Name: dii perfusion as a method for vascular visualization in ambystoma mexicanum
Uploaded: 2017-06-16T12:30:00.000+00:00
Duration: 6 min 5 s
Description: Watch this Scientific Journal Video about DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum at JoVE.com

This research was supported by the Brigham &#38; Women&#8217;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.

All axolotl experimentation was performed in accordance with Brigham and Women&#39;s Hospital&#39;s (BWH) Institutional Animal Care and Use Committee.
1. Set up Perfusion Experiment
<ol>
	<li>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.</li>
	<li>Set up the axolotl perfusion station.
	<ol>
		<li>Place the absorbent pad on a flat, level surface with the absorbent side facing up.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Make the dilutent solution with 0.7x PBS and 5% glucose in a 1:4 mixture.</li>
		<li>Mix 10 mL of diluent solution with 200 &#181;L 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.</li>
		<li>Fill a 50 mL conical tube with 0.7x PBS. 
		Note: PBS will be used for priming the loop and axolotl exsanguination.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Place the anesthetized axolotl supine on the paper towel inside the polystyrene foam frame.</li>
	</ol>
	</li>
</ol>
2. Opening the Axolotl Chest
<ol>
	<li>Use surgical forceps to pinch the skin along the central axis of the axolotl&#39;s chest, just below the line of the shoulders. Pull up.</li>
	<li>Use a scalpel to make a small incision where the skin has been pulled.</li>
	<li>Remove a square patch of skin over the chest in order to reveal two cartilage plates.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Carefully tear the connective tissue using forceps or the closed scissors in order to avoid cutting any major blood vessels.</li>
	<li>Lift each cartilage plate individually using the forceps and excise them with the surgical scissors.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Perfusion of the Axolotl
<ol>
	<li>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&#39;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.</li>
	<li>Turn on the peristaltic pump. 0.7x PBS should continue to flow through tubing.</li>
	<li>Insert the needle into the aorta.
	<ol>
		<li>Slide the forceps under the aortic arch and pull up slightly to allow for easy access.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Quickly lacerate one atrium with the scissors and allow blood to drain.
	<ol>
		<li>Flush with tricaine solution to prevent blood accumulation and clot formation in the chest cavity.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Ending the Perfusion and Visualization Preparation
<ol>
	<li>Stop the peristaltic pump and remove the needle from the axolotl aorta.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
5. Visualization the Perfused Axolotl
<ol>
	<li>Place the axolotl under a fluorescent confocal microscope.</li>
	<li>Turn the lights off as visualization of the DiI-stained vessels is impeded by light.</li>
	<li>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.</li>
</ol>

<ol>
	<li>Giuvarasteanu, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+electron+microscopy+of+vascular+corrosion+casts+-+standard+method+for+studying+microvessels.">Scanning electron microscopy of vascular corrosion casts - standard method for studying microvessels.</a> Rom J Morphol Embryo. 48 (3), 257-261 (2007).</li><li>Hasan, M. R., Herz, J., Hermann, D. M., Doeppner, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+perfusion+of+carbon+black+ink+allows+reliable+visualization+of+cerebral+vessels.">Intravascular perfusion of carbon black ink allows reliable visualization of cerebral vessels.</a> J Vis Exp. (71), e4374 (2013).</li><li> Minnich, B., Lametschwandtner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+Scanning+electron+microscopy+and+vascular+corrosion+casting+for+the+characterization+of+microvascular+networks+in+human+and+animal+tissues."> Scanning electron microscopy and vascular corrosion casting for the characterization of microvascular networks in human and animal tissues.</a> Microscopy: Science, Technology, Applications, and Education. 1, 29-39 (2010).</li><li> Honig, M., Hume, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+DiI+and+DiO:+versatile+fluorescent+dyes+for+neuronal+labelling+and+pathway+tracing."> DiI and DiO: versatile fluorescent dyes for neuronal labelling and pathway tracing.</a> Trends Neurosci. 13, 333-335 (1989).</li><li>Honig, M. G., Hume, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+carbocyanine+dyes+allow+living+neurons+of+identified+origin+to+be+studied+in+long-term+cultures.">Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures.</a> J Cell Biol. 103 (1), 171-187 (1986).</li><li>Schwartz, M., Agranoff, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outgrowth+and+maintenance+of+neurites+from+cultured+goldfish+retinal+ganglion+cells.">Outgrowth and maintenance of neurites from cultured goldfish retinal ganglion cells.</a> Brain Res. 206 (2), 331-343 (1981).</li><li>Li, Y., Song, Y., Zhao, L., Gaidosh, G., Laties, A. M., Wen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+labeling+and+visualization+of+blood+vessels+with+lipophilic+carbocyanine+dye+DiI.">Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI.</a> Nat Protoc. 3 (11), 1703-1708 (2008).</li><li>Kuo, T. H., Kowalko, J. E., DiTommaso, T., Nyambi, M., Montoro, D. T., Essner, J. J., Whited, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+TALEN-mediated+gene+editing+of+an+endogenous+locus+in+axolotl.">Evidence of TALEN-mediated gene editing of an endogenous locus in axolotl.</a> Regeneration. 2 (1), 37-43 (2015).</li><li>Brockes, J. P., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appendage+Regeneration+in+Adult+Vertebrates+and+Implications+for+Regenerative+Medicine.">Appendage Regeneration in Adult Vertebrates and Implications for Regenerative Medicine.</a> Science. 310 (5756), 1919-1923 (2005).</li><li>Smith, A. R., Wolpert, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerves+and+angiogenesis+in+amphibian+limb+regeneration.">Nerves and angiogenesis in amphibian limb regeneration.</a> Nature. 257 (5523), 224-225 (1975).</li></ol>

