- Email: [email protected]
Systemic administration of acromelic acid induces selective neuron damage in the rat spinal cord
Life Sciences, Vol. 49, pp. PL-91 - PL-96 Printed in the U.S.A.
Pergamon Press
PHARMACOLOGY LETTERS Accelerated Communication
S Y S T E M I C A D M I N I S T R A T I O N OF A C R O M E L I C A C I D I N D U C E S S E L E C T I V E N E U R O N D A M A G E IN THE R A T S P I N A L C O R D
Michiko
S h i n K w a k I, H i t o s h i Aizawa, Ishida*, a n d H a r u h i k o S h i n o z a k i *
N a t i o n a l I n s t i t u t e of N e u r o s c i e n c e , N a t i o n a l C e n t e r of N e u r o l o g y and Psychiatry (NCNP), 4-1-1, O g a w a h i g a s h i , K o d a i r a , T o k y o 187. *The T o k y o M e t r o p o l i t a n I n s t i t u t e of M e d i c a l S c i e n c e , 3 - 1 8 - 2 2 , H o n k o m a g o m e , B u n k y o - k u , T o k y o 113, Japan. (Submitted June 4, 1991; accepted July i0, 1991; received in final form July 23, 1991) Abstract. A single systemic administration of acromelic acid A (ACRO), a novel kainate analogue (kainoid), induces a series of characteristic behavioral changes in association with selective damage of interneurons in the caudal spinal cord in adult rats. When ACRO (5 mg/kg) was systemically administered, rats displayed forced extension of hindlimbs followed by frequent cramps and generalized convulsion. Most rats died during the convulsions without neuropathological change. Two rats developed long-lasting spastic paraparesis which persisted at least 3 months. Neuropathological changes were observed only in the rats with persistent paraparesis, in which neuron damage was identified selectively in small interneurons in the lumbosacral cord. The regional difference between kainate- and ACRO-induced neuron damage suggests the existence of plural kinds of kainate receptor subtypes. Introduction A c r o m e l i c a c i d is r e c e n t l y e x t r a c t e d f r o m a J a p a n e s e p o i s o n o u s m u s h r o o m , C l i t o c y b e a c r o m e l a l g a (ii) a n d a c r o m e l i c a c i d A (ACRO) (Fig. i) is the m o s t p o t e n t a m o n g k n o w n e x c i t a t o r y a m i n o a c i d s in the i s o l a t e d n e w b o r n rat s p i n a l c o r d (9,18). The d e p o l a r i z a t i o n i n d u c e d by A C R O in v i t r o is not a f f e c t e d by N M D A r e c e p t o r a n t a g o n i s t s but c o m p l e t e l y a b o l i s h e d by 6 - c y a n o - 7 - n i t r o q u i n o x a l i n e - 2 , 3 d i o n e (CNQX) (18). S i n c e s y s t e m i c a l l y a d m i n i s t e r e d k a i n a t e i n d u c e s s e l e c t i v e n e u r o n a l e x c i t a t i o n and d a m a g e in the l i m b i c s y s t e m of the rat (14), the p h a r m a c o l o g i c a l a n d s t r u c t u r a l h o m o l o g y w i t h k a i n a t e (Fig. i) i m p l i e s t h a t A C R O w o u l d i n d u c e a n e u r o n d a m a g e s i m i l a r to k a i n a t e in the m a m m a l i a n c e n t r a l n e r v o u s system. T h e s y s t e m i c e f f e c t s of A C R O w e r e h o w e v e r q u i t e d i s t i n c t f r o m t h o s e of k a i n a t e . The p r e s e n t s t u d y r e p o r t s the u n i q u e b e h a v i o r a l a n d n e u r o p a t h o l o g i c a l c h a n g e s in a d u l t rats i n d u c e d by the s y s t e m i c a d m i n i s t r a t i o n of ACRO. Our p r e l i m i n a r y r e s u l t s h a v e b e e n d e s c r i b e d elsewhere (12,17,18). Materials
and Methods
D u e to l i m i t e d a v a i l a b i l i t y of ACRO, we i n j e c t e d A C R O to rats in d o s e s n e a r the p r e s u m e d h a l f l e t h a l d o s e (LDs0). A C R O d i s s o l v e d in b u f f e r e d s a l i n e (pH 7.2) was a d m i n i s t e r e d s y s t e m i c a l l y in a s i n g l e d o s e of 2 to 5.5 m g / k g b o d y w e i g h t into 23 m a l e W i s t a r rats w e i g h ing 1 2 1 - 2 5 1 g (Table). T h r e e rats w e r e v e h i c l e - t r e a t e d . The rats l:To whom all the correspondence should be addressed. 0024-3205/91 $3.00 + .00 Copyright © 1991 Pergamon Press plc
PL-92
Selective Neuron Damage by Acromelic Acid
Vol. 49, No. 14, 1991
were left free in an e x p e r i m e n t a l field a f t e r the i n j e c t i o n a n d t h e i r b e h a v i o r s were c a r e f u l l y observed. On the 7th or 86th e x p e r i m e n t a l day, the rats were p e r f u s e d t r a n s c a r d i a l l y w i t h 200 ml of 3% p a r a f o r m a l d e h y d e a n d 0.5 % g l u t a r a l d e h y d e in 0.I M p h o s p h a t e b u f f e r (pH 7.4) u n d e r d e e p p e n t o b a r b i t a l a n e s t h e s i a . W h o l e b r a i n s a n d spinal cords w e r e d i s s e c t e d and b l o c k s of t i s s u e s were t r i m m e d and m o u n t e d in p a r a f f i n and cut into 3 or 5 ~tm sections. S e c t i o n s were s t a i n e d w i t h h e m a t o x y l i n a n d e o s i n (H.E.), K l 0 v e r - B a r r e r a (K.B.) and B o d i a n ' s s i l v e r stains.
