Experimental Neurology 238 (2012) 138–144
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Experimental modeling of recombinant tissue plasminogen activator effects after ischemic stroke Mohamad El Amki a, Dominique Lerouet a, Bérard Coqueran a, Emmanuel Curis b, Cyrille Orset c, Denis Vivien c, Michel Plotkine a, Catherine Marchand-Leroux a, Isabelle Margaill a,⁎ a b c
EA4475, “Pharmacologie de la Circulation Cérébrale”, UFR des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, Sorbonne Paris Cité, Paris, France EA4466, “Laboratoire de Biomathématiques”, UFR des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, Sorbonne Paris Cité, Paris, France INSERM UMR-S U919, Université Caen Basse-Normandie, GIP Cyceron, Caen, France
a r t i c l e
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Article history: Received 11 June 2012 Revised 26 July 2012 Accepted 7 August 2012 Available online 19 August 2012 Keywords: Cerebral ischemia Thrombin Tissue plasminogen activator rt-PA Hemorrhagic transformation
a b s t r a c t Recombinant tissue plasminogen activator (rt-PA) is currently the only approved drug for ischemic stroke treatment, with a dose of 0.9 mg/kg. Since the fibrinolytic activity of rt-PA has been reported in vitro to be 10-fold less potent in rodent than in human, in most in vivo experimental models of cerebral ischemia rt-PA is used at 10 mg/kg. The purpose of this study was to compare the effects of the “human” (0.9 mg/kg) and “rodent” (10 mg/kg) doses of rt-PA given at an early or a delayed time point in a mouse model of cerebral ischemia. Cerebral ischemia was induced by thrombin injection into the left middle cerebral artery of mice. Rt-PA (0.9 or 10 mg/kg) was intravenously administered 30 min or 4 h after the onset of ischemia. The degree of reperfusion after rt-PA was followed for 90 min after its injection. The neurological deficit, infarct volumes, edema and hemorrhagic transformations (HT) were assessed at 24 h. Reperfusion was complete after early administration of rt-PA at 10 mg/kg but partial with rt-PA at 0.9 mg/kg. Both doses given at 4 h induced partial reperfusion. Early administration of both doses of rt-PA reduced the neurological deficit, lesion volume and brain edema, without modifying post-ischemic HT. Injected at 4 h, rt-PA at 0.9 and 10 mg/kg lost its beneficial effects and worsened HT. In conclusion, in the mouse thrombin stroke model, the “human” dose of rt-PA exhibits effects close to those observed in clinic. © 2012 Elsevier Inc. All rights reserved.
Introduction Stroke is the third leading cause of death and the primary cause of adult disability worldwide (Feigin et al., 2009; Towfighi and Saver, 2011). Today, the only approved drug is the recombinant tissue plasminogen activator (rt-PA), a thrombolytic agent that improves outcomes in acute ischemic stroke patients by restoring cerebral blood flow (CBF) (The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995; Saver, 2006). Nevertheless, its use remains limited to less than 5% patients due to its narrow therapeutic window (initially 3 h after stroke onset, but later extended to 4.5 h; Hacke et al., 2008). Beyond this limit, rt-PA induces no more
Abbreviations: rt-PA, recombinant tissue plasminogen activator; HT, hemorrhagic transformation; CBF, cerebral blood flow; MCA, middle cerebral artery. ⁎ Corresponding author at: EA4475, “Pharmacologie de la Circulation Cérébrale”, UFR des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, Sorbonne Paris Cité, 4 avenue de l'Observatoire, 75006 Paris, France. Fax: +33 1 53 73 97 89. E-mail addresses:
[email protected] (M. El Amki),
[email protected] (D. Lerouet),
[email protected] (B. Coqueran),
[email protected] (E. Curis),
[email protected] (C. Orset),
[email protected] (D. Vivien),
[email protected] (M. Plotkine),
[email protected] (C. Marchand-Leroux),
[email protected] (I. Margaill). 0014-4886/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.expneurol.2012.08.005