The authors have nothing to disclose.

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&#8217;s blood is effectively drained and replaced with 0.7x PBS, followed immediately by DiI solution, and finally 4% PFA. This disrupts the axolotl&#8217;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.

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.

<table><tbody><tr><td>Peristaltic Pump&amp;nbsp;</td><td>Marshall Scientific&amp;nbsp;</td><td>RD-RP1</td><td></td></tr><tr><td>Perfusion tubing</td><td>Excelon Lab &amp;amp; Vacuum Tubing</td><td>436901705</td><td>size S1A</td></tr><tr><td>27g butterfly needle</td><td>EXELint Medical Products</td><td>26709</td><td></td></tr><tr><td>NaCl</td><td>AmericanBio</td><td>7647-14-5</td><td></td></tr><tr><td>KCl</td><td>AmericanBio</td><td>7747-40-7</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>AmericanBio</td><td>7558-79-4</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>AmericanBio</td><td>10049-21-5</td><td></td></tr><tr><td>Distilled water</td><td></td><td></td><td></td></tr><tr><td>HCl</td><td>AmericanBio</td><td>7647-01-0</td><td></td></tr><tr><td>Glucose</td><td>ThermoFischer</td><td>A2494001</td><td></td></tr><tr><td>1,1&amp;prime;-Dioctadecyl-3,3,3&amp;prime;,3&amp;prime;-tetramethylindocarbocyanine perchlorate</td><td>Sigma Aldrich</td><td>468495</td><td></td></tr><tr><td>Ethanol (100% vol/vol)</td><td>Sigma Aldrich</td><td>64-17-5</td><td></td></tr><tr><td>Surgical foreceps&amp;nbsp;</td><td>Medline</td><td>MDG0748741</td><td></td></tr><tr><td>Polystyrene foam frame</td><td></td><td></td><td>any polystyrene foam square with an axolotl-shaped&amp;nbsp; cut out</td></tr><tr><td>Surgical scissors</td><td>Medline</td><td>DYND04025</td><td></td></tr><tr><td>Scalpel&amp;nbsp;</td><td>Medline</td><td>MDS15210</td><td></td></tr><tr><td>Absorbent underpad</td><td>Avacare Medical</td><td>PKUFSx</td><td></td></tr><tr><td>Paper towels</td><td></td><td></td><td></td></tr><tr><td>Standard disposable transfer pipette</td><td>Fisherbrand</td><td>50216954</td><td></td></tr><tr><td>Clamp stand</td><td>Adafruit</td><td>291</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate</td><td>Sigma Aldrich</td><td>E10521</td><td>Tricaine powder</td></tr><tr><td>Adult axolotl</td><td></td><td></td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>AmericanBio</td><td>10034-99-8</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>C1016-100G</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>S5761-500G</td><td></td></tr><tr><td>Plastic tanks</td><td></td><td></td><td>Varying size appropriate for the axolotl</td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>30525-89-4</td><td></td></tr><tr><td>Axolotl</td><td></td><td></td><td></td></tr><tr><td>Leica Microscope</td><td>Leica</td><td>M165 FC</td><td></td></tr><tr><td>ET-CY3 Fluorescent Filter</td><td>Leica</td><td>M205FA/M165FC</td><td></td></tr><tr><td>MS-222</td><td></td><td></td><td></td></tr></tbody></table>

dii perfusion as a method for vascular visualization in ambystoma mexicanum

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&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-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&#8217;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.

