Effects of acute post-treatment with dipyridamole in a rat model of focal cerebral ischemia

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Effects of acute post-treatment with dipyridamole in a rat model of focal cerebral ischemia Lidia García-Bonilla a , Victoria Sosti a , Mireia Campos a , Anna Penalba a , Cristina Boada a , Mireia Sumalla a , Mar Hernández-Guillamon a , Anna Rosell a , Joan Montaner a,b,⁎ a

Neurovascular Research Laboratory, Institut de Recerca Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain Neurovascular Unit, Department of Neurology, Universitat Autònoma de Barcelona, Hospital Vall d'Hebron, Barcelona, Spain

b

A R T I C LE I N FO

AB S T R A C T

Article history:

Dipyridamole (DP) is a platelet inhibitor with known antithrombotic benefits in stroke

Accepted 2 December 2010

prevention. In addition to its anti-aggregant properties, recent studies have reported that DP

Available online 10 December 2010

promotes anti-inflammatory, anti-oxidative and neuroprotective effects. We aimed to test whether post-treatment with DP may exert protection after ischemic cerebral injury in the

Keywords:

rat. For this purpose, rats were subjected to 120 min or 90 min of middle cerebral artery

Anti-platelet

occlusion (MCAO) followed by 24 or 48 h of reperfusion, respectively. Either DP (100 mg/kg) or

MCAO

vehicle was administered i.v. at the onset of reperfusion; rats subjected to 90 min MCAO also

Pro-inflammatory cytokines

received additional doses of DP orally (60 mg/kg) at 24 and 36 h after ischemia. Matrix

Neuroprotection

metalloproteinases, extravasated hemoglobin content and IL-6, MIP-1α and MCP-1 cytokine level were examined in brain tissue by zymography, western blot and multiple ELISA, respectively. DP post-treatment led to a neurological improvement in both models (p < 0.05) and a significant reduction in the infarct volume of rats subjected to 90 min of ischemia, as compared to vehicle group (7.9% vs. 24.4%, p = 0.03). This neuroprotection was accompanied by a modest increase in expression of MMP-9 pro-form and a significant attenuation of MIP1α levels in the infarcted hemisphere. These results provide support for the development of novel therapies based on DP for acute treatment of stroke. In selected animals, intravenous administration of high dose DP induced an adverse hypotensive effect leading to rapid death. Thus, alternative ways of acute administration must be examined in order to avoid this unfavorable effect. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

Stroke remains a major cause of death and disability worldwide and is a major contributor to rising healthcare costs. Reperfusion therapy with tissue plasminogen activator (tPA) is efficient and improves clinical outcome in human

ischemic stroke. However, despite the high effectiveness of this treatment, tPA needs to be administered within 4.5 h after the onset symptoms (Anon., 1995; Hacke et al., 2008) and there is an associated risk of hemorrhagic transformation after tPA therapy (Anon., 1997). Therefore, it is desirable to develop new stroke treatments that are suitable for use in a broader stroke

⁎ Corresponding author. Neurovascular Research Laboratory, Neurovascular Unit, Institut de Recerca, Hospital Vall d'Hebron, Pg Vall d'Hebron 119-129, 08035 Barcelona, Spain. Fax: +34 934894015. E-mail address: [email protected] (J. Montaner). 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.12.005

