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The Antiapoptotic Effect of Remifentanil on the Immature Mouse Brain: An Ex Vivo Study

Tourrel, Fabien MD*; de Lendeu, Pamela Kwetieu PhD*; Abily-Donval, Lénaïg MD*; Chollat, Clément MD*; Marret, Stéphane MD, PhD*; Dufrasne, François PhD; Compagnon, Patricia MD; Ramdani, Yasmina*; Dureuil, Bertrand MD, PhD§; Laudenbach, Vincent MD, PhD*; Gonzalez, Bruno Jose PhD*; Jégou, Sylvie PhD*

doi: 10.1213/ANE.0000000000000159
Neuroscience in Anesthesiology and Perioperative Medicine: Research Report

BACKGROUND: The use of remifentanil in a context of potential prematurity led us to explore ex vivo the opioid effects on the immature mouse brain. Remifentanil enhances medullary glutamatergic N-methyl-D-aspartate (NMDA) receptor activity. Furthermore, in neonatal mouse cortex, NMDA was previously shown to exert either excitotoxic or antiapoptotic effects depending on the cortical layers. With the use of a model of acute cultured brain slices, we evaluated the potential necrotic and apoptotic effects of remifentanil, alone or associated with its glycine vehicle (commercial preparation of remifentanil, C.P. remifentanil), on the immature brain.

METHODS: Cerebral slices from postnatal day 2 mice were treated up to 5 hours with the different compounds, incubated alone or in the presence of NMDA. The necrotic effect was studied by measuring lactate dehydrogenase activity and 7-Aminoactinomycin D labeling. Apoptotic death was evaluated by measurement of caspase-3 activity and cleaved caspase-3 protein levels, using Western blot and immunohistochemistry. Extrinsic and intrinsic apoptotic pathways were investigated by measuring caspase-8, caspase-9 activities, Bax protein levels, and mitochondrial integrity.

RESULTS: C.P. remifentanil was ineffective on necrotic death, whereas it significantly reduced caspase-3 activity and cortical cleaved caspase-3 levels. C.P. remifentanil inhibited cortical Bax protein expression, caspase-9 activity, and preserved mitochondrial integrity, whereas it had no effect on caspase-8 activity. Its action targeted the neocortex superficial layers, and it was reversed by the opioid receptors antagonist naloxone and the NMDA antagonist MK801. Remifentanil and glycine acted synergistically to inhibit apoptotic death. In addition, C.P. remifentanil enhanced the antiapoptotic effect of NMDA, whereas it did not improve NMDA excitotoxicity in brain slices.

CONCLUSION: The present data indicate that at a supraclinical concentration C.P. remifentanil had no pronecrotic effect but exerted ex vivo antiapoptotic action on the immature mouse brain, involving the opioid and NMDA receptors, and the mitochondrial-dependent apoptotic pathway. Assessment of the impact of the antiapoptotic effect of remifentanil in in vivo neonatal mouse models of brain injury will also be essential to measure its consequences on the developing brain.

From the *Laboratory of Microvascular Endothelium and Neonate Brain Lesions, Rouen Institute for Biomedical Research (IRIB), University of Rouen, Cedex, France; Laboratory of Pharmaceutical Organic Chemistry, ULB, Brussels, Belgium; Departments of Pharmacology, and §Anesthetics and Intensive Care, Rouen University Hospital, Rouen, France.

Fabien Tourrel, MD, is currently affiliated with Département of Anesthetics and Intensive Care, Rouen University Hospital, Rouen, France; Lénaïg Abily-Donval, MD,* Clément Chollat, MD, Stéphane Marret, MD, PhD, and Vincent Laudenbach, MD, PhD, are currently affiliated with Department of Neonatal Paediatrics and Intensive Care, Rouen University Hospital, Rouen, France.

Funding: University of Rouen, Institut National de la Santé et de la Recherche Médicale (INSERM), Fonds Européen pour le Développement Régional (FEDER), Societe Francaise d’Anesthesie et de Reanimation (SFAR), Regional Platform for Cell Imaging, Conseil Regional de Haute-Normandie, La Fondation Motrice

The authors declare no conflicts of interest.

This report was previously presented, in part, at the 2010 and 2012 SFAR congress, 2009 and 2011 Journées Francophones de Recherche en Néonatologie (JFRN) congress, 2012 European Congress Perinatal Medicine, 2011 Colloque Société des Neurosciences

Reprints will not be available from the authors.

