XVII. European Stroke Conference
Nice, France

Oral Session:

Experimental studies II and Genetic disorders
Date: Thursday 15 May 2008  
Time: 16:30 - 16:40 -  Room: Euterpe
Chair: H. Chabriat, France and M. Endres, Germany

01
Axonal injury due to ischaemia: protection of axonal function in a new model induced in the rat spinal cord in vivo
F. Bei    K.J.Smith                                                        
 

Institute of Neurology

UNITED KINGDOM

Background: Ischaemic injury of the central white matter (IICWM) has been relatively little studied despite its contribution to the pathology of ischaemic stroke and vascular dementia. Research has been hampered by a lack of suitable experimental models. Methods: To develop a new model of IICWM, endothelin-1 (ET-1; 2.3 nmol; a potent vasoconstrictor) was injected bilaterally into the rat spinal grey matter. The local tissue perfusion was continuously monitored by laser Doppler flowmetry. Axonal function in the dorsal columns was assessed by continuously monitoring conduction. KB-R7943 (30 mg/kg; a selective blocker of the reverse-mode operation of the sodium-calcium exchanger) or vehicle was administered intra-arterially 1 hour before the intraspinal injection of ET-1. Results: Saline/control intraspinal injection had little or no effect on either local tissue perfusion or axonal conduction, but the injection of ET-1 reduced local tissue perfusion by more than 90% within minutes of application, accompanied by total blockade of axonal conduction. The local tissue perfusion gradually recovered to about 40% of baseline within 8 hours, and axonal conduction recovered to 18% of baseline by 4.5 hours after the injection of ET-1. Systemic pre-treatment with KB-R7943 increased the recovery of axonal conduction to 35% of baseline (35 +/- 5 %), compared with recovery from vehicle treatment (12 +/- 9 % of baseline; P < 0.0001). The drug treatment did not diminish the severity of the ischaemia per se. KB-R7943 did not affect the magnitude of axonal conduction in normal, sham-operated rats. Discussion: A new model of IICWM in vivo has been developed, resulting from the instraspinal injection of ET-1. Treatment with the sodium-calcium exchange blocker KB-7943 may provide a therapeutic approach to protect the function of the central white matter from ischaemic injury.

Experimental studies II and Genetic disorders
Date: Thursday 15 May 2008  
Time: 16:40 - 16:50 -  Room: Euterpe
Chair: H. Chabriat, France and M. Endres, Germany

02
Reduced Cardiac Baroreceptor Sensitivity Is Not Related To Increased Arterial Stiffness Following Acute Stroke In Treated Hypertensive Patients
N.S.Shah    J. Chernova    R.B.Panerai    J.F.Potter    T.G.Robinson                                   
 

Ageing & Stroke Medicine, University of Leicester

UNITED KINGDOM

Background Cardiac baroreceptor reflex sensitivity (BRS) is a short-term blood pressure regulator in health and disease state. Cardiac BRS is impaired following ischaemic stroke and predicts long-term death and disability. Impaired cardiac BRS may be due to afferent loop abnormalities due to increased large artery stiffness, impaired central processing of baroreceptor information or efferent loop abnormalities due to impaired blood pressure and heart rate control mechanism following stroke. We evaluated the relationship between large (aortic) artery stiffness and cardiac BRS during the acute phase of ischaemic or haemorrhagic stroke from a subgroup of COSSACS (Continue Or Stop post-Stroke Antihypertensive Collaborative Study). Methods Ninety four treated hypertensive stroke patients (51 male, mean age 72.4+/-12.5) were studied within 48 hours of onset. Cardiac BRS (determined by spectral and sequence analyses) and arterial stiffness estimated by the carotid to femoral pulse wave velocity (PWVcf) using applanation tonometry by SphygmocorTM were obtained. In addition to demographic data, NIHSS (National Institutes of Health Stroke Scale), casual blood pressure (BP) and body mass index were recorded at randomisation. Results At baseline, cardiac BRS values were inversely correlated with PWVcf (r = -0.24, P<0.05; r= -0.35, p=0.003 for spectral and sequence analysis, respectively). Age was significantly correlated with PWVcf (r = 0.50, p < 0.0001). In age, baseline NIHSS and mRS adjusted correlation, cardiac BRS was not related to PWVcf. In quantile regression models, taking into account the effect of all cardiovascular variables, and BP values, cardiac BRS was not related to PWVcf at baseline. Conclusions Arterial stiffness of the afferent loop involved in the baroreflex arc may account for, at least in part, the reduced cardiac BRS observed in acute stroke patients previously treated with antihypertensives, though age is a strong confounder and nullifies the relationship of cardiac BRS and PWVcf following acute stroke.

