**1. Introduction**

General anesthetics have been used in clinical practice since the middle of the XIXth century, but their molecular mechanisms of action on the nervous system remains poorly understood. The main targets of most inhalational anesthetic are pentameric ligand-gated ion channels such as inhibitory GABAA (-amino-butyric acid) receptors whose activity is potentiated by general anesthetics. However, the main targets of gaseous anesthetics like xenon or nitrous oxide are excitatory NMDA (N-methyl-D-aspartate) receptors and nicotinic acetylcholine receptors whose activity is inhibited by gaseous anesthetics (Campagna et al., 2003; Franks, 2008). The use of gaseous anesthetics became widely applicable in the nineteen-fifties, nitrous oxide being often administrated in complement of halogenated anesthetics. Xenon anesthetic properties have been described by Cullen in 1951 (Cullen et al., 1951), and is used in anesthesia since mid-2000 (Sanders et al., 2004; Sanders et al., 2005) in spite of its excessive cost, a major obstacle to its widespread use (Kennedy et al., 1992).

Anesthesia is a complex process that refers to several physiologically altered functions. Early stages of anesthesia such as amnesia and hypnosis required anesthetic concentrations lower than those required to produce deep sedation and reduction of motor and automomic responses to noxious stimuli (Campagna et al., 2003). Scales that assess the *in-vivo* potency of inhaled anesthetics in humans are based on the minimum alveolar anesthetic concentrations (MAC) that are associated with well-defined behavioural endpoints. Following this, MAC-awake defines the MAC that induces the first stages of anesthesia such as amnesia and hypnosis, and MAC-immobility defines the MAC that produces deep sedation and suppresses movement in response to a noxious stimuli.

Anesthesia mechanisms were for a long time though to be mediated by non-specific membranous perturbation (Trudell, 1977). This membranous theory was based on the Meyer-Overton rule that showed an almost perfect relationship between the anesthetic property of a chemical compound and its solubility in olive oil or benzene. However, more and more exceptions were found to the Meyer-Overton rule such as the non-immobilizers

Protein-Noble Gas Interactions Investigated by Crystallography

close xenon delivery systems are now being developed.

xenon potency (Cullen et al., 1951; Kennedy et al., 1992).

Gas Number

of electrons

1998; Quillin et al., 2000; Ruzicka et al., 2007)).

rises with pressure (Tilton et al., 1984).

GABA neurotransmission at the GABAA receptor (Abraini et al., 2003).

Polarizability (Å3)

cost-efficient strategy.

2000).

**pressure** 

induced brain hemorrages (Haelewyn et al., 2011).

on Three Enzymes - Implication on Anesthesia and Neuroprotection Mechanisms 287

(David et al., 2010) while nitrous oxide reduces ischemic brain damage but increases tPA-

Xenon is thus a very promising neuroprotective drug with few or no adverse side effects in models of acute ischemic stroke or perinatal hypoxia-ischemia (Homi et al., 2003; Ma et al., 2003; Abraini et al., 2005; David et al., 2008; Luo et al., 2008). Despite this, the widespread clinical use of xenon is limited by its scarceness and excessive cost of production, even if

Using a mixture of xenon and another anesthetic gas like nitrous oxide (Marassio et al., 2011), argon (David et al., submitted), or helium (David et al., 2009) could combine the efficiency of xenon and the low cost and availability of the second gas and is thought to be a

Argon is an inert gas which is easily available and has no narcotic nor anesthetic action at ambient pressure. It presents some mild to moderate neuroprotective properties (David et al., submitted). Argon, contrary to xenon and nitrous oxide, may act directly by potentiating

Krypton is significantly less potent as an anesthetic agent than xenon, consistently with the Meyer-Overton rule which shows that krypton anesthetic potency is four fold less than

Xenon, which has the highest solubility in lipids, also has the highest anesthetic potency (i.e. the lowest MAC-immobility) compared to krypton and argon (Table 1). Xenon also has the highest polarizability due to its high number of electron, compared to krypton and argon, so is predicted to be the gas which interact the most with proteins (Quillin et al.,

Ar 18 1.64 1.91 0.14 27 Kr 36 2.48 2.03 0.43 7.31 Xe 54 4.04 2.21 1.17 1.61 Table 1. Physical and anesthetic properties of argon, krypton and xenon (from (Koblin et al.,

**2. Determination of crystallographic structures of proteins under inert gases** 

To investigate the mechanism of interaction of gases with proteins, a structural approach using protein crystallography under gas pressure was developed. Xenon binds reversibly to proteins through non-covalent, weak energy van der Waals forces (Ewing et al., 1970). The first structures of protein – xenon complexes were solved in 1965 with myoglobin and haemoglobin under a xenon pressure of 2.5 bar, evidencing a xenon binding site in these two globins (Schoenborn, 1965; Schoenborn et al., 1965). At a pressure of 7 bar, four xenon binding sites were found in myoglobin indicating that the number of xenon binding sites

