ratio of fractional changes in D with respect to changes in CBF, signified by Ω. A value 0.81 ± 0.23 was calculated for Ω with the in vivo data presented here, which in turn corresponds to a supposition that the effective oxygen diffusivity of the capillary bed is not constant but presumably varies to meet local requirements in oxygen demand in a similar manner with both “deactivation” and “activation.”
Altered Association of Protein Tyrosine Kinases with Postsynaptic Densities after Transient Cerebral Ischemia in the Rat Brain
Authors: Cheung Herman H. , Takagi Norio , Teves Lucy , Logan Richard , Wallace M. Christopher , Gurd James W.
Published: November 2003

Transient cerebral ischemia results in an increase in the tyrosine phosphorylation of proteins associated with postsynaptic densities (PSDs). The authors investigated the possible mechanisms behind this increase by analyzing isolated PSDs for protein tyrosine kinase activity and for the presence of specific tyrosine kinases. Transient (15 minutes) global ischemia was produced in adult rats by four-vessel occlusion, and PSDs were isolated immediately after ischemia or after 20 minutes or 6 hours of reperfusion. Tyrosine phosphorylation of several PSD proteins, including the N-methyl-d-aspartate (NMDA) receptor subunits NR2A and NR2B, was enhanced relative to shams after 20 minutes of reperfusion and underwent a further increase between 20 minutes and 6 hours. The ability of intrinsic PSD tyrosine kinase to phosphorylate PSD proteins, including the NMDA receptor, increased threefold after ischemia. Whereas PSD-associated proline-rich tyrosine kinase 2 (PYK2) and gp145TrkB were elevated immediately after the ischemic event, increases in Src and Fyn were not apparent until 6 hours of reperfusion. The level of PSD-associated pp125FAK decreased after ischemia. The results demonstrate that ischemia results in selective changes in the association of protein tyrosine kinases with the PSD which may account for ischemia-induced increases in the tyrosine phosphorylation of PSD proteins.
A Temporal MRI Assessment of Neuropathology after Transient Middle Cerebral Artery Occlusion in the Rat: Correlations with Behavior
Authors: Virley David , Beech John S. , Smart Sean C. , Williams Steve C. R. , Hodges Helen , Hunter A. Jackie
Published: November 2003

The purpose of this study was to evaluate the temporal and spatial pathological alterations within ischemic tissue using serial magnetic resonance imaging (MRI) and to determine the extent and duration of functional impairment using objective behavioral tests after transient middle cerebral artery occlusion (tMCAO) in the rat. MRI signatures derived from specific anatomical regions of interest (ROI) were then appropriately correlated to the behavioral measures over the time course of the study (up to 28 days post-tMCAO). Sprague-Dawley rats (n = 12) were initially trained on the following behavioral tasks before surgery: bilateral sticky label test (for contralateral neglect); beam walking (for hindlimb coordination); staircase test (for skilled forelimb paw-reaching). Rats were then randomly assigned to receive either tMCAO (90 minutes, n = 6), by means of the intraluminal thread technique, or sham-control surgery (n = 6). Proton density, T2- and T2-diffusion-weighted MR images were acquired at 1, 7, 14, and 28 days post-tMCAO that were then smoothed into respective proton density, T2 relaxation, and apparent diffusion coefficient (ADC) maps. Apparent percent total lesion volume was assessed using T2W imaging. MR signatures were evaluated using the tissue maps by defining ROI for MCAO and sham-control groups, which corresponded to the caudate-putamen, forelimb, hindlimb, and lower parietal cortices both ipsilateral and contralateral to the occlusion site. Behavioral tests were undertaken daily from 1 to 28 days post-tMCAO. Results demonstrate that apparent percent lesion volume reduced from 1 to 7 days ( P < 0.05) but then remained constant up to 28 days for the MCAO group. Pathological changes in the temporal profile of T2 and ADC tissue signatures were significantly altered in specific ROI across the time course of the study ( P < 0.05 to < 0.001), reflecting the progression of edema to necrosis and cavitation. Both T2 and ADC measures of ischemic pathology correlated with parameters defined by each of the functional tests ( r ≥0.5, P < 0.05) across the time course. The staircase test revealed bilateral impairments for the MCAO group ( P < 0.001), which were best predicted by damage to the ipsilateral lower parietal cortex by means of hierarchical multiple regression analyses ( R2 changes ≥0.21, P < 0.03). Behavioral recovery was apparent on the beam walking test at 14 to 28 days post-MCAO, which was mirrored by MRI signatures within the hindlimb cortex returning to sham-control levels. This long-term study is the first of its kind in tracing the dynamic pathologic and functional consequences of tMCAO in the rat. Both serial MRI and objective behavioral assessment provide highly suitable outcome measures that can be effectively used to evaluate promising new antiischemic agents targeted for the clinic.