Experimental studies II and Genetic disorders
Date: Thursday 15 May 2008  
Time: 16:50 - 17:00 -  Room: Euterpe
Chair: H. Chabriat, France and M. Endres, Germany

03
Genetic variation in members of the leukotriene biosynthesis pathway confer an increased risk of ischaemic stroke - a replication study in two independent populations
S. Bevan    M. Dichgans    H. Wiechmann    A. Gschwendtner    T. Meitinger    H. Markus                            
 

St Georges, University of London

UNITED KINGDOM

Background The recent finding that genetic variants in 5-lipoxygenase activating protein (FLAP) and leukotriene A4 hydrolase (LTA4H) may confer an increased risk of ischaemic stroke has implicated the leukotriene family as potential mediators of cardiovascular disease. Using a case control replication methodology, all members of the leukotriene synthesis pathway and their receptors were examined for genetic variants which may act as risk factors for all ischaemic stroke and stroke subtypes. Methods A case control methodology using a UK stroke cohort (872 cases, 933 controls) was adopted, with additional FLAP genotyping and replication of positive findings undertaken in an independent stroke population from Germany (601 cases, 736 controls). 18 variants in FLAP, 2 variants in 5-LO, one variant in LTC4S, 4 variants in LTA4H, 4 variants in CYSLTR1, 2 variants in CYSLTR2 and 5 variants spanning LTB4R and LTB4R2 were examined in the UK cohort. Replication of variants in the two genes in which associations were detected in the UK population (LTC4S and LTB4R) was performed in the German cohort. Results Associations were identified with variants in FLAP, leukotriene C4 synthase (LTC4S) and the leukotriene B4 receptor (LTB4R1/2) complex. Differing risks were identified for ischaemic stroke subtypes. A variant in LTC4S was found to confer a 1.5 fold increase in risk of small vessel disease (RR 1.515 1.041-2.262 p=0.043) with replication in an independent cohort showing a similar risk (RR 1.687, 1.065-2.675, p=0.026). A haplotype in the LTB4R1/2 complex was found to confer a 2.3 fold increase in risk of cardioembolic stroke (RR 2.118, 1.194-3.760, p=0.01) and replication in a German cohort revealed a similar risk with a second distinct haplotype (RR 2.060, 1.162-3.665, p=0.013). Conclusions Genetic variation in leukotriene pathway members and their receptors confer an increased risk of ischaemic stroke in two independent populations. These risks show different magnitudes depending on ischaemic stroke subtype.

Experimental studies II and Genetic disorders
Date: Thursday 15 May 2008  
Time: 17:00 - 17:10 -  Room: Euterpe
Chair: H. Chabriat, France and M. Endres, Germany

04
NEUROPROTECTION AFTER ISCHAEMIC PRECONDITIONING IS MEDIATED BY TLR4.
J.M.Pradillo    I. García-Yébenes    D. Fernández-López    J. Caso    O. Hurtado    M. Sobrado    M.A.Moro    I. Lizasoain              
 

Department Pharmacology. School of Medicine. Universidad Complutense de Madrid

SPAIN

Background: It has been demonstrated that a short ischaemic event (ischaemic preconditioning; IPC) can result in a subsequent resistance to severe ischaemic injury (ischaemic tolerance; IT). We have recently demonstrated the role of immunity and in particular TLR4 in brain ischaemia (Caso et al., Circulation 2007). We have now used an in vivo model of IPC to investigate whether TLR4 signalling is involved in IT. Methods. Experiments were carried out on TLR4-deficient mice (C57BL/10ScNJ) and its respective control (C57BL/10ScSn). A period of 10-min temporary middle cerebral artery occlusion (tMCAO) was used for focal IPC, whereas sham surgery was used for control group. To evaluate the ability of IPC to induce IT, permanent MCAO was performed 48h after IPC. Stroke outcome was evaluated by determination of infarct volume and assesment of neurological scores. Brains were collected 24h after stroke and infarct volume was measured by TTC staining. 24h after IPC, MMP9, iNOS expression and NF-kappaB activation were studied by western blot and by ELISA kit (determining p65 subunit in the cellular nucleus). Results: IPC produced a reduction in infarct volume and a reduction on motor deficit measured by a neurological test in mice expressing TLR4 normally (ScSn: SHAM+MCAO: 35.4+/-3 vs IPC+MCAO: 13.9+/-2 mm3; 60% reduction, P<0.05). However, TLR4-deficient mice had significantly smaller reduction in infarct volume when compared with control mice (ScNJ: SHAM+MCAO: 28.2+/-2 vs IPC+MCAO: 23.1+/-2 mm3; 18% reduction, P<0.05). Western blot analysis of iNOS and MMP9 showed an upregulation in the expression of these proteins in both substrain mice, being higher in mice with TLR4 (ScSn). Same results were observed in the NF-kappaB activation. Conclusions. These data demonstrate that TLR4 signalling is involved in brain tolerance. The difference in the percentage of decrease in infarct volume produced by IPC between ScSn and ScNJ (60% vs. 18%), and the higher expression of iNOS, MMP9 and NF-kappaB activation in mice expressing TLR4, clearly supports these conclusions.

