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Chronic relapsing experimental allergic encephalomyelitis An experimental model of multiple sclerosis.

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Chronic Relapsing Experimental Allergic
Encephalomyelitis: An Experimental Model
of Multiple Sclerosis
H. M. Wiiniewski, M D , PhD, and A. B. K e i t h
A chronic relapsing f o r m of experimental allergic encephalomyelitis was induced by single sensitization of i m m a t u r e
strain 13 and Hartley g u i n e a pigs. Clinically, the disease was characterized b y remissions a n d relapses. Recent and old
demyelinating plaques, which are the two characteristic features of multiple sclerosis lesions, were also present. The
model described opens new avenues for the study of immunological mechanisms governing remissions and exacerbations i n multiple sclerosis and offers new opportunities f o r evaluating the efficacy of d r u g s for its treatment.
Wiiniewski HM, Keith AB: Chronic relapsing experimental allergc encephalomyelitis: an experimental
model of multiple sclerosis. Ann Neurol 1:144-148, 1977
The 1975 r e p o r t of t h e National Advisory C o m m i s sion o n Multiple Sclerosis [ 11 states that: “One obstacle i n the path of investigators seeking t h e cause of a n d
c u r e s a n d t r e a t m e n t for multiple sclerosis is t h e fact
that the disease occurs, so far as is yet k n o w n , only in
man,” and “ W h a t MS researchers need for t h e ultim a t e benefit of patients is a n animal m o d e l of multiple
sclerosis.” A t present, t h e closest approach t o a n
animal m o d e l of MS is a c u t e experimental allergic
encephalomyelitis (EAE). I n its n o r m a l course, howe v e r , EAE is n o t marked b y t h e unpredictable cycles
of exacerbation a n d remission so characteristic of MS.
H e r e w e r e p o r t t h e induction of chronic relapsing
EAE i n both strain 13 and H a r t l e y guinea pigs. T h e
progressive and multiphasic c o u r s e of EAE was prod u c e d by sensitization of i m m a t u r e animals and by
increasing the dose of Mycobucterium tuberculosis in
Freund’s adjuvant.
Materials and Methods
T h e Dunkin Hartley guinea pigs used in the experiment
came from our own randomly bred closed colony. This was
established 1 0 years ago and currently consists of 30 males
and 210 females. They are bred using a harem system of 1
male to 7 females. This colony has been built up from an
original stock of approximately 30 females and 5 males.
Strain 11, breeding stock was obtained from the National
Institute for iMedica1 Research, London, where the strain 13
guinea pigs are maintained on a breeding regimen of
brother-sister mating. The animals in our colony, consisting
of 30 females and 6 males, are allowed to breed randomly
within the same generation.
From the Medical Research Couricjl Demyeharing Diseases Unit,
Newcastle General Hospital, Newcastle upon Tyne, England.
Accepted for publication Aug 11, 1976.
144
PrepuratioTz of Antigen and Sensitization
To prepare the antigen, 10 g m guinea pig spinal cord was
homogenized in 10 ml of physiological saline plus 2 0 ml of
Freund’s adjuvant containing 10 mg per milliliter of M .
tuberrdosb (M37Ra). The animals in one group, consisting
of 52 Dunkin Hartley and 11 strain 13 guinea pigs of both
sexes and 18 to 21 days old, were sensitized by injecting 0.1
ml of the emulsion into the dorsum of all four feet; 30
Hartley guinea pigs were sensitized with the same dose
injected at four sites over the nuchal area. In addition, 20
strain 13 and 6 Hartley guinea pigs, also 18 to 21 days old,
were sensitized with an injection of 0.1 ml into the dorsum
of the two hind feet only (Table).The animals were housed
with their mothers up to the weaning period and given a diet
of guinea-pig pellets supplemented with cabbage and water
ad libitum.
Each animal was weighed and examined daily for neurological signs. The brains and spinal cords of the majority of
animals that died were rernovcd and fixed in 10?i; buffered
formalin. Moribund animals were anesthetized intraperitoneally with sodium pentobarbital and perfused through the
left ventricle with 5%, glutaraldehyde in an 0.1 M phosphate
buffer at pH 7.3. Brains and spinal cords together with roots
and dorsal root ganglia were removed for morphological
studies. Selected areas from the brain, spinal cord with
roots, and dorsal root ganglia were taken for electron microscopical studies. The rest ofthe material was embedded in
paraffin. Sections 1 y thick were embedded in Epon, stained
with IG; toluidine blue, and examined under the light
microscope to try to locate lesions for electron microscopy.
