複合管理 II

Many experiments require that a compound be administered into an animal via an oral or topical route, so that the method better mimics the natural intake of the experimental substance.

For oral dosing, the compound can be dissolved into the drinking water or incorporated into the food that the animals consume. Another, more precise method of oral dosing is oral gavage in which a large needle is used to place the compound directly into the stomach. On the other hand, a topical agent can be placed directly on the skin surface, and is generally intended to affect only the area to which it is applied. Each of these strategies has advantages and limitations, which we will discuss in this video. Following that we will demonstrate how to perform these compound administration techniques in lab animals, and some of their applications.

Before delving into protocols, let’s review the considerations, benefits, and shortcomings of these techniques. Oral dosing with tablets, capsules, or liquid is the most commonly used method of drug administration in humans. However, tablets and capsule are impractical for rodents, so the common oral dosing is accomplished by dissolving the compound in the drinking water or food.

When dosing via drinking water, sucrose is often added to the water to enhance palatability. However, it should be finely balanced to encourage normal water intake and not excessive drinking because of the sugary taste. A good rule is to start with a 10% sucrose solution, and then adjust depending on the intake. Another way to orally dose the animal is through specialized food. There is commercially available feed that has commonly dosed compounds, such as fenbendazole, incorporated into it. The rodent feed companies can also create specialized test diets to meet an experimental requirement.

The advantages with this form of oral dosing are that it is non-invasive, administers the compound continuously, and mimics human oral drug administration. However, it lacks precision because compound intake is dependent on the acceptance of the food or water, the stability of the compound, and the number of animals present in the cage. If exact dosing is not critical to the experiment, this non-invasive method is best for the well being of the animals.

For more precise oral administration, one can use oral gavage, which deposits the compound directly into the stomach at a specific time and at a specified volume. However, proper training is required to perform this technique because serious damage to the animal can occur if they are not properly restrained.

Coming to topical agents, these include creams, lotions, ointments, sprays, and gels. Typically, topical agents are intended to affect only the area to which they are applied. However, they can also be absorbed into the bloodstream, which may be intentional or unintentional. This absorption depends on: the condition of the skin, the surface area utilized, the concentration of the substance, the duration of contact, and the lipid solubility of the agent.

Now that we have discussed the background of oral and topical administration methods, let’s learn the protocols starting with oral administration via feed or water.

For dosing calculations, first you need to know the intended dose to be administered. Second, you have to determine the body weight. Rather than weighing individual animals, you can use the average weight of the mouse or rat strain for your calculations, unless otherwise specified. Third, you should be aware of the number of animals per cage, and fourth, you need to know the average daily water or food consumption per cage.

Remember, the formulated food or the drinking water with the dissolved compound may need to be replaced more frequently than standard food or water due to compound instability. Some compounds may be light sensitive or may precipitate over time.

Now, let’s learn how to orally administer precise doses by performing oral gavage in mice and rats.

First step is to select an appropriate needle for the procedure. These are either reusable made up of stainless steel or disposable with a flexible plastic shaft and silicone tip. For mice, 20-25 gauge straight or curved dosing needles that are 1.5 inches in length are appropriate. For rats, select 18-20 gauge straight or curved dosing needles that are 2-3 inches long. The correct needle gauge and syringe selection also depends on volume and viscosity. Review the Compound Administration I video to understand the impact of these factors. For dosing thick liquids a Luer lock syringe is preferred.

After making the selection, affix the syringe to the needle such that the graduations on the barrel can be read without turning the needle. This is important as once the needle has been placed into the esophagus any rotation can rupture the esophageal wall resulting in death of the animal. Next, fill the syringe with the correct volume of the solution to be administered.

This method should always be performed on conscious animals with intact swallowing reflexes to prevent accidental placement into the trachea. Using proper restraint technique is critical for this procedure. For mouse, grasp the skin at the base of the head and hold the scruff tightly. This ensures that the head has minimal opportunity for side-to-side movement. Also, make sure that the body is suspended in a straight line from the head to the tail; any twisting will impede the placement of the gavage needle into the esophagus. The hindquarters must be also stabilized to prevent body rotation.

To restrain a rat, grasp it over the shoulders using the index and middle fingers on each side of the neck. This will prevent the rat from turning its head from side to side. The thumb, third, and fourth fingers encircle the chest to prevent the animal from moving forward or backward.

Once you’ve properly restrained the animal, position the needle with the curve facing downward and in line with the natural curve of the neck. Then, introduce the needle into the mouth from the right or left side, through the diastemata and between the incisors and molars at an angle toward the back of the pharynx. Now that the needle is placed in the oral cavity, use it to tilt the head back until the nose is pointed up. At this point, the needle should slide easily into the esophagus. Once the needle is positioned in the stomach, gently deposit the compound. Then, remove the needle from the animal, making sure not to rotate it. It is important NOT to force the needle down. If you feel any resistance, remove it and try again. While performing the procedure, if the animal shows signs of cyanosis or respiratory distress or if there is solution flowing from its mouth, the dosing needle should be immediately removed and the animal should be released into its cage.

Lastly, let’s review the procedures for topical administration in lab animals. Start by anesthetizing the animal. This is commonly done using a fast acting inhalant, which allows quick animal recovery. For more details on anesthesia administration, please see another video in this collection.

Next, using a hair clipper remove all fur from the area of application. To avoid any cuts, place the clipper’s flat surface against the skin and shave in the direction opposite to that of the hair growth. Then, clean the skin with water and dry it with a gauze pad. Now, using a cotton-tipped applicator, apply the substance directly to the skin. For best results, apply several thin layers of ointment or cream, as opposed to a one-time application of a thick layer. To prevent accidental ingestion of topically applied compound due grooming, place a small collar or a shield to prevent the animal from reaching the affected body part. Furthermore, to prevent allogrooming from cage mates, the animals should be housed individually.

Now that you have an understanding of these alternate routes of administration, let’s see how they are being used in biomedical research today.

One of the important applications of oral gavage is precise administration of substances to study their direct effect on the gastrointestinal tract. Here, the researchers used this method to deliver a transformed probiotic yeast directly into the animal’s stomach. And then they dissected the Peyer’s patches, which are organized lymph nodules from small intestine, to study the adherence of the delivered microorganism.

The non-invasive oral route can be used to mimic the natural modus of food-borne infection. In this experiment, the scientist developed a model for oral transmission of Listeria monocytogenes in mice via ingestion of contaminated food. Following infection, the researchers harvested various organs like small intestine, colon, spleen, liver and gall bladder, to analyze spread of infection to these tissues.

Lastly, some researchers are interested in studying the mechanism by which UV radiation may lead to sunburns. Here, the scientists used topical route to apply a pharmacologic substance that induces production of epidermal melanin-a UV protective agent. Following that, they used a protocol that is similar to human studies to examine the protective effect of the topically applied agent against erythema induced by UV radiation.

You’ve just watched JoVE’s second installment of compound administration methods dealing with oral and topical dosing. You should now understand how one can orally dose the animal using feed or water, and how one can perform the more precise, oral gavage administration. Lastly, you should know the considerations and method for topical administration, and applications of these various techniques. As always, thanks for watching!