Prediction of soman-induced cerebral damage by distortion product otoacoustic emissions

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Toxicology 277 (2010) 38–48

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Toxicology

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Prediction of soman-induced cerebral damage by distortion productotoacoustic emissions

Pierre Carpentiera,∗, Benoit Pouyatosb, Frédéric Dorandeua, Pierre Campoc,Valérie Baillea, Annie Foquina, Agnès Jobd

a Institut de Recherche Biomédicale des Armées, Département de Toxicologie et Risques Chimiques, 24 Avenue des Maquis du Grésivaudan, BP 87,38702 La Tronche Cédex, Franceb Grenoble Institut des Neurosciences, INSERM U.836, Chemin Fortuné Ferrini, 38700 La Tronche, Francec Institut National de Recherche et de Sécurité, Département Polluants et Santé, Rue du Morvan, BP 27, 54519 Vandoeuvre Cédex, Franced Institut de Recherche Biomédicale des Armées, Antenne de la Tronche, 24 Avenue des Maquis du Grésivaudan, BP 87, 38702 La Tronche Cédex, France

a r t i c l e i n f o

Article history:Received 6 August 2010Accepted 24 August 2010Available online 9 September 2010

Keywords:RatOrganophosphorus compoundsOtoacoustic emissionsConvulsionsBrain damageAtropine/HI-6/Avizafone emergencytreatment

a b s t r a c t

The organophosphorus nerve agent soman is an irreversible cholinesterase (ChE) inhibitor that can pro-duce long-lasting seizures and seizure-related brain damage (SRBD) in which acetylcholine and glutamateare involved. Since these neurotransmitters play a key-role in the auditory function, it was hypothesizedthat a hearing test may be an efficient way for detecting the central effects of soman intoxication. In thepresent study, distortion product otoacoustic emissions (DPOAEs), a non-invasive audiometric method,were used in rats administered with soman (70 �g/kg). Four hours post-soman, DPOAE intensities weresignificantly decreased. They returned to baseline one day later. The amplitude of the temporary dropof the DPOAEs was well related to the severity of the intoxication. The greatest change was recorded inthe rats that survived long-lasting convulsions, i.e. those that showed the highest ChE inhibition in brainand severe encephalopathy. Furthermore, the administration, immediately after soman, of a three-drugtherapy composed of atropine sulfate, HI-6 and avizafone abolished the convulsions, the transient dropof DPOAEs at 4 h and the occurrence of SRBD at 28 h without modifying brain ChE inhibition. This showedthat DPOAE change was not directly related to soman-induced inhibition of cerebral ChE but rather to itsneuropathological consequences. The present findings strongly suggest that DPOAEs represent a promis-ing non-invasive tool to predict SRBD occurrence in nerve agent poisoning and to control the efficacy ofa neuroprotective treatment.

© 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

The organophosphorus (OP) compound, soman (O-1,2,2,-trimethylpropylmethyl-phosphonofluoridate), is a potent andirreversible inhibitor of both peripheral and central cholinesterases(ChEs). This “nerve agent” can cause generalized convulsiveseizures, respiratory distress, cardiovascular disorders and death(McDonough and Shih, 1997). In survivors, the development oflong-lasting seizure activity is related to dramatic brain damage(e.g. McDonough and Shih, 1997; Carpentier et al., 2000). Whilethe initiation of seizures is due to accumulation of acetylcholine(ACh), their maintenance, as well as the build up of seizure-relatedbrain damage (SRBD), is consensually attributed to glutamate (Glu)excitotoxicity (McDonough and Shih, 1997).

∗ Corresponding author. Tel.: +33 476 63 69 85; fax: +33 476 63 69 62.E-mail address: [email protected] (P. Carpentier).

Recent history proves that the potential for exposure to OPsmust still be considered as a major threat in the battlefield (e.g.the Gulf War in 1991) as well as following terrorist attacks (e.g.the Tokyo subway attack in 1995). In case of poisoning, a standardemergency treatment, often packaged in auto-injectors, is avail-able. It is composed of an anticholinergic drug (atropine sulfate: AS),which antagonizes the effect of excess ACh at muscarinic receptors,and an oxime, which acts as a ChE re-activator at the periph-eral level. In some countries, an anticonvulsant benzodiazepine,currently mostly diazepam, is also issued. In France, a new auto-injector has recently been licensed for use. The cartridge containsa freeze-dried combination of three drugs, which are made solublejust before i.m. injection: AS, pralidoxime methylsulfate and a pro-drug of diazepam (pro-diazepam or avizafone: AVZ). In contrastto diazepam, AVZ is water soluble and thus can be freeze-driedand stored in the same cartridge compartment as AS and prali-doxime. In vivo, AVZ undergoes rapid hydrolysis to give diazepam(Maidment and Upshall, 1990; Upshall et al., 1990). Early adminis-tration of the AS/pralidoxime/AVZ combination provides excellent

0300-483X/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.tox.2010.08.014


P. Carpentier et al. / Toxicology 277 (2010) 38–48 39

protection against the mortality, the convulsions and the SRBD pro-duced by even high doses of soman or sarin in rodents and primates(Taysse et al., 2003, 2006; Lallement et al., 2000, 2004). A similarcocktail in which pralidoxime was replaced by another oxime, HI-6, proved to be equally successful (Lallement et al., 1997; Myhreret al., 2006).

Due to the neuropathological effect of soman, there is a cru-cial need to monitor the evolution of SRBD over time and to assessthe neuroprotection afforded by medications. Assessment of SRBDthrough histopathology is time-consuming, requires animal sacri-fice and provides only snapshots of structural changes at a giventime. Above all, this invasive technique is not possible in man.Besides various other tools (clinical observations, analysis of EEGtracings and spectrum, measurement of blood ChE activity, mag-netic resonance imaging: review in Carpentier et al., 2008), we wererecently interested in a non-invasive audiometric method (Kempet al., 1990) based on the measurement of distortion product otoe-missions (DPOAEs). Our interest stemmed from the hypothesis thatsoman might induce changes in hearing that might reflect those inbrain since the same neurotransmitter systems (mainly ACh andGlu) are involved in both hearing and OP-induced seizures andSRBD (see references in Job et al., 2007). Otoacoustic emissions relyon the contractile properties of the cochlear outer hair cells (OHC)which can generate retrograde wave sounds whose intensity canbe captured and recorded by a sensitive microphone inserted intothe external auditory canal. DPOAEs were proved useful to evaluatethe cochlear function in laboratory and clinical settings followingnoise and/or ototoxic injuries (Harris, 1990; Mills et al., 1999; Joband Nottet, 2002; Lataye et al., 2003; Job et al., 2004; Pouyatos etal., 2005).