Address correspondence to Sylvie Jégou, PhD, ERI 28 Laboratory of Microvascular Endothelium and Neonate Brain Lesions, Rouen Institute for Biomedical Research (IRIB), University of Rouen, 76183 Rouen Cedex, France. Address e-mail to

Despite major improvements in the survival of premature infants, the rate of neurodevelopmental disabilities related to perinatal brain injury has not decreased significantly in developed countries.1 Premature infants born via urgent cesarean delivery under general anesthesia are at increased risk of hypoxic-ischemic and excitotoxic insults to the brain.2,3 Remifentanil is considered the most suitable opioid for obstetric anesthesia and analgesia as well as fetal immobilization during intrauterine surgery.4,5 It is a synthetic potent, ultrashort-acting μ opioid metabolized by nonspecific plasma and tissue esterases. These pharmacokinetic properties allow infusion for a short time period and a rapid recovery at the end of its administration.6,7 Its use is particularly relevant for anesthesia induction during cesarean delivery in pregnant women who are prone to gastric reflux and aspiration. Indeed, remifentanil and alfentanyl are the opioids with the fastest onset of action and are compatible with a rapid sequence induction, theoretically without increasing the risk of maternal inhalation.8,9 In addition, peripartum studies have suggested that remifentanil readily crosses the placenta10 and is also rapidly metabolized in neonates.11

Data concerning the impact of opioids on the developing brain are controversial. Clinical studies from the NEOPAIN trial have shown that severe intraventricular hemorrhage occurred more frequently in ventilated preterm neonates receiving open-label morphine.12 More recent investigations indicated that preemptive morphine analgesia in preterm neonates might result in subtle neurobehavioral differences in premature infants13 and altered some morphometric and behavioral variables in early childhood.14 Conversely, other reassuring results revealed that preterm or very preterm infants receiving brief or prolonged sedative and/or analgesia, respectively, did not exhibit subsequent neurological and neurodevelopmental disabilities.15,16 Although clinical investigations indicated that remifentanil seems to be an effective and safe opioid used in neonatal intensive care and anesthesia practice,5 no data report the effect of remifentanil on neonatal brains.

Previous reports demonstrated that an infusion of remifentanil, like other opioids, induces postoperative secondary hyperalgesia that involves the activation of the medullary glutamatergic N-methyl-D-aspartate (NMDA) receptor.17–21 More recent data evidenced that the tyrosine phosphorylation of the NR2B subunit of the NMDA receptor contributes to remifantanil-induced postoperative hyperalgesia.22 The cortical NMDA receptor, because of its high permeability toward Ca,2+ plays an important role in the development of neonatal brain lesions through the induction of an excitotoxic process.23,24 However, we demonstrated that NMDA can exert a dual effect on the immature postnatal day 2 (P2) neonatal mouse cortex: a necrotic death occurred in the deep cortical layers V to VI, contrasting with an antiapoptotic effect in the immature layers II to IV.25 Considering NMDA necrotic and antiapoptotic effects on the immature brain and that remifentanil can interact with the NMDA receptors, we investigated the potential effects of remifentanil and its clinically used formulation, C.P. remifentanil, on cell death in cortical slices from mouse neonates.

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NMRI (National Marine Research Institute) mice (Janvier, Le Genest Saint Isle, France) were kept in a temperature-controlled room (21°C ± 1°C) with an established photoperiod (the lights were on from 7 AM to 7 PM) with free access to food and tap water. Animal manipulations were performed in accordance with the European Communities Council Directives (86/609/EEC), the French National legislation (ethical approval no. 76–114), were approved by the local ethics committee (agreement N/02-09-06/15) and were under the supervision of authorized investigators (BJG, SJ authorizations no. 76.A.87 and 76.A.34 from the Ministère de l’Agriculture et de la Pêche).

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Remifentanil hydrochloride was synthesized and purified as previously described.17 The clinically used remifentanil formulation, C.P. remifentanil (Ultiva®, GlaxoSmithKline, France), contained 3.7 mM glycine per 50 μM remifentanil. NMDA and the NMDA receptor antagonist MK801 were purchased from Tocris (Bristol, United Kingdom). β-nicotinamide adenine dinucleotide (NADH), sodium pyruvate, glycine and naloxone hydrochloride, protease and phosphatase inhibitor cocktails, and rabbit anti-actin polyclonal antibody were from Sigma Aldrich (Saint-Quentin Fallavier, France). The Alexa Fluor® 488 donkey anti-rabbit IgG (A-21206) and anti-mouse IgG (A-21200) were from Invitrogen (Cergy Pontoise, France). The caspase-3 substrate Ac-DEVD-AMC, caspase-8 substrate Ac-LETD-AMC, and the caspase-9 substrate Ac-LEHD-AMC were obtained from Enzo Life Sciences (Plymouth Meeting, PA). Rabbit anti-caspase-3 (#9662), anti-cleaved caspase-3 (#9661), polyclonal antibodies, and cell lysis buffer (#9803) were purchased from Cell Signaling Technology (Boston, MA). Rabbit anti-Bax polyclonal antibody (ab57-14) was from Abcam (Cambridge, United Kingdom). ECL RPN 2108 kit for Western blot experiments was from GE Healthcare (Orsay, France). The 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1) mitochondrial probe was from Molecular Probes (Leiden, the Netherlands).

