Interactions between Nitrous Oxide 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 xenon, 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 serine 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 neuroprotection by the use of glutamatergic 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 psychotomimetic 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