We thank Dr Shinji Kanda for demonstration of cardiac perfusion with fixative solution in medaka, Ms Lourdes Carreon G Tan for help with medaka husbandry, and Mr Anthony Peltier for illustrations. This work was funded by NMBU and by the Research Council of Norway, grant number 248828 (Digital Life Norway program).

All animal handling was performed according to the recommendations for the care and welfare of research animals at the Norwegian University of Life Sciences, and under the supervision of authorized investigators.
1. Preparation of Instruments and Solutions
<ol>
	<li>Prepare DiI stock solution dissolving 5 mg of DiI crystal in 1.5 mL plastic tube with 1 mL of 96 % EtOH. Vortex for 30 s and keep covered using aluminum foil. 
	NOTE: The DiI stock solution can be conserved ...

<ol>
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The authors have nothing to disclose

Cardiac perfusion with DiI previously has been used to label blood vessels in several model species24, including teleost fish13.
As DiI is directly delivered to the endothelial cell membrane by perfusion in the vasculature, it is possible to increase the signal-to-noise ratio by increasing the DiI concentration in the fixative solution. In addition, the fluorophore provides intense staining when excited with minimal bleaching allowing ...

Blood vessels play an essential part of the vertebrate body as they provide the necessary nutrients, oxygen and hormonal signals to all organs. Also, since the discovery of their involvement in cancer development1, they have received much attention in clinical research. Although a number of publications have investigated the mechanisms allowing blood vessel growth and morphogenesis, and a large number of genes important for their formation have been identified2, a lot remains to be understood regarding the interaction between cells or tissues and the circulating blood.
Visualization of blood vasculature in the brain and pituitary is important. Neurons in the brain require a high supply of oxygen and glucose3, and the pituitary contains up to eight important hormone-producing cell types that use the blood flow to receive signal from the brain and send their respective hormones to different peripheral organs4,5. While in mammals, the portal system at the base of the hypothalamus named the median eminence, links the brain and the pituitary6, such a clear blood bridge has not been described in teleost fish. Indeed, in teleosts, preoptico-hypothalamic neurons directly project their axons into the pars nervosa of the pituitary7 and mostly innervate the different endocrine cell types directly8,9. However, some of these neurons have their nerve endings located in the extravascular space, in close vicinity to blood capillaries10. Therefore, the difference between teleost fish and mammals is not so clear, and the relationship between the blood vasculature and the brain and pituitary cells requires greater investigation in teleost fish.
Zebrafish has, in many aspects, an anatomically and functionally comparable vascular system to other vertebrate species11. It has become a powerful vertebrate model for cardiovascular research mostly thanks to the development of several transgenic lines where components of the vascular system are labeled with fluorescent reporter proteins12. However, exact circulatory system anatomy may vary between species, or even between two individuals belonging to the same species. Therefore, visualization of blood vessels may be of high interest also in other teleost species for which transgenesis tools do not exist.
Several techniques have been described to label blood vessels in both mammals and teleosts. These include in situ hybridization for vasculature-specific genes, alkaline phosphatase staining, microangiography, and dye injections (for a review see13). Fluorescent lipophilic cationic indocarbocyanine dye (DiI) was first used to study membrane lipids lateral mobility as it is retained in the lipid bilayers and can migrate through it14,15,16. Indeed, a molecule of DiI is composed of two hydrocarbon chains and chromophores. While the hydrocarbon chains integrate in the lipid bilayer cell membrane of the cells in contact with it, the chromophores remain on its surface17. Once in the membrane, DiI molecules diffuse laterally within the lipid bilayer which helps to stain membrane structures that are not in direct contact with the DiI solution. Injecting a DiI solution through cardiac perfusion, will therefore label all endothelial cells in contact with the compound allowing direct labelling of the blood vessels. Today DiI is also used for other staining purposes, such as single molecule imaging, fate mapping, and neuronal tracing. Interestingly, several fluorophores exist (with different wavelengths of emission) allowing the combination with other fluorescent labels, and the incorporation as well as the lateral diffusion of DiI can occur in both live and fixed tissues18,19.
Formaldehyde, discovered by Ferdinand Blum in 1893, has been used widely to the present day as the preferred chemical for tissue fixation20,21. It shows broad specificity for most cellular targets and preserves the cellular structure22,23. It also preserves the fluorescent properties of most fluorophores, and thus can be used to fixate transgenic animals for which targeted cells express fluorescent reporter proteins.
In this manuscript, a previous protocol developed to label blood vessels in small experimental mammalian models24&#160;has been adapted to the use in fish. The entire procedure takes only a couple of hours to perform. It demonstrates how to perfuse a fixative solution of formaldehyde containing DiI in the fish heart in order to directly label all blood vessels in the brain and the pituitary of the model fish medaka. Medaka is a small freshwater fish native to Asia, primarily found in Japan. It is a research model organism with a suite of molecular and genetic tools available25. Therefore, identification of blood vessels in this species as well as in others will allow to improve our understanding on how the brain and pituitary cells interact with blood vasculature in whole tissue or thick tissue slices.