H
HOOC=
H
N. ~._0
H
Acromelic acid A
Kainic acid FIG.
1.
For cytometry, sections of 5 ~m t h i c k n e s s w e r e cut f r o m the first sacral segment of the two p a r a p a r e t i c rats a n d of three c o n t r o l rats and s t a i n e d w i t h C r e s y l violet. All the n e u r o n s w i t h a v i s i b l e n u c l e o l u s in ten serial s e c t i o n s were counted. N e u r o n s were a r b i t r a r i l y d i v i d e d into three groups d e p e n d i n g on t h e i r diameter; large (over 20 ~m), i n t e r m e d i a t e (i0 to 20 ~m) a n d small (less than i0 ~tm) . D e g e n e r a t e d n e u r o n s were i d e n t i f i e d by p o o r s t a i n i n g a n d were not counted. M i c r o g l i a s with dark nuclei and a s t r o c y t e s w i t h l i g h t l y s t a i n e d n u c l e i were excluded. Ten serial s e c t i o n s of 5 ~m t h i c k n e s s were cut f r o m e a c h segment b e t w e e n the e i g h t h t h o r a c i c to the first c o c c y g e a l cord of the p a r a p a r e t i c rat s a c r i f i c e d on the 7th day a f t e r the i n j e c t i o n and s t a i n e d w i t h H.E., and the p o s i t i o n of d e g e n e r a t e d n e u r o n s were p l o t t e d on the h o r i z o n t a l axis of the g r a y matter. RESULTS The h a l f lethal dose (LD50) and the m i n i m u m lethal dose a p p e a r e d to be b e t w e e n 5 and 5.5 m g / k g and about 4 mg/kg, r e s p e c t i v e l y . We i n j e c t e d A C R O at a s u b c u t a n e o u s dose of 5 m g / k g in 16 rats and at v a r i o u s d o s e s in 7 rats (Table). At 5 mg/kg, A C R O r e p r o d u c i b l y i n d u c e d a series of b e h a v i o r a l changes, and two rats d e v e l o p e d l o n g - l a s t i n g s p a s t i c p a r a p a r e s i s . A f t e r the i n i t i a l s e d a t i v e stage w h i c h b e g a n s e v e r a l m i n u t e s after the i n j e c t i o n a n d l a s t e d for a p p r o x i m a t e l y 30 min, all rats b e g a n to move t h e i r tails like snake's a n d to b i t e them. Then, t h e i r h i n d l i m b s b e c a m e g r a d u a l l y e x t e n d e d a n d t h e i r b a c k slowly a n t e - f l e x e d w i t h e l e v a t i o n of the hip, w h i c h c o m p e l l e d the rats to walk on toes and led t h e m occasional falls. S u b s e q u e n t l y , rats were s u f f e r e d f r o m a t t a c k s of h i n d l i m b c r a m p w h i c h was i n i t i a l l y i n t e r m i t t e n t but later b e c a m e tonic. This h i n d l i m b cramp r e s e m b l e d s t r y c h n i n e - i n d u c e d t o n i c c o n v u l s i o n , e x c e p t for s p a r i n g the r o s t r a l h a l f of the b o d y a n d displaying righting reflex without respiratory difficulty. About
Vol. 49, No. 14, 1991
Selective Neuron Damage by Acromelic Acid
PL-93
1 h after the injection, ten rats were seized by generalized tonicc l o n i c c o n v u l s i o n s t h a t l a s t e d for 1 0 - 4 0 min. S i x r a t s d i e d d u r i n g t h e c o u r s e of t h e c o n v u l s i v e s e i z u r e . O f t h e f o u r s u r v i v i n g rats, two developed complete flaccid paraplegia which continued more than 2 h o u r s . T h e r a t s w e r e a l e r t a n d o c c a s i o n a l l y s q u e a k e d . On t h e f o l l o w i n g days, t h e r a t s d e v e l o p e d p a r a p a r e s i s of s e v e r e s p a s t i c i t y w h i c h was r e i n f o r c e d b y t o u c h i n g or p o k i n g . N o b l a d d e r b o w e l d i s t u r b a n c e was o b s e r v e d . The r a t s c o u l d m o v e u s i n g f o r e l i m b s a n d dragging their hindlimbs. The spastic paraparesis remained u n c h a n g e d u n t i l t h e r a t s w e r e s a c r i f i c e d for the h i s t o p a t h o l o g i c a l TABLE Behavioral
Changes
Sedation Hindlimb Extension Hindlimb Cramp Convulsion Death Persistent Paraparesis
Dosaqe
of A C R O
(mq/kq)
2 (i)
3 (i)
4(2)
5 (16)
1 0 0 0 0 0
1 1 0 0 0 0
2 2 1 1 1 0
16 16 16 I0 6 2
(N)
5.5
Numerals are the number of rats showing the listed behavioral changes after the injection of ACRO. (N): number of rats receiving each dose of ACRO, Convulsion: generalized tonic-clonic convulsion.
