Delayed treatment with lidocaine reduces mouse microglial cell injury and cytokine production after stimulation with lipopolysaccharide and interferon γ

Abstract

Background: Neuroinflammation is an important pathological process for almost all acquired neurological diseases. Microglial cells play a critical role in neuroinflammation. We determined whether lidocaine, a local anesthetic with anti-inflammatory property, protected microglial cells and attenuated cytokine production from activated microglial cells.

Methods: Mouse microglial cultures were incubated with or without 1 μg/mL lipopolysaccharide and 10 U/mL interferon γ (IFNγ) for 24 hours in the presence or absence of lidocaine for 1 hour started at 2, 3, or 4 hours after the onset of lipopolysaccharide and IFNγ stimulation. Lactate dehydrogenase release and cytokine production were determined after the cells were stimulated by lipopolysaccharide and IFNγ for 24 hours.

Results: Lidocaine dose-dependently reduced lipopolysaccharide and IFNγ-induced microglial cell injury as measured by lactate dehydrogenase release. This effect was apparent with lidocaine at 2 μg/mL (30.3% ± 5.8% and 23.1% ± 9.7%, respectively, for stimulation alone and the stimulation in the presence of lidocaine, n = 18, P = 0.025). Lidocaine applied at 2, 3, or 4 hours after the onset of lipopolysaccharide and IFNγ stimulation reduced the cell injury. This lidocaine effect was not affected by the mitochondrial K(ATP) channel inhibitor 5-hydroxydecanoate. Similar to lidocaine, QX314, a permanently charged lidocaine analog that usually does not permeate through the plasma membrane, reduced lipopolysaccharide and IFNγ-induced microglial cell injury. QX314 also attenuated the stimulation-induced interleukin-1β production.

Conclusions: Delayed treatment with lidocaine protects microglial cells and reduces cytokine production from these cells. These effects may involve action site(s) on the cell surface.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1

Expression of the microglial marker ionized calcium binding adaptor molecule 1 (Iba1) in the mouse C8-B4 cells. (A) Hoechst staining; (B) immunocytochemical staining for Iba1 and (C) merged image.

Fig. 2

Protective effects of lidocaine on lipopolysaccharide (LPS) and interferon γ (IFNγ)-induced cell injury. (A) Dose-response of the lidocaine effects. The mouse C8-B4 microglial cells were incubated with 1 µg/ml LPS and 10 U/ml IFNγ for 24 h. Cells were exposed to 2, 10 or 20 µg/ml lidocaine for 1 h at 2 h after the initiation of the LPS and IFNγ stimulation. Cell injury was quantified by lactate dehydrogenase (LDH) release assay. Results are mean ± S.D. (n = 15 – 18). (B) Time-window of delayed isoflurane treatment. The mouse C8-B4 microglial cells were incubated with 1 µg/ml LPS and 10 U/ml IFNγ for 24 h. Cells were exposed to 20 µg/ml lidocaine for 1 h at 3 or 4 h after the initiation of the LPS and IFNγ stimulation. Results are mean ± S.D. (n = 6 – 17). * P < 0.05 compared to control. ^ P < 0.05 compared to LPS plus IFNγ only.

Fig. 3

Protective effects of lidocaine or QX314 on lipopolysaccharide (LPS) and interferon γ (IFNγ)-induced cell injury. The mouse C8-B4 microglial cells were incubated with 1 µg/ml LPS and 10 U/ml IFNγ for 24 h. Cells were exposed to 20 µg/ml lidocaine or 25 µg/ml QX314 for 1 h at 2 h after the initiation of the LPS and IFNγ stimulation. Cell injury was quantified by lactate dehydrogenase (LDH) release assay. Results are mean ± S.D. (n = 6 for panel A and 36 for panel B). * P

Fig. 4

Effects of QX314 on lipopolysaccharide (LPS) and interferon γ (IFNγ)-induced cytokine production. The mouse C8-B4 microglial cells were incubated with 1 µg/ml LPS and 10 U/ml IFNγ for 24 h. Cells were exposed to 25 µg/ml QX314 for 1 h at 2 h after the initiation of the LPS and IFNγ stimulation. Cytokine concentrations in the culture medium during the 24-h stimulation were assayed. Results are mean ± S.D. (n = 6). * P

Fig. 5

Cell viability under control condition. The viability of mouse C8-B4 microglial cells was evaluated by the trypan blue exclusion test at various times after they were plated. A representative microscopic view is presented in panel A and the pooled results are in panel B. The arrow in panel A indicates a trypan blue-positive cell. Results are means ± S.D. (n = 6). * P