Journal of Cerebral Blood Flow and Metabolism
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Novel Therapeutic Strategy for Stroke in Rats by Bone Marrow Stromal Cells and <i>ex vivo</i> HGF Gene Transfer with HSV-1 Vector Occlusive cerebrovascular disease leads to brain ischemia that causes neurological deficits. Here we introduce a new strategy combining mesenchymal stromal cells (MSCs) and ex vivo hepatocyte growth factor (HGF) gene transferring with a multimutated herpes simplex virus type-1 vector in a rat transient middle cerebral artery occlusion (MCAO) model. Gene-transferred MSCs were intracerebrally transplanted into the rats' ischemic brains at 2h (superacute) or 24 h (acute) after MCAO. Behavioral tests showed significant improvement of neurological deficits in the HGF-transferred MSCs (MSC-HGF)-treated group compared with the phosphate-buffered saline (PBS)-treated and MSCs-only-treated group. The significant difference of infarction areas on day 3 was detected only between the MSC-HGF group and the PBS group with the superacute treatment, but was detected among each group on day 14 with both transplantations. After the superacute transplantation, we detected abundant expression of HGF protein in the ischemic brain of the MSC-HGF group compared with others on day 1 after treatment, and it was maintained for at least 2 weeks. Furthermore, we determined that the increased expression of HGF was derived from the transferred HGF gene in gene-modified MSCs. The percentage of apoptosis-positive cells in the ischemic boundary zone (IBZ) was significantly decreased, while that of remaining neurons in the cortex of the IBZ was significantly increased in the MSC-HGF group compared with others. The present study shows that combined therapy is more therapeutically efficient than MSC cell therapy alone, and it may extend the therapeutic time window from superacute to acute phase.
Journal of Cerebral Blood Flow and Metabolism - Tập 26 Số 9 - Trang 1176-1188 - 2006
boc-Aspartyl(OMe)-Fluoromethylketone Attenuates Mitochondrial Release of Cytochrome <i>c</i> and Delays Brain Tissue Loss after Traumatic Brain Injury in Rats The pathobiology of traumatic brain injury (TBI) includes activation of multiple caspases followed by cell death with a spectrum of apoptotic phenotypes. There are initiator (e.g. caspase-2, −8, and −9) and effector (e.g. caspase-3 and −7) caspases. Recently, caspase-2 and −8 have been shown to regulate cell death via provoking cytochrome c release from the mitochondria upstream of caspase-9. Here, we show that an intracerebral injection of the pan-caspase inhibitor boc-Aspartyl(OMe)-fluoromethylketone (BAF; 1 μmol) 1 min after TBI in rats reduces caspase-3-like activity, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and tissue damage, and cytochrome c release in ipsilateral cortex at 24 h versus vehicle. To investigate whether either caspase-2 and/or caspase-8 activation may contribute to cytochrome release, the effect of BAF treatment on caspase-2 and caspase-8 proteolysis was also examined. boc-aspartyl(OMe)-fluoromethylketone treatment inhibited proteolysis of caspase-2 but not caspase-8 24 h after TBI in rats versus vehicle. However, BAF with or without nerve growth factor (12.5 ng/hx14 days intracerebrally via osmotic pump) did not result in differences in motor function, Morris water maze performance, hippocampal neuron survival, nor contusion volume at 14 days. These data suggest that BAF treatment reduces acute cell death after TBI by inhibiting mitochondrial release of cytochrome c, possibly via a mechanism involving initiator caspases; however, BAF appears to delay cell death, rather than result in permanent protection.
Journal of Cerebral Blood Flow and Metabolism - Tập 27 Số 2 - Trang 316-326 - 2007
TNF Alpha and Fas Mediate Tissue Damage and Functional Outcome after Traumatic Brain Injury in Mice Tumor necrosis factor-alpha (TNFα) and Fas are induced after traumatic brain injury (TBI); however, their functional roles are incompletely understood. Using controlled cortical impact (CCI) and mice deficient in TNFα, Fas, or both (TNFα/Fas—/—), we hypothesized that TNFα and Fas receptor mediate secondary TBI in a redundant manner. Compared with wild type (WT), TNFα/Fas—/— mice had improved motor performance from 1 to 4 days ( P < 0.05), improved spatial memory acquisition at 8 to 14 days ( P < 0.05), and decreased brain lesion size at 2 and 6 weeks after CCI ( P < 0.05). Protection in TNFα/Fas—/— mice from histopathological and motor deficits was reversed by reconstitution with recombinant TNFα before CCI, and TNFα—/— mice administered anti-Fas ligand antibodies had improved spatial memory acquisition versus similarly treated WT mice ( P < 0.05). Tumor necrosis factor-alpha/Fas—/— mice had decreased the numbers of cortical cells with plasmalemma damage at 6h ( P < 0.05 versus WT), and reduced matrix metalloproteinase-9 activity in injured brain at 48 and 72 h after CCI. In immature mice subjected to CCI, genetic inhibition of TNFα and Fas conferred beneficial effects on histopathology and spatial memory acquisition in adulthood (both P < 0.05 versus WT), suggesting that the beneficial effects of TNFα/Fas inhibition may be permanent. The data suggest that redundant signaling pathways initiated by TNFα and Fas play pivotal roles in the pathogenesis of TBI, and that biochemical mechanisms downstream of TNFα/Fas may be novel therapeutic targets to limit neurological sequelae in children and adults with severe TBI.
