63. A free radical scavenger, edaravone, protects cerebral microvascular integrity after thrombolysis in a rat stroke model
T. Yamashita1, T. Kamiya1, K. Deguchi1, S. Nagotani1, T. Inaba2, H. Zhang1, K. Miyazaki1, A. Ohtsuka3, Y. Katayama2 and K. Abe1
1Department of Neurology, Okayama University, Okayama; 2Divison of Neurology, Second Department of Internal Medicine, Nippon Medical School, Tokyo; 3Department of Human Morphology, Okayama University, Okayama, Japan
Background: After stroke, the thrombolytic effect of tissue plasminogen activator (tPA) is beneficial, if it is given within a short time. However, delayed reperfusion with tPA sometimes causes hemorrhagic transformation (HT), strictly limiting its clinical use. An understanding of the mechanism underlying the HT and new therapeutic methods are both needed.
Methods: We used a spontaneously hypertensive rat model of middle cerebral artery occlusion (MCAO). Rats were treated with vehicle alone (n = 6), tPA alone (n = 16), or tPA plus edaravone, a free radical scavenger (n = 11). Edaravone (i.v., 3 mg/kg) was administrated every 1.5 h during the 4.5 h of MCAO, followed by tPA treatment (i.v., 10 mg/kg) at reperfusion. At 24 h after MCAO, the surviving rats were sacrificed.
Results: Administration of tPA alone significantly (P<0.05) worsened the survival rate compared with vehicle. However, treatment with edaravone plus tPA significantly (P<0.05) increased the survival rate, improved motor function, and significantly decreased HT. Immunostaining revealed that edaravone suppressed the tPA-induced lipid peroxidation and matrix metalloproteinase-9 (MMP-9) activation around cerebral microvessels. Moreover, electron microscopic analysis demonstrated that the basement membrane disintegrated and became detached from the astrocyte endfeet in rats treated with tPA alone, whereas treatment with edaravone plus tPA prevented the microvessels from dissociating.
Conclusions: Edaravone can protect the cerebral microvascular integrity, presumably by safeguarding the basement membrane from excess free radicals and MMP-9, leading to a decrease in HT, and an improved survival rate and neurological outcome.
93. Curcumin reduces matrix metalloproteinase-9 expression and ameliorates blood brain barrier dysfunction in stroke
N. Tyagi, S. Kundu, S. Givvimani, W. Gillespie, P. Mishra, L. Lominadze and S. Tyagi
Physiology and Biophysics, University of Louisville, Louisville, Kentucky, USA
Purpose: Curcumin, a yellow polyphenolic compound from the plant Curcuma ionga, is a commonly used spice and coloring agent with beneficial effects of anti-tumor, anti-inflammatory, and antioxidant activities. The objective of this study was to determine whether curcumin ameliorates blood-brain barrier (BBB) dysfunction in stroke by reducing matrix metalloproteinase in hyperhomocystemic mice.
Methods: Stroke was induced by a 1-h middle cerebral artery (MCA) occlusion using an intraluminal filament in all groups. Brain infarction was measured and neurological deficits were scored. BBB dysfunction was determined by examining brain edema and Evans Blue extravasation. Expression of collagen IV, the major component of basal lamina essential for maintenance of the endothelial permeability barrier, was quantitatively detected by Western blot and immunocytochemistry. Temporal relationship of expression of MMP-9 and its endogenous inhibitor, the tissue inhibitors of metalloproteinase-1 (TIMP-1), was determined by real-time PCR for mRNA and Western blot for protein during reperfusion.
Results: Brain edema and Evans Blue leakage were both significantly (P<0.01) reduced after stroke in the curcumin treated group as compared to hyperhomocystemic group, in association with reduced brain infarct volume and neurological deficits. Western blot analysis indicated that curcumin enhanced collagen IV expression and reduced the collagen loss after stroke. Immunocytochemistry demonstrated that collagen IV-labeled vessels were significantly (P<0.01) increased in curcumin treated mice. The ex vivo study also revealed a key role of MMP-9 in curcumin treated mice-strengthened collagen IV expression against I/R injury. TIMP-1 protein levels were significantly (P<0.01) increased by curcumin treatment.
Conclusion: These data demonstrate that curcumin effectively reduces brain injury by improving BBB function and enhancing basal lamina integrity in stroke. This study suggests a therapeutic role of dietary curcumin in patients with stroke.
115. Exposure of engineered nanoparticles from metals alters cerebral blood flow, blood-brain barrier permeability, brain edema and induces neurotoxicity in rats
H.S. Sharma1, A. Sharma1 and D.F. Muresanu2
1Surgical Sciences, Uppsala University Hospital, Uppsala, Sweden; 2Neurology, University of Medicine and Pharmacy, Cluj-Napoca, Romania
Influence of nanoparticles on brain function in vivo situations is still not well known. It appears that the magnitude and intensity of exposure to nanoparticles from the environment, food and/or water could affect neuronal functions and eventually may lead to neurotoxicity. This hypothesis was examined in present investigation using systemic administration of engineered nanoparticles from metals, i.e., Al, Ag and Cu (≈50 to 60 nm) on neurotoxicity in rats and mice. Intraperitoneal (50 mg/kg), intravenous (30 mg/kg), intracarotid (2.5 mg/kg) or intracerebroventricular administration (20 μg) of nanoparticles significantly altered the blood-brain barrier (BBB) function to Evans blue and radioiodine in several regions of the brain and spinal cord following 24 and 48 h after its administration. Marked decreases in local cerebral blood flow (CBF) and pronounced edema formation was seen in several brain and spinal cord areas associated with BBB leakage. Neuronal cell injuries, glial cell activation, heat shock protein (HSP 72 kD) expression and loss of myelinated fibers are quite common in these brain areas. These light microscopical changes were further confirmed using transmission electron microscopy. These pathological changes were most significant in mice compared to rats. Cu and Ag nanoparticles exerted marked effects on neuronal changes when administered in systemic circulation or into the brain ventricular spaces. Intraperitoneal administration was lest effective. Our results clearly demonstrate that that nanoparticles from metals by affecting regional CBF and BBB function are able to induce selective and specific neuronal, glial and myelin changes in the brain and spinal cord. This effect of nanoparticles on neurotoxicity, however, largely depends on the type of metals, route of administration and the species used.
117. Chronic exposure of nanoparticles exacerbate hyperthermia induced blood-brain barrier breakdown, cognitive dysfunction and brain pathology. Neuroprotecion by nanowired-antioxidant compound H-290/51
D.F. Muresanu1, A. Sharma2 and H.S. Sharma2
1Neurology, University of Medicine and Pharmacy, Cluj-Napoca, Romania; 2Surgical Sciences, Uppsala University Hospital, Uppsala, Sweden
There are reasons to believe that nanoparticles in the ambient air is translocated to the autonomic nervous system via circulation or to the CNS through sensory nerves in the respiratory tract, and induce serious cardiovascular or neurological effects. Thus, the translocated nanoparticles in humans can enter rapidly into the deeper brain structures in short exposure time and induce brain dysfunction. However, the possibility that chronic exposure of nanoparticles may alter stress reaction following hyperthermia and brain pathology is still not known. In this investigation, we examined the effects of chronic exposure of engineered nanoparticles from metals in rats on possible alterations and brain dysfunction in heat stress. For this purpose, engineered nanoparticles from Ag or Cu (≈50 to 60 nm) were administered (30 mg/kg, i.p.) once daily for 1 week in young male rats. On the 8th day these animals were subjected to 4 h heat stress at 38°C in a BOD incubator and stress reaction, blood-brain barrier (BBB) permeability and brain pathology were examined. Subjection of nanoparticle treated rats to heat stress showed exacerbation stress symptoms i.e., hyperthermia, salivation and prostration and exhibited greater BBB disruption, brain edema formation and brain damage compared to normal animals. This effect of enhanced brain pathology in heat stress was most marked in animals that received Ag nanoparticles compared to Cu treatment. Since nanoparticles of various sizes and different chemical compositions influence mitochondria to stimulate ROS overproduction and interfere with antioxidant defense mechanism, we used the effects of a potent antioxidant compound H-290/51 to induce neuroprotection in heat stress. Treatment with antioxidant compound H-290/51 either 30 or 60 mins after heat stress did not significantly attenuate hyperthermia induce brain pathology in nanoparticle treated rats. Whereas, administration of nanowirded-H-290/51 after 30 or 60 mins heat stress markedly attenuated BBB disruption and brain pathology in these groups. Our results show that chronic nanoparticles treatment exacerbate hypertehrmia induced brain pathology that is significantly attenuated by nanowired but not normal H-290/51 compound, not reported earlier. Taken together, these observations suggest that nano-wired drug delivery of H-290/51 is a promising approach to induce neuroprotection in hypertehrmia induced brain pathology.
