bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Tumor Necrosis Factor and Amelioration of Brain Infarction in Mice
Authors: Nawashiro Hiroshi , Martin David , Hallenbeck John M.
Published: November 2003

Tumor necrosis factor alpha (TNF- α) is expressed in the ischemic brain; however, its precise role is not fully understood. We studied the effect of the dimeric form of the type I soluble TNF receptor linked to polyethylene glycol (TNFbp) on focal cerebral ischemia in mice using a permanent middle cerebral arterial occlusion (MCAO) model. TNFbp was applied topically, intravenously, or intraperitoneally. TNFbp binds and inhibits TNF- α. The volume of cortical ischemic lesions was measured by means of 2,3,5-triphenyltetrazolium chloride 24 h after MCAO. TNFbp produced a significant reduction in the cortical infarct volume of vehicle-treated animals ( p < 0.001). The reduction in the volume of brain damage was 26% in animals that received 3 mg/kg of TNFbp topically. Further analysis of TNF- α inhibition following acute brain ischemia is indicated.
Changes of Biological Reducing Activity in Rat Brain following Closed Head Injury: A Cyclic Voltammetry Study in Normal and Heat-Acclimated Rats
Authors: Beit-Yannai E. , Kohen R. , Horowitz M. , Trembovler V. , Shohami E.
Published: November 2003

Reactive oxygen species (ROS) are normally generated in the brain during metabolism, and their production is enhanced by various insults. Low molecular weight antioxidants (LMWA) are one of the defense mechanisms of the living cell against ROS. The reducing capacity of brain tissue (total LMWA) was measured by cyclic voltammetry (CV), which records biological oxidation potential specific to the type of scavenger(s) present and anodic current intensity ( Ia), which depends on scavenger concentration. In the present study, the reducing capacity of rat brain following closed head injury (CHI) was measured. In addition, CV of heat-acclimated traumatized rats was used to correlate endogenous cerebroprotection after CHI with LMWA activity. Sham-injured rat brains displayed two anodic potentials: at 350 ± 50 mV ( Ia = 0.75 ± 0.06 μA/mg protein) and at 750 ± 50 mV ( Ia = 1.00 ± 0.05 μA/mg protein). Following CHI, the anodic waves appeared at the same potentials as in the sham animals. However, within 5 min of CHI, the total reducing capacity was transiently decreased by 40% (p < 0.01). A second dip was detected at 24 h (60%, p < 0.005). By 48 h and at 7 days, the Ia levels normalized. The acclimated rats displayed anodic potentials identical to those of normothermic rats. However, the Ia of both potentials was lower (60% of control, p < 0.001). The Ia profile after CHI was the direct opposite of the normothermic Ia profile: no immediate decrease of Ia and an increase from 4 h and up to 7 days (40–50%, p < 0.001). We suggest that the lowered levels of LMWA in the post-CHI period reflect their consumption due to overproduction of free radicals. The augmented concentration of LMWA found in the brain of the heat-acclimated rats suggests that these rats are better able to cope with these harmful radicals, resulting in a more favorable outcome following CHI.
Hypercapnia Reversibly Suppresses Low-Frequency Fluctuations in the Human Motor Cortex during Rest Using Echo–Planar MRI
Authors: Biswal B. , Hudetz A. G. , Yetkin F. Zerrin , Haughton Victor M. , Hyde James S.
Published: November 2003

Using magnetic resonance (MR) echo–planar imaging (EPI), we recently demonstrated the presence of low-frequency fluctuations (<0.1 Hz) in MR signal intensity from the resting human brain that have a high degree of temporal correlation ( p < 10–3) within and across associated regions of the sensorimotor cortex. These fluctuations in MR signal intensity are believed to arise from fluctuations in capillary blood flow and oxygenation. A substantial overlap between the activation map generated by bilateral finger tapping and temporally-correlated voxels from the sensorimotor cortex obtained during rest was observed. In the work reported here, we investigated whether respiratory hypercapnia, which is known to suspend spontaneous oscillations in regional cerebral blood flow, influences these low-frequency fluctuations. The magnitude of low-frequency fluctuations was reversibly diminished during hypercapnia, resulting in a substantial decrease of the temporal correlation both within and across contralateral hemispheres of the sensorimotor cortex. After the breathing mixture was returned to ambient air, the magnitude and spatial extent of the temporal correlation of low-frequency fluctuations returned to normal. Results of this study support the hypothesis that low-frequency physiological fluctuations observed by MR in the human cortex and spontaneous flow oscillations observed in early studies by laser–Doppler flowmetry (LDF) in the cortex of the rat are identical and are secondary to fluctuations in neuronal activity.
TNF-α Pretreatment Induces Protective Effects against Focal Cerebral Ischemia in Mice
Authors: Nawashiro Hiroshi , Tasaki Kaoru , Ruetzler Christl A. , Hallenbeck John M.
Published: November 2003

Cytokines are recognized to play an important role in acute stroke. Tumor necrosis factor- α (TNF) is one of the pro-inflammatory cytokines and is expressed in ischemic brain. We hypothesized that TNF might play a role in the regulation of tolerance to ischemia when administered prior to the ischemic episode. We studied the effects of pretreatment of TNF administered intravenously, intraperitoneally, or intracisternally in mice that were subjected to middle cerebral artery occlusion (MCAO) 48 h later. MCAO was performed in BALB/C mice by direct cauterization of distal MCA, which resulted in pure cortical infarction. A significant reduction in infarct size was noted in mice pretreated by TNF at the dose of 0.5 μg/mouse (p < 0.01) intracisternally. At the doses used in this study, administration of TNF by intravenous or intraperitoneal routes was not effective. Immunohistochemical analysis of brains subjected to 24 h of MCAO revealed a significant decrease in CD11b immunoreactivity after TNF pretreatment compared with control MCAO. Preconditioning with TNF affects infarct size in a time- and dose-dependent manner. TNF induces significant protection against ischemic brain injury and is likely to be involved in the signaling pathways that regulate ischemic tolerance.
Induction of Tumor Necrosis Factor-α in the Mouse Hippocampus following Transient Forebrain Ischemia
Authors: Uno Hisakazu , Matsuyama Tomohiro , Akita Hiroshi , Nishimura Hiroyuki , Sugita Minoru
Published: November 2003

