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.
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.
Visualization of vasculature plays a vital role in understanding the structure and function of organisms across many species. Starting in the 16th 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 pathogenesis1,2. Resins and waxes were colored with dyes such as India Ink or carmine red to allow for their easy visualization1,2. However, these techniques caused many issues because their high viscosities prevented full perfusion of the tissue of interest1. As the field became more sophisticated, the use of confocal and electron microscopes came into play, moving the perfusion techniques away from cast-molds and toward liquid perfusions of the vasculature, some of which allowed for the perfusion and imaging of blood vessels without destroying the initial tissue3. DiI, a fluorescent carbocyanine dye, is one such stain that allows for the perfusion of animals without damage to the vascular tissue.
Carbocyanine dyes are lipophilic dyes that incorporate into cell membranes upon contact. These dyes allow for easy and instantaneous staining of vascular endothelial cells, which can then be viewed under a fluorescent confocal microscope. DiI moves via lateral diffusion in the lipid membrane of cells, as shown in the labeling and tracing of neurons4. Chemically, the two alkyl chains of DiI give the dye its high affinity for cell membranes, while two conjugated rings from a fluorochrome which is responsible for emitting a red wavelength when excited by green fluorescent light filters4. DiI has been utilized in many capacities, including successful labeling of the plasma membrane and both anterograde and retrograde labeling in neurons5,6. DiI has previously been used in perfusion protocols while visualizing the vasculature of mice7.
Axolotls (Ambystoma mexicanum) are salamanders that live exclusively in brackish lakes near Mexico City, Mexico. These animals have become an important model for understanding regenerative processes as they can regenerate full limbs, tail (including nerve cord), portions of the heart and other internal organs, and portions of the eye as adults8,9. Additionally, with the recent application of genetic tools in axolotls, unprecedented insight into the molecules and cells driving these processes is now possible8. The successful regeneration of an entire limb requires an extensive revascularization process, which may play a significant role in regeneration beyond simply the traditional functions of blood vessels in providing oxygen and nutrients. Understanding revascularization in the context of tissue regeneration is imperative. Axolotl blood vessels have previously been visualized using India Ink, and while the results were intriguing, this process has not been revisited in subsequent decades10. We sought to adapt a DiI perfusion protocol developed for use in mammals to allow for a complete perfusion and visualization of the axolotl vasculature7. This protocol describes the steps taken to successfully perfuse and subsequently visualize the axolotl circulation with a DiI staining technique. This procedure will allow for precise visualization of patent blood vessels in homeostatic tissues, as well as in regenerating tissues, and provides a novel method for visualization and analysis of the revascularization process in the axolotl.
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
2. Opening the Axolotl Chest
3. Perfusion of the Axolotl
4. Ending the Perfusion and Visualization Preparation
5. Visualization the Perfused Axolotl
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 vascular network will appear. The DiI staining occurs in all body tissues when perfusion is successful, including the tail, limbs, gills, and eyes (Figure 2A, Figure 2B, Figure 2C, Figure 2D, repectively). Unsuccessful perfusions result in a lack of red-stained vasculature or in patchy staining of the vessels.
Figure 1: Schematic of the perfusion protocol. Axolotls successfully perfused with the lipophilic dye, DiI, demonstrate full staining of the vasculature upon imaging. 1: Full supine axolotl before perfusion experiment. 2: Opening the chest of the axolotl. 2: Axolotl with an open chest cavity. 3: Insertion of the 27 G butterfly needle into the aorta of the axolotl. 4: Tubing should first contain 0.7x PBS, then the DiI working solution, and lastly 4% PFA. 5: Fully perfused axolotls appear pink. Please click here to view a larger version of this figure.
Figure 2: Images of a Fully Perfused Axolotl. Images of the axolotl vasculature were taken using a fluorescent confocal microscope after successful perfusion with the DiI stain. 2A: Tail. 2B: Foot. 2C: Gills. 2D: Eye. Imaging is done using a confocal microscope with an green fluorescent emission filter cube. Magnification for images A, B, C, and D, are 1.74X, 2.16X, 1.18X, and 5.69X, respectively. Please click here to view a larger version of this figure.
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 and the axolotl required a revision of the protocol to fit the axolotl model.
