bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Mdm2 Expression in Rat Brain after Transient Middle Cerebral Artery Occlusion
Authors: Tu Yahong , Hou Sheng-Tao , Huang Zhigao , Robertson George S. , MacManus John P.
Published: November 2003

The negative regulator of p53 transactivation, Mdm2, increased in the ischemic territory after 90 minutes of transient middle cerebral artery occlusion in spontaneously hypertensive rats compared to sham controls. Increased mdm2 mRNA was detected by semiquantitative reverse transcriptase polymerase chain reaction by 6 hours of reperfusion in the ipsilateral hemisphere. In situ hybridization histochemistry was used to localize increases in mdm2 mRNA which occurred in neurons of ischemic cortex and dorsolateral striatum. The number of labeled neurons increased by approximately 20-fold and the cells displayed five-fold increases of mdm2 mRNA in the cortex. Immunohistochemical staining for Mdm2 revealed that its mRNA was efficiently translated in the ischemic cortex, but not striatum, by 8 to 24 hours of reperfusion. Western blotting confirmed 30- to 40-fold increases in the full-length protein of 90 kd at these time points without evidence of alternative splicing. Because Mdm2 is a negative regulator of the apoptosis promoting activity of p53, increased expression of Mdm2 may be a component of a repair response in injured neurons, and supports Mdm2 being an indicator of DNA damage in the brain early after an ischemic insult in a similar way to Gadd45.
Activation-Induced Resetting of Cerebral Oxygen and Glucose Uptake in the Rat
Authors: Madsen Peter L. , Linde Rasmus , Hasselbalch Steen G. , Paulson Olaf B. , Lassen Niels A.
Published: November 2003

In the clinical setting it has been shown that activation will increase cerebral glucose uptake in excess of cerebral oxygen uptake. To study this phenomenon further, this study presents an experimental setup that enables precise determination of the ratio between cerebral uptake of glucose and oxygen in the awake rat. Global CBF was measured by the Kety-Schmidt technique, and the ratio between cerebral uptake rates for oxygen, glucose, and lactate was calculated from cerebral arterial—venous differences. During baseline conditions, rats were kept in a closed box designed to minimize interference. During baseline conditions CBF was 1.08 ± 0.25 mL·g−1·minute−1, and the cerebral oxygen to glucose uptake ratio was 5.5. Activation was induced by opening the sheltering box for 6 minutes. Activation increased CBF to 1.81 mL·g−1·minute−1. During activation cerebral glucose uptake increased disproportionately to cerebral oxygen uptake, and the cerebral oxygen to glucose uptake ratio was 4.2. The accumulated excess glucose uptake during 6 minutes of activation amounted to 2.4 μmol/g. Activation was terminated by closure of the sheltering box. In the postactivation period, the cerebral oxygen to glucose uptake ratio rose to a maximum of 6.4. This response is exactly opposite to the excess cerebral glucose uptake observed during activation.
Characterization of Middle Cerebral Artery Occlusion Infarct Development in the Rat Using Fast Nuclear Magnetic Resonance Proton Spectroscopic Imaging and Diffusion-Weighted Imaging
Authors: Norris David G. , Hoehn-Berlage Mathias , Dreher Wolfgang , Kohno Kanehisa , Busch Elmar , Schmitz Bernd
Published: November 2003

A nuclear magnetic resonance study of the middle cerebral artery occlusion in the rat is presented. Experiments were performed on seven animals before and after occlusion, which occurred in situ. The emphasis in this study was on evaluating rapid proton spectroscopic imaging. Data were acquired with experimental durations of between 4 and 15 minutes for a 32 by 32 spatial matrix, with 64 spectroscopic data points per spatial element. The spectroscopic data were interleaved with diffusion-weighted nuclear magnetic resonance water images of the same slice. The study was terminated at about 6 hours after occlusion. The brains were then frozen in liquid nitrogen for biochemical imaging. The results showed that the signal from N-acetyl aspartate decreased and that of lactate increased within the infarcted region. The temporal course of these intensity changes varied between animals. Nineteen cortical spreading depressions (CSD) were observed by electrophysiologic monitoring during the experiments. Of these, 11 could be unambiguously detected in the lactate images, and a further 3 were on the threshold of detectability. As only a single slice could be examined, it is possible that the centers of depression for the remaining 6 CSD were outside the slice. To the authors' knowledge, this is the first report of the measurement of CSD using proton spectroscopic imaging. Thus, it is shown that this method is valuable not only in following the continuous evolution of proton metabolites with a good spatial and temporal resolution, but also in observing transient phenomena which are believed to play an important role in the expansion of the infarcted territory.
Diffusion of Radiotracers in Normal and Ischemic Brain Slices
Authors: Patlak Clifford S. , Hospod Frank E. , Trowbridge Sean D. , Newman George C.
Published: November 2003

Diffusion in the extracellular space (ECS) is important in physiologic and pathologic brain processes but remains poorly understood. To learn more about factors influencing tissue diffusion and the role of diffusion in solute-tissue interactions, particularly during cerebral ischemia, we have studied the kinetics of several radiotracers in control and hypoxic 450-μm hippocampal slices and in 1,050-μm thick slices that model the ischemic penumbra. Kinetics were analyzed by nonlinear least squares methods using models that combine extracellular diffusion with tissue compartments in series or in parallel. Studies with 14C-polyethylene glycol confirmed prior measurements of extracellular volume and that ECS shrinks during ischemia. Separating diffusion from transport also revealed large amounts of 45Ca that bind to or enter brain as well as demonstrating a small, irreversibly bound compartment during ischemia. The rapidity of 3H2O entry into cells made it impossible for us to distinguish intracellular from extracellular diffusion. The diffusion-compartment analysis of 3-O-methylglucose data appears to indicate that 5 mmol/L glucose is inadequate to support glycolysis fully in thick slices. Unexpectedly, the diffusion coefficient for all four tracers rose in thick slices compared with thin slices, suggesting that ECS becomes less tortuous in the penumbra.
Protective Effect of Apoptosis-Inhibitory Agent, N-Tosyl-l-Phenylalanyl Chloromethyl Ketone against Ischemia-Induced Hippocampal Neuronal Damage
Authors: Hara Akira , Niwa Masayuki , Nakashima Masaya , Iwai Tomohiko , Uematsu Toshihiko , Yoshimi Naoki , Mori Hideki
Published: November 2003

