(786d) Engineering the Gut-Liver-Brain Axis to Investigate Chemical Toxicity

The inter-organ crosstalk between the gut, liver and brain has been shown to affect many physiological functions. The accumulation of drugs, chemicals, toxins and their metabolites in the liver can reach the brain and result in impaired cognitive function. Brain diseases such as hepatic encephalopathy result from complex interactions along the gut-liver-brain axis. We have designed and assembled a multi-organ system to obtain fundamental insights into the intricate cross-talk between these three organs.

Primary intestinal tissues were surgically obtained from rat jejunum and cultured in medium containing antibiotics. Gram staining was performed to detect the presence of gut bacteria over a 3-day period. Minimal numbers of Gram-positive and Gram-negative bacteria were released over the 72 h period. Imaging was conducted to determine the viability of intestinal cells that were shed. Enterocyte and goblet cell phenotypes were confirmed via alkaline phosphatase activity and Alcian blue staining, respectively. The intestinal tissues expressed these markers even after 96 h.

Hepatocyte function in the integrated jejunum-liver models was measured through urea secretion and albumin production and was comparable to liver models alone. Acetaminophen, a well-known hepatotoxicant, was administered to integrated gut-liver cultures. Even at a 10mM concentration of acetaminophen, the decrease in urea production and albumin expression increased two-fold in the integrated cultures when compared to liver models only. These trends clearly demonstrate the enhanced sensitivity to acetaminophen exhibited by the integrated cultures, underscoring the need to combine organs.

The gut-liver-brain crosstalk was accomplished by exposing astrocyte cultures to spent medium from the integrated systems and liver models. Specifically, the spent culture medium was obtained from systems exposed to 10mM acetaminophen. The production of reactive oxygen species (ROS) in astrocytes was visualized using the DCFDA assay. ROS production was compared between astrocyte cultures treated with conditioned medium from the integrated gut-liver systems and liver models only. There was approximately 10-fold higher ROS in cultures treated with conditioned medium obtained from only liver models. This trend could be due to increased acetaminophen metabolism by the gut in the integrated cultures through the glucuronidation and sulfation pathways, thus decreasing the production of toxic metabolites from the cytochrome P450 2E1 (CYP2E1) mediated metabolism of this hepatotoxicant.

The design and assembly of multi-organ engineered tissues provides a powerful platform to investigate the interactions and crosstalk that occur during many physiological processes. We are currently focusing on elucidating the signaling pathways between these three organs to better understand how they function.