Chainllie Young

Potential of ketamine and midazolam, individually or in combination, to induce apoptotic neurodegeneration in the infant mouse brain

Recently, it was reported that anesthetizing infant rats for 6 h with a combination of anesthetic drugs (midazolam, nitrous oxide, isoflurane) caused widespread apoptotic neurodegeneration in the developing brain, followed by lifelong cognitive deficits. It has also been reported that ketamine triggers neuroapoptosis in the infant rat brain if administered repeatedly over a period of 9 h. The question arises whether less extreme exposure to anesthetic drugs can also trigger neuroapoptosis in the developing brain. To address this question we administered ketamine, midazolam or ketamine plus midazolam subcutaneously at various doses to infant mice and evaluated the rate of neuroapoptosis in various brain regions following either saline or these various drug treatments. Each drug was administered as a single one‐time injection in a dose range that would be considered subanesthetic, and the brains were evaluated by unbiased stereology methods 5 h following drug treatment. Neuroapoptosis was detected by immunohistochemical staining for activated caspase‐3. It was found that either ketamine or midazolam caused a dose‐dependent, statistically significant increase in the rate of neuroapoptosis, and the two drugs combined caused a greater increase than either drug alone. The apoptotic nature of the neurodegenerative reaction was confirmed by electron microscopy. We conclude that relatively mild exposure to ketamine, midazolam or a combination of these drugs can trigger apoptotic neurodegeneration in the developing mouse brain.

Apoptotic neurodegeneration induced by ethanol in neonatal mice is associated with profound learning/memory deficits in juveniles followed by progressive functional recovery in adults.

Administration of ethanol to rodents during the synaptogenesis period induces extensive apoptotic neurodegeneration in the developing brain. This neurotoxicity may explain the reduced brain mass and neurobehavioral disturbances in human Fetal Alcohol Syndrome (FAS). Here, we report binge-like exposure of infant mice to ethanol on a single postnatal day triggered apoptotic death of neurons from diencephalic structures that comprise an extended hippocampal circuit important for spatial learning and memory. The ethanol exposure paradigm yielding these neuronal losses caused profound impairments in spatial learning and memory at 1 month of age. This impairment was significantly attenuated during subsequent development, indicating recovery of function. Recovery was not associated with increased neurogenesis, suggesting plastic reorganization of neuronal networks compensated for early neuronal losses. We hypothesize that neuroapoptotic damage in homologous regions of human brain underlies cognitive deficits in FAS and the human brain of FAS victims has a similar capacity to effect functional recovery.

Subanesthetic doses of propofol induce neuroapoptosis in the infant mouse brain.

Drugs that block N-methyl-d-aspartate glutamate receptors or that promote &ggr;-aminobutyric acid type A inhibition trigger neuroapoptosis in the developing rodent brain. Propofol reportedly interacts with both &ggr;-aminobutyric acid type A and N-methyl-d-aspartate glutamate receptors, but has not been adequately evaluated for its ability to induce developmental neuroapoptosis. Here we determined that the intraperitoneal (i.p.) dose of propofol required to induce a surgical plane of anesthesia in the infant mouse is 200 mg/kg. We then administered graduated doses of propofol (25–300 mg/kg i.p.) and found that doses ≥50 mg/kg induce a significant neuroapoptosis response. We conclude that propofol induces neuroapoptosis at 1/4 the dose required for surgical anesthesia.

Isoflurane-induced Neuroapoptosis in the Developing Brain of Nonhypoglycemic Mice

