Brain Poster Session: Oxidative Mechanisms

41. Parkinson's disease related proteins induction after transient spinal cord ischemia in rabbits

M. Sakurai¹, T. Kawamura², H. Nishimura², H. Suzuki², F. Tezuka² and K. Abe³

¹Cardiovascular Surgery; ²National Hospital Organization Sendai Medical Center, Sendai; ³Neurology, Okayama University, Okayama, Japan

Background and aims: The mechanism of spinal cord injury has been thought to be related to the vulnerability of spinal motor neuron cells against ischemia. However, the mechanisms of such vulnerability are not fully understood.

Methods: We used a rabbit spinal cord ischemia model with use of a balloon catheter. The spinal cord was removed at 8 h, 1, 2, or 7 days after 15 min of transient ischemia, and histological changes were studied with hematoxylin-eosin staining. Western blot analysis for DJ-1, PINK1 and α-Synuclein, temporal profiles of DJ-1, PINK1 and α-Synuclein immunoreactivity, and double-label fluorescence immunocytochemical studies were performed.

Purpose: Because we previously reported that spinal motor neurons were lost probably by programmed cell death, we investigated a possible mechanism of neuronal death by immunohistochemical analysis for DJ-1, PINK1 and α-Synuclein.

Results: The majority of motor neurons were preserved until 2 days, but were selectively lost at 7 days of reperfusion. Western blot analysis revealed scarce immunoreactivity for DJ-1, PINK1 and α-Synuclein in the sham-operated spinal cords. However, they became apparent at 8 h after transient ischemia, which returned to the baseline level at 1 day. Double-label fluorescence immunocytochemical study revealed that both DJ-1andPINK1, and DJ-1 and α-Synuclein were positive at 8 h of reperfusion in the same motor neurons, which eventually die.

Conclusion: This study demonstrated that immunoreactivities for both DJ-1and PINK1, and DJ-1 and α-Synuclein were induced in the same motor neuron, which eventually die. The induction of DJ-1 and PINK1 proteins in motor neurons at the early stage of reperfusion may indicate oxidative stress, and the induction ofα-Synuclein may be implicated in the programmed cell death change after transient spinal cord ischemia.

81. Thiamine deficiency increases β-secretase activity and accumulation of β-amyloid peptides

Q. Zhang, J. Luo and Z.-J. Ke

Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

Thiamine pyrophosphate (TPP) and the activities of thiamine-dependent enzymes are reduced in Alzheimer′s disease (AD) patients. In this study, we analyzed the relationship between thiamine deficiency (TD) and amyloid precursor protein (APP) processing in both cellular and animal models of TD. In SH-SY5Y neuroblastoma cells overexpressing APP, TD promoted maturation of b-site APP cleaving enzyme 1(BACE1) and increased b-secretase activity which resulted in elevated levels of β-amyloid (Ab) as well as b-secretase cleaved C-terminal fragment (b-CTF). An inhibitor of b-secretase efficiently reduced TD-induced up-regulation of Ab and b-CTF. Importantly, thiamine supplementation reversed the TD-induced alterations. Furthermore, TD treatment caused a significant accumulation of reactive oxygen species (ROS); antioxidants suppressed ROS production and maturation of BACE1, as well as TD-induced Ab accumulation. On the other hand, exogenous Ab₁₋₄₀ enhanced TD-induced production of ROS. A study on mice indicated that TD also caused Aβ accumulation in the brain, which was reversed by thiamine supplementation. Taken together, our study suggests that TD could enhance Ab generation by promoting b-secretase activity, and the accumulation of Ab subsequently exacerbated TD-induced oxidative stress.

146. The protective role of PGC-1α in ischemia-induced hippocampal neuronal injury

S.-D. Chen¹, T.-K. Lin¹, D.-I. Yang², S.-Y. Lee¹, C.-W. Liou¹ and Y.-C. Chuang¹

¹Department of Neurology, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University, Kaohsiung; ²Institute of Brain Science, National Yang-Ming University, Taipei, Taiwan R.O.C.

Background and purpose: Transient global ischemia (TGI) is known to cause delayed hippocampal neuronal death. Excessive reactive oxygen species generation after TGI/reperfusion may contribute this TGI-induced hippocampal neuronal death. Activation of PGC-1α, a coactivator of peroxisome proliferator activated receptor gamma, is emerged as a master regulator in oxidative stress in hypoxic/ischemic condition. Uncoupling protein 2 (UCP 2) and superoxide dismutase 2 (SOD 2) are molecules important for counteracting excessive oxidative stress under certain circumstances including cerebral ischemia and may be regulated by PGC-1α. We studied hippocampal PGC-1α activation and the expression of UCP 2 and SOD 2 in a rat TGI model and explored if this signaling pathway is relevant to this delayed hippocampal neuronal death.

Methods: Rats were subjected to TGI by transient bilateral common carotid artery occlusion and systemic hypotension (mean arterial pressure to 35–40 mm Hg) for 10 min followed by restoration of blood flow to normal level. Hippocampal proteins were extracted and subjected to western blot for PGC-1α, UCP 2 and SOD 2 expression. Oxidized protein of hippocampus under ischemic condition was examined at various time points after reperfusion. Anti-sense oligodeoxynucleotide (ODN) for PGC-1α is used to investigate the role of PGC-1α over UCP 2 and SOD 2 expression and the effect over oxidized protein of hippocampus and neuronal survival under TGI/reperfusion. Apoptosis-related DNA fragmentation were evaluated for outcome measurement after ischemia/reperfusion. Surviving CA1 neuronal cells were counted under light microscopy.

