Acetate supplementation attenuates lipopolysaccharide-induced neuroinflammation

Abstract

Glyceryl triacetate (GTA), a compound effective at increasing circulating and tissue levels of acetate was used to treat rats subjected to a continual 28 day intra-ventricular infusion of bacterial lipopolysaccharide (LPS). This model produces a neuroinflammatory injury characterized by global neuroglial activation and a decrease in choline acetyltransferase immunoreactivity in the basal forebrain. During the LPS infusion, rats were given a daily treatment of either water or GTA at a dose of 6 g/kg by oral gavage. In parallel experiments, free-CoA and acetyl-CoA levels were measured in microwave fixed brains and flash frozen heart, liver, kidney and muscle following a single oral dose of GTA. We found that a single oral dose of GTA significantly increased plasma acetate levels by 15 min and remained elevated for up to 4 h. At 30 min the acetyl-CoA levels in microwave-fixed brain and flash frozen heart and liver were increased at least 2.2-fold. The concentrations of brain acetyl-CoA was significantly increased between 30 and 45 min following treatment and remained elevated for up to 4 h. The concentration of free-CoA in brain was significantly decreased compared to controls at 240 min. Immunohistochemical and morphological analysis demonstrated that a daily treatment with GTA significantly reduced the percentage of reactive glial fibrillary acidic protein-positive astrocytes and activated CD11b-positive microglia by 40-50% in rats subjected to LPS-induced neuroinflammation. Further, in rats subjected to neuroinflammation, GTA significantly increased the number of choline acetyltransferase (ChAT)-positive cells by 40% in the basal forebrain compared to untreated controls. These data suggest that acetate supplementation increases intermediary short chain acetyl-CoA metabolism and that treatment is potentially anti-inflammatory and neuroprotective with regards to attenuating neuroglial activation and increasing ChAT immunoreactivity in this model.

Figure 1

Plasma acetate level in rats treated with a single oral dose on glyceryl triacetate. Values represent the means ± SD (n = 5).

Figure 2

Time-dependent changes in the concentrations of brain acetyl-CoA (A) and free CoA (B) following a single oral dose of glyceryl triacetate in microwave fixed rat brain. Values represent the means ± SD in units of μg/g brain (n=9 per group). Statistical analysis was performed comparing the brain concentration of brain acetyl-CoA and free CoA in GTA-treated rats to rats treated with a single oral dose of water (6 g/kg) (*, P ≤ 0.05).

Figure 3

Quantification of activated microglia in control, LPS-treated, GTA-treated, and LPS+GTA-treated rats. Representative images of CD11b-positive microglia in control (panel A, scale bar is equal to 100 μm) and LPS-treated (panel B) rat brain. The bar graph (panel C) represents the averaged percent of total activated CD11b-positive microglia as determined from three independent measurements. A one-way analysis or variance was performed comparing the percentage change in activated microglia in the four different groups. All values represent the means ± SEM (n = 6 samples per group).

Figure 3

Quantification of activated microglia in control, LPS-treated, GTA-treated, and LPS+GTA-treated rats. Representative images of CD11b-positive microglia in control (panel A, scale bar is equal to 100 μm) and LPS-treated (panel B) rat brain. The bar graph (panel C) represents the averaged percent of total activated CD11b-positive microglia as determined from three independent measurements. A one-way analysis or variance was performed comparing the percentage change in activated microglia in the four different groups. All values represent the means ± SEM (n = 6 samples per group).

Figure 3

Quantification of activated microglia in control, LPS-treated, GTA-treated, and LPS+GTA-treated rats. Representative images of CD11b-positive microglia in control (panel A, scale bar is equal to 100 μm) and LPS-treated (panel B) rat brain. The bar graph (panel C) represents the averaged percent of total activated CD11b-positive microglia as determined from three independent measurements. A one-way analysis or variance was performed comparing the percentage change in activated microglia in the four different groups. All values represent the means ± SEM (n = 6 samples per group).

Figure 4

Quantification of reactive astroglia in control, LPS-treated, GTA-treated, and LPS+GTA-treated rats. Representative images of GFAP-positive astroglia in control (panel A, scale bar is equal to 100 μm) and LPS-treated (panel B) rat brain. The bar graph (panel C) represents the averaged percent of total reactive GFAP-positive astroglia as determined from three independent measurements. A one-way analysis or variance was performed comparing the percentage change in reactive astroglia in the four different groups. All values represent the means ± SEM (n = 6 samples per group).

Figure 4

Quantification of reactive astroglia in control, LPS-treated, GTA-treated, and LPS+GTA-treated rats. Representative images of GFAP-positive astroglia in control (panel A, scale bar is equal to 100 μm) and LPS-treated (panel B) rat brain. The bar graph (panel C) represents the averaged percent of total reactive GFAP-positive astroglia as determined from three independent measurements. A one-way analysis or variance was performed comparing the percentage change in reactive astroglia in the four different groups. All values represent the means ± SEM (n = 6 samples per group).

Figure 4

Quantification of reactive astroglia in control, LPS-treated, GTA-treated, and LPS+GTA-treated rats. Representative images of GFAP-positive astroglia in control (panel A, scale bar is equal to 100 μm) and LPS-treated (panel B) rat brain. The bar graph (panel C) represents the averaged percent of total reactive GFAP-positive astroglia as determined from three independent measurements. A one-way analysis or variance was performed comparing the percentage change in reactive astroglia in the four different groups. All values represent the means ± SEM (n = 6 samples per group).

Figure 5

Quantification cholinergic cell loss in control, LPS-treated, GTA-treated, and LPS+GTA-treated rats. Representative images of ChAT-positive cells found in the basal forebrain in control (panel A, scale bar is equal to 100 μm) and LPS-treated (panel B) rat brain. The bar graph (panel C) represents the averaged total number of activated ChAT-positive cell in the basal forebrain as determined from three independent measurements. A one-way analysis or variance was performed comparing the percentage change in activated microglia in the four different groups. All values represent the means ± SEM (n = 6 samples per group).

Figure 5

Quantification cholinergic cell loss in control, LPS-treated, GTA-treated, and LPS+GTA-treated rats. Representative images of ChAT-positive cells found in the basal forebrain in control (panel A, scale bar is equal to 100 μm) and LPS-treated (panel B) rat brain. The bar graph (panel C) represents the averaged total number of activated ChAT-positive cell in the basal forebrain as determined from three independent measurements. A one-way analysis or variance was performed comparing the percentage change in activated microglia in the four different groups. All values represent the means ± SEM (n = 6 samples per group).

Figure 5

Quantification cholinergic cell loss in control, LPS-treated, GTA-treated, and LPS+GTA-treated rats. Representative images of ChAT-positive cells found in the basal forebrain in control (panel A, scale bar is equal to 100 μm) and LPS-treated (panel B) rat brain. The bar graph (panel C) represents the averaged total number of activated ChAT-positive cell in the basal forebrain as determined from three independent measurements. A one-way analysis or variance was performed comparing the percentage change in activated microglia in the four different groups. All values represent the means ± SEM (n = 6 samples per group).