Dissection Manual

Dissection ManualDissection Manual1 Dissection ManualThe 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 ManualThe body of the rat consists of a head, neck, trunk and tail.3 Dissection Manual4 Dissection ManualMeasure Lung Volume Measure Lung Volume Pre-castingAnesthetizeAnesthetize and and Intubate Intubate Animal AnimalFlush Lungs with CO Gas Flush Lungs with CO2 Gas 2 CastingInjectInject Saline Saline into into Lungs LungsInjectInject Casting Casting Material Material into into Lungs LungsExciseExcise Lungs LungsDigestDigest Tissue Tissue Post-castingEvaluateEvaluate Cast Cast5 Dissection ManualMeasurements1. 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 ManualAnesthetizing1. 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 tail.7 Dissection Manual3. 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 ManualIntubating1. 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 Manual4. 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 Manual11 Dissection ManualCarbon Dioxide1. 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, becauseCO2 is more water soluble than oxygen or N2.12 Dissection ManualSaline1. 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. 13 Dissection ManualCasting Material14 Dissection Manual1. Determine the amount of casting material (Silastic E) needed. Use 3-5 lung volumes. Curing2. 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 15 Dissection Manual 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).16 Dissection Manual2. Place the cast in fresh 1M NaOH solution for another 24 hours to digest tissue.3. Dispose of the rat.Evaluation1. After the lung tissue has17 Dissection Manual 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.18 Dissection ManualSTEP 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.19 Dissection Manual 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 6:20 Dissection Manual 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 8:21 Dissection Manual 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!22

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