FIG.
2
A) Degenerated neuron with pyknotic nucleus (arrow) attached by microglias. H.E. Bar= i0 ~m. B) Degenerated neuron (arrow). Bodian's silver staining. C) Schematic diagram showing the position of degenerated neurons in the spinal segments. Each black circle represents one degenerated neuron appearing in one of 4 sections.
(3) 3 3 3 3 3 0
PL-94
Selective Neuron Damage by Acromelic Acid
Vol. 49, No. 14, 1991
a n a l y s i s on t h e 7 t h or 8 6 t h e x p e r i m e n t a l day. The o t h e r t w o surviving rats that transiently developed weak flaccid paraparesis a f t e r t h e c o n v u l s i o n s h o w e d n o r m a l b e h a v i o r o n t h e f o l l o w i n g days. T h e r a t s t h a t r e c e i v e d s m a l l e r d o s e s of A C R O d i s p l a y e d b e h a v i o r a l c h a n g e s t r a n s i e n t l y , a n d t h o s e r e c e i v e d 5.5 m g / k g d u r i n g t h e c o n v u l s i v e a t t a c k (Table).
died
H i s t o l o g i c a l c h a n g e s w e r e d e t e c t e d e x c l u s i v e l y in t w o r a t s t h a t developed persistent spastic paraparesis. There were numerous d e g e n e r a t e d n e u r o n s in the l u m b o s a c r a l s p i n a l s e g m e n t s of t h e rat s a c r i f i c e d on t h e 7 t h d a y a f t e r t h e i n j e c t i o n . D e g e n e r a t e d n e u r o n s were lightly eosinophilic with irregularly shrunken and strongly e o s i n o p h i l i c n u c l e i a n d s e v e r a l m i c r o g l i a s w e r e a t t a c h e d to t h e d e g e n e r a t e d n e u r o n s (Fig. 2a). In s i l v e r s t a i n i n g , n u m e r o u s a r g y r o p h i l i c g r a i n s w e r e o b s e r v e d in t h e c y t o p l a s m of t h e d e g e n e r a t e d neurons whose nuclei were intensely argyrophilic (Fig. 2b) . T h e degenerated n e u r o n s w e r e d i s t r i b u t e d in t h e c o r e p a r t of t h e g r a y m a t t e r in t h e l u m b o s a c r a l s e g m e n t s b u t not in t h e m o r e r o s t r a l s e g m e n t s (Fig. 2c) . The cytometry demonstrated that degenerated neurons were predomi n a n t l y s m a l l n e u r o n s (Fig. 3). In t h e s p i n a l c o r d of t h e p a r a p a r e t i c rat s a c r i f i c e d on t h e 8 6 t h e x p e r i m e n t a l day, no d e g e n e r a t e d
o Z
ioo
I
I
Iooo
Z
~o
> 20 ~ m
10 - 20 ~.m
Total
Laminae I & II
<10 ~ m
Cell Diameter
FIG.
3
Number of neurons in the first sacral segment in the paraparetic rat sacrificed 7 days (7 day) and 86 days (86 day) after the injection of ACRO (5 mg/kg). Columns with bars indicate the mean ± S.D. of the number of neurons appeared in ten sections. *:p<0.05, **:p<0.005, ***:p<0.001 by Student's t-test. Small neurons (diameter
Vol. 49, No. 14, 1991
Selective Neuron Damage by Acromelic Acid
PL-95
neurons were o b s e r v e d while small neurons were d e c r e a s e d in n u m b e r (Fig. 3). White columns, ventral roots and large a n t e r i o r horn cells were not a f f e c t e d in either p a r a p a r e t i c rats. These were the c h a r a c t e r i s t i c s o b s e r v e d t h r o u g h o u t the lumbar and sacral segments, while the c e r v i c a l and rostral t h o r a c i c segments were m o r p h o l o g i cally intact. The other structures were intact i n c l u d i n g the areas liable to s y s t e m i c kainate administration, such as the hippocampus, p y r i f o r m a n d e n t o r h i n a l cortices and a m y g d a l o i d nucleus (14), except for a some increase of the i m m u n o r e a c t i v i t y against GFAP in the s t r a t u m m o l e c u l a r e - l a c u n o s u m of CA1 and the CA4 in the parap a r e t i c rat s a c r i f i c e d on the 7th e x p e r i m e n t a l day.