Journal of Cerebral Blood Flow and Metabolism - Tập 27 Số 11 - Trang 1806-1818 - 2007
A Dynamic <i>in vitro</i> BBB Model for the Study of Immune Cell Trafficking into the Central Nervous System Although there is significant evidence correlating overreacting or perhaps misguided immune cells and the blood–brain barrier (BBB) with the pathogenesis of neuroinflammatory diseases, the mechanisms by which they enter the brain are largely unknown. For this purpose, we revised our humanized dynamic in vitro BBB model (DIV-BBBr) to incorporate modified hollow fibers that now feature transmural microholes (2 to 4 μm Ø) allowing for the transendothelial trafficking of immune cells. As with the original model, this new DIV-BBBr reproduces most of the physiological characteristics of the BBB in vivo. Measurements of transendothelial electrical resistance (TEER), sucrose permeability, and BBB integrity during reversible osmotic disruption with mannitol (1.6 mol/L) showed that the microholes do not hamper the formation of a tight functional barrier. The in vivo rank permeability order of sucrose, phenytoin, and diazepam was successfully reproduced in vitro. Flow cessation followed by reperfusion (Fc/Rp) in the presence of circulating monocytes caused a biphasic BBB opening paralleled by a significant increase of proinflammatory cytokines and activated matrix metalloproteinases. We also observed abluminal extravasation of monocytes but only when the BBB was breached. In conclusion, the DIV-BBBr represents the most realistic in vitro system to study the immune cell trafficking across the BBB.
Journal of Cerebral Blood Flow and Metabolism - Tập 31 Số 2 - Trang 767-777 - 2011
Microvascular Oxygen Tension and Flow Measurements in Rodent Cerebral Cortex during Baseline Conditions and Functional Activation Measuring cerebral oxygen delivery and metabolism microscopically is important for interpreting macroscopic functional magnetic resonance imaging (fMRI) data and identifying pathological changes associated with stroke, Alzheimer's disease, and brain injury. Here, we present simultaneous, microscopic measurements of cerebral blood flow (CBF) and oxygen partial pressure (pO2 ) in cortical microvessels of anesthetized rats under baseline conditions and during somatosensory stimulation. Using a custom-built imaging system, we measured CBF with Fourier-domain optical coherence tomography (OCT), and vascular pO2 with confocal phosphorescence lifetime microscopy. Cerebral blood flow and pO2 measurements displayed heterogeneity over distances irresolvable with fMRI and positron emission tomography. Baseline measurements indicate O2 extraction from pial arterioles and homogeneity of ascending venule pO2 despite large variation in microvessel flows. Oxygen extraction is linearly related to flow in ascending venules, suggesting that flow in ascending venules closely matches oxygen demand of the drained territory. Oxygen partial pressure and relative CBF transients during somatosensory stimulation further indicate arteriolar O2 extraction and suggest that arterioles contribute to the fMRI blood oxygen level dependent response. Understanding O2 supply on a microscopic level will yield better insight into brain function and the underlying mechanisms of various neuropathologies.
