Anesthetic-induced Neurotoxicity Research Articles

Glutamate Release and Neurologic Impairment After Intrathecal Administration of Lidocaine and Bupivacaine in the Rat

Local anesthetic-induced neurotoxicity is one of the potential causes of postspinal anesthesia neurologic injury. Many experimental and clinical studies have demonstrated that lidocaine is more neurotoxic than bupivacaine. The mechanisms of local anesthetic-induced neurotoxicity remain unclear. Glutamate is an excitatory amino acid and widely exists in the central nervous system. Overstimulation of the glutamate receptors may produce neuronal toxic effect. In this study, we used in vivo microdialysis to examine the glutamate release in cerebrospinal fluid (CSF) after intrathecal lidocaine and bupivacaine injection. Male Wistar rats were used. Administration of lidocaine (5 groups: normal saline, 2.5%, 5%, 10%, and 10% + MK-801 intrathecally injected) and bupivacaine (4 groups: normal saline, 0.25%, 0.5%, and 1% intrathecally injected) was performed in both microdialysis and postinjection neurologic sequelae studies. After intrathecal injection of the studied agents, the CSF dialysates were collected in 10-minute intervals for 2 hours. Cerebrospinal fluid glutamate concentrations were measured by high-performance liquid chromatography. In addition, tail-flick latencies were examined daily before and after microdialysis for 4 days. Intrathecal lidocaine concentration-dependently elevated glutamate release in CSF. Pretreatment with MK-801 significantly inhibited the glutamate release induced by 10% lidocaine. Intrathecal bupivacaine has no influence on glutamate release in CSF. The tail-flick latencies were significantly prolonged for 4 days after intrathecal lidocaine injection, and these effects were in a concentration-dependent manner. Pretreatment with MK-801 significantly reversed the 10% lidocaine-induced prolonged tail-flick latencies. There was no difference of the tail-flick latencies among the bupivacaine-treated groups. Intrathecal lidocaine caused a concentration-dependent increase of the CSF glutamate release and postinjection neurologic impairment; these effects can be reversed by MK-801. However, intrathecal bupivacaine shows no influence. We suggest that glutamate may be involved in the pathogenesis of lidocaine-induced spinal neurotoxicity.

Neurotoxicity of Adjuvants Used in Perineural Anesthesia and Analgesia in Comparison With Ropivacaine

Clonidine, buprenorphine, dexamethasone, and midazolam (C, B, D, M) have been used to prolong perineural local anesthesia in the absence of data on the influence of these adjuvants on local anesthetic-induced neurotoxicity. Therefore, the impact of these adjuvants on ropivacaine (R)-induced death of isolated sensory neurons was assessed. The trypan blue exclusion assay was used to assess death of sensory neurons isolated from adult male Sprague-Dawley rats. Drugs were applied, alone or in combination, for 2 or 24 hrs at 37°C. Neuronal viability was halved by 24-hr exposure to R (2.5 mg/mL), far exceeding the neurotoxicity of C, B, D, or M (at 2-100 times estimated clinical concentrations). Plain M at twice the estimated clinical concentration produced a small but significant increase in neurotoxicity at 24 hrs. After 2-hr exposure, high concentrations of B, C, and M increased the neurotoxicity of R; the combination of R + M killed more than 90% of neurons. Estimated clinical concentrations of C + B (plus 66 μg/mL D) had no influence on (i) R-induced neurotoxicity, (ii) the increased neurotoxicity associated with the combination of R + M, or (iii) the neurotoxicity associated with estimated clinical concentrations of M. There was increased neurotoxicity with 133 μg/mL D combined with R + C + B. Results with R reaffirm the need to identify ways to mitigate local anesthetic-induced neurotoxicity. While having no protective effect on R-induced neurotoxicity in vitro, future research with adjuvants should address if the C + B + D combination can enable reducing R concentrations needed to achieve equianalgesia (and/or provide equal or superior duration, in preclinical in vivo models).

