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

Summary

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.

Abstract

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.

Introduction

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 organs^4,5. 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 directly^8,9. 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 it^14,15,16. 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 tissues^18,19.

Formaldehyde, discovered by Ferdinand Blum in 1893, has been used widely to the present day as the preferred chemical for tissue fixation^20,21. It shows broad specificity for most cellular targets and preserves the cellular structure^22,23. 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 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.

Protocol

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

1. 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 in the dark at -20 °C for several months.
2. Prepare the fish holder (Figure 1A) by cutting a piece of polystyrene to 5 cm length, 3 cm width, and 2 cm thickness, and gluing it to a 9 cm-diameter plastic dish. Make a 3 cm boat-shaped hole with a scalpel blade in the polystyrene.
3. Prepare the perfusing system (Figure 2) by adding a 30-50 cm long plastic cannula at the extremity of the needle.
4. Prepare 40 mL of fresh 4% paraformaldehyde solution (PFA) by diluting 10 mL of 16% PFA with 30 mL of phosphate buffered saline solution (PBS) in a 50 mL plastic tube.
5. Prepare 10 mL of fixative/DiI solution by diluting 1 mL of DiI stock solution in 9 mL of freshly prepared 4% PFA in a 10 mL plastic tube. Keep in the dark until use.
   NOTE: The DiI crystals that are not dissolved can be kept in the stock solution tube, and new ethanol can be added to prepare new DiI stock solution (see step 1.1).
6. Prepare 50 mL of tricaine (MS-222) stock.
   1. Dissolve 200 mg of Tricaine powder in 48 mL of H₂O. Add 2 mL of 1 M Tris base (pH 9). Adjust to pH 7 with 1 M HCl and store at -20 °C.
7. Dilute 5 mL of Tricaine stock in 50 mL of clean water in a small glass.
8. Prepare several 5 cm glass pipettes (Figure 2) by stretching a glass capillary with a pipette puller following the manufacturer's instructions.

2. Dissection and Perfusion

NOTE: PFA is a toxic volatile compound, therefore the dissection and perfusion should be performed in a hood or in a ventilated room, and the user must be wearing a gas mask.

1. Prepare the dissection tools including small scissors, and one sharp and one strong forceps before dissection.
2. Fill the syringe with the prepared solution of PFA/DiI by placing it in the 50 mL tube and drawing up. Then place the needle with the cannula at the extremity of the syringe (Figure 2).
3. Fix the syringe to the bench using several pieces of tape placed in different directions, adjust the position of the microscope and the seat to obtain a good position for dissection and for pressing the syringe piston (Figure 1B).
   NOTE: In this protocol, the elbow is used to press down the syringe piston, but other possibilities options may be used, e.g., assistance from another person.
4. Euthanize the fish with an overdose of tricaine by placing the fish in the solution prepared in step 1.7. Wait 30 s and test the reflexes of the fish by grabbing its caudal fin with the forceps. The fish can be used when it has stopped responding to the stimuli.
5. Place the fish in the fish holder with its abdomen facing up and pin one needle into the extremity of the head and another one above the tail to keep the fish in place.
6. Open the anterior abdomen with the scissors by horizontally cutting the superficial layer of skin.
7. Using forceps, remove the skin above the heart until a clear access to the ventricle and the bulbus arteriosus is provided (Figure 3).
8. Add a glass pipette at the extremity of the capillary and break the end of the tip of the glass pipette.
   NOTE: The hole of the broken tip should be big enough to let the liquid out, but still small enough to easily enter the tissue. The glass pipette can be reused if not broken or clogged.
9. Bring the glass pipette close to the ventricle and add pressure to the syringe piston with the elbow to force the liquid out.
10. Pin the heart ventricle with the glass pipette while adding pressure to the syringe.
11. Directly after, perforate the sinus venosus with forceps to enable blood to leave the circulation.
    NOTE: The heart ventricle becomes more fragile because of the fixative. It also becomes less red as the blood is diluted with the fixative/DiI solution.
12. From the ventricle, adjust the angle of the glass pipette to find the entrance of the bulbus arteriosus. Bring the pipette opening inside the bulbus arteriosus as shown in Figure 2, and add more pressure to the syringe.
    NOTE: The bulbus arteriosus is transparent and the tissue is elastic. When replacing blood with DiI/fixative, the glass pipette inside the heart should become more visible and by adding pressure, the size of the bulbus arteriosus should expand. This step is crucial to make sure that enough pressure has been used to replace all the blood with the fixative/DiI solution, and that all blood vessels have been reached. Often when successful, the PFA provokes muscle contractions and the fish will be moving.
13. Continue adding pressure on the syringe for 30-60 s still keeping the glass pipette inside the bulbus arteriosus.
14. Remove the glass pipette and the needles from the fish.
15. Dissect the brain and the pituitary, and incubate tissues in fresh 4% PFA in PBS for 2 h in the dark at room temperature.
    NOTE: Dissection of brain and pituitary previously has been described in detail in two different ways by Fontaine et al.²⁶ and Ager-Wick et al.²⁷. Because of the fixation, the tissue becomes quite hard and the fragile link between the brain and the pituitary may be broken. After dissection, post fixation processing can also be performed overnight at 4 °C.
16. Rinse the tissue twice in PBS for 10 min before preparing for imaging.
    NOTE: The tissue can then be mounted between slide and cover slip with spacers in between and mounting medium for confocal imaging (see Figure 4), or sectioned with a vibratome as described in detail in Fontaine et al.²⁶ before mounting between slide and cover slip for imaging with a stereomicroscope (see Figure 4).

Results

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 ...

Discussion

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 ...

Materials

Name	Company	Catalog Number	Comments
16% paraformaldehyde	Electron Microscopy Sciences	RT 15711
5 mL Syringe PP/PE without needle	Sigma	Z116866-100EA	syringes
BD Precisionglide syringe needles	Sigma	Z118044-100EA	needles 18G (1.20*40)
borosilicate glass 10cm OD1.2mm	sutter instrument	BF120-94-10	glass pipette
DiI (1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)	Invitrogen	D-282
LDPE tube O.D 1.7mm and I.D 1.1mm	Portex	800/110/340/100	canula
Phosphate Buffer Saline (PBS) solution	Sigma	D8537-6X500ML
pipette puller	Narishige	PC-10
plastic petri dishes	VWR	391-0442
Super glue gel	loctite	c4356
tricaine (ms-222)	sigma	E10521-50G