Intravenous Microinjection of Nephrotoxins into Zebrafish Embryo: A Technique to Deliver Nephrotoxic Agents into the Bloodstream of Zebrafish Embryo

Overview

This video demonstrates the intravenous microinjection of nephrotoxins into the tail vein of a zebrafish embryo to study their chemical effects on renal function.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Solutions

1. Make a 50x stock solution of E3 embryo media by mixing 73.0 g NaCl, 3.15 g KCl, 9.15 g CaCl₂, and 9.95 g MgSO₄ in 5 L of distilled water, and store at RT.
2. Make a 50x stock solution of E3 embryo media by mixing 73.0 g NaCl, 3.15 g KCl, 9.15 g CaCl₂, and 9.95 g MgSO₄ in 5 L of distilled water, and store at RT.
3. For culturing of zebrafish embryos, dilute the 50x stock solution of E3 embryo media stock to a 1x working solution with distilled water, then add 200 μl of 0.05% methylene blue to every 1 L of 1x E3 to act as a fungicide, and store at RT.
4. Make a pigmentation blocking solution of E3 embryo media with 0.003% 1-phenyl-2-thiourea (PTU) by combining 40 mL of the 50x E3 stock with 0.06 mg PTU. Then bring the volume to a total of 2 L with distilled water.
5. Stir the E3/PTU O/N at RT to get the PTU powder into solution. Then, store the E3/PTU at RT.
   NOTE: The E3/PTU has a shelf life of approximately one week, and older solutions will be less effective at blocking pigmentation development in zebrafish larvae.
6. Make an anesthetic solution of 0.2% Tricaine (MS-222) by adding 1 g of Tricaine to 500 mL of distilled water and adjust the pH to 7.2 with 1 M Tris, pH 9.5.

2. Preparation of Tools

1. Prepare an embryo manipulator tool, used for positioning embryos for microinjection, by attaching a gel loading tip on the end of a 5" straight dissecting needle, and secure with tape or superglue.
2. Prepare microinjection needles using a needle puller, by first placing a fire-polished 10 cm borosilicate glass with filament into the instrument and secure the borosilicate glass by tightening the handles.
3. Pull the borosilicate glass to fashion fine-tapered needles. Use the following settings: heat 540, pull 245, velocity 200, and time 125.
4. Place needles inside a Petri dish, suspending them on a strip of modeling clay to protect the needle tips, and keep the dish covered to prevent dust accumulation.
5. To finish preparing the needle for microinjections, use a razor blade or fine forceps edge to cut the pulled end of the needle to create a sharp angular injection tip with a diameter of approximately 0.05–0.1 mm.
6. To make the microinjection tray, prepare a 1.5% agarose/E3 solution in a 100 mL Erlenmeyer flask and dissolve the agarose by heating the E3 to a boil in a microwave then allow to cool at RT for approximately 10 min.
7. Pour the cooled agarose/E3 solution into a Petri dish to make a foundation with a depth of approximately 0.5 cm. When this has solidified pour a second layer with a depth of approximately 0.3 cm and insert the prefabricated embryo well mold and allow the agarose to solidify at RT.
   NOTE: When inserting the mold into the top agar/E3 layer, placing the mold into the agar at an angle to decrease the number of air bubbles.
8. When the entire microinjection tray has set, lift the prefabricated embryo well mold out slowly and carefully using a scoopula, then fill the dish with E3 solution and store at 4 °C.

3. Microinjection of Nephrotoxin Solution

1. On the day of injection, prepare the desired nephrotoxin solution along with the appropriate vehicle control.
2. Vortex or mix gently to ensure the drug(s) is/are in solution, checking the sides of the tube and top of the water column.
   NOTE: Nephrotoxin solutions can be prepared to contain trace amounts of fluorescently conjugated dextran to monitor microinjection efficiency and also assess renal clearance at subsequent time points.
3. Load a trimmed microinjection needle with ~2–3 µL of the nephrotoxin by threading a fine gel loading tip into the back of the needle and suspend the needle vertically with a piece of tape to allow the solution to fill the trimmed needle tip by gravity.
4. Once the needle tip is full, secure the loaded needle in the micromanipulator.
5. Test the microinjection volume by placing a drop of mineral oil onto a micrometer slide and inject into the oil to evaluate the droplet size. An injection volume of 500 pl has a diameter of 0.1 mm.
6. Remove the dish of embryos from the incubator and anesthetize by adding approximately 5 mL of 0.2% Tricaine to the embryo dish.
7. To ensure complete anesthetization, gently touch embryo with the tip of the embryo manipulator tool. Lack of movement indicates sufficient anesthetization.
8. After successful anesthetization, transfer embryos to the injection mold with a transfer pipette.
9. Maneuver each embryo into a different well of the mold placing the head in the deepest area of the well such that the trunk rests along the depression and the tail sticks up out of the depression.
10. Insert the filled needle into the micromanipulator and position the needle tip next to an embryo.
11. Gently insert the needle into the tail vessel by moving the joystick forward and depress the foot pedal to deliver the microinjection.
    NOTE: Successful injection can be gauged by watching the liquid enter circulation. Further, co-injection of the nephrotoxicant with fluorescently conjugated dextran enables the researcher to verify successful injection subsequent to the procedure.
12. Gently pull back on the joystick to remove the needle from the embryo.
13. After injection, transfer the embryos to a clean dish and rinse to remove the Tricaine and replace with fresh E3/PTU.
14. Incubate the embryo to the desired time point to assess morphology, which can be documented by photography in methylcellulose mounting media, or process for experimental analysis as desired.
    NOTE: Recovery of embryos should be assessed to determine the percentage of individuals with edema, which commonly indicates renal injury.

Materials

Name	Company	Catalog Number	Comments
Sodium Chloride	American Bioanalytical	AB01915
Potassium Chloride	American Bioanalytical	AB01652
Magnesium Sulfate	Sigma-Aldrich	M7506
Calcium Chloride	American Bioanalytical	AB00366
N-Phenylthiourea (PTU)	Aldrich Chemistry	P7629
Ethyl 3-aminobenzoate (Tricaine)	Fluka Analytical	A5040
Borosilicate glass	Sutter Instruments Co.	BF100-50-10
Flaming/Brown Micropipette puller	Sutter Instruments Co.	Mo. P097
UltraPure Agarose	Invitrogen	15510-027
Falcon Diposable Petri Dishes, Sterile, Corning:
60 mm x 15 mm	VWR	25373-085
100 mm x 15 mm	VWR	25373-100
Microinjection tray 150 mm x 15 mm	VWR	25373-187
Low Temperature Incubator	Fischer Scientific	11 690 516DQ
Micro Dissecting Tweezer	Roboz Surgical Instruments Co.	RS-5010
Micrometer	Ted Pella, Inc.	2280-24