PHYSIOLOGICAL STUDIES IN ANAPHYLAXIS. II.

'
REACTION OF SMOOTH MUSCLE FROM GUINEA
PIGS RENDERED TOLERANT TO LARGE DOSES
OF SERUM
W. H. SCHULTZ
From the Division of Pharmacology, Hygienic Laboratory, Public Health and Marine
Hospital Service, Washington, D.C.

•¿ Receivedforpublication
November 28,1910

Recently2 it has been shown that smooth muscle contracts

quite readily when exposed to small quantities of serum. Further
more, this normal irritability may be greatly augmented by first
sensitizing the animal as is done in studies of anaphylaxis. Smooth
muscle from sensitized guinea-pigs, excised and treated with
serum, records a contraction curvemuch greater inextent than does
an unsensitized muscle preparation similar in every other respect.
Since there is present this peculiar reaction of smooth muscle
in sensitized animals it seemed probable that this supranormal
irritability might be reduced to normal or subnormal if the animals
were first rendered immune to relatively large doses of serum.
Three methods of rendering the animals immune to the pharmaco
logic action of serum are suggested: (1) The animal may be
injected subcutaneously with gradually increasing doses of serum,
the doses being given at relatively short intervals of time, having
the number of injections sufficient to make the total interval of
time between the initial or sensitizing dose and the final
dose sufficiently long so that whatever sensitization may be
set up is neutralized by the subsequent injection of serum.

1 Schultz, W. H.: I. The reaction of smooth muscle of the guinea-pig sensitized

withhorseserum. Journ.Pharmacol.and Exper. Therap.,1910,I,p.549.

2 Rosenau and Anderson seem to be the first to have demonstrated this point.

Rosenau and Anderson: Studies on hypersusceptibility and immunity. Bull. Hyg.

Lab.,U.S.P.H. and M.H.S.,1907,April.
222 W. H. SCHULTZ

In this way by the end of 20 days doses large enough to kill

a very sensitive guinea pig ought to have but little effect,
either because of an acquired immunity or an acquired toler
ance towards a foreign serum ; (2) The animal may be sensi
tized in the usual way and after 20 days sublethal doses of gradu
ally increasing size but small enough not to produce grave symp
toms are given, thus gradually neutralizing that which is neces
sary to high sensitization ; or, (3) highly sensitive animals may be
injected with sublethal doses large enough to produce grave symp
toms, still leaving them in a condition not to respond to other
large doses of serum within reasonable lengths of time. If smooth
muscle from animals desensitized by any of these three methc@1s
be tested it ought not to. react like that from a highly sensitized
guinea-pig provided the desensitization is complete and permanent.
In other words, if there is a condition of absolute immunity set
up when large doses of serum no longer kill, then muscle prepara
tions from such animals ought to respond in a manner similar to
that from normal ones, or possibly show even a subnormal irrita
bility. It will be seen, however, that neither of these conditions
obtains when the first method mentioned of rendering guinea-pigs
tolerant to large doses of serum is employed.
In this series of experiments guinea-pigs of known descent, raised
in our own pens, were used. A number of young pigs weighing
from 180 to 250 g. were divided into three groups, a, b, and c, and
kept in the same pen, bedded with hay, and fed on oats, cabbage,
and water. Group (a) were reserved for normal controls, group
(b) were sensitized by subcutaneous injections of 0.1 cc. of sterile
horse serum, and group (c) were injected with gradually increasing
doses of sterile serum, the doses for the first 21 injections being
at two-day intervals. The injections were started with 0.1 cc.
on July 1 and the dose doubled (0.1, 0.2, 0.4, 0.8, 1.6, 3.2) until
3.2 cc. was reached on July 14, after which 4 cc. was injected
peritoneally until July 24 when the irritability of the various
organs was tested. At the present time the results obtained with
smooth muscle from the intestinal tract will be reported. As a
rule each experiment consisted of from three to six tests with
tissue from two different animals, one animal, the control, being
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PHYSIOLOGICAL STUDIES OF ANAPHYLAXIS 223

either a normal animal or a sensitized one, the other being a de

sensitized (tolerant) guinea-pig. The desensitized pig had been
rendered so tolerant to serum as to react no more than does a
@ nonsensitized animal injected intravenously with cc., or pen
toneally with 4 or 5 cc. of serum. The following protocol rep
resents more in detail one type of experiment:

Experiment 115, a and b, August. 1, 1910

a = normal non-sensitized guinea-pig.
August 1. Wt. 321 ems., 10:7 a.m. 4 cc. intraperitoneal injection of horse serum.
12:51 p.m. decapitated. Small intestine tied off, excised, and placed in Howell's
solution, through which washed oxygen was slowly bubbling.
Segments 35 mm. long suspended in saline bath and treated with serum. (See
fig.1.)
Experiment 116 b.
Guinea-pig injected with gradually increasing doses of serum.
July 1, Wt. 199 gms. Subcutaneous injections of sterile horse serum. July 1,
0.1 cc., July 3, 0.2 cc., and intraperitoneal injections July 5, 0.4 cc., 7th 0.8 cc.,
9th, 1.6 cc., 11th, 2.0 cc., 13th, 2.5 cc., 15th, 3 cc, 17th, 3.5 cc., 19th, 4 cc., 21st,
23d, 25th, 27th, and August 1, 4 cc. respectively.
August 1, Wt. 315 gms. 115a and 115b, when injected intraperitoneally with
4 cc. of serum reacted about alike; without the cards it would have been im
possible to distinguish the control from the desensitized animal, since both animals
occasionally rubbed their noses, nawed their flanks, and yawned. Perhaps 1155
was on the whole more drowsy, but 115a examined its abdomen more frequently.
There were, however, no bucking movements, coughing or sneezing, no abnormal
excitementfollowedby subsequentparalysis and respiratory symptoms known
to be characteristic of anaphylactic shock. Only suchsymptoms were observed
as areseenwhen normal animalsareinjected forthefirst time with largedoses
ofserum.
August 1 at 12:49 115b was decapitated, the intestine tied off, excised, and placed
in oxygenated saline and a 35 mm. segment suspended in saline and tested
with serum (see fig. 1).

After this preliminary treatment segments of each of the excised

intestines, as nearly alike as possible, were suspended in a saline
bath kept at nearly a constant temperature. The light recording
levers were also as nearly alike as possible and arranged to record
the myogram of the control above or below that of the tested
segment, a slowlyrevolvingHarvard Kymograph being used
in obtaining the records on smoked paper. In the course of the
experiments it was soon learned that certain precautions, aside
 224 w. H. SCHULTZ

from changes in temperature and rate of oxygenation had to be

observed:
(1) The animals used for a given experiment had to be about
the same size, for as is well known the size of the alimentary
tract increases in proportion to the size of guinea pig, making the
small intestine of a 500 gm. pig much heavier and stronger than
that from a 300 gm. pig.
(2) Intestinal segments though taken from animals of approxi
mately the same size must be in similar states of physiological
activity. That is, both animals must be well and healthy, the
stomach and intestine should contain sufficient food to insure
the viscera of each being in about the same condition of digestion
and absorption, since there is considerable difference in the extent
of a myogram recorded by a segment excised during the height of
the digestive period and in one taken during the fasting stage or
period before feeding.
(3) Segments may be compared after standing in oxygen
ated Ringer for a number of hours. The best results are, however,
obtained soon after bleeding the animal. Hence all segments
were treated as soon as possible.
(4) Segments, in order to be comparable, must be taken from
approximately the same level of the alimentary canal.

1. EFFECT OF SINGLE INJECTIONS OF SERUM UPON THE NORMAL

IRRITABILITY OF THE INTESTINAL MUSCLE

@ A single intravenous injection of cc. of horse serum seems to

have no effect upon the normal irritability provided the muscle
be tested within a period of one or two hours after the injection.
And probably has no effect until the process of sensitization sets
in.
If, however, a sufficient time has passed since the first injection
of serum, the intestinal muscle shows a marked increase of irrita
bility, even though the animal, judged by the usual method,
shows no, or at best very mild, symptoms of anaphylaxis. Just
how long a time must elapse before this increased irritability can
be detected has not been worked out, but it is probably coincident
with the initial stages of anaphylaxis.
 PHYSIOLOGICAL STUDIES OF ANAPHYLAXIS 225

EXPLANATION OF FIGURES

Fia. 1. Small intestine from a nonsensitized guinea-pig and from a guinea-pig

rendered tolerant to horse serum.
Experiment liSa and 115b. August 1, 1910. Curve 115a from intestine of non
sensitized guinea-pig weighing 321 gms. Ether anaesthesia. Intestine removed
and placed in oxygenated saline 1:1 p.m. Segment 35mm. long, taken from duode
nal end about 12-15 in. from stomach, suspended in 10 cc. of Howell's solution at
36.4,°1:19 p.m.; 1:23 p.m., 1 cc. horse serum (36.4°)added to saline bath. Ratio
of lever arms 150 mm. to 30 mm. weight of 0.5 gms., 30 mm. from fulcrum on
writing arm.
Curve 1155 from 35 mm. segment of small intestine from a guinea pig tolerant to
horse serum. Guinea pig treated as follows. July 1, weight 199 gms.; subcutan
eous injections of horse serum July 1, 0.1 cc., July 3, 0.2 cc., July 5, 7, 9, 11,
13, 15 and 17, inclusive, intraperitoneal injections of 0.4, 0.8, 1.6, 2.0, 2.5, 3.0
and 3.5 cc. of horse serum respectively. On' each of the following days 4 cc.
of the same serum was injected intraperitoneally. July 19, 21, 22, 27, 29 and
August 1. August 1 Wt. 315 g. 10:9 a.m. received last injection; pig normal,
no abscesses, eats heartily. The larger doses of serum seem to affect this pig' less
than similar doses affected normal controls. 12:56 p.m., ether anaesthesia. Intes
tine removed and placed in oxygenated saline solution; 1:19 p.m. 35 mm. segment
suspended in 10cc. of saline at 36.4°C. along side of 115a. 1 23, 1 cc. horse serum
(36.4°)added to the saline bath in same manner as to control.
- -.

