LAT CHAPTER 11: Performing Common Technical Procedures

Milt

fish sperm

Coelom

fluid-filled body cavity lined with mesoderm

Blood Brain Barrier (BBB)

physiological barrier between the circulatory system and the central nervous system that establishes a privileged blood supply, restricting the flow of substances into the CNS; a tissue lining that separates the circulating blood from the brain and thus protects the brain tissue from many chemicals.

Urinary Catheterization

methods of placing a tube into the bladder to drain or collect urine; a urinary catheter is placed into the urethra of an animal to allow urine to flow out of the body. Bacterial flora of the urethra may cause complications. The bladder and proximal urethra are normally sterile; the distal urethra contains bacteria, making it impossible to avoid carrying some bacteria into the bladder even with aseptic technique. ANOTHER complicating factor is the internal location of the urethral office in females of most species. Seeing the urethral orifice is generally necessary for catheter insertion...

Gastric Gavage - Aquatic

May be performed on amphibians, but drugs are poorly absorbed through the stomach by these species. To gavage Xenopus, anesthetize the animal unit it ceases swimming and loses its righting reflex when placed on its back. Use a 1.5-inch gavage needle, introducing it at the corner of the mouth and gently guiding into the stomach. Don't use a needle that is too long as it may perforate the stomach. For fish, they should be fasted prior to gavage so the solution flows freely into the esophagus.

The gastric gavage dose rate for fish should not exceed what percentage of body weight?

1%

Which devices may an endotracheal tube be attached to?

- Anesthetic machine - Automatic ventilator - Ambu bag (manual ventilation)

Why is the inflated cuff that holds the endotracheal tube in place important?

- Ventilation via the tube can be controlled - Prevents the passage of fluids (secretions from the mouth and throat) into the lungs

What is the maximum IM injection volume for a mouse?

0.05 mL (50 microliters)

The total blood volume in an animal's body equals approximately what percentage of its total body weight?

6%

When performing a gastric gavage in a large animal, what two devices are needed?

A bite bar and a flexible stomach tube -- tube cannot enter trachea as this would cause death ... if tube enters the larynx, coughing will ensue, so the tube should be quickly removed.

Which device is used to place an endotracheal tube in large animals?

A laryngoscope, illuminates and exposes the larynx (the opening to the trachea)

Retroorbital Injections

A method for injection that may be used in rodents, but is generally discouraged because of the potential for eye injury. Animal must first be anesthetized and placed in later recumbency. The eyeball should protrude slightly and be kept moist with a topical ophthalmic anesthetic to prevent drying of the cornea. A 25-gague needle, bevel side down, is injected where the eyelids meet (medial canthus) at a 30-degree angle toward the midline. No greater than 150 microliters should be injected in adult mice, 10 microliters for neonates.

Retroorbital Sinus or Plexus

A method of blood collection from species with a large venous sinus (mice, hamsters, gerbils, ferrets, and guinea pigs) or a plexus posterior to the eye like the rat. Pigs may be bled from a venous sinus ventral to the eye. Anesthesia recommended, but the procedure may be performed on conscious animals using a topical anesthetic. May be used to inject small volumes (<0.15 mL). Generally discouraged b/c of the potential for eye injury. At the end of the procedure, the eyelid should be held closed so the punctured sinus may clot. Ophthalmic ointment may also be applied following the procedure. Repeated sampling should be minimized.

Why is a lancet preferred to a needle for blood collection from the maxillary vein?

A needle can be inserted too deeply into the tissues

How are females catheterized?

A vaginal speculum and good illumination are required

If the bladder has been punctured, what will appear in the syringe when aspirating prior to an IP injection?

A yellowish aspirate

Intramuscular Injections - Aquatic

Amphibians may be given this injection in the quadriceps muscle. The maximum volume is usually less than 0.5 mL, depending on the amphibian's size. For fish, use the dorsal epaxial or abdominal muscles. Avoid the lateral line and the ventral blood vessels. For zebrafish, only administer volumes less than 0.05mL/50g fish. Administer slowly to prevent leakage.

Which device is used to help place an endotracheal tube in small animals like mice and rats?

An otoscope, used to illuminate and magnify the view of the larynx

Hypodermis

Aso called a subcutaneous layer, this is a layer of *fat located under the dermis of the skin. The hypodermis helps to insulate the body and protects underlying muscles and other structures.

