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ASTROCYTES PROVIDE A SIGNALING CONDUIT FOR UPSTREAM (PIAL ARTERIOLAR) DILATION DURING CORTICAL NEURONAL ACTIVATION IN THE RAT Dale Pelligrino, Hao-Liang Xu Neuroanesthesia Research Department, University of Illinois at Chicago, Chicago, IL, USA BACKGROUND: The tight coupling between neuronal activity and blood flow is fundamental to brain function. Astrocytes play an important role in that process, via their capacity to “sense” neuronal activity and, in turn, transmit that information to parenchymal arterioles. In this study, we examined another aspect of astrocyte-related neurovascular coupling?the ability of astrocytes, in vivo, to act as a signaling conduit for the vitally important process of upstream vasodilation (represented by pial arterioles).

Pial arterioles are segregated from the underlying cortical neuropil by the presence of the astrocytic processes comprising the glia limitans (GL). We, therefore, postulated that, irrespective of the strength of cortical neuronal activation, whether excessive (bicuculline-induced seizure [BIS]) or physiologic (sciatic nerve stimulation [SNS]), the associated pial arteriolar dilation involves an astrocytic and GL-related signaling conduit. To test this, we topically applied (via a closed cranial window) L-alpha-aminoadipic acid (L-AAA), an astroglial toxin which selectively damages the GL (without affecting vascular smooth muscle, endothelium, or neurons). Two other hypotheses were addressed: a) inter-astrocytic signaling is dependent upon the gap junctional protein, connexin-43 (Cx43); and b) signaling from the GL to pial arterioles involves a diffusible mediator.

METHODS: Pial arteriolar diameter increases during BIS or SNS, in anesthetized rats, were measured in the absence or presence of L-AAA or Cx43 blockade (via topically-applied gap-27 peptide) and during increased rates of cortical suffusion (tests influence of diffusible mediator). In BIS experiments, EEG power increases were not altered by any experimental intervention, indicating that the changes in pial arteriolar responses were not due to changes in the level of cortical activation. RESULTS: The predominance of an astrocytic signaling conduit was indicated by data showing that pial arteriolar dilating responses to both neuronal activation paradigms were completely blocked following selective disruption of the superficial GL (fig. Gap-27 elicited a 60–75% reduction in BIS and SNS-induced dilations (fig.

Increasing the aCSF suffusion rate led to 40–50% reductions in BIS and SNS-related dilations (fig. CONCLUSIONS: Astrocytes and the GL play a key role in signaling upstream pial arteriolar dilation during both moderate and excessive cortical neuronal activation. The signaling process may involve Cx43-related gap junctions and/or hemichannels and a diffusible mediator arising from the GL. CHARACTERIZATION OF A NOVEL POTASSIUM CHANNEL, TWIK-2, CLONED FROM THE RAT MIDDLE CEREBRAL ARTERY Eric Lloyd, Sean Marrelli, Robert Bryan Department of Anesthesiology, Baylor College of Medicine, Houston, TX, USA Background and aims: recently a new family of ion channels, termed two-pore domain potassium channels (K2P), has been discovered and cloned. Although little is known about the expression and function of these channels in the vascular system, we have demonstrated that TWIK-2, a member of the K2P family, is highly expressed in rat middle cerebral artery (MCA) (AJP 291:H770, 2006).

The aim of this study was to characterize TWIK-2 and determine if its activation could dilate rat MCAs. Methods: TWIK-2 was cloned from rat MCA and expressed heterologously (COS and CHO cells) as a fusion protein to green fluorescent protein (GFP) for characterization. Electrical properties of TWIK-2 were determined using patch clamp methods in the whole cell configuration. MCAs were removed from rat brain, mounted on micropipettes in a vessel bath, pressurized, and perfused.

MCAs were magnified and the diameter recorded using image analysis software. Results: Direct fluorescence analysis by deconvolution microscopy in transiently transfected COS cells revealed that GFP-rTWIK-2 localized to the plasma membrane in a punctate pattern by 48 hours after transfection. Stable transfectants of GFP-rTWIK-2 were selected in CHO cells and analyzed by whole-cell electrophysiology.

Whole cell currents were 30-fold greater than the nontransfected control group, were non-rectifying in physiological K+, but were inwardly rectifying in symmetrical K+. The observed reversal potentials in different concentrations of extracellular K+ were similar to the theoretical reversal potentials. In physiological K+, membrane potential was −81 +/− 0.5 mv (n=13) for transfected and −51 +/− 2.5 mv (n=16) for control cells.

1 mM BaCl2 inhibited currents by 90 +/− 1% with a calculated IC50 of 86 uM. The TWIK-2 currents were minimally affected by 10 mM tetraethylammonium chloride (TEA), 3 mM 4-aminopyridine (4AP), and 10 uM glibenclimide (glib).

100 uM arachidonic acid increased the currents by 88 +/− 15% (n=6). In isolated pressurized rat MCA, arachidonic acid elicited robust dilations that were not blocked by 10 mM TEA, 3 mM 4AP, or 10 uM glib. Conclusions: TWIK-2 is a novel potassium channel expressed in cerebral arteries. It is characterized by a non-rectifying current in physiological K+ gradients and is resistant to most potassium channel inhibitors. We conclude that TWIK-2 is an important regulator of vascular tone. (RO1 NS 46666, NS 038660, and AHA Bugher Award 027011N). Introduction The cerebral blood flow (CBF) increase evoked by synaptic activity, or functional hyperemia, is critical for the brain's functional integrity.

A substantial component of the CBF response to activation is mediated by NMDA receptors. Tissue plasminogen activator (tPA), a protease involved in intravascular fibrinolysis, participates in NMDA receptor signaling, raising the possibility that tPA plays a role in functional hyperemia.

Therefore, we investigated whether tPA contributes to the increase in CBF evoked by somatosensory activation. Results The increase in somatosensory cortex blood flow produced by whisker stimulation was attenuated in tPA-/− mice (Figure A), but the response to topical application of acetylcholine was intact (tPA+/+: 22±3%; tPA-/−: 22±2%; p>0.05; ANOVA).

The attenuation in functional hyperemia in tPA-/− mice was rescued by exogenous tPA (rtPA) (Figure A). Functional hyperemia was not attenuated in mice lacking plasminogen (plg), the substrate of tPA (plg+/+: 24±3%; plg-/−: 23±1%; p>0.05). The NMDA receptor inhibitor MK-801 (10 µM; topical application) attenuated functional hyperemia in tPA+/+, but not in tPA-/− mice (Figure B), suggesting that the effect of tPA is mediated through the NMDA receptor. Consistent with this suggestion, the CBF increase elicited by topical NMDA (40 µM), but not kainate (10 µM), was reduced in tPA-/− mice (Figure C). The CBF increase produced by NMDA is mediated by neuronal nitric oxide synthase (nNOS), an enzyme functionally coupled to NMDA receptors through the scaffolding protein PSD-95.

A peptide that disrupts the interaction between nNOS and NMDA receptors (NR2B9c), but not its scrambled control (scNR2B9c), blocked the ability of rtPA to rescue functional hyperemia in tPA-/− mice (Figures A & B). NOC/OFQ ANTAGONIST POST INJURY TREATMENT PROTECTS AGAINST CEREBRAL CIRCULATORY COLLAPSE AND HISTOPATHOLOGY AFTER COMBINED PROLONGED HYPOTENSION AND BRAIN INJURY William M. Armstead, Xiao-Han Chen, Douglas H. Smith University of Pennsylvania, Philadelphia, PA, USA Introduction: In humans, traumatic brain injury (TBI) is often accompanied by hypovolemia and hypotension resulting from damage to other organ systems, which increases morbidity and mortality. Basic science animal studies have observed disturbed cerebral autoregulation during acute hypotension after TBI but few have investigated the effects of prolonged hypotension. The opioid NOC/oFQ contributes to impaired cerebral hemodynamics after fluid percussion brain injury (FPI) in the pig.