Paraffin sections were stained with hematoxylin and eosin;
the Bodian silver method was used for axons and the Loyez
method for myelin.
Address reprint requests to Dr Wisnieurski, NYS Institute for Basic
Research i n Mental Retardation, 1050 Forest Hill Rd, Staten Island, NY 10314.
Sumnzary of’ Remlts
Inoculum Route
Dorsum, Four Feet
(0.4 ml)
Dorsum, Two Hind Feet
(0.2 rnl)
Nuchal Area
(0.4 ml)
Hartley
(Hartiey; n = 30) ( n = 6)
Status of
EAE
Hartley
(n = 52)
(n
Acute EAE
Subacute relapsing
Chronic relapsing
No disease
30
5
5
7
4
7
10
12
6
18
2
11
2
Strain 13
=
11)
Results
Clin icul Obsewatiorrs
Acute monophasic EAE developed in 30 of the 52
Hartley guinea pigs inoculated in all four feet (see the
Table) and was characterized by weight loss and the
appearance of neurological signs, ie, various degrees
of paraparesis or tetraparesis. T h e average time of
onset of the disease was 18 days following sensitization, and survival time from the beginning of signs to
death was one to 11 days. Two episodes of the disease
occurred in 10 guinea pigs. However, the relapse was
after only eight to 11 days of remission. In these
animals with subacute relapsing EAE, the usual course
of the disease was as follows: weight loss and neurological signs for 24 to 72 hours, followed by almost
complete o r complete clearance of the neurological
deficit and weight gain. After the short period of
recovery (eight to 11 days) the animals again lost
weight, and neurological signs reappeared. During the
next four to 10 days their neurological and general
condition gradually deteriorated, and the animals died
or were perfused when moribund.
Chronic relapsing EAE developed in 1 2 guinea
pigs. The first episode of the disease was observed
between 12 and 30 days after sensitization, with neurological signs (paraparesis) lasting for one to eight
days. After this period, 10 of the 12 animals recovered completely. The remaining 2 , after an attack of severe paraparesis, showed persistent slight
residual weakness of the hind legs. In the majority of
these animals the remission period lasted for three to
four weeks. However, 2 guinea pigs had a prolonged
remission, ie, 56 and 84 days, respectively.
By and large, the clinical picture of the second
episode was similar to the first, although the neurological signs lasted longer: seven to 14 days. After this
period, 6 animals recovered completely and 5 showed
definite improvement; ie, after severe paraparesis
they were able to use their hind legs again, but after
exercise or when running, the hind legs showed incoordination and weakness and the animals again
started to drag their legs. In 1 animal, severe paralysis
of the hind legs gradually developed after the second
Strain 13
( n = 20)
episode of slight paraparesis, and the front legs also
became weak; 20 days after the second attack and 80
days from the day of sensitization severe respiratory
difficulties developed, and the animal was perfused.
The remission after the second attack lasted for 30
t u 60 days in the majority of animals. During this
remission period, 1 guinea pig was perfused for morphological studies. In 3 animals the second relapse of
EAE (severe paraparesis) developed overnight. T h e
other 7 animals showed gradual weakness of their
hind legs, which led to various degrees of paraparesis
and incontinence within two weeks.
N o n e of the animals recovered completely after the
third episode. Over a period of one to three months 5
guinea pigs gradually deteriorated, showing more
neurological signs (epileptic seizures, front leg involvement) and, usually due to secondary infections,
were perfused in moribund condition. In 3 guinea
pigs, after severe paraparesis and incontinence developed, no fluctuation of the neurological signs occurred, and they were perfused for morphological
studies three to five months after the second relapse.
Eight months after sensitization 2 animals are still
alive.
Of the 11 strain 13 guinea pigs inoculated in all four
feet (see the Table), 5 died or were perfused because
of their moribund state during the acute phase of
EAE. Chronic relapsing EAE of the type described in
the Hartley guinea pigs developed in 6, but the remission time between the recurrent episodes was longer
than in the Hartley guinea pigs. The neurological
symptoms were also longer after each episode. After
the third episode they all had general or focal epileptic
seizures. O n e of these guinea pigs was perfused for
morpliological study during the long (50-day) remission after the second episode. At the time of sacrifice
the animal showed no neurological signs. O n e guinea
pig from this group is still alive eight months after
sensitization but shows severe paraparesis, incontinence, and weakness of the front legs. This animal also
has general epileptic seizures.
