1. Subcutaneous injection
- Manually restrain the mouse via the one-handed restraint hold. Once grasped, allow the mouse to rest on a table or other secure surface. Be sure to avoid putting pressure on the body of the mouse.
- Rest the heel of the hand on the table to avoid undue weight on the mouse that can compromise respirations. Mice can also be restrained in a commercial device. For rats, it is best to use a commercial restraint device.
- Grasp the skin, and pull upward to create a tent of skin. If using a restraint device, forceps may be required to grasp the skin through the slot on the top.
- Insert the needle parallel to the spine, directed away from the head at the base of the skin fold of the tent. Direct the needle away from the head to avoid the possibility of punctures to the skull, as the skull bone of the mouse is very thin. Rats have the tendency to rear their head, which could cause the injection to be placed into the neck muscles.
- Insert the needle bevel up to allow a gentle glide into the skin. Note that the needle is inserted below the fingers grasping the skin. To improve safety when injecting biohazardous articles, the skin is grasped with forceps, thereby eliminating the possibility of needle stick injuries.
- Pull back the plunger to check for proper placement. If air is drawn into the syringe, it indicates that the tip of the needle is not positioned under the skin and will need to be withdrawn and repositioned. If there is back pressure when the plunger is pulled back, it indicates proper placement of the needle, and that the injection can proceed.
- Inject the substance slowly with a steady motion. To prevent loss of the injection article, pause after injection, slightly rotate the needle under the skin, and pinch the skin at the injection site while withdrawing the needle.
Figure 1. Subcutaneous injection in mice.
2. Intraperitoneal injection
- The one-handed manual restraint method is used for this injection technique in mice. Adjust the hand so that is it positioned high enough on the neck to prevent the mouse from turning.
- Grasp the loose skin at the nape of the neck between the thumb and index finger.
- Stabilize the hind quarters by either pinning the loose skin along the back between the second, third, and fourth fingers and the base of the thumb, or by placing the tail between the third and fourth fingers.
- The IP injection in the rat requires two people, one to restrain the rat and the other to perform the injection.
- Grasp the rat over the shoulders using the index and middle fingers on each side of the neck and the palm on the back. The thumb and the third and fourth fingers should encircle the chest to prevent forward or backward movement. The position of the fingers on each side of the neck prevents the rat from turning its head.
- Lift the rat and turn it to expose the ventral abdomen.
- Stabilize the hind quarters by grasping the feet and tail securely and extending them away from the body.
- Position the mouse or rat to expose the abdomen, ensuring that it faces upward.
- Tilt the animal with the head pointing downward at about a 30° angle, to allow the intestines to fall forward.
- Injection landmarks are as follows:
- The area to be injected is bordered cranially by an imaginary line extending horizontally across the body at the top of the hip (from flank to flank).
- The midline is the medial border, recognized as where hair growing in opposite directions meets. In hairless animals, the midline extends in a straight line from the xiphoid to the anus.
- The lateral border is a line from the top of the hip to the prepuce in the male, and from the top of the hip and following the teats in the female.
- This provides a triangular area for safe injection.
- Injection of an article within the landmarks
- Insert a needle perpendicular to the spine, off the midline, in the triangle as described above. Placing the needle at a 90° angle to the plane of the body allows for safe injection utilizing both sides of the abdomen. This is especially important with multiple injections, as it minimizes tissue trauma by allowing alternation of injection sites.
- When the needle is placed at a 90° angle, it will "pop" into the abdomen, allowing for easier determination of depth. This is also a visual and tactile cue that the needle is properly positioned.
- Aspirate the syringe to ensure placement within the peritoneal cavity and not within the urinary bladder, intestine, or vascular structures.
- Avoid injecting caudally in the male mouse to prevent administering the article into the scrotal sac. Avoid injecting into the teat of the female to prevent trauma.
Figure 2. The landmarks for intraperitoneal injection in mice.
3. Intramuscular injection
- Restraint for this technique for mice and rats requires either two people or the use of a restraint tube. Described here is a one-person method utilizing a restraining device.
- Place the animal in the restraint device.
- Once the animal is in the tube, grasp the tail to position the animal with the hind legs out of the tube.
- Grasp the skin of the flank at the cranial portion of the femur to extend the leg and prevent the stifle from bending.
- Position the restraint device so that it stands on the table with the animal's head pointed down, or lying on the table pointed toward the body of the technician to allow stabilization of the device while the injection is performed.
- Injection landmarks are as follows: both the mouse and rat leg are small with the gluteal muscles (the caudal thigh muscles) comprising the largest muscle mass in the hind limb; the second largest muscle mass in the hind limb is the gastrocnemius (the calf muscle). The injection is made from the caudal aspect of the leg.