To assess the role of tumor necrosis factor- α (TNF- α) in modulating the process of cerebral ischemic injury, we identified TNF- α-producing cells and studied the time course of TNF- α expression. Immunoreactivity for TNF- α appeared in white matter of the mouse hippocampus as early as 1.5 h following a 30-min global ischemic insult. Double staining for TNF- α and glial fibrillary acidic protein (GFAP) suggested that the TNF- α-positive cells are most likely microglia, not astrocytes. TNF- α immunostaining decreased at 6 and 24 h but increased again at 3 days, when pyramidal neurons showed degeneration. Adjacent-section staining for microglia and double staining with GFAP suggested that TNF- α-positive cells in the pyramidal cell layer were microglia and those in the white matter were astrocytes. By 5 days TNF- α immunostaining disappeared from these glial cells, while a number of microglia were accumulated in the degenerated hippocampal pyramidal layer. Pyramidal neurons never expressed TNF- α immunoreactivity. Western blotting confirmed biphasic TNF- α expression. Our findings suggest that early production of TNF- α by microglia may activate a cytokine network in postischemic brain resulting in TNF- α synthesis by astrocytes.
Inhibition of Nonselective Cation Channels Reduces Focal Ischemic Injury of Rat Brain
Authors: Hoehn-Berlage M. , Hossmann K.-A. , Busch E. , Eis M. , Schmitz B. , Gyngell M. L.
Published: November 2003

The effect of the novel inhibitor of receptor-activated and calcium store-operated nonselective cation channels, ( RS)-(3,4-dihydro-6,7-dimethoxyisoquinoline-1- γ1)-2-phenyl- N,N-di-[2(2,3,4-trimethoxyphenyl) ethyl]acetamide (LOE 908 MS), on focal cerebral ischemia was studied in halothane-anesthetized rats submitted to permanent suture occlusion of the right middle cerebral artery (MCA). The treated group (n = 7) received subcutaneous injections of 30 mg/kg LOE 908 MS (in 1 ml saline) 10 min after vascular occlusion and again after 3 h. The untreated group (n = 11) was injected subcutaneously with 1 ml saline at the same times. Evolution of infarct was monitored by electrophysiological recording of EEG and cortical steady potential and by diffusion-weighted magnetic resonance imaging during the initial 6 h of vascular occlusion. The hemodynamic, biochemical, and morphological changes were studied after 6 h by combining autoradiographic measurement of blood flow with histological stainings and pictorial measurements of ATP, glucose, and tissue pH. In the untreated animals, the ischemic lesion volume [defined as the region in which the apparent diffusion coefficient (ADC) of water declined to below 80% of control] steadily increased by ∼50% during the initial 6 h of vascular occlusion relative to the first set of data 10 min postocclusion. In the treated animals, in contrast, the ADC lesion volume declined by ∼20% during the same interval. Treatment also led to a significant reduction in the number of periinfarct depolarizations. After 6 h of vascular occlusion, blood flow was significantly higher in the treated animals, and the volume of ATP-depleted and morphologically injured tissue representing the infarct core was 60–70% smaller. The volume of severely acidic tissue, in contrast, did not differ, indicating that LOE 908 MS does not reduce the size of ischemic penumbra. These findings demonstrate that postocclusion treatment of permanent focal ischemia with LOE 908 MS delays the expansion of the infarct core into the penumbra for a duration of at least 6 h and therefore substantially prolongs the window of opportunity for the reversal of the ischemic impact in the peripheral parts of the evolving infarct.
Limited but Significant Protective Effect of Hypothermia on Ultra-Early-Type Ischemic Neuronal Injury in the Thalamus
Authors: Kawai Kensuke , Nakayama Hitoshi , Tamura Akira
Published: November 2003

We investigated the protective effect of hypothermia on ultra-early-type ischemic injury in the thalamic reticular nucleus of the rat. Cerebral ischemia was produced by 5 min of cardiac arrest followed by resuscitation. Rectal and cranial temperature during and after cardiac arrest was maintained at 37–38°C in the normothermic group and at 32–33°C in the hypothermic group. In the postischemic hypothermic group, temperature was maintained at 32–33°C starting 15 min after normothermic ischemia. Histological damage was evaluated quantitatively. While after 5 min of recirculation there was no difference in morphological changes in terms of neuronal halo formation, intraischemic hypothermia reduced the severity of the degenerative changes represented by vacuolated or dark neurons by 15 min. Postischemic hypothermia failed to show any evidence of protection by 30 min. The protective effect of intraischemic hypothermia remained significant when evaluated at 14 days after ischemia by volumetry of the lesion and neuronal density analysis, whereas postischemic hypothermia had no clear protective effect. These results suggest that the protective effect of intraischemic hypothermia applies to neurons susceptible to ultra-early-type injury, but the effect of postischemic hypothermia is limited because normothermic ischemia results in extensive degeneration in these neurons by 15 min.
Hyperglycemia Delays Terminal Depolarization and Enhances Repolarization after Peri-Infarct Spreading Depression as Measured by Serial Diffusion MR Mapping
Authors: Els Thomas , Röther Joachim , Beaulieu Christian , de Crespigny Alexander , Moseley Michael
Published: November 2003

