Author's Accepted Manuscript

SMTP-7, a new thrombolytic agent, decreases hemorrhagic transformation after transient middle cerebral artery occlusion under warfarin anticoagulation in mice Akira Ito, Kuniyasu Niizuma, Hiroaki Shimizu, Miki Fujimura, Keiji Hasumi, Teiji Tominaga www.elsevier.com/locate/brainres

PII: DOI: Reference:

S0006-8993(14)00911-1 http://dx.doi.org/10.1016/j.brainres.2014.07.004 BRES43635

To appear in: Brain Research

Received date:8 May 2014 Revised date: 18 June 2014 Accepted date: 1 July 2014 Cite this article as: Akira Ito, Kuniyasu Niizuma, Hiroaki Shimizu, Miki Fujimura, Keiji Hasumi, Teiji Tominaga, SMTP-7, a new thrombolytic agent, decreases hemorrhagic transformation after transient middle cerebral artery occlusion under warfarin anticoagulation in mice, Brain Research, http://dx.doi.org/10.1016/j.brainres.2014.07.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

SMTP-7, a new thrombolytic agent, decreases hemorrhagic transformation after transient middle cerebral artery occlusion under warfarin anticoagulation in mice

Akira Ito, M.D.,1 Kuniyasu Niizuma, M.D., Ph.D.,1 Hiroaki Shimizu, M.D., Ph.D., 1 Miki Fujimura, M.D., Ph.D.,1 Keiji Hasumi, Ph.D.,2,3 and Teiji Tominaga, M.D., Ph.D.1

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Department of Neurosurgery, Tohoku University Graduate School of Medicine, Sendai,

Japan 2

Department of Applied Biological Science, Tokyo University of Agriculture and

Technology, Tokyo, Japan 3

TMS Co., Ltd., Tokyo, Japan

Corresponding author: Kuniyasu Niizuma, MD, PhD Department of Neurosurgery, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan

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Phone: +81-22-717-7230 Fax: +81-22-717-7233 Email: [email protected]

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Abstract Stachybotrys microspora triprenyl phenol-7 (SMTP-7) is a new thrombolytic agent that exhibits anti-inflammatory effects. We previously demonstrated that the hemorrhagic transformation was fewer with SMTP-7 than with recombinant tissue plasminogen activator (rt-PA) following ischemia-reperfusion in animal models. We hypothesized that SMTP-7 may decrease hemorrhagic transformation after ischemia-reperfusion under the warfarin-treated condition. Transient middle cerebral artery occlusion (MCAO) was induced for three hours using an intraluminal suture in warfarin-treated mice to produce hemorrhagic transformation. Warfarin was administered orally for a 24-hour feeding period before MCAO through bottled drinking water (5 mg in 375 ml tap water), resulting in a mean INR of 5.6 ± 0.2. Mice were treated with vehicle, rt-PA, or SMTP-7 five minutes before reperfusion. Twenty percent of vehicle-treated and 50.0% of rt-PA-treated mice died 24 hours after reperfusion, while all SMTP-7-treated mice survived. Hemorrhagic severity in SMTP-7-treated mice was significantly lower than that in rt-PA-treated mice. Neurological deficit was significantly lower in SMTP-7-treated mice than vehicle- and rt-PA-treated mice. These results indicate that

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SMTP-7 decreases mortality, hemorrhagic transformation, and neurological deficits, and can be a safe thrombolytic agent following MCAO under the warfarin-treated condition.

Key words cerebral infarction, hemorrhagic transformation, focal ischemia, recombinant tissue plasminogen activator, thrombolysis, warfarin

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1. Introduction Intravenous (IV) recombinant tissue plasminogen activator (rt-PA) is the most widely used thrombolytic agent for acute ischemic stroke (Adams et al., 2007; Del Zoppo et al., 2009). However, approximately 2.4 to 8.8% of patients who receive IV rt-PA exhibit potentially life-threatening complications including hemorrhagic transformation (Group T.N.I.o.N.D.a.S.r.-P.S.S., 1995; Hacke et al., 1995; Hacke et al., 1998). Because warfarin is a risk factor of such hemorrhagic complications, warfarin-treated patients with a baseline international normalized ratio (INR) more than 1.7 are contraindicated for IV rt-PA (Albers et al., 2000; Group T.N.I.o.N.D.a.S.r.-P.S.S., 1995). Stachybotrys microspora triprenyl phenol-7 (SMTP-7) is a new low molecular weight fibrinolytic agent (Hu W et al., 2000). This compound, which has a vitamin E-like structure, promotes a conformational change in plasminogen, leading to plasminogen activator-catalyzed conversion of plasminogen to plasmin, fibrin binding to plasminogen, and fibrinolysis (Hasumi et al., 2010; Hu W et al., 2000). SMTP-7 has more gradual and milder fibrinolytic activity than rt-PA (Shibata et al., 2010). We previously demonstrated that the infarct volume (Akamatsu et al., 2011; Hashimoto et

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al., 2010; Miyazaki et al., 2011; Shibata et al., 2010) and severity of hemorrhagic transformation (Shibata et al., 2010) were lower with SMTP-7 than with rt-PA in animal embolic stroke models. Therefore, we hypothesized that SMTP-7 may decrease hemorrhagic transformation following ischemia-reperfusion even under the warfarin-treated condition. The purpose of this study was to investigate the efficacy and safety of SMTP-7 under the warfarin-treated condition. Three hours of ischemia-reperfusion was induced in mice under the warfarin-treated condition. Mice were treated with vehicle, rt-PA, or SMTP-7. Mortality, severity of hemorrhagic transformation, neurological deficits, infarct volume, severity of edema, pre-matrix metalloprotease-9 (MMP-9) and MMP-9 levels, degradation of collagen IV, and blood brain barrier (BBB) disruption 24 hours after reperfusion were assessed.

2. Results

2.1. No significant Difference in Physiological Parameters among the groups

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Physiological data are shown in Table 1. Under anesthesia with 1.2 to 1.5% isofluorane in 30% oxygen and 70% nitrous oxide at 37 ± 0.5͠, there was no significant difference in arterial pressure or blood gas among the three groups (n = 3, per group).

