Cardiac arrest and cardiopulmonary resuscitation (CA/CPR) increase the risk for affective disorders in human survivors. Postischemic anxiety- and depressive-like behaviors have been documented in animal models of CA/CPR; however, the stability of post-CA/CPR anxiety-like behavior over time and the underlying physiologic mechanisms remain unknown. The hypothalamic–pituitary–adrenal (HPA) axis and the corticotropin releasing factor (CRF) system may mediate the pathophysiology of anxiety and depression; therefore, this study measured CA/CPR-induced changes in CRF receptor binding and HPA axis negative feedback. Mice were exposed to CA/CPR or SHAM surgery and assessed 7 or 21 days later. Consistent with earlier demonstrations of anxiety-like behavior 7 days after CA/CPR, increased anxiety-like behavior in the open field was also present 21 days after CA/CPR. On postoperative day 7, CA/CPR was associated with an increase in basal serum corticosterone concentration relative to SHAM, but this difference resolved by postoperative day 21. The Dexamethasone Suppression Test showed that the CA/CPR group had enhanced negative feedback compared with SHAM controls at postoperative day 21. Furthermore, there was a gradual increase in CRF1 receptor binding in the paraventricular nucleus of the hypothalamus and bed nucleus of the stria terminalis, as well as a transient decrease of both CRF1, and CRF2A receptors in the dorsal hippocampus. Therefore, sustained changes in activity of the HPA axis and the CRF system after CA/CPR may contribute to the postischemic increase in affective disorders.
cardiac arrest; corticosterone; CRF; dexamethasone; HPA; ischemia
Positron emission tomography (PET) studies of the serotonin transporter (5-HTT) in the human brain are increasingly using the radioligand [11C]N, N-dimethyl-2-(2-amino-4-cyanophenylthio) benzylamine. A variety of models have been applied to such data in several published articles; however to date, these models have not been validated with test–retest data. We recruited 11 healthy subjects and conducted two identical scans on each subject on the same day. We considered four different models (one- and two-tissue compartment kinetic models, likelihood estimation in graphical analysis (LEGA; a bias-free alternative to the graphical method), and basis pursuit) along with fast noniterative approximations to the kinetic models. We considered four different outcome measures (total volume of distribution (VT), binding potential with (BP) and without (BP1), free-fraction adjustment, and specific-to-nonspecific equilibrium partition coefficient (BP2)). To assess the performance of each model, we compared results using six different metrics (percent difference (PD) and within-subject mean sum of squares for reproducibility, interclass coefficient for reliability, variance across subjects, identifiability based on bootstrap resampling of residuals for each method, and time stability analysis to determine minimal required scanning time). We considered analysis of both at the voxel level and at the region of interest (ROI) level and compared results from these two approaches to assess agreement. We determined that 100 mins of scanning time is adequate and that for ROI-level analysis, LEGA gives best results. Average PD is 5.51 for VT, 20.7 for BP, 17.2 for BP1, and 16.5 for BP2 across all regions. For voxel-level analysis we determined that the one-tissue compartment noniterative model is best.
bootstrap; compartment; kinetic; test–retest reproducibility; voxel
Neuroprotective therapy targeting the complement cascade may reduce injury associated with intracerebral hemorrhage (ICH). We investigated the role of C3a-receptor antagonist (C3aRA) after ICH in mice. Autologous whole blood was infused into the right striatum of mice that were treated with C3aRA or vehicle, using both a pre- and postinjury dosing regimen. Hematoma volume, brain water content, and inflammatory cell profile were assessed at 72 h post-ICH. Neurologic dysfunction was assessed by evaluating both spatial memory and sensorimotor capacity. Animals pretreated with C3aRA showed significantly improved neurologic function, brain water content, and granulocyte infiltration relative to vehicle-treated animals when assessed at 72 h. There was no significant difference in hemorrhagic/nonhemorrhagic ratio of microglial activation among all groups. Hematoma volumes were also not significantly different between C3aRA-treated and vehicle-treated animals. Administration of C3aRA beginning 6 h postinjury afforded significant amelioration of neurologic dysfunction as well as a reduction in brain water content. Treatment with C3aRA improved neurologic outcome while reducing inflammatory cell infiltration and brain edema formation after experimental ICH in mice. Results of this study suggest that the C3a receptor may be a promising target for therapeutic intervention in hemorrhagic stroke.
