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J Cereb Blood Flow Metab. 2010 April; 30(4): 718–728.
Published online 2010 January 20. doi:  10.1038/jcbfm.2009.275
PMCID: PMC2949169

Xenon is an inhibitor of tissue-plasminogen activator: adverse and beneficial effects in a rat model of thromboembolic stroke

Abstract

Preclinical evidence in rodents has proven that xenon may be a very promising neuroprotective agent for treating acute ischemic stroke. This has led to the general thinking that clinical trials with xenon could be initiated in acute stroke patients in a next future. However, an unappreciated physicochemical property of xenon has been that this gas also binds to the active site of a series of serine proteases. Because the active site of serine proteases is structurally conserved, we have hypothesized and investigated whether xenon may alter the catalytic efficiency of tissue-type plasminogen activator (tPA), a serine protease that is the only approved therapy for acute ischemic stroke today. Here, using molecular modeling and in vitro and in vivo studies, we show (1) xenon is a tPA inhibitor; (2) intraischemic xenon dose dependently inhibits tPA-induced thrombolysis and subsequent reduction of ischemic brain damage; (3) postischemic xenon virtually suppresses ischemic brain damage and tPA-induced brain hemorrhages and disruption of the blood–brain barrier. Taken together, these data indicate (1) xenon should not be administered before or together with tPA therapy; (2) xenon could be a golden standard for treating acute ischemic stroke if given after tPA-induced reperfusion, with both unique neuroprotective and antiproteolytic (anti-hemorrhaging) properties.

Keywords: tPA, xenon, acute ischemic stroke, thrombolysis, hemorrhages, brain damage

Acute ischemic stroke is one of the most common causes of death and long-term neurologic morbidity in the adult population. The primary cause of ischemic stroke is an acute and massive disruption of cerebral blood flow (CBF) through thromboembolism that leads to an oxygen and glucose deprivation in the brain. One of the major consequences is an excessive release of glutamate that leads to subsequent overstimulation of N-methyl--aspartate (NMDA) glutamatergic receptors, whose postsynaptic activation is known as a critical event in neuronal death induced by acute ischemic stroke and other excitotoxic diseases (Dirnagl et al, 1999; Lo et al, 2005). Two strategies have been pursued for the treatment of ischemic stroke: first, limitation of the primary vascular insult by early reperfusion; second, blockade of the neurotoxic cascade initiated by glutamate (Lee et al, 1999). Till today, the only therapy approved for the treatment of acute ischemic stroke is thrombolysis by the recombinant form of human tissue-type plasminogen activator (tPA), a serine protease that exerts its effect by converting the proenzyme plasminogen to the protease plasmin that is ultimately responsible for the degradation of the thrombus (Lijnen et al, 1980). Recombinant human tPA has benefited ischemic stroke patients if administered within 3 h of symptoms onset (The NINDS rtPA Stroke Study Group, 1995; Lees, 1999), but adverse effects particularly brain hemorrhages and disruption of the blood–brain barrier integrity have been reported (The NINDS rtPA Stroke Study Group, 1997) through tPA-mediated proteolytic processes (Yepes et al, 2009). In contrast, strategies of neuroprotection by the use of glutamatergic receptor antagonists have not yet been proven efficient in patients having hypoxic–ischemic insults, mainly because these compounds produce intolerable neurotoxic and psychotomimetic side effects (Olney et al, 1991; Davis et al, 2000). Taken together, this has led to the conclusion that methods of neuroprotection that also prevent tPA toxicity are now needed (Kaur et al, 2004).

Over the past decade, much attention has been paid to the potentially neuroprotective properties of the noble and remarkably tolerable anesthetic gas xenon. Preclinical evidence in rodents subjected to mechanical brain ischemia (and other hypoxic–ischemic insults) has proven that intraischemic or postischemic treatment with subanesthetic concentrations of xenon may be a very promising strategy with effective neuroprotective properties and no adverse side effects (David et al, 2003, 2008; Homi et al, 2003; Ma et al, 2003). This has led to the general thinking that clinical trials could be initiated in acute stroke patients in future. However, an unappreciated physicochemical property of xenon is that this gas also binds to the active site of a series of serine proteases within the S1 primary specificity pocket that specifically recognizes part of the substrate (Schiltz et al, 1995). Here, because the active site of serine proteases is structurally conserved, we have hypothesized and investigated whether xenon may modulate the catalytic efficiency of tPA and plasmin, and thereby alter the beneficial and/or adverse effects of tPA therapy in rats subjected to a thromboembolic middle cerebral artery occlusion (MCAO) model of acute ischemic stroke.

Materials and methods

Animals

Male Sprague–Dawley rats (n=50) were used. All animal use procedures were in accordance with the framework of the French legislation for biomedical experimentation and The European Communities Council Directive issued on 24 November 1986 (86/609/EEC). Before being used, rats were housed at 21±0.5°C, in Perspex home cages with free access to food and water. After surgery, they were housed individually. Light was maintained on a light/dark reverse cycle, with lights on from 20:00 to 08:00 hours.

Modeling of the Binding Site of Xenon Within the S1 Pocket of tPA and Plasmin

The structural superposition of the catalytic domain of tPA (PDB entry: 1A5H; Renatus et al, 1997) or plasmin (PDB entry: 1BUI; Parry et al, 1998) to elastase (PDB entry: 1C1M; Schiltz et al, 1995) was performed with the software PYMOL (DeLano Scientific, 2002, San Carlo, CA, USA). The root mean square deviation between the catalytic domain of tPA and elastase and that of plasmin and elastase was respectively 1.5 Å for 221 aligned carbon α (Cα) and 0.73 Å for 199 aligned Cα.

