TNFα Response to Systemic LPS Administration
To better understand the effects of systemic LPS on brain inflammation, we administered a single dose of LPS (5 mg/kg) i.p. to male C57BL/6 mice and followed liver, serum, and brain TNFα expression over time. LPS increased liver TNFα mRNA 140-fold that peaked within 30 min. This was followed by liver TNFα protein increase of ~29-fold that peaked at 60 min. Serum TNFα protein increased from 0 to ~6.452 ng/mL at 60 min after LPS treatment (see ). Brain showed an increase in TNFα mRNA (7,336%) and protein levels (653%) that both peaked at 60 min. Thus, while TNFα protein levels in response to systemic LPS peaked in the liver, serum, and brain at 1 h, brain TNFα mRNA synthesis occurred shortly after liver TNFα mRNA synthesis, very likely after the TNFα protein was able to enter the brain. These results support that liver-derived TNFα was the predominant source of the immediate TNFα response to systemic LPS (1-h post treatment) in the liver, serum, and brain, which then initiated the synthesis of TNFα in the brain.
Fig. 1 Effects of LPS treatment on liver, plasma, and brain TNFα. Male C57BL/6 mice were treated with saline or LPS (5 mg/kg) i.p. injection. At different time points following LPS or saline injection, mice were sacrificed and brain extracts, liver extracts, (more ...)
We also examined the long-term effects of systemic LPS injection from several hours until 10-months post treatment. Interestingly, serum TNFα declined to saline control levels by 9 h (, ). In liver, TNFα protein levels declined to 18% of peak values by 9 h after LPS (, ). Surprisingly, TNFα protein remained elevated in brain. At 1 week after LPS treatment TNFα levels were elevated in brain to ~319 ±20 pg/mg protein compared to control values of 65 ±5 pg/mg protein, a 490% increase similar to the 1-h peak induction. TNFα protein remained elevated at 14 days, 21 days, and even 10 months after LPS treatment (). In addition, 3 h after LPS treatment staining of microglia in cortex, hippocampus, and SN showed microglia with enlarged cytoplasm and cell bodies, irregular shape, and intensified Iba1 staining consistent with morphological activation of microglia (see ). In summary, these results support an association between early (1–9 h) TNFα production in the liver and blood, with chronic (10 months) microglial activation in the brain.
Fig. 2 Single LPS injection caused long-lasting elevated level of TNFα protein in mouse brain. LPS (5 mg/kg) was injected intraperitoneally in male C57BL/6 mice. At different time points following LPS or saline injection, mice were sacrificed and brain (more ...)
Fig. 3 Immunocytochemical analysis of microglia. C57BL/6 mice were sacrificed 3 h following saline or LPS (5 mg/kg) i.p. injection. Brain sections were immnostained with Iba1 specific microglial antibody. Activated microglia in substantia nigra, hippocampus (more ...)
TNFα Mediates Neuroinflammation in Response to Systemic LPS and TNFα Administration
Studies indicate that TNFα is transported into brain (Pan and Kastin, 2001
; Pan et al., 2006
) and that TNFα receptors are critical for transport of the TNFα protein from the periphery, across the blood brain barrier, and into the brain (Pan and Kastin, 2002
). To test the role of systemic TNFα on inflammation in the brain, we administered a single dose of LPS (5 mg/kg) i.p. or TNFα (0.25 mg/kg) i.p. to male wild type B6:129SF2/J mice (WT) or male mice deficient in the TNF Type 1 and Type 2 receptors (TNFR-KO). TNFα expression (brain, liver, and serum) and the up regulation of other pro-inflammatory genes (IL-1β, NFκB, and MCP-1) in the brain were determined.
Systemic LPS treatment increased TNFα mRNA and protein in the liver of both WT and TNFR-KO as expected, as LPS triggers an immune response independent of TNFα receptors (). LPS increased serum levels of TNFα in both WT and TNFR-KO mice. However, brain TNFα mRNA and protein increased only in the WT, but not in TNFR-KO mice, indicating that TNFα receptors are necessary for peripheral LPS to cause brain TNFα mRNA and protein production (). In a separate experiment, similar to , WT mice showed increased Iba1 (a specific marker for microglia) staining in several brain regions, such as SN, hippocampus, and cortex (images not shown) at 2-h post LPS-treatment. Iba1-IR cells showed increased cell size, irregular shape, and intensified Iba1 staining consistent with morphological changes in activated microglia. Iba1-IR cells in saline controls in WT mice showed resting microglial morphology. However, TNFR-KO animals did not show increased TNFα mRNA or protein synthesis in the brain (). These findings indicate that TNFα receptors are essential for systemic LPS administration to cause activation of microglia and TNFα synthesis within brain.