Drugs that suppress neuronal activity, including general anesthetics used in pediatric and obstetric medicine, trigger neuroapoptosis in the developing rodent brain. Exposure of infant rats for 6 hours to a combination of anesthetic drugs (midazolam, nitrous oxide, isoflurane) reportedly causes widespread apoptotic neurodegeneration, followed by lifelong cognitive deficits. Isoflurane, the dominant ingredient in this triple cocktail, has not been evaluated individually for apoptogenic potential. It was recently reported that (1) the minimum alveolar concentration (MAC) for anesthetizing infant mice with isoflurane is 2.26%, and; (2) that infant mice, without assisted respiration, maintain normal arterial oxygen values but become hypoglycemic when exposed to isoflurane 3% for 30 minutes, then 1.8% for 1 hour (1.46 MAC-hours). In the present experiments, infant mice were exposed to isoflurane at various sub-MAC concentrations and durations, and the brains were evaluated quantitatively 5 hours after initiation of anesthesia exposure to determine the number of neuronal profiles undergoing apoptosis. Blood glucose values were also determined under each of these conditions. All conditions tested (isoflurane at 0.75% for 4 h, 1.5% for 2 h, 2.0% for 1 h) triggered a statistically significant increase in neuroapoptosis compared with the rate of spontaneous apoptosis in littermate controls. Blood glucose determinations ruled out hypoglycemia as a potential cause of the brain damage. It is concluded that exposure to sub-MAC concentrations of isoflurane for one or more hours triggers neuroapoptosis in the infant mouse brain. These findings are consistent with other recent evidence demonstrating that brief exposure to ethanol, ketamine, or midazolam triggers neuroapoptosis in the developing mouse brain.

Anesthesia-induced developmental neuroapoptosis. Does it happen in humans?

THIS issue of the Journal contains a very exciting clinical report that describes the clinical efficacy of epidural sufentanil and neostigmine in laboring women. The authors examined the analgesic effects of different combinations of the two drugs without local anesthetic during the first stage of oxytocin-augmented labor. They found that a mixture of 10 g of sufentanil with 500 g of neostigmine seemed to be optimal based on onset, duration of action, and minimal motor block. Efficient labor analgesia without local anesthetics may have important consequences. Research conducted over the last 20 years has resulted in excellent analgesic recipes, typically combining a very low concentration of a long acting local anesthetic with a lipid-soluble opioid. Such combinations provide near-ideal clinical conditions: low pain scores, minimal motor block (allowing ambulation), stable hemodynamics (as a result of minimal sympathetic block), and essentially no measurable effects on obstetric outcome. Some might actually consider the search for better analgesic techniques to be pointless, given the efficacy of the current approaches. However, local anesthetics are imperfect drugs. Even at low doses, their use may have catastrophic consequences. Moreover, although cesarean deliveries resulting from motor block are uncommon, instrumental delivery is required more frequently when regional analgesia is used. In a recent meta-analysis, the rate of instrumental delivery was almost doubled in patients receiving low dose epidural local anesthetic solutions as compared with systemic opioid analgesia. There is thus a need for analgesic drugs devoid of motor block effect. That neostigmine (the only cholinesterase inhibitor clinically available for spinal use) is one such candidate may be surprising for most of us who have followed the 15-year history of research with this agent. After experimental studies that found that muscarinic receptors and cholinergic pathways are involved in the spinal control of pain, further studies confirmed the analgesic efficacy and the safety (i.e., the absence of neurotoxic complications) of intrathecal neostigmine in humans. However, enthusiasm rapidly declined because of the high incidence and sometimes extreme severity of nausea and vomiting. Because both spinally and epidurally administered drugs mainly act at the spinal level, it is thus difficult to understand the rationale for testing epidural neostigmine. Fortunately, such skepticism did not dissuade researchers who have now shown that after epidural administration, neostigmineinduced analgesia is not associated with emetic complications, thus rehabilitating our interest in cholinergic analgesic pathways. Several trials from various countries and using different acute/pain chronic settings have confirmed that the risk/benefit ratio is excellent with epidural neostigmine. Although epidural neostigmine cannot be used as the sole analgesic because its potency is limited, it can provide excellent pain relief when combined with an opioid. Because women are believed to experience better neostigmine-induced analgesia than men, obstetrics may represent an ideal setting for the drug. In addition, neostigmine is an old and inexpensive drug, thus facilitating its use in the current period of economic constraints. Should the results of Roelants and Lavand’homme lead us to the routine use of epidural neostigmine in all laboring women? Certainly not. Many questions remain unanswered, and additional studies are clearly needed. One major question lies in the safety of the drug. Although there are two trials (in three animal species) demonstrating the absence of neurotoxicity, the drug has been used in only a few hundred human subjects and it is likely that regulatory agencies in most countries will require much more data before concluding that epidurally injected neostigmine is truly safe. An additional concern lies in the analgesic potency of the drug. In the study presented in this issue of ANESTHESIOLOGY, the analgesia produced by the epidural sufentanilneostigmine combination was similar to that achieved with 20 g dose of sufentanil alone. Although this is an interesting result, it is of note that the dose of sufentanil used a comparator is the ED50, i.e., the dose which provides adequate analgesia in only 50% of patients. Clearly, before abandoning local anesthetics, one must ensure that neostigmine can reliably provide adequate analgesia in all patients. Moreover, as this was a singledose study, its efficacy for the whole duration of labor remains uncertain. Will the drug provide adequate analgesia when used as an infusion or will it require top-ups? Will tachyphylaxis occur? Will adverse effects occur (especially fetal effects secondary to placental transfer) after repeated administration and larger doses? Will sedation become a concern? Will muscle weakness become apparent after prolonged administration? These questions, only a few of those remaining, will require a substantial amount of added work to resolve. As with all good studies, questions remaining are more numerous than answers provided. Nevertheless, epidural neostigThis Editorial View accompanies the following article: Roelants F, Lavand’homme PM: Epidural neostigmine combined with sufentanil provides balanced and selective analgesia in early labor. ANESTHESIOLOGY 2004; 101:439–44.

