Agomelatine Attenuates Isoflurane-Induced Inflammation and Damage in Brain Endothelial Cells.

BACKGROUND AND PURPOSE: Neurotoxicity of anesthetics has been widely observed by clinicians. It is reported that inflammation and oxidative stress are involved in the pathological process. In the present study, we aimed to assess the therapeutic effects of agomelatine against isoflurane-induced inflammation and damage to brain endothelial cells. MATERIALS AND METHODS: MTT assay was used to detect cell viability in order to determine the optimized concentration of agomelatine. The bEnd.3 brain endothelial cells were treated with 2% isoflurane in the presence or absence of agomelatine (5, 10 µM) for 24 h. LDH release was evaluated and the ROS levels were checked using DHE staining assay. The expressions of IL-6, IL-8, TNF-α, VEGF, TF, VCAM-1, and ICAM-1 were evaluated using real-time PCR and ELISA. Real-time PCR and Western blot analysis were used to determine the expression level of Egr-1. RESULTS: The decreased cell viability promoted LDH release and elevated ROS levels induced by isoflurane were significantly reversed by the introduction of agomelatine in a dose-dependent manner. The expression levels of IL-6, IL-8, TNF-α, VEGF, TF, VCAM-1, and ICAM-1 were elevated by stimulation with isoflurane, which were significantly suppressed by the administration of agomelatine. The up-regulation of transcriptional factor Egr-1 induced by isoflurane was down-regulated by agomelatine. CONCLUSION: Agomelatine might attenuate isoflurane-induced inflammation and damage via down-regulating Egr-1 in brain endothelial cells.

Propofol Attenuates Isoflurane-Induced Neurotoxicity and Cognitive Impairment in Fetal and Offspring Mice.

BACKGROUND: Anesthesia in pregnant rodents causes neurotoxicity in fetal and offspring rodents. However, the underlying mechanisms and targeted treatments remain largely to be determined. Isoflurane and propofol are among commonly used anesthetics. Thus, we set out to investigate whether propofol can mitigate the isoflurane-induced neurotoxicity in mice. METHODS: Pregnant C57BL/6 mice at gestational day 15 (G15) were randomly assigned to 4 groups: control, isoflurane, propofol, and isoflurane plus propofol. Levels of interleukin (IL)-6 and poly-ADP ribose polymerase (PARP) fragment were measured in the brains of G15 embryos, and levels of postsynaptic density (PSD)-95 and synaptophysin were determined in the hippocampal tissues of postnatal day 31 (P31) offspring using Western blotting and immunohistochemical staining. Learning and memory functions in P31 offspring were determined using a Morris water maze test. RESULTS: Isoflurane anesthesia in pregnant mice at G15 significantly increased brain IL-6 (222.6% ± 36.45% vs 100.5% ± 3.43%, P < .0001) and PARP fragment (384.2% ± 50.87% vs 99.59% ± 3.25%, P < .0001) levels in fetal mice and reduced brain PSD-95 (30.76% ± 2.03% vs 100.8% ± 2.25%, P < .0001) and synaptophysin levels in cornu ammonis (CA) 1 region (57.08% ± 4.90% vs 100.6% ± 2.20%, P < .0001) and dentate gyrus (DG; 56.47% ± 3.76% vs 99.76% ± 1.09%, P < .0001) in P31 offspring. Isoflurane anesthesia also impaired cognitive function in offspring at P31. Propofol significantly mitigated isoflurane-induced increases in brain IL-6 (117.5% ± 10.37% vs 222.6% ± 36.45%, P < .0001) and PARP fragment (205.1% ± 35.99% vs 384.2% ± 50.87%, P < .0001) levels in fetal mice, as well as reductions in PSD-95 (49.79% ± 3.43% vs 30.76% ± 2.03%, P < .0001) and synaptophysin levels in CA1 region (85.57% ± 2.97% vs 57.08% ± 4.90%, P < .0001) and DG (85.05% ± 1.87% vs 56.47% ± 3.76%, P < .0001) in hippocampus of P31 offspring. Finally, propofol attenuated isoflurane-induced cognitive impairment in offspring. CONCLUSIONS: These findings suggest that gestational isoflurane exposure in mice induces neuroinflammation and apoptosis in embryos and causes cognitive impairment in offspring. Propofol can attenuate these isoflurane-induced detrimental effects.

The protective effect of trilobatin against isoflurane-induced neurotoxicity in mouse hippocampal neuronal HT22 cells involves the Nrf2/ARE pathway.

