Brain Poster Session: Inflammation

61. Pivotal role of cerebral interleukin-17-producing γδT cells in the late phase of ischemic brain injury

T. Shichita^1,2, H. Ooboshi², T. Kobayashi¹, Y. Sugiyama¹, H. Sugimori², D.J. Cua³, Y. Iwakura⁴, M. Iida² and A. Yoshimura¹

¹Department of Microbiology and Immunology, Keio University, School of Medicine, Tokyo; ²Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; ³Department of Immunology, DNAX Research Inc, Palo Alto, California, USA; ⁴Center for Experimental Medicine and Department of Cancer Biology, Institute of Medical Science, Tokyo University, Tokyo, Japan

Objectives: Recently, inflammatory cytokines and lymphocyte recruitment/activation have been implicated in the progression of cerebral ischemia-reperfusion (I/R) injury.^1,2 However, the roles of specific lymphocyte subpopulations and their cytokines in stroke remain to be clarified. Because both IL-17 and IL-23 are critical cytokine for the onset of experimental autoimmune encephalomyelitis (EAE) and modify many inflammatory responses in central nervous system, we examined the role of IL-23 and IL-17 in the evolution of brain infarction.

Methods: Transient focal ischemia using a suture occlusion model was applied to the following gene-deficient mice: IL-17^−/−, IL-23p19^−/−, IFN-γ^−/−, IL-6^−/−, and γδTCR^−/−. The infiltrating inflammatory cells in the ischemic brain were collected by Percoll density gradient centrifugation. The expression levels of IL-17, IL-23, and other neurotoxic factors in the infiltrating inflammatory cells and ischemic brain tissue were investigated by intracellular FACS or quantitative real time PCR.

Results: Infarct volume of IL-23-deficient mice was significantly smaller than wild-type at 1 to 7 days after ischemia, while that of IL-17 KO mice was smaller at 4 and 7 days but not at 1 day after ischemia. Disruption of IFN-γ or IL-6 gene did not affect infarct volume. The improvement of neurological deficits in IL-17 and IL-23 KO mice was also observed at day 4 but not at day 1. The expression of IL-23 in the brain increased 1 day after I/R, while IL-17-producing cells were infiltrated into the brain after day 3. IL-17-producing cells were not observed in the brain of IL-23 KO mice with I/R injury, suggesting that IL-17-producing cells induced by IL-23 are important effectors for the progression of ischemic brain damage. The expression of neurotoxic factors including IL-1β, TNFα, MMP-9, and ICAM-1 was extremely reduced in the ischemic brain by IL-17 and IL-23 deficiency. Intracellular cytokine staining indicated that γδT lymphocytes, but not CD4-positive helper T cells, were a major source of IL-17. Depletion of γδT lymphocytes by gene disruption or by anti- γδTCR antibody pre-treatment ameliorated the brain I/R injury to the levels seen in IL-17 deficient mice. Furthermore, a neuroprotective effect was also observed even when the administration of anti-TCRγδ antibody was delayed to 24 h after the induction of brain ischemia.

Conclusions: Our findings indicate that infiltrated γδT lymphocytes activated by IL-23 mainly produce IL-17 and contribute to the inflammatory responses to ischemic brain injury. We propose that γδT lymphocytes could be a therapeutic target for the later inflammatory events which amplify the initial damage in cerebral ischemia.

175. The effect of PPAR-γ during stroke recovery in the diabetic ob/ob mouse

R. Kumari, L. Willing, S. Patel and I. Simpson

Neural—Behavioral Sciences, Hershey Medical Centre, Penn State University, Hershey, Pennsylvania, USA

Background and aims: agonist to ameliorate stroke injury in genetically diabeticThe diabetic patient is 2 to 6 times more likely to experience a stroke and the subsequent morbidity and mortality is substantially greater than a euglycemic individual. PPAR-γ agonists are very effective in normalizing hyperglycemia and are widely used in the treatment of type II diabetes. Hence, we tested the effect of darglitazone, a selective peroxisome proliferators gamma activated receptor (PPAR-γ ob/ob mice.

Methods: Male ob/+ and ob/ob mice received darglitazone (1 mg/kg) in powdered chow or control chow for 7 days at 7 weeks of age. Blood glucose was measured before and on alternate days during treatment. Hypoxia/ischemic (H/I) insults were induced in 8 week old ob/ob and ob/+ mice by the ligation of the right common carotid artery followed by systemic hypoxia (8% oxygen: 92% N₂) for 24 mins. At 4, 8, and 24 h of recovery, blood samples were obtained by cardiac puncture for serum and plasma collection and measurement of corticosterone, cholesterol, triglycerides and VLDL. Brains were rapidly removed and frozen for histological examination, RNA isolation and in situ hybridization.

Results: Darglitazone significantly lowered the blood glucose level in ob/ob mice by day 3 and normalized elevated levels of triglycerides and VLDL in treated ob/ob mice, whereas cholesterol levels remained elevated. Hematoxylin and eosin staining was performed on brain sections at 24 h of recovery and infarct area was measured. Darglitazone treatment dramatically reduced the infarct area in ob/ob mice compared to the untreated group (29.54±12.29% to 3.32±1.65%) but was without significant effect on ob/+ mice (12.96±5.97% versus 7.65±2.89%), n = 6 to 8. Proinflamatory cytokines (IL-1β, TNF-α, IL6), mRNA determinations as well as in situ hybridization of bfl1 (microglia) and GFAP (astrocytes) at various times of recovery suggests that darglizatone promotes an initial activation of microglia in the ob/ob mice resulting in a transient increase in bfl1 and TNFa expression to control levels at 4 h of recovery, which were subsequently suppressed at 8 h and 24 h in ob/ob animals.

Conclusion: This study in ob/ob mice confirms our observations in the db/db mice that the microglial activation and proinflamatory responses are delayed and diminished in the diabetic animal and damage following H/I is greater. Moreover, treatment of the ob/ob mice with the PPAR-g agonist darglitazone, which restored euglycemia and lowered triglyceride levels, dramatically reduces infarct area in the diabetic mice. This reduction in infarct size was associated with an enhancement in the initial activation in microglial responsiveness to the ischemic insult, which appears to be associated with successful recovery. The challenge now is to determine whether this enhanced microglial response and the markedly reduced infarct are related and occur as a result of the restoration of euglycemia or to a specific interaction with darglitazone.

178. A sartan derivative with very low angiotensin II receptor affinity ameliorates ischemic cerebral damage through anti-oxidative and anti-inflammatory effects

S. Takizawa¹, E. Nagata¹, S. Takagi¹ and T. Miyata²

¹Division of Neurology, Department of Internal Medicine, Tokai University School of Medicine, Isehara; ²Center for Translational and Advanced Research on Human Disease, Tohoku University Graduate School of Medicine, Sendai, Japan

Aims: Angiotensin II receptor blockers (ARBs) have been reported to protect the brain against ischemic damage, but their hypotensive effect limits clinical use because blood supply to the penumbra is decreased. We synthesized a novel ARB-derivative, R-147176, which is 6700 times less potent than olmesartan in AT₁ binding inhibition, and therefore has a less anti-hypertensive effect, but has marked inhibitory effects on oxidative stress and advanced glycation. ¹ We evaluated the effect of R-147176, given orally or intravenously, on infarct volume in transient thread occlusion and photothrombotic models in rats. The anti-oxidative and anti-inflammatory properties were also investigated.

Methods: Transient focal ischemia was achieved through 2-h thread occlusion of the middle cerebral artery following 7-day reperfusion in Male Sprague-Dawley rats (n = 43) as described previously. ² Coronal brain sections were stained with hematoxylin and eosin for the measurement of infarct volume, as well as immunohistochemically with ED-1 and TUNEL. Levels of oxidatively modified proteins and the content of pentosidine were quantified. Permanent ischemia was induced by photothrombotic occlusion of middle cerebral artery (n = 16). Seven days later, infarct volume was measured from the sections stained with hematoxylin and eosin.