Delayed neuronal death in the gerbil hippocampal CA1 sector occurs 48 to 72 hours after severe forebrain ischemia. DNA fragmentation is observed in the hippocampal CA1 neurons at around that time. We show here that an inhibitor of proteolytic process of apoptosis, N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), protected hippocampal neuronal damage by inhibition of the DNA fragmentation in a dose-dependent manner and that TPCK induced an apoptosis-regulating molecule, Bcl-2 protein, in the surviving neurons. These results suggest the prevention of apoptosis-related DNA fragmentation by TPCK may be an attractive therapeutic strategy for preserving hippocampal neurons from ischemic insult.
Attenuation of Ischemic Inflammatory Response in Mouse Brain Using an Adenoviral Vector to Induce Overexpression of Interleukin-1 Receptor Antagonist
Authors: Yang Guo-Yuan , Liu Xiao-Hong , Kadoya Chitoshi , Zhao Yong-Jie , Mao Ying , Davidson Beverly L. , Betz A. Lorris
Published: November 2003

It has been demonstrated that administration of an interleukin-1 receptor antagonist protein (IL-1ra) reduces ischemic brain injury; however, the detrimental mechanism initiated by interleukin-1 (IL-1) in ischemic brain injury is unclear. In this study, we used mice that were transfected to overexpress human IL-1ra to elucidate the role of IL-1 in the activation of the inflammatory response after middle cerebral artery occlusion (MCAO). Myeloperoxidase (MPO) activity and immunohistostaining were used as a marker of polymorphonuclear leukocytes (PMNL) infiltration. Adenoviral vector (1 × 109 particles) was administered by injection into the right lateral ventricle in mice. Five days later, MCAO was performed on the mice using a suture technique. Permanent MCAO was achieved for 24 hours in the Ad.RSV IL-1ra-transfected, Ad.RSV lacZ-transfected, and saline (control) mice. Myeloperoxidase activity was quantified in each region and localization of MPO was determined by immunohistochemistry. After 2 hours of MCAO, the surface cerebral blood flow was reduced to 13.5% ± 3.4%, 10.75% ± 2.6%, and 10.9% ± 2.6% of baseline in the ischemic hemisphere in Ad.RSV IL-1ra-transfected, Ad.RSV- lacZ-transfected, and saline-treated mice, respectively. The MPO activity in the ischemic hemisphere in the Ad.RSV lacZ group was similar to that in the saline control group (cortex: 0.40 ± 0.22 versus 0.33 ± 0.11; basal ganglia: 0.46 ± 0.23 versus 0.49 ± 0.17; P > 0.05); however, it was significantly reduced in the Ad.RSV IL-1ra group (cortex: 0.18 ± 0.07; basal ganglia: 0.26 ± 0.15; P < 0.05). Myeloperoxidase immunohistochemistry showed that the massive accumulation of MPO-positive cells in the ischemic cortex, striatum, and corpus callosum regions was greatly attenuated in Ad.RSV IL-1ra-transfected mice. Our results indicate that Ad.RSV IL-1ra-transfected mice provide a useful tool to study the mechanism of action of IL-1. The MPO activity assay and immunostaining after 24 hours of focal ischemia were significantly reduced in IL-1ra gene-transfected mice, suggesting that IL-1 may play an important role in the activation of inflammatory cells during focal cerebral ischemia.
Calpain Mediates Eukaryotic Initiation Factor 4G Degradation during Global Brain Ischemia
Authors: Neumar Robert W. , DeGracia Donald J. , Konkoly Lynette L. , Khoury John I. , White Blaine C. , Krause Gary S.
Published: November 2003

Global brain ischemia and reperfusion result in the degradation of the eukaryotic initiation factor (eIF) 4G, which plays a critical role in the attachment of the mRNA to the ribosome. Because eIF-4G is a substrate of calpain, these studies were undertaken to examine whether calpain I activation during global brain ischemia contributes to the degradation of eIF-4G in vivo. Immunoblots with antibodies against calpain I and eIF-4G were prepared from rat brain postmitochondrial supernatant incubated at 37°C with and without the addition of calcium and the calpain inhibitors calpastatin or MDL-28,170. Addition of calcium alone resulted in calpain I activation (as measured by autolysis of the 80-kDa subunit) and degradation of eIF-4G; this effect was blocked by either 1 μmol/L calpastatin or 10 μmol/L MDL-28,170. In rabbits subjected to 20 minutes of cardiac arrest, immunoblots of brain postmitochondrial supernatants showed that the percentage of autolyzed calpain I increased from 1.9% ± 1.1% to 15.8% ± 5.0% and that this was accompanied by a 68% loss of eIF-4G. MDL-28,170 pretreatment (30 mg/kg) decreased ischemia-induced calpain I autolysis 40% and almost completely blocked eIF-4G degradation. We conclude that calpain I degrades eIF-4G during global brain ischemia.
Comparison of the Transient Equilibrium and Continuous Infusion Method for Quantitative PET Analysis of [11C]Raclopride Binding
Authors: Ito Hiroshi , Hietala Jarmo , Blomqvist Gunnar , Halldin Christer , Farde Lars
Published: November 2003