In a first study performed in rats (Job et al., 2007), DPOAEs weremeasured in a low frequency range (2.3–4.8 kHz) and, immobilitybeing required, under sodium pentobarbital anesthesia. To avoidthe possible influence of repetitive anesthesia, measurements weremade eight day before (baseline) and either 4 h or 24 h after expo-sure to a moderate dose of soman (45 �g/kg). This dose producedvarious symptoms, from almost none to long-lasting convulsions.Interestingly, the pre-soman DPOAE baseline was found to be thelowest in rats that, after the intoxication, displayed the highestbrain ChE inhibition, long-lasting convulsions and SRBD. This firstobservation suggested that DPOAEs might be used as a predictorof susceptibility to soman-induced convulsions and SRBD. The sec-ond main finding was that the intoxicated rats showed a decreaseof DPOAEs at 4 h post-challenge and the greatest drop was againrecorded in the rats displaying the most severe symptoms (i.e.long-lasting convulsions), the highest inhibition of cerebral ChE andextensive SRBD. At 24 h DPOAEs tended to normalize in all the rats.The temporary change of DPOAEs in the acute hours of the intox-ication suggested that this audiometric method might be a usefultool to non-invasively foresee the occurrence of SRBD.

The main purpose of the present study was to confirm thatDPOAEs could actually be predictive in soman poisoning. For this,two approaches were used: First, we investigated if our previousresults could be reproduced in different experimental conditions:(i) the rats were intoxicated with a higher dose of soman (70 �g/kg)to increase the percentage of animals with long-lasting convul-sions; (ii), the upper limit of the studied frequency range wasextended from 4.8 kHz to 12 kHz to reach frequency regions of besthearing in the rats; (iii), gaseous anesthesia with isoflurane was pre-ferred to sodium pentobarbital as it produces slighter anesthesiafollowed by fast recovery thus allowing repetitive pre- and post-challenge measurements in the same animals. Second, we wishedto demonstrate that the suppression of soman-induced seizuresand SRBD could be accompanied by the disappearance of the DPOAEdrop observed 4 h after the intoxication. With this aim, supple-mentary rats were intoxicated and immediately treated with an

anticonvulsant and neuroprotective combination of HI-6, AS andAVZ. In these animals, the impact of the therapy on DPOAEs, brainhistology and blood and brain ChE activities were assessed. Inaddition, the cochlear histology was investigated in some of theintoxicated rats.

2. Materials and methods

2.1. Animals

Adult male Wistar rats (300 g; Janvier, France; n = 126) served as subjects. Ani-mals were housed in a controlled environment (21 ± 2 ◦C; 12 h dark/light cycle withlight provided between 7.00 a.m. and 7.00 p.m.). Food and water were availablead libitum. Procedures were designed in accordance with the regulations regardingthe “protection of animals used in experimental and other scientific purposes” fromthe relevant Directives of the European Community (86/609/CEE). The protocolsreported herein were approved by the ethical committee of our institute.

2.2. Drugs, doses and injection routes

Soman (>97% pure as assessed by gas chromatography) was supplied by theCentre d’étude du Bouchet (Vert-le-Petit, France). Solutions were freshly preparedby diluting the initial solution (2 mg/mL in isopropanol) in ice-cold 0.9% (w/v) saline.Soman was administered subcutaneously at 70 �g/kg (∼0.8 LD50; 500 �L in saline).All the intoxications were performed between 9.30 a.m. and 11.30 a.m. to reducepossible circadian variations of cholinergic parameters (Elsmore, 1981). HI-6 dichlo-ride was generously provided by DRDC Suffield (Canada). Atropine sulfate (AS)and avizafone (AVZ) were purchased from Sigma–Aldrich (Saint Louis, MO, USA)and Neosystem (Strasbourg, France), respectively. The dose and i.m. route for HI-6(42 mg/kg), AS (14 mg/kg) and AVZ (3 mg/kg) were chosen according to a previousstudy on rats by Myhrer et al. (2006). The three drugs were mixed in saline beforeadministration (150 �L) in the hind leg. Isoflurane was from Belamont (France).

2.3. DPOAE

2.3.1. Experimental designThe first group (SOM) of rats (n = 31) was administered with soman only. The

second group (SOMTREAT) of rats (n = 22) was intoxicated and, within 1 min receivedthe treatment mixture composed of HI-6, AS and AVZ. Two control groups wereconstituted with sham-poisoned animals administered with saline (500 �L; s.c.)instead of soman. In the first control group (SAL; n = 13), no supplementary injectionwas performed after saline. In the second control group (SALTREAT; n = 20), therats were administered, 1 min after saline, with the therapeutic cocktail used in theSOMTREAT group.

The clinical observation was continuous for at least 8 h after the intoxication andon the following morning for at least 30 min. In our previous study (Job et al., 2007),the most significant DPOAE changes were detected only in the animals that expe-rienced long-lasting convulsions appearing within minutes after the intoxicationand lasting for at least the following 4 h. Therefore, in the present study, only twosymptom subgroups were considered in the SOM group and SOMTREAT group: ratswithout long-lasting convulsions (with either no signs at all, or mild symptoms, orrare and brief convulsive episodes within the first hour post-soman) noted C− andrats with long-lasting convulsions noted C+. All animals were weighed 1 h beforeand 24 h after the administration of soman or saline.

2.3.2. AnesthesiaAnesthesia was induced by placing the animals for approximately 3–5 min in a

Plexiglas box receiving 5% isoflurane in air at 0.3 L min−1 and then maintained (viaan adapted mask) with 1.5% isoflurane in air throughout the acquisition of DPOAEs.After the end of the anesthesia, the rats recovered apparently normal behaviorwithin 10–25 min.

2.3.3. DPOAE measurementsDPOAEs were measured in a thermoregulated and silent room using the DPOAE

GSI system (Grason-Stadler, Milford, NH, USA). During anesthesia, the probe wascarefully inserted into the right auditory canal via a neonatal tip, which allowedtight adaptation into the external auditory canal.

