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.

During metamorphosis, the sea lamprey liver loses the gall bladder and the entire biliary tree, an evolutionary mutant phenotype that mimics the human infant disease biliary atresia. Infant biliary atresia is a rare pediatric liver disease with severe medical complications^{4,5,6,7,8,9,10}, however the pathogenesis and etiology of biliary atresia are largely unknown⁴. Patients with biliary atresia die within two years after birth unless surgical intervention (Kasai procedure) is performed⁵. Subsequently, these patients require extensive clinical management and often liver transplantation ⁶. Many theories of biliary atresia etiopathogenesis have been proposed, such as viral infection, congenital malformation, autoimmune disease, and toxic insult. However, the contribution of each to the development of biliary atresia remains inconclusive^7,8,9,10.

Unlike infants that suffer pathological biliary atresia, sea lamprey undergo developmentally programmed biliary atresia without extensive necroinflammation, fibrosis or cirrhosis¹⁰. The animals may suffer transient cholestasis during this process¹⁰, but adapt to this developmental condition via de novo synthesis and secretion of bile salts in the intestine after developmental biliary atresia, in addition to known mechanisms such as reduction of bile salt synthesis in liver¹¹. This developmental process in sea lamprey provides the only known opportunity to examine the progression of biliary atresia.

A newly-developed method called “CLARITY” enables high-resolution imaging in complex mammalian nervous systems by transforming intact tissue into an optically transparent nanoporous hydrogel¹². Using sea lamprey liver and a modified CLARITY protocol, intact-tissue imaging of biliary degeneration can be documented throughout liver metamorphosis.

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 100 ml 10x PBS, add 1 ml Triton X-100, and bring the volume up to 1 L with distilled H₂O.
2. Make 1 L 10x 0.1M phosphate buffer (PB, pH 7.4): Weigh 26.2 g sodium phosphate (monobasic) and 115 g sodium phosphate (dibasic). 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 (pH 7.4): Take 100 ml 10x PB, and add 900 ml distilled H₂O.
   2. Make 1 L 0.1 M phosphate buffer (pH 7.4) with 0.1% Triton X-100: Take 100 ml 10x PB, add 1 ml Triton X-100, and bring the volume up to 1 L with distilled H₂O.
3. Make 400 ml hydrogel monomer solution (4% acrylamide, 0.25% bis-acrylamide, 4% paraformaldehyde, 0.0075% ammonium persulfate, 0.0005% saponin in 0.1 M PBS): Weigh 0.3 g ammonium persulfate and 0.2 g saponin. Add 210 ml distilled H₂O, 40 ml 40% acrylamide, 10 ml 2% bis-acrylamide, 40 ml 10x PBS (pH 7.4), 100 ml 16% paraformaldehyde, and mix well.
4. Make 4 L clearing solution (4% SDS in 0.2 M boric acid): Weigh 49.464 g boric acid and 160 g SDS. Dissolve in about 3.5 L distilled H₂O, adjust pH to 8.5 with NaOH, and bring the volume up to 4 L.
5. Make 1 L 80% glycerol solution: Measure 800 ml glycerol, add 200 ml distilled H₂O, and mix well.

2. Tissue Preparation

1. Prepare hydrogel monomer solution and aliquot 10 ml to each 15 ml centrifuge tube.
2. Dissect the whole tissue and fix in the hydrogel for 2 days at 4 °C. Make sure that the hydrogel solution is at least 10x the volume of the tissue.
3. Bring the hydrogel fixed tissue (in the 15 ml centrifuge tube) to a fume hood.
4. Polymerize the hydrogel fixed tissue by adding 5 µl TEMED, mix well, and let sit in the fume hood at room temperature for 2-3 hr.
5. Remove the excess gel thoroughly. Note: Extra gel will hinder the clearing process and the antibody penetration during staining.
6. Incubate the fixed tissue in 10 ml clearing solution in an incubator shaker set at 70 rpm and 50 °C for several days until the tissue becomes transparent. Change clearing solution at least once per day and remove the excess gel from the tissue when changing the clearing solution.
7. Rinse the tissue in 10 ml 0.1 M PB with 0.1% Triton X-100 at 70 rpm and 37 °C overnight. Repeat this step 3 times and remove the excess gel from the tissue when changing the buffer solution.
8. Incubate the clarified tissue in the primary antibody solution in PB with 0.1% Triton X-100 at 70 rpm and 37 °C for 2 days. Note: Make enough primary antibody solution to submerge the whole tissue.
9. Rinse the tissue in 10 ml PB with 0.1% Triton X-100 at 70 rpm and 37 °C overnight. Repeat this step 3 times and remove the excess gel from the tissue when changing the buffer solution.
10. Perform the following steps in a dark room or under dim light. Cover the samples with foil to prevent photo bleaching.
11. Incubate the clarified tissue with the fluorescent secondary antibody solution in PB with 0.1% Triton X-100 and the blocking serum at 70 rpm and 37 °C for 1 day.
12. Rinse the tissue in 10 ml PB with 0.1% Triton X-100 at 70 rpm and 37 °C overnight. Repeat this step 3 times and remove the excess gel from the tissue when changing the buffer solution.
13. Rinse the tissue in 10 ml PBS at 70 rpm and 37 °C overnight. Repeat this step 3 times and remove the excess gel from the tissue when changing the buffer solution.
14. Incubate the clarified tissue in 80% glycerol at 70 rpm and 37 °C overnight.
15. Store the fluorescent stained tissue at 4 °C prior to confocal microscopy.

Results

Several important developmental events occur in the hepatobiliary system during sea lamprey metamorphosis. The bile duct and the gall bladder undergo apoptosis and degenerate (Figure 1). Combining the modified clarification method and staining with liver cell marker cytokeratin 19 (CK19, present in both cholangiocytes and hepatocytes before and after metamorphosis¹³) and anti-apoptotic marker Bcl2 using confocal microscopy, the entire biliary system was captured along the Z-axis (Figur...

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