Lithium protects against anesthesia-induced developmental neuroapoptosis.

Background:Ethanol and anesthetic drugs trigger neuroapoptosis in the developing mouse brain. Recently, it was found that ethanol-induced neuroapoptosis is preceded by suppressed phosphorylation of extracellular signal-regulated protein kinase (ERK), and lithium counteracts both the phosphorylated ERK suppressant action and ethanol-induced neuroapoptosis. The current study was undertaken to address the following questions. (1) Do ketamine and propofol mimic ethanol in suppressing ERK phosphorylation? (2) If they do, does lithium prevent this suppressant action and also prevent these anesthetic drugs from triggering neuroapoptosis? Method:Postnatal day 5 mice were treated with propofol, ketamine, lithium, a combination of propofol or ketamine with lithium or saline, and their brains were prepared for Western blot analysis or histology. For Western blot, cytosolic lysates of caudate putamen were analyzed for expression of phosphorylated ERK and phosphorylated serine/threonine-specific protein kinase. For histology, brains were stained immunohistochemically with antibodies to activated caspase-3, and the density of activated caspase-3 positive cells was determined. Results:Ketamine and propofol suppressed phosphorylated ERK, and lithium counteracted both the phosphorylated ERK suppressant action and neuroapoptotic action of these anesthetic drugs. Conclusion:If further testing finds lithium to be safe for use in pediatric/obstetric medicine, administration of a single dose of lithium before anesthesia induction may be a suitable means of mitigating the risk of anesthesia-induced developmental neuroapoptosis.

Docosahexaenoic acid inhibits synaptic transmission and epileptiform activity in the rat hippocampus.

Docosahexaenoic acid (DHA) has been suggested to be required for neuronal development and synaptic plasticity. However, in view of the fact that DHA facilitates NMDA responses and blocks K+ channels, it might predispose the neurons to epileptiform bursting. By using extracellular recording of population spikes in the CA1 region of rat hippocampal slices, we tested this possibility by examining the effect of DHA on the epileptiform activity induced by bicuculline or in Mg2+‐free medium. When stimuli were delivered to the Schaffer collateral/commissural pathway every 20 or 30 sec, DHA had no significant effect on the epileptiform activity. However, when the frequency of stimulation was increased to 0.2 Hz, DHA attenuated the amplitude of the bursting activity induced by bicuculline to 57.5 ± 10.8% and those induced by Mg2+‐free ACSF to 65.8 ± 13.9% of control. DHA reduced the slope of field excitatory postsynaptic potential (fEPSP) to 77.1 ± 7.4% of baseline, without significant effect on the ratio of paired‐pulse facilitation (PPF). By intracellular recording of neurons in the stratum pyramidale of rat hippocampal slices, we found that DHA markedly inhibited the repetitive firing of action potentials elicited by depolarizing current pulses but did not affect the initial action potential. Thus, DHA may attenuate epileptic activity mainly through the frequency‐dependent blockade of Na+ channels. Synapse 37:90–94, 2000.

