Interactions between and Tissue Plasminogen Activator in a Rat Model of Thromboembolic Stroke

Benoît Haelewyn, Ph.D.,* He´le` ne N. David, Ph.D.,† Nathalie Colloc’h, Ph.D., D.Sc.,‡ Denis G. Colomb, Jr., Ph.D.,§ Jean-Jacques Risso, Ph.D., D.Sc.,ʈ Jacques H. Abraini, Ph.D., D.Sc., Psy.D.# Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/115/5/1044/452771/0000542-201111000-00027.pdf by guest on 25 September 2021

ABSTRACT What We Already Know about This Topic • Whether nitrous oxide, like , reduces of ischemic brain Background: Preclinical evidence in rodents has suggested damage in the setting of thrombolysis for thromboembolic that inert gases, such as xenon or nitrous oxide, may be prom- stroke is unknown. ising neuroprotective agents for treating acute ischemic stroke. This has led to many thinking that clinical trials could be initiated in the near future. However, a recent study has What This Article Tells Us That Is New shown that xenon interacts with tissue-type plasminogen ac- • In rats, when administrated during the ischemic period, nitrous tivator (tPA), a well-recognized approved therapy of acute oxide dose-dependently inhibited tPa-induced thrombolysis and subsequent reduction of ischemic brain damage. How- * Research Engineer and Head, Universite´ de Caen - Basse Nor- ever, in contrast to xenon, postischemic nitrous oxide in- mandie, Centre Universitaire de Ressources Biologiques, Caen, France. creased brain hemorrhage and barrier dysfunction. † Research Scientist, Universite´ Laval, Centre Hospitalier Universitaire Affilie´Hoˆtel-Dieu Le´vis, Le´vis, Que´bec, Canada; Universite´ Laval, Cen- tre de Recherche Universite´ Laval Robert-Giffard, Que´bec, Que´bec, Canada. ‡ Senior Research Scientist, Universite´ de Caen - Basse Nor- ischemic stroke. Although intraischemic xenon inhibits tPA- mandie, ERTi 1083, UMR 6232, Caen, France; Centre National de la induced thrombolysis and subsequent reduction of brain Recherche Scientifique, UMR 6232, Caen, France. § Lieutenant, United States Medical Services Corps, Navy Experimental Diving Unit, Panama damage, postischemic xenon virtually suppresses both isch- City, Florida. ࿣ Research Director, Institut de Recherche Biome´dicale emic brain damage and tPA-induced brain hemorrhages and des Arme´es, Antenne Toulon, Toulon, France. # Professor of Neuro- disruption of the blood–brain barrier. The authors investi- sciences, Universite´ de Caen - Basse Normandie, ERTi 1083, UMR 6232; Centre National de la Recherche Scientifique, UMR 6232; Universite´ gated whether nitrous oxide could also interact with tPA. Laval, Centre Hospitalier Universitaire Affilie´Hoˆtel-Dieu Le´vis; Methods: The authors performed molecular modeling of Universite´ Laval, Centre de Recherche Universite´ Laval Robert- nitrous oxide binding on tPA, characterized the concentra- Giffard, Que´bec. tion-dependent effects of nitrous oxide on tPA enzymatic Received from Universite´ de Caen - Basse Normandie, ERTi 1083, UMR 6232, Caen, France; Centre National de la Recherche Scientifique, and thrombolytic activity in vitro, and investigated the effects UMR 6232, Caen, France; Universite´ Laval, Centre Hospitalier Univer- of intraischemic and postischemic nitrous oxide in a rat sitaire Affilie´Hoˆtel-Dieu Le´vis, Le´vis, Que´bec, Canada; and Universite´ model of thromboembolic acute ischemic stroke. Laval, Centre de Recherche Universite´ Laval Robert-Giffard, Que´bec, Que´bec, Canada. Submitted for publication March 27, 2011. Accepted Results: The authors demonstrate nitrous oxide is a tPA inhib- for publication July 22, 2011. Supported by Universite´ de Caen, Caen, itor, intraischemic nitrous oxide dose-dependently inhibits tPA- France; the Centre National de la Recherche Scientifique, Paris, France; induced thrombolysis and subsequent reduction of ischemic the Direction Ge´ne´rale de l’Armement, Paris, France; and NNOXe Pharmaceuticals, Quebec City, Quebec, Canada. NNOXe Pharmaceu- brain damage, and postischemic nitrous oxide reduces ischemic ticals (Quebec City, Beubec, Canada) has patent applications on the brain damage, but in contrast with xenon, it increases brain use of inert gases and tPA for treating ischemia. The authors have hemorrhages and disruption of the blood–brain barrier. declared that no financial conflicts of interests exist. Lt. Colomb is a military service member, and this work was prepared as part of his Conclusions: In contrast with previous studies using me- official duties. The views expressed in this article are those of the chanical acute stroke models, these data obtained in a clini- author and do not reflect the official policy or position of the Depart- cally relevant rat model of thromboembolic stroke indicate ment of the Navy, Department of Defense, or the U.S. Government. that nitrous oxide should not be considered a good candidate Address correspondence to Dr. Abraini: ERTi 1083, UMR 6232, Universite´ de Caen, CNRS, CEA, Boulevard Henri Becquerel, Centre agent for treating acute ischemic stroke compared with xenon. CYCERON, 14074 Caen cedex, France, or Centre de Recherche Universite´ Laval Robert-Giffard, Quebec, Quebec, Canada. abraini@ cyceron.fr or [email protected]. Information on pur- CUTE ischemic stroke through thromboembolism re- chasing reprints may be found at www.anesthesiology.org or on the A mains a major cause of acute mortality and chronic masthead page at the beginning of this issue. ANESTHESIOLOGY’s morbidity. The most common approved therapy of acute articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue. ischemic stroke is thrombolysis by the recombinant form of Copyright © 2011, the American Society of Anesthesiologists, Inc. Lippincott the protease human tissue-type plasminogen activator 1 Williams & Wilkins. Anesthesiology 2011; 115:1044–53 (tPA). Although recombinant tPA has benefited ischemic

