Isoflurane preconditioning reduces mouse microglial activation and injury induced by lipopolysaccharide and interferon-gamma

Abstract

Activation and injury of microglial cells are involved in a broad range of brain diseases including stroke, brain infection and neurodegenerative diseases. However, there is very little information regarding how to reduce microglial reaction and preserve these cells to provide neuroprotection. Here, we showed that the incubation of C8-B4 mouse microglial cells with lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma) for 24 h decreased the viability of these cells. Pretreatment of these cells with 1%, 2% or 3% isoflurane, a commonly used volatile anesthetic, for 1 h at 30 min before the exposure to LPS plus IFNgamma attenuated the reduction of cell viability (preconditioning effect). LPS plus IFNgamma also activated these microglial cells to express inducible nitric oxide synthase (iNOS) and to induce accumulation of nitrite, a stable oxidation product of nitric oxide, in the incubation medium. Isoflurane preconditioning attenuated these LPS plus IFNgamma effects on the iNOS expression and nitrite accumulation. Aminoguanidine, an iNOS inhibitor, attenuated the LPS plus IFNgamma-induced glutamate release and decrease of microglial viability. Isoflurane preconditioning also reduced LPS plus IFNgamma-induced glutamate release. Exogenous glutamate decreased microglial viability. Finally, the isoflurane preconditioning-induced protection was abolished by chelerythrine, a protein kinase C inhibitor. These results suggest that LPS plus IFNgamma activates the iNOS-nitric oxide-glutamate pathway to induce microglial injury and that this activation is attenuated by isoflurane preconditioning. Protein kinase C may be involved in the isoflurane preconditioning effects.

Fig. 1. Lipopolysaccharide (LPS) plus interferon-γ (IFNγ) dose-dependently decreased cell viability

The mouse C8-B4 microglial cells were exposed to or were not exposed to various concentrations of LPS and IFNγ for 24 hr. The cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results are means ± S.D. (n = 12). * P < 0.05 compared with the control. ^ P < 0.05 compared with the corresponding cells exposed to the same concentrations of LPS plus 10 U/ml IFNγ.

Fig. 2. Isoflurane pretreatment reduced the lipopolysaccharide (LPS) plus interferon-γ (IFNγ)-induced decrease of cell viability

Panel A: The mouse C8-B4 microglial cells were exposed to or were not exposed to 10 ng/ml LPS plus 10 U/ml IFNγ for 24 hr or 5 mM glutamate for 24 hr. Panel B: The mouse C8-B4 microglial cells were pretreated with or without various concentrations of isoflurane. They were then exposed to 10 ng/ml LPS plus 10 U/ml IFNγ for 24 hr at 30 min after the isoflurane pretreatment. The cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results are means ± S.D. (n = 14 -18). * P < 0.05 compared with the control. ^ P < 0.05 compared with the corresponding cells exposed to LPS plus IFNγ only.

Fig. 3. Isoflurane pretreatment reduced the lipopolysaccharide (LPS) plus interferon-γ (IFNγ)-increased inducible nitric oxide synthase (iNOS) expression and nitrite production

The mouse C8-B4 microglial cells were pretreated with or without various concentrations of isoflurane. They were then exposed to 10 ng/ml LPS plus 10 U/ml IFNγ for 24 hr at 30 min after the isoflurane pretreatment. These cells and the cells that were not exposed to isoflurane and LPS plus IFNγ (normal culture) were harvested for Western analysis of iNOS expression (Panel A). The culture medium was collected for nitrite measurement (Panel B). Results are means ± S.D. (n = 8 - 9). * P < 0.05 compared with the corresponding cells exposed to LPS plus IFNγ only. Iso: isoflurane.

Fig. 4. The effects of D,L-threo-β-benzyloxyaspartate (TBOA), aminoguanidine (AG) and isoflurane pretreatment on the lipopolysaccharide (LPS) plus interferon-γ (IFNγ)-induced decrease of cell viability (panel A) and glutamate release (panel B)

The mouse C8-B4 microglial cells were pretreated with or without 2% isoflurane. They were then exposed to 10 ng/ml LPS plus 10 U/ml IFNγ for 24 hr at 30 min after the isoflurane pretreatment. Some cells were exposed to 10 ng/ml LPS plus 10 U/ml IFNγ in the presence of 10 μM TBOA or 10 μM AG for 24 hr. The cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The glutamate concentrations in the culture medium were measured by high performance liquid chromatography. Results are means ± S.D. (n = 6 - 31). * P < 0.05 compared with the control. ^ P < 0.05 compared with the corresponding cells exposed to LPS plus IFNγ only. Con: control.

Fig. 5. Attenuation of isoflurane preconditioning-induced protection by protein kinase C inhibition

The mouse C8-B4 microglial cells were pretreated with or without 2% isoflurane in the presence or absence of 10 μM chelerythrine. They were then exposed to 10 ng/ml lipopolysaccharide (LPS) plus 10 U/ml interferon-γ (IFNγ) for 24 hr at 30 min after the isoflurane pretreatment. The cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results are means ± S.D. (n = 14). * P < 0.05 compared with the control. ^ P < 0.05 compared with the corresponding cells exposed to LPS plus IFNγ only. & P < 0.05 compared with the cells pretreated with 2% isoflurane and then LPS plus IFNγ. Che: chelerythrine; Con: control; Iso: 2% isoflurane pretreatment.