intracerebral hemorrhage; mouse; complement; C3a receptor antagonist; C3a; C3a receptor
Caffeine, the most widely consumed psychoactive drug and a weak adenosine receptor antagonist, can be neuroprotective or neurotoxic depending on the experimental model or neurologic disorder. However, its contribution to pathophysiology and outcome in traumatic brain injury (TBI) in humans is undefined. We assessed serial cerebrospinal fluid (CSF) concentrations of caffeine and its metabolites (theobromine, paraxanthine, and theophylline) by high-pressure liquid chromatography/ultraviolet in 97 ventricular CSF samples from an established bank, from 30 adults with severe TBI. We prospectively selected a threshold caffeine level of ≥1 μmol/L (194 ng/mL) as clinically significant. Demographics, Glasgow Coma Scale (GCS) score, admission blood alcohol level, and 6-month dichotomized Glasgow Outcome Scale (GOS) score were assessed. Mean time from injury to initial CSF sampling was 10.77±3.13 h. On initial sampling, caffeine was detected in 24 of 30 patients, and the threshold was achieved in 9 patients. Favorable GOS was seen more often in patients with CSF caffeine concentration ≥ versus < the threshold (55.6 versus 11.8%, P = 0.028). Gender, age, admission CGS score, admission blood alcohol level, and admission systolic arterial blood pressure did not differ between patients with CSF caffeine concentration ≥ versus < the threshold. Increases in CSF concentrations of the caffeine metabolites theobromine and paraxanthine were also associated with favorable outcome (P = 0.018 and 0.056, respectively). Caffeine and its metabolites are commonly detected in CSF in patients with severe TBI and in an exploratory assessment are associated with favorable outcome. We speculate that caffeine may be neuroprotective by long-term upregulation of adenosine A1 receptors or acute inhibition of A2a receptors.
adenosine; alcohol; coffee; head injury; head trauma; theobromine
Endothelial progenitor cells (EPCs) may provide novel opportunities for therapeutic angiogenesis after ischemic diseases. However, it is unclear how the angiogenic potential of EPCs might be affected by an inflammatory environment. We examine how the potent cytokine interleukin-1β (IL-1β) affects angiovasculogenic responses in EPCs in culture. Mononuclear cells isolated from mouse spleen were plated on fibronectin-coated wells and grown in EGM-2MV media. Endothelial progenitor cells were phenotyped using multiple markers (UEA-Lectin, ac-LDL, CD133, CD34, vWillebrand Factor, Flk-1) and to identify the IL-1 Receptor-I. We quantified cell and colony counts and performed MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide) and Matrigel assays, in vitro, under control and IL-1β (10 ng/mL) conditions. Endothelial progenitor cells exposed to IL-1β increased in the number of cells and colonies compared with untreated cells, without any effect on cell metabolic integrity. Furthermore, IL-1β treatment augmented EPC angiogenic function, significantly increasing the number of vessel-like structures in the Matrigel assay. An early phosphorylation of ERK1/2 occurred after IL-1β stimulation, and this pathway was inhibited if IL-1 Receptor-I was blocked. Our results suggest that IL-1β is a potent stimulator of in vitro angiogenesis through ERK signaling in mouse EPCs. Further studies are warranted to assess how interactions between proinflammatory environments and EPC responses may be leveraged to enhance therapeutic angiogenesis.
angiogenesis; cytokine; MAP kinase; neuroinflammation; spleen; stroke
Warfarin-associated intracerebral hemorrhage (W-ICH) is a severe type of stroke. There is no consensus on the optimal treatment for W-ICH. Using a mouse model, we tested whether the rapid reversal of anticoagulation using human prothrombin complex concentrate (PCC) can reduce hemorrhagic blood volume. Male CD-1 mice were treated with warfarin (2 mg/kg over 24 h), resulting in a mean (±s.d.) International Normalized Ratio of 3.5±0.9. First, we showed that an intravenous administration of human PCC rapidly reversed anticoagulation in mice. Second, a stereotactic injection of collagenase was administered to induce hemorrhage in the right striatum. Forty-five minutes later, the animals were randomly treated with PCC (100 U/kg) or saline IV (n = 12 per group). Twenty-four hours after hemorrhage induction, hemorrhagic blood volume was quantified using a photometric hemoglobin assay. The mean hemorrhagic blood volume was reduced in PCC-treated animals (6.5±3.1 μL) compared with saline controls (15.3±11.2 μL, P = 0.015). In the saline group, 45% of the mice developed large hematomas (i.e., > 15 μL). In contrast, such extensive lesions were never found in the PCC group. We provide experimental data suggesting PCC to be an effective acute treatment for W-ICH in terms of reducing hemorrhagic blood volume. Future studies are needed to assess the therapeutic potential emerging from our finding for human W-ICH.
