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Acute Deg'eneratiTe Changes in the Neryous
System, as Illustrated by Snake-yenoni
Poisoning"

BY

WALTEE K. HUNTEE, M.D.

[Reprinted from the " Proceedings of the Royal Society of Medicine,"

July, 1910]

XoiiDoii

JOHN BALE, SONS & DANIELSSON, LTD.

OXFORD HOUSE

83-91, GREAT TITCHFIELD STREET, OXFORD STREET, W.

1910

Acute Degenerative Changes in the Nervous System, as
Illustrated by Snake-venom Poisoning.

By Walter K. Hunter, M.D.

For some years past I have been engaged, in conjunction with
Major Lamb, I.M.S., on an investigation into the action of venoms
of different species of poisonous snakes on the nervous system, and
our results have appeared from time to time in the Lancet,^ in a series
of six papers in all. This work has furnished me with a large number of
microscopic sections illustrative of acute degenerative changes in gan-
glion cells as a result of toxic poisoning, and I have ventured to think
some of these of sufficient interest to permit of me bringing them before
the notice of this Section. In doing so I wish to express the debt I owe
to Major Lamb for so freely placing at my disposal the tissues from the
animals killed in the course of this inquiry ; for their histological exami-
nation I myself am entirely responsible.

Monkeys were the animals used in these experiments. They were
injected with varying doses of one or other of the venoms ; the toxic
symptoms were observed and any corresponding changes in the nervous
system subsequently investigated. Perchloride of mercury was the
fixing agent almost invariably adopted, and the sections were stained
with thionin according to Nissl's method. Thirty-nine monkeys were in
this way examined, and by way of controls similar sections were prepared
from four normal monkeys.

The following were the venoms employed : (1) Cobra venom, (2)
venom of Enhydrina valahadien (common sea-snake), (3) venom of

' Lancet, 1904, i, p. 20, ii, p. 518, 1146; 1905, ii, p. 883; 1906, i, p. 1231 ; 1907, ii, p. 1017.

2 Hunter : Degenerative Changes in the Nervous System

Bungarus cwruleus (common krait), (4) venom of Bunga7-us fasciatus
(banded krait), and (5) venom of Dahoia rnssellii (chain viper). The
first fom- of these venoms had without doubt a direct action on the
central nervous system, and the symptoms produced by any one of them
had mucli the same characters as those produced by any one of the
other three. Large doses of any of the four vi^ould cause generahzed
convulsions, with death supervening in three to four minutes. But with
smaller doses death would be delayed several hours, and there would be
no stage of preliminary excitement ; indeed, the first symptom was now one
of drowsiness. Paralysis of the extremities would next appear, more
marked in the hind than in the fore limbs. Then the paralysis would
become so marked that the animal could not move. Respiration is next
involved, being first slow with inspiratory effort, and later it ceases
altogether. Whilst, therefore, there is a widespread paralysis with all
these venoms, the cause of death is paralysis of respiration.

The similarity in action of the first three venoms in particular
was specially marked, for it was almost impossible by observing the
symptoms to say with which of the three the animal had been injected.
It has been determined, however, that there are some minor differences.
For example, the venom of the common krait has a distinct action on
the vasomotor centre, causing paralysis of this centre and marked fall of
blood pressure, whilst cobra and sea-snake venoms have no such effect.
Again, cobra venom has a considerable action on the vagal cardio-inhibi-
tory centre, whilst the common-krait venom has only a slight action and
sea-snake venom none at all. The longest time that any animal lived
after receiving a minimal lethal dose of any of these three venoms was :
with cobra venom, six and half hours ; with sea-snake venom, six and
three-quarter hours; and with common-krait venom, sixteen and a half
hours.