We investigated the effect of hyperglycemia on the initiation and propagation of spreading depression-like peri-infarct ischemic depolarization (SD) induced by focal cerebral ischemia in rats. Peri-infarct SD were monitored during the initial 15 minutes after remotely induced middle cerebral artery occlusion (MCAO) using serial diffusion weighted magnetic resonance imaging. Maps of the apparent diffusion coefficient (ADC) were calculated and ADC decreases were monitored over time. Hyperglycemic rats (n = 6) had a significant prolongation of the time from induction of MCAO to the start of the ADC decrease as compared with normoglycemic control rats. The time to the maximal ADC decrease was significantly delayed and recovery of transient ADC declines in the area adjacent to the ischemic core was significantly faster in hyperglycemic rats. We conclude that hyperglycemia delays the terminal depolarization in the ischemic core and supports a faster repolarization in severely mal-perfused penumbral tissue after SD, which reflects the increased availability of energy substrates in the state of hyperglycemia.
Role of Nitric Oxide in Regulation of Brain Stem Circulation during Hypotension
Authors: Toyoda Kazunori , Fujii Kenichiro , Ibayashi Setsuro , Nagao Tetsuhiko , Kitazono Takanari , Fujishima Masatoshi
Published: November 2003

We tested the hypothesis that nitric oxide (NO) plays a role in CBF autoregulation in the brain stem during hypotension. In anesthetized rats, local CBF to the brain stem was determined with laser-Doppler flowmetry, and diameters of the basilar artery and its branches were measured through an open cranial window during stepwise hemorrhagic hypotension. During topical application of 10−5 mol/L and 10−4 mol/L Nω-nitro-L-arginine (L-NNA), a nonselective inhibitor of nitric oxide synthase (NOS), CBF started to decrease at higher steps of mean arterial blood pressure in proportion to the concentration of L-NNA in stepwise hypotension (45 to 60 mm Hg in the 10−5 mol/L and 60 to 75 mm Hg in the 10−4 mol/L L-NNA group versus 30 to 45 mm Hg in the control group). Dilator response of the basilar artery to severe hypotension was significantly attenuated by topical application of L-NNA (maximum dilatation at 30 mm Hg: 16 ± 8% in the 10−5 mol/L and 12 ± 5% in the 10−4 mol/L L-NNA group versus 34 ± 4% in the control group), but that of the branches was similar between the control and L-NNA groups. Topical application of 10−5 mol/L 7-nitro indazole, a selective inhibitor of neuronal NOS, did not affect changes in CBF or vessel diameter through the entire pressure range. Thus, endothelial but not neuronal NO seems to take part in the regulation of CBF to the the brain stem during hypotension around the lower limits of CBF autoregulation. The role of NO in mediating dilatation in response to hypotension appears to be greater in large arteries than in small ones.
Frequency Dependence of Cerebrovascular Impedance in Preterm Neonates: A Different View on Critical Closing Pressure
Authors: Michel Erik , Hillebrand Stefanie , vonTwickel Johanna , Zernikow Boris , Jorch Gerd
Published: November 2003

The nonproportional relationship between instantaneous arterial blood pressure (BP) and cerebral blood flow velocity (CBFv) is well explained by the concept of critical closing pressure (CCP). We aimed to determine the frequency response of the neonatal cerebrovascular system, and to establish the exact mathematical relationship between cerebrovascular impedance and CCP under physiologic conditions. In 10 preterm neonates (gestational age, 25–32 weeks; birth weight, 685–1,730 g; age 1–7 days) we Doppler-traced CBFv of the internal carotid artery. Blood pressure was traced simultaneously. Critical closing pressure was graphically determined. Cerebrovascular impedance was calculated as the square root of the ratio of the corresponding peaks in the power spectra of BP and CBFv at zero frequency, and at heart rate (H) and harmonics (xH). Uniformly, the impedance between H and 3H (2 to 6 Hz) was reduced about fivefold, compared with the impedance at zero frequency. The cerebrovascular system behaves like a high-pass filter, leading to a reduction of the DC (direct current) component of CBFv (analogous to current) relative to that of the driving force BP (analogous to voltage). The frequency response of cerebrovascular impedance reflects the ratio of CCP and DC BP. A mathematical derivation of this relationship is given matching the observed results. Thus, both the CCP and the impedance approach are valid.
Neuroprotective Effects of Inhibiting Poly(ADP-Ribose) Synthetase on Focal Cerebral Ischemia in Rats
Authors: Takahashi Kazushi , Greenberg Joel H. , Jackson Paul , Maclin Keith , Zhang Jie
Published: November 2003

Poly(adenosine 5′-diphosphoribose) synthetase (PARS) has been described as an important candidate for mediation of neurotoxicity by nitric oxide. In the current study, we demonstrate for the first time that in vivo administration of a potent PARS inhibitor, 3,4-dihydro 5-[4-1(1-piperidinyl) butoxy]-1(2H)-isoquinolinone, leads to a significant reduction of infarct volume in a focal cerebral ischemia model in the rat. Focal cerebral ischemia was produced by cauterization of the right distal middle cerebral artery (MCA) with bilateral temporary common carotid artery occlusion for 90 minutes. 3,4-Dihydro 5[4-(1-piperidinyl) butoxy]-1(2H)-isoquinolinone was dissolved in dimethyl sulfoxide and injected intraperitoneally. Animals were treated 2 hours before MCA occlusion (control, n = 14; 5 mg/kg, n = 7; 10 mg/kg, n = 7; 20 mg/kg, n = 7; 40 mg/kg, n = 7), and 2 hours after MCA occlusion (same doses as before treatment). Twenty-four hours after MCA occlusion, the total infarct volume was measured using 2,3,5-triphenyltetrazolium chloride. Inhibition of PARS leads to a significant decrease in the damaged volume in the 5 mg/kg–treated group (106.7 ± 23.2 mm3; mean ± SD, P < 0.002), the 10 mg/kg–treated group (76.4 ± 16.8 mm3, P < 0.001), and the 20 mg/kg–treated group (110.2 ± 42.0 mm3, P < 0.02) compared with the control group (165.2 ± 34.0 mm3). The substantial reduction in infarct volume indicates that the activation of PARS may play an important role in the pathogenesis of brain damage in cerebral ischemia through intracellular energy depletion.
Ischemic Brain Injury is Mediated by the Activation of Poly(ADP-Ribose)Polymerase
Authors: Endres Matthias , Wang Zhao-Qi , Namura Shobu , Waeber Christian , Moskowitz Michael A.
Published: November 2003