tPA and Plasmin Catalytic Activity Assay

The effects of xenon on the catalytic efficiency of tPA (alteplase), reteplase—a tPA-derived drug—and plasmin were assessed by the initial rate method. The recombinant form of human tPA in the form of Actilyse (Boehringer, Ingelheim, Germany) and murine tPA (ref. IRTPA; Innovative Research, Asbach, Germany) and their specific chromogenic substrate methylsulfonyl--phenyl-glycil-arginine-7-amino-4-methylcoumarin acetate (spectrozyme XF, ref. 444; American Diagnostica, Neuville-sur-Oise, France) were separately diluted in 1 ml of distilled water in 1.5 ml sterile tubes. Each tube containing 0.4 μmol/L of tPA or 10 μmol/L of the tPA substrate was closed, the cap of the tube was perforated with two holes using a needle (20 gauge × 32 mm in length), and saturated for 20 min at a flow rate of 60 to 80 ml/min with xenon of 25 to 75 vol% or medical air composed of 25% oxygen and 75% nitrogen through a needle that was introduced down to the bottom of the tube through one of the two holes previously perforated. Similar experiments were conducted with reteplase in the form of Rapilysin (Roche-Actavis, Le Plessis Robinson, France), a tPA-derived drug that lacks the fibronectin type I, EGF-like, and kringle 1 domains and thereby only possesses the kringle 2 and catalytic domains. Each tube containing 0.09 U reteplase or 10 μmol/L of the tPA substrate in 1 ml distilled water was closed, and saturated with 75 vol% xenon or medical air as stated above. For the plasmin experiments, 1 ml distilled water was saturated with xenon of 25 to 75 vol% or medical air as described above. Then, the recombinant form of human (ref. 411; American Diagnostica) and murine (ref. IRPLM; Innovative Research) plasmin and their specific chromogenic substrate H--norleucyl-hexahydrotyrosol-lysine-para-nitroanilide diacetate (spectrozyme PL, ref. 251; American Diagnostica) were separately diluted with distilled water previously saturated with xenon of 25 to 75 vol% or medical air up to a final concentration of 40 nmol/L plasmin and 500 μmol/L plasmin substrate. The catalytic efficiency of human and murine tPA (N=3, n=12, per concentration), reteplase (N=3, n=12, per concentration) and plasmin (N=3, n=9, per concentration) was assessed by incubating 50 μL of these serine proteases with 50 μL of their respective substrate at 37°C using a spectrofluorometer microplate reader.

In Vitro Thrombolysis Experiments

Male Sprague–Dawley mature rats weighing 600 to 650 g (n=6) were used. Whole-blood samples of 500 μl volume were transferred in preweighed sterile tubes of 1.5 ml, and incubated at 37°C for 3 h. Saline solution (45 ml) was prepared in a laboratory flask of 50 ml volume whose cap was drilled with two holes of 2 mm in diameter, and saturated for 30 mins with xenon of 25 to 75 vol% (with the remainder being oxygen at 25 vol% and nitrogen as needed) or with medical air at a flow rate of 80 ml/min through microtubing (2 mm in diameter) and a cylinder bubble stone that was introduced down to the bottom of the container through one of the two holes previously drilled. After clot formation and total serum removal, each tube was weighed to determine the clot weight. To reduce variability, we selected blood clots in the same weight range (0.260±0.056 g). Then, each tube was fully filled (including the cap) with saline solution containing 1 μg/ml of tPA in the form of Actilyse previously saturated with xenon of 25 to 75 vol% (n=11 to 16 per group) or medical air (n=8 to 12 per group), quickly closed to avoid xenon desaturation, and incubated at 37°C for an additional 90 min period. Then, the fluid was removed, and the tubes were weighed again to assess the percentage of clot lysis induced by tPA in the presence of medical air or xenon of 25 to 75 vol%. Particular attention was paid to avoid xenon desaturation by maintaining xenon at bubbling in saline solution while filling the tubes containing the blood clots with saline solution saturated with xenon.

MCAO Experiments with Intraischemic Xenon

The effects of xenon of 37.5 to 75 vol% on tPA-induced CBF reperfusion and subsequent reduction of ischemic brain damage were assessed in male Sprague–Dawley rats (n=24) weighing 250 to 275 g. Rats were subjected to MCAO-induced ischemia by administration of an autologous blood clot by the intraluminal method. At 24 h before the animals were subjected to ischemia, a whole caudal blood sample of 200 μl was withdrawn using PE-50 tubing, and allowed to clot at 37°C for 2 h. Then, the clot measuring approximately 0.35 mm in diameter and 120 mm in length was extruded from the catheter into a saline-filled Petri dish and stored at 4°C for 22 h before being used to induce thromboembolic acute ischemic stroke.

On the day of surgery, the rats were anesthetized with 5 vol% isoflurane in medical air, intubated, and ventilated artificially throughout the experiment using an anesthesia ventilator. Isoflurane was reduced at 2 vol% in medical air. Catheters were inserted into the femoral vein to allow injection of tPA or saline solution, and in the femoral artery for continuous monitoring of heart rate, diastolic, systolic, and mean arterial pressures, and for the periodic analysis of blood gases and pH. A midline neck incision was performed, and the right common carotid artery was exposed to perform coagulation of the proximal branches of the external carotid artery. Rats were placed prone in a stereotaxic frame, and a laser-Doppler flowmetry probe was positioned onto the right parietal bone—previously thinned with a saline-cooled dental drill (coordinates 1.7 mm posterior, 5.5 mm lateral from bregma)—to assess changes in CBF and successful induction of cerebral ischemia. The external carotid artery was incised to allow introduction of a PE-10 catheter. Changes in CBF were monitored continuously and expressed as a percentage from the CBF values recorded during a 20 min preocclusion period. A single clot measuring 40 mm in length was collected into additional PE-10 tubing that was connected to the PE-10 catheter previously introduced in the external carotid artery. Then, the PE-10 catheter was directed into the internal carotid artery up to 2 mm after the pterygopalatine—internal carotid artery bifurcation, and the clot was injected in a volume of 50 μL saline solution. To mimic clinical recommendations (Meyer and Rauch, 2000), rats (n=5) showing less than 50% and more than 85% reduction in CBF were strictly excluded from the study design. The interval between the clot collection and injection was less than 5 min. Following a 45 min period of occlusion during which all rats were given medical air, the PE-10 catheter was removed from the internal carotid artery to the external carotid artery and the rats were administered tPA in the form of Actilyse at the dose of 0.9 mg/kg in 1 ml saline solution (10% bolus and 90% perfusion over a 45 min period) with either medical air (n=5) or xenon at 37.5 (n=5), 50 (n=5), and 75 vol% (n=4). Then, after reperfusion has occurred, all animals were given medical air again. Sham-treated rats (n=5) were given medical air alone and saline solution. Rats were maintained normothermic at 37.5±0.5°C using a feedback-controlled thermostatic heating pad connected to a rectal probe all along the experiment. At the end of the experiment, the PE-10 catheter was removed, the external carotid artery stump tied off, and all the incisions were closed. Then, the animals were awaked and allowed moving freely in their home cage with food and water ad libitum for 24 h before being used for the histologic assessment of ischemic brain damage.

