Experimental allergic encephalomyelitis: Simple method for producing the localized form

Journal of Immunological Methods, 54 (1982) 355-360

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Elsevier Biomedical Press

Experimental Allergic Encephalomyelitis: Simple Method for Producing the Localized Form Seymour Levine i and Richard Sowinski Pathology Department, New York Medical College, Westchester County Medical Center, Valhalla, N Y 10595, U.S.A.

(Received 17 March 1982, accepted 30 April 1982)

In the past, the lesions of experimental allergic encephalomyelitis (EAE) have been induced to localize around brain tissue damaged by anoxia or direct physical or chemical attack. The procedure for producing the requisite antecedent brain injury has been simplified by use of a single subcutaneous injection of a neurotoxic chemical, thereby eliminating the need for surgery. The EAE lesions are concentrated in a very small area and can be obtained in one day after the passive transfer of a relatively small number of lymph node cells from immunized donors.

Key words: experimental allergic encephalomyelitis - - passive transfer

Introduction F o c a l b r a i n injuries i n d u c e the localization a r o u n d them of the lesions of e x p e r i m e n t a l allergic e n c e p h a l o m y e l i t i s ( E A E ) (Clark a n d Bogdanove, 1955). Electrical or t h e r m a l burns, i n t r a c e r e b r a l i m p l a n t a t i o n of chemicals, cerebral a n o x i a a n d ischemia are all effective p r o v i d e d that necrosis of b r a i n tissue is p r o d u c e d , w h e r e b y the b l o o d - b r a i n b a r r i e r is b r e a c h e d (reviewed b y Levine, 1974). T h e localized form o f E A E offers i m p o r t a n t a d v a n t a g e s over c o n v e n t i o n a l n o n l o c a l i z e d EAE. The t h r e s h o l d for d e v e l o p m e n t of E A E is lowered a n d the lesions develop r a p i d l y (5 d a y s after active i m m u n i z a t i o n , 1 d a y after passive transfer with cells f r o m i m m u n i z e d d o n o r s ) (Levine a n d W e n k , 1967; Levine a n d Hoenig, 1968; Levine a n d Sowinski, 1969). Localized E A E p r o d u c e d b y passive transfer is the o n l y w a y to p r o d u c e cellular infiltrates that are of e x a c t l y k n o w n a n d u n i f o r m age (Levine, 1970), a n d it is the o n l y w a y to p r o d u c e the n e u t r o p h i l i c form of E A E in which p o l y m o r p h o nuclears r e p l a c e m o n o n u c l e a r cells in the lesions (Levine a n d Hoenig, 1971). L o c a l i z e d E A E facilitates the transfer of disease across m a j o r a n d m i n o r h i s t o c o m -

I To whom correspondence should be addressed. 0022-1759/82/0000-0000/$02.75

© 1982 Elsevier Biomedical Press

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patibility barriers (Levine et al., 1967; Levine et al., 1970) and its speed makes it economical when materials for therapeutic trials are in short supply or transient in effect. The location of the lesions is precisely known in advance, a feature of importance when exacerbations of the disease are induced (Bogdanove and Clark, 1957). Despite all these advantages, the method is not widely used. Although none of the techniques for producing brain lesions are difficult, they all require some surgery or the use of asphyxiant gases, and this may be considered an obstacle. Therefore, we have developed a method for localization of EAE which requires nothing more than the subcutaneous injection of a readily available neurotoxic chemical.

Materials and Methods The aliphatic triamine chosen for these experiments is rapid in action and a single dose has the interesting property of producing necrotizing lesions at 3 very specific areas of the brain: dorsal medulla near the area postrema, hypothalamus adjacent to median eminence, and psalterium (ventral hippocampal commissure) near the intercolumnar tubercle (Levine and Sowinski, 1982). The triamine was purchased from Ames Laboratories, Milford, CT, under the name 3,3’-bis(methylamino)-N-methyldipropylamine, or from Aldrich Chemical Co., Milwaukee, WI, under the name 3,3’-methyliminobis-(N-methylpropylamine). The triamine is a liquid which was neutralized (w/v) with 10 ~01s. of 10% acetic acid and then diluted with saline to yield 20 mg/ml. Doses of 200 or 150 mg/kg were inoculated subcutaneously into rats a few days before EAE was produced. Passive transfer of EAE was done with female, 150-200 g, Lewis rats as donors and male, 250-350 g, Lewis rats as recipients. EAE was produced in the donors by injecting the right foot pad with 0.05 ml of an emulsion of 40% guinea pig spinal cord homogenate and an equal volume of Freimd’s complete adjuvant (Bay01 F-Arlacel A, 8.5 : 1.5, plus 4 mg/ml killed tubercle bacilli). Pertussis vaccine concentrate (0.1 ml, 20 billion organisms) was inoculated into the dorsum of the same foot as an ancillary adjuvant. Seven days later, the draining lymph nodes were harvested, processed into a cell suspension, washed and injected intravenously into the recipient rats that had been pretreated with the triamine. Recipient rats were killed 1 day after passive transfer. The brains were removed and fixed in Bouin’s fluid for 1 or 2 days. The area postrema was identified in the medulla at the caudal end of the fourth ventricle (‘calamus scriptorius’) after removing the overlying caudal end of cerebellum. The ventricular walls come together here in the midline at an acute angle that points caudally. The area postrema is located in the floor of this angle, between its vertex and the orifice of the central canal which is 1 mm anterior. With the aid of a good light, this area was removed in a 1 mm frontal block that was embedded in paraffin, cut at several levels and stained with hematoxylin and eosin. The desired area can also be obtained blindly by merely using the caudal end of the cerebellum as a guide to the underlying area postrema, or by cutting a mid-sagittal block of the medulla (since

