Human liver afferent and efferent nerves revealed by 3-D/Airyscan super-resolution imaging

Abstract

Neural regulation of hepatic metabolism has long been recognized. However, the detailed afferent and efferent innervation of the human liver has not been systematically characterized. This is largely due to the liver's high lipid and pigment contents, causing false negative (light scattering and absorption) and false positive (autofluorescence) results in in-depth fluorescence imaging. Here, to avoid the artifacts in 3D liver neurohistology, we embed the bleached human liver in the high-refractive-index polymer for tissue clearing and antifade 3D/Airyscan super-resolution imaging. Importantly, using the paired substance P (SP, sensory marker) and PGP9.5 (pan-neuronal marker) labeling, we detect the sensory nerves in the portal space, featuring the SP+ varicosities in the PGP9.5+ nerve bundles/fibers, confirming the afferent liver innervation. Also, using the tyrosine hydroxylase (TH, sympathetic marker) labeling, we identify: (1) condensed TH+ sympathetic nerves in the portal space, (2) extension of sympathetic nerves from the portal to the intralobular space, in which the TH+ nerve density is 2.6±0.7-fold higher than that of the intralobular space in the human pancreas, and (3) the TH+ nerve fibers and varicosities contacting the ballooning cells, implicating potential sympathetic influence on hepatocytes with macrovesicular fatty change. Finally, using the vesicular acetylcholine transporter (VAChT, parasympathetic marker), PGP9.5, and CK19 (epithelial marker) labeling with panoramic-to-Airyscan super-resolution imaging, we detect and confirm the parasympathetic innervation of the septal bile duct. Overall, our labeling and 3D/Airyscan imaging approach reveal the hepatic sensory (afferent) and sympathetic and parasympathetic (efferent) innervation, establishing a clinically related setting for high-resolution 3D liver neurohistology.

Loss of the brain-liver axis prevents hepatic steatosis in mice

“…3D imaging analysis of cleared liver tissue collected from ChAT Cre mice injected with AAV1-FLEX-tdTomato-T2A-sypGFP further conformed the presence of cholinergic nerve terminals around the portal vein and in a subset of hepatocytes (Fig. 1E, F, G, and Movie S1), consistent with a recent findings of parasympathetic cholinergic innervation of the bile duct in human liver tissues 14 .…”

“…In contrast to these prior findings, recent 3D imaging studies revealed a paucity of parasympathetic cholinergic innervation in the mouse liver 3,57 , indicating that hepatic metabolism is primarily regulated by the sympathetic nervous system. However, a recent elegant study by the Tang research group clearly demonstrated the presence of vAChT-positive nerve terminals in the bile duct of human liver tissues, suggesting that the liver receive parasympathetic cholinergic innervation 14 .…”

“…The parasympathetic cholinergic innervation to the liver may potentially exert a regulatory effect on bile acid synthesis and outflow from the liver, which could subsequently result in a decrease in lipolysis within the white adipose tissue. In fact, a recent 3D/Airyscan imaging study has demonstrated vesicular acetylcholine transporter (vAChT)-positive parasympathetic nerve terminals on the bile duct in human liver tissues 14 .…”