Poly(ADP-ribose)polymerase (PARP, EC 2.4.2.30), an abundant nuclear protein activated by DNA nicks, mediates cell death in vitro by nicotinamide adenine dinucleotide (NAD) depletion after exposure to nitric oxide. The authors examined whether genetic deletion of PARP (PARP null mice) or its pharmacologic inhibition by 3-aminobenzamide (3-AB) attenuates tissue injury after transient cerebral ischemia. Twenty-two hours after reperfusion following 2 hours of filamentous middle cerebral artery occlusion, ischemic injury was decreased in PARP−/− and PARP+/− mice compared with PARP+/+ litter mates, and also was attenuated in 129/SV wild-type mice after 3-AB treatment compared with controls. Infarct sparing was accompanied by functional recovery in PARP−/− and 3-AB–treated mice. Increased poly(ADP-ribose) immunostaining observed in ischemic cell nuclei 5 minutes after reperfusion was reduced by 3-AB treatment. Levels of NAD—the substrate of PARP—were reduced 2 hours after reperfusion and were 35% of contralateral levels at 24 hours. The decreases were attenuated in PARP−/− mice and in 3-AB–treated animals. Poly(ADP-ribose)polymerase cleavage by caspase-3 (CPP-32) has been proposed as an important step in apoptotic cell death. Markers of apoptosis, such as oligonucleosomal DNA damage, total DNA fragmentation, and the density of terminal deoxynucleotidyl transferase dUTP nick-end–labelled (TUNEL +) cells, however, did not differ in ischemic brain tissue of PARP−/− mice or in 3-AB–treated animals versus controls, although there were differences in the number of TUNEL-stained cells reflecting the decrease in infarct size. Thus, ischemic brain injury activates PARP and contributes to cell death most likely by NAD depletion and energy failure, although the authors have not excluded a role for PARP in apoptotic cell death at earlier or later stages in ischemic cell death. Inhibitors of PARP activation could provide a potential therapy in acute stroke.
Activity of Mitochondrial Respiratory Chain Enzymes after Transient Focal Ischemia in the Rat
Authors: Canevari Laura , Kuroda Satoshi , Bates Timothy E. , Clark John B. , Siesjö Bo K.
Published: November 2003

Previous results demonstrated that after 2-hour middle cerebral artery occlusion (MCAO) in the rat, 1- to 2-hour recirculation temporarily restored the bioenergetic state and mitochondrial function, but secondary deterioration took place after 4 hours. The authors measured the activity of mitochondrial respiratory chain complexes, citrate synthase, and glutamate dehydrogenase as possible targets of secondary damage. Focal and penumbral tissues were sampled in the control condition, after 2 hours of MCAO, and after 1, 2, or 4 hours of postischemic recirculation; two groups were treated with α-phenyl-N- tert-butyl-nitrone (PBN). Complex IV activity transiently decreased after MCAO, but after recirculation all measured activities returned to control values.
Profiles of Cortical Tissue Depolarization in Cat Focal Cerebral Ischemia in Relation to Calcium Ion Homeostasis and Nitric Oxide Production
Authors: Ohta Kouichi , Graf Rudolf , Rosner Gerd , Heiss Wolf-Dieter
Published: November 2003

Cortical depolarization was investigated in a topographic gradient of ischemic density after 1-hour transient middle cerebral artery occlusion in halothane-anesthetized cats. A laser Doppler flow probe, an ion-selective microelectrode, and a nitric oxide (NO) electrode measured regional CBF (rCBF), direct current (DC) potential, extracellular Ca2+ concentration ([Ca2+]o), and NO concentration in ectosylvian and suprasylvian gyri of nine animals. Recordings revealed 12 of 18 sites with persistent negative shifts of the DC potential, severe rCBF reduction, and a drop of [Ca2+]o characteristic for core regions of focal ischemia. Among these sites, two types were distinguished by further analysis. In Type 1 (n = 5), rapid, negative DC shifts resembled anoxic depolarization as described for complete global ischemia. In this type, ischemia was most severe (8.9 ± 2.5% of control rCBF), [Ca2+]o dropped fast and deepest (0.48 ± 0.20 mmol/L), and NO concentration increased transiently (36.1 ± 24.0 nmol/L at 2.5 minutes), and decreased thereafter. In Type 2 (n = 7), the DC potential fell gradually over the first half of the ischemic episode, rCBF and [Ca2+]o reductions were smaller than in Type 1 (16.2 ± 8.2%; 0.77 ± 0.41 mmol/L), and NO increased continuously during ischemia (53.1 ± 60.4 nmol/L at 60 minutes) suggesting that in this type NO most likely exerts its diverse actions on ischemia-threatened tissue. In the remaining six recording sites, a third type (Type 3) attributable to the ischemic periphery was characterized by minimal DC shifts, mild ischemia (37.2 ± 13.3%), nonsignificant alterations of [Ca2+]o, but decreased NO concentrations during middle cerebral artery occlusion. Reperfusion returned the various parameters to baseline levels within 1 hour, the recovery of [Ca2+]o and NO concentration being delayed in Type 1. An NO synthase inhibitor (NG-nitro-l-arginine, 50 mg/kg intravenously; four animals) abolished NO elevation during ischemia. In conclusion, even in the core of focal cerebral ischemia and reperfusion, different ischemic densities produce different types of cortical tissue manifesting distinctive chronological profiles of depolarization, Ca2+ influx, and NO synthesis.
Oxygen Deprivation Produces Delayed Inhibition of Long-Term Potentiation by Activation of NMDA Receptors and Nitric Oxide Synthase
Authors: Izumi Yukitoshi , Katsuki Hiroshi , Benz Ann M. , Zorumski Charles F.
Published: November 2003

