Effects of anesthetics on LPS-induced IL-1<i>β</i> protein expression and secretion in cultured glial cells.

<p>Primary cultured glial cells were exposed for 4 hours to LPS (1 µg/ml) with or without 1.5% isoflurane, 100 µM propofol, or 1 mM pentobarbital. (A) Whole cell lysates were analyzed for IL-1<i>β</i>, glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (IBA-1), and <i>β</i>-actin protein expression by immunoblot assay. Figures are representative of at least three independent experiments. (B) IL-1<i>β</i> protein concentration in cultured medium was measured with ELISA. Data are presented as mean ± SD (n = 3). *<i>P</i> < 0.05 versus control (Mann–Whitney U-test).</p

Effects of anesthetics on LPS-induced IL-1<i>β</i> upregulation in cultured glial cells.

<p>Primary cultured glial cells were exposed for 4 hours to LPS (1 µg/ml) in the presence of pentobarbital (A), midazolam (B), or ketamine (C) at the indicated concentrations, or in the presence of propofol (200 µM), ketamine (1 mM), pentobarbital (1 mM), midazolam (150 µM), or isoflurane (1.5%) for 2 (D), 8 (E), or 24 (F) hours. IL-1<i>β</i> mRNA was assayed with real-time RT-PCR. Data are presented as mean ± SD (n = 6). The expression levels of IL-1<i>β</i> were normalized to that of 18S and expressed relative to the control mean. *<i>P</i> < 0.05 versus control, # <i>P</i> < 0.05 versus LPS, <i>N.S.</i>, not significant (Mann–Whitney U-test).</p

Effect of anesthetics on LPS-induced IL-1<i>β</i> upregulation in BV-2, A-1, and THP-1 cells.

<p>BV-2 microglial cells were exposed to LPS (1 µg/ml) in the presence of the indicated concentrations of propofol (A) or isoflurane (B) for 4 hours. IL-1<i>β</i> mRNA was assayed with real-time RT-PCR. Data are presented as mean ± SD (n = 3). A-1 astrocytic cells, were exposed to LPS (1 µg/ml) in the presence of the indicated concentrations of pentobarbital for 4 hours. Human acute monocytic leukemia cells, or THP-1 cells, were exposed to LPS (1 µg/ml) in the presence of the indicated concentrations of pentobarbital or isoflurane for 4 hours. IL-1<i>β</i> expression levels were normalized to that of 18S and expressed relative to the control mean. *<i>P</i> < 0.05 versus control, <i>N.S.</i>, not significant (Mann–Whitney U-test).</p

Effects of propofol and isoflurane on proinflammatory cytokine expression in cultured glial cells.

<p>Primary cultured glial cells were exposed for 4 hours to lipopolysaccharide (LPS) (1 µg/ml) and propofol or isoflurane at the indicated concentrations. Interleukin (IL)-1<i>β</i> (A, B, C), IL-6 (D, E), and tumor necrosis factor-alpha (TNF-α) (F, G) mRNA were assayed with real-time RT-PCR. Data are presented as mean ± SD (n = 4). The expression levels of IL-1<i>β</i>, IL-6, and TNF-<i>α</i> were normalized to that of 18S, and were expressed relative to the control mean. *<i>P</i> < 0.05 versus control, N.S., not significant (Mann–Whitney U-test).</p

Mechanism underlying inhibitory effect of anesthetics on IL-1<i>β</i> upregulation in LPS-treated cultured glial cells.

<p>(A, B) Effect of anesthetics on nuclear factor-kappaB (NF-κB) and activator protein-1 (AP-1) transcription in LPS-treated cultured glial cells. Primary cultured glial cells were exposed to LPS (1 µg/ml) with or without 100 µM propofol, 1 mM pentobarbital, or 1.5% isoflurane for 4 h. NF-κB (A) and AP-1 (B) transcriptional activities were measured with an ELISA-based kit. Data are presented as mean ± SD (n = 3). *<i>P</i> < 0.05 versus control. No statistically significant difference was found between any two groups except with the control. (Mann–Whitney U-test) (C) Effect of anesthetics on activity of MAPK families in LPS-treated cultured glial cells. Primary cultured glial cells were exposed to LPS (1 µg/ml) with or without 1.5% isoflurane, 100 µM propofol, or 1 mM pentobarbital for 4 hours. Whole cell lysates were analyzed for extracellular signal-regulated kinase (ERK), phospho-ERK (p-ERK), phospho-JNK (p-JNK), JNK, phospho-p38 (p-p38), and p38 MAPK expression by immunoblot assay. Figures are representative of at least three independent experiments. (D, E) Effect of PD98059, an antagonist of ERK 1/2, on IL-1<i>β</i> and TNF-<i>α</i> expression in LPS-treated cultured glial cells. IL-1<i>β</i> and TNF-<i>α</i> mRNA was assayed with real-time RT-PCR. Primary cultured glial cells were exposed to LPS (1 µg/ml) and 50 µM PD98059 (PD) with or without 100 µM propofol or 1 mM pentobarbital, and harvested for 4 h. IL-1<i>β</i> and TNF-<i>α</i> mRNA was assayed with real-time RT-PCR. Data are presented as mean ± SD (n = 3). The expression levels of IL-1<i>β</i> and TNF-<i>α</i> were normalized to that of 18S and expressed relative to the mean of control. *<i>P</i> < 0.05 versus control, <i>N.S.</i>, not significant (Mann–Whitney U-test).</p

Effect of isoflurane on LPS-induced IL-1<i>β</i> expression in mice, and plasma adrenocorticotropic hormone (ACTH) levels.

