34. Asialoerythropoietin attenuates neuronal cell death in hippocampal CA1 region after transient forebrain ischemia in gerbil model
T. Yamashita, N. Nonoguchi, N. Ikeda, S. Kawabata, S. Miyatake and T. Kuroiwa
Neurosurgery, Osaka Medical College, Takatsuki, Japan
Objectives: In the central nervous system, recombinant human erythropoietin (rhEPO) attenuates ischemic injury in vitro and in vivo.
However, megadose of rhEPO is required to attain the protective effect, and that is afraid of accumulating thrombus and aggravating brain damage by up-regulating the red blood cell count and hyperactive platelet.
The nonerythropoietic asialoerythropoietin (asialoEPO),which is generated by total enzymatic desialylation of rhEPO, has extremely short plasma half life and has possibility of neuroprotective effect to the brain ischemia.
In this study, we showed that asialoEPO protects neurons against delayed neuronal death in the hippocampal pyramidal cells after transient forebrain ischemia.
Methods: 23 adult gerbils received 3 mins bilateral common carotid artery occlusion.
The drug (asialoEPO 10 U/g, rhEPO 10 U/g) or vehicle was injected intraperitoneally three times, 3 h before, just after, and 24 h after the insult.
Animals were allowed to survive for 7 days.
Learning ability test and retention test were carried out at day 6 and 7 respectively.
Results: In the training session, the vehicle injected group was received 7.5±2.56 footshocks in the learning trial. rhEPO (5.5±1.93 footshocks) and asialoEPO (4.86±1.07 footshocks) treated group were less than vehicle group statistically.
The latency of the vehicle treated animals to enter the dark compartment (37±54.3 secs) was significantly shorter than rhEPO (230±72.5 secs) and asialoEPO (264±71.9 secs) treated group.
AsialoEPO didn't affect bone marrow and didn't increase hemoglobin level.
The viable cells in CA1 field were counted by Nissl staining, the rhEPO treated group (103.57±78.91 cells/mm) and asialoEPO treated group (144.99±92.26 cells/mm) were significantly suppressed the neuronal cell death than vehicle treated group (19.53±10.72 cells/mm).
TUNEL (terminal deoxynucleotidyltransferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling) staining showed that rhEPO (33.40±23.0 cells/mm) and asialoEPO (25.61±41.96 cells/mm) injection caused a significant reduction than vehicle treatment (76.67±23.02 cells/mm) in the number of CA1 neurons 7 days after ischemia.
Conclusion: Multiple dosing of asialoEPO protects hippocampal CA1 neurons from ischemic damage by preventing from falling into apoptosis, and it does not affect erythropoiesis in the bone marrow.
48. Rutin protects against transient focal cerebral ischemia in rats
M. Khan1 and F. Islam1,2
1Department of Medical Elementology and Toxicology; 2Jamia Hamdard (Hamdard University), New Delhi, India
Background and purpose: Free radical induced neuronal damage is implicated in cerebral ischemia reperfusion (IR) injury and antioxidants are reported to have neuroprotective activity. The present study was designed to assess the neuroprotective mechanisms of rutin (Vitamin P), a free radical scavenger, through antioxidative, anti-inflammatory and anti-apoptotic pathways.
Methods: The middle cerebral artery of adult male wistar rat was occluded for 2 h and reperfused for 22 h. Rats were pretreated with Rutin (50 mg/kg) for 15 days. Infarct volume and neurological deficit scores were evaluated at several time points after ischemia. 24 h after MCAO, the brains were removed for assays of antioxidants, calcium, H2O2, DNA fragmentation, PARP, caspase-3, release of mitochondrial cytochrome c and p53.
Results: Neurological deficits were significantly decreased in rats treated with rutin. Rutin pre-treatment improved the antioxidant status, diminished the cytochrome-c expression, decrease DNA fragmentation and brain infarct. Furthermore, rutin markedly suppressed the p53 protein expression and calcium level.
Conclusions: These results indicate that rutin attenuates ischemic neuronal apoptosis by inhibiting cytochrome c release, increasing endogenous antioxidant enzymatic activities and reducing the p53 expression. Thus, rutin shows an excellent neuroprotective effect against ischemia/reperfusion brain injury.
103. Ketamine decreased ischemic neuronal damage by shortening the duration of ischemic depolarization; quantitative evaluation in gerbils
H. Taninishi, Y. Takeda, T. Sasaki, K. Shiraishi and K. Morita
Anesthesiology and Resuscitology, Okayama University Medical School, Okayama-city, Japan
Objectives: The effect of ketamine on ischemic neuronal damage was quantitatively evaluated. For this purpose, ischemia of four different intensities was induced in the same experimental model, and ischemic duration necessary for 50% neuronal damage (P50-ischemia) and duration of ischemic depolarization necessary for 50% neuronal damage (P50-depolarization) were determined.
Materials and methods: In 30% oxygen, sixty male gerbils were randomly assigned to a group receiving 1% halothane (H group; n = 30) and a group receiving 1 mg/kg/min of intravenous ketamine (K group; n = 30) Forebrain ischemia was initiated by occlusion of bilateral common carotid arteries for 3, 5, 7 or 10 mins (n = 6 in each group for 3 or 10 mins of ischemia, n = 9 in each group for 5 or 7 mins of ischemia). Duration of ischemic depolarization was recorded from the bilateral hippocampal CA1 regions. Histological outcome in the bilateral hippocampal CA1 region was evaluated five days after the ischemia. During the experimental period, cerebral and rectal temperatures were maintained at 37.0 °C±0.5 °C. Relationships of neuronal damage with ischemic duration and duration of ischemic depolarization were shown by probit curves, and P50-ischemia and P50-depolarization were determined. Statistical analysis was performed with multiple comparisons followed by Scheffe's test. A level of P<0.05 was considered to be significant.
Results: Duration of ischemic depolarization in the K group was shorter than that in the H group in each ischemic duration, and the difference in the case of 7 mins of ischemia was significant (K group versus H group: 7.50±1.91 versus 10.01±1.86 mins). With 5 and 7 mins of ischemia, the percentages of neuronal damage in the K group (21.2%±8.2% and 63.1%±24.7%, respectively) were significantly lower than those in the H group (50.9%±23.8% and 86.5%±19.9%, respectively). P-50 ischemia in the K group and that in the H group were 6.65 and 5.17 mins, respectively. P-50 depolarization in the K group and that in the H group were 7.23 and 6.69 mins, respectively.
Conclusion: Administration of ketamine decreased ischemic neuronal damage. Since duration of ischemic depolarization affects the degree of ischemic neuronal damage,1 reduction in the duration of ischemic depolarization was a factor that contributed to the decrease in neuronal damage by ketamine rather than factors occurring after the onset of ischemic depolarization.
109. Is there any uncoupling of nos during cerebral ischemia and reperfusion? (no production, hydroxyl radical metabolism in mice)
M. Yamazato1, N. Araki2, Y. Asano2, K. Hattori2, Y. Ito2, Y. Kato2, H. Nagoya2, T. Ohkubo2, T. Shimazu2 and K. Shimazu2
1Department of Neurology, Saitama Medical School, Medical Center, Saitama; 2Department of Neulorogy, Saitama Medical University, Moroyama, Japan
Objectives: The transformation of eNOS from a protective enzyme to uncoupled eNOS, which is a contributor of oxidative stress, has been observed in several in vitro models.1 The purpose of this study was to measure both hydroxyl radical and nitric oxide simultaneously in brain during cerebral ischemia and reperfusion, in order to analyze the uncoupling of eNOS.
Methods: Thirteen male C57BL/6 mice were used. Both NO production and hydroxyl radical metabolism were continuously monitored by in vivo microdialysis. Microdialysis probes were inserted into the bilateral striatum. The in vivo salicylate trapping method was applied for monitoring hydroxyl radical formation via 2,3 dihydroxybenzoic acid (2,3-DHBA), and 2,5 dihydroxybenzoic acid (2,5-DHBA). A laser Doppler probe was placed on the skull surface. Blood pressure, blood gases and temperature were monitored and maintained within normal ranges throughout the procedure. Forebrain cerebral ischemia was produced in 7 mice (ischemia group) by occlusion of both common carotid arteries for 10 mins. Six mice were used as sham group. Levels of nitric oxide metabolites, nitrite (NO2−) and nitrate (NO3−), in the dialysate were determined using the Griess reaction.
Results:
Blood Pressure: Ischemia group (73.9±13.5, 84.7±10.7, 79.9±12.4, 79.5±12.9 mm Hg, mean±s.d.) showed significantly higher than that of the sham group (48.1±22.9, 47.2±22.3, 46.4±27.0, 57.5±9.8) in 10 to 40 mins after reperfusion (P<0.05).
Cerebral blood flow: Ischemia group (16.2%±11.0%, 74.2%±22.2%, 68.2%±13.0%, 66.8%±10.6%) showed significantly lower than that of sham group (99.9±4.3, 98.4±11.7, 98.5±11.1, 99.8±7.7) in ischemia and 30 to 50 mins after reperfusion.
The level of NO2−; There was no significant differences between the two groups.
The level of total NO (NO2−+NO3−) [Figure 1]: Ischemia group (4.9±1.0, 5.0±1.0, 5.3±0.8, 5.2±1.2, 5.0±1.2, 5.7±2.6, 5.5±2.7, 5.5±2.2 μmol/L, mean±s.d.) showed significantly higher than that of sham group (3.7±0.98, 3.2±0.6, 3.7±0.9, 3.1±0.7, 2.9±0.8, 3.0±0.5, 3.0±0.7, 3.1±0.6) in 40 to 100 and 120 mins after reperfusion (P<0.05).
The level of 2,3DHBA [Figure 2]: Ischemia group (107.1±7.4%, mean±s.d.) showed significantly higher than that of sham group (99.9±3.2) in 10 mins after reperfusion (P<0.05).
The level of 2,5DHBA was no significant differences between the two groups.
Conclusion: Although NO production increased continuously after cerebral ischemia and reperfusion, hydroxyl radical increased temporarily after the start of reperfusion. These data suggests that uncoupling of NOS may play a little role in cerebral ischemia and reperfusion.
119. Neuroprotective effects of cerebrolysin in animal models of CNS injuries
H.S. Sharma1, S. Zimmermann-Meinzingen2, A. Sharma1 and D.F. Muresanu3
1Surgical Sciences, Uppsala University Hospital, Uppsala, Sweden; 2EBEWE Pharma, Unterach, Austria; 3Neurology, University of Medicine and Pharmacy, Cluj-Napoca, Romania
Previous reports from our laboratory suggest that a suitable combination of neurotrophic factors attenuates CNS pathologies following traumatic or metabolic insults to the brain or spinal cord. Since, Cerebrolysin (Ebewe Pharma, Austria) contains a mixture of neurotrophic peptides, we have undertaken a series of investigations to examine the neuroprotective efficacy of the drug in various animal models of CNS injuries.
We developed a new model of closed head injury (CHI) in which an impact of 0.224 N was applied on the right parietal bone under anesthesia by dropping a weight of 114.6g on the skull from a height of 20 cm through a guide tube. This concussive CHI resulted in profound edema formation and volume swelling at 5 h after the insult that was most pronounced in the contralateral cerebral hemisphere. The microvascular permeability disturbances to protein tracers were prominent in both the cerebral hemispheres and the underlying cerebral structures. Pretreatment with cerebrolysin (10, 20 or 40 μL/min for 10 mins) infused into the left lateral cerebral ventricle either 30 mins before or 30 mins after CHI significantly attenuated brain edema formation, volume swelling and brain pathology. This effect was most pronounced with 20 and 40 μL/min cerebrolysin infusions. On the other hand, no reduction in brain edema, BBB permeability or brain pathology was seen when cerebrolysin was administered 60 mins post-CHI.
Using a new model of spinal cord injury (SCI) in which an incision to the right dorsal horn at the T10–11 level induces profound disruptions of the blood-spinal coed barrier (BSCB) in rats after 5 h, the effects of topical application of cerebrolysin was evaluated. Topical application of cerebrolysin 5 mins after SCI (100, 2000 and 400 μL over 10 mins) over the traumatized cord markedly attenuated spinal cord edema formation at 5 h compared to untreated control group. This effect of cerebrolysin was dose related. In these animals cerebrolysin treatment in high doses (200 and 400 μL) significantly improved the motor functions and reduced the BSCB breakdown, edema formation and cell injury at 5 h. However, these potential beneficial effects of cerebrolysin were absent when the treatment was initiated 60 or 90 mins after SCI.
To further test the neuroprotective efficacy of cerebrolysin, we used a new model of hyperthermic brain injury (HBI). HBI was produced by subjection of animals to 4 h heat stress at 38°C that resulted in massive blood-brain barrier (BBB) disruption and brain edema formation. Pretreatment with cerebrolysin (5 or 10 mL/kg, i.p. 30 mins, before heat stress) markedly attenuated the BBB dysfunction and brain pathology. Interestingly, treatment with high dose of cerebrolysin (10 mL/kg) 30 mins after heat stress was also neuroprotective. However, administration of the drug 60 or 90 mins after the onset of heat stress was ineffective in reducing brain damage.
Taken together these observations suggest that an early intervention with cerebrolysin may have added therapeutic value for the treatment of various cases of CNS injuries.
123. Hemoglobin expression in neurons: dependency on PO2 and EPO
D.W. Schelshorn1, W. Kuschinsky1, A. Schneider2 and M.H. Maurer2
1Department of Physiology and Pathophysiology, University of Heidelberg; 2Sygnis Bioscience, Heidelberg, Germany
Hemoglobin (Hb) is the major protein in red blood cells and transports oxygen from the lungs to the organs. In invertebrates, neuronal Hb appears to serve as intracellular storage molecule for oxygen.
We describe here:
the cellular and area- specific distribution of Hb expression,
the relationship between Hb expression and local oxygenation,
the induction of neuronal Hb expression by erythropoietin (EPO).
Ad 1: Immunohistochemistry shows a specific expression of Hb in neurons of the cortex, hippocampus, olfactory bulb, and cerebellum of the rodent brain, but not in astrocytes and oligodendrocytes. The neuronal Hb distribution is distinct from the neuroglobin expression pattern on both cellular and subcellular levels.
Ad 2: Mice were injected with pimonidazole (Hypoxyprobe-1), a marker substance suitable to detect local oxygen pressures below 10 mm Hg. We verified this feature of pimonidazole by exposing neurons in culture to different degrees of hypoxia. With immunofluorescent double-staining (anti- Hb and anti-pimonidazole), we assessed the distribution of oxygen-deficient regions. A high pimonidazole staining which indicates a low pO2 was found in the somata of neurons of the CA3 and partly of the CA1 region of the hippocampus, as well as in Purkinje cells of the cerebellum. These cells also displayed weak Hb signals. They were often located in direct neighborhood of cells that had strong Hb signals but no indication of low oxygen levels. These findings indicate a reciprocal relationship between Hb expression and hypoxia, Hb -positive neurons being higher oxygenated than Hb- negative neurons.
Ad 3: EPO exerts neuroprotective actions in the CNS. We therefore asked whether EPO might induce Hb expression in neurons and, in parallel, might reduce brain hypoxic areas. Mice were injected with a reported neuroprotective dose and treatment scheme of EPO (5000 IU/kg) 24 h before the onset of hypoxia (8% O2) for 30 mins. In hippocampal sections the area stained by pimonidazole was determined. The hypoxic area increased from 3.8%±2.6% of the total hippocampus area during normoxia to 10.5%±2.0% (mean±s.d.) during hypoxia. The newly appearing hypoxic areas of pimonidazole binding encompassed both cells displaying weak as well as strong Hb signals. Hypoxia appeared in focally concentrated regions rather than evenly distributed throughout the hippocampus. EPO-treated mice showed a significantly reduced pimonidazole-positive area both under normoxic (0.3%±0.3%) and hypoxic (6.0%±1.5%, mean±s.d.) conditions. At the same time, the immunoreactivity for Hb was elevated in EPO-treated animals. Thus, EPO induces neuronal Hb expression and reduces zones of hypoxia both under physiological and hypoxic conditions. We therefore propose a novel and promising mechanism for EPO action in the brain, i.e. counteraction of cellular hypoxia by upregulation of neuronal hemoglobin expression.
We conclude that (1) Hb is expressed specifically by neurons in the brain, (2) local Hb expression is related to tissue oxygenation, (3) EPO can upregulate neuronal Hb expression resulting in an enhanced brain oxygenation under physiological and hypoxic conditions.