120. Inhibition of myosin light chain kinase prevents blood brain barrier breakdown after closed skull traumatic brain injury in the mouse
J. Rossi1,2, F. Patel2,3, M. Chrzaszcz2,3 and M. Wainwright2,3
1Department of Pediatrics, Division of Critical Care Medicine, Northwestern University, Feinberg School of Medicine; 2Center for Interdisciplinary Research in Pediatric Critical Illness and Injury, Children's Memorial Research Center; 3Department of Pediatrics, Division of Neurology, Northwestern University, Feinberg School of Medicine, Chicago, Illinois, USA
Background and aims: Disruption of the cytoskeleton contributes to breakdown of the blood brain barrier (BBB) after traumatic brain injury (TBI). We have previously shown that myosin light chain kinase (MLCK) plays a pivotal role in the compromise of barrier function following acute lung injury (Proc Natl Acad Sci 2003, 100:6322; AJP Lung 2007, 292:1327). The role of MLCK in BBB dysfunction after TBI is not known.
Methods: Adult C57Bl6 mice were subject to midline closed skull injury or sham operation using a stereotactically guided pneumatic compression device (Lloyd et al., J Neuroinflammation 2008, 5:28). Coronal sections from each animal (n = 8 per group) were prepared at 4- 24 h 3- and 5d recovery. To quantify extravasation of macromolecules following TBI, immunohistochemical (IHC) staining for IgG and albumin was performed. To quantify changes in MCLK, the substrate MLC and phosphorylated MLC (p-MLC) IHC was performed and the expression quantified in digitized images by blinded observers. To determine the functional role of MLCK in the mechanisms leading to compromise of the BBB after TBI, mice were treated with an inhibitor of MLCK (ML-7) 30 mins prior to TBI. After 24 h recovery BBB breakdown, MLCK, MLC and p-MLC expression were quantified.
Purpose: We tested the hypothesis that MLCK is up-regulated following TBI, leading to breakdown of the BBB and extravasation of macromolecules into brain parenchyma.
Results: Breakdown of the BBB begins at 4 h and continues until 5d recovery. At each time point there were significant increases (P<0.01 versus sham) in IgG and albumin in brain parenchyma at the impact site compared to sham controls. MLCK expression was increased compared to controls at 4 h, 24 h and 3d, with corresponding increases in p-MLC at 24 h and 3d (P<0.05 versus sham). The substrate MLC was significantly reduced at 24 h recovery (P<0.05 versus sham). Inhibition of MLCK with ML-7 prevented both the breakdown of the BBB at 24 h, as measured by albumin extravasation and the increase in MLCK in the injured animals.
Conclusions: Based on precedent for the important role of MLCK in the regulation of endothelial and epithelial barrier integrity in the lung, intestine and skin, we examined the role of MLCK in the mechanisms leading to breakdown of the BBB following TBI. Here, we show that expression of MLCK is increased after TBI, that this increase is associated with an increase in the enzyme product, pMLC, and with a decrease in the substrate MLC. Administration of an inhibitor of MLCK prior to TBI prevented both these changes as well as the increase in BBB permeability measured by extravasation of albumin. These findings support a role for MLCK in the mechanisms of BBB dysfunction following TBI. The effect of treatment with an inhibitor of MLCK implicates this pathway in the mechanisms by which TBI leads to breakdown of the BBB. Greater understanding of the mechanisms by which MLCK regulates BBB cytoskeletal integrity following TBI may advance the development of new therapeutic approaches to the prevention of cerebral edema.
213. Treating focal cerebral ischemia with BDNF gene delivered by recombinant herpes-simplex-virus vectors via intranasal olfactory pathway in rats
G. Xu1 and X. Liu2
1Neurology, Department of Neurology, Jinling Hosptial, Nanjing University School of Medicine; 2Jinling Hospital, Nanjing, China
Objective: Many growth factors such as brain-derived neurotrophic factor (BDNF) have been reported with effecacy of neurogenesis and neuroprotecton in amimal model of focal cerebral ischemia. But gene therapy for cerebrovascular disease in human still has many problems, of which lacking an effective delivery pathway to central nervous system (CNS) is a major hindrance for the clinical application of this promising strategy. This study was aimed to evaluate the feasibilty and efficacy of deliverying BDNF gene with recombinant herpes-simplex-virus vectors to CNS via intranasal olfactory pathway in rat with focal cerebral ischemia.
Method: Recombinant herpes-simplex-virus vector was constracted by deleting 6 viral genes, which included the coding sequence for an immediat early (IE) gene, ICP4. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) with a nylon filament in Sprague-Dawley rats. MCAO rats were randomized into three groups: BDNF, eGFP and control. Six hours after MCAO, recombinant herpes-simplex-virus vectors carrying BDNF gene or enhanced green fluorescent protein (eGFP) gene, a marker gene, or the void vectors were administrated intranasally. Bromodeoxyuridine (BrdU) was injected intraperitoneally twice daily on the fifth and sixth days for the purpose of neurogenesis measurement. On the seventh day and forteenth day after MCAO, the cerebral infarct volumes, neurogenesis, expression of BDNF and eGFP were assessed. Neurological outcomes were serially assessed by the rotated test after MCAO.
Results: Rats in BDNF group scored higher in neurological function tests 7 and 14 days after MCAO compared with eGFP and control groups (P<0.05). Rats administrated with BDNF gene has a trend of decreased infarct volume 14 days after MCAO compared with that of eGFP and control groups, but the differences did not reach the significant level (P = 0.065). Histological detections revealed that expression of BDNF in hippocampus and temporal lobe were significantly enhenced 7 and 14 days after MCAO in BDNF group in contrast to eGFP and control groups (P<0.05). Green fluorescence was also detected in hippocampus and temporal lobe of rats administrated with eGFP gene. The BrdU-positive cells in subventricular zone and hippocampus was significantly increased in BDNF gene treated rats than eGFP treated and control rats (P<0.05).
Conclusions: Recombinant herpes-simplex-virus vectors administrated via intranasal olfactory pathway can efficiently delivering BDNF gene into CNS and expressing. BDNF gene therapy is efficacious in treating focal cerebral ischemia in rats.