Cathepsin D Deficiency Induces Persistent Neurodegeneration in the Absence of Bax-Dependent Apoptosis

Neuronal ceroid lipofuscinosces/Batten disease (NCL) is a devastating group of neurodegenerative diseases caused by genetic disruptions in lysosomal function. Cathepsin D (CD) is a major lysosomal protease, and mutations in CD that render it enzymatically defective have been reported recently in subsets of NCL patients. The targeted deletion of CD in mice results in extensive neuropathology, including biochemical and morphological evidence of apoptosis and autophagic stress (aberrant autophagosome accumulation), effects that are similar to those observed in NCL. To determine the contribution of Bax-dependent apoptosis in this mouse model of NCL, combined Bax- and CD-deficient mice were generated. Morphological analysis of CD-deficient mouse brains indicated large numbers of pyknotic neurons and neurons with marked cytoplasmic swellings containing undigested lipofuscin. Cell death and apoptosis were evidenced by increases in terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) reactivity and activation of caspase-3, respectively. DeOlmos silver-positive neurons were abundant in CD-deficient brain and correlated with neuron loss, as indicated by significant decreases in NeuN (neuronal nuclear antigen)-positive neurons. Lysosome dysfunction and autophagic stress were apparent in CD-deficient brain as indicated by the accumulation of autofluorescent storage material and by increased levels of LC3-II (light chain 3-II, a selective autophagosome marker), respectively. Bax deletion significantly inhibited caspase-3 activation and hippocampal TUNEL reactivity but did not prevent the majority of CD deficiency-induced neuropathology, including the persistence of pyknotic neurons, elevated cortical TUNEL reactivity, lysosome dysfunction and autophagic stress, neurodegeneration, and neuron loss. Together, these results suggest that CD deficiency-induced neuropathology does not require Bax-dependent apoptosis and highlights the importance of caspase-independent neuron death and neurodegeneration resulting from the genetic disruption of lysosome function.

Potential of xenon to induce or to protect against neuroapoptosis in the developing mouse brain

