3.5.1. Subcutaneous Injection
Subcutaneous injection of cotton rats generally requires two people: one to restrain the animal and one to perform the injection. The cotton rat can be restrained using the methods described above. Short-term anesthetic restraint may be used to facilitate animal handling. The injection site(s) should be shaved using Wahl Peanut clippers or other appropriate clippers. The clipper operator should take care not to nick the skin. The shaved site should be swabbed with 70% ethanol prior to injection. A tuberculin syringe with a 25G x 5/8'' needle is used for the injection. The skin at the injection site should be lifted slightly for placement of the needle. The needle is inserted bevel side up under the skin and then the contents of the syringe injected. The correct placement of the needle can be checked by slightly pulling back on the plunger (this ensures it is not in a blood vessel), and the needle should move freely under the skin. The contents of the syringe should evacuate with minimal pressure exerted on the plunger. If the syringe contents feel hard to inject, the needle may be incorrectly placed either intradermally or intramuscularly. After the injection, a small raised area may be present at the site.
The injection site should be shaved as described above. A 0.3-mL insulin syringe with an attached 29G x 0.5'' needle is used for injection. The animal should be scruff-restrained as described earlier. The needle should be inserted as deep as possible into the tumor with the bevel side up. During injection, the needle is repositioned in a fan-like pattern as it is being withdrawn. After all of the syringe contents are evacuated and before completely withdrawing the needle, the syringe is twisted clockwise and withdrawn in a downward motion out of the tumor. This helps prevent leakage of the injectable out of the tumor.
Standard methods for intravenous injection in other rodent species do not work well in cotton rats. The tail veins are inaccessible because of the tail's coloration and the tendency for degloving. Cardiac puncture is effective for blood collection and injection, but is contraindicated because of the risk of lung puncture and exsanguination (12). The jugular vein is the preferred route for intravenous injection in the cotton rat. The vessel is easily accessible, and using this route of administration poses little risk of accidental death or injury to the animal. The cotton rat should be anesthetized using the long-term anesthetic protocol described above (Subheading 3.4.5.). When the animal is almost fully anesthetized, shave the ventral neck area with Wahl Peanut hair clippers from below the clavicles to midway on the neck. When the cotton rat is fully anesthetized, the injection site is swabbed with 70% ethanol. The animal should then be restrained on a rodent work stand. Loop the upper front teeth with the tooth loop (provided with the stand) and position the animal on the stand in dorsal recumbency. Place a small roll of gauze (approx 1/2'' in diameter) under the base of the cotton rat's neck (dorsal side) so that the head is slightly hyperextended. The jugular vein is located approximately at the midpoint of the clavicle. To facilitate access to the vessel, use Micropore surgical tape to tape down the front legs to the side arms of the stand. If the clavicle and the jugular vein cannot be visualized, it may be necessary to palpate the clavi-cle to determine the midpoint. A 0.3-mL insulin syringe with an attached 29G x 0.5'' needle (Terumo) is used for injection. Position the syringe at a 45° angle just cranial to the clavicle. Enter the skin with the needle bevel up, and pull back on the plunger gently as the needle is advanced. As the needle enters the vessel, blood will flow into the barrel of the syringe. If the needle has been advanced more than three-quarters of the way and no blood has entered the syringe, the needle has missed or advanced through the vein. Keep slight backpressure on the plunger, and slowly back the needle out of the animal. The needle may enter the vessel during withdrawal of the syringe. Once blood is flowing into the syringe barrel, the syringe contents may be slowly injected. The injector must keep the syringe position stable. Any repositioning of the needle may result in perivascular injection. If the syringe plunger becomes harder to advance or a raised area becomes visible at the injection site, the syringe may need to be repositioned and the patentcy of the needle checked. After injecting, remove the needle and hold off the vessel using mild pressure for approx 5-10 s. Gently remove the animal from the stand and remove the tooth loop. Recover the animal from anesthesia as described above (Subheading 3.4.5.).
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