Chronic relapsing experimental allergic encephalomyelitis An experimental model of multiple sclerosis.код для вставкиСкачать
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  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 . 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 2. Rainc CS, Wisnicwski H M , Dowling PC, er al: An ultrastructurd study of experimental demyelination and remyelination: 1V. 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