What is a potential benefit from using a vacutainer for blood collection?

Blood samples can be collected directly into a tube containing anticoagulant, or if the cells are not required, then a tube containing a gel that will separate the cells from the serum after centrifugation

For larger fish, how are repeated blood collections taken?

By using the tail puncture technique

How can the operator ensure that the gavage needle length will not puncture the stomach?

Check the needle length by measuring the needle from the corner of the mouth to the xiphoid process at the caudal end of the sternum

Substance Administration

Common routes of injection include intravenous (IV), intraperitoneal (IP), intramuscular (IM), subcutaneous (SC/SQ), and intradermal (ID). Intracerebral (IC), the retroorbital sinus, and the hollow cavity of a bone (intraosseous) are other routes of injection. Gastric gavage is another route of administration.

What are some factors that should be considered for the substance when administering it via immersion?

Consider the chemical activity and the rate of uptake relative to water chemistry, temperature, and light

Why is the choice of anticoagulant used for blood collection in amphibians important?

EDTA may cause hemolysis; an alternative is heparin or citrate, but clotting may occur with either agent.

If an endotracheal tube needs to be repositioned after the balloon has been inflated, what should be done first?

Deflate the balloon.

What is one way in which fish and amphibians differ from mammals?

Fish and amphibians do not possess a peritoneal cavity. Instead, the internal body cavity that houses abdominal and thoracic organs is called the coelom.

What should be done prior to performing an ICo injection on a fish so that the abdominal viscera is avoided?

Fish should be fasted 24 hours prior to the ICo injection

Cerebrospinal Fluid

Fluid in the space between the meninges that acts as a shock absorber that protects the central nervous system.

Bleb

Fluid-filled raised area under the skin; indicates that an ID injection was performed correctly

Blood Collection - Amphibians

For frogs and salamanders, the mid-ventral abdominal vein can be used for blood collection. Xenopus lacks both a tongue and a tail, and therefore lacks both lingual and tail veins, the two most common anatomical sites used for blood sampling in other amphibian species. Easiest method is to take blood by cardiocentesis (cardiac puncture), a terminal procedure only performed on anesthetized animals to be euthanized post-procedure.

Blood Collection - Zebrafish

For zebrafish, blood collection is challenging because of their small size, small blood volume, and lack of accessible peripheral vasculature. The caudal vein is commonly used, but has a small diameter. The vein must be cut with a scalpel at the base of the caudal fin to collect blood... but tissue fluid can contaminate the sample. For an non-contaminated sample, perform cardiocentesis. An alternative method uses a fine diameter glass pipette to extract blood from the dorsal aorta... minimally invasive. Several samples may be collected by this method... do not exceed 3 microliters for zebrafish.

How do the routes of injections rank from fastest to slowest rate of absorption?

IV > IP > IM > SQ > ID

What might go wrong when performing an IV injection in a rodent's tail vein?

If the needle is not correctly inserted into the vein, any substance injected can be lost in the surrounding tissues.

What is a drawback of using medicated feed with fish?

It is difficult to accurately quantify intake; fish may have compounds administered by mouth either by mixing the substance into food/gelatin, by encapsulating the substance into brine shrimp larvae, or by esophageal gavage.

What should you do if a mouse gasps when manually restrained?

Lessen the tension on the nape so the animal may breathe easily.

Why apply light pressure proximal to the injection site when performing an IV injection?

Light pressure from a tourniquet an assistant's hand causes the vein to distend with blood and become more visible. Note that veins are easily collapsible, so only light pressure is needed.

In which quadrant should intraperitoneal injections be performed?

Lower right quadrant of the abdomen to avoid bladder and cecum

How is proper placement of an endotracheal tube verified?

MUST be done before connecting the tube to a gas anesthesia machine or another initiating procedure... - Listen to the end of the tube for breath sounds, hold a light fiber/animal hair to the tube opening and observe for movement as animal breathes - Hold a stainless-steel surface up to the end of the tube, look for condensation indicating expiration - If the tube is clear, watch for alternating condesation and clearing within the tube itself

How may the BBB be bypassed?

Make injections through the skull of an anesthetized animal and into the cerebrospinal fluid (CSF) within the ventricles of the brain.

How is the endotracheal tube different for small animals?