Purpose: Drugs that suppress neuronal activity, including all general anesthetics that have been tested thus far (ketamine, midazolam, isoflurane, propofol, and a cocktail of midazolam, nitrous oxide and isoflurane), trigger neuroapoptosis in the developing rodent brain. Combinations of nitrous oxide and isoflurane, or ketamine and propofol, cause more severe neuroapoptosis than any single agent by itself, which suggests a positive correlation between increased levels of anesthesia and increased severity of neuroapoptosis. In contrast, there is evidence that the rare gas, xenon, which has anesthetic properties, protects against isoflurane-induced neuroapoptosis in the infant rat brain, while not inducing neuroapoptosis by itself. The present study was undertaken to evaluate the potential of xenon to induce neuroapoptosis or to protect against neuroapoptosis induced by isoflurane in the infant mouse brain.Methods: Seven-day-old C57BL/6 mice were exposed to one of four conditions: air (control); 0.75% isoflurane; 70% xenon; or 0.75% isoflurane +70% xenon for four hours. For histopathological evaluation of the brains, all pups were euthanized two hours later using activated caspase-3 immunohistochemical staining to detect apoptotic neurons. Under each condition, quantitative assessment of the number of apoptotic neurons in the cerebral cortex (CC) and in the caudate/putamen (C/P) was performed by unbiased stereology.Results: The combination of xenon + isoflurane produced a deeper level of anesthesia than either agent alone. Both xenon alone (p<0.003 in CC;p<0.02 in C/P) and isoflurane alone (p<0.001 in both CC and C/P) induced a significant increase in neuroapoptosis compared to controls. The neuroapoptotic response to isoflurane was substantially more robust than the response to xenon. When xenon was administered together with isoflurane, the apoptotic response was reduced to a level lower than that for isoflurane alone (p<0.01 in CP; marginally non-significant in CC).Conclusions: We conclude that xenon, in the infant mouse brain, has paradoxical properties. It triggers neuroapoptosis, and when combined with isoflurane, it increases the depth of anesthesia, and retains its own apoptogenic activity. However, it suppresses, rather than augments, isoflurane’s apoptogenic activity.RésuméObjectif: Les médicaments supprimant l’activité neuronale, y compris tous les anesthésiants généraux testés jusqu’à présent (kétamine, midazolam, isoflurane, propofol, et un cocktail de midazolam, de protoxyde d’azote et d’isoflurane) déclenchent la neuroapoptose dans le cerveau en développement des rongeurs. Des combinaisons de protoxyde d’azote de d’isoflurane, ou de kétamine et de propofol, provoquent une neuroapoptose plus grave que n’importe quel agent administré seul, ce qui suggère une corrélation positive entre des niveaux plus élevés d’anesthésie et une neuroapoptose plus grave. En revanche, il existe des données soutenant que le xénon, un gaz rare qui présente des propriétés anesthésiques, protège de la neuroapoptose induite par l’isoflurane dans le cerveau de rongeurs nourrissons, alors que seul, il n’induit pas de neuroapoptose. Cette étude a été menée dans le but d’évaluer le potentiel du xénon pour induire la neuroapoptose ou de protéger contre la neuroapoptose provoquée par l’isoflurance dans le cerveau de rongeurs nourrissons.Méthode: Des souris C57BL/6 de sept jours ont été exposées à un de quatre états : air (témoin) ; 0,75 % isoflurane ; 70 % xénon ; ou 0,75 % isoflurane + 70 % xénon pendant quatre heures. Afin de réaliser une évaluation histopathologique du cerveau, tous les petits ont été euthanasiés deux heures plus tard à l’aide d’une technique de coloration immunohistochimique de caspase-3 activée pour permettre de détecter les neurones apoptotiques. Dans chaque état, une évaluation quantitative du nombre de neurones apoptotiques dans le cortex cérébral (CC) et dans le noyau caudé / putamen (C/P) a été réalisée par stéréologie non biaisée.Résultats: La combinaison de xénon + isoflurane a provoqué un niveau d’anesthésie plus profond que lorsque les agents ont été administrés seuls. Le xénon seul (p<0,003 dans CC; p<0,02 dans C/P) et l’isoflurane seul (p<0,001 dans le CC et le C/P) ont provoqué une augmentation significative de neuroapoptose par rapport au groupe témoin. La réaction neuroapoptotique à l’isoflurane était considérablement plus puissante que la réaction au xénon. Lorsque le xénon a été administré avec l’isoflurane, la réaction apoptotique a diminué à un niveau plus bas que celui de l’isoflurane seul (p<0,01 dans CP; marginalement non significatif dans CC).Conclusion: Nous concluons que le xénon, dans le cerveau de rongeurs nourrissons, possède des propriétés paradoxales. Il déclenche la neuroapoptose et, lorsqu’il est combiné à l’isoflurane, approfondit l’anesthésie, et retient sa propre activié apoptogène. Toutefois il supprime plutôt qu’augmente l’activité apoptogène de l’isoflurane.

Excitotoxic Versus Apoptotic Mechanisms of Neuronal Cell Death in Perinatal Hypoxia / Ischemia

Hypoxic/ischemic (H/I) neuronal degeneration in the developing central nervous system (CNS) is mediated by an excitotoxic mechanism, and it has also been reported that an apoptosis mechanism is involved. However, there is much disagreement regarding how excitotoxic and apoptotic cell death processes relate to one another. Some authors believe that an excitotoxic stimulus directly triggers apoptotic cell death, but this interpretation is largely speculative at the present time. Our findings support the interpretation that excitotoxic and apoptotic neurodegeneration are two separate and distinct cell death processes that can be distinguished from one another by ultrastructural evaluation. Here we review evidence supporting this interpretation, including evidence that H/I in the developing CNS triggers two separate waves of neurodegeneration, the first being excitotoxic and the second being apoptotic. The first (excitotoxic) wave destroys neurons that would normally provide synaptic inputs or synaptic targets for the neurons that die in the second (apoptotic) wave. Since neurons, during the developmental period of synaptogenesis, are programmed to commit suicide if they fail to achieve normal connectivity, this explains why neuroapoptosis occurs following H/I in the developing CNS. However, it does not support the interpretation that H/I directly triggers apoptotic neurodegeneration. Rather, it documents that H/I directly triggers excitotoxic neurodegeneration, and apoptotic neurodegeneration ensues subsequently as the natural response of developing neurons to a specific kind of deprivation - loss of the ability to form normal synaptic connections.