Maiko Satomoto

Neonatal Exposure to Sevoflurane Induces Abnormal Social Behaviors and Deficits in Fear Conditioning in Mice

Background:Neonatal exposure to anesthetics that block N-methyl-d-aspartate receptors and/or hyperactivate &ggr;-aminobutyric acid type A receptor has been shown to cause neuronal degeneration in the developing brain, leading to functional deficits later in adulthood. The authors investigated whether exposure of neonatal mice to inhaled sevoflurane causes deficits in social behavior as well as learning disabilities. Methods:Six-day-old C57BL/6 mice were exposed to 3% sevoflurane for 6 h. Activated cleaved caspase-3 immunohistochemical staining was used for detection of apoptosis. Cognitive functions were tested by pavlovian conditioned fear test. Social behavior was tested by social recognition and interaction tests. Results:Neonatal exposure to sevoflurane significantly increased the number of apoptotic cells in the brain immediately after anesthesia. It caused persistent learning deficits later in adulthood as evidenced by decreased freezing response in both contextual and cued fear conditioning. The social recognition test demonstrated that mice with neonatal exposure to sevoflurane did not develop social memory. Furthermore, these mice showed decreased interactions with a social target compared with controls in the social interaction test, indicating a social interaction deficit. The authors did not attribute these abnormalities in social behavior to impairments of general interest in novelty or olfactory sensation, because they did not detect significant differences in the test for novel inanimate object interaction or for olfaction. Conclusions:This study shows that exposure of neonatal mice to inhaled sevoflurane could cause not only learning deficits but also abnormal social behaviors resembling autism spectrum disorder.

Isoflurane anesthesia induces biphasic effect on dopamine release in the rat striatum.

The effect of isoflurane anesthesia on changes in the extracellular concentrations of dopamine (DA) and its metabolites (3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)) modulated by pargyline, monoamine oxidase inhibitor, was studied using in vivo microdialysis techniques. A microdialysis probe was implanted into the right striatum of male SD rats. Each rat (n=5-6) was given saline or the same volume of 30 or 75 mg kg(-1) pargyline intraperitoneally with or without 1 h isoflurane anesthesia (1 or 3%). Isoflurane anesthesia increased the extracellular concentration of DA in high dose (3%) and increased the metabolite concentrations in a dose-dependent manner. Pargyline administration increased the extracellular concentration of DA and 3-MT, and decreased that of other metabolites. After 30 mg kg(-1) pargyline treatment, 1% isoflurane-induced DA release and increasing of 3-MT were preserved, whereas high dose isoflurane (3%) decreased the concentration of metabolites (DOPAC and HVA), despite of the increase by low dose isoflurane (DOPAC). When 75 mg kg(-1) pargyline was administered, isoflurane anesthesia decreased the concentration of DA and DOPAC. The isoflurane-induced 3-MT increase was preserved in all experiments. Our results suggest that isoflurane anesthesia induced biphasic effect on DA regulation probably by the potentiation of DA release and the inhibition of DA synthesis. Isoflurane might modulate DA homeostasis presynaptically.

Fentanyl attenuates the hemodynamic response to endotracheal intubation more than the response to laryngoscopy.

We examined the effectiveness of avoiding laryngoscopy in reducing the hemodynamic responses to orotracheal intubation during the induction of anesthesia. One hundred surgical patients who required orotracheal intubation were randomly allocated into four groups. The first and third groups underwent fiberoptic intubation, in which an anesthesiologist inserted the endotracheal tube into the trachea under TV monitoring through a bronchoscope, and the second and fourth groups underwent conventional orotracheal intubation using a rigid laryngoscope. The third and fourth groups were pretreated with 2 &mgr;g/kg fentanyl IV immediately before the induction of anesthesia. Blood pressure and heart rate were measured noninvasively. A significant reduction in hemodynamic response was seen in only the group treated with fentanyl and intubated using the fiberoptic technique. Without fentanyl, there was no significant difference in hemodynamic changes between the groups. We conclude that the administration of fentanyl suppresses the hemodynamic responses to endotracheal intubation more than it does to laryngoscopy. There was no significant difference in the hemodynamic responses to orotracheal intubation by fiberscopy and laryngoscopy without fentanyl pretreatment, whereas 2 &mgr;g/kg fentanyl significantly reduced the hemodynamic responses in the group intubated by fiberscopy.