Of the 3 0 Hartley guinea pigs sensitized with the
same dose (0.4 ml) of inoculum administered at four
Wiiniewski and Keith: Chronic Relapsing Experimental Allergic Encephalomyelitis
145
sites in the nuchal area (see the Table), 5 died two t o
three weeks after sensitization during the first episode
of the disease, and 7 succumbed during the second
episode, which was preceded by a short remission
(seven to 10 days). Of the remaining 18 guinea pigs, 6
had arelapse 30 to 60 days after the first attack, and 12
had a relapse 60 to 1 2 0 days after the first attack. O n e
died from pneumonia after the second relapse, and 1
was perfused. The rest of the animals ( 1 5 ) are alive
200 days after sensitization. They have all had one o r
two relapses, with 11 showing various degrees o f
paraparesis and incontinence and 4 showing minimal
weakness of the hind legs.
Among the 20 strain 13 guinea pigs immunized by
injection of 0 . 1 ml of inoculum into the dorsum of
each hind foot (Table), acute EAE developed in 18
within rwo to three weeks after sensitization. The
neurological signs (14 with severe, 4 with mild
paraparesis) lasted for one to six days; 1 guinea pig
died, and the rest recovered. At 7 0 days after sensitization 2 guinea pigs still had not shown any neurological signs; 7 had had one attack, and 11 had experienced two relapses after 3 4 to 5 5 days of remission. Of
6 Hartley guinea pigs sensitized with 0.1 ml in each
hind leg, 4 died after two to three weeks with symptoms of acute EAE; 1 recovered almost completely
after the acute episode, and 1 was left with mild residual paraparesis. These animals were alive and well
50 days after sensitization.
guinea pigs they were localized in the spinal cord.
Strain 1 3 guinea pigs showed inflammatory lesions
and plaques in the spinal cord, brain, and cerebellum
(Fig 2 ) . The areas most affected were the thoracic and
lumbosacral segments of the spinal cord. In some of
the animals the plaques were so large that they could
be seen with the naked eye. The recent lesions were
characterized by the presence of perivascular cuffs of
hematogenous cells, myelin-laden macrophages, and
demyelination. By and large, the old lesions were free
of inflammatory cells. In old plaques, remyelination
was a common phenomenon. In some large and
confluent plaques there was evidence, at the edges, of
active demyelination; in other areas, remyelination
was seen.
In Hartley guinea pigs a few lesions were found in
the cerebrum and cerebellum only o n rare occasions.
This contrasted with massive inflammation found in
the white matter and, to a lesser degree, in gray structures of the brain and cerebellum of the strain 13
guinea pigs. T h e brain lesions were characterized by
the presence of very broad cuffs of perivascular cells
that spilled out into the brain parenchyma. Most of the
inflammatory cells were large mononuclear cells of
the type found around the residual deposits of the
antigen in the foot dorsurn or around any other type of
chronic granulomatous lesions. Segmental demyelination was present in such areas, as well as evidence of
secondary (wallerian) demyelination.
niIo rphologicul 0 bserzwtio ns
Irrespective of the strain of animal and dose of antigen
used, animals that died during the acute stage of the
disease showed similar pathological changes. These
consisted of perivascular cuffs of hematogenous cells
in the leptomeninges and around some vessels in the
spinal cord and brain. In the majority of animals, the
number of affected vessels was small and the degree of
inflammation was very mild. In a few guinea pigs,
however, the inflammation was extensive and involved both white and gray matter. This was most
pronounced at the thoracic and lumbosacral level of
the spinal cord. Demyelination was present around
some vessels with inflammatory cuffs and was more
pronounced in animals with more extensive inflammatory lesions. In a few animals that showed neurological symptoms, no morphological evidence of EAE
was found in the material sampled. The 8 Hartley
guinea pigs with short remission between the two
episodes of EAE showed more pronounced inflammatory lesions; demyelination was also more extensive and more commonly found than in the
monophasic group.
T h e most striking feature in the animals with
chronic relapsing EAE was the presence of old and
recent demyelinating plaques (Fig 1). In Hartley
Discussion
T h e experiments reported demonstrate the successful
development of a chronic relapsing form of EAE in
guinea pigs. Clinically, the animals showed the classic
MS-like picture of remission and exacerbation. Morphologically. they showed the two characteristic features of MS lesions: recent and old demyelinating
plaques. In strain 13 guinea pigs a periventricular,
MS-like distribution of the lesions was observed, and,
as in MS, animals with a longer clinical history showed
more chronic than acute lesions.