Results: R-147176, given orally or intravenously (10 to 30 mg/kg/day), significantly reduced infarct volume (Figure 1B), albeit no influence on blood pressure lowering (Figure 1A), in both ischemia models. R-147176 significantly reduced the numbers of ED-1-positive cells in the penumbra and TUNEL-positive cells in the infarcted cortex and penumbra, compared with the vehicle-treated control. Protein carbonyl formation in the brain in the R-147176-treated group was significantly reduced compared with that in the vehicle-treated group.

Figure 1.

Conclusions: This ARB derivative, despite its significantly lower AT₁ receptor affinity and marginal anti-hypertensive effect, ameliorated ischemic cerebral damage through its anti-oxidative and anti-inflammatory effects. These findings may open a new avenue for the treatment of stroke.

244. Increased CD36 and inflammatory response are involved in exacerbated ischemic brain injury in diabetic condition

E. Kim¹, A. Tolhurst¹, Y. Bao¹ and S. Cho^1,2

¹Research/pre-clinic, Burke Medical Research Institute, White Plains; ²Neuroscience, Weill Cornell Medical College, New York, New York, USA

Background and aims: Diabetes, a chronic pro-inflammatory state in periphery, is a predisposing risk factor for stroke. Recent studies indicate that CD36 is an inflammatory mediator that contributes to tissue injury in cerebral ischemia (Cho et al, 2005; Kim et al, 2008). Furthermore, the expression of CD36 is up-regulated in the presence of high glucose and implicated as a novel marker for insulin resistance. The purpose of the study is to determine whether CD36 and associated inflammatory responses are involved in ischemic injury in experimental diabetic conditions.

Methods: Experimental diabetic conditions in mice were established by feeding normal chow (ND) or diabetogenic diet (DD) for 8 weeks and administrating vehicle (veh) or streptozotocin (STZ) 3 week after commencing the diets. This approach results in four groups of mice: ND/Veh, DD/Veh, ND/STZ, and DD/STZ. Fasting blood glucose levels were measured 7 week of diet. Following week, mice were subjected to 30 mins focal middle cerebral artery occlusion (MCAO) according to the methods previously described (Kim et al, 2008). Infarct volume (IV) and % swelling were measured in 3d-post ischemic brain. The expression of CD36, MCP-1, CCR2, TNFα, and IL-6 were compared between the most diabetic condition (DD/STZ) and ND/veh.

Results: Compared to ND/veh (32.8±2.6, n = 19), blood glucose levels were significantly increased in ND/STZ (247.9±25.5, P<0.01, n = 20) and DD/STZ (331.3±45.8 P<0.01, n = 11). Accordingly, IV was also increased in ND/STZ (42.7±2.0 P<0.05, n = 12) and DD/STZ (54.8±1.5, P<0.01, n = 11) compared to ND/veh (32.8±2.6, n = 20). The gene expression levels of inflammatory mediators were significantly elevated in the DD/STZ brain (CD36, 1.6±0.2; MCP-1, 2.1±0.2; CCR2, 1.7±0.2; TNFα, 1.4±0.1; IL-6, 2.0±0.4 fold versus ND/veh, P<0.05, n = 4 to 6). In addition, peritoneal macrophage CD36 protein expression was significantly increased in DD/STZ (2.1±0.5 fold versus ND/veh, P<0.05, n = 4 to 6).

Conclusions: The data demonstrate that diabetic condition exacerbates ischemic injury and increases the expression of inflammatory mediators in the brain/periphery. The study implicates that peripheral inflammatory status at the time of stroke and suggests an inclusion of risk factors in the experimental stroke models.

254. Rapid induction of chemokines in glia cells contributes to neutrophil recritment into cerebrospinal fluid in experimental klebsiella pneumoniae meningoencephalitis

C.-T. Chiu^1,2, S.-Y. Chen³, L.-L. Wen⁴, H.-P. Pao³ and J.-Y. Wang³

¹Neurosurgery, En Chu Kong Hospital; ²Graduate Institutes of Medical Sciences; ³Physiology, National Defense Medical Center; ⁴Clinical Laboratory, En Chu Kong Hospital, Taipei, Taiwan R.O.C.

Klebsiella pneumoniae (K. pneumoniae) infection of the brain was less reported than in other organs, but an increase in incidence and morbidity has been noted in Taiwan. The incidence of death (>30%) and long-term neurological sequelae remain high. We have previously demonstrated that glial cells are important cellular sources of proinflammatory cytokines in CSF in a rat of model of K. pneumoniae meningoencephalitis. The CXC chemokines such as growth related oncogene (GRO, CXCL1) and macrophage inflammation protein-2 (MIP-2, CXCL2) are chemoattractant for neutrophils while CC chemokines such as monocyte chemotractic protein-1 (MCP-1, CCL2) are chemoattractant for monocytes. We found that the increases of WBC counts in CSF and blood were time-dependent. Leukocytosis was observed in CSF at 8 h after K.p-infectiont with a predominance of PMNs. The concentrations of GRO (CXCL1) and MIP-2 (CXCL2) in CSF are much higher than those in serum at 4 and 8 h after K.p infection of the brain, suggesting a likely brain origin. After K.p. infection, the mRNA expression of chemokines (GRO, MIP-2, and MCP-1) in brain tissue increased significantly in a time-dependent manner. However, mRNA level of their receptors only showed slight changes with CXCR1 and CXCR2 mRNA level upregulated while the CCR2 mRNA expression rapidly suppressed compared to sham animals. Intracerebroventricular injection of reparixin (CXCR1 and CXCR2 antagonist) or vMIP-2 (viral macrophage inflammatory protein-2), a broad spectrum peptide antagonist of chemokine receptors, attenuated PMN recruitment into CSF. and also attenuated the elevated concentrations of GRO (CXCL1) in CSF and upregulation of gene expression of CXCR2, but not CXCR1, in brain tissue at 8 h after K.p infection. Taken together, our results suggest a rapid induction of chemokines and chemokine receptors in glial cells, which may play a role in PMN recritment into CSF and and subsequent cell/tissue injury following K. pneumonia infection of CNS.

389. Neuroinflammation extends brain tissue at risk to vital peri-infarct tissue: a double tracer [¹¹C]PK11195- and [¹⁸F]FDG-PET study

M.A. Dennin^1,2, M. Walberer^1,2, H. Backes², B. Neumaier², G.R. Fink¹, M. Schroeter^1,2 and R. Graf²

¹Department of Neurology, University Hospital of Cologne; ²Max-Planck-Institute for Neurological Research, Cologne, Germany

Background and aim: Focal cerebral ischemia elicits strong inflammatory responses involving activation of resident microglia and recruitment of peripheral monocytes/macrophages. These cells express peripheral benzodiazepine receptors (PBRs) and can be visualized by Positron Emission Tomography (PET) using [¹¹C]PK11195 that selectively binds to PBRs. Previous research suggests that transient ischemia in rats induces increased [¹¹C]PK11195 binding within the infarct core. We here investigated the expression of PBRs during permanent ischemia in rats.

Methods: Permanent cerebral ischemia in rats was induced by injection of macrospheres into the middle cerebral artery of Wistar rats (n = 5). Multimodal in vivo imaging 7 days after ischemia comprised:

• (i)

  Magnetic Resonance Imaging to assess localization and extent of the infarcts,

• (ii)

  [¹⁸F]-2-fluoro-2-deoxy-D-glucose ([¹⁸F]FDG)-PET to characterize local cerebral glucose transport and metabolism and

• (iii)

  [¹¹C]PK11195-PET to detect overexpression of PBRs depicting neuroinflammation. Immunohistochemistry ex vivo verified ischemic damage and characterized extent and location of neuroinflammation.