159. Brain ischemic postconditioning exerts its neuroprotective effect by modulating asic1a and NCX3 expression in the ischemic penumbra region
G. Pignataro, E. Esposito, O. Cuomo, R. Sirabella, R. Gala, P. Molinaro, G. di Renzo and L. Annunziato
Neuroscience, Federico II University of Naples, Naples, Italy
Background and aims: As clinical trials of pharmacological neuroprotective strategies in stroke have been disappointing, attention has turned to the brain's own endogenous strategies for neuroprotection. Two endogenous mechanisms have been characterized so far, ischemic preconditioning and ischemic postconditioning. However, preconditioning as a strategy to attenuate the pathophysiological consequences of ischemia-reperfusion injury can be applied on pretreatment situations, such as protection before cardiac bypass surgery. A non-pharmacological neuroprotective strategy to be applied after ischemia onset, however, remains illusive. Based on recent studies on the heart (Yellon et al, 2002) and proof of principle experiments in the brain (Burda et al, 2006; Pignataro et al, 2006, 2008; Wang et al, 2008; Zhao et al, 2006), a hypothesis has been offered that a modified reperfusion subsequent to a prolonged harmful ischemic episode may confer ischemic neuroprotection, a phenomenon termed postconditioning.
To study how this clinically relevant neuroprotective strategy exerts its effect many transcriptional mechanisms have been taken into consideration. In particular, the well known family of Mitogen Activated Kinases (MAPK) has been proposed as important mediator of this mechanism of neuroprotection.
Methods: Ischemic postconditioning has been induced in 8 weeks old SD male rats as previously described (Pignataro et al, 2008). Briefly, anaesthetized rats were subjected to 100 mins of middle cerebral artery occlusion by intraluminal suture method. After 10 mins of reperfusion, the filament was reintroduced and kept in situ for 10 more minutes. Protein and mRNA expression was evaluated at different time points by Western Blotting and RT-PCR, respectively.
Objectives: The main objective of this study was to evaluate the effect of MAPK activated by postconditioning on the expression of Acid Sensing Ionic Channels (ASIC) and Na+/Ca2+ exchangers (NCXs). These two plasmamembrane proteins are expressed in the CNS and are able to control the intracellular Na+ and Ca2+ homeostasis and involved in the progression of the ischemic lesion (Annunziato et al, 2004; Pignataro et al, 2004, 2008).
ASIC and NCX mRNA and protein expressions were investigated in the penumbra region of rats subjected to harmful ischemia or ischemic postconditioning and treated with specific MAPK inhibitors or with vehicle alone.
Results: Results of this study showed that NCX1, NCX3 and NCKX2 are up-regulated in those brain regions protected by postconditioning treatment, whereas ASIC1a expression is dramatically reduced in the same brain regions.
These changes in ASIC and NCX expression seem to be mediated by p-AKT. In fact, treatment with LY- 294002, a specific p-AKT inhibitor, was able to revert the postconditioning neuroprotective effect and to prevent variations in NCX and ASIC expression.
Conclusions: The results obtained in the present study indicate that NCX1, NCX3, NCKX2 and ASIC1a are involved in the neuroprotection mediated by p-AKT and elicited by ischemic postconditioning and may represent important targets in setting on a new strategy for stroke treatment.
187. Clarification of the anti-stress effects of angiotensin II AT1 receptor blockers and their mechanisms of action
M. Honda1,2, E. Sanchez-Lemus1 and J.M. Saavedra1
1Section on Pharmacology, National Institutes of Mental Health, NIH; 2JSPS Research Fellow in Biomedical and Behavioral Research at NIH, NIH, Bethesda, Maryland, USA
Objectives: Peripheral administration of Angiotensin II (Ang II) AT1 receptor blockers (ARBs) prevents the hypothalamus-pituitary-adrenal (HPA) axis stimulation during the psychological stress of isolation. We wished to determine, in models of acute psychological and physical stress, the effect of ARB administration on the HPA axis response and on the expression of brain Ang II receptors.
Methods: Adult male Wistar Hanover rats were treated for three days with s.c. administration of the ARB candesartan (1 mg/kg/day), followed by 2 h restraint in plastic cylinders, a model of psychological stress, or by 2 h of immobilization to a metal board, a model of psychological and physical stress.
Results: Peripherally administered candesartan significantly blocked all brain AT1 receptors including those in the PVN. There was increased plasma corticosterone, ACTH and aldosterone concentrations after restraint and immobilization stress. Candesartan did not prevent these increases. Both restraint and immobilization stresses increased AT1A mRNA expression selectively in the PVN, and candesartan prevented this upregulation produced by restraint stress. Acute restraint stress increased benzodiazepine-1(BZ-1) binding in cingulate, frontal, parietal cortex. Candesartan blocked these increases in the frontal and layer IV of parietal cortex. In the pituitary gland, significant AT1A mRNA increase and AT1B mRNA decrease were observed after immobilization stress. In adrenal zona glomerulosa, AT1 binding and AT1B mRNA decreased after restraint or immobilization stress. In adrenal medulla, AT1A and tyrosine hydroxylase mRNA increased after restraint and immobilization stress. However these changes were not prevented by ARB treatment.
Conclusions: Our results implicate ARBs in the PVN, pituitary and adrenal gland in the regulation of the response to psychological and physical stresses. There is a differential response of AT1 receptor subtypes during stress. Our results suggest that the control of the HPA axis stimulation by ARBs depends on the type and intensity of the stress challenge. On the other hand, our findings about the BZ-1 binding in some cortical areas suggest that the anti-anxiety properties of ARBs are a general phenomenon common to isolation and restraint stress. Our observations support the hypothesis of limited anti-stress effects of ARBs, depending on the stress type, intensity and duration.
234. Increasing OMEGA-3 intake with a diet rapeseed oil-enriched diet prevents from middle cerebral artery occlusion induced-damage
N. Blondeau1,2, B. Delplanque3, C. Gandin1, G. Agnani3, N. Simon-Rousseau4 and C. Heurteaux1,2
1Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UMR 6097, Valbonne; 2University of Nice—Sophia Antipolis, Nice; 3University of Paris Sud, Orsay; 4ONIDOL, Paris, France
Objectives: Cardiovascular and cerebral diseases are major public Health concerns in Western countries, whose populations have a severe deficiency in omega-3 intake, especially in alpha-linolenic acid (ALA). This is described as risk factors of coronary heart disease and stroke. The primary cause of stroke is an impairment of local blood flow resulting in neurovascular unit damages. Until now none of acute and preventive pharmacological therapies that have been tested demonstrates the capacity to preserve both arterial and neuronal function. Nevertheless, several studies suggest beneficial effects of diets rich in seafood and vegetable oils in cerebral diseases. The aim of this work was to evaluate preventive strategy with ALA on cerebral ischemia investigating particularly whether diet enriched with rapeseed oil, rich in ALA could prevent the damage induced by middle carotid artery occlusion (MCAO).
Methods:
To induce focal ischemia, we used the MCAO model in mice described as the closest to human. This intraluminal suture technique reliably induces neurological deficits and hemispheric infarcts restricted to the territory of the MCA 24 h after the onset of ischemia. The regional cerebral blood flow (CBF) was monitored by laser-Doppler flowmetry during ischemia and 15 mins after the filament withdrawal to assess the reperfusion level. Only animals whose cerebral blood flow during ischemia had decreased below 20% of the pre-ischemic level were selected for histological analyses. 24 h post-ischemia, we quantified the infarct volume on serial brain slices stained with Cresyl violet and the survival rate.
ALA supplementation was performed either by an ALA injection (500 μmol/kg) or by diet enriched with rapeseed oil (5%, 10%, 20%, provided by ONIDOL). As control, we used vehicle injection, palm oil enriched diet, lacking in ALA or regular diet (SAFE R03T-25).
Results: We had previously shown that single ALA injection preconditions the brain triggering tolerance in a rat model of global ischemia. We have now confirmed in mice that an ALA injection 3 days before a 60 mins MCAO reduced by half the infarct volume 24 h post-ischemia (P<0.05) and an ALA level increase might be a preventive strategy against focal ischemia. This led us to introduce the consumption of rapeseed oil (5%, 10%, 20%) in long-term diets in mice. A 5% rapeseed enriched diet (Rape-SS5) had no effect on the infarct volume. In contrast a 10% diet (Rape-SS10) drastically reduced the infarct volume by 45% (P<0.01) and a 20% rapeseed diet (Colza-SS20) by 30% (N.S). As control, a 5% palm oil diet, rich in palmitic acid failed to induce neuroprotective effects. Another parameter of evaluation, possibly of more importance concerns the reperfusion. 77.5% of the Rape-SS5 mice did not reperfused 30 mins after the filament withdrawal, contrary to the Rape-SS10 (33.3%). This outcome may not be interpreted as actual reduction of the infarct volume but may be essential in the prediction of the overall outcome.
Conclusion: Our results provide further evidence for a therapeutical value of alpha-linolenic acid enriched diet as preventive treatment of brain injury resulting from focal ischemia/reperfusion.
271. Pretreatment with ethyl eicosapentaenoic acid ameliorates ischemic brain damage in a rat transient focal ischemia model
M. Ueda, T. Inaba, Y. Nishiyama, S. Okubo, S. Suda, N. Kamiya, C. Nagata, H. Nagayama and Y. Katayama
Neurology, Nippon Medical School, Tokyo, Japan
Introduction: Long-chain n-3 polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are derived from marine products, have attracted considerable attention, because fish consumption is inversely related to stroke risk in a meta-analysis of cohort studies.1 In addition, a recent clinical controlled trial has shown that ethyl EPA (EPA-E) reduces stroke recurrence in Japanese hypercholesterolemic patients.2 The present study examined neuroprotective effects of EPA-E pretreatment using a rat transient focal ischemia model.
Methods: Under halothane anesthesia, 24 male Sprague-Dawley rats, weighing 250 to 300g, were subjected to 90 mins focal ischemia using an intraluminal suture technique following an overnight fast. EPA-E (100 mg/kg/day) or vehicle was orally administered once a day for seven days prior to ischemia induction. Rectal temperature was maintained at 37°C during ischemia and up to 2 h after reperfusion, and physiological parameters, including arterial blood pressure, arterial blood gases and blood glucose level, were determined several times during the surgical procedures. In addition, cortical blood flow was monitored during ischemia and up to 2 h after reperfusion using Laser-Doppler flowmetry. Neurological scores were assessed based on hemiparesis and abnormal posture using grading scale3 at 24 h after reperfusion, and then animals were decapitated (n = 7, each) or perfusion-fixed using 4% paraformaldehyde (n = 5, each) following blood examination for serum lipid profiles and fatty acid levels (EPA, DHA, gamma-linoleic acid, arachidonic acid). Infarct and edema volumes were determined using 2,3,5-triphenyltetrazolium chloride (TTC) stained brain sections (n = 7, each), and TUNEL staining and immunohistochemistry using antibodies against 8-hydroxydeoxyguanosine (8-OHdG) or 4-hydroxy-2-nonenal (4-HNE) were performed to examine apoptosis, oxidative DNA damage and lipid peroxidation (n = 5, each). Statistical significance was set at P<0.05.
Results: All physiological variables were within normal limits. Serum EPA levels, but not others, were significantly increased in the EPA group than the vehicle group. Cortical blood flow was decreased to approximately 20% of baseline during ischemia without any statistical differences between the groups. EPA-E treated rats showed significant reduction in infarct and edema volumes, as well as significantly improved neurological scores, compared with the vehicle treated animals. Immunohistochemistry revealed significant decrease in numbers of 8-OHdG, 4-HNE and TUNEL positive cortical cells in the EPA group than the vehicle group.
Conclusion: The present study showed that pretreatment with EPA-E (100 mg/kg/day) for 7 days ameliorated ischemic brain damage following transient focal ischemia without any cortical blood flow changes in rats, and that reduced oxidative stress might be involved in the neuroprotective mechanisms of EPA-E.
290. Type III phosphodiesterase inhibitor provide protective effects against post-stroke complications through cell signaling pathway of creb phosphorylation
T. Urabe, N. Zhang, N. Miyamoto, T. Watanabe, Y. Ueno, Y. Tanaka, R. Tanaka and N. Nattori
Neurology, Juntendo University School of Medicine, Tokyo, Japan
Background and aims: The number of patients afflicted with cerebral infarction is on the increase at present and stroke is a leading cause of disability worldwide, with no effective clinical treatment that enhances recovery. Post-stroke cognitive impairment, depressive disorders, and pneumonia are recognized complications in such patients. The present study was designed to assess the neuroprotective mechanisms of an inhibitor of type III phosphodiesterase (PDE III-I, cilostaqzol), through signaling pathways, which lead to activation of transcription factor cyclic adenosine monophosphate (cAMP) responsive element binding protein (CREB) phosphorylation using rat chronic cerebral hypoperfusion model.
Methods: Adult male Wister rats (8-week-old) weighing 250 to 270g underwent bilateral common carotid artery ligation (LBCCA). They were divided into the PDE III-I group (n = 167) and the vehicle (control) group (n = 167). Performance at the Morris water maze task and immunohistochemistry for 4-hydroxy-2-nonenal (HNE), GST-pi, Iba-1, phosphorylated CREB (p-CREB), Bcl-2, and cyclooxygenase (COX)-2 were analyzed at baseline and at days 3, 7, 14, 21, and 28 days after hypoperfusion. Elicited swallowing reflex, positive cultures of bacteria from the lung and immunohistochemistry for tyrosine hydroxylase (TH), substance P, and phosphorylated CREB (pCREB) were evaluated at baseline and at days 14, 28 and 42 after LBCCA. We also measured the levels of dopamine and substance P in the striatum.
Results: PDE III-I significantly improved spatial learning memory (6.8±2.3 secs; P<0.05) at 7 days after hypoperfusion. Cilostazol markedly suppressed accumulation of HNE-modified protein and loss of GST-pi-positive oligodendrocytes in the cerebral white matter during the early period after hypoperfusion (P<0.05). PDE III-I up-regulated p-CREB and Bcl-2 (P<0.05), increased COX-2 expression, and reduced microglial activation in the early period of hypoperfusion. PDE III-I treatment significantly improved the swallowing reflex by shortening the latency to swallowing and increasing the numbers of swallows (P<0.05) at 14 days of hypoperfusion. It decreased the positive culture of bacteria from the homogenized lung as well as the number of colony in the positive cultures. PDE III-I treatment markedly increased TH expression in the substantia nigra, and maintained dopamine (84.7%±2.3% versus 79.2%±4.1% control; P = 0.0512) and substance P levels (86.6%±7.9% versus 73.9%±6.5% control; P<0.05) in the striatum.
Conclusions: We have shown that PDE III-I provides protection against cerebral hypoperfusion-induced cognitive impairment and swallowing disturbance via white matter damage in a rat LBCCA model. The results suggest that PDE III-I has potential therapeutic and brain protective effects based on multi-target mechanism through cell signaling pathway of CREB phosphorylation, and may be helpful in the treatment of patients suffering from post-stroke complications.
307. Metformin attenuates Type 2 diabetes-induced brain oxidative imbalance
C. Carvalho1, S. Correia1, M. Santos1, R. Seiça2 and P. Moreira3
1Center for Neuroscience and Cell Biology and Department of Zoology, University of Coimbra; 2Faculty of Medicine, University of Coimbra; 3Center for Neuroscience and Cell Biology, Coimbra, Portugal
Type 2 diabetes is a well known metabolic disorder that usually occurs in people over 30 years old and is characterized by a relative insulin deficiency and an insulin resistance that leads to a state of hyperglycemia. There is considerable evidence that many biochemical pathways adversely affected by hyperglycemia are associated with the generation of reactive oxygen species (ROS), ultimately leading to increased oxidative stress in a variety of organs including the nervous system. The brain is one of the most important targets for ROS, due to its high levels of polyunsaturated fatty acids, high oxygen consumption, high content in transition metals (e.g., Fe2+) and poor antioxidant defenses. Metformin is a widely used drug for the management of type 2 diabetes. This oral antidiabetic agent is a biguanide derivative whose main actions are to reduce hepatic glucose production and increase glucose uptake. Due to these effects, metformin is considered an insulin sensitizer. Despite the success of this antidiabetic agent in the treatment of type 2 diabetes, its mechanisms of action remain unclear. Although some antioxidant properties have been attributed to metformin, the antioxidant action of this antidiabetic agent in brain tissue has not yet been evaluated. In this line, this study was designed to evaluate the effect of metformin treatment in the brain of diabetic Goto-Kakizaki (GK) rats (a nonobese, spontaneously type 2 diabetic model). For this purpose we compared brain homogenates obtained from untreated GK rats versus GK rats treated with metformin during a period of 4 weeks. Brain homogenates obtained from Wistar rats were used as control. Several parameters were analysed: lipid peroxidation [thiobarbituric acid reactive substances (TBARS) and malondialdehyde (MDA) levels] and protein oxidation (carbonyl groups) markers, H2O2 levels and non-enzymatic [reduced glutathione (GSH) and vitamin E] and enzymatic [glutathione peroxidase (GPx), glutathione reductase (GRed) and manganese superoxide dismutase (MnSOD)] antioxidant defenses. Compared to control rats, brain homogenates of GK rats presented a significant increase in the levels of MDA, GPx and GRed activities and a significant decrease in MnSOD activity supporting the idea that type 2 diabetes promotes an oxidative imbalance in brain tissue. However, metformin treatment normalized the majority of the parameters altered by diabetes. Besides its antihyperglycemic action, metformin induced a significant decrease in the levels of lipid peroxidation markers and GPx and GRed activities. Additionally, this antidiabetic agent promoted a significant increase in the levels of GSH and in the activity of MnSOD. These results indicate that metformin protects against diabetes-associated oxidative stress suggesting that metformin could be an effective neuroprotective agent.
