Delayed treatment with isoflurane attenuates lipopolysaccharide and interferon gamma-induced activation and injury of mouse microglial cells

Abstract

Background: Isoflurane pretreatment can induce protection against lipopolysaccharide and interferon gamma (IFNgamma)-induced injury and activation of mouse microglial cells. This study's goal was to determine whether delayed isoflurane treatment is protective.

Methods: Mouse microglial cells were exposed to various concentrations of isoflurane for 1 h immediately after the initiation of lipopolysaccharide (10 or 1000 ng/ml) and IFNgamma (10 U/ml) stimulation or to 2% isoflurane for 1 h at various times after initiation of the stimulation. Nitrite production, lactate dehydrogenase release, and cell viability measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were assessed after stimulation with lipopolysaccharide and IFNgamma for 24 h. Inducible nitric oxide synthase (iNOS) protein expression was quantified by Western blotting. The iNOS expression in mouse brain was also studied.

Results: Isoflurane applied 0 and 2 h after the initiation of lipopolysaccharide and IFNgamma stimulation improved cell viability. Isoflurane at 2%, but not at 1 or 3%, reduced the lipopolysaccharide and IFNgamma-induced nitrite production and decreased cell viability. Aminoguanidine, an iNOS inhibitor, also attenuated this decreased cell viability. Chelerythrine and bisindolylmalemide IX, protein kinase C inhibitors, abolished isoflurane effects on cell viability and iNOS expression after lipopolysaccharide and IFNgamma application. Isoflurane also decreased lipopolysaccharide-induced iNOS expression in mouse brain. Late isoflurane application to microglial cells reduced lipopolysaccharide and IFNgamma-induced lactate dehydrogenase release that was not inhibited by aminoguanidine.

Conclusions: These results suggest that delayed isoflurane treatment can reduce lipopolysaccharide and IFNgamma-induced activation and injury of microglial cells. These effects may be mediated by protein kinase C.

Fig. 1

Protective effects of isoflurane and aminoguanidine (AG) on cell viability. (A) Time-window of delayed isoflurane treatment. The mouse C8-B4 microglial cells were incubated with 10 ng/ml lipopolysaccharide (LPS) and 10 U/ml interferon γ (IFNγ) for 24 hr. Cells were exposed to 2% isoflurane for 1 hr at 0, 2, 4, 8, 16 and 23 hr after the initiation of the lipopolysaccharide and IFNγ stimulation. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results are mean ± S.D. (n = 10 – 15). ^* P < 0.05 compared to control. ^ P < 0.05 compared to lipopolysaccharide plus IFNγ only. Iso-post: 2% isoflurane post-treatment. (B) Dose-response of isoflurane effects on cell viability. The mouse C8-B4 microglial cells were incubated with 10 ng/ml lipopolysaccharide and 10 U/ml IFNγ for 24 hr. Cells were exposed to 1, 2, or 3% isoflurane for 1 hr immediately after the initiation of the lipopolysaccharide and IFNγ stimulation. Results are mean ± S.D. (n = 8). ^* P < 0.05 compared to control. ^ P < 0.05 compared to lipopolysaccharide plus IFNγ only. (C) AG effect. The mouse C8-B4 microglial cells were incubated with or without 10 ng/ml lipopolysaccharide and 10 U/ml IFNγ in the presence or absence of 10 μM AG for 24 hr. Results are mean ± S.D. (n = 9). * P < 0.05 compared to control. ^ P < 0.05 compared to lipopolysaccharide plus IFNγ only.

Fig. 2

Inhibition of isoflurane (Iso) protection by protein kinase C inhibition. The mouse C8-B4 microglial cells were incubated with 10 ng/ml lipopolysaccharide (LPS) and 10 U/ml interferon γ (IFNγ) for 24 hr. Cells were exposed to 2% isoflurane for 1 hr immediately after the initiation of the lipopolysaccharide and IFNγ stimulation. Chelerythrine chloride (Che, 2 μM) or bisindolylmaleimide IX (Bis IX, 10 μM) were present during isoflurane exposure. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results are mean ± S.D. (n = 15). ^* P < 0.05 compared to control. ^ P < 0.05 compared to lipopolysaccharide plus IFNγ only. # P < 0.05 compared with isoflurane and lipopolysaccharide plus IFNγ.