2.2. SMTP-7 Decreased Severity of Hemorrhage after MCAO under Warfarin-Treated Condition

Hemorrhagic severity scoring was quantified in TTC-stained slices from mice surviving 24 hours after reperfusion. Massive hemorrhage, such as that shown in Fig. 1B, was observed in all mice that died after 3 hours of MCAO. Warfarin-related hemorrhagic transformation was consistently observed in our model: All mice surviving 24 hours after reperfusion also developed hemorrhagic transformation after 3 hours of MCAO (Fig. 1C). The mean hemorrhagic severity score was 2.3 ±1.4 in vehicle-treated, 3.5 ± 0.5 in rt-PA-treated, and 1.3 ± 0.5 in SMTP-7-treated mice. Hemorrhagic severity scoring was significantly lower in SMTP-7-treated mice than in rt-PA-treated mice (p < 0.05). The pattern of hemorrhagic transformation in SMTP-7 treated mice showed a

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characteristic pattern. Only heterogeneous hemorrhage, but not parenchymal hemorrhage was observed in SMTP-7-treated mice.

2.3. SMTP-7 Decreased Mortality and Neurological Deficits

Mortality and neurological deficit scoring were evaluated 24 hours after reperfusion. Significant differences were observed in mortality among the groups (p < 0.01) (Fig. 2A). Twenty percent of vehicle- and 50% of rt-PA-treated mice died before the end of the experiment. In contrast, all SMTP-7-treated mice survived 24 hours after reperfusion. Neurological deficit scoring was significantly lower in SMTP-7-treated mice (1.1 ± 0.3) than in vehicle- (3.0 ± 1.8, p < 0.01) and rt-PA-treated (4.5 ± 1.7, p < 0.001) mice (Fig. 2B). The severity of neurological deficits was significantly higher in rt-PA-treated mice than in vehicle-treated mice (p < 0.05) (Fig. 2B).

2.4. SMTP-7 Slightly Decreased the Infarct Volume and Edema Index

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The infarct volume and edema index were quantified in TTC-stained slices from mice surviving 24 hours after reperfusion. No significant differences were observed in the infarct volume or edema index among the three groups (Fig. 3A and B). However, the infarct volume had a little tendency to be large in rt-PA-treated mice compared to SMTP-7- and vehicle-treated mice (Fig. 3A). SMTP-7 had a tendency to ameliorate the formation of edema following reperfusion (Fig. 3B).

2.5. SMTP-7 Inhibited the Activation of MMP-9

pro-MMP-9 and MMP-9 activity was evaluated by gelatin zymography (Fig. 4A–C). As described above, only mice surviving 24 hours after reperfusion were included. The activity of pro-MMP-9 was not significantly different between vehicle-treated (393.4 ± 63.1) and rt-PA-treated mice (512.3 ± 92.0) (Fig. 4B). MMP-9 activity was significantly lower in vehicle-treated mice (10.4 ± 1.0) than in rt-PA-treated mice (15.8 ± 1.3) (p < 0.05) (Fig. 4C). pro-MMP-9 and MMP-9 activity was significantly lower in SMTP-7-treated mice (pro-MMP-9: 88.9 ± 34.2 and MMP-9: 5.0 ± 1.6) than in vehicle-

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(pro-MMP-9, p < 0.01; MMP-9, p < 0.05) and rt-PA-treated (pro-MMP-9, p < 0.01; MMP-9, p < 0.001) mice (Fig. 4B and C).

2.6. SMTP-7 Ameliorated Damage to the Basal Membrane

Collagen IV is the main component of the basal membrane. The degradation of collagen IV was evaluated by immunohistochemistry. Fig. 5A shows representative micrographs of the boundary area of infarct in mice treated with vehicle, rt-PA, and SMTP-7, respectively. Morphologically, the basal membrane was damaged in both vehicle- and rt-PA-treated mice; however, this damage was more severe in rt-PA-treated mice (Fig. 5A). In contrast, the severity of damage to the basal membrane was less in SMTP-7-treated mice than in vehicle- and rt-PA-treated mice. Semi-quantitative analysis confirmed that the pixel intensity of collagen IV was significantly lower in rt-PA-treated mice (0.34 ± 0.004) and significantly higher in SMTP-7-treated mice (0.061 ± 0.002) (Fig. 5B, p < 0.001).

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2.7. SMTP-7 protected BBB disruption

The functional integrity of the BBB was evaluated by EB dye leakage 24 hours after reperfusion. We corrected the tissue content of EB dye by adopting the ratio of the ischemic hemisphere to the non-ischemic hemisphere (EB extravasation index, EBI). EB leakage was significantly higher in rt-PA-treated mice (4.1 ± 0.4) than in vehicle-treated (2.1 ± 0.9, p < 0.05) and SMTP-7-treated (1.5 ± 0.3, p < 0.01) mice (Fig. 6A and B).

3. Discussion In this study, we demonstrated that SMTP-7 decreased the severity of hemorrhagic transformation after 3 hours of MCAO under warfarin-treated condition. SMTP-7 inhibited MMP-9 activation, damage to the basal membrane, and BBB disruption. SMTP-7 also decreased neurological deficits, and mortality rate. Few experimental studies have investigated thrombolysis in acute ischemic stroke with warfarin treatment (Pfeilschifter et al., 2012; Pfeilschifter et al., 2011)

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because of the high mortality rate of the models with warfarin treatment. We confirmed that conventional MCAO (Clark et al., 1997) with long ischemic duration under warfarin treatment induced severe infarction, edema and hemorrhage. Most mice died by 24 hours after reperfusion (data not shown). Because of the high mortality rate, conventional MCAO was not suitable for our study. Involvement of the posterior cerebral artery (PCA) territory infarction was an important factor responsible for the high mortality in intraluminal MCAO model (Akamatsu et al., 2012). We modified a mouse MCAO model that did not involve the PCA territory infarction (Akamatsu et al., 2012), and developed a reproducible hemorrhagic infarction model in warfarin-treated mice. In the present study, we demonstrated that SMTP-7 was a thrombolytic agent with lower mortality rate even under the experimental warfarin-treated condition with severe INR prolongation. The severity of hemorrhagic transformation was significantly higher in rt-PA-treated mice than SMTP-7-treated mice. However, in contrast to previous studies (Hashimoto et al., 2010; Miyazaki et al., 2011; Shibata et al., 2010), no significant difference was observed in hemorrhagic severity between vehicle- and

