Experimental Techniques in Pre-clinical Studies 10-3-2015 Pharmacology Dept PESCP Page 1

Restraining techniques, routes of drug administration and blood withdrawal techniques

Purpose: The purpose of this event of this workshop is to inform and instruct personnel on the

safe and the least stressful way for restraining, injecting and blood withdrawal techniques on

experimental animals.

Restraining techniques

Restraint: Restraint is defined as the immobilization of a conscious animal in a fixed position

for a significant period of time while an examination or procedure is going on.

Proper restraint is one of the main factors in administering drug or blood withdrawal techniques

limiting the amount of stress and/or discomfort to the animal or the handler.

When working with lab animals the two types of restraint that are utilized most often are

physical and chemical

Chemical restraint:

Chemical restraint is recommended if there will be great distress or discomfort to the animal.

This can be achieved by using a gas anesthesia such as isoflurane or by a type of injectable

sedation such as Ketamine/Xylazine mix.

Physical restraint:

Basic/Four finger hold: Head between index and third/ring finger, thumb and knuckles along

body to support chest. Support tail and body with other hand.

One Hand Restraint Technique:

With the non-dominant hand, grab the base of the tail with the thumb and index finger to keep

the rat from running away, leaving the dormant hand free for use.

With the same hand, place the palm of the hand on the rat’s back. With the thumb and fingers

gather the loose skin along the dorsum including that along the dorsal neck. The thumb and

index finger are utilized to stabilize the head while the other three fingers stabilize the trunk.

Experimental Techniques in Pre-clinical Studies 10-3-2015 Pharmacology Dept PESCP Page 2

Be careful not to grasp too tight as this may compromised breathing.

Two Hand Restraint Techniques

With the non-dominant hand, grab the base of the tail with the thumb and index finger to keep

the rat from running away.

Next, with the other hand quickly and firmly grasp the rat/mice by the scruff of the neck.

With the tail in one hand and the scruff in the other, lift the animal between the palm and

the fingers.

Route of administration

Numerous routes have been documented in literature, as every route has both advantages as well

as disadvantages for instance, the absorption, bioavailability and metabolism of the substance.

The disposition and fate of the administered substance helps the scientists to choose the most

suitable route for administration.

a. Intra muscular route (IM)

Insert the needle into the large muscle mass posterior the femur. Requirements-Disposable

gloves, 1ml syringe, hypodermic needle- 22-30G. Max- 0.3 ml can be administered.

b. Subcutaneous injection (SC)

SQ injections are usually given in the scruff (access skin) of the neck or using the scruff back in

the hind quarters by tenting the skin. Requirements- syringe, hypodermic needle- 22-30G. Max -

5ml can be administered.

c. Intradermal injection (ID)

Given into the thick dermal skin layer. Needle directed at 20 to 30 degree angle into skin.

Requirements- 1ml syringe, hypodermic needle- 25-30G. Only small quantity of fluid 0.1 to 0.2

ml can be injected.

d.Intraperitoneal injection(IP)

Experimental Techniques in Pre-clinical Studies 10-3-2015 Pharmacology Dept PESCP Page 3

Insert needle into lower left/right quadrant of abdomen at a 30-degree angle to avoid hitting such

organs as liver, bladder, and cecum. Requirements- 1-3 ml syringe, hypodermic needle- 22-30G.

Max- 5ml can be administered.

e. Intravenous injection (IV)

Injection site- lateral vein, Insert the needle at 30 degree angle to skin. Requirements-

Restrainer, syringe, hypodermic needle- 25-30G. Max- 0.5ml. Dipping the tail in warm water

(45ºC) will dilate the vessels and facilitate injections.

f. Oral gavage route

Measure the distance from the tip of nose to the first rib. This is the length of needle that should

be used. Restrain the shoulders of the rat by your thumb and index finger, then support the lower

limbs with your right hand. Restrain the tail of the rat in between your ring finger and little

finger. Let the rat lying on your left palm and introduce the ball-tipped feeding needle from the

pharynx in to the esophagus when the rat is in the act of swallowing.

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Blood withdrawal techniques in rats

Blood volume- 6% of the body weight. For eg 200g rat-12ml. Weekly 1% of the body weight

blood can be collected. For eg 200g rat- 2ml blood can be withdrawn.

a. Blood collection by retro orbital sinus

This method may be used to collect large volumes of blood. This should be performed under

anesthesia. Requirement- Cotton, capillary tube and capillary tubes, eppendorff tubes.

b. Blood collection from saphenous vein

The saphenous Vein is located along the lower portion of the hind leg. This technique is used for

collecting small volumes of blood. Multiple samples collected in same day. Needle- 23G

c. Blood collection by lateral tail vein

Used to collect a small to medium volume of blood. Using a sterile scalpel blade, nick the lateral

tail vein as shown in this picture. After collection of blood, apply gentle pressure to stop

bleeding.

Experimental Techniques in Pre-clinical Studies 10-3-2015 Pharmacology Dept PESCP Page 5