Brain Poster Session: Experimental Cerebral Ischemia—Vascular and Endothelial

22. Effects of long-term administration of HMG-CoA reductase inhibitor, atorvastatin, on microvessels in the brain of stroke-prone spontaneously hypertensive rats

K. Nomura¹, T. Katsumata¹, N. Tanaka¹, M. Ghazizadeh², E. Jin², M. Fujiwara², S. Egawa², H. Shimizu², Y. Nishiyama¹, T. Otori¹, O. Kawanami² and Y. Katayama¹

¹Department of Neurology, Nippon Medical School, Tokyo; ²Department of Molecular Pathology, Institute of Development and Aging Sciences, Nippon Medical School, Kawasaki, Japan

Objective: The objective of this study was to determine whether the long-term administration of an HMG-CoA reductase inhibitor, atorvastatin, confers antioxidative effect in microvessels in the brain of stroke-prone spontaneously hypertensive rats (SHRSPs).

Method: Atorvastatin (20 mg/kg) or vehicle was orally administered to 8-week-old SHRSPs for 5 weeks. As normal controls, vehicle was orally administered to 8-week-old WKYs over the same period. The animals were decapitated and the brains were removed. The brains were then quickly frozen, and were sliced into horizontal sections of 6-mm-thickness using a cryostat. The markers for oxidative stresses on lipids (4HNE) and DNA (8OHdG) were immunohistochemically detected. The numbers of positive vessels in the 6 randomized 0.25 mm² areas in a horizontal section were counted. The ratio of positive vessels for each maker to all vessels (positive for von Wille brand factor) was calculated.

Results: The positive ratio of oxidative stress markers was significantly higher in the vehicle group than in the normal controls. Then the positive ratio was significantly lower in the atorvastatin group than in the vehicle group. Atorvastatin significantly reduced immunoreactivities for oxidative stress markers in microvessels. Lipids such as total cholesterol (T-cho), HDL-cholesterol (HDL-cho), LDL-cholesterol (LDL-cho), and triglyceride (TG) did not differ among the vehicle, the statin, and normal control groups.

Conclusions: The results suggest that statins may confer the antioxidative properties and have the protective effects in the endothelial cells, even in the brain microvessels.

35. Concomitant treatment with medroxyprogesterone and conjugated equine estrogens attenuates estrogen's protective effect on postischemic pial artery vasodilation to acetylcholine

C. Miyazaki¹, E. Zeynalov¹ and M. LIttleton-Kearney²

¹Anesthesia/Critical Care Medicine; ²Nursing/Anesthesia Critical Care Medicine, Johns Hopkins University, Baltimore, Maryland, USA

Background and aims: Chronic estrogen replacement partially preserves postischemic pial arteriole vasodilation to endothelium-dependent vasodilators. It is unclear if concomitantly administered equine estrogens (EE) and medroxyprogesterone (MP), hormone replacement therapy commonly prescribed for women, alters estrogen's effect on postischemic pial arterial vasodilation. We determined if:

• (1)

  EE preserves postischemic pial artery dilation to acetylcholine (Ach),

• (2)

  concomitant administration of MP and EE alters the effect of EE alone on postichemic Ach-evoked dilation,

• (3)

  natural hormone depletion associated with aging depresses postischemic pial artery Ach-evoked dilation, and

• (4)

  concomitantly administered EE and MP has the same effect on postischemic pial artery dilation in young ovariectomized (OVX) and aged reproducively senescent rats (RS).

Methods: Young OVX (2 to 3.5 months) or aged RS female rats (20 to 22 months) were used for these studies. The young groups included; OVX, EE-treated (EE) and EE plus MP (EEMP). The aged groups included; RS, EE-treated (RSEE) and EE plus MP (RSEEMP). The hormones were administered via gavage daily for 1 to 1.5 months. Drug dosages, based on normal dosages for women (EE-0.625 mg; MP-2.5 mg), were weight-adjusted for rat. Rats were subjected to 15 mins forebrain ischemia. Pial artery responses to the endothelium-dependent vasodilator Ach (10 μmol/L) were examined through closed cranial windows before and 1 h postischemia.

Purpose: To determine if concomitant EE and MP treatment alters estrogen's beneficial effect on postischemic pial arteriole vasodilatory capacity in response to Ach.

Results: Compared to the young, preischemic pial artery dilation was markedly reduced in the aged groups (P = 0.001). Postischemic dilation to Ach was profoundly depressed in OVX and in the RS groups (P = 0.001) compared to preischemia. EE preserved the postischemic pial artery response in the EE and the RSEE groups. EE combined with MP attenuated the effect of EE in the young and the aged rats (P = 0.001). Because we found that aging alters pial artery vasodilatory capacity we evaluated the magnitude (delta) of the postischemic reduction in Ach-evoked vasodilation between the young and aged groups. The change between the pre and the postischemic response was similar between the OVX and the RS rats. However, the magnitude of the reduction in postischemic response to Ach was greater in the EEMP (25.3±4.7) compared to the REEMP (14.1±3.0) animals (P = 0.001).

Pre and postischemic dilation

	Preischemia	Postischemia
OVX (n = 6)	24.7±5.0	8.1±1.2
EE (n = 6)	24.4±2.9	22.2±3.4
EEMP (n = 5)	26.6±5.8	1.3±2.0
RS (n = 6)	14.4±2.0	0.64±1.2
RSEE (n = 6)	14.4±2.4	13.2±1.2
RSEEMP (n = 6)	13.5±0.8	−0.5±3.1

Conclusions: Aging alters the ability of pial arterioles to dilate in response to Ach. Postischemic pial artery vasodilatory dysfunction in both young OVX and RS rats is attenuated by chronic EE administration, but not if MP is combined with EE. Concomitantly administered MP and CE may interfere with estrogen's ability to protect the cerebrovasculature during early reperfusion.

95. Essential role of interleukin-6 for post-stroke angiogenesis

K. Gertz¹, G. Kronenberg¹, T. Baldinger¹, M. Balkaya¹, C. Werner², U. Laufs² and M. Endres¹

¹Experimental Neurology, Charité—University Medicine Berlin, Berlin; ²Department of Internal Medicine, University of Saarland, Homburg, Germany

Background: There is a paucity of studies investigating longer-term endpoints after brain ischemia. The pleiotropic cytokine interleukin (IL)-6 mediates multiple effects which may be both beneficial and detrimental to the injured brain. Here, we pursued the hypothesis that IL-6 is an essential part of the regenerative long-term response of the brain to cerebral ischemia—and in particular plays a role for post-stroke angiogenesis.