anticoagulation; intracerebral hemorrhage; mouse model; warfarin
NRG1 signaling has multiple functions in neurons and glia. The data in this study demonstrate that NRG1 may also possess significant signaling and cytoprotective properties in human brain endothelial cells (BMECs). NRG1 mRNA and protein expression are present in these cells, and NRG1 receptors erbB2 and erbB3 are phosphorylated in response to NRG1. NRG1 triggers clear biological responses in BMECs – elevated phospho-Akt levels, increased ring formation in a Matrigel assay, and decreased cell death after oxidative injury with H2O2. These data suggest that NRG1 signaling is functional and cytoprotective in BMECs.
blood-brain barrier; erbB; neuregulin; neuroprotection; neurovascular unit; oxidative stress
Mitochondria are known to be central to the cell's response to ischemia, because of their role in energy generation, in free radical generation, and in the regulation of apoptosis. Heat shock protein 75 (Hsp75/Grp75/mortalin/TRAP1) is a member of the HSP70 chaperone family, which is targeted to mitochondria. Overexpression of Hsp75 was achieved in rat brain by DNA 7transfection, and expression was observed in both astrocytes and neurons. Rats were subjected to 100 mins middle cerebral artery occlusion followed by assessment of infarct volume, neurological score, mitochondrial function, and levels of oxidative stress at 24 h reperfusion. Overexpression of Hsp75 reduced infarct area from 44.6%±21.1% to 25.7%±12.1% and improved neurological outcome significantly. This was associated with improved mitochondrial function as shown by protection of complex IV activity, marked reduction of free radical generation detected by hydroethidine fluorescence, reduction of lipid peroxidation detected by 4-hydroxy-2-nonenol immunoreactivity, and increased preservation of ATP levels. This suggests that targeting mitochondria for protection may be a useful strategy to reduce ischemic brain injury.
Grp75; mitochondria; mortalin; oxidative stress; stroke; TRAP1
Electron paramagnetic resonance imaging (EPRI) is a new modality for visualizing O2 distribution in tissues, such as the brain following stroke or after administration of drugs of abuse. We have recently shown that 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl  is a pro-imaging agent that can cross the blood-brain barrier. After hydrolysis by esterases, the anion of 3-carboxy-2,2,5,5-tetramethyl-1-tetramethyl-1-pyrrolidinyloxyl  is trapped in brain tissue. In the present study, we investigated the feasibility of using this to map the changes of O2 concentration in mouse brain following focal ischemia. The decrease in tissue O2 concentration in the ischemic region of mouse brain was clearly visualized by EPRI. The hypoxic zone mapped by EPRI was spatially well-correlated with the infarction area in the brain imaged by diffusion-weighted MRI. Finally, we observed a decrease in the size of the hypoxic region, when the mouse breathed higher levels of O2. This finding suggests that EPRI with specifically designed nitroxides is a promising imaging modality for visualizing O2 distribution in brain tissue, especially in an ischemic brain. We believe that this imaging method can be used for monitoring the effects of therapeutic intervention, aimed at enhancing brain O2 supply, which is crucial in minimizing brain injury following stroke.
electron paramagnetic resonance imaging; oxygen; nitroxides; stroke
Neuroprotective properties of ketosis may be related to the up-regulation of hypoxia inducible factor 1 (HIF-1α), a primary constituent associated with hypoxic angiogenesis and a regulator of neuroprotective responses. The rationale that the utilization of ketones by brain results in elevation of intracellular succinate, a known inhibitor of prolyl-hydroxylase (the enzyme responsible for the degradation of HIF-1α) was deemed as a potential mechanism of ketosis on the up-regulation of HIF-1α. The neuroprotective effect of diet-induced ketosis (3 weeks of feeding a ketogenic diet), as pretreatment, on infarct volume, following reversible middle cerebral artery occlusion (MCAO) and the up-regulation of HIF-1α was investigated. The effect of beta-hydroxybutyrate (BHB), as a pretreatment via intraventricular infusion (4 days of infusion prior to stroke) was also investigated following MCAO. HIF-1α and Bcl-2 (anti-apoptotic protein) protein levels, and succinate content were measured. A 55–70% reduction in infarct volume was observed with BHB infusion or diet-induced ketosis, respectively. HIF-1α and Bcl-2 protein levels increased 3-fold with diet-induced ketosis; BHB infusions resulted in increases in these proteins. As hypothesized, succinate content increased by 55% with diet-induced ketosis and 4-fold with BHB infusion. We conclude, the biochemical link between ketosis and the stabilization of HIF-1α is through the elevation of succinate, and both HIF-1α stabilization and Bcl-2 up-regulation play a role in ketone induced neuroprotection in brain.