With the fourth venom, that of the banded krait, the symptoms
differed in some respects from the other three. With a large dose there
were convulsions and death in a few minutes, due in this case to intra-
vascular thrombosis ; with a smaller dose the symptoms were those of
cobra-venom poisoning — that is, paralysis of the limbs and later of
respiration, only with the krait venom death was much longer delayed,
one of our airimals not dying till forty-four hours after injection. But
with a still smaller dose death might be delayed for twelve days. When
this was so there would be an interval of two to six days when there
were no symptoms except that the animal seemed depressed and off
its food. Then it would begin to emaciate till the emaciation assumed

Neurological Section

3

the characters of muscle atrophy with corresponding muscle weakness ;
and before death, in some of the animals, there seemed to be more or
less complete paralysis. The symptoms suggested, therefore, an acute
and progressive muscular atrophy.

The fifth venom investigated — that of Daboia russellii — showed little
action on the nervous system. In large doses it produced an extensive
intravascular thrombosis, the animal dying in a few minutes. With
smaller doses the animal might live for several days, and then there
is no such thrombosis, and no paralysis of the limbs or of respiration.
Death, indeed, seemed to be due to cardiac syncope : Rogers holds that
it depends on a vasomotor paralysis of central origin.

That, then, is a brief resume of the symptoms produced by the
several venoms, and now we come to consider the histological changes
met with as a result of their action on the nervous system. And first
in regard to Daboia venom. Briefly, it had no recognizable action
on the motor ganglion cells of the cortex, pons, medulla or cord, even in
an animal that lived for sixty hours after injection of the venom. The
suggestion that death was due to vasomotor paralysis made us pay
very special attention to the "vasomotor area" in the medulla. And
so we examined this area by means of serial sections in four of the
monkeys. In two the sections were cut transversely, and in two in
longitudinal direction. For comparison serial sections were cut through
the corresponding areas of four normal monkeys. But careful com-
parison of the sections from the normal monkeys with those from the
animals killed with the venom failed to show in the latter any certain
signs of even an early degeneration in ganglion cells. Histological
examination, therefore, was negative as regards giving evidence of
Daboia venom having a selective action on the ganglion cells in that
part of the bulb within which the vasomotor centre is supposed to be
situated. The sections, then, from the monkeys killed with Daboia
venom fail to supply us with specimens of degenerative lesions in
ganglion cells ; yet they are not without their value, for they serve
as controls with which to compare the sections from the animals
killed with the other venoms.

Now as to the other four venoms, we have just seen that they
each produce symptoms of a widespread action on the central nervous
system ; and histological examination confirms this by showing a corre-
spondingly extensive degenerative change in the motor ganglion cells of
cortex, pons, medulla and cord. The type of cell degeneration varies
little with these several venoms, only they do not all produce their

^.'.'.ASGOW
! '--iVERSITY

4 Hunter: Degenerative - Changes in the Nervous System

histological effect with equal rapidity. Thus, in an animal dying one and
a third hours after injection of sea-snake venom, the ganglion cells showed
undoubted chromatolytic changes ; whereas in the animals dying two
hours after cobra venom, or four and a half hours after the venom of
the common krait, their cells presented little abnormality. Speaking
generally, however, the longer the animal lived after injection, the more
marked were the histological changes, and hence within certain limits
these changes were in inverse proportion to the dose of the venom. In
other words, the histological changes in the cells seemed to depend less
on the quantity of venom than on the length of time it had to act. The
action of certain venoms, too, seems much more enduring than that of
others. For example, after injection of a small dose of cobra venom, if
the animal does not die within two to three days at the longest, recovery
invariably takes place, and recovery, too, from any paralysis that may
have developed is very rapid. With the venom of Bmigarus fasciatus,
on the other hand, symptoms need not appear till six days after injec-
tion, and death may be delayed till six days later still. If, then, the
changes in the ganglion cells are in proportion to the length of time the
animal lives, it was thought that by studying the cells in animals dying
at varying intervals, one might be able to trace the stages of this
degenerative process, for we have sections showing changes in the cells
from animals dying one and one-third hours, four hours, six hours, six
and three-quarter hours, ten hours, twelve hours, fourteen and a-half
hours, forty-four hours, three and a-half days, five days, six days, eight
days, and ten days after injection. It is, however, only generally true
that the changes in the cells are in proportion to the time the animal
lived, for the degenerative process in the animals that lived for days was
rather less acute than in those that died in a few hours ; and so one
finds examples of more extensive disintegration of the ganglion cell in
those animals that died in six to seven hours than in the monkey that
lived for ten days.