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Preparation and Treatment of Cerebral Slices from Postnatal Day 2 Mice

Brain slices were obtained from P2 mice. Excitotoxic and hypoxic-ischemic cortical lesions induced at this developmental stage presented similarities with those observed in preterm infants.26–29 Pups were killed by decapitation and their brains rapidly dissected to isolate the cerebral hemispheres. Meninges covering the brain were carefully removed, and the brain was immediately placed into ice-cold artificial cerebrospinal fluid (aCSF) containing (in mM): NaCl, 125; KCl, 3; CaCl2, 2; NaH2PO4, 1.2; NaHCO3, 26; D-Glucose 10; pH 7.4. Frontal sections (250 μm) were cut at 4°C by using a vibratome VT1000S (Leica Microsystems, Nanterre, France). For each pup, 3 to 4 slices were collected through the median forebrain from the rostral level of the junction of corpus callosum. For each slice, the 2 hemispheres were rapidly separated and individually transferred into 24-well plates containing aCSF (200 μL) and incubated for a 30-minute recovery period at 37°C in a humidified incubator under controlled atmosphere of 5% CO2/95% air. After recovery, hemislices were washed with fresh aCSF and treated for 3 or 5 hours at 37°C. For each pair of hemisections, one was incubated in aCSF alone (control), and the other was incubated in aCSF supplemented with the different drugs. Thus, the controlateral hemisphere was considered an internal control of the treated hemisphere. For comparison of different incubation times (0, 3, and 5 hours) on enzyme activities, hemislice processed just after recovery time was considered control (t0).

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Remifentanil Half-Life in the Ex Vivo Model

The stability of remifentanil in our ex vivo experiment was investigated by combining reverse-phase high-performance liquid chromatography analysis with tandem mass spectrometry. Remifentanil (50 μM) was incubated as described above, in the absence or presence of brain P2 slices for 5 hours. An aliquot of incubation medium was collected each hour and immediately frozen. Samples were concentrated using a 0.21 × 30 mm POROS R2/20 (Applied Biosystems, les Ulis, France) and analyzed on a 0.21 × 50 mm Alltima HP C18 HL, 3 μm (Grace, Epernon France) at a constant flow rate (200 μL/min). The mobile phase consisted of a gradient established over 5 minutes with 0.2% formic acid and 2 mM ammonium formate in acetonitrile. The concentration of remifentanil was calculated using a calibration curve. The half-life of remifentanil incubated in medium alone or in the presence of brain slices was 1 hour 50 minutes and 1 hour 36 minutes, respectively.

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Lactate Dehydrogenase Activity Assay

Lactate dehydrogenase (LDH) activity in the extracellular medium was used as an index of cell lysis.30 After hemibrain slice exposure to aCSF alone or with C.P. remifentanil (10–240 μM) or NMDA (6.25–400 μM), 30 μL incubation medium was collected and mixed with 100 μL freshly made solution containing 0.5 mM NADH. Conversion of NADH to its oxidized form NAD+ was initiated by adding 20 μL of 5 mM sodium pyruvate. LDH activity was then monitored for 15 minutes by measuring the decrease of NADH absorbance using spectrophotometry at 340 nm with a Chameleon plate reader (ScienceTec, les Ulis, France).

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Caspase Activity Assays

For caspase-3 activity measurements, each hemibrain slice was resuspended in 200 μL hypotonic lysis buffer, and 90 μL homogenate was incubated at 30°C with caspase buffer containing 50 μM caspase-3 substrate Z-DEVD-AMC. For caspase-8 and caspase-9 activities measurements, 4 hemibrain slices were pooled in 300 μL hypotonic lysis buffer, and 90 μL homogenate was incubated with buffer containing 50 μM caspase-8 substrate Ac-LETD-AMC or caspase-9 substrate Ac-LEHD-AMC. As a positive control, caspase-3 activity was evaluated in the same preparation. Fluorescence intensity was quantified every 5 minutes for 2 hours at excitation/emission wavelengths of 485/520 nm, respectively, using a Chameleon plate reader (Hidex, Turku, Finland).

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Visualization of Cell Death

Cell death associated with plasma membrane permeability was visualized after 5 hours of treatment using 7-Aminoactinomycin D (7-AAD) a noncell-permeant DNA intercalator. Briefly, hemibrain slices were washed with phosphate buffer saline (PBS) at 37°C and incubated for 5 minutes with 15 μM 7-AAD (producing red fluorescence in the nuclei of dead cells). Sections were then washed 3 times for 5 minutes in PBS at 37°C and fixed overnight at 4°C with 4% paraformaldehyde (PFA) in PBS as in previous immunohistochemical studies. Dead cells were visualized using a fluorescence videomicroscope system DMI 6000B Leica (Rueil-Malmaison, France) at excitation/emission wavelengths of 530 and 585 nm, respectively.