Regulatory changes in cytokine permeation across the blood–brain barrier (BBB) may have crucial roles in central nervous system (CNS) autoimmune disease. Accordingly, we examined the interactions of interleukin (IL)-15 with the cerebral vasculature after induction of experimental autoimmune encephalomyelitis (EAE). In contrast to the influx of 125I-IL15 from blood to the CNS in normal mice and the persistence of IL15 influx in the spinal cord of EAE mice, influx was reduced in the EAE brain. Analyses of disappearance kinetics, FITC (fluorescein isothiocyanate)-albumin space, and delivery of IL15 by in situ perfusion, all indicate that the changes were not caused by BBB disruption but by the rapid availability (high volume of distribution) of IL15 and albumin. Although there was no significant change in the BBB permeation of IL15 in either direction in EAE mice, there was an upregulation of its specific receptor, IL15Rα, and an increased in situ production of IL15 mRNA that showed regional variation in both basal and EAE states. Overall, for IL15, its increased cerebral vascular space in the brain was equally as important as its persistent influx across the blood–spinal cord barrier, indicating that it is fully capable of activating the upregulated IL15Rα in the brain along with the intrinsic CNS source of IL15 in EAE mice.
autoimmunity; blood–brain barrier; blood-spinal cord barrier; EAE; IL15
We hypothesized that urokinase plasminogen activator (uPA) contributes to age-dependent early hyperemia after fluid percussion brain injury (FPI) by activating extracellular signal-related kinase (ERK) mitogen-activated protein kinase (MAPK), leading to histopathologic changes in the underlying cortex. Both cerebrospinal fluid (CSF) uPA and phosphorylation of CSF ERK MAPK was increased at 1 min after FPI in newborn pigs, but was unchanged in juvenile pigs. uPA and phosphorylated ERK MAPK, detectable in sham piglet brain by immunohistochemistry, was markedly elevated and associated with histopathology 4 h after FPI in the newborn but there was minimal staining and histopathology in the juvenile. EEIIMD, a peptide derived from PA inhibitor-1 that does not affect proteolysis, blunted FPI-induced phosphorylation of ERK MAPK. FPI produced pial artery dilation and increased cerebral blood flow at 1 min after insult in the newborn, but not in the juvenile. Antilipoprotein-related protein (LRP) antibody, EEIIMD, a soluble uPA antagonist, and the ERK MAPK antagonist U 0126 inhibited FPI-associated hyperemia. These data indicate that uPA is upregulated after FPI and produces an age-dependent early hyperemia followed by histopathology through an LRP- and ERK MAPK-dependent pathway.
cerebral circulation; newborn; plasminogen activators; signal transduction
Irreversible translation arrest occurs in reperfused neurons that will die by delayed neuronal death. It is now recognized that suppression of protein synthesis is a general response of eukaryotic cells to exogenous stressors. Indeed, stress-induced translation arrest can be viewed as a component of cell stress responses, and consists of initiation, maintenance, and termination phases that work in concert with stress-induced transcriptional mechanisms. Within this framework, we review translation arrest in reperfused neurons. This framework provides a basis to recognize that phosphorylation of the alpha subunit of eukaryotic initiation factor 2 is the initiator of translation arrest, and a key marker indicating activation of neuronal stress responses. However, eIF2 alpha phosphorylation is reversible. Other phases of stress-induced translation arrest appear to contribute to irreversible translation arrest specifically in ischemic vulnerable neuron populations. We detail two lines of evidence supporting this view. First, ischemia, as a stress stimulus, induces irreversible co-translational protein misfolding and aggregation after 4 to 6 h of reperfusion, trapping protein synthesis machinery into functionally inactive protein aggregates. Second, ischemia and reperfusion leads to modifications of stress granules (SGs) that sequester functionally inactive 48S preinitiation complexes to maintain translation arrest. At later reperfusion durations, these mechanisms may converge such that SGs become sequestered in protein aggregates. These mechanisms result in elimination of functionally active ribosomes and preclude recovery of protein synthesis in selectively vulnerable neurons. Thus, recognizing translation arrest as a component of endogenous cellular stress response pathways will aid in making sense of the complexities of postischemic translation arrest.