Methods: We investigated the influence of IL-6 on long-term outcome in a well-characterized model of mild ischemic stroke. To do so, IL-6 knockout versus wildtype mice (C57Bl6 background) were subjected to 30 mins of middle cerebral artery occlusion (MCAo) followed by up to four weeks of reperfusion. Proliferating cells were pulse-labelled with bromodeoxyuridine (BrdU). Functional outcome was assessed by Morris water maze. Following sacrifice we determined ischemic lesion size and performed immunohistological studies. Additionally, we counted vessel density via Evans blue perfusion signal and measured VEGF-levels in serum. Because endothelium-derived nitric oxide (NO) is known to be essential for the growth of new vessels, we also measured protein levels of endothelial nitric oxide synthase (eNOS) in brain, aorta and myocardial tissue. In wildtype mice we evaluated time-dependent regulation of inflammatory cytokines IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha and the IL-6-receptor (IL-6R) as well as gp130.

Results: While there was a significant up-regulation of TNF-alpha and IL-1beta mRNA at early time-points after ischemia, both IL-6R and gp130, and to a lesser extent also IL-6 itself, showed a delayed up-regulation up to four weeks after MCAo/reperfusion. Whereas in wildtype mice, eNOS and VEGF protein levels were significantly elevated at two days after MCAo/reperfusion, this effect was absent in IL-6 deficient animals. At four weeks after MCAo IL-6 knockout mice showed significantly lower BrdU+ cell density in ischemic striatum. Importantly, not only the number of BrdU/Iba-1 double labelled cells was reduced within the ischemic lesion of IL-6 deficient mice, but also the number of newly generated endothelial cells (2.6-fold decrease compared to wildtype mice; P<0.05). In addition, while in wildtype animals the density of perfused microvessels within the ischemic lesion was significantly increased compared to the contralateral side this effect was completely absent in IL-6 knockout mice. Interestingly, this failure in post-stroke angiogenesis in IL-6 knockout mice was associated with significant long-term functional and cognitive impairment assessed with Morris water maze. In the place task, latencies to find the platform were significantly longer in IL-6-deficient mice. In the probe trial, IL-6-deficient animals visited the target zone more frequently in the latter 60 secs of the trial, indicative of a distinct deficit in strategy-switching. In addition, at 4 weeks after MCAo IL-6 knockout mice presented larger lesions compared to wildtype mice while acute lesion size at two days was not significantly different.

Conclusion: We demonstrate that loss of IL-6 results in impaired long-term histological and functional protection four weeks after cerebral ischemia. IL-6 augments inflammation and angiogenesis during the chronic phase after brain ischemia, and this is associated with improved recovery. These effects may in part been mediated by eNOS.

112. Normobaric hyperoxia attenuates blood brain barrier disruption by inhibiting matrix metalloproteinase-9 in transient focal cerebral ischemia

W Liu¹, J. Hendren¹, R. Sood², Q. Chen¹, X. Qin¹ and K.J. Liu^1,2

¹College of Pharmacy; ²Department of Neurology, University of New Mexico, Albuquerque, New Mexico, USA

Objectives: Using in vivo electron paramagnetic resonance oximetry, we have shown that normobaric hyperoxia (NBO) treatment during ischemia can maintain penumbral tissue oxygenation at the physiological level, which leads to a significant reduction in stroke lesion volumes in ischemic stroke.^1,2 In this study, we evaluated the effect of NBO on blood brain barrier (BBB) damage and the underlying mechanisms using a rat model of transient focal cerebral ischemia.

Methods: Rats were subjected to NBO (95% O₂) or normoxia (21% O₂) during 90 mins filament occlusion of the middle cerebral artery (MCAO), followed by 3 or 22.5 h of reperfusion. Infarction volume was evaluated by MRI apparent diffusion coefficient (ADC) analysis and 2,3,5-triphenyltetrazolium chloride (TTC) stain. BBB damage was assessed by measuring Evan's blue extravasation or the permeability coefficient with MRI in ischemic brain. Western blot and immunohistochemistry were used to analyze the protein levels of tight junction protein occludin and NADPH oxidase in the cerebral microvessels. Matrix metalloproteinase-9 (MMP-9) was analyzed by in situ and gel gelatin zymography.

Results: NBO treatment during ischemia significantly reduced infarction volume, Evan's blue extravasation, BBB permeability and brain edema in the ischemic brain after 90-min MCAO with 3 or 22.5 h of reperfusion. At the early reperfusion time point (3 h), we observed that Evan's blue leakage was accompanied by increased gelatinolytic activity and reduced immunostaining for tight junction protein occludin. Moreover, incubation of brain slices or isolated microvessels with purified MMP-9 revealed specific degradation of occludin. At late reperfusion time point (22.5 h), MMP-9 induction concurrently occurred with the upregulation of NADPH oxidase catalytic subunit gp91^phox in the ischemic cerebral microvessels. Gel gelatin zymography identified that MMP-9, but not MMP-2, was the major gelatinotylic enzyme in the cerebral microvessels. Inhibition of NADPH oxidase with apocynin reduced MMP-9 increase, supporting a causal link between NADPH oxidase-derived superoxide and MMP-9 induction in the BBB microvasculature at late reperfusion (22.5 h). Importantly, NBO treatment significantly inhibited MMP-9 induction in the ischemic cerebral microvessels under both experimental stroke conditions.

Conclusions: Our results indicate that NBO treatment protects BBB integrity in focal cerebral ischemia, and its inhibitory actions on NADPH oxidase and MMP-9-mediated tight junction disruption may represent important mechanisms for this protection.

Keywords: Normobaric hyperoxia, cerebral ischemia, matrix metalloproteinases, NADDPH oxidase, occludin.