Journal of Cerebral Blood Flow and Metabolism - Tập 31 Số 4 - Trang 1051-1063 - 2011
A Phase-Contrast MRI Study of Physiologic Cerebral Venous Flow Although crucial in regulating intracranial hydrodynamics, the cerebral venous system has been rarely studied because of its structural complexity and individual variations. The purpose of our study was to evaluate the organization of cerebral venous system in healthy adults. Phase-contrast magnetic resonance imaging (PC-MRI) was performed in 18 healthy volunteers, in the supine position. Venous, arterial, and cerebrospinal fluid (CSF) flows were calculated. We found heterogeneous individual venous flows and variable side dominance in paired veins and sinuses. In some participants, the accessory epidural drainage preponderated over the habitually dominant jugular outflow. The PC-MRI enabled measurements of venous flows in superior sagittal (SSS), SRS (straight), and TS (transverse) sinuses with excellent detection rates. Pulsatility index for both intracranial (SSS) and cervical (mainly jugular) levels showed a significant increase in pulsatile blood flow in jugular veins as compared with that in SSS. Mean cervical and cerebral arterial blood flows were 714 ± 124 and 649 ± 178 mL/min, respectively. Cerebrospinal fluid aqueductal and cervical stroke volumes were 41 ± 22 and 460 ± 149 μL, respectively. Our results emphasize the variability of venous drainage for side dominance and jugular/epidural organization. The pulsatility of venous outflow and the role it plays in the regulation of intracranial pressure require further investigation.
Journal of Cerebral Blood Flow and Metabolism - Tập 29 Số 6 - Trang 1208-1215 - 2009
Astrocyte-Specific Expression of Aquaporin-9 in Mouse Brain is Increased after Transient Focal Cerebral Ischemia Aquaporin-9 (AQP9) is a new member of the aquaporin family of water-selective channels mainly expressed in liver and testis, presenting the characteristic of also being permeable to various solutes, particularly lactate. Recent data have shown the presence of AQP9 on tanycytes in the rat brain. In the current study, the authors show the expression of AQP9 in astrocytes in the mouse brain and changes in its expression after cerebral ischemia. Indeed, in control mouse, the AQP9 immunolabeling is present on astrocytic processes bordering the subarachnoid space and ventricles. The labeling also is observed on astrocytes in the white matter, hippocampus, hypothalamus, and lateral septum. After focal transient ischemia, an increase of the immunolabeling is detected on astrocytes in periinfarct areas. This AQP9 distribution study in mouse brain suggests a role of AQP9 in water homeostasis in the central nervous system. Furthermore, the overexpression of AQP9 on astrocytes surrounding an ischemic lesion suggests that AQP9 may also play a role in the regulation of postischemia edema and, in view of its permeability to monocarboxylates, in the clearance of lactate from the ischemic focus.
Journal of Cerebral Blood Flow and Metabolism - Tập 21 Số 5 - Trang 477-482 - 2001
Intracerebral Hirudin Injection Attenuates Ischemic Damage and Neurologic Deficits without Altering Local Cerebral Blood Flow There has been considerable interest in the use of thrombin inhibitors to reduce the occurrence of stroke or to potentiate tissue plasminogen activator-induced reperfusion. However, there is growing evidence that thrombin may also have extravascular effects that influence ischemic brain injury. Male Sprague-Dawley rats were subjected to either 90 minutes of temporary middle cerebral artery (MCA) occlusion or sham operation to examine thrombin and protease activated receptor-1 (PAR-1) expression. In another set of rats, the MCA was occluded for 90 minutes and 10 U of hirudin or the same volume of vehicle was injected into the caudate followed by reperfusion for up to 28 days, to test the effects of local thrombin inhibition on ischemic damage, neurologic outcome and cerebral blood flow (CBF). Thrombin immunoreactivity was increased in the ischemic caudate at 4 and 24 hours, whereas PAR-1 expression was unchanged. Hirudin reduced infarct volume in the caudate at 24 hours (79 ± 41 vs. 115 ± 20 mm3 , P < 0.05) and resulted in a larger residual tissue volume in the caudate at 28 days (17.6 ± 3.9 vs. 11.8 ± 6.3 mm3 , P < 0.05). Hirudin treatment also had a beneficial effect on body weight and ameliorated neurologic deficits tested by forelimb placing and forelimb use asymmetry during 28 days survival. These beneficial effects of hirudin were not associated with improved regional CBF during reperfusion. These results suggest that, in addition to their effects on coagulation and circulation, thrombin inhibitors also have direct neuroprotective properties and may be considered in stroke therapy.