Results: PGC-1α expression increased in 1 to 4 h after ischemia/reperfusion. UCP 2 expression increased in 4 to 48 h after ischemia/reperfusion. SOD 2 expressions increased in 4 to 48 h after ischemia/reperfusion. Increased oxidized protein of hippocampus under ischemic condition was detected at 1–24 h after reperfusion. Anti-sense ODN for PGC-1α pretreatment 24 h before TGI showed decreased PGC-1α, UCP 2 and SOD 2 expression which was accompanied by a heightened protein oxidation and enhanced hippocampal CA1 neuronal damage based on apoptosis-related DNA fragmentation.and the surviving neuronal counts.

Conclusions: This study may provide a molecular evidence for the PGC-1α/ UCP 2 and SOD 2 neuroprotective cascade in ischemic brain injury.

360. Down-regulation of Casein kinase 2 triggers ischemic neuronal death via AKT inhibition and ROS production after oxidative insults in mice

G.S. Kim, J.E. Jung, K. Niizuma and P.H. Chan

Department of Neurosurgery, Stanford University School of Medicine, Stanford, California, USA

Objectives: Casein kinase 2 (CK2) plays a pivotal role in cell proliferation, cell cycle control and apoptosis by phosphorylating its various substrates. ² But the role of CK2 and changes in its subunit levels and activity after oxidative insults in the brain are not well known.

Methods: In this study, we employed in vivo transient focal cerebral ischemia (tFCI) in models of 45 min transient middle cerebral artery occlusion, ³ subarachnoid hemorrhage (SAH) and in vitro oxygen glucose deprivation (OGD) to investigate the role of and changes in CK2 subunits following oxidative stress.

Results: We found that CK2 protein activity in the damaged cortex region from the ipsilateral hemisphere was reduced compared with the contralateral hemisphere (1 versus 0.7±0.1, P<0.05). Also, the protein levels of catalytic subunits CK2α and CK2α and phosphorylation of CK2β in the cortex region damaged by ischemic injury were markedly decreased 24 h after ischemic injury and reperfusion (CK2α: 1 versus 0.35±0.09, P<0.01; CK2α′: 1 versus 0.44±0.19, P<0.05; pCK2β: 1 versus 0.3±0.12, P<0.01), whereas the regulatory subunit CK2β was not changed after ischemia and reperfusion as assessed by Western blotting. We also observed similar changes in the CK2 subunits in the SAH and OGD models, which are known to induce oxidative stress in neuronal cells. Immunofluorescent staining revealed that CK2α- and phospho-CK2β-positive cells were significantly diminished in the penumbra and ischemic core region in the injury area compared with the non-injured area. Treatment with MG132, a proteasome inhibitor, blocked CK2α down-regulation in a dose-dependent manner after tFCI and in the OGD model. The protein levels of CK2α in SOD1 transgenic mice did not decrease after ischemic injury compared with wild-type mice. CK2α-positive cells did not colocalize with cleaved poly(ADP-ribose) polymerase and terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling in the injured cortex. Moreover, administration of a CK2-specific pharmacological inhibitor, tetrabromocinnamic acid (TBCA), ¹ into the ventricle facilitated neuronal cell death and increased infarct volume (Veh versus TBCA: 17.0%±7.24 versus 34.3%±12.8, P<0.05) after tFCI by regulating AKT phosphorylation and reactive oxygen species production.

Conclusions: Oxidative stress induced by tFCI causes a reduction in CK2 activity via the loss of catalytic subunits α and α′ and CK2β phosphorylation, and this down-regulation of CK2 activity sensitizes neuronal cells to death after oxidative injury.

630. NADPH oxidase activity is higher in cerebral versus systemic arteries of four animal species: role of Nox2

A.A. Miller¹, G.R. Drummond¹, T.M. De Silva¹, A.E. Mast¹, H. Hickey², J.P. Williams³, B.R. Broughton¹ and C.G. Sobey¹

¹Department of Pharmacology, Monash University; ²Department of Pharmacology, Melbourne University; ³Howard Florey Institute, Melbourne, VIC, Australia

Objectives: Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide serve as important cell signalling molecules for the regulation of normal vascular function. Moreover, a wealth of evidence suggests that excessive generation of vascular ROS plays a key role in the pathogenesis of several vascular diseases such as hypertension and stroke. NADPH oxidases are now believed to be a major physiological source of ROS within cerebral and systemic vasculatures. We previously reported that NADPH oxidase activity is profoundly greater in intracranial cerebral versus systemic arteries of the rat. ¹ . In the present study we firstly confirmed our finding in rats and then tested whether the activity of NADPH oxidase is also greater in intracranial cerebral arteries than in systemic arteries of other animal species, i.e. mouse, rabbit and pig. Secondly, using Nox2 deficient mice we evaluated the involvement of Nox2-containing NADPH oxidase in any such regional differences.