Experimental studies II and Genetic disorders
Date: Thursday 15 May 2008  
Time: 17:10 - 17:20 -  Room: Euterpe
Chair: H. Chabriat, France and M. Endres, Germany

05
Implant for Augmentation CBF Clinical Trial-1 (ImPACT-1). Safety and Efficacy of the Ischemic Stroke System (ISS*) in the treatment of Acute Ischemic Stroke – An interim analysis
N. Borenstein    D. Khurana     S. Kaul    A. Csányi     N. Ichaporia     D. Schneider     D. Tanne     Y. Solberg     M. Fisher        
 

Tel Aviv Medical Center

ISRAEL

Background: In rat stroke models, sphenopalatine ganglion (SPG) stimulation up to 24 hours after stroke onset augments cerebral blood flow, reduces infarct volume and improves neurological deficits. Presented are preliminary safety and efficacy data of SPG stimulation with ISS in patients with acute ischemic stroke (AIS). Design: This is an ongoing multi-center, multi-national open label study recruiting patients with AIS in the anterior vasculature, NIHSS 7-20, treatment initiated within the first 24 hours following onset. The ISS is implanted adjacent to the SPG with a minimally invasive oral procedure (15 min., local anesthesia). The therapeutic regimen consists of 3hr/d stimulation up to 7 days. Primary endpoint is incidence of AE and SAE, secondary is the efficacy of SPG stimulation as measured by mRS and NIHSS at day 90. Efficacy results are compared to an historical control population composed of the placebo group in two large previous AIS trials, the NINDS rt-PA studies I and II. Results: To date 62 patients have been enrolled. A small number of AE and SAE were observed in a few patients, none of them deemed related to the treatment. The mean 90 day mRS score of the Intend To Treat (ITT) patients cohort in the study is 2.53 and that of the Per Protocol (PP) patients cohort is 2.31, both of which are significantly lower than the mean 90 day mRS of the matched NINDS controls, which is 3.47 (CMH test p=0.0031 and p=0.0006, respectively). Moreover, both ITT and PP cohorts have a significantly higher rate of favorable NIHSS outcome as compared to the controls (ITT: 50% and PP: 56.3% vs. 23.6% for the NINDS controls, p=0.0015 and p=0.0002 respectively, using Chi square). Conclusion: This interim analysis suggests that SPG stimulation appears to be safe and efficacious for the treatment of AIS when initiated within 24hr after symptom onset. ImPACT-1 is still enrolling. In 2008 a larger multi-national multi-center pivotal study (ImPACT-24) is planned to commence. * investigational device limited by Federal law to investigational use

Experimental studies II and Genetic disorders
Date: Thursday 15 May 2008  
Time: 17:20 - 17:30 -  Room: Euterpe
Chair: H. Chabriat, France and M. Endres, Germany

06
Additive Effects of Statin and Dipyridamole on Cerebral Blood Flow and Stroke Protection
H.H.Kim    N. Sawada    H.S.Lee    Z. Zhou    M.A.Moskowitz                                   
 

Brigham & Women's Hospital and Harvard Medical School

USA

Recent studies suggest that dipyridamole (DP) may exert stroke protective effects beyond platelet inhibition. The purpose of this study is to determine whether statin and dipyridamole (DP) could enhance stroke protection through nitric oxide (NO)-dependent vascular effects. Mice were pre-treated with DP (10-60 mg/kg, q12 hr, 3 d) alone or in combination with a statin (simvastatin, 0.1 to 20 mg/kg/d, 14 d) before transient intraluminal middle cerebral artery occlusion. Although simvastatin (1 mg/kg/d, 14 d) increased endothelial NO synthase (eNOS) activity by 25% and DP (30 mg/kg, q12 hr, 3 d) increased aortic cGMP levels by 55%, neither statin nor DP alone, at these sub-therapeutic doses, increased absolute cerebral blood flow (CBF) or conferred stroke protection. However, the combination of sub-therapeutic doses of simvastatin and DP increased CBF by 50%, decreased stroke volume by 54%, and improved neurological motor deficits, all of which were absent in eNOS-/- mice. In contrast, treatment with ASA (10 mg/kg/d, 3 d) did not augment the neuroprotective effects of DP. These findings indicate that statin and DP exert additive NO-dependent vascular effects. These results suggest that the combination of statin and DP may have greater benefits in stroke protection than statin alone through vascular protection.