Isoflurane anesthesia inhibits clozapine- and risperidone-induced dopamine release and anesthesia-induced changes in dopamine metabolism was modified by fluoxetine in the rat striatum: An in vivo microdialysis study

Previously, we have reported that halothane anesthesia increases the extracellular concentrations of dopamine (DA) metabolites in the rat striatum using in vivo microdialysis techniques, and we have suggested that volatile anesthetics affect DA release and metabolism in various ways. The present investigation assesses the effect of isoflurane, widely used in clinical anesthesia, on DA release and metabolism. A microdialysis probe was implanted in the striatum of male Sprague-Dawley rats (n=5-7 per group). After recovery, the probe was perfused with modified Ringers solution and 40 microl of dialysate were injected into a high performance liquid chromatograph every 20 min. The rats were given saline or the same volume of 10 mg kg(-1) clozapine, risperidone, fluoxetine or citalopram. After the pharmacological treatment, the rats were anesthetized with 1.0% or 2.5% isoflurane for 1h. The data were analyzed using two-way analysis of variance (ANOVA). For each drug with significant (p<0.05) drug-time interactions, the statistical analysis included one-way ANOVA and Newman-Keuls post hoc comparisons. A high concentration of isoflurane (2.5%) anesthesia increased the extracellular concentration of DA metabolites during emergence from anesthesia. The levels of DA metabolites increased in an isoflurane concentration-dependent manner. Isoflurane attenuated DA release induced by clozapine and risperidone. Fluoxetine, but not citalopram, antagonized the isoflurane-induced increase in metabolites. The results of current investigation suggest that isoflurane enhances presynaptic DA metabolism, and that the oxidation of DA might be partially modulated by the activities of the dopaminergic-serotonergic pathway at a presynaptic site in the rat striatum.

Inhibiting NADPH oxidase protects against long-term memory impairment induced by neonatal sevoflurane exposure in mice

BACKGROUND Neonatal exposure to anaesthetics such as sevoflurane has been reported to result in behavioural deficits in rodents. However, while oxidative injury is thought to play an underlying pathological role, the mechanisms of neurotoxicity remain unclear. In the present study, we investigated whether the NADPH oxidase inhibitor apocynin protects against long-term memory impairment produced by neonatal sevoflurane exposure in mice. METHODS Postnatal day six mice were divided into four groups; (1) non-anaesthesia, (2) intraperitoneal apocynin (50 mg kg(-1)) treatment, (3) 3% sevoflurane exposure for 6 h, and (4) apocynin treatment combined with sevoflurane exposure. Superoxide concentrations and NADPH oxidase expression in the brain were determined using dihydroethidium fluorescence and immunoblotting, respectively. Cleaved caspase-3 immunoblotting was used for the detection of apoptosis, and cytochrome c immunoblotting was performed to evaluate mitochondrial function. Long-term cognitive impairment was evaluated using the fear conditioning test in adulthood. RESULTS Sevoflurane exposure increased concentrations of superoxide (109%) and the NADPH oxidase subunit p22phox (39%) in the brain, and apocynin abolished these increases. Neonatal sevoflurane exposure caused learning deficits in adulthood. Apocynin also maintained long-term memory function in mice given neonatal sevoflurane exposure, and it reduced apoptosis and decreased cytochrome c concentrations in the brains of these mice. CONCLUSIONS Apocynin reduces neuronal apoptosis and protects against long-term memory impairment in mice, neonatally exposed to sevoflurane by reducing superoxide concentrations. These findings suggest that NADPH oxidase inhibitors may protect against cognitive dysfunction resulting from neonatal anaesthesia.