Cristina Carvalho has a PhD fellowship from the Fundação para a Ciência e a Tecnologia (SFRH/BD/43965/2008).
329. Histological and neurobehavioral outcomes in isoflurane preconditioned ischemic male mouse brain may be dihydrotestosterone dose-specific
W. Zhu1, L. Wang2, J. Palmateer1, N. Libal1, P. Herson1, P. Hurn1 and S. Murphy1
1Anesthesiology and Peri-Operative Medicine, Oregon Health and Science University, Portland, Oregon, USA; 2Neurology, Affiliated Drum Tower Hospital, Nanjing University, Nanjing, China
Objective: Isoflurane preconditioning (IsoPC) neuroprotection in experimental stroke is male-specific and testosterone-dependent.1,2 Testosterone can be converted via 5α-reductase to the more potent androgen and pure androgen receptor agonist, dihydrotestosterone (DHT). We hypothesized that DHT would have similar or greater neuroprotective effects than testosterone on histological and neurobehavioral outcomes in IsoPC ischemic male brain.
Methods: C57BL/6 mice were castrated (CAST) and received no hormone or subcutaneous DHT pellets (0.5, 1.5, 5 mg) 7 days before preconditioning. Male and CAST+DHT mice were preconditioned for 4 h with air (sham preconditioning, Sham PC) or 1.0% IsoPC 24 h before 2 h of middle cerebral artery occlusion. At 22 h reperfusion, cortical and striatal infarct volumes (% contralateral structure) were determined by digital image analysis of coronal brain slices stained with 2,3,5-triphenyltetrazolium chloride. Based on infarct outcomes, the lowest neuroprotective DHT dose (0.5 mg) was used to assess neurobehavioral outcomes in sham PC and IsoPC CAST+DHT mice (n = 13 to 15 per group) for 9 days following ischemia. Paw preference test (PPT), locomotor activity in the open field (OF), passive avoidance (PA) and novel object recognition task (ORT) were done to analyze forelimb asymmetry, spontaneous locomotor activity and cognitive function.
Results: IsoPC decreased infarct volumes in male and CAST+0.5 DHT mice but had no effect in CAST, CAST+1.5 DHT and CAST+5 DHT groups (Table 1). Long-term behavioral assessment revealed that IsoPC in CAST and CAST+0.5 DHT prevented impairment in PPT. In addition, 0.5 mg DHT in the absence of IsoPC improved PPT outcome. In contrast, IsoPC reduced impairment in ORT in CAST mice, but this effect was blocked in CAST+0.5 DHT mice. OF and PA outcomes were equivalent among groups.
Conclusions: Histological protection from IsoPC in experimental stroke is androgen-dependent, with intact males being protected and neuroprotection being lost in androgen-deficient CAST mice. Furthermore, IsoPC neuroprotection relative to histological outcomes is DHT-dependent and dose-specific, with IsoPC reducing infarct volumes only in CAST+0.5 DHT mice and having no effect in CAST mice treated with higher DHT doses. However, IsoPC neuroprotection appears to be DHT-independent relative to neurobehavioral outcomes. Neurobehavioral studies showed that IsoPC minimized contralateral forelimb impairment in CAST mice regardless of 0.5 mg DHT treatment status. In addition, 0.5 mg DHT alone improved forelimb asymmetry regardless of preconditioning status. Finally, IsoPC improved cognitive function in CAST mice, with the effect being blocked by 0.5 mg DHT. Further studies are needed in ischemic male brain to clarify whether DHT has a causal or independent role in IsoPC neuroprotection relative to histological and neurobehavioral outcomes.
337. Progesterone promotes a dose-specific neuroprotective response to isoflurane preconditioning in ischemic ovariectomized female mouse brain
W. Zhu, N. Libal, P. Hurn and S. Murphy
Anesthesiology and Peri-Operative Medicine, Oregon Health and Science University, Portland, Oregon, USA
Objective: The response to isoflurane preconditioning (IsoPC) in focal stroke is sexually dimorphic, with male and ovariectomized female mice being protected by IsoPC, but IsoPC neuroprotection is lost in females and estradiol-treated ovariectomized mice.1,2 We evaluated the progesterone (PROG) dose relationship in the ischemic sensitivity of IsoPC female mouse brain.
Methods: Young adult C57BL/6 female mice were ovariectomized (OVX) and received no hormone or subcutaneous PROG pellets (7.5 or 15 mg) 7 to 8 days before preconditioning and experimental stroke. Intact females, OVX, OVX+7.5 mg PROG, and OVX+15 mg PROG were preconditioned for 4 h with air (sham preconditioning, Sham PC) or 1.0% IsoPC. Mice then underwent 2 h of middle cerebral artery occlusion via intraluminal filament 24 h after preconditioning. Laser-Doppler flowmetry (LDF) was used to monitor cortical perfusion. Brains were collected at 22 h reperfusion. Cortical and striatal infarct volumes (% contralateral structure) were determined by digital image analysis of 2 mm thick coronal brain slices stained with 2,3,5-triphenyltetrazolium chloride.
Results: Relative LDF changes were equivalent among groups. In contrast to corresponding Sham PC groups, IsoPC increased cortical and striatal infarct volumes in intact females (Table). However, IsoPC decreased cortical and striatal infarct volumes in OVX and OVX+PROG (7.5 or 15 mg) mice, with the greatest reduction in infarction volumes being seen in OVX+15 mg PROG group (Table).
Conclusions: IsoPC worsened ischemic injury in young female mice, but ovariectomizing young females and thus significantly decreasing endogenous female sex steroid levels resulted in a neuroprotective response to IsoPC, suggesting that one or both female sex steroids (estradiol, progesterone) may be responsible for the response to IsoPC observed in young ischemic female brain. Furthermore, progesterone promoted IsoPC neuroprotection in female ischemic brain in a dose-specific manner, with IsoPC reducing infarct volumes comparably in OVX and OVX mice treated with 7.5 mg progesterone but having the greatest neuroprotective effect in OVX mice treated with 15 mg progesterone. More studies are needed to further delineate the role of progesterone in the brain's response to IsoPC as well as determine the mechanisms underlying how progesterone might promote a protective response to IsoPC in female ischemic brain.
342. Protective effects of lithium supplementation on aluminium-induced functional and structural alterations in rat brain
P. Bhalla and D.K. Dhawan
Department of Biophysics, Panjab University, Chandigarh, India
Background and aims: The present study was conducted to assess the role of lithium in conditions where aluminium (Al) toxicity leads to the chain of molecular events which can initiate and promote neurodegeneration.
Methods and purpose: To carry out the various investigations, Al was administered at a dose of 100 mg/kg b.wt./day whereas lithium was supplemented in diet (1.1g/Kg diet, daily) for the period of two months.
Results: Ca2+ ATPase activity was observed to be decreased in Al treated animals. Whereas, a significant increase in Ca2+ influx (via the voltage-operated calcium channels) and in the levels of cAMP were observed following Al treatment which were decreased with lithium co-administration. Further, a significant increase in the levels of phopholipase C (PLCÝ1) was observed following Al treatment which was normalized following lithium supplementation. The nitric oxide synthase enzyme activity and the levels of L-citrulline were also found to be significantly increased in different brain regions (cerebrum and cerebellum) after Al treatment, which were significantly decreased following lithium supplementation. The DNA damage caused in the cell as a result of Al treatment was examined by single cell gel electrophoresis as well as DNA fragmentation studies and a significant increase in the DNA damage was observed which was found to be significantly improved upon lithium supplementation. Alterations in the neuronal histoarchitecture and ultra structure were also observed following Al treatment. Lithium supplementation greatly restored normalcy in the cerebrum and cerebellum layers with no loss of cerebral or purkinje cell layer as evident by light microscopy. Further, lithium supplementation to Al treated rats resulted in appreciably thwarting the ultrastructural changes with regard to integrity of the cells as a whole as well as the cell organelles as observed by transmission electron microscopy. The study demonstrates that lithium, an antidepressant drug, has the potential in containing or reversing the Al-induced functional and structural changes as evidenced by oxidative stress, DNA damage and altered calcium homeostasis as well as disordered signal cascade in experimental animals.
Conclusion: Al has been linked to neurodegeneration for decades and thus based on the neuroprotective effect of lithium in the brain in this animal system, we suggest that lithium should be considered and be exploited further for the prevention and therapy of brain disorders.
385. Neuroprotective effects of indole-3-propionic acid via reducing oxidative stress in the ischemic hippocampus
1Department of Anatomy and Cell Biology, College of Veterinary Medicine, Seoul National University, Seoul; 2Department of Anatomy and Neurobiology, College of Medicine, Hallym University, Chuncheon, South Korea
Background and aims: One of acceptable hypotheses is that cellular events involving reactive oxygen species (ROS) mediated oxidative damage may evoke neuronal damage. In normal condition, the ROS produced as by-products of mitochondrial electron transport chain are quenched by antioxidants and converted to non-toxic compounds by free radical scavenging enzymes. However, in aging and related neurodegenerative diseases, the accumulation of free radicals in tissues is found and can result in cell dysfunction and death. Since oxidative stress is frequently associated with mitochondrial dysfunction, antioxidants are able to penetrate intracellular compartments and ensure protection close to the ROS production site should exhibit a higher bioavailability than those remaining in the extracellular compartment or cytoplasmic membrane. Tryptophan-derived indole compounds have been widely investigated as antioxidants and as free radical scavengers. In these compounds, indole-3-propionic acid (IPA) is a deamination product of tryptophan and possesses a heterocyclic aromatic ring structure such as melatonin. It has been reported that IPA is equivalent to melatonin in scavenging free radicals and protecting against oxidative damage.
Methods: We used the progeny of male Monglian Gerbils (6 months of age). IPA (10 mg/kg) treatment for 15 days before transient ischemia. After induction of transient forebrain ishcemia, we performed the the following activity to elucidate the protective effects of IPA; Spontaneous motor activity, Cresyl violet staining, Fluoro-Jade B (F-J B) histofluorescence staining, Immunohistochemistry for NeuN, GFAP, S-100, vimentin, Iba-1, Isolectin B4, HNE and 8-OhdG and Western blot for GFAP and Iba-1.
Purpose: There is no report on the effects of IPA against ischemic damage in vivo. In this study, therefore, we examined neuroprotective effects of IPA and its anti-oxidative effects in the hippocampus after 5 mins of transient forebrain ischemia in gerbils.
Results: We investigated the neuroprotective effects of IPA against ischemic damage and anti-oxidative effects in the hippocampal CA1 region after 5 mins of transient forebrain ischemia. The repeated oral administration of IPA (10 mg/kg) for 15 days before ischemic surgery protected neurons from the ischemic damage. In this group, the number of cresyl violet positive neurons in the CA1 was 56.8% compared to that in the sham group. In the vehicle-treated group, glial fibrillary acidic protein (GFAP)-, S-100- and vimentin-immunoreactive astrocytes, and Iba-1 and isolectin B4 (IB4)-immunoreactive microglia were activated 4 days after ischemia/reperfusion, whereas in the IPA-treated ischemic group, GFAP, S-100, Iba-1 and IB4 immunoreactivity was distinctly lower, but not in vimentin immunoreactivity, than those in the vehicle-treated ischemic groups. The administration of IPA significantly decreased 4-HNE, a marker for lipid peroxidation, levels in ischemic hippocampal homogenates compared to that in the vehicle-treated ischemic groups at various time points after ischemia/reperfusion. In addition, in the IPA-treated ischemic groups, DNA damage, using immunostaining for 8-hydroxy-2′-deoxyguanosine, in pyramidal neurons in the ischemic CA1 was significantly lower than that in the vehicle-treated ischemic groups.
Conclusions: IPA protects neurons from ischemic insult with deceases of lipid peroxidation and DNA damage in the ischemic hippocampal CA1 pyramidal neurons.
387. Preconditioning or postconditioning with sevoflurane can induce neuroprotection against focal cerebral ischemia in rats
H. Wang1, S. Lu2, Q. Yu1, H. Gao2, W. Liang1, J. Chen2,3 and Y. Gao2
1Department of Anesthesiology, Huashan Hospital, Fudan University; 2State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, China; 3Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
Background and purpose: The phenomenon of ischemic tolerance in the heart was first reported by Murry et al in 1986,and that of ischemic preconditioning (PC) in the brain was reported by Kitagawa et al four years later. Since then, studies on the mechanism and pharmacological agents to mimic ischemic preconditioning have been intense. Zhao et al elucidated firstly that ischemic postconditioning could improve the outcomes of cardio ischemia/reperfusion insult in 2003. Recent data demonstrate that the PC effect of sevoflurane (Sevo), a comprehensively used volatile anesthetics clinically, is valid in vitro. It's unknown that the impact of Sevo pretreatment or post-administration in vivo and the precise mechanism. In this study, we preconditioned or postconditioned with Sevo to investigate the neuroprotective effect of Sevo against ischemic neuronal injury.
Methods: Male Sprague-Dawley rats were allocated to sham, vehicle, Sevo PC, Sevo postconditoning groups (n = 8 to 10/group). In the PC groups, the Sevo 1 and Sevo 2 groups received 0.5 or 1 minimum alveolar concentration (MAC) Sevo PC in air 30 min/d after 15 mins equilibrium for 4 consecutive days, whereas postconditioning animals inhaled 1MAC Sevo in air once for 30 mins at 10 mins, 30 mins or 2 h after reperfusion. Animals were subjected to focal ischemia for 60 mins by filament occlusion of the middle cerebral artery with the monitor of regional cerebral blood flow at 24 h after Sevo pretreatment or before Sevo post-intervention. Rectal temperature was servo-controlled at 37°C to 38°C and physiological parameters were detected and maintained according to baseline status throughout the experiment. Neurologic assessment was performed 3 days after ischemia. The volume of cerebral infarction were determined by image analysis of cresyl voilet-stained coronal brain sections. Animals were euthanized at 0, 6, 12 and 24 h after reperfusion for detecting caspase3, caspase9 and apoptosis inducing factor (AIF) by immunofluorescence and Western blotting tests.
Results: Infarction volume was less in the Sevo 1 and Sevo 2 groups (13.6%±13.3% and 13.9±5.7 respectively, mean±s.d.) than in vehicle group (46.6±9.3, P<0.01and P<0.001), but there were no significant difference between Sevo 1and Sevo 2 groups (P>0.05). In comparison with the vehicle, the post-administration of sevoflurane at 10 and 30 mins after reperfusion significantly decreased infarction volume (19.8±14.3, 26.4±18.3, P<0.05). Similarly, the sevoflurane pre- and post-treatment at 10 and 30 mins after reperfusion groups demonstrated better neurologic function than the vehicle group (P<0.05). Brains with ischemic insult without Sevo-treatment showed increase in caspase3, caspase9 and AIF, otherwise, Pre- or post-intervention with Sevo restored the levels of those factors.
Conclusion: The repeated preconditioning of sevoflurane may provide neuroprotection against focal cerebral ischemia in dose-independent manner and the effect of sevoflurane postconditioning is available in a short therapeutic window. Suppression of apoptosis responses may contribute to neuroprotection of sevoflurane.