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			<td height="21" style="height:21px;width:432px;">16% paraformaldehyde</td>
			<td style="width:171px;">Electron Microscopy Sciences</td>
			<td style="width:123px;">RT 15711</td>
			<td style="width:152px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">5 mL Syringe PP/PE without needle</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:123px;">Z116866-100EA</td>
			<td style="width:152px;">syringes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:432px;">BD Precisionglide syringe needles</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:123px;">Z118044-100EA</td>
			<td style="width:152px;">needles 18G (1.20*40)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">borosilicate glass 10cm OD1.2mm</td>
			<td style="width:171px;">sutter instrument</td>
			<td style="width:123px;">BF120-94-10</td>
			<td style="width:152px;">glass pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">DiI (1,1&#8242;-Dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindocarbocyanine perchlorate)</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:123px;">D-282</td>
			<td style="width:152px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:432px;">LDPE tube O.D 1.7mm and I.D 1.1mm</td>
			<td style="width:171px;">Portex</td>
			<td style="width:123px;">800/110/340/100</td>
			<td style="width:152px;">canula</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Phosphate Buffer Saline (PBS) solution</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:123px;">D8537-6X500ML</td>
			<td style="width:152px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">pipette puller</td>
			<td style="width:171px;">Narishige</td>
			<td style="width:123px;">PC-10</td>
			<td style="width:152px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">plastic petri dishes</td>
			<td style="width:171px;">VWR</td>
			<td style="width:123px;">391-0442</td>
			<td style="width:152px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Super glue gel</td>
			<td style="width:171px;">loctite</td>
			<td style="width:123px;">c4356</td>
			<td style="width:152px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">tricaine (ms-222)</td>
			<td style="width:171px;">sigma</td>
			<td style="width:123px;">E10521-50G</td>
			<td style="width:152px;"></td>
		</tr>
	</tbody>
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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.

This protocol demonstrates a step by step procedure to label blood vessels in the medaka brain and pituitary, and at the same time fix the tissue. After labelling by cardiac injection of a fixative solution containing DiI into the heart, blood vessels can be observed on slices using a fluorescent stereomicroscope (Figure 4) or on whole tissue using a confocal microscope (Figure 5). Either on the thick tissue slice or on the whole tissue, the architecture of the ...

Watch this Scientific Journal Video about Labeling of Blood Vessels in the Teleost Brain and Pituitary Using Cardiac Perfusion with a DiI-fixative at JoVE.com

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

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 ...