A New Phase in Anesthetic-induced Neurotoxicity Research

A New Phase in Anesthetic-induced Neurotoxicity Research

Xenon Pretreatment Attenuates Anesthetic-induced Apoptosis in the Developing Brain in Comparison with Nitrous Oxide and Hypoxia

Administration of certain general anesthetics to rodents during the synaptogenic phase of neurodevelopment produces neuronal injury. Preconditioning (pretreatment) can reduce tissue injury caused by a severe insult; the authors investigated whether pretreatment strategies can protect the developing brain from anesthetic-induced neurotoxicity. Seven-day-old Sprague-Dawley rats were pretreated with one of the following: 70% xenon, 70% nitrous oxide, or 8% hypoxia for 2 h; 24 h later, rats were exposed to the neurotoxic combination of 70% nitrous oxide and 0.75% isoflurane for 6 h. Cortical and hippocampal neuroapoptosis was assessed using caspase-3 immunostaining. Separate cohorts were maintained for 40 days at which time cognitive function with trace fear conditioning was performed. In other pretreated cohorts, rat cortices were isolated for immunoblotting of caspase-3, Bcl-2, cytochrome C, P53, and mitogen-activated protein kinases. To obviate physiologic influences, organotypic hippocampal slices harvested from postnatal rat pups were cultured for 5 days and exposed to the same conditions as obtained for the in vivo studies, and caspase-3 immunostaining was again the measured outcome. Xenon pretreatment prevented nitrous oxide- and isoflurane-induced neuroapoptosis (in vivo and in vitro) and cognitive deterioration (in vivo). Contrastingly, nitrous oxide- and isoflurane-induced neuroapoptosis was exacerbated by hypoxic pretreatment. Nitrous oxide pretreatment had no effect. Xenon pretreatment increased Bcl-2 expression and decreased both cytochrome C release and P53 expression; conversely, the opposite was evident after hypoxic pretreatment. Although xenon pretreatment protects against nitrous oxide- and isoflurane-induced neuroapoptosis, hypoxic pretreatment exacerbates anesthetic-induced neonatal neurodegeneration.

Anesthetic-Induced Oxidative Stress and Potential Protection

Prolonged exposure of developing mammals to general anesthetics affects the N-methyl-D-aspartate (NMDA)–type glutamate or γ-aminobutyric acid (GABA) receptor systems and enhances neuronal toxicity. Stimulation of immature neurons by NMDA antagonists or GABA agonists is thought to increase overall nervous system excitability and may contribute to abnormal neuronal cell death during development. Although the precise mechanisms by which NMDA antagonists or GABA agonists cause neuronal cell death are still not completely understood, up-regulation of the NMDA receptor subunit NR1 may be an initiative factor in neuronal cell death. It is increasingly apparent that mitochondria lie at the center of the cell death regulation process. Evidence for the role of oxidative stress in anesthetic-induced neurotoxicity has been generated in studies that apply oxidative stress blockers. Prevention of neuronal death by catalase and superoxide dismutase in vitro, or by M40403 (superoxide dismutase mimetic) in vivo, supports the contention that the involvement of reactive oxygen species (ROS) and the nature of neuronal cell death in rodents is mainly apoptotic. However, more evidence is necessary to in order verify the role of the NMDA receptor subunit NR1 and ROS in anesthetic-induced neurodegeneration.

CON: The Toxic Effects of Anesthetics in the Developing Brain: The Clinical Perspective