Several approaches have been applied for quantification of D2 dopamine receptors in positron emission tomography studies using [11C]raclopride. Initial approaches were based on analyses of data obtained after rapid bolus injection of [11C]raclopride. A continuous infusion paradigm has more recently been applied. The current study compares these approaches in healthy men. Two positron emission tomography measurements were performed in each of six healthy men, the first with rapid bolus injection and the second with continuous infusion of [11C]raclopride. In rapid bolus injection, the binding potential was calculated by the following methods. One approach is the kinetic analysis using the standard three-compartment model. Another is to define a transient equilibrium at the moment when the specific binding reaches its maximum. In continuous infusion, binding potential was calculated by using time-activity data at equilibrium condition. All methods gave almost identical binding potential, representing cross-validation of these methods. The continuous infusion method can provide “true” equilibrium condition. The kinetic analysis is a sophisticated approach but requires determination of an arterial input function. The transient equilibrium method thus is suitable for routine clinical research, since it does not require determination of an arterial input function.
Experimental Hypoxemic Hypoxia: Effects of Variation in Hematocrit on Magnetic Resonance T2*-Weighted Brain Images
Authors: Lin Weili , Paczynski Richard P. , Celik Azim , Hsu Chung Y. , Powers William J.
Published: November 2003

T2*-weighted gradient echo magnetic resonance images of rat brain were obtained dynamically during acute hypoxemic hypoxia to investigate the relations between changes in cerebral blood oxygen saturation(ΔYb), blood hematocrit (Hct), and R2* (ΔR2*). Images from hypoxemic rats with normal Hct (42.8% ± 2.33%; n = 12) were compared with those from hypoxemic rats with mild (33.4% ± 1.88%; n = 8) or moderate (27.14% ± 2.7%; n = 10) reduction of Hct. A linear relation between ΔYb and ΔR2* was obtained for all three groups. However, the slopes of the linear regressions were statistically different from one another ( P < 0.001), with the slopes of the regression lines increasing inversely with Hct; that is, the slope for normal Hct is less than the slope for mildly reduced Hct, which is less than the slope for moderately reduced Hct. These data suggest that for any given reduction in the oxygen saturation of cerebral blood, the ΔR2* will be of a lesser magnitude when the hemoglobin concentration is reduced; the data are consistent with existing theoretical models of deoxyhemoglobin content-dependent effects in T2*-weighted magnetic resonance imaging.
Stimulation of Tyrosine Phosphorylation of a Brain Protein by Hibernation
Authors: Ohtsuki Toshiho , Jaffe Howard , Brenner Michael , Azzam Nabil , Azzam Rita , Frerichs Kai U. , Hallenbeck John M.
Published: November 2003

Mammalian hibernation is a state of natural tolerance to severely decreased brain blood flow. As protein tyrosine phosphorylation is believed to be involved in the development of resistance to potentially cell-damaging insults, we used immunoblotting for the phosphotyrosine moiety to analyze extracts from various tissues of hibernating and nonhibernating ground squirrels. A single, hibernation-specific phosphoprotein was detected in the brain, but not in any other tissue tested. This protein, designated pp98 to reflect its apparent molecular weight, is distributed throughout the brain, and is associated with the cellular membrane fraction. The presence of the protein is tightly linked to the hibernation state; it is not present in contemporaneously assayed animals that are exposed to the same cold temperature as the hibernators, is present for the duration of a hibernation bout (tested from 1 to 14 days), and disappears within 1 hour of arousal from hibernation. The close association of pp98 with the hibernation state, its presence in cellular membranes, and the known properties of membrane phosphotyrosine proteins suggest that it may transduce a signal for adaptation to the limited availability of oxygen and glucose and low cellular temperature that characterizes hibernation in the ground squirrel.
Prolonged Therapeutic Window for Ischemic Brain Damage Caused by Delayed Caspase Activation
Authors: Fink Klaus , Zhu Jinmin , Namura Shobu , Shimizu-Sasamata Masao , Endres Matthias , Ma Jianya , Dalkara Turgay , Yuan Junying , Moskowitz Michael A.
Published: November 2003

Apoptotic cell death is prominent in neurodegenerative disorders, such as Alzheimer's disease and Huntington's disease, and is found in cerebral ischemia. Using a murine model of delayed cell death, we determined that cleavage of zDEVD-amino-4-trifluoromethyl coumarin (zDEVD-afc) in brain homogenate, a measure of caspase activation, increased initially 9 hours after brief (30 minutes) middle cerebral artery occlusion along with caspase-3p20 immunoreactive cleavage product as determined by immunoblotting. zDEVD-afc cleavage activity was blocked by pretreatment or posttreatment with the caspase-inhibitor N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl-ketone (zDEVD-fmk), and ischemic damage was reduced when the drug was injected up to 9 hours after reperfusion. The protection was long lasting (21 days). Hence, the period before caspase activation defined the therapeutic opportunity for this neuroprotective agent after mild ischemic brain injury. Prolonged protection after caspase inhibition plus the extended treatment window may be especially relevant to the treatment of neurodegenerative disorders.
Effect of Xenon-Induced Flow Activation on Xenon-Enhanced Computed Tomography Cerebral Blood Flow Calculations
Authors: Obrist Walter D. , Zhang Zihong , Yonas Howard
Published: November 2003