- Locate the gluteal muscle mass posterior to the femur. The bone can be palpated and the large muscle is easily felt.
- Note that the midline of the leg from the posterior aspect runs from the point of the hock to the tail. The hair often has a ridge where it comes together, as it grows in opposite directions from the lateral and inner surfaces.
- Injections are made toward the lateral aspect of the thigh off the midline. This reduces the likelihood of damaging the nerves and blood vessels located on the medial surface of the leg.
- Injections made into the gastrocnemius are done from the posterior aspect and with careful consideration of the location of the lateral saphenous vein.
- Injection of an article within the landmarks
- Insert the needle perpendicularly to the femur off the midline as described above, to a maximum depth of 5 mm for the gluteal muscle and a maximum of 3 mm for the gastrocnemius.
- To avoid trauma to the muscle, extraneous movement of the needle in the tissue must be prevented. The syringe should be held in a manner that does not require repositioning of the hand to perform the injection.
- Aspirate the syringe to ensure placement within the muscle and not in a blood vessel.
- Inject the material in a steady, fluid motion. Do not inject rapidly as to allow slow expansion of the muscle. Rapid injection will cause tissue trauma.
Figure 3. Intramuscular injection into the gluteal muscle in rats.
4. Intravenous injection utilizing the tail vein
- Restraint of the animal is dependent upon whether the animal is anesthetized or awake.
- Use a cylindrical restraint tube for conscious mice or rats, except for the hairless or nude animals. Due to the oily nature of the hairless animals, it is difficult to reposition and remove them from the acrylic restrainers, as their skin tends to adhere to the rigid plastic. Thus, a flexible plastic cone is used.
- Other injection devices include lighted platforms, heated platforms, and rigid plastic cones where the mouse is kept in the cone strictly with tension on the tail.
- Anesthetized mice may not need restraint.
- Warm either the entire body or just the tail to ensure vasodilation of the tail vessels.
- Warm the whole body with an electrical heating pad set on medium or a circulating water blanket.
- Place the animal in the restraint device, wrapped in the heat source.
- Observe the animal closely, and remove it from the heat source as soon as the blood vessels dilate. Whole body warming can also be achieved with the use of a heat lamp directed into a cage. When using a heat lamp the animals must be closely observed, as it is easy to overheat them.
- When whole body warming is not feasible, heating the tail alone can be achieved using warm compresses from a heated towel, hot water bottle, or submerging the tail into warm water. Caution must be employed to avoid burning the tail because hot water bottles, warmed towels, and warm water do not have temperature controls. It is common for the heat sources to be overheated initially. These heat sources also cool down quickly.
- The use of tissue oil is another way to enhance the visualization of the vessels.
- Dip a cotton-tipped applicator into the tissue oil, and apply the oil well away from the portion of the tail that is gripped for stabilization. The tissue oil causes the vessels to appear more clearly defined.
- There are nontoxic oils commercially available that contain a chemical in the formulation that creates warming of the skin and vasodilation with evaporation. These oils are applied, and after a few moments, when the vessels are visible, the excess oil is wiped away with a cotton ball.
- All oils can make the tail more difficult to hold.
- When using oils, the injection must be made immediately upon visualization of the vessels.
- Injection landmarks are as follows:
- The lateral tail veins are located on the sides of the tail. There are also vessels that run along the dorsal and ventral midlines of the tail that are not suitable for injection purposes.
- The animal in the restrainer can be rotated so that the lateral vessels are facing up for ease of injection. Alternatively, the tail can be turned to bring the vessels into a more acceptable position for injection.
- The vessels are very superficial. They become more superficial as they reach the base of the tail.
- Injection of an article into the tail vein requires skill. To avoid trauma to the blood vessel, extraneous movement of the needle must be prevented. The syringe is held in a manner that does not require repositioning of the hand to perform the injection.
- Position the tail so that the vessel is visible and held under tension. Do not apply excessive tension to the tail, or the vessel can be stretched and the blood flow diminished.
- Place the needle directly over the vessel as far distally as possible.
- Apply slight pressure and slide the needle into the tail parallel to the vertebrae. Avoid angling the needle downward, as the needle will transect the vessel. The needle should be visible in the vessel as it is advanced at least 2 mm into the lumen.
- Inject the material in a slow, fluid motion to avoid rupture of the vessel.
- Pay special attention to the tail during injection. If properly inserted in the vessel, the injection material flowing into the vessel is felt with no resistance. The blood vessel should blanch as the blood is pushed out by the injection material.
- If the needle is not in the vessel, there will be strong resistance when injecting. If injecting with force, the material will fill the subcutaneous space and the tail will balloon. Stop immediately, as material designed to be injected intravenously may be caustic to the surrounding tissues. Withdraw the needle, and attempt another injection more cranially on the tail.