The process of degeneration, as far as can be made out, is something
as follows (see Plate, figs. 6 to 26) : In the first place the cell stains
with basic dyes more intensely than normal, and one was forced to
recognize that the sections from the paralysed monkeys took longer to
decolorize than those from the control animals. This staining, while
deeper, is possibly rather more difl'use than normal, and it gives the
appearance of deeply-stained tigroid bodies in a less deeply-stained
protoplasm. The tigroid bodies next appear as if they were being dis-
solved in the cell protoplasm, and they suggest the idea of pieces of

Neurological Section

5

metal being acted on by an acid medium. The chromatic granules are
now smaller than normal, but there is never the dust-like chromatolysis
that one sees in the more chronic degenerations ; neither is there
enlargement nor globular deformity of the cell itself. The granules
and diffuse staining next disappear, and leave a skeleton cell with its
margin, reticulum, and nucleus well differentiated, though rather deeply
stained. The cell reticulum has sometimes a granular appearance,
but in any case it stains well with the basic dye. Later on the cell
stains much more faintly, and is then the typical " ghost " cell. During
almost any of these stages vacuoles may appear in the body of the cell,
and its margins frequently become indented, as if little pieces had been
bitten out of them. Finally, the cell begins to disintegrate, and portions
entirely disappear, leaving behind little more than the nucleus, with
perhaps some adherent stroma attached to it. Vacuolation, as well as
disintegration of the cell, is seen mostly in the pale (ghost cell) stage,
but in the most acute degenerations it is frequently met with while
the cell is still deeply stained. During the whole of this degenerative
process the nucleus, at least in a large proportion of the cells, seems to
be little affected otherwise than is shown by a varying intensity and
diffuseness of its staining. It remains central in the vast majority of
the cells, and shows little change in size or shape. The nucleolus, too,
shows little change. In the more rapid degenerations, for instance, with
cobra or sea-snake venom, the Nissl granules seem to be more or less
uniformly affected throughout the whole cell, and hence it could not be
said that the chromatolysis was either " peripheral " or " perinuclear."
With the more slowly-acting venoms, on the other hand — e.g., Bmigarus
casruleus venom — a proportion of the cells suggested that the chroma-
tolysis was, to begin with, perinuclear, and that it extended from thence
outwards, for in these cells the perinuclear area was devoid of granules,
whilst granules were still present at the periphery of the cell.

There was nothing specially distinctive in the histological appear-
ances in the monkeys killed v^ith the venom of banded krait, and which
lived from five to ten days after injection. Such a length of time should
permit of extreme changes developing in the ganglion cells ; also it
might almost give time for trophic symptoms to appear in the muscles
and other tissues. General emaciation was very noticeable in these
animals, some of them losing from 20 to 25 per cent, of their weight
in a few days ; but it is very doubtful if this emaciation can in any way
be related to the changes in the anterior horn cells. We do not know
on what day the degeneration in these cells first appeared, but in

6 Hunter : Degenerative Changes m the Nervous System

Monkey No. 19 signs of paresis were not noted till the fifth day, and
paralysis was not complete till two days later. Neither were we able to
show that the venom was having a selective action, affecting certain
ganglion cells before others. In all the animals killed with this venom
practically all the cells in the anterior horns of the cord were entirely
free from grannies. Some cells were faintly stained, and some deeper
stained, skeletons, and many showed considerable disintegration of their
protoplasm. It could not, however, be determined that there was any
actual loss in the number of cells in the anterior horns, and there was
little difference in the appearances of the cells in the monkey that died
in ten days from those of the animal that died in three and a half days
after injection. It is, of course, difficult to be certain as to what is the
change in the cell that constitutes loss of function. In some of the
monkeys killed with cobra venom — e.g., Monkey No. 10 — there was
definite paralysis, but no recognizable structural change in the ganglion
cells of the motor cortex or cord ; and, on the other hand, it is
recognized that there may be almost complete loss of Nissl granules
without paralysis or other loss of function.