411. The effect of PDE3 inhibitor cilostazole on CRE activity in human neuronal cell line
T. Sasaki1, Y. Yagita2, Y. Terasaki1, E. Omura-Matsuoka1, N. Ohyama1, Y. Sugiyama2, S. Okazaki2, S. Sakoda2 and K. Kitagawa2
1Division of Stroke Center, Department of Internal Medicine; 2Department of Neurology, Osaka University Graduate School of Medicine, Osaka, Japan
Background: Cilostazole is therapeutically approved as antiplatelet drug for use in patients with ischemic stroke. Recently, Cilostazole has been shown to have neuroprotective activities and inhibit white matter damage. Cilostazole generates various downstream biological activities. Among these pleiotropic effects, the activation of cAMP responsive element binding protein (CREB)—CRE cascade has been reported to contribute to their neuroprotective activity in animal model. In order to directly examine the relationship, we have chosen to utilize a cell culture-based assay using human neuronal cell lines.
Subjects and methods: We used M03.13 (human oligodendrocyte cell line), SH-SY5Y (human neuroblastoma cell line), and 1321N1 (human astrocyte cell mine). To directly detect the CRE transcription activity, we have transfected adeno-CRE reporter to these cell lines and measured the CRE activity using luciferase assays. M03.13 were cultured in DMEM+10% fetal bovine serum (FBS)+antibiotics, and then replaced with the DMEM medium without FBS to differentiate to mature oligodendrocyte. SH-SY5Y were maintained in DMEM+10% fetal bovine serum (FBS) including 10 μmol/L all-trans retinoic acid to differentiate to neuronal lineage. Cilostazole (1, 3, 10, 30 μmol/L), the type χ phosphodiesterase (PDE) inhibitor rolipram (30 μmol/L), and adenylate cyclase activator forskolin (5 μmol/L) were administered for 24 h.
Results: In M03.13, cilostazole significantly increased the CRE transcription activities with dose dependent manner. On the other hand, cilostazole did not significantly enhance the CRE activity in both SH-SY5Y and 1321N1, in contrast to be treated by rolipram and forskolin.
Conclusion: The effect of cilostazole on CRE activity was different between human cell types. Activation of cAMP-CREB signaling in oligodendrocyte by cilostazole may be one of the mechanisms of their protective effects and may have implications for therapeutic intervention to white matter damage via the activation of CREB-CRE signaling.
423. Protective effect of hisperidin on transient focal cerebral ischemia in male wistar rats
S. Raza and F. Islam
Department of Toxicology, Hamdard University, New Delhi, India
Introduction: The adult brain consumes 3 to 4 mL O2/min per 100g tissue, and this can represent up to 20% of all inhaled oxygen. When cerebral circulation is altered, oxygen consumption in the ischemic zone is dramatically reduced, thus inducing significant neuronal injury (Chan 2001). An imbalance between oxidant and antioxidant has been postulated to lead the neurodegeneration in cerebral ischemia-reperfusion injury (Mao-Tsun Lin, 2002).
Hypothesis: In this study, we examined whether Hisperidin, an antioxidant can prevent or slowdown neuronal injury in cerebral ischemia.
Lipid peroxidation.
Antioxidant enzymes activity.
Materials and methods: Animals were pretreated with oral Hisperidin (25 mg/kg for 15 days). The middle cerebral artery of adult male Wistar rats was occluded for 1 h and reperfused for 22 h (Longa et al 1989).
Results: Hisperidin was found a good antioxidant in up-regulating the antioxidant status, lowering the TBAR's level and recovering results close to baseline.
Discussion: The activity of antioxidant enzymes and content of GSH was decreased significantly in MCAO group as compared with sham. The rats of MCAO+Hisperidin group have shown a significant protection in the activity of above-mentioned antioxidant enzymes and content of glutathione when compared with MCAO group. Increase in lipid peroxidation after MCAO in ipsilateral and contralateral hemisphere of brain was observed which was reduced by Hisperidin treatment. Further Histopathological changes and its attenuation in drug treated animal confirm the result.
Conclusion: These results suggest the neuroprotective potential of Hisperidin in cerebral ischemia and is mediated through its antioxidant activity.
427. Neuroprotective effects of metaplexis japonica on global and focal cerebral ischemia rat models
D. Lee1, N.R. Pandit1, N. Jamarkattel1, J.-G. Kim2, M.-Y. Kim1, M. Song1, J. Park2, D. Lim1, J.Y. Kim1, Y. Bu1 and H. Kim1,2
1Herbal Pharmacology, College of Oriental Medicine, Kyung Hee University; 2Korea Institute of Science and Technology for East Medicine (KISTEM), Neumed Co. Ltd, Seoul, South Korea
Objectives: Metaplexis japonica (Apocynaceae) is a perennial herb, extensively used in traditional chinese medicine like consumptive disease, impotence, seminal emission, white vaginal discharge, agalactia, scroful. The purpose of our study was to evaluate the protective effect of M. japonica and its different fractions against in vitro and in vivo ischemia.
Methods: In the present study, we fractionated the 70% EtOH extract of M. japonica with different polarity solvents and administered the resulting fractions at a dose 100 mg/kg to the rats and subjected to the middle cerebral artery occlusion (MCAo) rat model and four-vessel occlusion (4-VO) model of global ischemia. Oxygen-glucose deprivation followed by reoxygenation was used to investigate the effects of M. japonica in neuronal cell line. We used the intraluminal suture method induced by the transient MCAo for 2 h followed by 24 h reperfusion in rats. After 24 h of MCAo, the infarction volume was measured by TTC staining. Functional effects of M. japonica and its fractions were investigated by Rota-rod and balance beam test 20 h after MCAo. 4-vessel occlusion was induced for 10 mins followed by 7 d reperfusion.
Results: The effect on neuronal damage was measured 7 days after ischemia. Infarct volume after rats were subjected to 2 h MCAo and 24 h reperfusion was found as 33.0%±1.6% in vehicle treated group whereas M. japonica (30 and 100 mg/kg) showed 27.2%±3.6% and 16.1%±4.0% (P<0.01), respectively. Hexane, EtOAc and BuOH fractions treated group showed 24.9%±5.2%, 12.6%±2.9% (P<0.001) and 19.2%±4.2% (P<0.01) infract volume respectively.
Conclusions: M. japonica and its fractions protected the hippocampal neuronal cell death, reduced brain damage and improved behavioral deficits in stroke rat model.
Acknowledgments: This work was supported by a grant (PF 0320201–00) from Plant Diversity Research Center of 21st Century Frontier Research Program (Ministry of Science and Technology, Korea), and by grants from the Second Stage of Brain Korea 21 Project (Ministry of Education, Korea).
464. Rosuvastatin protects cultured cortical neurons against oxygen-glucose deprivation and glutamate excitotoxicity
F. Domoki1, B. Kis2, T. Gáspár3, F. Bari1, J.A. Snipes3 and D.W. Busija3
1Department of Physiology, University of Szeged, Szeged, Hungary; 2Department of Radiology Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; 3Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina, USA
Objectives: Statins are known to exert pleiotropic effects unrelated to the inhibition of cholesterol biosynthesis. We examined if rosuvastatin (RST) preserved viability of cultured neurons after oxygen-glucose deprivation (OGD) and glutamate (L-glut) excitotoxicity.
Methods: Primary rat cortical neurons were isolated from E18 rat fetuses.1 Cultures 5 to 7 days in vitro (DIV) were treated with RST and/or mevalonate, geranylgeranyl pyrophosphate (GGPP), and cholesterol for 1 to 3 days. On 8 DIV, the cultures were exposed to 3 h OGD or L-glut (200 mmol/L, 1 h). Neuronal viability was determined 24 h after completion of the experiments. Additional experiments were performed to determine whether perillic acid, a non-specific inhibitor of both geranylgeranyl transferase GGT 1 and Rab GGT, and the GGT 1-specific inhibitor, GGTI-286, would reproduce the neuroprotective effect of RST.
Results: Three-day pretreatment with RST (5 μmol/L) increased neuronal survival after OGD from 44%±1% to 82%±2%*, and after L-glut (0.5 μmol/L RST) from 56%±1% to 89%±4%* (% of untreated controls, mean±s.e.m., n = 24 to 96, *P<0.05). One-day RST treatment was not protective against either stress. RST-induced neuroprotection against OGD was abolished by mevalonate coapplication; however, RST-induced protection against L-glut was not sensitive to mevalonate. These data indicate that inhibition of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMGCoA) reductase is involved in the mechanism of protection against OGD but not L-glut. The neuroprotective effect of RST against OGD was also abolished by GGPP but not by cholesterol coapplication, and was replicated by perillic acid but not by GGTI-286. RST and perillic acid but not GGTI-286 reduced neuronal ATP and membrane Rab3a protein levels, and they reduced post-OGD reactive oxygen species (ROS) levels determined with fluorescent detection of hydroethidin-ethidium conversion. Neuronal glutathione (GSH) and antioxidant protein (Mn superoxide dismutase (SOD), CuZnSOD, GSH peroxidase, catalase) levels were unaffected by RST. In RST-treated neurons, L-glut-evoked increases in intracellular Ca2+ levels—determined with Fluo4 AM fluorescence/confocal microscopy—were reduced by ∼50%, and similar reduction in L-glut-induced ROS generation was observed.
Conclusions: RST elicits delayed preconditioning-like neuroprotection in cultured neurons against OGD and L-glut excitotoxicity. However, the mechanism of protection is markedly different against the two stresses. The neuroprotective mechanism of RST against OGD involves inhibition of HMGCoA reductase leading to depletion of GGPP and decreased geranylgeranylation of proteins which are probably not isoprenylated by GGT 1. Reduced neuronal ATP levels and ROS production after OGD may be directly involved in the mechanism of neuroprotection. In contrast, RST-induced neuroprotection against excitotoxic stress appears to be independent of HMGCoA reductase inhibition. Instead, L-glut results in reduced neuronal Ca2+ influx and subsequent reduction in ROS generation in RST-treated neurons that can contribute to the neuroprotection.
Grant support: National Institute of Health (HL30260, HL65380, HL77731), National Scientific Research Fund of Hungary (OTKA K68976, K63401). Ferenc Domoki was supported by tthe János Bólyai Research Scholarship of the Hungarian Academy of Sciences.
496. Post-synaptic signaling components and caveolin-1 decrease with age in membrane/lipid rafts and synaptosomal membrane fractions isolated from murine brains
B.P. Head1, J. Bonds1, D.M. Roth1, J. Peart2, H.H. Patel1 and P.M. Patel1
1Anesthesia, VA/UCSD, San Diego, California, USA; 2Heart Foundation Research Center, Griffith University, Goldcoast, QLD, Australia
Objective: Spines/synaptic densities decreases in aged brains.1,2 Recent work has demonstrated that membrane/lipid rafts (MLRs) are essential for the development and stabilization of synapses and that synaptic residents involved in neurotransmission and neurotrophic signaling (e.g., NMDARs, AMPARs, Trk receptors, p75NTR, SFKs), are compartmentalized to MLRs and that their expression and localization to these discrete microdomains is dependent upon the cholesterol binding/scaffolding protein caveolin (Cav).3–8 MLRs, enriched in cholesterol, glycosphingolipids, and sphingomyelin, are essential for development/stabilization of synapses. We have previously demonstrated that Cav-1 expression is necessary for NMDAR-mediated signaling and neuroprotection against oxygen-glucose deprivation (OGD) in primary neurons in vitro.3 We therefore assessed whether there exists age-related changes in synaptic signaling components within MLRs and synaptosomes (Syn).
Methods: Sucrose density fractionation methods were used to isolate MLRs and Syn from hippocampi of wild type (WT) C57BL/6 mice [young (Yg, 3 to 6 months), mature (Mt, 12 months), and aged (Ag, 24 months)] and Cav-1 KO mice (Yg). For MLRs, tissue was homogenized in 150 mmol/L sodium carbonate (pH 11.0), and mixed with 1 mL of 80% sucrose to generate 2 mL of 40% sucrose. Above the 40% layer, 6 mL of 35% and 4 mL of 5% sucrose were layered and centrifuged at 175,000g for 3 h at 4°C. Samples were removed in 1 mL aliquots and MLRs were found in buoyant fractions 4 to 5 (5/35% interface). For isolation of synaptosomes, tissue was homogenized in 5 mL of solution A (0.32 mol/L sucrose, 0.5 mmol/L CaCl2, 1 mmol/L NaHCO3, 1 mmol/L MgCl2) containing protease and phosphatase inhibitors and then centrifuged for 10 mins at 1000g at RT to remove large debris. Supernatant was layered onto 4 ml of 1.2 mol/L sucrose and centrifuged at 160,000g for 15 mins. Synaptosomes (interface between the 1.2/0.32 mol/L) were mixed with 4 mL of 0.32 mol/L sucrose, layered onto 4 mL of 0.8 mol/L sucrose, and centrifuged at 160,000g for 15 mins to yield a pellet enriched in synaptosomes. Pellet was resuspended in 1 mL of lysis buffer containing protease and phosphatase inhibitors and used for immunoprecipitation (IP) and/or immunoblot analysis.
Results: PSD-95 (post-synaptic density marker), the ionotropic glutamate receptors NMDAR2A (NR2A), NR2B, AMPAR, and Cav-1 were detected in MLRs from Yg brains, with less detection in MLRs from Mt and Ag brains. Cav-1 and PSD-95 IPs of MLRs showed a decrease in NR2A, NR2B, AMPAR, PSD-95 and Cav-1 in both IPs from Mt and Ag MLRs compared to Yg. There was also a significant decrease in PSD-95, NR2A, NR2B, and Cav-1 in Syn and PSD-95 IPs of Syn with age. Hippocampi Syn from Cav-1 KO mice (Yg) showed reduced PSD95, NR2A, and Cav-1 compared to WT (Yg).
Conclusions: These data indicate that Cav-1 expression and its ability to organize signaling complexes may be a key determinant of age-related changes in synaptic signaling in addition to changes that result from certain neurological disorders (i.e., Alzheimer’, Parkison’, and Prion disease).
497. TRPM2 channels contribute to male-specific cortical neuron cell death following oxygen-glucose deprivation
P.S. Herson, S. Verma, Y.-F. Yang, E. Oyarazabel and P.D. Hurn
Department of Anesthesiology and Peri-Operative Medicine, Oregon Health and Sciences University, Portland, Oregon, USA
Objective: Excessive production of reactive oxygen species (ROS), termed oxidative stress, is a major contributor to ischemia-induced neuronal cell death in vitro and in vivo. Data from our group and others have demonstrated that oxidative stress activates the transient receptor potential-M2 (TRP-M2) non-selective cation channel resulting in cell death,1,2 suggesting that TRPM2 may participate in ischemia-induced neuronal cell death. However, no direct link between TRPM2 channel activation and ischemic neuronal death has been established. Therefore, we examined the effect of genetic and pharmacological inhibition of TRPM2 channels on neuronal survival in vitro following oxygen-glucose deprivation.
Methods: Male and female neurons were cultured separately from cortex of embryonic day 18 (E18) C57BL/6 mice. The TRP inhibitors, 2-Aminoethoxy diphenyl borate (2-APB), N-(p-Amylcinnamoyl)anthranilic acid (ACA) or clotrimazole were applied to the culture medium 20 minsprior to OGD and maintained throughout OGD and re-oxygenation (ReOX). Cortical cultures were infected with 3rd generation pseudo-type lentivirus containing shRNA, or empty vector, and expressing green fluorescent protein (GFP) by applying 1 to 2 μL virus (>106 infective particles/mL) directly to the culture media (1 mL). Cells were visualized by fluorescent microscopy (infected cells green). Forty-eight hoursafter transduction, cultures were exposed to oxygen-glucose deprivation (OGD) for 2 h after which normoxia was re-established in culture media(ReOx). Cell death was assayed 24 h after ReOx by detecting the release of lactate dehydrogenase (LDH) and cell survival assayed using colorimetric assay of MTT conversion to formazan.
Results: Exposure to the TRP inhibitor 2-ADP (100 μmol/L) significantly protected male cortical neurons from injury, decreasing cell death from 17.3%±2.3% (n = 3) to 7.1%±1.8% (n = 3; P<0.05). In contrast, the application of 2-ADP had no effect on survival of female cortical neurons (n = 2). Similar results were obtained with two additional TRP channel inhibitors, clotrimazole and ACA. Infection of cultures with shRNA lentivirus produced efficient knockdown of TRPM2 as determined by quantitative RT-PCR, and resulted in significantly reduced male neuronal cell death, decreasing from 28.9%±4.5% (n = 7) to 18.3%±6.5% (n = 7). No further neuroprotection was observed in male neurons infected with shRNA and additionally exposed to TRP channel inhibitors. Consistent with TRP inhibitor data, no protection was observed in female neurons infected with TRPM2 shRNA lentivirus.