199. Alpha4-Integrin inhibition is neuroprotective after experimental brain ischemia in mice

A. Liesz¹, S. Karcher¹, C. Sommer² and R. Veltkamp¹

¹Neurology, University of Heidelberg, Heidelberg; ²Neuropathology, University of Mainz, Mainz, Germany

Objectives: Inflammatory cascades play a detrimental role in the acute phase after ischemic stroke. ¹ Invading inflammatory cells are an important source of proinflammatory cytokine release and secondary infarct growth.^1,2 Alpha4-Integrins are adhesion molecules on leukocytes. Blocking of these integrins by specific antibodies prevents the migration of leukocytes into the brain ³ which represents a potent therapy in experimental encephalomyelitis ⁴ and clinical multiple sclerosis. ⁵ Therefore, we investigated the effect of Alpha4-Integrin inhibition on brain ischemia outcome and its impact on postischemic brain inflammation and systemic immune parameters.

Methods: Permanent focal cerebral ischemia was induced by transtemporal middle cerebral artery occlusion (MCAO). Mice were treated 24 h before MCAO with a single intraperitoneal dose of 300 μg Anti-Alpha4-Integrin, controls received IgG2 isotype. Impact of antibody treatment on circulating lymphocyte counts was analyzed by flow cytometry. Infarct volume and behavioural deficits (‘Corner Test’, ‘Cylinder Test’) were determined at several time points after MCAO. Brain cytokine expression was analyzed by RT-PCR, serum cytokine levels were measured by ELISA. Leukocyte brain invasion (CD45⁺, CD3⁺, B200⁺, DX5⁺, MPO⁺ and Foxp3⁺ cells) and microglial activation (IBA-1⁺ cells) was determined by immunohistochemistry.

Results: Anti-Alpha4-Integrin treatment significantly reduced the infarct size 7d after MCAO (11±1.7 mm³) compared to control treatment (14±1.9 mm³, n = 10 per group, P<0.01). Correspondingly, behavioural assessment revealed a significantly improved outcome in the treatment group at 3d (P = 0.015) and 7d (P = 0.012) after MCAO. Anti-Alpha4-Integrin injection did not alter the number of circulating B-, T_Helper-, T_Effector- and NK-cells in the blood 3d after treatment compared to control mice. We investigated the brain invasion of diverse leukocyte subsets after ischemia, including T-, B-, NK-cells and neutrophilic granulocytes as well as the activation of resident IBA-1⁺ microglial cells, in treated mice and control animals 5d after MCAO. Alpha4-Integrin inhibition significantly (n = 5, P<0.05) reduced cerebral expression of the proinflammatory cytokines TNF-alpha, IL-6 and Interferon-gamma at 24 h and 3d after MCAO compared to controls. In contrast, expression of the neuroprotective, anti-inflammatory cytokines IL-10 and TGF-beta was not affected by the therapy. Furthermore, Anti-Alpha4-Integrin injection did not alter the serum concentrations of the main pro- and anti-inflammatory cytokines in naïve mice and at 24 h and 5d after MCAO, respectively.

Conclusions: To our knowledge, this study shows for the first time that Alpha4-Integrin inhibition significantly reduces infarct size and behavioural deficits after experimental brain ischemia. The present data suggests that this neuroprotection is mediated by reduced cerebral neurotoxic cytokine release whereas systemic immune system function and cytokine homeostasis is not altered.

293. Gelatin-siloxane hybrid scaffolds with vascular endothelial growth factor induces brain tissue regeneration

M. Takamiya¹, H. Zhang¹, K. Deguchi¹, K. Tsuru², V. Lukic¹, T. Yamashita¹, S. Nagotani¹, S. Hayakawa²; A. Osaka², T. Kamiya¹ and K. Abe¹

¹Department of Neurology; ²Biomaterial Laboratory, Okayama University, Okayama, Japan

Background and aims: In the brain after infarction or trauma, the tissue becomes pannecrotic and forms a cavity. In such situation, a scaffold is necessary to produce new tissue. In this study, we implanted a new porous gelatin-siloxane hybrid derived from gelatin and 3-(glycidoxypropyl) trimethoxysilane (gelatin-GPTMS) scaffolds into a brain defect, and investigated whether it makes a new brain tissue. In addition, vascular endothelial growth factor (VEGF) was added on gelatin-GPTMS scaffolds and its effect on tissue regeneration was examined.

Figure 1

Whole brain (A) and HE stained coronal brain section (B) with gelatin-GPTMS scaffold implantation. The hybrid scaffold (arrow heads) kept the integrity of brain shape (A, B). The inset in panel A shows the scaffold observed by scanning electron microscopy. Representative gelatin-GPTMS scaffold implanted coronal brain sections, without (C-a, and C-c) and with (C-b and C-d) VEGF. The panels C-c and C-d were from rectangles in panels C-a and C-b, respectively. The white dotted line indicates the boundary between the newly formed tissue and the original tissue (C-c and C-d). Panel C-e shows quantitative analysis of the newly formed tissue volume in the gelatin-GPTMS scaffold (*P<0.05).

Results: At 30 days after the implantation, the marginal territory of the scaffolds became occupied by newly formed tissue. Immunohistochemical analysis revealed that the new tissue was constituted by endothelial, astroglial and microglial cells, some of which were labeled for bromodeoxyuridine (BrdU). Addition of VEGF promoted numbers of these cells.

Conclusions: The present study demonstrated that combination of gelatin-GPTMS scaffolds and VEGF was preferable for brain regeneration.

303. Enhanced contractile response of vascular contractile receptors following distal focal permanent occlusion

M.N.P. Rasmussen¹, M. Hornbak¹ and L. Edvinsson^1,2

¹Department of Clinical Experimental Research, Glostrup Research Institute, Glostrup University Hospital, Glostrup, Denmark; ²Department of Medicine, Institute of Clinical Sciences, University Hospital of Lund, Lund, Sweden

Objectives: Previous investigations have revealed that cerebral ischemia is accompanied by changes in the receptor expression in cerebral vasculature. It has been shown that following experimental cerebral ischemia levels of the vasoconstrictors endothelin (ET), 5-hydroxytryptamine (5-HT) and angiotensin (Ang II) are increased.^1,2 We have previously demonstrated a time-dependent up-regulation of the contractile ET_B, AT₁ and 5-HT₁ receptors in cerebral arteries following subarachnoid haemorrhage and transient middle cerebral artery (MCA) occlusion at both mRNA, protein and functional levels.^3,4 This up-regulation leads to enhanced vasoconstriction and show correlation with reduction in regional cerebral blood flow (rCBF) and infarct volume. The present study was designed to investigate if small permanent vascular insult without visible ischemic damage leads to altered expressional profile of contractile receptors in the smooth muscle cells of the cerebral artery. To test the hypothesis we employed a model of focal permanent occlusion by ligation of the distal part of the MCA. This additionally gives us the opportunity to investigate vessel segments both upstream and downstream to an occlusion. The knowledge obtained from this study will further the understanding of how cerebral arteries participate in the events that take place following a stroke.