In the context of the neurovascular unit concept, this study addressed the hypothesis that NOC/oFQ contributes to the transition from reversible cerebrovascular dysregulation to the cerebral circulatory collapse and histopathology that occurs during combined prolonged hypotension and FPI. Methods: FPI (2 atm) was produced in pigs (4 weeks old) equipped with a closed cranial window and CBF determined by radiolabeled microspheres. Acute (10 min) hypotension (45% decrease in arterial blood pressure) was induced at 60 and 240 min post FPI, prolonged hypotension (225 min) was induced at 15 min post FPI, while the NOC/oFG antagonist [F/G]NOC/oFQ(1-13)NH2 (NA) (1 mg/kg iv) was administered 30 min post FPI. Results: CSF NOC/oFQ was increased significantly more by prolonged hypotension and FPI compared to acute hypotension and FPI or normotensive FPI. CBF and pial artery diameter decreased after normotensive FPI.

Pial artery dilation (PAD) was blunted at 60,120, and 240 min during acute hypotension and at 60 min post FPI for prolonged hypotension. In contrast, at 120 and 240 min post FPI and prolonged hypotension, PAD during hypotension was reversed to vasoconstriction. In the pons, CBF was decreased at 60, but not at 120, 240 min post FPI during acute hypotension, but decreased at 60, 120, and 240 min post FPI during prolonged hypotension. In the cerebrum, CBF was decreased more during prolonged compared to acute hypotension at all timepoints post FPI. Hypercapnia (PaCO2 55 or 75 mm Hg) and topical acetylcholine (10-7, 10-5 M) induced PAD was blunted significantly greater during prolonged hypotension compared to acute hypotension or normotension at 60 min post FPI, but was reversed to vasoconstriction during prolonged hypotension at 120 min post FPI. Post FPI administration of the NOC/oFQ antagonist NA blocked the reversal of PAD to vasocontriction, the CBF dysregulation in the cerebrum and pons, and the reversal of PAD to vasoconstriction with hypercapnia and acetylcholine administration during prolonged hypotension at 120, 240 min post FPI.

H + E staining of the CA3 hippocampus demonstrated normal neurons in sham control animals, considerable degenerating neurons in FPI and prolonged hypotension animals, but no degenerating neurons in injured animals post treated with NA. In the cortex, significant hemorrhage with edema and high density of degenerating neurons were noted in untreated FPI and prolonged hypotension animals, but small hemorrhage and edema with few degenerating neurons in injured animals that received NA. Conclusions: These data indicate that NOC/oFQ contributes to the transition from reversible dysregulation to circulatory collapse and histopathology after combined prolonged hypotension and FPI. These observations suggest a novel pharmacotherapeutic intervention for the treatment of CNS disorders resulting from complex hypovolemic TBI origin. IN VIVO MULTIPHOTON MICROSCOPY OF OLFACTORY GLOMERULI REVEALS ASTROCYTIC CONTROL OF NEUROVASCULAR COUPLING VIA MULTIPLE GLUTAMATERGIC PATHWAYS Gabor C.

Petzold, Dinu F. Albeanu, Tomokazu Sato, Venkatesh N.

Murthy Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA Background and aims: Glutamate is centrally involved in the signaling pathways underlying neurovascular coupling. However, the relevance of presynaptic release, postsynaptic actions and extrasynaptic activity of glutamate have remained controversial. Recent studies have suggested that astrocytic metabotropic glutamate receptors (mGluR) are involved in stimulation-evoked changes of cerebral blood flow (CBF), but the physiological relevance of this and other astrocytic pathways have remained undefined. The aim of this study was to noninvasively investigate these pathways in vivo using physiological stimuli.

Methods: We imaged mice expressing a reporter of neurotransmitter release called synaptopHluorin (spH) in synapses made by the sensory neurons of the olfactory system in vivo using two-photon microscopy. With this probe, presynaptic glutamate release by olfactory sensory synapses in specialized regions called glomeruli can be visualized following physiological odor stimulation (Fig. Simultaneously, we monitored erythrocytic velocity, flow and linear density within glomeruli by injecting Texas Red dextran into the tail vein and obtaining line scans along the axis of glomerular capillaries (Fig. Results: We found that CBF increases in glomeruli activated by physiological stimulation with odorants. The CBF increase showed a wide dynamic range (~5–70%), and was highly correlated with the amount of presynaptic glutamate release (Fig. Surprisingly, odor-evoked CBF increase was not affected by the suppression of postsynaptic activity of the principal output neurons using antagonists of AMPA and NMDA receptors. Glutamate is likely to mediate neurovascular coupling by acting on astrocytes since blockers of astrocytic mGluR-5 reduced the odor-induced CBF increase.

Cyclooxygenase (COX) inhibitors partially prevented the effects of mGluR-5 antagonists, indicating the involvement of the previously described phospholipase-A signaling pathway. Unexpectedly, the CBF increase was also inhibited by blockade of glutamate uptake by the broad-spectrum glutamate transport inhibitor DL-threo-β-Benzyloxyaspartic acid and by the astrocyte-specific glutamate transport inhibitor dihydrokainate. This inhibition remained unchanged after pretreatment with COX inhibitors, indicating that astrocytic mGluR and glutamate uptake affect neurovascular coupling via separate pathways. Conclusions: We have shown that neurovascular coupling in olfactory glomeruli involves presynaptic release of glutamate, but not its postsynaptic actions.

Our data also indicate that glutamate increases CBF by acting on astrocytes via separate pathways: stimulation of mGluR and subsequent COX activation, and uptake by astrocytic glutamate transporters. Background The cerebral microvasculature is a highly interconnected network of vessels, which delivers oxygen and other nutrients to brain tissue. Brain tissue is permeated with these vessels, from which no region of brain tissue is very far. The high level of connectivity provides a degree of ‘redundancy’, whereby no individual micro-vessel is crucial for oxygen supply to any region of brain tissue. Even at a higher level in the vascular tree, the effects of reductions or stoppages in blood flow can be ameliorated by this vessel connectivity, although, without detailed knowledge of the structural and connectivity properties of the microvasculature, the regional response to changes in blood flow cannot be estimated accurately.

However, recent experimental work has led to the characterisation of the cerebral microvasculature in terms of vessel lengths, diameters and connectivity (Cassot et al., Microcirculation, 13: 1-18, 2006). This enables the response of the microvascular flow, and consequent tissue oxygen concentration, to be analysed under changing input conditions. Methods Two and three-dimensional mathematical models of a section of the microvasculature have been constructed, using both regular patterns for vessel distribution and an approach based on random node allocation. The former model has vessels all of the same length, initially with each node connected to its 4 (in 2-D) and 6 (in 3-D) nearest neighbours: the three-connectedness of the microvasculature is obtained by removal of connections based on the flow patterns. These flow patterns are obtained by allocating sources and sinks to individual nodes, to simulate the supplying arterial and draining venous vessels. The latter model generates random nodes and connects the vessels in a way that obtains the correct length distribution and average connectivity. The blood flow through the network is then calculated by solving the pressure-resistance equations in matrix form and the oxygen concentration in the blood vessels is calculated from the mass transport equation.

The tissue oxygen concentration is solved using the convection-diffusion equation for a given metabolic consumption. Results and conclusions The response of the network to decreases in blood flow has been examined to investigate how the connectivity of the network provides redundancy. It is found that the greater the connectivity, the more robust the network is to local decreases in supplying blood flow. This is also heavily dependent upon the initial flow patterns, as the blood flow in the network can alter significantly upon a local decrease if the initial flow patterns are particularly directional. However, even under extreme conditions, the network is able to provide a high level of ‘protection’ by adjusting the flow patterns and this may play a key role in the response to decreases in cerebral blood flow. It is hoped that this will help in our understanding of the response of the cerebral vasculature in diseased states such as stroke and dementia as well as interpreting the low frequency oscillations that play an important role in resting state networks.

ABNORMAL DIAZOXIDE INDUCED VASODILATION IN CEREBRAL ARTERIES FROM ZUCKER OBESE RATS WITH INSULIN RESISTANCE Prasad Katakam, James A. Snipes, Anna Busija, David W. Busija Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston Salem, NC, USA Diazoxide, a mitochondrial KATP channel activator, elicits arterial vasodilation by increasing the generation of mitochondria-derived reactive oxygen species (ROS) that stimulate Ca2+ sparks and Ca2+ activated K+ channels (KCa). We evaluated the diazoxide induced vasodilation in serotonin preconstricted isolated cerebral arteries from 11-13 weeks old Zucker obese (ZO) and lean (ZL) rats by videomicroscopy. The ZO rats were insulin resistant with hyperinsulinemia but were euglycemic and normotensive. Diazoxide induced vasodilation (% maximal relaxation) was diminished in ZO (5.4±5%, p.