Since then, many structures of protein-xenon complexes were solved, with xenon used as a heavy atom in isomorphous replacement phasing method (MIR), because xenon has a high

van der Waals radius (Å) Solubility in lipids

MACimmobility (bar)

predicted to be potent anesthetics which indeed produced amnesia but not anesthesia or the eniantomers which have the same solubility in lipids but different anesthetic potencies (Campagna et al., 2003). Since the mid-1980, general anesthetics are though to act by disrupting protein functions (Franks et al., 1984; Franks et al., 1994). Chemical compounds that cross the blood brain barrier are generally soluble in lipids and could explain the Meyer-Overton rule (Franks, 2008). Anesthetics act mainly at ionotropic receptors which play a key role in regulation of ions' concentration on each side on the cytoplasmic membrane (Campagna et al., 2003). For a review of the major targets of general anesthetics and their effects, see (Yamakura et al., 2000).

Xenon and nitrous oxide have been shown in 1998 to be effective inhibitors of the NMDA receptor (Franks et al., 1998; Jevtovic-Todorovic et al., 1998) which gave them potentially interesting neuroprotective properties for treating major brain insults such as cerebral ischemia. Cerebral ischemia is indeed a major health problem, constituting the third cause of mortality and the first cause of morbidity in industrialized countries. Cerebral stroke is provoked by an acute interruption of the cerebral blood flow, leading to an oxygen and glucose deprivation for the brain, inducing dramatic dysfunctions in the excitatory glutamatergic neurotransmission. The resulting toxic accumulation of glutamate leads to an over-stimulation of glutamatergic receptors like the NMDA receptors, this process is called glutamate excitotoxicity (Choi et al., 1988; Dirnagl et al., 1999; Lo et al., 2005). The use of NMDA receptor antagonists to block the neurotoxic cascade initiated by the glutamate has not yet been proven to be clinically efficient in humans, because of the intrinsic neurotoxicity of these chemical compounds (Olney et al., 1991). The only treatment today is the use of a thrombolytic agent, the tissue-type plasminogen activator (tPA), which degrades the insoluble fibrin clots (NINDS, 1995). However, this treatment is only applicable in a few number of cases, due to the hemorrhagic risk which shorten the therapeutic window (Lee et al., 1999; Ahmed et al., 2010). The research of new neuroprotective drugs thus constitutes a major therapeutic goal. Inert gases are a new class of therapeutic agents which have a remarkably safe clinical profile and readily cross the blood brain barrier. Moreover, they have low solubility in blood, which is advantageous in terms of rapid inflow and washout. Gases have thus the great advantages to present a reducing risk of neurotoxic side effects, compared to chemical neuroprotective drugs, especially at the low concentrations used for neuroprotection.

Nitrous oxide and xenon reduce ischemic neuronal death in an *in-vivo* model of transient cerebral ischemia in rats and decrease the NMDA-induced Ca2+ influx on neuronal cell cultures studied by *in-vitro* calcium video microscopy (David et al., 2003). These two gases produce the same effect than memantine, a low-affinity antagonist of NMDA receptor which is already used in clinics for neurodegenerative disease treatments (David et al., 2006). Investigations in rodents have confirmed that xenon at subanesthetic concentrations of about 50 vol% provides maximal neuroprotection, even when given 2 to 4 h after the insult onset (Ma et al., 2005; Dingley et al., 2006; David et al., 2008). Nitrous oxide (Haelewyn et al., 2008) and argon (David et al., submitted) which are less expensive gases than xenon, possess mild-to-moderate neuroprotective properties against excitotoxic insults and hypoxicischemic injuries.

However, xenon and nitrous oxide inhibit tPA-induced thrombolysis, preventing their use during the intra-ischemic period. When administrated after the reperfusion, xenon has beneficial effect by suppressing ischemic brain damage and tPA-induced brain hemorrhages

predicted to be potent anesthetics which indeed produced amnesia but not anesthesia or the eniantomers which have the same solubility in lipids but different anesthetic potencies (Campagna et al., 2003). Since the mid-1980, general anesthetics are though to act by disrupting protein functions (Franks et al., 1984; Franks et al., 1994). Chemical compounds that cross the blood brain barrier are generally soluble in lipids and could explain the Meyer-Overton rule (Franks, 2008). Anesthetics act mainly at ionotropic receptors which play a key role in regulation of ions' concentration on each side on the cytoplasmic membrane (Campagna et al., 2003). For a review of the major targets of general anesthetics