Results: The rate constant K1 of [¹⁸F]FDG was used to describe the transport of [¹⁸F]FDG from blood into brain tissue as a surrogate marker for cerebral blood flow, and the rate constant Ki to assess [¹⁸F]FDG metabolization. Based on those two parameters we identified four distinct regions of the ischemic brain. The ‘infarct core’ was characterized by a significant decrease in [¹⁸F]FDG transport (K1 = 0.042±0.011 mL/ccm/min) compared to ‘contralateral cortex’ (K1 = 0.099±0.002 mL/ccm/min, P<0.01) as well as by a significantly decreased [¹⁸F]FDG metabolic rate constant (Ki = 0.009±0.002 min⁻¹) compared to the contralateral side (Ki = 0.020±0.003 min⁻¹, P<0.01). Adjacent to the core, an ‘infarct margin’ of tissue was defined as showing reduced [¹⁸F]FDG transport of K1<0.08 mL/ccm/min, with a regular [¹⁸F]FDG metabolic rate constant (Ki = 0.015±0.002 min⁻¹). The ‘peri-infarct zone’ adjacent to the infarct margin was characterized by preserved [¹⁸F]FDG transport (K1 = 0.099±0.009 mL/ccm/min) and an increased [¹⁸F]FDG metabolic rate constant (Ki = 0.032±0.003 min⁻¹) that was 60% higher than in the contralateral hemisphere (Ki = 0.020±0.003 min⁻¹, P<0.01). Increased [¹¹C]PK11195 binding (mean standard uptake value 1.93±0.49) was found exclusively in this normo-perfused peri-infarct zone, co-localizing with the increase of the [¹⁸F]FDG metabolic rate constant. Contrasting with previous data for transient ischemia, no [¹¹C]PK11195 binding was found in the infarct core. Overexpression of PBRs detected in vivo co-localized with the accumulation of activated microglia and macrophages assessed immunohistochemically.

Conclusion: These results suggest that after permanent focal ischemia, neuroinflammation occurs in the normoperfused peri-infarct zone together with increased energy demand. This combination of inflammatory processes with metabolic disturbances is likely to put even normoperfused peri-infarct tissue at risk of secondary damage.

390. Comparison between lectin-like oxidized low density lipoprotein receptor 1 expression and preoperative echogenic findings of vulnerable carotid plaque

A. Saito¹, M. Fujimura², T. Inoue², H. Shimizu¹ and T. Tominaga¹

¹Neurosurgery, Tohoku University, Graduate School of Medicine; ²Neurosurgery, Konan Hospital, Sendai, Japan

Background and aims: Lectin-like oxidized low density lipoprotein 1 (LOX1) is an important cell surface receptor for the progression of atherosclerosis. LOX1 expressions in coronary atherosclerotic lesions and other systemic arterial plaques have been reported, however, the detail role of LOX1 in atherosclerotic formation of carotid plaques is unclear. Moreover, the relationships of LOX1 and atherosclerotic factors for the vulnerability of carotid plaque and preoperative echogenic findings have not been clarified.

Methods: LOX1, matrix metalloproteinase (MMP) -2, 9 and tissue inhibitor of MMP (TIMP)-2 expressions were immunohistochemically analyzed using carotid endarterectomy specimens obtained from 14 patients. Groups were divided into stable plaque group A and vulnerable plaque group B by preoperative echogenic findings of carotid plaques. Endothelial immunoreactive cells of LOX1 were calculated as endothelial index and the immunohistochemical findings of LOX1 and MMPs were compared between two groups.

Purpose: Our purpose is to clarify the relationship between LOX1 expression in carotid plaques and the vulnerable plaque formation comparing the LOX1 immunohistochemical expression in plaque specimen with the preoperative echogenic plaque findings.

Results: LOX1 was remarkably expressed, especially in smooth muscle cells in vulnerable plaque and colocalized in MMP-9 positive cells and apoptotic cells. All LOX1, MMP-2,9 and TIMP2 were remarkably expressed in the subendothelial layer in group B compared with group A. The endothelial LOX1 index was 63.75±4.92 in group A and 83.0±5.02 in group B (P = 0.02). The endothelial MMP-2 index was 24.38±5.50 in group A and 32.83±6.79 in group B (P = 0.01). The endothelial MMP-9 index was 46.13±6.31 in group A and 59.17±2.14 in group B (P = 0.002). The endothelial TIMP-2 index had no significant difference between two groups (P = 0.14).

Conclusions: LOX-1 expression was correlated to the echogenic findings of the vulnerability carotid plaques. LOX-1 may play an important role in the progression of vulnerable carotid plaque and might regulate vulnerable plaque formation in cooperation with MMPs and TIMP-2. Endothelial MMP-2 might suppress TIMP-2 activation in vulnerable plaques.

413. Platelet depletion with an anti-gpib-antibody improves the functional outcome after focal cerebral ischemia in mice

S.M. Sonanini^1,2, S.M. Krieg^1,2 and N. Plesnila^1,2,3

¹Department of Neurosurgery; ²Institut for Surgical Research, University of Munich Medical Center—Großhadern, Munich, Germany; ³Royal College of Surgeons in Ireland, Dublin, Ireland

Objectives: Following reperfusion from focal cerebral ischemia platelets adhere to the cerebrovascular endothelium. This interaction is mediated by interaction of the glycoprotein (GP) Ib receptor with endothelial von Willebrand factor ¹ and occurs within the time window of post-ischemic cell death, i.e. 2 to 8 h after reperfusion. The pathophysiological role of this interaction is yet unclear. Therefore the aim of the current study was to address the role of platelets for the development of reperfusion injury after experimental ischemic stroke.

Methods: C57/Bl6 mice (n = 36) were randomized to 6 groups (n = 6 each) and were subjected to 45 mins of transient middle cerebral artery occlusion (tMCAO). ⁵ Complete blockade of GPIbα and >95% depletion of circulating platelets was achieved by intraperitoneal injection of 2 μg/g body weight of a monoclonal anti-GPIbα Fab antibody at reperfusion (t = 0 h) or 1, 3, 5 or 7 h thereafter. ² 24 h after tMCAO, functional outcome was evaluated by using a modified 19-point neurological severity score (NSS). Infarct volume, neuronal cell loss, and the degree of intracerebral haemorrhage (ICH) were quantified by Nissl staining.

Results: All treated groups showed an improved functional outcome after 24 h as compared to controls, particularly groups R+5 h and R+7 h with a 55% and a 76% better NSS, respectively (R+5h: 6.3±1.0; R+7h: 3.3±0.3 versus control: 13.9±0.9; P<0.05). Compared with controls, ischemic lesions were reduced by a maximum of 52% (R+7 h) (R+0h: 30.4±3.1 mm³; R+1h: 35.5±2.5 mm³; R+3h: 31.7±2.3 mm³; R+5h: 36.4±3.9 mm³; R+7 h. 25.7±1.1 mm³ versus control: 49.3±1.7 mm³; P<0.05). After 5 and 7 h reperfusion less necrotic neurons could be observed (R+5h: 57.1±12.4 cells; R+7h: 50.7±6.9 cells versus control: 144±21.9 cells; P<0.05). Despite functional and histological improvements a significant number of animals showed ICH (R+0h: n = 2; R+1h: n = 3; R+3h: n = 5; R+5h: n = 2; R+7h: n = 1 versus control: n = 0; P>0.05).

Conclusions: These data indicate that—despite the increased risk of ICH—blockade of GPIbα, a major platelet adhesion receptor, has an important impact on functional and histopathological outcome following reperfusion from focal cerebral ischemia. Thus, early inhibition of platelet adhesion with an GPIbα antibody may offer a novel treatment strategy for cerebral ischemia.