beneficial effects. Furthermore, clinical data showed that whatever its administration time, rt-PA increases the risk of hemorrhagic transformation (HT) (Saqqur et al., 2008; Wardlaw et al., 1997). Clinical practice also showed that rt-PA does not induce recanalization in all ischemic patients. Therefore, the research challenge is to develop a strategy that could increase the therapeutic window of rt-PA, reduce its toxicity and improve thrombolysis. To achieve this goal, STAIR (Stroke Treatment Academic Industry Roundtable) recommendations highlight the need for models mimicking as precisely as possible clinical stroke (Saver et al., 2009). Recently, Orset et al. (2007) described a new mouse model of thromboembolic stroke based on in situ thrombin injection into the middle cerebral artery (MCA), which allows to study the thrombolytic effect of rt-PA. In this model, the early infusion of 10 mg/kg rt-PA improves brain damage, while its delayed administration has no neuroprotective effect and increases HT (García-Yébenes et al., 2011; Orset et al., 2007). In 1981, Korninger and Collen (1981) showed in vitro that the rat's fibrinolytic system is 10-fold less sensitive than the human one, leading to use rt-PA at 10 mg/kg in rodents instead of 0.9 mg/kg in human in most in vivo stroke studies. The only in vivo study with 0.9 mg/kg of rt-PA was recently provided in rats by Haelewyn et al. (2010) and showed that this dose is as efficient as 10 mg/kg to reduce brain
M. El Amki et al. / Experimental Neurology 238 (2012) 138–144
infarct volume and edema when administered early (45 min) after MCA occlusion by autologous blood clot injection. However, there is still no experimental data about the mouse's fibrinolytic system compared to the human one and the effects of a treatment with rt-PA at 0.9 mg/kg in murine stroke models. In this context, the purpose of this work was to evaluate, in the thrombin model of stroke, the effects of the low dose of rt-PA (0.9 mg/kg; “human”) compared to the high dose (10 mg/kg; “rodent”), after an early or delayed infusion, on reperfusion and on post-ischemic outcomes with a particular interest in HT.
Methods Animals All animal experiments were performed on male Swiss albino mice (25–32 g, Janvier, Le Genest-St-Isle, France) in compliance with the European Community Council Directive of 24 November 1986 (86/609/EEC) and the French regulations (D2001-486) regarding the protection of animals used for experimental and other scientific purposes. Protocols were approved by the ethical regional committee for animal experimentation (P2.CM.152.10). Animals were housed under standard conditions with a 12 h light/dark cycle and allowed access to food and water ad libitum. Animals were randomly assigned to experimental groups and treated in a blinded manner. Experiment analysis was realized by an investigator who was blinded to the treatment groups.
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Thrombolytic effect of rt-PA In order to evaluate the thrombolytic effect of rt-PA, CBF was recorded for 90 min after its administration (n = 4–6 mice/group). Mice receiving rt-PA 30 min after ischemia were continuously under anesthesia, while mice treated at 4 h were allowed to awaken 30 min after thrombin injection and then re-anesthetized just before rt-PA or saline infusion for CBF recording. Three levels of reperfusion were defined according to the final CBF recorded: complete reperfusion (grade A: CBF≥75% of baseline level), partial reperfusion (grade B: 50%≤CBFb 75% or grade C: 25%≤CBFb 50% of baseline level) and no reperfusion (grade D: CBFb 25% of baseline level). Details regarding number of animals per group and exclusion criteria are provided in Table 1. Effects of rt-PA on post-ischemic outcomes In a second set of experiment, ischemic mice treated with saline or rt-PA (0.9 or 10 mg/kg) 30 min or 4 h after ischemia and shamoperated mice were used to evaluate outcomes 24 h after ischemia onset (n = 9–10 mice/group). Treatment was administered in awake animals. Details regarding number of animals per group and exclusion criteria are provided in Table 2. Functional outcome Neurological deficit was evaluated using a global neurological score modified from Wahl et al. (1992). Contralateral sensimotor functions were examined by assessing 3 items:
Induction of focal cerebral ischemia Mice were intraperitoneally anesthetized with ketamine (50 mg/kg) and xylazine hydrochloride (6 mg/kg). Ischemia was induced by the occlusion of the left MCA according to Orset et al. (2007). Briefly, mice were placed in a stereotaxic device, the skin between the left eye and ear was incised, and the temporal muscle was retracted. After craniotomy and dura excision, a glass pipette (calibrated at 15 mm/μl; Assistent ref. 555/5; Hoecht, Sondheim-Rhoen, Germany) was introduced into the lumen of the MCA and 1 μl of purified human alpha-thrombin (0.75 UI; HCT-0020, Haematologic Technologies Inc., USA) was injected to induce the formation of a clot in situ. The pipette was removed 10 min after the beginning of thrombin injection. The same surgical procedure was performed in sham-operated mice, except the introduction of the pipette into the MCA. CBF was monitored in the MCA region by laser Doppler flowmetry (Moor instruments, Ltd, UK) after the beginning of thrombin injection, and a clot was defined as stable when CBF rapidly falls to at least 50% of the baseline level (Astrup et al., 1977; Sekhon et al., 1995) and remains below this value for 30 min. Mice with less than a 50% drop or with spontaneous reperfusion (transient CBF drop to less than 50% returning spontaneously above 50% during the 30 min of recording) were excluded. Body temperature was monitored throughout surgery by a rectal probe and maintained at 37 ± 0.5 °C with a homeothermic blanket control unit (Harvard Apparatus, Edenbridge, Kent, U.K.). After surgery, mice were returned to their home cage and fed with mashed lab chow.
Protocol of rt-PA administration Ischemic mice received intravenously (tail vein; 10% bolus, 90% perfusion during 30 min) isotonic saline or rt-PA (0.9 or 10 mg/kg; Actilyse; dissolved and diluted in saline) 30 min or 4 h after ischemia. Compared to the clinical therapeutic window of rt-PA, 4 h is considered as a late treatment in this model (García-Yébenes et al., 2011).
– The abnormal postures after tail suspension (contralateral forelimb flexion and thorax twisting). – The forelimb and hindlimb grasping reflexes performed onto a metallic wire (0.4 mm diameter). – The forelimb and hindlimb hanging reflexes (the paw was gently pulled down, and after release, was checked for replacing). To these sensorimotor scores, we added the grip and the string tests described by Hall (1985). Grip score was measured as the length of time that mice remained on a metal bar for a maximum of 30 s. String test evaluated the manner of mice to hang and move on the string. The scores for each item were then summed and used as a global neurological score which maximum was 16 (Table 3). The lower the neurological score the more severe the deficit. Hemorrhagic transformation Immediately after functional outcome assessment, mice were anesthetized with sodium pentobarbitone (60 mg/kg; ip) and transcardially perfused with isotonic saline. Brains were quickly removed and frozen in isopentane and then stored at − 40 °C. Table 1 Thrombolytic effect of rt-PA: distribution of ischemic mice. Treatment time
30 min
4h
Total of animals
n = 25
n = 17
n=1 n=2
n=1 n=2
1/6 2/6 1/6
0/4 1/4 0/5
Excluded animals CBF drop b 50% Spontaneous reperfusion Number of mice (deada/included) Ischemia + saline Ischemia + 0.9 mg/kg rt-PA Ischemia + 10 mg/kg rt-PA
a All animals died before treatment, except 1 mouse in the saline group and 1 in 10 mg/kg rt-PA group.