PRESSURE AND FLOW REGULATION WITHIN THE BRAIN -FACTS, MYTHS AND MISSING LINKS Mare Czosnyka, Ivan Timofeev, Piotr Smielewski, Andrea Lavinio, Peter Hutchinson, Peter Kirkpatrick, Jonn Pickard Academic Neurosurgery, Addenbrooke's Hospital, Cambridge, UK Objective: Continuous assessment of cerebral autoregulation using time- or frequency- domain correlation, phase shift- and transmission between slow waves of MCA blood flow velocity and cerebral perfusion pressure (CPP) or arterial pressure (AP) have been introduced a decade ago. We intend to review clinical applications of two indices describing cerebral autoregulation and pressure reactivity following head injury. Material and Method: We studied nearly 300 ventilated head injured patients. TCD was recorded digitally along with ICP and AP daily within baseline periods (no interventions) from 10 minutes to 2 hours.

ICP and AP were recorded and analysed continuously during whole period of intensive care. Moving correlation coefficient (3 min window), called Mx index, was calculated between low-pass filtered (0.05 Hz) signals of FV and CPP or AP. Similar index, expressing Pressure Reactivity (PRx), was calculated as moving correlation coefficient between AP and ICP.

Results:Facts: Mx correlated strongly with PRx index. Both indices correlated significantly with static autoregulation. Mx showed good agreement with transient hyperaemic response test, leg-cuff test, phase-shift methods, and CO2 reactivity. Poor autoregulation and pressure-reactivity proved to be independent predictor of fatal outcome following head injury. Both indices were significantly disturbed by intracranial hypertension (ICP>25 mmHg). Autoregulation was worse for low CPP (CPP105 mmHg) than for normal CPP (between 60 and 100 mmHg).

Asymmetry of Mx (left-right) was associated with asymmetry of CT scanautoregulation was worse at the side of contusion and at the side of brain expansion causing midline shift. Both Mx and PRx after head injury worsens with age, probably contributing to worse outcome in elderly patients. Mx showed deterioration of cerebral autoregulation in large (>4 ug/ml) plasma concentration of propofol. PRx correlated with a state of low CBF and CMRO2 revealed using PET. PRx is more robust than Mx (no need of fixation of external probes) and seems to be helpful in optimization of CPP-oriented therapy.

Myths: It is probably not true that Mx in head injury patients can be calculated using only AP instead of CPP. Both indices are able to reflect transient changes of autoregulation, like during plateau waves of ICP. Indices can be quite noisy and decent time for averaging (30 minutes minimum) in individual cases is required to assess whether given patient at given time autoregulates or not. Thresholds for functional and disturbed autoregulation dramatically depend on arterial tension of CO2?therefore comparison between patients cannot be performed without comparing their PaCO2 concentrations. Missing Links: Discrepancies between PRx and Mx are still poorly understood. Whether restoration of failing autoregulation can improve outcome, remains to be demonstrated prospectively. Can ‘optimal CPP’ therapy be understood as a consensus between CPP-protocol and Lund-concept?

Can autoregulation after head injury be improved with statins or EPO, like in SAH? Conclusion: PRx and Mx can be used in many clinical scenarios for continuous monitoring of cerebrovascular regulation, being possibly helpful in predicting outcome and optimizing therapeutical strategies. Background and aims Neurons are susceptible to hypoxia and ischemia. However, endogenous adaptive responses aim at protecting the tissue from hypoxic-ischemic injury.

Angiogenesis and neuroprotection are two such adaptive responses, and both processes seem to be governed by the same hypoxia-induced growth factors, of which vascular endothelial growth factor (VEGF) is a prominent example. VEGF is upregulated during hypoxia and ischemia and improves outcome after stroke. New neurons are generated continuously in the subventricular zone (SVZ) and dentate gyrus of the adult brain. Neuropathological processes, including cerebral ischemia, can enhance neurogenesis, as can growth factors and other physiological stimuli. VEGF has been implicated in the hypoxia-induced regulation of neural stem cells (NSC), but it is unknown whether VEGF can enhance migration of newborn neurons toward sites of ischemic injury, where they might be able to replace neurons that undergo ischemic death.

Results In V1 mice, brains examined 7-28 days after MCAO showed markedly increased SVZ neurogenesis, chains of neuroblasts extending from the SVZ to the peri-infarct cortex, and an increase in the number of newly generated cortical neurons at 14-28 days after ischemia. In concert with these effects, VEGF overexpression reduced infarct volume and improved postischemic motor function. NSC isolated from the SVZ zone of V1 mice expressed more VEGF and differentiated faster into more mature neuronal cells. Furthermore, VEGF overexpression promoted NSC survival and proliferation under hypoxic conditions and protected NSC from apoptosis, induced by growth factor depletion.

Finally, V1 NSC showed an increased phosphorylation of p44/42 MAP kinase and Akt, accompanied by reduced apoptosis under hypoxic conditions which was reversed when the PI3-kinase/Akt pathway was blocked by Wortmannin. VCAM-1 TARGETED INTRACAROTID DELIVERY OF NEURAL STEM CELLS IMPROVES FUNCTIONAL OUTCOME AFTER EXPERIMENTAL STROKE Raphael Guzman, Alejandro De Los Angeles, Samuel Cheshier, Jason Liauw, Stanley Huang, Raymond Choi, Tonya Bliss, Gary Steinberg Department of Neurosurgery, Stanford University, Stanford, CA, USA Background: In acute injury of the brain, transplantation of neural progenitor cells (NPC) is aimed at replacing damaged neural cells. Intravascular delivery of NPC after stroke has been limited by the low efficiency of transendothelial migration. VCAM-1 is one of the endothelial adhesion molecules known to be upregulated early after stroke and is responsible for the firm adhesion of CD49d (VLA-4) expressing inflammatory cells. We hypothesized that selecting CD49d positive NPC would improve their homing to the injured brain and improve functional outcome after stroke. Methods: Mouse NPC's were analyzed for the expression of the surface integrin CD49d by FACS analysis, immunohistochemistry and static adhesion assay. On the day of intraarterial injection, a CD49d enriched (>90%) and depleted (.

SUPPRESSED BRAIN ANGIOGENESIS DURING MODERATE HYPOXIA IN CYCLOOXYGENASE-2 DEFICIENT MICE Joseph C. LaManna, Xiaoyan Sun, Mary P. Modesitt, Constantinos P. Tsipis Department of Anatomy, Case Western Reserve University, Cleveland, OH, USA Background and Aims: Exposure to chronic moderate hypoxia induces angiogenesis in tissues through cooperation of the HIF-1 transcription factor-vascular endothelial growth factor and the Cyclooxygenase-2(COX-2)-angiopoietin 2 signaling pathways(,, ).

This study demonstrated the significance of COX-2 in hypoxia-induced brain angiogenesis. Methods: male, 3-month old COX-2 knockout (KO, -/−), wild type (WT) and heterozygote (HET) mice were exposed to hypobaric hypoxia (0.4 ATM, ~8% oxygen) for 3 weeks. Their littermates were kept normoxic in the same location.

Mice were fixed for immunohistochemical staining of GLUT-1, an indicator of brain capillaries. Capillary density (N/mm2) was estimated through the full depth of the frontal cortex from 5 sections (4 µm). Results: Under normoxic conditions there was no difference in capillary density of KO mice compared to their WT and HET littermates. As expected, hypoxic exposure significantly (*) increased the capillary density in the WT and HET mice, compared to that of their normoxic littermates (491 ± 45, n = 4 vs.

416 ± 39, n = 3, mean ± SD, and 496 ± 41, n = 5 vs. 393 ± 16, n = 3, respectively). However, the capillary density in COX-2 -/− mice was unchanged (424 ± 24 vs. 401 ± 13, n = 3 each). Conclusions: Our data suggest that the COX-2 signaling pathway is required for cerebrovascular adaptation to prolonged moderate hypoxia.