Anesthesiology, V 115 • No 5 1044 November 2011 CRITICAL CARE MEDICINE

stroke patients if given within 4.5 h of symptoms,2–4 adverse used for surgery, all rats were housed individually. Light was effects, primarily brain hemorrhages and disruption of the maintained on a light–dark reverse cycle, with lights on from blood–brain barrier integrity through tPA-mediated proteo- 8:00 PM to 8:00 AM. lytic processes, have been reported.3–7 In contrast, strategies of by the use of receptor an- Modeling of the Binding Site of Nitrous Oxide within the tagonists to counteract ischemia-induced overstimulation of S1 Pocket of tPA the N-methyl-D-aspartate (NMDA) glutamatergic receptors, We performed the structural superposition of the catalytic whose postsynaptic activation is known as a critical event in domain of tPA (Protein Data Bank entry: 1A5H)30 to elas- neuronal death induced by acute ischemic stroke,8–10 have not tase in complex with nitrous oxide with the software PyMOL been proven efficient in patients experiencing ischemic insults,

(DeLano Scientific, San Carlos, CA). The root mean square Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/115/5/1044/452771/0000542-201111000-00027.pdf by guest on 25 September 2021 primarily because these compounds produced intolerable neu- deviation between the catalytic domain of tPA and elastase rotoxic and side effects.11,12 Taken together, was 1.5 Å for 221 aligned carbon ␣ (C␣). this has led to the conclusion that methods of neuroprotection 13 that could prevent tPA toxicity are needed. tPA Catalytic Activity Assay with Nitrous Oxide During the past decade, much attention has been paid to We assessed the effects of nitrous oxide on the catalytic effi- the potentially neuroprotective properties of the remarkably ciency of tPA by using the initial rate method. The recombinant safe anesthetic gases xenon and nitrous oxide. Both gases form of human tPA (Actilyse௡; Boehringer Ingelheim, Ingel- share many pharmacologic properties as antagonists of the heim am Rhein, Germany) and murine tPA (ref. IRTPA; 14–17 NMDA and nicotinic acetylcholine receptor, activators Innovative Research, Novi, MI) and their specific chromogenic ϩ 18 of the TREK-1 two-pore–domain K channel, and en- substrate methylsulfonyl-D-phenyl-glycil-arginine-7-amino-4- 19,20 zyme inhibitors. Preclinical evidence in rodents has methylcoumarin acetate (Spectrozyme௡ XF, product 444; proven that these gases may have effective neuroprotective American Diagnostica, Stamford, CT) were separately diluted 14 properties by reducing excitotoxic neuronal death, me- in 1 ml distilled water in 1.5-ml sterile tubes. Each tube contain- chanical middle cerebral artery occlusion (MCAO)-induced ing 0.4 ␮M tPA or 10 ␮M tPA substrate was saturated for 20 21–25 brain damage, neonatal hypoxic–ischemic brain dam- min at a flow rate of 60–80 ml/min with nitrous oxide of 25–75 26,27 age, and cardiopulmonary bypass-induced neurologic vol% or medical air composed of 25% oxygen and 75% nitro- 28 and neurocognitive dysfunction, with no adverse side ef- gen as described previously.29 We assessed the catalytic effi- fects when used at subanesthetic concentrations. This has led ciency of human and murine tPA (N ϭ 3, n ϭ 12, per concen- to many thinking that clinical trials could be initiated in tration) in the presence of air or nitrous oxide by incubating 50 acute stroke patients in the near future. However, a recent ␮l tPA with 50 ␮l substrate at 37°C using a spectrofluorometer study performed in a rat model of thromboembolic stroke microplate reader. has shown that xenon inhibits tPA by binding directly within its catalytic site, thereby producing adverse effects by reduc- In vitro Thrombolysis Experiments with Nitrous Oxide ing tPA-induced thrombolysis and, as a consequence, throm- We used male Sprague-Dawley mature rats weighing 600–650 bolysis-induced reduction of brain damage when adminis- g(n ϭ 6). Whole blood samples of 500 ␮l volume were trans- tered during the intraischemic period and beneficial effects ferred in preweighed 1.5-ml tubes and incubated at 37°C for by suppressing both ischemic brain damage and recombinant 3 h. Saline solution was saturated for 30 min with nitrous oxide tPA-induced brain hemorrhages and disruption of the 29 of 25–75 vol% (with the remainder being oxygen at 25 vol% blood–brain barrier when given after reperfusion. Because and nitrogen as needed) or with medical air as described previ- nitrous oxide shares pharmacologic and neuroprotective ously.29 Briefly, after clot formation and total serum removal, properties similar to that of xenon in excitotoxic-ischemic 14,21–23 each tube was weighed to determine the clot weight. We se- models, we hypothesized that nitrous oxide may lected blood clots in the same weight range (0.260 Ϯ 0.056 g) to modulate the catalytic efficiency of tPA and thereby alter the reduce variability. Each tube was filled with saline solution con- beneficial and/or adverse effects of recombinant tPA therapy taining 1 ␮g/ml tPA in the form of Actilyse௡ saturated with 25 in a rat model of thromboembolic ischemia. vol% nitrous oxide (n ϭ 16) or air (n ϭ 12), 37.5 vol% nitrous oxide (n ϭ 16) or air (n ϭ 15), 50 vol% nitrous oxide (n ϭ 12) Materials and Methods or air (n ϭ 8), or 75 vol% nitrous oxide (n ϭ 16) or air (n ϭ 15), Animals and incubated at 37°C for a 90-min period. The fluid was re- We used male Sprague-Dawley rats. All animal-use experi- moved, and the tubes were weighed again to assess the percent- ments were approved by a local ethics committee (Caen, age of clot lysis induced by tPA in the presence of medical air or France) in accordance with the French legislation for bio- nitrous oxide of 25–75 vol%. medical experimentation and the European Communities Council Directive of November 24, 1986 (86/609/EEC). MCAO Experiments with Intraischemic Nitrous Oxide Rats were housed at 21 Ϯ 0.5°C in acrylic home cages with Male Sprague-Dawley rats weighing 250–275 g were used to free access to food and water in groups of four. After being assess the effects of nitrous oxide on cerebral blood flow