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Western Blot Analysis

Hemislices were incubated for 5 hours with aCSF or 50 μM C.P. remifentanil. Cortices were rapidly dissected, and total cellular proteins from 4 hemicortices per brain were extracted using 300 μL cell lysis buffer containing 1% Triton X-100, 20 mM Tris/HCl, 1 mM EDTA, 1 mM EGTA, 1 μg/mL leupeptin and supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), 1% protease and phosphatase inhibitor cocktails. The homogenate was centrifuged (20,000g, 4°C, 15 minutes), and the supernatant proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 15% acrylamide gel and transferred to nitrocellulose Amersham Hybond-ECL membranes by electroelution. Membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 and 5% bovine serum albumin at room temperature for 1 hour and incubated overnight at 4°C in blocking buffer with antibodies against caspase-3 or Bax (dilution 1:1000). After washes, they were incubated with a secondary antibody conjugated to horseradish peroxidase at room temperature for 1 hour. Peroxidase labeling was detected using the ECL Western blotting detection system. To check equal protein loading, membranes were stripped and reprobed with actin antibody. Autoradiographic films were quantified using ImageQuant TL analysis system (GE Healthcare). Commercial markers (Seablue prestained standard, Invitrogen) were used as molecular weight standards.

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Cleaved Caspase-3 and Bax Immunohistochemistry

Hemibrain slices previously incubated in aCSF or 50 μM C.P. remifentanil were fixed with 4% PFA, incubated overnight at 4°C with rabbit antibodies directed against cleaved caspase-3 or Bax (dilution 1:200 in PBS containing 1% bovine serum albumin and 0.1 % Triton X-100). Sections were then rinsed twice with PBS for 20 minutes and incubated with the same incubation buffer containing an Alexa Fluor® 488 donkey anti-rabbit IgG. Cell nuclei were visualized by incubating the slices with 1 μg/mL Hoechst 33258 in PBS. Fluorescent signals were observed with a Leica DMI 6000B microscope (Philips Rearch, Eindhoven, Netherlands). The specificity of the immunoreactions was controlled by substituting the primary antibodies by PBS.

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Electron Microscopy

Ultrastructural studies were performed according to standardized protocols. Briefly, cortex obtained from hemibrain slices previously exposed to aCSF alone or 50 μM C.P. remifentanil for 5 hours were fixed in 2.5% glutaraldehyde solution, postfixed with osmium tetroxide, and embedded in resin epoxy. Semithin sections were stained with toluidine blue. Ultrathin sections (70 nm) were contrasted with uranyl acetate and lead citrate and examined under a PHILIPS CM10 electron microscopy.

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Visualization and Quantification of Pyknotic Nuclei

Hemislices from 6 P2 mice were treated for 5 hours in aCSF alone (control condition) or in the presence of 50 μM C.P. remifentanil. Five minutes before the end of the incubation, sections were treated for 5 minutes with 1 μg/mL Hoechst, washed twice with PBS for 5 minutes, and fixed overnight with 4% PFA. TIFF format images of the Hoechst positive nuclei were acquired, and the proportion of pyknotic nuclei in the regions of interest (ROIs) was quantified using the Metamorph image analysis station (Roper Scientific). Ten ROIs per section were considered. Data were expressed as the ratio of pyknotic nuclei over the total number of nuclei.

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Visualization and Quantification of Mitochondrial Integrity

Mitochondrial membrane potential was assessed using the ratiometric probe JC-1. In healthy cells, the intact membrane potential allows the lipophilic dye JC-1 to enter into the mitochondria where it accumulates and aggregates, producing an intense orange signal. In cells where the mitochondrial membrane potential collapses, the JC-1 remains monomeric and stays in the cytoplasm, where it emits green fluorescence. Brain hemislices from P2 mice were treated for 5 hours at 37°C in the absence or presence of 50 μM C.P. remifentanil, incubated for 30 minutes with 3 μL/mL JC-1, and finally washed twice for 5 minutes with PBS at the same temperature. Fluorescence was visualized immediately without previous fixation at the 485-nm excitation and 530 nm (green) emission wavelengths and at 550-nm excitation and 590 nm (orange) emission wavelengths. Green and orange signals were acquired and saved in TIFF format using a computer-assisted image analysis station Metamorph. Fluorescence intensity profiles corresponding to monomeric, and aggregated JC-1 were used to quantify the 590/530 ratio. For each fluorescent signal, a background level was defined in a negative region of the images. The quantification was performed in the superficial layers of the frontoparietal cortex on 10 to 12 ROIs from 2 hemislices per animal, and 6 mice were analyzed.