Long-term exposure to isoflurane may induce long-term developmental neurotoxicity and cognitive impairments in the neonatal brains. Trilobatin, a leaf extract from the Chinese traditional sweet tea Lithocarpus polystachyus Rehd, possesses various biological properties including anti-inflammatory and anti-oxidant properties. Our study aimed to explore the neuroprotective effect of trilobatin on isoflurane-induced neurotoxicity in mouse hippocampal neuronal HT22 cells. The effects of trilobatin on cell viability, LDH release, apoptosis, and caspase-3/7 activity in isoflurane-induced HT22 cells were explored by CCK-8, LDH release assay, flow cytometry analysis, and caspase-3/7 activity assay, respectively. Oxidative stress was evaluated by measuring the levels of reactive oxygen species (ROS) and malonyldialdehyde (MDA) and activities of superoxide dismutase (SOD) and catalase (CAT). The expression of nuclear erythroid-2 related factor 2 (Nrf2), nuclear Nrf2, heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase 1 (NQO1) was determined by western blot and qRT-PCR. Results suggested that exposure to isoflurane significantly reduced cell viability and increased LDH release, apoptotic rate and caspase-3/7 activity in HT22 cells, which were abolished by trilobatin. Trilobatin reversed isoflurane-induced increase of ROS and MDA levels and reduction of SOD and CAT activities in HT22 cells. Additionally, trilobatin promoted the nuclear translocation of Nrf2 as well as the mRNA and protein expression of HO-1 and NQO1 in HT22 cells exposed to isoflurane. Nrf2 knockdown attenuated the effects of trilobatin on isoflurane-induced viability reduction, LDH release, apoptosis, and oxidative stress in HT22 cells. Overall, trilobatin protected HT22 cells against isoflurane-induced neurotoxicity via activating the Nrf2/antioxidant response element (ARE) pathway.

Neuroprotective effect of DUSP14 overexpression against isoflurane-induced inflammatory response, pyroptosis and cognitive impairment in aged rats through inhibiting the NLRP3 inflammasome.

OBJECTIVE: Postoperative cognitive dysfunction (POCD) is a common complication after general anesthesia in the elderly people. Dual-specificity phosphatase 14 (DUSP14, also known as MKP6) has been implicated in the pathogenesis of various inflammatory diseases. However, the exact role and mechanism of DUSP14 in POCD remains unclear. MATERIALS AND METHODS: An isoflurane exposure induced POCD aged rat model was successfully constructed. The pathological changes of hippocampal tissues of aged rats were detected by Nissl staining. Evaluation of learning and memory abilities in aged rats was measured using Morris water maze task test. The DUSP14 level was detected by immunohistochemistry (IHC) assay, quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. Levels of brain injury markers [S-100ß and neuron specific enolase (NSE)] and inflammatory cytokines [interleukin (IL)-1ß (tumor necrosis factor (TNF)-α and IL-6] were detected using Enzyme Linked Immunosorbent Assay (ELISA) or qRT-PCR. The apoptosis of hippocampal nerve cells was assessed by Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Western blot assay was used to measure the expression of proteins related to apoptosis, pyroptosis and NOD-like receptor family pyrin domain-containing 3 (NLRP3)-Caspase-1 pathway. RESULTS: Isoflurane exposure led to brain injury, inflammatory response, cognitive dysfunction in aged rats and decreased the expression of DUSP14. Overexpression of DUSP14 could inhibit apoptosis, inflammation, pyroptosis, brain tissue damage, and improve cognitive dysfunction of aged rats after isoflurane anesthesia. Further mechanism studies revealed that DUSP14 may play a neuroprotective effect on POCD by regulating NLRP3 inflammasome-mediated pyroptosis. CONCLUSIONS: DUSP14 may effectively protect against isoflurane-induced neuro-inflammation, brain damage and cognitive dysfunction, indicating that DUSP14 may be a potential predictor and therapeutic target for POCD.

Efecto antagonista del flumazenil sobre el isoflurano en la emersión de la anestesia general / Antagonist effect of flumazenil on isofluran in reversion of general anesthesia

INTRODUCTION AND OBJECTIVES: Isoflurane, an inhalational general anesthetic widely used in medical practice, belonging to the group of volatile liquids together with desflurane and sevoflurane, with various properties including sedation, hypnosis and anesthesia of patients undergoing treatment. surgical acts. Volatile inhalational anesthetics (halogenated) as mechanism of action, has the property of increasing inhibitory synaptic transmission at postsynaptic level by potentiating ion channels regulated by ligand activated by alpha-aminobutyric acid (GABA). Flumazenil is a benzodiazepine antagonist belonging to the group of imidazobenzodiazepine. It is currently known that there is no specific drug capable of antagonizing the effects of halogenates that allow the rapid and complete recovery of general anesthesia, for this reason this work focuses its efforts on demonstrating whether flumazenil has the ability to reverse the actions of the patient. isoflurane and allow an early restoration of the level of consciousness. MATERIAL AND METHODS: The study to be performed is a clinical type of longitudinal, prospective, unicentric and double blind. The sample will be formed by patients who are going to be subjected to a balanced general anesthesia. The sample will be divided into 2 large groups: group C (control) and group F (Flumazenil). At the end of the surgery, the mixture will be administered according to the selected group in a random manner (Flumazenil 0.25 mg or 0.9% solution in a 20 cc syringe) and the time of extubation, recovery time of the level of consciousness, time of discharge UCPA and hemodynamic state (FC, TAM and SO2). RESULTS: The flumazenil group showed a significantly shorter time from injection to extubation than the placebo group (p = 0.007). Differences in terms of shorter times needed to achieve Aldrete of 9 points in the flumazenil group (P = 0.04) were observed as were shorter anesthetic arousal times represented by a Ramsey 2. Heart rate, mean arterial pressure and saturation they had similar values between the 2 groups. CONCLUSION: The study showed that a single dose of 0.25 mg of flumazenil administered at the end of the surgical act, just after completing all surgical stimulation was beneficial (P = 0.007) in the context of extubation times and shorter anesthetic arousal times.