<p>Ten-week-old BALB/c mice were exposed to 0.5% isoflurane for 4 hours after LPS treatment (5 mg/kg) (n = 6). IL-1<i>β</i> mRNA in the hypothalamus (A) and cortex (B) was assayed with real-time RT-PCR analysis. IL-1<i>β</i> expression levels were normalized to that of 18S and expressed relative to the mean of control mice. IL-1<i>β</i> protein concentration (pg/ml) in the brain (C) and plasma (D) was quantified with ELISA. Data are presented as mean ± SD. Serum ACTH concentration (pg/ml) was quantified with ELISA. Data are presented as mean ± SEM. *<i>P</i> < 0.05 versus control; <i>N.S.</i>, not significant (Mann–Whitney U-test).</p

Effect of isoflurne on mRNA expression of HIF target genes.

<p>(A) HIF-1 and HIF-2 have unique, as well as common, target genes. HIF-1 specifically regulates glycolytic genes, including lactate dehydrogenase A (LDHA), phosphoglycerate kinase (PGK), as well as carbonic hydrase-9 (CA IX) whereas HIF-2 exclusively regulates POU transcription factor Oct-4, cyclin D1, and transforming growth factor α (TGF-α). Other hypoxia-inducible genes, such as vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT1), and EPO are regulated by both HIF-1 and HIF-2. (B, C) 6-week-old BALB/c mice were exposed to 10% O₂ (hypoxia) for 3 hours with or without 0.5% isoflurane and compared with controls. Control mice were exposed to air without isoflurane (normoxia). (D) 6-week-old BALB/c mice were exposed to 0.5% or 1.0% isoflurane in air for 3 hours. Data are presented as mean ± SD (n = 6). The expression levels of EPO, VEGF, LDHA and GLUT1 were assayed using real-time RT-PCR and normalized to that of 18S and expressed relative to the mean of mice exposed to air without isoflurane (normoxia).</p

Effect of various anesthetics on hypoxia-induced EPO upregulation in the brain.

<p>(A, B) 6-week-old BALB/c mice were exposed to 10% O₂ (hypoxia) in the presence of 0.5% sevoflurane, halothane or isoflurane for 3 hours. (C) 6-week-old mice were exposed to 10% O₂, 90% N₂O (hypoxia- N₂O) for 3 hours and compared with 10% O₂, 90% N₂ (hypoxia- N₂). (D–F) 6-week-old mice were exposed to 10% O₂ with pentobarbital (D), ketamine (E) or propofol (F). In the all experiments, control mice were exposed to air without anesthetics (normoxia). EPO mRNA in the brain (A, C–F) and kidney (B) was assayed with real-time RT-PCR. Data are presented as mean ± SD (n = 6). The expression levels of EPO were normalized to that of 18S and expressed relative to the mean of control mice. *<i>P</i><0.05, **<i>P</i><0.01 versus control, <i>N.S.</i>; not significant (Mann-Whitney U-test).</p

Effect of age and species on hypoxic EPO induction in mice brains.

<p>(A) 1-week and (B) 16-week-old C57BL/6N CrSlc mice were exposed to 10% O₂ (hypoxia) in the presence of 0.5% isoflurane for 3 hours. Control mice were exposed to air without anesthetics (normoxia). EPO mRNA in the brain was assayed with real-time RT-PCR. Data are presented as mean ± SD (n = 3–5). The expression levels of EPO were normalized to that of 18S and expressed relative to the mean of control mice. *<i>P</i><0.05 versus control (Mann-Whitney U-test).</p

Effect of various anesthetics on EPO expression in primary cultured astrocytes.

<p>Primary cultured astrocytes were exposed to 1% O₂ (hypoxia) in the presence of indicated concentrations of isoflurane (A), pentobarbital (B), ketamine (C), propofol (D) or morphine (E) for 4 hours. In the all experiments, control was exposed to 20% O₂. EPO mRNA was assayed with real-time RT-PCR. Data are presented as mean ± SD (n = 4). The expression levels of EPO were normalized to that of 18S and expressed relative to the mean of control. *<i>P</i><0.05, **<i>P</i><0.01 versus control, <i>N.S.</i>; not significant (Mann-Whitney U-test).</p