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Statistical Analysis

Statistical analyses were performed using PRISM (Graphpad San Diego, CA). The data were plotted as mean ± SEM. Minimum sample sizes were calculated according to our previous study using the same experimental model.25 For a given condition, the number of control and treated hemislices was the same. Each sample size was specified in the figure legends. Differences between groups were assessed by 1-way analysis of variance test followed by a post hoc multiple comparison Dunnett test or unpaired t test depending on the experimental design. Because sample sizes were too small to test the distribution, a conservative approach was chosen: for each test, a P-value of 0.01 or less was considered statistically significant. Results from analyses were reported with corresponding 95% confidence intervals (CIs) when P < 0.15. Each experiment was independent of the others.

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Effect of C.P. Remifentanil on Necrotic and Apoptotic Cell Death

Action of C.P. remifentanil on P2 cerebral slices was evaluated by quantifying LDH and caspase-3 activities representative of cell lysis30 and apoptotic31 cell death, respectively. Treatment of slices with aCSF alone increased LDH and caspase-3 activities after 3- (P = 0.0063, 95% CI, 100.4 to 606.6] for LDH; P = 0.4 for caspase-3) and 5-hour incubation times (P < 0.0001, 95% CI, 321.1–827.4 for LDH and P < 0.0001, 95% CI, 804.9–1242 for caspase-3), (Fig. 1A), suggesting a basal excitotoxic and apoptotic process in control slices. As described before, a 5-hour treatment with 400 μM NMDA simultaneously increased LDH activity (P < 0.0001, 95% CI, 15.5–41.1; Fig. 1B) and inhibited caspase-3 activity (P = 0.0012, 95% CI, −44.5 to −9.3; Fig. 1C).25 When compared with the control condition (aCSF alone), exposure to graded concentrations of C.P. remifentanil from 10 to 240 μM for 5 hours did not significantly modify LDH activity (Fig. 1B). In contrast, from the dosage 50 μM, C.P. remifentanil significantly inhibited caspase-3 activity (P = 0.0079, 95% CI, −37.4 to −4.5 for 50 μM; P = 0.0015, 95% CI, −39.2 to −7.8 for 240 μM; Fig 1C). Because quantification of LDH and caspase-3 activities are not representative of cortical lamination, necrotic and apoptotic death were also visualized in P2 brain slices using the fluorescent probe 7-AAD and cleaved (potentially activated) caspase-3 immunohistochemistry, respectively. At P2, the development of cortical layers is not totally achieved, and deep layers V to VI are more mature than superficial layers II to IV that remain immature with neurons still migrating.32 In control conditions, a 7-AAD signal was homogeneous in the neocortex (Fig. 1D, right) while cleaved caspase-3-labeled cells were mainly localized in the superficial II to IV (Fig. 1D, left). When cortical slices were exposed to 50 μM C.P.remifentanil, in agreement with that of the quantification of LDH and caspase-3 activities, the distribution pattern of 7-AAD labeling was not modified (Fig. 1E, right) whereas a decrease of cleaved caspase-3-immunoreactivity was found in the superficial layers II to IV (Fig. 1E, left). To further confirm the presence of apoptotic cells in these layers, electron microscopic observation and double labeling with Hoechst and cleaved caspase-3 antibody were performed. Typical pyknotic nuclei presenting condensed chromatin were more readily found by electronic microscopy in aCSF-treated slices (Fig. 2A, left) than in C.P. remifentanil-treated slices (Fig. 2A, right). At high magnification, cleaved caspase-3 immunolabeled cells contained condensed nucleus, indicating that they were engaged in apoptotic death (Fig. 2B, upper), whereas cleaved caspase-3 negative cells exhibited nucleus with normal appearance (Fig. 2B). A comparison of nucleus morphology by Hoechst counterstaining at high magnification in control and C.P. remifentanil conditions indicated that C.P. remifentanil significantly decreased the proportion of pyknotic nuclei in superficial layers (P = 0.0043, 95% CI, −4.7 to −1.1; Fig. 2C).

Figure 1

Figure 1

Figure 2

Figure 2

Caspase-3 is an executioner protease involved in different apoptotic cascades.31 To identify the signaling pathway(s) associated with the antiapoptotic effect of C.P. remifentanil, we quantified caspase-8 and caspase-9 activities, 2 initiator caspases of the extrinsic and intrinsic pathways, respectively. Exposure of neonatal brain slices to 50 μM C.P. remifentanil for 3 hours inhibited caspase-9 as well caspase-3 activities (P= 0.0007, 95% CI, −54.3 to −16.3 and P= 0.0014, 95% CI, −38.9 to −10.4, respectively; Fig. 3A). Conversely, C.P. remifentanil had no effect on caspase-8 activity (Fig. 3A), suggesting that C.P remifentanil interacts with the mitochondrial pathway.