However, COX-2 is apparently not necessary for establishing capillary density under normoxic conditions. ISOLATION AND CHARACTERIZATION OF INJURY-INDUCED NEURAL STEM/PROGENITOR CELLS FROM POST-INFARCT AREA IN MICE Takayuki Nakagomi 1, Akihiko Taguchi 2, Orie Saino 1, Masatoshi Fujikawa 1, Yoshihiro Fujimori 1, Tomoyuki Nishizaki 3, Tomohiro Matsuyama 1 1Institute for Advanced Medical Science, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan, Hyogo, Japan, 2Department of Cerebrovascular Disease, National Cardiovascular Center, Osaka, Japan, Osaka, Japan, 3Department of Physiology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan. Methods Permanent focal cerebral ischemia was produced in male 6-week-old SCID mice (Clea Japan Inc.) by ligation and disconnection of the distal portion of the left MCA (Taguchi, et al., 2004). The infarct area of this model was limited to the ipsilateral cerebral cortex. For the formation of neurosphere-like cell clusters, cell suspensions were made from ipsilateral post-infarct area or contralateral cerebral cortex on day 7 after stroke. These cell suspensions were incubated in medium promoting formation of neurosphere-like cell clusters. Briefly, cells were incubated in 6 cm2 tissue culture flasks with DMEM/F12 containing EGF, bFGF and N2 supplement.

Roger Troutman Patch Micro Korg Vocoder. The total number of cell clusters exceeding 80 µm in diameter was counted every other day for 1 month using a phase contrast microscope. To examine whether these clusters give rise to generation of secondary spheres, neurosphere-like cell clusters were dissociated by passage, and incubated again. Neurosphere-like cell clusters after 6 days in culture were characterized by immunohistochemistry.

Further, differentiation was induced in the medium by omitting EGF and bFGF for 7 days. The samples were subjected to the analysis by RT-PCR and immunohistochemistry. Neurosphere-like cell clusters after 14 days in culture were characterized by using FACS. Patch-cramp test also was performed for the functional analysis on differentiated neural cells. Results After 6 days in culture, formation of neurosphere-like cell clusters in cultures derived from the stroke-affected area was observed.

These clusters continued to grow up to day 16 in culture. The total number of cell clusters also was increased (about 50 per dish). These cell clusters revealed the capability of self-renewal and again formed neurosphere-like cell clusters. In contrast, no cell clusters were seen in cell suspensions from the contralateral cerebral cortex. On immunohistochemistry, multiple proliferating cells (Ki67 antigen-positive) were observed in neurosphere-like cell clusters.

Cell clusters also expressed the neural stem cell marker (nestin) and notch1. Virtually all of the nestin-positive cells expressed notch1.

FACS analysis revealed that ~40% of the cells from the clusters were notch1-positive. Differentiated cells expressed nestin, a marker of neurons (Tuj1, MAP2), as well as astrocytes (GFAP) and oligodendrocyte markers (O4). Expression of transcripts for nestin, MAP-2, GFAP and PLP (a marker for oligodendrocytes) was confirmed by RT-PCR. Patch-cramp analysis showed that some of the isolated cells developed the properties of voltage-gated Na+ channels, indicating the differentiation of neural stem cells to functional neurons. INHIBITING DEGRADATION OF HYPOXIA-INDUCIBLE FACTOR INCREASES NEURAL STEM CELLS IN MOUSE FOCAL ISCHEMIA Tsuyoshi Uesugi 1, Shunya Takizawa 1, Yuko Izuhara 2, Yuko Morita 1, Shigeharu Takagi 1, Toshio Miyata 2 1Division of Neurology, Department of Internal Medicine, Tokai University School of Medicine, Kanagawa, Japan, 2Institute of Medical Science, Tokai University School of Medicine, Kanagawa, Japan Background and purpose: Hypoxia-inducible factor (HIF) is a regulatory factor of transcription that is induced in hypoxic cells. It activates target genes involved in compensation for ischemia, including erythropoietin, VEGF, and glut-1.

We examined the cerebroprotective effects of TM6008, a novel inhibitor of the HIF-degrading enzyme prolylhydroxylase (PHD), on infarct volume and intrinsic neural stem cells in mice with permanent focal ischemia. Methods: The experiments were performed on C57BL/6J mice weighing 20 grams. Briefly, animals were anesthetized using 2.0% isoflurane anesthesia with a mixture of 30% oxygen and 70% nitrous oxide, and the left middle cerebral artery was permanently occluded by cauterizing. Mice were divided into two groups: vehicle (0.2 ml/day of milk alone) and TM6008-treated (200 mg/kg/day of TM6008 + 0.2 ml/day of milk) groups. Mice in both groups were given 50 mg/kg/day of BrdU for 2 days before sacrifice on day 7 or day 14 after occlusion. In all animals, the rotor rod test and blood sampling were performed. Coronal brain sections were fixed with 4% paraformaldehyde, and stained with hematoxylin and eosin.

Infarct volume was measured, and immunohistochemical reactions for HIF-1 and BrdU were performed. Results: No significant difference of rotor rod test results was seen between the TM6008-treated and vehicle groups at any time point. At day 7 after occlusion, there was no statistically significant difference of infarct volume, HIF-1 or BrdU-positive cells between the two groups.

At day 14, however, TM6008 significantly reduced brain atrophy and the number of BrdU-positive cells, compared with the vehicle group. Conclusions: Inhibition of PHD by TM6008 increases intrinsic neural stem cells in the infarcted hemisphere of mice with permanent focal ischemia, accompanied with a reduction of infarct volume. Increasing HIF-1 by blocking its degradation is a novel strategy to treat cerebral infarction, at least in the subacute phase, by promoting neurogenesis. CIRCULATING ENDOTHELIAL PROGENITOR CELLS AS THE PATHOGENETIC MARKER OF MOYAMOYA DISEASE Kon Chu, Keun-Hwa Jung, Soon-Tae Lee, Eun-Cheol Song, Dong-In Sinn, Jeong-Min Kim, Hee-Kwon Park, Manho Kim, Jae-Kyu Roh Stroke and Neural Stem Cell Laboratory, Department of Neurology, Stem Cell Research Center, Seoul National University Hospital, Seoul, South Korea Backgrounds: Endothelial progenitor cells (EPCs) derived from bone marrow are believed to support the integrity of the vascular endothelium. It has reported that EPC mobilization increases following cerebral ischemia, whereas some data have suggested that the number and function of EPCs correlate inversely with cardiovascular risk factors, outcome and the presence of stroke. Moyamoya disease is an unusual from of chronic cerebrovascular occlusive disease followed by the formation of characteristically abnormal vessel.

Objectives: In this study, we attempted to examine the EPC number and function in moyamoya disease. Methods: The stage of steno-occlusive lesions on angiogram was determined according to the modified staging system. MMD patients (stage II-IV) undergoing chronic stroke was included. EPCs were isolated from the peripheral venous blood of 20 patients with moyamoya disease and 20 age-matched healthy volunteers. EPC was counted as colony-forming units (CFU) at the 7-day culture, and the yield of outgrowth endothelial cells were measured during the 3 months of culture.

EPC function was measured by Matrigel plate assay. Results: The number of CFU after 7-day culture was significantly reduced in patients with moyamoya disease versus control subjects (12.34±8.66 versus 70.6±15.35, p. HYPOXIA-INDUCED DAMAGE AND REPAIR IN ADULT MOUSE BRAIN Marc Uy, Aaron Hirko, Renee Dallasen, Nicole Brandon, Yan Xu Department of Anesthesiology, University of Pittsburgh, Pittsburgh, PA, USA Background and aims: Hypoxia is associated with a number of clinical conditions such as asthma, altitude sickness, carbon monoxide poisoning, and stroke. It has been postulated that mild hypoxia alone does not cause neuronal death, and hypoxia preconditioning in rodents has been shown to significantly reduce injury in focal and global ischemia. A possible protective mechanism through preconditioning is the pre-activation of neuronal stem cells in the adult brain tissue.

We report here an investigation of the long-term physiological responses to a 30-min episode of respiratory hypoxia with 5% O2 in mice, with the ultimate goal of understanding the mechanisms of brain injury and recovery after hypoxia. Download Songs Of Raw Star Song By Darshan more. Method: Male CD1 mice, weighing 20-30 g, were anesthetized with pentobarbital. Animals were transorally intubated and mechanically ventilated.