MCAO Experiments with Postischemic Xenon

The effects of xenon of 50 vol% on ischemic brain damage were assessed in male Sprague–Dawley rats (n=15) weighing 250 to 275 g. Rats were subjected to MCAO-induced ischemia by administration of an autologous blood clot that was prepared as described above.

Rats were anesthetized with 2 vol% in medical air, and allowed breathing spontaneously throughout the surgical intervention. On day −1 before the main surgical protocol, a catheter was inserted into the femoral vein, tunneled subcutaneously and exteriorized at the neck to allow subsequent injection of tPA or saline solution in awake animals. Then 1 day later, a midline neck incision was performed, and the right common carotid artery was exposed. After coagulation of the branches of the external carotid artery, the external carotid artery was incised to allow introduction of a PE-10 catheter into the internal carotid artery up to 2 mm after the pterygopalatine–internal carotid artery bifurcation, and the clot was injected in a volume of 50 μL saline solution. After 1 min, an additional volume of 50 μL saline solution was injected. The interval between the clot collection and injection was less than 5 mins. The animals were maintained normothermic at 37.5±0.5°C using a feedback-controlled thermostatic heating pad connected to a rectal probe all along the experiment. Following injection of the blood clot, the PE-10 catheter was removed from the internal carotid artery, the external carotid was stump tied off, and the common carotid artery was ligatured. Then, all the incisions were closed, and the animals were awaked and allowed moving freely in their home cage. Following a 45 min period of ischemia, the ligature of the common carotid artery was removed, and the rats were administered tPA in the form of Actilyse at the dose of 0.9 mg/kg in 1 ml saline solution (10% bolus and 90% perfusion over a 45 min period). After tPA infusion had ended, the animals were treated for 3 h in a closed chamber of 10 liter volume, fitted with a viewing window, with either medical air or xenon at 50 vol% at a flow rate of 10 liter/min (n=5 per group). This allows maintenance of carbon dioxide levels at 0.03 vol% and humidity between 60% and 70%. Sham rats (n=4) were administered saline solution and medical air. After treatment, the rats were allowed moving freely in their home cage with food and water ad libitum for 48 h before being used for the histologic assessment of brain hemorrhages, disruption of the blood–brain barrier integrity, and ischemic brain damage.

Gas Pharmacology

Oxygen, nitrogen, and xenon of medical grade were bought from Air Liquide Santé (Paris, France). Gas mixtures containing 75 vol% nitrogen and 25 vol% oxygen (medical air) or xenon at 25 to 75 vol%, with the remainder being oxygen at 25 vol% completed with nitrogen when necessary, were obtained using computer-assisted calibrated flowmeters and gas analyzers, and used or administered as described above according to a masked and randomized procedure.

Assessment of Infarct Size

Rats were killed by decapitation under isoflurane anesthesia, 24 h (intraischemic experiments) or 48 h (postischemic experiments) after the onset of brain ischemia, which are time conditions that have been shown to allow obtaining consolidated and reliable infarct volume in several comparative studies using magnetic resonance imaging, triphenyltetrazolium chloride, thionin, and neuronal nuclei immunochemical staining techniques (Henninger et al, 2006; Haelewyn et al, 2008a). Then, the brain was rapidly removed, frozen in isopentane, and placed at −80°C. Coronal brain sections (20 μm) were cryostat-cut, mounted on slides, stained with thionin, and digitized on a computer. Volumes of brain infarction were analyzed with an image analyzer (ImageJ software; Scion corp., Frederick, MD, USA). The lesion areas were delineated by the pallor of staining in the necrotic tissue compared with the surrounding healthy tissue; then, infarction volumes were calculated by integration of the infarct surfaces over the whole brain, corrected for tissue edema, and expressed in mm3 of infarction volume.

Assessment of Brain Hemorrhages and Disruption of the Blood–Brain Barrier Integrity

At 48 h after the onset of brain ischemia, rats treated with postischemic xenon at 50 vol% were administered Evans blue intravenously (4% in 1 mL saline solution over a 30 sec period). The brain was removed and sliced as described above. While cutting the unstained brain still mounted on the cryostat, macrophotographs were taken, digitized on a computer, and analyzed using an image analyzer (ImageJ software; Scion Corp.). Disruption of the blood–brain barrier integrity was assessed as follows: the volume of the brain showing Evans blue extravasation was calculated, multiplied by the extravasation optical density, and then divided by the total infarction volume. Brain hemorrhages were delineated as blood evident at the macroscopic level. For each rat, the ratio of hemorrhagic transformation was calculated by dividing the total surface of hemorrhages by the total surface of infarction in the slices that showed hemorrhages. Finally, coronal sections were used to assess infarct size as described above.

Statistical Analysis

Data are given as mean±s.e.m. All data were analyzed using the Kruskal–Wallis nonparametric analysis of variance and/or the post hoc Mann–Whitney nonparametric unpaired U-test. The level of significance was set at P<0.05.

