Bile Acid Signaling Is Involved in the Neurological Decline in a Murine Model of Acute Liver Failure

Abstract

Hepatic encephalopathy is a serious neurological complication of liver failure. Serum bile acids are elevated after liver damage and may disrupt the blood-brain barrier and enter the brain. Our aim was to assess the role of serum bile acids in the neurological complications after acute liver failure. C57Bl/6 or cytochrome p450 7A1 knockout (Cyp7A1(-/-)) mice were fed a control, cholestyramine-containing, or bile acid-containing diet before azoxymethane (AOM)-induced acute liver failure. In parallel, mice were given an intracerebroventricular infusion of farnesoid X receptor (FXR) Vivo-morpholino before AOM injection. Liver damage, neurological decline, and molecular analyses of bile acid signaling were performed. Total bile acid levels were increased in the cortex of AOM-treated mice. Reducing serum bile acids via cholestyramine feeding or using Cyp7A1(-/-) mice reduced bile acid levels and delayed AOM-induced neurological decline, whereas cholic acid or deoxycholic acid feeding worsened AOM-induced neurological decline. The expression of bile acid signaling machinery apical sodium-dependent bile acid transporter, FXR, and small heterodimer partner increased in the frontal cortex, and blocking FXR signaling delayed AOM-induced neurological decline. In conclusion, circulating bile acids may play a pathological role during hepatic encephalopathy, although precisely how they dysregulate normal brain function is unknown. Strategies to minimize serum bile acid concentrations may reduce the severity of neurological complications associated with liver failure.

Supplemental Figure S1

Farnesoid X receptor (FXR) signaling protein is elevated during liver failure. A: Representative immunoblot for apical sodium-dependent bile acid transporter (ASBT), FXR, and small heterodimer partner (SHP) during indicated stages of neurological decline in the cortex of vehicle and azoxymethane (AOM)–treated mice. β-Actin is used as a loading control. B: Quantification of cortical ASBT, FXR, and SHP protein from immunoblot analyses in vehicle and AOM mice on wild-type (WT) and Cyp7A1^−/− genetic backgrounds. ^∗P < 0.05 versus vehicle-treated mice. N = 4 (B).

Figure 1

Bile acids are elevated in the brain after liver failure. A: Total bile acid quantification in the frontal cortex of mice treated with vehicle or azoxymethane (AOM). B: Relative fluorescence in the cortex of vehicle or AOM-treated mice injected with cholyl-lysyl-fluorescein (CLF). Data are expressed as means ± SEM (A and B). n = 4 (A and B). ^∗P < 0.05 versus vehicle-injected mice. RFU, relative fluorescence units.

Figure 2

Reduction of circulating bile acids is protective against acute liver failure–induced neurological impairment. A: Serum bile acid concentrations in mice that ingest a control or cholestyramine-supplemented diet. B: Neurological score of azoxymethane (AOM)–treated mice that consume control (black) or cholestyramine-supplemented (gray) diet. C: Time to coma in hours for AOM-treated mice that were fed either control or cholestyramine diets. D: Hematoxylin and eosin staining of AOM-treated mice fed with control or cholestyramine diets. ^∗P < 0.05 versus control diet mice. n = 5 (A–C).

Figure 3

Knockout of cytochrome p450 7A1 (Cyp7a1) reduces circulating bile acids and neurological decline associated with acute liver failure. A: Wild-type (WT) and Cyp7a1^−/− mice serum bile acid levels. B: Neurological decline of azoxymethane (AOM)–treated WT (black) and Cyp7a1^−/− (gray) mice. C: Time to coma in hours for AOM-treated WT and Cyp7a1^−/− mice. D: Representative hematoxylin and eosin staining in WT and Cyp7a1^−/− mice treated with AOM. ^∗P < 0.05 versus WT mice. n = 5 (A–C).

Figure 4

Enrichment of bile acid pools differentially affects the neurological decline associated with acute liver failure. A: Neurological decline in azoxymethane (AOM)–treated mice that were fed diets supplemented with cholic acid, deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), or a control diet. B: Time to coma in hours for hepatic encephalopathy mice that consumed diets supplemented with cholic acid, DCA, UDCA, or a control diet. C: Hematoxylin and eosin histochemistry in control or AOM-treated mice that ingested diets supplemented with cholic acid, DCA, UDCA, or a control diet. ^∗P < 0.05 versus control diet–fed mice. n = 5 (A and B).

Figure 5

Farnesoid X receptor (FXR) signaling is elevated in brain during acute liver failure. A: Apical sodium-dependent bile acid transporter (ASBT), FXR, and small heterodimer partner (SHP) cortical mRNA expression during the time course of neurological decline after azoxymethane (AOM) injection. B: Fluorescence immunoreactivity of ASBT and FXR (red) costained with neuronal marker NeuN (green) in the cortex. The arrows indicate colocalization of ASBT and NeuN immunoreactivity (top panel) and FXR and NeuN immunoreactivity (bottom panel). C: Immunoblots for ASBT, FXR, and SHP in wild-type (WT) and Cyp7a1^−/− mice treated with control or AOM. β-Actin is used as a loading control. ^∗P < 0.05 versus vehicle-treated mice. n = 6 (A). Scale bar = 25 μm (B).

Figure 6

Primary neurons transduce farnesoid X receptor (FXR) signaling in response to deoxycholic acid (DCA). A: Immunocytochemistry for FXR (red) of primary neurons treated with 10 μmol/L DCA, 10 μmol/L guggulsterone, or both. DAPI (blue) is used as a nuclear marker. B: Relative small heterodimer partner (SHP) mRNA expression in primary neurons treated with 10 μmol/L DCA, 10 μmol/L guggulsterone, or both. ^∗P < 0.05 versus basal primary neurons. n = 4 (B). Scale bar = 25 μm (A).

Figure 7

Neurological decline during acute liver failure is exacerbated via farnesoid X receptor (FXR) signaling. A: FXR immunhohistochemisty in the cortex of mice after intracortical infusion with FXR-mismatched morpholino or FXR Vivo-morpholino. B: Immunoblot against FXR with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as a loading control in FXR-mismatched morpholino or FXR Vivo-morpholino mice. C: Neurological decline in azoxymethane (AOM) mice infused with FXR-mismatched morpholino or FXR Vivo-morpholino. D: Time to coma in hours of AOM mice infused with FXR-mismatched morpholino or FXR Vivo-morpholino. E: Hematoxylin and eosin histochemistry in vehicle and AOM mice treated with FXR-mismatched morpholino or FXR Vivo-morpholino. ^∗P < 0.05 versus FXR-mismatched morpholino mice. n = 5 (C and D).