Journal of Cerebral Blood Flow and Metabolism - Tập 24 Số 2 - Trang 159-166 - 2004
Respiratory Chain Inhibition Induces Tolerance to Focal Cerebral Ischemia The authors show that the inhibitor of the succinate dehydrogenase, 3-nitroproprionic acid (3-NPA), which in high doses and with chronic administration is a neurotoxin, can induce profound tolerance to focal cerebral ischemia in the rat when administered in a single dose (20 mg/kg) 3 days before ischemia. Infarcts were approximately 70% and 35% smaller in the 3-NPA preconditioned groups of permanent and transient focal cerebral ischemia, respectively. This regimen of 3-NPA preconditioning neither induced necrosis, apoptosis, or any other histologically detectable damage to the brain, nor did it affect behavior of the animals. 3-NPA led to an immediate (1-hour) and long-lasting (3-day) decrease in succinate dehydrogenase activity (30% reduction) throughout the brain, whereas only a short metabolic impairment occurred (ATP decrease of 35% within 30 minutes, recovery within 2 hours). The authors found that 3-NPA induces a burst of reactive oxygen species and the free radical scavenger dimethylthiourea, when administered shortly before the 3-NPA stimulus, completely blocked preconditioning. Inhibition of protein synthesis with cycloheximide given at the time of 3-NPA administration completely inhibited preconditioning. The authors were unsuccessful in showing upregulation of mRNA for the manganese superoxide dismutase, and did not detect increased activities of the copper-zinc and manganese superoxide dismutases, prototypical oxygen free radicals scavenging enzymes, after 3-NPA preconditioning. The authors conclude that it is possible to pharmacologically precondition the brain against focal cerebral ischemia, a strategy that may in principal have clinical relevance. The data show the relevance of protein synthesis for tolerance, and suggests that oxygen free radicals may be critical signals in preconditioning.
Journal of Cerebral Blood Flow and Metabolism - Tập 19 Số 11 - Trang 1229-1237 - 1999
Tolerance-Inducing Dose of 3-Nitropropionic Acid Modulates bcl-2 and bax Balance in the Rat Brain: A Potential Mechanism of Chemical Preconditioning Many studies have reported ischemia protection using various preconditioning techniques, including single dose 3-nitropropionic acid (3-NPA), a mitochondrial toxin. However, the cellular signal transduction cascades resulting in ischemic tolerance and the mechanisms involved in neuronal survival in the tolerant state still remain unclear. The current study investigated the mRNA and protein expression of the antiapoptotic bcl-2 and the proapoptotic bax, two antagonistic members of the bcl-2 gene family, in response to a single dose of 3-NPA, to global cerebral ischemia–reperfusion, and to the combination of both 3-NPA-pretreatment and subsequent global cerebral ischemia–reperfusion. Brain homogenates of adult Wistar rats (n = 25) were analyzed for bcl-2 and bax mRNA expression using a new highly sensitive and quantitative polymerase chain reaction (PCR) technique that allows real-time fluorescence measurements of the PCR product (LightCycler; Roche Diagnostics, Mannheim, Germany). Animals for mRNA analysis received 3-NPA (20 mg/kg, intraperitoneal; “chemical preconditioning”) or vehicle (normal saline), and were either observed for 24 plus 3 hours or were subjected to 15 minutes of global cerebral ischemia 24 hours after the pretreatment and observed for 3 hours of reperfusion. Immunohistochemistry was applied to serial brain sections of additional rats (n = 68) to determine amount and localization of the respective Bcl-2 and Bax protein expression in various brain areas. One set of animals was injected with 3-NPA and observed for 3, 12, 24, and 96 hours; a second set was exposed to 15 minutes global cerebral ischemia, 3, 12, and 24 hours reperfusion; and a third set was pretreated with 3-NPA or saline 24 hours before the ischemic brain insult and observed for 96 hours of reperfusion. The authors found single dose 3-NPA treatment to be associated with an elevated bcl-2:bax ratio (increased bcl-2 expression, decreased bax expression), both on the transcriptional (mRNA) and the translational (protein) level. The differential influence of 3-NPA was maintained during early recovery from global cerebral ischemia (3 hours), when 3-NPA pretreated animals showed higher bcl-2 and lower bax mRNA levels compared with rats with saline treatment. Respective changes in protein expression were localized predominately in neurons vulnerable to ischemic damage. Compared with baseline, Bcl-2 protein was significantly higher in surviving neurons at 96 hours after the insult, whereas Bax protein remained unchanged. However, at this late time of postischemic recovery (96 hours), the protein expression pattern of surviving neurons was not different between animals with and without 3-NPA pretreatment. To the authors' knowledge, the current study is the first report on the differential expression of pro-and antiapoptotic genes after a single, nonlethal dose of 3-NPA. The current results suggest alterations in the balance between pro-and antiapoptotic proteins as a potential explanation for the reported protection provided by chemical preconditioning using 3-NPA in rats.
Journal of Cerebral Blood Flow and Metabolism - Tập 20 Số 10 - Trang 1425-1436 - 2000
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