Results

Modeling of the Binding Site of Xenon within the S1 Pocket of tPA and Plasmin

First, we investigated whether xenon could bind to tPA. Because serine proteases share a similar structure around their identical catalytic triad and can be therefore easily structurally superposed, we performed modeling of the possible binding of xenon to the catalytic domain of tPA and of plasmin by superposing the crystallographic structure of elastase in complex with xenon to the crystallographic structure of the human tPA catalytic domain in complex with the bis-benzamidine inhibitor tPA Stop and to the crystallographic structure of the human plasmin catalytic domain. As shown in Figure 1A, the structure of the catalytic domain of tPA in complex with tPA Stop reveals that an extremity of this inhibitor binds within the specificity pocket S1. The xenon atom fits within the S1 pocket of tPA at the position of one extremity of the tPA inhibitor tPA Stop. Similarly, the xenon atom fits within the S1 pocket of plasmin (Figure 1B). These data suggest that xenon could reduce the catalytic efficiency of tPA and plasmin by binding within the S1 specificity pocket of these enzymes.

Figure 1
(A) Modeling of xenon binding to tissue-plasminogen activator (tPA). Xenon is predicted to bind within the primary specificity pocket S1 where the tPA inhibitor tPA Stop binds. (B) Modeling of xenon binding within the primary specificity pocket S1 of ...

Xenon Dose Dependently Inhibits the Catalytic Efficiency of tPA In Vitro

Next, we investigated the effects of xenon on the catalytic efficiency of human and murine tPA by the initial rate method. The recombinant form of human and murine tPA, and their specific chromogenic substrate were diluted in distilled water and saturated with xenon of 25 to 75 vol% (with the remainder being 25 vol% oxygen completed with nitrogen when necessary) or medical air composed of 25% oxygen and 75% nitrogen. The addition of xenon into the medium inhibited the catalytic efficiency of human and murine tPAs in a dose-dependent manner. Although xenon at 25 vol% had no effect, xenon at higher concentrations of 37.5, 50, and 75 vol% inhibited the catalytic efficiencies of human and murine tPAs in a similar manner (human tPA: P<0.0001, IC50=34.85±0.26 vol% murine tPA: P<0.0001, IC50=37.39±0.22 vol% Figure 1C). We also examined the effect of xenon at 75 vol% on the catalytic efficiency of reteplase, a tPA-derived drug that lacks the fibronectin type I, EGF-like, and kringle 1 domains and thereby possesses only the kringle 2 and catalytic domains. We found that xenon inhibited the catalytic efficiency of reteplase in a similar manner than that of tPA (P<0.0001; see inset in Figure 1C). Together with the modeling of the binding site of xenon to tPA described above, these data strongly support that xenon would exert its effect by binding within the catalytic domain of tPA and tPA-derived drugs. In addition, similar inhibiting effects of xenon were obtained with human and murine plasmin and their specific chromogenic substrate. Although xenon at concentrations of 50 and 75 vol% inhibited the catalytic efficiency of both human and murine plasmin, xenon at 37.5 vol% inhibited only human but not murine plasmin (human plasmin: P<0.0001 to 0.01, IC50=39.97±0.94 vol% murine plasmin: P<0.0001 to 0.001, IC50=45.45±0.003 vol% Figure 1D). Taken together, all of these data suggest that xenon could reduce the thrombolytic efficiency of tPA (and plasmin) in a dose-dependent manner.

Xenon Dose Dependently Inhibits the Thrombolytic Efficiency of tPA In Vitro

Next, to examine whether xenon may inhibit tPA-induced thrombolysis, we studied the effect of this noble gas at concentrations of 25 to 75 vol% on the thrombolytic efficiency of tPA in whole-blood samples drawn from male mature rats. After clot formation and total serum removal, each tube was filled with saline solution containing a clinically relevant concentration of 1 μl/ml of tPA in the form of Actilyse previously saturated with xenon or medical air. We found that xenon at concentrations of 37.5, 50, and 75 vol% reduced tPA-induced thrombolysis in a dose-dependent manner by 45%, 51%, and 75% as compared with control samples saturated with medical air (P<0.0001; IC50=35.28±5.77 vol% Figure 2A). No evidence of clot lysis was found in whole-blood samples treated with saline solution and medical air. Scatter plot analyses of the inhibiting effects of xenon on both tPA-induced thrombolysis and the catalytic efficiency of tPA and plasmin (Figure 2B) indicate that the inhibiting effects of xenon on thrombolysis are likely to be due to its effects on tPA (R2=0.9655) rather than on plasmin (R2=0.6365) that is ultimately responsible for thrombolysis.

Figure 2
(A) Dose-dependent inhibition by xenon of tissue-type plasminogen activator (tPA)-induced brain damage in vitro. Xenon of 25 to 75 vol% inhibits tPA-induced thrombolysis in blood clots obtained from whole-blood samples (IC50=35.28±5.77 ...

In Vivo Intraischemic Xenon Inhibits tPA-Induced Thrombolysis and Subsequent Reduction of Brain Damage