Conclusions: Sexual dimorphism in ischemic injury after stroke is well documented and the effect of sex steroids in outcome has been well established. Using sex-specific cortical neuronal cultures, we report a gender-specific role for TRPM2 channel activation in neuronal damage following in vitro ischemia. These data indicate that differential TRPM2 channel regulation following ischemia contributes to sex-specific outcome and that inhibition of TRPM2 channels may provide a novel neuroprotective strategy for improving stroke outcomes in men.
499. Involvement of HIF pathway in the protective mechanisms of aged garlic extract in ischemia
C.D. Gomez-Martinez1, P. Aguilera2, M. Espinoza-Rojo3, P.D. Maldonado2, A. Ortiz-Plata4 and M.E. Chanez-Cardenas2
1Universidad Autonoma de Guerrero; 2Patologia Vascular Cerebral, Instituto Nacional de Neurologia y Neurocirugia; 3Laboratorio de Biologia Molecular, Universidad Autonoma de Guerrero; 4Neuropatologia, Instituto Nacional de Neurologia y Neurocirugia, Mexico, Mexico
Ischemia and reperfusion generate oxidative stress hence the use of antioxidants diminish disease progression. Aged garlic extract (AGE) is an odorless garlic presentation with antioxidant properties. In middle cerebral artery occlusion (MCAO) model in rats the administration of AGE at the onset of reperfusion (R) significantly reduced the size of the infarct area. The treatment with AGE prevented the increase in nitrotyrosine positive cells and the decrease in antioxidant enzymes activity induced by MCAO. Several antioxidant compounds like vitamin E and quercetin decrease damage induced by ischemia partially through the hypoxia inducible factor (HIF) pathway. In order to determine if this pathway is involved in the protective mechanism of AGE, we evaluated the expression of HIF-1 alpha and its target genes hemooxygenase-1 (HO-1) and glucose transporter 1 and 3 (GLUT-1 and-3).
Methods: Rats were subjected to MCAO for 2 h, treated with 1.2 mL/kg/i.p. of AGE at the beginning of reperfusion (R) and sacrificed after 2 h (2I/2R group). Brain damage was determined measuring the infarct area by 2,3,5-triphenyltetrazolium chloride staining and evaluating motor impairment.1 Total RNA was obtained from frontoparietal cortex and concentration was determined spectrophotometrically. Five micrograms of RNA were used to synthesize the cDNA. TaqMan probes were used for detection of HIF-1alfa, HO-1, GLUT-1 and -3 mRNA. 18S ribosomal RNA was used as an internal control. Amplification was performed utilizing an ABI PRISM 7500 sequence detection system from Applied Biosystems. Data were calculated utilizing the DCt method. Data are expressed as mean-fold change±s.d. and analyzed by one-way analysis of variance (ANOVA) followed by a post hoc Tukey test (SPSS 13.0). P<0.05 was considered statistically significant.
Results: AGE reduced the infarct area in 2I/2R+AGE group by 70%. However, this treatment itself was not associated with a noticeable improvement in the neurological outcome. HIF-1 alpha mRNA in 2I/2R group (0.62±0.04-fold) showed an increase with the AGE treatment (1.84±0.12-fold). After this result we decided to analyze the response of HIF-1 target genes HO-1, GLUT-1 and -3. HO-1 mRNA showed an increase after 2I/1R and 2I/2R (16.23±4.19 and 48.93±20.70-fold respectively). 2I/2R+AGE group did not induce any significantly change (61.24±18.35-fold). After 1 h R, GLUT mRNA increased (GLUT-1, 2.43±0.77 and GLUT-3, 3.16±0.48 -fold) and returned to basal level after 2 h R (GLUT-1, 1.03±0.33 and GLUT-3, 1.33±0.15 fold). AGE treatment after 2 h R induces an increase in GLUT-3 mRNA in control+AGE and 2I/2R+AGE groups (6.62±2.95 and 3.44±1.42 -fold, respectively).
Conclusions: The increase observed in HO-1 and GLUT-1 mRNA in the 2I/1R group could be associated to the HIF-1 alpha protein stabilization or by activation of other transcription factors as part of brain endogenous protective mechanisms. Interestingly, HIF-1 alpha and GLUT-3 mRNA was increased by AGE treatment. Since HIF-1 alpha and its target genes could contribute to the establishment of tolerance in the brain, it is possible that AGE is contributing to protect brain through the activation of this pathway.
501. Ischemia causes delayed and sustained removal of functional synaptic SK2 channels
P.S. Herson1, D. Allen1,2, M. Kuroiwa1, C. Bond2, J. Maylie3, R. Lujan4 and J.P. Adelman2
1Department of Anesthesiology and Peri-Operative Medicine; 2Vollum Institute; 3Department of Obstetrics and Gynecology, Oregon Health and Sciences University, Portland, Oregon, USA; 4Departmento de Ciencias Medicas, Universidad de Castilla-La Mancha, Albacete, Spain
Objective: We have shown that synaptic SK2 channel activity in CA1 neurons dampens glutamate-mediated excitability and Ca2+ influx through NMDAr,1 and that the induction of long term potentiation (LTP) results in the immediate endocytosis of synaptic SK2 channels from potentiated CA1 spines.2 In addition, our recent data indicates that pharmacological enhancement of SK channel activity protects CA1 neurons against cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Therefore, we utilized electrophysiology and immuno-EM techniques to examine the effect of CA/CPR on synaptic SK channels.
Methods: C57BL/6 male mice underwent 8 mins. CA/CPR, as described previously.3 For electrophysiology experiments, transverse hippocampal slices were prepared using our published protocols.2 Current-clamp recordings were obtained to record excitatory post-synaptic potentials (EPSPs) from visualized CA1 neurons, with solutions; ACSF solution (mM): 119 NaCl, 2.5 KCl, 1 NaH2PO4, 26.2 NaHCO3, 1.3 MgCl2, 2.5 CaCl2, 10 Dextrose, and aerated with 95% O2/5% CO2; SR95531 (5 μmol/L) and CGP55845 (2 μmol/L) present to reduce GABAA and GABAB contributions, respectively; Internal pipette solution (mmol/L): 140 KMeSO4, 8 NaCl, 1 MgCl2, 10 HEPES, 2 Mg-ATP, 0.4 Na2-GTP, and 20 μmol/L EGTA, pH 7.3. Post-embedding immunohistochemistry and electron microscopy (immuno-EM) were performed as described previously2 and radial distance from the post-synaptic density (PSD) was analyzed.
Results: The effect of ischemia on synaptic SK2 channels was examined by recording evoked subthreshold EPSPs from CA1 neurons in acute hippocampal slices. Blocking SK2 channels with apamin significantly increased the amplitude of EPSPs in slices from mice taken 15 mins after CA/CPR (197%±28% (n = 4) of baseline; P<0.05) equivalent to the effect of apamin in sham-operated control mice (196%±29% (n = 9); P<0.05). The effect of apamin was significantly reduced at 3 h post CA/CPR (129%±4% (n = 4); P<0.05 compared to Sham controls), and reduction observed at 3 h was prevented by injection of mice with a protective dose of 1-EBIO (16 mg/kg) 30 mins prior to CA/CPR (190%±12% (n = 3); P<0.05 compared to CA/CPR+vehicle). We performed immuno-EM labeling to determine the precise sub-spine distribution of SK2 channels in hippocampal slices from sham mice and 3 h after CA/CPR. Analysis of radial distance revealed that SK2 immunogold particles were predominantly observed in the synapse (PSD) in sham mice (80%) and that 3 h after CA/CPR very few SK2 immunogold particles were observed in the PSD (8%), while most were observed to be intracellular within spines (92% >20 nm from PSD).
Conclusions: The electrophysiology data presented indicates that CA/CPR causes a functional loss of synaptic SK2 channels at a time-point between 15 mins and 3 h of resuscitation. Consistent with this functional data, post-embedding immuno-EM data shows that CA/CPR causes endocytosis of synaptic SK2 channels. Therefore, our data indicates that ischemia causes a physical and functional loss of synaptic SK2 channels, which may underly the increased sensitivity to excitotoxicity of hippocampal CA1 neurons.
S. Hou1, S. Jiang1, B. Zurakowski1, A. Desbois1 and J. Kappler2
1Institute for Biological Sciences, National Research Council Canada, Ottawa, ON, Canada; 2Institut für Physiologische Chemie, Rheinische Friedrich-Wilhelms-Universität Bonn, Bonn, Germany
Background and aims: Calpains belong to a highly conserved family of calcium-dependent proteases which cleave a large number of substrates including cytoskeletal elements such as spectrins, and important regulatory proteins such as CDK-5 and collapsin mediator proteins (CRMPs). In the central nervous system, calpains are widely expressed and their activities are modulated by an endogenously-expressed inhibitory protein, calpastatin. Calpain activity can be inhibited by synthesized inhibitors, such as ALLN and calpeptin, which also provide potent neuroprotection against excitotoxicity. CRMPs are important brain-specific proteins with distinct functions during normal development and under disease conditions. CRMPs, also referred to as TOAD-64 (turned on after division 64 kDa), Ulip (UNC-33-like protein) or DRP (dihydropyrimidinase-related phosphoprotein), are a family of five cytosolic proteins discovered a decade ago with a role in collapsing and repelling growth cones. CRMPs can mediate the collapse of growth cones through the regulation of actin and tubulin cytoskeletons. In the present study, we provided evidence to show that all CRMP family members are targeted for cleavage by calpain activated in ischemic mouse brain.
Methods: All procedures for cerebral ischemia produced by middle cerebral artery occlusion (MCAO) using C57/black mice were approved by the local Animal Care Committee. Under temporary isofluorane anesthesia, mice were subjected to MCAO using an intraluminal filament as previously described. After 1 h of MCAO, the filament was withdrawn, blood flow restored to normal by laser Doppler flowmetry and wounds sutured. Animals were sacrificed after 24 h of reperfusion. Brain infarction was measured and brain tissues were subjected to protein extraction for Western blotting, and immunostaining analyses to determine changes of CRMPs.
Results: Among the five CRMPs, the expressions of CRMP1, CRMP3 and CRMP5 were the most abundant in the cerebral cortex and that all CRMPs were targeted for cleavage by ischemia-activated calpain. Subcellular fractionation analysis showed that cleavage of CRMPs by calpain occurred not only in the cytoplasm but also in the synaptosomes isolated from ischemic brains. Moreover, synaptosomal CRMPs appeared to be at least one fold more sensitive to cleavage compared with those isolated from the cytosolic fraction in an in vitro experiment, suggesting that synaptosomal CRMPs are critical targets during cerebral ischemia-induced neuronal injury. Finally, the expression of all CRMPs was co-localized with TUNEL positive neurons in the ischemic mouse brain which further supports the notion that CRMPs may play an important role in neuronal death following cerebral ischemia.
Conclusions: Collectively, these studies demonstrated that CRMPs are targets of calpains during cerebral ischemia and they also highlighted an important potential role that CRMPs may play in modulating ischemic neuronal death.
535. Critical role of serine-specific phosphorylation in the activation of HSP27 and its neuroprotective effect against ischemic neuronal injury
R. Stetler1,2,3, G. Cao1,2,3, G. Yanqin3, F. Zhang1,2, S. Wang1,2, Z. Weng1, P. Vosler1, L. Zhang1 and J. Chen1,2,3
1Neurology, University of Pittsburgh School of Medicine; 2Geriatric Research, Education and Clinical Center, Veterans Affairs Pittsburgh Health Care System, Pittsburgh, Pennsylvania, USA; 3State Key Laboratory of Medical Neurobiology, Fudan University School of Medicine, Shanghai, China
Background and aims: Other than thrombolytic therapy, an effective neuroprotective treatment for stroke is currently unavailable. Abnormal activation of cell death programs likely contributes to the pathogenesis of ischemic brain injury, as suppression of apoptosis via alteration of signaling pathways of death program promotes neuronal survival and improves functional recovery in rodent stroke models. HSP27 is a member of the small heat shock protein family, a group of ubiquitous stress proteins that are expressed in virtually all organisms. The expression of HSP27 is induced in the brain after cerebral ischemia, and overexpression of HSP27 has been found to be a potent neuroprotective molecule. In addition to its known function as a protein chaperone, recent studies suggest that HSP27 has apoptosis-suppressing effects in many cell death models, including ischemia. In nonneuronal systems, HSP27 gains anti-apoptotic properties after undergoing serine-specific phosphorylation. We have recently demonstrated that HSP27 interacts with and suppresses activated ASK1 via the N-terminal region of HSP27 and the kinase domain of ASK1. The N-terminal region of HSP27 contains critical serine residues required for phosphorylation; however, the precise mechanism underlying the effects of HSP27 phosphorylation on neuroprotection against ischemic insults is not fully understood.
Methods: In order to address the effects of HSP27 phosphorylation on neuroprotection against ischemic insults, we have used both an in vitro cortical culture model (oxygen/glucose deprivation) as well as the in vivo transient focal ischemia model. We created a transgenic mouse line overexpressing either wildtype HSP27 or an HSP27 phosphorylation mutant where three critical serine residues (at positions 15, 78 and 82) were mutated to alanines to yield a non-phosphorylatable mutant (HSP27-ala). Assessment of ASK1 activity, activation of downstream kinases, mitochondrial cell death signaling and cell survival were compared between transgenic strains and wildtype mice.
Purpose: In this study, we sought to determine if phosphorylation of HSP27 is required for inhibition of ASK1 activity and cell death signaling following cerebral ischemia.
Results: We have found that tg-HSP27 mice, but not tg-HSP27-ala mice, exhibit decreased infarct area following focal cerebral ischemia. Wildtype tg-HSP27 undergoes rapid phosphorylation and shifts to lower molecular weight oligomeric structures following ischemic injury, whereas the non-phosphorylatable mutant tg-HSP27-ala shifts to larger oligomers. The overexpression of HSP27 was neuroprotective in cortical cultures exposed to OGD, but HSP27-ala had no effect on cell survival. HSP27, but not HSP27-ala, physically interacted with activated ASK1 and suppressed downstream kinase signaling and mitochondrial cell death signaling following ischemic insults. In a cell-free system, neither purified recombinant HSP27 nor HSP27-ala was capable of inhibiting activated ASK1; however, when phosphorylated in vitro, phospho-HSP27 effectively inhibited ASK1 activity in a dose-dependent manner.
Conclusions: Our results suggest that phosphorylation of HSP27 is required for neuroprotection and suppression of ASK1 activity following ischemic injury. The interaction between phosphorylated HSP27 and activated ASK1 demonstrates a novel mechanism of HSP27 neuroprotection and provides a tangible basis for the development of small molecule inhibitors targeting ASK1 activity based on an HSP27 phosphorylation-specific structure.
544. CART inhibits neurotoxic effect of Aβ by affecting Aβ metabolism and Aβ-induced oxygen stress in vivo and in vitro
Y. Xu1, L. Zhang1, F.N. Niu2 and H. Jiang1
1Department of Neurology, The Affiliated Drum Tower Hospital of Nanjing University Medical School; 2Department of Neurology, Drum Tower Hospital of Nanjing Medical University, Nan Jing, China
Background and aims: The accumulation and deposition of Amyloid β protein (Aβ) (neuritic plaques) in the brain are the key pathological features of Alzheimer's disease (AD) even through its mechanism has not been completely understood. β-site amyloid precursor protein cleaving enzyme (BACE1) cleaves the amyloid precursor protein (APP) to promote Aβ generation while neprilysin (NEP) functions as one of the most important Aβ degradation proteases. Current therapeutic guideline on AD includes blocking Aβ overproduction and its neurotoxicity such as oxygen stress. Our previous work indicated that CART (cocaine and amphetamine regulated transcript) had neuroprotection on stroke. The aims of this study are:
To further investigate the neuroprotective role of CART on AD through inhibiting neurotoxicity induced by Aβ in vitro and in vivo.
To explore a potential mechanism of the neuroprotection by CART.
Methods: Aβ treats primary cortex neurons as an AD model in vitro. B6C3-Tg APP transgenic mouse serves as an AD model (10 months old) in vivo. Then CART or vehicle was injected into mouse lateral ventricles in APP mouse. CART was added into the primary neurons 24 h after Aβ treatment 24 h. Neuronal viability or neuronal cell death was assayed by MTT or Fluorescence Activated Cell Sorter (FACS) analysis. Learning/memory impairment of the mice was tested by Water maze behavioral test. The levels of mRNAs or proteins from cells and brains were measured by real time PCR or Western blot. Mitochondrial depolarization and reactive oxygen species (ROS) production were measured by fluorescence Microscopy and FACS analysis.
