NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
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punctures a marked infiltration into the areolar tissues of a purple blood-like
fluid, and that this infiltration contains few red blood corpuscles.
In experimental observations on animals, especially on horses and donkeys,
the abscess which sometimes forms, when a sub-lethal dose of cobra venom is
given, contains thin red-coloured pus. Further, the mucous discharges are
sometimes stained red. Thus, I have seen a marked red-stained discharge
from the rectum of an ass which had received a little over the minimum lethal
dose of cobra venom. As I have said, however, in animal experiments with
cobra poison all observers are agreed that repeated microscopic examinations
of the blood of animals, which die from cobra venom intoxication, show an
entire absence of any appreciable structural change in the red blood cells. The
probable explanation of this fact is, either that, when a large dose has been
injected, death takes place so rapidly that such a marked destruction of the
cells has not had time to take place as to be demonstrable by microscopic
examination, or that, when a smaller dose has been given and death is delayed,
the quantity of venom injected is not large enough to cause any extensive
destruction of the red cells.
While this fact is undoubted, Cunningham 8 has shown in several experi-
ments that, if the blood of an animal which has received a large dose of cobra
venom, either intravenously or subcutaneously, be withdrawn from the body
after death and allowed to clot, the serum which exudes from the clot is of a
red colour ; also that, if a fowl has been the animal employed, many free nuclei
of the red cells are found on examining the blood at death ; there has been, in
fact, a considerable solution of the bodies of the red cells. Further, this
observer has demonstrated that, if the dose of venom injected into a fowl is a very
large one, namely, I00 to 300 milligrammes, the blood at death contains a
great abundance of free nuclei, and the remaining red cells appear deformed ;
he has also shown that, if the specimen be allowed to stand, at the end of a
few hours complete destruction of the red cells will have taken place. I have
been able to confirm these statements of Cunningham. These observations,
therefore, conclusively show that cobra venom does have a certain amount of
hæmolytic action in vivo, even when it is injected in moderate doses. When,
however, a quantity of venom, which will bring about this effect in a marked
degree, is injected into an animal, the animal dies so rapidly that there has
been time for complete destruction of only a comparatively few cells to take
place. It would appear, therefore, from â priorî reasons, that the hæmolysis
which results from this action of cobra venom can have little or no connection
with the nervous symptoms which are always observed in cases of intoxication
with this poison, and cannot be a factor in producing the fatal result. An oppo-
site opinion is, however, strongly held by Cunningham.
4
The destructive action of cobra venom on the red blood cells in vitro has
been investigated by several observers. Thus, Cunningham, 9 by drawing blood
directly into solutions of cobra venom, found that the serum which exuded
from any clot which formed was dark red or brownish in colour. He demon-
strated this phenomenon both with mammalian bloods and with fowl's blood.
Kanthack,I0 Stephens,II and Myers 1 2
have also demonstrated the marked hæmo-
lytic action of cobra venom in vitro. These observers further noticed that the
blood cells of different animals were of different susceptibility to this hæmolysing
poison, and that this susceptibility of the blood cells bore no relation to the
general susceptibility of the animals to subcutaneous injection of the poison.
The most convenient and most reliable method to demonstrate and to
estimate this hæmolytic action in vitro, the method which I have employed
in all my experiments, is stated as follows.
