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A HYPERACUTE FORM OF ALLERGIC ENCEPHALOMYELITIS SEYMOUR LEVINE, M.D., AND EUGENE J. WENK, M.A. From the Pathology Department, New York Medical College Center for Chronic Disease, Bird S. Coler Hospital, Welfare Island, N.Y. Acute necrotizing hemorrhagic encephalopathy is a rare disease of man characterized by abrupt onset of neurologic symptoms, rapid progression of paralysis, and fatal outcome.' Usually the onset is pre- ceded by an upper respiratory infection. Histologically, there are hem- orrhages, perivascular infiltrates of polymorphonuclear leukocytes, fi- brinous exudation, edema and necrosis. Another neurologic disease that is preceded by a potentially immunizing event is acute disseminated encephalomyelitis (post-infectious, post-exanthematous, or post-vac- cinal). Although the inflammatory lesions in this disease are also peri- vascular, other histologic features, as well as the prognosis, differ from acute necrotizing hemorrhagic encephalopathy. Nevertheless, it has been suspected that the two diseases are related.2 The present paper describes a reproducible, uniform, experimental model for human acute necrotizing hemorrhagic encephalopathy. This new experimental syndrome is actually a hyperacute form of experi- mental allergic encephalomyelitis (EAE). Ordinary EAE has been ac- cepted for many years as a laboratory model for acute disseminated encephalomyelitis.3 The pathogenetic links between the new hyperacute form and ordinary EAE provide evidence for the close nosologic relation- ship of the two human diseases mentioned above. The discovery of the hyperacute form was an outgrowth of work on the production of EAE without the aid of Freund's adjuvants.4 Previous reports have demonstrated that a single large dose of aqueous central nervous system (CNS) tissue homogenate without adjuvant produced ordinary EAE in the highly susceptible inbred Lewis strain of rats.4 This procedure did not work as well on rats of the Fischer 344 strain unless their susceptibility was increased by pretreatment with pertussis vaccine. It has been known for some years that intraperitoneal (IP) vac- cination of mice and rats with Bordetella pertussis organisms enhances the encephalitogenic effects of CNS tissue-adjuvant emulsions admin- This investigation was supported by Grant No. 3I7-2 from the National Multiple Sclerosis Society, and by a gift from Dr. Earl J. Halligan, Medical Director, St. Francis Hospital, Jersey City, N.J. Accepted for publication, February 23, I965. 6I

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  • A HYPERACUTE FORM OF ALLERGICENCEPHALOMYELITIS

    SEYMOUR LEVINE, M.D., AND EUGENE J. WENK, M.A.

    From the Pathology Department, New York Medical College Center for ChronicDisease, Bird S. Coler Hospital, Welfare Island, N.Y.

    Acute necrotizing hemorrhagic encephalopathy is a rare disease ofman characterized by abrupt onset of neurologic symptoms, rapidprogression of paralysis, and fatal outcome.' Usually the onset is pre-ceded by an upper respiratory infection. Histologically, there are hem-orrhages, perivascular infiltrates of polymorphonuclear leukocytes, fi-brinous exudation, edema and necrosis. Another neurologic disease thatis preceded by a potentially immunizing event is acute disseminatedencephalomyelitis (post-infectious, post-exanthematous, or post-vac-cinal). Although the inflammatory lesions in this disease are also peri-vascular, other histologic features, as well as the prognosis, differ fromacute necrotizing hemorrhagic encephalopathy. Nevertheless, it has beensuspected that the two diseases are related.2The present paper describes a reproducible, uniform, experimental

    model for human acute necrotizing hemorrhagic encephalopathy. Thisnew experimental syndrome is actually a hyperacute form of experi-mental allergic encephalomyelitis (EAE). Ordinary EAE has been ac-cepted for many years as a laboratory model for acute disseminatedencephalomyelitis.3 The pathogenetic links between the new hyperacuteform and ordinary EAE provide evidence for the close nosologic relation-ship of the two human diseases mentioned above.The discovery of the hyperacute form was an outgrowth of work on

    the production of EAE without the aid of Freund's adjuvants.4 Previousreports have demonstrated that a single large dose of aqueous centralnervous system (CNS) tissue homogenate without adjuvant producedordinary EAE in the highly susceptible inbred Lewis strain of rats.4This procedure did not work as well on rats of the Fischer 344 strainunless their susceptibility was increased by pretreatment with pertussisvaccine. It has been known for some years that intraperitoneal (IP) vac-cination of mice and rats with Bordetella pertussis organisms enhancesthe encephalitogenic effects of CNS tissue-adjuvant emulsions admin-

    This investigation was supported by Grant No. 3I7-2 from the National MultipleSclerosis Society, and by a gift from Dr. Earl J. Halligan, Medical Director, St. FrancisHospital, Jersey City, N.J.

    Accepted for publication, February 23, I965.

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  • LEVINE AND WENK

    istered several days later and by a different route.5"6 Discussors at arecent symposium 7 suggested that enhancement of EAE in Fischer 344rats by prior IP pertussis vaccination might be a type of adjuvant effect.They assumed that pertussis organisms or fractions reached the site ofinoculation of CNS antigen (or its draining lymph node) by way of thecirculation, and acted as an adjuvant. In point of fact, pertussis organ-isms are known to have adjuvant properties since they can replace themycobacteria in Freund's adjuvant (mineral oil, emulsifier and myco-bacteria). It was not known, however, whether pertussis organismscould reach the sites of antigen deposition in adequate amounts follow-ing intraperitoneal injection, and whether they had adjuvant propertiesin the absence of an oil. In order to investigate the latter problem, weinjected a mixture of aqueous CNS tissue with aqueous pertussis vac-cine. It was in this manner that the hyperacute form of EAE was firstproduced. A preliminary report has appeared.10

    METHODSExperiments were performed on female or male Lewis rats (Microbiological As-

    sociates, Inc., Bethesda, Md.) or other strains and species as designated below. Ratswere fed Purina Laboratory Chow and tap water ad libitum. They were housed inmetal hanging cages in groups of 6.The adjuvant was aqueous commercial pertussis vaccine stated to contain approxi-

    mately 6o billion organisms per ml or concentrated vaccine that had 3oo billion or-ganisms per ml. The antigen was usually frozen spinal cord tissue from rats or guineapigs, heated to 600 C for 45 minutes immediately before use. For intraperitonealinoculation, weighed aliquots of tissue were passed back and forth through a 20-gaugeneedle from a syringe to a vial that contained sterile, pyrogen-free saline and pertussisvaccine. Each dose was 3 ml in volume; it contained 200 mg tissue (wet weight)and o.6 ml commercial pertussis vaccine or O.I2 cc pertussis vaccine concentrate (36billion organisms). In the case of footpad inoculations, the volume of inoculum waslimited by the small size of the foot. Therefore, 8 parts of whole spinal cord tissuewere homogenized directly with 2 parts of pertussis vaccine concentrate; no salinewas added. The mixture was injected intradermally in a dose of 0.25 ml (200 mg wetweight of tissue and i5 billion organisms). This volume was distributed equallyamong 5 of the 6 cutaneous thickenings (pads) on the sole of the right hind foot.Footpad, but not intraperitoneal injections, were performed with the aid of lightether anesthesia. Precautions for sterility were observed.Advanced EAE was manifested as weakness, ataxia, paralysis and urinary reten-

    tion. The earliest indications of disease were paralysis and complete loss of tonus ofthe distal end of the tail or of the entire tail. The animals were sacrificed at onsetof clinical disease, or within 2 to 9 days of onset by exsanguination from neck ves-sels under ether anesthesia. Rats without clinical signs were sacrificed after 8 to 22days, except as indicated. Central nervous tissue was fixed in Bouin's solution, othertissues in acetate-buffered formalin. Paraffin sections were stained by hematoxylinand eosin, phosphotungstic acid-hematoxylin (PTAH), and by the luxol fast blue-periodic acid-Schiff-hematoxylin stains.