Fig. 3

Effects of isoflurane (Iso) and protein kinase C inhibition on inducible nitric oxide synthase (iNOS) expression in microglial cells. The mouse C8-B4 microglial cells were incubated with 10 ng/m lipopolysaccharide (LPS) and 10 U/ml interferon γ (IFNγ) for 24 hr. Cells were exposed to 2% isoflurane for 1 hr immediately [Iso(0 h)] (panel A), 8 hr [Iso(8 h)] (panel A) or 2 hr [Iso(2 h)] (panel B) after the initiation of the lipopolysaccharide and IFNγ stimulation. Chelerythrine chloride (Che, 2 μM) or bisindolylmaleimide IX (Bis IX, 10 μM) was present during isoflurane exposure. These cells and the cells that were not exposed to isoflurane or lipopolysaccharide plus IFNγ (normal culture) were harvested for Western blotting. Results are mean ± S.D. (n = 9 for panel A and 12 for panel B). ^* P < 0.05 compared to lipopolysaccharide plus IFNγ only. ^ P < 0.05 compared with isoflurane and lipopolysaccharide plus IFNγ.

Fig. 4

Dose-response of isoflurane effects on nitrite production. The mouse C8-B4 microglial cells were incubated with 10 ng/ml lipopolysaccharide (LPS) and 10 U/ml of interferon γ (IFNγ) for 24 hr. Cells were exposed to 1, 2, or 3% isoflurane for 1 hr immediately after the initiation of the lipopolysaccharide and IFNγ stimulation. The culture medium was collected for nitrite measurement. Results are mean ± S.D. (n = 15). ^* P < 0.05 compared to control. ^ P < 0.05 compared to lipopolysaccharide plus IFNγ only.

Fig. 5

Effects of isoflurane (Iso) on inducible nitric oxide synthase (iNOS) expression in mice. C57Bl/6 mice received an intraperitoneal injection of 4 mg/kg lipopolysaccharide (LPS) and exposed to 1.5% isoflurane for 30 min immediately [Iso(0 h)] or 2 hr [Iso(2 h)] after the injection. Cerebral cortex was harvested at 6 hr after lipopolysaccharide injection for Western analysis. Results are mean ± S.D. (n = 7 – 8). ^* P < 0.05 compared to control. ^ P < 0.05 compared with lipopolysaccharide alone group.

Fig. 6

Protective effects of isoflurane on lipopolysaccharide (LPS) and interferon γ (IFNγ)-induced cell injury. (A) Time-window of delayed isoflurane treatment. The mouse C8-B4 microglial cells were incubated with 1000 ng/ml lipopolysaccharide and 10 U/ml IFNγ for 24 hr. Cells were exposed to 2% isoflurane for 1 hr at 0, 2, 4, 8, 16 and 23 hr after the initiation of the lipopolysaccharide and IFNγ stimulation. Cell injury was quantified by lactate dehydrogenase (LDH) release assay. Results are mean ± S.D. (n = 20 – 42). ^* P < 0.05 compared to control. ^ P < 0.05 compared to lipopolysaccharide plus IFNγ only. Iso-post: 2% isoflurane post-treatment. (B) Time-course of LDH release. The mouse C8-B4 microglial cells were incubated with or without 1000 ng/ml lipopolysaccharide and 10 U/ml IFNγ for 0, 2, 4, 8, 16 and 23 hr. The incubation solution and the cells were harvested for LDH activity assay. The actual mean values for each time point (n = 4 – 6) from the experiments and the 95% interval are presented. (C) Aminoguanidine (AG) effect. The mouse C8-B4 microglial cells were incubated with or without 1000 ng/ml lipopolysaccharide and 10 U/ml IFNγ in the presence or absence of 10 μM AG for 24 hr. Results are mean ± S.D. (n = 18 – 24). ^* P < 0.05 compared to control.