Guidelines for Techniques with Rodents (Duke University)
HANDLING & RESTRAINT
MOUSE: Mice are small rodents that are hard to grasp. Care should be taken to avoid getting bit or causing harm to the animal. One method of moving mice from one cage to another is by grasping the skin behind the neck with a pair of forceps. When using this method be careful not to grasp too hard. Restraining the mouse can be done by grasping the base of the tail with one hand and with the other grasp the loose skin behind its neck. Take extra precaution to avoid getting bitten. When you have a firm grasp you may secure the tail in the same hand you have the scruff in to accomplish a one handed restraint. Care should be taken not to excite any of these animals. Slow deliberate movements will make the job of restraining much easier. Noise should also be kept to a minimum.
RAT: Rats can inflict a painful bite. DO NOT grasp the rat by the scruff, as it will react violently to this type of restraint. First grab the rat by the base of the tail and lift out of the cage and place on a soft surface. Hard smooth surfaces can make the rat tense. Secondly, gently pet the rats – this will calm it. Place your index and middle fingers along side the rat’s head and your thumb and ring fingers under its forelegs. Use your index and middle fingers to secure its head and the remaining fingers to support the chest. Hold the tail and support the lower body with your other hand. Be careful not to squeeze hard as this may interfere with the rat’s ability to breathe. Care should be taken not to excite any of these animals. Slow deliberate movements will make the job of restraining much easier. Noise should also be kept to a minimum.
GUINEA PIG: Guinea pigs are curious, easy to handle animals. They are not aggressive by nature. Do not grasp the guinea pig by the loose skin. Body hair is easily pulled out and the guinea pig will often object when handled in this manner. Calmly grasp it with one hand under the chest and use your other hand to support its hindquarters. Care should be taken not to excite any of these animals. Slow deliberate movements will make the job of restraining much easier. Noise should also be kept to a minimum.
SEX DETERMINATION
MOUSE: Restrain the mouse and lift the base of the tail. Sex is most easily determined by ano-genital distance. Males normally have a greater distance between the anus and urogenital openings. Male mice also have a larger genital papilla.
RAT: Procedure same as in the mouse.
GUINEA PIG: Both male and female guinea pigs display similar ano-genital distances. The female has a separate urethal orifice, a vaginal membrane, a perineal sac, and an anus; the male has a penis, a larger perineal sac, and an anus. The penis lies just under the skin and can be inverted with gentle pressure. The testes and penis are palpable in adults.
ORAL GAVAGE
MOUSE: Restrain the mouse and measure the gavage tube from the tip of the nose to the last rib. This is the length you must insert the tube. With the use of the tube push the mouse’s head slightly upward and back to straighten the esophagus. Position tube to the right or left of the mouth and slowly pass the tube watching for the swallowing reflex. The tube should pass freely into the esophagus. DO NOT FORCE. When desired length of insertion is achieved, inject solution. Observe mouse after the procedure for signs of distress, such as gasping or frothing of the mouth.
RAT: Procedure same as in the mouse.
GUINEA PIG: The guinea pig has a small palatal ostium that is easily damaged. For this reason this procedure is not recommended in the guinea pig.
SPECIAL NOTE: When using a plastic tube, care should be taken to ensure animal does not bite down and sever the tube. An artificial device to hold the mouth open can be used.
INJECTION TECHNIQUES - GENERAL OVERVIEW
MOUSE: Injection sites should be cleaned with a suitable disinfectant, typically isopropyl alcohol. Sterile syringes and needles must be used for all injections. The one time use of disposable supplies insures aseptic techniques and sharp needles. Always select the smallest gauge needle possible to limit tissue trauma and injection discomfort. A 25-30 gauge needle is recommended for use in a mouse. Before injecting, check for correct placement by pulling back on the plunger of the syringe to create a vacuum. This is known as aspiration.
RAT: Same overview as the mouse except it is recommended that 21-30 gauge needles be used.
GUINEA PIG: Same overview as the mouse except it is recommended that 22-30 gauge needles be used.