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population and that could be used alone or in combination with reperfusion therapies. These treatment modalities, including neuroprotective therapies, should mitigate the harmful effects of an ischemic episode and reduce cellular injury or death. Dipyridamole (DP) is a platelet inhibitor with anti-thrombotic benefit in preventing the recurrence of stroke, particularly when combined with low-dose aspirin (Dengler et al., 2010; Diener et al., 1996; von Maxen et al., 2006). It inhibits the cellular uptake and metabolism of adenosine, triggering an increase in adenosine plasma levels and corresponding strong reduction in platelet activation (Born and Cross, 1963). This increase in plasma adenosine levels is likely to be one of the main mechanisms by which DP might play a significant role in the prevention of stroke (Kim and Liao, 2008; Muller et al., 1990). Adenosine, acting via A2A receptors, stimulates adenosine adenylyl cyclase in platelets and increases intracellular levels of cyclic adenosine monophosphate, which strongly reduces platelet activation (Born and Cross, 1963). Other mechanisms by which DP might be beneficial in stroke include its inhibitory effects on guanosine monophosphate phosphodiesterase (PDE), leading to blocking of platelet aggregation and vasodilatation (Schoeffter et al., 1987) and increases in prostacyclin (PGI22) production leading to vasodilatation, via inhibition of PDE in the vascular wall (Kim and Liao, 2008; Kim et al., 2008). In addition to its platelet inhibitory effects, DP has additional pharmacological actions. DP scavenges oxygen and peroxy free radicals, thereby inhibiting oxidative tissue damage (Chakrabarti et al., 2005; Iuliano et al., 1995) and exerts anti-inflammatory effects both indirectly, via adenosine and PGI2, and directly via inhibition of platelet–monocyte interactions by suppressing monocyte chemoattractant protein-1 (MCP-1) production and reducing matrix metalloproteinase-9 (MMP-9) (Weyrich et al., 2005). Taken together these pharmacologic effects could potentially account for the neuroprotective properties of DP in acute ischemic stroke. In fact, recent studies have shown the benefit of DP pre-treatment in reducing the infarct volume using murine models of stroke (Aldandashi et al., 2007; Kim et al., 2008). In the current study our aim was to determine whether acute post-treatment with DP, a closer experimental paradigm to clinical stroke therapy, offers benefits to ischemic brain damage. In particular, we examined the ability of DP to reduce ischemic lesion size and improve neurological function following induction of transient (120 min or 90 min) model focal ischemia in rats. We report neurological improvement in rats in response to post-stroke treatment with DP and provide evidence to suggest that these effects may be mediated at least in part by anti-inflammatory mechanisms.

after 40 min administration in naïve rats by fluorospectrometry assay. Lower levels of DP were observed at the 24 h time-point, however they were still above effective concentrations in both naïve (1.52 ± 0.44 μg/ml) and MCAO (1.89 ± 0.52 μg/ml) rats and of a similar profile to levels generated in human subjects treated with Aggrenox (Derendorf et al., 2005). Furthermore, we detected DP in the brain of naïve rats (2.34 ± 0.30 μg/ml mg−1) by fluorospectrometry, demonstrating that the drug penetrates the cerebral parenchyma. A decrease in the mean arterial pressure (MAP) was observed immediately after drug administration (Table 1), which recovered in the following 5 min. The detected drop in MAP was more prominent in naïve rats than in MCAO rats, as indicated in the table. Since the drop and the recovery in MAP were similar using either a rate of 200 μl/min infusion or a 3time lower rate of 66 μl/min (data not shown), this effect most likely did not depend on the rate of infusion. We selected a 200 μl/min rate in an attempt to avoid larger periods of anesthesia. No animals died in this pilot study.

2.2. DP improves neurological outcome and reduces infarct volume Rats treated with vehicle and undergoing 120 min of ischemia (120-tMCAO) did not show a statistically significant change in neurological score at 24 h reperfusion with respect to baseline scores (1 h), (4 [3–5.25] and 4 [4–5.255]; p = 0.87; 24 h vs. baseline). However, an improvement in neurological function was detected in rats treated with DP (2.5 [2–4] and 4 [3.25–4.75]; p = 0.004) (Fig. 1A). Moreover, when neurological scores were compared between both groups at 24 h, the DP-treated animals displayed better scores than vehicle treated animals (2.5 [2–4] and 4 [3–5.25]; p = 0.028; DP vs. vehicle). All animals subjected to 90 min of ischemia (90-tMCAO model) showed a significant improvement in neurological deficit over the reperfusion time of 48 h (Fig. 1B). Additionally, the neurological recovery observed in DP-treated rats was greater after both 24 and 48 h reperfusion as compared with the vehicle group: ((5.5 [4.25-6] vs. 5 [4.75-6] at baseline); (2.5 [2–3.5] vs. 3.5 [3–4.5] at 24 h; p < 0.05) and (2 [1.5–3] vs. 3 [3–4] at 48 h; p < 0.05); DP vs. vehicle). The administration of DP in 120-tMCAO model triggered a non-significant reduction in the total infarct volume, as compared to the vehicle-treated group (24.05 ± 5.20% vs. 29.19 ± 5.65%; p = 0.63) (Fig. 2A). In contrast, treatment with DP in rats subjected to 90-tMCAO led to significantly smaller infarct volume than the corresponding vehicle control group (7.9 ± 2.82% vs. 24.4 ± 4.14%; p = 0.03). The reduction in total

Table 1 – Mean arterial pressure (MAP).