236. Tobacco smoke: a critical etiological factor for vascular impairment at the blood-brain barrier
L. Cucullo1, T. Sathe2, M. Hossain1, V. Fazio1, E. Rapp3 and D. Janigro1,4
1Cell Biology; 2Cleveland Clinic; 3Flocel Inc; 4Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio, USA
Objectives: Tobacco smoke (TS) is a critical etiological factor for vascular impairment1 and the pathogenesis and progression of a variety of neuroinflammatory diseases (e.g., Alzheimer's disease and multiple sclerosis2,3). However, the exact pathophysiology of TS at the brain microvascular level has yet to be unveiled. For this purpose, we first assessed the effect of chronic TS exposure on human brain microvascular endothelial (HBMEC) and monocytes (THP-1) cell cultures and then evaluated in vitro the physiological response to TS of a human BBB model. This was assessed under normal and pathological hemodynamic changes (flow-cessation/reperfusion). Since many vascular adverse effects of smoking are related to the exposure to reactive oxidative substances of which TS is highly enriched, we tested the hypothesis that a concomitant antioxidant (vitamins E and C) supplementation can protect the BBB.
Methods: A concentrated TS solution was prepared from 2R4F research cigarettes using a Borgwaldt RM2 apparatus. TS was quantified as puffs/mL (p/mL), and diluted to the desired concentration (0.008, 0.016, and 0.032 p/mL) prior use.
Vitamin E and C (Fischer Scientific) were diluted to a final physiological concentration of 40 and 80 μmol/L respectively.
The specific effects of TS on endothelial cells and monocytes were initially assessed in static culture systems (12 well plates). Cell morphology and viability were assessed via inverted light microscopy. The release of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and matrix metalloproteinases 2 and 9 was assessed by ELISA. The immune response of the endothelial and THP-1 cells was assessed by FACS.
The effect of tobacco smoke exposure on the BBB and the protective efficacy of vitamin E and C were assessed in a well established dynamic in vitro BBB model (DIV-BBB) that closely mimics the physiological characteristics and response of that in vivo.4
Results: TS decreased endothelial cell viability only at high concentrations (≥0.016 puffs/mL) while not significantly affecting that of monocytes. At low concentration (0.008 puffs/mL) TS induced the expression of the vascular endothelial adhesion molecules VCAM-1, P-selectin and E-selectin, the differentiation of THP-1 cells into macrophages, the release of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, and that of activated matrix metalloproteinase-2 and -9. In addition, TS worsened BBB failure after flow-cessation/reperfusion (Fc/Rp). The coadjuvant administration of physiological concentrations of either vitamin E or C significantly decreased the pro-inflammatory activity of TS and reduced the loss of BBB integrity after reperfusion in smoke-exposed BBB modules.
Conclusions: TS is a strong vascular inflammatory primer that directly affects the BBB endothelium and the immune cells and can significantly worsen the BBB damage caused by other vascular inflammatory insults. Our data also suggest that supplementation of antioxidant vitamins can significantly reduce this risk.
289. The roles of tissue inhibitor of metalloproteinases 1 and 2 during cerebral ischemia-reperfusion injury
M. Gomi, M. Fujimoto, T. Aoki, M. Hayase, T. Marumo, M. Nishimura and Y. Takagi
Neurosurgery Department, Kyoto University, Kyoto, Japan
Background: Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that are involved in a variety of cellular activities. Enhanced MMPs can cause vasogenic edema and hemorrhagic transformation after cerebral ischemia, and affect the extent of ischemic injury. Active and latent MMPs are stringently regulated by endogenous tissue inhibitor of metalloproteinase (TIMPs). We hypothesized that the endogenous TIMPs were essential to protect against blood-brain barrier (BBB) disruption after ischemia by regulating the activities of MMPs.
Materials and methods: C57BL/6 male mice, TIMP-1(−/−) mice and TIMP-2(−/−) mice were used. Cerebral ischemia was induced by using the standard intraluminal middle cerebral artery occlusion method.
Results: In Wild-type (WT) mice, TIMP-1 mRNA expression was quite low under normal conditions, and was gradually increased until 24 h after reperfusion. MMP-2 was up regulated at 3 h, and MMP-9 was at 3 and 24 h after ischemia. In TIMP-1(−/−) mice, MMP-9 protein expression and gelatinolytic activity were significantly more augmented after cerebral ischemia than those in WT mice at 24 h. TIMP-2 gene deletion mice exhibited no significant difference in MMP expressions. Evans blue leakage was used to estimate the extent of postischemic vasogenic edema. In TIMP-1(−/−) mice and TIMP-2(−/−) mice, evans blue extravasation was significantly increased when compared with WT mice. We evaluated DNA fragmentation by TUNEL staining and cleaved caspase-3 expression. At 24 h after ischemia, there was a significant increase of TUNEL-positive cells in the cerebral cortex in TIMP-1(−/−) mice and TIMP-2(−/−) mice compared with WT mice, but no change in the caudate putamen. The expression of cleaved caspase-3 in the cerebral cortex in TIMP-1(−/−) mice was significantly more enlarged than in WT mice. The infarct area was augmented in TIMP-1(−/−) mice than in WT mice at 24 h after ischemia, but TIMP-2(−/−) mice also exhibited a slightly exacerbated infarct area although this difference did not reach statistical significance.
Conclusion: These results suggest that TIMP-1 inhibits MMP-9 activity and can play a neuroprotective role in cerebral ischemia.
330. Cyclooxygenase-1 and 2 differently modulate lipopolysaccharide- induced blood-brain barrier disruption via a matrix metalloproteinase mechanism
S. Aid1, A.C. Silva2, E. Candelario-Jalil3, S.-H. Choi1, G.A. Rosenberg3 and F. Bosetti1
1BPMS/NIA, NIH; 2Cerebral Microcirculation Unit, NINDS/NIH, Bethesda, MD; 3Department of Neurology, University of New Mexico/Health Sciences Center, Albuquerque, New Mexico, USA
Objectives: Cyclooxygenases (COX) -1 and -2 play a key role in inflammation and are the pharmacological targets of non-steroidal anti-inflammatory drugs. We recently demonstrated that the expression of pro-inflammatory cytokines and chemokines is reduced in COX-1 null (−/−) mice, and increased in COX-2−/− mice compared to their wild-type controls after intracerebroventricular (icv) lipopolysaccacharide (LPS) injection.1,2 Since chemokines are involved in leukocyte recruitment into the inflamed brain, we hypothesized that COX-1 and COX-2 deletion will differentially modulate blood-brain barrier (BBB) permeability in response to LPS.
Methods: icv LPS (5 μg) was injected in COX-1 and COX-2 null and wild type mice. Twenty-four hours later, BBB permeability was quantified using gadolinium-enhanced magnetic resonance imaging (MRI). Gelatin zymography and fluorometric assay were used to measure the activity of matrix metalloproteinase (MMP)-9 and MMP-3, respectively. Gene expression level was assessed using quantitative real-time PCR.
Results: We found that LPS-induced BBB disruption was increased in COX-2−/− versus their respective wild-type mice. In the hippocampus and cortex of LPS-treated mice, MMP-3 activity was significantly decreased in COX-1−/− mice, whereas in COX-2−/− mice the activity of both MMP-9 and MMP-3, known to mediate BBB breakdown, were increased. The gene expression of the leukocyte attracting chemokine CXCL10, the endothelial marker ICAM-1, and the macrophage marker CD45 were also found increased in the whole brain of COX-2−/− versus COX-2+/+ mice after LPS. The above genes, except for ICAM-1, were found decreased in the whole brain of COX-1−/− versus COX-1+/+ mice after LPS.
Conclusion: Altogether, these results indicate for the first time that COX-2−/− mice have increased BBB disruption in response to LPS and suggest that COX-2 selective inhibition may be detrimental to BBB integrity during inflammation.