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rt-PA-treated mice in our study. This may be because of the timing to evaluate hemorrhagic severity. We evaluated hemorrhagic severity on mice only surviving 24 hours after reperfusion; mice exhibiting more severe hemorrhage had already died and were excluded. Therefore, neurological deficit scoring that includes “death” may more accurately reflect the effect of drugs. Actually, neurological deficit scoring was significantly higher in rt-PA-treated mice than in vehicle-treated mice. IV rt-PA is an effective therapy in acute ischemic stroke, but its efficacy is limited by hemorrhagic complications. Previous studies have indicated that the adverse effect of thrombolysis by rt-PA is partially caused by the cytotoxicity of rt-PA itself, as well as reperfusion injury (Hacke et al., 1999; Wang et al., 1998).Wang et al. first reported that rt-PA activates MMP-9 in the brain endothelium, leading to degradation of the basal membrane and BBB disruption (Wang et al., 2003). Subsequent studies have suggested that the t-PA - MMP-9 pathway is responsible for BBB disruption and subsequent hemorrhagic transformation (Lapchak et al., 2000; Pfefferkorn and Rosenberg, 2003; Sumii, 2002). Thus, rt-PA is an important drug in acute ischemic stroke; however, it is a two-edged blade. BBB disruption through the upregulation of

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MMP-9 followed by degradation of the basal membrane after cerebral ischemia-reperfusion is responsible for the development of hemorrhagic transformation (Fujimura et al., 1999; Gasche et al., 2001; Pfefferkorn and Rosenberg, 2003; Sumii, 2002). The basal membrane is mainly degraded in ischemic brains (Tagaya et al., 1997; Yamashita et al., 2009). In this study, gelatin zymography revealed that pro-MMP-9 and MMP-9 activation was significantly lower in SMTP-7-treated mice than in rt-PA- and vehicle-treated mice. In other words, SMTP-7 inhibited the upregulation of MMP-9 after ischemia-reperfusion in warfarin-treated mice. Consistent with results obtained by zymographic analysis, SMTP-7 suppressed the degradation of collagen IV, which is the main constituent element of the basal membrane, suggesting that SMTP-7 protected the BBB. Functional breakdown of BBB was examined by EB dye leakage into the brain tissue. EB leakage was significantly higher in rt-PA-treated mice, resulting in severe hemorrhagic transformation. In contrast, SMTP-7 protected BBB and ameliorated hemorrhagic transformation. We consider the mechanisms why SMTP-7 inhibited hemorrhagic transformation after severe focal ischemia and reperfusion under the

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warfarin-treated condition as follows. First, SMTP-7 never increases cytotoxic endogenous t-PA. SMTP-7 does not affect the enzymatic activity of t-PA, but promotes t-PA-induced plasminogen activation by approximately 100-fold (Hu et al., 2012). Therefore, SMTP-7 does not activate the t-PA - MMP-9 pathway. Second, SMTP-7 has anti-inflammatory and anti-oxidative effects (Miyazaki et al., 2011; Shibata et al., 2010; Shibata et al., 2011). Acute ischemia-reperfusion and subsequent chemokine responses lead to the recruitment of some inflammatory cells into the ischemic brain tissue (Charo and Ransohoff, 2006). Inflammatory cells release proteolytic enzymes, including MMP-2 and MMP-9, which cause BBB disruption (Charo and Ransohoff, 2006). Furthermore, neutrophils produce oxidative stress in brain tissue (Kilic et al., 2005). We previously reported that SMTP-7 directly neutralized IL-1 induced VCAM-1 expression, reactive oxygen species (ROS) generation, and leukocyte-endothelial interaction (Miyazaki et al., 2011). Finally, SMTP-7 increased local fibrinolysis only and did not affect systemic fibrynolytic activity because its action is to alter the conformation of plasminogen, leading to its proteolytic activation when the plasminogen activator is available, especially in a thrombus or embolus (Hasumi et al.,

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2010; Shibata et al., 2010). Although SMTP-7 decreased hemorrhagic transformation and improved neurological deficits after MCAO under the warfarin-treated condition, it did not significantly reduce the infarct volume and edema. It may be because long duration of ischemia (3 hours) upregulated endogenous tPA, and its neurotoxic effects prevented the significant reduction of the infarct volume and edema. Wang et al., reported that endogenous tPA more increased after 3 hours of MCAO compared to 2 hours (Wang et al., 1998). Injection of tPA after 2 hours of MCAO increased the infarct volume because of its neurotoxicity (Wang et al., 1998). In contrast, administration of tPA after 3 hours of MCAO did not increase the infarct volume because of the ceiling effect of endogenous tPA (Wang et al., 1998).

4. Conclusion SMTP-7 decreased hemorrhagic transformation and improved neurological deficits after MCAO under the warfarin-treated condition with severe INR prolongation. In contrast, IV rt-PA induced significantly higher hemorrhagic transformation and led to a poor

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outcome. Based on these results, we believe that SMTP-7 can be a safe thrombolytic agent for warfarin-treated patients who are contraindicated for IV rt-PA.

5. Experimental Procedure 5.1. Mouse Model of Oral Anticoagulation

Eight- to nine-week-old male C57BL/6J mice (Japan SLC, Shizuoka, Japan) were used in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80- 23, revised 1996). All procedures were approved by the animal care facility of Tohoku University Graduate School of Medicine. Following a previously described protocol, we administered warfarin orally through bottled drinking water (Foerch et al., 2008; Pfeilschifter et al., 2011; Pfeilschifter et al., 2011). Briefly, a 5 mg coumadin tablet (warfarin sodium, crystalline, 0556-0172-70, Bristol Myers Squibb, Munich, Germany) was dissolved in 375 ml tap water. This dose corresponds to warfarin uptake of 2.00 mg/kg per mouse for a 24-hour feeding period, assuming water consumption is 15 ml/100 g per 24 hours (Foerch et al., 2008;

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Pfeilschifter et al., 2011; Pfeilschifter et al., 2011). In this study, we measured a mean INR of 5.6

0.2 (n = 3) by CoaguChek (410350009, Roche, Basel, Switzerland).