Anesthesiology 2011; 115:1044–53 1045 Haelewyn et al. Nitrous Oxide and tPA Interactions

reperfusion induced by tPA and subsequent reduction of MCAO Experiments with Postischemic Nitrous Oxide ischemic brain damage. Nitrous oxide was used at 75 vol%, a Male Sprague-Dawley rats weighing 250–275 g were used to concentration shown in a recent dose-effect study to provide assess the effects of nitrous oxide at 75 vol% on ischemic maximal neuroprotection in rats subjected to MCAO-in- brain damage. Rats were anesthetized with in duced ischemia.23 Rats were subjected to MCAO-induced medical air and subjected to thromboembolic MCAO-in- ischemia by administration of an autologous blood clot by duced ischemia by administration of an autologous blood the intraluminal method. Twenty-four hours before the an- clot, as described above, while breathing spontaneously imals were subjected to MCAO-induced ischemia, a whole throughout the surgical intervention. After injection of the caudal blood sample of 200 ␮l was withdrawn and allowed to blood clot, the animals were awakened and allowed to move freely in their home cages. After a 45-min period of ischemia, clot at 37°C for 2 h. The clot was extruded from the catheter Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/115/5/1044/452771/0000542-201111000-00027.pdf by guest on 25 September 2021 into a saline-filled petri dish and stored at 4°C for 22 h before the ligature of the common carotid artery was removed, and ௡ being used the day after to induce thromboembolic ischemia. the rats were given tPA in the form of Actilyse , as described On the day of surgery, the rats were anesthetized with 2 above, through a catheter that had been inserted into the vol% isoflurane in medical air, intubated, and ventilated ar- femoral vein and exteriorized at the neck on the day before tificially. Catheters were inserted into the femoral vein to the main surgical protocol. After tPA infusion, the rats were allow injection of tPA or saline solution and in the femoral treated for3hinaclosed chamber with either medical air (n ϭ 5) or nitrous oxide at 75 vol% (n ϭ 4) at a flow rate of artery for continuous monitoring of heart rate, diastolic, sys- one volume chamber per minute. Such a flow rate allows tolic, and mean arterial pressures and for the periodic analysis maintaining carbon dioxide concentrations at 0.03 vol%. of blood gases and pH. A midline neck incision was per- Sham rats (n ϭ 4) were treated with saline solution and formed, and the right common carotid artery was exposed to medical air. After treatment, the rats were allowed to move perform coagulation of the proximal branches of the external freely in their home cages and provided food and water ad carotid artery. A laser-doppler flowmetry probe was posi- libitum. Brain damage was assessed 48 h after the onset of tioned onto the right parietal bone (previously thinned) to ischemia. To mimic clinical recommendations, rats showing assess successful induction of cerebral ischemia and monitor less than 50% reduction in cerebral perfusion [sham rats changes in cerebral blood flow while the rat was being placed (saline ϩ medical air), n ϭ 1; control rats (tPA ϩ medical prone. Changes in cerebral blood flow were monitored con- air), n ϭ 2] were excluded from the study design. tinuously and expressed as a percentage from the cerebral blood flow values recorded during a 20-min preocclusion NMDA and NMDA Plus tPA-induced Neuronal Death with period. A single clot measuring 40 mm in length was injected Postinsult Nitrous Oxide in a volume of 50 ␮l saline solution through a polyethyl- On the day of surgery, rats were anesthetized with ene-10 catheter directed into the internal carotid artery up to (1.5%) in medical air and mounted on a stereotaxic apparatus 2 mm after the pterygopalatine-internal carotid artery bifur- 29 with the incisor bar set 3.9 mm below the horizontal zero. Dur- cation, as detailed previously. After a 45-min period of ing surgery, body temperature was kept at 37 Ϯ 0.5°C. A burr occlusion during which all rats were given medical air, the hole was drilled and a micropipette (ϳ10 ␮m at the tip) was catheter was removed from the internal carotid artery to lowered into the right striatum (anterior, 0.6 mm; lateral, 3.0 the external carotid artery, and the rats were given tPA in the mm; ventral, 5.8 mm, from bregma) to allow injection of 50 ௡ form of Actilyse at 0.9 mg/kg (a dose shown to be as clini- nmol of NMDA alone or in combination with 3 ␮g tPA in the cally relevant as that of 10 mg/kg, which is often used in form of Actilyse௡ in 1 ␮l phosphate buffered saline solution (pH 31 rodents ) in 1 ml saline solution (10% bolus plus 90% 7.4) over a 2-min period. After an additional 5-min period, the perfusion over a 45-min period) with either medical air (n ϭ micropipette was removed, and the rats were returned to their 5) or 75 vol% nitrous oxide (n ϭ 5). Then, all animals were home cages with free access to food and water. Sixty minutes given medical air again. Sham-treated rats (n ϭ 5) were given after administration of NMDA or NMDA plus tPA, the rats medical air and saline solution. Rats were maintained in a were treated for 3 h with medical air or nitrous oxide as de- normothermic state. After surgery, the polyethylene-10 cath- scribed above for rats subjected to ischemia. The number of eter was removed, and the rats were returned to their home animals was n ϭ 8 per group. Brain damage was assessed 24 h cages and allowed to move freely and provided food and after NMDA or NMDA plus tPA injection. water ad libitum. Brain damage was assessed 24 h after the onset of ischemia. To mimic clinical recommendations, rats Gas Pharmacology that showed more than 85% reduction (n ϭ 0) or less than Oxygen, nitrogen, and nitrous oxide of medical grade were pur- 50% reduction in cerebral perfusion as calculated over a 15- chased from Air Liquide Sante´ (Paris, France). Gas mixtures min period before treatment [sham rats (saline ϩ medical containing 75 vol% nitrogen and 25 vol% oxygen (medical air) air), n ϭ 1; control rats (tPA ϩ medical air), n ϭ 2; nitrous or nitrous oxide at 25–75 vol%, with the remainder being oxy- oxide-treated rats (tPA ϩ nitrous oxide), n ϭ 1] were ex- gen at 25 vol% completed with nitrogen when necessary, were cluded from the study design. obtained using computer-driven calibrated flowmeters and gas