Experimental studies II and Genetic disorders
Date: Thursday 15 May 2008  
Time: 17:30 - 17:40 -  Room: Euterpe
Chair: H. Chabriat, France and M. Endres, Germany

07
STATINS MIGHT HELP CLEARANCE OF EXCESS EXTRACELLULAR GLUTAMATE IN STROKE
T. Gasull    V. Guirao    J. Ponce    A. Davalso                                          
 

Fundacio Institut Investigacio Ciencies Salut Germans Trias i Pujol

SPAIN

Background: Excitotoxicity by overactivation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors initiates damage in stroke. The widely prescribed drugs statins have been reported to protect from excitotoxicity in neurons in culture, and we recently found that simvastatin reduce the association of NMDA receptors to the membrane functional domains called lipid rafts without altering its total expression. However, the effect of statins on the other players of the excitotoxic game still remains to be elucidated. To provide new light into this subject we studied the effect of simvastatin on the expression and the association to lipid rafts of the neuronal glutamate transporter 3 (EAAT3) and its negative regulator, the glutamate transport associated protein (GTRAP). Methods: Primary neuronal cultures were treated with simvastatin for 5 days, total protein and protein of the lipid rafts fractions from control and simvastatin-treated neurons were obtained, and Western blots were performed using specific anti-EAAT3 and anti-GTRAP antibodies. Results: Sustained treatment with simvastatin increased EAAT3 total expression and decreased the total expression of GTRAP (Figure 1). However, simvastatin did not change the percentage of EAAT3 or GTRAP associated to lipid rafts. Discussion: Pre-treatment with simvastatin increases the expression of the neuronal glutamate transporter and decreases the expression of its inhibitor. Therefore, by improving the capacity of neurons to uptake glutamate, statins might reduce excitotoxicity during stroke. This effect might underlay the benefits reported for stroke patients in statins before the onset of cerebral ischemia. Figure 1.

http://www.eurostroke.org/ni_graphics/g_aid3034.htm
 

Experimental studies II and Genetic disorders
Date: Thursday 15 May 2008  
Time: 17:40 - 17:50 -  Room: Euterpe
Chair: H. Chabriat, France and M. Endres, Germany

08
Maximizing neuroprotection by minocycline in cell culture and following intracerebral hemorrhage in mice
M. Xue    E. Mikliaeva    V.W.Yong                                                 
 

Hotchkiss Brain Institute and University of Calgary

CANADA

The brain injury following intracerebral hemorrhage (ICH) involves inflammatory responses and neuronal death. Minocycline (MO) has anti-inflammatory and anti-apoptotic properties, and it provides histological protection in ICH, although contradictory results have also been noted due to the dose and route of administration used for MO. We hypothesize that high doses of MO applied locally to the proximity of ICH would be more efficacious. In culture, human fetal neurons (HFN) were exposed to human blood, and immunostaining was used to examine neuronal survival. In mice, 10 µl of autologous blood mixed with MO or saline was injected into the right striatum of CD-1 mice. Other mice received MO injected intracerebrally (IC) into the hematoma but treatment was initiated 1h after ICH. ICH mice injected intraperitoneally (IP) with MO was used as comparison group. 24h after ICH, brain were evaluated for lesion areas, neuronal death and inflammation. Other groups of mice survived 2 weeks for behavioral tests. In culture, concentration-dependent neurotoxicity was observed when HFN were exposed to blood. MO added 5 min before blood reduced its neurotoxicity and there was a concentration-dependent effect from 10 to 40 mg/ml of MO. In vivo, 24h following ICH, the histopathological changes were significantly reduced by MO applied IC plus IP compared to each separate route of administration. Moreover, the reduction of ICH-inflicted brain damage and neuronal death was achieved by MO in a concentration-dependent manner when MO was applied IC. Behavioral tests over 14d show significant improvement in mice treated with MO applied IC plus IP compared to IP administration. Our results have relevance to the application of MO in humans to maximize recovery from certain neurological insults.