Sulpicio G. Soriano, MD, FAAP‡ “All models are wrong, some models are useful” This quote by the statistician George E. P. Box seems to have relevance for the current preeminent controversy in pediatric anesthesiology, namely, developmental neuroapoptotic cell death after an anesthetic exposure in the immature brain. Worldwide, general anesthetics and sedatives are used in hundreds of thousands of neonates and infants every year during surgical operations, invasive procedures, and imaging studies. The possibility of anesthesiainduced neuronal cell loss, as suggested by animal models, during an otherwise uneventful procedure has sparked vigorous discussions among anesthesiologists about the safety of anesthesia in human newborns and infants. These concerns were recently addressed at the March 29, 2007, public hearing of the Anesthesia and Life Support Drugs Advisory Committee of the Food and Drug Administration (transcript available at http://www. fda.gov/ohrms/dockets/ac/07/transcripts/2007-4285t1.pdf). Although the exact mechanism of general anesthesia is not entirely understood, alterations of synaptic transmission involving -aminobutyric acid type A (GABAA) and N-methyl-d-aspartate (NMDA) glutamate receptors, to varying degrees, seem to play an important role. Because GABA and NMDA-mediated neuronal activity is essential for normal mammalian brain development, exposure to anesthetics could potentially interfere with brain maturation, learning, and neurocognitive function. Concerns about the effects of general anesthetics on neuronal structure and neurocognitive function were first raised more than two decades ago. In a series of studies, chronic subanesthetic exposure of pregnant rats to halothane led to delayed synaptogenesis and behavioral abnormalities in their pups. More recently, the potential for ketamine to cause increased neuronal cell death was documented in rat pups. However, although ketamine is rarely used for pediatric anesthesia, general anesthetics routinely used in pediatric practice have subsequently also been implicated not only in producing widespread neuronal cell death, but also in leading to long-term cognitive impairment in adult animals exposed to neonatal anesthesia. A 6-h exposure to a combination of isoflurane, nitrous oxide, and midazolam led to widespread apoptotic brain cell death in 7-day-old rats. When animals were examined in adulthood, many tests of behavior and attention remained normal. However, several tests of spatial learning and memory demonstrated impairment in adult animals that were exposed to the anesthetic cocktail as neonates, compared with their unanesthetized littermates. Several groups of investigators have now confirmed the neurotoxic effects of various anesthetics in a variety of in vivo and in vitro developing animal models. From the *Departments of Anesthesia and Pediatrics, Cincinnati Children’s Hospital Medical Center and University of Cincinnati College of Medicine; and †Institute of Pediatric Anesthesia, Cincinnati Children’s Research Foundation, Cincinnati, Ohio; and ‡Department of Anaesthesia, Children’s Hospital Boston and Harvard Medical School, Boston, Massachusetts. Accepted for publication March 5, 2008. Address correspondence and reprint requests to Dr. Andreas Loepke, Department of Anesthesia, Cincinnati Children’s Hospital Medical Center, ML2001, 3333 Burnet Ave., Cincinnati, OH 45229. Address e-mail to Andreas.Loepke@cchmc.org. Copyright © 2008 International Anesthesia Research Society DOI: 10.1213/ane.0b013e3181733ef8

General anesthetic-induced neurotoxicity: an emerging problem for the young and old?

A growing body of evidence from cells, rodents, and sub-human primates suggests that general anesthetics can be neurotoxic to the developing and senescent brain. We review this evidence and put the studies into perspective for the practicing clinician. Studies indicate that a variety of general anesthetics, which act primarily as gamma-amino-butyric acid receptor modulators and N-methyl-D-aspartic acid glutamate receptor antagonists, produce apoptotic neurodegeneration in the developing rodent and nonhuman primate brain. Vulnerability to this neurotoxicity is greatest during the period of synaptogenesis and presumably reflects disruption of the normal balance between excitation and inhibition during a critical period of brain development. Moreover, in the rodent, the neurodegeneration is associated with cognitive impairment into adulthood. Recent data also reveal that general anesthesia produces enduring cognitive impairment in aged but not young rodents and that halothane and isoflurane increase the generation and toxicity of amyloid beta, a protein strongly implicated in the pathogenesis of Alzheimer's disease. The meaning of these experimental results for human surgical patients is unclear, however, because human studies are lacking. General anesthetics produce neurotoxicity and enduring cognitive impairment in young and aged animals but it is premature to change clinical practice because the issue has not been adequately studied in humans.

Neurotoxicity of Lidocaine Involves Specific Activation of the p38 Mitogen-activated Protein Kinase, but Not Extracellular Signal–regulated or c-jun N-Terminal Kinases, and Is Mediated by Arachidonic Acid Metabolites

