Peripheral Administration of Tumor Necrosis Factor-Alpha Induces Neuroinflammation and Sickness but Not Depressive-Like Behavior in Mice

Abstract

Clinical observations indicate that activation of the TNF-α system may contribute to the development of inflammation-associated depression. Here, we tested the hypothesis that systemic upregulation of TNF-α induces neuroinflammation and behavioral changes relevant to depression. We report that a single intraperitoneal injection of TNF-α in mice increased serum and brain levels of the proinflammatory mediators TNF-α, IL-6, and MCP-1, in a dose- and time-dependent manner, but not IL-1β. Protein levels of the anti-inflammatory cytokine IL-10 increased in serum but not in the brain. The transient release of immune molecules was followed by glial cell activation as indicated by increased astrocyte activation in bioluminescent Gfap-luc mice and elevated immunoreactivity against the microglial marker Iba1 in the dentate gyrus of TNF-α-challenged mice. Additionally, TNF-α-injected mice were evaluated in a panel of behavioral tests commonly used to study sickness and depressive-like behavior in rodents. Our behavioral data imply that systemic administration of TNF-α induces a strong sickness response characterized by reduced locomotor activity, decreased fluid intake, and body weight loss. Depressive-like behavior could not be separated from sickness at any of the time points studied. Together, these results demonstrate that peripheral TNF-α affects the central nervous system at a neuroimmune and behavioral level.

Figure 1

Peripheral TNF-α administration induces the release of immune mediators in serum and brain. Time course of serum (left) and brain concentrations (right) of interleukin- (IL-) 1β, IL-6, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-10, and monocyte chemoattractant protein-1 (MCP-1) as measured at 2 h, 6 h, and 24 h after i.p. TNF-α injection. Note that serum concentrations are shown as pg/mL while brain levels are expressed in pg/mg protein. Dashed lines indicate the detection limit of the measured analyte. Graphs are plotted as mean + SEM (n = 6-7 per group). Data were analyzed by ANOVA followed by independent samples t-test. (∗) 0.1 < P < 0.05; ^∗ P < 0.05; ^∗∗ P < 0.01; ^∗∗∗ P < 0.001 compared to 0 μg/kg TNF-α.

Figure 2

TNF-α activates astrocytes in a dose- and time-dependent manner. Intraperitoneal injection of TNF-α caused a clear bioluminescent signal in the brain of Gfap-luc mice, as shown in representative images taken at 6 h after injection (a). This signal peaked at 6 h and then gradually waned over time (b). The color scale indicates the number of photons emitted from the animal per second. The graph is plotted as mean ± SEM (n = 7 per group). Data were analyzed by rmANOVA followed by independent samples t-test. BL: baseline. ^∗ P < 0.05; ^∗∗ P < 0.01; ^∗∗∗ P < 0.001 compared to 0 μg/kg TNF-α.

Figure 3

TNF-α increases Iba1 immunoreactivity in the dentate gyrus. TNF-α (250 μg/kg, i.p.) caused a strong upregulation of the microglial activation marker Iba1 in the hippocampal dentate gyrus at 24 h after administration. Representative images (10x) (a) and image quantifications of n = 8 per group (b). Graph is plotted as mean + SEM. Data were analyzed by ANOVA followed by independent samples t-test. ^∗ P < 0.05 compared to vehicle.

Figure 4

TNF-α causes sickness, but no clear depressive-like behavior. Systemic injection of TNF-α caused body weight loss ((a)-(b)), reduced locomotor activity in the open field test ((c)-(d)), and decreased total fluid intake in the sucrose preference test (g). Measures of behavioral despair in the forced swim test were not affected by administration of TNF-α ((e)-(f)). A high dose of TNF-α did decrease sucrose preference in the SPT (h) but this can be considered biologically irrelevant due to the overlapping time course of sickness. The dashed line in the sucrose preference test indicates the chance level (50%) for sucrose preference. Please note that the y-axis does not start at 0 for the forced swim and sucrose preference test data. Graphs are plotted as mean ± SEM (n = 10 per group, except n = 8 for 1000 μg/kg TNF-α). Data were analyzed by rmANOVA followed by independent samples t-test. ^∗ P < 0.05 compared to 0 μg/kg TNF-α.

Figure 5

Peripheral TNF-α administration reduces total fluid intake (a) but does not induce clear anhedonia (b) in the sucrose preference test. Separate groups of naive animals were injected i.p. with TNF-α and tested in the SPT at either 6 h or 24 h. Dashed line indicates chance level for sucrose preference. Graphs are plotted as mean + SEM (n = 10 per group). Data were analyzed by ANOVA followed by independent samples t-test. ^∗ P < 0.05; ^∗∗ P < 0.01 compared to 0 μg/kg TNF-α at the same time point.