Hypoxia was induced by adjusting pO2 to 38 mmHg while the animals were paralyzed with pancuronium to prevent spontaneous gasping. Control animals were administered a normal pO2 =138 mmHg for 30 minutes. Body temperature was held constant at 36.8 ± 0.4°C. After the hypoxia, ventilation with room air (pO2 =138 mmHg) was continued until spontaneous breathing was restored. The heart rate, femoral artery pulse distention, blood O2 saturation (spO2), breath distention, and rectal temperature were recorded continuously before, during, and after the hypoxia. Diffusion-weighted MRI, apparent diffusion coefficient (ADC) maps, and angiography were acquired at 14.1 T.

Final histology included Nissl staining and NeuN, GFAP, and vimentin immunohistostaining. The percent of damaged neurons and the density of GFAP+ and vimentin+ cells were analyzed. Results and conclusions: Normal spO2 with room air ventilation was 96 ± 3%. During the 30-min hypoxia, spO2 steadily declined from 82 ± 6% (averaged the first 5 min) to 65 ± 7% (averaged for the last 5 min). While most animals had normal ADC in the hippocampal CA1 region, some did show a significant decrease in ADC values the second day after the hypoxia, followed by an overshoot in the subsequent days. This is a typical response to ischemia explained by the initial cytosolic edema followed by the interstitial edema in response to injuries. The animals with MRI abnormalities also showed visible neuronal damage in Nissl stained sections.

There is a rather strong correlation between the percent of damaged neurons in the CA1/CA3 regions and the density of vimentin+ cells in the same and adjacent regions (r2 =0.90). This result might indicate an influx of neural and astrocytic precursors in response to the cellular damage.

At the short and intermediate time points examined after hypoxia, the vimentin+ cells have astrocytic morphology and are NeuN —. At a later stage, these cells are NeuN+ and resemble neurons in structure and orientation. Thus, our results suggest that similar to developing brain, the radial glia in the adult brain from the subventricular zone and dentate gyrus also migrate and transdifferentiate into neurons in response to injury after prolonged hypoxia and reoxygenation.

(Funded by NIH R01NS/HL036124). Results We generated multipotential neural progenitors from ES cells by SDIA method. These cells could differentiate in vitro into neurons, glia, and oligodendrocytes, thus indicating them to be neural stem cells. The cells transplanted into the ischemic brain became located widely around the ischemic area. Moreover, the transplanted cells differentiated into various types of neurons and glias. In the case of primate ES cells, the cells transplanted into the ischemic brain became located widely around the ischemic area.

Moreover, the transplanted cells differentiated into various types of neurons and glias. These cells expressed HuC, HuD, GFAP and TuJ1. At 28days after transplantation, over 10 times more cells in the graft were labeled with Fluorogold (FG) by stereotactic focal injection of FG into anterior thalamus on the grafted side when compared with the number at 14 days. In the case of mouse ES cells, at 2 weeks after ischemia, the transplanted cells occupied 18.8+2.5% of the hemispheric area. By 4 weeks after it, this percentage rose to 26.5+4.0%. At 4 weeks after tranplantation, green fluorescent protein (GFP)-positive transplanted cells showed a mature neuronal morphology.

In addition, we investigated the expression of differentiation markers and that of various neurotransmitters. Transplanted cells were immunopositive for neuronal nuclei (NeuN), tubulin-III (TuJ1), and glial fibrillary acidic protein (GFAP). Of the GFP-positive cells, 33.3+11.5% were glutamate decarboxylase (GAD)-positive, 13.3+5.8% were glutamate-positive, 2.1+2.5% were tyrosine hydroxylase (TH)-positive, 1.8+2.0% were serotonin-positive, and 0.4+0.2% were choline acetyltransferase (ChaT)-positive ones. Background and aims Mild cognitive impairment (MCI) is a transitional state between normal ageing and dementia, especially early Alzheimer's disease (AD). Patients with MCI have an increased risk of developing AD and early detection of these patients is of great importance in the effort to treat patients at an early stage of the disease.

Beta amyloid (Aβ) depositions are one of the major histopathological hallmarks of AD and the presence of Aβ at an early stage of the disease has been reported. Recently the Pittsburgh Compound-B (PIB) labeled with 11C has been demonstrated to image brain Aβ using Positron Emission Tomography (PET) and to discriminate between AD patients and healthy controls. In this study we aim to investigate amyloid depositions in patients with MCI and relate that to parameters as cerebral glucose metabolism (CMRglc), CSF measurements, as well as neuropsychological investigations. The results from the MCI patients were compared with previous studies of AD patients and healthy controls. Methods 21 MCI patients were recruited from the Geriatric clinic, Karolinska University Hospital, Stockholm, Sweden, and studied with PIB-PET and FDG-PET at Uppsala Imanet AB, Uppsala, Sweden.

The patients also underwent thorough clinical investigations including neuropsychological investigations and CSF sampling of Aβ, total Tau and phosphorylated Tau. For the FDG examinations, parametric maps of CMRglc were generated and normalized to whole brain. For the PIB examination, mean uptake values of the regions-of-interest obtained in a late time interval were normalized to the corresponding uptake in a reference region. Results The total group of MCI patients had significantly higher PIB retention in cortical brain areas compared to healthy controls. Seven MCI patients that later converted to AD showed significant higher PIB retention compared to healthy controls and non-converting MCI patients respectively. The PIB retention in the MCI converters was comparable to AD patients. Our results showed significant correlation between CSF Aβ, total Tau, phosphorylated Tau, episodic memory and PIB retention, while no significant correlations were observed between global cognition or CMRglc and PIB retention.

Conclusions Seven MCI patients that later converted to AD showed significant higher PIB retention compared to healthy controls and non-converting MCI patients respectively. These findings show that it is possible to find amyloid depositions in MCI patients and that PIB probably could serve as a diagnostic marker able to discriminate patients with an increased risk of developing AD. The negative correlation between CMRglc and PIB retention that has previously been observed in patients with mild AD was not present in the MCI group.

This suggests that the parallel decline in CMRglc and cognitive performance might occur after the deposition of amyloid. NORMAL DATABASE OF DOPAMINERGIC NEUROTRANSMISSION SYSTEM IN LIVING HUMAN BRAIN MEASURED BY PET Hiroshi Ito, Hidehiko Takahashi, Ryosuke Arakawa, Harumasa Takano, Tetsuya Suhara Clinical Neuroimaging Team, Molecular Neuroimaging Group, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan Introduction: The central dopaminergic system is of interest in the pathophysiology of schizophrenia and other neurodegenerative diseases. Both pre and postsynaptic dopaminergic functions can be estimated by PET with several radiotracers. However, the integrated database of both pre and postsynaptic dopaminergic functions including receptors, transporter, and endogenous neurotransmitter synthesis has not been reported. In the present study, we have built the normal database of pre and postsynaptic dopaminergic functions in the living human brain using PET. Methods: To measure the dopamine D1 receptor binding, striatal and extrastriatal dopamine D2 receptor binding, dopamine transporter binding, and endogenous dopamine synthesis, PET scans were performed on healthy men after intravenous injection of [C-11]SCH23390, [C-11]raclopride, [C-11]FLB457, [C-11]PE2I, or L-[C-11]DOPA, respectively (10 healthy men for each tracer).

The binding potential (BP) was calculated by the reference tissue model method on a voxel by voxel basis with use of the cerebellum as a reference region. For L-[C-11]DOPA study, the dopamine synthesis index (I) was calculated on a voxel by voxel basis with use of the occipital cortex as a reference region from the ratio of area under the time-activity curve of brain regions to a reference region (I = ratio? All PET images were anatomically standardized using SPM2, and database was built for each radiotracer. Gray matter images were segmented and extracted from all anatomically standardized MR images using the SPM2 and used for partial volume correction. Results: These databases allow to compare regional distributions of the striatal and extrastriatal dopamine D1 and D2 receptor binding, dopamine transporter binding, and endogenous dopamine synthesis.

The BP and I values of each region-of-interest are given in table 1. In the striatum, greatest bindings to dopamine D1, D2 receptors and transporters and greatest dopamine synthesis were observed. For the limbic system, relatively high bindings of dopamine D1 and D2 receptors and relatively high dopamine synthesis were observed in the amygdala and anterior cingulate.

