Lithium Protects against Anesthesia-induced Developmental Neuroapoptosis

Abstract

TRANSIENT exposure of infant rodents to several classes of drugs, including N -methyl-d-aspartic acid antagonists and γ-aminobutyric acid-A agonists, triggers widespread neurodegeneration in the developing brain.1–6The cell death process triggered by these drugs displays all of the classic ultrastructural characteristics of apoptosis,3,7,8and it is mediated by the Bax-dependent mitochondrial intrinsic pathway involving cytochrome-c release and activation of caspases 9 and 3.9–11The window of vulnerability to these agents coincides with the developmental period of rapid synaptogenesis,1,2also known as the brain growth spurt period, which occurs primarily during the first 2 weeks after birth in mice and rats but extends from about midgestation to several years after birth in humans.12Ethanol, which has both N -methyl-d-aspartic acid antagonist and γ-aminobutyric acid-A–mimetic properties, induces widespread neurodegeneration in the developing brain.1,3,6Zhong et al. recently reported that a single dose of lithium (6 mEq/kg) coadministered with ethanol to infant mice protects against ethanol-induced neuroapoptosis.13Further, it was hypothesized that the protective effect of lithium might be mediated by action of lithium on the glycogen synthase kinase 3 signaling system; however, no evidence for an interaction between either ethanol or lithium and the glycogen synthase kinase system was found.Recent work in our laboratory has demonstrated that lithium suppresses the programmed cell death process that occurs naturally in the developing mouse brain and has confirmed the findings of Zhong et al. that lithium powerfully protects against ethanol-induced neuroapoptosis.14To explore the mechanism of action of lithium, we focused on kinase signaling systems (extracellular signal-regulated kinase [ERK), serine/threonine-specific protein kinase [Akt], Jun N -terminal kinase [JNK]) that are believed to play a regulatory role in cell survival. We found that very rapidly (within 30 min) after ethanol administration, there is a marked suppression of ERK phosphorylation and that lithium stimulates ERK phosphorylation and prevents ethanol from suppressing this phosphorylation process.14Ethanol also suppressed phosphorylated Akt, but lithium did not counteract this effect. We also found that ethanol activates the JNK system; but this does not explain the neurotoxic action of ethanol because JNK activation did not occur in the same neuronal populations that are killed by ethanol.The current study was undertaken to determine whether anesthetic drugs suppress ERK and/or Akt phosphorylation, whether lithium counteracts this suppressant action, and whether lithium protects against anesthesia-induced developmental neuroapoptosis. The anesthetic drugs focused on in this study were ketamine, an agent that interacts primarily with N -methyl-d-aspartic acid glutamate receptors, and propofol, an agent that interacts primarily with γ-aminobutyric acid-A receptors but also possibly interacts with N -methyl-d-aspartic acid glutamate receptors.15In the first set of experiments, we sought to determine whether anesthetic drugs mimic ethanol in suppressing phosphorylation of ERK and Akt, and if they do, whether lithium counteracts this suppressant action. For this purpose, postnatal day 5 (P5) C57/Bl6 mouse pups were treated with vehicle, ketamine (40 mg/kg, subcutaneous), propofol (50 mg/kg, intraperitoneal), lithium (6 mEq/kg, intraperitoneal), or a combination of ketamine (40 mg/kg) or propofol (50 mg/kg) and lithium (6 mEq/kg). These anesthetic dosing regimens were used because they have been shown to induce a significant neuroapoptosis response in C57BL6 infant mice.14,16This dose of lithium was chosen because it was the dose Zhong et al. 13used in their original study showing that lithium protects against ethanol-induced neuroapoptosis. Pups were killed 120 min after administration of drug, brains were collected, and cytosolic extracts of caudate putamen were prepared for Western blot analysis of phosphorylated ERK 1/2 and phosphorylated Akt.In a second set of experiments, we evaluated the ability of lithium (6 mEq/kg) to prevent apoptotic neurodegeneration induced by ketamine or propofol. For experiments with ketamine, P5 mouse pups were injected with saline vehicle, ketamine (40 mg/kg), lithium (6 mEq/kg), or a combination of ketamine (40 mg/kg) and lithium (6 mEq/kg). For experiments with propofol, P5 mouse pups were injected with intralipid vehicle, propofol (50 mg/kg), lithium (6 mEq/kg), or a combination of propofol (50 mg/kg) and lithium (6 mEq/kg). Pups were killed 5 h after drug administration, and brain tissue was prepared for immunohistochemical staining with antibodies against activated caspase-3.We undertook a third set of experiments to determine whether a lower dose of lithium would be protective against the neuroapoptogenic actions of ketamine and propofol. Zhong et al. 13reported that blood lithium levels after administra