The acute and delayed effects of anoxia on synaptic transmission and long-term potentiation (LTP) were examined in the CA1 region of rat hippocampal slices. Oxygen deprivation for 20 minutes completely but reversibly depressed excitatory postsynaptic potentials mediated by both N-methyl-d-aspartate receptors (NMDAR) and non-NMDAR. Although LTP was reliably produced by a single tetanus delivered 30 minutes after reoxygenation, LTP could not be induced when a tetanus was delivered 70 to 100 minutes after reoxygenation. A tetanus delivered 100 minutes after reoxygenation produced lasting synaptic enhancement when 100 μmol/L d,l-amino-phosphonovaleric acid (APV), a competitive NMDAR antagonist, was administered during the period of oxygen deprivation. The delayed effects of oxygen deprivation were not blocked when APV was administered after oxygen deprivation. Similarly, the delayed effects on LTP induction were overcome by inhibitors of nitric oxide synthase when the nitric oxide synthase inhibitors were administered during anoxia, but not when administered after oxygen deprivation. These results suggest that untimely activation of NMDAR and nitric oxide release during anoxia produce delayed inhibition of LTP induction and may be involved in the memory defects that occur subsequent to cerebral hypoxia.
Subcellular Localization and Duration of μ-Calpain and m-Calpain Activity after Traumatic Brain Injury in the Rat: A Casein Zymography Study
Authors: Zhao Xiurong , Posmantur Rand , Kampfl Andreas , Liu Shi-Jie , Wang Kevin K. W. , Newcomb Jennifer K. , Pike Brian R. , Clifton Guy L. , Hayes Ronald L.
Published: November 2003

Casein zymographic assays were performed to identify changes in μ-calpain and m-calpain activity in naive, sham-injured, and injured rat cortex at 15 minutes, 3 hours, 6 hours, and 24 hours after unilateral cortical impact brain injury. Cortical samples ipsilateral and contralateral to the site of injury were separated into cytosolic and total membrane fractions. Marked increases in μ-calpain activity in cytosolic fractions in the ipsilateral cortex occurred as early as 15 minutes, became maximal at 6 hours, and decreased at 24 hours to levels observed at 15 minutes after injury. A similar temporal profile of cytosolic μ-calpain activity in the contralateral cortex was observed, although the increases in the contralateral cortex were substantially lower than those in the ipsilateral cortex. Differences were also noted between cytosolic and total membrane fractions. The detection of a shift in μ-calpain activity to the total membrane fraction first occurred at 3 hours after traumatic brain injury and became maximal at 24 hours after traumatic brain injury. This shift in μ-calpain activity between the two fractions could be due to the redistribution of μ-calpain from the cytosol to the membrane. m-Calpain activity was detected only in cytosolic fractions. m-Calpain activity in cytosolic fractions did not differ significantly between ipsilateral and contralateral cortices, and increased in both cortices from 15 minutes to 6 hours after injury. Relative magnitudes of m-calpain versus μ-calpain activity in cytosolic fractions differed at different time points after injury. These studies suggest that traumatic brain injury can activate both calpain isoforms and that calpain activity is not restricted to sites of focal contusion and cell death at the site of impact injury but may represent a more global response to injury.
Reduced Ischemic Brain Injury in Interleukin-1β Converting Enzyme—Deficient Mice
Authors: Schielke Gerald P. , Yang Guo-Yuan , Shivers Brenda D. , Betz A. Lorris
Published: November 2003

A variety of recent studies suggest a role for both inflammatory cytokines such as interleukin-1 beta (IL-1β), and apoptosis in ischemic brain injury. Because IL-1β converting enzyme (ICE) is required for the conversion of proIL-1β to its biologically active form, and has homology with proteins that regulate apoptosis in invertebrates, we studied the effect of cerebral ischemia on brain injury in mutant mice deficient in the ICE gene (ICE knockout [KO] mice). Focal cerebral ischemia, produced by occlusion of the middle cerebral artery, resulted in brain edema (increased water and sodium content) at 4 hours and a histologically defined brain lesion at 24 hours. Both of these markers of brain injury were significantly reduced in the ICE KO mice as compared to wild-type C57BL/6 mice. Regional cerebral blood flow, determined using the flow tracer, N-isopropyl [methyl 1,3-14C] p-iodoamphetamine (14C-IMP), was similar in the two strains of mice, indicating that the reduced brain injury in the KO mice was not a result of a lesser degree of ischemia. These data show that ICE contributes to the development of ischemic brain damage, and that it plays a role at an early time in the pathologic process. Although the mechanism of this effect is uncertain, our results suggest that pharmacologic inhibition of ICE may be a useful treatment for stroke.
Attenuation of Delayed Neuronal Death after Mild Focal Ischemia in Mice by Inhibition of the Caspase Family
Authors: Endres Matthias , Namura Shobu , Shimizu-Sasamata Masao , Waeber Christian , Zhang Lin , Gómez-Isla Teresa , Hyman Bradley T. , Moskowitz Michael A.
Published: November 2003