Among the neocortical regions, highest binding to dopamine D2 receptors was observed in the temporal cortex, while D1 receptor binding was uniform. The binding to dopamine transporter was very low in the neocortical regions. These distributions were in good agreement with those in the human post mortem studies. The partial volume correction did not change regional distribution of pre and postsynaptic dopaminergic functions in cerebral cortices. Conclusions: Although a limited spatial resolution of PET scanner might hamper observation of pre and postsynaptic dopaminergic functions in detail, such database must be useful to understand physiology of neurotransmission in the living human brain. These database can also be used to investigate regional abnormality of dopaminergic neurotransmission in neuropsychiatric diseases.

Methods 10 healthy adult volunteers were studied. We measured cerebral blood flow velocity (Vmca) with transcranial Doppler ultrasonography and changes in concentrations of total haemoglobin (delta[HbT]=delta[HbO2]+delta[HHb]), haemoglobin difference (delta[Hbdiff]=delta[HbO2]-delta[HHb]) and delta[oxCCO] with BBS using optodes fixed to the right side of the forehead. Inspired oxygen concentration (FiO2) was increased using high flow oxygen via a tight fitted face mask and end tidal carbon dioxide (EtCO2) was increased by adding carbon dioxide to the inspired gases.

Measured variables were compared with baseline using non-parametric ANOVA. Discussion We demonstrate a significant increase in delta[oxCCO] during hyperoxia and hypercapnea in the healthy adult brain. This suggests that oxidation of the mitochondrial electron transfer chain occurs during two physiological paradigms that increase oxygen delivery, as shown by the rise in delta[Hbdiff], via different mechanisms. Hyperoxia increases arterial oxygen content and is associated with a decrease in cerebral blood flow whereas hypercapnea increases cerebral blood flow without affecting arterial oxygen content. In-vivo studies in animals confirm that cerebral CCO can be further oxidised by increases in oxygen delivery (Springett et al Adv. 2003; Quaresima et al Biochim Biophys Acta 1998).

Our data support the conclusion that, at normoxic normocapnea, cerebral CCO is not fully oxidised in the human adult and that further oxidation is possible by increasing oxygen delivery. These findings have implications for the monitoring and treatment of brain injury.

ACUTE FUNCTIONAL RECOVERY OF CEREBRAL BLOOD FLOW FOLLOWING FOREBRAIN ISCHEMIA IN THE RAT Chao Zhou 1, Tomakazu Shimazu 2, Turgut Durduran 1,3, Daniel Y. Kimberg 2, Guoqiang Yu 1, Xiao-Han Chen 4, John A.

Detre 3, Arjun G. Yodh 1, Joel H. Greenberg 2 1Department of Physics and Astronomy, University of Pennsylvania, Philadelphia, PA, USA, 2Department of Neurology, University of Pennsylvania, Philadelphia, PA, USA, 3Department of Radiology, University of Pennsylvania, Philadelphia, Philadelphia, PA, USA, 4Department of Neurosurgery, University of Pennsylvania, Philadelphia, Philadelphia, PA, USA Background: Following complete cerebral ischemia the post-ischemic blood flow response to functional activation is severely attenuated for several hours. However, little is know about the spatial and temporal extent of the blood flow response in the acute post-ischemic period following incomplete cerebral ischemia.

Methods: We used laser speckle flowmetry, which provides two-dimensional high spatial and temporal resolution blood flow images, to examine the response of relative cerebral blood flow (rCBF) to controlled vibrissae stimulation during the first few hours following temporary incomplete ischemia. Male Sprague-Dawley rats (n=10) were prepared under halothane anesthesia and then switched to α-chloralose.

The bone overlying the right somatosensory cortex was thinned for the laser speckle imaging. Prior to ischemia the left vibrissae (B1-3, C1-3, D1-3) were stimulated for 3 sec at 5 Hz every minute over a 10-minute period while the rCBF data was collected every 200 msec. Fifteen minutes of transient forebrain ischemia was induced by bilateral carotid arterial occlusion along with controlled hypotension (mean arterial blood pressure lowered to 45-50 mmHg by slowly withdrawing blood from a femoral arterial catheter).

This reduced cerebral blood flow to 6.6±1.8% (mean±SE) of the pre-ischemic level. The shed blood was re-infused, the carotid ties were released, and the rCBF response to vibrissae stimulation was repeated at 20, 30, 60, 120, and 180 mins after the end of the ischemia. RCBF images were analyzed using a modified general linear model (GLM) with VoxBo (). Statistical maps from the analysis were used to determine the spatial extents of the activation at different time points with a fixed Bonferroni-corrected threshold (pth=0.01). Results: The activation area was significantly reduced 20 mins into reperfusion compared to prior to ischemia (49±12%, p.

IMAGING BETA AMYLOID DEPOSITION IN VIVO: QUANTITATIVE COMPARISON OF [18F]FDDNP AND [11C]PIB Nelleke Tolboom 1,2, Maqsood Yaqub 1, Wiesje van der Flier 2, Ronald Boellaard 1, Gert Luurtsema 1, Albert D. Windhorst 1, Frederik Barkhof 3, Philip Scheltens 2, Adriaan A. Lammertsma 1, Bart N.M. Van Berckel 1 1Department of Nuclear Medicine and PET Research, VU University Medical Centre, Amsterdam, The Netherlands, 2Department of Neurology and Alzheimer Centre, VU University Medical Centre, Amsterdam, The Netherlands, 3Department of Radiology, VU University Medical Centre, Amsterdam, The Netherlands. Methods In this ongoing study, dynamic 90 minutes 3D [11C]PIB and [18F]FDDNP scans were acquired on the same day using an HR+ (Siemens) PET scanner. During both scans, continuous on-line and discrete manual sampling was performed to derive a metabolite corrected arterial plasma input curve.

For each subject, volumes of interest (VOI) were defined on an individually acquired, co-registered T1 weighted structural MRI scan using an automated procedure. Preliminary analysis of time-activity curves was performed using the simplified reference tissue model with cerebellum grey matter as reference tissue. Binding potential (BP) was used as outcome measure, in particular for areas previously associated with AD: frontal, parietal and temporal cortex. Results At present, three AD patients, three patients with Mild Cognitive Impairment (MCI) and three age matched normal controls were included. Results are summarised in figures 1 and 2. [11C]PIB showed good contrast between AD patients and normal controls, as described previously.

In addition, the range of BP values in MCI patients was broader, probably due to the known heterogeneity of this group. [18F]FDDNP provided less contrast between AD patients and normal controls. In AD patients, [11C]PIB BP was in general tenfold higher than [18F]FDDNP BP.

EFFECTS OF EARLY LIFE STRESS ON 5-HT1A RECEPTOR AVAILABILITY IN TWO YEARS OLD RHESUS MONKEYS MEASURED BY [18F]FPWAY AND PET Simona Spinelli 1, Richard E. Carson 2, Cristina S. Barr 1, Stephen J. Suomi 3, Markus Heilig 1, J.

Dee Higley 1, Lixin Lang 4, Elliot Stein 5, Svetlana I. VALIDITY AND VALUE OF QUANTITATIVE SPECT RECONSTRUCTION IN A MULTI-CENTER CLINICAL STUDY USING I-123 RADIOPHARMACEUTICALS Iida Hidehiro, Watabe Hiroshi, Nakazawa Mayumi, Hayashida Kohei Quantitative SPECT Working Group, Japan Introduction: SPECT has a potential to provide parametric functional images in a “quantitative” manner, as has been widely done with PET in vivo, using several tracers typically labeled with I-123. Due to availability of equipments, as well as well-established delivery/transportation of radio-ligands, SPECT has an advantage in applying it to large-scale clinical studies. It has however been considered that accuracy and inter-institutional reproducibility of SPECT are limited for quantitative assessment of functional parametric images, largely attributed to a lack of general consensus of reconstruction procedures (attenuation/scatter correction) as well as, in case of I-123, significant amount of penetration through collimator from high-energy photons. We have recently developed a novel method to reconstruct SPECT images from existing projection data including appropriate corrections for scatter and attenuation in the object, head contour detection, as well as for collimator-dependent penetration. This study was aimed at evaluating validity and impact of our approach for Multicenter clinical study.

