Cholestasis alters brain lipid and bile acid composition and compromises motor function in neonatal piglets

Abstract

Infants with neonatal cholestasis are prone to neurodevelopmental deficits, however, the underlying pathogenesis is unclear. Lipid malabsorption and accumulation of potentially neurotoxic molecules in the blood such as bile acids are important yet relatively unexplored pathways. Here, we developed a translational piglet model to understand how the molecular bile acid and lipid composition of the brain is affected by this disease and relates to motor function. Piglets (8-days old) had bile duct ligation or sham surgery and were fed a formula diet for 3 weeks. Alongside sensory-motor deficits observed in bile duct-ligated animals, we found a shift toward a more hydrophilic and conjugated bile acid profile in the brain. Additionally, comprehensive lipidomics of the cerebellum revealed a decrease in total lipids including phosphatidylinositols and phosphatidylserines and increases in lysophospholipid species. This was paralleled by elevated cerebellar expression of genes related to inflammation and tissue damage albeit without significant impact on the brain transcriptome. This study offers new insights into the developing brain's molecular response to neonatal cholestasis indicating that bile acids and lipids may contribute in mediating motor deficits.

Keywords: bile acids; brain; cholestasis; lipids; motor skills.

FIGURE 1

Representative images of macroscopic and histological liver pathology from the SHAM‐operated (SHAM, a–c) and bile duct‐ligated (BDL, d–f) group on day 26. Images b, c, e and f are from the highlighted area in images a and d. The left panel shows the liver of a SHAM (a) and BDL (d) piglet, where cystic dilation of the biliary tree and gall bladder are seen in image d. The middle and right panel show cytokeratin staining for bile duct epithelium and masson's trichrome staining for collagen in a SHAM (b, c) and BDL (e, f) piglet, respectively. 2× objective. Scale bars are 500 μm.

FIGURE 2

Motor function tests: (a) beam test, (b) neuromotor score, (c) open field test—time spent in zone 4 of the arena (entrance zone) and (d) absolute stance time in SHAM‐operated (SHAM, n = 10–11) and bile duct‐ligated (BDL, n = 10–11) piglets on day 15 and 25. *p < 0.05. Data are presented as means ± SD.

FIGURE 3

Lipid profile of the cerebellum: (a) Total lipids, (b) lipid species (c) lipid classes and (d) total fatty acyls in SHAM‐operated (SHAM, n = 8–11) and bile duct‐ligated (BDL, n = 8–11) piglets on day 26. *p < 0.05, **p < 0.01, ***p < 0.001. Benjamini‐Hochberg‐adjusted p values are reported in b, c, and d. Data are presented as means ± SD (a, c, d) or fold change of the BDL relative to SHAM group (b). ACar, acylcarnitine; CE, cholesteryl ester; Cer, ceramide; CL, cardiolipin; DAG, diacylglycerol; DMPE, dimethyl‐phosphatidylethanolamine; GM1, monosialotetrahexosylganglioside; GM3, monosialodihexosylganglioside; HexCer, hexosyl ceramide; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPC O‐, lysoalkylphosphatidylcholine; LPE, lysophosphatidylethanolamine; LPE O‐, lysoalkylphosphatidylethanolamine; LPI, lysophosphatidylinositol; LPS, lysophosphatidylserine; MLCL, monolysocardiolipin; MMPE, monomethylphosphatidylethanolamine; NEFA, non‐esterified fatty acyl, phosphatic acid, phosphatidylcholine; PC O‐, alkylphosphatidylcholine; PE, phosphatidylethanolamine; PE O‐, alkylphosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SHexCer, sulfatides hexosyl ceramide; SM, sphingomyelin; ST, cholesterol; TAG, triacylglyceride.

FIGURE 4

Relative bile acid levels in plasma (a, b) and cerebellum tissue (c, d) in SHAM‐operated (SHAM) and bile duct‐ligated (BDL) piglets on day 26. Data are provided as the percentage of individual bile acids out of the total amount of bile acids measured by LC–MS/MS. Low‐abundance bile acids are grouped as “other” and their absolute values can be seen in Table 1. CDCA, chenodeoxycholic acid; HCA, hyocholic acid; HDCA, hyodeoxycholic acid; G‐: glycine conjugated; T‐, taurine conjugated.

FIGURE 5

Day 26 C4 (7‐alpha‐hydroxy‐4‐cholesten‐3‐one) levels in plasma and cerebellum tissue (a), and day 5 and 26 plasma fibroblast growth factor 19 (FGF‐19) levels (b) in SHAM‐operated (SHAM, n = 11) and bile duct‐ligated (BDL, n = 11) piglets. ***p < 0.001. Data are presented as means ± SD.

FIGURE 6

Relative qPCR gene expression in the cerebellum of SHAM‐operated (SHAM, n = 11) and bile duct‐ligated (BDL, n = 11) piglets on day 26. Statistical analysis was performed on the absolute data. *p ≤ 0.05, **p < 0.01, ***p < 0.001.