Computer simulations of stable xenon (sXe) uptake curves were used to evaluate the effect of xenon-induced flow activation on CBF calculations by xenon-enhanced computed tomography, Estimates of flow activation were based on repeated transcranial Doppler measurements of blood velocity during 4,5 minutes of sXe inhalation, The synthetic curves were generated from a generalized Kety equation that included time-varying blood flow activation, In contrast to the peak 35% increase in blood flow velocity during sXe inhalation, a standard analysis of the flow-varying synthetic curves revealed only minor 3% to 5% increases in calculated CBF. It is concluded that brief xenon inhalations can provide blood flow estimates that contain minimal bias from activation.
Inductions of 3-l-Nitrotyrosine in Motor Neurons after Transient Spinal Cord Ischemia in Rabbits
Authors: Sakurai Masahiro , Fukuyama Naoto , Takizawa Shunya , Abe Koji , Hayashi Takeshi , Shinohara Yukihito , Nakazawa Hiroe , Tabayashi Koichi
Published: November 2003

The induction and distribution of 3-l-nitrotyrosine (NO2-Tyr) were examined with HPLC and immunohistochemistry in rabbit spinal cords after 15 minutes of transient ischemia until 7 days of the reperfusion. After the 15-minute ischemia, there was a significant decrease of neurologic scores in the ischemic group compared with the sham-operated control group at 7 days of reperfusion ( P = 0.0017), and the majority of motor neurons was selectively lost at 7 days of reperfusion ( P = 0.0039). NO2-Tyr was transiently induced at 8 hours of reperfusion in the ventral part of the spinal cord (0.47% ± 0.86%, NO2-Tyr/total tyrosine; P = 0.0021), but was not induced at any time point of reperfusion in the dorsal part of the spinal cord. Strong immunoreactivity for NO2-Tyr was selectively induced in large pyramidal motor neurons at 8 hours of reperfusion and was still weakly present until 7 days of reperfusion. (There may be a difference in sensitivity between the two techniques.) These results suggested that protein tyrosine nitration by nitric oxide plays a role in the selective motor neuron cell damage after transient spinal cord ischemia.
Cytosolic Redistribution of Cytochrome C after Transient Focal Cerebral Ischemia in Rats
Authors: Fujimura Miki , Morita-Fujimura Yuiko , Murakami Kensuke , Kawase Makoto , Chan Pak H.
Published: November 2003

Recent in vitro cell-free studies have shown that cytochrome c release from mitochondria is a critical step in the apoptotic process. The present study examined the expression of cytochrome c protein after transient focal cerebral ischemia in rats, in which apoptosis was assumed to contribute to the expansion of the ischemic lesion. In situ labeling of DNA breaks in frozen sections after 90 minutes of middle cerebral artery (MCA) occlusion showed a significant number of striatal and cortical neurons, which were maximized at 24 hours after ischemia, exhibiting chromatin condensation, nuclear segmentation, and apoptotic bodies. Cytosolic localization of cytochrome c was detected immunohistochemically in the ischemic area as early as 4 hours after 90 minutes of MCA occlusion. Western blot analysis of the cytosolic fraction revealed a strong single 15-kDa band, characteristic of cytochrome c, only in the samples from the ischemic hemisphere. Western blot analysis of the mitochondrial fraction showed a significant amount of mitochondrial cytochrome c in nonischemic brain, which was decreased in ischemic brain 24 hours after ischemia. These results provide the first evidence that cytochrome c is being released from mitochondria to the cytosol after transient focal ischemia. Although further evaluation is necessary to elucidate its correlation with DNA fragmentation, our results suggest the possibility that cytochrome c release may play a role in DNA-damaged neuronal cell death after transient focal cerebral ischemia in rats.
Deficiency of Intercellular Adhesion Molecule 1 Attenuates Microcirculatory Disturbance and Infarction Size in Focal Cerebral Ischemia
Authors: Kitagawa Kazuo , Matsumoto Masayasu , Mabuchi Takuma , Yagita Yoshiki , Ohtsuki Toshiho , Hori Masatsugu , Yanagihara Takehiko
Published: November 2003

Recent evidence has shown crucial roles for cell-adhesion molecules in inflammation-induced rolling, adhesion, and accumulation of neutrophils in tissue. Intercellular adhesion molecule-1 (ICAM-1) is one of these adhesion molecules. Previous studies have shown marked reduction in the size of infarction after focal cerebral ischemia by depletion of granulocytes and administration of the antibody against ICAM-1. In the present study we investigated the role of ICAM-1 in the size of ischemic lesions, accumulation of granulocytes, and microcirculatory compromise in focal cerebral ischemia by using ICAM-1–knockout mice. Ischemic lesions were significantly mitigated in knockout mice after permanent and transient focal ischemia, even though the number of granulocytes in the infarcted tissue was almost the same between knockout and wild-type mice. Depletion of granulocytes further decreased the size of ischemic lesions after transient focal ischemia in ICAM-1–knockout mice. Microcirculation was reduced after focal ischemia, but it was better preserved in the cerebral cortex of knockout mice than that of wild-type mice. The present study demonstrated that ICAM-1 played a role in microcirculatory failure and subsequent development and expansion of infarction after focal cerebral ischemia. However, it is highly unlikely that ICAM-1 played a key role in accumulation of granulocytes after focal cerebral ischemia.
Quantitation of Microvascular Plasma Perfusion and Neuronal Microtubule-Associated Protein in Ischemic Mouse Brain by Laser-Scanning Confocal Microscopy
Authors: Zhang Zhenggang , Davies Kenneth , Prostak Jonathan , Fenstermacher Joseph , Chopp Michael
Published: November 2003