No cuff; the tapered tube seats itself in the larynx to seal the airway around the tube.

How are catheters sized?

On the French scale, abbreviated Fr. An 8 Fr catheter measures 2.7 mm around the outside diameter.

What volume may be injected at a single site for ID injections?

Only a small volume, 0.1-0.2 m; multiple injections are necessary for a larger quantity

Which health parameter should be monitored while an animal is anesthetized?

Respiration

Intraperitoneal Injections

Substances injected directly into the abdominal cavity; Almost exclusively performed in lab animals and hospital settings. Avoid puncturing the bladder and cecum and abdominal midline. 30-degree angle ALWAYS aspirate syringe prior to injection to confirm proper needle placement

How would a sterile urine sample be acquired?

The bladder may be tapped, a procedure called cystocentesis.

Why are tail snips controversial in older animals?

The cartilage of the tail is replaced by bone. Anesthesia is recommended.

Which body area is commonly used for injections for small volumes in mice and rats?

The lateral tail vein, located by swabbing the area with alcohol or by applying proximal pressure

What must be done before removing an endotracheal tube?

The cuff must be deflated by aspirating air through the injection port. When the pilot balloon has collapsed, it is then safe to withdraw the endotracheal tube.

Saphenous Vein

The longest vein in the body, it drains the leg, thigh, and dorsum of the foot; carries blood from the leg to the groin; on the lateral side of the lower rear leg and may be used for blood collection in both large and small laboratory animals. Anesthesia is not necessary, but appropriate restraint is important. Shave and prep site with antiseptic. For large animals, the vein is cannulated with a needle and blood is collected into a syringe/vial. For small animals, a hypodermic needle or lancet is used to puncture the vein, but NOT to cannulate it. Blood is collected as it wells onto the skin surface, either wicked up by a capillary tube or dripped into a small vial.

How is venous blood collection unlike IV injections?

The proximal pressure on the vein is maintained throughout the blood collection to keep the vein distended to pool blood to be drawn. Pressure is released once the required volume is withdrawn.

What does the rate of absorption of an injected substance depend upon?

The route of injection

Why are guinea pigs and chinchillas unique for gastric gavage procedures?

The soft palate is attached to the base of the tongue, which forms a membrane between the mouth and the pharynx. A small hole in the membrane (palatal ostium) allows food, water, and air to pass through. Damage to this membrane must be prevented.

Gamete Collection

The terms stripping and milking are used to refer to collecting either ova from females or sperm from males without harming them. Gametes may be cryopreserved.

For gastric gavage in a large animal, what is done with the stomach tube following administration of the solution?

The tube is folded or kinked to prevent aspiration of any remaining liquid during the withdrawal of the tube

Technical Procedures in Aquatic Species

These procedures focus on the two most common aquatic laboratory species, the zebrafish (Danio rerio) and the African clawed frog (Xenopus laevis).

Why are zebrafish rarely given percutaneous injections?

They have a small body size and therefore only a small volume can be given of a test substance. **The exception is VIE tags for ID.

Why may silicone grease be applied onto the skin when collecting blood from the skin's surface?

To help the blood well up and to minimize clotting both after disinfection and prior to vein puncture

Heart & Jugular Bleeds (Cardiac Puncture)

This method of blood collection is used when a large volume of blood is needed from small animals; performed as a terminal procedure; euthanized at the end of the blood collection. 1) The heart is accessed through the intercostal spaces (between the ribs) with the animal on its side OR 2) penetrate the diaphragm to reach the heart as the needle is directed cranially between the xiphoid process and the ribcage OR 3) introduce the needle from the top of the rib cage and into the right ventricle. Rats, dogs, pigs, and ruminants (sheep, goats) are routinely bled from the jugular vein. It is easy to access and allows for a large volume of blood to be collected.

How do most fish and many amphibians perform gas exchange?

Through either gills or skin; both contain rich capillary beds that allow gas exchange to occur, but also make the animals more sensitive to chemicals in the water.

Why is pressure applied once the IV needle is removed?

To allow the puncture site to form a clot (hemostasis) which prevents bleeding into the surrounding tissues (hematoma) and also keeps injected material from leaking out of the vein.

Why perform IM injections in either the quadriceps or biceps?

To avoid the sciatic nerve, the femoral vein, artery, and nerve that are located deep within tissues caudal/medial to the femur.