Present e x a m i n a t i o n d i s c l o s e d the s e l e c t i v e d e g e n e r a t i o n of i n t e r n e u r o n s w h i c h r e s u l t e d in p e r s i s t e n t paraparesis. The p r e f e r e n c e of A C R O for the spinal neurons is unique among other kainoids w h i c h have a p r e f e r e n c e for the limbic system. A single systemic a d m i n i s t r a t i o n of kainate induces 'wet dog shakes' and c h a r a c t e r i s t i c limbic motor seizures with d e g e n e r a t i o n of neurons and tissue n e c r o s i s in the limbic system but not in the spinal cord (14). The effects of systemic a d m i n i s t r a t i o n of domoate, a n o t h e r kainoid, are quite similar to those of kainate w i t h p r e f e r e n c e for i n d u c i n g h e a d s c r a t c h i n g b e h a v i o r (19). On the other hand, spastic p a r a p a r e s i s with inducing selective d e g e n e r a t i o n of l u m b o s a c r a l i n t e r n e u r o n s have never b e e n o b s e r v e d after systemic a d m i n i s t r a t i o n of k a i n a t e or domoate. The m o r p h o l o g i c a l a l t e r a t i o n of the d e g e n e r a t e d i n t e r n e u r o n s in the rat w i t h p e r s i s t e n t p a r a p a r e s i s closely r e s e m b l e s that of the h i p p o c a m p a l CA1 p y r a m i d a l cells seen after s y s t e m i c a d m i n i s t r a t i o n of kainate (14) and 'ischemic cell change' (3). Furthermore, severe r i g i d o - s p a s t i c p a r a p a r e s i s a s s o c i a t e d with e x t e n s i v e i n t e r n e u r o n damage in the l u m b o s a c r a l cord is also induced by spinal ischemia in the dog, cat and rat (5,7). The p a t h o l o g i c a l s i m i l a r i t y suggests the p r e s e n c e of a common m e c h a n i s m u n d e r l y i n g the d e l a y e d n e u r o n a l death after b r a i n ischemia (i0) and neuron damage i n d u c e d by the kainoids. Since it seems reasonable to assume that A C R O acts p r i m a r i l y on kainate r e c e p t o r s a c c o r d i n g to p h a r m a c o l o g i c a l studies in vitro (9,15,18), the m a r k e d regional d i f f e r e n c e in n e u r o n a l e x c i t a t i o n and damage b e t w e e n kainate and A C R O would be e x p l a i n e d by a s s u m i n g d i f f e r e n t subtypes of kainate receptors for A C R O and kainate. A C R O causes a significant d e p o l a r i z a t i o n in the spinal dorsal root fibers of the n e w b o r n rat slightly more e f f e c t i v e l y than k a i n a t e (16). Since dorsal root C-fibers of immature rats are e f f e c t i v e l y d e p o l a r i z e d by kainate and domoate but weakly by N M D A or A M P A (1,3), A C R O p o s s e s s e s a c o n s i d e r a b l y h i g h a f f i n i t y for k a i n a t e receptors. A C R O is, however, c o n s i d e r a b l y less potent t h a n kainate in i n h i b i t i n g [3H]kainate b i n d i n g to the rat spinal cord s y n a p t i c m e m b r a n e s (2). Above e v i d e n c e suggests that there is a k a i n a t e r e c e p t o r subtype in the rat spinal cord r e s p o n d i n g p r e f e r e n t i a l l y to A C R O (tentatively called "ACRO receptor"). A human d i s e a s e analogous to the p a r a p a r e t i c rats i n d u c e d by A C R O is 'stiffman syndrome', a p r o g r e s s i v e n e u r o l o g i c a l d i s e a s e c h a r a c t e r i z e d by slowly a s c e n d i n g r i g i d o - s p a s t i c i t y with p a i n f u l
PL-96
Selective Neuron Damage by Acromelic Acid
Vol. 49, No. 14, 1991
spasms of limb a n d axial m u s c u l a t u r e (6). The salient n e u r o p a t h o l o g i c a l c h a n g e of s t i f f m a n s y n d r o m e is the m a r k e d loss of spinal i n t e r n e u r o n s a n d e x t e n s i v e g l i o s i s s p a r i n g b o t h large m o t o n e u r o n s in the v e n t r a l h o r n s a n d the c o r t i c o s p i n a l t r a c t (8,20). Since e x c i t a t o r y a m i n o acids are i m p l i c a t e d as a p a t h o g e n e s i s of seveal n e u r o l o g i c a l d i s e a s e s a f f e c t i n g the spinal c o r d i n c l u d i n g a m y o t r o p h i c l a t e r a l s c l e r o s i s (ALS), G u a m A L S - p a r k i n s o n - d e m e n t i a c o m p l e x a n d n e u r o l a t h y r i s m (13), the close s i m i l a r i t y of c l i n i c o p a t h o l o g i c a l p i c t u r e d e s e r v e s s c r u t i n y of the m e c h a n i s m u n d e r l y i n g n e u r o n d a m a g e i n d u c e d by ACRO. Acknowledaements We t h a n k to Prof. H a r u h i s a S h i r a h a m a for the g e n e r o u s gift of A C R O a n d t o Mrs K u m i k o M i u r a for e x c e l l e n t t e c h n i c a l a s s i s t a n c e . This study was p a r t i a l l y s u p p o r t e d by a G r a n t - i n - A i d for S c i e n t i f i c R e s e a r c h a n d a G r a n t - i n - A i d for S c i e n t i f i c R e s e a r c h on P r i o r i t y A r e a s f r o m the M i n i s t r y of Education, S c i e n c e a n d C u l t u r e of Japan. References i. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12.