INTRODUCCIÓN Y OBJETIVOS: El isoflurano un anestésico general inhalatorio usado ampliamente en la práctica médica, perteneciente al grupo de los líquidos volátiles junto con el desflurano y sevoflurano, con variadas propiedades entre las que se encuentran la sedación, hipnosis y anestesia de los pacientes sometidos a actos quirúrgicos. Los anestésicos inhalatorios volátiles (halogenados) como mecanismo de acción, tiene la propiedad de aumentar la transmisión sináptica inhibidora a nivel postsináptico potenciando los canales iónicos regulados por ligando activados por ácido alfa-aminobutírico (GABA). El flumazenil es un antagonista benzodiazepínico perteneciente al grupo de los imidazobenzodiazepina. Se conoce actualmente que no existe un fármaco específico capaz de antagonizar los efectos de los halogenados que permitan la recuperación rápida y completa de la anestesia general, por tal motivo este trabajo centra sus esfuerzos en demostrar si el flumazenil tiene la capacidad para revertir las acciones del isoflurane y permitir un restablecimiento temprano del nivel de conciencia. MATERIALES Y MÉTODOS: El estudio a realizar es de tipo clínico de corte longitudinal, prospectivo, unicéntrico y doble ciego. La muestra se conformará por pacientes que vayan a ser sometidos a anestesia general balanceada. Se procederá a dividir la muestra en 2 grandes grupos: grupo C (control) y grupo F (flumazenil). Al final de la cirugía se administrará la mezcla según grupo seleccionado de manera al azar (flumazenil 0,25 mg o solución 0,9% en una jeringa de 20 cc) y se valorará el tiempo de extubación, tiempo de recuperación del nivel de conciencia, tiempo de alta de la UCPA y estado hemodinámico (FC, TAM y SO2). RESULTADOS: El grupo de flumazenil presentó un tiempo desde la inyección hasta la extubación significativamente más bajo que el grupo placebo (p = 0,007). Se observaron diferencias en términos de tiempos más bajos necesario para alcanzar Aldrete de 9 puntos en el grupo flumazenil (P = 0,04) al igual que tiempos de despertar anestésico más cortos representados por un Ramsey 2. La frecuencia cardíaca, presión arterial media y la saturación tuvieron valores similares entre los 2 grupos. CONCLUSIÓN: El estudio demostró que una única dosis de 0,25 mg de flumazenil administrado al final del acto quirúrgico, justo después de culminar toda estimulación quirúrgica fue beneficiosa (P = 0,007) en el contexto de tiempos de extubación y tiempos de despertar anestésico más cortos.

Neuroprotective effects of dexmedetomidine against isoflurane-induced neuronal injury via glutamate regulation in neonatal rats.

BACKGROUND: Considerable evidences support the finding that the anesthesia reagent isoflurane increases neuronal cell death in young rats. Recent studies have shown that dexmedetomidine can reduce isoflurane-induced neuronal injury, but the mechanism remains unclear. We investigated whether isoflurane cause neurotoxicity to the central nervous system by regulating the N-methyl-D-aspartate receptor (NMDAR) and excitatory amino acid transporter1 (EAAT1) in young rats. Furthermore, we examined if dexmedetomidine could decrease isoflurane-induced neurotoxicity. METHODS: Neonatal rats (postnatal day 7, n=144) were randomly divided into four groups of 36 animals each: control (saline injection without isoflurane); isoflurane (2% for 4 h); isoflurane + single dose of dexmedetomidine (75 µg/kg, 20 min before the start of 2% isoflurane for 4 h); and isoflurane + dual doses of dexmedetomidine (25 µg/kg, 20 min before and 2 h after start of isoflurane at 2% for 4 h). Six neonates from each group were euthanatized at 2 h, 12 h, 24 h, 3 days, 7 days and 28 days post-anesthesia. Hippocampi were collected and processed for protein extraction. Expression levels of the NMDAR subunits NR2A and NR2B, EAAT1 and caspase-3 were measured by western blot analysis. RESULTS: Protein levels of NR2A, EAAT1 and caspase-3 were significantly increased in hippocampus of the isoflurane group from 2 h to 3 days, while NR2B levels were decreased. However, the -induced increase in NR2A, EAAT1 and caspase-3 and the decrease in NR2B in isoflurane-exposed rats were ameliorated in the rats treated with single or dual doses of dexmedetomidine. Isoflurane-induced neuronal damage in neonatal rats is due in part to the increase in NR2A and EAAT1 and the decrease in NR2B in the hippocampus. CONCLUSION: Dexmedetomidine protects the brain against the use of isoflurane through the regulation of NR2A, NR2B and EAAT1. However, using the same amount of dexmedetomidine, the trend of protection is basically the same.

Carbon monoxide incompletely prevents isoflurane-induced defects in murine neurodevelopment.

