DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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^1,2. 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 neurons^5,6. 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 adults^8,9. 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.

Protokół

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 no reflexive movement when the foot is gently squeezed.
   Caution: Though tricaine is an anesthetic specifically used for aquatic organisms, direct skin contact with the tricaine solution should be avoided.
2. Set up the axolotl perfusion station.
   1. Place the absorbent pad on a flat, level surface with the absorbent side facing up.
   2. Cut a hole in the polystyrene foam frame that is the appropriate size and shape for the anesthetized axolotl to lay in the supine position. Place the frame on the absorbent pad.
      Note: Some extra paper towels can be placed immediately under the frame for additional absorbency.
   3. Load the peristaltic pump with the perfusion tubing. Set the pump to a flow rate of 0.7 ml/min, flowing in the clockwise direction.
   4. Make the dilutent solution with 0.7x PBS and 5% glucose in a 1:4 mixture.
   5. Mix 10 mL of diluent solution with 200 µL of the DiI stock solution in a 50 mL conical tube. Cap and mix by inversion. Cover this tube with aluminum foil paper to protect the working solution from exposure to light.
      Note: Volumes should be changed according to the size of the axolotl proportionally. These values are for an approximately 15 cm axolotl (snout-to-tail length). Animals of this size may not have reached full sexual maturity so animal sex may not be determined at this time.
   6. Fill a 50 mL conical tube with 0.7x PBS.
      Note: PBS will be used for priming the loop and axolotl exsanguination.
   7. Attach the 27-gauge butterfly needle to the exit end of the perfusion tubing. Fold the butterfly wings onto each other and place in the clamp stand.
   8. Place the free end of the perfusion tubing in the 50 mL conical tube filled with 0.7x PBS and run the perfusion pump until the entire tubing is filled with solution. Pause the pump once the entire tubing is filled with PBS.
      Note: Be sure the tubing is free of air bubbles at all times, as these will cause air emboli in the axolotl and prevent full perfusion.
   9. Place a paper towel in the axolotl-shaped mold in the polystyrene foam frame. Using a transfer pipette, soak the towel with tricaine solution.
      Note: Cut a small square in the middle of the paper towel to allow for the drainage of fluids during the perfusion procedure.
   10. Place the anesthetized axolotl supine on the paper towel inside the polystyrene foam frame.

2. Opening the Axolotl Chest

1. Use surgical forceps to pinch the skin along the central axis of the axolotl's chest, just below the line of the shoulders. Pull up.
2. Use a scalpel to make a small incision where the skin has been pulled.
3. Remove a square patch of skin over the chest in order to reveal two cartilage plates.
   1. Remove the skin to open a window over the thoracic cavity large enough to clearly see the heart and approximately 5 mm of the aorta branching off of the heart.
4. Carefully tear the connective tissue using forceps or the closed scissors in order to avoid cutting any major blood vessels.
5. Lift each cartilage plate individually using the forceps and excise them with the surgical scissors.
6. Carefully pinch the pericardium with the forceps, pull up, and puncture it using the surgical scissors; this incision should be just deep enough to puncture the very thin pericardium and should be large enough to allow for removal of the pericardium. Take care not to cut the heart.
7. Delicately remove the pericardium to expose the heart and the aorta.
   Note: Using a transfer pipette, periodically flush the chest cavity and the gills with tricaine solution to keep the area clear and keep the axolotl anesthetized.

3. Perfusion of the Axolotl

1. Place the clamp stand with the loaded butterfly needle next to the polystyrene foam frame, such that the arm of the clamp can easily be manipulated to insert the needle into the axolotl aorta. Point the needle's tip toward the rostral aspect of the animal during insertion and keep the needle parallel to the aorta to avoid puncturing it through the opposite side.
2. Turn on the peristaltic pump. 0.7x PBS should continue to flow through tubing.
3. Insert the needle into the aorta.
   1. Slide the forceps under the aortic arch and pull up slightly to allow for easy access.
   2. Maneuver the needle-clamp combination such that the needle runs along the length of the aorta, pointing up towards the head. Insert the needle while using the forceps for support behind the aorta.
      Note: The needle should be inserted deep enough into the aorta to ensure that it will not slip out during perfusion. This may be about 5 mm for a 15 cm axolotl. Ensure that the needle is perfectly in line with the aorta to avoid complete puncture of the vessel. Through-and-through punctures can cause massive hemorrhage and decrease success rates of perfusion. Successful insertion can be confirmed by visible enlargement of the atria of the heart.
4. Quickly lacerate one atrium with the scissors and allow blood to drain.
   1. Flush with tricaine solution to prevent blood accumulation and clot formation in the chest cavity.
5. Perfuse the axolotl with about 20-30 mL of PBS. The animal should change from light pink in color to white in a successful perfusion.
6. Pause the peristaltic pump and move the free end of the tubing into the 15 mL tube of DiI solution. Restart the pump, take care to avoid creating any air bubbles in the tubing.
7. Perfuse the axolotl with the entire working stock of DiI.
   Note: In a successful perfusion, the axolotl with change color to the bright pink of the DiI. This will be most noticeable in the gills.
8. Pause the pump after the perfusion with DiI is complete and place the free end of the tubing into 4% Paraformaldehyde (PFA) solution to fix the tissue. Restart the pump and perfuse at least 10 mL of PFA.
   Caution: PFA is toxic and should be handled and disposed of appropriately. Gloves and safety glasses should be worn, and solutions should be made inside a fume hood. Perfusion of the axolotl with PFA to fix the tissue results in the death of the animal.

4. Ending the Perfusion and Visualization Preparation

1. Stop the peristaltic pump and remove the needle from the axolotl aorta.
2. Place the axolotl on a plastic plate.
   Note: Using one half of a large Petri dish works well and allows for pouring a small amount of Tricaine or PBS onto the axolotl to keep its skin wet and improve visualization quality.
3. Dispose of all used materials in the appropriate waste bins. Clean surgical tools with 70% ethanol, disinfect using a glass-bead sterilizer between animals, and sterilize by autoclaving following the procedure. Flush tubing with the PBS solution and then drain, dry fully, and store for further use.

5. Visualization the Perfused Axolotl

1. Place the axolotl under a fluorescent confocal microscope.
2. Turn the lights off as visualization of the DiI-stained vessels is impeded by light.
3. Use a green fluorescence emission filter cube (e.g. ET-CY3) with the confocal microscope to visualize the vasculature of the axolotl. Use excitation light of wavelength 545 nm.
   Note: To obtain an image of high quality, the following parameters can be used: exposure for 1.1 s, gain of 1x, saturation of 1.0, magnification of 2X.

Wyniki

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...

Dyskusje

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...

Materiały

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