Mechanical allodynia but not thermal hyperalgesia is impaired in mice deficient for ERK2 in the central nervous system

Summary Mice deficient in ERK2 in the central nervous system exhibited altered responses in pain models, indicating a predominant role of ERK2 in pain plasticity. ABSTRACT Extracellular signal‐regulated kinase (ERK) plays critical roles in pain plasticity. However, the specific contribution of ERK2 isoforms to pain plasticity is not necessarily elucidated. Here we investigate the function of ERK2 in mouse pain models. We used the Cre‐loxP system to cause a conditional, region‐specific, genetic deletion of Erk2. To induce recombination in the central nervous system, Erk2‐floxed mice were crossed with nestin promoter‐driven cre transgenic mice. In the spinal cord of resultant Erk2 conditional knockout (CKO) mice, ERK2 expression was abrogated in neurons and astrocytes, but indistinguishable in microglia compared to controls. Although Erk2 CKO mice showed a normal baseline paw withdrawal threshold to mechanical stimuli, these mice had a reduced nociceptive response following a formalin injection to the hind paw. In a partial sciatic nerve ligation model, Erk2 CKO mice showed partially restored mechanical allodynia compared to control mice. Interestingly, thermal hyperalgesia was indistinguishable between Erk2 CKO and control mice in this model. In contrast to Erk2 CKO mice, mice with a targeted deletion of ERK1 did not exhibit prominent anomalies in these pain models. In Erk2 CKO mice, compensatory hyperphosphorylation of ERK1 was detected in the spinal cord. However, ERK1 did not appear to influence nociceptive processing because the additional inhibition of ERK1 phosphorylation using MEK (MAPK/ERK kinase) inhibitor SL327 did not produce additional changes in formalin‐induced spontaneous behaviors in Erk2 CKO mice. Together, these results indicate that ERK2 plays a predominant and/or specific role in pain plasticity, while the contribution of ERK1 is limited.

Pentobarbital inhibits L-DOPA-induced dopamine increases in the rat striatum: An in vivo microdialysis study.

Pentobarbital is reported to inhibit ketamine-induced dopamine (DA) release in the rat nucleus accumbens. The accumbens is a part of the limbic dopaminergic system in the brain, and the dopaminergic neural activity of other components may also be sensitive to pentobarbital. We investigated the effect of pentobarbital administration on DA release in the striatum known as DA-rich basal ganglia, and the interaction between pentobarbital and L-DOPA, using in vivo microdialysis techniques. Male SD rats were implanted microdialysis probe into the right striatum. The probe was perfused with modified Ringers solution and dialysate was directly injected to an HPLC. Every group of rats was consisted of six to seven animals. In the first experiment, rats were given saline, 25 and 50 mg kg(-1) pentobarbital. The second, each rat was given a local administration of 2 and 5 microg ml(-1) of L-DOPA with perfusate. Finally, other sets of rats were given 5 microg ml(-1) of L-DOPA and 25, 50, or 100 mg kg(-1) pentobarbital. Pentobarbital anaesthesia decreased the extracellular concentration of DA, and local administration of L-DOPA significantly increased DA concentration. Pretreatment with pentobarbital diminished the L-DOPA-induced DA increase. The results of the present investigation demonstrate that administration of pentobarbital might inhibit dopaminergic neural activity not only in the nucleus accumbens but also in the rat striatum. Pentobarbital anaesthesia antagonizes DA increase induced by L-DOPA and suggests the inhibition of metabolism of L-DOPA. The results of some animal experiments on dopaminergic activity under pentobarbital anaesthesia should be reconsidered.

Therapeutic effects of intravenous administration of bone marrow stromal cells on sevoflurane-induced neuronal apoptosis and neuroinflammation in neonatal rats.

