INJECTION OF BLOOD VESSELS.
Injection of blood vessels may be performed on small animals, or on
individual human organs after removal from the body. The object is to
fill the vessels with a coloured fluid which will solidify afterwards.
It is possible in the same organ to inject the arteries with a red
medium, the veins blue, and secretory ducts, such as bile ducts, yellow
or blue.
The most convenient basis for an injection mass is gelatine, as its
solutions liquefy at a temperature of about 100° F., and solidify a
little below that point, and when solidified cut readily, and do not
tend to become brittle. The various masses are prepared as follows:--
*Red injection mass* (Woodhead’s formula) consists of gelatine softened
by mixture with water and coloured by carmine.
(1) Carmine 4 grms.
Liq. ammoniæ B.P. 8 grms.
Distilled water 150 c.c.
Dissolve the carmine in the ammonia in a mortar. Pour on the water. Mix
thoroughly and filter.
(2) Gelatine 10 grms.
Distilled water 50 c.c.
Allow it to stand in the cold water until the water is absorbed and the
gelatine has become soft.
Warm (1) almost to boiling point over a Bunsen burner, and add the
gelatine slowly. Stir thoroughly and add a ten per cent. solution
of acetic acid until the solution becomes slightly acid. This will
be shewn by the mass assuming a darker and duller colour. A little
salicylic acid may be added to preserve it.
*Blue injection mass.*--To the gelatine mass (2) prepared as above, and
liquefied by heat, add instead of carmine
Soluble Prussian blue 5 grms.
Distilled water 60 c.c.
Every trace of alkali must be kept away from the mass during and after
the preparation. Sections of injected organs should be mounted in
Farrant’s solution slightly acidulated with formic or acetic acid. With
every care, however, the blue colour is apt to fade in the course of
time.
*Green injection mass.* Robin’s formula (modified).
(1) Arseniate of soda (sat. sol.) 80 c.c.
Glycerine 50 "
(2) Sulphate of copper (sat. sol.) 40 "
Glycerine 50 "
Mix and add one part to three parts of the gelatine mass made as for
the red and blue injections.
*Method of injection.*--In injecting the vessels of tissues it is
necessary that the organ or the entire animal, as the case may be,
shall be kept during injection at a temperature well above that at
which the gelatine mass will melt, otherwise the gelatine will “set”
in the arteries and will never reach the capillaries. This warming
is effected by immersing the animal in a water bath. The liquefied
gelatine is forced into the artery by a syringe or by air pressure.
It is essential that the pressure be uniform and steady. This is so
much more easily managed with air pressure that this method is strongly
recommended to the beginner. But, whatever method be adopted, perfect
results can only be obtained with certainty after long practice.
Sometimes too high pressure will be employed and the vessels give way,
at others the injection may not reach the capillaries at all. The most
scrupulous attention to details is essential.
By far the most effective apparatus for injecting is the modification
of Ludwig’s constant pressure apparatus devised by Fearnley.[2]
Although the apparatus appears complicated, the various parts are
easily obtained and it would be easy to improvise a substitute for the
water bath.
[2] “Practical Histology,” (Macmillan & Co.).
The apparatus which is shewn in figures 10 and 11 consists of a
bath deep enough to contain the animal, and a vessel containing the
injection fluid. The bath is kept at a temperature of about 110° by an
ordinary Bunsen burner. A large Wolff’s bottle (20–40 oz.) with three
necks, is fitted with three india-rubber stoppers perforated by glass
tubes. Through the central stopper a glass tube connected by a rubber
tube, with an ordinary Higginson’s syringe, passes almost to the bottom
of the bottle. From one of the other necks a rubber tube passes to an
ordinary mercurial manometer, while from the third a tube passes to
the flask containing the liquefied injection mass, which is immersed
in the water bath. This flask is also firmly stoppered, and should be
about half filled with injection material. The delivery tube from the
large Wolff’s bottle should only just come through the cork. Another
glass tube passes down almost to the bottom of the flask, and is
connected by a rubber tube with the cannula inserted into the artery.
It will be evident from figure 11 that when water is pumped by the
Higginson’s syringe into the Wolff’s bottle the pressure there will
be raised (as indicated by the manometer). This increase of pressure
will equally affect the air inside the bottle containing the injection
fluid, and the fluid will be forced out along the tube and through the
cannula into the artery.
[Illustration: Fig. 10.--Fearnley’s arrangement for injecting blood
vessels. (Reproduced by permission of Messrs. Macmillan, from
Fearnley’s _Practical Histology_).]
[Illustration: Fig. 11.--Scheme shewing distribution of pressure in
Fearnley’s Injection Apparatus (from Fearnley’s _Practical Histology_).]
Before using the apparatus a clamp should be placed on the exit tube
of the vessel containing the injection fluid, and the pressure should
be raised to see that the apparatus is everywhere air-tight. Any leaks
should be sealed before the actual injection is commenced.
If an isolated organ is to be injected, a cannula of glass or brass
should first be inserted into the artery and securely tied in position.
The organs, if cold, must be soaked in water at 120° F. for about half
an hour and then transferred to a water bath.
In the case of injecting an entire animal, such as a rabbit, rat, or
guinea pig, the injection is best made a few minutes after death. The
animal may be chloroformed, and then bled to death by opening a large
vein. As soon as death has occurred incise the skin over the thorax
in the middle line. Cut through the costal cartilages to the right of
the sternum, and through the junction of the manubrium and body of
the sternum. These incisions being for most part through non-vascular
parts will not lead to escape of fluid during injection. The sternum
being forcibly raised towards the left, the pericardium will be exposed
and must be carefully divided. An incision must be made into the left
ventricle, and a cannula passed up into the aorta and firmly secured
by a ligature passed round the aorta with the assistance of forceps
or an aneurism needle. Any blood is cleaned away and the animal is
then placed in the water bath for about ten minutes. The tube from the
bottle containing the injection fluid is then filled by gentle pressure
on the syringe, and clamped when full. Its end is then placed on the
cannula and secured there by a ligature. The pressure should be raised
by squeezing the syringe until the manometer registers one inch. The
clamp should then be removed and the injection commenced. The pressure
should be raised very gently and constantly by working the syringe, and
the condition of the gums, lips, and eyes of the animal observed. The
gums will soon shew a pink tinge. The best indications are obtained by
watching the effect on the small vessels of the sclerotic. When these
are completely filled, which will be in about five to ten minutes
according to the rate at which the air pressure has been increased,
the injection may be stopped. This result will be obtained, under good
conditions, before the manometer indicates a pressure of five inches.
The aorta should now be ligatured, and the animal placed in cold water
frequently renewed until it is thoroughly cooled. The organs may then
be removed and placed in methylated spirit and hardened. Sections are
afterwards cut and mounted in the usual way.