Methods: NADPH (100 μmol/L)-stimulated superoxide production by basilar (BA), middle cerebral (MCA) and common carotid (CA) arteries, and thoracic aorta (AO) from rat (Sprague Dawley, n = 6), mouse (C57Bl/6J, WT, n = 68; and Nox2−/−, n = 17), rabbit (New Zealand White, n = 6) and pig (Sus scrofa, n = 6) was measured using lucigenin (5 μmol/L)-enhanced chemiluminescence. Basal superoxide and hydrogen peroxide production by mouse cerebral arteries (pooled BA and MCA), AO and CA, was measured using L-012 (100 μmol/L)-enhanced chemiluminescence and Amplex Red fluorescence, respectively. Protein expression of Nox2 and superoxide dismutase (SOD) isoforms 1–3 in mouse cerebral arteries (pooled BA and MCA), AO and CA was measured using Western Blotting. Confocal immunofluorescence was used to localize Nox2 in mouse MCA, AO and CA.

Results: In rats, WT mice, rabbits and pigs, NADPH-stimulated superoxide production by cerebral arteries was up to 40-fold greater than in AO and CA (n = 3−12, P<0.05). In WT mice, basal superoxide and hydrogen peroxide production by cerebral arteries was ∼9-fold and ∼2.5–fold higher (n = 4–10, P<0.05), respectively, than AO and CA, and was associated with ∼40% greater expression of Nox2 (n = 4, P<0.05). By contrast, SOD1, SOD2 and SOD3 protein expression did not differ between artery types (n = 6–8). Nox2 immunofluorescence was localized to the endothelium, and to a lesser extent the adventitia of MCA, AO and CA and appeared to be more intense in the endothelium of MCA than AO or CA (n = 3). In Nox2−/– mice, NADPH-stimulated superoxide production was reduced by ∼35% versus WT mice (n = 5, P<0.05), whereas Nox2 deletion did not affect superoxide production in AO or CA.

Conclusions: In summary, the findings of this study reveal that NADPH oxidase activity is greater in intracranial cerebral versus systemic arteries of several animal species. Furthermore, we provide evidence that higher cerebrovascular Nox2 activity may, in part, account for these regional differences.

673. Reactive oxygen species generation and Bcl-2 downregulation mediated apoptosis in human Glioma U87 cells by demethoxycurcumin

R. Kumar and P.M. Luthra

Dr B R Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India

Objective: In efforts to find novel approaches to inhibit proliferation and induce apoptosis in human glioma cells, we examined the action of naturally occurring compound demethoxycurcumin on human glioma U87 cell line. The protooncogene Bcl-2 inhibited reactive oxygen species (ROS) production and prevented apoptosis by regulating the cellular redox potential.^1–3 In this work, demethoxycurcumin induced ROS generation and Bcl-2 downregulation mediated apoptosis in human glioma U87 cells has been studied.

Methods: Apoptosis in U87 cells was induced by treating the cells with or without demethoxycurcumin (12.5 μg/ml and 25 μg/ml) for 2, 4, 6, 12, 24 and 48 h. The net intracellular generation of ROS was assessed by dichorofluorescein diacetate (H₂DCF-DA). Proliferation inhibition, chromatin condensation and DNA fragmentation was carried by MTT assay, nuclear staining and DNA ladder method respectively. Bcl-2 protein expression was measured by western blotting. The distribution of cells in the different phases of the cell cycle was carried by flow cytometery.

Results: Demethoxycurcumin exhibited high level of ROS generation in U87 cells in dose and time dependent manner (Figure 1) and was associated with collapse of mitochondrial membrane potential (Δψm), proliferation inhibition, chromatin condensation and DNA fragmentation and reduced expression of Bcl-2. In cell cycle analysis demethoxycurcumin showed G2/M arrest at 12.5 μg/ml concentration after 24 h treatments, however at 48 h significant increase in sub-G1 apoptotic fraction was observed.

Figure 1

ROS production during demethoxycurcumin-induced apoptosis. Human glioma U87 cells were exposed to 12.5 μg/ml and 25 μg/ml Demethoxycurcumin for 2, 4 and 6 h. ROS production was detected H2DCFH-DA selective for H₂O₂. The results were presented as a percentage of the fluorescence intensity compared with the control sample.

Conclusions: We have demonstrated for the first time that the induction of apoptosis associated with ROS production and down regulation of Bcl-2 in glioma U87 cells is mediated through demethoxycurcumin, meriting its further evaluation in vivo.

709. Interaction of caveolin-1, cholesterol and nitric oxide synthases in hypoxic human SK-N-MC neuroblastoma cells

J. Shen ^1,2

¹School of Chinese Medicine; ²Research Centre of Heart, Brain, Hormone & Healthy Aging, University of Hong Kong, Hong Kong, Hong Kong S.A.R.

Objectives: Interaction between nitric oxide (NO) and caveolins appears to be a potential cellular signal pathway in ischemic brain injury. To elucidate the interaction of NO and caveolins in hypoxic neural cells, we investigated the expressions of caveolin-1 and caveolin-2 and NO production in human SK-N-MC neuroblastoma cells exposed to different periods of hypoxia.