Inhibitors of apoptosis and of excitotoxic cell death reduce brain damage after transient and permanent middle cerebral artery occlusion. We compared the neuroprotective effects of two caspase family inhibitors with the N-methyl-d-aspartate receptor antagonist (+)-MK-801 hydrogen maleate (MK-801) in a newly characterized cycloheximidesensitive murine model of transient middle cerebral artery occlusion (30 minutes) in which apoptotic cell death is prominent. Ischemic infarction, undetected by 2,3,5-triphenyltetrazolium chloride staining at 24-hour reperfusion, featured prominently in the striatum at 72 hours and 7 days on hematoxylin-eosin—stained sections. Markers of apoptosis, such as oligonucleosomal DNA damage (laddering) and terminal deoxynucleotidyl transferase—mediated dUTP-biotin nick-end labeling (TUNEL)–positive cells first appeared at 24 hours and increased significantly at 72 hours and 7 days after reperfusion. The TUNEL-labeled cells were mostly neurons and stained negative for glial (GFAP, glial fibrillary acid protein) and leukocyte specific markers (CD-45). The caspase inhibitors, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD.FMK; 120 ng intracerebroventricularly) or N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (z-DEVD.FMK; 480 ng intracerebroventricularly) decreased infarct size and neurologic deficits when administered 6 hours after reperfusion. The extent of protection was greater than in models of more prolonged ischemia or after permanent occlusion, and the therapeutic window was extended from 0 to 1 hours after 2-hour middle cerebral artery occlusion to at least 6 hours after brief ischemia. Also, z-VAD.FMK and z-DEVD.FMK treatment decreased oligonucleosomal DNA damage (DNA laddering) as assessed by quantitative autoradiography after gel electrophoresis. By contrast, MK-801 protected brain tissue only when given before ischemia (3 mg/kg intraperitoneally), but not at 3 or 6 hours after reperfusion. Despite a decrease in infarct size after MK-801 pretreatment, the amount of DNA laddering did not decrease 72 hours after reperfusion, thereby suggesting a mechanism distinct from inhibition of apoptosis. Hence, 30 minutes of reversible ischemia augments apoptotic cell death, which can be attenuated by delayed z-VADPMK and z-DEVD.FMK administration with preservation of neurologic function. By contrast, the therapeutic window for MK-801 does not extend beyond the time of occlusion, probably because its primary mechanism of action does not block the development of apoptotic cell death.
A Reproducible Model of Middle Cerebral Artery Occlusion in Mice: Hemodynamic, Biochemical, and Magnetic Resonance Imaging
Authors: Hata Ryuji , Mies Günter , Wiessner Christoph , Fritze Klaus , Hesselbarth Daniel , Brinker Gerrit , Hossmann Konstantin-Alexander
Published: November 2003

A reproducible model of thread occlusion of the middle cerebral artery (MCA) was established in C57 Black/6J mice by matching the diameter of the thread to the weight of the animals. For this purpose, threads of different diameter (80 to 260 μm) were inserted into the MCA of animals of different weights (18 to 33 g), and the success of vascular occlusion was evaluated by imaging the ischemic territory on serial brain sections with carbon black. Successful occlusion of the MCA resulted in a linear relationship between body weight and thread diameter ( r = 0.46, P < 0.01), allowing precise selection of the appropriate thread size. Laser-Doppler measurements of CBF, neurological scoring, and 2,3,5-triphenyltetrazolium chloride staining confirmed that matching of animal weight and suture diameter produced consistent cerebral infarction. Three hours after MCA occlusion, imaging of ATP, tissue pH, and cerebral protein synthesis allowed differentiation between the central infarct core, in which ATP was depleted, and a peripheral penumbra with reduced protein synthesis and tissue acidosis but preserved ATP content. Perfusion deficits and ischemic tissue alterations could also be detected by perfusion- and diffusion-weighted magnetic resonance imaging, demonstrating the feasibility of dynamic evaluations of infarct evolution. The use of multiparametric imaging techniques in this improved MCA occlusion model opens the way for advanced pathophysiological studies of stroke in gene-manipulated animals.
Effect of Decreased Glucose Concentration on Cerebrovascular Tone In Vitro
Authors: Swafford Melba W.G. , Raty Sally , Childres William F. , Bryan Robert M.
Published: November 2003

We studied the effect of decreased glucose concentration on cerebrovascular tone in vitro. Segments of rat middle cerebral arteries (MCA) were isolated, cannulated at both ends with glass micropipettes, and pressurized to 85 mm Hg. Decreasing the glucose in the extraluminal bath and luminal perfusate from 5.5 mmol/L to 1.0 or 0.5 mmol/L for 1.5 hours each had no significant effect on the diameter of the arteries. When all the glucose was removed from the extraluminal bath and luminal perfusate for 1.5 hours, the MCA dilated by 23% [252 ± 24 (SD) μm to 311 ± 7 μm ( P < .5, n = 7)]. This dilation was 80% of the maximum dilation produced by removal of Ca+2 from the bathing solutions. Neither removal of the endothelium nor inhibition of the ATP-sensitive K channels with 10−5 mol/L glibenclamide altered the response of the isolated MCA to the removal of glucose. We conclude that rat MCA are relatively more resistant to substrate limitation compared to the brain as a whole.
Reperfusion after Thrombolytic Therapy of Embolic Stroke in the Rat: Magnetic Resonance and Biochemical Imaging
Authors: Busch Elmar , Krüger Karsten , Allegrini Peter R. , Kerskens Christian M. , Gyngell Michael L. , Hoehn-Berlage Mathias , Hossmann Konstantin-Alexander
Published: November 2003

