Stress injuries and autophagy in mouse hippocampus after chronic cold exposure

Abstract

Cold exposure is an external stress factor that causes skin frostbite as well as a variety of diseases. Estrogen might participate in neuroprotection after cold exposure, but its precise mechanism remains unclear. In this study, mice were exposed to 10°C for 7 days and 0-4°C for 30 days to induce a model of chronic cold exposure. Results showed that oxidative stress-related c-fos and cyclooxygenase 2 expressions, MAP1LC3-labeled autophagic cells, Iba1-labeled activated microglia, and interleukin-1β-positive pyramidal cells were increased in the hippocampal CA1 area. Chronic cold exposure markedly elevated the levels of estrogen in the blood and the estrogen receptor, G protein-coupled receptor 30. These results indicate that neuroimmunoreactivity is involved in chronic cold exposure-induced pathological alterations, including oxidative stress, neuronal autophagy, and neuroimmunoreactivity. Moreover, estrogen exerts a neuroprotective effect on cold exposure.

Keywords: autophagy; chronic cold exposure; estrogen; hippocampal CA1 area; microglial cells; nerve regeneration; neural regeneration; neuroimmunoreactivity; oxidative stress.

Figure 1

Cold-induced cellular oxidative stress, inflammatory injury, and autophagy in the hippocampal CA1 area (immunofluorescence assay). (A, B) c-fos-immunoreactive cells (red) in the control group (A) and cold exposure group (B); (C, D) COX2-immunoreactive cells (red) (arrows) in the control group (C) and cold exposure group (D). The pyramidal layer is visualized by DAPI counterstaining (blue); (E, F) MAP1LC3-immunoreactive cells (red) in the control group (E) and cold exposure group (F). After cold exposure, the numbers of c-fos-immunoreactive cells, COX2-immunoreactive cells, and MAP1LC3-immunoreactive cells are increased. Scale bars: 50 μm. COX2: Cyclooxygenase 2; DAPI: 4′,6-diamidino-2-phenylindole.

Figure 2

Expression of oxidative stress-related proteins after chronic cold exposure. (A, B) The numbers of c-fos- (A) and COX2-immunoreactive cells (B) in the CA1 area after chronic cold exposure (immunofluorescence assay). (C) Western blots of c-fos, COX2, and NF-κB expression in the mouse hippocampus. (D) Semi-quantitative analysis of relative proteins by western blot assay. Relative protein expression is represented as the optical density ratio of targeted protein to β-actin. Data are expressed as the mean ± SD. **P < 0.01, vs. control group (independent-sample t-test). NF-κB: Nuclear factor kappa B; COX2: cyclooxygenase 2.

Figure 3

Expression of autophagy-related proteins after chronic cold exposure. (A) Quantitation of MAP1LC3-immunoreactive cells in the hippocampal CA1 area after chronic cold exposure (immunofluorescence assay). (B) Western blots of MAP1LC3 expression in the mouse hippocampus. (C) Semi-quantitative analyses of MAP1LC3 (western blot assay). Data are expressed as the mean ± SD. **P < 0.01, vs. control group (independent-sample t-test). Relative protein expression is represented as the optical density ratio to β-actin.

Figure 4

Cold exposure increases neuroimmunoreactivity and estrogen receptor GPR30 expression (immunofluorescence assay). (A, B) Iba1-labeled microglial cells (red) in the control group (A) and cold exposure group (B). The pyramidal layer is visualized by DAPI counter-staining (blue). The number of microglial cells (red) was significantly increased after chronic cold exposure. (C, D) IL-1β-immunoreactive cells (red) in the control group (C) and cold exposure group (D). The number of IL-6-immunoreactive cells (red) was significantly increased after chronic cold exposure. (E, F) IL-6-immunoreactive cells (red) in the control group (E) and cold exposure group (F). Cold exposure induced an increase of IL-6-immunoreactive cells. (G, H) GPR30-immunoreactive cells (red) in the control (G) and cold exposure (H) groups. GPR30 expression was increased after cold exposure. Scale bars: 50 μm. DAPI: 4′,6-Diamidino-2-phenylindole; IL: interleukin; GPR30: G protein-coupled receptor 30.

Figure 5

Expression of neuroimmunoreactivity-related proteins after chronic cold exposure. (A–C) Quantitation of Iba1- (A), IL-1β- (B), and IL-6- (C) immunoreactive microglial cells in the hippocampal CA1 area after chronic cold exposure (immunofluorescence assay). (D) Western blots of Iba1 and CD11b expressions in the mouse hippocampus. (E) Semi-quantitative analyses of western blot assay. Relative protein expression is represented as the optical density ratio to β-actin. Data are expressed as the mean ± SD. **P < 0.01, vs. control group (independent-sample t-test). IL: Interleukin.

Figure 6

Estrogen levels and GPR30 expression after chronic cold exposure (immunofluorescence assay). (A) Estrogen levels in the blood after cold exposure detected by immunoassay. (B) Alterations of GPR30-immunoreactive cells in the hippocampal CA1 area after chronic cold exposure detected by immunofluorescence assay. Data are expressed as the mean ± SD. **P < 0.01, vs. control group (independent-sample t-test); GPR30: G protein-coupled receptor 30.