BACKGROUND: Commonly used anesthetics have been shown to disrupt neurodevelopment in preclinical models. It has been proposed that such anesthesia-induced neurotoxicity is mediated by apoptotic neurodegeneration in the immature brain. Low dose carbon monoxide (CO) exerts cytoprotective properties and we have previously demonstrated that CO inhibits isoflurane-induced apoptosis in the developing murine brain. Here we utilized anti-apoptotic concentrations of CO to delineate the role of apoptotic neurodegeneration in anesthesia-induced neurotoxicity by assessing the effect of CO on isoflurane-induced defects in neurodevelopment. METHODS: C57Bl/6 mouse pups underwent 1-hour exposure to 0ppm (air), 5ppm, or 100ppm CO in air with or without isoflurane on postnatal day 7. Cohorts were evaluated 5-7weeks post exposure with T-maze cognitive testing followed by social behavior assessment. Brain size, whole brain cellular content, and neuronal density in primary somatosensory cortex and hippocampal CA3 region were measured as secondary outcomes 1-week or 5-7weeks post exposure along with 7-day old, unexposed controls. RESULTS: Isoflurane impaired memory acquisition and resulted in abnormal social behavior. Low concentration CO abrogated anesthetic-induced defects in memory acquisition, however, it also resulted in impaired spatial reference memory and social behavior abnormalities. Changes in brain size, cellular content, and neuronal density over time related to the age of the animal and were unaffected by either isoflurane or CO. CONCLUSIONS: Anti-apoptotic concentrations of CO incompletely prevented isoflurane-induced defects in neurodevelopment, lacked concentration-dependent effects, and only provided protection in certain domains suggesting that anesthesia-related neurotoxicity is not solely mediated by activation of the mitochondrial apoptosis pathway.

Erythropoietin diminishes isoflurane-induced apoptosis in rat frontal cortex.

BACKGROUND: During the brain growth spurt, anesthetic drugs can cause cellular and behavioral changes in the developing brain. The aim of this study was to determine the neuroprotective effect of erythropoietin after isoflurane anesthesia in rat pups. METHODS: A total of 42, 7-day-old Wistar rats were divided into three groups. Control group (GC; n = 14): Rats breathed 100% oxygen for 6 h; Isoflurane group (GI; n = 14): Rats were exposed to 1.5% isoflurane in 100% oxygen for 6 h; Isoflurane + erythropoietin group (GIE; n = 14): 1000 IU·kg(-1) (intraperitoneal; IP) Erythropoietin was administered after isoflurane anesthesia. Each group was divided into two groups for pathology and learning and memory tests. Silver, caspase-3, and fluoro-jade C staining were used for detecting apoptotic cells in frontal cortex, striatum, hippocampus, thalamus, and amygdala. Morris water maze was used to evaluate learning and memory. RESULTS: There was a significant increase in apoptotic cell count after isoflurane anesthesia in the frontal cortex when compared with control group (29.0 ± 9.27 vs 3.28 ± 0.75 [P = 0.002], 20.85 ± 10.94 vs 2.0 ± 0.81 [P = 0.002] and 24.57 ± 10.4 vs 5.14 ± 0.69 [P = 0.024] with silver, caspase-3, and fluoro-jade C staining, respectively). The apoptotic cell count in the frontal cortex was significantly higher in GIE than GC with caspase-3 staining (9.14 ± 3.13 vs 2.0 ± 0.81, P = 0.002). The apoptotic cell count in GIE was significantly reduced in the frontal cortex when compared with GI (4.0 ± 0.81 vs 29.0 ± 9.27 [P = 0.002], 9.14 ± 3.13 vs 20.85 ± 10.94 [P = 0.04] and 4.0 ± 1.63 vs 24.57 ± 10.4 [P = 0.012] with silver, caspase-3, and fluoro-jade C staining, respectively). CONCLUSIONS: A total of 1000 IU·kg(-1) IP erythropoietin diminished isoflurane-induced neuroapoptosis. Further experimental studies have to be planned to reveal the optimal dose and timing of erythropoietin before adaptation to clinical practice.

α2-Adrenergic stimulation of the ventrolateral preoptic nucleus destabilizes the anesthetic state.

The sleep-promoting ventrolateral preoptic nucleus (VLPO) shares reciprocal inhibitory inputs with wake-active neuronal nuclei, including the locus ceruleus. Electrophysiologically, sleep-promoting neurons in the VLPO are directly depolarized by the general anesthetic isoflurane and hyperpolarized by norepinephrine, a wake-promoting neurotransmitter. However, the integration of these competing influences on the VLPO, a sleep- and anesthetic-active structure, has yet to be evaluated in either brain slices in vitro or the intact organism. Single-cell multiplex RT-PCR conducted on both isoflurane-activated, putative sleep-promoting VLPO neurons and neighboring, state-indifferent VLPO neurons in mouse brain slices revealed widespread expression of α2A-, α2B- and α2C-adrenergic receptors in both populations. Indeed, both norepinephrine and the highly selective α2 agonist dexmedetomidine each reversed the VLPO depolarization induced by isoflurane in slices in vitro. When microinjected directly into the VLPO of a mouse lightly anesthetized with isoflurane, dexmedetomidine increased behavioral arousal and reduced the depressant effects of isoflurane on barrel cortex somatosensory-evoked potentials but failed to elicit spectral changes in spontaneous EEG. Based on these observations, we conclude that local modulation of α-adrenergic activity in the VLPO destabilizes, but does not fully antagonize, the anesthetic state, thus priming the brain for anesthetic emergence.