Results: Neuronal survival by MTT after 24 Aβ treatment was higher in CART-treated cultures (0.279±0.039, n = 3, each in triplicate) compared to vehicle-treated cultures (0.199±0.029 n = 3 in triplicate, P<0.05). Similar results were obtained using FACS analysis. Water maze behavioral test indicated that CART administration shortened the mean escaping latency of the APP mice compared to vehicle-treated mice from 67.91±19.45 to 59.33±17.95 (n = 10 per group, P<0.05). CART suppressed mRNA and protein expression of BACE1 and Aβ, while increased expression of NEP in the cortex and hippocampus of APP brain (BACE1 mRNA from 0.215±0.013 to 0.139±0.019; BACE1 protein from 1.210±0.015 to 0.723±0.022; Aβ mRNA from 0.237± 0.022 to 0.119±0.038; Aβ protein from 1.068±0.122 to 0.661±0.093; NEP mRNA from 0.579±0.028 to 0.721±0.013; NEP protein from 0.059±0.014 to 0.075±0.034. P<0.05). Finally, CART reduced Mitochondrial depolarization and ROS induced by Aβ (from 5806.33±51.67 to 4999±22.73, P<0.05).
Conclusions: The findings suggest that CART is an important neuroprotectant against neurotoxicity induced by Aβ in vitro and in vivo. The mechanism of this neuroprotection is in part linked to reducing overproduction by affecting on generation, degradation of Aβ and blocking oxygen stress toxicity of Aβ. Thus, CART and its mechanism of action may serve as a novel therapeutic strategy against AD-related brain damage.
553. Neurotoxic roles of microglial NF-KappaB and P2Y12 in an in vitro ischemia model
C. Webster1, H. Ma1, R. Giffard2 and M. Yenari1
1Neurology, UCSF-SFVAMC; 2Anesthesia and Neurosurgery, Stanford University School of Medicine, San Francisco, California, USA
Following cerebral ischemia insults, microglia, brain immune cells, have been shown to become activated, initiating a complex inflammatory cascade via the key inflammatory player, nuclear transcription factor kappa B (NFkappaB), which plays a dual role in neuroprotection as well as exacerbating neurotoxicity. There is evidence that NFkappaB is activated by extracellular nucleotides, which have been shown to be generated by cells following necrotic insults such as ischemia. Here, we aim to establish the role of microglial NFkappaB as neurotoxic or neuroprotective following the in vitro ischemic model, oxygen glucose deprivation (OGD). Furthermore, P2Y12, a G-protein coupled purinergic receptor that is activated by extracellular ATP or ADP, also the target for the anti-coagulant drug clopidogrel, has been shown to be involved in microglial chemotaxis following injury, and may activate NFkappaB via the G-protein coupled PKC pathway. Thus, we investigate the role of P2Y12 in microglial migration and neuron cell death following OGD. To determine the role of microglia following in vitro ischemia, we co-cultured microglial cells or primary microglia with neurons and astrocytes (1:10:10) or microglia plus neurons (1:10), as well as astrocyte/neuron (1:1) and neuron only mixtures, and subjected these mixed cell cultures to OGD and reperfusion (as previously described) or control conditions, and measured neuron viability via MAP-2 ELISA. When microglial cells (either BV2 or primary) were co-cultured with both astrocytes and neurons, they increased neuron cell death following OGD two-fold (n = 6, P<0.05) relative to cultures of neurons alone, neuron/astrocyte, neuron/microglial cells, or no OGD controls. We also show that knocking down either NFkappaB (p65 or p50) or P2Y12 in microglial cells, via siRNA transfection, significantly protected neurons in the triple culture model (neurons, BV2, astrocytes) following OGD compared to control triple cultures exposed to OGD (n = 6, p50 P<0.05, p65 P<0.05, P2Y12P<0.05). Furthermore, microglial cells in the triple cultures formed clusters around neurons and astrocytes following OGD with a four fold higher cluster score compared to their no OGD controls (n = 6, NFkappaB p65 <0.01, P2Y12P<0.01). This data taken together suggests that astrocytes are required for microglial mediated neuron cell death following in vitro ischemia, and that the NFkappaB pathway in microglia, which seems to involve the purinergic receptor P2Y12, significantly contributes to microglial migration toward injured neurons, and neurotoxicity in this model.
554. Effect of plant extracts on ischaemia and reperfusion induced cerebral injury
R. Shri, K.S. Bora and S. Arora
Phamaceutical Sciences and Drug Research, Punjabi University, Patiala, India
Stroke is the rapidly developing loss of brain functions due to a disturbance in the blood vessels supplying blood to the brain. This can be due to ischemia or due to a hemorrhage.
Ischemic stroke is a syndrome characterized by rapid onset of neurological injury due to interruption of blood flow to the brain.3 Free radicals have been implicated in cerebral ischemia and reperfusion-induced neuronal injury.4,5 Levels of antioxidant enzymes fall during ischemia while free radical production increases during reperfusion. Many synthetic anti-oxidants have shown neuroprotective effect in ischemia and reperfusion-induced cerebral injury.6,7 Naturally occurring plant contents such as phenolic compounds, carotenoids, ascorbic acid, thiols, and tocopherols have also shown anti-oxidant activity.8,9
Objective: The present study has been designed to investigate the neuroprotective effect of extracts of two plants possessing antioxidant activity viz Allium cepa and Ocimum basilicum on Ischemia and Reperfusion induced cerebral ischemia in Swiss albino mice.
Methods: Global cerebral ischemia was induced by bilateral carotid artery occlusion for 10 mins followed by reperfusion for 24 h.10 Cerebral infarct size was estimated using triphenyltetrazolium chloride (TTC) staining.11 Elevated plus maze was employed to estimate short term memory. Degree of motor incoordination was evaluated using inclined beam walking test. Mitochondrial Thiobarbituric acid reactive substances (TBARS) assay was employed as an index for oxidative stress.
Results: Global cerebral ischemia followed by reperfusion produced a significant impairment in short term memory, motor coordination and a significant increase in TBARS. Pretreatment with methanolic extract of outer scales and edible portion of A.cepa (100 and 200 mg/kg, p.o.) and methanolic and ethyl acetate extracts of leaves of O.basilicum (100 and 200 mg/kg, p.o.) reduced cerebral infarct size, attenuated impairment in short term memory and motor coordination along with a marked decrease in mitochondrial TBARS. The effect was more significant with methanolic extract of outer scales of A.cepa and ethyl acetate extract of leaves of O.basilicum.
Conclusions: Pretreatment with test extracts may scavenge reactive oxygen species and consequently attenuate ischaemia and reperfusion induced cerebral injury.
575. Brain lipid peroxidation, radical scavenging effects and antitumor activity of taiwanese botany—YY-24 on human glioblastoma cells
L.L. Yang1, Y.W. Yu1 and W.T. Huang2
1Department of Pharmcognosy, College of Pharmacy, Taipei Medical University, Taipei; 2Division of Hemato-oncology/Chi-Mei Medical Center, Tainan, Taiwan R.O.C.
In the nervous system, reactive oxidative species (ROS) can cause neurodegenerative disorders, like Alzheimer′s disease (AD), Huntington′s diease (HD), Parkinson′s disease (PD), amytrophic lateral sclerosis (ALS) and ataxia telangiectasia. Overproduction of ROS results in oxidative stress that can be an important mediator of damage lipids, proteins, and nucleic acids. Antioxidants prevent cellular damage by scavenging and inhibition of free radicals and prevention of pathologies. Oxidative stress has been implicated in the pathogenesis, including aging, atherosclerosis, diabetes mellitus, chronic inflammation and cancers. Gliomas are the most common type of primary brain tumor. Nearly two-thirds of gliomas are highly malignant lesions that account for a disproportionate share of brain tumor-related morbidity and mortality. Despite many technologic advances in neuroimaging, neurosurgery, and radiation therapy, there has been little improvement in survival for patients with malignant glioma. Approximately 17,000 primary brain tumors are diagnosed every year, and of those, about 60% are gliomas. Tumors of gliol origin such as glioblastoma multiforme comprise the majority of human brain tumors. In this paper, 30 kinds of Taiwanese Botany extracts were isolated from herbal medicine in our laboratory and their effects on 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, superoxide anion radical scavenging, superoxide anion radical inhibition, ferrous ions chelating activities, ferric ion reducing antioxidant power (FRAP), lipid peroxidation, cell proliferation and potential cytotoxicity. The level of lipid peroxidation (LPO) was expressed as thiobarbituric acid reactive substances (TBARS). LPO product of MDA(TBA)2 was induced by ferrous chloride and ferrous ammonium sulfate respectively on Wistar rat liver mitochondria and brain homogenate. Otherwise, the human malignant glioblastoma cell lines of GBM 8401 (astrocytoma-glioblastoma, grade III) and U-87 MG (astrocytoma- glioblastoma, grade IV) were used. We established screening program employing the 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay system utilizing human malignant glioblastoma cells. One of the most potential compounds, YY-24 exhibited a significantly antioxidant and brain homogenate lipid peroxidation. It had exhibited a dose dependent and time dependent manner in human malignant glioblastoma cells. The IC50 value of YY-24 after 24 h were 175.03±9.88 and 200.00±0.02 μg/mL in GBM 8041 and U-87 MG, respectively. In addition, we had observed the apoptotic bodies in GBM 8401 and U-87 MG. YY-24 may medicate the cytotoxicity via apoptosis in GBM 8401 and U-87 MG. Furthermore, we well evaluated the mechanism of apoptosis by western blot in the future.
583. Carbamylated erythropoietin (I-125-CEPO) as potential radiotracer for imaging neuroprotection in cerebral ischemia
A. Thiel1,2, P. Rosa-Neto1, E. Schirrmacher3, H. Schipper1,2 and R. Schirrmacher2,3
1Neurology and Neurosurgery, McGill University; 2Neurological Sciences, SMBD Jewish General Hospital and Lady Davis Institute for Medical Research; 3Cyclotron Unit, McConnell Brain Imaging Centre, Montreal, QC, Canada
Objectives: Erythropoietin (EPO) and its derivatives have recently been shown to be neuroprotective in animal models of stroke, trauma and encephalitis1 as wll as in a pilot study in human stroke.2 This neuroprotective effect is thought to be mediated by the Epo-ßc-heteroceptor complex which is expressed in low concentration in the normal brain but is up-regulated in situations where the brain is in need for neuroprotection.3 This study seeks to demonstrate that uptake of I-125-labelled carbamylated EPO (CEPO), a derivative which is neuroprotective but not erythropoietic, can be detected in ischemic brain tissue using ex-vivo autoradiography in a permanent rat middle cerebral artery occlusion (MCAO) model.
Methods: In experiment 1 male Sprague-Dawley (SD) rats were injected with 1.25 μg of I-125-CEPO and sacrificed at 0.5, 1, 2, 3 and 4 h after injection. Radioactivity in blood-samples was measured as well as in 7 consecutive slices of the forebrain using autoradiography. In experiment 2 male SD-rats underwent a permanent right MCAO-occlusion procedure4 and were injected with I-125-CEPO1, 2 and 3 h after onset of ischemia. All animals were sacrificed 2 h after injection and blood-radioactivity as well as tissue radioactivity were measured using the same procedures as in control animals. Standardized uptake values (SUV) were calculated for both hemispheres and within a standard region- of-interest (ROI) placed within the maximum tracer uptake and a contralateral mirror ROI.
Results: In control animals, elimination of radioactivity from the blood followed a two-parameter exponential decay function with an elimination half-life of 1.7 h. Tracer activity in the brain followed the same kinetic. No focal uptake outside blood vessels was observed. Mean global SUV was 0.0024±0.00140 g/μL. An increase of radiotracer activity in the ischemic hemisphere was observed in the MCAO animals. This accumulation was highest when the tracer was injected 3 h post ischemia onset. This increase in the ischemic hemisphere was 30% of global uptake. In a standard ROI placed in the activity maximum within the ischemic hemisphere, an increase of 90% relative to the unaffected mirror ROI was found 3 h after onset of ischemia (ipsilateral SUVMAX = 5.66e-3 g/μL; mean contralateral SUV = 2.59e-3±0.734e-3., see Figure).
Conclusion: We demonstrate binding of I-125-CEPO in the ischemic hemisphere 3 h after onset of ischemia. No significant binding was observed In control animals up to 4 h after tracer injection. This study demonstrates the feasibility of using macromolecular substances to image neuroprotective systems and provides important data for the development of PET-based in vivo imaging methods for the EPO-receptor.
587. IGF-1 attenuates estrogen-mediated neurotoxicity in a stroke injury model in acyclic female rats
F. Sohrabji and A. Selvamani
Neuroscience and Experimental Therapeutics, Texas A&M Health Science Center, College Station, Texas, USA
Previous work from our lab has shown that middle cerebral artery occlusion (MCAo) results in a larger cortical-striatal infarct in acyclic (reproductive senescent) females as compared to mature adult females that have normal, but lengthened, estrous cycles. Moreover, estrogen treatment to ovariectomized senescent females significantly increased infarct size, while estrogen replacement to ovariectomized mature adult females was neuroprotective. In order to understand the dimorphic role of estrogen in the mature adult and senescent brain, we examined reproductive age-related changes in IGF-1, a peptide that has been shown to:
be neuroprotective in stroke models,
decrease with advancing age and
downregulated by estrogen treatment in older females.
We found that circulating levels of IGF-1 were reduced in reproductive senescent females and further reduced by estrogen at both ages. We therefore tested whether IGF-1 administered to reproductive senescent females immediately after MCAo would decrease tissue damage in this group. Estrogen-treated and estrogen-deprived senescent females were subject to endothelin-1 mediated MCA occlusion and simultaneously infused with IGF-1 via a cannula implanted in the ventricle. At 7 days post-MCAo, animals were terminated and their brains analyzed for infarct size by TTC staining. As we reported previously, estrogen treatment increased infarct size in this group, while IGF-1 treatment completely abolished estrogen mediated neurotoxicity. Interestingly, IGF-1 treatment had no effect on the control (no estrogen) group, which suggests that IGF-1 interacts with estrogen to improve cell survival in the cortex. This is further supported by our second study where mature adult females were infused with the IGF-1 antagonist, JB-1. Here, too, JB-1 had no affect on the control (no estrogen) group, but abolished estrogen′s neuroprotective effect on infarct size in this group. Cross-talk between estrogen and IGF-1 signaling pathways have been implicated in other adult neurodegenerative disease models and may also be implicated in stroke injury. Our findings support the hypothesis that aging- and estrogen-related changes in IGF-1 might be responsible for the significantly higher cortical damage in the reproductive senescent rats compared to their mature adult counterparts, and the disparate actions of estrogen in younger versus older females.
591. Brain lipid peroxidation, radical scavenging effects and antitumor activity of taiwanese botany—YY-07 on human glioblastoma cells
Y.W. Yu1, W.T. Huang2 and L.L. Yang1
1Department of Pharmcognosy, College of Pharmacy, Taipei Medical University, Taipei; 2Division of Hemato-oncology/Chi-Mei Medical Center, Tainan, Taiwan R.O.C.