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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55740/55740fig1.jpg" /> 
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. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55740/55740fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55740/55740fig2.jpg" /> 
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. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55740/55740fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum at JoVE.com

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

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JoVE Journal

Biology

9.9K Views.  Harvard Medical School and Brigham and Women's Hospital. 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.

Video: DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult forms has revealed the genetic basis of some of these differences. Less is known about how the populations differ at post-larval stages (at the onset of feeding). Such studies may provide insight into how cavefish survive through adulthood in their natural environment. Methods for comparing post-larval development in the laboratory require standardized aquaculture and feeding regimes. Here we describe how to raise fish on a diet of nutrient-rich rotifers in non-recirculating water for up to two-weeks post fertilization. We demonstrate how to collect post-larval fish from this nursery system and perform whole-mount immunostaining. Immunostaining is an attractive alternative to transgene expression analysis for investigating development and gene function in A. mexicanus. The nursery method can also be used as a standard protocol for establishing density-matched populations for growth into adults.

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

Genetics

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Developmental Biology

Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The cerebral vasculature dynamically responds to various pathophysiological states and it exhibits considerable differences between strains and under conditions of genetic manipulations. Essentially, a reliable technique for intracranial vessel staining is essential in order to study the pathogenesis of ischemic stroke. Until recently, a set of different techniques has been employed to visualize the cerebral vasculature including injection of low viscosity resin, araldite F, gelatin mixed with various dyes1 (i.e. carmine red, India ink) or latex with2 or without3 carbon black. Perfusion of white latex compound through the ascending aorta has been first reported by Coyle and Jokelainen3. Maeda et al.2 have modified the protocol by adding carbon black ink to the latex compound for improved contrast visualization of the vessels after saline perfusion of the brain. However, inefficient perfusion and inadequate filling of the vessels are frequently experienced due to high viscosity of the latex compound4. Therefore, we have described a simple and cost-effective technique using a mixture of two commercially available carbon black inks (CB1 and CB2) to visualize the cerebral vasculature in a reproducible manner5. We have shown that perfusion with CB1+CB2 in mice results in staining of significantly smaller cerebral vessels at a higher density in comparison to latex perfusion5. Here, we describe our protocol to identify the anastomotic points between the anterior (ACA) and middle cerebral arteries (MCA) to study vessel variations in mice with different genetic backgrounds. Finally, we demonstrate the feasibility of our technique in a transient focal cerebral ischemia model in mice by combining CB1+CB2-mediated vessel staining with TTC staining in various degrees of ischemic injuries.

Analysis of rodent cerebrovascular anatomy plays an important role in experimental stroke research. In this context, intravascular perfusion with colored latex has been considered as a standard tool for several years. However, this technique implies distinct technical limitations, which undermine its reproducibility. Here, we describe a simple method to visualize cerebral vessels in a reproducible manner. Injection of a mixture of two commercially available carbon black inks through the left myocardial ventricle results in adequate filling of cerebral vessels with high contrast visualization. We have successfully applied this technique to identify anastomotic points between cerebral vascular territories of mice with different genetic backgrounds. We finally give evidence that this novel and simple method for vessel staining can be combined with triphenyltetrazolium chloride (TTC) staining - a widely used tool to observe and analyze infarct volumes in mice.

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The ...

Neuroscience

Preparing a Microfluidics-Based Vascularized Human Micro-Tumor Model

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

Author Spotlight: Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

Cancer Research

Labeling of Blood Vessels in the Teleost Brain and Pituitary Using Cardiac Perfusion with a DiI-fixative

Blood vessels innervate all tissues in vertebrates, enabling their survival by providing the necessary nutrients, oxygen, and hormonal signals. It is one of the first organs to start functioning during development. Mechanisms of blood vessel formation have become a subject of high scientific and clinical interest. In adults however, it is difficult to visualize the vasculature in most living animals due to their localization deep within other tissues. Nevertheless, visualization of blood vessels remains important for several studies such as endocrinology and neurobiology. While several transgenic lines have been developed in zebrafish, with blood vessels directly visualized through expression of fluorescent proteins, no such tools exist for other teleost species. Using medaka (Oryzias latipes) as a model, the current protocol presents a quick and direct technique to label blood vessels in brain and pituitary by perfusing through the heart with fixative containing DiI. This protocol allows improvement of our understanding on how brain and pituitary cells interact with blood vasculature in whole tissue or thick tissue slices.

