Visualization of hepatobiliary excretory function by intravital multiphoton microscopy

Abstract

Intravital imaging of hepatobiliary excretion is vital for elucidating liver metabolism. In this work, we describe a novel method to observe the intravital dynamics of the uptake, processing, and excretion of an organic anion, 6-carboxyfluorescein diacetate (6-CFDA) in the hepatobiliary system. This is achieved by the use of multiphoton microscopy and an intravital hepatic imaging chamber. The high-quality images show sequential uptake and processing of 6-CFDA from the hepatocytes and the subsequent excretion into bile canaliculi within approximately 50 min. This is a promising technique to study intravital hepatic physiology and metabolism.

Quantitative intravital microscopy of hepatic transport

“…[34][35][36][37][38][39][40][41][42] As outlined below, we have adapted and extended the approaches used in these studies into quantitative assays of hepatocyte transport.…”

“…Previous studies have verified that its efflux is mediated by Mrp2 and Mrp3 57 and 6-CFDA has been used to quantify hepatocyte transport in vitro 56 and in vivo. 37,38 The results of studies using 6-CFDA to assay the effect of rifampin on canalicular secretion are summarized in Figure 4. Figure 4A shows an example of a projected image volume collected from the liver of a vehicle-treated living rat, 6 min after intravenous injection of 6-CFDA.…”

In vivo dynamic metabolic imaging of obstructive cholestasis in mice

“…Ligation of cystic duct was to avoid overdistension of gall bladder, which would push the underlying liver surface away from the central light path of the hepatic imaging window on the next day's imaging experiments. Following CBDL, the mice were installed with hepatic imaging window on the upper abdomen as previously described (9). They recovered awaiting imaging experiments the next day.…”

“…They recovered awaiting imaging experiments the next day. The hepatic imaging window used in the present study is modified from previous one (9), and hence the second generation window. This new window consists of only one doughnut-shaped titanium ring (Fig.…”

“…We have successfully developed a technique capable of examining hepatobiliary excretory function in living mice by using multiphoton microscopy through a newly designed hepatic imaging window (9). A nonfluorogenic substance carboxyfluorescein diacetate (CFDA) was used as the substrate.…”