We attempted, but not with much success, to get some observations
on this subject by examining the ganglion cells of monkeys that had
recovered after being paralysed. The first of these monkeys was
injected with cobra venom and it showed signs of paralysis in two hours
ten minutes. Antivenomous serum was then given intravenously, and
paralysis was still present fifty minutes later. The dose of antivenom
was repeated and one and a half hours later all signs of paralysis had
gone. The animal was at once killed. On examining the ganglion
cells of the motor cortex and anterior horns of the cord it was found
that quite 50 per cent, of the cells showed some deficiency of their
Nissl bodies, and many of them had these bodies fragmented, giving a
much finer granulation to the cell protoplasm than was normal. In
some cells the granulation was quite dust like, but all gradations were
found between these and the cells with Nissl bodies of normal size.
Generally, the cells had the appearance of a much more chronic chroma-
tolysis than seemed to be present in other cases of cobra poisoning ; also
there was httle diffuse staining of the cell, and the staining was not so
intense as in the cases not treated with antivenom. Two other monkeys
were similarly injected, first with venom, and when paralysis was com-
plete with antivenom one animal was killed twenty hours and the other
forty-four hours after signs of paralysis had disappeared. In both of
these the ganglion cells were practically normal. The Nissl bodies in a

Neurological Section

7

proportion of the cells were possibly slightly deficient in number and not
quite so large as in a normal animal, but, on the other hand, some of the
adjacent granules seemed to have become fused together, forming masses
of chromatic material considerably larger than normal. In none of the
cells was there any appearance of fragmentation of granules, as in the
first animal.

In these three monkeys I take it that there had been at least slight
chromatic changes in the ganglion cells before the antivenom had been
injected, for other animals dying paralysed in two hours after injection
with the same venom showed such slight changes. The second animal
would seem to show that the cells return to their normal within twenty
hours after the paralysis disappears. But it is doubtful what interpre-
tation one should give to the appearances in the first of the three. It
may be that they represent a regeneration of the chromatic material.
This, however, is not probable, for though changes had almost certainly
commenced in the cells before the antivenom had been given, these
changes must have been slight and not nearly so marked as those seen
in the animal after recovery from its paralysis. It is more likely that
the antivenom inhibited the venom and so modified the degenerative
reaction in the cell, for the appearances were such as one associates with
a much more chronic chromatolysis than that produced by ordinary
cobra venom.

The connective-tissue elements (glia cells) of the grey matter seem to
play an entirely secondary part in the degenerative changes produced by
snake venoms. These glia cells may be slightly increased in number
round the ganglion cell in its earliest stages of chromatolysis, but this
increase is not in the least considerable, and it is not till vacuolation
comes on and the cell begins to disintegrate that they are seen to cluster
definitely round the disappearing ganglion cell. During this later stage,
and sometimes also at an earlier stage, the glia cells are found indenting
the margins and sometimes they are right inside the body of the cell.
It is doubtful if there is any definite increase in the number of the glia
cells throughout the rest of the grey matter, and they did not seem
more numerous in animals living eight to ten days than in those dying
in a few hours. The individual cells surrounding the effete ganglion
cells are distinctly increased in size as compared with ordinary glia cells,
and there seems little doubt that they are derived from pre-existing glia
cells and that they are neuroclastic in function.