To avoid any circadian variations, all DPOAE measurements were always per-formed between 13:30 h and 16:30 h. Baseline DPOAEs were measured 24 h beforesoman (or saline in controls). Post-soman (or post-saline in controls) DPOAEs wereperformed on each animal 4 h and 28 h post-challenge. DPOAEs were investigatedin a frequency range covering from 2601 Hz to 9832 Hz for f1 and from 3175 Hz to11988 Hz for f2 over 24 frequencies.

Conditions of measurements were selected from pilot experiments on control(saline-treated) rats. In these, DP-grams were obtained from each animal usingthree different protocols of stimulation (expressed in decibel sound pressure level:dB SPL): 70/70 dB SPL; 60/50 dB SPL; 55/35 dB SPL with either 5 runs (n = 6) or 3runs (n = 14) applied in succession in the same animal at the three different timepoints. For each stimulation protocol, no significant change was ever detected in


40 P. Carpentier et al. / Toxicology 277 (2010) 38–48

Fig. 1. Mean DP-grams in control Wistar rats anesthetized with isoflurane. The dia-grams show the mean (±SEM) DPOAE levels in response to five runs using a 60/50 dBSPL protocol applied from 3.2 kHz to 11.9 kHz (f2) to the right ear of six rats. The threeupper curves represent the average responses obtained 24 h before the administra-tion of saline and 4 h and 28 h after in the same animals, respectively. The curves arealmost indistinguishable from each other (with responses below 15 dB SPL under7 kHz followed by a sharp increase at higher frequencies, exceeding 30 dB SPL fromabout 9 kHz and above) evidencing the good reproducibility of the measurementsin spite of the succession of runs and anesthesia. Also note the good separation fromthe noise floor (NF).

the DP-grams (Wilk’s Lambda test) evidencing an excellent reproducibility of themeasurements and proving that neither the repetition of the stimulations nor thesuccession of three periods of anesthesia over a two-day-interval exerted majorinfluence on the DPOAEs. The stimulus protocol 60/50 dB SPL, with a ratio f2/f1 setat 1.22, was finally selected because it showed reproducible responses and goodseparation from the noise floor (Fig. 1).

In the final experiments, three successive DP-grams were collected for each rat ateach time point. In a first approach, the average response for the 24 frequencies wascalculated. However, since DPOAE change appeared restricted to the 3.5–5.0 kHzregion (see the Section 3), a secondary calculation was performed limited to the 10frequencies that covered this low frequency range.

After the second post-challenge DPOAE measurements at 28 h the animals thatwere not kept for histology (see Section 2.4) were euthanized with an i.p. adminis-tration (500 �L) of Dolethal® (sodium pentobarbital, 182.2 mg/mL; Vétoquinol SA,Lure, France).

2.4. Brain histology in the SALTREAT and SOMTREAT groups

Four rats from the SALTREAT group and six from the SOMTREAT group were ran-domly selected after the 28-h DPOAE measurement. They were deeply anesthetizedwith sodium pentobarbital (80 mg/kg; i.p.) and transcardially perfused with hep-arinized saline followed by fixative (formaldehyde 4%; acetic acid 3%). The perfusedanimals were kept for at least 1 h at 4 ◦C. The brain was then removed, post-fixed(formaldehyde 4%) and processed for embedding in paraffin. Coronal sections werestained with hemalun-phloxin (H&P) for the detection of eosinophilic (damaged)cells. Four coronal planes were chosen from a stereotaxic atlas of the rat brain(Paxinos and Watson, 1986) for light microscopy examination. They contain brainstructures known to be especially sensitive to convulsive doses of soman (Carpentieret al., 1990) such as the hippocampus, the amygdala, the thalamus and the cortices(coronal plane: −3.8 mm posterior to the bregma) but also structures that belong tothe auditory pathways, namely the auditory cortex (bregma −3.8 mm), the medialgeniculate body (bregma −5.8 mm), the ventral and dorsal cochlear nuclei and theinferior colliculus (bregma −9.3 mm and −11.3 mm). Observations were comparedto those previously made in intoxicated rats receiving no emergency treatment (Jobet al., 2007).

2.5. Cochlear histology in the SOM group

The effects of soman on cochlea were explored. Deep anesthesia with sodiumpentobarbital (80 mg/kg; i.p.) was induced at the end of the DPOAE experimentin four rats of the SOM group (2 C+ and 2 C−) and in two controls from the SALgroup. The rats were then fixed by transcardial perfusion with a brief flush of0.08 M sodium cacodylate buffer (pH 7.4) followed by 300 mL of a fixative solutioncomposed of 3% glutaraldehyde, 2% formaldehyde, 1% acroleine and 2.5% dimethyl-sulfoxide (DMSO) in 0.08 M sodium cacodylate buffer. Then, the tympanic bullaewere removed, opened and the cochlea extracted. Post-fixation was carried out byslow perilymphatic perfusion of the tri-aldehydic fixative mixture through the fen-estra. Following the primary 24-h fixation, cochleae were washed in the sodium

cacodylate buffer, post-fixed 1 h in OsO4 2%, washed again and finally stored in 70%ethanol. The samples were then sent to the Institut National de Recherche et deSécurité (Nancy, France) for further treatment and analysis. The right cochleae weredissected in 70% ethanol at room temperature. The trimmed tissue was mounted onmicroscope slides. The organ of Corti was dissected away in three pieces, mountedin glycerine and surface-observed through a light microscope. The left cochleaewere immersed at room temperature in 0.7 M ethylene diamine tetraacetic acid(pH 7.8) for 8 h, then dehydrated in ascending concentrations of ethanol up to 100%,and finally infiltrated with resin (Epon/araldite). Semi-thick (2.5 �m) sections werestained with cresyl violet and observed through light microscopy.

2.6. Blood and brain ChE assays in SOMSAL and SOMTREAT groups

To test the effect of the HI-6/AS/AVZ treatment on soman-induced inhibitionof blood and brain ChE, an independent experiment was undertaken in which sup-plementary rats were intoxicated and treated with the three-drug therapy as in theDPOAE study (SOMTREAT group; n = 20). Other animals were also administered withsoman but received saline instead of the treatment (SOMSAL group; n = 20).