Subclinical carbon monoxide limits apoptosis in the developing brain after isoflurane exposure.

BACKGROUND: Volatile anesthetics cause widespread apoptosis in the developing brain. Carbon monoxide (CO) has antiapoptotic properties, and exhaled endogenous CO is commonly rebreathed during low-flow anesthesia in infants and children, resulting in subclinical CO exposure. Thus, we aimed to determine whether CO could limit isoflurane-induced apoptosis in the developing brain. METHODS: Seven-day-old male CD-1 mouse pups underwent 1-hour exposure to 0 (air), 5, or 100 ppm CO in air with or without isoflurane (2%). We assessed carboxyhemoglobin levels, cytochrome c peroxidase activity, and cytochrome c release from forebrain mitochondria after exposure and quantified the number of activated caspase-3 positive cells and TUNEL positive nuclei in neocortex, hippocampus, and hypothalamus/thalamus. RESULTS: Carboxyhemoglobin levels approximated those expected in humans after a similar time-weighted CO exposure. Isoflurane significantly increased cytochrome c peroxidase activity, cytochrome c release, the number of activated caspase-3 cells, and TUNEL positive nuclei in the forebrain of air-exposed mice. CO, however, abrogated isoflurane-induced cytochrome c peroxidase activation and cytochrome c release from forebrain mitochondria and decreased the number of activated caspase-3 positive cells and TUNEL positive nuclei after simultaneous exposure with isoflurane. CONCLUSIONS: Taken together, the data indicate that CO can limit apoptosis after isoflurane exposure via inhibition of cytochrome c peroxidase depending on concentration. Although it is unknown whether CO directly inhibited isoflurane-induced apoptosis, it is possible that low-flow anesthesia designed to target rebreathing of specific concentrations of CO may be a desired strategy to develop in the future in an effort to prevent anesthesia-induced neurotoxicity in infants and children.

Volatile anesthetic isoflurane inhibits LTP induction of hippocampal CA1 neurons through α4ß2 nAChR subtype-mediated mechanisms.

BACKGROUND AND PURPOSE: Volatile anesthetic isoflurane contributes to postoperative cognitive dysfunction and inhibition of long-term potentiation (LTP), a synaptic model of learning and memory, but the mechanisms are uncertain. Central neuronal α4ß2 subtype nicotinic acetylcholine receptors (nAChRs) are involved in the induction of LTP in the hippocampus. Isoflurane inhibits α4ß2 nAChRs at concentrations lower than those used for anesthesia. Therefore, we hypothesized that isoflurane-inhibited LTP induction of hippocampal CA1 neurons via α4ß2 nAChRs subtype inhibition. METHODS: Transverse hippocampal slices (400µm thick) were obtained from male rats (6-8 weeks old). Population spikes were evoked using extracellular electrodes by electrical stimulation of the Schaffer collateral-commissural pathway of rat hippocampal slices. LTP was induced using high frequency stimulation (HFS; 100Hz, 1s). Clinically relevant concentrations (0.125-0.5mM) of isoflurane with or without nicotine (nAChRs agonist), mecamylamine (nAChRs antagonist), 3-[2(S)-2-azetidinylmethoxy] pyridine (A85380) and epibatidine (α4ß2 nAChRs agonist), dihydro ß erythroidine (DHßE) (α4ß2 nAChRs antagonist) were added to the perfusion solution 20min before HFS to test their effects on LTP by HFS respectively. RESULTS: A brief HFS induced stable LTP in rat hippocampal slices, but LTP was significantly inhibited in the presence of isoflurane at concentrations of 0.125-0.5mM. The inhibitive effect of isoflurane on LTP was not only reversible and could be prevented by nAChRs agonist nicotine and α4ß2 nAChRs agonist A85380 and epibatidine, but also mimicked and potentiated by nAChRs antagonist mecamylamine and α4ß2 nAChRs antagonist DHßE. CONCLUSIONS: Inhibition of α4ß2 nAChRs subtype of hippocampus participates in isoflurane-mediated LTP inhibition.

Isoflurane and sevoflurane increase interleukin-6 levels through the nuclear factor-kappa B pathway in neuroglioma cells.

