70

Laboratory

Animals

(1986) 20.70-72.

A better method for terminal bleeding of mice

A. J.-H. ADEGHE & J. COHEN
Department of Zoology and Comparative Physiology, University of Birmingham, P. O. Box 363, Birmingham, B/52TT, United Kingdom
Summary A new method for exsanguinating mice is described. The procedure is carried out under COranaesthesia and involves venepuncture of the caudal (inferior) vena cava and gentle aspiration of blood into a syringe. It consistently gives large volumes of blood (1,60-250 mllmouse), free from haemolysis and contamination. The technique is easy, quick and reliable. of adequate volumes of normal sera or antisera (antispermatozoal in our laboratory). Some techniques are designed specifically to yield small volumes of blood (Stoltz & Bendall, 1975) and so enable a serial study of desired parameters. Other techniques, intended to yield larger volumes of blood, have various limitations, e.g. cardiac puncture gives results which are inconsistent qualitatively (Cubitt & Barrett, 1978) and quantitatively, and most are found unreliable (e.g. Lewis, Thacker, Mitchell & Baer, 1976). It is important that any technique used to obtain blood should not allow contamination by any secretion or tissue fluids or cause significant haemolysis. Table 1 compares various techniques for mouse-bleeding. This report describes a new method for collecting consistently large volumes of unadulterated blood from mice.
methods for mouse-bleeding 1976)

Keywords: Mice; Bloodletting

Many techniques exist for bleeding mice (CunliffeBeamer, 1983). Small or larger volumes of blood may be required, depending on the use for which it is intended. While small volumes (up to 05 ml) would suffice for many haematologic or biochemical studies, larger amounts are needed for preparation
Table I. Comparison of various

I. Fro/II lail blood vessels (e.g. Stoltz & Bendall.

1975: Fields & Cunningham.

Yields small amounts of blood U5 ml). Larger volumes (up to O'71ml) may be obtained by heparinization/warming up the mouse to 45C - (Lewis el /II . 1(76). Disadvantages of this method arc: (a) requires several special gadgets. (b) warming up to 4SoC is discomforting to the mouse, (c) tail necrosis is a problem after several repeats. 2. From dors/llme/alarsal vein (Nobunaga. Nakamura 8:. Imamichi. 1966)

Gives small volumes and the vein may not be readily visible. 3. Cardiac pllllC/IIre (e.g. Cubitt & Barrett. 1978; Frankenberg. 1979)

Variable volumes of blood; found unsatisfactory by several workers; blood likely to be haemolysed (Lewis et /II 1976) and contaminated by tissue fluids. especially when performed as a closed (blind) technique. 4. From Ihe orbital Vel/Oil.\'sill liS (Stone. 1954)

Involves puncture of ophthalmic venous plexus by a microcapillary glass tube. Angle of insertion of tube is critical; many trials required for acquisition of skill: blood unsuitable for some biochemical studies (e.g. LDH levcls) bccause of haemolysis. Furthermore the procedure is distasteful to many workers. 5. Severillg diJferenl blood vessels The method of Young & Chambers (1973) on brachial vessels. gives volumes of I {1-l'S ml. but has the disadvantage of exposing blood to surrounding tissues. The jugular vein, carotids, femoral vessels. and abdominal aorta have been used by different workers: generally. volumes are less than 10 ml.

n.

From tile caudal (il/ferior)

vena cava (present

report) Consistently /985. yields 16(1--250 mlimoLise of whole blood free from any

By venepuncture and gentle aspiration. significant haemolysis. Received 10 July /985. Accepled /8 September

Terminal

bleeding of mice

71

Materials and methods The materials required are: a small vein set (Portex Ltd., Kent, UK; also called scalp-vein set) which consists of a 23-G 'butterfly' needle attached to a narrow transparent tubing, and a 5-ml syringe, connected to the Luer fitting of the small vein set. The mouse is anaesthetized by carbon-dioxide inhalation (Green, 1979) and laid on a dissecting board on its back, with the limbs secured onto the board. The abdomen is swabbed with 70% alcohol and opened by a midline longitudinal incision about 2 cm long. The skin flaps are pulled aside, the peritoneum is opened and the abdominal viscera displaced to expose the vertebral column. Next, the caudal (inferior) vena cava, lying to the right of the vertebral column and the abdominal aorta, is identified and dissected free of surrounding fascia. Air is drawn into the syringe through the attached small vein set, up to about the 05 ml mark, to free the piston and reduce resistance within the syringe. The needle is then carefully introduced into the inferior vena cava, starting from just above the confluence of the common iliac veins, until about 05 cm is in the lumen. As soon as the vena cava is punctured blood rises into the small vein set, aided by capillary action. Further gentle suction is applied with the syringe until blood flow into the syringe ceases (about a minute). At the end of the procedure, the mouse has usually died from exsanguination but notwithstanding the diaphragm and heart are cut with scissors or the neck dislocated. The technique was used on 20 mice (12 male, 8 female) aged 3-6 months and of different strains (Birmingham strains of Strong-A (related to A/J strain). C57BL/6, C57BL/10 and IStrong-A xC57BLllO] FI). The samples of blood were pooled, allowed to clot over 24 h at 4C and then centrifuged at 1500 g for preparation of serum.