In all these animals, retro-orbital blood samples were drawn under short anes-thesia (5% isoflurane in 0.3 L min−1 air) 24 h prior to soman and 4 h post-intoxicationusing heparinized capillaries. The samples were immediately diluted (1/10) insaponin (1% in distilled water), frozen in liquid nitrogen and stored at −80 ◦C. Forbrain ChE assay, the rats were sacrificed by decapitation 4 h after the intoxication.The brains (without the cerebellum) were immediately homogenized in Tris buffer(pH 7.4)/saccharose 0.32 M and centrifuged (15 min, 100 × g). Assays were per-formed in duplicate (whole blood) and triplicate (brain) on supernatants accordingto the method of Ellman et al. (1961) using an automated procedure (Hitachi 704; KitReagent MPR2 124117, Roche Molecular Biochemical) with final acetylcholine con-centration set at 0.95 mM and absorbance measured at 412 nm. Average whole bloodChE activity was expressed in IU/L and post-soman inhibition was expressed as a per-cent of the pre-soman activity in the same animals. Average brain ChE activity wasexpressed in nmol min−1 mg−1 protein (proteins were quantified using the Pierce®

BCA protein Kit 23227, Pierce Biotechnology, Rockford, IL, USA) and post-somaninhibition was expressed as a percent of the average control activity measured in thesham-treated rats (n = 26; 206 ± 19 nmol min−1 mg−1 protein) used in our previouswork (Job et al., 2007).

2.7. Statistics

Data are presented as mean ± SEM. Statistical analyses were performed usingSPSS v.10.0 software (Chicago, IL). The level of significance was set at p = 0.05 for allthe tests.

Multivariate analyses of variance (MANOVA) with a between-subject factor“groups” and a within-subject factor “frequencies” were used for comparing DPOAEintensities in the various groups and subgroups at the different time points (SAL vsSOM-C− vs SOM-C−; SAL vs SALTREAT; SALTREAT vs SOMTREAT). Homogeneity ofvariance was checked with Levene’s test.

The 24-h-survival rate in SOM group was compared to that in the SOMTREATgroup using a 2 × 2 cross table. Statistical significance was tested by Fisher exact test(one tail).

The average loss of the body weight (as a percent of the pre-challenge bodyweight) recorded 24 h post-challenge in the different groups and subgroups werecompared using an analysis of variances (ANOVA). Homogeneity of variances waschecked with Levene’s test. Bonferroni post hoc test was used to specify whichgroups were statistically different from the others.

Due to variance inequality, soman-induced ChE inhibition (expressed as % ofcontrol) in either the whole blood or the brain of the SOMSAL group was comparedto those of the SOMTREAT group using the Mann and Whitney test.

3. Results

3.1. Clinical observations and body weight loss

In the SAL group, the animals recovered very fast after each ofthe three periods of anesthesia. They all survived the experimentsand did not lose weight (Table 1).

In the SOM group (n = 31), the intoxication with 70 �g/kgproduced toxic signs in all animals (chewing, hypersalivation,prostration, fasciculation, tremors). However, 13 rats (41.9%) ratsdid not experience long-lasting convulsions and were gatheredin one SOM-C− subgroup for further analysis. All these animalssurvived the experiment, recovered relatively well and showedonly a non-significant loss of body weight at 24 h (Table 1). Incontrast, 18 rats (58.06%) displayed long-lasting convulsions andconstituted a SOM-C+ subgroup. In almost all of them, convulsionsstarted 5–30 min post-challenge. Respiratory distress and proba-



Table 1Clinical effects of soman (70 �g/kg) in the DPOAE experiment.

Groups Loss of body weight at 24 h (% ± SEM) Number of deaths at 28 h (%)

SAL (n = 13) −0.1% ± 0.3 (n = 13) 0 (0%)SOM (n = 31) Total (n = 31) −10.2% ± 1.5a (n = 25) 8 (25.8%)

SOM-C+ (n = 18) −17.4% ± 0.3b (n = 12) 8 (44.4%)SOM-C− (n = 13) −3.6% ± 0.9 (n = 13) 0 (0%)

SALTREAT (n = 20) −1.8% ± 0.9 (n = 20) 0 (0%)SOMTREAT (n = 22) −7.5% ± 1.1c (n = 20) 2 (9.09%)

SAL: rats administered with saline; SOM: rats intoxicated with soman; SOM-C+: intoxicated rats with long-lasting convulsions; SOM-C−: intoxicated rats with short-lastingconvulsions; SALTREAT: rats administered with saline and HI-6/AS/AVZ; SOMTREAT: rats administered with soman and HI-6/AS/AVZ. The loss of body weight measured at24 h post-challenge with soman (or saline) is expressed as a percent of the body weight measured 1 h before administration of saline or soman.

a Not statistically tested.b Body weight in the SOM-C+ subgroup significantly different from that in the SAL, SALTREAT and SOMTREAT groups (p < 0.001).c Body weight in the SOMTREAT group significantly different from that in the SAL and SALTREAT groups (p < 0.001) and from that in the SOM-C− subgroup (p < 0.05). The

difference of mortality between the SOM and the SOMTREAT groups does not reach statistical significance.

ble pulmonary edema (as suggested by the presence of bloodymouth secretion) induced the death of six animals within thefirst 3 h. In survivors, convulsions lasted for hours (except duringthe anesthesia performed at 4 h for the first post-soman DPOAEmeasurement). Afterwards, the animals progressively fell into anapparently comatose state. One day later, the survivors displayeddacryohemorrhea, were unable to feed and to move properly and,compared to the SAL group, showed a highly significant (p < 0.001)drop in body weight (Table 1). Two additional animals died beforethe second post-challenge DPOAE acquisition (Table 1).

In the SALTREAT group (n = 20), administration of the tri-therapy(HI-6/AS/AVZ) produced rapid loss of righting ability and the ratsthen stayed immobile for 1–2 h. Four animals experienced res-piratory disorders with labored breathing and throat rattle thatcould last until the first DPOAE measurement. Afterwards, respi-ration normalized and the rats progressively became more alert.All the animals survived and showed apparently normal behaviorand minimal loss of body weight (Table 1).