Although, this INR was too longer to mimic clinical situations, we adopted this warfarin dose to emphasize the safety of SMTP-7 under warfarin anticoagulation. Details of this mouse model of oral anticoagulation can be found elsewhere (Foerch et al., 2008; Pfeilschifter et al., 2011; Pfeilschifter et al., 2011).

5.2. Experimental Groups and Drug Treatments

Mice were randomly assigned to three groups: vehicle- (n = 20), SMTP-7- (n = 15) and rt-PA-treated group (n = 30). Vehicle, rt-PA, or SMTP-7 was administered to examine the effect of different thrombolytic agents after MCAO under the warfarin-treated condition. Normal saline was injected intravenously as a bolus at a dose of 10 ml/kg, 5 minutes before reperfusion for vehicle treatment. rt-PA (Grtpa, 128-61398-8, Mitsubishi Tanabe Pharma, Osaka, Japan) was dissolved in water at 2 mg/ml concentration and administered intravenously at a dose of 10 mg/kg with 1 mg/kg as an initial bolus 5

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minutes before reperfusion followed by continuous intravenous infusion (Baby Bee syringe drive, MD-1001, Bee Hive controller, MD-1020, Bioanalytical Systems, West Lafayette, USA) of 9 mg/kg over 20 minutes (Sumii, 2002). SMTP-7 (lot N01YH) provided by TMS (TMS Co., Ltd., Tokyo, Japan) was dissolved in normal saline at a concentration of 2 mg/ml and administrated intravenously as a bolus at a dose of 10 mg/kg, 5 minutes before reperfusion. Previously, we investigated optimal dose of SMTP-7 for thrombolysis in a mice embolic stroke model and found out that 10 mg/kg was the most effective (Shibata et al., 2010). Vehicle- and SMTP-7-treated mice remained canulated for 20 minutes after the bolus injection as a sham procedure.

5.3. Focal Cerebral Ischemia

To mimic the clinical situations, we designed the present experiment whereby drugs were administrated 5 minutes before reperfusion by pulling out an intraluminal suture from its origin in the middle cerebral artery so that drug-associated damage could be assessed under similar conditions of reperfusion (Yamashita et al., 2009). We

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previously reported a mouse intraluminal middle cerebral artery occlusion (MCAO) model that did not involve the posterior cerebral artery (PCA) territory (Akamatsu et al., 2012). With slight modifications to this model, we developed a reproducible hemorrhagic infarction model under the warfarin-treated condition. Briefly, we used silicon-coated 6-0 nylon monofilaments (602123PK5Re, Doccol, Redlands, CA, USA); the diameter and length of the silicon-coated tip were 0.23 ± 0.02 mm and 1.5 to 2.0 mm, respectively. After a 24-hour warfarin feeding period, mice were anesthetized with 1.2 to 1.5% isofluorane in 30% oxygen and 70% nitrous oxide and breathed spontaneously. Rectal temperature during all surgical procedures was maintained at 37 ± 0.5͠ using a feedback-regulated heating pad (BWT- 100, Bio Research Center, Nagoya, Japan). A mouse in a prone position was fixed in a head holder (SG-4N, Narishige, Tokyo, Japan), and the scalp was shaved and cut with surgical scissors to expose the thin skull over the bilateral cerebral and cerebellar hemispheres. The surface of the skull was covered with a slipcover over a thin layer of saline to prevent drying. The baseline cerebral blood flow (CBF) values were recorded through the intact skull for 1 minute using laser speckle flowmetry (LSF) (OMEGAZONE, Omegawave, Tokyo,

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Japan). During CBF measurement, the skull surface was diffusely illuminated by 780 nm laser light. Scattered light was filtered and detected using a charge-coupled device camera positioned above the head. The filter detected only scattered light with perpendicular polarization to the incident laser light. Raw speckle images were used to calculate the speckle contrast, which corresponds to the number and velocity of moving red blood cells and is directly related to CBF. Signal processing was performed using the algorithm developed by Forrester et al (Forrester et al., 2002). This experimental settings allowed us to measure the relative CBF of the dorsal surface of the cerebrum. Color-coded CBF images were obtained in high-resolution mode (638 × 480 pixels; 1 image/second). The mouse was then placed a supine position. The neck was incised at the midline between the manubrium and the jaw, and the left common carotid artery (CCA) was carefully separated from the vagus nerve. The superior thyroid artery was cauterized and cut. The external carotid artery (ECA) was ligated with a 7-0 nylon suture near its bifurcation into the lingual and maxillary arteries and cauterized distally to the suture. The occipital artery was cauterized and cut. The pterygopalatine artery was then exposed and isolated. A collar suture at the origin of the ECA was prepared

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using a 5-0 silk suture, the internal carotid artery (ICA) was closed using a vascular clip (MH-1-20, BEAR Medic, Tokyo, Japan), and the CCA was temporarily ligated with a 7-0 nylon suture. The silicone-coated suture was introduced into the arteriotomy hole in the ECA and was advanced into the CCA bifurcation. After removing the vascular clip and cutting the ECA, the suture was advanced distally into the ICA. Once the tip of the inserted suture reached the ICA, the collar suture of the ECA was tightened to avoid bleeding from the arteriotomy hole, and the suture of the CCA was unfastened to restore blood flow from CCA. The intraluminal suture was inserted into the ICA 9.0 ± 0.5 mm from the CCA bifurcation until mild resistance was felt. The collar suture of the ECA stump was tightened securely around the inserted filament. The wound was closed with 6-0 nylon sutures, and the intraluminal suture was hidden in the wound. The CBF value was recorded using LSF for 1 minute and confirmed the decreasing CBF value in the MCA territory without the PCA territory. The mouse was then allowed to regain consciousness. The mouse was reanesthetized two hours and forty minutes after induction of ischemia, fixed in the head holder in a prone position, and CBF value was recorded. Mice in which CBF values were spontaneously restored were excluded form

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further experiments. The mouse was then replaced in a supine position and the cervical wound was reopened. Left jugular vein was exposed and cannulated by PE-10 tubing (427400, Becton Dickinson, New Jersey, USA) for the administration of each drugs. The drugs administration procedure was described above. Three hours after the induction of ischemia, the intraluminal suture was withdrawn gently and slowly to achieve the reperfusion and the ECA was ligated with the collar suture. After drug administration, the wound was closed and the mouse was positioned in a prone position. The recovery of CBF value was confirmed and the mouse was awakened to allow for a 24-hour survival period.