This study discusses a method of DiI perfusion of the axolotl in order to successfully visualize the vasculature. Differences in the anatomy and physiology between the salamander and rodent demand alterations in major aspects of the perfusion, including location of needle insertion, method of perfusion, and the reagents used. In order to achieve a successful perfusion, we limited the damage done to the vasculature of the axolotl. While opening the chest cavity, care was taken to fully expose the heart and aorta while avoiding any damage or lacerations to major blood vessels. Limiting the use of the surgical scissors prevented accidental clipping of major vessels while small incisions maintained control over the exposure of the heart and aorta. Success rates of perfusions also increased when the DiI needle was inserted through the aorta, rather than directly into the chambers of the heart. The axolotl, unlike the mouse, has a three chambered heart, containing only one ventricle with significantly less musculature than that of the mouse. Because of these differences, the location of needle insertion had to be moved to the more stable aorta. The aorta was determined to be the optimal location for insertion of the perfusion needle as it is large enough for puncture by a 27 G needle and has limited movement. Movement was minimized in order to avoid accidental removal or slipping of the perfusion needle or through-and-through puncture of the aorta. Cardiac perfusions using the ventricle as an insertion point proved to have a much lower rate of success than those with an aortic insertion point. Erroneous puncture of the vasculature often resulted in the formation of emboli or prevented perfusion, resulting in very low rates of successful vascular labeling. By using a clamp stand to hold the butterfly needle during perfusion, we have decreased its movement, therefore increasing the rate of successful perfusions. Additionally, due to the delicacy of the axolotl tissue, when compared to the mouse, a peristaltic perfusion pump was necessary, as opposed to the manual perfusion previously used. The use of this pump allowed for a hands-free approach to the axolotl perfusion to minimize mistaken puncture of the thin tissues. Perfusions were unsuccessful for many additional reasons, including through-and-through puncture, clotting, and embolism. In the case that the needle was inserted into the aorta and a second puncture was created through the posterior wall, the DiI solution would flow directly into the chest cavity rather than pass through the systemic circulation. Additionally, once blood exited the vasculature, it quickly formed a blood clot which could impede perfusion. Clots and air bubbles could also form in the vasculature, causing emboli which preclude successful perfusion. Lastly, this protocol incorporated reagents adjusted to fit the axolotl osmolality, which differs significantly from that of the mammal. Adaptation of this protocol and the significant changes made to fit the axolotl model will assist in the pursuit of understanding the process of revascularization of tissues during regeneration.
DiI, which is pink in color, will perfuse the animal and give it a bright pink hue. Successfully perfused axolotls became bright pink to the naked eye, with highly vascularized regions appearing more intensely stained. Perfused animals viewed with a fluorescent confocal microscope using a green filter can be visualized in the red-orange emission spectrum. Vasculature was best visualized in thinner tissues that minimized accidental DiI staining of non-vascular tissues. Perfusion of the tissue with 4% Paraformaldehyde (PFA) immediately after DiI perfusion should be done to fix the tissue.
DiI perfusions are end-point experiments for the axolotl. During the procedure, all of the animal’s blood is effectively drained and replaced with 0.7x PBS, followed immediately by DiI solution, and finally 4% PFA. This disrupts the axolotl’s ability to engage in the vital act of gas exchange and it loses the ability to oxygenate its body tissues. Due to this end-point nature, each perfusion captures only a single time-point of vascular growth, and the animal cannot be further perfused at a later time. Due to this time-limiting factor, multiple animals must be used in order to describe a time course of vascular development.
This DiI protocol, and the modifications applied to improve it, can be used to successfully label and visualize the vasculature of the axolotl. Since the axolotl is an essential model organism for the study of regeneration, successful perfusions open up opportunities to interrogate the process of angiogenesis during regeneration. The axolotl is a model organism for the study of regeneration because it is a neotenous animal and therefore retains a remarkable ability to regenerate throughout adulthood8. The revascularization process of regenerating tissues, however, is not well understood, therefore the adaptation of the DiI perfusion to the axolotl system presents opportunities to understand regeneration that were not available with the mammalian model. The perfusion of the axolotl using DiI is a novel technique for the study of revascularization of regenerating tissue in this animal model, therefore, this protocol can be further used to understand organogenesis during development and angiogenesis during disease as well as be utilized as an important tool during the study of regeneration.
The authors have nothing to disclose.
This research was supported by the Brigham & Women’s Hospital and the March of Dimes. The authors would like to thank all of the members of the Whited Lab for their support and advice.
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 |