538. Role of NADPH oxidase in post-ischemia brain inflammation: a temporal profile study

H. Chen¹ and P. Chan^1,2,3

¹Department of Neurosurgery; ²Department of Neurology and Neurological Sciences; ³Program in Neurosciences, Stanford University School of Medicine, Palo Alto, California, USA

Objectives: In response to ischemia, the brain transcribes and expresses inflammation proteins, which activate downstream pathways and cause further detrimental effects. ¹ SOD1 transgenic mice showed significantly less NF-kB-pathway activation, which suggests oxidative stress is important for the brain inflammation process after cerebral ischemia. ² NADPH oxidase (NOX) is a resource of reactive oxygen species generation in the brain after cerebral ischemia. ³ Previous studies have revealed that NOX inhibition is neuroprotective after focal ischemia. ⁴ However, the role of NOX in greatly developed inflammation is not clear. Our hypothesis is that NOX is involved in the post-ischemic inflammation process and that inhibition of NOX will ameliorate inflammation and delayed infarction.

Methods: We subjected wild-type (WT) mice, gp91^phox−/− (gp91 KO) mice, and NOX inhibitor apocynin-treated mice (Apo) to 60 or 75 mins of focal ischemia followed by 3 h, 24 h, 3 d and 7 d of reperfusion. We measured the brain infarction volume, inflammation-related pathways and protein expression (COX-2, ICAM-1, ERK), inflammation cell infiltration (neutrophils and microglia), and cytokine mRNA levels (IL-1b, IL-6, iNOS).

Results: There was a significantly higher infarction volume at 3 d than at 24 h of reperfusion, which indicates a continuous evolution of cell death that leads to delayed, larger infarction. In addition, a larger area of infarction was observed in the WT mice than in the Apo and gp91 KO mice at both 24 h and 3 d of reperfusion. Furthermore, the WT mice exhibited higher mortality than the gp91 KO mice at 7 d of reperfusion. At 3 h of reperfusion, there was little ICAM-1 and COX-2 protein expression. However, at 24 h of reperfusion, ICAM-1 and COX-2 expression increased and was more significant in the WT mice than in the Apo and gp91 KO mice. An ELISA revealed more neutrophil infiltration in the WT mice than in the gp91 KO or apocynin-treated mice, and there was abundant microglia activation at 24 h of reperfusion in the WT mice. The inflammation cytokine gene expression studies showed that at 24 h of reperfusion, only IL-1b mRNA levels increased by 2.3 fold. However, at 72 h of reperfusion, the levels of IL-1b, IL-6 and iNOS mRNA all were significantly increased. Furthermore, inhibition of NOX alleviated increases in the cytokine mRNA level. Oxidative stress is known to be involved in neuronal death via ERK signaling focal ischemia. We hypothesize that ERK signals are involved in gp91 KO-mediated neuronal protection. At 24 h of reperfusion, there was significantly more ERK phosphorylation in the WT mice than in the Apo and gp91 KO mice.

Conclusions:

• (1)

  The brain exhibits post-ischemic temporal patterns of inflammation progress, with minimal inflammation at 3 h and a robust inflammation process at 1 to 3 d of reperfusion.

• (2)

  NOX activation contributes to the progress of post-ischemic inflammation and subsequent delayed brain damage.

556. Fas ligand mediated activation of NF-kB and MiR-155 pathway in experimental ischemia

X. Zhang¹, Y. Xu¹ and F.N. Niu²

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: Inflammatory response accompanying apoptosis is one of the main pathological changes in ischemic brain injury. CD95 (Fas) and its natural ligands, CD95 ligand (Fasl), is involved in the pathological effect. Previous studies indicated that Fas ligation was a major cause of secondary inflammatory reaction in the penumbra after stroke through the activation of nuclear factor kB (NF-kB). MiR-155 is a NF-kB depend small non-coding RNAs whose target gene might be TNF-α. The activation by TNF-α leads to apoptosis and inflammation which form a lethal threshold and course neuron cell to die in penumbra. Fasl mutation mice (gld mice) were strongly resistant towards stroke-induced damage. However, its mechanism has not been completely understood. The aim of this study is to explore wether a potential mechanism of the neurotoxicity by Fasl is through activation of the NF-kB and miR-155 pathway. Deficiency of Fasl and its reaction in stoke may serve as a novel therapeutic strategy for stroke.

Methods: Gld and wild type (WT) mice were subjected to 2 h MCAO and sacrificed at 24 h of reperfusion. Brains were then removed, stained with 2,3,5-triphenyltetrazolium chloride (TTC) for infarct size measurement or dissected and quickly frozen for real time PCR and western blot analysis. In the in vitro model of neuronal oxygen-glucose deprivation (OGD), we pretreated neuron with MG-132, an inhibitor of NF-kB. Real-time PCR of miR-155 were performed according to standard protocols. The protein expression of TNF-α was assayed by Western blot.

Results: In vivo, total infarct size at 24 h after MCAO in gld mice (15.02±5.22%) was significantly smaller than WT mice (24.19±5.59%, n = 6 per group, P<0.05). Real time PCR revealed that the miR-155 and TNF-α were down-regulated in the cortex of brain in gld mice compared to that of WT mice at 24 h after MCAO (miR-155 decreased by 0.75±0.04 fold as compared to WT mice, TNF-α decreased from 1.15±0.13 to 0.80±0.04, P<0.05). In vitro, neuronal viability after 24 h were also significantly higher in gld (40.07±9.85%, n = 3 in triplicate) than WT neurons (24.95±7.89%, n = 3 in triplicate, P<0.05), and miR-155 and TNF-α were also down-regulated in gld neuronal cell as in vivo. However, the levels of miR-155 remained unchanged following OGD treatment if pretreated with MG-132, suggesting that miR-155 was clearly dependent on the level of NF-kB activity. Furthermore, pretreatment with anti-miR-155 resulted in decreased expression of TNF-a in primary neurons after OGD in WT but not gld animals.

Conclusions: Our results indicated that deficiency of FasL inhibited the secondary inflammatory injury of stroke-related damage through the regulation of NF-kB and miR-155. These results offer new approaches in stroke treatment.

560. Smaller cerebral infarct size in females versus males is reperfusion-dependent and associated with less inflammation and Nox2-containing infiltrating T lymphocytes

V.H. Brait¹, K.A. Jackman¹, H. Diep¹, B.R.S. Broughton¹, A.K. Walduck², G.R. Drummond¹ and C.G. Sobey¹

¹Pharmacology, Monash University, Clayton; ²Pharmacology, The University of Melbourne, Parkville, VIC, Australia

Background and aims: Infarct size is typically smaller in females than in males following cerebral ischemia-reperfusion, the underlying mechanisms of which are not well understood. The aims of this study were:

• (1)

  to test the importance of reperfusion in the protection of females following ischemic stroke, and

• (2)

  to assess the involvement of local inflammation and the peripheral immune system.

Methods: In male (n = 99) and female (n = 91) C57Bl6/J mice, cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 0.5 h followed by reperfusion for 23.5 h (ischemia-reperfusion; I-R). Control mice were subjected to sham surgery. In order to assess the importance of reperfusion, in some mice MCAO was maintained for 24 h with no reperfusion (I-NR). Brains were used to quantitate infarct volume, or for western blotting or immunofluorescence, and blood and spleens were removed for fluorescent activated cell sorting analysis or T lymphocyte proliferation assays. We assessed whether outcomes after cerebral I-R were associated with changes in expression of the pro-inflammatory proteins Nox2, cyclooxygenase-2 (Cox-2), or vascular cell adhesion molecule-1 (VCAM-1) protein, differences in circulating lymphocyte and splenocyte populations, or T lymphocyte function.