Fig. 4 Comparison of LPS-induced changes in TNFα in TNFR1/R2+/+ (WT) and TNFR1/R2−/− (KO) mice. TNFα mRNA and protein were measured 1 h after saline or LPS (5 mg/kg) i.p. injection. (A) LPS activated liver TNFα mRNA (left (more ...)
To confirm the role of systemic TNFα in brain inflammation, WT and TNFR-KO mice were treated with TNFα (0.25 mg/kg, i.p.). TNFα treatment resulted in a 132-fold increase in liver TNFα mRNA and a 27-fold increase in liver TNFα protein in WT, but not in TNFR-KO mice (). The injection of TNFα increased serum levels of TNFα in both WT and TNFR-KO (). Importantly, systemic TNFα injection induced brain TNFα mRNA and protein in WT, not in TNFR-KO mice (). Further, TNFα induced brain synthesis of other pro-inflammatory factors, such as monocyte chemotactic protein 1 (MCP-1) as well as NF-κB p65 subunit, interleukin-1β (IL-1β) in WT mice. However, TNFα failed to induce production of the pro-inflammatory factors in TNFR-KO mice (see ). Thus, systemic TNFα induces pro-inflammatory cytokine production in brain, serum, and liver, as well as induces brain microglial activation.
Fig. 5 Effects of systemic TNFα on liver, serum and brain TNFα in TNFR1/R2+/+ (WT) and TNFR1/R2−/− (KO) mice. Animals were sacrificed 1 h following saline or TNFα (0.25 mg/kg) i.p. injection. TNFα mRNA and protein (more ...)
Fig. 6 Systemic TNFα increases IL-1β, NF-κB, and MCP-1 expression in TNFR1/R2+/+ (WT) mice. TNFR1/R2+/+ (WT) and TNFR1/R2−/− (KO) mice were injected with saline or TNFα (0.25 mg/kg) intraperitoneally. Brain mRNA (more ...)
A comparison of TNFα and LPS indicates these two inflammatory activators are remarkably similar. Our findings suggest that systemic LPS acts by inducing TNFα synthesis in the liver to increase serum TNFα, which then activates brain microglia to induce TNFα, other cytokines, and neuroinflammatory protein synthesis. Taken together, these findings suggest that an acute systemic injection of LPS or TNFα activates brain microglia through TNFα receptors that initiate sustained activation of brain cytokine synthesis and neuroinflammation.
Peripheral Inflammation Induces a Delayed and Progressive Loss of Dopaminergic Neurons in the Substantia Nigra
The substantia nigra (SN) is densely populated with microglia (Kim et al., 2000
; Lawson et al., 1990
) and it is hypothesized that this brain region is particularly susceptible to inflammatory insult. To discern the effects of chronic neuroinflammation (sustained increase in brain TNFα for at least 10 months) on neuron survival in the SN, we measured the number of dopamine neurons, as indicated by the number of tyrosine hydroxylase immunoreactive (TH-IR) cells within the SN, after a single LPS i.p. treatment. Previous reports using intranigral injection of LPS demonstrated that the LPS-induced decline of TH-IR cells is due to cell loss, as evidenced by a decline in the over-all number of neurons in the SN (Gao et al., 2002
). As shown in , LPS injection caused a delayed and progressive loss of nigral TH-IR neurons. Comparison of the number of nigral TH-IR neurons between the LPS-treated and saline-treated mice showed no significant loss of TH-IR neurons within the first 4 months after LPS injection (). However, significant loss of TH-IR neurons started to manifest between 7 and 10 months after LPS treatment. At 7 months, a 23% loss was observed. The LPS-induced loss of nigral TH-IR neurons further progressed over time and a 47% loss was shown at 10 months (). Using double labeled (TH-IR and NeuN-IR) staining, we have previously reported that inflammation-induced loss of TH-IR cells in the nigra represents neuronal death, rather a down regulation of TH expression (Gao et al., 2002
). Changes in TH-IR cells were more pronounced in SN than in the adjacent ventral tegmental area (VTA) DA neurons (). Interestingly, staining for microglia using F4/80 antibody indicated that the SN contains much more microglia than the adjacent VTA (). Cell loss in other brain regions was not determined. Thus, the SN may be inherently susceptible to inflammatory insult when compared to other regions of the basil ganglia due to the presence of increased microglia. These studies support the hypothesis that an acute increase in systemic TNFα activates neuroinflammatory processes within brain that are sustained for long periods, which leads to delayed and progressive degeneration of DA neurons.
Fig. 7 Delayed and time-dependent loss of nigral dopaminergic neurons following LPS i.p. injection. Male C57BL/6 mice were treated with saline or LPS (5 mg/kg, i.p.) and then maintained under normal conditions for the indicated time points. Mice were sacrificed (more ...)