Methods: Human neuroblastoma SK-N-MC cells were grown as a monolayer in minimum essential medium (MEM, Sigma) supplemented with fetal bovine serum (10%, v/v), glutamine (10 mg/ml) and antibiotics (penicillin and streptomycin, 10 mg/ml). For hypoxic treatment, confluent monolayers in multiple-well plates were placed in a modular incubator chamber. The cells were consistently incubated with 2% O₂ plus 5% CO₂ balanced with N₂ gas for 8, 15, 24 and 36 hrs at 37°C respectively. To understand the interactions of caveolin-1, cholesterol and nitric oxide synthases in the hypoxic SK-N-MC cells, the cells were treated with different reagents to change the contents of NO and cholesterol.

Results: The expression of caveolin-1 mRNA and protein was transiently upregulated by 15 hrs of hypoxia but downregulated by 24 hrs or longer exposure to hypoxia, whereas the expression of caveolin-2 mRNA down-regulated by hypoxia. In normoxic SK-N-MC cells, both S-Nitroso-N-acetyl-DL-penicillamine (SNAP, NO donor) and 3-morpholinosydnonimine (SIN-1, peroxynitrite donor) up-regulated the expression of caveolin-1. Moreover, in the SK-N-MC cells exposed to 15 hrs of hypoxia, pretreatments of N^G-nitro-L-arginine methyl ester (L-NAME, a non-selective NOS inhibitor), 1400W (a inducible NOS inhibitor) or FeTMPyP (a peroxynitrite decomposition catalyst) attenuated the increase of caveolin-1 expression, but there were no obvious changes in the expression of caveolin-1 by incubation of 7-nitroindazole (7-NI, a selective neural NOS inhibitor) and l-N5-(1-iminoethyl)-ornithine (L-NIO, a selective endothelial NOS inhibitor). Meanwhile, hypoxia treatment induced intracellular cholesterol accumulation. Pretreatments of methyl-β-cyclodextrin (MCD) and methyl-β-cyclodextrin-cholesterol (MCD-CHOL) and oleic acid inhibited and enhanced the expression of caveolin-1 respectively. Those results suggest that the upregulation of caveolin-1 could be associated with iNOS-induced reactive nitrogen species production and lipid accumulation in the hypoxic neural cells. Furthermore, we compared the expression of eNOS, iNOS, and nNOS and NO production in wild-type and caveolin-1 over-expressed SK-N-MC cells after 15 hrs of hypoxia treatment. Exposure to 15 hrs of hypoxia inhibited eNOS expression but induced iNOS expression and NO production in the wild-type cells. However, over-expression of caveolin-1 prevented the loss of eNOS but downregulated the expression of iNOS and inhibited NO production.

Conclusion: The results suggest that augmentation of caveolin-1 in response to hypoxia stimulation could be a physiological regulating mechanism to inhibit iNOS-induced NO production. Overall, the complex interactions of reactive nitrogen species and caveolin-1 could be an important signal pathway in the modulation of NO production in hypoxic neural cells.

Acknowledgement: This work was supported by Hong Kong RGC GRF grant No. 7495/04 M and No. 7748/08 M.

724. Important role of NADPH oxidase in striatal neuronal injury after severe transient global ischemia in mice

H. Yoshioka^1,2, K. Niizuma¹, M. Katsu¹, N. Okami¹, G.S. Kim¹, P. Narasimhan¹, H. Kinouchi² and P.H. Chan¹

¹Neurosurgery, Stanford University School of Medicine, Stanford, California, USA; ²Neurosurgery, University of Yamanashi, Chuou, Japan

Objectives: Reactive oxygen species (ROS) have been implicated in neuronal injury after cerebral ischemia. ¹ NADPH oxidase has been well characterized in immune cells to produce mainly ROS as superoxide radicals for host defense. The oxidase is a complex enzyme that consists of the membrane subunit cytochrome b₅₅₈ (gp91^phox and p22^phox) and multiple cytosolic subunits (p47^phox, p67^phox, p40^phox, and Rac-1). Upon stimulation, the cytosolic subunits migrate to the plasma membrane, whereby a functional complex generating ROS is formed. Recent studies suggest that NADPH oxidase can serve as a major source of ROS in the central nervous system^2,3; however, the roles of NADPH oxidase in neuronal death after transient global cerebral ischemia (tGCI) are not well understood. We hypothesized that oxidative stress generated by NADPH oxidase in ischemic brain cells could lead to neurovascular injury. The purpose of the present study is to test this hypothesis in a newly developed model of tGCI in mice.

Methods: Gp91^phox−/− (gp91 KO) mice with a C57BL/6 background and their respective wild-type (Wt) littermates were subjected to tGCI by bilateral common carotid artery occlusion (BCCAO) for 22 min (we chose this timing because preliminary studies have shown reproducible striatal damage without high mortality). Ischemic neuronal injury was evaluated in the striatum 3 days after ischemia by cresyl violet staining. Western blot analyses of samples from the striatum were performed to investigate the expression and translocation of NADPH oxidase subunits. Localization of NADPH oxidase was examined by double immunofluorescence. For the detection of oxidative protein damage after ischemia, the carbonyl groups introduced into proteins were measured.

Results: Striatal neurons of the Wt mice 3 days after 22 min of BCCAO showed severe damage with high reproducibility in this model. Western blot analysis showed the upregulation of gp91^phox and an increase in the cytosolic subunits (p47^phox, p67^phox, Rac-1) in the membrane fraction after ischemia. A double immunofluorescent study revealed that p67^phox–positive cells colocalized with neuron-specific nuclear protein or lectin-positive cells in the striatum, suggesting NADPH oxidase expressed in neurons and vascular endothelial cells. The carbonyl groups introduced into proteins by oxidative stress increased after ischemia in the Wt mice, and this increase was suppressed in the gp91 KO mice. Furthermore, striatal neuronal injury was significantly reduced in the gp91 KO mice compared with the Wt mice.