    RESULTS

    Hyperacute EAE was produced by intraperitoneal or footpad inocu-lations of a mixture of spinal cord tissue and pertussis vaccine. Clinical

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    signs occurred in all animals 6 to 9 days after inoculation and lesionsexhibited a predominance of neutrophils (Table I). Spinal cord homog-enate without adjuvant produced ordinary rather than hyperacuteEAE, even if pertussis vaccine was administered simultaneously but bya different route (Table I); the onset of clinical disease was later, and

    TABLE IREQUIREMENT FOR PRODUCTION OF HYPERACUTE EAE

    (CNS ANTIGEN MIXED WITH PERTUSSIS VACCINE)

    EAEAntigen* Pertussist Day of onset$ Histologic type

    G. pig cord IP IP mixed with antigen 7 7 7 7 8 8 8 8 8 8 9 9 Hyperacute" " " Foot Foot" " " 6 6 6 7 7 7 7 7 7 Hyperacute

    Sub-"" " Subcut cut" " " I4I4 Ordinary

    " " " IP None 8 IO II ii - Ordinary" " " Foot IP I2 12 - - - Ordinary" " nerve IP IP mixed with antigen 9 IO IO IO iO i Ordinary" " " Foot Foot" " " 9 9 9 9 9 9 OrdinaryRat kidney IP IP " " " - - - - - - NoneNone IP ----- None

    *Antigens, 200 mg wet weight; frozen nerve and kidney microtomed before homogenizing.o.6 ml commercial vaccine diluted to 3.0 ml with saline for all IP and subcutaneous inoculations;

    0.05 ml concentrated vaccine for footpad inoculations.* In this and subsequent Tables, each figure represents a single female Lewis rat with clinical

    signs, each hyphen represents a single rat without signs. In nerve groups, clinical signs were due toneuritis as well as EAE.

    Intraperitoneal.

    neutrophils were not frequent in the lesions. Peripheral nerve mixed withpertussis vaccine caused EAE probably due to the antigenic overlapbetween CNS and peripheral antigens 11`3; here again it was ordinaryrather than hyperacute in histologic character. Pertussis vaccine with-out any tissue or mixed with non-neural tissue (kidney) did not yieldCNS lesions of any type.

    Histologic Features of Hyperacute EAE. The severity of lesions par-alleled the severity of clinical signs. Fully developed lesions occurredin rats that had signs for 24 hours or more. These consisted of a hyper-acute inflammatory process involving meninges, gray and white matterat all levels of the neuraxis, severe in spinal cord, severe, moderate orslight in brain stem and cerebellum, and slight in cerebrum. The conusmedullaris of the cord was a site of predilection. Cauda equina rootsand filum terminale were often involved. The inflammation was vascularand perivascular, with preferential involvement of small veins (Fig. I ).Lesions became confluent frequently in the meninges and occasionally inthe parenchyma.

    Neutrophils were present in enormous numbers (Figs. I, 4, 7 and 8).Two other types of inflammatory cells were observed. One of these had

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    relatively large, pale nuclei with scanty chromatin. The irregular, elon-gated, indented, often lobulated or reniform nuclei indicated that thesecells were monocytes. The third, least frequent, cell type had a smaller,darker, round or oval nucleus with more compact chromatin. This cellwas a lymphocyte. All three cell types were seen in vessel lumens, linedup along the endothelium, in the walls, and in the inflammatory in-filtrate.

    These observations suggested that the inflammatory cells were derivedfrom the blood stream (as has been shown by Kosunen, Waksman andSamuelsson in ordinary EAE). Some "monocytes," however, may havebeen activated microglia. The inflammatory cells surrounded the vesselsand extended deep into the parenchyma in large numbers. The threetypes of cells occurred in any combination and in very variable propor-tions; neutrophils, however, usually outnumbered the others by far.Many inflamed vessels were surrounded by pale, edematous zones (Fig.i). Neutrophils wandered into the parenchyma, even beyond the edem-atous zone. Sometimes the vessel walls were necrotic and stronglyeosinophilic. More frequently narrow zones of the perivascular paren-chyma were deeply eosinophilic (Fig. i). The eosinophilic materialstained strongly with PTAH, indicating that it was fibrin (Figs. 3, 4 and5). The fibrin was concentric or eccentric, or even restricted to oneside of a vessel (Fig. 3). Frequently, vessel lumens were occluded bythrombi (Figs. 6, 7 and 8). The pale perivascular zones were demyeli-nated (Fig. 9). They contained periodic acid-Schiff (PAS) positiveedema fluid which formed large pools that dissected along the junctionof gray and white matter, or separated bundles of myelinated fibers(Fig. io) or extended along the central canal. The relation of edemafluid to blood vessels of origin was lost.On occasion, edema fluid surrounded a small vessel which had few

    or no inflammatory cells in or around it. It seemed that vascular per-meability might be increased selectively for cells or for plasma, or permitboth fluid and cellular components to pass. The central canal wasdistended with eosinophilic, PAS positive fluid that resembled plasma(Fig. i i). The subarachnoid space contained similar fluid, in whichthe PTAH stain revealed the presence of fibrin (Fig. I 2). The meningesand the subpial parenchyma were infiltrated with many inflammatorycells (Figs. I2 and I3). Small areas of necrosis and large areas ofmyelomalacia were observed (Fig. 13). Hemorrhages were numerous,but small, and usually had no obvious relation to blood vessels (Fig.I4).In the most severe examples of hyperacute EAE, the spinal cord

    was studded with thrombosed, necrotic vessels, each surrounded by

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    enormous numbers of neutrophils that completely obscured the under-lying parenchyma. With confluence of lesions, there were large fieldsin which virtually nothing was visible but inflammatory cells (Fig. I4).In contrast, rats with ordinary EAE had a predominately mononuclearinflammatory reaction. Neutrophils were uncommon; fibrinous exuda-tion was rare; vascular thrombosis and necrosis were not observed (Fig.2).

    Onset of Clinical Signs. Two large groups of female Lewis rats werechallenged with guinea pig cord homogenate-pertussis vaccine mixtureby the intraperitoneal route. Signs were detected in a few rats during themorning or afternoon of the sixth day after injection (Table II); the

    TABiE IICLINICAL ONSET OF HYPERACUTE EAE (CUMULATIVE PERCENT)

    Time of Examination Exp. i (90 rats) Exp. 2 (77 rats)

    Day + 6 8 AM 6% o%2PM 13 38PM 7

    Day + 7 2 AM 88AM 36 122PM 40 348 PM 84 64

    (Terminated)Day + 8 2AM 8I

    8AM 942 PM 968 PM IOO

    Female Lewis rats, 200 mg guinea pig cord mixed with o.6 ml pertussis vaccine intro-duced intraperitoneally. The second experiment was similar to the first except for morefrequent observations and longer surveillance.

    signs were mild (tail only). During the seventh day, the rate of ap-pearance of new cases accelerated, and Y4 of the sick rats had hind limbas well as tail weakness when their illness was first discovered. By theevening of the seventh day or morning of the eighth day, almost all therats had signs of EAE. At the end of the eighth day, the incidence ofdisease was ioo per cent (Table II, Exp. 2). It is possible that apparentonset of hyperacute EAE on the ninth day or later, in a few animals inother experiments, was sometimes due to lack of examination late onthe eighth day.

    The Early Lesion and Course of Hyperacute EAE. No CNS lesionswere found in 6 rats killed on the fifth day after injection. Three of ioasymptomatic rats killed on the sixth day after inoculation had lesions.The lesions were small inflammatory infiltrates in nerve root or lepto-meninges, with or without subpial extension. The infiltrates were pre-

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    dominantly mononuclear but some neutrophils were present. Elevenrats with mild clinical signs of EAE were sacrificed on the same day,and all had lesions. Lesions were somewhat more numerous than in theasymptomatic rats. The meninges and subpial region were always af-fected, but there were also lesions deep in parenchyma. Neutrophils werestill in the minority except for small foci in two specimens in whichneutrophils predominated and fibrinous exudation was present.