13. 14. 15. 16.
17. 18.
19. 20.
S.G. A G R A W A L A N D R.H.EVANS, Br. J. Pharmac. 87 3 4 5 - 3 5 5 (1986). H. A I Z A W A AND S. KWAK, Neurosci. Res., in p r e s s (1991). A.W. B R O W N AND J . B . B R I E R L E Y , J. Neurol. Sci. 16 59-84 (1972). R.H. EVANS, S.J.EVANS, P.C.POOK, AND D . C . S U N T E R , Br. J. Pharmac. 91 531-537 (1987). S. G E L F A N AND I.M.TARLOV, J. Physiol. 146 594-617 (1959). E.E. GORDON, D . M . J A N U S Z K O , AND L.KAUFMAN, Am. J. Med. 42 5 8 2 - 5 9 9 (1967). S. HADZOVIC, Iugoslav. Physiol. P h a r m a c o l . A c t a ~ 97-100 (1970). D.A. HOWELL, A.J.LEES, AND P . J . T O G H I L L , J. Neurol. N e u r o s u r g . P s y c h i a t . 42 773-785 (1979). M. I S H I D A AND H. SHINOZAKI, B r a i n Res. 474 3 8 6 - 3 8 9 (1988). T. KIRINO, B r a i n Res. 239 57-69 (1982). K. KONNO, K. HASHIMOTO, Y. OHFUNE, H. S H I R A H A M A A N D T. M A T S U M O T O , J. Am. Chem. Soc. ii0 4 8 0 7 - 4 8 1 5 (1988). S. KWAK, H. AIZAWA, M. ISHIDA, Y. G O T O H AND H. SHINOZAKI, A m i n o Acids: Chemistrv. B i o l o a v a n d M e d i c i n e . G . L u b e c a n d G . A . R o s e n t h a l (eds), 318-326, ESCOM, L e i d e n (1990). J.W. OLNEY, Ann. Rev. Pharmacol. Toxicol. 30 47-71 (1990). J.E. SCHWOB, T.FULLER, J.L.PRICE, AND J.W.OLNEY, N e u r o s c i e n c e 991-1014 (1980). H. SHINOZAKI, P r o g r e s s N e u r o b i o l . 30 3 9 9 - 4 3 5 (1988). H. SHINOZAKI, E x c i t a t o r v A m i n o A c i d Receptors~ P. K r o g s g a a r d L a r s e n a n d J.J. H a n s e n (eds), in press, Ellis Howard, C h i c h e s t e r (1991) H. SHINOZAKI, I S H I D A M., G O T O H Y. AND K W A K S., B r a i n Res. 503 3 3 0 - 3 3 3 (1989). H. SHINOZAKI, I S H I D A M., G O T O H Y. AND K W A K S, A m i n o Acids: C h e m i s t r y . B i o l o g y a n d Medicine. G . L u b e c a n d G . A . R o s e n t h a l (eds), 281-293, ESCOM, L e i d e n (1990). G.R. STEWART, C.F. ZORUMSKI, M.T. PRICE, AND J.W. OLNEY, Exp. Neurol. ii0 127-138 (1990). A.M. WHITELY, M.SWASH, AND H.URICH, B r a i n 99 27-42 (1976).