In an exposition of the technique of calculating distribution volumes from laser-scanning confocal microscopic (LSCM) data, three-dimensional images of the distribution of one or two fluorescent markers in mouse brain specimens were generated by LSCM and processed by a system developed for morphometric analysis of fixed and stained serial brain histologic samples. To determine the volume of perfused cerebral capillaries, one of two fluorescent plasma markers, either fluorescein isothiocyanate (FITC)-dextran or Evans blue, was intravenously administered to mice subjected to 1 hour of embolic middle cerebral artery (MCA) occlusion (n = 9) and to mice that were not operated on (n = 3); after 1 minute of circulation, brains were removed, immersion-fixed, and processed for LSCM. In some of these animals (n = 5), the volume of endogenous microtubule-associated protein-2 (MAP2) fluorescence was also determined using immunohistochemical staining. For mice that were not operated on, this methodology yielded highly localized volumes of (1) microvascular plasma, which agree with those determined for rodents by other techniques, and (2) MAP2 expression, which appears physiologically and morphologically reasonable. After 1 hour of MCA occlusion, the MAP2 volumes of distribution were less than 10% of normal in the ipsilateral hemisphere in which plasma perfusion essentially ceased. In conclusion, precise colocalization and quantitation of early ischemic neuronal damage and cerebral plasma perfusion deficit can be done with this three-dimensional, microphysiologic and microanatomic methodology.
Selective Cerebral Hematocrit Decrease in the Centrum Semiovale after Carotid Artery Occlusion: A PET Study
Authors: Yamauchi Hiroshi , Fukuyama Hidenao , Nagahama Yasuhiro , Katsumi Yukinori , Hayashi Takuya , Okazawa Hidehiko , Yonekura Yoshiharu
Published: November 2003

The centrum semiovale may be susceptible to hypoperfusion as a result of carotid artery occlusion. Recent studies suggest that the cerebral hematocrit decreases with diminished cerebral perfusion pressure. To investigate whether the effect of carotid artery occlusion on the hematocrit in the centrum semiovale is different from that in the cerebral cortex, seven patients with unilateral carotid artery occlusion were studied with positron emission tomography. The distributions of the red blood cell and plasma volumes were assessed using carbon monoxide labeled with oxygen 15 and human serum albumin-dithiosemicarbazone tracers labeled with copper 62, respectively. The CBF and CMRO2 were also measured with the 15O steady-state technique. The calculated values for the hematocrit in the centrum semiovale ipsilateral to the arterial occlusion were significantly decreased compared with those in any of the other regions examined (the overlying cortical region and the contralateral cortex and centrum semiovale). This decrease in hematocrit, which resulted from a more pronounced increase in plasma volume than in red blood cell volume, was associated with a decrease in CBF and an increase in the oxygen extraction fraction. Hemodynamic disturbance caused by carotid artery occlusion may induce selective decrease of hematocrit limited to the centrum semiovale.
Experimental Axonal Injury Triggers Interleukin-6 mRNA, Protein Synthesis and Release into Cerebrospinal Fluid
Authors: Hans Volkmar H. J. , Kossmann Thomas , Lenzlinger Philipp M. , Probstmeier Rainer , Imhof Hans-Georg , Trentz Otmar , Morganti-Kossmann Maria C.
Published: November 2003

Diffuse axonal injury is a frequent pathologic sequel of head trauma, which, despite its devastating consequences for the patients, remains to be fully elucidated. Here we studied the release of interleukin-6 (IL-6) into CSF and serum, as well as the expression of IL-6 messenger ribonucleic acid (mRNA) and protein in a weight drop model of axonal injury in the rat. The IL-6 activity was elevated in CSF within 1 hour and peaked between 2 and 4 hours, reaching maximal values of 82,108 pg/mL, and returned to control values after 24 hours. In serum, the levels of IL-6 remained below increased CSF levels and did not exceed 393 pg/mL. In situ hybridization demonstrated augmented IL-6 mRNA expression in several regions including cortical pyramidal cells, neurons in thalamic nuclei, and macrophages in the basal subarachnoid spaces. A weak constitutive expression of IL-6 protein was shown by immunohistochemical study in control brain. After injury, IL-6 increased at 1 hour and remained elevated through the first 24 hours, returning to normal afterward. Most cells producing IL-6 were cortical, thalamic, and hippocampal neurons as confirmed by staining for the neuronal marker NeuN. These results extend our previous studies showing IL-6 production in the cerebrospinal fluid of patients with severe head trauma and demonstrate that neurons are the main source of IL-6 after experimental axonal injury.
Hyperglycemia and Focal Brain Ischemia
Authors: Gisselsson Lars , Smith Maj-Lis , Siesjö Bo K.
Published: November 2003

The influence of hyperglycemic ischemia on tissue damage and cerebral blood flow was studied in rats subjected to short-lasting transient middle cerebral artery (MCA) occlusion. Rats were made hyperglycemic by intravenous infusion of glucose to a blood glucose level of about 20 mmol/L, and MCA occlusion was performed with the intraluminar filament technique for 15, 30, or 60 minutes, followed by 7 days of recovery. Normoglycemic animals received saline infusion. Perfusion-fixed brains were examined microscopically, and the volumes of selective neuronal necrosis and infarctions were calculated. Cerebral blood flow was measured autoradiographically at the end of 30 minutes of MCA occlusion and after 1 hour of recirculation in normoglycemic and hyperglycemic animals. In two additional groups with 30 minutes of MCA occlusion, CO2 was added to the inhaled gases to create a similar tissue acidosis as in hyperglycemic animals. In one group CBF was measured, and the second group was examined for tissue damage after 7 days. Fifteen and 30 minutes of MCA occlusion in combination with hyperglycemia produced larger infarcts and smaller amounts of selective neuronal necrosis than in rats with normal blood glucose levels, a significant difference in the total volume of ischemic damage being found after 30 minutes of MCA occlusion. After 60 minutes of occlusion, when the volume of infarction was larger, only minor differences between normoglycemic and hyperglycemic animals were found. Hypercapnic animals showed volumes of both selective neuronal necrosis and infarction that were almost identical with those observed in normoglycemic, normocapnic animals. When local CBF was measured in the ischemic core after 30 minutes of occlusion, neither the hyperglycemic nor the hypercapnic animals were found to be significantly different from the normoglycemic group. Brief focal cerebral ischemia combined with hyperglycemia leads to larger and more severe tissue damage. Our results do not support the hypothesis that the aggravated injury is caused by any disturbances in CBF.
Changes in the Diffusion of Water and Intracellular Metabolites after Excitotoxic Injury and Global Ischemia in Neonatal Rat Brain
Authors: Dijkhuizen Rick M. , de Graaf Robin A. , Tulleken Kees A. F. , Nicolay Klaas
Published: November 2003

