Figure 9

In vivo imaging of hepatobiliary toxicity: passive hepatic congestion. (A) Widefield fluorescence DAPI/UV, control liver, 20 dpf, showing the normal in vivo appearance of hepatic vasculature (black arrowhead). Vasculature appears dark (non fluorescent). Epithelium of parenchyma appears light gray. (B and C) At 4 μM ANIT, 24 hrs post exposure, modest dilation of hepatic vasculature was observed throughout the liver (black arrowhead). Image B is DAPI/UV (autofluorescence), image C is TRITC (autofluorescence). (D) At 8 μM ANIT, 24 hrs, marked dilation of the intrahepatic vasculature was observed (DAPI/UV). (E) In vivo confocal imaging, acquired at 48 hrs post ANIT exposure, confirmed dilation of intrahepatic vasculature (black arrowhead) was a pan-hepatic response, occurring uniformly throughout the liver. Vasculature is dark gray, hepatocytes (H) appear green. (F) Transmission electron micrograph (8 μM, 20 dpf, 48 hrs post exposure) of an intrahepatic vessel in ANIT treated medaka, revealing abnormal sinusoid/endothelial cell morphology (S). A single red blood cell can be seen in the sinusoid lumen. Endothelium is highly attenuated. In tandem with morphological changes were changes to cardiovascular function; decreasing heart rate and motility along with increase in vasodilation, in medaka exposed to 0.5 μM to 8 μM ANIT. Control heart rates averaged 129 bpm to 140 bpm (n = 12). Heart rate decreased with increasing ANIT concentrations. At 8 μM ANIT heart rate (means) was observed to be 118 bpm at 6 hrs post exposure, 73 bpm at 24 hrs post exposure, and 61 bpm at 48 hrs post exposure. At dosage regimes above 4 μM (acute and chronic), vasodilation of the hepatic vasculature was evident. Dilation of sinusoids, hepatic vein, and hepatic portal vein, were all observed. Sinusoid diameters: control sinusoids averaged 7.4 μm. At 8 μM ANIT, 48 hrs post exposure, sinusoid diameter averaged 15.3 μm.