Methods: Focal permanent occlusion is obtained by a distal ligation of the MCA in male Wistar rats. Artery access is achieved by craniotomy and occlusion is verified by laser-Doppler. ⁵ Controls consist of un-ligated left MCA and sham operated rats. After 24 h, MCA segments located upstream and downstream to the ligature are investigated for expressional changes of ET, AT and 5-HT receptors at protein (immunohistochemistry) and functional (myograph) level. ⁶ TTC stainings are performed to examine brains for ischemic damage.

Results: The contractile responses to S6c (selective ET_B agonist) and 5-CT (5-HT₁ agonist) are significantly stronger in the right occluded MCA (85.1±27.2%, and 59.1±9.4%, respectively) compared with the left MCA (11.8±4.3% and 27.7±6.7%, respectively), and for S6c also the MCA from sham operated rats (21.3±9.7%) 24 h after occlusion. Changes were observed in the vessel segments downstream to the occlusion and not in the upstream. TTC staining showed no indication of ischemic damage in any rat groups.

Conclusion: A small focal vascular occlusion without tissue ischemia leads to increased contractile function of ET_B and 5-HT_1B receptors. Since in the upstream segments, receptor expression was unaltered it does not appear as change in shear stress is the crucial factor. In addition, the changes seen in the downstream segments, without noticible tissue damage, indicate that it is not the ischemic condition per se which is responsible for an increased receptor expression/sensitivity.

326. Pre and postischemic estrogen receptor expression in isolated cerebral microvessels after acute or prolonged periods of hypoestrogenicity

E. Zeynalov¹, C. Miyazaki¹ and M. Littleton-Kearney^1,2

¹Johns Hopkins University School of Medicine; ²Johns Hopkins University School of Nursing, Baltimore, Maryland, USA

Objectives: Estrogen receptors (ERs) in the brain modulate post-ischemic neo-vascularization, play a role in suppressing mitochondrial free radical formation, and may improve vasoreactivity in the brain in the presence of circulating estrogen. These effects are achieved when estrogen replacement therapy (ERT) is initiated early after loss of endogenous estrogen production, but after a time of prolonged hypoestrogenicity estrogen replacement therapy may be ineffective or even harmful relative to stroke prevention and treatment. It is unclear how short-term or prolonged estrogen deprivation effects ER expression in the healthy or postischemic cerebral microvasculature. The current study was designed to assess:

• (1)

  basal and

• (2)

  postischemic cerebral microvascular ER expression in estrogen depleted and in 17β-estradiol-treated rats after short-term or prolonged estrogen depletion.

Methods: Three groups of sexually mature female rats were used: ovariectomized (OVX), OVX plus early estrogen-treated (E2) and OVX plus delayed estrogen-treated (DE2). Estrogen treated rats were recovered for either 1 or 10 weeks postovariectomy then subcutaneously implanted with 17-β-estradiol pellets (0.05 mg). After 2 weeks of ERT rats were subjected to 15-mins global 4 vessel occlusion ischemia and 3-h reperfusion. All surgical manipulations were performed under 1.5% isoflurane anesthesia, and all physiological variables were maintained within normal range. At the end of each experiment, blood was collected for estrogen levels, and brains were perfused and harvested for microvessel isolation. Each cortex was homogenized, then filtered 3 times through a 60-μm nylon mesh. Microvessel isolation purity was confirmed by microscopic imaging and ER protein expression was evaluated using Western blotting techniques. The band densities were quantified and expressed as a ratio to actin.

Results: Immunoblots for Von Willebrand Factor were used to confirm microvessel enrichment. ER-α expression was lower in the OVX (0.006±0.01) when compared to the E2 (0.183±0.15) and DE2 (0.192±0.10) groups. Early ischemia/reperfusion increased the microvascular ER-α expression in the OVX (0.358±0.12) group, but not in the E2 (0.111±0.05) or DE2 (0.190±0.03) groups. There were no differences in ER-β expression among groups under basal OVX 0.147±0.05; E2 0.093±0.07; DE2 0.139±0.07, or postischemic OVX 0.181±0.11; E20.084±0.06; DE2 0.152±0.08 conditions.

Conclusions: Compared to levels in healthy brain, transient global cerebral ischemia markedly icreases ER-α expression in isolated cerebral microvessels of estrogen-depleted female rats. This effect is notobserved in estrogen-treated rats regardless if ERT isinitiated within 1 week or delayed for 10 weeks after ovariectomy. We conclude that the timing of ERT has no effect on basal or postischemic ER-α or ER-β expression. Further studies are needed to determine the mechanism of the difference between basal and postischemic ER-α expression in estrogen-depleted animals.

354. Normobaric hyperoxia reduces the neurovascular complications associated with delayed tissue plasminogen activator treatment in transient focal cerebral ischemia

W. Liu¹, J. Hendren¹, X.-J. Qin¹ and K.J. Liu^1,2

¹College of Pharmacy; ²Department of Neurology, University of New Mexico, Albuquerque, New Mexico, USA

Background and purpose: A major limitation of tissue plasminogen activator (tPA) thrombolysis for ischemic stroke is the narrow time window for safe and effective therapy. ¹ Delayed tPA thrombolysis increases the risk of cerebral hemorrhage and mortality, which, in part, is related to neurovascular proteolysis mediated by matrix metalloproteinases (MMPs). We recently showed that normobaric hyperoxia (NBO) treatment reduces MMP-9 expression and blood brain barrier (BBB) disruption in the ischemic brain.^2,3 Therefore, we hypothesized that NBO could increase the safety of delayed tPA thrombolysis in stroke.

