Blood collection and administration of fluids and drugs (rat):

Administration of Fluids and Drugs (Overview):

Gastro-Intestinal Tract:

• Oral or per os (po)- - through the mouth
• Gavage - - into the stomach via a feeding needle

Parenteral Administration; General Considerations

• Intravenous (iv) directly into the vascular system through a vein
• Intraperitoneal (ip) - - into the abdominal cavity
• Subcutaneous (sc) - - under the skin
• Intramuscular (im) - - into a muscle

Blood Collection:

• Site Preparation
• Lateral tail vein venipuncture in mice
• Ventral tail artery
• Toenail clip; Tail clip
• Cardiac puncture
• Orbital sinus venipuncture

Administration of Fluids and Drugs (Overview):

When drugs, vaccines, injectable anesthetics or other agents are to be administered, one of several different routes may be selected. The route is governed by the nature of the agent being administered, the animal, and the purpose of the administration, among other factors.

Gastro-Intestinal Tract:

Oral: Substances may be administered orally by addition to the food or the drinking water. The amount of drug consumed by an animal will vary between each individual. If the drug imparts an unpleasant taste, affecting palatability, the drug will be consumed in decreased amounts. Environmental conditions such as the ambient temperature will also affect both water and food consumption. The general rule of thumb is that 5 grams of food and 10 mls of water will be consumed daily per 100 grams of body weight.

Gavage: If it is necessary to administer exact amounts of a substance gastric feeding needles should be used. Entry normally may be obtained without anesthesia using hand restraint. Feeding needles with a ball tip helps prevent introduction of the needle into the trachea and prevents trauma to the oral cavity (picture). Flexible or plastic tubes may be bitten or chewed and are not recommended for rodents. Feeding needles are inserted through the mouth into the stomach or lower esophagus. Placing the needle next to the rat so that the end of the needle is adjacent to the last rib and marking the position on the needle that is adjacent to the tip of the nose will determine the distance the feeding needle should be advanced into the oral cavity to insure administration of the compound into the stomach (picture). Care must be taken that the tube or needle does not enter the trachea or puncture the esophagus or stomach, therefore knowledge of the oropharyngeal anatomy is necessary. In most cases, introduction of the tube toward the rear of the mouth will induce swallowing and the tube will readily enter the esophagus. A violent reaction (coughing, gasping) usually follows accidental introduction of the tube into the larynx or trachea. With the mouse restrained in one hand the feeding needle is introduced in the space between the incisors and the beginning of the molars (diastema)(future picture). If introduced from the left diastema the needle should be directed caudally toward the right ramus of the mandible. As the needle approaches the pharynx the mouse will usually swallow allowing introduction into the esophagus. Using the feeding needle to gently extend the neck facilitates introduction into the stomach (future video). With the stomach tube fitted to a syringe or aspirator, materials may be administered or withdrawn as required.

Parenteral Administration: General Considerations

• Intravenous (iv) - - directly into the vascular system through a vein
• Intra-arterial (ia) - - directly into the vascular system through an artery
• Intraperitoneal (ip) - - into the abdominal cavity
• Subcutaneous (sc) - - under the skin
• Intramuscular (im) - - into a muscle
• Intradermal (id) - - between layers of skin

General Considerations: Parenteral routes of administration involve injections into various compartments of the body. Sites used for collection of blood from veins may also be used for intravenous administration. Intraperitoneal administration is one of the most frequently-used parenteral routes in rodents. Other locations are the musculature and subcutis. Materials given intramuscularly must be in very small volumes and is generally not recommended unless necessary. Absorption by this route is more rapid than from subcutaneous administration. Regardless of the route used, it is essential that the subject be securely restrained to prevent unnecessary struggling by the animal and to avoid injury to personnel by dislodged needles.

The investigator should know the physiological and chemical properties of the substance that he/she plans to inject. Considerable tissue damage and discomfort can be caused by irritating vehicles, drugs or solutions when injected into animals. The use of the foot pad as an injection site for antigens with or without adjuvant is discouraged since it is a needless and painful procedure. More suitable sites for antigen injections are subcutaneously in the axilla or lateral thoracic wall, deep in large muscle masses, or into the popliteal lymph node.

Demonstration/instruction sessions can be arranged with the ACU.

Intravenous:

Equipment: 20-25 g needle, 1-3 ml syringe, rat holder, warming lamp.

Volume: The volume injected IV into an adult rat should not exceed 0.5 ml.

The lateral veins (future picture) of the tail are the most frequently-used veins. Best results are obtained if the tail is immersed in warm water for 5 to 10 seconds or the rat warmed for 5 to 15 minutes in the cage with a warming lamp with a 40 to 100 watt bulb. The veins can be seen when the tip of the tail is lifted and rotated slightly in either direction. The tip of the needle can be followed visually as it penetrates the vein. Trial injection verifies proper needle placement. Also, accurate placement can be confirmed when the vessel is visually flushed when the compound is administered. The formation of a bleb at the site indicates improper placement of the needle. A second attempt can be performed by removing the needle and trying a site on the same vessel in a more proximal location on the tail. Practice is essential. Younger rats are easier to inject since the skin tends to thicken in adults making accurate needle placement difficult.

The lateral saphenous vein (future picture) on the lateral aspect of the hindleg may also be used for intravenous injection. Light anesthesia and/or confinement in a cylindrical rat holder is usually necessary. Other sites that have been utilized are the sublingual vein and dorsal penile vein in anesthetized rats.

Prolonged IV administration/sampling can be accomplished by jugular or femoral vein catheterization, requiring surgical implantation.