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Table 2 Effects of rt-PA on post-ischemic outcomes: distribution of ischemic mice. Treatment time
30 min
4h
Total of animals
n= 37
n= 44
n= 0 n= 2
n= 2 n= 3
0/10 1/9 5/10
2/10 4/10 4/9
Excluded animals CBF drop b 50% Spontaneous reperfusion Number of mice (deada/included) Ischemia + saline Ischemia + 0.9 mg/kg rt-PA Ischemia + 10 mg/kg rt-PA a
Spearman correlation test was used to evaluate the differences in grades of reperfusion. Infarct volume and edema: differences were evaluated by ANOVA followed by Student t test. Neurological deficit and HT: differences between groups were evaluated by non-parametric Kruskall–Wallis analysis with subsequent comparison by Mann–Whitney U test. Differences were statistically significant for Pb 0.05, after Bonferroni corrections for multiple comparisons when necessary. All tests were performed with GraphPad Prism software (GraphPad prism, Prism 5 for Mac, version 5.0).
All animals died after treatment, except 1 mouse in each 0.9 mg/kg rt-PA group.
Results Brains were coronally sectioned at 13 levels from 6.5 to 0.5 mm anterior to the interaural line (each 500 μm interval), according to a stereotaxic brain atlas (Paxinos and Franklin, 2001). Microscopic, macroscopic and total hemorrhagic scores were visually quantified on each level, directly into the cryostat, as previously described by Haddad et al. (2008).
Infarct volume and brain edema One slice (20 μm thick) was also collected at each level and stained with cresyl violet to quantify cortical infarct volume: infarct area was outlined, measured on each slice using a computer image analysis system (Image J version 1.41), and then, multiplied by the ratio of the surface of the infarcted cortex (ipsilateral) to the intact cortex (contralateral) to correct the lesion for brain edema (Golanov and Reis, 1995). The infarct volume was calculated by linear integration of infarcted areas. Brain swelling, indicating edema, was also calculated according to the following formula: [(ipsilateral cortex volume − contralateral cortex volume)/contralateral cortex volume] × 100.
Statistical analysis Data are expressed as means ± SEM. Repeated measures one-way analysis of variance (ANOVA) followed by Student t test was used to evaluate differences in CBF.
Table 3 The global neurological score. Behavioral test
Score
Forelimb flexion
0–2
0: flexion 1: mid flexion 2: no flexion Thorax twisting 0: twisting 1: mid twisting 2: no twisting Grasping reflex 0: no reflex 1: reflex (for each contralateral paw) Leg hanging reflex 0: no reflex 1: reflex (for each contralateral paw) Grip 0: 0 s 1: 1–10 s 2: 11–20 s 3: 21–30 s String 0: fall down during the 30 s period 1: hang on during 30 s without using the 4 paws 2: hang on using the 4 paws at least 5 s 3: hang on using the 4 paws and the tail at least 5 s 4: hang on using the 4 paws and the tail and move at least 5 s 5: reach one of the vertical rods during the 30 s period Global neurological score 0–16
0–2
0–2 0–2
Thrombolytic effect of rt-PA A total of 42 mice were used in this experiment (Table 1). Six mice were excluded: 2 mice had less than a 50% drop in CBF and 4 showed spontaneous reperfusion during the first 30 min. Three mice died before treatment and 2 after treatment. Early administration Thrombin injection into the MCA led to an immediate and similar CBF drop in the 3 groups of mice before treatment (residual blood flow of saline treated animals: 9±2% of baseline level, 9±2% in 0.9 mg/kg rt-PA treated mice and 13±2% in 10 mg/kg rt-PA group, Fig. 1A). The injection of saline 30 