2.

Results

2.1.

Intravenous administration of DP

First, we sought to test if DP at a dose of 100 mg/kg (i.v.) reached the effective plasma concentration of 1 μg/ml (Eisert, 2002). We detected a high level of DP (189.90 ± 29.56 μg/ml)

MAP Baseline DP infusion a b

Naïve rats

MCAO rats

89.27 ± 3.87 71.61 ± 4.11 a

97.40 ± 2.31 90.55 ± 1.99 b

Significantly different from baseline (p < 0.05). Significantly different from DP-treated naïve rats (p < 0.05).

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Fig. 1 – Effect of dipyridamole (DP) on neurological score (scale 0–9) after 1 h, 24 h or 48 h of middle cerebral artery occlusion (MCAO) in rats subjected to 120 min (A) (vehicle, n = 10; DP, n = 12) or 90 min (B) (vehicle, n = 10; DP, n = 8) of transient ischemia. ANOVA followed by Newman–Keuls Multiple Comparison post hoc test was used to determine statistical differences. Box plots represent median and range. ##p < 0.01, ###p < 0.001, statistical significances between 1 h vs. 24 h or 48 h. *p < 0.05 statistical significance vehicle vs. DP.

infarct was mostly due to lower infarction in the cortical area (1.58 ± 1.16% vs. 15.38 ± 3.28%; p = 0.019) (Fig. 2B).

2.3.

Molecular mechanisms of the benefit of DP treatment

Next, we studied whether the previously described antiinflammatory mechanisms of DP are associated with its

neuroprotective effects in stroke, mainly observed in rats subjected to 90-tMCAO. Levels of pro-form (pro-) and active (a-) cleaved forms of MMP-9 and MMP-2 protein were measured by gel zymography in brain tissue. Results are expressed as the increase of the level in the ipsilateral hemisphere with respect to contralateral hemisphere (n-fold; Fig. 3). Higher pro-MMP-9 levels were detected in the vehicle

Fig. 2 – Effect of dipyridamole (DP) on cerebral infarct volume. Total, cortical and subcortical infarct size are expressed as percentage (%) of the ipsilateral hemisphere in rats subjected to 120 min of middle cerebral artery occlusion (MCAO) and 24 h of reperfusion (120-tMCAO) (A) or 90 min of occlusion and 48 h of reperfusion (90-tMCAO) (B). Representative images of TTC staining: TTC-unstained areas correspond to infarcted brain tissue. Note that rats underwent 90-tMCAO and DP treatment showed only infarction in striatum area without cortical involvement. Mann–Whitney test was used to determine statistical differences. Bars represented the mean ± SEM.*p < 0.05, statistical significances vehicle vs. DP group.

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Fig. 3 – Matrix metalloproteinases (MMPs) and extravasated hemoglobin levels. Representative zymography (A) and levels of MMP-9 pro-forms (B), MMP-9 cleaved (a-MMP9) (C) and MMP-2 pro-forms (D) in the ipsilateral (IP) with respect to contralateral (CL) hemisphere (IP/CL) of animals subjected to 90 min of middle cerebral artery occlusion (MCAO). Levels of extravasated hemoglobin in the IP and CL hemispheres of 90-tMCAO rats (E). Bars represented the mean ± SEM. C+, positive control; vehicle (n = 4); dipyridamole (DP) (n = 4). Non-statistical significances were found.

group (2.97 ± 1.02) as compared to the DP-treated group (1.61 ± 0.49), although it did not reach statistical significance (Fig. 3B). A slight increase (less than two-fold) was observed in a-MMP-9 levels in both groups in response to ischemia (1.77 ± 0.22, vehicle; 1.40 ± 0.38, DP; Fig. 3C). There was virtually no change in MMP-2 pro-form levels after ischemia plus DP treatment (1.31 ± 0.09 and 1.19 ± 0.13; vehicle and DP group; Fig. 3D), while the cleaved form was not detected. Extravasation of hemoglobin (Hb) into the cerebral parenchyma was evaluated in the ipsilateral and contralateral hemispheres as a measure of blood–brain barrier (BBB) disruption. The level of Hb detected in the brains of sham animals, and the contralateral hemisphere of MCAO rats was equally very low. Extravasated Hb increased in the ipsilateral hemisphere with respect to the contralateral hemisphere of rats subjected to 90 min ischemia (Fig. 3E). In response to DP treatment, the amount of Hb in the cerebral parenchyma