Conclusions: These results suggest an important role for NADPH oxidase in ROS production and striatal neuronal death after severe transient forebrain ischemia. We propose that NADPH oxidase is a therapeutic target for neurovascular damage in ischemic stroke.

730. Measurements of glial metabolic fluxes with ¹¹C-acetate using positron emission and an adapted NMR-based metabolic modeling approach

B. Lanz¹, M.T. Wyss^2,3, B. Weber^2,3, A. Buck² and R. Gruetter^1,4,5

¹Laboratory of Functional and Metabolic Imaging, Ecole Polytechnique Fédérale de Lausanne, Lausanne; ²PET Center, Division of Nuclear Medicine, University Hospital Zurich; ³Institute of Pharmacology and Toxicology, University of Zurich, Zurich; ⁴Department of Radiology, University of Lausanne, Lausanne; ⁵Department of Radiology, University of Geneva, Geneva, Switzerland

Objectives: Acetate brain metabolism has the particularity to occur specifically in glial cells. Labeling studies, using acetate labeled either with ¹³C (NMR) or ¹¹C (PET), are governed by the same biochemical reactions and thus follow the same mathematical principles. In this study, the objective was to adapt an NMR acetate brain metabolism model to analyse [1-¹¹C]acetate infusion in rats.

Methods: Brain acetate infusion experiments were modeled using a two-compartment model approach used in NMR.^1–3 The [1-¹¹C]acetate labeling study was done using a beta scintillator. ⁴ The measured radioactive signal represents the time evolution of the sum of all labeled metabolites in the brain. Using a coincidence counter in parallel, an arterial input curve was measured. The ¹¹C at position C-1 of acetate is metabolized in the first turn of the TCA cycle to the position 5 of glutamate (Figure 1A). Through the neurotransmission process, it is further transported to the position 5 of glutamine and the position 5 of neuronal glutamate. After the second turn of the TCA cycle, tracer from [1-¹¹C]acetate (and also a part from glial [5-¹¹C]glutamate) is transferred to glial [1-¹¹C]glutamate and further to [1-¹¹C]glutamine and neuronal glutamate through the neurotransmission cycle.

Figure 1

(A) Brain metabolic model used for [1¹¹-C]acetate infusion experiment. A similar model ² was used for ¹³C NMR studies following [2-¹³C]acetate infusion, with adapted label positions (i.e. glutamate and glutamine C4 followed by C3). The dotted arrows show the fluxes taking place in the second tum of the TCA cycle. The orange arrows show the loss of label in to CO². (B) Typical fit of beta-probe data to the sum of the metabolites described in the model of Figure 1A). The colored lines show the respective contribution of each metabolite to the total measured radioactivity. The red dotted line represents the fit with a standard two-pool model.

Results: The standard acetate two-pool PET model describes the system by a plasma pool and a tissue pool linked by rate constants. Experimental data are not fully described with only one tissue compartment (Figure 1B). The modified NMR model was fitted successfully to tissue time-activity curves from 6 single animals, by varying the glial mitochondrial fluxes and the neurotransmission flux Vnt.

A glial composite rate constant Kgt^g = Vgt^g/[Ace]_plasma was extracted. Considering an average acetate concentration in plasma of 1 μmol/g⁵ and the negligible additional amount injected, we found an average Vgt^g = 0.08±0.02 (n = 6), in agreement with previous NMR measurements. ¹

The tissue time-activity curve is dominated by glial glutamate and later by glutamine (Figure 1B). Labeling of neuronal pools has a low influence, at least for the 20 mins of beta-probe acquisition. Based on the high diffusivity of CO₂ across the blood-brain barrier; ¹¹CO₂ is not predominant in the total tissue curve, even if the brain CO₂ pool is big compared with other metabolites, due to its strong dilution through unlabeled CO₂ from neuronal metabolism and diffusion from plasma.

Conclusion: The two-compartment model presented here is also able to fit data of positron emission experiments and to extract specific glial metabolic fluxes. ¹¹C-labeled acetate presents an alternative for faster measurements of glial oxidative metabolism compared to NMR, potentially applicable to human PET imaging. However, to quantify the relative value of the TCA cycle flux compared to the transmitochondrial flux, the chemical sensitivity of NMR is required. PET and NMR are thus complementary.

921. Biodistribution of [⁶¹Cu]-oxinate in fibrosarcoma tumors for pet imaging

A.R. Jalilian¹, S. Zolghadri², R. Faghihi², J. Garousi¹, H. Yousefnia², A. Majdabadi¹ and F. Bolourinovin¹

¹Nuclear Medicine Research Group, Agricultural, Medical and Industrial Research School, Karaj; ²Department of Nuclear Engineering, School of Engineering, Shiraz University, Shiraz, Iran

Objectives: This work was conducted for production of an radiolabeled anticancer copper complex, i.e. ⁶¹Cu-oxinate as a potential PET in oncology.