The reduction of the apparent diffusion coefficient (ADC) of brain tissue water in acute cerebral ischemia, as measured by diffusion-weighted magnetic resonance imaging, is generally associated with the development of cytotoxic edema. However, the underlying mechanism is still unknown. Our aim was to elucidate diffusion changes in the intracellular environment in cytotoxic edematous tissue. The ADC of intracellular metabolites was measured by use of diffusion-weighted 1H-magnetic resonance spectroscopy after (1) unilateral N-methyl-D-aspartate (NMDA) injection and (2) cardiac arrest-induced global ischemia in neonatal rat brain. The distinct water ADC drop early after global ischemia was accompanied by a significant reduction of the ADC of all measured metabolites ( P < 0.01, n = 8). In the first hours after excitotoxic injury, the ADC of water and the metabolites taurine and N-acetylaspartate dropped significantly ( P < 0.05, n = 8). At 24 and 72 hours after NMDA injection brain metabolite levels were diminished and metabolite ADC approached contralateral values. Administration of the NMDA-antagonist MK-801 1.5 hours after NMDA injection completely normalized the water ADC but not the metabolite ADC after 1 to 2 hours (n = 8). No damage was detected 72 hours later and, water and metabolite ADC had normal values (n = 8). The contribution of brain temperature changes (calculated from the chemical shift between the water and N-acetylaspartate signals) and tissue deoxygenation to ischemia-induced intracellular ADC changes was minor. These data lend support to previous suggestions that the ischemia-induced brain water ADC drop may partly be caused by reduced diffusional displacement of intracellular water, possibly involving early alterations in intracellular tortuosity, cytoplasmic streaming, or intracellular molecular interactions.
An Intrathecal Bolus of Cyclosporin a before Injury Preserves Mitochondrial Integrity and Attenuates Axonal Disruption in Traumatic Brain Injury
Authors: Okonkwo David O. , Povlishock John T.
Published: November 2003

Traumatic brain injury evokes multiple axonal pathologies that contribute to the ultimate disconnection of injured axons. In severe traumatic brain injury, the axolemma is perturbed focally, presumably allowing for the influx of Ca2+ and initiation of Ca2+-sensitive, proaxotomy processes. Mitochondria in foci of axolemmal failure may act as Ca2+ sinks that sequester Ca2+ to preserve low cytoplasmic calcium concentrations. This Ca2+ load within mitochondria, however, may cause colloid osmotic swelling and loss of function by a Ca2+-induced opening of the permeability transition pore. Local failure of mitochondria, in turn, can decrease production of high-energy phosphates necessary to maintain membrane pumps and restore ionic balance in foci of axolemmal permeability change. The authors evaluated the ability of the permeability transition pore inhibitor cyclosporin A (CsA) to prevent mitochondrial swelling in injured axonal segments demonstrating altered axolemmal permeability after impact acceleration injury in rat. At the electron microscopic level, statistically fewer abnormal mitochondria were seen in traumatically injured axons from CsA-pretreated injured animals. Further, this mitochondrial protection translated into axonal protection in a second group of injured rats, whose brains were reacted with antibodies against amyloid precursor protein, a known marker of injured axons. Pretreatment with CsA significantly reduced the number of axons undergoing delayed axotomy, as evidenced by a decrease in the density of amyloid precursor protein-immunoreactive axons. Collectively, these studies demonstrate that CsA protects both mitochondria and the related axonal shaft, suggesting that this agent may be of therapeutic use in traumatic brain injury.
Pinealectomy Aggravates and Melatonin Administration Attenuates Brain Damage in Focal Ischemia
Authors: Kilic Ertugrul , ÖUzdemir Yasemin Gürsoy , Bolay Hayrunnisa , Keleştimur Haluk , Dalkara Turgay
Published: November 2003

Large infarcts develop in pinealectomized rats subjected to middle cerebral artery occlusion, which was attributed to loss of antioxidant action of melatonin. However, melatonin also has vascular actions, and pinealectomy may induce hypertension. The authors investigated (I) whether hemodynamic factors contribute to infarct development in pinealectomized rats, (2) whether melatonin administration can reverse the unfavorable effect of pinealectomy on infarct formation, and (3) whether melatonin can reduce the infarct volume in nonpinealectomized rats subjected to focal transient ischemia (2 hours middle cerebral artery occlusion, 22 hours reperfusion). Rats were pinealectomized 3 months before ischemia to eliminate any possible action of pinealectomy-induced hypertension on stroke. Blood pressure and regional CBF values during ischemia and reperfusion were not significantly different between pinealectomized and sham-operated rats, suggesting that pinealectomy-induced increase in infarct was not related to hemodynamic factors. The infarct volume resumed to the level of sham-operated rats on melatonin administration. Injection of melatonin (4 mg/kg) before both ischemia and reperfusion reduced infarct volume by 40% and significantly improved neurologic deficit scores in pinealectomized as well as sham-operated rats subjected to middle cerebral artery occlusion. These data suggest that physiologic melatonin release as well as exogenously given melatonin has a neuroprotective action in focal cerebral ischemia.
Low Brain Intracellular Free Magnesium in Mitochondrial Cytopathies
Authors: Barbiroli Bruno , Iotti Stefano , Cortelli Pietro , Martinelli Paolo , Lodi Raffaele , Carelli Valerio , Montagna Pasquale
Published: November 2003

