Central and systemic endotoxin challenges exacerbate the local inflammatory response and increase neuronal death during chronic neurodegeneration

Abstract

The contribution of inflammation to the progression of neurodegenerative diseases such as Alzheimer's, Parkinson's, and prion diseases is poorly understood. Brain inflammation in animal models of these diseases is dominated by chronic microglial activation with minimal proinflammatory cytokine expression. However, these inflammatory cells are "primed" to produce exaggerated inflammatory responses to subsequent lipopolysaccharide (LPS) challenges. We show that, using the ME7 model of prion disease, intracerebral challenge with LPS results in dramatic interleukin-1beta (IL-1beta) expression, neutrophil infiltration, and inducible nitric oxide synthase expression in the brain parenchyma of prion-diseased mice compared with the same challenge in normal mice. Systemic inflammation evoked by LPS also produced greater increases in proinflammatory cytokines, pentraxin 3, and inducible nitric oxide synthase transcription in prion-diseased mice than in control mice and induced microglial expression of IL-1beta. These systemic challenges also increased neuronal apoptosis in the brains of ME7 animals. Thus, both central and peripheral inflammation can exacerbate local brain inflammation and neuronal death. The finding that a single acute systemic inflammatory event can induce neuronal death in the CNS has implications for therapy in neurodegenerative diseases.

Figure 1.

Neuropathological and inflammatory changes after intracerebral LPS. a-c, Tomato lectin staining for microglial cells in brains from NBH+LPS (a), ME7+saline (b), and ME7+LPS (c) animals. d-f, IL-1β immunostaining in NBH+LPS (d), ME7+saline (e), and ME7+LPS (f) brains. MBS-1 immunostaining for infiltrating neutrophils in NBH+LPS (g, j), ME7+saline (h, k), and ME7+LPS (i, l) brains is shown at 10× (g, h, i) and 40× (j, k, l), respectively. m-o, Immunostaining for iNOS in NBH+LPS (m), ME7+saline (n), and ME7+LPS (o) brains. Scale bar: (in a) a-f, j-o, 100 μm; (in g) g, h, i, 500 μm.

Figure 2.

The impact of systemic challenge with LPS on cytokine and inflammatory gene transcription in ME7 and NBH mice. LPS produced greater IL-1β (a), TNFα (b), IL-6 (c), and iNOS (e) mRNA increases in ME7 animals than in NBH animals. The difference between the IL-1β, TNFα, IL-6, and iNOS expression in NBH+LPS and ME7+LPS was statistically significant (p < 0.001, p < 0.01, and p < 0.05, respectively). All ME7 groups were statistically significantly different from both NBH groups in TGFβ1 transcription (d), but LPS (intraperitoneal) did not produce increases in TGFβ1 in either NBH or ME7 animals. Black bars represent mean ± SEM. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ANOVA. NBH+LPS, n = 4; ME7+saline, ME7+LPS, n = 5. Sal, Saline.

Figure 3.

Impact of systemic challenge with LPS on CNS acute phase reactant transcription expression in ME7 and NBH animals. PTX3 (a), α₂-AP (b), C3 (c), and SAA (d) mRNA were all increased by peripheral LPS injection, but only PTX3 showed a statistically significant increase in the ME7+LPS group relative to NBH+LPS. All data are presented as mean ± SEM. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ANOVA. NBH+LPS, n = 4; ME7+saline, ME7+LPS, n = 5. Sal, Saline.

Figure 4.

Neuropathological and inflammatory changes after intraperitoneal LPS. a-c, Tomato lectin staining for microglial cells in brains from NBH+LPS (a), ME7+saline (b), and ME7+LPS (c) animals. d-f, IL-1β immunostaining in NBH+LPS (d), ME7+saline (e), and ME7+LPS (f) brains (inset illustrates clear IL-1β-positive staining in cells with microglial morphology at 63× magnification). Immunostaining for cyclooxygenase-2 in NBH+LPS (g), ME7+saline (h), and ME7+LPS (i) brains is shown at 40× (g, h, i) and 63× (inset), respectively. j, TUNEL-positive staining in three cells (arrows) in a single field in the thalamus of an ME7+LPS brain. Inset, Hoescht 33352-stained nucleus showing shrunken and condensed chromatin (left) compared with normal (right). TUNEL-positive DAB staining in a CA3 neuron is shown (k), as identified by morphology and location, with failure to clearly double stain for NeuN (VIP chromagen). l, m, Activated caspase-3-positive cells showing neuronal morphology. An activated caspase-3-positive neuron (o), double stained using immunolabeling for neurofilament heavy chain (p) with overlay (q) in a 1 μm section by confocal microscopy is shown. n shows TOPRO-3 nuclear stain. Scale bars: (in a) a-i, 100 μm; (in m) l, m, 80 μm; n-q, 50 μm.