was lower, as compared to vehicle, although this reduction was not statistically significant (9948 ± 3016 Int. × mm2 vs. 16,920 ± 5091 Int. × mm2). The level of IL-6, MIP-1α and MCP-1 inflammatory cytokines was measured by multiple ELISA in plasma (pg/ml) and brain homogenates (pg/μg) obtained from 90-tMCAO rats at 48 h. Plasma level of IL-6 and MIP-1α was undetected in all samples; the plasma level of MCP-1 did not differ among sham and MCAO rats treated either with DP or vehicle (data not shown). Cytokine levels in the ipsilateral hemisphere of MCAO rats were elevated with respect to both the contralateral hemisphere and the ipsilateral hemisphere of sham animals, as shown in Fig. 4. Interestingly, we found that DP treatment led to a significant reduction in the level of MIP-1α, as compared with the vehicle group (9.33 ± 5.10 pg/μg vs. 80.88 ± 17.33 pg/μg, DP vs. vehicle; p = 0.02; Fig. 4C). A reduction in IL-6

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Fig. 4 – Effect of dipyridamole (DP) on levels of IL-6 (A), MCP-1 (B) and MIP-1α (C) in the ipsilateral (IP) and contralateral (CL) hemisphere of animals subjected to 90 min of middle cerebral artery occlusion (MCAO) and treated with DP or vehicle (pg/μg of cerebral homogenates). Protein levels in the IP of sham animals are also shown. Mann–Whitney test was used to determine statistical differences between vehicle and DP group. *p < 0.05, statistical significances. Bars represented the mean ± SE. Vehicle (n = 4), DP (n = 4).

(29.31 ± 10.22 pg/μg vs. 73.73 ± 31.56 pg/μg, DP vs. vehicle; p = 0.20) and MCP-1 (138.0 ± 66.75 pg/μg vs. 337.80 ± 71.12 pg/μg, DP vs. vehicle; p = 0.20) were also observed, although this did not reach statistical significance.

2.4.

Mortality

Overall mortality for the entire study was 18.4% (Fig. 5). The majority of deaths occurred during DP infusion or shortly

Fig. 5 – Mortality during dipyridamole (DP) infusion (A) and mortality as a result of ischemia–reperfusion injury (B). Bars represented the number of animals subjected to 120 min or 90 min of middle cerebral artery occlusion (MCAO) (120-tMCAO and 90-tMCAO) that survived or died (dark fill) in different groups. Mortality rate is also expressed as a percentage within bars. Fisher's exact test was used to determine statistical differences. *p < 0.05, statistical significances vehicle vs. DP group. Below, mean arterial blood pressure (MAP) during DP infusion in both naïve and MCAO surviving animals treated with DP (C) and in selected MCAO animals that died after DP infusion (D). Time at 0 min indicates the moment when DP starts to be infused; after 10 min, DP is completely administered.

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(5–10 min) after (Fig. 5A). Four ischemic rats subjected to 120-tMCAO model and 5 rats subjected to 90-tMCAO model died after i.v. administration of DP (p = 0.072 and p = 0.047, respectively). These rats had a large drop in MAP during drug infusion that did not recover in the following minutes, in contrast to surviving rats treated with DP (Fig. 5C). Therefore, these deaths were most likely due to the acute hypotensive effects of DP. One animal subjected to 120 min of ischemia in the vehicle group died before 24 h, while no animals in the DP group died (Fig. 5B). In the 90-tMCAO model, all vehicle-animals survived and 3 animals treated with DP died before 48 h. This cannot be explained by differences in brain injury, as a significant difference in ischemic injury was not observed between the vehicle and DP group in both models (p = 1.00 and p = 0.22, 120-tMCAO and 90-tMCAO models, respectively).

3.