SUBCUTANEOUS (SQ) INJECTION
MOUSE: The mouse should be restrained in the normal manner. With your fingers, lift the skin to make a “tent”. Disinfect the injection site and insert needle into the subcutaneous tissue. Aspirate prior to making the injection. Proper placement should yield no aspirate. Inject.
* Most common injection site is the loose skin around the neck and shoulder area.
RAT: Restrain the rat by grasping the base of the tail with one hand and with the other, flatten the rat against the table. With your fingers, lift the skin to make a “tent”. Disinfect injection site and insert needle into the subcutaneous tissue. Aspirate. You should not aspirate anything. Inject.
* Most common injection site is the loose skin around neck and shoulder area.
GUINEA PIG: The guinea pig should be restrained and the injection site disinfected. With your fingers, lift the skin to make a “tent”. Insert needle into the subcutaneous tissue and aspirate. You should not aspirate anything. Inject.
* Most common injection site is the loose skin around neck and shoulder area.
***The recommended needle size for SQ injections is 23-25 gauge.
INTRAMUSCULAR (IM) INJECTION
MOUSE: IM injections are not recommended due to the mouse’s lack of muscle mass. Injection may cause discomfort and local tissue irritation.
RAT: Restrain rat by either holding rat against your body and isolating the rear leg or restrain the rat in the traditional manner and grasp the hind leg and hold securely. Disinfect injection site and insert needle into the caudal thigh muscle. You first must isolate the caudal thigh muscle to prevent injection into the ischiatic nerve. Injection into the nerve may cause discomfort and lameness. Aspirate and inject. If blood is aspirated, you must reposition the needle.
GUINEA PIG: Restrain the guinea pig and isolate the caudal thigh muscles. Disinfect injection site and insert the needle. Aspirate. Prior to making the injection be sure to inject into the caudal thigh. Placement of the needle too far laterally can result in damage to the ischiatic nerve. Another injection site is the lumbar muscles. To administer an IM injection here, outline the lumbar muscles with your thumb and second finger, using your index finger locate the vertebral column for orientation. Insert needle lateral to the midline, avoiding the spine.
***The recommended needle size for an IM injection is 25 gauge.
INTRADERMAL (ID) INJECTIONS
MOUSE: ID injections are not commonly performed in the mouse due to limited clinical application.
RAT: ID injections are not commonly performed in the rat due to limited clinical application.
GUINEA PIG: Restrain the guinea pig by using either physical or chemical restraint. Shave or Nair the injection site and disinfect. Isolate the injection site by pinching or stretching skin. Insert the needle bevel up just under the surface of the skin and inject. A distinct bleb should form.
***The recommended needle size for an ID injection is 25 gauge.
INTRAPERITONEAL (IP) INJECTION
MOUSE: Restrain the mouse and tilt so that the head is facing downward and its abdomen is exposed. Disinfect injection site and insert the needle cranially into the abdomen at a 30-45 degree angle caudal to the umbilicus and lateral to the midline.
Aspirate:
* Greenish-brown aspirate indicates needle penetration into the intestines
* Yellow aspirate indicates needle penetration into the bladder
If any fluid is aspirated, your solution is contaminated and must be discarded and the procedure repeated with a new syringe and needle. If nothing is aspirated, inject. The recommended needle size for IP injections in the mouse is 25-27 gauge.
RAT: Procedure same as in the mouse. The recommended needle size for IP injections in the rat is 25 gauge.
GUINEA PIG: Procedure same as in the mouse. The recommended needle size for IP injections in the guinea pig is 23-25 gauge.
INTRAVENOUS (IV) INJECTION
MOUSE (Tail Vein): Restrain the mouse with physical or chemical restraint. Rotate the tail slightly to visualize vein. Disinfect injection site and insert needle (27-30 gauge) into the vein at a slight angle. You will not be able to aspirate, instead inject slowly and watch for clearing of the lumen. Incorrect positioning will result in a slight bulge in the tail. If this occurs, remove needle and repeat process proximal to previous site. Upon completion remove needle and apply pressure to injection site.