In the nervous system, reactive oxidative species (ROS) can cause neurodegenerative disorders, like Alzheimer′s disease (AD), Huntington′s disease (HD), Parkinson′s disease (PD), amytrophic lateral sclerosis (ALS) and ataxia telangiectasia. Overproduction of ROS results in oxidative stress that can be an important mediator of damage lipids, proteins, and nucleic acids. Antioxidants prevent cellular damage by scavenging and inhibition of free radicals and prevention of pathologies. Oxidative stress has been implicated in the pathogenesis, including aging, atherosclerosis, diabetes mellitus, chronic inflammation and cancers. Gliomas are the most common type of primary brain tumor. Nearly two-thirds of gliomas are highly malignant lesions that account for a disproportionate share of brain tumor-related morbidity and mortality. Approximately 17,000 primary brain tumors are diagnosed every year, and of those, about 60% are gliomas. Tumors of gliol origin such as glioblastoma multiforme comprise the majority of human brain tumors. In this paper, 30 kinds of Taiwanese botany extracts were isolated from herbal medicine in our laboratory and their effects on 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, superoxide anion radical scavenging, superoxide anion radical inhibition, ferrous ions chelating activities, ferric ion reducing antioxidant power (FRAP), lipid peroxidation, cell proliferation and potential cytotoxicity. The level of lipid peroxidation (LPO) was expressed as thiobarbituric acid reactive substances (TBARS). LPO product of MDA (TBA)2 was induced by ferrous chloride and ferrous ammonium sulfate respectively on Wistar rat liver mitochondrias. and brain homogenates. We established screening program employing the 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay system utilizing the human malignant glioblastoma cell lines of GBM 8401 (astrocytoma-glioblastoma, grade III) and U-87 MG (astrocytoma-glioblastoma, grade IV) cells. One of the most potential compounds, YY-07 exhibited a significantly antioxidant and brain homogenate lipid peroxidation inhibition. It had also exhibited a dose dependent and time dependent manners in human malignant glioblastoma cells. The IC50 value of YY-07 after 24 h were 81.09±1.26 and 83.75±2.17 μg/mL in GBM 8041 and U-87 MG, respectively. In addition, we had observed the apoptotic bodies in YY-07 treated GBM 8401 and U-87 MG. YY-07 may medicate the cytotoxicity via apoptosis in GBM 8401 and U-87 MG. Furthermore, we well evaluated the mechanism of apoptosis by western blot in the future.
763. Novel mechanism of neuroprotection of ginseng against oxidative stress
S. Dore and Y.-S. Kim
Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
Objectives: Ginseng, the root of Panax ginseng (Araliaceae), which has been reported to have antioxidant properties and neuroprotective effects against transient and permanent ischemic damage, is used in complementary and alternative medicine. We have postulated that some of ginseng′s associated neuroprotective action might be mediated by the induction of heme oxygenase 1 (HO1) enzyme. Heme oxygenase is the only enzyme that can degrade heme (iron protoporphyrin-IX) to release free iron, carbon monoxide, and biliverdin/bilirubin. In the present study, we examined the neuroprotective actions of ginseng in primary neuronal cells and the potential biological mechanisms.
Methods: Primary cortical neurons isolated from 17-day-old embryos of timed-pregnant mice were plated onto poly-d-lysine-coated 24-well plates at a high density in high-glucose neurobasal medium supplemented with B27. To investigate the neuroprotective effects of ginseng, we pretreated neuronal cells with ginseng for 7 h. Pretreatment is required to increase intracellular antioxidant components and endogenous antioxidant proteins. Then we changed the medium to that without antioxidant and exposed the cells to 10 μmol/L tert-butyl hydroperoxide (t-BuOOH) for 16 h. t-BuOOH was freshly diluted in the culture medium immediately before use. The cell viability was measured by MTT assay. Experiments were performed in triplicate and reproduced three times with different primary culture batches.
Results: We found that pretreatment of primary neuronal cells with ginseng (0, 25, 75, 100, 200, and 400 μg/mL) for 7 h significantly increased the expression of HO1 in a dose-dependent manner and attenuated cell death induced by t-BuOOH (10 μmol/L) cytotoxicity (P<0.05). The HO inhibitor SnPPIX (5 μmol/L) inhibited the neuroprotective effects of ginseng, and the protein synthesis inhibitor cycloheximide (5 μg/mL) inhibited the neuronal protective effects of ginseng against t-BuOOH-induced oxidative stress. Moreover, we did not detect a significant cytoprotective effect of ginseng in neuronal cultures derived from HO1 knockout mice. Ongoing work is being performed in in vivo models of focal and global ischemia.
Conclusions: Together, our results suggest that the neuroprotective effects of ginseng could be mediated at least partially by HO1 activity and its by-products. This mechanism of action could potentially address the preventive properties associated with ginseng, which might not necessarily occur by its direct antioxidant properties, but rather by stimulation of an endogenous system capable of resisting various stress stimuli associated with age, acute, and/or chronic neurodegenerative conditions.
(This work was supported by a NIH-NCCAM grant.)
764. Carbon monoxide is protective against permanent focal brain ischemia
B. Wang and S. Doré
Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
Objectives: Historically, carbon monoxide (CO) gas has been considered to be toxic. We have recently shown that CO can be neuroprotective in transient focal cerebral ischemia [Zeynalov E, Doré S, Neurotox. Res. In Press]. Using in vivo and in vitro approaches, we demonstrated that the rate-limiting enzyme for the endogenous synthesis of CO, heme oxygenase, is highly neuroprotective against various types of insults. Our long term goals are to determine whether low levels of CO would also limit histological and functional outcomes in the permanent focal ischemia and determine the optimal therapeutic window.
Methods: Two-month-old male C57BL/6 mice (22 to 28g) were subjected to the permanent middle cerebral artery occlusion model (pMCAO). Mice were then immediately exposed to room air or 250ppm CO for 18 h. Mice were maintained for 7d after surgery. Neurological deficits were assessed at 1 and 7d after pMCAO using the sticky tape task. To measure infarct volume, brains were harvested, sliced into 50-μm thick sections, collected at 500-μm intervals, and stained with luxol fast blue/cresyl violet. In a separate cohort, blood levels of carboxyhemoglobin (COHb) and methemoglobin (MetHb) were determined. In addition to these standard measures and to determine whether low levels of CO exposure are sufficient to increase the inducible heme oxygenase-1 within the brain, immunohistology and Western blots were performed. All animal protocols were in accordance to NIH guidelines and approved by the JHU Animal Care and Use Committee.
Results: Mice that received 18 h exposure to 250ppm CO beginning immediately after pMCAO developed a significantly smaller infarct size than control mice exposed to air only (P<0.05; n = 12). Under this experimental protocol, mice developed no obvious signs of intoxication or histological changes. The animals exposed to CO had a trend toward shorter time to remove the sticky tape from the paws of the ischemic side compared to animals exposed to air. Moreover, in the other mouse cohort, levels of COHb were only transiently increased after CO exposure, from 4.3% to 17.1%, no detectable changes were observed in the MetHb levels (n = 6/group), and no anatomical changes following H&E staining. Finally, for the CO-induced HO1 expression, a significant increase at 48 h and 7d after 18h-CO exposure as compared to controls was observed.
Conclusions: A brief exposure to low levels of CO limits brain damage following permanent ischemia. Together, these results support the hypothesis that CO per se has neuroprotective actions and that its effects on stroke do not only result from a decrease in ischemic-reperfusion induced injury. Physiological amounts of CO play a key role in many cytoprotective mechanisms. Low concentrations of exogenous CO have anti-inflammatory/anti-apoptotic effects, which would protect tissues and organs against a variety of hypoxic/ischemic insults. CO can also mediate vascular signaling leading to vasodilatation, improved collateral blood flow, and improved brain perfusion. Development of a protocol to maintain CO within optimal and safe levels could be a novel therapeutic tool to fight brain damage and improve neurological outcome associated with stroke and other age/vascular-related neurodegenerative disorders.
(Supported by a GEMI award, and NIH grants).
780. Proteinase—activated receptor TYPE I (PAR1) is necessary for neuroprotective action of activated protein C and thrombin
L. Gorbacheva1, S. Strukova1, V. Pinelis2 and G. Reiser3
1The Lomonosov Moscow State University; 2The Scientific Centre for Children's Health, Moscow, Russia; 3Institute for Neurobiochemistry, Medical Faculty of Otto-von-Guericke University, Magdeburg, Germany
It is known, that serine proteinases participate in regulatory process in CNS. Glutamate (Glu)- induced citotoxicity is an important mechanism in ischemic brain injury and neuron death. Our early findings suggest that at low concentrations (up to 10 nmol/L) thrombin has a protective effect on cortical and hippocampal neurons against Glu-induced toxicity (Gorbacheva et al, 2006). However, the role of PARs and activated protein C (APC) in regulation of neurons survival is not clearly.
Studies were performed using 8 to 10-day primary culture of neurons from cortex obtained from brain of 1 to 3 day-old Wistar rats. Neuron viability was determined by morphological method using fluorescent probes (Hoechst 33342, Ethidium bromide and Syto-13), and biochemical method with MTT-test. Statistical treatment of data was performed using one-way analysis of variance with GraphPrism software within multiple groups.
At 24 h after Glu exposure (100 μmol/L, 30 mins) there was a increase in death of cortical neurons, reaching 30%. Preincubation of cell culture with APC at concentrations from 0.05 to 10 nmol/L (0.05; 0.1; 1.0; 10.0 nmol/L) reduced the Glu-induced neuronal death to 15.1%; 10.1%; 13.5% and 10.0%, accordingly. These findings suggest that at low concentrations (up to 10 nmol/L) APC has a protective effect on cortical neurons against Glu-induced toxicity. 0.1 nmol/L APC is the most effective neuroprotactive concentration in cortical neurons, but such concentration in hippocampal culture of neurons was 0.05 nmol/L APC. Thus, the cortical neurons sensitivity to APC is some higher than in hippocampal cells.
The presence of proteolytic activity is absolutely necessary for displaying the neuroprotective effects of APC and thrombin, as 10 nmol/L APC or thrombin, inactivated by PMSF did not protect cortical neurons from Glu-cytotoxicity.
To analyze the role of PAR-1 and PAR-3 in the neuroprotective effect of APC, cells were preincubated for 30 mins with blocking and control antibodies to these receptors prior to addition of APC and glutamate. We showed that APC at low doses via PAR-1 protected rat hippocampal and cortical neurons against glutamate-induced cell death.
Thrombin was shown to protect hippocampal rat neurons against Glu-induced death via PAR1 activation. Because PAR1 antagonist Mrp(Cha) (100 μmol/L) abolish the protective effect of thrombin and peptide-agonist PAR1 (PAR1AP) reduced the neuronal death induced by Glu.
Moreover, activation of PAR-1 by thrombin, the principal PAR-1 activator, triggers proinflammatory events, enhances endothelial permeability and initiates apoptosis (Feistritzer and Riewald 2005). We showed that pretreatment cells with APC before the high concentration of thrombin protected cells from death. These results point to PAR-1-desensitization by APC before the action of thrombin.
Taken together, our results demonstrate PAR-1-dependent neuroprotective effects of APC and thrombin at excitotoxicity. These findings can be of importance for developing novel therapeutic strategies to treat neurodegenerative disorders, where the modulation of PAR1-activity can used.
782. Alloxan induced neuroprotection in cultured cortical neurons exposed to oxygen glucose deprivation is mediated through nmda receptor antagonism
W.J. Costain, I. Rasquinha, A. Burgess, J. Slinn and J.S. Tauskela
Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada
Objectives: Protein O-GlcNAcation is a stress-induced post-translational modification of intracellular proteins.1 A variety of neuronal proteins have been shown to harbor O-GlcNAc modifications,2 which are dynamically regulated by two enzymes, OGT (O-GlcNAc transferase) and O-GlcNAcase. Protein O-GlcNAcation is proposed to be involved in mediating cellular responses to injurious conditions and is likely to play a protective role in mediating cellular stress responses.3 The objective of this study was to assess the role of O-GlcNAcation in mediating neuronal cell death in response to oxygen glucose deprivation (OGD) and NMDA excitotoxicity.
Methods: E18 rat primary cortical neurons were cultured and studied on day 14. Neurotoxicity was performed by subjecting neurons to OGD (1 hour) or NMDA (10 to 300 μm) and viability was determined using propidium iodide (PI) staining. Intracellular Ca++ concentrations were determined in individual neurons using Fura-2, and in neuronal populations using Fluo-4 AM fluorescence. Neurotoxicity was compared in the absence and presence of PUGNAC (0 to 300 μmol/L) and alloxan (0.3 to 3.0 mmol/L). Western blots were performed in primary cortical neuronal homogenates using anti-O-GlcNAc (CTD110.6).
Results: We examined the effect of blocking OGT and O-GlcNAcase on OGD-induced cell death in primary cortical neurons. Western blot analysis revealed that OGD induced only moderate changes in O-GlcNAcation. However, O-GlcNAcase inhibition with PUGNAC induced a marked increase in protein O-GlcNAcation but did not impart a neuroprotective effect against OGD. OGT inhibition, with alloxan, had no overt effect on protein O-GlcNAcation, but produced a significant neuroprotective effect against OGD (68% reduction in cell death) and NMDA toxicity. Alloxan inhibited NMDA-mediated neuronal death and Ca++ currents in a dose dependent manner consistent with NMDA receptor blockade.
Conclusions: This study demonstrates that inhibition of O-GlcNAcase was not neuroprotective against OGD in cultured primary cortical neurons. Further, it was shown that alloxan-mediated neuroprotection was likely to be due to inhibition of NMDA receptor mediated excitotoxicity and not inhibition of OGT activity.
783. Docosahexaenoic acid-mediated neuroprotection in focal cerebral ischemia in rats: characterization by sequential magnetic resonance imaging, behavior, and histopathology
N.G. Bazan1, L. Khoutorova1, T. Niemoller1, K.D. Atkins1, A. Obenaus2, P. Hayes2, E. Titova2 and L. Belayev1
1Neuroscience, Louisiana State University, New Orleans, LA; 2Radiation Medicine, Loma Linda University School of Medicine, Loma Linda, California, USA
Introduction: Docosahexaenoic (DHA 22:6n-3) acid is the precursor of NPD1, which inhibits oxidative stress-mediated proinflammatory gene induction and promotes cell survival (Bazan, 2005). DHA involved in memory formation, excitable membrane function, neuronal signaling and promotes neurogenesis both in vitro and in vivo, and has been implicated in neuroprotection (Bazan, 2003). We have recently showed DHA therapy to confer histological protection in the focal cerebral ischemia in rats (Belayev et al, 2005). We have now used diffusion-weighted magnetic resonance imaging (DWI) in conjunction with behavior and histological methods to expand our understanding of this therapeutic approach.
Methods: Sprague-Dawley rats were anesthetized with isoflurane/nitrous oxide and mechanically ventilated; rectal and cranial temperatures were regulated at 36°C to 37.5°C. Rats received 2 h middle cerebral artery occlusion (MCAo) by retrograde insertion of an intraluminal suture (Belayev et al, 1996). Susceptibility-weighted (SWI) and T2-weighted (T2WI) MRI was carried out on a 4.7T magnet at 24, 72 h, and 7 days after MCAo. Animals were treated with either DHA (n = 5), 5 mg/kg, or saline vehicle (n = 4) i.v. at 1 h after reperfusion. Neurological status was evaluated before MCAo and after treatment (at 24, 72 h, and 7 days), a grading scale of 0 to 12 was employed, as previously described. Brains were perfusion-fixed on day 7 and infarct volumes were determined.
Results: All animals showed similar values for rectal and cranial temperature, arterial blood gases, and plasma glucose during and after MCAo. DHA treatment significantly improved neurological scores compared to saline treatment at 72 h (3.3±0.6 versus 7.3±1.0) and 7 days (2.7±0.3 versus 18.0±1.0, respectively, P<0.05). Lesion volumes computed from T2WI images and by histopathology agreed closely with one another, but histological infarct volume was underestimated by approximately 2.5-fold. DHA treatment significantly reduced subcortical infarct volumes computed from T2WI images compared to the Alb treatment on day 1 (62±10 versus 123±13 mm3) and day 3 (26±5 versus 80±20 mm3), but not on day 7 (12±2 versus 55±39 mm3). Cortex was completely protected by DHA treatment and none of the rats had cortical infarction compared to the saline treated rats.
Conclusions: DHA experimental therapy improves neurological and histological outcome following focal cerebral ischemia and might provide the basis for future therapeutics in patients suffering ischemic stroke.
This study was supported by NS23002 and NS046741 (NGB).
836. Neuroprotective effect of genistein and daidzein is dependent on PPARγ activation
O. Hurtado1, G. Torregrosa2,3, J.B. Salom2,3, F.J. Miranda3, E. Alborch2,3, M. Sobrado1, I. Lizasoain1 and M.A. Moro1
1Departamento de Farmacología, Facultad de Medicina, Universidad Complutense de Madrid (UCM), Madrid; 2Centro de Investigación, Hospital Universitario La Fe; 3Departamento de Fisiología, Universitat de València, Valencia, Spain
Objectives: Phytoestrogens are a group of plant-derived compounds which include mainly isoflavones (genistein, daidzein, glycitein, equol and biochanin A), lignans, coumestans, flavonoids, stilbenes and mycotoxins. Phytoestrogens prevent neuronal damage1 and improve outcome in experimental stroke;2 however, mechanism of their neuroprotective action has not been fully elucidated. In this context, Peroxisome proliferator-activated receptor-γ (PPARγ) agonists have been shown to exert antiinflammatory effects in several settings, including the central nervous system (CNS).3 The aim of this study is to determine whether phytoestrogen-induced neuroprotection is brought about by selective stimulation of PPARγ.
