A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

Summary

Sea lamprey lose the gall bladder and bile ducts during metamorphosis, a process similar to human biliary atresia. A new fixation and clarification method (CLARITY) was modified to visualize the entire biliary tree using laser scanning confocal microscopy. This method provides a powerful tool to study biliary degeneration.

Abstract

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey (Petromyzon marinus) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called “CLARITY” was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.

Introduction

Sea lamprey develop through three distinct life stages^1,2. Larval sea lamprey (L) spend most time in burrows as benthic filter feeders. After going through seven metamorphic stages of dramatic changes in external morphology and reorganization in internal organs³, the resulting juveniles (JV) enter a parasitic stage during which they feed on blood and tissue fluids from host fish, increasing the body mass more than 100 times. After 1.0 to 1.5 years feeding on the host fish in the ocean or large lakes, adults cease feeding during the early spring and migrate into rivers to spawn and then die^1,2.

Protocol

1. Solution Preparation

1. Make 1 L 10x 0.1 M phosphate buffer saline (PBS, pH 7.4): Weigh 26.2 g sodium phosphate (monobasic), 115 g sodium phosphate (dibasic), and 87.66 g NaCl. Dissolve in about 800 ml distilled H₂O, adjust pH, and bring the volume up to 1 L with distilled H₂O.
   1. Make 1 L 0.1 M phosphate buffer saline (pH 7.4): Take 100 ml 10x PBS, and add 900 ml distilled H₂O.
   2. Make 1 L 0.1 M phosphate buffer saline (pH 7.4) with 0.1% Triton X-100: Take 10.......

Discussion

This protocol is modified from a new method called “CLARITY”¹², which crosslinks intact tissue with polyacrylamide to form a nanoporous hydrogel, and then strips away the plasma membrane of the tissue to achieve optical transparency and macromolecular permeability. “CLARITY” allows intact-tissue imaging of long-range projection and local circuit wiring in the nervous system. This new method can be used to visualize the entire biliary system in sea lamprey liver during metamorphosis. It .......

Materials

Name	Company	Catalog Number	Comments
40% acrylamide	Bio-Rad	161-0140
2% bis-acrylamide	Bio-Rad	161-0142
TEMED	Bio-Rad	161-0800
ammonium persulfate	Sigma	A3678-25G
boric acid	Sigma	B7901-1KG
saponin	Sigma	47036
sodium dodecyl sulfate	Sigma	L337-500G
sodium phosphate (monobasic)	Sigma	04269-1KG
sodium phosphate (dibasic)	Sigma	S5136-1KG
Triton X-100	Sigma	X100-500ML
glycerol	Sigma	G9012-500ML
16% paraformaldehyde	Electron Microscopy Sciences	15710-S
NaOH pellets	EMD	SX0590-3
15 ml centrifuge tubes	Any brand
dissecting tools	Any brand