    Sixteen of 27 asymptomatic rats killed on the seventh post-inoculationday had lesions, a considerably greater proportion than had been foundin clinically-well rats on the sixth day. However, the character and in-tensity of these early, pre-symptomatic lesions were unchanged. Thirty-five rats with clinical signs were killed on the seventh day and all hadlesions. About half of these animals had mild clinical signs with moderatenumbers of lesions in which the proportion of neutrophils varied fromIO to 50 per cent. The remaining rats had severe clinical signs with verymany fibrin-impregnated lesions in which neutrophils predominated.Even these spinal cords, however, had some fields in which the infiltrateswere exclusively mononuclear, or in which the only abnormality wasmargination of mononuclear cells in vessel lumens. The disease pro-gressed rapidly; mild weakness made its appearance in as short a timeas I hour; severe paralysis was observed in rats known to have ap-peared perfectly well 5 hours before. The rapidity of progress was themore remarkable because pre-symptomatic lesions showed no increaseof severity from the sixth to the seventh day. In other words, the lesionstypical of hyperacute EAE (abundance of neutrophils, fibrin impregna-tion and edema) did not develop in an occult fashion in clinically-wellrats, but probably appeared at the same time as clinical signs.

    Six rats that developed mild signs (limp tail) on the evening of theseventh day were not sacrificed until the next morning. At that time, theyall had severe paralysis. The lesions had increased in number and se-verity. Fibrin-impregnated lesions, swarming with neutrophils, were ob-served in all or many low-power fields. Exclusively mononuclear lesionswere no longer present. Thrombi, hemorrhages, focal necrosis and con-fluence of lesions were noted. Death occurred frequently after I or 2days of sickness, but in I experiment 4 rats survived until 3, 3, 4 and7 days after onset. Remissions have not yet been observed.Route and Site of Sensitization. Hyperacute EAE was produced read-

    ily after either intraperitoneal or hind footpad inoculation of pertussisvaccine-spinal cord mixtures (Tables I and III). In contrast, the sub-cutaneous route elicited only ordinary EAE (Table I). Intradermalinoculations in neck or footpads of forelimb yielded intermediate results(Table III). Some of the rats had hyperacute lesions, although these

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    were not usually as characteristic as those that followed intraperitonealor hind foot challenge.The predilection of ordinary EAE lesions for the lower portions of

    the rat's neuraxis has been noted in other investigations.6',4 The distribu-tion of lesions of hyperacute EAE followed the same pattern. A ratwhose spinal cord was riddled with lesions often had no abnormality oronly a minor meningeal infiltrate in the forebrain. Shiina and Iida have

    TABLE IIIPRODUCTION OF HYPERACUTE EAE (EFFECT OF ROUTE AND SITE OF INJECTION)

    Histologic type of EAE*Route Site Day of onset of EAE Hyperacute Ordinary

    Intraperitoneal 7 7 7 8 8 8 8 8 8 9/9 0/9Intradermal Hind footpads 7 7 8 8 9 12 4/6 2/6

    49 Fore footpads 7 8 8 8 8 8 9 12 4/I0 6/ioNeck I2 13 13 I3 I3 14 I4 14 I4 15 2/10 8/IO

    Female CD F rats injected with a mixture of 200 mg guinea pig cord homogenate and o.o5 mlpertussis vaccine concentrate. Dose 0.25 ml administered IP, or intradermally divided among 5 sitesin right hind footpads, right forelimb footpads, or dorsal and ventral neck.

    * Numerator represents number of rats with EAE of designated type. Denominator representstotal number of rats.

    suggested that the localization of lesions is determined by the site ofinoculation.15 However, there was no evidence of increased forebrain in-volvement after forepaw or neck inoculation compared to hind footinoculation.

    Attempts to Produce Hyperacute EAE with other Adjuvants. Thehistologic appearance of the hyperacute disease produced by inoculationof a mixture of CNS tissue and pertussis vaccine suggested that it was aform of EAE of exceptional severity. Therefore, an attempt was madeto produce this type of disease with other types of intense antigenicstimulation. CNS tissue in Freund's complete adjuvant or in Freund'sadjuvant with increased content of killed mycobacteria was injected intothe highly susceptible Lewis rats with or without pretreatment withpertussis vaccine.6 A high incidence of EAE was obtained and the onsetwas very early, 6 to ii days after challenge (Table IV). A few neu-trophils were present in the lesions in almost every animal, and in 2instances there were focal subpial accumulations. In no case, however,was there the massive neutrophil infiltration and fibrinous exudationthat is characteristic of hyperacute EAE.The failure of Freund's adjuvant to produce hyperacute EAE in-

    dicated that aqueous pertussis vaccine had some unique adjuvant prop-erty. This property was lost, however, when the mixture of pertussisorganisms and CNS tissue was emulsified in oil. All the rats that re-

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    ceived this emulsion developed EAE (Table IV), but it was ordinaryrather than hyperacute EAE.

    Heating to IOO1 for i hour destroyed the adjuvant quality of aqueouspertussis vaccine for the production of the hyperacute disorder (TableIV). On the contrary, washing the organisms 3 times with saline had no

    TABLE IVUNSUCCESSFUL ATTEMPTS TO PRODUCE HYPERACUTE EAE WITH ADJUVANTS OTHER THAN AQUEOUS

    PERTUSSIS VACCINE

    Dose EAEPretreatment Adjuvant G. pig cord Route Day of onset Histologic type

    - Complete Freund's 20 mg Foot 8 9 9 IO II OrdinaryPertussis " " 20 Foot 8 8 9 9 9 IO Ordinary

    D-5, IPPertussis Fortified Freund's 20 Foot 6 6 8 8 8 IO Ordinary

    D-5, IP- Pertussis and Bayol-

    Arlacel ioo Foot 13 13 14 14 15 I9 Ordinary- Pertussis and Bayol-

    Arlacel 200 IP 10 10 - - - - Ordinary- Pertussis, heated 200 IP 10 - - - - - Ordinary- Pertussis, supernate 200 IP 7 7 7 7 7 8 Intermediate- Pertussis, washed 200 IP 7 7 7 7 8 8 Hyperacute- Pertussis, whole 200 IP 7 7 7 7 8 8 Hyperacute

    Pretreatment: aqueous pertussis vaccine, o.6 ml diluted to 3.0 ml with saline, injected IP 5days before challenge.

    Adjuvants: Complete Freund's, 8.5 parts Bayol F, i.5 parts Arlacel A, with killed tuberclebacilli 4 mg/ml. Fortified Freund's, same except tubercle bacilli 49 mg/ml. Pertussis and Bayol-Arlacel, pertussis concentrate incorporated into cord homogenate which was then emulsified inBayol:Arlacel 85:I5; dose of concentrate 0.025 ml (footpad) or O.I2 ml (intraperitoneal). Pertussis,heated, o.6 ml commercial vaccine per dose, io0oC for i hour. Pertussis, supernate, o.6 ml commercialvaccine per dose, organisms removed by 3 centrifugations. Pertussis, washed, o.6 ml commercialvaccine per dose, organisms obtained by centrifuging, followed by 3 washes with saline and reconsti-tution to original volume with saline. Pertussis, whole, o.6 ml per dose of unaltered commercialvaccine.

    effect. The supernate recovered from pertussis vaccine after removalof bacilli by centrifugation had an intermediate degree of reactivity.

    Adrenalectomy and Hyperacute EAE. Adrenalectomy has increasedthe incidence and severity of ordinary EAE in rat strains of low suscep-tibility."6 It was of interest to observe the effect of adrenalectomy onhyperacute EAE. Control Lewis rats challenged with spinal cord-per-tussis vaccine mixture developed hyperacute EAE on the seventh toninth days, as expected (Table V). Rats with bilateral adrenalectomywere maintained on saline and challenged with the same mixture; thesedeveloped EAE or died on the fifth and sixth days. The symptoms weresevere and often attended by convulsions.