- After successful injection, withdraw the needle and apply pressure to the injection site to insure good hemostasis before returning the animal to the cage.
Figure 4. Tail vein injection in mice.
Compound administration is often an integral component of an animal study, and many factors need to be evaluated to assure that the compound is delivered correctly and in a humane manner. The two principal administration routes are enteral-via the digestive tract, and parenteral-outside the digestive tract. The main difference is, if the compound is given via the enteral route it undergoes hepatic metabolism BEFORE entering the blood stream. Whereas, via any parenteral route, like intravenous or intramuscular, the substance skips this first pass through the liver, usually resulting in a higher bioavailability.
In this first video of the series on compound administration, we'll start by discussing the factors affecting the choice of route of administration in general. And then we'll review the most common parenteral injections methods, including subcutaneous abbreviated as SC or SubQ, intraperitoneal or IP, intramuscular or IM, and intravenous AKA IV.
Let's begin by reviewing a few things that one should bear in mind before preforming any type of compound administration procedure. The first step is to prepare the solution or suspension to be injected, and the first fundamental consideration for substance preparation is sterility. To prevent introducing pathogens into the animal, it is important that the injection material as well as the needles and syringes to be used are sterile.
The second consideration is physiological compatibility. A dosing solution, whether administered enterally or parenterally, must be physiologically buffered to suitable pH for the compound to be properly absorbed, and to prevent tissue injury. The third factor is the viscosity of the injection article, which plays a critical role in needle selection. Usually, 20 to 30 gauge needles are used in parenteral administration procedures for mice and rats. The hubs of these needles are usually color coded for ease of identification.
The solution should have enough fluidity to pass through at least one of these needles. If the solution can pass through more than one, then, in general, the choice is the smallest gauge feasible. The next factor is administration volume that affects syringe selection. Similar to needles, the smallest possible syringe needed for accurate dosing with correct graduations, should be selected. The needle gauge and the administration volume are also dependent on the route, and the species, size, and age of the animal being administered. See Table 1 below to review the values related to the routes discussed in this video. The final important consideration is the absorption rate, which varies significantly with each route of administration and may play a role in selecting the best-suited method.
Now, let's talk about the peculiar characteristics of the routinely employed injection methods.
SC or SubQ Injection places the material between the skin layers and the muscle in a virtual space created by lifting the skin. This allows for safe injection of larger volumes, but the absorption rate is slower than other routes, providing a more sustained effect. During IP administration, the compound is injected directly into the peritoneal cavity. This is another common method used to deliver large volumes of solution. Although an IP injection is considered parenteral administration, the absorption mechanism is actually more similar to oral dosing.
An IM injection delivers a compound directly into the gluteal or the gastrocnemius muscle. A substance injected IM is rapidly absorbed due to the abundant number of vessels within the muscle tissue, which might make it a preferred route in some cases. Improper or repeated injection in the muscle can cause nerve damage resulting in paralysis or muscle necrosis. Lastly, an IV injection into the tail vein of the animal is the most effective route of administration, as the substance is directly introduced into the circulatory system. Note that the compound ought to be injected into one of the lateral caudal tail veins located on the sides of the tail. There is a vessel that runs along the ventral midline of the tail, which is not suitable for injection purposes.
Now that we have discussed the background, let's learn the procedures, starting with SC injections. For mice, pick the animal by its tail and allow it to rest on another secure surface, like wire-bar lid. Then, manually restrain the animal by lifting the skin around the neck, forming a tent. Next, set the animal on the table with the back feet resting on the surface and rest the heel of your hand on the table to avoid undue weight on the animal that may compromise breathing.
To inject, pick up the syringe with the solution to be injected, and direct the needle parallel to the spine and away from the head to avoid the possibility of punctures to the skull. Then, insert the needle with bevel facing upwards, which allows for a gentle glide into the skin. Pull back the plunger to check for proper needle placement. If there is backpressure when the plunger is pulled back, then the needle is in the correct position. If air is drawn into the plunger, then the needle will need to be repositioned.
After confirming that the needle is positioned properly, inject slowly with a steady motion. Pause after the injection and rotate the needle slightly under the skin to prevent loss of the injection article. Then, pinch the skin at the injection site and withdraw the needle. This same method can be used in weanling rats.
For adult rats, first the animal has to be restrained using a device, the procedure for which is discussed in the "Basics of Handling and Restraint" video of this collection. Then, one can perform the SC injection in the same manner as done in mice.
