Download SOP No - Griffith University

Griffith University
Animal Ethics Manual- Laboratory
STANDARD OPERATING PROCEDURE (SOP)
SOP No:
L-4
SUBJECT:
Isolated (in vitro) Perfused Rodent Heart Preparation.
POLICY:
For anaesthesia/euthanasia of wild type or transgenic rodents
(mouse/rat/guinea pig/rabbit) and subsequent surgical removal
and mechanical maintenance of entire isolated hearts. On the
GUAEC application form Project Classification is 1.3.1.(B).
PRECAUTIONS:
Personal Protective Equipment (Lab coat/gown, gloves, eye
protection and masks) should be worn by technician.
Sharps and biological fluid (blood/urine/tissue) hazards apply
and all risk assessments need to be in place.
EQUIPMENT:
Surgical table and appropriate instruments.
22-30g needles, 1-2.5 ml syringes.
Suitable injectable or inhalation anaesthetic agent as
appropriate for species (see Ref. 1) Examples:
Pentabarbitone (NembutalTM, LethabarbTM), Isoflurane,
Ketamine/Xylazine, Fentanyl/Fluanisone, Zoletil/Xylazine.
Mechanical support / perfusion system.
Animal carcass storage.
PROCEDURE:
Many minor variations to the equipment and perfusion
conditions exist. First described in 1895 by Langendorff (Ref
2) and known as the “Landendorff” Perfused Heart Model.
Reviewed at http://www.usouthal.edu/ishr/help/hearse/ (Ref 3)
and Neely (Ref 4) and Ross (Ref 5) are key references.
ANIMAL PREPARATION FOR SURGERY.
Species, gender, age, weight, diet and other conditions may vary according to
AEC approved project protocol and all pre-surgical animal procedures must
comply with the Code (Ref 6). The anaesthetic regime must be terminal and
surgery conducted using a pain free level of analgesia /anaesthesia.
1. Procedure is to take place in a quiet, clean environment,
separate from other animals. Refer to Physical Containment
(PC) guidelines for location classification appropriate for
species.
2a. To inject anaesthetic of required dosage (mg/kg body weight)
intraperitoneally, restrain the animal by a method appropriate for
the species.
2b. For inhalation anaesthetic in a fume extracted environment,
induce anaesthesia with appropriate gas mixture. Monitor
animals carefully in cage or fume hood for adequate depth of
anaesthesia.
1
3. Begin surgery immediately upon the loss of species specific
reflexive and pain responses (e.g. pedal reflex, blink reflex).
4.
Open the skin with a midline incision from neck to midabdomen. A sternotomy (open rib cage) is performed. Excise
the heart by blunt dissection of connecting vessels and place it
into ice cold crystalloid buffer (salts/water). This can take an
experienced technician 30-60 seconds.
5. Euthanasia by exsanguination (blood loss) will occur.
6. Seal carcass and transfer to dedicated -18oC+ freezer storage
and holding area (unless protocol specifies secondary uses of
tissues). Permanently remove or destroy carcass using local
Hazardous Waste Procedure.
PERFUSED HEART PREPARATION
1. Transfer the heart quickly to external perfusion. Reconnect it to
a warm, pressurised and gassed crystalloid buffer (or donour
blood, if approved by protocol) until heart function returns and
defined experiments can proceed. See diagram below.
2. The isolated heart can experience mechanical, physical or
pharmacological treatments depending on the AEC approved
protocol. Treatment periods may be from 10 minutes to 6 hours
and heart tissue may be dissected, preserved, stored or
disposed (see 6. above). The heart can also be liberated of its
component individual cells (myocytes) and further cell based
experiments conducted.
Perfused heart system (Figure 1.) and a perfused rat heart (below).
2
RECOMMENDATIONS:
The procedure must be conducted by technicians previously certified competent in
small animal care, restraint, IP injections and species specific anaesthesia /
euthanasia techniques or, under the direct supervision of such a competent person.
Pre-surgical animal care and welfare or pre-treatment can modify ethical
considerations or project classification 1.3.1. (B) “euthanasia to obtain tissues or
samples…”.
DATE ISSUED:
REVISED:
June 2008
2011
Dr G Harrison
CHAIR OF GUAEC
REFERENCES
1. Flecknell PA. Laboratory Animal Anaesthesia, 2nd Ed. 1996. Academic Press. ISBN10: 0122603613
2. Langendorff O. Untersuchungen am uberlebenden Saugethierherzen. Pflugers
Archives fur die Gesamte Physiologie des Menschen and der Tiere 1895; 61:291332.
3. Sutherland FJ, Hearse DJ. The isolated blood and perfusion fluid perfused heart.
http://www.usouthal.edu/ishr/help/hearse/
4. Neely JR, Liebermeister H, Battersby EJ and Morgan HE. Effect of pressure
development on oxygen consumption by isolated rat heart. American Journal of
Physiology 1967; 212:H804-H814.
5. Ross BD. Perfusion Techniques in Biochemistry: Chapter 5. Clarendon Press.
Oxford 1972.
6. NHMRC (2004) Australian code of practice for the care and use of animals for
scientific purposes (7th edition). Australian Government.
3