To examine whether xenon may actually decrease thrombolysis in vivo, we investigated the effects of intraischemic xenon on tPA-induced CBF reperfusion and subsequent reduction of brain damage in rats subjected to thromboembolic brain ischemia. At 45 mins after the onset of brain ischemia, rats were administered tPA in the form of Actilyse. In line with clinical studies in rtPA-treated patients (Christou et al, 2000; Delgado-Mederos et al, 2007), we found that rats injected with tPA at 0.9 mg/kg - a dose shown to be as clinically relevant than that of 10 mg/kg that is often used in rodents (Haelewyn et al, in press)–exhibited full cerebral reperfusion 40 mins after tPA injection. All physiologic parameters including arterial fell within normal range values (Table 1). Sham rats treated with saline solution and medical air showed no CBF reperfusion and had a volume of ischemic brain damage of 391±33 mm3 as evaluated 24 h after the onset of ischemia, a time condition shown to allow obtaining consolidated and reliable infarct volumes in previous method studies (see Materials and methods). As expected, control rats treated with tPA and medical air exhibited full CBF reperfusion (P<0.01) and a reduced volume of brain damage of 186±11 mm3 as compared with sham-treated rats (P<0.01). Contrasting with this latter finding, we found that rats treated with tPA and intraischemic xenon exhibited a dose-dependent reduction in CBF reperfusion. Xenon at 37.5, 50, and 75 vol% reduced CBF reperfusion by 51%±11% (P<0.05), 59%±12% (P<0.05), and 86%±9% (P<.02), respectively, as compared with controls (Figures 3A and 3B). In addition, as it might be expected from the effects of intraischemic xenon on CBF, we found that rats treated with tPA and intraischemic xenon at 37.5, 50, and 75 vol% had greater volumes of brain damage of 317±25 mm3 (P<0.01), 330±63 mm3 (P<0.01), and 383±23 mm3 (P<0.02) than control animals treated with tPA and medical air (Figures 3C–3E).

Figure 3
(A and B) Time-course (A) and cumulated (B) dose-dependent inhibitory effects of intraischemic xenon on tissue-type plasminogen activator (tPA)-induced thrombolysis. Rats treated with tPA and intraischemic xenon at 37.5 vol% ([open triangle]), 50 vol% ...
Table 1
Mean values of arterial PO2, SaO2, PCO2, pH, and body temperature in rats treated with medical air or xenon at 37.5 , 50 , or 75 vol%

In Vivo Postischemic Xenon Suppresses Ischemic Brain Damage and Hemorrhages

Aside its beneficial thrombolytic action, it is well known that tPA also possesses proteolytic properties that can produce adverse effects, particularly disruption of the blood–brain barrier integrity and brain hemorrhage. Therefore, because xenon can inhibit tPA, we studied the effects of postischemic xenon on brain hemorrhages, disruption of the blood–brain barrier integrity, and ischemic brain damage in rats subjected to thromboembolic brain ischemia. At 45 mins after the onset of brain ischemia, rats were administered intravenous tPA in the form of Actilyse at 0.9 mg/kg. Then, 15 min after tPA infusion has ended rats were treated with either medical air (controls) or xenon at 50 vol%, a concentration shown to provide maximal postischemic neuroprotection with no risk of potentially adverse effects in rats subjected to mechanical MCAO (David et al, 2008). To allow comparison with this former study, we performed histologic analysis 48 h (instead of 24 h) after the onset of brain ischemia, a time condition shown to allow obtaining reliable infarct volumes using thionin staining (Haelewyn et al, 2008a). Sham rats treated with saline solution and medical air had a mean volume of ischemic brain damage of 388±42 mm3, and further exhibited brain hemorrhages and disruption of the blood–brain barrier. Compared with sham-treated rats, control rats treated with tPA and medical air had similar levels of brain hemorrhages and disruption of the blood–brain barrier integrity, despite reduced volume brain damage (126±26 mm3, P<0.01; Figures 4A–4D). Contrasting with these findings, we found that rats treated with tPA and postischemic xenon at 50 vol% exhibited virtual suppression of both brain damage (10.9±1.4 mm3, P<0.01) and disruption of the blood–brain barrier (P<0.01), and further showed no brain hemorrhages (P<0.01) as compared with sham-treated rats and control rats treated with tPA and medical air (Figures 4A–4D).

Figure 4
(A) Effect of postischemic xenon at 50 vol% on middle cerebral artery occlusion (MCAO)-induced brain damage. (B) Typical examples of brain damage in rats treated with saline and medical air, tissue-type plasminogen activator (tPA) and medical ...

Discussion

Over the past decade, preclinical evidence in animal models of mechanical acute brain ischemia and hypoxia–ischemia has proven that xenon may be a promising agent with effective neuroprotective properties and no adverse effects when administered at subanesthetic concentrations (David et al, 2003, 2008; Homi et al, 2003; Ma et al, 2003). However, none has previously investigated the effects of xenon in models of thromboembolic stroke and animals treated with tPA, the only approved therapy of acute ischemic stroke today.

Multiple studies have indicated a pleiotropic role for tPA that is highly dependent on the compartment on which tPA exerts its effect (Yepes et al, 2009). tPA is found in the intravascular space, the neurovascular unit (the interface between the blood and the brain), and the brain parenchyma. In the intravascular space, the most important role of tPA is to promote thrombolysis. Therefore, recombinant tPA is used for the treatment of patients who have acute ischemic stroke. However, beside this beneficial effect, there is a growing body of evidence from human and animal studies that tPA also has deleterious proteolytic (hemorrhaging) and proneurotoxic effects in the neurovascular unit and the brain parenchyma. tPA favors cerebral-ischemia-induced disruption of the blood–brain barrier and hemorrhagic transformation (The NINDS rtPA Stroke Study Group, 1995; Lees, 1999; Kidwell et al, 2008), and further increases ischemia-induced neuronal death (Tsirka et al, 1995; Wang et al, 1998).