226 W. H. SCHULTZ

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PHYSIOLOGICAL STUDIES OF ANAPHYLAXIS 227

2. THE EFFECT OF REPEATED INJECTIONS OF GRADUALLY IN

CREASING DOSES OF SERUM

It seems to be quite generally accepted that in order to produce

in guinea-pigs a condition of anaphylaxis a certain interval of
time must elapse after the sensitizing dose. So far as is known
there are no abrupt changes resulting in complete anaphylaxis.
The present data support the idea that the changes in irritability
as tested by the second injection are gradual, finally reaching a

maximum in from 10 to 20 days and then very slowly falling off.

If the ordinates represent the degree of irritability and the abscissa
the number of days since the sehsitizing dose, a curve may be plot
ted representing the changes in irritability towards the second
dose of serum. The ascending limb of such a curve will be gradual
but much steeper than that representing the return to normal.
Indeed, the descending limb remains, after the first preliminary
drop, almost flat so slowly do the conditions responsible for ana
phylaxis disappear. Now so far as the gross symptoms are con
cerned irregularities may be introduced into the irritability curve.
For example, relatively large doses injected subcutaneously soon
after the sensitizing dose greatly hinder the conditions necessary
to a vigorous response of the organism to a subsequent injection
of serum provided too long an interval has not elapsed between the
last two doses. Whatever it is that is affected by these so-called
immunizing2 doses of serum one thing is certain, that there is a
condition set up inhibiting either the extra or acquired irritability
of the cells, or else neutralizing the medium through which they
are stimulated. This is true of the intact animal, but is it true
of tissues when removed from the body?
Figure 1 illustrates the results of a crucial test which ought to
throw some light upon this vexing problem. In this series of
experiments of which 115a, b, and c were members, a was a normal
control, b injected with increasing doses of serum reacting less,
when judged by the gross body symptoms, to the 4 cc. injections
than to the 2 cc. ones; still never at the most did it show true ana
phylactic symptoms, whereas c, the sensitized animal, died within
5 minutes from anaphylactic shock. Judging from the gross reac
228 W. H. SCHULTZ

tion of 115b one would imagine the animal well protected against
large doses of serum. The myogram, however, shows the smooth
muscle to be still hypenirnitable, in fact it reacts very much like
that from a sensitized animal. It matters not whether the final
dose of serum be injected into the abdominal cavity or into the
circulation, for the muscle of this group of pigs reacted much as
did that from sensitized animals. These results would seem to
indicate that at least in animals tolerant to serum there is still
present that- hyperirnitability of smooth muscle so characteristic
in sensitized animals. Since the smooth muscle of the intestine
is hyperirritable towards serum it would hardly be correct to say
that there is not present at least a high degree of latent sensi
tivity. Yet by reason of the absence of the usual characteristic
anaphylactic symptoms some chemical or physical process seems
to be responsible for protecting the pig from the injection of what
is ordinarily a lethal dose of serum.

CONCLUSIONS

1. Single intravenous or intrapenitoneal injections of horse

serum seem to have no effect upon the normal irritability of in
testinal muscle towards the same serum provided the muscle be
tested within a period of one or two hours after the injection.
2. If, after the first injection of foreign serum, a long enough
interval elapse, intestinal muscle shows a supranormal degree of
irritability towards the same serum. The time necessary to ac
quire this increased irritability is probably coincident with that
required for the process known as sensitization in anaphylactic
animals.
3. Guinea-pigs may be rendered tolerant to large doses of
foreign serum by injecting increasingly large doses of it, at
intervals of two days, for a period of 20 to 30 days. The gross
body reflexes and the cardiac and respiratory reactions differ
markedly from those of a sensitized animal, but intestinal smooth
muscle continues to show a supranormal irritability towards
a serum similar to that of smooth muscle from a sensitized animal.
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PHYSIOLOGICAL STUDIES OF ANAPHYLAXIS 229

4. The tolerance induced by repeated injections of foreign

serum resembles tolerance acquired towards certain chemical
substances familiar to Pharmacologists. As to immunity, it seems
impossible by repeated injections to initiate a condition of abso
lute immunity, since certain tissues not only remain irritable to
the serum but acquire a supranormal irritability to it.