of pooled blood from the 20 mice was 420 ml which yielded 180 ml of serum free from any significant haemolys,is. It was found important to watch out for, and avoid, premature deaths from COT narcosis as when performed on a dead mouse 5 min of apparent death) smaller volumes were obtained. Discussion This method has several advantages. It consistently yields large volumes of blood free from haemolysis and contamination and there is no prior exposure to tissue fluids before collection. It is easy and quick to perform and because the vena cava is relatively a big vessel, venepuncture is done accurately with very little chance of perforation. The variation of yield from mouse to mouse (160-25 ml) in our series is not unexpected as the total blood volume in mice may vary with age (Masters, Leslie & Kaldor, 1972), strain (Kano & Mizuma, 1974) and weight. The technique should find special use in commercial production of mouse serum (normal or immune) and may also find application in other smalliaboratory rodents, e.g. rats and hamsters. However the following points about the technique should be noted: (a) Very gentle suction only should be applied to the syringe as sudden or forceful aspiration causes haemolysis of red blood cells and damage to the vessel wall, and must be avoided. (b) If perforation of the vena cava occurs and there is spillage of blood around the surrounding tissues, that blood should be considered contaminated and unsuitable for some uses. (c) COz-anaesthesia is unsuitable when blood samples are to be obtained for blood gas measurements. Acknowledgements We wish to acknowledge the assistance of Ms April Dorsey and express our gratitude to Mrs Jean Hill for typing the manuscript. The study was done under a Commonwealth Medical Award No. NIGERIA M12/84 made by the Commonwealth Scholarship Commission in the UK, to Dr A. J. H. Adeghe.

Results The caudal vena cava was found easy to locate and clean. The whole procedure, from skin incision to complete aspiration of blood, took about 2 min for each mouse and the volume obtained ranged from 160 to 250 ml with a mean of 2\0 ml. The volume

References Cubitt, J. G. K. & Barrett, C. P. (1978). A comparison of serum calcium levels obtained by two methods of cardiac puncture in mice. Laboratory Animal Science 28. 347. Cunliffe-Beamer, T. L. (1983). Biomethodology and surgical techniques. In The Mouse in Biomedical Research, Vol. 3. (Eds H. L. Foster, J. K. Small & J. G. Fox). pp. 408-410. London: Academic Press.

Fields, B. T. Jr. & Cunningham. D. R. (1976). A tail artery technique for collecting one-half millilitre of blood from a mouse. Laboratory AI/ill/al Sciel/ce 26, 505-506. Frankenberg, L. (1979). Cardiac puncture in the mouse through the anterior thoracic aperture. Laboratory Animals 13, 311-312.

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Green, C. J. (1979). Animal Anaesthesia. Laboratory Animal Handbooks 8, pp. 147-154, London: Laboratory Animals Ltd. Kano, K. & Mizuma, Y. (1974). Comparison of the total blood volume in four inbred strains of mice. Ex-

Adeghe & Cohen

Nobunaga, T., Nakamura, K. & Imamichi, T. (1966). A method for intravenous injection and collection of blood from rats and mice without restraint and anaesthesia. Laboratory Animal Care 16, 4()-49. Stoltz, D. R. & Bendall, R. D. (1975). A simple technique for repeated collection of blood samples from mice.

perimelllal Animals 23, 123-127.

Lewis, V. J., Thacker, W. L., Mitchell, S. H. & Baer, G. M. (1976). A new technique for obtaining blood from mice. Laboratory Animal Science 26, 211-213. Masters, A. M., Leslie, A. J. & Kaldor, 1. (1972). Red cell and plasma volume development in newborn rats measured with double label. American Journal of

Laboratory Animal Science 25, 353-354.

Stone, S. H. (1954). Method for obtaining venous blood from the orbital sinus of the rat or mousc. Science, New

York 119, 110.

Young, L. & Chambers, T. (1973). A mouse bleeding techniquc yielding consistent volume with minimal haemolysis. Laboratory Animal Science 23, 428-430.

Physiology 222, 49-54.

Eine bessere

Methode fUr den terminalen & J. COHEN

AderlaB Illei Mausen

A. J.-H. ADEGHE

Zusammenfassung Es wird eine neue Methode zum Entbluten von Mausen beschrieben. Das Verfahren wird unter CO~-Anasthesie ausgeflihrt und unfaBt die Venenpunktion der Vena cava inferior (posterior) und die behutsame Aspiration von Blut

in cine Spritze. Es liefert gleichbleibend groBe Mengen Blm (160-250 mIlMaus), frei von Hamolyse und Kontamination. Die Methode ist einfach, schnell und zuverlassig.