brain ischemia and reperfusion; CA1; cellular stress; cotranslational aggregation; stress granules; translation arrest or protein synthesis inhibition
Using modified oxygen needle microelectrodes and intravital videomicroscopy, measurements were made of tissue oxygen tension (PO2) profiles near cortical arterioles and transmural PO2 gradients in the pial arterioles of the rat. Under control conditions, the transmural PO2 gradient averaged 1.17 ± 0.06 mm Hg/μm (mean ± s.e., n = 40). Local arteriolar dilation resulted in a marked decrease in the transmural PO2 gradient to 0.68 ± 0.04 mm Hg/μm (P < 0.001, n = 38). The major finding of this study is a dependence of the transmural PO2 gradient on the vascular tone of the pial arterioles. Using a model of oxygen transport in an arteriole and experimental PO2 profiles, values of radial perivascular and intravascular O2 fluxes were estimated. Our theoretical estimates show that oxygen flux values at the outer surface of the arteriolar wall are approximately 10−5 mL O2/cm2 per sec, independent of the values of the arteriolar wall O2 consumption within a wide range of consumption values. This also means that PO2 transmural gradients for cerebral arterioles are within the limits of 1 to 2 mm Hg/μm. The data lead to the conclusion that O2 consumption of the arteriolar wall is within the range for the surrounding tissue and O2 consumption of the endothelial layer appears to have no substantial impact on the transmural PO2 gradient.
cortical microvessels; O2 transport model; oxygen microelectrodes; tissue PO2 profiles; transmural PO2 gradient
The mechanisms underlying neurologic deficits and delayed neuronal death after ischemia are not fully understood. In the present study, we report that transient cerebral ischemia induces accumulation of ubiquitinated proteins (ubi-proteins) in postsynaptic densities (PSDs). By immuno-electron microscopy, we demonstrated that ubi-proteins were highly accumulated in PSD structures after ischemia. On Western blots, ubi-proteins were markedly increased in purified PSDs at 30 minutes of reperfusion, and the increase persisted until cell death in the CA1 region after ischemia. In the resistant DG area, however, the changes were transient and significantly less pronounced. Deposition of ubi-proteins in PSDs after ischemia correlates well with PSD structural damage in the CA1 region as viewed by electron microscopy. These results suggest that the ubiquitin-proteasome system fails to repair and remove damaged proteins in PSDs. The changes may demolish synaptic neurotransmission, contribute to neurologic deficits, and eventually lead to delayed neuronal death after transient cerebral ischemia.
Ubiquitin; Brain ischemia; Postsynaptic density; Electron microscopy
Accurate identification of ischemic penumbra will improve stroke patient selection for reperfusion therapies and clinical trials. Current magnetic resonance imaging (MRI) techniques have limitations and lack validation. Oxygen challenge T2* MRI (T2* OC) uses oxygen as a biotracer to detect tissue metabolism, with penumbra displaying the greatest T2* signal change during OC. [14C]2-deoxyglucose (2-DG) autoradiography was combined with T2* OC to determine metabolic status of T2*-defined penumbra. Permanent middle cerebral artery occlusion was induced in anesthetized male Sprague-Dawley rats (n=6). Ischemic injury and perfusion deficit were determined by diffusion- and perfusion-weighted imaging, respectively. At 147±32 minutes after stroke, T2* signal change was measured during a 5-minute 100% OC, immediately followed by 125 μCi/kg 2-DG, intravenously. Magnetic resonance images were coregistered with the corresponding autoradiograms. Regions of interest were located within ischemic core, T2*-defined penumbra, equivalent contralateral structures, and a region of hyperglycolysis. A T2* signal increase of 9.22%±3.9% (mean±s.d.) was recorded in presumed penumbra, which displayed local cerebral glucose utilization values equivalent to contralateral cortex. T2* signal change was negligible in ischemic core, 3.2%±0.78% in contralateral regions, and 1.41%±0.62% in hyperglycolytic tissue, located outside OC-defined penumbra and within the diffusion abnormality. The results support the utility of OC-MRI to detect viable penumbral tissue following stroke.
