Protective effects of 7-nitroindazole on ketamine-induced neurotoxicity in rat forebrain culture

Abstract

Ketamine, a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, is used as a pediatric anesthetic for surgical procedures. Recent data suggest that anesthetic drugs may cause neurodegeneration during development. The purpose of this study was to determine the dose and temporal response of ketamine using newborn rat forebrain cultures and also to determine if co-administration of 7-nitroindazole, a nitric oxide synthase (NOS) inhibitor, could protect or reverse ketamine-induced cell death. Neural cells collected from the rat forebrain were incubated for 24 h with 1, 10 or 20 μM ketamine alone or with ketamine plus 1, 5, 10 or 20 μM 7-nitroindazole. Ketamine (10 μM) caused an increase in DNA fragmentation and elevated immunoreactivity to nitrotyrosine, a marked reduction in the expression of the neuronal marker polysialic acid neural cell adhesion molecule (PSA-NCAM) and in mitochondrial metabolism, as well as an increased Bax/BCL-XL ratio. No significant effect was observed in the release of lactate dehydrogenase (LDH). Ketamine-induced neurotoxic effects were effectively blocked by 7-nitroindazole (10 μM). These data indicate a role for nitric oxide in the enhanced degeneration induced by ketamine in vitro and also suggest that blocking neuronal nitric oxide synthase (nNOS) may help reduce the risk of ketamine in pediatrics.

Introduction

Ketamine, a non-competitive N-methyl-d-aspartate (NMDA) receptor ion channel blocker, is used as a pediatric anesthetic. Ketamine is a non-barbiturate, dissociative anesthetic used during short diagnostic and surgical procedures in infants and toddlers. Recent data (Ikonomidou et al., 1999, Jevtovic-Todorovic et al., 2003, Scallet et al., 2004, Slikker et al., 2005, Slikker et al., 2007, Wang et al., 2005, Wang et al., 2006) suggest that anesthetic drugs may cause enhanced neurodegeneration. The window of vulnerability to ketamine coincides with the developmental period of synaptogenesis, also known as the brain growth spurt period, which in rodents occurs primarily after birth. In order to better determine if the neurotoxicity of ketamine in the developing rat has clinical relevance and to dissect underlying mechanisms and intracellular pathways that mediate cell death responses, ketamine needs to be examined in an appropriate in vitro preparation that parallels in vivo conditions during development.

The blockade of NMDA receptors is known to cause neurotoxicity in some instances, but the underlying mechanisms involved in such effects are unknown. The administration of non-competitive NMDA receptor antagonists such as ketamine, phencyclidine (PCP) and MK-801 to rats during a critical period of development results in neurotoxicity/neurodegeneration in several major brain areas (Ikonomidou et al., 1999, Scallet et al., 2004). Our previous studies have shown that repeated administration of PCP to young animals results in a sensitized locomotor response in rats subjected to later PCP challenge (Johnson et al., 1998). This sensitization is associated with apoptotic cell death and an increase in NMDA receptor NR1 subunit mRNA and NMDA receptor immunoreactivity in rat forebrain (Hanania et al., 1999, Wang et al., 1999). Previous in vitro studies have also demonstrated that repeated ketamine administration produces an apparent increase in the NMDA receptor NR1 subunit expression. Co-administration of NR1 antisense oligonucleotides specifically prevents synthesis of NMDA receptor NR1 protein and subsequently blocks the neuronal loss induced by ketamine (Wang et al., 2005, Wang et al., 2006). In addition, PCP-induced neurodegeneration and associated deficits in prepulse inhibition can be attenuated by treatment with a superoxide dismutase mimetic, M40403 (Wang et al., 2003), suggesting an important role of superoxide anions in NMDA antagonist-induced apoptosis and behavioral alterations.

It has been postulated that the continuous exposure of developing brains to ketamine causes selective cell death by a mechanism that involves a compensatory up-regulation of NMDA receptor subunits (Wang et al., 2005, Wang et al., 2006). Associated with Ca²⁺ influx is an increase in reactive oxygen species (ROS) that appears to originate in the mitochondria (Johnson et al., 1998, Slikker et al., 2005). This Ca²⁺ loading by the mitochondria beyond its buffering capacity reduces the membrane potential and disrupts electron transport, which results in the increased production of the reactive free radical superoxide anion (O₂⁻) (Slikker et al., 2005, Wang et al., 2000). Meanwhile, the increased intracellular free calcium [Ca²⁺] is a potent activator of neuronal nitric oxide synthase (nNOS). Active nNOS generates nitric oxide (NO), a multi-faceted second messenger, which permeates neuronal cell membranes to affect specific tissue regions. Therefore, a significant challenge of this study was to determine whether increased nNOS activity and increased generation of peroxynitrite are key regulatory steps in the apoptotic process induced by NMDA antagonist administration, including ketamine.

The α-2,8-linked sialic acid polymer on neural cell adhesion molecules (PSA-NCAM), an important regulator of cell surface interactions (Muller et al., 1996), is a neuronal specific marker known to be an NMDA-regulated molecule important in synaptogenesis during development (Bruses and Rutishauser, 1998, Wang et al., 2005). The purposes of this study were to determine the robustness of ketamine-induced neurotoxicity using rat forebrain cultures by examining neuropathological and neurobiological outcomes, and also to determine if co-administration of 7-nitroindazole, a selective nNOS inhibitor, could protect or reverse ketamine-induced cell death.