齧歯動物の処理と抑制技術

Rodents are widely used model organisms to investigate diverse scientific questions, and it has been demonstrated that even minimal handling is stressful for these animals.

Handling for simple tasks such as cage changing may cause an increase in heart rate, blood pressure, and serum corticosterone levels, and these fluctuations can continue for several hours. The methods of restraint required for common injection and blood withdrawal procedures can also cause physiological changes, which can potentially affect the scientific data. Therefore, training for proper handling of mice and rats is required to minimize these effects on animals.

In this video, we will first review the basic techniques for handling rodents, and then we'll discuss the rodent restraint procedures including manual techniques and methods involving usage of restraining devices.

Let's begin by discussing the basics of rodent handling. One of the most common methods for moving either an adult mouse or rat is by lifting the animal by its tail. While doing this, it is imperative that the tail be grasped at the base near the rump. If the animal is lifted by the end of its tail, the skin can be pulled off due to the body weight and may lead to an injury resulting in tail amputation. Importantly, moving an animal from one place to another should be done quickly and steadily. Suspending the animal in air for any length time will not only cause distress, but will also cause the animal to twist or struggle, possibly causing harm to the tail. Also, the handler should not place a hand under the animal, because it may try to grasp it for security, resulting in a bite.

Another method for handling adult rodents is to use forceps. Types of forceps commonly used are long-dressing forceps or tongue forceps with rubber, plastic, or silicone tubing placed over the ends to provide a cushion. The forceps can grasp the mouse either at the scruff of the neck or at the base of the tail. The use of forceps minimizes cross contamination from animal to animal, and it is quicker and less expensive than changing gloves between each animal cage. Beginners will need to be trained on how tightly to grasp the mice with forceps, especially at the scruff as it could interfere with breathing. When using the forceps on the tail, care must be taken to place the forceps near the base of the tail to avoid injury. Remember to store them facedown in 70% ethanol between handling of different groups of animals.

Also, one can use tubes made of a non-porous material to transfer animals. These tubes can be added to a cage as a place for the animal to shelter or seek safe refuge. Most mice or rats would willingly enter the tubes when their cage is opened, or they can be easily guided into the tube. When the animal is inside, cover the open ends with your palm and fingers. Next, to encourage the animal to leave, gently tip the tube upward in the new cage. Animals are easily conditioned to this method of cage changing, making this a quick and easy process for moving animals from one place to another.

Some institutions prefer that animals-especially rats-be lifted by their body, even while routine handling. Adult rats are gently grasped around the thorax, lifted, and quickly placed into another cage. It is important that animals are habituated to this method of handling from an early age. When working with young rats, placing a hand over the rat's back will cause it to jump into the palm of the handler allowing it to be grasped. A second hand may be needed to prevent the rat from squirming from the grasp.

When changing cages with neonate mice or rats, it is often necessary to remove them while keeping the nest intact. Use two hands to scoop the nest and pups from the bottom of the cage into a new cage. Hold your fingers close together to avoid dropping the pups. Once the nest has been moved to a new cage, it is important to verify that all the pups are present. Alternatively, a plastic scoop may be used to move neonate rodents in their nests. While doing this, it is important that you place your hand over the top of the scoop to prevent neonates from wiggling or jumping out. Compared to the individual handling of the pups, scooping the entire nest is less distressing to both the pups and the dams.

Now that we have discussed the basics of rodent handling, let's delve into detailed procedures for rodent restraint. First we will outline how to restrain these animals manually. Mice and young rats can be restrained using the one handed or the two-handed technique. However, for adult rats, full body restraint using the T-rex grip or the forelimb crisscross method, are the preferred techniques.

Let's begin by learning the two-handed restraint method. First, place the animal on a surface they can grip, such as the wire bar lid. With your dominant hand, hold the tail at the base and gently tug the mouse backwards. This causes the animal to hold onto the surface and allow its body to be stretched. Next, gently pin the rodent with your non-dominant index finger and thumb positioned at the base of the skull, and grasp the loose skin on the neck to lift up the animal. Use your remaining fingers to stabilize the animal with its back against the base of your thumb. You can secure the hindquarters by pinning the tail with your ring finger against the palm of your hand.

For the one-handed restraint method, again place the animal on a surface that it can grip. Hold the tail between the third and fourth fingers of your non-dominant hand and apply gentle backward traction. With your index finger and thumb of the same hand, pin the rodent over the shoulders. Gently slide your thumb and index finger forward and grasp the loose skin at the neck and lift the animal. Use your middle finger to stabilize the animal along the back by pinning the dorsal skin against the base of the thumb.