RAT (Tail or Saphenous Vein): Tail injection procedure same as in the mouse. For the saphenous vein, restrain the rat with the use of anesthesia. Extend the hind leg and shave hair to expose lateral saphenous vein. Disinfect the injection site and apply tourniquet-like pressure to the upper portion of the leg. Insert needle into the vein and aspirate. Release tourniquet pressure and inject. Upon completion remove needle and ensure proper hemostasis. The recommended needle size for IV injections in the rat is 22-25 gauge.
GUINEA PIG (Saphenous Vein): Same procedure as for the rat, but much more difficult to visualize in the guinea pig. The recommended needle size for IP injections in the guinea pig is 25 gauge.
***Be sure there are no air pockets or bubbles in the solution to be injected, as this can kill small animals.
BLOOD WITHDRAWAL TECHNIQUES - GENERAL OVERVIEW
MOUSE: Withdrawal sites should be cleaned with a suitable disinfectant. Sterile syringes and needles must be used for all withdrawals. The one time use of disposable supplies insures aseptic techniques and sharp needles. Always select the smallest gauge needle possible to limit tissue trauma and discomfort. A 25-30 gauge needle is recommended for use in a mouse. Check for correct placement by pulling back on the plunger of the syringe to create a vacuum. This is known as aspiration.
RAT: Same overview as the mouse except it is recommended that 21-30 gauge needles be used.
GUINEA PIG: Same overview as the mouse except it is recommended that 22-30 gauge needles be used.
TAIL ARTERY AND VEINS
MOUSE: The tail arteries and veins may be used to collect small amounts of blood. First warm the animal in an incubator or under an incandescent light. The animal must be watched closely during this procedure to ensure they don’t overheat or receive thermal burns to their extremities, particularly their ears. There are two blood collection techniques that can be used.
* Restrain mouse, disinfect withdrawal site and insert a 25 gauge needle into the vein and use a capillary tube to collect the blood from the hub.
* Restrain mouse in a rodent restrainer, disinfect withdrawal site and locate vein. Using a razor blade, knick tail vein and collect in a blood collection tube or capillary tube. This method should only be used every 1-2 weeks for a limited number of collections.
Upon completion of either blood withdrawal technique, ensure proper hemostasis.
RAT: The tail arteries and veins may be used to collect small amounts of blood. First you must warm the animal in an incubator or under an incandescent light. Restrain rat in a rodent restrainer, disinfect withdrawal site and insert a 25 gauge needle into the vein. Use a capillary tube to collect the blood from the hub. Upon completion apply pressure until bleeding stops.
GUINEA PIG: This method of blood collection is not recommended in the guinea pig, as the guinea pig has no tail.
PERIORBITAL VENOUS SINUS - ORBITAL VENOUS PLEXUS
MOUSE (periorbital venous sinus): This method must be done on an anesthetized mouse and is only recommended to be done at weekly intervals using alternate eyes. Microhematocrit tubes or Pasteur pipettes may be used to collect blood. Push the upper and lower eyelids apart to protrude the globe. Insert the tube into the medial canthus of the eye. Apply slight downward pressure while rotating the tube to pass it through the conjunctiva and into the periorbital sinus. When the tube “pops” through, back tube out slightly to allow blood to flow. When collection is complete, close both eyelids and apply pressure with a piece of gauze until bleeding stops.
RAT (orbital venous plexus): This method must be done on an anesthetized rat and is only recommended to be done at weekly intervals using alternate eyes. Microhematocrit tubes or Pasteur pipettes may be used to collect blood. Push the upper and lower eyelids apart to protrude the globe. Insert the tube into the dorsal portion of the bony orbit. Apply slight downward pressure while rotating the tube to pass it through the conjunctiva and into the venous plexus. When the tube “pops” through, back tube out slightly to allow blood to flow. When collection is complete, close both eyelids and apply pressure with a piece of gauze until bleeding stops.
GUINEA PIG: This method of blood collection is not recommended in the guinea pig.