The venom is dissolved in a solution of common salt, isotonic 1 3
for the parti
-cular blood under observation. This solution of venom is made of a strength of
c.c..=2 milligrammes, namely, 0.2 per cent. From this original solution a suc
-cession of two fold dilutions, made with salt solution of the same strength as the
original solution of venom, is prepared in a series of test tubes, so that each dilution
contains half the amount of poison of the one just above it in the series. A conve-
nient quantity, namely, 0 5 c.c., of each dilution is then measured into a series of
small test tubes. To each tube a measured quantity of fresh blood is added by
means of a graduated capillary pipette. The quantity of blood I found most suit
-able to add to each tube was 0.005 c.c. In these operations care must be taken
to avoid, as far as possible, any bacterial contamination. Ordinary precautions,
however, of having sterile tubes, sterile salt solution and sterile pipettes are all
that are requisite. Control tubes of salt solution and blood alone are prepared in
all experiments. The mixtures are allowed to stand at laboratory temperature
(about 25° C.) for 20 to 24 hours. It is then observed in which tubes complete
laking has taken place, in which partial laking and in which no laking at all. When
there is any doubt from the naked eye appearances as to whether complete
or only partial hæmolysis has taken place, a microscopic examination will soon
settle the point. Working in this way, I have been able to confirm the general
results got by the observers mentioned above ; and, further, to estimate fairly
accurately the hæmolysing power of cobra venom towards the blood of several
varieties of animals. But, inasmuch as the results obtained lead to the same
conclusions as were arrived at by the observers mentioned above, it is unneces
-sary to detail these experiments. It is, however, well to point out that slight
variations occur, from time to time, in the minimum amount of venom which
will produce hæmolysis of the blood of any particular species of animal. Such
variations were also noticed by Kanthack, but they are never so marked as to
5
invalidate the general results. They are, in fact, quite explicable on the ground
of experimental error.
In view of Martin's hypothesis, mentioned on page I, it is important to be
able to put on record some experiments which bear on the effect which heating
solutions of cobra venom has on the hæmolytic action of this poison. In Table I
of the protocols are shown the results of such experiments. From this Table
it will be seen that, while heating solutions of cobra venom so as to precipitate
all the coagulable proteids does not destroy the hæmolysing power of the venom,
such heating does cause a diminution of this power, and that this diminution is
more marked if the solution which is heated be a comparatively weak solution.
Thus, two solutions of cobra venom were made of 1 per cent. and 0.I per cent.
strength respectively. These were heated in the same water bath for half
an hour at 73° C. The coagulated proteids were then filtered off. The
hæmolysing power of the same sample of venom, unheated, and of these
two heated solutions, was estimated after the method I have described above.
A reference to the protocols will show that the greatest dilution of venom, in
which complete or almost complete hæmoloysis was obtained in each instance,
was as follows:—
(1) Unheated venom.—Complete hæmolysis : 0. 5 c.c.=0 . 015 milligramme.
(2)
I per cent. heated solution.—Nearly complete hæmolysis : 0 .5 c.c.=
0 . 0312 milligramme.
(3)
0 I per cent. heated solution.—Complete hæmolysis 0 5 c.c.=0 25
milligramme.
This result is in harmony with, although not so striking as, the fact pointed
out by Hanna and myself, 14 namely, that, while the power of daboia venom to
cause intravascular thrombosis is not completely destroyed, but only diminished,
by heat i ng a I per cent . s ol ut i on of t he poi s on f or hal f an hour at 75° C. , heat i ng
a 0.I per cent. solution of this venom for the same length of time at the same tem-
perature completely destroys this clotting power. Further, it lends no support
to Martin's theory.
The action of daboia venom on the red blood corpuscles in vivo
and in vitro .
There can be no doubt that daboia venom has a marked destructive
action on the red blood corpuscles in vivo, and that the results of this action
are much more manifest to a clinical observer than is the case in bites from
the cobra. Thus, in the recorded cases of daboia bites in man one reads of a
large blood-stained extravasation all around the site of the punctures, of an
exudation of a reddish-brown fluid from the wounds, of a dark-stained fluid
oozing from the various mucous orifices of the body—from the nose, from the
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bowel, from the mouth,—and of marked hæmaturia or hæmoglobinuria. Actual
hæmorrhages from mucous surfaces are also commonly seen in cases of
daboia bite.