Methods: Cu-61 was prepared from natural zinc target irradiation by 22 MeV protons (150 uA) via nuclear reaction with a yield of 3.33 mCi/μAh. In order to obtain the best labeling method optimization reactions were performed for pH, temperature and concentration and finally best solid phase extraction method. Biodistribution of the tracer was studied in normal and fibrosarcoma bearing mice.

Results: At the optimized conditions, ITLC showed radiochemical purity more than 97% with a specific activity of 0.06 Ci/mM. This was kept unchanged even with presence of human serum as well as room temperature for 5 h.

conclusion: Biodistribution of the tracer in fibrosarcoma bearing mice demonstrated significant tumor uptake after 2 h. This tracer can be used in the detection of some brain tumors using PET scan.

948. Mild hypothermia attenuates oxidative/nitrosative stress and cytotoxic brain edema in experimental acute liver failure

C. Bemeur, W. Jiang, P. Desjardins and R.F. Butterworth

Neuroscience Research Unit, CHUM, St-Luc Hospital, University of Montreal, Montreal, QC, Canada

Objectives: Encephalopathy and brain edema are serious neurological complications of acute liver failure (ALF). The precise pathophysiologic mechanisms responsible have not been fully elucidated but it has been suggested that oxidative/nitrosative stress is involved. ² In the present study, we evaluated the role of oxidative/nitrosative stress in the pathogenesis of hepatic encephalopathy and brain edema in rats with ALF resulting from hepatic devascularization and mice with ALF resulting from azoxymethane hepatotoxicity. ¹ We also studied the effect of hypothermia, a treatment previously shown to delay the progression of encephalopathy and the onset of brain edema, on ALF-induced oxidative stress. ³

Methods: ALF animals were sacrificed at precoma and coma stages of encephalopathy along with their appropriate controls. Hypothermic ALF animals were sacrificed in parallel with normothermic comatose ALF animals. Cerebral GSH/GSSG ratio were assessed spectrophotometrically. Nitrite/nitrate levels in plasma and brain were measured using the Griess reaction and expression of nitric oxide synthase (NOS) isoforms and heme oxygenase-1 (HO-1) were measured using real-time quantitative PCR and western blot analysis.

Results: Cerebral GSH/GSSG ratio was significantly decreased in ALF comatose animals compared to the controls. Increased nitrite/nitrate levels were observed in the plasma and frontal cortex in ALF animals at coma stage of encephalopathy compared to controls. Increased expression of HO-1 protein and mRNA was observed in the frontal cortex of ALF animals at both precoma and coma stages of encephalopathy. Significant increases in expression of endothelial and inducible nitric oxide synthase (NOS) mRNA isoforms also occured at precoma and coma stages of encephalopathy. Expression of the neuronal nitric oxide synthase isoform (nNOS) was not altered by ALF. Hypothermia normalized GSH/GSSG ratio and nitrite/nitrate levels in brain and significantly attenuated HO-1, eNOS and iNOS expression.

Conclusions: These results suggest that, oxidative/nitrosative stress participates in the pathogenesis of brain edema and its complications in ALF and that the beneficial effect of hypothermia depends on its ability to inhibit oxidative/nitrosative stress-related mechanisms.

977. Hyperbaric oxygen stimulates the transcription rate of the antioxidant enzyme cupper/zinc-superoxide dismutase in rat brain cortex tissue

S. Oter¹, S. Sadir¹, B. Uysal¹, M. Ozler¹, K. Simsek², H. Ay², H. Yaman³, R. Ogur⁴, R.J. Reiter⁵, T. Topal¹ and A. Korkmaz¹

¹Physiology; ²Undersea and Hyperbaric Medicine; ³Biochemistry; ⁴Public Health Gulhane Military Medical Academy, Ankara, Turkey; ⁵Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA

Objectives: HBO therapy is a treatment method known to cause oxidative stress ¹ while having beneficial effects in several pathophysiological processes where free radical damage is contributory. ² Moreover, antioxidant enzyme activities are usually increased with HBO exposure,^3,4 which may be a compensatory response to enhanced production of reactive oxygen species. Another hypothesis suggests that HBO has the ability to stimulate antioxidant enzyme synthesis directly. ⁵ Evidence for both these potential explanations is not completely understood. Therefore, taken the brain as one of the main target organs affected by HBO exposure, we aimed to elucidate this matter via detecting the transcription rate of cupper/zinc-superoxide dismutase (Cu/Zn-SOD) in rats.

Methods: eventy-two male Sprague-Dawley rats were divided into 6 (n = 12 for each) groups: control, HBO, HBO+Glutathione (GSH) and HBO+SOD, HBO+melatonin, and melatonin. HBO was administered once for 2 h at 3 atmospheres pressure. GSH, SOD, and melatonin were administred intraperitoneally 1 h before HBO exposure. Right after exposure, their brain cortexes were harvested immediately and following biochemical assay was performed: malondialdehyde (MDA) and protein carbonyl (PCO) levels, catalase, glutathione peroxidase and SOD activities, and reduced/oxidized glutathione (GSH/GSSG) ratio. Additionally, the transcription rate of Cu/Zn-SOD mRNA was determined via RT-PCR.

The reason for establishing study groups with antioxidants (GSH, SOD, melatonin) was to distinguish a potential effect of HBO on the transcription rate of SOD with an indirectly stimulated effect via enhanced radical production. Melatonin itself is known to induce antioxidant enzymes expression; ⁶ therefore, the melatonin only group will give the ability to compare the strongivity of the results.