5.4. 2, 3, 5-TriphenylTerazolium Chloride (TTC) Staining

Only mice surviving until 24 hours after reperfusion were evaluated (n = 6, per group). Mice were given an overdose of isoflurane 24 hours after reperfusion, and then transcardially perfused with cold saline. Brains were removed immediately and cut into 5 serial 2-mm-thick coronal sections. These sections were incubated in a 1 % solution of

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2, 3, 5-triphenyltetrazolium chloride (TTC) (17779-10XX10MLML-FF, Sigma-Aldrich, St Louis, Mo, USA) for 10 min at 37°C. Images were captured using a digital camera (GR DIGITAL III, 173240, Ricoh, Tokyo, Japan).

5.5. Hemorrhagic Severity Scoring

The severity of cerebral hemorrhage was quantified in TTC-stained sections as described previously: non-hemorrhage (Score 0); hemorrhagic infarction Type 1 (HI-1), defined as heterogeneous small petechiae, generally along the boundary of the infarct (Score 1); hemorrhagic infarction Type 2 (HI-2), with more confluent petechiae within the infarcted area (Score 2); parenchymal hemorrhage Type 1 (PH-1), characterized by hematoma covering less than 30 % of the infarct (Score 3); and parenchymal hemorrhage Type 2 (PH-2) with dense hematoma in more than 30 % of the infarct (Score 4) (Aronowski et al., 2003; Berger et al., 2001; Kasahara et al., 2012). Examples of each type are demonstrated in Fig. 1A.

5.6. Neurological Deficit Scoring

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Neurological deficits were assessed 24 hours after reperfusion in each group (vehicle, n = 20; SMTP-7, n= 15; rt-PA, n = 30) with a 6 points neurological deficit scoring (0 = no deficit, 1 = failure to extend left forepaw, 2 = circling to the left, 3 = falling to the left, 4 = ‘‘barrel rolling’’, 5 = unable to move spontaneously, 6 = dead) (Pfeilschifter et al., 2011; Pfeilschifter et al, 2011). Mice surviving at 24 hours after reperfusion (vehicle, n = 16; SMTP-7, n= 15; rt-PA, n = 15) were assigned to further examinations.

5.7. Infarct Volume and Edema Index

Unstained areas were measured on TTC-stained sections by Image J, version 1.46r (National Institutes of Health) as the infarct volume. The edema index was calculated as (ipsilateral hemispheric volume - contralateral hemispheric volume) / contralateral hemispheric volume x 100 (Garcia-Yebenes et al., 2011).

5.8. Gelatin Zymography

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Gelatin zymography was performed on samples from ischemic hemispheres to evaluate the activity of MMP-9 24 hours after reperfusion. Mice surviving until 24 hours after reperfusion (n = 3, per group) were given an overdose of isoflurane and transcardially perfused with cold saline. The ipsilateral ischemic hemisphere was rapidly removed and homogenized on ice in 1 ml of lysis buffer (N-PER Neuronal Protein Extraction Reagent, 87792, Thermo scientific, MA, USA) containing protease inhibitor cocktail (Complete, Mini, EDTA-free in EASYPack, 5892791, Roche, Basel, Switzerland) and phosphatase inhibitor cocktail (PhosSTOP Phosphatase Inhibitor Cocktail, 4906845, Roche, Basel, Switzerland). Homogenates were centrifuged at 10,000 g for 10 minutes at 4°C. To analyze the MMP-9 activity, protein extraction of the tissue was performed as previously described with little modifications(Fujimura et al., 1999). The supernatants were recovered and incubated for 60 min with gelatin-sepharose 4B (17-0956-01, GE Healthcare, Uppsala, Sweden), with constant shaking at 4°C. After incubation, the samples were centrifuged at 500 g for 2 min. The pellets were washed with a buffer containing 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM CaCl2, 0.05% BRIJ-35, 0.02% NaN3. After a second centrifugation, the pellets were resuspended in

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elution buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM CaCl2, 0.05% BRIJ-35, 0.02% NaN3, 10% dimethylsulfoxide) for 30 min. The samples were subjected to gelatin zymography with Gelatin Zymo-Electrophoresis Kit (AK47, Primary Cell, Hokkaido, Japan) according to the manufacturer's directions. Images were captured and analyzed by ChemiDoc MP ImageLab PC system (BioRad, CA, USA).

5.9. Immunohistochemistry of Collagen IV

Nine surviving mice were examined (n = 3, per group). Twenty-four hours after reperfusion, mice were given an overdose of isoflurane and transcardially perfused with cold saline and followed by 4 % paraformaldehyde. After fixation, the forebrain was embedded in O.C.T. compound (Tissue-Tek, Sakura Finetek Japan, Tokyo, Japan) and frozen. Sections were cut in the coronal plane at 10 μm using a cryostat (Tissue-Tek Cryo3, Sakura Finetek Japan, Tokyo, Japan). Sections were incubated with anti-collagen IV (1 : 400, ab6586, Abcam, Cambridge, UK). Immunohistochemistry was performed with the avdin-biotin technique, and nuclei were counterstained with

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hematoxylin. The basal membrane was identified by its morphology, and staining intensity was measured using Image J, version 1.46r (National Institutes of Health).

5.10. Evaluation of BBB Permeability

BBB permeability was assessed by fluorescent detection of extravasated Evan's blue (EB) dye, as described previously (Li et al., 2011; Tang et al., 2009). We assessed BBB permeability in 10 mice (Vehicle group, n = 4; SMTP-7 group, n = 3; rt-PA group, n = 3) based on slight modifications to this method. Briefly, under isoflurane anesthesia, 2 % of EB dye in saline was administered from left jugular vein at a dose of 3 ml/kg 3 hours before euthanasia. Mice were given an overdose of isoflurane 24 hours after the reperfusion, and then transcardially perfused with cold saline to remove intravascular EB dye. Brains were removed immediately and dissected into left (ischemic) and right (non-ischemic) hemispheres. Each hemisphere was homogenized in 1 ml of 50% trichloroacetic acid solution. After centrifugation at 10,000 g for 30 min, the supernatants were collected, and the concentration of EB dye was determined with a

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spectrophotometer (SpectraMax 190 Absorbance Microplate reader, Molecular Devices, CA, USA) at 620 nm for absorbance. EB dye extravasation was expressed as the ratio of absorbance intensity in the ischemic hemisphere to that in the non-ischemic hemisphere (EB extravasation Index, EBI) (Chen et al., 2006).