    Although adrenalectomy had accelerated the onset of clinical disease,

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    histologic lesions were surprisingly mild, usually limited to a few in-filtrates in the meninges. Neutrophils predominated in most lesions,but not to the extent noted in control rats with hyperacute EAE. Fi-brinous exudate was not observed. The histologic appearance wasreminiscent of the very early pre-symptomatic lesions of intact animals

    TABLE VACCELERATION OF ONSET OF HYPERACUTE EAE BY ADRENALECTOMY

    Day of death EAE(without

    Antigen Adjuvant Surgery EAE lesions) Day of onset Histologic tyl

    G. pig cord Pertussis None 7 7 7 7 7 8 8 8 9 Hyperacut"9 it it cc Adrenalectomy 3 5 666 5 6 6 6 6 6 6 6 See text" " c None None 11 11 13 13 I315 - - - Ordinary

    it "9 cc is Adrenalectomy 6 6 I8 20 II 12 13 I3 14 15 I8 Ordinary

    Male Lewis rats bilaterally adrenalectomized on the day of injection with 200 mg guinea pcord, with or without admixture of pertussis vaccine, in right footpad.

    described in a previous section. It was concluded that adrenalectomizedrats responded with inordinately severe symptoms to relatively mildCNS lesions, so that they died or had to be sacrificed before they coulddevelop the full-blown histologic picture of hyperacute EAE.

    Four adrenalectomized rats died on the fifth and sixth days and hadno CNS lesions. The coincidence of their deaths with the appearanceof EAE symptoms and lesions in the other adrenalectomized animalssuggested a relationship. Perhaps some mild CNS lesions were not de-tected, or perhaps they died because of a constitutional effect of theimmune reaction that was active at this particular time.

    Although adrenalectomy accelerated the onset of hyperacute EAEafter challenge with spinal cord-pertussis vaccine mixture, it had noeffect on the onset or severity of ordinary EAE after challenge with200 mg plain spinal cord homogenate without adjuvant (Table V).However, half the adrenalectomized rats that received exceptionallylarge doses of spinal cord homogenate (400 to 8oo mg) without anyadjuvant had focal accumulations of neutrophils under the spinal piaand even around an occasional parenchymal vessel. Only one of themexhibited the massive neutrophil response characteristic of hyperacuteEAE. Up to the present, therefore, regular production of hyperacuteEAE requires the use of aqueous pertussis vaccine as adjuvant.

    Quantitative Considerations. Ordinary EAE has been produced byaqueous CNS homogenates without the aid of any adjuvant, providedsufficiently large doses were injected.4 CNS tissue emulsified in Freund'sadjuvant produced ordinary EAE with only I/200 to I/400 the dose

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    required when adjuvants were omitted.4 Was CNS antigen conserved insimilar fashion when aqueous pertussis vaccine was the adjuvant? TableVI shows that this was not the case. Two hundred mg rat cord in salineproduced ordinary EAE; 200 mg rat cord in pertussis vaccine producedhyperacute EAE. Forty mg rat cord in either saline or pertussis vaccineresulted in much less EAE, of the ordinary variety in both cases. Lowerdoses were completely ineffective despite admixture with pertussis vac-cine.The term "adjuvant" is correctly applied to aqueous pertussis vac-

    cine because it accelerated, intensified and altered development of EAEfollowing its injection with CNS tissue antigen. Nevertheless, its adju-

    TABLE VIDOSE OF CNS ANTIGEN REQUIRED FOR PRODUCTION OF HYPERACUTE EAE

    Dose of cord EAEantigen Adjuvant Day of onset Histologic type Histologic severity*

    200 mg None 7 9 10 II II I2 Ordinary 4.040 Ordinary I.7200 Pertussis 8 8 9 II Hyperacute 4.040 9 II - - Ordinary 2.110 ----- None oI -- --- None 0

    Lewis female rats inoculated IP with rat cord homogenate suspended in saline or in aqueouspertussis vaccine as indicated.

    * Group average, graded individually from zero to four.

    vant property was limited. Its effect on the type of EAE was lost if thedose of CNS antigen was suboptimal; it did not increase the encephali-togenic potency of small doses of CNS antigen.

    Type of Antigen in Relation to Hyperacute EAE. In experiments re-corded above, hyperacute EAE was produced with either homologous(rat) or heterologous (guinea pig) mammalian cord as antigen. It hasbeen shown that mammalian, avian, reptilian and amphibian spinal cordsemulsified in Freund's complete adjuvant produced ordinary EAE inrats, while fish cord was nonencephalitogenic.17 An attempt was made toproduce hyperacute EAE with non-mammalian CNS tissue. Two hun-dred mg amphibian (Necturus, salamander, Amphiuma) or fish (carp)spinal cord was mixed with pertussis vaccine and injected in the foot-pads of 4 and 6 rats respectively. No clinical signs of EAE were detected.Mild histologic lesions were found in single rats that received salamanderor Amphiuma cord. These were not of the hyperacute variety.

    Guinea pig peripheral nerve mixed with pertussis vaccine caused bothallergic neuritis and EAE. This may be due to the antigenic overlap be-

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    tween peripheral and central neural antigens.11-13 The EAE lesions wereof ordinary rather than hyperacute variety (Table I). Neuritis has beenproduced previously only with the aid of oily adjuvants.-113

    These results suggested that the type of antigen as well as the doseand route must be optimal in order for pertussis vaccine adjuvant toproduce hyperacute EAE.

    Hyperacute EAE in Various Strains and Species. The CD F (Fischer344) strain of rats was almost as susceptible to hyperacute EAE as theLewis strain. The onset of disease occurred 7 or 8 days after intra-peritoneal inoculation in all 9 CD F rats, and almost as good resultswere obtained after footpad inoculation (Table III). In another ex-periment on I2 CD F rats, the onset after intraperitoneal inoculationwas a little later, 7 to io days. In both experiments, the lesions weretypical of the hyperacute process.The inbred Lewis and CD F strains are known to be highly susceptible

    to ordinary EAE produced without the aid of adjuvants.4 The random-bred Hemlock Hollow Wistar strain is distinctly less susceptible, al-though it responds well when Freund's adjuvants are employed.4' Eightto ii days after intraperitoneal inoculation with guinea pig cord-per-tussis vaccine mixture, clinical signs of EAE appeared in 9 of ii Hem-lock Hollow Wistar rats. These signs and the histologic lesions weremilder than those noted in Lewis or CD F rats, and only 2 of them hadthe features of hyperacute EAE.

    Thirteen female Swiss-Webster mice (25 to 30 g) were inoculatedintraperitoneally with Y%o or % the volume of guinea pig cord-pertussisvaccine mixture employed for rats. Nine female Hartley guinea pigs(430 to 500 g) were inoculated intraperitoneally with 2 or 4 times thedose for rats. No EAE signs or lesions occurred in the guinea pigs, andonly 2 of the mice had a few minor lesions.These results suggest that hyperacute EAE can be produced with

    regularity only in those highly susceptible strains in which ordinary EAEcan be produced by aqueous CNS homogenates without any adjuvant.In order for pertussis vaccine to exert its unique adjuvant effect, thehost as well as the amount and type of antigen and route must be opti-mum.