Anesthesiology 2011; 115:1044–53 1046 Haelewyn et al. CRITICAL CARE MEDICINE

analyzers, and used or administered as described above accord- (SEM), were analyzed using one-way parametric analysis of ing to a blinded and randomized procedure. variance and unpaired Student t test with two-tailed testing. In addition, the inhibitory concentration of nitrous oxide

Assessment of Brain Damage producing half of the maximal effect (IC50) was also calcu- Rats were killed by decapitation under isoflurane anesthesia. lated. In vivo data, given as median value and the twenty-fifth Brain damage was assessed 24 h after injection of NMDA or to seventy-fifth percentiles, were analyzed using the Kruskal- NMDA plus tPA or after the onset of ischemia for intraisch- Wallis nonparametric analysis of variance and Mann–Whit- emic experiments and 48 h after the onset of brain ischemia ney nonparametric unpaired U-test with two-tailed testing. for postischemic experiments to allow “better” comparison The level of significance was set at P Ͻ 0.05. The number of with previous mechanical and thromboembolic stud- animals per group for in vivo studies was assessed by perform- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/115/5/1044/452771/0000542-201111000-00027.pdf by guest on 25 September 2021 ies.23,24,29 Comparative studies using multiple staining tech- ing power analysis through an online software** with the niques32,33 have shown that assessment of infarct size at 24 h following variables taken from a previous study with xenon at is a time condition sufficient to obtain consolidated infarct 50 vol% (based on the fact that the minimum anesthetic volumes (i.e., whose assessment would be similar if per- concentration ratio of the effects of xenon and nitrous oxide formed one or several days later), thereby allowing reliable indicates that nitrous oxide at 75 vol% is as potent as xenon comparison between the data obtained at 24 h or 48 h after at 50 vol%): mean Ϯ SD for tPA ϭ 166 Ϯ 52 mm3; mean Ϯ the onset of brain ischemia. The brain was removed and SD for xenon at 50 vol% ϭ 330 Ϯ 140 mm3; ␣ error level frozen in isopentane. Coronal brain sections (20 ␮m) were (confidence level) ϭ 0.05; ␤ error level (statistical power) ϭ cryostat-cut, mounted on slides, stained with thionin, and 0.25. Rats that died during MCAO-induced ischemia and digitized on a computer. The lesion areas were delineated by those that died before being used for histologic analysis [in- the pallor of staining in the necrotic tissue compared with the traischemic experiments: sham rats (saline ϩ medical air), surrounding healthy tissue; then volumes of brain damage n ϭ 1; control rats (tPA ϩ medical air), n ϭ 2; nitrous were estimated by integration over the whole brain of the oxide-treated rats (tPA ϩ nitrous oxide), n ϭ 1; postischemic infarct surfaces calculated with the ImageJ௡ software ana- experiments: sham rats (saline ϩ medical air), n ϭ 2; control lyzer (Scion Corp., Frederick, MD), corrected for tissue rats (tPA ϩ medical air), n ϭ 4; nitrous oxide-treated rats edema and expressed in cubic millimeters. (tPA ϩ nitrous oxide), n ϭ 3] were excluded from statistical data. No data were excluded from statistical analysis from in Assessment of Brain Hemorrhages and Disruption of the vitro studies or in vivo NMDA and NMDA ϩ tPA studies. Blood–Brain Barrier Integrity Forty-eight hours after the onset of brain ischemia, rats were Results given intravenously (4% in 1-ml saline solution over a 30-s period). The brain was removed and sliced as described Modeling of the Binding Site of Nitrous Oxide within the above. While the unstained brain still mounted on the cryostat S1 Pocket of tPA was cut, macrophotographs were made and analyzed with a First, we investigated whether nitrous oxide could bind to tPA. computer using the ImageJ௡ software analyzer. We assessed Xenon binds within the S1 pocket of elastase,35 and nitrous brain hemorrhages and disruption of the blood–brain barrier oxide binds to elastase at the same location.36 Because all serine integrity as described previously.29,34 Evans blue extravasation proteases share a similar structure around their identical catalytic was delineated and multiplied by the extravasation optical den- triad and thus can be easily structurally superposed, we per- sity; the ratio of extravasation was calculated for each rat by formed modeling of the possible binding of nitrous oxide to the dividing the volume of the brain showing extravasation as ob- catalytic domain of tPA by superposing the crystallographic tained by integration over the whole brain of the surfaces show- structure of elastase in complex with nitrous oxide to the crys- ing Evans blue extravasation by the total infarction volume, tallographic structure of the human tPA catalytic domain in corrected for tissue edema. Brain hemorrhages were delineated complex with the bis-benzamidine inhibitor tPA Stop. As as blood evident at the macroscopic level. For each