min after ischemia induction did not affect CBF, while 0.9 mg/kg rt-PA led to a significant increase 40 min after rt-PA administration (66 ± 21% of baseline level, P b 0.05 versus saline group). On the other hand, 10 mg/kg rt-PA infusion led to a rapid but partial reperfusion at 20 min (51 ± 14%, P b 0.05 versus saline group) which was complete 20 min later. At the end of CBF recording, 0.9 mg/kg rt-PA induced no reperfusion in 17% of mice (grade D), partial reperfusion in 50% of mice (33% grade C and 17% grade B) and complete reperfusion in 33% of mice (grade A), while 10 mg/kg rt-PA treated mice exhibited partial (33% grade B) and complete (67% grade A) reperfusion (Fig. 1C). Reperfusion increased significantly in a dose-dependent manner (P b 0.05): the more the dose of rt-PA increased, the higher the reperfusion degree was. Delayed administration As illustrated in Fig. 1B, thrombin injection led to an immediate and similar CBF drop in saline group and mice post-treated with rt-PA at 0.9 mg/kg or 10 mg/kg (residual blood flow: 11 ± 4%, 11 ± 3% and 20 ± 7% of baseline level, respectively). Four hours after ischemia induction, the injection of saline did not affect CBF. Administration of 0.9 or 10 mg/kg rt-PA induced a slow reperfusion, which stabilized to less than 50% of baseline level with both doses. At the end of CBF recording, no difference in the grade of reperfusion between both rt-PA treated groups was observed (Fig. 1D). Effects of rt-PA on post-ischemic outcomes at 24 h
0–3
0–5
A total of 91 mice (included 10 sham-operated mice) were used in this experiment (Table 2). Seven mice were excluded: 2 had less than a 50% drop in CBF and 5 showed spontaneous reperfusion during the first 30 min. Two mice died before treatment and 14 after treatment (without significant differences between groups). Statistical analysis (ANOVA) did not show any significant differences in the residual blood flow between the 6 animal groups (early and late administration). Functional outcome Thrombin injection induced a similar neurological deficit in mice receiving saline 30 min or 4 h after ischemia (7.7 ± 0.9 and 7.1 ± 1.2 versus 15.6 ± 0.2 in sham-operated mice, P b 0.001; Fig. 2).
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Fig. 1. Cerebral blood flow (CBF) after an early (A) or delayed (B) administration with low or high rt-PA doses. CBF was measured by laser Doppler in ischemic mice treated with saline or rt-PA 30 min (n = 6 per group) or 4 h (n = 4–5 per group) after ischemia induction. The degree of reperfusion of early (C) and delayed (D) treated groups was defined according to the last CBF measure. Data are expressed as mean ± SEM. *P b 0.05, **P b 0.01 and ***P b 0.001 versus saline; †P b 0.05 versus 0.9 mg/kg rt-PA.
Early treatment with both doses of rt-PA led to a significant functional recovery compared to the corresponding saline group (10.9 ± 1.3 with 0.9 mg/kg rt-PA, P b 0.05; 11.7 ± 0.9 with 10 mg/kg rt-PA, P b 0.01), while delayed administration of rt-PA had no effect whatever the dose. Cortical lesion and edema Ischemia induced a cortical infarction of 18.5 ± 1.9 mm 3 and 16.5 ± 1.9 mm 3 in mice treated with saline either 30 min or 4 h after clot formation (Figs. 3A and C). Administration of rt-PA at 0.9
Fig. 2. Neurological deficits after an early or delayed administration with low or high rt-PA dose evaluated 24 h after ischemia. Ischemic mice were treated with saline or rt-PA 30 min or 4 h after ischemia induction (n = 9–10 per group). Data are expressed as mean ± SEM. †††P b 0.001 versus sham; *P b 0.05 and **P b 0.01 versus saline.