The effect of thrombolytic therapy was studied in rats submitted to thromboembolic stroke by intracarotid injection of autologous blood clots. Thrombolysis was initiated after 15 minutes with an intracarotid infusion of recombinant tissue-type activator (10 mg/kg body weight). Reperfusion was monitored for 3 hours using serial perfusion- and diffusion magnetic resonance imaging, and the outcome of treatment was quantified by pictorial measurements of ATP, tissue pH, and blood flow. In untreated animals, clot embolism resulted in an immediate decrease in blood flow and a sharp decrease in the apparent diffusion coefficient (ADC) that persisted throughout the observation period. Thrombolysis successfully recanalized the embolized middle cerebral artery origin and led to gradual improvement of blood flow and a slowly progressing reversal of ADC changes in the periphery of the ischemic territory, but only to transient and partial improvement in the center. Three hours after initiation of thrombolysis, the tissue volume with ADC values less than 80% of control was 39 ± 22% as compared to 61 ± 20% of ipsilateral hemisphere in untreated animals (means ± SD, P = .03) and the volume of ATP-depleted brain tissue was 25 ± 31% as compared to 46 ± 29% in untreated animals. Recovery of ischemic brain injury after thromboembolism is incomplete even when therapy is started as early as 15 minutes after clot embolism. Possible explanations for our findings include downstream displacement of clot material, microembolism of the vascular periphery, and events associated with reperfusion injury.
Autoregulatory Vasodilation of Parenchymal Vessels is Impaired during Cerebral Vasospasm
Authors: Yundt Kent D. , Grubb Robert L. , Diringer Michael N. , Powers William J.
Published: November 2003

Impaired CBF autoregulation during vasospasm after aneurysmal subarachnoid hemorrhage (SAH) could reflect impaired capacity of distal vessels to dilate in response to reduced local perfusion pressure or simply indicate that the perfusion pressure distal to large arteries in spasm is so low that vessels are already maximally dilated. Autoregulatory vasodilation can be detected in vivo as an increase in the parenchymal cerebral blood volume (CBV). Regional CBV, CBF, and oxygen extraction fraction in regions with and without angiographic vasospasm obtained from 29 positron emission tomography studies performed after intracranial aneurysm rupture were compared with data from 19 normal volunteers and five patients with carotid artery occlusion. Regional CBF was reduced compared to normal in regions from SAH patients with and without vasospasm as well as with ipsilateral carotid occlusion ( P < .0001). Regional oxygen extraction fraction was higher during vasospasm and distal to carotid occlusion than both normal and SAH without vasospasm ( P < .0001). Regional CBV was reduced compared to normal in regions with and without spasm, whereas it was increased ipsilateral to carotid occlusion ( P < .0001). These findings of reduced parenchymal CBV during vasospasm under similar conditions of tissue hypoxia that produce increased CBV in patients with carotid occlusion provide evidence that parenchymal vessels distal to arteries with angiographic spasm after SAH do not show normal autoregulatory vasodilation.
Brain Correlates of Memory Processes in Patients with Dementia of Alzheimer's Type: A Spect Activation Study
Authors: Cardebat Dominique , Démonet Jean-François , Puel Michèle , Agniel Alain , Viallard Gerard , Celsis Pierre
Published: November 2003

Task-induced changes in regional cerebral blood flow (rCBF) during verbal episodic memory activation were compared in 17 right-handed patients with dementia of the Alzheimer's type (DAT) and 20 healthy volunteers. Regional cerebral blood flow was assessed using single photon emission computed tomography (SPECT) and an injection of 133Xe (xenon, isotope of mass 133) in 21 regions of interest (ROI) during rest, passive listening to 36 words, and memorizing of a 12-word list repeated three times. In healthy subjects, memory—listening comparison showed activation of a distributed system involving several left-sided ROI, especially the posterior inferior frontal region. In patients with DAT, the same pattern of activation was found for listening—rest comparison, and no significant changes were found in memory—listening comparison. During listening compared with rest, significant activation was observed in left-sided hypoperfused regions. A significant correlation between memory performance and rCBF recorded in patients with DAT during the memory task was found only in the right lateral frontal region, a region that was not hypoperfused significantly in patients. The involvement of this region might relate to either retrieval effort or actual performance of patients with DAT on the memory task.
Apolipoprotein E and the CNS Response to Injury
Authors: Laskowitz Daniel T. , Horsburgh Karen , Roses Allen D.
Published: November 2003

Apolipoprotein E (apoE) is a multifunctional protein with an expanding role in the neurobiology of disease. Although originally described in the context of cholesterol metabolism, interest in the neurobiology of apoE has intensified following the association between apoE genotype and risk of developing Alzheimer's disease. Recent clinical observations also suggest that apoE genotype may influence recovery after a variety of neurological insults. Thus, in addition to the study of disease-specific mechanisms by which apoE may modulate susceptibility of developing Alzheimer's disease, there has been an increasing focus on its role in modulating the CNS response to acute injury. Although the neurobiology of apoE in the injured brain remains incompletely defined, there is evidence to suggest neurotrophic, immunomodulatory, and antioxidant effects.
Propentofylline Potentiates Induced Ischemic Tolerance in Gerbil Hippocampal Neurons Via Adenosine Receptor
Authors: Kawahara Nobutaka , Ide Takafumi , Saito Nobuhito , Kawai Kensuke , Kirino Takaaki
Published: November 2003