5.11. Statistical Analysis

All values were given as the mean ± SD. Statistical significance was assessed with a one-way ANOVA and subsequent Tukey’s multiple comparison test for infarct volume, edema index, gelatin zymography, pixel intensity of collagenΦ and EBI. For non-parametric neurological deficit scoring and hemorrhagic severity scoring, we used a Kruskal-Wallis test and subsequent Dunn’s multiple comparison test. And mortality was assessed with 2 test. Values of p < 0.05 were considered statistically significant. Graph Pad Prism 5.03 (Graph Pad Software, La Jolla, CA, USA) was used for all statistical analysis.

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Acknowledgements

This work was supported in part by a Grant-in-Aid from the Japan Brain Foundation (KN) and a grant from the Japan Science and Technology Agency (TT, KH).

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REFERENCES Adams, H.P., Jr., del Zoppo, G., Alberts, M.J., Bhatt, D.L., Brass, L., Furlan, A., Grubb, R.L., Higashida, R.T., Jauch, E.C., Kidwell, C., Lyden, P.D., Morgenstern, L.B., Qureshi, A.I., Rosenwasser, R.H., Scott, P.A., Wijdicks, E.F., 2007. Guidelines for the early management of adults with ischemic stroke: a guideline from the American Heart Association/American Stroke Association Stroke Council, Clinical Cardiology Council, Cardiovascular Radiology and Intervention Council, and the Atherosclerotic Peripheral Vascular Disease and Quality of Care Outcomes in Research Interdisciplinary Working Groups: The American Academy of Neurology affirms the value of this guideline as an educational tool for neurologists. Circulation. 115,, e478–534. Akamatsu, Y., Saito, A., Fujimura, M., Shimizu, H., Mekawy, M., Hasumi, K., Tominaga, T., 2011. Stachybotrys microspora triprenyl phenol-7, a novel fibrinolytic agent, suppresses superoxide production, matrix metalloproteinase-9 expression, and thereby attenuates ischemia/reperfusion injury in rat brain. Neurosci Lett. 503,, 110–4. Akamatsu, Y., Shimizu, H., Saito, A., Fujimura, M., Tominaga, T., 2012. Consistent focal cerebral ischemia without posterior cerebral artery occlusion and its real-time monitoring in an intraluminal suture model in mice. J Neurosurg. 116,, 657–64. Albers, G.W., Bates, V.E., Clark, W.M., Bell, R., Verro, P., Hamilton, S.A., 2000. Intravenous tissue-type plasminogen activator for treatment of acute stroke: the Standard Treatment with Alteplase to Reverse Stroke (STARS) study. JAMA. 283,, 1145–50. Aronowski, J., Strong, R., Shirzadi, A., Grotta, J.C., 2003. Ethanol plus caffeine (caffeinol) for treatment of ischemic stroke: preclinical experience. Stroke. 34,, 1246–51. Berger, C., Fiorelli, M., Steiner, T., Schabitz, W.R., Bozzao, L., Bluhmki, E.,

31

Hacke, W., von Kummer, R., 2001. Hemorrhagic transformation of ischemic brain tissue: asymptomatic or symptomatic? Stroke. 32,, 1330–5. Charo, I.F., Ransohoff, R.M., 2006. The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med. 354,, 610–21. Chen, C.H., Toung, T.J., Sapirstein, A., Bhardwaj, A., 2006. Effect of duration of osmotherapy on blood-brain barrier disruption and regional cerebral edema after experimental stroke. J Cereb Blood Flow Metab. 26,, 951–8. Clark, W.M., Lessov, N.S., Dixon, M.P., Eckenstein, F., 1997. Monofilament intraluminal middle cerebral artery occlusion in the mouse. Neurol Res. 19,, 641–8. Del Zoppo, G.J., Saver, J.L., Jauch, E.C., Adams, H.P., Jr., 2009. Expansion of the time window for treatment of acute ischemic stroke with intravenous tissue plasminogen activator: a science advisory from the American Heart Association/American Stroke Association. Stroke. 40,, 2945–8. Foerch, C., Arai, K., Jin, G., Park, K.P., Pallast, S., van Leyen, K., Lo, E.H., 2008. Experimental model of warfarin-associated intracerebral hemorrhage. Stroke. 39,, 3397–404. Forrester, K.R., Stewart, C., Tulip, J., Leonard, C., Bray, R.C., 2002. Comparison of laser speckle and laser Doppler perfusion imaging: measurement in human skin and rabbit articular tissue. Med Biol Eng Comput. 40,, 687–97. Fujimura, M, G.Y., Morita-Fujimura, Y, Massengale, J, Kawase, M, Chan, PH, 1999. Early appearance of activated matrix metalloproteinase-9 and blood–brain barrier disruption in mice after focal cerebral ischemia and reperfusion. Brain Res. 842,, 92–100. Garcia-Yebenes, I., Sobrado, M., Zarruk, J.G., Castellanos, M., Perez de la Ossa, N., Davalos, A., Serena, J., Lizasoain, I., Moro, M.A., 2011. A mouse model of hemorrhagic transformation by delayed tissue plasminogen activator administration after in situ thromboembolic stroke. Stroke. 42,, 196–203. 32