Figure 3

Figure 3

Western blot analysis using an antibody directed against caspase-3 revealed that 50 μM C.P. remifentanil strongly decreased expression of the activated fragment (17 KDa) in mouse cortices, confirming that C.P. remifentanil exerts antiapoptotic action (P= 0.0004, 95% CI, −47.8 to −19.9; Fig. 3B). Genes from the Bcl-2 family play a key role in initiating the mitochondrial apoptotic pathway.33 Quantification of proapoptotic Bax expression by Western blot showed that treatment of slices with 50 μM C.P. remifentanil reduced Bax levels in cortex extracts. However, this decrease was not statistically significant (P= 0.0249, 95% CI, −69.6 to −06.2; Fig. 3B). The visualization of Bax signal was performed by immunohistochemistry. In control slices, superficial immature layers II to IV were intensively labeled with Bax antibody (Fig 3C, left). Treatment of slices with 50 μM C.P. remifentanil for 5 hours markedly decreased the Bax signal in the cortical layers (Fig. 3C, right). Because Bax and caspase-9 activation are associated with induction of the mitochondrial apoptotic pathway, we visualized and quantified mitochondrial integrity in the immature layers II to IV by using the ratiometric probe JC-1. A dotted red signal was representative of the aggregated form of JC-1 into healthy mitochondria (Fig. 4A), while green fluorescence visualized the cytosolic form of the probe (Fig. 4B). At low magnification, overlap of the aggregated and monomeric forms of JC-1 showed that 50 μM C.P. remifentanil increased the red fluorescent signal, resulting in a significant increase of the JC-1 590/530 ratio (P < 0.0001, 95% CI, 98.9–199.5; Fig. 4, D and E) and indicating that C.P. remifentanil preserved mitochondrial integrity.

Figure 4

Figure 4

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Involvement of Opioid and NMDA Receptors in the Antiapoptotic Effect of C.P. Remifentanil

Remifentanil has a high affinity for μ-opioid receptors. To assess whether the opioid receptors antagonist naloxone was able to reverse the effect of C.P. remifentanil, 50 μM C.P. remifentanil was coincubated with naloxone (Fig. 5A). Naloxone incubated alone did not affect caspase-3 activity, conversely, at dosages of 1 or 2 mM; it, respectively, partially or totally abolished C.P. remifentanil-induced caspase-3 inhibition.

Figure 5

Figure 5

Previous studies indicated that medullary NMDA receptor activation contributes to C.P. remifentanil-induced postoperative hyperalgesia.17,21,22 Furthermore, we previously showed that NMDA decreased apoptosis in P2 brain slices.25 To assess the implication of NMDA receptors in the antiapoptotic action of C.P. remifentanil, we investigated the effect of 50 μM C.P. remifentanil in the presence of the NMDA antagonist MK801 (Fig. 5B). As described previously, 20 μM MK801 used alone likely increased the activity of caspase-3 (P= 0.0116, 95% CI, 02.9–26.4). In the presence of 20 μM MK801, C.P. remifentanil lost its inhibitory effect on caspase-3 activity.

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Effect of C.P. Remifentanil in the Presence of NMDA

We have shown that exposure of P2 brain slices to NMDA exerts a dual effect in the neocortex: an increase of necrotic and excitotoxic cell death in deep mature layers and a decrease of apoptosis in superficial immature layers. NMDA, respectively, increased and reduced LDH and caspase-3 activities in a dose-dependent manner.25 To evaluate the effect of C.P. remifentanil at different levels of NMDA receptor activation in immature brain, we quantified LDH and caspase-3 activities after a 5 hours incubation of brain slices with an ineffective dosage of C.P. remifentanil (25 μM) in the presence of graded concentrations of NMDA (Fig. 6). As described previously,25 NMDA increased LDH and decreased caspase-3 activities in a dose-dependent manner. The addition of 25 μM C.P. remifentanil did not modify LDH activity measured in the presence of 6.25 to 100 μM NMDA. Furthermore, the significant increase in LDH activity induced by 400 μM NMDA (P= 0.010, 95% CI, 10.1–46.5) was reduced in the presence of C.P. remifentanil. Conversely, coadministration of 25 μM C.P. remifentanil with ineffective dosages of NMDA (6.25–25 μM) markedly inhibited caspase-3 activity (P = 0.0007, 95% CI, −38.2 to −14.4 for 6.25 μM, P = 0.006, 95% CI, −54.1 to −13.9 for 25 μM). In addition, 25 μM C.P. remifentanil enhanced the antiapoptotic effect of 100 μM NMDA (P = 0.006, 95% CI, −25.9 to −6.7). Altogether, these data indicated that C.P. remifentanil enhances the antiapoptotic action of NMDA without improving its excitotoxicity.

