Consecutive intra-gingival injections of lipopolysaccharide and butyric acid to mice induce abnormal behavior and changes in cytokine concentrations

Abstract

Background: Periodontopathic bacteria such as Porphyromonas gingivalis produce several metabolites, including lipopolysaccharide (LPS) and n-butyric acid (BA). Past work suggested that periodontal infection may cause cognitive impairment in mice.

Aims: To elucidate the mechanisms by which metabolites such as LPS and BA, resulting from Porphyromonas gingivalis activity, induce immunological and physiological abnormalities in mice.

Methods: In the present work, 28 male ICR mice were placed in an open-field arena and the total distance (cm/600 s) they covered was recorded. Based on their moving distances, mice were divided into 4 groups (n = 7) and injected the following substances into their gingival tissues for 32 consecutive days: saline (C), 5 mmol/L of BA (B), 1 μg/mouse of LPS (L), and BA-LPS (BL) solutions. Distances covered by mice were also measured on days 14 and 21, with their habituation scores considered as "(moving distance on day 14 or 21)/(moving distance on day 0)". Afterwards, mice were dissected, and hippocampal gene expression and the concentrations of short-chain fatty acids, neurotransmitters and cytokines in their blood plasma and brains were analyzed. In addition, mouse brain and liver tissues were fixed and visually assessed for histopathological abnormalities.

Results: Group BL had significantly higher habituation scores than C and B on day 14. LPS induced higher habituation scores on day 21. LPS induced significant decreases in the mRNA levels of interleukin (IL)-6 and brain-derived neurotrophic factors, and an increase in neurotrophic tyrosine kinase receptor type 2. In both plasma and brain, LPS induced a significant acetate increase. Moreover, LPS significantly increased acetylcholine in brain. In plasma alone, LPS and BA significantly decreased monocyte chemoattractant protein 1 (MCP-1). However, while LPS significantly decreased tyrosine, BA significantly increased it. Lastly, LPS significantly decreased IL-6 and tumor necrosis factor in plasma. No histopathological abnormalities were detected in liver or brain tissues of mice.

Conclusion: We showed that injections of LPS and/or BA induced mice to move seemingly tireless and that both LPS and BA injections strongly induced a reduction of MCP-1 in blood plasma. We concluded that LPS and BA may have been crucial to induce and/or aggravate abnormal behavior in mice.

Keywords: Abnormal behavior; Lipopolysaccharide; Mouse model; Porphyromonas gingivalis; n-Butyric acid.

Fig. 1

Experimental design of this study. TST: Tail suspension test, FST: Forced swim test

Fig. 2

Effect of lipopolysaccharide (LPS) and/or n-butyric acid (BA) injection on mouse behavior. Closed triangles and open bars: Control (group C). Open squares and gray bars: BA injection (group B). Closed squares or slash bars: LPS injection (group L). Closed circles or closed bars: BA and LPS injection (group BL). a Total moving distance on days 0, 14, and 21. b Habituation score calculated by the total distance on days 0, 14, and 21. The asterisks represent significant differences (P < 0.05) between groups. c Immobility time in TST. Panel d: Immobility time in FST

Fig. 3

Effect of lipopolysaccharide (LPS) and/or butyrate injection on hippocampal mRNA expression. For further details, see Fig. 2

Fig. 4

Effect of lipopolysaccharide (LPS) and/or butyrate injection on the concentrations of cytokines in blood plasma. For further details, see Fig. 2