Here, we show that xenon is an inhibitor of the catalytic efficiency of tPA that produces either adverse antithrombolytic or beneficial antiproteolytic (antihemorrhaging) effects depending on whether it is administered together with tPA during the ischemic period or after tPA therapy during the postischemic period. Thus, when administered during the ischemic period, we showed that xenon dose dependently reduces tPA-induced CBF reperfusion and thereby decreases and even suppresses the benefits of tPA therapy in term of reduction of ischemic brain damage. This is at odds with previous findings that have reported effective neuroprotection with intraischemic xenon in rodents subjected to mechanical brain ischemia (Homi et al, 2003). However, although xenon was administered before and during the entire ischemic period in this former study, in the present study intraischemic xenon was only administered after a 45 min period of ischemia according a more clinically relevant model of acute thromboembolic stroke whose results clearly indicate that the antithrombolytic properties of intraischemic xenon overcome its neuroprotective action. Alternatively, contrasting with the adverse effects of intraischemic xenon, we found that postischemic xenon virtually suppresses ischemic brain damage and tPA-induced brain hemorrhages and disruption of the blood–brain barrier. Because tPA is known to increase excitotoxin and ischemia-induced neuronal degeneration (Tsirka et al, 1995; Wang et al, 1998) by potentiating the NMDA receptor signaling (Nicole et al, 2001), it is likely that the ability of xenon at inhibiting the catalytic efficiency of tPA may have also contributed to its neuroprotective action in addition of its well-documented pharmacologic properties at the NMDA glutamatergic receptor and other additional neuronal targets and signaling pathways of potentially neuroprotective interest (Franks et al, 1998; Yamakura and Harris, 2000; Gruss et al, 2004; Dinse et al, 2005; Colloc'h et al, 2007; Dickinson et al, 2007; Cattano et al, 2008; Luo et al, 2008; Bantel et al, 2009). Taken together, these findings confirm and extend previous data on postischemic xenon neuroprotection (David et al, 2008) by showing that postischemic xenon has unique neuroprotective and antiproteolytic properties that allow blocking both tPA toxicity and ischemic brain damage, conditions shown to allow obtaining full neurologic recovery (Haelewyn et al, 2008c).

Compatibility between treatments is a necessary and inalienable condition to allow combining multiple therapeutic strategies. Taken together, our findings indicate that the use of xenon as a neuroprotective agent for treating acute ischemic stroke should obey well-defined guidelines. Because xenon dose dependently inhibits the thrombolytic properties of tPA, we assume as an ethical principle of caution that xenon, or at least xenon at concentrations around or higher than 35 to 40 vol%, should not be administered in acute ischemic stroke patients before reperfusion has occurred due to the risk of inhibiting the benefits of tPA therapy or the possible occurrence of endogenous fibrinolysis in patients who are not or cannot be treated with tPA. Support for this is the fact that xenon at such concentrations strongly inhibits human plasmin in addition of its effects on tPA (see Figure 1). Then, once CBF would be restored spontaneously or following tPA-induced thrombolysis, we propose that xenon may be used, even at high concentrations above 35 to 40 vol% if necessary, both to reduce the risk of tPA-induced brain hemorrhages and disruption of the blood–brain barrier and to provide efficient neuroprotection. However, in order not to favor reocclusion, a phenomenon shown to occur in 10% to 15% of ischemic stroke patients after tPA-induced reperfusion (mean reocclusion time: 41±43 min after tPA bolus) (Rubiera et al, 2005), the use of xenon at concentrations that reduce the thrombolytic properties of tPA could require to be delayed according to a benefit–risk medical evaluation for the patient. If such, this delay should not hamper the neuroprotective potential of xenon, suggested to have a possible therapeutic window of about 6 to 8 h (David et al, 2008).

During the past decade, studies on the manipulation of various inhaled inert gases have led to the conclusion that the use of such agents may be a promising therapy for treating acute ischemic stroke. Apart xenon, nitrous oxide that shares many pharmacologic properties with xenon (Jevtović-Todorović et al, 1998; Yamakura and Harris, 2000; David et al, 2006) has been also shown to possess neuroprotective action in rats subjected to excitotoxic insults or mechanical MCAO-induced ischemia (Jevtović-Todorović et al, 1998; David et al, 2003; Abraini et al, 2005; Haelewyn et al, 2008a, 2008b). Importantly, in line with previous findings that have shown that nitrous oxide increases disruption of the blood–brain barrier (Johansson and Linder, 1978; Remsen et al, 1999), results from our laboratory in rats subjected to the same experimental protocols than those used in this study indicate that postischemic nitrous oxide, unlike postischemic xenon, does not inhibit tPA-induced brain hemorrhages and disruption of the blood–brain barrier, and does not reduce ischemic brain damage in a similar amplitude than postischemic xenon (B Haelewyn et al, unpublished observations). This provides a definitive advantage to xenon compared with nitrous oxide as a future possible neuroprotectant, and further indicates that the subtle molecular mechanisms of action of xenon and nitrous oxide differ from each other.

In conclusion, this study provides multiple evidences that xenon inhibits tPA. With no doubt, this constitutes an important factor that should be considered in future clinical trials and possible therapeutic use of xenon in acute ischemic stroke patients. Although intraischemic xenon should be avoided due to the risk of inhibiting the benefits of tPA therapy, postischemic xenon could constitute a golden standard with unique neuroprotective and antiproteolytic (antihemorrhaging) properties allowing blocking both excitotoxic processes and tPA toxicity. Beside acute ischemic stroke, xenon has been also proven to be efficient at providing tissue protection in other types of ischemia, such as heart attack and renal ischemia (Preckel et al, 2000; Hartlage et al, 2004; Ma et al, 2009). Therefore, similar considerations should be taken into account for using xenon in combination with tPA or tPA-derived drugs for the treatment of such ischemic diseases.

Acknowledgments

This research was supported by NNOXe Pharmaceuticals, the French Ministry of Defence, the University of Caen, and the CNRS. HND and JHA performed the catalytic activity assay experiments that allowed discovering the inhibiting effects of xenon on the catalytic efficiency of tPA and plasmin. BH performed the in vitro and in vivo experiments on the effects of xenon on tPA-induced thrombolysis and MCAO-induced brain damage. NC performed the modeling of the binding of xenon on tPA and plasmin. JJR and JHA designed the experiments. JHA wrote the paper.

Footnotes

Disclosure/conflict of interest

HND and BH are NNOXe Pharmaceuticals scientists. NNOXe Pharmaceuticals has patent applications on the use of xenon and other gases for treating ischemia. The authors declared that no other competing interests exist.