Results: O clearly increased oxidative stress indices MDA and PCO. SOD activity as well as its mRNA transcription rate was also increased with HBO exposure. Melatonin and GSH were able to reduce the oxidative action of HBO. Nevertheless, neither SOD nor GSH could block the mRNA enhancing effect of HBO for Cu/Zn-SOD. Interestingly, melatonin increased the SOD mRNA transcription itself, but limited (not blocked) the increasing effect of HBO. The Cu/Zn-SOD mRNA expression rates of all study groups are demonstrated in Figure.

Figure 1

Cortical SOD mRNA Expression

Conclusion: The present findings support the suggestion that HBO have the ability to stimulate directly the mRNA transcription of SOD in rat′s brain cortex. The limiting effect of melatonin indicates a molecular interaction and has to be further elucidated.

Grant: This study was supported by the ‘Scientific & Technological Research Council of Turkey’ with the grant Nr.105S060.

984. Changes in nitric oxide synthase (NOS), cyclooxygenase-2 (COX-2) and superoxide dismutase (SOD) protein levels in chronic cerebral hypoperfusion in rats

A. Institoris^1,2, E. Mracsko², M. Hugyecz², A. Matyas³, Z. Sule³, E. Farkas² and F. Bari²

¹Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina, USA; ²Department of Physiology, University of Szeged; ³Department of Anatomy, University of Szeged, Szeged, Hungary

Chronic cerebral hypoperfusion (CCH) is a condititon which contributes to cognitive decline during senescence and in several types of dementia. Its experimental model is the permanent bilateral common carotid artery occlusion (2VO) in rats. Following 2VO, the slow but progressive restoration of cerebral blood flow is accompanied with ischemic neuronal damage, neuroinflammation and cognitive dysfunction. The nitric oxide synthase (NOS) and cyclooxygenase (COX) enzyme systems participate in the maintenance of cerebral perfusion, meanwhile under acute ischemia these prooxidant enzymes aggrevate oxidative damage via producing neurotoxic free radicals. However, the temporal characteristics of oxidative stress and the involvement of these enzyme systems in CCH have not yet been described.

Our objective was to detect the expression of endothelial (e)NOS, inducible (i)NOS, neuronal (n)NOS (nNOS), COX-2 enzyme and manganase (Mn)SOD with Western blot analysis and immunocytochemistry (ICC) in the cerebral cortex and in the hippocampus in a temporal manner following 2VO.

Male Wistar rats were exposed to the 2VO (n = 41) or SHAM operation (n = 42). Alternatively, some rats were not operated on (naive) (n = 20). Western blot analysis was carried out from tissue samples of the hippocampus and frontal cortex taken 1 day, 3 days, 1 week, 3–6—and 12 months following surgery. ICC detection of eNOS, nNOS and MnSOD was performed on paraformaldehyde-fixed brain samples taken 3–6—and 12 months after 2VO.

In the hippocampus, the expression of eNOS enzymes significantly increased (P<0.01), COX-2 expression only mildly increased, while iNOS and nNOS levels decreased at the early phase of CCH. Three months after 2VO, lower eNOS production could be detected (P<0.05). In the cortex, the early upregulation of eNOS and nNOS was followed by a reduction of COX-2 and eNOS production at 3 months of CCH. Finally, eNOS level increased again at 12 months after 2VO. There was no change in the MnSOD enzyme level in any of the observed regions. ICC failed to show significant changes.

The significant elevation of eNOS in the early stage of CCH is probably an important compensatory mechanism against the reduced perfusion in both areas, while the lower eNOS expression at the later phase of CCH might be a consequence of capillary degeneration. The early suppression of iNOS production indicates that the enzyme is probably not the origin of reactive oxygen species. The early activation of nNOS following 2VO in the cortex may reflect the effort of ischemic neurons to obtain more oxygen and glucose by releasing the vasodilator NO. The elevation of nNOS one year after 2VO might indicate synaptic remodeling in the hippocampus. Decreased levels of COX-2 in the cortex 3 months after 2VO are probably due to the loss of COX-2-positive neurons. The constant level of MnSOD suggests that the degree of oxidative stress is not remarkable in this model or other antioxidant mechanisms are induced against oxidative agents. In summary, the NOS system is substantially affected by CCH, however, the exact role of these prooxidant enzymes in the 2VO model remains to be elucidated.

1012. Hyperbaric oxygen induced oxidative stress in rats' brain cortex tissue with relation to the pressure/duration range of the treatment

S. Oter¹, K. Simsek², S. Sadir¹, M. Ozler¹, B. Uysal¹, T. Topal¹, H. Ay² and A. Korkmaz¹

¹Physiology; ²Undersea and Hyperbaric Medicine, Gulhane Military Medical Academy, Ankara, Turkey

Objective: The toxicity risk of hyperbaric oxygen (HBO) has long been of interest.^1,2 Since having a high rate of blood flow and oxygen consumption, the brain is one of the most sensible organs to hyperoxic injury. ³ During the last decade, our laboratory has been focused on pressure-related and exposure time-related correlation of HBO exposure in a series of experimental studies performed with rats; another study set elucidated the persistence of oxidative stress markers following HBO procedure. The results of have been published previously.^4–6 This presentation will summarize the outcome of our lab′s recent work.