Results: After I-R, males had a larger infarct volume than females (55±11 versus 24±6 mm³, n = 7 to 8; P<0.05), whereas there was no gender difference following I-NR (88±11 versus 88±9 mm³, n = 8 to 9). Expression of Nox2, Cox-2 and VCAM-1 was each significantly increased in the ischemic hemisphere of males after I-R, but was unchanged in females. Immunofluorescence studies indicated more Nox2 protein and T lymphocytes (CD3+) in the infarct zone in males than in females following I-R, and revealed Nox2 to be largely co-localized with CD3. In the spleen, there were significantly lower levels of T and B lymphocytes following I-R in both males and females, whereas preliminary studies indicated no significant change in circulating lymphocyte levels in either gender. In addition, T lymphocyte proliferation in vitro was not different between males and females following I-R.

Conclusions: Thus, following cerebral ischemia the smaller cerebral infarct volume observed in females is reperfusion-dependent. Furthermore, less brain inflammation occurs in females than males, and is associated with less infiltration of Nox2-expressing T lymphocytes into the brain in females after I-R. We speculate that Nox2-derived superoxide from infiltrating T lymphocytes contributes to the greater infarct volume in males versus females following cerebral I-R.

609. Differential modulation of the cellular immune system in a mouse model of intracerebral hemorrhage

S. Illanes, A. Liesz and R. Veltkamp

Neurology, University of Heidelberg, Heidelberg, Germany

Background and purpose: A stroke-induced immunodeficiency syndrome encompassing innate and adaptive immune cells develops in murine large ischemic stroke models. ¹ Intracerebral hemorrhage (ICH) accounts for 15% of acute stroke, and is associated with high mortality and morbidity rates. ² Experimental studies have identified a number of immunological factors that are involved in the pathophysiology of ICH, including leukocyte brain infiltration, initiation of the complement cascade and microglial activation. ³ However, there are no present studies characterizing the systemic immune response after experimental ICH. Our objective was to analyze lymphocytes subpopulation in blood and lymphatic organs in an experimental model of intracerebral hemorrhage (ICH) in mice.

Methods: In C57Bl/6 mice, ICH was induced by injection of 10 μL (n = 17), 30 μL (n = 17) or 50 μL (n = 17) of blood in the left striatum. Sham operation (n = 17) served as control group. Differential leukocyte counts were performed in blood. Lymphocytic subpopulations were further characterized by FACS in blood, spleen, lymph node, and thymus day 3 after ICH. We compared absolute numbers of T_helper cells (CD3⁺CD4⁺), T_effector cells (CD3⁺CD8⁺), B cells (B220⁺), T_regulatory cells (CD4⁺CD25⁺Foxp3⁺) and NK cells (PanNK⁺).

Results: Blood lymphocyte subpopulations were reduced in ICH compared to Sham, with a reduction of 5.7% (P = 0.79) in the 30 μL-ICH group and a reduction of 64% (P = 0.19) in the 50 μL-ICH group. In spleen the total number of splenocytes had an increase of 10.7% (P = 0.27) in the ICH-30 μL group and a significant reduction of 55% (P = 0.0026) of the total splenocytes number compared to Sham in the ICH-50 μL group. In thymus we found a reduction in the thymocyte counts number of 17.9% (P = 0.11) in the 30 μL-ICH group and a significant reduction of 68% (P = 0.0001) in the 50 μL-ICH group. The most remarkable findings were seen for the T_regulatory cells with an increase number of 57.3% (P = 0.0001) compeering to Sham in the 30 μL-ICH group in spleen, 270.4% (P = 0.0001) in thymus and 23.5% (P = 0.15) in lymph nodes. However in the 50 μ-ICH group the T_regulatory cells were similar to Sham values in spleen, thymus and lynph nodes.

Conclusion: Hemorrhage size is a major determinator of post-stroke systemic immune modulation. Different Intracerebral blood volumes have a different impact on lymphocyte subpopulations, tending to induce proinflammatory reaction in mild and moderate hemorrhage size and a severe immunosuppression in large hemorrhagic stroke.

641. Pioglitazone down-regulates interleukin-1β and up-regulates interleukin-1 receptor antagonist in the peri-infarct frontoparietal cortex of rats exposed to cerebral ischemia

J. Culman, T. Glatz, I. Stöck, P. Gohlke and Y. Zhao

Institute of Experimental and Clinical Pharmacology, University Hospital of Schleswig-Holstein, Kiel, Germany

Objectives: Interleukin-1 (IL-1) upon binding to the IL-1 receptor I (IL-1RI) activates the transcription of a number of pro-inflammatory cytokines and neurotoxic mediators which exert deleterious effects and exacerbate neuronal injury after ischemic stroke. Interleukin-1 receptor antagonist (IL-1ra) is the natural antagonist which inhibits the effects of IL-1 on target cells. A number of studies have demonstrated that systemic or central treatment with the peroxisome-proliferator-activated receptors gamma (PPARγ) agonists ameliorates brain injury after cerebral ischemia. We studied the effects of pioglitazone, a selective PPARγ agonist, applied intracerebroventricularly (ICV) on the expression of IL-1β, IL-1RI and IL-1ra in the peri-infarct frontoparietal cortex of rats exposed to cerebral ischemia.

Methods: Pioglitazone (3 nmol/h) or vehicle (controls) were infused ICV via osmotic minipumps in male Wistar rats, over a 5-day period before, and 24 or 48 h after transient middle cerebral artery occlusion (MCAO) for 90 mins. The induction of IL-1β, IL-1RI and IL-1ra in the frontoparietal cortex adjacent to the ischemic core was studied by immunohistochemistry and Western blot at both time points after MCAO. Cortical cell cultures were used to ascertain the role of the PPARγ in the regulation of IL-1ra expression in neurons.

Results: Activation of cerebral PPARγ reduced the infarct size at both time points (by 37% and 32%, respectively). Pioglitazone infused ICV abolished IL-1β protein levels and decreased the number of IL-1 beta positive cells in the frontoparietal cortex adjacent to the ischemic core, the expression of the IL-1RI was not affected. Pioglitazone up-regulated IL-1ra protein in the peri-infarct cortical tissue and increased the numbers IL-1ra positive cells in this area at both time points after MCAO. In primary cortical cell culture, the selective PPARγ antagonist, GW-9662, failed to reverse the pioglitazone-induced up-regulation of IL-1ra protein, indicating that this effect was not mediated by PPARγ.

Conclusions: The activity of the IL-1 system depends on the balance between IL-1 and its antagonist, IL-1ra. Inhibition of IL-1β expression und the up-regulation of IL-1ra by pioglitazone shifted the balance between agonist/antagonist ligands towards reduction of post-ischemic inflammation. The suppression of post-ischemic inflammation in brain tissue limits the progression of ischemic injury and improves the recovery from ischemic stroke.

666. Correlations between immune inflammatory processes in atherosclerosis and the bioelectrical activity and hemodynamics of the brain

S. Kuznetsova, V. Kuznetsov and I. Palamarchuk

Cerebrovascular Pathology, Institute of Gerontology AMS Ukraine, Kyiv, Ukraine

Background: That the patients with cerebral atherosclerosis show the dysfunction of brain structures, vascular tone disorders and reduced overall speed of cerebral hemodynamics and that the atherogenesis represents a process of immune and inflammatory disturbances is the common knowledge (del Zoppo GJ, 2008; Caplan LR 2008; Ross R 1999; Libby P, 2002).

Our recent investigations have been focused on the neuroimmune reactions in the patients with cerebral atherosclerosis. We found the imbalance of main lymphocyte subpopulations, the high level of circulating immune complexes and the high activity of autoimmune reactions in response to neuro antigens (S.M. Kuznetsova, M.I. Lisiany and I.S. Palamarchuk, 2008).