    Passive Transfer. Ordinary EAE has been transferred passively withlymph node cells from sensitized donors, provided that the donors weresplenectomized, or the recipients were made immunologically "tolerant"of donor cells, or recipients and donors were histocompatible.1122 Inthe highly inbred Lewis strain utilized in this study, passive transfer ofordinary EAE has been accomplished by Paterson without the need forancillary measures.22a On the contrary, attempts to reproduce the disease

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    with serum from sensitized animals have been uniformly unsuccessful.23The results obtained with cells and serum from Lewis rats with

    hyperacute EAE have been analogous to those mentioned above. Medi-astinal lymph nodes were obtained from rats with early hyperacuteEAE. The nodes were minced with scissors in sterile saline, and a cellsuspension was prepared with the aid of a cytosieve. Cell suspensionsfrom i to 8 donors were injected intravenously, slowly, in single or di-vided doses, into normal recipients. Some recipients sacrificed 5 daysafter injection, and all recipients killed at later times, had histologiclesions of EAE (Table VII). Several recipients had clinical signs of EAE

    TABiE VIIPASSIVE TRANSFERS WITH LYMPH NODE CELLS FROM DONORS WITH HYPERACUTE EAE

    EAE in recipientsDonor/recipient Sacrifice (days after Clinical (day Histologic

    Transfers ratio first transfer) of onset) score*

    Live cells, multiple 5 5 6 5 3 5 8 3 3 4 5 5 5 6 6 7 8 9 -- 7-- 0 2 3 3 4 1 2Live cells, single I I I 5 7 7 0 2 2tLive cells, single 2 3 ? 5 ? 5 5 5 6 6 88 ------ 2 0 1 1 1 3

    Live cells, single 3 4 3 7 7 7 5 5 7 3 4 2Live cells, single,

    splenectomizeddonors 5 4 7 7 5 - 3 3

    Killed cells, single 1 3 4 4 7 7 7 7 ---- 0 0 0 0

    Two experiments on adult Lewis rats injected with guinea pig cord-pertussis vaccine mixture IP.Cell suspensions prepared in saline from mediastinal lymph nodes shortly after onset of EAE, in-jected intravenously into normal Lewis recipients. Killed cells: portions of cell suspensions injectedinto other rats of same experiment were heated to 480 for 20 minutes.

    * Scored from o to 4 according to number and intensity of lesions (ordinary EAE). Figuresrepresent single rats, arranged in same order as in other columns.

    'Weanlings (all other rats were adults).

    as early as 5 days after passive transfer. Although the lymph node cellscame from rats with hyperacute EAE, the passively induced disease hadlesions of ordinary EAE only. Heat-killed cells caused no lesions in re-cipients. Splenectomy of donors did not influence the results.

    Injection of large amounts of serum from rats before the onset ofhyperacute EAE, or early or late in the disease, failed to cause lesionsin adult or weanling recipients (Table VIII).Induced Localization of Hyperacute EAE. Lesions of ordinary EAE

    can be caused to localize in regions of traumatic, anoxic or chemicalbrain damage.2v26 Induced localization is associated with and is propor-tional to the disruption of the blood-brain barrier.2+27 There is a lower-ing of the threshold for development of EAE lesions in the damagedarea.27

    72 Vol. 47, No. z

  • ALLERGIC ENCEPHALOMYELITIS

    A total of 96 female Lewis rats received implants of sterile graphitein the right cerebral hemisphere (frontal region), o to 8 days after theintraperitoneal injection of guinea pig cord-pertussis vaccine mixture.Most of the animals were sacrificed 24 hours after they received the im-plant. All rats that had lesions of hyperacute EAE in spinal cord and

    TABLE VIIIUNSUCCESSFUL SERUM TRANSFERS FROM DONORS WITH HYPERACUTE EAE

    Sacrifice (daysDonors* Recipients* Serum volumet after first transfer)t

    Advanced EAE I7 37 ml I ICDF, HH Wistar 40 IEarlyEAE 254I 702646 14455Early EAE Weanlings I7 IPre-symptomatic 25 17 1 4Pre-symptomatic Weanlings 17 IEarly EAE Pertussis I7* I9t 2 2

    pretreatedSplenectomized, Pertussis 25 2

    early EAE pretreated

    * Donors: adult rats (Lewis, except as indicated) injected with guinea pig cord-pertussisvaccine mixture IP. Serum from donors injected intravenously into Lewis recipients (adult,except as indicated). None of the recipients had clinical or histologic evidence of EAE.

    t Each figure represents a single rat, arranged in same order in both columns.s Single injection (all other transfers given in divided doses).

    hindbrain also had lesions clustered around the implant ("induced local-ization"). There were few or no lesions in the left cerebral hemisphereor in the right hemisphere at sites distant from the implant. Some ratskilled 7 days after the beginning of the experiment, and all rats killedat earlier times, had no EAE lesions in spinal cord or hindbrain. Never-theless, there were lesions around the implant in most rats that weresacrificed after 7, 6 or 5 days and in 2 of 4 rats after 4 days. The in-duced lesions were well developed in rats killed I2 or 24 hours after thegraphite was implanted, and were just detectable after 6 hours. Thelesions consisted of inflammatory cells, mostly neutrophils, in andaround walls of vessels adjacent to the implant. Control animals that hadreceived intraperitoneal injections of saline or of pertussis vaccine with-out CNS tissue had neutrophils in the lumens and walls of some vesselsnear the implant, but few or no inflammatory cells in the perivasculartissue.

    Extraneural Tissues in Hyperacute EAE. After intraperitoneal in-jection of CNS tissue-pertussis adjuvant mixture, the omentum had adense inflammatory infiltrate with large and small mononuclears, epi-thelioid cells, neutrophils and a fibrinous exudate. After injection in thefootpad, there was a similar local inflammatory reaction in the skin and

    73fuly, I965

  • subcutis, with many foci of suppuration. The draining lymph nodes(mediastinal or popliteal) contained many epithelioid cell granulomas,large numbers of neutrophils, and severe hyperplasia. Many sinusoidswere occluded by thrombi. These lesions were similar to those that fol-lowed pertussis vaccine alone.28 There were excessive numbers of neu-trophils in splenic red pulp and pulmonary alveolar septa. The bloodleukocyte count rose from an average of IO,400 per cmm before treat-ment to 46,ooo per cmm 7 days later. Neutrophils increased from 20 to29.5 per cent (2,OO to I3,600 cells per cmm). Large and small lympho-cytes were also increased in number. Similar hematologic values wereobtained when the pertussis and spinal cord were administered byseparate routes, or the latter omitted entirely. Therefore, these resultswere caused only by the pertussis vaccine.

    Karyorrhexis of lymphocytes in splenic white pulp and of thymocyteswas observed. Liver, pancreas, genitalia, adrenals, intestines, skeletalmuscle and heart had no significant abnormalities.

    DIscuSSION

    Several authors have recorded the occurrence of polymorphonuclearneutrophils in lesions of EAE. In monkeys especially, neutrophils werenumerous and associated with fibrinous exudation, necrosis, and rarethrombi; lesions of this character predominated in monkeys dying orsacrificed during the first few days after appearance of symptoms, andwere sporadic in occurrence or focal in distribution thereafter.2833 Moreor less similar lesions of sporadic occurrence have been recorded fordogs,83435 cats,86 rabbits,37 guinea pigs,38839 rats40 and mice.41 The hy-peracute syndrome described in this paper differed in degree and inuniformity. It occurred in all or almost all rats in a regular and predict-able manner, and the neutrophil infiltrates and fibrinous exudates werenumerous and widespread. Because of these features, the hyperacuteform of EAE is a better laboratory model than ordinary EAE for hu-man acute necrotizing hemorrhagic encephalopathy. Furthermore, thevery same features make the hyperacute syndrome a useful tool for theinvestigation of pathogenetic mechanisms in EAE. The uniformity of theexperimental results may be due to the genetic uniformity of the inbredrats.