451. Intracarotid injection of dehydrocholic acid (DHC) induces cerebral ischemia and blood brain-barrier disruption
E.J. Kang1,2, S. Major1,2, D. Jorks2, A. Friedman3 and J.P. Dreier1,2
1Center for Stroke Research Berlin (CSB); 2Experimental Neurology, Charité—University Medicine Berlin, Berlin, Germany; 3Faculty of Health Sciences, Ben-Gurion University, Beer-Sheva, Israel
Objectives: Intracarotid application of the bile salt DHC has been used to open the blood brain-barrier (BBB) in the ipsilateral hemisphere.1,2 This observation has drawn attention as a possible means for therapeutic studies of drug delivery into the central nervous system (CNS). However, DHC's mechanism of action is unknown. Here we studied the effects of intracarotid application of DHC on regional cerebral blood flow (rCBF), intracortical direct current (DC) potential and extracellular potassium concentration ([K+]o) in a rat cranial window preparation.
Methods: Male Wistar rats (n = 6; 250 to 350 g) were anaesthetized with thiopental-sodium (100 mg/kg, intraperitoneally), tracheotomised, and artificially ventilated. End-tidal CO2 concentration was adjusted to approximately 35 mm Hg. Body temperature was maintained at 38.0°C with a heating pad. Systemic arterial pressure was monitored via the left femoral artery. Evans blue was administered via the left femoral vein to evaluate BBB opening. The right external carotid artery was cannulated in a retrograde manner for intracarotid injection of DHC. An open parietal window was implanted to place two K+-sensitive microelectrodes and a laser-Doppler flowmetry (LDF) probe. Epidural DC potential and rCBF were measured by an Ag/AgCl electrode and a second LDF probe at a closed frontal window.
Results: Intracarotid application of DHC (17.5%, 1 mL) induced an increase of rCBF to 373±90% followed by a decrease to 33±16% after 65±85 secs in 5 of 6 experiments. A shallow increase of the [K+]o from 3.0 to 9.8±6.3 mmol/L started simultaneously with the decrease of rCBF typical of cerebral ischemia. This was accompanied by ictaform epileptic field potentials with an amplitude of −3.4±2.3 mV, a spike duration of 64.8±5.5 ms, and spike frequency of 1.2±0.8 Hz in 5 experiments. Moreover, an increase of systemic arterial pressure accompanied the decrease of rCBF typical of a compensatory systemic hypertension. 45±28 secs after onset of the rCBF decrease we observed a sharp saddle-shaped negative intracortical DC shift of −20.3±1.9 mV accompanied by a sharp rise of [K+]o to 49.1±10.6 mmol/L typical of cortical spreading depolarisation (CSD). The latency of this intracortical DC shift was 20±20 secs between the rostral and the caudal microelectrodes indicating a spread typical of CSD between them. The DC negativity and rise of [K+]o did not recover for an observation period of at least 60 mins.
Conclusions: Spigelman et al1 previously described a BBB disruption in response to intracarotid DHC (17.5%, 1 mL) in the rat. No routine histology was performed. Here we show that this procedure induces prolonged cerebral ischemia. The BBB disruption may either be a direct effect of DHC or secondary to the ischemia. However, the procedure is not suitable for drug delivery into the CNS. Bile acids act as detergents. The ischaemia might be the consequence of severe damage to the cerebrovascular endothelium which will be investigated further.
475. Human brain imaging and radiation dosimetry of [11C]-N-desmethyl-loperamide, a positron emission tomographic radiotracer to measure the function of P-glycoprotein
N. Seneca1,2, S.S. Zoghbi2, J.-S. Liow2, W. Kreisl2, P. Herscovitch3, K. Jenko2, R.L. Gladding2, A. Taku2, V.W. Pike2 and R.B. Innis2
1Clinical Research and Exploratory Development, F. Hoffmann-La Roche Ltd., Basl, Switzerland; 2Molecular Imaging Branch, NIMH; 3PET Department, Clinical Center/NIH, Bethesda, Maryland, USA
P-glycoprotein (P-gp) is a membrane-bound efflux pump that limits the distribution of drugs to several organs of the body.1 At the blood-brain barrier, P-gp blocks the entry of both loperamide and its metabolite N-desmethyl-loperamide (dLop) and thereby prevents central opiate effects. Animal studies have shown that [11C]dLop, in comparison to [11C]loperamide, is a promising radiotracer because it generates negligible radiometabolites that enter brain.2,3
Objectives: The purposes of this study were to determine if [11C]dLop is a substrate for P-gp at the blood-brain barrier in humans and to measure the distribution of radioactivity in the entire body so as to estimate radiation exposure.
Methods: Brain PET scans were acquired in four healthy subjects for 90 mins and included concurrent measurements of the plasma concentration of parent radiotracer. Time-activity data from the whole brain were quantified using a one-tissue compartment model to estimate the rate of entry (K1) of radiotracer into brain. Whole-body PET scans were acquired in eight healthy subjects for 120 mins.
Results:Brain imaging. After injection of [11C]dLop, the concentration of radioactivity in brain was low (∼15% SUV) and stable after ∼20 mins. In contrast to the low uptake in brain, pituitary and choroid plexus accumulated high concentrations of radioactivity about 650% and 280% SUV, respectively. Similar to brain, uptake in the pituitary and choroid plexus was relatively rapid and stable until the end of the scanning session. Plasma concentration of [11C]dLop declined rapidly over time, but the percentage composition of plasma was unusually stable, with parent radiotracer constituting 85% of total radioactivity after ∼5 mins. The rate of brain entry was very low (K1 = 0.009±0.002 mL × cm−3 × min−1). Whole-body imaging. As a measure of radiation exposure to the entire body, the effective dose of [11C]dLop was 7.8±0.6 mSv/MBq.
Conclusions: The low brain uptake of radioactivity is consistent with [11C]dLop being a substrate for P-gp in humans and confirms that this radiotracer generates negligible quantities of brain-penetrant radiometabolites. The low rate of brain entry (K1) is consistent with P-gp rapidly effluxing substrates while they transit through the lipid bilayer. The current data reports baseline brain uptake of [11C]dLop in healthy humans and future studies after P-gp blockade will further confirm that [11C]dLop is acting as an active substrate for P-gp in humans. Based on our animal studies which showed that brain uptake of [11C]dlop was high after inactivation of P-gp by either genetic knockout or pharmacological inhibition,2–4 we hope to translate these animal studies to humans. The radiation exposure of [11C]dLop is similar to that of many other 11C-radiotracers. In conclusion, [11C]dLop is a promising radiotracer to study the function of P-gp at the blood-brain barrier, where impaired function would allow increased uptake into brain.
484. Cytochrome P450 expression at the human blood-brain barrier: focus on CYP3A4 and its role in carbamazepine brain metabolism
C. Ghosh1, M. Hossain1, J. Gonzalez-Martinez2,3, L. Cucullo1, D. Janigro1,3 and N. Marchi1
1Cell Biology, Cleveland Clinic Lerner College of Medicine; 2Neurological Surgery, Cleveland Clinic; 3Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio, USA
Objectives: Drug delivery to the brain may be hampered by the overexpression of multiple drug resistance proteins in blood-brain barrier (BBB) endothelial cells.1,3,4 We hypothesized that BBB endothelial cells may also act as a metabolic barrier or as a system capable of pro-drug transformation. Thus, while cytochrome P450 (CYPs) iso-enzymes play a major role in the metabolism of AED in the liver, it is not clear whether these enzymes are also functionally expressed in human BBB endothelial cells and possibly involved in drug response or resistance.2,4
Methods: Primary endothelial cultures were obtained from brain specimens of patients affected by drug resistant epilepsy (EPI-EC) and resected aneurisms domes (ANE-EC). A human-derived cell line of brain microvascular endothelial cells was used as controls (HBMEC). Transcript levels of CYPs were assessed by cDNA microarrays.5 Results were confirmed by immunohistochemistry on resected epileptic brain tissue. In vitro, control EC were exposed to laminar flow and the levels of CYP3A4 evaluated. HPLC analysis was performed to assess the functional relevance of CYPs BBB expression on carbamazepine metabolism.