References

  • Abraini JH, David HN, Lemaire M. Potentially neuroprotective and therapeutic properties of nitrous oxide and xenon. Ann NY Acad Sci. 2005;1053:289–300. [PubMed]
  • Bantel C, Maze M, Trapp S. Neuronal preconditioning by inhalational anesthetics: evidence for the role of plasmalemmal adenosine triphosphate-sensitive potassium channels. Anesthesiology. 2009;110:986–995. [PMC free article] [PubMed]
  • Cattano D, Valleggi S, Ma D, Kastsiuchenka O, Abramo A, Sun P, Cavazzana AO, Natale G, Maze M, Giunta F. Xenon induces transcription of ADNP in neonatal rat brain. Neurosci Lett. 2008;440:217–221. [PubMed]
  • Christou I, Alexandrov AV, Burgin WS, Wojner AW, Felberg RA, Malkoff M, Grotta JC. Timing of recanalization after tissue plasminogen activator therapy determined by transcranial Doppler correlates with clinical recovery from ischemic stroke. Stroke. 2000;31:1812–1816. [PubMed]
  • Colloc'h N, Sopkova de Oliveira Santos J, Retailleau P, Vivarès D, Bonneté F, Langlois d′Estainto B, Gallois B, Brisson A, Risso JJ, Lemaire M, Prangé T, Abraini JH. Protein crystallography under xenon and nitrous oxide pressure: comparison with in vivo pharmacology studies and implications for the mechanism of inhaled anesthetic action. Biophys J. 2007;92:217–224. [PubMed]
  • David HN, Ansseau M, Lemaire M, Abraini JH. Nitrous oxide and xenon prevent amphetamine-induced carrier-mediated dopamine release in a memantine-like fashion and protect against behavioral sensitization. Biol Psychiatry. 2006;60:49–57. [PubMed]
  • David HN, Haelewyn B, Rouillon C, Lecoq M, Chazalviel L, Apiou G, Risso JJ, Lemaire M, Abraini JH. Neuroprotective effects of xenon: a therapeutic window of opportunity in rats subjected to transient cerebral ischemia. FASEB J. 2008;22:1275–1286. [PubMed]
  • David HN, Leveille F, Chazalviel L, MacKenzie ET, Buisson A, Lemaire M, Abraini JH. Reduction of ischemic brain damage by nitrous oxide and xenon. J Cereb Blood Flow Metab. 2003;23:1168–1173. [PubMed]
  • Davis SM, Lees KR, Albers GW, Diener HC, Markabi S, Karlsson G, Norris J. Selfotel in acute ischemic stroke: possible neurotoxic effects of an NMDA antagonist. Stroke. 2000;31:347–354. [PubMed]
  • Delgado-Mederos R, Rovira A, Alvarez-Sabín J, Ribó M, Munuera J, Rubiera M, Santamarina E, Maisterra O, Delgado P, Montaner J, Molina CA. Speed of tPA-induced clot lysis predicts DWI lesion evolution in acute stroke. Stroke. 2007;38:955–960. [PubMed]
  • Dickinson R, Peterson BK, Banks P, Simillis C, Martin JC, Valenzuela CA, Maze M, Franks NP. Competitive inhibition at the glycine site of the N-methyl--aspartate receptor by the anesthetics xenon and isoflurane: evidence from molecular modeling and electrophysiology. Anesthesiology. 2007;107:756–767. [PubMed]
  • Dinse A, Föhr KJ, Georgieff M, Beyer C, Bulling A, Weigt HU. Xenon reduces glutamate-, AMPA-, and kainate-induced membrane currents in cortical neurones. Br J Anaesth. 2005;94:479–485. [PubMed]
  • Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci. 1999;22:391–397. [PubMed]
  • Franks NP, Dickinson R, de Sousa SL, Hall AC, Lieb WR. How does xenon produce anaesthesia. Nature. 1998;396:324. [PubMed]
  • Gruss M, Bushell TJ, Bright DP, Lieb WR, Mathie A, Franks NP. Two-pore-domain K+ channels are a novel target for the anesthetic gases xenon, nitrous oxide, and cyclopropane. Mol Pharmacol. 2004;65:443–452. [PubMed]
  • Haelewyn B, Alix P, Maubert E, Abraini JH. NMDA-induced striatal brain damage and time-dependence reliability of thionin staining in rats. J Neurosci Methods. 2008a;168:479–482. [PubMed]
  • Haelewyn B, David HN, Rouillon C, Chazalviel L, Lecocq M, Risso JJ, Lemaire M, Abraini JH. Neuroprotection by nitrous oxide: facts and evidence. Crit Care Med. 2008b;36:2651–2659. [PubMed]
  • Haelewyn B, Risso JJ, Abraini JH. Human recombinant tissue-plasminogen activator (Alteplase): why not to used the “human” dose for stroke studies in rats J Cereb Blood Flow Metab(in press) [PMC free article] [PubMed]
  • Haelewyn B, Rouillon C, Risso JJ, Abraini JH. Functional (neurologic) recovery following transient focal cerebral ischemia in the rat requires at least 80% of ipsilateral cortical and subcortical integrity. Exp Neurol. 2008c;213:238–240. [PubMed]
  • Hartlage MA, Berendes E, Van Aken H, Fobker M, Theisen M, Weber TP. Xenon improves recovery from myocardial stunning in chronically instrumented dogs. Anesth Analg. 2004;99:655–664. [PubMed]
  • Henninger N, Küppers-Tiedt L, Sicard KM, Günther A, Schneider D, Schwab S. Neuroprotective effect of hyperbaric oxygen therapy monitored by MR-imaging after embolic stroke in rats. Exp Neurol. 2006;201:316–323. [PubMed]
  • Homi HM, Yokoo N, Ma D, Warner DS, Franks NP, Maze M, Grocott HP. The neuroprotective effect of xenon administration during transient middle cerebral artery occlusion in mice. Anesthesiology. 2003;99:876–881. [PubMed]
  • Jevtović-Todorović V, Todorović SM, Mennerick S, Powell S, Dikranian K, Benshoff N, Zorumski CF, Olney JW. Nitrous oxide (laughing gas) is an NMDA antagonist, neuroprotectant and neurotoxin. Nat Med. 1998;4:460–463. [PubMed]