BACKGROUND: Isoflurane can increase pro-inflammatory cytokine interleukin (IL)-6 levels. However, the up-stream mechanism remains unknown. Nuclear factor-kappa B (NF-κB) promotes the generation of pro-inflammatory cytokines. We examined the effects of isoflurane and sevoflurane on the NF-κB signalling pathway and its association with IL-6 levels in cultured cells. METHODS: H4 human neuroglioma cells (H4 cells), and mouse primary neurones and microglia were treated with 2% isoflurane or 4.1% sevoflurane for 6 h, for analysis of IL-6 and NF-κB. Pyrrolidine dithiocarbamate (an NF-κB inhibitor) or 2-deoxy-d-glucose (2-DG) (an inhibitor of glucose glycolysis) was applied 1 h before anaesthetic treatment. RESULTS: Isoflurane or sevoflurane treatment increased the levels of IL-6 [isoflurane: 410% (54); sevoflurane: 290% (24)], the nuclear levels of NF-κB [isoflurane: 170% (36); sevoflurane: 320% (30)], and the transcription activity of NF-κB in H4 cells. Moreover, isoflurane enhanced the transcription activity of NF-κB in mouse microglia, but not primary neurones. Finally, pyrrolidine dithiocarbamate and 2-DG attenuated isoflurane-induced increases in IL-6 and NF-κB, and the transcription activity of NF-κB. CONCLUSIONS: These studies in H4 cells suggest that the NF-κB signalling pathway could contribute to isoflurane or sevoflurane-induced neuroinflammation. This could lead to the targeted intervention of anaesthetic-induced neuroinflammation.

Potentiation of GABAA receptor activity by volatile anaesthetics is reduced by α5GABAA receptor-preferring inverse agonists.

BACKGROUND: Animal studies have shown that memory deficits in the early post-anaesthetic period can be prevented by pre-treatment with an inverse agonist that preferentially inhibits α5 subunit-containing γ-aminobutyric acid type A (α5GABA(A)) receptors. The goal of this in vitro study was to determine whether inverse agonists that inhibit α5GABA(A) receptors reduce anaesthetic potentiation of GABAA receptor activity. METHODS: Cultures of hippocampal neurones were prepared from Swiss white mice, wild-type mice (genetic background C57BL/6J and Sv129Ev) and α5GABA(A)receptor null mutant (Gabra5-/-) mice. Whole-cell voltage clamp techniques were used to study the effects of the α5GABA(A) receptor-preferring inverse agonists L-655,708 and MRK-016 on anaesthetic potentiation of GABA-evoked currents. RESULTS: L-655,708 (50 nM) reduced sevoflurane potentiation of GABA-evoked current in wild-type neurones but not Gabra5-/- neurones, and produced a rightward shift in the sevoflurane concentration-response plot [sevoflurane EC50: 1.9 (0.1) mM; sevoflurane+L-655,708 EC(50): 2.4 (0.2) mM, P<0.05]. Similarly, L-655,708 (50 nM) reduced isoflurane potentiation of GABA-evoked current [isoflurane: 4.0 (0.6) pA pF(-1); isoflurane+L-655,708: 3.1 (0.5) pA pF(-1), P<0.01]. MRK-016 also reduced sevoflurane and isoflurane enhancement of GABA-evoked current [sevoflurane: 1.5 (0.1) pA pF(-1); sevoflurane+MRK-016 (10 nM): 1.2 (0.1) pA pF(-1), P<0.05; isoflurane: 3.5 (0.3) pA pF(-1); isoflurane+MRK-016 (1 nM): 2.9 (0.2) pA pF(-1), P<0.05]. CONCLUSIONS: L-655,708 and MRK-016 reduced the potentiation by inhaled anaesthetics of GABAA receptor activated by a low concentration of GABA. Future studies are required to determine whether this effect contributes to the memory preserving properties of inverse agonists after anaesthesia.

Is hydrogen sulfide-induced suspended animation general anesthesia?

Hydrogen sulfide (H(2)S) depresses mitochondrial function and thereby metabolic rates in mice, purportedly resulting in a state of "suspended animation." Volatile anesthetics also depress mitochondrial function, an effect that may contribute to their anesthetic properties. In this study, we ask whether H(2)S has general anesthetic properties, and by extension, whether mitochondrial effects underlie the state of anesthesia. We compared loss of righting reflex, electroencephalography, and electromyography in mice exposed to metabolically equipotent concentrations of halothane, isoflurane, sevoflurane, and H(2)S. We also studied combinations of H(2)S and anesthetics to assess additivity. Finally, the long-term effects of H(2)S were assessed by using the Morris water maze behavioral testing 2 to 3 weeks after exposures. Exposure to H(2)S decreases O(2) consumption, CO(2) production, and body temperature similarly to that of the general anesthetics, but fails to produce a loss of righting reflex or muscle atonia at metabolically equivalent concentrations. When combined, H(2)S antagonizes the metabolic effects of isoflurane, but potentiates the isoflurane-induced loss of righting reflex. We found no effect of prior H(2)S exposure on memory or learning. H(2)S (250 ppm), not itself lethal, produced delayed lethality when combined with subanesthetic concentrations of isoflurane. H(2)S cannot be considered a general anesthetic, despite similar metabolic suppression. Metabolic suppression, presumably via mitochondrial actions, is not sufficient to account for the hypnotic or immobilizing components of the anesthetic state. Combinations of H(2)S and isoflurane can be lethal, suggesting extreme care in the combination of these gases in clinical situations.

Trehalose protects from aggravation of amyloid pathology induced by isoflurane anesthesia in APP(swe) mutant mice.