Methods: Thirty six male Sprague Dawley rats were exposed to NBO (95%O₂) or normoxia (21%O₂) during 5-h filament occlusion of the middle cerebral artery, followed by 19-h reperfusion. Thirty min before reperfusion, saline or tPA were continuously infused to rats over 1 h. Successful MCAO was confirmed with 2,3,5-triphenyltetrazolium chloride (TTC). Outcome parameters were neurological score, mortality rate, brain edema, hemorrhage volume and MMP-9. Hemorrhage was quantified with a hemoglobin spectrophotometry method. Edema was evaluated as hemispheric enlargement. MMP-9 was measured by gelatin zymography.

Results: Regardless of saline or tPA treatment, NBO-treated rats showed a relatively smaller infarct area on the TTC-stained brain section than normoxic rats. In normoxic rats, delayed tPA treatment at 4.5 h after stroke onset resulted in high mortality, more severe neurological deficits, increased hemorrhage volumes and augmented MMP-9 induction in the ischemic BBB microvasculature compared with saline. Rats treated with combined NBO and tPA showed significant reductions in tPA-associated mortality, brain edema, hemorrhage and MMP-9 augmentation, as compared with tPA alone.

Conclusions: These results suggest that early NBO treatment may represent an important strategy to increase the safety of delayed tPA thrombolysis in ischemic stroke.

Keywords: Tissue plasminogen activator; cerebral hemorrhage; matrix metalloproteinases; oxygen; stroke.

416. Progression of platelet—endothelium interactions after acute stroke in mice

S.M. Sonanini^1,2, S.M. Krieg^1,2, S.W. Kim³ and N. Plesnila^1,2,3

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

Objectives: Secondary to their function in haemostasis, platelets (Plt) are supposed to have pro-inflammatory properties.^3,4 As secondary neuronal damage after ischemia is accompanied by neuroinflammatory activity, especially leukocyte-endothelium-interactions (LEI), ² the aim of the current study was to characterize the progression of Plt-activation and adhesion after focal transient cerebral ischemia.

Methods: For cerebral ischemia (I) C57/Bl6 mice were subjected to 45 mins of occlusion of the middle cerebral artery (tMCAO). After 1, 3, 5, 12, or 24 h (n = 6 each) of reperfusion (R) mice were anesthetized, intubated, and mechanically ventilated. Blood was harvested from donor mice by cardiac puncture and Plts were separated and stained with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). A total of 100 × 106 Plt were injected i.v. Plt-endothelium interactions (PEI) in cortical cerebral vessels of the ischemic penumbra were investigated by intravital fluorescence microscopy (IVM) for 2 h.

Results: Key physiologic parameters like body temperature, blood pressure, blood pH, pO2, and pCO2 were within the physiologic range during tMCAO and IVM. In sham operated animals only physiologic rolling of Plt along the cerebrovascular endothelium was observed (3.4±1.0 Plt/100 μm/mins). After I/R a significant (P<0.05) increase of TEI could be observed in postcapillary venules with a peak at R = 5 h (rolling Plt: 12.97±0.74 Plt/100 μm/mins; sticking Plt: 1.31±0.17 Plt/100 μm/mins). In arterioles significant rolling Plt was already noticed 3 h after ischemia (1.36±0.24 Plt/100 μm/mins versus sham: 0.19±0.10 Plt/100 μm/mins). 12 and 24 h after I/R PEI returned to baseline values.

Conclusion: Platelet-endothelium interactions (PEI) occur in the vessels of the ischemic penumbra during the time window of neuronal cell death (2 to 8 h after ischemia) and show the same time course as the adhesion of leukocytes. ² Since Plt do not only interact with the endothelium but also with leukocytes ⁴ our data suggest that Plt may be part of the inflammatory response which leads to reperfusion damage following cerebral ischemia. ¹

476. Therapeutic window of arginine vasopressin V₁ receptor inhibition following transient focal cerebral ischemia in mice

S.W. Kim^1,2, R. Trabold² and N. Plesnila^1,2

¹Royal College of Surgeons in Ireland, Dublin, Ireland; ²Laboratory of Experimental Neurosurgery, Department of Neurosurgery and Walter-Brendel-Center for experimental Medicine, University of Munich Medical Center—Grosshadern, Ludwig-Maximilians-University, Munich, Germany

Objectives: Previously we showed that inhibition of arginine vasopressin (AVP) V₁ but not V₂ receptors have beneficial effects following experimental stroke when treatment was initiated before the onset of ischemia. ¹ Since such a pre-treatment protocol has no clinical relevance, the current study was designed to investigate the therapeutic window of AVP V₁ receptor inhibition following transient cerebral ischemia.

Methods: Male C57/BL6 mice were subjected to 60 mins occlusion of the middle cerebral artery by an intraluminal filament (MCAo) followed by 23 h of reperfusion. 15, 75, 180, or 360 mins after MCAo, 500 ng of the AVP V₁ receptor antagonist [deamino-Pen(1), O-Me-Tyr(2), Arg(8)]-vasopressin were injected into the left ventricle (n = 8 each). Twenty-four hours after MCAo infarct volume and brain edema formation were quantified. Mortality and neurological function were evaluated in separate groups over 7 days.

Results: Mice treated with the AVP V₁ receptor antagonist immediately after the MCAo had less brain edema (P<0.01) and 42% smaller infarct volumes as compared to vehicle treated animals (P<0.05). This neuroprotection resulted in significantly improved mortality and neurological function (P<0.05). Although brain edema formation was also reduced when treatment started up to 75 mins after MCAo no further beneficial effects were observed on the other assessed parameters.

Conclusions: Together with our previous results ¹ the current findings demonstrate that vasopressin V1 receptors play an important role in the pathophysiology of ischemic stroke, most likely through the inhibition of brain edema formation. Therefore inhibition of AVP V1 receptors may have a particularly high clinical potential when applied shortly before or after reperfusion, e.g. in patients receiving thrombolytic therapy by rtPA.