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the area postrema is an unpaired midline structure in the rat). In any event, blocks on either side of the area postrema can be taken also, inasmuch as the lesions are often extensive. For the present work, a 1 m m frontal slice through the median eminence of the hypothalamus, and a 1 m m mid-sagittal block through the anterior part of the forebrain (to reveal the psalterium and intercolumnar tubercle) were processed along with the medulla, but these additional areas are not necessary for the proposed procedure (see Results). For comparison, thermal injuries were produced in the right cerebral hemisphere of other recipient rats with a soldering iron (Levine and Hoenig, 1968). These large areas of necrosis were embedded as 4 or 5 frontal slices encompassing most of the cerebrum, each slice about 2 m m thick, and only a single level of each was examined. EAE scores were based on the density of EAE lesions on the perimeter of the necrotizing lesions. Rats scored 4 + had lesions along virtually the entire perimeter whereas 1 + implied from 1 to 5 widely scattered lesions on the entire slide, and 2 + and 3 + were intermediate. Slides were randomized and scored without knowledge of whether EAE cells had been given, or their amount.

Results Rats given the triamine alone developed the expected lesions of medulla, hypothalamus and psalterium. These were either edematous or necrotizing, but neither type was accompanied by an inflammatory reaction except for a few polymorphonuclear leukocytes in areas of necrosis. Rats given the passive transfer alone did not have EAE lesions in the areas under study or anywhere else, as 1 day is not sufficient time to develop nonlocalized EAE. Recipient rats given the triamine and the passive transfer developed perivascular mononuclear inflammatory infiltrates typical of EAE. The EAE infiltrates were located along the perimeter of the triamine lesion (Fig. 1). Only necrotizing triamine lesions induced the localization of EAE; the milder edematous lesions were ineffective. The larger dose of triamine produced more severe necrosis than the smaller dose, and the number of localized EAE lesions varied correspondingly. There was much more EAE in medulla than in hypothalamus or psalterium even when necrosis seemed to be of equal severity. Although the triamine lesions were in the neighborhood of area postrema, median eminence and intercolumnar tubercle, these structures themselves usually escaped the chemical injury and only the area postrema had a very variable development of inflammatory infiltrates. Administration of triamine either 2 or 3 days before passive transfer was effective, but a 1-day interval gave poor localization of EAE despite extensive necrosis (Table I). Similarly, recent thermal injuries are ineffective for localization (Levine and Hoenig, 1968). Localization of EAE was dependent on the number of passively transferred cells (Table I). A donor:recipient ratio as low as 1:8 (approximately 4 X 107 cells) produced good EAE lesions, but half that dose yielded only minimal or questionable

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Fig. 1. T h e a r e a p o s t r e m a is in the m i d l i n e of the d o r s a l s u r f a c e of the m e d u l l a o b l o n g a t a (top). Below it is a p a l e a r e a o f n e c r o t i c tissue d u e to a d m i n i s t r a t i o n o f the t r i a m i n e 3 d a y s b e f o r e the passive t r a n s f e r . A d j a c e n t to the p e r i m e t e r of the n e c r o t i c z o n e a r e n u m e r o u s E A E lesions c h a r a c t e r i z e d b y d a r k l y s t a i n e d p e r i v a s c u l a r i n f l a m m a t o r y infiltrates. T h e s e lesions d e v e l o p e d in 1 d a y a f t e r t r a n s f e r of E A E cells. H e m a t o x y l i n - e o s i n , × 55. TABLE I LOCALIZATION OF EAE AROUND TRIAMINE LESIONS OF MEDULLA OR THERMAL INJURIES OF CEREBRAL HEMISPHERE ONE DAY AFTER PASSIVE TRANSFER Brain injury