In the case of experimental injection of daboia venom into animals, while
both Wall 1 5
a n d C u n n i n g h a m
1 6
n o t e t h a t s a n i o u s d i s c h a r g e s a r e c o m m o n s e q u e l æ
of such injections, those observers do not particularly state that any destruction
of the red blood corpuscles has taken place: a perusal of their works, in fact,
leaves one with the idea that these discharges are stained with the whole blood
and not with hæmoglobin only. It is, however, easy to demonstrate that
marked hæmolysis results when daboia venom is injected into animals intra-
venously or subcutaneously. Two examples out of many, of which I have
records, will be sufficient to put forward here. The first experiment was made
along with Professor Martin of Melbourne, to whom I am indebted for much
valuable assistance.
(I) A horse of 293 kilos in weight received into the jugular vein 3 c.c. of a
I p e r c e n t . s o l u t i o n o f d a b o i a v e n o m ,i
. e
., 30 milligrammes or about 0 I milli-
gramme per kilo. Convulsions began 50 seconds after the injection and re
- t u r n e d a t i n t e r v a l s t i l l t h e h o r s e d i e d . D e a t h t o o k p l a c e I 8 m i n u t e s a f t e r t h e
venom had been run into the vein. Not long before death a bright reddish-
stained fluid collected at the nostrils. A microscopic examination of this fluid
showed it to contain very few red cells, Samples of blood were drawn from the
jugular vein seven minutes before death and at death. Two hours after with-
drawal both these samples were still only softly clotted. The red corpuscles
had by this time sedimented, leaving a dark red serum which contained only
an occasional red cell but plenty of blood platelets.
(2) A female donkey, which weighed 89 kilos, received under the skin of
the left shoulder 20 milligrammes of daboia venom, i.e ., about 0 22 milligramme
per kilo. This animal died about 40 hours after the injection. The post-
mortem examination, made immediately after death, showed the following condi-
tion. Around the site of the inoculation there was a large bloody extravasation.
On the front of the thorax at the root of the neck, and also in the abdominal
wall, namely, two dependent parts, there were large œdematous swellings. On
cutting into these swellings a dark port-wine coloured fluid escaped. Under
the microscope this fluid was seen to contain very few red cells. After this
exudate had been centrifugalised, the supernatant fluid remained dark in colour
and contained no red corpuscles. The blood collected from a large vein clotted
only slightly after standing for an hour. A sample of blood was centrifugalised
immediately after it had been withdrawn from the body. The supernatant plasma,
which microscopic examination showed to be quite free from red cells, was
clear and of a very dark port-wine colour. After the blood had stood for a few
7
hours a firm clot had formed, and the serum which had exuded was of the same
colour as the whole plasma got by centrifugalisation. The urine collected from
the bladder was dark in colour like porter. It contained exceedingly few red
blood corpuscles, only two or three being visible in a microscopic field.
I have been able to produce similar symptoms and evidence of marked
hæmolysis by injections of this venom into monkeys, rabbits, and pigeons.
These observations, therefore, show positively that daboia venom has a very
marked and intense destructive action in vivo on the red blood corpuscles
of most animals, and that the effects of this action are important factors in the
production of the symptoms observed and of the fatal result. To anyone who
has worked with both cobra and daboia venoms the marked difference which
exists between their hæmolytic actions in vivo must be apparent. The hæmo-
lysis produced by cobra venom might easily escape observation, while in the
case of daboia poison it is a most prominent feature of the intoxication.
Such being the case, one would expect to find that a marked hæmolytic
action with daboia venom would be easy to demonstrate in vitro, similar to
the hæmolytic action which I have mentioned is easily got in vitro with cobra
venom. This surmise, however, does not hold good. Thus Stephens, 17 as a
result of a few experiments only, has come to the conclusion that, while daboia
venom dissolved in saline solution has no hæmolysing power in vitro on the
red cells of men, this poison has a marked destructive power on the human red
corpuscles if it is dissolved in normal horse serum. On the other hand,
Cunningham 1 8
f o u n d t h a t , i f t h e b l o o d o f a f o w l i s r u n i n t o s o l u t i o n s o f d a b o i a
venom, the serum which exudes from the clot, which, he remarks, is always soft and
tremulous, is dark and red in colour, showing that a considerable destruction
of the red cells has taken place. Using a similar technique to that which I
have described above in the experiments with cobra venom, I have found that
it is possible to demonstrate that daboia venom dissolved in saline solution does
produce in vitro laking of the blood of many animals. This action, how-
ever, is somewhat variable. Care has to be taken that the salt solution is not
of too great a strength ; that, in fact, it is just slightly stronger than the solution
which would of itself produce a certain amount of laking. In Table II of the
protocols the results of five series of experiments of this description are detailed.