Gasche, Y., Copin, J.C., Sugawara, T., Fujimura, M., Chan, P.H., 2001. Matrix metalloproteinase inhibition prevents oxidative stress-associated blood-brain barrier disruption after transient focal cerebral ischemia. J Cereb Blood Flow Metab. 21,, 1393–400. Group T.N.I.o.N.D.a.S.r.-P.S.S., 1995. Tissue plasminogen activator for acute ischemic stroke. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. N Engl J Med. 333,, 1581–7. Hacke, W., Kaste, M., Fieschi, C., Toni, D., Lesaffre, E., von Kummer, R., Boysen, G., Bluhmki, E., Hoxter, G., Mahagne, M.H., et al., 1995. Intravenous thrombolysis with recombinant tissue plasminogen activator for acute hemispheric stroke. The European Cooperative Acute Stroke Study (ECASS). JAMA. 274,, 1017–25. Hacke, W., Kaste, M., Fieschi, C., von Kummer, R., Davalos, A., Meier, D., Larrue, V., Bluhmki, E., Davis, S., Donnan, G., Schneider, D., Diez-Tejedor, E., Trouillas, P., 1998. Randomised double-blind placebo-controlled trial of thrombolytic therapy with intravenous alteplase in acute ischaemic stroke (ECASS II). Second European-Australasian Acute Stroke Study Investigators. Lancet. 352,, 1245–51. Hacke, W., Brott, T., Caplan, L., Meier, D., Fieschi, C., von Kummer, R., Donnan, G., Heiss, W.D., Wahlgren, N.G., Spranger, M., Boysen, G., Marler, J.R., 1999. Thrombolysis in acute ischemic stroke: controlled trials and clinical experience. Neurology. 53,, S3–14. Hashimoto, T., Shibata, K., Nobe, K., Hasumi, K., Honda, K., 2010. A Novel Embolic Model of Cerebral Infarction and Evaluation of Stachybotrys microspora Triprenyl Phenol-7 (SMTP-7), a Novel Fungal Triprenyl Phenol Metabolite. Journal of Pharmacological Sciences. 114,, 41–49. Hasumi, K., Yamamichi, S., Harada, T., 2010. Small-molecule modulators of zymogen activation in the fibrinolytic and coagulation systems. FEBS J. 277,, 3675–87. Hu, W., Narasaki, R., Nishimura, N., Hasumi, K., 2012. SMTP (Stachybotrys microspora triprenyl phenol) enhances clot clearance in a pulmonary embolism model in rats. Thromb J. 10,, 2. 33

Hu, W., Hasumi K, 2000. Activation of fibrinolysis by SMTP-7 and -8, novel staplabin analogs with a pseudosymmetric structure. J Antibiot (Tokyo). 53,, 241–247. Kasahara, Y., Nakagomi, T., Matsuyama, T., Stern, D., Taguchi, A., 2012. Cilostazol reduces the risk of hemorrhagic infarction after administration of tissue-type plasminogen activator in a murine stroke model. Stroke. 43,, 499–506. Kilic, E., Kilic, U., Matter, C.M., Luscher, T.F., Bassetti, C.L., Hermann, D.M., 2005. Aggravation of focal cerebral ischemia by tissue plasminogen activator is reversed by 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor but does not depend on endothelial NO synthase. Stroke. 36,, 332–6. Lapchak, P.A., Chapman, D.F., Zivin, J.A., Hsu, C.Y., 2000. Metalloproteinase Inhibition Reduces Thrombolytic (Tissue Plasminogen Activator)-Induced Hemorrhage After Thromboembolic Stroke Editorial Comment. Stroke. 31,, 3034–3040. Li, M., Zhang, Z., Sun, W., Koehler, R.C., Huang, J., 2011. 17beta-estradiol attenuates breakdown of blood-brain barrier and hemorrhagic transformation induced by tissue plasminogen activator in cerebral ischemia. Neurobiol Dis. 44,, 277–83. Miyazaki, T., Kimura, Y., Ohata, H., Hashimoto, T., Shibata, K., Hasumi, K., Honda, K., 2011. Distinct effects of tissue-type plasminogen activator and SMTP-7 on cerebrovascular inflammation following thrombolytic reperfusion. Stroke. 42,, 1097–104. Pfefferkorn, T., Rosenberg, G.A., 2003. Closure of the blood-brain barrier by matrix metalloproteinase inhibition reduces rtPA-mediated mortality in cerebral ischemia with delayed reperfusion. Stroke. 34,, 2025–30. Pfeilschifter, W., Spitzer, D., Czech-Zechmeister, B., Steinmetz, H., Foerch, C., 2011. Increased risk of hemorrhagic transformation in ischemic stroke occurring during warfarin anticoagulation: an experimental study in mice. Stroke. 42,, 1116–21. Pfeilschifter, W., Bohmann, F., Baumgarten, P., Mittelbronn, M., Pfeilschifter, J., Lindhoff-Last, E., Steinmetz, H., Foerch, C., 2012. Thrombolysis 34

with recombinant tissue plasminogen activator under dabigatran anticoagulation in experimental stroke. Ann Neurol. 71,, 624–33. Shibata, K., Hashimoto, T., Nobe, K., Hasumi, K., Honda, K., 2010. A novel finding of a low-molecular-weight compound, SMTP-7, having thrombolytic and anti-inflammatory effects in cerebral infarction of mice. Naunyn Schmiedebergs Arch Pharmacol. 382,, 245–53. Shibata, K., Hashimoto, T., Nobe, K., Hasumi, K., Honda, K., 2011. Neuroprotective mechanisms of SMTP-7 in cerebral infarction model in mice. Naunyn Schmiedebergs Arch Pharmacol. 384,, 103–8. Sumii, T., 2002. Involvement of Matrix Metalloproteinase in Thrombolysis-Associated Hemorrhagic Transformation After Embolic Focal Ischemia in Rats. Stroke. 33,, 831–836. Tagaya, M., Liu, K.F., Copeland, B., Seiffert, D., Engler, R., Garcia, J.H., del Zoppo, G.J., 1997. DNA scission after focal brain ischemia. Temporal differences in two species. Stroke. 28,, 1245–54. Tang, J., Li, Y.J., Mu, J., Li, Q., Yang, D.Y., Xie, P., 2009. Albumin ameliorates tissue plasminogen activator-mediated blood-brain barrier permeability and ischemic brain injury in rats. Neurol Res. 31,, 189–94. Pfeilschifter, W., Spitzer, D., Pfeilschifter, J., Steinmetz, H., Foerch, C., 2011. Warfarin Anticoagulation Exacerbates the Risk of Hemorrhagic Transformation after rt-PA Treatment in Experimental Stroke: Therapeutic Potential of PCC. PLoS ONE 6,, e26087. Wang, X., Lee, S.R., Arai, K., Lee, S.R., Tsuji, K., Rebeck, G.W., Lo, E.H., 2003. Lipoprotein receptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator. Nat Med. 9,, 1313–7. Wang, Y.F., Tsirka, S.E., Strickland, S., Stieg, P.E., Soriano, S.G., Lipton, S.A., 1998. Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice. Nat Med. 4,, 228–31. Yamashita, T., Kamiya, T., Deguchi, K., Inaba, T., Zhang, H., Shang, J., Miyazaki, K., Ohtsuka, A., Katayama, Y., Abe, K., 2009. Dissociation 35

and protection of the neurovascular unit after thrombolysis and reperfusion in ischemic rat brain. J Cereb Blood Flow Metab. 29,, 715–25.

