Collecting blood from mice and rats is necessary for a wide variety of scientific studies, and researchers have developed different methods to achieve specific experimental goals.

In the first installment, we discussed the general blood withdrawal consideration and reviewed the retro-orbital eye bleed, tail snips and nicks, as well as intra-cardiac blood collection methods. Here, we will outline the procedures for blood collection from facial, submandibular, saphenous, and femoral veins. These methods are less invasive and do not require anesthesia, which makes them methods-of-choice when the use of anesthetics may confound blood results or other data.

Let's begin with the procedure for obtaining a blood sample from a murine facial vein. On the mouse, there is an easily accessible facial vein that runs across the cheek. Start by choosing the proper equipment for the procedure. Basically, you need a small centrifuge tube for blood collection and a lancet, which is available in different sizes. Selection of correct lancet is dependent on the age, sex, and bodyweight of the animal and size of sample to be collected. See text protocol for details on lancet selection.

Begin the procedure by restraining the animal using the scruffing technique. Proper restraint minimizes side-to-side movement of the head and helps in ensuring accurate and safe venipuncture with the lancet. If the grip is too tight, the blood flow may be restricted resulting in a reduced volume collected. Once the animal is restrained, locate the approximate area of the facial vein by measuring the length of the eye below the lateral canthus and the width of the eye caudally. With the tip of the lancet gently feel for the point at which the jawbone ends. For better accuracy in puncturing the vessel, you may want to position the mouse on its side.

Now, at the insertion site, hold the lancet perpendicular to the skin surface with the tip facing slightly toward the nose. This is critical, because if the lancet tip is directed caudally, the ear canal will be pierced causing bleeding from the ear. To puncture the vein, apply a firm push and pierce the skin up to the lancet shoulder. Upon removal, the blood will begin to flow. To assist the flow, position the animal with the head lower than the heart. To stop bleeding, blot the puncture site to achieve hemostasis and prevent excessive blood loss. Lastly, release pressure on the scruff and return the animal to its cage. Note that the collection volumes will vary, but it is imperative NOT to exceed the maximum volume for survival blood collection. See the text protocol of the Blood Withdrawal One video for details on recommended maximum volume of collection.Serial samples can be taken by alternating the side used. Neither side should be used more often than every 5-7 days.

Now let's review the submandibular vein bleed method. Although this is very similar to the technique for the facial vein bleed, there are variations in equipment and subtle differences in the bleeding procedure.

The submandibular vein runs along the lower jaw of the mouse and converges with the facial vein into the jugular vein. Instead of a lancet, this method is performed using needles. But similar to lancets, selection of needle gauge is dependent on the age, sex, and animal's weight and sample size - see text protocol for details.

To start, scruff the animal in the same manner as for the facial vein bleed so that there is minimal side-to-side movement of the head. Recall - overly tight grip may decrease blood collection volume. While restraining the mouse, imagine a line from the corner of the mouth across the face and a line from the lateral canthus of the eye. The intersection point of these lines is the approximate area of the submandibular vein. This coincides with a small hairless dimple found caudal to the corner of the mouth and slightly below the jaw line.

For better accuracy, place the animal in lateral recumbency (3.6.1). Next, hold the needle perpendicular to the surface of the skin and insert it with a firm push. DO NOT insert the needle beyond the bevel tip, as insertion depth greater than that may result in trauma to the muscles, nerves and other vessels that are in the head, neck, and oral cavity. Upon removal of the needle, the blood will begin to flow. Like for facial vein bleed, position the mouse with the head lower than the heart to assist with the blood flow. Finally, blot the puncture site to achieve hemostasis and release pressure on the scruff to return animal to its cage.

Now, let's learn how to collect blood from the saphenous vein. This vein is a superficial vessel that runs dorsally and then laterally across the tarsal joint.

The equipment that you need for this procedure includes a restraining device, which could be a flexible plastic one for rats or, for mice a modified 50-milliliter plastic conical tube can be used. Note that the tube end is cut off to allow a breathing hole, and a half-inch wide and 2-inches long slot is cut from the cap end. The edges are covered in clot tape for animal's safety. This procedure also requires a tourniquet -- manufactured using a 3 cc syringe and a length of 0-2 non-absorbable suture, triple antibiotic ointment with a swab -- to be used as a moisture proof barrier between the skin and the blood droplet, a hair clipper, a 22 gauge needle for venipuncture, and hematocrit tube for blood collection.