Results: EPI-EC and ANE-EC displayed up regulated mRNA levels for Cyp3A4, Cyp2J2, Cyp4A11, Cyp2E1 and Cyp11b compared to control. An increased mRNA level of CYPs was paralleled with an increase in drug-transporter transcripts. Laminar flow induced mRNA levels of a broad spectrum of enzymes: Cyp33, Cyp2J2, CARS-Cyp, Cyp4A11, Cyp2C9, Cyp3A4, Cyp2E1, Cyp11b, Cyp2A6, Cyp1B1 and Cyp2C. Among these enzymes, Cyp3A4, Cyp2C9, Cyp2A6 and Cyp2J2 are involved in the metabolic conversion of AEDs. In particular, CYP3A4 levels were increased in EPI and ANE endothelial cells and up-regulated by shearing forces.5 Immunohistochemical analysis confirmed BBB expression of CYP3A4 in dysplastic human epileptic brain while CYP3A4 was lower in specimens of well organized cortex. In vitro, CYP3A4 expression positively correlated with the extent of carbamazepine metabolism.
Conclusions: Our results reveal the functional expression of P450 enzymes at the human BBB. Among these enzymes, the levels of CYP3A4 determined the amount of CBZ metabolized. These results reveal a novel powerful molecular mechanism of AED metabolism by the BBB.
518. Flair of early BBB disruption in acute experimental stroke
E. Henning, A. Martin, S. Warach and L. Latour
Stroke Diagnostics and Therapeutics Section, NINDS/NIH, Bethesda, Maryland, USA
Introduction: In acute stroke patients, extravasation of Gd-contrast appears hyperintense on FLAIR in CSF spaces such as the sulci and lateral ventricles. This observation is thought to be indicative of early blood-brain barrier (BBB) disruption and has been associated with reperfusion, hemorrhagic transformation, and worse clinical outcome. Unfortunately, the mechanisms of barrier disruption and movement of Gd-contrast to the CSF space remain poorly understood and cannot be investigated in the clinic. In this study, we employed FLAIR to identify the same phenomenon early after experimental stroke in spontaneously hypertensive rats (SHR).
Methods:N = 10 male SHR, 360±10 grams, were divided into two groups. Group 1 (N = 5) received 50±6 mins of right middle cerebral artery occlusion (MCAO) followed by reperfusion. Group 2 (N = 5) received sham surgery. Imaging was performed using a 7.0T/30 cm imaging spectrometer equipped with ±45 G/cm gradients. DW-EPI and GRE were performed at baseline (30-mins post-MCAO). DW-EPI, GRE, T2, and FLAIR imaging were performed at 30-mins, 1 h, and 1.5 h post-reperfusion. At 1 and 1.5 h post-reperfusion, Gd-DTPA (0.2 mL @ 0.5 mol/L, i.v.) was administered. Additional FLAIR images were acquired 10 and 20 mins post-contrast for assessment of Gd-DTPA extravasation. VOIs were drawn on ADC maps for stroke volume calculation and on FLAIR images at baseline for delineation of the lateral ventricles and healthy parenchyma. VOIs drawn on FLAIR were projected to post-reperfusion time points in order to track any observed signal enhancement.
Results: Lesion volume at baseline was 260±20 mm3. At 30-mins post-reperfusion, lesion volume decreased significantly to 50±20 mm3 (P<0.01). This decrease continued through 1 h (14±7 mm3, P<0.01) and 1.5 h (9±6 mm3, P<0.01) post-reperfusion. There was no difference in FLAIR signal at baseline between stroke and sham groups for the contralateral (P = 0.28) and ipsilateral (P = 0.32) ventricles. At 1 h post-reperfusion (∼2 h after stroke onset), there was significant post-contrast FLAIR signal enhancement for the ipsilateral ventricle (2.31±1.04, P<0.01) in comparison to sham (0.58±0.04). There were no differences for the contralateral ventricle (P = 1.00). These findings were consistent at 1.5 h post-reperfusion. Additional FLAIR enhancement was observed in N = 1 animal in the parenchyma and N = 1 animal in the meninges, both ipsilateral to stroke.
Conclusions: In this study, we have shown that FLAIR permits delineation of barrier disruption as early as 1 h post-reperfusion following acute experimental stroke. These observations are identical to what has been observed in the clinic and are within the time frame of tPA administration. The methods presented herein provide an avenue to investigate:
Gd extravasation as a marker of BBB disruption;
the relationship between Gd extravasation, reperfusion, tPA, and hemorrhagic transformation;
novel therapies targeting the BBB.
573. Hsp70 overexpression attenuates ischemia-induced disruption of tight junction proteins following experimental stroke
Z. Zheng, J.Y. Kim and M.A. Yenari
UCSF-SFVAMC, San Francisco, California, USA
Background and aims: We previously showed that microglia potentiate ischemic injury to blood brain barrier (BBB) components, and that overexpression of Hsp70 protects against stroke, in part, through an anti-inflammatory mechanism. Recent studies indicate that increases in BBB permeability following brain ischemia may be associated with alterations in neurovascular proteolytic activity, particularly thought the matrix metalloproteinases (MMPs). In this study, we evaluated the effects of Hsp70 overexpression on several BBB-related tight junction proteins in a previously validated transient middle cerebral artery occlusion (tMCAO) model in Hsp70 transgenic mouse (Tg). To determine whether this effect is mediated through Hsp70 expression within the brain or within circulating cells, we also studied bone marrow chimeras, where wildtype (Wt) mice received bone marrow from Tg mice (Tg chimera) or Wt mice as a control.
Methods: The mice were subject to 2 h brain ischemia, followed by 24 h reperfusion. IgG immunostaining was used to assess BBB integrity. Tight junction proteins, claudin-5, collagen 4 and ZO-I were assessed in cortical tissue ipsilateral to MCAo by using western blots and semi-quantitative densitometry was performed on digitized images using Taphone software.
Results: Increases in parenchymal IgG immunoreactivity were observed in both Wt and Tg mice, indicating the disruption of BBB, but the area and intensity of IgG immunostaing among transgenic mice were significantly decreased compared to wt mice, even when normalized to infarct size (P<0.01). Similar patterns were observed in the bone marrow chimeras, with decreased BBB disruption in the Tg chimeras. Marked reduction of claudin-5, ZO-1 was evident in Wt mice. In contrast, Tg transgenic mice and Tg chimeras showed significant preservation of claudin-5 and ZO-1 proteins (P<0.05). There were trends showing less disruption of collagen 4 in Tg mice and Tg chimeras, but this did not reach statistical significance.
Conclusions: Our data suggest that Hsp70 overexpression attenuates exacerbation of BBB permeability through the conservation of tight junction proteins, claudin-5 and ZO-1, and this effect appears to be mediated by Hsp70 overexpression in circulating leukocytes.