The authors studied, by in vivo phosphorus magnetic resonance spectroscopy (31P-MRS), the occipital lobes of 19 patients with mitochondrial cytopathies to clarify the functional relation between energy metabolism and concentration of cytosolic free magnesium. All patients displayed defective mitochondrial respiration with low phosphocreatine concentration [PCr] and high inorganic phosphate concentration [Pi] and [ADP]. Cytosolic free [Mg2+] and the readily available free energy (defined as the actual free energy released by the exoergonic reaction of ATP hydrolysis, i.e., ΔGATPhyd) were abnormally low in all patients. Nine patients were treated with coenzyme Q10 (CoQ), which improved the efficiency of the respiratory chain, as shown by an increased [PCr], decreased [Pi] and [ADP], and increased availability of free energy (more negative value of ΔGATPhyd). Treatment with CoQ also increased cytosolic free [Mg2+] in all treated patients. The authors findings demonstrate low brain free [Mg2+] in our patients and indicate that it resulted from failure of the respiratory chain. Free Mg2+ contributes to the absolute value of ΔGATPhyd. The results also are consistent with the view that cytosolic [Mg2+] is regulated in the intact brain cell to equilibrate, at least in part, any changes in rapidly available free energy.
Noninvasive Assessment of Changes in Cytochrome-c Oxidase Oxidation in Human Subjects during Visual Stimulation
Authors: Heekeren Hauke R. , Kohl Matthais , Obrig Hellmuth , Wenzel Rüdiger , von Pannwitz Wolfram , Matcher Steven J. , Dirnagl Ulrich , Cooper Chris E. , Villringer Arno
Published: November 2003

In this study the authors used a whole-spectrum near-infrared spectroscopy approach to noninvasively assess changes in hemoglobin oxygenation and cytochrome- c oxidase redox state (Cyt-Ox) in the occipital cortex during visual stimulation. The system uses a white light source (halogen lamp). The light reflected from the subject's head is spectrally resolved by a spectrograph and dispersed on a cooled charge-coupled device camera. The authors showed the following using this approach: (1) Changes in cerebral hemoglobin oxygenation (increase in concentration of oxygenated hemoglobin, decrease in concentration of deoxygenated hemoglobin) in the human occipital cortex during visual stimulation can be assessed quantitatively. (2) The spectral changes during functional activation cannot be completely explained by changes in hemoglobin oxygenation solely; Cyt-Ox has to be included in the analysis. Only if Cyt-Ox is considered can the spectral changes in response to increased brain activity be explained. (3) Cytochrome- c oxidase in the occipital cortex of human subjects is transiently oxidized during visual stimulation. This allows us to measure vascular and intracellular energy status simultaneously.
Age-Dependent Increase in Ischemic Brain Injury in Wild-Type Mice and in Mice Lacking the Inducible Nitric Oxide Synthase Gene
Authors: Nagayama Masao , Aber Tracy , Nagayama Tomiko , Ross M. Elizabeth , Iadecola Constantino
Published: November 2003

The authors investigated the influence of age on the outcome of cerebral ischemia in wild-type mice and in mice with a deletion of the inducible nitric oxide synthase (iNOS) gene. The middle cerebral artery was permanently occluded in iNOS-null mice and in wild-type (C57BL/6) controls aged 4, 8, 16, and 24 weeks. Infarct volume was determined in thionin-stained brain sections 4 days after permanent middle cerebral artery occlusion. No differences in forebrain volume were found among wild-type and iNOS-null mice at the ages studied (P> 0.05). In C57BL/6 mice (n = 5 to 6/group), neocortical infarct volume corrected for swelling was 28 ± 5 mm3 in 4-week-old mice, 28 ±3 at 8 weeks, 35 ±4 at 16 weeks, and 37 ± 6 at 24 weeks (mean ± SD). iNOS-null mice (n = 5 to 6/group) had smaller infarcts than wild-type controls at all ages ( P < 0.05). However, the magnitude of the reduction was greater in 4-week-old (–29% ± 10%) or 8-week-old mice (–24% ± 8%), than in 16-week-old (–14% ± 10%) or 24-week-old mice (–11% ± 6%). Neurologic deficit scores improved significantly between 24 and 96 hours in 4- and 8-week-old iNOS-null mice compared with age-matched wild-type mice ( P < 0.05). However, in 16- or 24-week-old iNOS-null mice, neurologic deficits did not improve ( P > 0.05). The authors conclude that in iNOS–/– and in wild-type mice, the size of the infarct produced by occlusion of the middle cerebral artery is larger in older than in younger mice. However, the reduction in infarct volume observed in iNOS-null mice is age-dependent and is greatest at 1 to 2 months of age. Therefore, age is a critical variable in studies of focal cerebral ischemic damage, both in wild-type mice and in mouse mutants.
Kynurenine Hydroxylase Inhibitors Reduce Ischemic Brain Damage: Studies with (m-Nitrobenzoyl)-Alanine (mNBA) and 3,4-Dimethoxy-[-N-4-(Nitrophenyl)Thiazol-2YL]-Benzenesulfonamide (Ro 61-8048) in Models of Focal or Global Brain Ischemia
Authors: Cozzi A. , Carpenedo R. , Moroni F.
Published: November 2003