The overall goal of this procedure is to label blood vessels in the brain and pituitary of teleost fish, and we use cardiac perfusion with a fixative containing DiI in the model species medaka. So this technique which is simple and efficient will help answer important questions regarding the vascular anatomy in the brain and pituitary of teleost fish as well as the relationship between neurons or endocrine cells and blood vessels. Some advantages of this technique include a fast and easy simple preparation, and its adaptability to the teleost fish species.To prepare a fish holder, cut a piece of polystyrene to a five centimeter length, a three centimeter width, and a two centimeter thickness, and glue the polystyrene to a nine centimeter diameter plastic dish. Then use a scalpel to make a three centimeter boat shaped hole in the polystyrene. To prepare the perfusion system, add a 30 to 50 centimeter long plastic cannula at the extremity of the perfusion system needle.To prepare five centimeter glass pipettes, use a pipette puller to stretch individual glass capillaries according to the manufacturer's instructions. Before beginning the dissection, load a 10 milliliters syringe with the prepared DiI-fixative solution and place a needle with a cannula at the extremity of the syringe. Then use several pieces of tape placed in different orientations to fix the syringe to the bench, and adjust the position of the microscope and the seat to obtain a good position for dissection and for pressing the syringe piston with the elbow.Next, place a euthanized fish in the fish holder abdomen side up and secure the head and tail with pins. Use scissors to horizontally cut the superficial layer of skin to open the anterior abdomen, and use forceps to remove the skin above the heart until a clear access to the ventricle and the bulbus arteriosus is achieved. Add a glass pipe at the extremity of the capillary and break the tip of the pipette.Bring the glass pipe close to the ventricle and add elbow pressure to the syringe piston to force the liquid out while pinning the heart ventricle with the pipette. Then immediately perforate the sinus venosus with forceps to release the blood from circulation. From the ventricle, adjust the angle of the glass pipette to locate the entrance of the bulbus arteriosus, and move the pipette opening inside the bulbus arteriosus.Then add pressure to the syringe for an additional 30 to 60 seconds while keeping the pipette inside the bulbus arteriosus. For optimal labeling of all the blood vessels it is critical to perfuse into the bulbus arteriosus long enough to replace all the blood with the DiI-fixative solution. After about 60 seconds of perfusion, when no more blood leakage is observed, remove the glass pipette and needles from the fish, and harvest the brain and pituitary into fresh 4%paraformaldehyde for two hours at room temperature in the dark.Then rinse the tissues two times in fresh PBS for 10 minutes per wash before preparing the samples for imaging. After cardiac injection of a fixative solution containing DiI as demonstrated, the labeled blood vessels can be observed on tissue slices by fluorescent stereo microscopy or on whole tissue by confocal microscopy. Note that if the solution is injected in the heart ventricle instead of the bulbus arteriosus or if the solution is injected with too low of a pressure and/or for too short a period, an inadequate labeling of the vessels will be observed.In addition, when imaging the same tissue with the same imaging parameters, a decrease in the labeling intensity can be observed after several days, with the signal appearing more spread-out within the tissue sample. Tissue slices from transgenic lines within which the cells of interest express fluorescent reporter proteins can be further labeled for specific proteins of interest using standard immunofluorescence protocols allowing the investigation of interactions between blood vessels and other cell types. It is important to reach the bulbus arteriosus.When this is achieved, adding pressure to the syringe piston should induce an expansion of the bulbus confirming a successful procedure. This technique can be used to investigate the interaction between endocrine cells and blood vessels in the teleost pituitary in general, but maybe more specifically to investigate possible differences or similarities between the portal systems in teleost and mammals. Because this political use toxic and volatile compounds precautions such as using a gas mask and a ventilated room should always be taken when performing these procedures.

labeling-blood-vessels-teleost-brain-pituitary-using-cardiac

Research

JoVE Journal

Bioengineering

7.4K Görüntüleme.  Norwegian University of Life Sciences. Bu işlemin genel amacı beyin ve teleost balık hipofiz kan damarları etiket etmektir, ve biz model tür medaka DiI içeren bir fiksatif ile kardiyak perfüzyon kullanın. Yani basit ve verimli olan bu teknik, teleost balıklarının beyindeki damar anatomisi ve hipofiz ile nöronlar veya endokrin hücreler ve kan damarları arasındaki ilişki ile ilgili önemli soruların yanıtlanmasına yardımcı olacaktır. Bu tekniğin bazı avantajları hızlı ve kolay basit hazırlık ve teleost b...