Xenon and nitrous oxide have been shown in 1998 to be effective inhibitors of the NMDA receptor (Franks et al., 1998; Jevtovic-Todorovic et al., 1998) which gave them potentially interesting neuroprotective properties for treating major brain insults such as cerebral ischemia. Cerebral ischemia is indeed a major health problem, constituting the third cause of mortality and the first cause of morbidity in industrialized countries. Cerebral stroke is provoked by an acute interruption of the cerebral blood flow, leading to an oxygen and glucose deprivation for the brain, inducing dramatic dysfunctions in the excitatory glutamatergic neurotransmission. The resulting toxic accumulation of glutamate leads to an over-stimulation of glutamatergic receptors like the NMDA receptors, this process is called glutamate excitotoxicity (Choi et al., 1988; Dirnagl et al., 1999; Lo et al., 2005). The use of NMDA receptor antagonists to block the neurotoxic cascade initiated by the glutamate has not yet been proven to be clinically efficient in humans, because of the intrinsic neurotoxicity of these chemical compounds (Olney et al., 1991). The only treatment today is the use of a thrombolytic agent, the tissue-type plasminogen activator (tPA), which degrades the insoluble fibrin clots (NINDS, 1995). However, this treatment is only applicable in a few number of cases, due to the hemorrhagic risk which shorten the therapeutic window (Lee et al., 1999; Ahmed et al., 2010). The research of new neuroprotective drugs thus constitutes a major therapeutic goal. Inert gases are a new class of therapeutic agents which have a remarkably safe clinical profile and readily cross the blood brain barrier. Moreover, they have low solubility in blood, which is advantageous in terms of rapid inflow and washout. Gases have thus the great advantages to present a reducing risk of neurotoxic side effects, compared to chemical neuroprotective drugs,

Nitrous oxide and xenon reduce ischemic neuronal death in an *in-vivo* model of transient cerebral ischemia in rats and decrease the NMDA-induced Ca2+ influx on neuronal cell cultures studied by *in-vitro* calcium video microscopy (David et al., 2003). These two gases produce the same effect than memantine, a low-affinity antagonist of NMDA receptor which is already used in clinics for neurodegenerative disease treatments (David et al., 2006). Investigations in rodents have confirmed that xenon at subanesthetic concentrations of about 50 vol% provides maximal neuroprotection, even when given 2 to 4 h after the insult onset (Ma et al., 2005; Dingley et al., 2006; David et al., 2008). Nitrous oxide (Haelewyn et al., 2008) and argon (David et al., submitted) which are less expensive gases than xenon, possess mild-to-moderate neuroprotective properties against excitotoxic insults and hypoxic-

However, xenon and nitrous oxide inhibit tPA-induced thrombolysis, preventing their use during the intra-ischemic period. When administrated after the reperfusion, xenon has beneficial effect by suppressing ischemic brain damage and tPA-induced brain hemorrhages

and their effects, see (Yamakura et al., 2000).

especially at the low concentrations used for neuroprotection.

ischemic injuries.

(David et al., 2010) while nitrous oxide reduces ischemic brain damage but increases tPAinduced brain hemorrages (Haelewyn et al., 2011).

Xenon is thus a very promising neuroprotective drug with few or no adverse side effects in models of acute ischemic stroke or perinatal hypoxia-ischemia (Homi et al., 2003; Ma et al., 2003; Abraini et al., 2005; David et al., 2008; Luo et al., 2008). Despite this, the widespread clinical use of xenon is limited by its scarceness and excessive cost of production, even if close xenon delivery systems are now being developed.

Using a mixture of xenon and another anesthetic gas like nitrous oxide (Marassio et al., 2011), argon (David et al., submitted), or helium (David et al., 2009) could combine the efficiency of xenon and the low cost and availability of the second gas and is thought to be a cost-efficient strategy.

Argon is an inert gas which is easily available and has no narcotic nor anesthetic action at ambient pressure. It presents some mild to moderate neuroprotective properties (David et al., submitted). Argon, contrary to xenon and nitrous oxide, may act directly by potentiating GABA neurotransmission at the GABAA receptor (Abraini et al., 2003).

Krypton is significantly less potent as an anesthetic agent than xenon, consistently with the Meyer-Overton rule which shows that krypton anesthetic potency is four fold less than xenon potency (Cullen et al., 1951; Kennedy et al., 1992).

Xenon, which has the highest solubility in lipids, also has the highest anesthetic potency (i.e. the lowest MAC-immobility) compared to krypton and argon (Table 1). Xenon also has the highest polarizability due to its high number of electron, compared to krypton and argon, so is predicted to be the gas which interact the most with proteins (Quillin et al., 2000).


Table 1. Physical and anesthetic properties of argon, krypton and xenon (from (Koblin et al., 1998; Quillin et al., 2000; Ruzicka et al., 2007)).