769. Transforming growth factor-β (TGF-β) signaling is involved in the regulation of blood-brain barrier (BBB) functional integrity during peripheral inflammatory pain
P.T. Ronaldson, K.M. Demarco, L. Sanchez-Covarrubias, C.M. Solinsky and T.P. Davis
Department of Pharmacology, University of Arizona, Tucson, Arizona, USA
Objectives: Our laboratory has shown altered BBB functional integrity during λ-carrageenan-induced peripheral inflammatory pain (CIP) characterized by changes in tight junction (TJ) protein expression and sucrose permeability.1–4 However, the intracellular signaling mechanisms involved have not been elucidated. One pathway that may be involved is the transforming growth factor-β (TGF-β) system, which is known to regulate endothelial cell function. Of particular interest are processes mediated by activin receptor-like kinase 5 (ALK5), a TGF-β receptor that may decrease vascular permeability.5 The objectives of this study were to investigate, in vivo, the role of TGF-β/ALK5-mediated signaling on (i) TJ protein expression in brain microvessels and (ii) BBB permeability to 14C-sucrose. Both objectives were evaluated in the context of CIP.
Methods: CIP was induced in female Sprague-Dawley rats (200 to 250 g) by subcutaneous injection of 3% λ-carrageenan into the plantar surface of the right hind paw. Control animals were injected with 0.9% saline instead of λ-carrageenan. SB431542 (1.5 mg/kg, i.p.), a selective ALK5 inhibitor, was administered 30 mins prior to footpad injection. Similarly, human recombinant TGF-β1 (12.5 ng/kg, i.p.) was injected 30 mins prior to CIP or control treatment. After 3 h, animals were sacrificed, blood samples collected, and brain microvessels isolated. ELISA was used to measure serum TGF-β1 concentrations. Protein expression of TGF-β receptors (i.e., ALK5), TJ proteins (i.e., claudin-3, claudin-5, occludin, ZO-1), and intracellular TGF-β signaling molecules (i.e., Smad2–4) were determined using immunoblot analysis. Brain permeability to 14C-sucrose (10 μCi/20 mL perfusate) was assessed using the in situ brain perfusion technique.
Results: During CIP, serum TGF-β1 and ALK5 protein expression were reduced. Brain permeability to 14C-sucrose was increased and expression of TJ proteins (i.e., claudin-5, occludin, ZO-1) were altered after 3 h CIP. Pharmacological inhibition of ALK5 with SB431542 further enhanced brain uptake of 14C-sucrose, increased TJ protein expression (i.e., claudin-3, claudin-5, occludin, ZO-1), and decreased nuclear expression of TGF-β/ALK5 signaling molecules (i.e., Smad2, Smad3), suggesting a role for TGF-β/ALK5 signaling in the regulation of BBB functional integrity. Interestingly, administration of exogenous TGF-β1 prior to CIP activated the TGF-β/ALK5 pathway and reduced BBB permeability to 14C-sucrose.
Conclusions: Our data show that TGF-β/ALK5 signaling is involved in the regulation of BBB functional integrity. Overall, these observations suggest that complex mechanisms are involved in controlling the dynamic nature of the BBB and may represent a novel target for pharmacological manipulation of brain microvascular permeability.
This work was supported by NIH grants R01-NS42652 and R01-DA12684 to TPD.
879. Identification of neuroinflammation-associated proteins and glycoproteins in human brain endothelial cells using mass spectrometry-based quantitative proteomics
A. Haqqani1, J. Mullen1, W. Zhang1, P. Couraud2 and D. Stanimirovic1
1Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada; 2Université Paris Descartes, Paris, France
The luminal surface of brain vascular endothelium is decorated with proteins, glycoproteins and proteoglycans that form a thick coating called glycocalyx. The glycocalyx is involved in various vascular functions including endothelial permeability, leukocyte adhesion/emigration and vascular blood flow. Recent work has shown that the true interface between the endothelium and flowing blood is the endothelial glycocalyx rather than the endothelial plasma membrane. The glycocalyx is significantly altered in response to ischemia/reperfusion or various inflammatory stimuli; for example, most of the endothelial surface and adhesion molecules that are up-regulated as hallmarks of cerebrovascular inflammation are glycosylated proteins. However, the complex nature of the brain endothelial glycocalyx, has so far hindered deeper understanding of its molecular alterations in pathological conditions.
Objectives: To identify proteins and glycoproteins present in the membranes of human brain endothelial cells (HBEC) and their quantitative changes in response to inflammatory stimulus using advanced (glyco)proteomics and bioinformatics tools.
Methods: Total membrane extracts were isolated from Immortalized HBEC (HCMEC/D3 obtained from P. Couraud) exposed to 100 U/mL IL-1b for 16 h by ultracentrifugation and sucrose gradient. The membranes were then treated with N-linked deglycosylating enzyme (PNGase F) and fractionated using cation exchange chromatography. Each fraction was analyzed by label-free mass spectrometry-based quantitative proteomics using nanoLC-MS and MS/MS followed by a suite of bioinformatics tools. Three biological replicates and two technical replicates were carried out. The quantitative variability of the proteomic analyses was <10% for technical replicates and <20% for biological replicates.
Results: 10,608 high-quality unique peptide sequences have been identified in the total HBEC membranes, corresponding to 2817 proteins (Table 1). More than 400 proteins were found to be glycoproteins as determined by PNGase F treatment (e.g., TMEM30A, various solute carrier family and ABC transporters). While most (68%) of the PNGase F-responsive proteins are known glycosylated proteins, many novel N-glycosylated proteins were identified. In addition, >750 proteins showed differential expression in response to IL-1b treatment (>2.5-fold up- or down-regulated, P<0.05, 3 biological and 2 technical replicates). The majority of these proteins have not been previously reported as IL-1b-responsive, and >10% were of unknown function. Classification of the proteins using Panther Classification System showed their functions/processes consistent with pathophysiology of neuroinflammation.
Number and examples of proteins up- or down-regulated in response to IL-1β in HCMEC/D3 total membranes
Total
Upregulated
Downregulated
Examples
Total
2817
390
378
Signal transduction
317
37
26
IL22RA1, erbB-2
Adhesion molecules and tight junctions
192
21
20
ICAM1, integrins, ALCAM, PECAM1, ZO1, ZO2
ECM proteins
154
16
6
MMPs, ECM2
Transporters
140
21
19
SLC1A5, ABCs
Immunity and defense
126
18
9
HSPs, GSH synthase
MMPs and other proteases
67
11
8
MMP9
Protein traffic
38
3
1
MHC antigens associated proteins
33
7
3
Chemokines and cytokines
26
6
2
Angiogenesis
23
3
1
Unclassified/unknown category
298
35
32
Conclusion: These analyses identified many proteins of yet unknown function as well as previously unknown glycoproteins that respond to inflammatory stimuli in brain endothelium. The data suggest an important role for glycocalyx in physiological and pathological responses of brain endothelium.
900. MR characterization of smart contrast agents: toxicity, stability, and permeability
D. Coman1,2, M. Gattas-Sethi3,4, H.K.F. Trubel5,6, P. Herman1,2,7, F. Hyder1,2,3, G. Kiefer8 and F. D'errico3,4
1Diagnostic Radiology; 2Quantitative Neuroscience with Magnetic Resonance (QNMR); 3Biomedical Engineering; 4Therapeutic Radiology, Yale University, New Haven, Connecticut, USA; 5Department of Pediatrics, University Witten/Herdecke, HELIOS-Klinikum Wuppertal; 6Bayer HealthCare Research Center, Wuppertal, Germany; 7Semmelweis University, Budapest, Hungary; 8Macrocyclics, Dallas, Texas, USA
Introduction: The measurement of ex vivo kinetic inertness, in vivo blood-brain barrier permeability and in vivo cytotoxicity are three important steps in assessing the feasibility of using any smart contrast agent (SCA) for any ulterior biological studies. Over the past decade, a new non-invasive method for simultaneous measurements of temperature and pH was developed, based on the strong dependence on temperature and pH of the proton chemical shifts from the complex between the thulium ion and the macrocyclic chelate 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetra (methylene phosphonate) or TmDOTP5−.1,2,4 More recently, another temperature probe was introduced, for which the macrocyclic chelate is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethyl-1,4,7,10-tetraacetate or TmDOTMA−.3 In the present work, we measured ex vivo kinetic inertness, in vivo blood-brain barrier permeability and in vivo cytotoxicity of TmDOTP5− and TmDOTMA− agents.