Intraperitoneal:

Equipment: Syringe and 20-25 g, 1 to 1.5-inch needle, preferably with a short bevel.

Volume: The volume injected IP into an adult rat should not exceed 10 ml.

The rat is grasped as previously described, and held in dorsal recumbency in a head-down position. The injection is made in the lateral aspect of the lower left quadrant (future picture). The use of a short bevel needle inserted through the skin and musculature and immediately lifted against the abdominal wall, aids in avoiding puncture of the abdominal viscera. Utilizing the needle cover which has been cut 1 to 1.5 cm from the end will limit the depth of injection (future picture) . Immobilizing the left leg is also essential in reducing this risk. Restraint is best accomplished with a second person holding the rat in a head-down stretched-out position; light anesthesia is recommended (future picture). Rapid injection, especially with a large syringe, may cause discomfort and tissue damage and should be avoided.

Subcutaneous:

Equipment: Syringe and 20-25 g, 1/2 to 3/4-inch needle.

Volume: The volume injected SQ into an adult rat should not exceed 10 ml.

This route is frequently used as an alternative to intramuscular injections in the rat. The site usually chosen is the loose skin between the shoulder blades. Alternatively, the ventral abdomen is commonly used, employing one handed restraint (future picture). The needle is inserted through the skin and advanced 5 to 10 mm through the subcutaneous tissue to prevent leakage from the site. Rat skin tends to be thick and difficult to penetrate. Care should be taken to avoid accidental human injections.

Intramuscular:

Equipment: Syringe and < 21 g, 1/2-inch needle.

Volume: The volume injected intramuscular in the adult rat should not exceed 0.3 ml.

This route is usually not used and is not recommended because of the small muscle mass available and the danger of damaging vital structures. However, when it is used, the back and hind leg muscles (quadricep; posterior thigh) (future picture) are the usual sites selected.

Blood Collection:

The amount of blood needed and other factors will govern the method and sites of collection. Descriptions of the various techniques for venipuncture in different species is available in the Animal Care Unit (400 ML; 335-7985) in text and videotape format. Proper insertion of the needle into a vein or other part of the vascular system is normally the most difficult part of the procedure. Certain guidelines can be given, but only practice provides proficiency. Veins may be expected to roll, collapse, or shift, making entrance difficult. A precise, careful introduction of the needle is best and several attempts may be required. Starting at distal sites will allow repeat attempts more proximally. The needle is inserted parallel to the vein and the tip directed into the lumen along the longitudinal axis. When withdrawing blood from a vein, aspiration should be slow so the vessel does not collapse.

Site Preparation:

The area of injection or incision should be cleaned with alcohol. Some procedures will require sedation or anesthesia; others may be carried out without anesthesia provided suitable restraint is used. In order to better visualize veins dilation can be accomplished by immersing the tail in warm water for 5 to 10 seconds or by warming the animal with a low-wattage light bulb for 5 to 15 minutes prior to venipuncture. This also aids by providing additional light.

Lateral tail vein venipuncture in rat:

Equipment: scalpel blade, 25 to 30 gauge needle

The rat is restrained (picture, picture) using a mechanical device. The veins may be seen laterally near the base of the tail but good illumination and dilation will normally be required. Dipping the tail in warm water will help dilate the vessels. A small blood sample may be collected by capillary action using a microhematocrit tube inserted into the hub of a small needle previously placed into the tail vein (future picture). Blood pooling can be induced by placing a small rubber band around the base of the tail.

Ventral tail artery

Equipment: 22 gauge needle, 3 cc syringe

The rat should be anesthetized and placed in dorsal recumbency. Bleeding from the tail may be increased by warming it in water at 40C to 50C. Induce arterial dilatation by applying pressure 1 to 2 inches from the tip of the tail with a finger. Remove the syringe plunger and place the needle bevel up into the tail artery entering at a 20 to 30 degree angle (future picture)⁶. If placed properly the syringe will immediately begin to fill with blood. If blood flow is slow or stops, slowly withdraw the needle to re-establish flow. Retries should always be done with a new needle in a more proximal location on the tail. Pressure on the site may be necessary to cease blood flow after needle withdrawal.

Toe clipping or tail clipping to obtain blood samples: Clipping toes is an unacceptable method of blood collection. Tail clipping is not a preferable method for blood collection.

Cardiac puncture:

Equipment: 0.90 to 0.50 mm needle

Cardiac puncture represents an accepted method of blood collection from rats when more than a few drops are required. However, this method also carries considerable risk to the animal and occasionally deaths occur. Therefore, it is not recommended as a repetitive blood sampling procedure. Animals must be anesthetized and restrained in dorsal recumbency. The needle is inserted under the xyphoid cartilage slightly to the left of midline (picture). The needle is advanced at a 20 to 30 degree angle from the horizontal axis to the sternum to enter the heart. Aspirate lightly while advancing. Blood should be withdrawn slowly, and the amount must be limited (up to 4 ml in an adult rat) unless euthanasia is planned.

Orbital sinus venipuncture in rats:

Equipment: Capillary tubes

Blood collection from the orbital sinus of rats is frequently used. Bleeding requires that the tube be directed into the orbital sinus (future picture) which surrounds the globe. In the rat, the capillary tube is inserted in the medial canthus with gentle rotation while directing the tube caudally and towards the midline (picture, picture). Knowledge of the location of the venous structures of the orbit of the rat aids in establishing a successful peri-orbital bleeding technique. Pressure should be applied after blood collection to prevent hematomas. 0.5 ml of blood can be obtained weekly using this method. Anesthesia is required for all peri-orbital bleeding procedures.