Intravenous Administration of Recombinant Adeno-Associated Virus Vectors in a Rat Model

Samenvatting

Source: Grames, M. S., et al. Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction. J. Vis. Exp. (2017)

This video demonstrates the tail vein injection of recombinant adeno-associated virus (AAV) in rats. After injection into the lateral tail vein, the virus enters the bloodstream, circulates through the heart, and reaches the brain, where it enters neurons and facilitates target protein production for neurotherapeutic applications.

Protocol

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

1. Prepare the rat

1. Turn the oxygen up to 1 L/min and set the isoflurane anesthesia to 5%. Ensure that the gas anesthesia is flowing to the induction chamber by checking all connecting hoses and stopcock orientations.
2. Place the rat in the gas anesthesia induction chamber for about 3 min until the rat is unresponsive.
3. Ensure adequate depth of anesthesia by pinching the toe. There should be no movement in the foot or leg after the toe pinch.
4. Reduce the isoflurane anesthesia to 2% for maintenance and adjust the stopcock so that the anesthesia flows to the anesthesia mask.
5. Place the rat's nose in the anesthesia mask and adjust the rat so that it is lying on its side.
6. Identify the lateral tail vein.
   NOTE: The lateral tail veins lie at approximately 10 and 2 o'clock, with the dorsal top of the tail at 12 o'clock. With the rat lying on its side, the lateral tail vein should be facing up.
7. Wipe the injection area with the alcohol preparation pad. The injection site is two-thirds down the length of the tail.

2. Inject the vector

1. Firmly hold the tail slightly above the injection area with one finger directly over the lateral tail vein; this will dilate the vein.
2. With the bevel facing up, align the needle with the visible tail vein in the same direction.
3. Pierce the skin over the tail vein while taking great care to avoid the other hand holding the tail and pressing on the tail vein.
4. Release pressure from the tail vein to allow for normal blood flow.
5. Move the needle into the vein. To ensure that the needle has punctured the tail vein, pull back slightly on the plunger. If the needle is in the vein, blood will flow into the needle.
   1. Reposition the needle as needed. Then, to stabilize the needle, move the hand above the injection site to below the injection site and hold the end of the syringe against the tail.
6. Slowly inject the vector into the tail (approximately 20 µL/s).
   NOTE: The vein may lose color as the vector flows through it. Signs that the solution was injected outside the vein include blebbing, blanching of the skin, or resistance when injecting the vector. With practice, extra-vascular exposure can be minimized.
7. After the vector has been administered, remove the needle from the rat and press a gauze pad against the injection site to help stem the bleeding for 30 - 60 seconds.

3. Clean-up

1. Turn off the isoflurane anesthesia and oxygen. Monitor the rat until it regains consciousness, then return it to its cage.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
1-mL syringe	BD	309626	Comes with a 25g needle attached. Replace with 27-30g needle for injection
30g needle	BD	305106
Isoflurane	Piramal Healthcare	66794-013-25	Isoflurane USP 250 mL
Anesthesia mask	Kopf Instruments	906
Gauze	Henry Schein	100-2524	2" x 2" (5 cm x 5 cm)
Gas anesthesia vaporizer	SurgiVet	4214322	Classic T3
Gas anesthesia scavenger	SurgiVet	32373B10	Enviro-PURE
Heating pad	Sears	17280
70% Ethanol	Decon Laboratories, Inc.	2401	140 proof
70% Isopropanol	VWR	89108-160