Pharmacologic inhibition of the p38 mitogen-activated protein kinase (MAPK) leads to a reduction in lidocaine neurotoxicity in vitro and in vivo. The current study investigated in vitro the hypotheses that lidocaine neurotoxicity is specific for dorsal root ganglion cells of different size or phenotype, involves time-dependent and specific activation of the p38 MAPK, that p38 MAPK inhibitors are only effective if applied with local anesthetic, and that p38 MAPK activation triggers activation of lipoxygenase pathways. The authors used primary sensory neuron cultures and pheochromocytoma cell line cultures to detect time-dependent activation of the p38 MAPK or related pathways such as extracellular signal-regulated kinases and c-jun N-terminal kinases. Cells were divided by size or by immunoreactivity for calcitonin gene-related peptide or isolectin B4, indicative of nociceptive phenotype. The authors also investigated whether arachidonic acid pathways represent a downstream effector of the p38 MAPK in local anesthetic-induced neurotoxicity. All types of dorsal root ganglion cells were subject to neurotoxic effects of lidocaine, which were mediated by specific activation of the p38 MAPK but not extracellular signal-regulated kinases or c-jun N-terminal kinases. Neuroprotective efficacy of p38 MAPK inhibitors declined significantly when administered more than 1 h after lidocaine exposure. Activation of p38 MAPK preceded activation of arachidonic acid pathways. Neurotoxicity of lidocaine, specific activation of p38 MAPK, and neuroprotective effects of a p38 MAPK inhibitor were further confirmed in pheochromocytoma cell line cultures. Specific and time-dependent activation of the p38 MAPK is involved in lidocaine-induced neurotoxicity, most likely followed by activation of lipoxygenase pathways.

The Neurotoxic Effects of Amitriptyline Are Mediated by Apoptosis and are Effectively Blocked by Inhibition of Caspase Activity

Oral tricyclic antidepressants, widely used as adjuncts in the treatment of chronic pain, block sodium channels in vitro and nerve conduction in vivo. However, toxicity of amitriptyline has been observed after neural application. We therefore investigated the mechanism and possible prevention of amitriptyline neurotoxicity. To assess dose-dependent neurotoxicity of amitriptyline, we incubated neuron cultures from adult rat dorsal root ganglia with amitriptyline and quantified neuronal survival. Additionally, we investigated accepted markers of apoptosis (mitochondrial membrane potential, cytosolic cytochrome c, and activated caspase-3) and co-incubated amitriptyline with an inhibitor of caspase activity, z-vad-fmk, to assess the effect on cell survival. We found a dose-dependent neurotoxic effect of amitriptyline. Neurons incubated with amitriptyline exhibited loss of mitochondrial membrane potential, release of cytochrome c into the cytoplasm, and activation of caspase-3. Co-incubation with z-vad-fmk substantially improved neuronal survival in culture. In conclusion, amitriptyline-induced neurotoxicity is mediated by apoptosis and is attenuated by inhibition of caspase activity, suggesting that inhibition of apoptotic pathways may be efficient at alleviating local anesthetic-induced neurotoxicity. In vivo studies will have to corroborate whether the co-injection of anti-apoptotic drugs with local anesthetics decreases neurotoxic side effects.

Mitigation of Direct Neurotoxic Effects of Lidocaine and Amitriptyline by Inhibition of p38 Mitogen-activated Protein Kinase In Vitro and In Vivo

Local anesthetic-induced direct neurotoxicity (paresthesia, failure to regain normal sensory and motor function) is a potentially devastating complication of regional anesthesia. Local anesthetics activate the p38 mitogen-activated protein kinase (MAPK) system, which is involved in apoptotic cell death. The authors therefore investigated in vitro (cultured primary sensory neurons) and in vivo (sciatic nerve block model) the potential neuroprotective effect of the p38 MAPK inhibitor SB203580 administered together with a clinical (lidocaine) or investigational (amitriptyline) local anesthetic. Cell survival and mitochondrial depolarization as marker of apoptotic cell death was assessed in rat dorsal root ganglia incubated with lidocaine or amitriptyline either with or without the addition of SB203580. Similarly, in a sciatic nerve block model, the authors assessed wallerian degeneration by light microscopy to detect a potential mitigating effect of MAPK inhibition. Lidocaine at 40 mm/approximately 1% and amitriptyline at 100 microm reduce neuron count, but coincubation with the p38 MAPK inhibitor SB203580 at 10 mum significantly reduces cytotoxicity and the number of neurons exhibiting mitochondrial depolarization. Also, wallerian degeneration and demyelination induced by lidocaine (600 mm/approximately 15%) and amitriptyline (10 mm/approximately 0.3%) seem to be mitigated by SB203580. The cytotoxic effect of lidocaine and amitriptyline in cultured dorsal root ganglia cells and the nerve degeneration in the rat sciatic nerve model seem, at least in part, to be mediated by apoptosis but seem efficiently blocked by an inhibitor of p38 MAPK, making it conceivable that coinjection might be useful in preventing local anesthetic-induced neurotoxicity.