The effect of propentofylline, an adenosine uptake inhibitor, on ischemic tolerance was investigated in the gerbil global ischemia model. Propentofylline was administered 24 hours after short preconditioning ischemia, and animals were subjected to 5-minute ischemia 24 hours thereafter. Propentofylline at a dose of 20 mg/kg intraperitoneally, but not at a dose of 10 mg/kg, significantly potentiated the protective effect of preconditioning ischemia in the CA1 hippocampal neurons. This effect was completely abolished by simultaneous administration of theophylline (20 mg/kg), an adenosine receptor blocker. This finding suggests the involvement of adenosine receptor for the development of ischemic tolerance.
“Facilitated” Amino Acid Transport is Upregulated in Brain Tumors
Authors: Miyagawa Tadashi , Oku Takamitsu , Uehara Hisao , Desai Revathi , Beattie Bradley , Tjuvajev Juri , Blasberg Ronald
Published: November 2003

The goal of this study was to determine the magnitude of “facilitated” amino acid transport across tumor and brain capillaries and to evaluate whether amino acid transporter expression is “upregulated” in tumor vessels compared to capillaries in contralateral brain tissue. Aminocyclopentane carboxylic acid (ACPC), a non-metabolized [14C]-labeled amino acid, and a reference molecule for passive vascular permeability, [67Ga]-gallium-diethylenetriaminepentaacetic acid (Ga-DTPA), were used in these studies. Two experimental rat gliomas were studied (C6 and RG2). Brain tissue was rapidly processed for double label quantitative autoradiography 10 minutes after intravenous injection of ACPC and Ga-DTPA. Parametric images of blood-to-brain transport (K1ACPC and K1Ga-DTPA, μL/min/g) produced from the autoradiograms and the histology were obtained from the same tissue section. These three images were registered in an image array processor; regions of interest in tumor and contralateral brain were defined on morphologic criteria (histology) and were transferred to the autoradiographic images to obtain mean values. The facilitated component of ACPC transport (∂KlACPC) was calculated from the K1ACPC and K1Ga-DTPA data, and paired comparisons between tumor and contralateral brain were performed. ACPC flux, K1ACPC, across normal brain capillaries (22.6 ± 8.1 μL/g/min) was >200-fold greater than that of Ga-DTPA (0.09 ± 0.04 μL/g/min), and this difference was largely (~90%) due to facilitated ACPC transport. Substantially higher K1ACPC values compared to corresponding K1DTPA values were also measured in C6 and RG2 gliomas. The ∂K1ACPC values for C6 glioma were more than twice that of contralateral brain cortex. K1ACPC and ∂K1ACPC values for RG2 gliomas was not significantly higher than that of contralateral cortex, although a ~2-fold difference in facilitated transport is obtained after normalization for differences in capillary surface area between RG2 tumors and contralateral cortex. K1ACPC, ∂K1ACPC, and K1DTPA were directly related to tumor cell density, were higher in regions of “impending” necrosis, and the tumor/contralateral brain ACPC radio-activity ratios (0 to 10 minutes) were very similar to that obtained with 0 to 60 minutes experiments. These results indicate that facilitated transport of ACPC is upregulated across C6 and RG2 glioma capillaries, and that tumors can induce upregulation of amino acid transporter expression in their supporting vasculature. They also suggest that early imaging (e.g., 0 to 20 minutes) with radiolabeled amino acids in a clinical setting may be optimal for defining brain tumors.
Neuroprotective Efficacy and Mechanisms of Novel Pyrrolopyrimidine Lipid Peroxidation Inhibitors in the Gerbil Forebrain Ischemia Model
Authors: Oostveen Jo A. , Dunn Edwige , Carter Donald B. , Hall Edward D.
Published: November 2003

A brief period of bilateral carotid occlusion (BCO)-induced forebrain ischemia in gerbils triggers neuronal degeneration and the subsequent expression of amyloid precursor protein (APP), b-amyloid protein (b-AP), and apolipoprotein E (APO-E) in the selectively vulnerable CA, region of the hippocampus. The increase in immunoreactivity is secondary to the postischemic degeneration of the CA1 neurons and is largely astrocyte-derived as evidenced by a simultaneous increase in glial fibrillary acidic protein (GFAP) staining. Oxygen radical-induced lipid peroxidation has been strongly suggested to play a role in postischemic neuronal damage and Alzheimer's disease. Recent literature suggests a possible link between early oxidative stress and APP overexpression. Therefore, the present investigation examined the effect of two novel brain-penetrating pyrrolopyrimidine lipid peroxidation inhibitors (PNU-101033E and PNU-104067F) on CA1 neurodegeneration and the subsequent increase in APP, b-AP, APO-E, and GFAP immunostaining at 4 days after a 5-minute episode of forebrain ischemia. Using an antibody for lipid peroxidation–derived malondialdehyde (MDA)-modified proteins, the authors also examined the effects of PNU-104067F on MDA immunostaining 2 days after ischemia, before completion of the neuronal loss. At 2 days, the authors also evaluated microglial activation using an antibody to surface major histocompatibility complex class II antigen expressed by activated microglia. Gerbils were treated at 30 mg/kg orally 30 minutes before the BCO and 2 hours after ischemia, followed by daily dosing for the next day (microglia and MDA) and the successive 3 days for APP, b-AP, APO-E, and GFAP immunostaining. APP and APO-E staining was significantly suppressed by 50% and 66%, respectively, with either compound. b-AP immunoreactivity was decreased 56% with both compounds, and GFAP expression was significantly decreased 53% (PNU-101033E) and 60.5% (PNU-104067F). There was a concomitant partial sparing of the CA1 hippocampal neurons by both PNU-101033E and PNU-104067F ( P<.01) as determined by cresyl violet histochemistry. PNU-104067F significantly inhibited lipid peroxidation-derived MDA immunostaining and microglia activation ( P<.05) at 48 hours after ischemia. Brain-penetrable lipid peroxidation inhibitors may provide attenuation of various glial response proteins after ischemic injury, probably secondary to neuronal protection.