Discussion

We showed for the first time that DP reduces infarct size and improves neurological outcome when administered after stroke in rats. Since assaying multiple doses and shorter ischemia results in a better protection, the protective effect is related to the drug dose and/or the ischemia severity. Animal models of transient cerebral ischemia mimic the clinical situation in which the occluded artery is recanalized, i.e., after thrombolytic stroke therapy, and thereby, the neurological symptoms might be partially recovered. Interestingly, our results show that DP treatment induced a better neurological recovery and reduced the infarct volume when compared with vehicle. These beneficial effects were most apparent when ischemia was less severe (90 min of occlusion instead of 120 min). Furthermore, the reduction in infarction was associated with a decrease in the cortical lesion, as expected. As we used a stroke model that induces proximal occlusion of the MCA, the ipsilateral striatum represents the core of the lesion and the ipsilateral cortex represents the penumbra—the primary area to be rescued (Jokivarsi et al., 2010). If this experimental paradigm is translated to the clinical setting, it is tempting to speculate that DP could be combined with existing thrombolytic drugs to improve the current standard stroke therapy. In fact, DP given as a preventive treatment provided additional benefits in reducing infarct volume and improving perfusion deficit when used in combination with tPA in a stroke embolic model in rats (Aldandashi et al., 2007). Whether DP post-treatment in combination with thrombolytic therapy would represent the best acute therapy for stroke patients warrants further investigation. Cerebral ischemia initiates a cascade of brain damage associated with local inflammatory reactions which further contributes to tissue injury during reperfusion (Aronowski et al., 1997). Whereas CBF restoration is essential to avoid cell degeneration, reperfusion of the occluded vessel triggers production of reactive oxygen species (ROS) either by reperfusion with oxygenated blood or generation within brain and immune cells (Chan, 2001). Diverse inflammatory mediators contribute to the pathophysiology of cerebral

ischemia/reperfusion injury (Barone and Feuerstein, 1999). ROS may stimulate ischemic cells to secrete a variety of cytotoxic agents including cytokines, MMPs, nitric oxide (NO) and further ROS which induce additional cell damage as well as disruption of the BBB and subsequent brain edema (Wang et al., 2007). Previous investigations have demonstrated that an abnormal expression of MMP-2 (gelatinase A) or MMP-9 (gelatinase B) occurs after cerebral ischemia, contributing to brain injury and BBB breakdown (Romanic et al., 1998; Rosenberg et al., 1996). Pharmacological or genetic inhibition of MMP-9 significantly decreases infarct size and the risk of hemorrhagic complications (Asahi et al., 2001; Sumii and Lo, 2002). We have previously described that MMP-9 is elevated in brain tissue after both hemorrhagic and ischemic stroke in humans (Rosell et al., 2006) and that hemorrhagic complications and the infiltration to the ischemic tissue by peripheral blood neutrophils filled with MMP-9 is related to basal lamina degradation, compromising BBB integrity (Rosell et al., 2008). The present results show a downtrend regulation of both MMP-9 pro-form levels and decreased extravasated hemoglobin into brain parenchyma in DP treated rat that might be consistent with a beneficial effect of DP in reducing BBB disruption. We also detected a decrease in IL-6, MCP-1 and especially MIP-1α levels, following DP treatment. IL-6 is a pleiotropic cytokine up-regulated in the ischemic brain (Clark et al., 1999; Legos et al., 2000) and recently, it has been reported that its neutralization ameliorates cerebral ischemic damage in a rat model of MCAO (Tuna et al., 2009). Moreover β-chemokines, including MIP-1α and MCP-1, have been widely implicated as potential modulators of the acute inflammatory response in the brain, where they mediate microglial cell activation and leukocyte infiltration after brain ischemia (Kim et al., 1995; Nishi et al., 2005; Takami et al., 1997; Yi et al., 2007). Direct evidence of the deleterious role of chemokines comes from studies showing that MCP-1 deficient mice display a significant reduction in infarct size after focal cerebral ischemia (Hughes et al., 2002) and in vitro experiments reporting inhibition of MCP-1 formation in response to treatment with DP (Weyrich et al., 2005). In the present study, the decrease in cytokine levels observed in brain homogenates from rats receiving DP suggests that it might act to modify secondary inflammatory mechanisms of ischemic damage. However, the mechanism by which DP might act to reduce the inflammatory response following cerebral ischemic injury still needs to be elucidated. In addition to leukocytes, both platelet recruitment and intravascular aggregation to the postischemic endothelium can lead to further vessel reocclusion early during reperfusion (Massberg et al., 1999). Using a rat model of MCA occlusion, the antiplatelet agents acetylsalicylic acid and triflusal have been reported to reduce the formation of microemboli when administered after occlusion (Heye et al., 1991). Therefore we cannot dismiss that this mechanism may play an additional role in the protection observed in our study. The main limitation in our study was the mortality that was observed in rats immediately after DP administration. While we tried to establish the best route for administering a high dose infusion of DP, intravenous administration