ADC; CBF; imaging; LCMRglu; MCAO; rat
Magnetic resonance imaging (MRI) with oxygen challenge (T2* OC) uses oxygen as a metabolic biotracer to define penumbral tissue based on CMRO2 and oxygen extraction fraction. Penumbra displays a greater T2* signal change during OC than surrounding tissue. Since timely restoration of cerebral blood flow (CBF) should salvage penumbra, T2* OC was tested by examining the consequences of reperfusion on T2* OC-defined penumbra. Transient ischemia (109±20 minutes) was induced in male Sprague-Dawley rats (n=8). Penumbra was identified on T2*-weighted MRI during OC. Ischemia and ischemic injury were identified on CBF and apparent diffusion coefficient maps, respectively. Reperfusion was induced and scans repeated. T2 for final infarct and T2* OC were run on day 7. T2* signal increase to OC was 3.4% in contralateral cortex and caudate nucleus and was unaffected by reperfusion. In OC-defined penumbra, T2* signal increased by 8.4%±4.1% during ischemia and returned to 3.25%±0.8% following reperfusion. Ischemic core T2* signal increase was 0.39%±0.47% during ischemia and 0.84%±1.8% on reperfusion. Penumbral CBF increased from 41.94±13 to 116.5±25 mL per 100 g per minute on reperfusion. On day 7, OC-defined penumbra gave a normal OC response and was located outside the infarct. T2* OC-defined penumbra recovered when CBF was restored, providing further validation of the utility of T2* OC for acute stroke management.
ADC; CBF; imaging; MCAO; T2*
Accurate imaging of the ischemic penumbra is a prerequisite for acute clinical stroke research. T2* magnetic resonance imaging (MRI) combined with an oxygen challenge (OC) is being developed to detect penumbra based on changes in blood deoxyhemoglobin. However, inducing OC with 100% O2 induces sinus artefacts on human scans and influences cerebral blood flow (CBF), which can affect T2* signal. Therefore, we investigated replacing 100% O2 OC with 40% O2 OC (5 minutes 40% O2 versus 100% O2) and determined the effects on blood pressure (BP), CBF, tissue p2, and T2* signal change in presumed penumbra in a rat stroke model. Probes implanted into penumbra and contralateral cortex simultaneously recorded p2 and CBF during 40% O2 (n=6) or 100% O2 (n=8) OC. In a separate MRI study, T2* signal change to 40% O2 (n=6) and 100% O2 (n=5) OC was compared. Oxygen challenge (40% and 100% O2) increased BP by 8.2% and 18.1%, penumbra CBF by 5% and 15%, and penumbra p2 levels by 80% and 144%, respectively. T2* signal significantly increased by 4.56%±1.61% and 8.65%±3.66% in penumbra compared with 2.98%±1.56% and 2.79%±0.66% in contralateral cortex and 1.09%±0.82% and −0.32%±0.67% in ischemic core, respectively. For diagnostic imaging, 40% O2 OC could provide sufficient T2* signal change to detect penumbra with limited influence in BP and CBF.
CBF; hyperoxia; oxygen challenge; penumbra; p2; T2*
Methamphetamine (METH), a potent stimulant with strong euphoric properties, has a high abuse liability and long-lasting neurotoxic effects. Recent studies in animal models have indicated that METH can induce impairment of the blood brain barrier (BBB), thus suggesting that some of the neurotoxic effects resulting from METH abuse could be the outcome of barrier disruption. Here we provide evidence that METH alters BBB function via direct effects on endothelial cells and explore possible underlying mechanisms leading to endothelial injury. We report that METH increases BBB permeability in vivo, and exposure of primary human microvascular endothelial cells (BMVEC) to METH diminishes tightness of BMVEC monolayers in a dose- and time-dependent manner by decreasing expression of cell membrane associated tight junction (TJ) proteins. These changes were accompanied by enhanced production of reactive oxygen species, increased monocyte migration across METH-treated endothelial monolayers, and activation of myosin light chain kinase (MLCK) in BMVEC. Anti-oxidant treatment attenuated or completely reversed all tested aspects of METH induced BBB dysfunction. Our data suggest that BBB injury is caused by METH-mediated oxidative stress, which activates MLCK and negatively affects the TJ complex. These observations provide a basis for antioxidant protection against brain endothelial injury caused by METH exposure.