The bloods used were those of man, monkey, rabbit, guinea-pig, and pigeon re-
spectively.
The strength of the salt solution employed for each variety of blood was
previously determined by actual experiment. This strength is in each instance
indicated in the protocols.
A glance at this table will show that, while the control tube in every in-
stance showed no hæmolysis, a marked and even complete laking took place
B
8
in many of the venom tubes, It will also be seen that the action is somewhat
variable, that is to say, the hæmolysis obtained in a tube containing a weak
solution of venom is sometimes more complete than in tubes containing stronger
solutions. Thus, it has often been observed in one series of tubes that while
the stronger solutions of venom have only a slight or no destructive action on
the red cells, the result of the action of . the weaker solutions is an absolutely
complete hæmolysis. Such a result is well shown in the series of experiments
with the blood of a rabbit tabulated in Table III of the protocols.
This variability in the hæmolytic action of daboia venom in vitro is in
marked contrast to the results obtained with cobra venom. This, however, is
a point to which I shall return.
When a weak solution ( viz., 0.1 per cent.) of daboia venom is heated so as
to coagulate the coagulable proteids, the hæmolytic property, which the
unheated venom has in vitro, is completely destroyed.
In Table IV of the protocols two parallel series of experiments are detailed
in order to demonstrate this face. In these observations a 0.1 per cent. solution
of venom in 0.6 per cent. saline solution was divided into two portions. One por-
tion was left unheated, while the other portion was heated in a water bath for half
an hour at 75° C. The coagulated proteids were then filtered off. The hæmoly-
sing power of each portion, heated and unheated, was then determined in the
manner I have already described, the blood of the same rabbit being used in
each instance. It will be seen from this table that, while the unheated solution
showed a well marked haemolytic action in many of the tubes, no laking was
observed in any of the tubes containing the dilutions of the heated venom.
We may pause here for a moment to sift the data contained in the obser-
vations and experiments which I have put forward above, with the view of sum-
marising the differences which exist between the hæmolysing action of cobra
venom and that of daboia venom.
Firstly, as regards the hæmolysis which these venoms produce in vivo,
it is important to note that cobra venom, howsoever introduced into the body,
exerts only a slight destructive action on the red cells, so that, as a rule, no
symptoms result. Indeed the results of this particular action of the poison
have generally escaped observation. On the other hand, the hmolytic action
of daboia venom i n v i v o i s w e l l m a r k e d a n d l e a d s t o a t r a i n o f s y m p t o m s ,
which are very prominent to even a casual observer.
Secondly, when the hæmolysing power of these two poisons is estimated
side by side in vitro, using exactly the same technique in each case, we have
seen that, while this action of cobra venom is well marked and regular, varying
according to the strength of the venom solution used, no matter from what
species of animal the blood is taken, the destructive effect on the red cells
9
of daboia venom, under similar circumstances, varies considerably and often
apparently does not depend on the strength of the poison solution with which
the blood is brought in contact. This action of daboia venom in vitro is,
in fact, by no means so definite as the corresponding action of cobra venom.
Thirdly, we have seen that heating a 0 I per cent. solution of cobra venom,
while diminishing, does not completely destroy, the hmolysing property of the
poison; and that heating a solution of daboia venom of the same strength and
in exactly the same manner has the effect of completely destroying the hæmoly-
tic power which the unheated solution possessed.