Nerve fibres from the peripheral as well as from the central nervous
system were staineal in various ways so as to demonstrate any change

8 Hunter : Degenerative Changes in the Nervous System

in their myeline or axis cylinders. The peripheral nerves examined
were the vagus in the cervical region, the median near the elbow, and
the posterior tibial from the lower leg. In all the animals the sections
were stained (1) with osmic acid, according to the method of Marchi, or
that of Busch ; (2) with thionin or Congo red, according to Nissl's
method ; and (3) with haematoxylin and eosin, or van Gieson's method.
In none of the animals was there a genuine degenerative reaction with
the Marchi or Busch method. In some sections there was slight
" blackening" of some of the fibres, but I could not satisfy myself that
this was not an artefact, and in any case the axis cylinders in these
fibres were shown to remain intact. There was not even a definite
" Marchi reaction " in the monkeys that lived for eight and ten days
after injection of the Bungarus fasciatus venom. It is, of course,
difficult to know what effect chromatolysis in a ganglion cell has on its
nerve fibre, but in any case the absence of a secondary degeneration in
the nerve fibres in these animals can be explained on the supposition
that the degenerating ganglion cells had not lost their trophic functions
till, say, forty-eight hours or so before the death of the animal. With
the other stains there seemed to be, in a number of the monkeys, a
slight increase of the internodal nuclei of the peripheral nerves. This
was perhaps most definite in the animals that lived longest — for example,
in the monkey that lived for ten days. With the view of showing the
earliest recognizable degeneration in myeline and axis cylinders, nerve
fibres from certain of the monkeys — those killed with sea-snake and
with common-krait venom — were also stained by Donaggio's method.
With this stain {see Plate, figs. 27 to 31) it seemed definitely shown
that a considerable proportion of fibres, of the central as well as the
peripheral nervous system, gave the appearances of an early degenera-
tion. These fibres stained much more intensely and resisted much
longer the decolorizing agents than do the nerve fibres of a normal
animal. In the brain and spinal cord the deep staining seemed to
affect chiefly the axis cylinders. These structures, in addition to their
deep staining, were in places wavy in outline, and occasionally showed
slight swellings so as to form at intervals spindle-like thickenings. But
the myeline also retained the stain more tenaciously than normal,
though less uniformly than the axis cylinders. This patchy staining
of the myeline seems to give the fibre an uneven outline, constrictions
alternating with apparent swellings. In some of the fibres the myeline
and axis cylinders stain the same colour and with the same intensity, so
that one could not be distinguished from the other, the two seeming to

Neurological flection

9

have merged into one. In the peripheral nerves and nerve-roots some-
thing of the same appearances vi^ere to be seen ; but as a rule the deep
staining of the axis cylinders was less in evidence, and what stained
most intensely seemed to be a fragmented myeline, as represented by
deep-stained granules, most often arranged at the margin of the fibre,
but sometimes scattered through its whole diameter. This granular
appearance was most marked in the monkeys that lived for the longer
periods, whilst in Monkey No. 39, which lived for one and a third
hours, there was less granulation visible and more of the deep-stained
axis cylinder. I take it, then, that the first change in the nerve fibres
in snake-venom poisoning shows itself by a deeper staining of the axis
cylinders, and that later this quality passes to the myeline, which later
still may show some signs of fragmentation. There is therefore an
early degeneration in these nerve fibres, but it seems to be chiefly
chemical in nature, for there was little structural alteration to be
demonstrated. The axis cylinders are in places somewhat swollen, and
the myeline uneven in outline, with rarely some granular disintegration.
But there was no definite Marchi reaction and certainly no evidence of
Wallerian degeneration, even in the animals living for eight and ten
days after injection.