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provoked the irreversible deregulation of the blood pressure in selected animals stroke animals, triggering a rapid death. The hypotensive effects of DP, together with the fact that cerebral auto-regulation in systemic arterial pressure is impaired after acute stroke (Immink et al., 2005), were probably responsible of the immediate fatal effect of the drug administration in selected rats. Although we cannot rule out a selection bias in animals treated with DP, the deleterious hypotensive effect of DP is transient and therefore independent of its long-term effects on brain tissue and other measures of outcome. An optimal method of acute administration of DP therefore must be established in order to avoid this lethal side effect. In conclusion our data support administration of DP after experimental cerebral ischemia for protection of the brain from ischemic injury. We observed a reduction in neurological impairment and decreased brain injury, mediated at least in part by inhibition of post-stroke cerebral inflammation. Since DP is currently widely used in the clinic for secondary stroke prevention, the transition to clinical trials examining its effectiveness when administered soon after stroke onset seems feasible.

4.

Experimental procedures

All procedures were approved by the Ethics Committee of the Research Institute of Vall d'Hebron Hospital (protocol number 3551) and were conducted in compliance with the Spanish legislation and in accordance with the Directives of the European Union and Guide for the Care and Use of Laboratory Animals. Experiments were performed in male SD-OFA rats (250–300 g) from Charles Rivers Laboratories (Wilmington, MA, USA). Rats were kept in a temperature-controlled environment (22 ± 2 °C and 5 ± 15% humidity) on a 12-h light: 12-h dark cycle. Food and water were available ad libitum.

4.1. Intra-arterial suture occlusion of the middle cerebral artery (MCAO) Infarction in the territory of the middle cerebral artery (MCA) was induced by extracranial vascular occlusion, as described in detail elsewhere (Longa et al., 1989). Animals were anesthetized under spontaneous respiration with isoflurane (2%) maintenance (Abbot Laboratories, Kent, UK) and body temperature was maintained at 37 °C with a temperature controlled heating pad attached to a rectal probe during surgery. Continuous laser-Doppler flowmetry (LDF; Moor Instruments, Devon, England) was used to monitor regional cerebral blood flow (rCBF) in the cortex supplied by the MCA to ensure accurate occlusion and reperfusion. One day before induction of the ischemia, a small burr hole in the right parietal bone (2 mm posterior and 3.5 mm lateral to bregma) was drilled leaving the intact dura and a cannula was fixed to the skull with cyanacrylate and dental cement for LDF probe placement and cortical rCBF assessment. The right common carotid artery was exposed through a midline incision in the ventral cervical skin and it was carefully dissected from the tissue using microsurgical technique. The occipital and superior thyroid branches of the external carotid artery were cauterized and the pterygo-palatine branch of the internal

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carotid artery was irreversibly occluded. The external carotid artery was tied and cut and then, a heat-blunted 4-0 nylon monofilament was inserted into the external carotid artery and advanced into the internal carotid artery 18 to 20 mm, blocking the origin of MCA. Only animals with rCBF reduction >75% upon MCA occlusion and an increase >80% of rCBF upon reperfusion, with respect to their baseline register, were included. Sham-operated rats were subjected to the same surgical procedure without occlusion of the MCA.

4.2.

Experimental design

Before conducting experiments, a pilot study was conducted to test whether DP administration reached an effective concentration in the plasma and whether it was detected in brain parenchyma after 24 h (naïve control rats, n = 10; MCAO rats, n = 6). A total of 84 rats were used to test the actions of DP post-treatment in stroke injury. Eight animals were excluded after applying the following criteria: i) inappropriate occlusion (i.e. <75% reduction) (n = 0); ii) death during ischemic period (n = 1); iii) spontaneous reperfusion (n = 0); iv) unsatisfactory reperfusion (i.e. <80% reperfusion; n = 4) and v) occurrence of subarachnoid hemorrhage, detected after removing monofilament (n = 3). Animals were subjected to 120 min of MCAO and 24 h of reperfusion (120-tMCAO; n = 30) or 90 min of MCAO and 48 h of reperfusion (90-tMCAO; n = 30) and randomly allocated to experimental groups (vehicle or DP) using a computergenerated randomization list. Rats subjected to 120-tMCAO model received a single dose of DP (100 mg/kg i.v.; Boehringer Ingelheim Pharmaceuticals Inc., Ingelheim, German) or saline administered at the onset of reperfusion. DP was diluted in saline (15 mg/ml) with tartaric acid (7.5 mg/ml; Sigma-Aldrich Inc., Barcelona, Spain) for administration and infused via right femoral vein at 200 μl/min flow rate (10 min of infusion). Furthermore, we established that lowering isoflurane anesthesia from 2% to 1.5% 1 min before drug infusion allowed a prompt recovery in the decrease in arterial pressure induced by DP. Arterial pressure, rCBF and temperature were monitored during drug administration. In addition, rats of the 90-tMCAO model received additional oral doses of DP (60 mg/kg p.o.; 15 mg/ml) diluted in 1 ml of water with tartaric acid (7.5 mg/ml) at 24 and 36 h after MCAO.