481. Leukocyte adhesion to cerebral endothelium is sufficient to induce post-ischemic cell death

S.W. Kim^1,2, H. Kataoka³ and N. Plesnila^1,2

¹Royal College of Surgeons in Ireland, Dublin, Ireland; ²Laboratory of Experimental Neurosurgery, Department of Neurosurgery and Walter-Brendel-Center for Experimental Medicine, University of Munich Medical Center—Grosshadern, Ludwig-Maximilians-University, Munich, Germany; ³The Department of Neurosurgery, Kyoto University Graduate School of Medicine, Kyoto, Japan

Objectives: Despite the fact that leukocytes are known to play a pivotal role in brain damage after cerebral ischemia, the pathomechanism of leukocyte-induced cell death is not well understood. The aim of the current study was to investigate the temporal and causal relationship between leukocyte-endothelium interactions (LEI), leukocyte migration into brain parenchyma, and neuronal cell death following reperfusion from focal cerebral ischemia.

Methods: ICAM-1^−/− or C57BL/6 mice were subjected to permanent middle cerebral artery occlusion (MCAo) or 60 mins MCAo followed by 23 h of reperfusion. LEI were examined by intravital fluorescent microscopy 40, 60, 90 and 120 mins after permanent ischemia and 4, 4.5, 5, 5.5 and 6 h after reperfusion following transient ischemia. Animals were sacrificed 12 h after onset of permanent ischemia and 24 h after the onset of transient ischemia for determination of infarct volume and tissue leukocyte counts.

Results: Deletion of Intercellular Adhesion Molecule-1 (ICAM-1), a leukocyte adhesion molecule, reduced leukocyte adhesion to cerebral endothelium by 30% (P<0.05) and decreased the infarct volume by 48% (P<0.05), but did not affect the number of leukocytes in the brain parenchyma.

Conclusions: Our results suggest that the transmigration of leukocytes into post-ischemic brain may not be directly related to neuronal cell death as previously anticipated but that already the intravascular interaction of leukocytes with the cerebral endothelium may be sufficient for the induction of post-ischemic cell death. Further studies will be necessary to identify the transendothelial signaling pathways responsible for this novel phenomenon.

559. Intramicrovascular behavior of platelets in murine brain after subarachnoid hemorrhage examined by intravital fluorescent microscopy

N. Tanahashi¹, T. Saitoh², K. Hattori¹ and N. Araki³

¹Neurology, Saitama International Medical Center, Saitama Medical University, Hidaka City; ²Neurosurgery, Medical Center, Saitama Medical University, Kawagoe City; ³Neorology, Saitama Medical University, Iruma-gun, Japan

Background and aims: The mechanisms of early brain injury after subarachnoid hemorrhage (SAH) are not fully understood. We examined platelet behavior in cerebral microvessels of a murine model of subarachnoid hemorrhage using intravital fluorescence microscopy.

Methods: We induced SAH using endovascular sutures in 16 C57BL/6J mice. Twelve mice underwent sham operations (n = 12). A cranial window was prepared in the right parietal region. Platelets obtained from donor mice were labeled with a fluorescent dye (carboxyfluorescein iodoacetate succinimidyl ester; CSFE) in vitro. Labeled platelets were intravenously administered at 3 h after SAH and then platelet behavior in the brain microvessels was observed using intravital fluorescence microscopy.

Results: Platelet rolling and adhesion were observed in the cerebral microvessels of SAH mice. We found significantly more platelets rolling in the pial veins (1712±840/mm²/30 secs) and arteries (244±299/mm²/30 secs) of SAH than in those of sham operated mice (904±979 and 15±26/mm²/30 secs, respectively; P<0.01). Similarly, significantly more platelets adhered to the pial veins (81±44/mm²) and arteries (30±45/mm²) of SAH than sham operated mice (56±79 and 3±6/mm², respctively; P<0.01) More rolling platelets were identified in the pial veins than arteries f the SAH group (1712±840 versus 244±299/mm²/30 secs; P<0.05). More platelets adhered to pial veins than to those in pial arteries but the difference did not reach significance. We classified the SAH mice into two groups based on the severity of hemorrhage and compared platelet numbers and behavior. More platelets were rolling and adhesive in the group with severe, than with mild SAH, but the difference did not reach significance (P<0.01). Intravascular microthrombus formation in the pial veins occasionally resulted in vessel occlusion in mice with severe SAH.

Conclusions: Platelets apparently became activated in murine cerebral microvessels and interacted with the endothelium during the acute phase of subarachnoid hemorrhage resulting in cerebral microcirculatory disturbance.

626. Endothelial dysfunction abrogates the beneficial effects of normobaric hyperoxia on cerebral perfusion and tissue outcome after focal cerebral ischemia

H.K. Shin^1,2, P.L. Huang³, K.W. Hong², C.D. Kim² and C. Ayata⁴

¹Division of Meridian and Structural Medicine, School of Oriental Medicine, Pusan National University; ²MRC for Ischemic Tissue Regeneration, Pusan National University, Yangsan-si, South Korea; ³Cardiovascular Research Center and Cardiology Division; ⁴Department of Neurology and Radiology, Massachusetts General Hospital & Harvard Medical School, Charlestown, Massachusetts, USA

Background and aims: Improving brain tissue oxygenation via 100% O₂ inhalation is a potential therapeutic strategy in acute stroke. We have previously showed that normobaric hyperoxia (NBO) improves cerebral blood flow (CBF) and ameliorates tissue injury in focal ischemia. However, the mechanism of improved CBF and its contribution to tissue protection are unclear. One potential mechanism for hyperoxia to augment CBF is by enhancing endothelial nitric oxide synthase (eNOS) function in ischemic cortex. Stroke often occurs in individuals with vascular risk factors associated with endothelial dysfunction (e.g., hyperlipidemia). It is, therefore, possible that the efficacy of NBO in acute stroke is diminished in patients with endothelial dysfunction.

Methods: The spatiotemporal evolution of ischemic CBF deficit was studied during 60 mins distal middle cerebral artery occlusion (dMCAO) using high resolution laser speckle flowmetry noninvasively through intact skull in eNOS knockout (eNOS^−/−) and hypercholesterolemic ApoE knockout (ApoE^−/−) mice (20 to 30 g, male). ApoE^−/− were fed high-fat ‘western’ diet for 8 weeks starting at 4 weeks of age. Normoxic mice (30% O₂) were compared to NBO mice (100% O₂). The area of severe CBF deficit (≤20% residual CBF) was measured by thresholding. 47 h after reperfusion, indirect infarct volume was measured using TTC staining.

Purpose: To test whether eNOS function is critical for the neuroprotective effects of NBO.