methamphetamine; blood brain barrier; oxidative stress; tight junction; monocyte
This study aimed at combining an iron-based, steady-state, Vessel Size Index MRI approach (VSI MRI), and a Gd-based, Dynamic Contrast Enhanced MRI approach (DCE MRI) to characterize tumoral microvasculature. Rats bearing an orthotopic glioma (C6, n=14 and RG2, n=6) underwent DCE MRI and combined VSI and DCE MRI four hours later, at 2.35T. Gd-DOTA (200 μmol of Gd/kg) and ultrasmall superparamagnetic iron oxide (USPIO) (200 μmol of iron/kg) were used for DCE and VSI MRI, respectively. C6 and RG2 glioma were equally permeable to Gd-DOTA but presented different blood volume fractions and VSI, in good agreement with histological data. The presence of USPIO yielded reduced Ktrans values. The Ktrans values obtained with Gd-DOTA in absence and in presence of USPIO were well correlated for the C6 glioma but not for the RG2 glioma. It was also observed that, within the time frame of DCE MRI, USPIO remained intravascular in the C6 glioma while it extravasated in the RG2 glioma. In conclusion, VSI and DCE MRI can be combined provided that USPIO does not extravasate with the time frame of the DCE MRI experiment. The mechanisms at the origin of USPIO extravasation remain to be elucidated.
Animals; Brain Neoplasms; blood supply; diagnosis; Cell Membrane Permeability; Contrast Media; Dextrans; diagnostic use; pharmacokinetics; Ferrosoferric Oxide; diagnostic use; pharmacokinetics; Gadolinium; diagnostic use; pharmacokinetics; Glioma; diagnosis; pathology; Heterocyclic Compounds; diagnostic use; pharmacokinetics; Iron; diagnostic use; pharmacokinetics; Magnetic Resonance Imaging; methods; Magnetite Nanoparticles; Male; Microvessels; Neovascularization, Pathologic; diagnosis; Organometallic Compounds; diagnostic use; pharmacokinetics; Rats; Rats, Wistar; blood volume; dynamic contrast-enhanced MRI (DCE MRI); brain tumors; ultrasmall superparamagnetic iron oxide particles (USPIO); vessel size index (VSI).
Here, we show that oxygen and glucose deprivation (OGD) causes increased small ubiquitin-like modifier (SUMO)-1 and SUMO-2/3 conjugation to substrate proteins in cultured hippocampal neurones. Surprisingly, the SUMO protease SENP-1, which removes SUMO from conjugated proteins, was also increased by OGD, suggesting that the neuronal response to OGD involves a complex interplay between SUMOylation and deSUMOylation. Importantly, decreasing global SUMOylation in cultured hippocampal neurones by overexpression of the catalytic domain of SENP-1 increased neuronal vulnerability to OGD-induced cell death. Taken together, these results suggest a neuroprotective role for neuronal SUMOylation after OGD.
ischemia; neuronal culture; oxygen and glucose deprivation (OGD); posttranslational modification; SENP-1; SUMO
Medium spiny neurons (MSNs) constitute most of the striatal neurons and are known to be vulnerable to ischemia; however, the mechanisms of the vulnerability remain unclear. Activated forms of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase (NOX), which require interaction between cytosolic and membrane-bound subunits, are among the major sources of superoxide in the central nervous system. Although increasing evidence suggests that NOX has important roles in neurodegenerative diseases, its roles in MSN injury after transient global cerebral ischemia (tGCI) have not been elucidated. To clarify this issue, C57BL/6 mice were subjected to tGCI by bilateral common carotid artery occlusion for 22 minutes. Western blot analysis revealed upregulation of NOX subunits and recruitment of cytosolic subunits to the cell membrane at early (3 to 6 hours) and late (72 hours) phases after tGCI. Taken together with immunofluorescent studies, this activation arose in MSNs and endothelial cells at the early phase, and in reactive microglia at the late phase. Pharmacological and genetic inhibition of NOX attenuated oxidative injury, microglial activation, and MSN death after tGCI. These findings suggest that NOX has pivotal roles in MSN injury after tGCI and could be a therapeutic target for brain ischemia.