Why should blood be withdrawn slowly when collecting blood from the heart?

To prevent the heart from collapsing

Why is lidocaine sprayed onto the larynx during endotracheal intubation?

To prevent tissue swelling due to any trauma caused by inserting the tube through the larynx

Why may a urinary catheter be used in species including male cats?

To unblock the urethra

Why must you be sure not to inject too much fluid into a muscle?

Too large a volume would damage the tissue

Substance Administration - Aquatic

Two routes of administration unique to aquatic species are immersion and intracoelomic injections.

Which device is used for sampling for jugular bleeds in large animals?

Vacutainers -- blood flows in without needing to be under pressure

How is the gastric gavage needle designed to prevent injury to the animal's esophageal lining?

With a round, bulbous tip

What should you do if any aspirate is seen when aspirating prior to an IP injection?

Withdraw the needle, discard it, and start the procedure again. DO NOT REUSE NEEDLE/SYRINGE -- could cause contamination/injury/infection.

Why is gastric gavage difficult to perform on Xenopus?

Xenopus may regurgitate when stressed or if noxious substances are administered via gavage

Retroorbital Sinus

a complex network of blood vessels behind the eyeball.

Percutaneous Injections

a method to inject a substance into amphibians; injected just under the skin of the back, cranial to the cloacal papilla and medial to the lateral midline. Use a small (22-28) gauge needle. Inject a volume less than 0.5 mL.

Tail Snip (Transection)

a method used to collect several drops of blood from mice or rats. The end of the tail is prepped with disinfectant before 1-2mm of the tip is snipped off using a scalpel blade or scissors. A gauze sponge should be used to control any bleeding. Tissue glue may also be used.

Hematoma

a solid swelling of clotted blood within the tissues; a bruise

Subcutaneous Injections

are very similar to intramuscular injections except that they deliver medications into the layers of the skin rather than into the muscle. This results in a slightly slower absorption than with intramuscular injections. These injections are placed between the skin and the underlying muscle -- in the hypodermis. Avoid performing an IM injection or passing through the tented skin. Also prevent needlestick injuries to your body. Large volumes of non-irritating fluids may be administered by this method b/c the skin is only loosely adhered to underlying tissues.

Intramuscular Injections

generally result in lower but longer lasting blood concentrations than with intravenous administration; given into a large muscle mass, usually the hind limb... either the quadriceps (anterior to the femur) or the biceps (lateral to the femur). May also be given in the back/shoulder muscles. Aspirate prior to injection to confirm that the needle tip is not inside a vessel. Use another site for IM injection if blood is aspirated.

Visible Implant Elastomer (VIE)

an ID tag injected percutaneously; it is a small amount of nontoxic silicone dye that is injected under the skin in multiple locations (by the tail, under the eye, or by the pectoral fin)

Intradermal Injections

are administered into the dermis, just below the epidermis, at a shallow angle (10-20 degrees). This is NOT a painful procedure, but animals are usually anesthetized to immobilize them b/c it is difficult to accurately perform in a conscious animal. A 25 gauge or smaller hypodermic needle is often used with a 1 mL syringe. The needle is advanced only until its bevel is inserted within the skin. Routinely performed on NHPs for tuberculosis testing

Sagittal Suture

between parietal bones

Facial or Maxillary Vein

blood collection from this source is easy and safe for mice and rats and is preferred to retroorbital bleeding. Anesthesia is not required. To collect blood, a needle/lancet punctures the vessel behind the mandible and in front of the ear canal. The puncture is made at a 30-degree angle. Blood should flow immediately. Maintain gentle pressure at the puncture site until hemostasis occurs following blood collection.

Arterial Blood Collection

frequently collected in rabbits and pigs via the central ear (auricular) artery, easily seen along the center of the ear lobe or pinna. This example of blood collection may also be performed on dogs or NHPs, but from the femoral artery rather than the auricular artery. Similar to venous blood collection, except that proximal pressure is not applied to the artery because the artery is always filled with sufficient blood for collection.

Laryngoscope

hollow tube with a light and camera used to visualize or operate on the larynx; consists of a handle, long blade, and a light at the blade tip. The blade has smooth edges as to not traumatize the tissues.