There is an open controversy about the role of surgery and anesthesia in the pathogenesis of Alzheimer's disease (AD). Clinical studies have shown a high prevalence of these procedures in subjects with AD but the interpretation of these studies is difficult because of the co-existence of multiple variables. Experimental studies in vitro and in vivo have shown that small molecular weight volatile anesthetics enhance amyloidogenesis in vitro and produce behavioral deficits and brain lesions similar to those found in patients with AD. We examined the effect of co-treatment with trehalose on isoflurane-induced amyloidogenesis in mice. WT and APP(swe) mice, of 11 months of age, were exposed to 1% isoflurane, 3 times, for 1.5 hours each time and sacrificed 24 hours after their last exposure to isoflurane. The right hemi-brain was used for histological analysis and the contra-lateral hemi-brain used for biochemical studies. In this study, we have shown that repetitive exposure to isoflurane in pre-symptomatic mature APP(swe) mice increases apoptosis in hippocampus and cerebral cortex, enhances astrogliosis and the expression of GFAP and that these effects are prevented by co-treatment with trehalose, a disaccharide with known effects as enhancer of autophagy. We have also confirmed that in our model the co-treatment with trehalose increases the expression of autophagic markers as well as the expression of chaperones. Cotreatment with trehalose reduces the levels of ß amyloid peptide aggregates, tau plaques and levels of phospho-tau. Our study, therefore, provides new therapeutic avenues that could help to prevent the putative pro-amyloidogenic properties of small volatile anesthetics.

2-Deoxy-D-glucose attenuates isoflurane-induced cytotoxicity in an in vitro cell culture model of H4 human neuroglioma cells.

BACKGROUND: ß-Amyloid protein (Aß) accumulation and caspase activation have been shown to contribute to Alzheimer disease neuropathogenesis. Aß is produced from amyloid precursor protein through proteolytic processing by aspartyl protease ß-site amyloid precursor protein-cleaving enzyme (BACE). The inhaled anesthetic isoflurane has been shown to induce caspase activation and increase levels of BACE and Aß. However, the underlying mechanisms and interventions of the isoflurane-induced neurotoxicity remain largely to be determined. The glucose analog 2-deoxy-d-glucose (2-DG) has neuroprotective effects. Therefore, we sought to determine whether 2-DG can reduce caspase-3 activation and the increase in the levels of BACE and reactive oxygen species (ROS) induced by isoflurane. METHODS: H4 human neuroglioma cells were treated with saline or 2-DG (5 mM) for 1 hour followed by a control condition or 2% isoflurane for 6 hours. The levels of caspase-3 cleavage (activation), BACE, cytosolic calcium, and ROS were determined. Two-way analysis of variance was used to assess the interactions of 2-DG and isoflurane on caspase-3 activation, and levels of BACE and ROS. RESULTS: In H4 human neuroglioma cells, 2-DG reduced the caspase-3 activation (477% vs 186%, F = 8.68; P = 0.019) and the increase in BACE levels (345% vs 123%, F = 42.24; P = 0.0002) induced by isoflurane. 2-DG decreased the levels of cytosolic calcium and ROS (100% vs 66%, F = 1.94; P = 0.014). CONCLUSIONS: These results suggest that 2-DG may decrease oxidative stress and increase cytosolic calcium levels, thus attenuating isoflurane-induced neurotoxicity.

GABAergic excitotoxicity injury of the immature hippocampal pyramidal neurons' exposure to isoflurane.

BACKGROUND: Certain anesthetics exhibit neurotoxicity in the brains of immature but not mature animals. γ-Aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, is excitatory on immature neurons via its action at the GABAA receptor, depolarizing the membrane potential and inducing a cytosolic Ca2+ increase ([Ca2+]i), because of a reversed transmembrane chloride gradient. Recent experimental data from several rodent studies have demonstrated that exposure to isoflurane during an initial phase causes neuronal excitotoxicity and apoptosis. GABAA receptor-mediated synaptic voltage-dependent calcium channels' (VDCCs) overactivation and Ca2+ influx are involved in these neural changes. METHODS: We monitored [Ca2+]i using Fluo-4 AM fluorescence imaging. Using whole-cell patch clamp techniques, IVDCC (voltage-dependent calcium channel currents) were recorded from primary cultures of rat hippocampal neurons (5-day culture) exposed to isoflurane. To further investigate the neurotoxicity of high cytosolic-free calcium after isoflurane in a dose- and time-dependent manner, the possibility of increased caspase-3 levels was evaluated by Western blot and quantitative real-time polymerase chain reaction. Statistical significance was assessed using the Student t test or 1-way analysis of variance followed by the Tukey post hoc test. RESULTS: Under control conditions, isoflurane enhanced the GABA-induced [Ca2+]i increase in a dose-dependent manner. Dantrolene and nicardipine markedly inhibited this enhancement mediated by isoflurane. Moreover, in Ca2+-free media, pretreatment with isoflurane did not show any influence on the caffeine-induced increase of [Ca2+]i. Similarly, using whole-cell recording, isoflurane increased the peak amplitude of IVDCC in the cultured neurons from rat hippocampus by depolarization pulses. Isoflurane (0.25, 0.5, 0.75, and 1 minimum alveolar concentration [MAC]) potentiated IVDCC peak current amplitude by 109.11%±9.03%, 120.56%±11.46%, 141.33%±13.87%, and 146.78%±15.87%, respectively. To analyze variation in protein levels, the effect of treatments with isoflurane on caspase-3 activity was dose- and time-dependent, reaching a maximal caspase-3 activity after exposure to 1 MAC for 6 hours (P<0.001). However, in the mRNA levels, hippocampal caspase-3 mRNA levels began to be significantly increased in isoflurane-treated developing rat hippocampal neurons after 6 hours of exposure to 0.25 MAC isoflurane (P<0.001). CONCLUSIONS: Isoflurane-mediated enhancement of GABA-triggered [Ca2+]i release results from membrane depolarization with subsequent activation of VDCCs and further Ca2+-induced Ca2+ release from the ryanodine-sensitizing Ca2+ store. An increase in [Ca2+]i, caused by activation of the GABAA receptor and opening of VDCCs, is necessary for isoflurane-induced calcium overload of immature rat hippocampal neurons, which may be involved in the mechanism of an isoflurane-induced neurotoxic effect in the developing rodent brain.