The third restraint method we will discuss is the T-rex grip- a two-handed restraint method for rats. First, place the rat on a solid surface, like a mat, that it can grip. While holding the base of the tail with one hand, place your other hand over the rat's shoulder with your index and middle finger split on either side. This is to restrict the side-to-side head movement. Once the head is stabilized, encircle the body behind the forelegs with the middle and ring fingers and the thumb. Avoid compressing the chest so that it does not compromise the animal's breathing. Finally, stabilize the tail and hindquarters by grasping the base of the tail WITHOUT holding onto the hind feet, if you plan to administer an oral gavage; or WITH holding onto the hind feet, if the intention is to inject the animal.

Lastly, to restrain a rat using the forelimb crisscross method, place the animal on a surface it can grip. Then with your dominant hand, hold the tail at the base and apply gentle backward traction so that the animal's body elongates. Next, with your other hand, grasp the animal directly behind the shoulders, with your fingers on one side and thumb on the other side of the chest, and slide the hand forward. This motion will force the animal's forelegs to cross under its chin, creating a physical barrier that prevents the animal from moving its head down. Take care to avoid compressing the chest, which will lead to difficulty in breathing.

Now that we have reviewed the manual restraint techniques, let's learn how to restrain rodents using specialized devices.

The first device that we'll learn about is the Broome-style restraint device. It has a slot that runs along the full length of the device and a plastic nosepiece, which is inserted to hold the animal in place. Start by loosening the screw to remove the nosepiece. Then, orient the restraint tube such that the slit is facing up. Grasp the animal at the base of the tail and gently pull the animal into the restraint device, hindquarters first. Once the animal is inside, slide the plastic nosepiece back into the tube to prevent it from opening. Place the nosepiece such that the animal's nose is centered in the opening. Do not position it so tightly that the animal cannot breathe.

Another example of a restraint device is the flat-bottomed rodent restrainer, which is a half cylinder with openings that allow access from the top and the bottom of the device. It also has slots in which a rigid plastic gate can be inserted to hold the animal in place. To use this restrainer, grasp the rodent by the base of the tail with your dominant hand and place it on a smooth surface. Using your other hand, hold the flat-bottomed restraint tube so that the open end is at the animal's head at a 45° angle to the surface. Place the tube over the animal's head, and immediately tap the rump to prevent the animal from backing up. Once it is in the device, slide the plastic gate into the appropriate slot to secure the animal's position.

The third type of device that we'll discuss is tube restraints. These are Plexiglass cylinders with one closed end containing holes for air circulation, and a slit at the open end. To use a tube restraint, grasp the animal and place it on a smooth surface. Next, place the open end of the tube at the animal's head at a 45° angle. As soon as the rodent enters the tube, tap the rump to prevent it from backing up. Finally, occlude the opening by stuffing a large latex glove, which will grip the Plexiglass surface and prevent the animal from exiting the tube.

Lastly, we will discuss how to use flexible restrainers, which are disposable flexible plastic cones available in different sizes. Selection of a cone depends on the animal size. Ideally, it should be long enough to extend 2-3 inches beyond the animal's rump. Like previous methods, place the animal on a smooth surface and place restraint cone at a 45° angle over the animal's head. As soon as the animal enters the cone, grasp the open end and seal it to force the animal to the end. Secure the opening by folding the plastic to one side of the tail and applying a binder clip to the folded plastic being careful to avoid clamping the tail or skin.

After discussing the basics of handling and different restraining techniques, let's review why having an understanding of these procedures is necessary for any in vivo scientist.

The knowledge and rationale behind these basic handling procedures is at the core of maintaining animal's health. From the time the animals are born, they are handled for simple, day-to-day activities like weighing, inspection and cage changing, which ought to be performed irrespective of the scientific objective.

The restraining procedures are also useful for marking purposes. For example, in order to perform procedures like ear punching the animal need to be restrained by scruffing. On the other hand, a restraint device should be used when inserting a microchip in rats so that they can be tagged permanently.

Lastly, the manual restraining methods are used for common injection procedures like subcutaneous and intraperitoneal administration. And the knowledge of restrain devices is useful when performing intravenous administration, which demands minimal animal movement during the injection.

You've just watched JoVE's introduction to basic handling and restraint techniques for rodents. These procedures require a confident and firm, but gentle touch. Tentative approaches can result in handler bites, while aggressive handling can cause injury to the rodent. Therefore, it is important to learn these techniques thoroughly and find a balance of an assertive yet gentle grip. As always, thanks for watching!