Inhibition of p75 Neurotrophin Receptor Attenuates Isoflurane-mediated Neuronal Apoptosis in the Neonatal Central Nervous System

Abstract

A CONVENTIONAL premise of general anesthesia is that anesthetics produce a nontoxic and reversible state of unconsciousness. However, recent evidence has indicated that exposure of neonatal animals to anesthetics triggers widespread neurodegeneration, leading to persistent memory and learning abnormalities during adulthood.1–3Specifically, exposure of postnatal day 5–10 (PND5-10) rat pups to a combination of isoflurane, nitrous oxide, and midazolam resulted in substantial neurodegeneration in the hippocampus and neocortex1as well as impaired electrophysiologic and behavioral function in the hippocampus weeks to months after exposure.1,3Such neurotoxicity has now been demonstrated for ketamine, midazolam,4diazepam, pentobarbital,5thiopental,3nitrous oxide,1and propofol.3,6–8The pattern of neonatal anesthetic neurotoxicity is similar to that produced by ethanol, exposure to which in utero is associated with the development of fetal alcohol syndrome. These observations have raised concerns about the potentially adverse impact of general anesthesia in the human fetus, neonate, and infant.3,9A key element of anesthetic neurotoxicity is that the brain is most susceptible to injury during the period of synaptogenesis.10An important signaling mechanism that contributes to synaptogenesis and to consolidation and maturation of synapses is the neurotrophin brain-derived neurotrophic factor (BDNF).11BDNF has dual function in that it can enhance neuronal survival or cause neuronal apoptosis through activity at either the tropomyosin receptor kinase B (TrkB) or neurotrophic receptor (p75NTR), respectively.12–14BDNF is secreted from synaptic vesicles as a proneurotrophin (proBDNF) that is proteolytically cleaved in the synaptic cleft by plasmin to a mature form (mBDNF).15Plasminogen, the precursor to plasmin, is cleaved by tissue plasminogen activator (tPA), a protease released from presynaptic vesicles.16Mature BDNF (mBDNF) preferentially binds to TrkB receptors to promote neuronal survival and synaptogenesis.15In the absence of tPA, proBDNF is uncleaved and binds with high affinity to p75NTR, resulting in reduced synaptogenesis, withdrawal of dendritic spines, and neuronal apoptosis.15Thus tPA can serve to control which effect of BDNF is predominant.17The mechanisms by which anesthetics mediate neurotoxicity are not clear. What is common among the anesthetics that have proven neurotoxicity in experimental studies is a profound suppression of neuronal activity; this has the potential to adversely affect the tPA-plasminogen-plasmin system. The release of tPA is activity-dependent,18and it is therefore conceivable that anesthetics might suppress neuronal activity, reduce tPA release, and enhance proBDNF signaling via the p75NTR, thereby leading to loss of dendritic filopodial spines and synapses and subsequent neuronal apoptosis. The current study, using in vitro neuronal cultures and in vivo mouse pups, was conducted to test this hypothesis.All studies performed on animals were approved by the Veteran Affairs San Diego Institutional Animal Care and Use Committee and conform to relevant National Institutes of Health guidelines.Neonatal mouse neurons (The Jackson Laboratory, Bar Harbor, ME) were isolated using a papain dissociation kit (Worthington Biochemical, Lakewood, NJ) as previously described.19Mix cortical and hippocampal neurons were isolated from 1-day-old pups (PND1) and grown in culture for 5 to 21 days in vitro (DIV). Neurons were cultured in Neuobasal A media supplemented with B27 (2%), 250 mm GLUTMax1, and penicillin/streptomycin (1%). Cells were cultured on poly-d-lysine/laminin (2 μg/cm2)–coated plates or coverslips at 37°C in 5% CO2for 5, 14, or 21 days before experiments. Cleaved-caspase 3 (Cell Signaling, Danvers, MA) and/or caspase-activated DNase (CAD)–positive cells (Santa Cruz Biotech, Santa Cruz, CA) were used to determine apoptosis via immunoblot and/or immunofluorescence and deconvolution microscopy. Antibodies to phospho-JNK and GAPDH were obtained from Cell Signaling and Imgenex (San Diego, CA), respectively. Cell death was normalized to total caspase-3 or the neuronal marker doublecortin (Abcam, Cambridge, MA) or NeuN (Chemicon/Millipore, Billerica, MA) of cells imaged. TAT-Pep5 was purchased from CalBiochem (Gibbstown, NJ).Neurons were placed in a plexiglass chamber within an incubator and exposed to 1.4% isoflurane delivered from a calibrated vaporizer in a gas mixture of 5% CO2, 21% O2, balance nitrogen gas at a flow rate of 2 l/min. The concentration of isoflurane was continuously monitored by a Datex Capnomac (DRE Medical, Inc., Louisville, KY).Animals were exposed to isoflurane (1.4%; air flow served as the carrier) at a flow rate of 2 l/min for 4 h. Body temperature was monitored with an 8 mm, 22-gauge temperature probe (Mon-a-therm 6510; Mallinckrodt Medical, Inc., St. Louis, MO) that was affixed to the dorsal neck. The temperature in the incubator was maintained at 37°C; this results in