Propofol inhibits desflurane-induced preconditioning in rabbits.

OBJECTIVES: The authors tested the hypothesis that ischemic and desflurane-induced preconditioning are blocked by propofol. DESIGN: A prospective, randomized, vehicle-controlled study. SETTING: A university research laboratory. SUBJECTS: New Zealand white rabbits (n = 52). METHODS: Pentobarbital-anesthetized rabbits were subjected to 30 minutes of coronary artery occlusion followed by 3 hours of reperfusion. Rabbits received 0.0 (control) or 1.0 minimum alveolar concentration of desflurane (30 minutes' duration and a 30-minute memory period) or ischemic preconditioning (5 minutes of ischemia and a 30-minute memory period) in the absence or presence of propofol (10 mg/kg/h intravenously) or its vehicle (10% Intralipid emulsion; B Braun, Melsungen, Germany). The myocardial infarct size was measured with triphenyltetrazolium staining. Statistical analysis was performed with 1-way and 2-way analysis of variance when appropriate, followed by a post hoc Duncan test. Data are mean ± standard deviation. RESULTS: Myocardial infarct size was 56% ± 8% in control animals (n = 7). Desflurane significantly (p < 0.05) reduced the infarct size to 37% ± 6% (n = 7). Desflurane-induced preconditioning was blocked by propofol (65% ± 10%, n = 7) but not by its vehicle (45% ± 11%, n = 5). Propofol and its vehicle alone had no effect on the infarct size (62% ± 8% [n = 6] and 58% ± 3% [n=5], respectively). Ischemic preconditioning reduced infarct size in the absence or presence of propofol to 24% ± 7% (n = 7) and 29% ± 12% (n = 6). CONCLUSION: Desflurane-induced preconditioning markedly reduced infarct size and was blocked by propofol, whereas ischemic preconditioning was not blocked by propofol. The results suggest an important interference between propofol and anesthetic-induced preconditioning and might explain some contradictory findings in studies in humans.

Dexmedetomidine attenuates isoflurane-induced neurocognitive impairment in neonatal rats.

BACKGROUND: Neuroapoptosis is induced by the administration of anesthetic agents to the young. As alpha2 adrenoceptor signaling plays a trophic role during development and is neuroprotective in several settings of neuronal injury, the authors investigated whether dexmedetomidine could provide functional protection against isoflurane-induced injury. METHODS: Isoflurane-induced injury was provoked in organotypic hippocampal slice cultures in vitro or in vivo in postnatal day 7 rats by a 6-h exposure to 0.75% isoflurane with or without dexmedetomidine. In vivo, the alpha2 adrenoceptor antagonist atipamezole was used to identify if dexmedetomidine neuroprotection involved alpha2 adrenoceptor activation. The gamma-amino-butyric-acid type A antagonist, gabazine, was also added to the organotypic hippocampal slice cultures in the presence of isoflurane. Apoptosis was assessed using cleaved caspase-3 immunohistochemistry. Cognitive function was assessed in vivo on postnatal day 40 using fear conditioning. RESULTS: In vivo dexmedetomidine dose-dependently prevented isoflurane-induced injury in the hippocampus, thalamus, and cortex; this neuroprotection was attenuated by treatment with atipamezole. Although anesthetic treatment did not affect the acquisition of short-term memory, isoflurane did induce long-term memory impairment. This neurocognitive deficit was prevented by administration of dexmedetomidine, which also inhibited isoflurane-induced caspase-3 expression in organotypic hippocampal slice cultures in vitro; however, gabazine did not modify this neuroapoptosis. CONCLUSION: Dexmedetomidine attenuates isoflurane-induced injury in the developing brain, providing neurocognitive protection. Isoflurane-induced injury in vitro appears to be independent of activation of the gamma-amino-butyric-acid type A receptor. If isoflurane-induced neuroapoptosis proves to be a clinical problem, administration of dexmedetomidine may be an important adjunct to prevent isoflurane-induced neurotoxicity.