Dissection Manual

1 Dissection Manual

The species, also known as the common rat are originated from

Southeast Asia. Their emigration spread globally through out Europe,

Africa and eventually reached South and North America. Rats are well known for their similarities in both physiological and anatomical features with human beings. Moreover, their high reproductive rate and the ease of maintenance in a laboratory setting have made them ideal candidates for medical research and diagnostic purposes. Thus, the use of rats in the anatomy lab as a model for small animals provides the students with a fundamental understanding of the typical mammalian anatomy. The purpose of this manual is to present a clear, easy- to-follow procedure in order to enhance the learning experience and obtain a lung cast.

2 Dissection Manual

The body of the rat consists of a head, neck, trunk and tail.

3 Dissection Manual

4 Dissection Manual

Measure Lung Volume Measure Lung Volume Pre-casting

AnesthetizeAnesthetize and and Intubate Intubate Animal Animal

Flush Lungs with CO Gas Flush Lungs with CO2 Gas 2 Casting

InjectInject Saline Saline into into Lungs Lungs

InjectInject Casting Casting Material Material into into Lungs Lungs

ExciseExcise Lungs Lungs

DigestDigest Tissue Tissue Post-casting

EvaluateEvaluate Cast Cast

5 Dissection Manual

Measurements

1. Place an animal on a scale and measure its weight. The weight generally ranges from 200 to 300 grams.

2. Approximate lung volume by multiplying rat’s weight by 0.35%

6 Dissection Manual

Anesthetizing

1. Prior to administering anesthetizing doze, the animal is placed under

a heating lamp for approximately 5 to 10 minutes. Heating increases

the blood flow dilating the veins. In the adult rat the large vein in the

tail is most accessible. The lateral veins along the sides of the tail

are most accessible, therefore are chosen as the route of

intravenous injection.

2. Restrain the rat as shown in the diagram below to gain access to its

7 Dissection Manual

3. Administer an anesthetizing dose of Sodium Pentobarbital through

the lateral vein. The dose is ~ 0.5 mg. Drug is provided by RIT’s

Biology department. After injecting an animal with Sodium

Pentobarbital, verify that the animal is dead.

8 Dissection Manual

Intubating

1. Place a rat on its back in dissection tray. Pin legs down if necessary.

2. Spray or swap alcohol on rat’s chest to mat down fur.

3. Carefully cut from throat to ribcage with scissors. Use tweezers to

pull away muscles and membrane around trachea. Make sure not to

cut through an artery, or the blood flow will make the procedure

more difficult. And more importantly, do not accidentally puncture

the lungs. Wear gloves throughout the entire procedure.

9 Dissection Manual

4. Make a small incision in trachea inferior to the 3rd-5th tracheal ring.

Cut off 5 to 6 inches of tubing (Inside Diameter = 0.0567” = 0.144 cm).

Cut one end to a 45° tip. Carefully feed tipped end (cut at 45°) into

trachea and tie off with suture.

10 Dissection Manual

Carbon Dioxide

1. Connect tracheal tube to a Carbon

Dioxide (CO2) tank.

2. Regulate CO2 output with a

pressure relief valve and keep the

pressure to 30 cm H2O or lower

(0.427 psi). Inflate and deflate lungs

10 times. CO2 replaces O2 and N2 in the lungs. Carbon Dioxide helps produce a bubble-free cast, because

CO2 is more water soluble than oxygen or N2.

Saline

1. Calculate the amount of degassed saline for the procedure. Find

0.35% of rat’s body weight. Saline is used

to dissolve CO2 gas in the lungs.

2. Slowly inject saline into lungs through

tracheal tubing by using 5-10 cm column

of saline (equivalent pressure is 0.07 to

0.14 psi). Total infusion time is

approximately 15-30 minutes.

3. To accommodate for the long saline injection time, use a Braintree

Scientific model BSP-99M programmable syringe pump. The pump

can handle syringe as large as 50 CC and as small as 5 CC. Adjust

the flow of saline to 10 cubic centimeters per minute.