Methods: Animal studies: Sprague-Dawley rats were tracheotomized and artificially ventilated (70% N2O, 30% O2). During the animal preparation, isoflurane (1 to 2%) was used for induction. The anesthetized rats were prepared with renal ligation as previously described.4 CBF was measured by a laser Doppler flowmetry probe and pO2 was measured by an oxygen fluorescence probe.
Cytotoxicity: The colony-forming ability of the CCL16 Chinese hamster lung cells in the cultures exposed to the SCAs was compared with that of cells from untreated cultures to calculate the relative surviving fraction.
Results: Kinetic stabilities of the SCAs in blood extracts from the sagittal sinus were estimated by the intensity of their 1H NMR resonances over time. For both TmDOTP5− and TmDOTMA−, the results indicate that there is no change in the intensity of the methyl group resonance from day 1 to day 59. Our observations suggest that TmDOTP5− does cross the blood-brain barrier quite efficiently, which allows quantifying brain temperature and pH.4 Concomitant 1H signals of TmDOTP5− in blood plasma and cerebral spinal fluid (CSF) confirm that the majority of the in vivo MR signals emanate from tissue.4 A plausible delivery path into the extracellular space may be through the fenestrated vessels of the circumventricular organs. The in vivo cytotoxicity tests consistently showed survival rates between 90 and 95%. Our electrophysiological measurements suggest that an infusion dose of 1 to 2 mmol/kg results in stable systemic physiology without affecting normal brain function. Localized changes in CBF and pO2 during forepaw stimulation in α-chloralose anesthetized rats are nearly identical before and after SCAs infusion.
Conclusion: Our results indicate that these two SCAs are kinetically stable, they cross the blood-brain barrier, they clearly exclude acute toxicity on Chinese hamster lung cells and they demonstrate that the brain's activity is unaffected by their presence in the extracellular space.
Acknowledgements: Supported in part by a pilot grant from P30 NS052519 of the QNMR Program.
993. Inhibition of myosin light chain kinase ameliorates brain edema formation after traumatic brain injury
C. Ricken1, C. Kuhlmann2, H. Luhmann2, K. Engelhard1, C. Werner1 and S. Thal1
1Department of Anesthesiology; 2Department of Physiology, Johannes Gutenberg-University, Mainz, Germany
Introduction: The role of the endothelial contractile apparatus in the process of brain edema formation after brain trauma is not clarified. Myosin light chain kinase (MLCK) regulates the contraction of endothelial cells through phosphorylation of regulatory myosin light chains (MLC). The integrity of the blood brain barrier (BBB) may therefore be influenced by the phosphorylation state of the MLC. In endothelial cells hypoxia results in an up-regulation of MLCK and leads to increased phosphorylation of MLCs. This mediates a cytoskeletal rearrangement, which may result in blood-brain-barrier (BBB) dysfunction.1 The present study investigates the effect of selective MLCK inhibition on brain edema formation and histological outcome after experimental brain trauma.
Methods: Male C57Bl/6 mice were anesthetized with midazolam, fentanyl and medetomidine and were subjected to controlled cortical impact brain injury (CCI). Mice were treated with intraperitoneal injection of the selective MLCK inhibitor ML-7 (1 mg/kg) or vehicle solution (0.9% NaCl) 1 h prior to and 6 h after trauma. Brain water content was determined 24 h after CCI in animals randomized to:
vehicle+sham surgery;
vehicle+CCI;
ML-7+sham surgery;
ML-7+CCI (n = 7 per group).
Brain contusion volume was determined 15 mins and 24 h post CCI in two separate set (small trauma: depth 1 mm; large trauma: depth 1.5 mm) of animals randomized to:
vehicle+CCI and
ML-7+CCI (n = 8 per group).
Statistics: ANOVA on RANKS, P<0.05.
Results: 24 h after CCI brain water content increased significantly in vehicle treated animals (CCI: 79.95%±0.69) in comparison to sham animals (78.64%±0.46). ML-7 significantly reduced edema formation (CCI: 78.97%±0.53). Brain damage expanded significantly between 15 mins and 24 h after CCI in vehicle and ML-7 treated animals. Independent of trauma size, brain contusion volume was not influence by ML-7 application (1.0 mm: vehicle = 22.3 mm3±3.7; ML-7 = 22.4 mm3±3.4; 1.5 mm: vehicle = 44.6 mm3±10.9; ML-7 = 41.1 mm3±6.8).
Conclusion: The present study demonstrates that inhibition of MLCK prevents brain edema formation 24 h following trauma. The data are in accordance to a previous study, where ML-7 significantly reduced brain water content in focal cerebral ischemia.1 Despite experimental evidence for the detrimental effects of brain edema formation after brain trauma, prevention of brain edema formation did not result in reduction of contusion volume or improved neurological outcome. In this model increase of intracranial pressure and impairment of cerebral perfusion might have been not severe enough to demonstrate a histological or neurological effect of brain edema reduction by MLCK inhibition. The results suggest that brain edema formation depends on the phosphorylation state of MLC. This might present an important mechanism of BBB leakage after head trauma.
997. Endothelial nitric oxide synthase mediates blood-brain barrier injury in the thiamine-deficient mouse brain
É. Beauchesne, P. Desjardins, A.S. Hazell and R.F. Butterworth
Neurosciences Research Unit, CHUM, St-Luc Hospital, University of Montreal, Montreal, QC, Canada
Objectives: Wernicke's Encephalopathy (WE) in humans is a chronic metabolic disorder caused by thiamine deficiency (TD) characterized clinically by ophthalmoplegia, ataxia and confusion. TD leads to cerebral impaired oxidative metabolism, due to the inhibition of α-ketoglutarate dehydrogenase (α-KGDH), a rate-limiting enzyme of the TCA cycle. Neuropathologic consequences of TD include selective neuronal cell death and petechial hemorrhagic lesions in medial thalamus. Blood-brain barrier (BBB) breakdown plays an important role in TD pathogenesis.2 Among early changes in TD, increased expression of the endothelial isoform of nitric oxide synthase (eNOS) occurs selectively in vulnerable regions of the brain.3 We hypothesized that eNOS induction in TD leads to oxidative/nitrosative stress, tight junction protein (TJP) alterations and BBB breakdown.
Methods: TD was induced in C57BL6 and eNOS−/− mice by feeding with a thiamine-deficient diet and by treatment with the thiamine antagonist pyrithiamine. Pair-fed control (PFC) mice (from both strains) were fed the same diet and treated with thiamine. Experiments were conducted in medial thalamus versus frontal cortex (spared area) from the brains of PFC and TD (WT and eNOS−/−) mice. Cresyl violet was used for histological evaluation, and oxidative/nitrosative stress was assessed by measuring levels of NOx (nitrites/nitrates) and by heme oxygenase-1 (HO-1) immunohistochemistry. BBB integrity was assessed by measuring immunoglobulin-G (IgG) extravasation, expression of TJPs occludin, zonula occludens-1 and -2 (ZO-1, ZO-2), as well as activity of the related matrix-metalloproteinases (MMPs).