The above phenomena seem to be related with transcranial duplex density of atherosclerotic plaques and the dominant hemisphere of functional and cerebral hemodynamic disorders. We decided to look into this problem at a more specific angle of view, namely, to study immune changes in atherosclerosis and at CNS dysfunction.

Objective: The pattern of correlations between immune inflammatory processes and brain bioelectrical activity/hemodynamics in cerebral atherosclerosis (CA).

Methods: The study involved the group of CA patients, altogether 15 men and women at age range 50 to 65 years. Ten age-matched healthy subjects made the control group. EEG was recorded on Neurofax EEG 1100k (Nihon Kohden, Japan); extra- and transcranial scanning of major head and neck arteries (EN VISIOR, Philips); immunological investigation using monoclonal antibodies (Sorbent Service, Russia; Becton Dickinson, USA).

Results and discussion: The EEG recordings evidenced for diencephalic disturbances in both hemispheres, with signs of central-parietal-limbic and reticular structures dysfunction being noted for right hemisphere. In the CA patients, median frequency of the EEG spectrum in the right occipital-parietal leads correlated with the pulse index value (2.44±0.13) in the basilar artery (r = −0.65, P<0.05) and with an increased adhesive activity of the neutrophils (r = −0.66, P<0.05). Pulse index in the basilar artery correlated with the level of circulating immune complexes (117.0±31.04 in nominal units) of the blood (r = −0.65, P<0.05). Total frequency of the EEG spectrum in the right central leads correlated with the level of antibodies to NSE (r = 0.55, P<0.05) and with LBFV in the left ICA (r = 0.80, P<0.05). Theta rhythm power in the right occipital leads correlated with LBFV in the right ICA (r = 0.61, P<0.05) and with the level of sensibilization of the neutrophils to the main myelin protein (r = −0.56, P<0.05).

Conclusion: There exist statistical correlations in cerebral atherosclerosis between cerebral hemodynamics and neuroimmune indices and the bioelectric activity of the brain. The brain bioelectrical activity in the right hemisphere correlates with the high activity of autoimmune reactions (the adhesive activity of the neutrophils, the level of sensibilization of the neutrophils to the main myelin protein, the antibodies to NSE) in the patients with cerebral atherosclerosis.

686. Pretreatment with the heme-oxigenase inhibitor zinc protoporphyrin-IX 24 h before ischemia enhances inflammation and increases infarct volume

I. Pérez de Puig, R. Gorina, A. Martín, S. Rojas, T. Santalucía and A.M. Planas

Brain Ischemia and Neurodegeneration, Institute of Biomedical Research (IIBB)-CSIC-IDIBAPS, Barcelona, Spain

Objectives: Heme-oxygenase (HO) is an enzymatic system responsible for heme degradation. ¹ Two HO isoenzymes have been described: an inducible form, HO-1, and a constitutive form HO-2. HO-1 mediates the anti-inflammatory effects of IL-10. ² Expression of HO-1 is induced in focal brain after ischemia,^3–5 and several lines of evidence suggest that it exerts a protective action against ischemic damage.^6,7 However, other findings suggest that HO-1 expression may be harmful. ⁸ Zinc proto-porphyrin-IX (ZnPP) is a naturally occurring compound that acts as a competitive inhibitor of HO. ZnPP has negative effects in several inflammatory conditions. However, previous studies showed that administration of ZnPP 30 mins prior to transient middle cerebral artery occlusion (MCAO) or shortly after reperfusion, is protective against ischemic damage.^9,10 An inhibitory action of ZnPP on IL-1beta has been reported and may underlie the beneficial effects of ZnPP. ¹¹ Here we tested whether systemic administration of ZnPP induced the expression of proinflammatory cytokines in the brain and whether the time of administration affected the outcome of cerebral ischemia.

Methods: We used a model of permanent MCAO with craniotomy in adult male FVB mice. ZnPP was given i.p. either 24 h (ZnPP-24 h) or 30 mins (ZnPP-0.5 h) before ischemia. The time course expression of TNF-alpha, IL-1beta, and HO-1 mRNA before and after ischemia was examined. Neutrophil infiltration was assessed with myeloperoxidase, and infarct volume was measured at day 4.

Results: ZnPP-0.5 h reduced infarct volume, in agreement with previous reports. In contrast, ZnPP-24 h had the opposite effect as it significantly increased infarct volume. Systemic administration of ZnPP specifically induced IL1beta and HO-1 mRNA expression in the control brain at 24 h, but not at 0.5 h. Therefore, the ZnPP-24 h group, but not the ZnPP-0.5 h group, had higher IL1beta and HO-1 than vehicle group at the time that ischemia was induced. The expression of TNF-alpha, IL1beta and HO-1 mRNA was increased at 4 and 8 h postischemia. At the latter time points, the ZnPP-24 h group showed noticeably higher TNF-alpha mRNA expression than the vehicle group, and exacerbated neutrophil infiltration also.

Conclusions: These findings evidence that ZnPP had dual effects depending on the time of administration. The negative effect of ZnPP-24 h is likely mediated by exacerbation of the proinflammatory burden driven by ischemia.

Acknowledgement: IP has has a predoctoral fellowship from AGAUR (Generalitat de Catalunya). Supported by the Spanish Ministry of Education and Science (CICYT) and by the European Community's Seventh Framework Programme (FP7/2007-201, grant no 201024, European Stroke Network).

737. Endogenous immunomodulation after experimental focal ischemia: multitargeted cerebroprotection by regulatory T cells is the key

R. Veltkamp¹, E. Suri-Payer², C. Veltkamp³, C. Sommer⁴, S. Rivest⁵ and A. Liesz¹

¹Neurology, University Heidelberg; ²Immunogenetics, German Cancer Research Center; ³Internal Medicine IV, University Heidelberg, Heidelberg; ⁴Neuropathology, University Mainz, Mainz, Germany; ⁵Molecular Endocrinology, Laval University, Quebec, QC, Canada

Background and objectives: Systemic and local inflammatory processes have come into the focus of translational cerebrovascular research because they play an important, mainly detrimental role in the pathophysiology of ischemic stroke.^1,2 To date, little is known about endogenous counterregulatory mechanisms preventing excessive inflammation after ischemia although they may represent powerful targets for therapy. CD4⁺CD25⁺Foxp3⁺ regulatory T lymphocytes (T_reg) are key immunoregulators under physiological conditions and in various systemic and CNS inflammatory diseases. ³ However, their functional role in cerebral ischemia is largely unknown. Herein, we report the impact of Treg on outcome in various murine stroke models, and identify their protective pathways and targets.

Methods: In most experiments, focal ischemia was induced by permanent transtemporal MCAO; effect on outcome was also studied after 30 or 90 mins filament-MCAO. Infarct sizes and behavioural deficits were measured after 1, 3, and 7d. Mice were depleted of Treg either by CD25-specific antibodies or by selective transfer of CD4⁺CD25⁻ T cells into lymphocyte deficient RAG^−/− mice. Cerebral cytokine expression was measured by RT-PCR, serum cytokine concentrations were quantified by ELISA. Leukocyte brain invasion and microglial activation was determined by immunohistochemistry. Lymphocyte subpopulations and quantitative intracellular cytokine production was analyzed by flow cytometry. Recombinant IL-10 was injected intraperitoneally (i.p.) or intracerebroventricularly (i.c.v.), in other experiments TNF-alpha or Interferon-gamma were neutralized by antibodies.