Pergamon Press
PHARMACOLOGY LETTERS Accelerated Communication
S Y S T E M I C A D M I N I S T R A T I O N OF A C R O M E L I C A C I D I N D U C E S S E L E C T I V E N E U R O N D A M A G E IN THE R A T S P I N A L C O R D
Michiko
S h i n K w a k I, H i t o s h i Aizawa, Ishida*, a n d H a r u h i k o S h i n o z a k i *
N a t i o n a l I n s t i t u t e of N e u r o s c i e n c e , N a t i o n a l C e n t e r of N e u r o l o g y and Psychiatry (NCNP), 4-1-1, O g a w a h i g a s h i , K o d a i r a , T o k y o 187. *The T o k y o M e t r o p o l i t a n I n s t i t u t e of M e d i c a l S c i e n c e , 3 - 1 8 - 2 2 , H o n k o m a g o m e , B u n k y o - k u , T o k y o 113, Japan. (Submitted June 4, 1991; accepted July i0, 1991; received in final form July 23, 1991) Abstract. A single systemic administration of acromelic acid A (ACRO), a novel kainate analogue (kainoid), induces a series of characteristic behavioral changes in association with selective damage of interneurons in the caudal spinal cord in adult rats. When ACRO (5 mg/kg) was systemically administered, rats displayed forced extension of hindlimbs followed by frequent cramps and generalized convulsion. Most rats died during the convulsions without neuropathological change. Two rats developed long-lasting spastic paraparesis which persisted at least 3 months. Neuropathological changes were observed only in the rats with persistent paraparesis, in which neuron damage was identified selectively in small interneurons in the lumbosacral cord. The regional difference between kainate- and ACRO-induced neuron damage suggests the existence of plural kinds of kainate receptor subtypes. Introduction A c r o m e l i c a c i d is r e c e n t l y e x t r a c t e d f r o m a J a p a n e s e p o i s o n o u s m u s h r o o m , C l i t o c y b e a c r o m e l a l g a (ii) a n d a c r o m e l i c a c i d A (ACRO) (Fig. i) is the m o s t p o t e n t a m o n g k n o w n e x c i t a t o r y a m i n o a c i d s in the i s o l a t e d n e w b o r n rat s p i n a l c o r d (9,18). The d e p o l a r i z a t i o n i n d u c e d by A C R O in v i t r o is not a f f e c t e d by N M D A r e c e p t o r a n t a g o n i s t s but c o m p l e t e l y a b o l i s h e d by 6 - c y a n o - 7 - n i t r o q u i n o x a l i n e - 2 , 3 d i o n e (CNQX) (18). S i n c e s y s t e m i c a l l y a d m i n i s t e r e d k a i n a t e i n d u c e s s e l e c t i v e n e u r o n a l e x c i t a t i o n and d a m a g e in the l i m b i c s y s t e m of the rat (14), the p h a r m a c o l o g i c a l a n d s t r u c t u r a l h o m o l o g y w i t h k a i n a t e (Fig. i) i m p l i e s t h a t A C R O w o u l d i n d u c e a n e u r o n d a m a g e s i m i l a r to k a i n a t e in the m a m m a l i a n c e n t r a l n e r v o u s system. T h e s y s t e m i c e f f e c t s of A C R O w e r e h o w e v e r q u i t e d i s t i n c t f r o m t h o s e of k a i n a t e . The p r e s e n t s t u d y r e p o r t s the u n i q u e b e h a v i o r a l a n d n e u r o p a t h o l o g i c a l c h a n g e s in a d u l t rats i n d u c e d by the s y s t e m i c a d m i n i s t r a t i o n of ACRO. Our p r e l i m i n a r y r e s u l t s h a v e b e e n d e s c r i b e d elsewhere (12,17,18). Materials
and Methods
D u e to l i m i t e d a v a i l a b i l i t y of ACRO, we i n j e c t e d A C R O to rats in d o s e s n e a r the p r e s u m e d h a l f l e t h a l d o s e (LDs0). A C R O d i s s o l v e d in b u f f e r e d s a l i n e (pH 7.2) was a d m i n i s t e r e d s y s t e m i c a l l y in a s i n g l e d o s e of 2 to 5.5 m g / k g b o d y w e i g h t into 23 m a l e W i s t a r rats w e i g h ing 1 2 1 - 2 5 1 g (Table). T h r e e rats w e r e v e h i c l e - t r e a t e d . The rats l:To whom all the correspondence should be addressed. 0024-3205/91 $3.00 + .00 Copyright © 1991 Pergamon Press plc
PL-92
Selective Neuron Damage by Acromelic Acid
Vol. 49, No. 14, 1991
were left free in an e x p e r i m e n t a l field a f t e r the i n j e c t i o n a n d t h e i r b e h a v i o r s were c a r e f u l l y observed. On the 7th or 86th e x p e r i m e n t a l day, the rats were p e r f u s e d t r a n s c a r d i a l l y w i t h 200 ml of 3% p a r a f o r m a l d e h y d e a n d 0.5 % g l u t a r a l d e h y d e in 0.I M p h o s p h a t e b u f f e r (pH 7.4) u n d e r d e e p p e n t o b a r b i t a l a n e s t h e s i a . W h o l e b r a i n s a n d spinal cords w e r e d i s s e c t e d and b l o c k s of t i s s u e s were t r i m m e d and m o u n t e d in p a r a f f i n and cut into 3 or 5 ~tm sections. S e c t i o n s were s t a i n e d w i t h h e m a t o x y l i n a n d e o s i n (H.E.), K l 0 v e r - B a r r e r a (K.B.) and B o d i a n ' s s i l v e r stains.
H
HOOC=
H
N. ~._0
H
Acromelic acid A
Kainic acid FIG.
1.