It seems, therefore, justifiable to conclude from these three facts that, as
far as the hæmolysing substances of these venoms are concerned, we are
dealing with substances different but resembling one another, and that in this
respect, as in all other respects, cobra venom and daboia venom, although at
first sight presenting a superficial resemblance, yet differ materially from one
another.
The action of cobra and daboia venoms on the blood
coagulability in vivo.
It has been known for some time that many snake venoms have a well
marked action on the coagulability of the blood plasma in vivo . To this fact
attention was first drawn by Fontana, 19 who more than a hundred years ago
noticed that the blood remained fluid in animals dead of viper-bite. Brainard 19
and. Weir Mitchell 1 9
have also shown that rattle-snake venom has a marked ac-
tion on the coagulability of the blood. The former observer noticed that in
animals which died immediately after a rattle-snake bite, the blood was clotted ;
while, if death was delayed for any length of time, the blood remained fluid in the
vessels. Weir Mitchell, while confirming those observations, explained the
differences by the hypothesis that in cases of very rapid death the poison had
not had time to affect the blood. In Australia, 19 observed the same
fluidity of the blood to follow the injection of the venom of some Australian
snakes.
Further, the continued fluidity of the blood after death from the bite of the
Indian viperine snakes has been frequently noted by numerous observers in this
country.
The work, however, of Martin of Melbourne 20 with the venoms of the two
Australian snakes (Hoplocephalus and Pseudechis) put this problem on a firmer
basis. This careful observer has shown that the venoms of these two species of
snakes produce, when injected intravenously in sufficient amount, such a marked
increase in the blood coagulability as to lead to an extensive intravascular throm-
bosis, but that, if the quantity injected is not sufficient to cause death from this
B 2
10
thrombosis, then a marked deficiency or negative phase of the blood coagula-
bility follows a short-lived phase of increased coagulability. Here, then, was the
probable explanation of the results obtained by the American investigators with
crotalus venom.
Martin, further, put forward the hypothesis that all snake venoms prob-
ably contained, in greater or less quantity, the same proteid substance which
brought about this most remarkable effect on the blood coagulability, one
venom differing from another in the amount of this constituent. Along with
Dr. Hanna 21 I was, however, able to show that this hypothesis was not a sufficient
explanation as far as our Indian snakes were concerned. For we proved beyond
a doubt that, while the effect of the action of daboia venom on the blood coagu-
lability was apparently the same as Martin had described with regard to the
venom of the Australian species, cobra venom did not at any rate, even when
injected intravenously in very large quantity, produce intravascular clotting.
Again, in a paper on "Some effects upon the blood produced by the injection
of the venom of Pseudechis," Martin 22 critically sifts the available data, in an
endeavour to find an explanation of the significance of the phenomena which he
described. He arrives at the conclusion that the great changes which snake
venoms bring about on the blood coagulability are in all probability due to
nucleo-proteids set free from various cells, especially the red blood corpuscles,
as a result of their destruction by the venom, and he compares the phenomena he
observed in using snake venom with the phenomena which Wooldridge described,
as resulting from an injection of nucleo-proteids directly into the blood stream
of an animal. It is unnecessary to follow this observer in the close reasoning
which runs through this paper. It is, however, necessary to point out that at
the close of the paper he sums up as follows :
—
"Finally, I say that, although I believe that the very striking facts, which I have
hereto observed, do warrant the hypothesis advanced in this paper, namely,
that the phenomena of coagulation following the injection of snake venom are
identical as regards their immediate cause with those described by my friend
the late Dr. Wooldridge, as consequent upon the injection of tissue-fibrinogens,
I am yet quite prepared to entertain a different explanation, should further
observation reveal facts discordant with this theory."
The observations which I have now to bring forward appear to me to reveal
facts discordant with this theory, so discordant, indeed, that I am of opinion
that Martin's hypothesis can no longer stand, and that the problem of the action
of snake venoms on the coagulability of the blood is a much more complicated
problem than this observer supposed, and will have to be reconsidered in the
light of these new facts. It is also evident that no generalisation can be made,
founded on observations with one variety of snake venom, but that every variety