Cardiopulmonary Block Harvest from Rabbit: A Surgical Procedure to Retrieve Functional Heart-Lung Block from Rabbit Model

Overview

In this video, we show a procedure to surgically harvest a functional cardiopulmonary block from a rabbit model. The harvested block can be used for further physiological, pharmacological or biochemical studies.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Equipment and preparation of apparatus.

1. Equipment arrangement:
   1. Set up an operating table with size according to the weight of the rabbit.
   2. Mount the cover of the artificial thorax on the steel column with the glass chamber underneath and the ventilator with a roller pump by the sides.
   3. Ensure that the cover is easily inclined to have the tracheal cannula in line with the trachea to allow a faster connection.
2. Artificial thorax:
   NOTE: It is an essential part of the system. It consists of a water-jacketed glass chamber sealed by a special cover. The cover works as the organ holder with the connections to cannulate the trachea and vessels embedded in it.
   1. Set up a venturi jet operated by compressed air to generate the negative pressure inside the artificial thorax.
      NOTE: The ventilation control module (VCM) allows separate adjustments of inspiratory and end-expiratory pressures as well as respiration rate and the ratio of inspiratory duration to total cycle duration.
3. Apparatus:
   1. Ensure that a normally working apparatus consists of a main steel column mounted on a base plate holding the artificial thorax, with the pneumotachometer and weight transducer located above it and behind the preheating coil with a bubble trap.
   2. Connect one differential pressure transducer to the pneumotachometer and another to the chamber pressure. Set a different couple of pressure transducers behind the thorax to measure perfusion and venous pressures.
   3. Connect the changeover stock below the oxygenator with a level electrode and the ventilation system beside the apparatus.

2. Surgical extraction of the cardiopulmonary block.

1. Anesthesia:
   1. Use a combination of a sedative (xylazine) and a barbiturate (pentobarbital).
      NOTE: Different anesthetic cocktails can be used with no effect on experimental outcomes.
   2. First, sedate the healthy New Zealand rabbits with a single intramuscular injection of xylazine hydrochloride (3-5 mg/kg). Ensure that the rabbits remain calm and relaxed to allow further manipulation after a few minutes of the injection.
   3. Following sedation, use the marginal (lateral) ear veins as access to anesthetize the rabbits with an intravenous injection of pentobarbital sodium (28 mg/kg).
2. Monitoring:
   1. To avoid insufficient anesthesia or excessive depression of cardiac and respiratory functions, monitor the following parameters. To assess the depth of anesthesia, perform a toe pinch test.
   2. Ensure that the mucous membrane is pink. Blue or gray shades indicate hypoxia.
   3. Ensure that the heart rate is between 120-135 beats/min, and that the body temperature does not drop below 36.5 °C.
3. Animal placement:
   1. Shave the rabbit's torso and place the animal on the operating table in supine position. Place the ventilation system near the table, behind the rabbit's head, to permit connecting the cannulae quickly after tracheotomy to avoid tissular damage.
4. Incision and tracheotomy:
   1. Dissect the skin with a ventral median line incision of 3-5 cm from the diaphragm up to the neck.
   2. With the operating scissors, cut the anterior 2/3 of the trachea between two cartilage rings to insert the tracheal cannula through the tracheal fibrous membrane.
   3. Insert a 5 mm (outer diameter; OD) tracheal cannula through the tracheal fibrous membrane and use a 4-0 silk suture to fix it carefully.
   4. Place either forceps or tweezers underneath the trachea to ensure the cannula did not bend against the trachea.
5. Positive-pressure ventilation:
   1. As long as the lungs remain outside the artificial thorax, use a small species respiration pump to ventilate a positive pressure in order to avoid lung collapse during the surgery.
   2. Initiate ventilation through the tracheal cannula connected to the respiration pump quickly after tracheotomy and before the thorax is opened.
   3. Set the tidal volume at 10 mL/kg.
      NOTE: Depending on the experiment setup and artificial thorax model, provide positive-pressure ventilation by either the same ventilation pump used to provide negative-pressure or a different one, granting a quick re-cannulation.
6. Thoracotomy and exsanguination:
   1. To access the thoracic cavity, use a scalpel or scissors to open the thorax wall and perform a medial sternotomy up to the upper third of the thorax.
   2. Hold the thorax halves open by two retractors. Several lung flaps usually surround the heart.
   3. Localize the superior and inferior vena cava and refer them with threads.
   4. Prior to the exsanguination of the animal, identify the right ventricle and inject 1000 UI/kg of heparin.
   5. Immediately after the injection, ligate the superior and inferior vena cava with the pre-looped thread and perform exsanguination.
7. Heart-lung harvest:
   1. Harvest the cardiopulmonary block gently and quickly. Use direct digital dissection or spring scissors to separate the connective tissue so as to remove the lungs from the thorax.
   2. Dissect the vasculature in the area, as well as the esophagus.
   3. Cut through the manubrium sterni to extend the medial sternotomy towards the tracheal cannula, releasing the trachea on both sides from connecting tissue.
   4. Now, resect the trachea above the tracheal cannula. Gently pull up the cannula in a craniocaudal axis as the dorsal fixation of the trachea and lungs is resected.