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Figure Legends Fig. 1– Hemorrhagic severity scoring 24 hours after reperfusion. (A) The severity of cerebral hemorrhage was divided to 5 types: non-hemorrhage; score 0, hemorrhagic infarction Type 1 (HI-1); score 1, hemorrhagic infarction Type 2 (HI-2); score 2, parenchymal hemorrhage Type 1 (PH-1); score 3, parenchymal hemorrhage Type 2 (PH-2); score 4. There was no non-hemorrhage (score 0) in this study. (B) Example of the dead mice brain. Massive parenchymal hematoma was observed in all dead mice. (C) The severity of hemorrhagic transformation was higher in rt-PA-treated mice than in SMTP-7-treated mice. The severity of hemorrhagic transformation was slightly higher in rt-PA-treated mice than in vehicle-treated mice. The severity of hemorrhagic transformation was also slightly less in SMTP-7-treated mice than in vehicle-treated mice (*p < 0.05. n = 6, per group).

Fig. 2– Mortality and Neurological deficit scoring 24 hours after reperfusion. (A) All SMTP-7-treated mice survived 24 hours after reperfusion. In contrast, 20% of vehicle-treated and 50% of rt-PA-treated mice died. A significant difference was

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observed. (B) Neurological deficit scoring was significantly better in SMTP-7-treated mice than in vehicle- and rt-PA-treated mice. The severity of neurological deficits was significantly higher in rt-PA-treated mice than in vehicle-treated mice (*p < 0.05, **p < 0.01, ***p < 0.001. Vehicle, n = 20; SMTP-7, n = 15; rt-PA, n = 30).

Fig. 3– The infarction volume and edema index 24 hours after reperfusion. (A) No significant difference was observed in the infarct volume among the groups. (B) No significant difference was observed in the edema index among the groups (n = 6, per group).

Fig. 4– pro-matrix metalloproteinase (MMP-9) and MMP-9 levels 24 hours after reperfusion. (A) The photograph of gelatin zymography. pro-MMP-9 and MMP-9 were detected as 105 and 97 kDa, respectively. (B) pro-MMP-9 levels were significantly lower in vehicle-treated mice than in rt-PA treated mice. pro-MMP-9 levels were significantly lower in SMTP-7-treated mice than in vehicle- and treated mice. (C) Semi-quantitative analysis of MMP-9 level. MMP-9 levels were significantly lower in

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vehicle-treated mice than in rt-PA treated mice. MMP-9 levels were also significantly lower in SMTP-7-treated mice than in vehicle- and rt-PA-treated mice (*p < 0.05, **p < 0.01, ***p < 0.001. n = 3, per group).

Fig. 5– Degradation of collagen IV following ischemia reperfusion. (A) Representative micrographs of the boundary area of infarct in vehicle-, SMTP-7- and rt-PA-treated mice, respectively (upper: low magnification, lower: high magnification, Scale bars = 100 μm). Morphologically, the basal membrane was damaged in both vehicle- and rt-PA-treated mice. In contrast, the severity of damage to the basal membrane was less in the SMTP-7-treated. The interstitial spaces were dilated in the vehicle- and rt-PA treated mice. (B) The pixel intensity of collagen IV was significantly lower in rt-PA-treated mice. SMTP-7 significantly inhibited the degradation of collagen IV (*p < 0.05, ***p < 0.001. n = 3, per group).

Fig. 6– Blood-brain barrier (BBB) permeability 24 hours after reperfusion. (A) BBB permeability was evaluated by Evan’s blue (EB) dye leakage. Representative

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photographs of EB dye leakage in vehicle-, SMTP-7- and rt-PA-treated mice, respectively. (B) The EB extravasation index (EBI) was used for semi-quantitative analysis. EBI was significantly higher in rt-PA-treated mice than in vehicle- and SMTP-7-treated mice (*p < 0.05, **p < 0.01. Vehicle group, n = 4; SMTP-7 group, n = 3; rt-PA group, n = 3).

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Table 1 Physiolosical parameters MABP (mmHg)

pH

PaCO2 (mmHg)

PaO2 (mmHg)

Vehicle

54.0 ± 2.2

7.38 ± 0.02

30.6 ± 4.5

128.0 ± 8.5

rt-PA

55.3 ± 0.9

7.37 ± 0.03

34.1 ± 1.2

123.3 ± 8.4

SMTP-7

55.7 ± 2.6

7.38 ± 0.02

32.4 ± 1.3

133.3 ± 25.4

Vehicle

52.3 ± 1.2

7.36 ± 0.11

32.9 ± 4.3

127.0 ± 11.8

rt-PA

55.0 ± 2.4

7.26 ± 0.03

34.6 ± 1.3

131.0 ± 15.1

SMTP-7

56.0 ± 0.8

7.32 ± 0.03

35.0 ± 2.3

137.3 ± 26.8

Vehicle

56.7 ± 2.6

7.32 ± 0.04

37.4 ± 4.0

125.3 ± 7.3

rt-PA

56.0 ± 2.4

7.30 ± 0.05

36.9 ± 1.3

129.7 ± 10.9

SMTP-7

55.7 ± 1.2

7.32 ± 0.08

35.6 ± 1.7

127.3 ± 10.7

Before MCAO

After MCAO

After reperfusion

MABP, mean arterial blood pressure, MCAO, middle cerebral artery occlusion

41

z

SMTP-7 is a new thrombolytic agent that exhibits anti-inflammatory effects

z

We hypothesized SMTP-7 decrease hemorrhagic transformation

z

MCAO was induced in warfarin-treated mice to produce hemorrhagic transformation

z

SMTP-7 decreased mortality, hemorrhagic transformation and neurological deficits

42

Figure1

Figure2

Figure3

Figure4

Figure5

Figure6