In the SOMTREAT group (n = 22), the symptoms noted afterthe administration of soman followed by the tri-therapy cocktailappeared very different from those observed in the SOM groupon at least three points: (i) none of these animals showed pro-fuse salivation, (ii) none experienced any convulsions, and (iii) the28-h mortality was reduced, although not significantly, from 25.8%(SOM group) to 9.0% (Table 1). However, eight rats showed severerespiratory disorders. Two of them died within the first 4 h of theexperiment while the six others exhibited noisy breathing for hoursand showed poor clinical status at 24 h (prostration, eye bleed-ing). The remaining animals showed lighter clinical responses: afteran initial 20–120 min phase of fasciculations, tremors, and loss ofrighting ability, they remained ataxic for a supplementary dura-tion of about 30–120 min. Afterwards, they slowly recovered andwere found in good, almost normal clinical status the day after.In this group, the average loss of body weight at 24 h signifi-cantly exceeded the ones in the SAL, SALTREAT and SOM-C− groupsbut appeared significantly lower than that of the SOM-C+ animals(Table 1).

3.2. DPOAE measurements in the SOM group

3.2.1. Baseline DPOAEs in relation to post-somansymptomatology

As illustrated in Fig. 2, rats of the SOM-C+ subgroup exhibitedbaseline DPOAEs that tended to be lower (about −3 dB SPL) thanthose of the SOM-C− animals. This difference, which did not reachstatistical significance, was detectable only within the lowest fre-quency range (3.0–5.5 kHz).

3.2.2. Post-challenge DPOAE changesFour hours after the intoxication, a decrease, limited to the

3.0–5.5 kHz frequency range, of the DPOAE responses was recordedin the SOM group compared to the pre-soman baseline. The DPOAEdrop was the smallest (about 4 dB SPL) in the SOM-C− rats and thegreatest (about 7 dB SPL) in the SOM-C+ rats (Fig. 2). The DPOAElevels appeared significantly lower than those observed in the SALgroup in which there were no detectable changes at the same timepoint (MANOVA; F(2.35) = 7.87; p = 0.002). At the 28 h time point,DPOAEs in the SOM group no longer differed from those in the SALgroup (Fig. 2).

3.3. DPOAE measurements in the SOMTREAT group

Although the noise floor was increased by the noisy breath-ing induced by the treatment (Section 3.1), DPOAE measurementsin the SALTREAT and SOMTREAT groups were achieved inalmost all the animals. Whatever the time point, no statis-tical difference of the DPOAE responses was ever detectedbetween either the SALTREAT group and the SAL groupor between the SOMTREAT group and the SALTREAT group(Fig. 3).

3.4. Brain histology in the SALTREAT and SOMTREAT groups

In the four coronal planes examined, no eosinophilic cellswere ever detected in any brain structures of the SALTREATor SOMTREAT rats sacrificed 28 h post-challenge (Fig. 4).These included areas that are usually damaged after a con-vulsive dose of soman (the hippocampus, the amygdala, thethalamus, the cortices) and other structures that are compo-nents of the central auditory pathways (mainly the medialgeniculate body, the inferior colliculus, the ventral and dor-sal cochlear nuclei, the lateral lemniscus and the superiorolive).

3.5. Cochlear histology in the SAL and SOM groups

In rats, sacrificed 28 h post-challenge, from either the SALgroup, or the SOM-C− and the SOM-C+ subgroups, surfaceexamination and classical histology did not reveal any alter-ation of either the inner or outer hair cells of the organ ofCorti (Fig. 5a–d). The only detectable damage was observedwithin the spiral ganglion of the SOM-C+ group (Fig. 5e–f).The damaged cells, in which the nucleus was sometimes notvisible, appeared strongly retracted and surrounded by large vac-uoles.



Fig. 2. DPOAEs recorded in the SAL group (non-intoxicated controls) and in the SOM-C− (intoxicated rats with short-lasting convulsions) and SOM-C+ (intoxicated rats withlong-lasting convulsions) subgroups. The left column shows the complete DP-grams averaged from three runs over 24 frequencies from about 3.0 kHz to 12 kHz (f2). The rightcolumn shows the average DPOAE response calculated over the 10 lowest frequencies (3.0–5.5 kHz). (a and d) Show that baseline DPOAE responses within the 3.0–5.5 kHzrange were lowest in the SOM-C+ subgroup. (b and e) Show the significant decrease (MANOVA test; p < 0.01) of the DPOAE responses recorded within the same low frequencyrange 4 h after soman, the DPOAE drop being the greatest in the SOM-C+ subgroup. (c and f) show that DPOAEs normalized 24 h after the intoxication.

3.6. Whole blood and brain ChE activities in SOMSAL andSOMTREAT groups 4 h post-challenge

Clinically, post-soman symptoms in the SOMSAL group weresimilar to those observed in the SOM group used in the DPOAE

experiment (see Section 3.1). Thus a majority (65%) of the animalsexperienced long-lasting convulsions constituting a SOMSAL-C+subgroup (Table 2). The other SOMSAL rats (35%) did not showlong-lasting convulsions and were gathered in a SOMSAL-C− sub-group. In the great majority of the SOMTREAT rats (85%), symptoms



Fig. 3. DPOAEs recorded in the SALTREAT group (controls administered with saline and HI-6/AS/AVZ) and in the SOMTREAT group (rats administered with soman andHI-6/AS/AVZ). As in Fig. 2, the left column shows the complete DP-grams and the right column the average DPOAE responses calculated within the 3.0–5.5 kHz frequencyrange. The diagrams show that, in the presence of the three-drug therapy, no significant change of the DPOAE responses could be observed in the two groups 4 h or 28 hpost-challenge. Note the difference in the scale of the Y-axis in (d–f) as compared to Fig. 2d–f.

were also comparable to those described in the equivalent groupof the DPOAE study (see Section 3.1). However, in contrast withthe previous experiment in which no animals experienced long-lasting convulsions, 15% of the present rats displayed obvioussigns of fully developed convulsive activity. Hence, as shownin Table 2, two subgroups (SOMTREAT-C+; SOMTREAT-C−) were

considered that did not exist in the previous DPOAE experi-ment.