Figure 6

Figure 6

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Synergistic Effect of Remifentanil and Glycine

Each 1-mg C.P. remifentanil vial contains 15 mg glycine as adjunct. In an attempt to determine the differential role of remifentanil and glycine to the antiapoptotic effect of C.P. remifentanil, we tested the effect of 50 μM synthetic remifentanil supplemented or not with 3.6 mM glycine (Fig. 7A). Incubation of brain slices with 50 μM remifentanil or 3.6 mM glycine alone had no impact on apoptotic activity. In contrast, the combination of both compounds likely inhibited caspase-3 activity (P = 0.0155, 95% CI, −38.8 to −3.5) in the same order of magnitude as 50 μM C.P. remifentanil (P = 0.0025, 95% CI, − 35.2 to −6.8), suggesting a glycine-dependent effect. Although 50 μM remifentanil was devoid of action in basal condition (Fig. 7A), coincubation of 50 μM remifentanil with 12.5 μM NMDA exerted a significant antiapoptotic effect (Fig. 7B, P = 0.0005, 95% CI, −42.3 to −11.1), indicating that remifentanil by itself was active in a NMDA receptor activation condition.

Figure 7

Figure 7

In an attempt to confirm the glycine implication, we tested the effect of 25 μM synthetic remifentanil supplemented or not with 1.8 mM glycine in the presence of 12.5 μM NMDA (Fig. 7B). Incubation of brain slices with 25 μM remifentanil or 1.8 mM glycine alone had no impact on apoptotic activity, whereas the addition of 25 μM remifentanil and 1.8 mM glycine (25 μM C.P. remifentanil) significantly inhibited caspase-3 activity (P = 0.0002, 95% CI, −54.4 to −16.2), indicating that remifentanil, glycine, and NMDA likely act synergistically.

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Several studies focusing on the use of remifentanil and its effect on parturients have been conducted during general anesthesia for cesarean delivery.34–37 Conversely, the impact of remifentanil exposure on the neonatal brain remains unstudied. The only study data concern the clinical state of neonates, such as Apgar scores, and hemodynamic and respiratory variables.35–37 By using a model of brain organotypic slices from P2 neonate mice, we investigated this unexplored aspect of remifentanil known to interact with NMDA receptors.17,21,22,38 At this developmental stage, intracortical injection of glutamate analog was shown to produce brain damage mimicking that observed in human preterm neonates.26,29 Even if different receptors are involved in glutamate-induced excitotoxicity, the NMDA receptor is considered to play an important role in the development of excitotoxic lesions.23,39 In our model, incubation of brain slices during 5 hours in aCSF alone significantly increased cell lysis and apoptosis, suggesting that this model can be considered an acute cerebral injury model. The current study provided as evidence for the first time that C.P. remifentanil exerts an antiapoptotic effect on the neonatal cortex in the aggressive conditions imposed by our experimental model. However, because apoptosis is a physiological phenomenon, it is currently not possible to determine the in vivo effect of this pharmacological limitation of apoptosis by C.P. remifentanil.40

In the hyperalgesia model, it has been shown that C.P. remifentanil increases the activity of NMDA receptors through a μ-opioid receptor-initiated cascade.17–21 Presently, we found that the inhibitory effect of C.P. remifentanil was reversed by the opioid receptor antagonist naloxone and the NMDA antagonist MK801. These data suggest the implication of NMDA receptors in the antiapoptotic effect of C.P. remifentanil, and the role of opioid receptors as a trigger of an intracellular pathway are able to positively regulate the activity of NMDA receptors.

We previously showed in our model that NMDA exerts a strong antiapoptotic effect on immature layers II to IV of the immature cortex (where neurons are still migrating at this stage)32 and that NMDA receptor blockade promotes apoptosis in the superficial layers via the proapoptotic protein Bax induction and mitochondrial pathway activation.25 We found here that C.P. remifentanil did not modify the activity of caspase-8 that is associated with the extrinsic apoptotic pathway. Conversely, C.P. remifentanil decreased caspase-9 activity as well as cleaved caspase-3 and Bax protein expression in the superficial cortex. Furthermore, C.P. remifentanil preserved mitochondrial integrity. All these data suggest that C.P. remifentanil inhibits the intrinsic mitochondrial-dependent apoptotic pathway. NMDA was previously shown to promote strong excitotoxic cell death in neonatal cortical slices, especially in deep cortical layers (containing postmigrating and differentiated neurons).25 Surprisingly, C.P. remifentanil had no effect on the necrotic process. NMDA receptors are heteromeric complexes consisting of NR1, NR2 (2A–2D), and NR3 subunits. The NR1 subunit is essential for functional NMDA receptor channels whereas the NR2 subunits modulate channel functional properties.41 It has been reported that phosphorylation of NR2B tyrosine 1472, which is associated with enrichment of NMDA receptor synaptic expression,42 may be involved in upregulation of NMDA receptor function by remifentanil.22 Further experiments in our ex vivo model will be needed to determine whether C.P. remifentanil can differently modulate phosphorylation of NMDA receptors both in superficial and deep layers of the immature cortex.