For cytometry, sections of 5 ~m t h i c k n e s s w e r e cut f r o m the first sacral segment of the two p a r a p a r e t i c rats a n d of three c o n t r o l rats and s t a i n e d w i t h C r e s y l violet. All the n e u r o n s w i t h a v i s i b l e n u c l e o l u s in ten serial s e c t i o n s were counted. N e u r o n s were a r b i t r a r i l y d i v i d e d into three groups d e p e n d i n g on t h e i r diameter; large (over 20 ~m), i n t e r m e d i a t e (i0 to 20 ~m) a n d small (less than i0 ~tm) . D e g e n e r a t e d n e u r o n s were i d e n t i f i e d by p o o r s t a i n i n g a n d were not counted. M i c r o g l i a s with dark nuclei and a s t r o c y t e s w i t h l i g h t l y s t a i n e d n u c l e i were excluded. Ten serial s e c t i o n s of 5 ~m t h i c k n e s s were cut f r o m e a c h segment b e t w e e n the e i g h t h t h o r a c i c to the first c o c c y g e a l cord of the p a r a p a r e t i c rat s a c r i f i c e d on the 7th day a f t e r the i n j e c t i o n and s t a i n e d w i t h H.E., and the p o s i t i o n of d e g e n e r a t e d n e u r o n s were p l o t t e d on the h o r i z o n t a l axis of the g r a y matter. RESULTS The h a l f lethal dose (LD50) and the m i n i m u m lethal dose a p p e a r e d to be b e t w e e n 5 and 5.5 m g / k g and about 4 mg/kg, r e s p e c t i v e l y . We i n j e c t e d A C R O at a s u b c u t a n e o u s dose of 5 m g / k g in 16 rats and at v a r i o u s d o s e s in 7 rats (Table). At 5 mg/kg, A C R O r e p r o d u c i b l y i n d u c e d a series of b e h a v i o r a l changes, and two rats d e v e l o p e d l o n g - l a s t i n g s p a s t i c p a r a p a r e s i s . A f t e r the i n i t i a l s e d a t i v e stage w h i c h b e g a n s e v e r a l m i n u t e s after the i n j e c t i o n a n d l a s t e d for a p p r o x i m a t e l y 30 min, all rats b e g a n to move t h e i r tails like snake's a n d to b i t e them. Then, t h e i r h i n d l i m b s b e c a m e g r a d u a l l y e x t e n d e d a n d t h e i r b a c k slowly a n t e - f l e x e d w i t h e l e v a t i o n of the hip, w h i c h c o m p e l l e d the rats to walk on toes and led t h e m occasional falls. S u b s e q u e n t l y , rats were s u f f e r e d f r o m a t t a c k s of h i n d l i m b c r a m p w h i c h was i n i t i a l l y i n t e r m i t t e n t but later b e c a m e tonic. This h i n d l i m b cramp r e s e m b l e d s t r y c h n i n e - i n d u c e d t o n i c c o n v u l s i o n , e x c e p t for s p a r i n g the r o s t r a l h a l f of the b o d y a n d displaying righting reflex without respiratory difficulty. About
Vol. 49, No. 14, 1991
Selective Neuron Damage by Acromelic Acid
PL-93
1 h after the injection, ten rats were seized by generalized tonicc l o n i c c o n v u l s i o n s t h a t l a s t e d for 1 0 - 4 0 min. S i x r a t s d i e d d u r i n g t h e c o u r s e of t h e c o n v u l s i v e s e i z u r e . O f t h e f o u r s u r v i v i n g rats, two developed complete flaccid paraplegia which continued more than 2 h o u r s . T h e r a t s w e r e a l e r t a n d o c c a s i o n a l l y s q u e a k e d . On t h e f o l l o w i n g days, t h e r a t s d e v e l o p e d p a r a p a r e s i s of s e v e r e s p a s t i c i t y w h i c h was r e i n f o r c e d b y t o u c h i n g or p o k i n g . N o b l a d d e r b o w e l d i s t u r b a n c e was o b s e r v e d . The r a t s c o u l d m o v e u s i n g f o r e l i m b s a n d dragging their hindlimbs. The spastic paraparesis remained u n c h a n g e d u n t i l t h e r a t s w e r e s a c r i f i c e d for the h i s t o p a t h o l o g i c a l TABLE Behavioral
Changes
Sedation Hindlimb Extension Hindlimb Cramp Convulsion Death Persistent Paraparesis
Dosaqe
of A C R O
(mq/kq)
2 (i)
3 (i)
4(2)
5 (16)
1 0 0 0 0 0
1 1 0 0 0 0
2 2 1 1 1 0
16 16 16 I0 6 2
(N)
5.5
Numerals are the number of rats showing the listed behavioral changes after the injection of ACRO. (N): number of rats receiving each dose of ACRO, Convulsion: generalized tonic-clonic convulsion.
FIG.
2
A) Degenerated neuron with pyknotic nucleus (arrow) attached by microglias. H.E. Bar= i0 ~m. B) Degenerated neuron (arrow). Bodian's silver staining. C) Schematic diagram showing the position of degenerated neurons in the spinal segments. Each black circle represents one degenerated neuron appearing in one of 4 sections.
