A Neonatal Rodent Model of Retroorbital Vein Injection

Retro-orbital injection is a specialized injection method used to administer compounds into the venous circulation. Animal research is an important step towards clinical work. As part of animal research, there is the need to administer different types of compounds via different routes.

Some of these routes include subcutaneous, intraperitoneal, and intravenous. Intravenous injection is the most commonly used route in neonates that are in the neonate intensive care unit. However, in rodent research, particularly in adult rodents, IV injections are routinely performed on the tail vein.

In neonates, it is extremely difficult to perform a tail vein injection successfully, and reproducibly. Therefore, the retro-orbital route can be used as an easier alternative. First, we will demonstrate to using a dye, trypan blue, so that the viewer can clearly see the dye entering the venous circulation.

The neonatal rat is placed under full anesthesia, with its head laying on its side, resting above a light source, so the eye and lateral vasculature is exposed and visible. The needle is inserted at the front of the eye socket, the equivalent of the medial canthus. Inject the solution in a gentle, smooth, and fluid motion.

To better reproduce this procedure, it is important to understand the orbital venous anatomy. In the rat, there is a network of several veins just below the eye, which allow easy and direct access to the venous plexus. In neonatal albino rat, it is also visible both the superficial temporal and facial veins.

This procedure can be performed in the exact same manner. In neonatal mice. Use a needle with 29 to 31 gauge around 0.30 millimeters.

For accurate volumes, draw up the solution to be injected from a pipetted volume. Place the animals on a flat surface in lateral recumbency. Induce full-body Isoflurane anesthesia.

5%induction, 3%maintenance. Check the depth of anesthesia using the pull withdrawal reflex method. With its head facing to the right, administer the injection into the right retro-orbital sinus.

Insert the needle bevel down at the front of the eye socket, the equivalent of the middle canthus, at an angle of approximately 40 degrees. This angle allows the needle to be directed to the back of the eye orbit. Inject in a gentle, smooth, and fluid motion.

Wait for a brief moment before withdrawing the needle, slowly, to avoid back flow. Use a new sterile syringe for each animal to avoid contamination. Place the pup in the recovery box, rested on a protected warming device, around 35 to 37 degrees Celsius.

Wait for the recovery, and check for any signs of distress before returning the pup to the dam. This technique was used to administer the tracer biotin-dextran into the vasculature of the brain of animals that have undergone germinal matrix hemorrhage. Animals that received a saline injection as a control show no visible staining of the vasculature within the brain.

Successful retro-orbital injection of the biotin-dextran tracer, allowed assessment of its presence in the brain vasculature within 10 minutes after retro-orbital administration. This technique was then used to detect the vascular leakage of the tracer at the individual blood vessel level in the GMH injured animals. This result allows, for example, for the quantification of the injury by quantifying the amount of blood vessels that are leaking following GMH brain injury.

Additionally, this procedure can also be performed without a light source, making it a simple, straightforward, and reproducible technique that allows compound administration directly into the venous circulation of neonatal rats and mice. When performed correctly, this procedure should not result in any adverse effects. This root of administration allows injections of antibodies, cells, and other compounds directly into the venal circulation of neonatal animals.

This route is of greater clinical relevance when compared to other routes, such as intraperitoneal or subcutaneous injections.