Methods: A total of 162 male Sprague-Dawley rats were used for the abovementioned experimental sets. The approved and clinically used maximal pressure/duration range, 3 ATA/2 hours, ⁷ was chosen as HBO exposure limit. After HBO treatment procedures, brain tissues of the rats were harvested immediately and their cortexes were reserved to be used for biochemical assay. Thiobarbituric acid reactive substances (TBARS), indicator of lipid peroxidation, were measured in all tissue samples. Antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were also determined. In some of the experiments nitrite-nitrate values (NO_x) were measured in order to evaluate nitric oxide production.

Results: TBARS levels as well as SOD activities of brain cortex samples represented significant pressure-related increasing courses, estimated to be positively correlated with each other. A relation of HBO-induced oxidative action with exposure time was also found for TBARS and SOD, whereas GSH-Px activity increased independently. NO_x increased only at the maximal exposure level of 3 ATA for 2 h. Anyway, all of the aforesaid oxidative stress and antioxidant defense indices returned to their baseline levels within 90 min at the most. The collective outcome was summarized in the table.

	HBO exposure pressure (at 2 h duration for each) 4			HBO exposure time (at 3 atm pressure for each) 6			Time after HBO treatment (with 3 atm for 2 h) 5
	1 atm	2 atm	3 atm	30 mins	60 mins	120 mins	30 mins	60 mins	90 mins
TBARS	**	**	**	**	**	**	**	—	—
SOD	—	**	**	—	**	**	**	**	—
GSH-Px	n.d.	n.d.	n.d.	**	**	**	**	**	—
NOX	n.d.	n.d.	n.d.	—	—	**	**	—	—

HBO & Oxidative Stress: **Significvantly different.

Conclusion: The combined conclusion of these studies proves the oxidative effect of HBO, but also supports its safety as a treatment modality within its approved therapeutic limits. Especially, the increased activities of antioxidant enzymes and the relative short persistence time of oxidant molecules support this debate. However, to avoid unwanted oxidative side-effects as low pressure/duration ranges as possible should be used in clinical applications.

Grant: These studies were supported by the Gulhane Military Medical Academy Research and Progress Center with grants Nr. AR-2003/37 and AR-2005/25.

1068. Stress induced anxiogenesis and brain oxidative injury in rats: influence of age, sex and emotionality

A. Ray, A. Chakraborty and K. Gulati

Department of Pharmacology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India

In biology and medicine, stress is defined as any external or internal stimulus capable of disrupting the physiological homeostasis and the ability to cope with such aversive inputs are crucial determinants of health and disease. A variety of stressful situations are known to induce complex neurobehavioral changes, the mechanisms of some of which are poorly understood. Anxiety is one such syndrome in which is regulated by the brain and has an impact on various body systems like neuroendocrine, cardiovascular and immune systems. The CNS, in addition to being the seat for interactive neural networking, is also highly susceptible to oxidative injury, and in view of this the present study evaluated the possible role of prooxidant/antioxidant balance in stress induced neurobehavioral and anxiogenic responses in rats. Further, interactions between both ROS and RNS were also evaluated during such stress induced anxiogenic responses. In addition, the role of physiological variables like age, sex and emotionality status was also assessed on stress susceptibility and their relationship with oxidative injury in the brain was studied. Restraint stress (RS), a universally model of behavioral stress, was used as the experimental stressor, and anxiety was measured by the elevated plus maze (EPM) test. Pharmacological modulation of stress induced behavioral responses were assessed and brain homogenates were assayed for oxidative and nitrosative stress markers, for corroborative purposes. Exposure to RS resulted in neurobehavioral suppression in the EPM test which was evidenced by a reduction in the number of open arm entries (OAE) and open arm time (OAT) during a 5 min period, and was indicative of an anxiogenic response. Pretreatment with the conventional anxiolytic, diazepam, attenuated this behavioral suppression, suggesting an anxiolytic response of the drug, which was, however, also accompanied by sedation. On the other hand, pretreatment with antioxidants (ascorbic acid, alpha tocopherol and melatonin) and NO mimetics (L-arginine and isosorbide dinitrate), showed clear-cut antagonizing effects on RS-induced EPM activity suppression. RS induced anxiogenic effects were accompanied by elevations in brain MDA levels and reductions in GSH and NOx activity, all of which were differentially reversed by the antioxidants and NO mimetics. Interestingly, in a parallel set of experiments, it was observed that RS induced anxiogenic responses were greater in:

• (a)

  male as compared to female rats, and

• (b)

  old as compared to young rats.

Further, ‘high emotional’ rats were more susceptible to RS induced anxiogenic effects, as compared to ‘low emotional’ rats. Concurrent assay of brain homogenates showed that brain oxidative injury was greater in:

• (a)

  males than in females,

• (b)

  old than in young, and

• (c)

  ‘high emotional’ than ‘low emotional’ rats, which were susceptible to reversal by antioxidants and NO mimetics.

These results are suggestive of the involvement of brain oxidative injury and ROS-RNS interactions during stress induced anxiogenic behavior. In addition, it is inferred that age, sex and emotionality status may play a crucial role in deciding the extent of stress induced anxiogenesis and oxidative injury.