Results: The area of severely ischemic cortex was significantly larger in eNOS^−/− compared to wild type (WT) controls, and remained larger throughout the imaging period (75 mins). In WT mice NBO (15 mins after dMCAO) significantly improved CBF and reduced the area of ischemic cortex by ∼45% compared to normoxic WT (n = 8 and 11, respectively), as previously reported. In eNOS^−/− mice, however, NBO had the opposite effect: the area of CBF deficit rapidly and markedly increased after NBO (42% increase versus normoxic eNOS^−/−, P<0.01; n = 5 and 9, respectively). In WT mice, NBO decreased infarct volume (indirect method) by ∼45% compared to normoxic WT. In contrast, NBO increased infarct volume by ∼10% in eNOS^−/− compared to normoxic group (P>0.05). As in eNOS^−/− mice, NBO rapidly increased the area of CBF deficit by ∼30% in ApoE^−/− compared to normoxic group (n = 5 and 9, respectively; P<0.05). The neuroprotective effect of NBO was also diminished in ApoE^−/− compared to WT (∼35% reduction in infarct volume versus normoxic ApoE^−/−, P>0.1).

Conclusions: The beneficial effects of NBO on both ischemic tissue perfusion and outcome are abolished by endothelial dysfunction, suggesting that the neuroprotective effects of NBO are critically dependent on eNOS function. These data have direct implications for patient care, and suggest that while may improve tissue perfusion and outcome in younger stroke patients, it may be detrimental in older patients with multiple vascular risk factors associated with severe endothelial dysfunction. Current and future clinical trials of NBO in acute stroke need to address this issue by subgroup analyses.

This work was supported by the Korea Research Foundation Grant (KRF-2008-314-E00477, Shin), the MRC program of MOST/KOSEF (R13-2005-009, Kim) and NIH (NS055104, Ayata; NS048426, Huang).

728. Dipyridamole confers protection in acute focal cerebral ischemia in the rat

L. Garcia-Bonilla, V. Sosti, M. Hernandez-Guillamon, A. Rosell and J. Montaner

Neurovascular Research Laboratory, Institut de Recerca Vall d'Hebron, Barcelona, Spain

Dipyridamole (DP) is a platelet inhibitor that has been previously shown to have antithrombotic benefit in the prevention of stroke, particularly when combined with low dose of aspirin. Recent experimental studies have reported as well that pre-treatment with DP promotes anti-inflammatory, antioxidative and neuroprotective effects in cerebral ischemia. We aimed to determine whether post-treatment with DP, an experimental paradigm more close to clinical stroke therapy, can also exert neuroprotection. Male Sprague-Dawley rats (n = 48) were anesthetized with isoflurane and subjected to 120 mins (group 1) or 90 mins (group 2) of transient middle cerebral artery occlusion (MCAo). Reperfusion was allowed during the 24 or 48 h follow-up (group 1 and 2, respectively). Animals were treated at the onset of reperfusion with either i.v. DP (100 mg/Kg) or vehicle. Rats in group 2 also received vehicle or DP dose (60 mg/Kg) orally at 24 and 36 h after MCAo. DP was detected by fluorospectrometry 40 mins after administration in treated rats (190±30 microg/mL, mean±s.e.). The concentrations found in plasma (1.52 ±0.44 microg/mL, mean±s.e.) and in the ischemic rat brain homogenates (2.34 ±0.30 microg/mL, mean±s.e.) after 24 h were higher than the DP effective concentration (1 microg/mL). Primary endpoints were infarct volume, evaluated using 2,3,5-triphenyltetrazolium chloride staining, and neurological outcome measured at 60 mins, 24 and 48 h following MCAo. DP treated animals subjected to 120 mins MCAo showed a significantly neurological improvement at 24 h when compared with neurological score after 60 mins of occlusion (P = 0.0006). A slight non-significant trend in the infarct size was observed compared to the vehicle-treated group (24.5% versus 29.19%, P = 0.6282). However, when a shorter period of ischemia (90 mins) and a long-lasting DP treatment were conducted, a significant reduction in the infarct volume was observed (7.9% versus 24.4%, P = 0.0381). That was accompanied with a significant improvement in the neurological score (P = 0.0065). DP protects from ischemic injury when given after MCA occlusion, suggesting that development of new acute therapies based on or combined with DP treatment might be beneficial for stroke patients.

811. The distribution of collateral circulations after a mini-ischemia in rat cortex evaluated by optical imaging

W. Luo, P. Li, S. Chen and Q. Luo

Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology (HUST), Wuhan, China

Objectives: The cerebral collateral circulation is recruited as a subsidiary vascular network to stabilize the local cerebral blood flow (CBF) when the principal supplying arteries are constricted or occluded, and is crucial for the pathophysiology and outcome of acute cortical ischemia. Current understanding of collateral circulation still remains sparse, largely due to prior limitations of spatial or/and temporal resolution in methods to evaluate these diminutive redistributive routes of cerebral blood flow especially in leptomeningeal anastomoses that connected cortical arteries. The leptomeningeal anastomoses can be divided into 2 types according to their position and original source: inter-arterial and intra-arterial anastomoses, which connect the adjacent branches from same common artery and from different arteries, respectively. The aim of the present report was to evaluate the priority and kinetics of developing inter-arterial or intra-arterial anastomoses after mini-ischemia in rat cortex by optical imaging techniques with the utility of high spatiotemporal resolution, including laser speckle imaging and optical imaging of intrinsic signal.

Methods: Adult male Sprague-Dawley rats (n = 10) were anesthetized with α-chloralose/urethane and the parietal-temporal cortex was exposed through craniectomy mini-ischemia (3 to 4 mm in diameter) was formed in rat cortex by ligating one or several small branches of middle cerebral artery (MCA) with short pieces of 11–0 suture passed through the dura and under the vessels. The animals were divided into two groups. One group (n = 6) were subjected to cerebral blood flow measurement using laser speckle flowmetry. The animals in another group were adopted for tracing changes in cerebral blood volume after ischemia in rat cortex by optical imaging of intrinsic signal at a wavelength of 550 nm, which is an isoabsorptive point of oxy and deoxy-hemoglobin.