    Hyperacute EAE was produced only with an aqueous inoculum; CNStissue emulsified in oil, with either pertussis organisms or tubercle bacilli,produced only ordinary EAE. Therefore, the production of hyperacuteEAE probably depended, above all, on the fact that aqueous CNS tissuehomogenates were encephalitogenic for Lewis and Fischer 344 rats.4Oily adjuvants enhance immunologic responses by protecting the antigen

    74 LEVINE AND WENK Vol. 47, No. z

  • ALLERGIC ENCEPHALOMYELITIS

    from destruction, but they probably also retard absorption. On the otherhand, aqueous depots of antigen permit rapid absorption which may beessential for production of hyperacute EAE. The footpad and intra-peritoneal routes favor rapid absorption into lymphatics, and were ef-fective in producing hyperacute EAE. Large doses of inoculum mayhave made up for excessive destruction of antigen in the aqueous depot.A susceptible strain of rats, an effective type and adequate dose of

    antigen, an aqueous adjuvant, and an appropriate route of injection,these were all necessary conditions for production of hyperacute EAE.How did the aqueous suspension of pertussis organisms aid in the pro-duction of hyperacute EAE ? The suppurative and granulomatous in-flammatory response evoked locally and in draining lymph nodes bypertussis bacilli may have provided a suitable cellular environment forsensitization, and may have increased absorption into lymphatics. Itmay be significant that polymorphonuclear leukocytes were present inlarge numbers not only at the site of inoculation and in draining lymphnodes, but also in blood, spleen and lungs (as well as in the CNS lesions).Therefore, both the local and systemic effects of pertussis vaccine mayhave been important.

    In the present experiments, pertussis vaccine played the role of adju-vant; in the absence of CNS antigen, pertussis vaccine produced localinflammatory lesions at inoculation sites and draining lymph nodes andsystemic leukocytosis, but no CNS disease (Table I). It must be re-corded, however, as a confusing but possibly relevant fact, that pertussisinfections and vaccinations have produced encephalopathy in humans,including a case of acute necrotizing hemorrhagic encephalopathy.42

    Passive transfer of disease with living lymph node cells indicatedthat hyperacute EAE was a hypersensitive response. The fact that therecipients had ordinary EAE lesions proved that the hyperacute syn-drome was merely a variant of EAE. Additional evidence included thefollowing: lesions of ordinary EAE often accompanied those of hy-peracute type; very early lesions in rats sacrificed before onset of thehyperacute disease were similar to ordinary EAE; lesions were peri-vascular and exhibited predilection for spinal cord and for veins in bothconditions; focal disruption of blood-brain barrier induced localizationof both diseases. In both syndromes, the inoculum had to contain ner-vous tissue but mammalian tissue was more effective than non-mam-malian, and CNS tissue was more effective than peripheral nerve. Inboth, the intradermal route was more effective than the subcutaneous.Both diseases had an incubation period and were enhanced by adrenal-ectomy. The host range suitable for hyperacute EAE was more narrowthan for ordinary EAE, but the rat strains that developed the hyperacute

    July, I965 75

  • LEVINE AND WENK

    disease were those distinguished by exceptional susceptibility to ordi-nary EAE. Finally, if any one of the experimental conditions (host,type of antigen, dose of antigen, and route of inoculation) was notoptimum for development of hyperacute EAE, then ordinary EAE wasthe outcome.

    In view of the essential unity of the hyperacute and ordinary varietiesof EAE, how can one account for the unique features of the former?In the terminology of Waksman43 and Gell," hyperacute EAE mightrepresent a "vasculonecrotic" reaction superimposed upon the "perivas-cular island" and "invasive-destructive" lesions that are typical of thedelayed hypersensitivities. Waksman has recognized that "when a vas-culonecrotic change is present it may transcend in apparent importanceother elements of the reaction, as in haemorrhagic, necrotizing form ofallergic encephalomyelitis ... 43 It is possible that the edema,necrosis, thrombosis, exudation of neutrophils and fibrin that character-ize hyperacute EAE were merely indicators of parenchymal damage dueto an unusually severe immunologic injury.38 According to this hypo-thesis, hyperacute and ordinary EAE differed only quantitatively, andthe qualitative differences in the histologic patterns were merely sec-ondary consequences of tissue damage. It is thought that tissue damagein ordinary EAE is a direct consequence of the influx of lymphoidcells from the blood stream into the CNS.14'38 If this is true, one mightexpect an overabundance of lymphoid cells, or a moderate number ofparticularly potent lymphoid cells, to precede the appearance of neutro-phils. This was not the case. The very early lesions of hyperacute EAEexhibited mononuclear cells, but these were few in number, and even thefully established lesions often contained very few mononuclears. In nocase was there evidence of transition from a severe mononuclear infil-trate to a hyperacute lesion. Also, not all lesions of hyperacute EAEwere severe; some lesions had only a few neutrophils and monocytes,little or no fibrin, and no evidence of necrosis. Such observations werenot easily reconciled with the concept of mononuclear infiltrate as aneccessary preliminary for tissue damage and subsequent developmentof hyperacute lesions.

    In analyzing the pathogenesis of EAE, it is necessary to considerthe possibility that lesions of a given age in the early phase of the diseasemight differ histologically from lesions of the same age in a late phaseof the disease. It is possible that lesions observed in the pre-symptomaticstage of hyperacute EAE were composed of mononuclear cells becausethe disease was in an early stage of development and the concentrationof circulating pathogenic agent was low. As the disease evolved and thepathogenic agent increased in quantity, it is possible that even a veryearly lesion might be of hyperacute character.

    76 Vol. 47, No. z

  • ALLERGIC ENCEPHALOMYELITIS

    The morphologic evidence suggests the existence of a qualitativedifference between hyperacute and ordinary EAE. The resemblanceof the lesions of hyperacute EAE to the Arthus reaction suggests thepresence of a humoral antibody. Failure to transfer EAE passivelywith serum cannot be considered conclusive. This speculation in no waynegates the importance of lymphoid cells, whose role in EAE has beenestablished conclusively by labeling and by passive transfer experi-ments.'14'1822 The possibility of combined action of cells and antibodiescannot be ruled out. At the moment, however, these suggestions arepurely speculative.

    There are morphologic resemblances between hyperacute EAE andthe combination of ordinary EAE with the Shwartzman phenomenon.45In our experiments, it is conceivable that the endotoxin of B. pertussisorganisms might cause a Shwartzman reaction to be superimposed uponthe lesions of ordinary EAE. Actually, the time relationships are notappropriate for the classical Shwartzman phenomenon, since CNS le-sions do not develop until 6 or 7 days after the B. pertussis organismsare injected. Recent experiments have provided more direct evidenceagainst participation of a Shwartzman-like mechanism: heat (800 C for30 minutes) impairs the adjuvant effect of pertussis vaccine (whereasendotoxin is heat-stable); neither typhoid vaccine nor endotoxins pre-pared from B. pertussis or E. coli produce hyperacute EAE when mixedwith CNS antigen.46

    SUMMARY

    A hyperacute form of allergic encephalomyelitis (EAE) was pro-duced by the injection of a mixture of aqueous spinal cord homogenateand aqueous pertussis vaccine in certain highly susceptible rat strains.The disease had short incubation period, high incidence, rapid progres-sion, and fatal outcome. It was accelerated by adrenalectomy. The peri-vascular lesions contained large numbers of neutrophils, as well as mono-nuclear leukocytes, fibrin and edema fluid. The lesions localized in areaswhere the blood-brain barrier was disrupted.

    Hyperacute EAE was produced by a mixture of nervous tissue andpertussis vaccine only when the strain of rats, type of nervous tissueantigen, dose of antigen, and route of inoculation were optimum; whenany of these conditions was not optimum, then the mixture producedordinary EAE. Passive transfer of lymph node cells from donor ratswith hyperacute EAE produced ordinary EAE in recipients.The unique histologic features and course of hyperacute EAE seemed

    to be related to the nature of the adjuvant and the aqueous character ofthe inoculum. Hyperacute EAE is a reproducible laboratory model forhuman acute necrotizing hemorrhagic encephalopathy.