Casting Material

1. Determine the amount of casting material (Silastic E) needed. Use 3-5

lung volumes. Curing

2. Mix casting material with

its catalyst using a

stirrer. Use 10:1

mass ratio.

3. With programmable syringe pump, slowly inject Silastic E through

tracheal tubing and into the lungs. Filling time is between 60-90

minutes (20 CC/min). The long injection time avoids excessive

pressure inside the lungs and is within working time of the casting

material. Silastic E displaces saline in the lungs and allows saline to

diffuse through the pleural membrane.

Uncured Properties Color white Density [gm/cm3] 1.12  When casting material is fully Viscosity [poises] 1200 Cured Properties injected into the lungs leave Tensile Strength [psi] 700 Temp. Stability [F] 500 Shrinkage after a week [%] 0.1

everything intact. Do not pull tubing out of trachea or syringe out of

tubing. Cover the dissection tray with plastic lid and allow Silastic E to harden for the next 24Excision hours at room temperature.

1. Carefully excise the lungs from the thorax (chest cavity).

2. Place the cast in fresh 1M NaOH solution for another 24 hours to

digest tissue.

3. Dispose of the rat.

Evaluation

1. After the lung

tissue has

digested for 24 hours, carefully peel off remaining tissue with

tweezers.

2. Neutralize the cast with a basic solution of HCl and wash in soap and

water. Allow to dry for 5 minutes.

3. Evaluate the cast.

STEP 1:

 Weigh the animal after administering an anesthetizing dose.

STEP 2:

 Use steel mesh gloves to handle a rat.

 Make sure the restraining device is secured to the work surface.

STEP 3:

 If unable to locate tail vein after exposure to heat lamp, first give

small inter-muscular injection to knock the animal out, and then

follow with a larger injection directly in the heart.

 Be careful to not puncture the lungs if giving an injection directly in

the heart.

 Try to leave the ribcage intact; it helps keeping the lungs in their

natural shape when inflated.

 Do not cut into the lungs while exposing the trachea. If this happens,

the lungs will collapse.

 If there is an excessive bleeding, use a Kimwipe or paper towel to

absorb blood.

 Rotate hose while inserting trachea tube to help guide it in.

 Have one person tie off intubation hose with suture string, while the

other is holding the hose in the trachea.

STEP 4:

 Ensure inlet pressure to secondary low pressure regulator does not

exceed 30 psi (It could damage secondary regulator). Practice

controlling pressure with regulator before actually working with the

rat to prevent potential lung deformation.

STEP 5:

 Be sure to pinch off intubation hose while switching to saline to

prevent CO2 from escaping.

 Use degassed saline.

 While injecting saline, make sure you don’t have any air bubbles in

the tubing.

 Use BSP-99M flow rate chart to determine correct setting.

STEP 6:

 Do not use tubing longer than 4-6 inches. This will increase the

injection time to several hours.

 Use a high accuracy digital scale to determine amount of Silastic E

and catalyst.

 Use exactly 10:1 mass ratio.

 Excessive hardener causes the silicone rubber to cure faster, but

there is more shrinkage.

 Too little hardener results in longer curing time and sticky rubber.

 The highly viscous casting agent can cause the hose to separate

from the syringe. Securely attach it to the syringe pump with a clamp

or a rubber band.

STEP 7:

 Keep rat at room temperature and out of direct sunlight during the

curing time to slow decomposition process.

 Cover the tray with a lid.

 Do not lift the lid for the next 24 hours.

STEP 8:

 Remove heart first, then slowly and carefully lung connective tissue.

It may be helpful to remove the entire ribcage to facilitate excision.

Be careful to avoid stomach and other digestive organs.

 Double bag rat in thick plastic bags, clearly label contents, and place

in non-food storage freezer. Contact local humane association for

disposal.

STEP 9:

 Remove any remaining tissue with tweezers; leave the cast in NaOH

solution to digest tissue for several more hours if necessary.

 Pour HCl in the same container as NaOH and lungs to fully

neutralize. Rinse lungs for several minutes in tap water and carefully

clean with mild detergent.

 Neutralized solution is safe to pour down drain. Do pH test just to

make sure!