Results: In the medial thalamus of TD-WT mice, we observed increased levels of NOx and that HO-1 immunostaining was co-localized with eNOS in vessel walls, showing the presence of oxidative/nitrosative stress in thalamic vessels. BBB breakdown was demonstrated by the presence of hemorrhagic lesions, an increase in BBB permeability to IgG, a loss in expression of occludin, ZO-1 and ZO-2, and a concomitant increase in MMP-9 activity. No such changes were observed in frontal cortex. eNOS gene deletion prevented NOx increase and led to attenuation of neuronal cell death, confirming the neurotoxic role of eNOS-derived nitric oxide (NO) in TD.1 Moreover, HO-1 immunostaining was eliminated in vessels, and BBB breakdown was prevented as shown by absence of haemorrhages, prevention of IgG extravasation, normalization of TJP expression and prevention of the increase in MMP-9 activity in the medial thalamus of TD-eNOS−/− mice, compared to TD-WT mice.
Conclusions: These data demonstrate that eNOS-derived NO is a major factor leading to oxidative/nitrosative stress, alterations of the cerebrovascular endothelial cells and BBB breakdown in TD.
1042. Evaluation of blood-brain barrier permeabilities of clinical PET tracers using in vitro model of human adult blood-brain barrier
A. Mabondzo1, M. Bottlaender2 and J.R. Deverre2
1Insitut de Biologie et de Technologie de Scalay, CEA/Service de Pharmacologie et d'Immunoanalyse, Gif sur Yvette; 2I2BM, CEA, Orsay, France
Background and objectives: The development of drugs targeting the central nervous system (CNS) requires precise knowledge of their brain penetration and, ideally, this information should be obtained as early as possible. The physical transport and metabolic blood-brain barrier (BBB) is highly complex and numerous in vitro models have been designed to study kinetic parameters in the CNS. In vitro BBB models must be carefully assessed for their capacity to reflect accurately the passage of drugs into the CNS in vivo. In this study, we report for the first time the evaluation of BBB permeabilities of compounds studied in human subjects by PET and using the in vitro human BBB model, in order to reassess the predictive power of the in vitro system.
Materials and methods: Six clinical PET tracer with different molecular size ranges and degree BBB passage were used (2 of them [18F]-FDOPA and [18F]-FDG are ligand of amino acid and glucose transporters, respectively). The k1 and k2 in human brain of those 6 tracers were available. The human BBB model was a coculture of primary human BECs and astrocytes as previously described (Mégard, 2002; Josserand et al., 2006). Before drug permeation screening, the integrity of the in vitro system was evaluated.
Results: Our findings indicate that the in vitro co-culture model of human BBB has important features of the BBB in vivo (low paracellular permeation, well developed tight junctions, functional expression of efflux transporters) and is suitable for discriminating between CNS and non-CNS compounds. We evaluated drug permeation into the human brain using PET imaging in parallel to the assessment of drug permeability across the in vitro model of the human BBB. 2-[18F]fluoro-A-85380 and [11C]-raclopride show absent or low cerebral uptake with the distribution volume under 0.6, while [11C]-flumazenil, [11C]-befloxatone, [18F]-FDOPA and [18F]-FDG show a cerebral uptake with the distribution volume above 0.6. The in vitro human BBB model discriminates the compounds in the same way as in vivo human brain PET imaging analysis. We cast new light on the close relationship between in vitro and in vivo pharmacokinetic data (r2: 0.90, P<0.001).
Discussion and conclusion: Past in vivo-in vitro studies often did not have good correlations for substances with transporters. This is probably because experiments have been performed with too much material in vitro, saturing transporters and so obscurancing that contribution. Using the radioactive labeled probes and the small amounts of compounds, this problem could be avoid. This first double study in human subjects demonstrates a close relationship between the assessment of in vitro human BBB passage and in vivo human brain penetration. These findings which can be extended to peptides, proteins, viruses and with substances with high degrees of protein binding in vivo or other non BBB factors dictating their penetration need to be specifically studies. The in vitro human BBB model offers the possibility of subtle discrimination between different BBB permeabilities and transport mechanisms. This in vitro human BBB model may help to test compounds of pharmaceutical importance for CNS diseases.
1080. Blood-brain barrier Na-K-Cl cotransporter and Na/H exchanger: therapeutic targets for ischemia-induced brain Na uptake and edema formation
Y.-J. Chen, T.I. Lam, S.E. Anderson, J.H. Walton and M.E. O'donnell
Physiology and Membrane Biology, University of California, Davis, California, USA
Objectives: Ischemia-induced brain edema is a major cause of morbidity and mortality in stroke although much is unknown about the processes involved. Previous studies have shown that during the early hours of cerebral ischemia, edema forms in the presence of an intact blood-brain barrier (BBB) by mechanisms involving increased secretion of Na, Cl and water across the barrier from blood into brain. The BBB ion transporters responsible for this have been largely unknown although luminal Na transporters appear to be rate limiting in the process. Our previous studies have provided evidence that a luminal BBB Na-K-Cl cotransporter participates in ischemia-induced cerebral edema formation. Among our findings are the observations that Na-K-Cl cotransporter activity of cerebral microvascular endothelial cells (CMEC) is stimulated by hypoxia, aglycemia and arginine vasopressin (AVP), three prominent factors present in ischemia, with a time course and sensitivity consistent with ischemia-induced edema formation. In addition, inhibition of the BBB Na-K-Cl cotransporter by intravenous administration of bumetanide significantly attenuates edema formation and infarct in the rat permanent middle cerebral artery occlusion (MCAO) model of ischemic stroke. In recent studies we have found evidence that a BBB Na/H exchanger is also stimulated by hypoxia, aglycemia and AVP and, further, that intravenous administration of the Na/H exchange inhibitor HOE642 also reduces edema formation and infarct in permanent MCAO. We also found that both NHE1 and NHE2 isoforms of the Na/H exchanger are present in cultured cerebral microvascular endothelial cells (CMEC). The present study had two goals: (1) to investigate NHE isoforms present in BBB in situ; and (2) to determine whether bumetanide and HOE642 reduce the brain Na uptake that underlies edema in ischemic stroke.
Methods: Perfusion-fixed brains of rats subjected to 90 mins of permanent left MCAO were evaluated for the presence of NHE1 and NHE2 Na/H exchanger isoforms in BBB in situ using immunoelectron microscopy (immunoEM) and antibodies that specifically recognize NHE1 and NHE2 isoform proteins. For brain Na uptake studies, rats were given bumetanide (30 mg/kg), HOE642 (30 mg/kg) or vehicle i.v. 20 mins before, or 1 or 2 h after initiation of left MCAO. Localized 23Na magnetic resonance spectroscopy (MRS) with the shift reagent DyTTHA was then used to assess total and extravascular brain [Na] in selected regions of interest during up to 5 h of MCAO.
Results: ImmunoEM revealed that both NHE1 and NHE2 isoforms are present predominantly in the luminal BBB membrane in situ, whether normoxic or ischemic cortex. For 23Na MRS studies, the ipsilateral/contralateral brain ratios of both total [Na] and extravascular [Na] increased linearly following induction of ischemia. Both bumetanide and HOE642 significantly reduced brain Na uptake and infarct whether given before or after the start of MCAO.
Conclusions: These findings support the hypothesis that the BBB Na/H exchanger, possibly both NHE1 and NHE2 isoforms, participates in ischemia-induced edema formation and that intravenous bumetanide and HOE642 are both effective in reducing brain Na uptake, edema and infarct even when given after the onset of ischemia.
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