global cerebral ischemia; medium spiny neuron, microglia; NADPH oxidase; oxidative stress; striatum
Human immunodeficiency virus (HIV)-associated infection involves the entry of virus-bearing monocytes into the brain, followed by microglial activation, neuroinflammation, and upregulated arachidonic acid (AA) metabolism. The HIV-1 transgenic (Tg) rat, a noninfectious HIV-1 model, shows neurologic and behavioral abnormalities after 5 months of age. We hypothesized that brain AA metabolism would be elevated in older HIV-1 Tg rats in vivo. Arachidonic acid incorporation from the plasma into the brain of unanesthetized 7-to-9-month-old rats was imaged using quantitative autoradiography, after [1-14C]AA infusion. Brain phospholipase (PLA2) activities and eicosanoid concentrations were measured, and enzymes were localized by immunostaining. AA incorporation coefficients k* and rates Jin, measures of AA metabolism, were significantly higher in 69 of 81 brain regions in HIV-1 Tg than in control rats, as were activities of cytosolic (c)PLA2-IV, secretory (s)PLA2, and calcium independent (i)PLA2-VI, as well as prostaglandin E2 and leukotriene B4 concentrations. Immunostaining of somatosensory cortex showed elevated cPLA2-IV, sPLA2-IIA, and cyclooxygenase-2 in neurons. Brain AA incorporation and other markers of AA metabolism are upregulated in HIV-1 Tg rats, in which neurologic changes and neuroinflammation have been reported. Positron emission tomography with [1-11C]AA could be used to test whether brain AA metabolism is upregulated in HIV-1-infected patients, in relation to cognitive and behavioral disturbances.
arachidonic acid; brain imaging; eicosanoids; HIV-1; phospholipase A2
Accurate identification of ischemic penumbra will improve stroke patient selection for reperfusion therapies and clinical trials. Current magnetic resonance imaging (MRI) techniques have limitations and lack validation. Oxygen challenge T2∗ MRI (T2∗ OC) uses oxygen as a biotracer to detect tissue metabolism, with penumbra displaying the greatest T2∗ signal change during OC. [14C]2-deoxyglucose (2-DG) autoradiography was combined with T2∗ OC to determine metabolic status of T2∗-defined penumbra. Permanent middle cerebral artery occlusion was induced in anesthetized male Sprague-Dawley rats (n = 6). Ischemic injury and perfusion deficit were determined by diffusion- and perfusion-weighted imaging, respectively. At 147±32 minutes after stroke, T2∗ signal change was measured during a 5-minute 100% OC, immediately followed by 125 μCi/kg 2-DG, intravenously. Magnetic resonance images were coregistered with the corresponding autoradiograms. Regions of interest were located within ischemic core, T2∗-defined penumbra, equivalent contralateral structures, and a region of hyperglycolysis. A T2∗ signal increase of 9.22%±3.9% (mean±s.d.) was recorded in presumed penumbra, which displayed local cerebral glucose utilization values equivalent to contralateral cortex. T2∗ signal change was negligible in ischemic core, 3.2%±0.78% in contralateral regions, and 1.41%±0.62% in hyperglycolytic tissue, located outside OC-defined penumbra and within the diffusion abnormality. The results support the utility of OC-MRI to detect viable penumbral tissue following stroke.
ADC; CBF; imaging; LCMRglu; MCAO; rat
Magnetic resonance imaging (MRI) with oxygen challenge (T2∗ OC) uses oxygen as a metabolic biotracer to define penumbral tissue based on CMRO2 and oxygen extraction fraction. Penumbra displays a greater T2∗ signal change during OC than surrounding tissue. Since timely restoration of cerebral blood flow (CBF) should salvage penumbra, T2∗ OC was tested by examining the consequences of reperfusion on T2∗ OC-defined penumbra. Transient ischemia (109±20 minutes) was induced in male Sprague-Dawley rats (n = 8). Penumbra was identified on T2∗-weighted MRI during OC. Ischemia and ischemic injury were identified on CBF and apparent diffusion coefficient maps, respectively. Reperfusion was induced and scans repeated. T2 for final infarct and T2∗ OC were run on day 7. T2∗ signal increase to OC was 3.4% in contralateral cortex and caudate nucleus and was unaffected by reperfusion. In OC-defined penumbra, T2∗ signal increased by 8.4%±4.1% during ischemia and returned to 3.25%±0.8% following reperfusion. Ischemic core T2∗ signal increase was 0.39%±0.47% during ischemia and 0.84%±1.8% on reperfusion. Penumbral CBF increased from 41.94±13 to 116.5±25 mL per 100 g per minute on reperfusion. On day 7, OC-defined penumbra gave a normal OC response and was located outside the infarct. T2∗ OC-defined penumbra recovered when CBF was restored, providing further validation of the utility of T2∗ OC for acute stroke management.
ADC; CBF; imaging; MCAO; T2*