Two kynurenine hydroxylase inhibitors, ( m-nitrobenzoyl)-alanine (mNBA) and 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfonamide (Ro 61-8048), have been tested as neuroprotective agents on brain lesions induced by bilateral carotid occlusion in gerbils or by middle cerebral artery occlusion in rats. The percentage of lesioned pyramidal neurones found in the hippocampal CA1 region of gerbils subjected to bilateral carotid occlusion for 5 minutes decreased from 92 ± 10% in vehicle-treated animals to 7 ± 6% after mNBA (400 mg/kg intraperitoneally, three times at 1, 30, and 180 minutes after occlusion) or to 10±11% after Ro 61-8048 (40 mg/kg intraperitoneally, three times). A significant reduction in infarct volumes also was found when the kynurenine hydroxylase inhibitors were given to rats after permanent middle cerebral artery occlusion (from 207 ± 111 mm3 in vehicle-treated rats to 82 ± 18 and to 62 ± 57 mm3 in rats treated with mNBA, 400 mg/kg intraperitoneally, or with Ro 61-8048, 40 mg/kg intraperitoneally, respectively). The administration of mNBA (400 mg/kg intraperitoneally) or Ro 61-8048 (40 mg/kg intraperitoneally) to gerbils with a dialysis probe in their dorsal hippocampus or to rats with a dialysis probe in their parietal cortex significantly increased kynurenic acid concentration in the dialysates. The data suggest that inhibition of kynurenine hydroxylase could be a new avenue to reduce neuronal loss in brain ischemia.
Breakdown of Calcium Homeostasis in Relation to Tissue Depolarization: Comparison between Gray and White Matter Ischemia
Authors: Kumura E. , Graf R. , Dohmen C. , Rosner G. , Heiss W. D.
Published: November 2003

In vitro studies suggest that ischemic injury of cerebral white matter is mediated by nonsynaptic cellular mechanisms, such as Ca2+ entry into axons through reversal of the Na+-Ca2+ exchanger. The authors investigated extracellular Ca2+ concentration in relation to tissue depolarization (direct current potential) in vivo using ion-selective electrodes in cortical gray and subcortical white matter of α-chloralose-anesthetized cats during 120 minutes of global cerebral ischemia. On induction of ischemia, regional CBF, as measured by hydrogen clearance, ceased. The direct current potential decreased rapidly within minutes in gray matter and with little time delay in white matter. Extracellular Ca2+ concentration decreased just as quickly in gray matter. In white matter, in contrast, extracellular Ca2+ increased in the first 20 to 30 minutes, and a delayed and much slower decline, compared with gray matter, was observed thereafter, reaching a minimal level only about 60 minutes after occlusion. Our results suggest that smaller and delayed transmembrane shifts of Ca2+ are correlates of delayed ischemic membrane dysfunction in central white matter tracts, which may be explained by a lack of synaptic mechanisms.
The Effect of Transient Global Ischemia on the Interaction of Src and Fyn with the N-Methyl-d-Aspartate Receptor and Postsynaptic Densities: Possible Involvement of Src Homology 2 Domains
Authors: Takagi Norio , Cheung Herman H. , Bissoon Nankie , Teves Lucy , Wallace M. Christopher , Gurd James W.
Published: November 2003

Transient ischemia increases tyrosine phosphorylation of N-methyl-d-aspartate (NMDA) receptor subunits NR2A and NR2B in the rat hippocampus. The authors investigated the effects of this increase on the ability of the receptor subunits to bind to the Src homology 2 (SH2) domains of Src and Fyn expressed as glutathione-S-transferase–SH2 fusion proteins. The NR2A and NR2B bound to each of the SH2 domains and binding was increased approximately twofold after ischemia and reperfusion. Binding was prevented by prior incubation of hippocampal homogenates with a protein tyrosine phosphatase or by a competing peptide for the Src SH2 domain. Ischemia induced a marked increase in the tyrosine phosphorylation of several proteins in the postsynaptic density (PSD), including NR2A and NR2B, but had no effect on the amounts of individual NMDA receptor subunits in the PSD. The level of Src and Fyn in PSDs, but not in other subcellular fractions, was increased after ischemia. The ischemia-induced increase in the interaction of NR2A and NR2B with the SH2 domains of Src and Fyn suggests a possible mechanism for the recruitment of signaling proteins to the PSD and may contribute to altered signal transduction in the postischemic hippocampus.
Early Appearance of Activated Matrix Metalloproteinase-9 after Focal Cerebral Ischemia in Mice: A Possible Role in Blood—Brain Barrier Dysfunction
Authors: Gasche Yvan , Fujimura Miki , Morita-Fujimura Yuiko , Copin Jean-Christophe , Kawase Makoto , Massengale Justin , Chan Pak H.
Published: November 2003

During cerebral ischemia blood–brain barrier (BBB) disruption is a critical event leading to vasogenic edema and secondary brain injury. Gelatinases A and B are matrix metalloproteinases (MMP) able to open the BBB. The current study analyzes by zymography the early gelatinases expression and activation during permanent ischemia in mice (n = 15). ProMMP-9 expression was significantly ( P < 0.001) increased in ischemic regions compared with corresponding contralateral regions after 2 hours of ischemia (mean 694.7 arbitrary units [AU], SD ± 238.4 versus mean 107.6 AU, SD ± 15.6) and remained elevated until 24 hours (mean 745,7 AU, SD ± 157.4). Moreover, activated MMP-9 was observed 4 hours after the initiation of ischemia. At the same time as the appearance of activated MMP-9, we detected by the Evan's blue extravasation method a clear increase of BBB permeability, Tissue inhibitor of metalloproteinase-1 was not modified during permanent ischemia at any time. The ProMMP-2 was significantly ( P < 0.05) increased only after 24 hours of permanent ischemia (mean 213.2 AU, SD ± 60.6 versus mean 94.6 AU, SD ± 13.3), and no activated form was observed. The appearance of activated MMP-9 after 4 hours of ischemia in correlation with BBB permeability alterations suggests that MMP-9 may play an active role in early vasogenic edema development after stroke.