We particularly investigated consistency and reproducibility of quantitative values for I-123, at different SPECT systems from different manufacturers. Materials and methods: The Windows program can handle original projection data to analyze and/or DICOM format obtained from 5 manufactures with essentially 7 different operation systems.

Twenty-five institutions provided a series of phantom data for 28 collimators according to a given protocol. Nine are symmetric fanbeam, and rest parallel beam collimators. Experiments were done for a radioactivitycalibrated point source of I-123 in air to determine the absolute sensitivity of the system and to determine the penetration factor for each collimator. Additional experiments were done using a uniform cylindrical phantom filled with the radioactivity mentioned above, and single/multiple rod sources placed in cylindrical water.

The scatter/penetration correction was based on the previously proposed transmission dependent convolution subtraction method, and the attenuation correction was implemented during the ordered-subset EM reconstruction. Dynamic SPECT studies were carried out in 9 institutions on 64 patients using I-123 iodo-amphetamine (IMP) to assess quantitative CBF images at rest and during Diamox challenge.

Inter- and intra-institutional reproducibility was then evaluated. Results and discussion: Scatter fraction was consistent among different collimators from different manufacturers as predicted, but the penetration varied largely dependent on the collimator design and manufacturers. Our approach successfully compensated for the penetration and the scatter as well as the attenuation, as demonstrated in reconstructed images of the water-filled single/multiple rod source phantoms, except for one fanbeam collimator. Parallel beam collimator tends to have small penetration than fanbeam. Absolute concentration of the uniform cylindrical phantom was also consistent among institutions, although it was significantly smaller than the true value (0.88+/−0.05). Clinical data also demonstrated that CBF values obtained with I-123 IMP at rest and during Diamox were reproducible among institutions. Normal CBF values obtained in 2 institutions showed also no significant difference.

Conclusion: These data suggested that QSPECT could be a useful tool for quantitative mapping using I-123 compounds, allowing a large scale clinical evaluation using SPECT. MICROGLIA PROVIDE NEUROPROTECTION AND ENGAGED IN CLOSE CELL-CELL CONTACT WITH NEURONS AFTER ISCHEMIA Jens Neumann 1, Matthias Gunzer 2, Oliver Ullrich 3, Klaus Dinkel 4, Klaus G. Reyman 1,4 1Project Group Neuropharmacology, Leibniz Institute for Neurobiology, Magdeburg, Germany, 2German Research Centre for Biotechnology, Immunodynamics, Braunschweig, Germany, 3Institute of Immunology, University Hospital Magdeburg, Magdeburg, Germany, 4Institute for Applied Neuroscience (FAN GGmbH), Magdeburg, Germany Many neurological insults are accompanied by a marked acute inflammatory reaction, involving the activation of microglia. Using a model of exogenous application of fluorescence-labeled BV2 microglia in pathophysiologically relevant concentrations onto organotypic hippocampal slice injury. Neuronal cell death after oxygen-glucose deprivation (OGD) was determined by propidium iodide incorporation and Nissl staining. Migration and interaction with neurons were analyzed by time resolved 3-D two-photon microscopy.

We show that microglia protect against OGD-induced neuronal damage and engage in close physical cell-cell contact with neurons in the damaged brain area. Neuroprotection and migration of microglia were not seen with integrin regulator CD11a-deficient microglia or HL-60 granulocytes. The induction of migration and neuron-microglia interaction deep inside the slice was markedly increased under OGD conditions. Lipopolysaccharide-prestimulated microglia failed to provide neuroprotection after OGD. Pharmacological interference with microglia function resulted in a reduced neuroprotection.

Microglia proved to be neuroprotective even when applied up to 4 h after OGD, thus defining a??protective time window.?? In acute injury such as trauma or stroke, appropriately activated microglia may primarily have a neuroprotective role. Anti-inflammatory treatment within the protective time window of microglia would therefore be counterintuitive. Methods & Results Recombinant adenovirus expressing Stat3 wild type (St3.Wt), Stat3 dominant negative type (St3.F) and LacZ were constructed as described previously. In vitro: Rat cultured neurons were exposed to adenovirus vector expressing St3.Wt(AdSt3.Wt), St3.F(AdSt3.F) or LacZ(AdLacZ) for 2h and incubated with fresh medium up to 72h. Then, neurons were co-cultured with rat cultured microglia in two different ways. • direct co-culture: microglia were added directly to 24 well plates containing neurons.

• insert co-culture: microglia were seeded into cell culture inserts that were placed on the top of the 24 well plates containing neurons. At 48 hr after co-culture, cells were homogenized in lysis buffer and immunoblotted with antibody against Stat3, p-Stat3(Tyr-705) and MAP2.

In vivo: A fine glass micropipette was introduced into the left caudoputamen in the rats. Twenty microliters of adenovirus vectors, AdSt3.Wt, AdSt3.F or AdLacZ were administered over a 20 min period. Two days later, the left middle cerebral artery (MCA) of the infected animal was occluded. One day after MCA occlusion, infarct volume was evaluated using TTC staining.

Results As shown in Figs A and B, overexpression of St3.Wt induced p-Stat3 and cell death in neurons after direct co-culture with microglia but not after insert co-culture with microglia. These results suggested that direct contact of microglia with neurons expressing St3.Wt induced stat3 activation and cell death in neurons. Moreover, in vivo experiment revealed that the infarct volume of the AdSt3.Wt-treated group was significant larger than that of AdLacZ-treated group while the infarct volume of the AdSt3.F-treated group was significant smaller than that of AdLacZ-treated group suggesting deteriorative effects of Stat3 on the ischemic brain. CORTICAL SPREADING DEPRESSION AND PERI-INFARCT DEPOLARISATION IN HUMAN ISCHEMIC STROKE Christian Dohmen 1,2, Oliver Sakowitz 3, Martin Fabricius 4, Bert Bosche 5, Thomas Reithmeier 5, Ralf-Ingo Ernestus 5, Gerrit Brinker 5, Jens Dreier 6, Johannes Woitzik 7, Antony J.

Materials and Methods We measured CBF and CMRO2 in 6 normal subjects (2 males and 4 females, mean age=33.376.6 years old) by PET and the 15O steady-state inhalation, while we estimated CMRGlc in the other 6 subjects (6 males, 22.871.9) by PET and 2-deoxy-2-[F-18]fluoro-D-glucose according to the method developed by Phelps et al with standard rate constants and lumped constant of 0.42. We analyzed these data statistically by applying SPM99. We also analyzed absolute values of CBF, CMRO2, CMRGlc, and their relationships. Discussion In supply-demand correlation, hippocampus is characterized by having excessive supply of glucose and oxygen, pons having excessive supply of oxygen, cerebellum having excessive supply of glucose. In glucose-oxygen correlation, cerebral cortices showed excessive glucose consumption for oxygen utilization. Shurr hypothesized that the end product of glycolysis would be lactate.

Our results may indicate that there is a lactate reserve in cerebral cortices excessively produced as a substrate for TCA cycle. TOWARD A FUNCTIONAL METABOLOMICS OF BRAIN ENERGY METABOLISM AND NEUROTRANSMITTER TRAFFICKING Paul K. Maciejewski Magnetic Resonance Research Center, Yale University, New Haven, CT, USA Although it is widely accepted that glucose is the primary substrate that supports brain function, fundamental questions about pathways and compartmentalization of metabolic processes that accomplish glucose catabolism in brain tissue remain unanswered.

Furthermore, a basic understanding of the role of glucose in brain function must recognize that glucose not only serves as a basic energy substrate for brain but also as a substrate for the de novo synthesis of chemical neurotransmitters such as glutamate and GABA. Metabolomics, an emerging field of biological investigation, is interested in not only the identification and characterization of small-molecule metabolites that are present in biological systems, but also in the identification and characterization of their role within the context of those systems (i.e., what might be called a “functional metabolomics:). Metabolites are material elements that are consumed and produced in metabolic processes. Fluxes of metabolites into and out of metabolic processes provide an indication of the function of metabolites within the context of living systems. Consequently, a functional metabolomics of brain energy metabolism and neurotransmitter trafficking ought to consist of the identification and characterization of metabolic processes that participate in these basic metabolic functions in brain quantified in terms of fluxes of metabolites through those processes. In principle, brain energy metabolism and other brain metabolic functions (e.g., neurotransmitter synthesis and neurotransmitter transport processes) can be investigated in vivo using isotopic label-tracer methods implemented using Mass Spectroscopy (MS) or Magnetic Resonance Spectroscopy (MRS). However, unlike other metabolomics technologies that identify metabolites and characterize systems in terms of profiles of metabolites, label-tracer methods depend on detailed a priori knowledge about metabolic pathways, knowledge which is used to explain the transfer of label from one metabolite to another.