  • Johansson BB, Linder LE. Cerebrovascular permeability to protein in the rat during nitrous oxide anaesthesia at various blood pressure levels. Acta Anaesthesiol Scand. 1978;22:463–466. [PubMed]
  • Kaur J, Zhao Z, Klein GM, Lo EH, Buchan AM. The neurotoxicity of tissue plasminogen activator. J Cereb Blood Flow Metab. 2004;24:945–963. [PubMed]
  • Kidwell CS, Latour L, Saver JL, Alger JR, Starkman S, Duckwiler G, Jahan R, Vinuela F, Kang DW, Warach S. Thrombolytic toxicity: blood brain barrier disruption in human ischemic stroke. Cerebrovasc Dis. 2008;25:338–343. [PubMed]
  • Lee JM, Zipfel GJ, Choi DW. The changing landscape of ischaemic brain injury mechanisms. Nature. 1999;399:A7–14. [PubMed]
  • Lees KR. ECASS-II: intravenous alteplase in acute ischaemic stroke. European Co-operative Acute Stroke Study-II. Lancet. 1999;353:65–66. [PubMed]
  • Lijnen HR, Hoylaerts M, Collen D. Isolation and characterization of a human plasma protein with affinity for the lysine binding sites in plasminogen. Role in the regulation of fibrinolysis and identification as histidine-rich glycoprotein. J Biol Chem. 1980;255:10214–10222. [PubMed]
  • Lo EH, Moskowitz MA, Jacobs TP. Exciting, radical, suicidal: how brain cells die after stroke. Stroke. 2005;36:189–192. [PubMed]
  • Luo Y, Ma D, Ieong E, Sanders RD, Yu B, Hossain M, Maze M. Xenon and sevoflurane protect against brain injury in a neonatal asphyxia model. Anesthesiology. 2008;109:782–789. [PubMed]
  • Ma D, Lim T, Xu J, Tang H, Wan Y, Zhao H, Hossain M, Maxwell PH, Maze M. Xenon preconditioning protects against renal ischemic-reperfusion injury via HIF-1alpha activation. J Am Soc Nephrol. 2009;20:713–720. [PubMed]
  • Ma D, Yang H, Lynch J, Franks NP, Maze M, Grocott HP. Xenon attenuates cardiopulmonary bypass-induced neurologic and neurocognitive dysfunction in the rat. Anesthesiology. 2003;98:690–698. [PubMed]
  • Meyer JS, Rauch G. Why emergency XeCT-CBF should become routine in acute ischemic stroke before thrombolytic therapy. Keio J Med. 2000;49 (Suppl 1:A25–A28. [PubMed]
  • Nicole O, Docagne F, Ali C, Margaill I, Carmeliet P, MacKenzie ET, Vivien D, Buisson A. The proteolytic activity of tissue-plasminogen activator enhances NMDA receptor-mediated signaling. Nat Med. 2001;7:59–64. [PubMed]
  • Olney JW, Labruyere J, Wang G, Wozniak DF, Price MT, Sesma MA. NMDA antagonist neurotoxicity: mechanism and prevention. Science. 1991;254:1515–1518. [PubMed]
  • Parry MA, Fernandez-Catalan C, Bergner A, Huber R, Hopfner KP, Schlott B, Gührs KH, Bode W. The ternary microplasmin—staphylokinase–microplasmin complex is a proteinase–cofactor–substrate complex in action. Nat Struct Biol. 1998;5:917–923. [PubMed]
  • Preckel B, Mullenheim J, Moloschavij A, Thamer V, Schlack W. Xenon administration during early reperfusion reduces infarct size after regional ischemia in the rabbit heart in vivo. Anesth Analg. 2000;91:1327–1332. [PubMed]
  • Remsen LG, Pagel MA, McCormick CI, Fiamengo SA, Sexton G, Neuwelt EA. The influence of anesthetic choice, PaCO2, and other factors on osmotic blood–brain barrier disruption in rats with brain tumor xenografts. Anesth Analg. 1999;88:559–567. [PubMed]
  • Renatus M, Bode W, Huber R, Stürzebecher J, Prasa D, Fischer S, Kohnert U, Stubbs MT. Structural mapping of the active site specificity determinants of human tissue-type plasminogen activator. Implications for the design of low molecular weight substrates and inhibitors. J Biol Chem. 1997;272:21713–21719. [PubMed]
  • Rubiera M, Alvarez-Sabín J, Ribo M, Montaner J, Santamarina E, Arenillas JF, Huertas R, Delgado P, Purroy F, Molina CA. Predictors of early arterial reocclusion after tissue plasminogen activator-induced recanalization in acute ischemic stroke. Stroke. 2005;36:1452–1456. [PubMed]
  • Schiltz M, Fourme R, Broutin I, Prangé T. The catalytic site of serine proteinases as a specific binding cavity for xenon. Structure. 1995;3:309–316. [PubMed]
  • The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group Tissue plasminogen activator for acute ischemic stroke. N Engl J Med. 1995;333:1581–1587. [PubMed]
  • The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group Intracerebral hemorrhage after intravenous t-PA therapy for ischemic stroke. Stroke. 1997;28:2109–2118. [PubMed]
  • Tsirka SE, Gualandris A, Amaral DG, Strickland S. Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator. Nature. 1995;377:340–344. [PubMed]
  • Wang YF, Tsirka SE, Strickland S, Stieg PE, Soriano SG, Lipton SA. Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice. Nat Med. 1998;4:228–231. [PubMed]
  • Yamakura T, Harris RA. Effects of gaseous anesthetics nitrous oxide and xenon on ligand-gated ion channels. Comparison with isoflurane and ethanol. Anesthesiology. 2000;93:1095–1101. [PubMed]
  • Yepes M, Roussel BD, Ali C, Vivien D. Tissue-type plasminogen activator in the ischemic brain: more than a thrombolytic. Trends Neurosci. 2009;32:48–55. [PubMed]

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