A New Best Practice for Validating Tail Vein Injections in Rat with Near-infrared-Labeled Agents

This method of validating and assessing the injection accuracy provides an important quality control step and a crucial stage in animal studies for facilitating consistent and reproducible tail vein injections. Assessing the injection via fluorescence imaging immediately after it's performed offers the advantage of determining whether your agent has entered the blood circulation in its intended quantity. Tail vein injection is a hands-on manipulation that benefits from visual demonstration and that can be enhanced by observing the clearance of the injected agent from the injection site.

Demonstrating the procedure with Muzamil Saleem will be Brooke Deal, a PhD student from my laboratory. Prior to tail vein injection, position an anesthetized animal laterally in a near-infrared fluorescence imager with the injection site on the tail exposed to establish a fluorescence baseline. After the image has been acquired, transfer the animal to the surgical bench in the prone position and dilate the tail vasculature by submerging it in warm water.

After approximately one minute, orient the tail so the lateral side is in view. The lateral tail vein should appear dark in color. Sterilize the length of the vein with alcohol pads.

Position the tail laterally and insert a 25 to 27-gauge needle bevel side up and parallel to the lifted tail into the selected lateral tail vein starting at the distal coccygeal vertebrae region of the tail and moving more proximal if proper needle placement fails as necessary. In a successful needle insertion, blood flashback should be observed in the rim of the needle. If no flashback is apparent, slowly move the needle tip without removing it from the tail to find vein insertion.

When the needle has been properly placed, insert a syringe with the injectable material into the rim of the needle and depress the plunger to deliver the contents into the vein. Alternatively, a catheter with a blood flow indicator can be used. A prompt flashback in the indicator segment of the catheter will be observed following correct placement.

Slight back pressure can then be used to pull blood into the syringe to confirm proper placement in the vessel before injecting. Again, no resistance will be felt. Once all the material has been delivered, remove the needle and apply pressure with a sterile gauze for at least one minute to ensure clotting.

Mark the site of injection with a pen to ensure that the injection site will be visible on the white light channel of the acquired image. To assess the post-injection fluorescence signal at the site of injection, return the animal to the near-infrared imaging stage and orient the animal on its lateral side to expose the injection site on the lateral tail for imaging as demonstrated. To analyze the baseline and post-injection injection site fluorescence, regions of interest can be drawn in the near-infrared imager software on the tail near the injection site before and around the area of fluorescence after the tail vein injection.

Successfully-delivered injections will demonstrate nearly equally-low levels of fluorescent signal before and after the injection. Images in which the fluorescence is visible throughout the length of the tail are not acceptable and should be removed from the analysis. Any behavioral, surgical, or ex vivo molecular techniques can be performed as normal following this procedure.

This technique provides quality control for a tail vein injection, revealing the fast clearance by venous blood flow of the properly-injected agent, or pooling at the site of injection if the vein is missed. The reagent used in this method are not hazardous, however, the appropriate precautions should be taken if the fluorescence-based agent is incorporated with a hazardous material.