Experimental studies II and Genetic disorders
Date: Thursday 15 May 2008  
Time: 17:50 - 18:00 -  Room: Euterpe
Chair: H. Chabriat, France and M. Endres, Germany

09
Mitochondrial haplogroup H1 is protective for ischemic stroke
A. Rosa    B.V.Fonseca    T. Krug    H. Manso    L. Gouveia    I. Matos    G. Lopes    J.M.Ferro    A.M.Vicente    S.A.Oliveira
and the PORTuguese Stroke GENetics (PORTSGEN) Group

Instituto Gulbenkian de Ciência

PORTUGAL

Background - The genetic contribution to stroke is well established but it has proven difficult to identify the genes and the disease-associated alleles mediating this effect, possibly because only nuclear genes have been intensely investigated so far. Mitochondrial DNA (mtDNA) has been implicated in several disorders having stroke as one of its clinical manifestations. The aim of this case-control study was to assess the contribution of mitochondrial polymorphisms and haplogroups to ischemic stroke risk and age-at-onset. Methods - We genotyped 28 mtDNA single nucleotide polymorphisms (SNPs) defining the major European haplogroups in 534 ischemic stroke patients and 499 controls collected in Portugal, and tested their allelic and haplogroup association with ischemic stroke risk and age-at-onset. Results - Haplogroup H1 was found to be significantly less frequent in stroke patients than in controls (OR=0.61, 95% CI=0.45-0.83, p=0.005), when comparing each clade against all other haplogroups pooled together. Conversely, the pre-HV/HV and U mtDNA lineages emerge as potential genetic factors conferring risk for stroke (OR=3.14, 95% CI=1.41-7.01, p=0.004, and OR=2.87, 95% CI=1.13-7.28, p=0.022, respectively). SNPs 3010GA, 7028CT and 11719GA strongly influence ischemic stroke risk, their allelic state in haplogroup H1 corroborating its protective effect. None of the tested haplogroups or polymorphisms was found to strongly associate with the age-at-onset of ischemic stroke. Discussion - Our data suggests that mitochondrial haplogroup H1 has an impact on ischemic stroke risk, but mtDNA does not influence its age-at-onset.

Experimental studies II and Genetic disorders
Date: Thursday 15 May 2008  
Time: 18:00 - 18:10 -  Room: Euterpe
Chair: H. Chabriat, France and M. Endres, Germany

10
NXY-059 Efficacy Meta-analysis in individual Animals with Stroke (NEMAS)
P.M.Bath    L.J.Gray    A.R.Green                                                 
For NEMAS Collaborators

Institute of Neuroscience, University of Nottingham, UK

UNITED KINGDOM

Background NXY-059 was neuroprotective in experimental stroke but ineffective in a large clinical trial. In the first ever meta-analysis using individual animal data, we assessed preclinical stroke studies of NXY-059. Methods Studies from AstraZeneca (AZ) and PubMed searches. Individual data for each animal (or published summary if unavailable) were obtained from the lead author of each study and/or AZ. Multilevel models compared NXY-059 with control taking account of heterogeneity between trials. Infarct volume and neurological impairment were standardised to reflect different species and scales. Standardised mean difference (SMD) and 95% confidence intervals (95% CI) are presented. Results 21 studies (11 laboratories) involved 534 animals (NXY-059 322, control 212) from 3 species (mice 9, rats 489, marmosets 36). 7 studies were unpublished (6 were neutral). Individual data were available for 445 animals. Investigators from 4 studies (89 animals), all academic, did not share data. Studies variably used randomisation (8, 38%), and blinding to treatment of the surgeon (7, 33%) and outcome assessor (10, 48%). NXY-059 was associated with reductions in total (SMD -1.2, 95% CI -1.5 to -0.8), cortical (SMD -1.1, 95% CI -1.5 to -0.7) and subcortical (-1.3, 95% CI -1.6 to -0.9) lesion volume; efficacy was seen in models of transient and permanent ischaemia, up to 180 minutes post occlusion. 18 (7%) animals receiving NXY-059 had no lesion versus receiving control (p=0.001). Neurological impairment was reduced with NXY-059 (SMD -1.1, 95% CI -1.3 to -0.8). There was no difference in death rate. Conclusion NXY-059 reduced lesion volume and impairment in experimental stroke models. Absence of lesions in some animals given NXY-059 could reflect neuroprotection or bias. Preclinical stroke studies must embody the principles of clinical trials (randomisation, blinding), and be followed by a meta-analysis using individual animal data.