Spontaneous relapses of EAE or experimental allergic neuritis [2-51 have been reported, but they
occur in only a small proportion of sensitized animals.
A chronic disseminated form of EAE in immature
strain 13 and Hartley guinea pigs was described by
Stone and Lerner [GI.However, their report stressed a
slow, uninterrupted, progressive deterioration from
the onset of clinical signs to paralysis, debility, and
death. Although Stone and his colleagues [7] later
reported relapses in strain 13 animals, the number so
affected was low. Why so few were affected is not
clear. However, it is possible that the differences in
route of injection (foot dorsum versus nuchal area),
the smaller volume of antigen (0.4 and 0.2 ml versus
0.5 ml), and the lesser amount o f M . tuberculosis (2 mg
146 Annals of Neurology
Vol 1 No 2
February 1977
A
C
B
versus 2.5 mg) used by us created more favorable
conditions for a relapsing form instead of a chronic,
progressively downhill form of EAE.
I t is known that the amount of antigen-adjuvant
mixture used and the site and manner of its inoculation influence the course of the disease. This was again
confirmed by our experiments. In the Hartley guinea
pigs, administration of the antigen into the nuchal
areas appears to produce good relapsing EAE with
minimal mortality during the first attack. T h e strain 13
guinea pigs inoculated only in the dorsum of the hind
feet also had a low death rate during the acute stage o f
EAE.
Although the clinical course of the disease in
Hartley and strain 13 guinea pigs was similar, there
were substantial morphological differences between
the two strains. In the Hartley guinea pigs with
chronic relapsing EAE, n o lesions, or only minimal
ones, were found in the brain and cerebellum with just
a few exceptions. This contrasted with the findings in
strain 13 animals, in which the most extensive inflammation, reminiscent of the chronic granulomatous
type of lesion, was observed in the brain and cerebellum three to five months after sensitization and usually after the third clinical episode of the disease. Also,
Wiiniewski and Kcith: Chronic Relapsing Experimencal Allergic Eticrphalomyrlitis
147
also observed in our guinea pigs: animals killed during
clinical remission showed extensive inflammation and
demyelination, and some guinea pigs that died during
the acute phase of EAE and had severe paraparesis and
respiratory difficulties displayed minimal if any morphological changes.
EAE is an autoimmune disease induced by a welldefined antigen, the myelin basic protein, and in
which tissue lesion and demyelination occur in the
presence of immunocornpetent cells. Although the
cause of MS is unknown, the sequence of events leading to dernyelination in MS appears to be similar to
that seen in EAE [8]. If this is the case, the chronic
relapsing form of EAE presented here will allow us to
study the immunological mechanism involved in rhe
pathogenesis of MS. Relapsing EAE also appears to be
the best laboratory model for evaluating the efficacy
ol drugs for treatment o f MS.
A
The authors thank Mr E. A. Caspary and D r R. Eastinan for discussions and constructive criticism. We also appreciate the expert
technical, photographic, and secretarial assistance of Mr A. Oakley,
Miss Jacqueline Bristow, Miss Carol Coulcer, and Miss Margaret
Herron.
References
in the spinal cords of animals with chronic disease,
active lesions were more often found in the strain 13
animals than in the Hartley guinca pigs.
It has been known since the days of Charcot that in
MS there is a great disparity between anatomical
change and disturbance of function. This disparity
between clinical signs and morphological changes was
148
Annals of Neurology
Vol 1 No 2
February 1977
I Report and Recommendations by rhe National Advisory Commission on Multiple Sclerosis. (DHEW Publication N o . ( N I H )
74-534, 1974.) Vol2. Washington, DC, Department of Health,
Education and Welfare, 1974
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4. Wisniewski H M , IjrosroK SW, Carter H , et al: Recurrenr experimental allergic polygangliorad~ruloneuritis:multiple demyelinaring episodes in Rhesus monkey sensitized with rabbit
sciaric nerve myelin. Arch Neurol 30:34?-358, 1974
5. Ferraro A, Cazzullo CL: Chronic experimental allergic encephalomyelitis in monkeys. J Neuropathol Exp Neuroi
7~235-260,1948
6. Stone SH, Lerner EM 11: Chronic disseminated allergic encephalomyelitisinyineapigs. Ann N Y Acad Sci 122:227-241,
1965
Snyder D H , Valsamls MP, Scone SH, e t al: Progressive demyelination and reparative phenomena in chronic expcrimentai
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8 . Adams CWM: T h e onset and progression of the lesion in
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I .
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