4.3.

Determination of plasma and brain DP Levels

For determination of DP plasma levels, blood samples were obtained from naïve and MCAO rats in EDTA vials at baseline, 40 min and 24 h after DP administration. Plasma was immediately separated by centrifugation at 500g and stored at −20 °C until use. In order to test if DP was detectable in the cerebral parenchyma (due to crossing of the BBB), animals were deeply anesthetized and sacrificed by transcardial perfusion with cold saline at 24 h. DP was extracted from brain tissue by homogenization (1:4) followed by sonication in a 50% of Tri-Chloro-Acetic Acid (TCA, Sigma-Aldrich) solution. Afterwards, homogenates were centrifuged at 12,000g during 20 min (4 °C) and supernatants were saved. Fluorescence was measured in duplicate plasma samples (100 μl) and brain homogenate supernatants (100 μl) by

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fluorospectrometry assay (405 nm excitation and 535 nm emission). DP standards at 0–10 μg/ml diluted in 0.9% NaCl with 50 μM of tartaric acid were used for determination of DP concentration in samples.

4.4.

Infarct volume

Infarct volume was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich) staining (Bederson et al., 1986). Animals were sacrificed and brains were removed, sliced in 2-mm-thick sections and stained with 2% TTC for 10 min at 37 °C, followed by an overnight fixation with 4% paraformaldehyde. Images of the stained slices (anterior and posterior views) were captured using a CanoScan 4200F scanner (Canon USA Inc., NY, USA) and infarcted, contralateral and ipsilateral areas were outlined in both views and quantified using an image analysis system (Scion Image v4.02, Scion Corporation, Maryland, USA). Infarct volume was measured by a researcher blinded to the treatment protocol and calculated by integration of infarcted areas, considering the average of anterior and posterior views. Correction for brain edema was made by multiplying the infarct area by the ratio of the contralateral area to the ipsilateral area (Burguete et al., 2006). Infarct volume data are expressed as the relative percentage of ipsilateral hemisphere.

4.5.

Neurological deficit

Rats were assessed using a 9-point neurological deficit scale, as previously described (Perez-Asensio et al., 2005). Four consecutive tests were conducted: (I) spontaneous activity (moving and exploring = 0, moving without exploring = 1, no moving or moving only when pulled by the tail = 2); (II) left drifting during displacement (none = 0, drifting only when elevated by the tail and pushed or pulled = 1, spontaneous drifting = 3, circling without displacement, or spinning = 4), (III) parachute reflex (symmetrical = 0, asymmetrical = 1, contralateral forelimb retracted = 2), and (IV) resistance to left forepaw stretching (stretching not allowed = 0, stretching allowed after some attempts = 1, no resistance = 2). Neurological score was assessed in a blinded manner at 1 h, 24 h and 48 h after occlusion.

4.6.

Brain sample preparations

Rats were deeply anesthetized and transcardially perfused with ice-cold saline. Afterwards, ipsilateral and contralateral hemispheres were briefly dissected and kept frozen (−80 °C) until use. Cerebral tissue was homogenized 1:5 with lysis buffer containing 50 mM Tris–HCl pH 7.6, 150 Mm NaCl, 5 mM CaCl2, 0.05% Brij-35, 0.02% NaN3, 1% Triton X-100, 1 mM PMSF and 7 μg/ml aprotinin, and centrifuged at 12,000g for 15 min at 4 °C. Total protein determinations were measured by BCA assay (Thermo Fisher Scientific Inc., IL, USA).

4.7.