E A E cells

Interval (days)

N o . of r a t s

L o c a l i z e d E A E score

None Triamine Triamine Triamine Triamine Triamine Triamine Triamine Thermal Thermal Thermal Thermal Thermal

1:1a none 1:4 1 :4 1:1 1:4 1 :8 1 : 16 none 1: 1 1 :4 1:8 1 : 16

_ 1 2 3 3 3 3 3 3 3 3

4 4 4 7 10 11 4 10 2 6 10 4 3

0 0 1.0 2.7 3.8 3.4 1.5 0.6 0 3.8 2.7 2.3 0.3

b b b b b b b

a D o n o r : r e c i p i e n t ratio. E a c h d o n o r p r o v i d e d 2 - 3 > ( 108 cells f r o m d r a i n i n g l y m p h nodes. Five passive transfer experiments are summarized. b D o s e s of 150 o r 200 m g / k g are p o o l e d ; r a t s w i t h n o n - n e c r o t i z i n g lesions e x c l u d e d . Scores refer o n l y to E A E lesions a r o u n d t r i a m i n e n e c r o s i s in m e d u l l a .

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inflammatory foci. The same quantitative results were obtained with EAE localized around thermal injuries. It should be remembered that the same histologic score implies many more EAE lesions for the large thermal injuries than for the small triamine lesions, because the scores are based on density of EAE lesions rather than absolute counts.

Discussion Triamine lesions of the brain were as effective as thermal injuries for localizing EAE. The location of triamine lesions in the medulla was convenient because of the small, easily identified target, but this location may also be responsible for some mortality. The effects of the chemical may vary with age, size and strain of rat, and may depend on environmental temperature, diet, etc. Therefore, we recommend administration of 2 dose levels (150 and 200 m g / k g ) to be sure of getting necrotizing but nonlethal lesions. An interval of 2 or 3 days before the EAE transfer was optimum. In our hands, as few as 4 X l07 cells (total count) produced localized EAE in 1 day. However, our cell suspensions were only approximately 50% viable by trypan blue exclusion. Procedures that produce better viability would probably succeed with even lower total counts. Localized EAE does not produce clinical signs. However, it does make it possible to identify and count every EAE lesion, inasmuch as they are concentrated in 3 small areas. For purposes other than total counts, there is no reason to study the hypothalamus or psalterium, as the best EAE lesions are in the medulla. Probably any neurotoxic chemical that produces brain necrosis could be used to localize EAE. In a preliminary experiment, 4 rats were given a single subcutaneous injection of 200 m g / k g of dipiperidinoethane, 3 days before passive transfer. When killed 1 day after transfer, localized EAE was present, not in medulla and hypothalamus, but in amygdaloid nucleus and pyriform cortex which are the usual sites of predilection for the necrotizing effects of this particular chemical (Levine and Sowinski, 1980). The ease of inducing localization of passive EAE by a single subcutaneous injection of the triamine or other chemicals should increase the exploitation of the numerous advantages of localized EAE.

Acknowledgements Supported by a research grant from the National Multiple Sclerosis Society. We thank Mr. R. Lindsay for the photomicrograph.

References Bogdanove, L.H. and G. Clark, 1957, J. Neuropathol. Exp. Neurol. 16, 57. Clark, G. and L.H. Bogdanove, 1955, J. Neuropathol. Exp. Neurol. 14, 433.

360 Levine, Levine, Levine, Levine, Levine, Levine, Levine, Levine, Levine, Levine,

S., 1970, J. Neuropathol. Exp. Neurol. 29, 6. S., 1974, Acta Neuropathol. 28, 179. S. and E.M. Hoenig, 1968, J. Immunol. 100, 1310. S. and E.M. Hoenig, 1971, Am. J. Pathol. 64, 13. S. and R. Sowinski, 1969, Am. J. Pathol. 56, 97. S. and R. Sowinski, 1980, J. Neuropathol. Exp. Neurol. 39, 56. S. and R. Sowinski, 1982, J. Neuropathol. Exp. Neurol. 41, 54. S. and E.J. Wenk, 1967, J. Immunol. 99. 1277. S., E.J. Wenk and E.M. Hoenig, 1967, Transplantation 5, 534. S., R. Sowinski, E.M. Hoenig and R. Gruenewald, 1970, Proc. Soc. Exp. Biol. Med. 135, 569.