In whole blood, pre-soman ChE control activity (Table 2) wassimilar to that established in our former work (Job et al., 2007). Inthe SOMSAL group, an average inhibition of almost 78% was mea-sured 4 h after soman administration with no significant difference



Fig. 4. Light micrographs (H&P stain) of the dorsal hippocampus (CA1 sector) (A–C), the piriform cortex (D–F), the medial geniculate nucleus (G–I), the inferior colliculus(J–L) and the ventral cochlear nucleus (M and N) from rats belonging to different experimental groups: left column (A, D, G, J, M) and central column (B, E, H, K, N): brainstructures from rats of the SALTREAT group (controls administered with saline and HI-6/AS/AVZ) and the SOMTREAT group (rats administered with soman and HI-6/AS/AVZ),respectively: in both groups, no damage was ever observed in any of the examined regions. For comparison, the microphotographs of the right column (C, F, I, L, O) show thehistopathological consequences of soman-induced long-lasting convulsions in the same structures of rats sacrificed 24 h after administration of soman in the absence of anytreatment. In these animals from our previous work (Job et al., 2007), note that, although a lower dose of soman was used (45 �g/kg vs 70 �g/kg in the present study), thedorsal hippocampus (C), the piriform cortex (F), and the medial geniculate nucleus (I) were profoundly damaged (dramatic edema, numerous eosinophilic and condensedcells (arrows)). By contrast, the inferior colliculus (L) and the ventral cochlear nucleus remained free of any lesions. cc: corpus callosum; pyr: pyramidal layer of hippocampus(CA1); pir: piriform cortex (pyramidal layer); amg: amygdala.

between the SOMSAL-C+ subgroup and the SOMSAL-C− subgroup(Table 2). In the SOMTREAT group, ChE inhibition (about 69%) wassignificantly lower (p < 0.001) than in the SOMSAL group (Table 2).

Compared to controls, brain ChE inhibition in the whole SOM-SAL group was about 83% 4 h after the administration of soman(Table 2). The inhibition was significantly higher by 16% in the

SOMSAL-C+ subgroup compared to the SOMSAL-C− subgroup. Thebrain ChE inhibition measured in the whole SOMTREAT groupwas not significantly different from the whole SOMSAL groupevidencing that the three-drug therapy had no major influenceon soman-induced brain ChE inhibition. In the SOMTREAT group,the probable relationship between a larger central inhibition and



Fig. 5. Light micrographs of the cochlea. (a–d) Surface preparations (a, b;×20) and semi-thin sections (c, d) of the organ of Corti from animals sacrificed 28 h after administrationof either saline (SAL group, a, c) or soman (SOM-C+ subgroup with long-lasting convulsions, b, d): no cellular alterations of any sort could be detected in either case. (e–g)Sections of the spiral ganglion of rats from the SAL group (control administered with saline; e; ×5) and the SOM-C+ subgroup (intoxicated rats with long-lasting convulsions;f, g; ×5): note the absence of damage in the SAL animal (e) and the presence of a few (f) or numerous (g) retracted cells, some having lost their nucleus and with clearlydamaged cytoplasm, surrounded by large vacuoles (arrows) in two SOM-C+ rats. OHC: outer hair cells; IHC; inner hair cells.

Table 2Whole blood and brain ChE activities 4 h after soman administration (70 �g/kg).

ChE sites Groups Subgroups n Control ChE activity Post-treatment ChE activity (% inhibition)

Blood SOMSAL Total 19 2538.4 ± 53.7 563.4 ± 19.0 (77.8% ± 0.5)C+ 12 2559.5 ± 62.6 592.5 ± 24.6 (76.9% ± 0.5)C− 7 2502.1 ± 103.8 513.5 ± 19.6 (79.3% ± 0.9)

SOMTREAT Total 19 2356.5 ± 69.8 719.4 ± 32.4 (68.7% ± 1.7b)C+ 3 2255.0 ± 216.5 690.0 ± 55.6 (69.1 ± 2.0)C− 16 2375.6 ± 74.9 725.0 ± 37.5 (68.69% ± 2.1)

Brain CONTR – 26 206.0 ± 19 –SOMSAL Total 18 – 34.7 ± 4.4 (83.1% ± 2.1)

C+ 12 – 24.1 ± 3.4 (88.2% ± 1.3c)C−a 6 – 55.8 ± 4.3 (72.8% ± 2.1)

SOMTREAT Total 16 – 40.3 ± 7.4 (80.4% ± 3.6)C+ 3 – 12.4 ± 3.3 (93.7% ± 1.5)C−a 13 – 46.8 ± 8.1 (77.4% ± 4.0)

Blood ChE: activity expressed in IU/L; post-challenge inhibition in percent of pre-challenge activity. Brain ChE: activity expressed in nmol min−1 mg−1 protein; post-challengeinhibition as a percent of control activity in sham-treated rats. SOMSAL: rats administered with soman and saline; SOMTREAT: rats administered with soman and HI-6/AS/AVZ;CONTR: sham-treated rats from our former work (Job et al., 2007).

a For technical reasons, one measurement of the brain ChE inhibition in the SOMSAL group and three in the SOMTREAT group were discarded from the statistics.b Blood inhibition in the whole SOMTREAT group significantly different from that in the whole SOMSAL group (p < 0.001).c Brain inhibition in the SOMSAL-C+ subgroup significantly different from that in the SOMSAL-C− subgroup (p < 0.001). Due to the anticonvulsant effect of the tri-therapy

and the low number of C+ animals, brain inhibition in the SOMTREAT-C+ subgroup could not be compared to that in the SOMTREAT-C− subgroup.



initiation of convulsions cannot be studied owing to the use ofthe anticonvulsant therapy. Moreover, only three animals showedlong-lasting convulsions and were classified in the SOMTREAT-C+subgroup (against 13 in the SOMTREAT-C− subgroup). Therefore,no statistical comparison could be performed.

4. Discussion

The present study describes the effects of a convulsive doseof soman in the absence or the presence of treatment composedof HI-6, AS and AVZ, components of the next generation Frenchauto-injector for emergency self-treatment of nerve agent poison-ing. We report the impact of intoxication and therapy on cochlearfunction (as monitored by DPOAEs) and histology as well as onclinical symptoms, cerebral histology, and ChE inhibition in bloodand brain. We confirm that DPOAEs can be considered as a predic-tive tool for soman-induced SRBD and provide the first evidence ofsoman-induced cochleotoxicity.