Results: The blood flow and diameter in the intra-arterial anastomoses were enhanced immediately after the ligation of one branch of middle cerebral artery and recovered to baseline level as arterial recirculation was performed. Whereas the communicative flow through the posterior cerebral artery-middle cerebral artery anastomoses was not significant enough to be determined. A step-size increase in local blood volume initially occurs in the cortical regions proximal to the adjacent unligated branches of MCA, and then forms a ring-shape zone surrounding the ischemic core. It is interesting that a gradual decrease in blood flow was indicated in the medial part of the parietal cortex, which supplied by anterior cerebral artery of posterior cerebral artery.

Conclusions: This is the evidence that in some case intra-arterial anastomoses were the primary routes to restore the blood flow into the ischemic territory during the acute phase of focal ischemia, whereas the inter-arterial anastomoses was not well established shortly after the ligation of the branches originating from MCA. This finding suggests the inter-anastomoses would be a potential therapeutic target when a sudden occlusion of the MCA was encountered, although the underlying mechanism is in need of further investigations.

Keywords: Collateral circulation, anastomoses, optical intrinsic signals, laser speckle imaging.

861. Environmental enrichment promotes the resolution of post stroke mild cognitive impairment in rats

Y. Wang^1,2, B. Bontempi³, X. Leinekugel³, P. Weinstein¹ and J. Liu¹

¹Neurosurgery, UCSF, San Francisco, California, USA; ²Neurosurgery, Beijing Tiantan Hospital, Capital University of Medical Science, Beijing, China; ³Centre de Neurosciences Intégratives et Cognitives, CNRS UMR 5228 and Université Bordeaux 1, Bordeaux, France

Objective: While motor impairment due to ischemic damage of motor pathways is a hallmark of clinical and experimental stroke, post-stroke cognitive dysfunction can be observed without direct injury to brain regions supporting cognitive functions. The hippocampus plays a crucial role in learning and memory processes, however this structure is typically spared in both human stroke syndromes, and animal models of stroke. Here we examine whether hippocampal hypoactivity and memory impairments could result from damage to remotely connected cortical regions and whether post-ischemic hippocampal dysfunction can be reversed by environmental enrichment (EE).

Methods: Spatial memory in the Barnes maze and multi-channel in vivo electrophysiological recordings of cortical and intrahippocampal transmission were assessed in adult rats following distal middle cerebral artery occlusion (dMCAO). Imaging of the activity-dependent gene c-fos, a sensible proxy for neuronal activity, was used to map the brain regions exhibiting neuronal dysfunction immediately following spatial exploration or performing memory task. The effects of EE on hippocampal neuronal activity, neurogenesis and spatial memory were examined. The anterograde tracer biotinylated dextranamine (BDA) was injected iontophoretically into the parietal cortex of intact rats to reveal topographical projections between the cortex and hippocampus.

Results: Ischemic rats exhibited cortical damage and impaired hippocampal-dependent spatial learning at 5 weeks post-ischemia while hippocampal structural integrity was preserved. Contrasting with the expected reduction of ipsilateral cortical activity, functional spontaneous and evoked local field potentials recorded from hippocampal CA1 pyramidal layer and stratum radiatum were not disrupted following reperfusion in the ischemic rats. Fos imaging in dMCAO rats exploring a novel environment or submitted to memory testing revealed widespread, but region-specific, hypoactivations in hippocampus and connected brain regions of the hippocampal formation remote from the ischemic damage, including the entorhinal and perirhinal cortices. This hippocampal hypofunction and spatial memory deficits were reversed by rearing in the EE for 4 weeks, which also stimulated neurogenesis. Anatomical tracing with BDA demonstrated that parietal cortex project extensively to the parahippocampal regions, which include the perirhinal cortex.

Conclusions: Our results indicate that the experimental stroke-induced mild cognitive impairment is attributed to hippocampal hypofunction. Considering the absence of hippocampal damage in dMCAO rats, the, post-stroke cognitive deficit could be the consequence of a reduced source of cortical information (i.e. deactivation) or increased inhibition through the parahippocampal region that acts as a key gateway for information exchange between cortical areas and the hippocampus. EE reversed this diaschisis-like hippocampal hypofunction and associated spatial memory deficits. Thus, EE serves as a rehabilitative therapy to restore memory function by promoting the resolution of hippocampal diaschisis via an array of plasticity mechanisms that may include hippocampal neurogenesis.

1011. Agmatine reduces brain edema by regulating the expression of aquaporins and matrix metalloproteinases after collagenase induced intracerebral hemorrhage in rats

J.Y. Kim¹, S.H. Kim², J.H. Kim³, W.T. Lee³, K.A. Park³ and J.E. Lee¹

¹Anatomy, BK21 Project for Medical Science, Yonsei University, College of Medicine, Seoul; ²Neurosurgery, Ajou University, School of Medicine, Suwon; ³Anatomy, Yonsei University, College of Medicine, Seoul, South Korea

Objectives: we investigate the effect of agmatine on the expression of matrix metalloproteinases (MMPs) and aquaporines after collagenase induced intracerebral hemorrhage in rat.

Methods: ICH was induced on adult male Sprague-Dawley rats (280 to 300 g) by stereotactic injection of collagenase type VII 0.5 μL using 32-Guage needle on right caudate putamen (0.2 mm Anterior and 3 mm Lateral to bregma, 6 mm ventral for dura). ¹ Agmatine (100 mg/kg, intraperitoneally) was administrated at 6 h, it has delayed initial treatment at 6 h to a more clinically relevant time, ² and 1 to 7days after ICH onset. Brains were cut into 2 mm thick coronal sections in a cutting block, the sections were used for evaluated western blot, immunohistochemistry and swelling volume analysis.

Results: Agmatine treatment significantly increased the immunoreactivity of AQP4 and MMP9 in the peri-hematoma region. Swelling volume was clearly stabilized at 3 day to 7day in agmatine treatment group. Western blot analysis showed that agmatine treatment decreased the expression of MMP2 and AQP9, correlated with brain edema as water channels. The expression of MMP2 and AQP9 were reduced in agmatine treatment group. Agmatine could attenuate brain edema through lessening water content by suppression of the expression of aquaporins and also play a critical role in maintaining the integrity of the BBB.

Conclusions: These results may suggest agmatine as a novel therapeutic material for vasogenic and cytosolic edema including traumatic brain injury and intracerebral hemorrhage.