Foley Catheter

indwelling catheter inserted through the urethra and into the bladder that includes a collection system allowing urine to be drained into a bag; the catheter can remain in place for an extended period. Has a balloon in the tip and a syringe port on the other end. Once the catheter (with balloon uninflated) is passed through the urethra and its tip reaches the bladder, a syringe is connected to the port to inject fluid or air, which inflates the balloon. The inflated balloon holds the catheter tip in the bladder, preventing it from slipping out.

Intracranial Injections

may take place in a stereotaxic apparatus; may be done freehand, particularly on neonatal animals with thinner skull bones. Identify the sagittal suture and points at its cranial (bregma) and caudal (lambda) ends

Endotracheal Intubation

placement of a tube through the mouth into the pharynx, larynx, and trachea to establish an airway; used when extended anesthesia is required (surgery). Provides an airway in which gas anesthetics or oxygen can be administered. For the tube to first be passed into the trachea, the animal is anesthetized with a short-acting injectable anesthetic. This is IMPOSSIBLE on a conscious animal. The tube is connected to a gas line from an anesthesia machine, maintaining anesthesia. Once proper placement is confirmed, a syringe filled with air is used to inflate a cuff that will hold the endotracheal tube in place. The cuff is located near the tip within the trachea. Once the cuff is inflated, the tube should not be moved or else the tracheal lining will be damaged.

Intracoelomic Injections

referred to as ICo. Insert needle in the lower right quadrant. Aspirate syringe so that no blood is seen -- if blood is aspirated, remove, discard, restart because the spleen may have been punctured. More common to be performed on amphibians than fish. If performed on fish, care must be taken to not penetrate the gut. ICo injections should be administered along the midline, cranial to the anus (urogenital pore) and caudal to the pectoral fins. Avoid the urogenital pore.

Immersion

state of being deeply engrossed; absorption; the administration of test agents/medication/anesthesia to an immersed animal. Aquatic animals are placed in water containing a substance (ex. anesthetic MS-222) which is then absorbed through the gills (branchial absorption) or the skin in larval fish and all amphibians (cutaneous absorption). This is the preferred method of anesthetizing fish.

Palatal Ostium

structure in the soft palate that divides oropharynx and the remainder of the pharynx; sensitive to trauma but must be bypassed to view epiglottis; present in guinea pigs and chinchillas.

Cystocentesis

surgical puncture of the urinary bladder, usually to collect urine; performed by passing a hypodermic needle through the surgically prepared abdomen and into the bladder. The animal should be tranquilized and the skin site for needle penetration may be locally numbed.

Gastric Gavage - Terrestrial

the administration of a solution directly into the stomach by intubation or injection down the esophagus. Effective for rodents/rabbits because they are physiologically unable to regurgitate stomach content. The needle may be rigid or flexible, curved or straight. Do NOT use force -- gravity and the weight of the needle are sufficient to advance the needle into the stomach. If any resistance is felt, STOP and remove the needle. Slight resistance will be encountered at the pharynx, usually causing the animal to swallow, which advances the needle/tube.

Intravenous Injections

the needle is inserted into a blood vessel; fast absorption and distribution of the substance within the body. Some substances must be administered by this method as they would otherwise irritate tissues when given outside the vessels. The injection site may be shaved/plucked/prepped with antiseptic to disinfect the skin and make the vessel easier to see. When completed, pressure is applied over the injection site as the needle is withdrawn.

Hemostasis

to stop or control bleeding

Catheter

tube for injecting or removing fluids; usually flexible with a tapered end. A sterile lubricating jelly should be applied to the tip before it is advanced through the urethra and into the bladder.

If the cecum/intestines have been punctured, what will appear in the syringe when aspirating prior to an IP injection?

A greenish-brown fluid

Blood Collection - Terrestrial

This is a common procedure to aid in the diagnosis of disease or to support data generation. Collection limit: a volume equal to 1% of the animal's total body weight can be safely taken every 2 weeks without causing side effects associated with blood loss (anemia).

Why apply pressure to an artery for several minutes following the withdrawal of the needle?

To prevent hemorrhage and hematoma formation; arteries take longer to clot than veins

Peritoneum

a multilayered membrane that protects and holds the organs in place within the abdominal cavity

When performing maxillary blood collection in white rats, which landmark should be used?

A gray spot on the jawline, directly below the lateral canthus (corner) of the eye; the puncture site is approximately 0.5 cm behind the gray spot.