Methods: Primary cultures of rat mixed cortical cells and oxygen and glucose deprivation (OGD) were performed as described:4 Culture medium was replaced by OGD solution. OGD cells were transferred to an anaerobic chamber containing a gas mixture of 95%N2/5%CO2 at 37°C. The time of exposure to OGD was 150 mins. In another set of experiments, cultures were exposed to the specific PPARγ antagonist5 T0070907 (Tularik, San Francisco, CA, USA) for 24 h. As a marker of necrotic tissue damage, LDH (lactate dehydrogenase) activity released from damaged cells was determined 24 h after OGD. For preparation of nuclear extracts cells were collected 24 h after treatment with phytoestrogens in phosphate-buffered saline. The homogenate was centrifuged at 4°C, 12,000g. Supernatant was discarded and nuclear extracts were prepared as described.6 PPARγ activity was assessed in nuclei using PPARγ Transcription Factor Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA).
Results: Administration of genistein or daidzein 24 h before, during and after exposure to OGD decreased lethal OGD-induced LDH release measured 24 h after the ischemia and caused neuroprotection in pure neuronal cultures (LDH: 92%, 88% and 72% versus 100% OGD with 0.05, 0.5 and 5 μmol/L genistein and 46%, 45% versus 100% OGD with daidzein 0.5 and 5 μmol/L respectively) but not in mixed cultures. The PPARγ antagonist T0070907 (1 μmol/L) inhibited the protective effect caused by the administration of 5 μmol/L genistein or daidzein. To check the role of genistein and daidzein in PPARγ activation, we next quantified PPARγ transcriptional activity by the ability of nuclear proteins to bind a specific oligonucleotide containing the PPRE consensus sequence using an assay kit. Indeed, both phytoestrogens induced an increase in PPARγ activation in pure neuronal cultures: 129% and 125% versus control (100%) with 0.5 and 5 μmol/L genistein and 122% and 117% versus control (100%) with 0.5 and 5 μmol/L daidzein.
Conclusions: These results demonstrate that genistein and daidzein could be used as preventive agents in cerebral ischemia and suggest that these phytoestrogens act as agonists of PPARγ nuclear receptor.
Supported by RETICS-RENEVAS from ISCIII. RD06/0026/0005.
856. Trehalose induces neuroprotection via autophagy activation in cerebral ischemia
J.-S. Sunwoo, H.-K. Park, K. Chu, K.-H. Jung, S.-T. Lee, J.-J. Bahn, M. Kim, S.-K. Lee and J.-K. Roh
Neurology, Seoul National University Hospital, Seoul, South Korea
Objective: Trehalose is a non-reducing disaccharide found in many organisms including plants and yeast. By inducing autophagy and chemical chaperone, trehalose protects mammalian cells against neurodegenerative diseases, although it is not endogenously synthesized in mammalian cells. Recently, it is known that the autophagy induced by mild hypoxia is neuroprotective by modulating the biogenesis of mitochondria and the is related to the molecules like PGC-1 alpha, BNIP3, and Beclin-1. Here, we investigated whether the addition of trehalose could enhance the neuroprotection against the ischemia.
Methods: Using cell viability assay (WST-1) and flow cytometry (FACS), we measured the difference of cell viability of PC 12 cell line under oxygen-glucose deprivation (OGD), between trehalose treatment group (0.1, 1, 10 mmol/L) and controls. The protein levels of molecules related to the autophagy and ischemia were evaluated before and after trehalose treatment. To find the neuroprotection of trehalose in vivo models, we measured the infarcted volume, modified limb placing tests (MLPT) and hemispheric atrophy after 90 mins—transient MCA occlusion (MCAO) by thread insertion.
Results: The cell viability assay and FACS showed the trehalose addition enhanced the cell viability after OGD significantly and this neuroprotective effect was dose-dependent. The trehalose treatment (2% of drinking water, 7 days) reduced the infarcted volume one day after MCAO. Trehalose also improved hemispheric atrophy and the MLPT over 30 days after MCAO. We found that the increased protein expression of PGC-1alpha, BNIP-3, Beclin-1 and LC3-II after trehalose treatment, suggesting that enhanced hypoxia-induced mitochondrial autophagy. The amount of Hsp70 increased after addition of trehalose and this fact indicated that trehalose also enhanced the survival-related molecules. FACS and TTC staining showed that the inhibitors of BNIP3 (Ibuprofen) and autophagy (Bafilomycin, Wortmannin) reduced the cell viability and increased the infarcted volume of trehalose treatment group.
Conclusions: Trehalose exerted the neuroprotection against ischemic injury. The beneficial effect is associated with the pathway of hypoxia-induced mitochondrial autophagy. The modulation of mitochondrial autophagy can be; one of the new therapeutic strategies for stroke treatment.
858. Tolerance to oxygen nutrient deprivation in the hippocampal slides of the naked mole rat
T. Nathaniel1, F. Umesiric2, A. Saras3 and F. Olajuyigbe4
1Center for Health and Natural Sciences, Marywood University, Scranton, PA; 2Chemistry, University of AlbertaToledo, Toledo, OH; 3Department of Molecular and Cell Biology, University of California, Irvine, 111 Life Sciences Addition, Berkeley, California, USA; 4Science, College of Education, Ondo, Nigeria
Hypoxia tolerance in the naked mole rats (Heterocephalus glaber), represents a unique physiological phenomenon characterized by the capability to regulate oxygen demand to attenuate energetically costly response to hypoxia. Several aspects of tolerance to hypoxia in the naked mole rat is consistent with a state of neuroprotection, however, it remains to be established if such protective capability is expressed in the brain cells of mole rats subjected to hypoxia insults. The objective of this study was determine whether evidence of tolerance to oxygen nutrient deprivation exists in the chronic cultures of the naked mole rats hippocampal slices. We used oxygen nutrient deprivation (OND) an in vitro model of hypoxia tolerance to determine neuronal survival in the hippocampal slices from mole rats and Rattus sp. At 4 h OND, followed by 20 h recovery, slices of neurons in CA1 of mole rats resist cell death, while slices of Rattus sp were susceptible to OND with a significant decrease in CA1 neurons after 4 to 24 h in culture. These results demonstrate that hippocampal slices of mole rats kept in hypoxia condition, consistently tolerate OND right from the onset time point in cultured slices, and maintained for 24 h.
896. Losartan alleviates global cerebral ischemia induced memory dysfunction associated with apoptosis in mouse
K.F. Ahamed1, M. Reddy Chaparapu1, R. Srinivas1, V. Ravichandiran1 and P.T.-H. Wong2
1Department of Pharmacology, Vels University, Chennai, India; 2Department of Pharmacology, Young Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
Objective: A mouse model Global cerebral ischemia (GCI) mimics the vascular dementia, through cholinergic dysfunction, neuronal apoptosis and memory impairment. Previous studies on losartan indicated that reduced GCI -induced delayed neuron death. However the effect of Losartan on GCI induced vascular dementia has not yet established. In this study, treatment of losartan (10 mg/kg) after ischemia for 45 days, we evaluated the long-term influence of losartan on behavior and biochemical changes.
Methods: Global cerebral ischemia (GCI) was induced by bilateral common carotid artery occlusion (BCCAo) for 30 mins followed by reperfusion for 45 days. Learning and memory was assessed in step through passive avoidance task. Western blot analysis was carried out to express the apoptotic markers in brain.
Results: Step-through passive avoidance tests suggested that ischemic mice showed decreases in step-through latency throughout the entire learning trial and memory session, which suggested learning and memory impairment. Additionally, Acetyl cholinesterase (AChE) activity also increased in the frontal cortex and hippocampus of ischemic mice compared with sham control. However, losartan significantly alleviated GCI induced antrograde memory deficits and increased AChE activity in the frontal cortex and hippocampus of ischemic mice. Expression of apoptotic markers were present in the neurons of the ischemic mice, losartan reduced the expression apoptotic markers in ischemic mouse brain.
Conclusion: Our results indicate that neuroprotection by losartan is partly related to modulation of apoptosis and protection of the cholinergic neurons. Early rational losartan interventions may be one of the most promising therapeutic approach for cerebrovascular and neurodegenerative diseases.
959. Neuroprotective effects of hydrogen gas on brain in three types of stress models: a 31P-NMR study
C. Kuroki, O. Tokumaru, H. Koga and I. Yokoi
Department of Neurophysiology, Oita University Faculty of Medicine, Oita, Japan
It has recently been reported that hydrogen gas (H2) acts as a radical scavenger by reducing the hydroxyl radical and reduces acute oxidative stress on brain in ischemia-reperfusion stress (Ohsawa et al, Nat. Med. 2007). We measured high-energy phosphates in rat brain slices by phosphorus nuclear magnetic resonance (31P-NMR) and evaluated neuroprotective effects of H2 using a hypoxic stress model, an ischemia-reperfusion model and a high-K+ stress model. Brain slices were superfused with well-oxygenated (PO2≈500torr) artificial cerebrospinal fluid (ACSF) equilibrated with (H2+ group) or without 4% H2 gas (H2- group) at 27.5°C. In the hypoxic stress model, slices were superfused with hypoxic ACSF (PO2≈120torr) for 2 h. In the ischemia-reperfusion model, perfusion was stopped for 1 h. In the high-K+ stress model, slices were superfused with ACSF containing 60 mmol/L K+ for 1 h. In the hypoxic stress model, phosphocreatine (PCr) level of H2+ group recovered to 81% relative to the pre-stress level, whereas that of H2- group was 85% (n.s.). There was no significant difference in γ-ATP levels. In the ischemia-reperfusion model, PCr levels after stress were not significantly different between the two groups (H2+: 45%, H2−: 47%; P>0.05). But γ-ATP level of H2+ group 1 to 2 h after stress was significantly higher than that of H2- group (H2+: 60%, H2−: 39%; P<0.05). In the high-K+ stress model, PCr level of H2+ group at the early stage of the stress was significantly higher than that of H2− group (H2+: 71%, H2−: 54%; P<0.05). Our result in the ischemia-reperfusion model is consistent with the previous report that H2 may exert neuroprotective effects on brain slices against strong oxidative stress by scavenging hydroxyl radical. But we observed the significant difference of PCr level in the high-K+ stress model, because the high-K+ stress has distinct mechanisms from oxidative stress, this difference is in conflict with the previous report.
Conclusion: Our result showed that H2 may have neuroprotective effects on brain slices from a viewpoint of energy metabolism. In addition, our results indicated that careful consideration must be given to neuroprotective mechanisms other than that H2 acts as a radical scavenger by reducing the hydroxyl radical.
971. Cyclic pattern of 17β-estradiol pretreatment protects the hippocampal ca1 region against cerebral ischemia
N. Hirsch1, I. Saul1, K. Dave1, R.A. Defazio1, H. Bramlett2, M. Perez-Pinzon1 and A. Raval1
1Department of Neurology, University of Miami; 2Department of Neurological Surgery, Miami Project to Cure Paralysis, University of Miami, Miami, Florida, USA
Objectives: Chronic 17β-estradiol treatment improves pathophysiological outcome after brain ischemia in experimental animal models.1–3 In contrast to chronic 17β-estradiol treatment strategy, our recent study demonstrated that a single 17β-estradiol bolus 48 h prior to ischemia induces neuroprotection in the hippocampal CA1 region in slice culture and rat models of global cerebral ischemia.4,5 Based on these results, we hypothesized that cyclic 17β-estradiol treatment provides neuroprotection against cerebral ischemia in the rat model.
Method: Normal cycling female rats were ovariectomized (OvX) and 7 days later an injection schedule of 17ß-estradiol (5 μg/Kg; i.p) or vehicle (oil) was started. Rats were injected with 17ß-estradiol/oil at an interval of every 48 or 72 h for 4 cycles over the period of one month. Forty eight or seventy two hours following the last hormone treatment rats were exposed to cerebral ischemia produced by 10 mins of bilateral carotid occlusion and systemic hypotension (50 mm Hg). Seven days after ischemia, rat brains were fixed for histopathological assessment. Hippocampal sections at the level of 3.8 mm posterior to bregma were examined for normal neurons. Results are expressed as mean±s.e.m. Statistical significance was determined with an ANOVA test followed by a Bonferroni's post-hoc test.
Results: The number of normal neurons per slice in the CA1 hippocampal region in naïve rats was 1100±45 (n = 4). The ischemic insult in OvX rats decreased the number of normal neurons to 18% of naïve (192±10, n = 6, P<0.05). Intermittent estradiol-17β treatment to ovariectomized rats prior to cerebral ischemia increased the number of normal neurons to 51% (559±13, n = 7, dose 5 μg/Kg/every 48 h for 21 days) and 41% (446±14, n = 7, dose 5 μg/Kg/every 72 h for 21 days) as compared to OvX group (P<0.05). Vehicle treatment did not show any significant difference in the number of normal neurons versus OvX groups. Female rats receiving sham-OvX showed no difference from OVX in neuron loss after ischemia.
Conclusion: A cyclic pattern of 17β-estradiol bolus treatment conferred protection against ischemia in ovariectomized rats. This study emphasizes the need to investigate a cyclical estrogen hormone replacement regimen to promote improved cardio- and cerebro-vascular health and reduce stroke/cerebral ischemia incidents in post-menopausal women while avoiding the known side effects of chronic estradiol treatment.
Grant support: AHA-SDG-National Center #0730089N and James and Esther King Biomedical Research Program, Florida Department of Health 07KN-10.
1051. Genome-wide expression analysis of ischemic preconditioning in adult hippocampal organotypic slice cultures
E. Benardete
Neurosurgery, SUNY Downstate Medical Center, Brooklyn, New York, USA
Objectives: Adult rat hippocampal slice cultures become resistant to ischemic injury by prior exposure to a sublethal ischemic insult. This process, called ischemic preconditioning (IPC), which also occurs in vivo, triggers protective cellular pathways. We sought to characterize the changes in gene expression that occur with IPC. We also sought to determine whether NMDA-receptor blockade with (RS)-3-(2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) would inhibit IPC and change the pattern of gene expression associated with IPC.
Methods: Adult hippocampal slice cultures were obtained as previously described.1 Briefly, hippocampal slices were prepared from P20–P30 Sprague-Dawley rats. Slices were then cultured on filter inserts for two weeks at 32 C. Slices were then shifted to 37 C for 2 to 3 days. Slices were exposed to 5 mins. of oxygen-glucose deprivation (OGD) in a specially designed chamber. Total RNA was then isolated from slices at 3, 6, and 12 h following OGD. Total RNA was also isolated from slices undergoing mock OGD and OGD in the presence of 100 umol/L CPP. Total RNA was then reverse-transcribed into cRNA and used to probe genome-wide expression arrays, Rat 230 2.0 (Affymetrix, Santa Clara, CA). Raw hybridization data was the processed using robust microarray analysis (RMA) and analyzed using GeneSifter (Geospiza, Inc., Seattle, WA), GeneSpring 7.0 (Agilent, Inc., Santa Clara, CA), and Ingenuity Pathway Analysis (Ingenuity Systems, Inc., Redwood City, CA) software. Changes in individual genes were confirmed using real-time PCR (RT-PCR) with primers from Applied Biosystems Inc. (Foster City, CA).
Results: Genes were identified based on statistically significant changes in gene expression levels relative to controls at 3, 6, and 12 h post-IPC. Heat maps and hierarchical clustering were used to identify and sort individually regulated genes. These genes were then placed into Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The mitogen-activated protein kinase (MAPK) pathway, the Toll-like receptor pathway, and apoptosis pathway were statistically significantly regulated across all time points. In addition, at 3 h post-IPC, the Wnt signaling and at 6 h post-IPC, the ErbB signaling pathways were identified as significantly regulated. Changes in individual transcripts at 3, 6, and 12 h were confirmed using RT-PCR. CPP failed to block IPC and did not significantly alter the levels of most transcripts. However, certain genes were markedly down-regulated by NMDA-receptor blockade by CPP, such as prostaglandin-endoperoxide synthase 2 (COX-2).
Conclusions: Endogenous mechanisms of neuroprotection are important targets for therapeutic approaches to stroke and brain injury. Genome-wide expression analysis is a useful technique to identify candidate molecules involved in IPC. These data expand results from other studies that suggest a role for the MAPK, Toll-like receptor, and apoptosis pathways. Further experiments will focus on blocking these pathways in vitro to confirm their roles in IPC and looking at these molecular markers in clinical subjects.
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