Next, we will learn how to perform an IP injection. In mice, use the two-handed restraint technique to manually restrain the animal, again described in the "Basics of Handling and Restraint" video. Ensure that your non-dominant hand is positioned high enough on the neck to prevent the animal from turning. Stabilize the hindquarters by placing the tail between the third and fourth fingers or by pinning the skin firmly between the remaining fingers and the base of the thumb.
Next, position the animal to expose its abdomen. Tilt the animal with the head pointing downward at a 30° angle to allow the intestines to fall forward. The injection landmark can be outlined like this: draw an imaginary line that extends horizontally across the body, at the top of the hip, from flank to flank. Then draw the medial border, or the midline, along the line where the hair growing in opposite directions meets. Lastly, imagine the lateral border, which is a line from the top the hips to the prepuce in males and following the teats in females. This provides the triangular area for safe IP injection.
The next method that we'll discuss is IM injection. Restraint in this technique for both mice and rats requires either two people or the use of a restraint tube. Here, we'll describe the one-person method utilizing a restraining device.
First, place the animal in the tube and with the hind legs out, pull the tail to position the animal. Next, grasp the skin of the flank at the cranial portion of the femur to extend the leg and prevent the stifle from bending. Then, position the restraint device to allow visualization of the injection site.
To identify the gluteal landmark, locate the gluteal muscle mass posterior to the femur. The bone can be palpated and the large muscle is easily felt. Note the midline that runs from the point of the hock to the tail. You can often see the ridge where the hair from the lateral and inner surfaces come together growing in opposite directions. Typically, injections are made toward to lateral aspect off the midline. The gastrocnemius is the calf muscle and injection into this muscle is also best performed from the posterior aspect.
For the gluteal muscle, at the located landmark, insert the needle to a maximum depth of about 5 mm. Avoid repositioning the syringe during the injection to prevent muscle damage. Next, aspirate to ensure that the placement is within the muscle, not a blood vessel. And lastly, inject the material in a slow and steady fashion, since rapid injection will cause tissue trauma. Remove the needle in a perpendicular fashion following the same route as that of the insertion. For gastrocnemius muscle, inset the needle to a maximum depth of 3 mm and perform the injection in the same manner as for the gluteal muscle.
Lastly, let's learn how to perform an IV injection in the tail veins of rodents. The same method is applicable for both mice and rats.
Place the animal in a cylindrical restraint tube and warm the entire body of the animal by using an electrical heating pad set to MEDIUM for about 2-5 minutes. Continue to warm the animal, until the blood vessels become dilated, that is noticeably larger. Remember, the lateral caudal tail veins are located on the sides of the tail. The artery that is in the ventral midline is not suitable for injection purposes. Rotate the restraint device and position the tail such that the lateral tail veins are facing up and held under tension. Do not apply excessive tension or the vessel can be stretched and the blood flow diminished.
Place the needle bevel-up directly over the blood vessel as distally as possible, and apply slight pressure to slide the needle into the tail parallel to the spine. Inject the material in a slow, fluid motion and notice that the vessel blanches as blood is pushed out by the injection material. If the needle is not properly positioned in the vessel, there will be strong resistance when injecting, and if you inject with force the material will fill the subcutaneous space and the tail will balloon. Stop immediately as the material - that is designed to be injected intravenously - may be caustic to the surrounding tissues. Withdraw the needle and attempt another injection more cranially on the tail. After successful injection, withdraw the needle and apply pressure to the injection site to insure good hemostasis before returning the animal to the cage.
Now that you're familiar with common injection methods, let's look at some applications of these routes of administration, other than drug delivery.
In several experiments, mice are injected with a specific pathogen to study infection. Here, the researchers used the subcutaneous route to introduce antibiotic resistant bacteria that causes lesions, and the size of these lesions served as a readout for the pathogen's virulence. Various scientists are interested in studying distribution and survival of stem cells following systemic delivery. In this study, the investigators performed tail vein injection of genetically tagged neural stem cells in a multiple sclerosis animal model and located the distribution of the injected cells to spinal cord and brain regions.
In another experiment, researchers injected fluorescently tagged myoblasts intramuscularly in an animal model of muscular dystrophy. Following that, bioluminescence was performed to analyze successful implantation of the stem cells. Lastly, injections can also be used to generate animal models. These scientists perfomed intraperitoneal injection of Dimethylnitrosamine - a potent liver toxin - in male Wistar rats to generate an animal model of liver fibrosis, which was then be used to study the development of the liver disease.
You've just watched JoVE's first installment on compound administration discussing the commonly employed parenteral injections. Remember, the optimal delivery route is based on several factors including the pH, volume, and viscosity of the injected solution. And each technique has advantages and disadvantages, which must be considered in relation to the experimental needs. As always, thanks for watching!