and 10 mg/kg 30 min after ischemia was associated with a 65% or 77% reduction in infarct volume (6.5 ± 1.7 mm 3 and 4.2 ± 1.2 mm 3, respectively; P b 0.001), while both doses did not modify infarct volume when administered 4 h after ischemia induction. Ischemia led to a cortical edema of 15.4 ± 2.0% and 13.8 ± 1.4% when saline was administered at 30 min and 4 h (Figs. 3B and C). Early treatment with 0.9 or 10 mg/kg rt-PA reduced brain edema by 64% (5.5 ± 1.3%, P b 0.001) and 76% (3.7 ± 0.1%, P b 0.001), respectively, while a delayed administration had no effect. Hemorrhagic transformations In ischemic mice treated with saline 30 min after ischemia, microscopic and macroscopic hemorrhagic scores were significantly increased compared to sham-operated mice (12.7 ± 0.9 versus 2.3 ± 0.5 and 7.3 ± 2.2 versus 0.3 ± 0.3, respectively; P b 0.01; Figs. 4A and B). Accordingly, the total hemorrhagic score was 6.7-fold higher in the ischemic group than in sham-operated mice (P b 0.001; Fig. 4C). Early administration of both doses of rt-PA did not modify these hemorrhagic scores. Saline mice treated 4 h after ischemia also showed increased microscopic and macroscopic hemorrhagic scores compared to shamoperated mice (8.7 ± 1.7 versus 2.3 ± 0.5 and 8.5 ± 3.6 versus 0.3 ± 0.3, respectively; P b 0.01 and P b 0.05) and the total hemorrhagic score was 5.7-fold higher than in sham-operated mice (P b 0.001). Delayed administration of 0.9 mg/kg rt-PA did not affect microscopic score, but increased the macroscopic score (36.6 ± 8.6, P b 0.05), and consequently, the total hemorrhagic score (×3; P b 0.01). In 10 mg/kg rt-PA-treated mice, both microscopic and macroscopic scores were significantly increased (30.2± 3.5 and 52.9 ±11.9, respectively; P b 0.01 and P b 0.001), resulting in a 5-fold increase in total hemorrhagic score (Pb 0.001).
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Fig. 3. Cortical lesion (A) and edema (B) after an early or delayed administration with low or high rt-PA dose evaluated 24 h after ischemia. Ischemic mice were treated with saline or rt-PA 30 min or 4 h after ischemia induction (n = 9–10 per group). Representative brain lesions, stained by cresyl violet, are presented in (C) for each treated ischemic group. Data are expressed as mean ± SEM. ***P b 0.001 versus saline.
Discussion We hereby show that, despite some differences in thrombolysis efficiency (when administered early), the clinical dose of rt-PA (0.9 mg/kg) and the dose of 10 mg/kg, commonly used in preclinical stroke studies in rodents, induce similar beneficial or deleterious effects depending on the administration time in a murine model of cerebral ischemia. To our knowledge, this is the first demonstration that delayed administration
of 0.9 mg/kg rt-PA induces no neuroprotection and, most importantly, increases HT, like the commonly used dose of rt-PA in rodents (10 mg/kg) does. The evaluation of the thrombolytic effect of an early administration of rt-PA showed that the dose of 10 mg/kg induces a rapid and complete recovery of CBF, while mice treated with 0.9 mg/kg rt-PA exhibited slower and partial reperfusion (CBF finally stabilized at approximately 60% of baseline level). In rats, Haelewyn et al. (2010) also
Fig. 4. Hemorrhagic score after an early or delayed administration with low or high rt-PA dose. Microscopic (A), macroscopic (B) and total hemorrhagic scores (C) were evaluated 24 h after ischemia (n = 9–10 per group). Ischemic mice were treated with saline or rt-PA 30 min or 4 h after ischemia induction. Data are expressed as mean± SEM. †P b 0.05; ††P b 0.01 and †††P b 0.001 versus sham; *P b 0.05; **Pb 0.01 and ***P b 0.001 versus saline.