    July, z965f 77

  • 78 LEVINE AND WENK Vol. 47, No. z

    REFERENCES

    i. ADAMS, R. D.; CAMMERMEYER, J., and DENNY-BROWN, D. Acute necrotizinghemorrhagic encephalopathy. J. Neuropath. Exp. Neurol., 1949, 8, I-29.

    2. RUSSELL, D. S. The nosological unity of acute haemorrhagic leucoencephalitisand acute disseminated encephalomyelitis. Brain, I955, 78, 369-376.

    3. ADAMS, R. D. A Comparison of the Morphology of the Human DemyelinativeDiseases and Experimental "Allergic" Encephalomyelitis. In: "Allergic" En-cephalomyelitis. Proceedings of a Symposium: Experimental "Allergic" En-cephalomyelitis and its Relation to Other Diseases of Man and Animals.KIES, M. W., and ALVORD, E. C., JR. (eds.). Charles C Thomas, Springfield,Ill., I959, I83-209.

    4. LEVINE, S., and WENK, E. J. Induction of experimental allergic encephalo-myelitis in rats without the aid of adjuvant. Ann. N.Y. Acad. Sci. 165, 122,209-2 26.

    5. LEE, J. M., and OLITSKY, P. K. Simple method for enhancing development ofacute disseminated encephalomyelitis in mice. Proc. Soc. Exp. Biol. Med.,I955, 89, 263-266.

    6. LEVINE, S., and WENK, E. J. Studies on the mechanism of altered susceptibil-ity to experimental allergic encephalomyelitis. Amer. J. Path., I96I, 39, 4I9-44I.

    7. Conference on Research in Demyelinating Diseases, New York, Jan. 2o-22,I964. New York Academy of Sciences and National Multiple Sclerosis So-ciety.

    8. WIENER, S. L.; TINKER, M., and BRADFORD, W. L. Experimental meningoen-cephalomyelitis produced by Hemophilus pertussis. Arch. Path., I959, 67,694-699.

    9. SHAW, C.-M.; ALVORD, E. C., JR.; FAHLBERG, W. J., and KIES, M. W. Substi-tutes for the mycobacteria in Freund's adjuvants in the production of experi-mental "allergic" encephalomyelitis in the guinea pig. J. Immun., I964, 92,28-40.

    IO. LEVINE, S., and WENK, E. J. Allergic encephalomyclitis: a hyperacute form.Science, I964, I46, I68I-I682.

    II. WAKSMAN, B. H., and ADAMS, R. D. A comparative study of experimental al-lergic neuritis in the rabbit, guinea pig, and mouse. J. Neuropath. Exp. Neurol.,I956, 15, 293-33I.

    I2. WAKSMAN, B. H. Experimental Immunologic Disease of the Peripheral Ner-vous System. In: Mechanisms of Demyelination. ROSE, A. S., and PEARSON,C. M. (eds.). McGraw-Hill Book Co., New York, Toronto, London, I963,170-I98.

    I3. LEVINE, S., and WENK, E. J. Allergic neuritis induced in rats without the useof mycobacteria. Proc. Soc. Exp. Biol. Med., I963, II3, 898-900.

    I4. KOSUNEN, T. U.; WAKSMAN, B. H., and SAMUELSSON, I. K. Radioautographicstudy of cellular mechanisms in delayed hypersensitivity. II. Experimentalallergic encephalomyelitis in the rat. J. Neuropath. Exp. Neurol., I963, 22,367-380.

    I5. SHIINA, T., and IIDA, T. Experimental studies on paralysis after antirabies vac-cination. I. Histological studies on acute demyelinating encephalomyelitisin guinea pigs. Jap. J. Microbiol., I958, 2, I87-I96.

    i6. LEVINE, S.; WENK, E. J.; MULDOON, T. N., and COHEN, S. G. Enhancement ofexperimental allergic encephalomyelitis by adrenalectomy. Proc. Soc. Exp.Biol. Med., I962, III, 383-385.

  • July, z965 ALLERGIC ENCEPHALOMYELITIS 79

    I 7. LEVINE, S., and WENK, E. J. Encephalitogenic potencies of nervous system tis-sues. Proc. Soc. Exp. Biol. Med., I963, II4, 220-222.

    I8. PATERSON, P. Y. Transfer of allergic encephalomyelitis in rats by means oflymph node cells. J. Exp. Med., I960, iiI, II9-I36.

    I9. KoPROWSKI, H.; PARAF, A., and JERVIS, G. A. Allergic encephalitis, with allits implications towards suppression of natural tolerance. In: La toleranceacquise et la tolerance naturelle a l'egard de substances antigeniques definies.Colloques Internationaux du Centre National de la Recherche Scientifique,I963, ii6, 339-357.

    20. STONE, S. H. Transfer of allergic encephalomyelitis by lymph node cells ininbred guinea pigs. Science, I96I, I34, 6I9-620.

    2I. PATERSON, P. Y., and DIDAKOW, N. C. Transfer of allergic encephalomyelitisusing splenectomized albino rats. Proc. Soc. Exp. Biol. Med., I96I, io8, 768-77I.

    22. ASTR6M, K.-E., and WAKSMAN, B. H. The passive transfer of experimental al-lergic encephalomyelitis and neuritis with living lymphoid cells. J. Path. Bact.,I962, 83, 89-IO6.

    22a. PATERSON, P. Y. Cells, Antibodies and Auto-immune Disease. In: Cell-BoundAntibodies. Conference of the National Academy of Sciences, National Re-search Council held May io, I963. Proceedings. AMos, B., and KoPROWSKI,H., (eds.). Wistar Institute Press, Philadelphia, I963, I01-I05.

    23. CHASE, M. W. A Critique of Attempts at Passive Transfer of Sensitivity toNervous Tissue. In: "Allergic" Encephalomyelitis. Proceedings of a Sym-posium: Experimental "Allergic" Encephalomyelitis and Its Relation to OtherDiseases of Man and Animals. KIEs, M. W., and ALVORD, E. C., JR. (eds.).Charles C Thomas, Springfield, Ill., I959, 348-374.

    24. CLARK, G., and BOGDANOVE, L. H. The induction of the lesions of allergicmeningoencephalomyelitis in a predetermined location. J. Neuropath. Exp.Neurol., I955, 14, 433-437-

    25. LEVINE, S. Localization of allergic encephalomyelitis in lesions of cyanideencephalopathy. J. Neuropath. Exp. Neurol., I960, 19, 238-247.

    26. LEVINE, S.; ZIMMERMAN, H. M.; WENK, E. J., and GONATAS, N. K. Experi-mental leukoencephalopathies due to implantation of foreign substances.Amer. J. Path., I963, 42, 97-II7.

    27. LEVINE, S., and WENK, E. J. Effect of induced localization on incidence anddistribution of experimental allergic encephalomyelitis. Neurology, Minneap.,I960, IO, IO90-IO95.

    28. LEVINE, S., and GRUENEWALD, R. Ascites in the rat produced by pertussis vac-cine. Exp. Molec. Path., I962, I, I04-II2.

    29. WOLF, A.; KABAT, E. A., and BEZER, A. E. The pathology of acute disseminatedencephalomyelitis produced experimentally in the Rhesus monkey and itsresemblance to human demyelinating disease. J. Neuropath. Exp. Neurol.,I947, 6, 333-357.

    30. KABAT, E. A.; WOLF, A., and BEZER, A. E. Experimental Studies on Acute Dis-seminated Encephalomyelitis in Rhesus Monkeys. In: Multiple Sclerosis andthe Demyelinating Diseases. Proceedings of the Association, Dec. io and ii,I948, New York. Research Publications of the Association for Research inNervous and Mental Disease, Williams and Wilkins Co., Baltimore, I950,Vol. 28, II3-I32.

    3I. MORGAN, I. M. Allergic encephalomyelitis in monkeys in response to injectionof normal monkey nervous tissue. J. Exp. Med., 1947, 85, I3I-140.