This a prior knowledge about metabolic pathways is provided by metabolic models, that is, by detailed, mathematical descriptions of processes that are presumed to explain the transfer of label from label-enriched precursor substrates to other metabolites. However, existing metabolic models tend to focus on single pathways and do not accommodate the complexity of metabolism and the likelihood that specific metabolic functions may be accomplished via a multiplicity of functionally redundant pathways. This narrow focus limits the design and interpretation of existing in vivo label-tracer studies of metabolism. Here I will introduce a new “functional metabolomics” approach to the investigation of biochemical pathways for brain energy metabolism and neurotransmitter trafficking in vivo that consists of: 1) a rational method for the specification of alternative, hypothetical biochemical pathways that in principle accomplish these basic metabolic functions in brain, and 2) a method for the design of isotopic label-tracer experiments to determine the extent to which these alternative, hypothetical pathways actually accomplish these specific metabolic functions in vivo. The approach integrates information derived from cellular and molecular biology, metabolomics and proteomics with concepts and techniques derived from engineering, mathematics, and computational sciences.

(Supported by NIH NS-044316). Background Traumatic brain injury (TBI) metabolism research has indicated that despite increased levels of glucose, conventional cerebral glucose metabolism rarely rises to the expected supra-normal levels to compensate for the injury-induced decrease in oxidative metabolism. In fact, it has been well established that increased plasma glucose (PlGluc) is detrimental following TBI. These observations suggest the activation of alternative glucose metabolic pathways. Investigations around a novel metabolite, propylene glycol (PG), a compound that we recently identitifed in cerebrospinal fluid (CSF) of TBI patients, suggested that increased PlGluc results in an increased rate of glycation.

Glycation is a deleterious metabolic process resulting in the production of toxic PG, advanced glycation end products (AGEs), and methylglyoxal. Both, methylglyoxal and AGEs, have been shown to affect physiological aging, and neurodegenerative diseases, such as Alzheimer's. We hypothesize that the methylglyoxal pathway is likely to be activated following brain injury.

As a result, glucose might be involved in unique pathophysiological mechanisms following TBI. Results Statistical analysis of PlGluc levels and PG, an end-product of glycation, revealed a significant correlation (r=0.432; p=0.001). PlGluc higher than 160mg/dl resulted in significantly higher overall PG concentrations (PGGluc160=4.38mM; p=0.004).

Additionally, propylene glycol correlated significantly with CSF lactate concentrations (r=0.408; p=0.002), which was expected since PG can very easily be converted to lactate. Most interestingly, the GC/MS analysis of CSF revealed the presence of methylglyoxal. Methylglyoxal was significantly higher in the CSF of TBI patients when compared to the CSF of non-injured patients. On average, TBI patients had 56+/−14ng/g of sample of methylglyoxal present in their CSF, whereas the non-injured patients only had 9.4+/−8.0ng/g of sample (p=0.05). Conclusion While there have been numerous studies on glucose metabolism following TBI, to our knowledge, no studies in TBI research have examined the methylglyoxal pathway.

This process can be activated after glucose initiates glycation under conditions of hyperglycemia and the presence of free radicals. This pathway has been recently investigated in diabetes research. Considering the physiological similarities between hyperglycemia in diabetes and TBI with regards to the presence of free radicals and the increased susceptibility to developing neurodegenerative diseases, glycation may be an important pathophysiological mechanism following TBI. The detection of PG and methylglyoxal in the CSF of TBI patients suggests the presence of glycation reactions in the injured brain under conditions of high PlGluc.

We speculate that free radicals inhibit glycolysis at the glyceraldehydes-3-phosphate (G3P) step. G3P then gets metabolized to methylglyoxal, AGEs, and PG, all of which can have detrimental effects on the injured brain. In light of these data, we believe that it is of utmost importance to monitor PlGluc and glycation marker levels closely in severely head injured patients to prevent further metabolic distress and cell damage. INHIBITION OF THE MYOSIN LIGHT CHAIN KINASE PREVENTS HYPOXIA/STROKE-INDUCED BLOOD BRAIN BARRIER DISRUPTION IN VITRO AND IN VIVO R. Tamaki 2,3, C. Kuhlmann 1, V. Lessmann 1, H.

Luhmann 1, Oliver Kempski 2 1Institute of Physiology and Pathophysiology, Johannes Gutenberg University, Mainz, Germany, 2Institute for Neurosurgical Pathophysiology, Johannes Gutenberg University, Mainz, Germany, 3Department of Neurosurgery, Nara Medical University, Nara, Japan Background: Increased mortality after stroke is associated with development of brain edema. The aim of the present study was to examine the contribution of endothelial myosin light chain (MLC) phosphorylation to hypoxia/stroke-induced blood brain barrier (BBB) opening. Methods: Measurements of Trans-Endothelial-Electrical-Resistance (TEER) were performed to analyze BBB integrity in an in-vitro co-culture model (bovine brain microvascular endothelial cells (BEC) and rat astrocytes). Brain water content was analyzed in rats 24 hrs after venous stroke induction using a two-vein occlusion (TVO) model. Dihydroethidium was used to monitor intracellular generation of reactive oxygen species (ROS) in BEC. MLC phosphorylation was detected by immunostainings using a confocal laser-scanning microscope.

Results: Hypoxia induced in-vitro by a combined oxygen-glucose deprivation caused a decrease of TEER values by more than 40%, which was prevented by the application of the MLC-kinase inhibitor ML-7 (10 µmol/l). ML-7 also significantly reduced the elevated brain water content invivo after TVO (80.184± 0.540%, n=8) to 79.714 ± 0.337% (n=10) (sham-op: 78.793± 0.326%, n=8).

The NAD(P)H-oxidase inhibitor apocynin (500 µmol/l) prevented the hypoxia-induced TEER decrease. Hypoxia-dependent ROS generation was completely abolished by apocynin and by the Ca2+ chelator BAPTA (10 µmol/l). Interestingly, the hypoxia-dependent decrease of TEER was also blocked by BAPTA. Inhibition of Ca2+ signaling (BAPTA), ROS-formation (apocynin), or the MLC-kinase (ML-7) completely abolished the hypoxia-induced increase of MLC-phosphorylation. Conclusions: Our data demonstrate for the first time, that MLC phosphorylation contributes to hypoxia/stroke-induced BBB disruption in-vivo and in-vitro. The hypoxia-induced increase of intracellular Ca2+ increases the activity of the NAD(P)H-oxidase.

The resulting ROS generation is responsible for an increase of MLC phosphorylation that activates the endothelial contractile machinery leading to paracellular gap formation. Background Our recent studies have shown that the water channel aquaporin-4 (AQP4) is induced 1h after stroke onset. The role of AQP4 in the evolution of cytotoxic and vasogenic oedema after stroke remains debated.

The absence of AQP4 in KO-mice reduces the early cytotoxic oedema formation and in contrast aggravates the later vasogenic oedema. A second aquaporin, AQP9, is induced at a later time-point than AQP4 after stroke onset, and the function of AQP9 is predicted to be in relation with brain energy metabolism. Thrombin at high doses is known to induce oedema while preconditioning is induced by a single injection of a low dose of thrombin (TPC).

TPC attenuates lesion size and oedema after middle cerebral artery occlusion (MCAo). This preconditioning protocol is a useful model to understand endogenous mechanisms of protection after brain ischemia and increase our knowledge of the roles of aquaporins in oedema development. We studied the expression of AQPs in ischemic mouse brains after TPC in correlation with the reduction in oedema formation. Results In TPC animals, oedema formation assessed by measurement of the hemispheric enlargement is significantly reduced at 1h (4.5 ± 2% vs 11.0 ± 5% in controls, p.

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