rat, hemor- shown in figure 1A, the structure of the catalytic domain of tPA rhagic transformation was calculated as the sum of the in complex with tPA Stop reveals that an extremity of this in- hemorrhagic ratio obtained by dividing the total surface of hem- hibitor binds within the specificity pocket S1. The nitrous oxide orrhages by the total surface of infarction in the slices that atom fits within the S1 pocket of tPA at the position of one showed hemorrhages. Finally, coronal sections were used to as- extremity of the tPA inhibitor tPA Stop. This suggests that ni- sess infarct size as described above. trous oxide could reduce the catalytic efficiency of tPA by bind- ing within the S1 specificity pocket. Statistical Analysis Data were analyzed using Statview software (SAS Institute, Nitrous Oxide Dose-dependently Inhibits the Catalytic Ϯ Cary, NC). In vitro data, given as mean the SE of the mean Efficiency of tPA in vitro ** Available at: www.dssresearch.com/toolkit/sscalc/size_a2.asp. Next, we investigated the effects of nitrous oxide on the catalytic Accessed May, 24, 2011. efficiency of the recombinant form of human and murine tPA

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Fig. 1. Modeling of nitrous oxide (N2O) binding to tissue plasminogen activator (tPA). Nitrous oxide is predicted to bind within the primary specificity pocket S1, where the tPA inhibitor tPA Stop binds (A). Dose-dependent inhibition by nitrous oxide of ϭ Ϯ ϭ Ϯ Ͻ 25–75 vol% of the catalytic efficiency of humans (IC50 32.14 0.26 vol%) and murine (IC50 35.12 0.13 vol%) tPA. * P 0.001 versus nitrous oxide of 25–75 vol% (B). Dose-dependent inhibition by nitrous oxide of 25–75 vol% of tPA-induced brain ϭ Ϯ Ͻ Ͻ thrombolysis in vitro in blood clots obtained from whole blood samples (IC50 27.13 1.04 vol%). ** P 0.01, * P 0.05, # P Ͻ 0.1 versus controls treated with medical air (C). Data are mean Ϯ SEM. Control experiments were performed with medical air instead of nitrous oxide. by the initial rate method. Human and murine tPA and their fig. 1C). No evidence of clot lysis was found in whole blood specific chromogenic substrate were diluted in distilled water samples treated with saline solution and medical air. and saturated with nitrous oxide of 25–75 vol% (with the re- mainder being 25 vol% oxygen completed with nitrogen when In vivo Intraischemic Nitrous Oxide Inhibits tPA-induced necessary) or medical air composed of 25% oxygen and 75% Thrombolysis and Subsequent Reduction of Brain nitrogen. The addition of nitrous oxide into the medium inhib- Damage ited the catalytic efficiency of human and murine tPA in a dose- To examine whether nitrous oxide may actually decrease throm- dependent manner. Nitrous oxide of 25–75 vol% inhibited the bolysis in vivo, we investigated the effects of intraischemic ni- catalytic efficiencies of human and murine tPA in a similar man- trous oxide on tPA-induced cerebral blood flow reperfusion and Ͻ ϭ Ϯ ner (human tPA: P 0.0001, IC50 32.14 0.26 vol%; subsequent reduction of brain damage in rats subjected to Ͻ ϭ Ϯ murine tPA: P 0.0001, IC50 35.12 0.13 vol%; fig. 1B). thromboembolic brain ischemia. Nitrous oxide was used at 75 This suggests that nitrous oxide could reduce the thrombolytic vol%, a concentration shown in a recent dose-effect study to efficiency of tPA. provide maximal neuroprotection in rats subjected to MCAO- induced ischemia.23 At 45 min after the onset of brain ischemia, Nitrous Oxide Dose-dependently Inhibits the rats were administered tPA in the form of Actilyse௡. In line with Thrombolytic Efficiency of tPA in vitro the findings of clinical studies of tPA-treated patients,37,38 we Next, to examine whether nitrous oxide may inhibit tPA- found that rats injected with tPA exhibited full cerebral reper- induced thrombolysis, we studied the effect of this inert gas fusion 40 min after tPA injection. All physiologic parameters, at concentrations of 25–75 vol% on the thrombolytic effi- including arterial pressure, decreased to within normal range ciency of tPA in whole blood samples drawn from male ma- values (table 1). Sham rats treated with saline solution and med- ture rats. After clot formation and total serum removal, each ical air showed no cerebral blood flow reperfusion and had a tube was filled with saline solution containing a clinically volume of ischemic brain damage of 395 mm3 (twenty-fifth to relevant concentration of 1 ␮l/ml tPA in the form of seventy-fifth percentiles: 370–402 mm3). As expected, control Actilyse௡ previously saturated with nitrous oxide or medical rats treated with tPA and medical air exhibited full cerebral air. We found that nitrous oxide at concentrations of 25–75 blood flow reperfusion (P Ͻ 0.01) and a reduced volume of vol% reduced tPA-induced thrombolysis in a concentration- brain damage of 167 mm3 (twenty-fifth to seventy-fifth percen- Ͻ ϭ Ϯ 3 Ͻ dependent manner (P 0.001; IC50 27.13 1.04 vol%; tiles: 160–209 mm ) compared with sham-treated rats (P