Background Sevoflurane exposure during the early postnatal period causes neuroinflammation and neuronal apoptosis in rodents. Bone marrow stromal cells (BMSCs) have been shown to protect and repair the damaged central nervous system, for example in ischemic stroke models. In this study, we investigated whether intravenous administration of BMSCs ameliorated neurodegeneration, induced by sevoflurane exposure, in neonatal rats. Methods Sprague-Dawley rat pups (postnatal day 7) were exposed to 2% sevoflurane for 6 h (vehicle group, n = 7). BMSCs were administered 30 min after induction of sevoflurane anesthesia (BMSCs group, n = 7). The pups were exposed to carrier gas only, as a negative control (mock anesthesia group, n = 4). We assessed the therapeutic effects of BMSC treatment by measuring expression of the pro-inflammatory cytokine interleukin-6 (IL-6), and levels of cleaved caspase-3, in brain tissues immediately following sevoflurane anesthesia. Results Analysis of the cleaved caspase-3 bands revealed that levels of activated caspase-3 were elevated in the vehicle group compared with the mock anesthesia group, indicating that a single exposure to sevoflurane at subclinical concentrations can precipitate neuronal apoptosis. BMSC treatment did not suppress apoptosis induced by sevoflurane exposure (compared with the vehicle group). The vehicle group had higher proinflammatory cytokine IL-6 protein levels compared with the mock anesthesia group, indicating that sevoflurane exposure induces IL-6 expression. BMSC treatment suppressed sevoflurane-induced increases in IL-6 expression, indicating that these cells can inhibit the neuroinflammation induced by sevoflurane exposure (vehicle group vs. BMSC group). Conclusions Intravenous administration of BMSCs reduces neuroinflammation, but does not attenuate apoptosis induced by sevoflurane exposure.

Anesthesia-induced neurotoxicity in an animal model of the developing brain: mechanism and therapies

Children are being exposed to an increasingly greater variety of anesthetics with advances in pediatric and obstetric surgery. Recent animal and retrospective human data suggest that the general anesthetics commonly used in pediatric medicine could be damaging to the developing brain when used at clinical concentrations. In vivo primate and rodent models have shown that neonatal exposure to clinical concentrations of anesthetics causes neural apoptosis and long-term cognitive impairment. Many general anesthetics, such as isoflurane, sevoflurane, barbiturates, benzodiazepines, ketamine, propofol, and nitrous oxide, cause adverse changes in the neonatal rodent and primate brain. Animal and human data suggest an association between general anesthesia during the neonatal period and long-term cognitive impairment. Cohort studies involving humans have recently been started. The window of vulnerability to these neurotoxic effects of anesthetics is restricted to the period of synaptogenesis, also known as the “brain growth spurt” (BGS) period. To minimize the risk of neurodegeneration, it is necessary to study both the mechanism of neurotoxicity and preventative medicine. Neonatal anesthetic exposure affects many mechanisms of neurotoxicity. Mechanisms of anesthetic-induced neurotoxicity seem to involve altered expression of ligand-gated ion channels, disturbance of intracellular calcium homeostasis, and the mitochondria-mediated apoptotic pathway. Several agents reportedly help to prevent anesthesia-induced neurotoxicity, including hydrogen, melatonin, apocynin, and ketorolac, and should thus be co-administered with anesthetics. After anesthesia, only environmental enrichment can improve learning deficits due to anesthesia-induced neurotoxicity. Further studies of environmental enrichment (Wu et al., 2016) after anesthesia are necessary to develop preventative and therapeutic strategies for anesthesia-induced neurotoxicity.

The determinants of propofol induction time in anesthesia.

Background The required dose of anesthetics is generally smaller in patients with low cardiac output (CO). A high CO decreases the blood concentration of anesthetics during induction and maintenance of anesthesia. However, a high CO may also shorten the delivery time of anesthetics to the effect site, e.g. the brain. We assessed the time required for induction of anesthesia with propofol administered by target-controlled infusion (TCI), and investigated factors that modify the pharmacodynamics of propofol. Methods After measuring CO and blood volume (BV) by dye densitometry, propofol was infused using TCI to simulate a plasma concentration of 3 µg/ml. After infusion, the time taken to achieve bispectral index (BIS) values of 80 and 60 was determined. Age, sex, lean body mass (LBM), and cardiovascular parameters were analyzed as independent variables. The dependent variables were the time taken to achieve each BIS value and the plasma concentration of propofol (Cp) 10 min after the commencement of infusion. Results Multiple regression analysis revealed that a high CO significantly reduced the time taken to reach the first end point (P = 0.020, R2 = 0.076). Age and LBM significantly prolonged the time taken to reach the second end point (P = 0.001). Cp was negatively correlated with BV (P = 0.020, R2 = 0.073). Conclusions Cardiac output was a statistically significant factor for predicting the time required for induction of anesthesia in the initial phase, whereas, age and LBM were significant variables in the late phase. The pharmacodynamics of propofol was intricately altered by CO, age, and LBM.