Zymography

Standard gel zymography was used to measure levels of MMP2 and MMP-9 in brain samples. Thirty micrograms of total protein were loaded and resolved by 10% Novex® Zymogram

Tris-glycine gel (Invitrogen, CA, USA) electrophoresis, according to the manufacturer's instructions (Invitrogen). Gels were incubated with developing buffer (Invitrogen) at 37 °C for 3 days. Finally, the gels were stained with 0.5% Coomassie blue R-250 (Sigma-Aldrich) for 1 h and then destained appropriately in 10% acetic acid and 30% methanol. Band intensities of total MMP-9 and MMP-2 (including pro- and cleaved forms) were quantified by densitometry techniques using Image Quantity One version 4.6.0 software package (BioRad Laboratories, Inc., Barcelona, Spain) and expressed as a ratio between the level found in ipsilateral hemisphere with respect to contralateral.

4.8.

Wester blot

Hemoglobin protein content and β-actin were detected by western blot in brain samples. Briefly, equal protein amounts (35 μg) were resolved by 8% sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Separated proteins were transferred onto a PVDF membrane using a Mini TransBlot®Electrophoretic Transfer Cell (Bio-Rad Laboratories) during 1 h at 100 V. Non-specific binding was blocked with 10% non-fat milk and 0.05% tween-phosphate buffer saline (TBS) before membranes were incubated overnight at 4 °C with anti-hemoglobin antibody (St Cruz, CA, USA) at 1:1000 dilution or for 1 h at room temperature (RT) with anti-β-actin 1/10000, in 5% non-fat milk and 0.05% TBS. Secondary antibodies (Chemicon, IL, USA) anti-rabbit-HRP or anti-mouse-HRP respectively, were diluted 1:2000 (5% non-fat milk; 0.05% TBS) and incubated at RT for 1 h. Before and after incubations, membranes were rinsed 3 times (10 min each) with 0.05% TBS. The substrate reaction was developed with chemiluminescent reagent Immobilion (Millipore, MA, USA) and visualized with a luminescent image analyzer (Las-3000, Fujifilm, CT, USA). Scanned western blot bands were quantified with the Image Quantity One software package (BioRad Laboratories).

4.9.

Multiple ELISA (Searchlight)

SearchLight Custom Rat 3-plex (Aushon BioSystems Inc., MA, USA) was used to measure interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α) levels in rat plasma and brain homogenates. This assay consists of multiplexed sandwich enzyme-linked immunosorbent assay and was used for the simultaneous quantitative measurement of 3 proteins in each sample. Plasma samples (50 μl, diluted 1/5) and brain homogenates (50 μl, 25 μg total protein) were assayed twice and the mean value of both measurements was used. The mean intraassay coefficient of variation was < 15% for all measured molecules. The enzyme-substrate reaction produced a luminescent signal that is detected whit a cooled CCD camera (Thermo Fisher Scientific Inc., IL, USA). The image obtained was analyzed by ArrayVision version 8.0 software (Imaging Research Inc., CA, USA). The array standard curves and concentration in samples were given in pg/ml units. The sensitivity limit for each molecule was: IL-6, 12.5 pg/ml; MCP-1, 0.8 pg/ml and MIP-1α, 0.39 pg/ml, as provided by the manufacturer. The resulting protein concentration for each molecule was expressed in pg/μg of total protein.

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4.10.

Statistical analysis

Data were analyzed using GraphPad Prims_v4 software. Statistical significance for intergroup differences was assessed by Student's t-test and ANOVA for parametric data (Kolmogorov–Smirnov). Paired t-test was also used to determinate statistical differences in neurological score into each group. For non-parametric data, Mann–Whitney test was assayed. Statistical significance for intergroup differences in mortality was assessed by the Fisher's exact test. A p value < 0.05 was considered statistically significant at a 95% confidence level.

Acknowledgments This work is supported in part by a research grant from Boehringer Ingelheim Pharmaceuticals and from the Instituto de Salud Carlos III (FIS 08/0481). We thank Wolfgang Eisert for critical review of the paper. L.G-B., M.H-G. and A.R. are supported by Post-Doctoral Fellowships from the FIS. The Neurovascular Research Laboratory takes part in the European Stroke Research Network (EUSTROKE, 7FP Health F2-2008202213) and the Spanish Stroke Research Network RENEVAS (RD06/0026/0010). We appreciate the assistance of Dr. Katherine Jackman for proofreading this document.

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