Results: Depletion of T_reg led to delayed infarct growth in models of MCA-coagulation and brief (30 mins) reversible MCAO. In contrast, anti-CD25 treatment did not exacerbate the already extensive lesions after 90 mins filament-MCAO. Corresponding to infarct volumes, behavioural outcome was significantly worse at day 3 and 7 in T_reg depleted mice. T_reg depletion resulted in highly increased expression of cerebral proinflammatory cytokines TNF-alpha, IL-1beta and Interferon-gamma, while i.c.v. neutralization of these cytokines in anti-CD25 treated mice reversed infarct volumes to values of control mice. Activated microglia and invading T lymphocytes were identified as the predominant cellular sources of TNF-alpha and Interferon-gamma, respectively. Anti-CD25 injection was associated with significantly increased number of invading granulocytes and activated (IBA-1+) microglia 24 h after MCAO; quantitative analysis of intracellular Interferon-gamma production in brain invading T cells 5d after ischemia revealed multifold higher values in Treg depleted animals. Serum concentration of TNF-alpha and Interferon-gamma were significantly elevated at all measured time points from 6 h to 7d after ischemia in anti-CD25 treated mice. Furthermore, i.c.v. (but not i.p.) administration of IL-10 prevented infarct enlargement in T_reg depleted mice and significantly lowered the postischemic overexpression of cerebral proinflammatory cytokines after T_reg depletion.

Conclusions: Treg are potent endogenous cerebroprotective modulators in models of cortical and subcortical stroke. IL-10 is their key mediator by suppressing proinflammatory cytokine expression and by targeting innate and adaptive immune cells.

Supported by Else-Kröner-Fresenius Stiftung (R.V.).

779. Brain perihematoma gene profile following spontaneous human intracerebral hemorrhage

A. Rosell¹, E. Cuadrado¹, I. Ferández-Cadenas¹, P. Delgado¹, T. García¹, M. Ribó¹, M. Hernández-Guillamon¹, A. Ortega-Aznar² and J. Montaner¹

¹Neurovascular Research Laboratory, Vall d'Hebron Research Institut; ²Neuropathology, Vall d'Hebron Hospital, Barcelona, Spain

Objectives: Spontaneous intracerebral hemorrhage (ICH) occurs from spontaneous rupture of an intracranial vessel. It represents about 15% of all strokes and is associated with high mortality rates and dreadful functional outcome. Neuroimaging studies have demonstrated a dynamic process where hematomas might expand over time and are associated with cerebral edema and perihematomal injury. Our aim was to identify the biological pathways activated in perihematomal areas by determining differences in the gene expression profile when compared to contraleteral healthy brain areas.

Methods: Twelve brain samples were obtained from four deceased patients who suffered a spontaneous ICH, including perihematomal tissue (PH) and the corresponding contralateral white (CW) and grey (CG) matter. Importantly, brain tissue was obtained within the first 6 h after death to preserve RNA integrity and to avoid post-mortem autolysis. After isolation, total RNA was analyzed for quality (Agilent-BioAnalyzer). Finally, samples were processed using the Affymetrix GeneChip platform for analysis of over 47,000 transcripts (human U133 Plus 2.0). Paired samples from the same individual were used for comparisons between the 3 studied areas (PHvsCW, PHvsCG and CWvsCG). In order to deal with the multiple testing issues, P-values were adjusted using the Benjamini and Hochberg method. Microarray Analysis Suite 5.0 was used to process array iamges and the Ingenuity Pathway Analysis System was used to model and analyze the ontology pathways of the differentially expressed genes.

Results: Quality control parameters (standard Affymetrix parameters and a Probe-Level Model) and plots indicated that the quality of the arrays was good and with a relatively low background. Over 4000 genes expressed and showing some variability were screened: of these 359 were differentially expressed in the PHvsCW group and over 2000 in both PHvsCG and CWvsCG groups (adj.P.Val <0.05 for all groups). All genes showing significant differences in their expression between CWvsCG groups were discarded since they most likely represent inherent differences due to different cellular populations and physiological functions. Finally, we identified 472 genes in the PH areas displaying a different expression pattern with a fold change between −3.74 and +5.15 when compared to the contralateral areas (309 were over-expressed and 163 were under-expressed). Of them, 6.5% of the genes represented differences between PHvsCW, 82.5% between PHvsCG and 11% between PH and both control areas. Bioinformatics analysis revealed important gene interactions in 8 key networks of cell death, cell signaling interactions, antigen presentation, cell-mediated immune response, immune cell trafficking, leukocyte-extravasation signaling pathways. Besides, gene overexpression related to cellular growth, development and proliferation was also found to be activated in the perihematomal areas. The top genes which appeared most significantly overexpressed in the PH areas codify for cytokines, chemokines, blood coagulation factors or cell growth and proliferation factors while the underexpressed codify for proteins involved in axonal guidance, cell cycle or neurotrophins.

Conclusions: The genetic-expression analysis of perihematomal areas after hemorrhagic stroke reveals changes in multiple pathological pathways but also in brain repair processes. This perihematomal area with putative secondary edema injury might also undergo endogenous repair mechanisms of therapeutical interest.

850. Downregulation of inflammatory gene expression by rosiglitazone through A 5-lipoxigenase dependent mechanism in experimental stroke

I. Ballesteros¹, M. Sobrado¹, J. Vivancos², F. Nombela², J.M. Pradillo¹, I. Lizasoain¹ and M.A. Moro¹

¹Pharmacology, Universidad Complutense de Madrid; ²Neurology, Hospital Universitario La Princesa, Madrid, Spain

Background: PPARg is a ligand-activated transcription factor belonging to the nuclear receptors superfamily. The PPARg agonist rosiglitazone (RSG) is neuroprotective in experimental acute stroke and reduces inflammation. We have recently shown that RSG induces 5-lipoxygenase (5-LO) expression in ischemic rat brain.^1,2 Since 5-LO plays a key role in inflammatory responses and its inhibition abolishes RSG induced neuroprotection, we aimed to study the 5-LO dependent mechanisms which underlie RSG neuroprotective efects in experimental stroke.

Methods: Focal permanent ischemia was induced by middle cerebral artery occlusion (MCAO) in Fisher rats. RSG was administered 10 mins after MCAO and BWA4C (selective 5-LO inhibitor) was administered 3 h after MCAO. mRNA levels of inflammatory mediators (COX-2, TNFalpha, MMP9 and iNOS) were measured by real time PCR (5 h after MCAO). Protein levels were also determined by western blot (18 h after MCAO).

Results: RSG inhibited the expression of COX-2, iNOS, and mature forms of TNF-α and MMP9. More interesting, the 5-LO inhibitor BWA4C reversed RSG-induced inhibition of the gene expression of COX-2, iNOS and TNF-α. For MMP9, reversal of expression was observed only at the mRNA level.

Conclusions: inhibition of inflammatory gene expression is a mechanism involved in the neuroprotective actions of 5-LO induction by RSG, suggesting that 5-LO by-products mediates RSG-induced neuroprotection.

898. Th1-type modulation of the peripheral immune response exacerbates ischemic brain damage

A. Denes, N. Rothwell, R. Grencis and N. Humphreys

Faculty of Life Sciences, University of Manchester, Manchester, UK

Objectives: Peripheral infection and inflammation have been associated with pathological processes in the CNS including autoimmune, neurodegenerative disorders and cerebral ischemia. To date, the exact mechanisms by which the peripheral immune response can modify central inflammatory conditions and damage formation in stroke have not been revealed. Our aim was to investigate the effect of the Th1- and Th2-polarized peripheral immune responses on focal cerebral ischemia in mouse. To achieve this, we used a well-established model of parasitic infection of the gut. This model enables us to modulate the nature of the immune response, making it possible to evaluate the effects of Th1 and Th2 type immune responses on the ischemic brain damage in the same model system.

