The antiapoptotic effect of remifentanil on the immature mouse brain: an ex vivo study.
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.


CONCLUSIONS
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
F
Tourrel
Pamela Kwetieu
PK
de Lendeu
Lénaïg
L
Abily-Donval
Clément
C
Chollat
Stéphane
S
Marret
François
F
Dufrasne
Patricia
P
Compagnon
Yasmina
Y
Ramdani
Bertrand
B
Dureuil
Vincent
V
Laudenbach
Bruno Jose
BJ
Gonzalez
Sylvie
S
Jégou
D000701:Analgesics, Opioid / Q000493:pharmacokinetics / Q000494:pharmacology*; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D001921:Brain / Q000166:cytology* / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000648:ultrastructure; D004357:Drug Synergism; D005998:Glycine / Q000494:pharmacology; D006207:Half-Life; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051379:Mice; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects / Q000648:ultrastructure; D010880:Piperidines / Q000493:pharmacokinetics / Q000494:pharmacology*; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects; D000077208:Remifentanil; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D000701:Analgesics, Opioid; D010880:Piperidines; D016194:Receptors, N-Methyl-D-Aspartate; D051028:bcl-2-Associated X Protein; D007770:L-Lactate Dehydrogenase; D053148:Caspase 3; D000077208:Remifentanil; D005998:Glycine