4.1. Clinical and histological effects of soman in the absence ofany therapy

As expected, the use of a 70 �g/kg s.c. dose of soman producedmore severe clinical effects compared to those previously observedwith 45 �g/kg in the same rat strain (Job et al., 2007). For instance,in the DPOAE study, the 24-h-mortality rate increased from 5% tomore than 25% in the SOM group. As in other studies on soman(e.g. Rickett et al., 1986; Carpentier et al., 1994, 2000), acute deathwas related to extreme respiratory distress. The percentage of ratsthat showed long-lasting convulsions also augmented from 18%to 58% (SOM group in the DPOAE experiment) or 65% (SOMSALgroup in the ChE experiment). The observed convulsions appearedwell related to actual EEG seizures owing to the body weight lossmeasured at 24 h, a recognized indicator of long-lasting seizureactivity (McDonough et al., 1989; Carpentier et al., 2000; Job etal., 2007). Thus, the survivors of the SOM-C+ subgroup showed themost important loss in body weight which appeared almost identi-cal whether the animals were given 45 �g/kg of soman (−16.5%; Jobet al., 2007) or 70 �g/kg (−17.4%; the present study). This suggeststhat long-lasting convulsions, more than the dose that producesthem, are the preponderant factor for soman-induced debilitation.

In the present study, brain histology was not studied in the SOMgroup. However, since long-lasting convulsions and the accom-panying severe loss of body weight are always associated withcerebral lesions in soman poisoning (e.g. McDonough et al., 1989;Carpentier et al., 1990, 2000), it is reasonable to believe that braindamage also occurred in the present SOM-C+ animals with topo-graphical distribution similar to that observed in our previous study(Job et al., 2007).

In all animals of the present SOM group, including those of theSOM-C+ subgroup, outer or inner hair cell loss was never found inthe organ of Corti. However, the spiral ganglion – i.e. the first extra-cochlear relay of the afferent auditory pathway – showed a numberof damaged cells in the SOM-C+ rats. Indeed, these lesions, in addi-tion to those observed in the cortical and thalamic parts of theauditory system (Job et al., 2007), may induce hearing dysfunctionafter intoxication by a convulsive dose of soman.

4.2. Clinical and histological effects of soman in the presence ofHI-6/AS/AVZ

Administration of the three-drug therapy in naive rats(SALTREAT group) was followed by marked, although transient,motor incapacitation and respiratory disorders. These effects areprobably due to AVZ since benzodiazepines are known to causemotor and respiratory impairments (e.g. Taysse et al., 2003, 2006).

Compared to the intoxicated groups receiving no medication(SOM group, SOMSAL group), the percentage of rats with long-lasting convulsions, the number of deaths and the loss of bodyweight in the SOMTREAT groups were considerably reduced. Sim-ilar protection has been reported in several animal models (mice,rats, guinea-pigs, non-human primates) of soman or sarin intox-ication (Lallement et al., 1997, 2004; Taysse et al., 2003, 2006;Myhrer et al., 2006). However, the protection did not appear com-plete since some animals did convulse (ChE study) and could showprofound respiratory disorders and incomplete clinical recoveryat 24 h, including a body weight loss exceeding that observed incontrols. Subsisting respiratory debilitation after the emergencythree-drug treatment in the intoxicated rats could be again, as innaïve rats, attributed to the presence of AVZ. In contrast with ASthat tends to counteract soman-induced respiratory changes (Kassaand Fusek, 1998, 2000; Carpentier et al., 1994, 2000; Taysse et al.,2006), AVZ is known to potentiate the central depressant action ofthe nerve agent (Taysse et al., 2006). On the other hand, no SRBDwas ever detected in the brain of the SOMTREAT rats. Interest-ingly, the same treatment administered to rats three times within30 min after intoxication by doses of soman higher than those in thepresent study (3 × LD50 or 4 × LD50) was successful in protectingthe hippocampal subfield CA1, but failed to protect other sensitivestructures such as the amygdala or the piriform cortex (Myhrer etal., 2006). The neuroprotective efficacy of the tri-therapy thereforeappears strongly dependent upon the severity of the intoxication.As shown in various experiments on rats or guinea-pigs, the pres-ence and dose of AS are very important factors for AVZ to fullyexpress its anticonvulsant and neuroprotective activities in somanpoisoning (Clement and Broxup, 1993; Taysse et al., 2006). All in all,our findings show that the association HI-6/AS/AVZ provided excel-lent protection against the lethal, convulsive and neuropathologicalconsequences of soman administration and must be favorably con-sidered as an emergency treatment in the medical managementof OP poisoning (Lallement et al., 1997, 2004; Taysse et al., 2003,2006).

4.3. DPOAE studies

4.3.1. Baseline DPOAEs in the SOM groupAs previously (Job et al., 2007), the data obtained in the present

study show that the lowest baseline DPOAEs within the 3–5.5 kHzfrequency range 24 h prior to the intoxication were those which,after the intoxication, experienced long-lasting convulsions, SRBDand the highest inhibition of brain ChE. In the previous study, base-line DPOAEs of rats with long-lasting convulsions were significantlydifferent from those that showed the weakest symptoms but notsignificantly different from those with intermediate symptoms.In the present study, the difference between baseline DPOAEs inSOM-C+ and SOM-C− animals did not reach statistical significance.Indeed, differences in individual susceptibility to poisoning wereless prominent owing to the higher dose of soman and the sub-sequent global increase of the intoxication severity. Nevertheless,the present study supports our previous proposition that DPOAEsmight serve as a predictor of susceptibility to soman exposure andmight help detecting individuals that are the most sensitive tosoman poisoning and the most prone to develop severe convul-sions and SRBD after a low or mild dose of soman. Moreover, itwas recently demonstrated that low DPOAEs in otherwise normalhearing ears represented a risk marker for acute acoustic traumasubsequent to noise exposure (Job et al., 2009). Therefore, DPOAEability to simultaneously predict the vulnerability of a subject tonoise exposure and to the central effects of soman might appearuseful for soldier selection since, in a war context, impulse noisedue to firearms may be associated to nerve gas exposure.



4.3.2. Post-challenge DPOAEs in the SOM groupAt the 4 h time point, DPOAE responses within the 3–5.5 kHz

frequency range were decreased in the SOM group compared tocontrols. The amplitude of decrease was not significant in theSOM-C− rats but highly significant in the SOM-C+ animals whichexperienced long-lasting convulsions, the largest inhibition of brainChE and SRBD. In spite of different experimental conditions (anes-thesia, dose of soman. . .), the present results fully reproduced thoseobserved at the same time point in our previous work (Job et al.,2007). They strengthened the conclusion that DPOAEs measuredwithin the early hours of soman intoxication might serve as a non-invasive tool allowing to predict SRBD occurrence. At the 28 h timepoint, DPOAEs in the SOM-C+ subgroup recovered control levelsevidencing that the drop seen at the 4 h time point was a transientphenomenon, the duration of which remains to be determined.

