DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Summary

Using a lipophilic 1,1'-Dioctadecy-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

Abstract

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1’-Dioctadecy-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl’s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

Introduction

Visualization of vasculature plays a vital role in understanding the structure and function of organisms across many species. Starting in the 16^th century with Leonardo da Vinci, models and graphic representations of the circulation have been studied¹. Using waxes and rubber molds, tissues were perfused to create three-dimensional models of the vasculature, which allowed for the study of organogenesis and pathogenesis^1,2. Resins and waxes were colored with dyes such as India Ink or carmine red to allow for their easy visualization^1,

Protocol

All axolotl experimentation was performed in accordance with Brigham and Women's Hospital's (BWH) Institutional Animal Care and Use Committee.

1. Set up Perfusion Experiment

1. Place an adult axolotl in a plastic container filled with 0.1% tricaine solution (MS222) for 15-20 min or until completely anesthetized. Ensure that the container is filled with enough tricaine solution such that the axolotl is completely submerged.
   Note: All procedures must be performed in accordance with institutional animal care guidelines. At BWH, an axolotl is considered fully anesthetized when it fails a foot-pinch test, meaning there is....

Results

With DiI staining, the vasculature of the axolotl can be easily visualized. Blood vessels of animals perfused with the lipophilic dye are immediately visible under a fluorescent confocal microscope. Figure 1.1-1.5 is a schematic representation of the perfusion protocol. After perfusion with the bright pink dye, a successfully perfused axolotl will appear pink. Using a green fluorescent filter on a confocal microscope a red emission of the vas.......

Discussion

Visualization of the vasculature of the axolotl can be successfully accomplished via perfusion with the lipophilic carbocyanine dye, DiI. In this study, we describe a novel protocol for the perfusion of the axolotl with DiI using a peristaltic pump. We also show the subsequent visualization of the axolotl vasculature using a fluorescent confocal microscope. This protocol was an adaptation of the rodent DiI perfusion protocol seen in Li et al.⁷, however major differences between the rodent.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Peristaltic Pump	Marshall Scientific	RD-RP1
Perfusion tubing	Excelon Lab & Vacuum Tubing	436901705	size S1A
27g butterfly needle	EXELint Medical Products	26709
NaCl	AmericanBio	7647-14-5
KCl	AmericanBio	7747-40-7
Na2HPO4	AmericanBio	7558-79-4
NaH2PO4	AmericanBio	10049-21-5
Distilled water
HCl	AmericanBio	7647-01-0
Glucose	ThermoFischer	A2494001
1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate	Sigma Aldrich	468495
Ethanol (100% vol/vol)	Sigma Aldrich	64-17-5
Surgical foreceps	Medline	MDG0748741
Polystyrene foam frame			any polystyrene foam square with an axolotl-shaped  cut out
Surgical scissors	Medline	DYND04025
Scalpel	Medline	MDS15210
Absorbent underpad	Avacare Medical	PKUFSx
Paper towels
Standard disposable transfer pipette	Fisherbrand	50216954
Clamp stand	Adafruit	291
Ethyl 3-aminobenzoate methanesulfonate	Sigma Aldrich	E10521	Tricaine powder
Adult axolotl
MgSO4	AmericanBio	10034-99-8
CaCl2	Sigma Aldrich	C1016-100G
NaHCO3	Sigma Aldrich	S5761-500G
Plastic tanks			Varying size appropriate for the axolotl
Paraformaldehyde	Sigma Aldrich	30525-89-4
Axolotl
Leica Microscope	Leica	M165 FC
ET-CY3 Fluorescent Filter	Leica	M205FA/M165FC
MS-222