Table 1. Range of Physiologic Values of Arterial PaO2,SaO2,PaCO2, Arterial pH, and Body Temperature during Ischemia in Rats Treated with Medical Air and Rats Subjected to Intraischemic Nitrous Oxide at 75 vol%

—PaO2 (mmHg) PaCO2 (mmHg) SaO2 (%) Arterial pH Temp. (°C) Air 75–100 36–40 95–97 7.37–7.46 37.1–37.6

N2O75 75–90 35–41 95–97 7.39–7.52 37.4–37.6

ϭ ϭ ϭ PaCO2 arterial carbon dioxide partial pressure; PaO2 arterial oxygen partial pressure; SaO2 arterial hemoglobin saturation of oxygen; Temp ϭ body temperature.

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Fig. 2. Time course of the inhibitory effects of intraischemic nitrous oxide (N2O) on tissue plasminogen activator (tPA)-induced thrombolysis expressed as a percentage change of cerebral blood flow (CBF) baseline values. Rats treated with tPA and intraischemic nitrous oxide at 75 vol% had decreased tPA-induced reperfusion compared with control rats treated with medical air and tPA. Sham-treated rats treated with saline (Sal) and medical air showed no reperfusion (A). Cumulated (T to T=) inhibitory effects of intraischemic nitrous oxide on tPA-induced thrombolysis. Nitrous oxide at 75 vol% inhibited tPA-induced thrombolysis by 51 Ϯ 7% (B). Effects of intraischemic nitrous oxide on middle cerebral artery occlusion (MCAO)-induced brain damage. Rats treated with tPA and intraischemic nitrous oxide at 75 vol% had increased volume of brain damage compared with controls rats treated with tPA and medical air. The lower part of each histogram represents the volume of cortical (CX) brain damage, whereas the upper part of each histogram represents the volume of subcortical (SCX) brain damage (C). Data are median value Ϯ twenty-fifth to seventy-fifth percentiles. # P Ͻ 0.001 versus sham-treated rats; * P Ͻ 0.05 versus controls (tPA ϩ medical air).

