Age and duration of inflammatory environment differentially affect the neuroimmune response and catecholaminergic neurons in the midbrain and brainstem

Abstract

Neuroinflammation and degeneration of ascending catecholaminergic systems occur early in the neurodegenerative process. Age and the duration of a pro-inflammatory environment induced by continuous intraventricular lipopolysaccharide (LPS) differentially affect the expression profile of pro- and anti-inflammatory genes and proteins as well as the number of activated microglia (express major histocompatibility complex II; MHC II) and the integrity and density of ascending catecholaminergic neural systems originating from the locus coeruleus (LC) and substantia nigra pars compacta (SNpc) in rats. LPS infusion increased gene expression and/or protein levels for both pro- and anti-inflammatory biomarkers. Although LPS infusion stimulated a robust increase in IL-1ß gene and protein expression, this increase was blunted with age. LPS infusion also increased the density of activated microglia cells throughout the midbrain and brainstem. Corresponding to the development of a pro-inflammatory environment, LC and SNpc neurons immunopositive for tyrosine-hydroxylase (the rate-limiting synthetic enzyme for dopamine and norepinephrine) decreased in number, along with a decrease in tyrosine-hydroxylase gene expression in the midbrain and/or brainstem region. Our data support the concept that continuous exposure to a pro-inflammatory environment drives exaggerated changes in the production and release of inflammatory mediators that interact with age to impair functional capacity of the SNpc and LC.

Keywords: Aging; Alzheimer's disease; Cytokines; Locus coeruleus; Microglia; Neuroinflammation; Parkinson's disease; Rat; Substantia nigra.

Figure 1

Brainstem cytokines levels and gene expression. (A) LPS exposure significantly (*p<0.05) increased in IL-1β levels and a far lesser increase in IL-1α levels. The duration of the LPS infusion was independently responsible with a significant (#p<0.001) increase in the brainstem level of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-12, IL-13 and GM-CSF. The age of the rat was independently responsible for an increase in IFNγ levels in the23 mo rats infused with LPS and for a blunting of the LPS effects on IL-1β in the 9 and 23 mo rats (†p<0.05). (B) LPS significantly (*p<0.001) increased the gene expression of the fractalkine receptor, IL1β, TGFβ, TLR4, TNFα and XcT. The duration of the LPS infusion was responsible with a significant (#p<0.01) increase in gene expression of GLT1 and TGFβ. In contrast, the duration of the LPS infusion was responsible with a significant decrease (#p <0.01) in the gene expression of TH, TNFα and BDNF within the brainstem and DBH gene expression within the hippocampus. The age of the rat was independently responsible for a significant (†p<0.001) decrease in the gene expression of BDNF and XcT. Each biomarker represents a minimum of 6 rats/group.

Figure 2

Photomicrographs of double-immunostained sections showing TH-IR cells and MHC II-IR microglia in the LC. (A) 3 mo aCSF 56 days; (B) 23 mo LPS 56 days. Scale bar for A & B = 200 μm. Inset shows the morphology of a typical microglia (section was counterstained with cresyl violet); scale bar for inset = 2.5 μm. The density of MHC II-IR microglia in the LC was significantly increased by the LPS infusion after 56 days of exposure. Only MHC II-IR microglia with their characteristic bushy morphology and ramified processes are visible in B. The density of darkly-stained TH-IR cells and their fibers (A) in the LC was significantly decreased by the LPS infusion (B, C). The effects of LPS were not influenced by the age of the rat or the duration of the LPS exposure (*p<0.01 vs. 3 mo aCSF 21). The histological analyses in C represent a minimum 5 rats/group.

Figure 3

Photomicrographs or MHC II-IR and SNpc cells and density quantification of TH-IR cells in the SNpc. (A) 3 mo aCSF 21 days; (B) 3 mo LPS 21 days; (C) 23 mo aCSF 21 days; (D) 23 mo LPS 21 days. Scale bar: 400 μm. Aging significantly decreased the density of TH-IR cells in the SNpc. The number of TH-IR cells in the SNpc of young and middle-aged, but not aged, rats was significantly greater on day 56 than day 21. Many MHC II-IR microglia with their characteristic bushy morphology and ramified processes are visible in B surrounding and within the region of the SNpc; very few darkly stained TH-IR cells are present. (*p<0.01 vs. 3 mo aCSF 21; +p<0.05 vs aCSF and LPS 56). Data from the 21 and 56 day aCSF groups were collapsed for representation. The histological analyses represent a minimum 5 rats/group.