Methods: Male C57BL/6 mice were infected orally with a high or low dose of Trichuris muris eggs, to induce Th2- or Th1-polarized peripheral immune responses, respectively. Post-infection intervals were selected on the basis of previous experiments to obtain the peak of Th2- and Th1-type responses. Infected mice and age/weight matched controls were subjected to 45 mins of middle cerebral artery occlusion (MCAo) or sham surgery. 24 and 48 h after reperfusion, blood was taken and brains were perfusion-fixed or homogenized. Unfixed spleen, bone marrow, liver samples and mesenteric lymph nodes were collected. Ischemic and blood brain barrier damage as well as the number of apoptotic neurons in the brain were determined.

Results: Simultaneous measurement of 16 different cytokines by cytometric bead array in the periphery, analysis of bone marrow cells by flow cytometry and evaluation of anti-parasite IgG levels by ELISA confirmed appropriate Th1- and Th2-type response in infected mice. The Th1-type response significantly exacerbated ischemic- and BBB damage as well as neuronal death compared to control mice. Proinflammatory cytokine levels were elevated in the lymph nodes compared to control- and Th2-type mice. Anti-parasite IgG1 levels showed a negative correlation with the ischemic damage in mice with Th1-type response. IL-6, KC, MCP-1, G-CSF and RANTES in brain homogenates of Th1-shifted mice showed sustained elevation after MCAo compared to controls. RANTES levels were significantly higher in plasma, liver and blood cells of sham Th1-polarized mice compared to control sham animals. Ischemic brain damage was not augmented in mice with Th2-polarized response compared to controls, but showed positive correlation with the peripheral levels of proinflammatory cytokines and anti-parasite IgG2a antibodies.

Conclusions: Our results show that a peripheral Th1-type immune response can significantly exacerbate ischemic brain damage in mice. Analysis of multiple cytokines in plasma and several peripheral organs indicate augmented inflammation in these mice after MCAo and identifies RANTES as the most likely candidate to mediate peripheral Th1-type response induced changes to the brain.

988. Beta 1 adrenergic activation offers neuroprotection after ischemia in organotypic hippocampal slices by inducing anti-inflammation, anti-apoptosis and activating oxidative defense

T. Markus¹, T. Wieloch², T. Cronberg³, S. Hansson⁴, C. Cilio⁵ and D. Ley¹

¹Pediatrics; ²Experimental Brain Research; ³Neurology; ⁴Obstetrics and Gynecology, Lund University, Lund; ⁵Cellular Autoimmunity, Malmo University Hospital, Malmo, Sweden

Objectives: Adrenergic agonists have potent anti-inflammatory ¹ and anti-epileptic effects. ² We evaluated the neuroprotective and anti-inflammatory effects of beta adrenergic agonists in a previously developed model of perinatal brain inflammation and ischemia using immature organotypic hippocampal slices. ³ To assess the level of inflammation we measure the levels of pro- and anti-inflammatory cytokines released by the hippocampal slice culture and to evaluate the neuronal cell death we measure levels of incorporation of propidium iodide (PI) in cells of the neuronal regions of the hippocampus. We further characterize the mechanisms of induced anti-inflammation and neuroprotection by beta adrenergic stimulation in hippocampal slice cultures by analyzing profiles of gene expression using microarray technology.

Methods: Hippocampal slices from balb/c mice were obtained at postnatal day 6 and grown in vitro up until in vitro day 9. To induce inflammation hippocampal slices were exposed to lipopolysaccharide (LPS) during 24 h followed by 15 mins of in vitro ischemia, induced by exposure to oxygen and glucose deprivation (OGD). To study the modifying effects of beta adrenergic agonists on inflammation and ischemia, hippocampal slices were exposed to the beta1 adrenergic agonist dobutamine during the 24 h of LPS exposure. Neuronal cell death was determined with PI which was measured by fluorescent light microscopy. Inflammation was assessed by measuring cytokine release using cytometric bead array (Becton Dickinson). Microglial activation and neuronal depletion was studied by immunohistochemistry. Gene expression profiles were studied by microarray technology, using slides which were printed with 33 k oligos. A commercial mouse RNA (Stratagene) was used for quantification of specific gene transcripts in culture samples.

Results: Exposure to LPS+OGD resulted in extensive cell death in neuronal sub-regions CA1, -2, -3 and the dentate gyrus. Co-incubation with beta1 adrenergic agonist (50 μM) during LPS exposure conferred complete protection from cell death in all neuronal subregions (P<0.001). The neuroprotective effect of beta1 agonist was associated with decreased levels of inflammatory cytokines in medium and an inhibition of morphological microglial activation. Gene expression analysis revealed a suppressed inflammatory activation, increased defense against oxidative damage, and increased expression of anti-apoptotic mediators in association with beta1 adrenergic neuroprotection after induced inflammation and ischemia.

Conclusions: Enhanced signaling through the beta1 adrenoceptor may enable neuroprotection in the context of perinatal inflammation and ischemia. Differences in endogenous adrenergic input could explain varied individual vulnerability to inflammatory and ischemic insults. Expression profiling of endogenous neuroprotective strategies might aid in the development of neuroprotective drug discovery.

Chronic activation of the cannabinoid type 2 receptor (CB2) is neuroprotective in ischemic stroke by decreasing proinflamatory cytokines

J.G. Zarruk, D. Fernandez-Lopez, J. Pradillo, V.G. Romera, I. Lizasoain and M.A. Moro

Pharmacology, Universidad Complutense de Madrid, Madrid, Spain

Background: Ischemic Stroke continues to be one of the main causes of death worldwide. ¹ The cannabinoid system has been of great interest as a possible pharmacological approach to treat neurodegenerative diseases and stroke.^2,3 Cannabinoid type 2 receptor (CB2) is expressed predominantly by cells of the immune system, including microglia in the central nervous system. ⁴ The selective activation of CB2 has been shown to be neuroprotective after transient Middle Cerebral Artery Oclussion (MCAO) in mice, associated with the attenuation of rolling and adhesion of leucocytes. ⁵ However, the effect of selective chronic activation of CB2 on proinflammatory cytokines after permanent MCAO has not been studied yet.

Objectives: To determine the neuroprotective effect of CB2 chronic activation and the expression of proinflamatory cytokines after experimental ischemic stroke in mice treated with a selective CB2 agonist compared with vehicle.

Methods: ICR male mice were divided in two groups according to the pharmacological treatment. Five days prior to the MCAO and two days after MCAO, animals received a daily intraperitoneal dose of either the selective CB2 agonist JWH-133 (5 mg/kg) or vehicle. Neurological symptoms were evaluated using the modified Neurological Stroke Scale (mNSS) 24 and 48 h after MCAO. To determine infarct volume, brains were removed 48 h after MCAO, cut into 7 coronal brain slices 1 mm thick and stained with 2% TTC (2,3,5-triphenyltetrazolium chloride). Another group of animals, chronically treated with JWH-133 or vehicle (5 days prior to MCAO), was sacrificed 8 h after MCAO to determine the brain expression of Tumor Necrosis Factor α (TNF-α), Interleukin 6 (IL6), Interleukin 1β (IL-1β) and RANTES chemokine.

Results: The chronic administration of JWH-133 significantly improved the neurological impairment after MCAO at 24 and 48 evaluated with the mNSS and decreased brain infarction compared with the vehicle treated group (P<0.05). Permanent ischemia in the untreated animals (vehicle) was associated with an increase in the expression of TNF-α, IL-6, IL1β and RANTES chemokine. In contrast, the selective CB2 agonist significantly decreased the expression of these proinflamatory chemokines.

Conclusions:

• (1)

  Chronic administration of JWH-133 serves as a neuroprotective treatment, reducing neurological symptoms and infarct volumen in experimental permanent ischemia.

• (2)

  A decreased expression of TNF-α, IL-6, IL1β, and RANTES chemokine, could explain the neuroprotective mechanism of the chronic activation of CB2.

Acknowlegments: Zarruk JG is Supported by the Programme Alban, the EU Programme of High Level Scholarships for Latin America, scholarship No. E07D400805CO'.