0.01). In contrast to this latter finding, we found that rats treated hemorrhages (P Ͻ 0.01) and disruption of the blood–brain with tPA and intraischemic nitrous oxide showed a reduction in barrier (P Ͻ 0.05) compared with control rats treated with cerebral blood flow reperfusion of 42% (twenty-fifth to seventy- tPA and medical air (fig. 3A–D). fifth percentiles: 35–48%; P Ͻ 0.05) compared with control animals treated with tPA and medical air (fig. 2A–B). As might In vivo Postinsult Nitrous Oxide Reduces NMDA- and be expected from this effect, we found that rats treated with tPA NMDA Plus tPA-induced Neuronal Death and intraischemic nitrous oxide at 75 vol% had greater volumes Finally, we investigated the effects of nitrous oxide on 3 of brain damage (308 mm ; twenty-fifth to seventy-fifth percen- NMDA-induced neuronal death when given 1 h after 3 Ͻ tiles: 291–457 mm ; P 0.05) than did control animals treated NMDA or NMDA plus tPA injection. Administration with tPA and medical air (fig. ). of NMDA in the striatum led to a volume of excitotoxic neuronal death of 18.6 mm3 (twenty-fifth to seventy-fifth In vivo Postischemic Nitrous Oxide Reduces Ischemic percentiles: 17.3–19.5 mm3). As expected from previous Brain Damage but Increases Brain Hemorrhages data,39,40 rats injected with NMDA plus tPA showed greater We then studied the effects of postischemic nitrous oxide on brain damage than did those injected with NMDA alone, brain hemorrhages, disruption of the blood–brain barrier with a volume of neuronal death of 33.2 mm3 (twenty-fifth integrity, and ischemic brain damage in rats subjected to to seventy-fifth percentiles: 29.6–36.8 mm3;PϽ 0.001). thromboembolic brain ischemia. Forty-five minutes after the We found that nitrous oxide reduced NMDA-induced brain onset of brain ischemia, rats were given intravenous tPA in damage by approximately 25%, so that rats treated with ni- the form of Actilyse௡. Fifteen minutes after tPA infusion trous oxide and NMDA had a lower volume of brain damage ended, rats were treated with medical air (controls) or nitrous (13.8 mm3; twenty-fifth to seventy-fifth percentiles: 12.6– oxide at 75 vol%. Sham rats treated with saline solution and 16.4 mm3; P Ͻ 0.05) than did rats that were given NMDA medical air had a mean volume of ischemic brain damage of and medical air. Nitrous oxide further decreased neuronal 3 383 mm (twenty-fifth to seventy-fifth percentiles: 350– death produced by coinjection of NMDA plus tPA by more 3 420 mm ) and exhibited brain hemorrhages and disruption than 40% so that rats treated with nitrous oxide and NMDA of the blood–brain barrier. Compared with sham-treated plus tPA had a lower volume of brain damage (19.2 mm3; rats, control rats treated with tPA and medical air had similar twenty-fifth to seventy-fifth percentiles: 16.3–21.9 mm3; ratios of brain hemorrhages and disruption of the blood– P Ͻ 0.001) than did rats that were given with NMDA plus brain barrier integrity, despite a reduced volume of brain tPA in medical air (fig. 4). damage (99 mm3; twenty-fifth to seventy-fifth percentiles: 92–134 mm3;PϽ 0.01; fig. 3A–D). Rats treated with tPA Discussion and postischemic nitrous oxide at 75 vol% had reduced brain damage (49 mm3; twenty-fifth to seventy-fifth percentiles: During the past decade, preclinical studies have proven that 40–59 mm3; P Ͻ 0.02) but exhibited greater ratios of brain inert gases such as xenon and nitrous oxide at subanesthetic

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Fig. 3. Effect of postischemic nitrous oxide (N2O) at 75 vol% on middle cerebral artery occlusion (MCAO)-induced brain damage (A). Effects of postischemic nitrous oxide at 75 vol% on MCAO-induced disruption of the blood-brain barrier as assessed using Evans blue extravasation and MCAO- and tissue plasminogen activator (tPA)-induced brain hemorrhages (B). Typical examples of brain damage in rats treated with saline (Sal) and medical air, tPA and medical air, or tPA and nitrous oxide at 75 vol%. Slices in the background are those shown in D below (C). Examples of Evans blue extravasation and brain hemorrhages in brain slices (those shown in the background of B) treated with saline and medical air, tPA and medical air, or tPA and nitrous oxide at 75 vol% (D). Data are median value Ϯ twenty-fifth to seventy-fifth percentiles. # P Ͻ 0.05 versus sham-treated rats; * P Ͻ 0.01–0.05 versus control rats (tPA ϩ medical air). Sub-Cx ϭ subcortex. concentrations may have effective neuroprotective properties lack of neuroprotective action of intraischemic nitrous oxide with no adverse side effects.14,21–28 However, a recent study almost certainly can be attributed to nitrous oxide inhibiting performed in a rat model of thromboembolic stroke has the enzymatic activity of tPA—that is the basic mechanism shown that xenon interacts directly with tPA, the most com- responsible for its thrombolytic action—by binding within mon approved therapy of acute ischemic stroke. Interactions the S1 pocket of tPA at the position of the specific tPA of xenon with tPA produce adverse effects by reducing tPA- inhibitor tPA Stop, which is shown in the current study. induced thrombolysis and, as a consequence, thrombolysis- Next, in contrast to the adverse effect of intraischemic induced reduction of brain damage when administered dur- nitrous oxide and in agreement with previous reports in rats ing the intraischemic period and beneficial effects by subjected to mechanical ischemia,21,23 we found that nitrous suppressing ischemic brain damage and tPA-induced brain oxide given after ischemia reduces ischemic brain damage at hemorrhages and disruption of the blood–brain barrier the cortical but not the subcortical level. Such a dichotomic when given after reperfusion.29 Because nitrous oxide has effect of postischemic nitrous oxide has been attributed to pharmacologic and neuroprotective properties similar to that the striatum (which constitutes the main part of the subcor- of xenon in excitotoxic-ischemic models,14,21–23 we hypoth- tical areas that are damaged in the MCAO ischemic model) esized that nitrous oxide may modulate the thrombolytic and being difficult to protect against stroke because it experiences proteolytic effects of tPA. complete blood flow cessation during ischemia because of its First, we found that nitrous oxide dose-dependently in- lack of collateral vasculature compared with the cortex.41 In hibits the catalytic (enzymatic) and thrombolytic efficiency addition, the dichotomic effect of postischemic nitrous oxide of tPA in vitro. As expected from these results, we showed at the cortical and subcortical level could result at least in part that nitrous oxide, when administered with tPA during the from nitrous oxide increasing the cerebral metabolic rate in intraischemic period, reduces tPA-induced thrombolysis and subcortical areas, such as the striatum and the thalamus,42 a thereby decreases and even suppresses the benefits of tPA condition that could “prolong” ischemia-induced disruption therapy in terms of reduction of ischemic brain damage. The in oxygen and glucose even after reperfusion. In addition, as