To restrain a mouse, place it into the tube, nose first. Then, gently guide the hind leg into the slot and stabilize it using the index finger and thumb. Subsequently, shave the hair from the lateral aspect of the leg from the hock to the stifle. Next, smear a very thin layer of the triple antibiotic ointment to the hairless area. Following that, apply the tourniquet as far cranially as possible and tighten it. The saphenous vessel running across the outer surface from the knee to the ankle will begin to fill and will become raised and easy to visualize.

Next, hold a 22-gauge needle directly over the blood vessel and perpendicular to the surface of the skin. And puncture the vessel, being careful not to insert the needle too deeply and puncturing muscle or bone. The blood will bead up on the surface of the leg for collection into a hematocrit tube. Once the blood has been collected, loosen the tourniquet and apply pressure over the puncture for hemostasis. After the bleeding has stopped, remove the animal from the restraint and return it to its cage. Volumes collected with this method are between 10 to 150 μL, depending upon the frequency of sampling. And the sample quality is variable, as it may contain tissue products. No more than 4 blood samples should be taken within a 24-hour period from the same leg.

Lastly, we will learn how to collect blood from the femoral vein, which is another option for blood collection on a rat. The femoral vein runs on the medial aspect of the hind leg from the groin to the knee joint before crossing the knee and becoming the lateral saphenous vein, making it easily accessible.

The advantage to femoral vein bleed is that a larger volume is more easily collected than from the saphenous vein. The disadvantage is that this procedure, in a conscious animal, requires two people. The equipment needed for this procedure are similar to the saphenous vein method, except that it does not require the tourniquet, and the preferable restraining device is a flexible cone. To select the correct cone, measure it against the animal's body length and then make an oval hole cut at the level of the thigh.

To restrain the animal, place it into the tube, nose first. Then, fold the end of the cone and close it using a small binder clip to prevent the animal from exiting. Now pull the hind leg through the oval opening to gain access to the femoral vein. Don't grip too tightly as it might exacerbate hematoma formation. Next, shave off the inner surface of the leg from the groin to the knee, and apply thin layer of triple antibiotic ointment to the hairless area. Subsequently, the restraint person should occlude the femoral vein and grasp the rat with inner surface of the leg facing the person who is going to draw blood. Holding the 22-gauge needle perpendicular to the blood vessel, directly puncture the vein and collect the blood in hematocrit tubes as it beads on the skin surface. The puncture should be as close to the knee as possible to allow for future sampling anterior to this site. Also, ensure that the depth of the puncture is no deeper than the length of the bevel of the needle. To assist the blood flow, position the rat with the leg lower than the heart. Finally, release pressure on the leg and apply pressure on the puncture site to achieve hemostasis and prevent excessive blood loss. Note that significantly more amount of blood can be collected using this procedure.

After discussing the basics of blood collection techniques, let's review some examples of why these procedures may be useful for scientific research

Blood is often analyzed to confirm the immune response during vaccine development. Here, researchers delivered a peptide vaccine to animals suffering from bladder cancer. Next, they collected blood via the submandibular vein and separated the serum to detect the levels of different cytokines, which serve as indicators of vaccine effectiveness.

Blood collection is also commonly performed to test efficacy of a treatment by studying biomarkers of the human disease, like glucose levels in diabetes. These researchers were interested in testing the efficacy of a novel gene therapy delivered via the tail vein in diabetic animals. Following injection, these investigators collected blood from the saphenous vein at several time points to analyze the effect of different treatment protocols on blood glucose level.

Lastly, for some experiments, it is important to know the basic status of the animal, such as levels of inflammation or stress. In this example, blood plasma was collected from a rat before and after a stressful event. And as you can see, the corticosterone, or the stress hormone, level is higher in animals ten minutes following the stressful event.

You've just watched JoVE's second installment on blood withdrawal techniques for mice and rats. After watching both these videos, you should have a better understanding of the considerations and procedures for blood collection from these animals and how they are being used in biomedical research today. As always, thanks for watching!