Materials

Name	Company	Catalog Number	Comments
2-Stop Tygon E-Lab Tubing, 3.17 mm ID, 12/pack, Black/White	Hugo Sachs Elektronik (HSE)	73-1864
Adapter for Positive Pressure Ventilation on IPL-4	Hugo Sachs Elektronik (HSE)	73-4312
Alternative Pressure-Free Gas Supply for IPL-4: To supply the trachea with gas mixture different from room air during negative ventilation	Hugo Sachs Elektronik (HSE)	73-4309
Base Unit for the Rabbit to Fetal Pig Isolated Perfused Lung	Hugo Sachs Elektronik (HSE)	73-4138
Bovine serum A2:D41albumin lyophilized powder	sigma	3912	500 g
Differential Low Pressure Transducer DLP2.5, Range +- 2.5 cmH2O, HSE Connector	Hugo Sachs Elektronik (HSE)	73-3882
Differential Pressure Transducer MPX, Range +- 100 cmH2O, HSE Connector	Hugo Sachs Elektronik (HSE)	73-0064
Ethanol absolute HPLC grade	Caledon
Harvard Peristaltic Pump P-230 (Complete with Control Box and P-230 Motor Drive)	Hugo Sachs Elektronik (HSE)	70-7001
Heated Linear Pneumotachometer 0 to 10 L/min flow range	Hugo Sachs Elektronik (HSE)	59-9349
Heater Controller for Single Pneumotachometer 230 VAC, 50 Hz	Hugo Sachs Elektronik (HSE)	59-9703
Heparin	PISA		5000 UI
IPL-4 Core System for Isolated Rabbit to Fetal Pig Lung, 230 V	Hugo Sachs Elektronik (HSE)	73-4296
Lauda Thermostatic Circulator, Type E-103, 230 V/50 Hz, 3 L Bath Volume, Temperature Range 20 to 150°C	Hugo Sachs Elektronik (HSE)	73-0125
Negative Pressure Ventilation Control Option with Pressure Regulator for IPL-4	Hugo Sachs Elektronik (HSE)	73-4298
New Zeland rabbits
PISABENTAL (Pentobarbital sodium)	PISA	Q-7833-215
PLUGSYS Case, Type 603* 7	Hugo Sachs Elektronik (HSE)	73-0045
PLUGSYS TCM Time Counter Module	Hugo Sachs Elektronik (HSE)	73-1750
PLUGSYS Transducer Amplifier Module (TAM-A)	Hugo Sachs Elektronik (HSE)	73-0065
PLUGSYS Transducer Amplifier Module (TAM-D)	Hugo Sachs Elektronik (HSE)	73-1793
PLUGSYS VCM-4R Ventilation Control Module with Pressure Regulator	Hugo Sachs Elektronik (HSE)	73-1755
PROCIN (Xylacine clorhydrate)	PISA	Q-7833-099
Pulmonary Artery Cannula for Rabbit with Basket, OD 4.6 mm	Hugo Sachs Elektronik (HSE)	73-4161
Scalpel knife
Servo Controller for Perfusion (SCP)	Hugo Sachs Elektronik (HSE)	73-2806
Surgical gloves No. 7 1/2
Surgical gloves No. 8
Tracheal Cannula for Rabbit, OD 5.0 mm	Hugo Sachs Elektronik (HSE)	73-4163