1. Intracardiac injection

1. Landmarks and positioning: Position the mouse or rat either in right lateral recumbency (with the left side facing upward) or in dorsal recumbency, and identify the landmarks.
   1. Position the heart approximately level with the point of the elbow and just to the left of the sternum.
   2. Insert the needle between the ribs at the point of the elbow.
   3. In an animal in dorsal recumbency, insert the needle into the chest parallel to the table.
   4. In an animal in lateral recumbency, insert the needle into the chest perpendicular to the table.
2. Injection of the article
   1. Ensure there is minimal movement of the needle once it is inserted into the heart to prevent the muscle from being torn and causing bleeding into the pericardial sac, as this compromises cardiac function.
   2. Aspirate the syringe to determine proper placement. There is often a flash of blood into the hub of the syringe.
   3. Hold the syringe so that the hand is not repositioned on the syringe for injection once the needle has been positioned in the heart.
   4. Inject the article in a slow and steady manner.
   5. Withdraw the needle slowly to minimize bleeding.
   6. Mice that experience respiratory distress, seizure, cyanosis, prolonged ataxia, or other sequelae related to injection trauma must be evaluated by the veterinarian staff and euthanized according to their recommendation.

Figure 1. Intracardiac injection in mice.

2. Intravenous injection utilizing the retro-orbital plexus

1. Injection landmarks and positioning
   1. Place the animal on a flat surface in lateral recumbency.
   2. Insert the needle into the medial canthus of the eye at a 45° angle to the nose.
   3. The needle depth must be sufficient to penetrate the conjunctival tissues and advance behind the ocular orbit and into the ocular plexus. When placing the needle, it should not encounter the bone at the back of the orbit.
   4. Place the index finger on the top of the head with the thumb on the jaw.
   5. Gently pull back and downward to tighten the skin and protrude the eyeball.
   6. Take care not to apply pressure on the trachea and restrict air flow.
2. Injection
   1. Place the needle behind the eye and not intra-ocularly.
   2. Ensure that the needle has minimal movement once inserted into the retro-orbital plexus, or the vessels will rupture causing bleeding and loss of the agent into the tissues behind the eye. Hold the syringe so that the hand is not repositioned on the syringe for injection once the needle has been positioned in the retro-orbital plexus.
   3. Do not aspirate with the syringe, as you will collapse the vessels.
   4. Inject the article in a slow and steady manner.
   5. Withdraw the needle and apply light pressure to the eye to control bleeding and to provide hemostasis.
   6. Mice that experience swelling, conjunctivitis, or other sequelae related to injection trauma must be evaluated by the veterinary staff and treated or euthanized according to their recommendation

Figure 2. Retro orbital injection in mice.

3. Footpad Injection

1. For restraint, place the animal in a restraint tube with one hind foot isolated, and extend by grasping the skin above the stifle.
2. Wipe the foot with water or alcohol to remove debris prior to injecting.
3. Inject subcutaneously into the center of the hind foot forming a small bleb at the injection site. To avoid the blood vessel that runs the length of the foot, inject just off the midline parallel to the vessel.

Figure 3. Footpad injection in mice and rats.