Nerve terminals were examined in monkeys killed with Daboia
venom, in those killed with sea-snake venom, as well as in those killed
with common-krait venom. The nerve terminals were fixed and stained
in osmic acid. With the first of these three venoms the terminals were
practically normal. With the second a considerable proportion of the
fibres were also normal, but others had their myeline rather wavy in
outline and segmented at frequent intervals. In a few other fibres still
the myeline seemed entirely fragmented and to be represented by small
granules occupying the whole diameter of the fibre. With the venom of
the common krait, especially in Monkey No. 31, which lived for sixteen
and a half hours, the same sort of granular appearance was to be seen,
but a larger proportion of fibres was affected. Some few fibres stained
diffusely, showing no differentiation of myeline or axis cylinder, and
they looked as if their contents were in soh;tion. The nerve terminals
would seem then to show in a proportion of their fibres the appear-
ances of a commencing degeneration.

10 Huuter : Degenerative Cha7iges in the Nervous System

DESCRIPTION OF PLATE.

Figs. 1, 2, 3, and 4. — From monkeys killed with Daboia venom. These cells may be regarded
as being normal.

Figs. 5 and 6. — From Monkey No. 36, which died in six and three-quarter hours after injec-
tion of sea-snake venom. Shows some fragmentation of the Nissl granules and the
whole cell rather diffusely stained.

Fig. 7. — From Monkey No. 36, which died in six and three-quarter hours after injection of
sea-snake venom. Cell paler with disappearance of the Nissl granules from a part
of the cell.

Figs. 8, 9, and 10. — From same monkey as fig. 7. Show deep-staining reticulum, but almost
complete loss of granules. In fig. 8 there is a neurophage at the margin of the cell.

Fig. 11. — From Monkey No. 37, which died six and three-quarter hours after injection of sea-
snake venom. Shows loss of Nissl granules as well as vacuolation of nucleus.

Fig. 12. — From same animal as fig. 11. Shows pale cell with no appearance of nucleus of
limiting membrane.

Fig. 13. — From Monkey No. 38, which died four hours after injection of sea-snake venom.

Shows a skeleton cell in the process of disintegrating ; there is vacuolation and a
neurophage visible.

Fig. 14. — From same monkey as fig. 13. Shows later stage of disintegration.

Figs. 15 and 16. — From same monkey as figs. 13 and 14. Shows little more than nucleus

surrounded by neurophe cells.
Figs. 17, 18, and 19. — Prom Monkey No. 39, which died in one and a third hours after

injection of sea-snake venom. Shows Nissl bodies breaking up and disappearing ;

vacuolation in fig. 19.

Figs. 20 and 21. — From Monkey No. 33, which died in ten hours after injection of common-
krait venom. Fig. 20 shows fragmented granules ; in fig. 21 the granules have
disappeared.

Pig. 22. — From Monkey No. 31, which died in sixteen and a half hours after injection of
common-krait venom. Shows cell deeply stained, with sort of reticulum, but no
granules.

Pigs. 23 and 24.— From Monkey No. 18, which died in six days after injection of banded-
krait venom. Fig. 23 shows a few granules ; in fig. 24 they have almost disap-
peared, and there is a vacuole and a neurophe cell.

Figs. 25 and 26. — Prom Monkey No. 20, which died in ten days after injection of banded-
krait venom. Show " ghost " cells without granules.

Fig. 27. — From Monkey No. 37, which died six and three-quarter hours after injection of sea-
snake venom. Shows nerve fibre irregular in outline with axis cylinder and myeline
not differentiated.

Fig. 28. — Prom Monkey No. 36, which died in six and three-quarter hours after injection
of sea-snake venom. Shows axis cylinder deeply stained and differentiated from
foam-like myeline.

Fig. 29. — From Monkey No. 39, which died one and a third hours after injection of sea-snake
venom. Shows axis cylinder deeply stained, with myeline faintly indicated.

Figs. 30 and 31. — From Monkey No. 32, which died in twelve hours after injection of
common-krait venom. Fig. 30 shows both axis cylinder and myeline ; fig. 31 shows
spindle-shaped swelling of axis cylinder.

Neiiroloqical Section

11

Acute degenerative changes in the nervous system, as illustrated by snake-venom poisoning.

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