The efficient dosage of C.P. remifentanil (50 μM) in the present study was comparable with that previously used in an experimental study of the involvement of C.P. remifentanil in hyperalgesia.17 However, this concentration is far from clinical dosages, which range from 10 to 100 nM.43 Thus, additional experiments using an in vivo model will be necessary to get closer to clinical conditions with clinically relevant dosages of C.P. remifentanil.

During acute cerebral injury, extracellular levels of glutamate varied throughout the brain tissue. For example, in a sheep model of neonatal hypoxic-ischemic lesions, a positive correlation has been shown between the glutamate efflux and the extension of the cerebral lesion.44 Furthermore, in a rodent neonatal hypoxic-ischemic model, extracellular concentrations of excitatory amino acids, including glutamate, were found to be higher in infarcted and border zone areas compared with the undamaged cortex.45 To study the effect of C.P. remifentanil at different levels of NMDA receptor activation, we incubated C.P. remifentanil in the presence of graded concentrations of NMDA. In our model, NMDA was previously shown to exert necrotic or antiapoptotic effects depending on the cortical layers.25 In the present study, we found that C.P. remifentanil enhances the NMDA-induced antiapoptotic effect, which occurs in superficial layers. Conversely, the association of C.P. remifentanil with graded NMDA concentrations did not improve NMDA-induced excitotoxicity in deeper layers. These data strongly support a functional interaction between activation of opioid receptors by C.P. remifentanil and NMDA receptors specifically in superficial layers of the neonatal cortex.

C.P. remifentanil contains glycine, which is a coagonist of NMDA receptors. Because previous reports had different conclusions concerning the contribution of glycine in activation of NMDA receptors by C.P. remifentanil,17,38 it was essential to evaluate remifentanil, glycine, or the association of both in our model. In basal conditions, we showed that 50 μM remifentanil is ineffective on caspase-3 activity, whereas its association with glycine significantly inhibits the apoptotic process. In the presence of a low dosage of NMDA, NMDA receptors were probably slightly activated. In this configuration, 50 μM remifentanil and endogenous glycine seemed to be sufficient to reduce apoptotic death. Moreover, the addition of a lower dosage of remifentanil or glycine alone did not modify cortical caspase-3 activity, whereas remifentanil associated with glycine at the same dosage exerted a significant antiapoptotic effect. Taken together, these data suggest that remifentanil and glycine likely synergistically activate NMDA receptors in the immature cortex.

In conclusion, by using a model of acute brain organotypic slices from neonatal mice, the present study demonstrated that at a supraclinical concentration C.P. remifentanil has no necrotic effect but exerts antiapoptotic action on the immature cortex, involving both opioid and NMDA receptors, and the apoptotic mitochondrial pathway. Furthermore, remifentanil and glycine synergistically inhibited the apoptotic process. The assessment of the impact of the antiapoptotic effect of remifentanil in in vivo neonatal mouse models of brain injury will also be essential to measure its consequences on the developing brain.

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Name: Fabien Tourrel, MD.

Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.

Attestation: Fabien Tourrel has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Pamela Kwetieu de Lendeu, PhD.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Pamela Kwetieu de Lendeu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Lénaïg Abily-Donval, MD.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Lénaïg Abily-Donval has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Clément Chollat, MD.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Clément Chollat has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Stéphane Marret, MD, PhD.

Contribution: This author helped design the study.

Attestation: Stéphane Marret approved the final manuscript.

Name: François Dufrasne, PhD

Contribution: This author helped Synthesis of remifentanil

Attestation: François Dufrasne approved the final manuscript

Name: Patricia Compagnon, MD.

Contribution: This author helped in vitro remifentanil pharmacokinetics.

Attestation: Patricia Compagnon approved the final manuscript.

Name: Yasmina Ramdani.

Contribution: This author helped electronic microscopy study.

Attestation: Yasmina Ramdani approved the final manuscript.

Name: Bertrand Dureuil, MD, PhD.

Contribution: This author helped design the study.

Attestation: Bertrand Dureuil approved the final manuscript.

Name: Vincent Laudenbach, MD, PhD

Contribution: This author helped design the study

Attestation: Vincent Laudenbach approved the final manuscript

Name: Bruno Jose Gonzalez, PhD.

Contribution: This author helped design and conduct the study and analyze the data.

Attestation: Bruno Jose Gonzalez has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Sylvie Jégou, PhD.

Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.

Attestation: Sylvie Jegou has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

This manuscript was handled by: Gregory J. Crosby, MD.

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