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    32. FERRARO, A., and RoizIN, L. Neuropathologic variations in experimental al-lergic encephalomyelitis. Hemorrhagic encephalomyelitis, perivenous enceph-alomyelitis, diffuse encephalomyelitis, patchy gliosis. i. Neuropath. Exp.Neurol., 1954, I3, 60-89.

    33. JACOTOT, H.; GUILLON, J. C.; VIRAT, B., and VALLUE, A. L'encephalomyeliteallergique experimentale chez le porc. Rev. Franc. Allerg., I962, 2, I27-132.

    34. HALPERN, B. N.; BERTRAND, I., and LHERMITTE, F. L'encephalomyelite al-lergique experimentale. Presse Med., I950, 58, 684-687.

    35. THOMAS, L.; PATERSON, P. Y., and SMITHWICK, B. Acute disseminated en-cephalomyelitis following immunization with homologous brain extracts.I. Studies on the role of a circulating antibody in the production of the con-dition in dogs. J. Exp. Med., I950, 92, I33-I52.

    36. PATERSON, P. Y. Organ-Specific Tissue Damage Induced by MammalianTissue-Adjuvant Emulsions. In: Cellular and Humoral Aspects of the Hyper-sensitive States. A Symposium Held at the New York Academy of Medicine.LAWRENCE, H. S. (ed.). Paul B. Hoeber, Inc., New York, I959, 469-503.

    37. CONDIE, R. M., and GOOD, R. A. Experimental Allergic Encephalomyelitis:Its Production, Prevention, and Pathology as Studied by Light and ElectronMicroscopy. In: The Biology of Myelin. KoREY, S. R. (ed.). Paul B. Hoeber,Inc., New York, I959, 32I-384.

    38. WAKSMAN, B. H., and ADAMS, R. D. A histologic study of the early lesion inexperimental allergic encephalomyelitis in the guinea pig and rabbit. Amer. J.Path., I962, 41, I35-I62.

    39. COLOVER, J. Experimental allergic encephalomyelitis and its immunochemis-try. Proc. Roy. Soc. Med., I958, 51, 745-747.

    40. WAKSMAN, B. H. Discussion of the Reactions of Different Species to Injec-tion of Brain-Containing Vaccines. In: "Allergic" Encephalomyelitis. KIES,M. W., and ALvoRD, E. C., JR. (eds.). Charles C Thomas, Springfield, Ill.,I959, I42-I44.

    4I. OLITSKY, P. K., and YAGER, R. H. Experimental disseminated encephalo-myelitis in white mice. J. Exp. Med., I949, 90, 2I3-224.

    42. MoosY, J.; WOLF, A., and COWEN, D. Acute hemorrhagic leukoeacephalitis.Its relationship to the demyelinating diseases. Amer. J. Path., I954, 30, 642-643.

    43. WAKSMAN, B. H. A Comparative Histopathological Study of Delayed Hyper-sensitive Reactions. In: Ciba Foundation Symposium on Cellular Aspects ofImmunity. WOLSTENHOLME, G. E. W., and O'CONNOR, M. (eds.). Little,Brown & Co., Boston, I960, 280-322.

    44. GELL, P. G. H. Cytologic Events in Hypersensitivity Reactions. In: Cellularand Humoral Aspects of the Hypersensitive States. New York Academy ofMedicine, Section on Microbiology, Symposium #9, I959. LAWRENCE, H. A.(ed.). Paul B. Hoeber, Inc., New York, I959, 43-66.

    45. WAKSMAN, B. H., and ADAMS, R. D. Studies of the effect of the generalizedShwartzman reaction on the lesions of experimental allergic encephalo-myelitis. Amer. J. Path., I957, 33, I3I-I53.

    46. LEVINE, S.; WENK, E. J., and IOVINE, L. Hyperacute allergic encephalo-myelitis: Mechanism of adjuvant effect of pertussis vaccine. Fed. Proc., I965,24, 242.

    We are indebted to Louis Iovine for technical assistance, to Dr. J. A. McMillen ofLederle Laboratories and Dr. H. B. Devlin of Parke, Davis and Co. for generous gifts ofpertussis vaccine, and to Morris Moritz for photography.

  • ALLERGIC ENCEPHALOMYELITIS

    [ Illustrations follow ]

    July, I965 8i

  • LEVINE AND WENK

    LEGENDS FOR FIGURES

    Unless otherwise indicated photomicrographs were prepared from sections stainedwith hematoxylin and eosin.

    Figures i and 2: Comparison of hyperacute and ordinary experimental allergicencephalomyelitis (EAE).FIG. i. Hyperacute EAE in spinal cord. Vein wall is edematous. Perivascular pa-

    renchyma has narrow dark eosinophilic zone surrounded by broad, pale, edema-tous area with many neutrophils. X 460.

    FIG. 2. "Ordinary" EAE in spinal cord. The vein wall contains many lymphoidcells, and there are a few similar cells in the perivascular parenchyma. Thistypical lesion of ordinary EAE occurred in a recipient of mediastinal lymph nodecells from rats with hyperacute EAE. X 460.

    82 Vol. 47, No. z

  • ALLERGIC ENCEPHALOMYELITIS

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  • LEVINE AND WENK

    Figures 3 to 8: Fibrin and thrombi in hyperacute EAE.FIG. 3. Impregnation of perivascular parenchyma with darkly stained fibrin. Fibrin

    is much more abundant adjacent to one side of the wall (above) than to theother. Reticulated pattern of fibrin is apparent on right side. Inflammatory cellsare relatively scanty, and are predominantly neutrophils. Phosphotungstic acidand hematoxylin (PTAH) stain. X 280.

    FIG. 4. The entire circumference of a vein and of an entering capillary (below) areimpregnated with fibrin. There are many neutrophils in and around the vein.PTAH stain. X 950.

    84 Vol. 47, No. I

  • July, I965 ALLERGIC ENCEPHALOMYELITIS 85

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    FIG. 5. Two perivascular lesions in white matter exhibit heavy deposits of fibrinand many leukocytes. PTAH stain. X 200.

    FIG. 6. A leptomeningeal vein is occluded by a thrombus. The lumen near top ispatent and contains erythrocytes and leukocytes. X 950.

    FIG. 7. The venule lumen is partly occluded by thrombus and leukocytes. Fibrinand leukocytes infiltrate the adjacent white matter. X 400.

    FIG. 8. The vessel lumen is completely occluded by a thrombus. Fibrin is visiblebelow the vessel, and leukocytes are present on all sides. X 400.

    5

    7

    6

    8

    86 Vol. 47, No. z

  • July, 1965 ALLERGIC ENCEPHALOMYELITIS 87

    9

    .i a;'

    11

    Figures g to II: Demyclination and edema in hyperacute EAE.FIG. 9. Perivascular demyelination in white matter of spinal cord (longitudinal sec-

    tion). Luxol fast blue, PAS and hematoxylin stain. X I 20.FIG. IO. Pools of PAS-positive edema fluid (homogeneous gray in photograph, pink

    in stained section) separate the bundles of myelinated fibers. Luxol fast blue,PAS and hematoxylin stain. X I30.

    FIG. I I. The central canal is distended by PAS-positive spinal fluid. Luxol fast blue,PAS and hematoxylin stain. X 950.

  • LEVINE AND WENK

    12

    Figures I 2 to 14: Fibrin, necrosis and hemorrhage in hyperacute EAE.FIG. I 2. The leptomeninges (left) contain abundant darkly-stained strands of

    fibrin. The sub-pial parenchyma has an inflammatory exudate in which neutro-phils predominate. PTAH stain. X 460.

    FIG. I3. Leptomeninges (left) and sub-pial parenchyma have a dense leukocyticinfiltrate. Severe necrosis is apparent in the adjacent white matter (below).X I30.

    FIG. I4. Conus medullaris (nerve roots on each side). There are several hemorrhagesand diffuse inflammation. X 200.

    88 Vol. 47, No. I