Remarkably, soman-induced decrease of DPOAE response waslimited to the lowest frequency range (about 3–5.5 kHz) withinwhich the hearing of rats is rather poor and the size of the DPOAEresponses is extremely low relative to the levels that are gener-ated at higher tone frequencies (see Fig. 1). The higher frequencyzone around 12 kHz is the region of best hearing in the rat but isalso especially vulnerable to a variety of factors (e.g. age, noise) andhearing pathologies (Emmerich et al., 2005). Therefore, the fact thatsoman-induced DPOAE changes were limited to the low frequencyrange, within which interactions with changes from other sourceswould be minimal, could be considered as a potential advantagewith the view to use DPOAEs as a specific diagnostic tool for OPpoisoning.

The mechanisms responsible for the acute decrease of theDPOAE responses within the first hours of soman poisoning remainto be determined. The irreversible damage detected in the cere-bral auditory pathways and spiral ganglion cells could hardly beconsidered as the cause of a typically transient event such asthe observed DPOAE changes. Possible causes may involve (i) theblockade of cholinergic receptors on the middle ear muscles thatmove the tympano-ossicular chain, (ii) the impact of soman onthe cholinergic, glutamatergic and/or GABAergic systems involvedin the auditory function at the central and cochlear levels (Puelet al., 2002; Paludetti et al., 2001; Neill et al., 2005; Pujol et al.,1985, 1986), (iii) changes of the ionic status of cochlear fluids,(iv) the hypothermia induced by soman (Clement, 1993) sinceDPOAEs were shown to decrease during hypothermia (Borin andCruz, 2008), or (v) the seizure activity which, although declining,is still present at the 4 h time point (e.g. Carpentier et al., 1990,2000). However, in contrast to seizure activity which is abolishedby anesthetics (e.g. Testylier et al., 2007), the DPOAE change wasstill observable under isoflurane (the present study) or pentobar-bital (Job et al., 2007), suggesting that the two phenomena aresomewhat independent. In case of anesthesia, this constitutes atheoretical advantage for DPOAE measurement over EEG recordingas a potential marker of SRDB.

4.3.3. Post-challenge DPOAEs in the SOMTREAT groupThe transient decrease of the DPOAE response observed 4 h

after soman intoxication in the SOM group was not detected inthe SOMTREAT rats that did not experience long-lasting convul-sions and were free from any brain damage. The present studyalso showed that brain ChE inhibition in this group was unchangedcompared to that measured in the SOM group. The latter obser-vation is not surprising since neither AS nor AVZ have knowninteraction with ChE. Besides, HI-6 can re-activate OP-inhibitedChE at the periphery as confirmed by the decrease of ChE inhibitionobserved in the present study in whole blood. However, this oximeis unable to re-activate cerebral ChE in spite of some brain pene-tration (Cassel and Fosbraey, 1996). All in all, the findings obtainedfrom the SOMTREAT group help to specify the conclusions drawn

from the SOM group: DPOAEs do not seem directly dependent uponbrain ChE inhibition, but are rather related to the main consequenceof this inhibition, i.e. the SRBD. DPOAEs might therefore be a usefulnon-invasive tool to assess the neuroprotective efficacy of a medicaltreatment.

4.4. Additional comments on the ChE study

In the present SOMSAL group, whole blood ChE inhibition mea-sured 4 h after the administration of 70 �g/kg of soman (77.8%) isvery close to that previously found (Job et al., 2007) with 45 �g/kg(78%). This observation is reminiscent of that of Jimmerson et al.(1989) showing that all doses of soman from 33 �g/kg to 99 �g/kgrapidly induced an inhibition of plasma and erythrocyte ChE activ-ities that equally exceeded 70% in all cases. According to theseauthors, “above a certain threshold of blood concentration suffi-cient to saturate the available binding sites in the blood (the variousesterases), augmenting the dose of soman does not further increaseblood ChE inhibition”. This also explains why, in our present andprevious studies (Job et al., 2007) no difference in whole blood inhi-bition was ever detected between C+ and C− rats. Indeed, wholeblood and plasma ChE activities are most often considered as anindex of exposure to ChE inhibitors rather than as an index ofcentral toxicity for which red blood cell AChE would have pro-vided a better indicator (Lotti, 1995; Jimmerson et al., 1989; Younget al., 2001). Confirming other studies (Job et al., 2007 and refstherein), brain ChE inhibition induced by soman 4 h after intoxica-tion was well correlated to the symptom severity and remains thebest biomarker of soman neurotoxicity. Unfortunately, such a mea-surement is invasive and therefore impossible for use in humans.

5. Conclusions

The present study strengthens the results of our previous work(Job et al., 2007) and provides supplementary evidence suggestingthat DPOAEs may become a novel non-invasive tool to predict thecentral neuropathological effects of soman and may serve to assessthe neuroprotective efficiency of a medication. Further experi-ments remain to be performed to more firmly establish the realvalue of the DPOAE technique in experimental fields or clinical set-tings. For instance, there is a crucial need to define the duration ofthe transient post-soman decrease of the DPOAE response and totest the method in other animal models and under various otherintoxication paradigms. In addition, the present work reports, forthe first time, evidence of OP-induced cochlear neurotoxicity andconfirms the interest of the HI-6/AS/AVZ cocktail as an emergencymedication for OP poisoning.

Conflict of interest statement

The authors declare that there is no conflict of interest.

Acknowledgments

This work was supported by grants from the Direction Généralede l’Armement (DGA; Contract No. 08co502). The authors wouldlike to thank the Service of Microscopy (Dr. E. Gentilhomme, Mr.C. Castellarin) and the Biological Laboratory of our Institute (Dr.A. Alonso, Ms. J. Denis) for helpful technical expertise in histologyand cholinesterase activity measurement, respectively. We wish tothank Dr. F.J. Hemming for correction of the English manuscript.

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Prediction of soman-induced cerebral damage by distortion product otoacoustic emissions

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