The article describes a quick protocol for labeling blood vessels in a teleost fish by cardiac perfusion of DiI diluted in fixative, using medaka (Oryzias latipes) as a model and focusing on brain and pituitary tissue.

Blood vessels innervate all tissues in vertebrates, enabling their survival by providing the necessary nutrients, oxygen, and hormonal signals. It is one ...

Bioengineering

2D and 3D Echocardiography in the Axolotl (Ambystoma Mexicanum)

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model species such as zebrafish and salamanders that are capable of intrinsic heart regeneration hold promise for future regenerative therapies for human patients. To evaluate the outcome of cardioregenerative experiments it is imperative that heart function can be monitored. The axolotl salamander (A. mexicanum) represents a well-established model species in regenerative biology attaining sizes that allows for evaluation of cardiac function. The purpose of this protocol is to establish methods to reproducibly measure cardiac function in the axolotl using echocardiography. The application of different anesthetics (benzocaine, MS-222, and propofol) is demonstrated, and the acquisition of two-dimensional (2D) echocardiographic data in both anesthetized and unanesthetized axolotls is described. 2D echocardiography of the three-dimensional (3D) heart can suffer from imprecision and subjectivity of measurements, and to alleviate this phenomenon a solid method, namely intra/inter-operator/observer analysis, to measure and minimize this bias is demonstrated. Finally, a method to acquire 3D echocardiographic data of the beating axolotl heart at a very high spatiotemporal resolution and with pronounced blood-to-tissue contrast is described. Overall, this protocol should provide the necessary methods to evaluate cardiac function and model anatomy, and flow dynamics in the axolotl using ultrasound imaging with applications in both regenerative biology and general physiological experiments.

2D and 3D Echocardiography in the Axolotl Ambystoma Mexicanum

Here we present echocardiography protocols for two-dimensional and three-dimensional image acquisition of the beating heart of the axolotl salamander (Ambystoma mexicanum), a model species in heart regeneration. These methods allow for longitudinal evaluation of cardiac function at a high spatiotemporal resolution.

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model ...

Medicine

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

Blood Perfusion in African Clawed Frogs for Transcriptomics and Proteomics

Effective Rapid Blood Perfusion in Xenopus

Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol in Xenopus, aimed at a consistent and drastic reduction of blood within all tissues, is defined. Perfusion is carried out by inserting a needle directly into the ventricle of the heart and pumping heparinized phosphate-buffered saline (PBS) through the vascular system. The procedure can be completed in approximately 10 min per animal. The blood is dominated by a few highly abundant proteins and cell types, creating numerous issues as these proteins mask most other molecules and cell types of interest. The reproducible characterization of adult Xenopus tissues with quantitative proteomics and single-cell transcriptomics will benefit from applying this protocol prior to organ sampling. The protocols for tissue sampling are defined in companion papers. These procedures are aimed at the standardization of practices across Xenopus of different sex, age, and health status, specifically X. laevis and X. tropicalis.

Author Spotlight: Effective Rapid Blood Perfusion in Xenopus  

Presented here is an effective rapid blood perfusion protocol to prepare tissue samples from African clawed frogs for transcriptomics and proteomics studies.

Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol ...

Assessing Vessel Perfusion via Angiography: A Technique to Introduce Contrast Agent in Descending Aorta to Visualize Blood Vessel Perfusion in Rabbit Model

Source: Sligar, A. D et al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/58964">Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits.</a> J. Vis. Exp. (2019).
This video describes the technique for performing angiography by injecting a contrast agent into the descending aorta to visualize blood vessel perfusion in a rabbit model. This technique helps to understand the perfusion in blood vessels with blockages and after treatment during recovery.

Source: Sligar, A. D et al., Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits. J. Vis. Exp. (2019).
This video describes the technique for ...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Summary

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Chapters in this video

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

Labeling of Blood Vessels in the Teleost Brain and Pituitary Using Cardiac Perfusion with a DiI-fixative

2D and 3D Echocardiography in the Axolotl (Ambystoma Mexicanum)

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

Effective Rapid Blood Perfusion in Xenopus

Assessing Vessel Perfusion via Angiography: A Technique to Introduce Contrast Agent in Descending Aorta to Visualize Blood Vessel Perfusion in Rabbit Model