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brain regions at concentrations to 1 minimum anesthetic concentration,45–47 whereas in contrast, nitrous oxide at concentrations as low as 0.5 minimum anesthetic concentra- tion is well known to increase cerebral blood flow and cere- bral blood flow velocity,48,49 conditions that could favor dis- ruption of the blood–brain barrier, particularly during reperfusion. The impact of isoflurane administration on the findings of the current study and the possibility that postischemic nitrous oxide at 75 vol% may have offered neuroprotection Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/115/5/1044/452771/0000542-201111000-00027.pdf by guest on 25 September 2021 by reducing the rats’ brain temperature should be consid- ered. During the intraischemic experiments, rats were main- Fig. 4. Effects of nitrous oxide (N2O) on N-methyl-D-aspartate tained anesthetized after tPA injection for 45 min with a (NMDA)-induced neuronal death and tissue plasminogen ac- combination of isoflurane and nitrous oxide. In contrast, tivator (tPA)-induced potentiation of NMDA-induced neuronal during postischemic experiments, rats were given isoflurane death. Administration of nitrous oxide 1 h after intrastriatal injection of NMDA or coinjection of NMDA plus tPA de- and nitrous oxide separately. Differences in neuroprotection creases both NMDA-induced neuronal death and tPA-in- against MCAO-induced brain damage between postisch- duced potentiation of NMDA-induced neuronal death. Data emic nitrous oxide alone and intraischemic nitrous oxide- are expressed as the median value and twenty-fifth to sev- isoflurane, as reported in this study and as can be concluded enty-fifth percentiles. ** P Ͻ 0.001, * P Ͻ 0.05 versus rats from independent studies,21,23,25,50 have been attributed to a # Ͻ treated with NMDA and medical air; P 0.001 versus rats proapoptogenic interaction between isoflurane and nitrous treated with NMDA plus tPA and medical air. oxide51 because neither nitrous oxide nor isoflurane pro- reported previously,23 we found that postinsult nitrous oxide duces apoptosis when given alone in the adult rat. In addi- decreased NMDA-induced neuronal death and postinsult tion, it is possible that isoflurane could have contributed to nitrous oxide decreased to a greater extent the potentiation neuroprotection in the postischemic experiments of this by tPA of NMDA-induced neuronal death, a finding that to study because this volatile anesthetic has been shown to be a 52–54 our knowledge is reported here for the first time. Therefore, neuroprotectant preconditioning agent in the adult rat, it is likely that inhibition of residual tPA by nitrous oxide although adverse apoptogenic effects have been reported in 55,56 after reperfusion played a major role in the ability of post- the developing brain. Alternatively, the possibility that ischemic nitrous oxide to reduce ischemia-induced cortical 75 vol% postischemic nitrous oxide in freely moving animals brain damage in addition to its well known pharmacologic may offer neuroprotection by reducing the rats’ brain tem- 23 antagonistic action at the NMDA receptor,15,16 whose post- perature by 1°C has been ruled out previously because synaptic activation is known as a critical event in excitotoxic nitrous oxide at a lower concentration of 50 vol% has neu- 23 neuronal death. However, additional pharmacologic proper- roprotective action without altering brain temperature, the ties of nitrous oxide at other neuronal targets of potentially brain temperature must be decreased to at least 34°C to 57 neuroprotective interest, such as the nicotinic acetylcholine provide neuroprotection in adult rats, and a reduction of ϩ receptor17 and the TREK-1 two-pore–domain K chan- brain temperature to 35°C is not sufficient to produce neu- 27 nel,18 could have contributed to the neuroprotective action roprotection in rat pups. of postischemic nitrous oxide at the cortical level. Alterna- Because compatibility between treatments is an inalien- tively, in contrast with its beneficial effects on ischemic cor- able condition for allowing combination therapeutic strate- tical brain damage and NMDA- and NMDA-plus-tPA–in- gies, the interactions of nitrous oxide with tPA shown in this duced neuronal death, we found that postischemic nitrous study clearly caution against using nitrous oxide for treating oxide increases tPA-induced brain hemorrhages and disrup- acute ischemic stroke patients; this is in contrast to our pre- tion of the blood–brain barrier. This effect, which is in good vious findings in rats subjected to mechanical brain ischemia. agreement with previous data showing that nitrous oxide However, the current report has several possible limitations increases cerebrovascular permeability to proteins and pro- inherent in the study design and methodology. Intraischemic duces disruption of the blood–brain barrier,43,44 opposes and postischemic rats were killed at different times after the that of xenon being shown to reduce dramatically ischemic onset of ischemia and exposed to different isoflurane-nitrous brain damage and tPA-induced brain hemorrhages and dis- oxide protocols with tPA. However, as discussed in Materials ruption of the blood–brain barrier.29 Difference between the and Methods and elsewhere in the text, each nitrous oxide effects of postischemic xenon and postischemic nitrous oxide group was compared with its own tPA control group, and we on tPA-induced brain hemorrhages and disruption of the are confident, based on our in silico and in vitro catalytic and blood–brain barrier are likely to occur at the vascular, rather thrombolytic experiments, that differences between intrais- than the parenchymal, level. This could be because xenon has chemic and postischemic nitrous oxide result from the effect no effect or even decreases cerebral blood flow in specific of nitrous oxide at inhibiting competitively the enzymatic

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