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observed a slower reperfusion with the low dose of rt-PA, but this dose induced a total reperfusion. Besides species differences, this discrepancy might be linked to the blood clot composition in both models, and thus to a different resistance to lysis. Indeed, Niessen et al. (2003) demonstrated, in a model of cerebral ischemia induced by clot injection, that thrombolysis with rt-PA is more efficient on clots forming spontaneously than on thrombin-induced clots. Thrombin converts soluble fibrinogen into strands of fibrin and also activates factor XIII, which increases the stability of the fibrin clot. Thus, the more clot thrombin content is important, the more fibrin network is tight. This might explain that, in our model, the direct injection of thrombin into the MCA leads to the formation of a more compact clot than in the Haelewyn's model, induced by autologous clots prepared ex vivo. Several authors reported that, in stroke patients, recanalization after rt-PA is either complete, partial or none (Christou et al., 2000; Molina et al., 2004; Ribo et al., 2006). This variability seems to be reproduced by the low dose of rt-PA in our study, although this should be ascertained with larger sample sizes. Thus this low dose of rt-PA might be of particular interest to evaluate strategies to associate with rt-PA for improving thrombolysis. Despite partial reperfusion with 0.9 mg/kg rt-PA at this early time point, this dose reduced to the same extent the neurological deficit, infarct volume and brain edema 24 h after ischemia than the dose of 10 mg/kg did. These results suggest that reperfusion above 60% of baseline level is sufficient to reduce brain lesion and edema, which could explain functional outcome improvement. At a late stage after stroke onset, both doses of rt-PA induced slow and partial reperfusion below 40% of baseline level. Low reperfusion grade might result from clots becoming more resistant to fibrinolysis over time, which is in line with experimental studies demonstrating that old clots have more fibrin cross-linking, are more compacted and more difficult to dissolve (del Zoppo et al., 1992; Molina et al., 2004; Zhang et al., 2001). Moreover, Zhang et al. (2001) observed that a fibrin-rich clot undergoes a time-dependent transformation into a platelet-rich one. It is worth noting that activated platelets can release endogenous t-PA inhibitors like plasminogen activator inhibitor-1 (PAI-1), protease nexin-1 (PN-1) and α2‐antiplasmin, which in turn reduce the thrombolytic effect of rt-PA (Boulaftali et al., 2011; Zhang et al., 2001). Besides the age of the clot, the “no-reflow” phenomenon may also contribute to this low reperfusion grade after the late administration of rt-PA. This phenomenon, due to several factors including (1) extrinsic compression from edema, endothelial swelling and endothelial microvillus formation, and (2) intravascular obstruction, related to the activation of endothelium and coagulation, leukocyte and platelet adhesion (del Zoppo and Mabuchi, 2003; del Zoppo, 2008), is indeed closely linked to the duration of ischemia (Hossmann, 1993). Hemorrhagic transformation is a common and potentially serious complication of brain infarction occurring in 6 to 43% of ischemic stroke patients in the absence of revascularization therapy (Khatri et al., 2007). In our model, spontaneous microscopic and macroscopic hemorrhages were observed throughout the ischemic cortex 24 h after ischemia. Of note, in the same thrombin stroke model, García-Yébenes et al. (2011) also detected modest hemorrhage areas using an equivalent visual hemorrhagic score, which was confirmed by increased intraparenchymal hemoglobin level (Drabkin assay). By contrast, Orset et al. (2007) did not detect any evidence of brain hemorrhage using the perls’ prussian blue coloration. These discrepancies might be due to differences in the method of evaluation. Early administration of both doses of rt-PA did not modify the spontaneous post-ischemic HT in our model, while delayed treatment worsened them. Despite the fact that hemorrhages with the high dose of rt-PA was more important, but still not significantly different from the low dose, our major finding is that rt-PA related-hemorrhages were also observed with the “human” dose of rt-PA. Moreover, this HT aggravation after delayed rt-PA administration, together with
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the low reperfusion rate, might explain its lack of neuroprotective effect on infarct volume, brain edema and neurological deficit.
Conclusion Our study shows that the dose of rt-PA used in stroke patients (0.9 mg/kg) closely mimics the two faces of the thrombolytic in the mouse model of cerebral ischemia induced by thrombin injection: on the one hand, early thrombolysis is associated with beneficial effects (The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995), and on the other hand, delayed treatment leads to an aggravation of HT and a loss of neurologic improvement (Hacke et al., 2004). Furthermore, in view of the various cascades triggered by rt-PA, it might be more relevant from a clinical perspective to use in our mouse model the dose administered to human. These experimental conditions could be more appropriate for the evaluation of therapy to combine with rt-PA in order to improve thrombolysis, to reduce HT and to increase its therapeutic window.
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