Anesthetics and brain toxicity

Recent experimental data from rodent studies have demonstrated accelerated neurodegeneration in rat pups exposed to commonly used anesthetic drugs. These provocative findings certainly question and undermine the safe use of anesthetic drugs, particularly in pediatric anesthesia, and have prompted many to investigate the neurotoxic effect of anesthetic drugs on the developing brain. This review will address the scientific evidence for the anesthetic-induced neurotoxicity and its applicability in humans. Several investigators have shown that prolonged administration of anesthetic drugs, including ketamine, isoflurane, nitrous oxide and midazolam, produced increased neurodegeneration in 7-day-old rat pups. The combination of the latter three drugs led to altered learning behavior in adulthood. Despite these unequivocal findings in rodents, similar changes cannot be reproduced in other species. Furthermore, withholding anesthesia during painful procedures in neonatal rats resulted in significant long-term aberrant responses to sensory stimulation and pain thresholds. Taken together, these studies question the applicability of these data to the anesthetic management of the neonate. Further investigations in this area are needed before withholding anesthetics in the anesthetic management of pediatric surgical patients.

Graded, irreversible changes in crayfish giant axon as manifestations of lidocaine neurotoxicity in vitro.

High concentrations of lidocaine induce irreversible conduction block with little effect on resting membrane potential (Em). We assumed the mechanism of persistent neurologic deficit caused by local anesthetics may result from neural death, as represented by the loss of Em. We investigated the effects of lidocaine on Em and action potential (AP) in single crayfish giant axons in vitro. Axons were perfused with two doses of lidocaine for either 15 or 30 min, and they were continuously washed. No axons exposed to 80 mM lidocaine for 30 min showed recovery of AP and Em. Those exposed to 40 mM for 30 min and 80 mM for 15 min showed a return to baseline for Em, but no recovery of AP. Those exposed to 40 mM lidocaine for 15 min showed full recovery of Em and AP immediately after washing. The membrane depolarization was significantly greater during exposure to 80 mM lidocaine for 30 min than in other groups. We conclude that lidocaine has a direct neurotoxic effect on crayfish giant axons and that the generation of AP is more vulnerable than the maintenance of Em. The irreversibility of AP and Em is dose- and time-dependent. Highly concentrated lidocaine induced an irreversible conduction block and a complete loss of resting membrane potential in crayfish giant axons in vitro. Our results may represent a possible explanation for various grades of local anesthetic-induced neurotoxicity in clinical cases if the same toxicity occurs in mammalian nerves in vivo.

Graded, Irreversible Changes in Crayfish Giant Axon as Manifestations of Lidocaine Neurotoxicity In Vitro

High concentrations of lidocaine induce irreversible conduction block with little effect on resting membrane potential (Em).We assumed the mechanism of persistent neurologic deficit caused by local anesthetics may result from neural death, as represented by the loss of Em. We investigated the effects of lidocaine on Em and action potential (AP) in single crayfish giant axons in vitro. Axons were perfused with two doses of lidocaine for either 15 or 30 min, and they were continuously washed. No axons exposed to 80 mM lidocaine for 30 min showed recovery of AP and Em. Those exposed to 40 mM for 30 min and 80 mM for 15 min showed a return to baseline for Em, but no recovery of AP. Those exposed to 40 mM lidocaine for 15 min showed full recovery of Em and AP immediately after washing. The membrane depolarization was significantly greater during exposure to 80 mM lidocaine for 30 min than in other groups. We conclude that lidocaine has a direct neurotoxic effect on crayfish giant axons and that the generation of AP is more vulnerable than the maintenance of Em. The irreversibility of AP and Em is dose- and time-dependent. Implications: Highly concentrated lidocaine induced an irreversible conduction block and a complete loss of resting membrane potential in crayfish giant axons in vitro. Our results may represent a possible explanation for various grades of local anesthetic-induced neurotoxicity in clinical cases if the same toxicity occurs in mammalian nerves in vivo. (Anesth Analg 1998;86:569-73)