(3) 3 3 3 3 3 0
PL-94
Selective Neuron Damage by Acromelic Acid
Vol. 49, No. 14, 1991
a n a l y s i s on t h e 7 t h or 8 6 t h e x p e r i m e n t a l day. The o t h e r t w o surviving rats that transiently developed weak flaccid paraparesis a f t e r t h e c o n v u l s i o n s h o w e d n o r m a l b e h a v i o r o n t h e f o l l o w i n g days. T h e r a t s t h a t r e c e i v e d s m a l l e r d o s e s of A C R O d i s p l a y e d b e h a v i o r a l c h a n g e s t r a n s i e n t l y , a n d t h o s e r e c e i v e d 5.5 m g / k g d u r i n g t h e c o n v u l s i v e a t t a c k (Table).
died
H i s t o l o g i c a l c h a n g e s w e r e d e t e c t e d e x c l u s i v e l y in t w o r a t s t h a t developed persistent spastic paraparesis. There were numerous d e g e n e r a t e d n e u r o n s in the l u m b o s a c r a l s p i n a l s e g m e n t s of t h e rat s a c r i f i c e d on t h e 7 t h d a y a f t e r t h e i n j e c t i o n . D e g e n e r a t e d n e u r o n s were lightly eosinophilic with irregularly shrunken and strongly e o s i n o p h i l i c n u c l e i a n d s e v e r a l m i c r o g l i a s w e r e a t t a c h e d to t h e d e g e n e r a t e d n e u r o n s (Fig. 2a). In s i l v e r s t a i n i n g , n u m e r o u s a r g y r o p h i l i c g r a i n s w e r e o b s e r v e d in t h e c y t o p l a s m of t h e d e g e n e r a t e d neurons whose nuclei were intensely argyrophilic (Fig. 2b) . T h e degenerated n e u r o n s w e r e d i s t r i b u t e d in t h e c o r e p a r t of t h e g r a y m a t t e r in t h e l u m b o s a c r a l s e g m e n t s b u t not in t h e m o r e r o s t r a l s e g m e n t s (Fig. 2c) . The cytometry demonstrated that degenerated neurons were predomi n a n t l y s m a l l n e u r o n s (Fig. 3). In t h e s p i n a l c o r d of t h e p a r a p a r e t i c rat s a c r i f i c e d on t h e 8 6 t h e x p e r i m e n t a l day, no d e g e n e r a t e d
o Z
ioo
I
I
Iooo
Z
~o
> 20 ~ m
10 - 20 ~.m
Total
Laminae I & II
<10 ~ m
Cell Diameter
FIG.
3
Number of neurons in the first sacral segment in the paraparetic rat sacrificed 7 days (7 day) and 86 days (86 day) after the injection of ACRO (5 mg/kg). Columns with bars indicate the mean ± S.D. of the number of neurons appeared in ten sections. *:p<0.05, **:p<0.005, ***:p<0.001 by Student's t-test. Small neurons (diameter
Vol. 49, No. 14, 1991
Selective Neuron Damage by Acromelic Acid
PL-95
neurons were o b s e r v e d while small neurons were d e c r e a s e d in n u m b e r (Fig. 3). White columns, ventral roots and large a n t e r i o r horn cells were not a f f e c t e d in either p a r a p a r e t i c rats. These were the c h a r a c t e r i s t i c s o b s e r v e d t h r o u g h o u t the lumbar and sacral segments, while the c e r v i c a l and rostral t h o r a c i c segments were m o r p h o l o g i cally intact. The other structures were intact i n c l u d i n g the areas liable to s y s t e m i c kainate administration, such as the hippocampus, p y r i f o r m a n d e n t o r h i n a l cortices and a m y g d a l o i d nucleus (14), except for a some increase of the i m m u n o r e a c t i v i t y against GFAP in the s t r a t u m m o l e c u l a r e - l a c u n o s u m of CA1 and the CA4 in the parap a r e t i c rat s a c r i f i c e d on the 7th e x p e r i m e n t a l day.
Present e x a m i n a t i o n d i s c l o s e d the s e l e c t i v e d e g e n e r a t i o n of i n t e r n e u r o n s w h i c h r e s u l t e d in p e r s i s t e n t paraparesis. The p r e f e r e n c e of A C R O for the spinal neurons is unique among other kainoids w h i c h have a p r e f e r e n c e for the limbic system. A single systemic a d m i n i s t r a t i o n of kainate induces 'wet dog shakes' and c h a r a c t e r i s t i c limbic motor seizures with d e g e n e r a t i o n of neurons and tissue n e c r o s i s in the limbic system but not in the spinal cord (14). The effects of systemic a d m i n i s t r a t i o n of domoate, a n o t h e r kainoid, are quite similar to those of kainate w i t h p r e f e r e n c e for i n d u c i n g h e a d s c r a t c h i n g b e h a v i o r (19). On the other hand, spastic p a r a p a r e s i s with inducing selective d e g e n e r a t i o n of l u m b o s a c r a l i n t e r n e u r o n s have never b e e n o b s e r v e d after systemic a d m i n i s t r a t i o n of k a i n a t e or domoate. The m o r p h o l o g i c a l a l t e r a t i o n of the d e g e n e r a t e d i n t e r n e u r o n s in the rat w i t h p e r s i s t e n t p a r a p a r e s i s closely r e s e m b l e s that of the h i p p o c a m p a l CA1 p y r a m i d a l cells seen after s y s t e m i c a d m i n i s t r a t i o n of kainate (14) and 'ischemic cell change' (3). Furthermore, severe r i g i d o - s p a s t i c p a r a p a r e s i s a s s o c i a t e d with e x t e n s i v e i n t e r n e u r o n damage in the l u m b o s a c r a l cord is also induced by spinal ischemia in the dog, cat and rat (5,7). The p a t h o l o g i c a l s i m i l a r i t y suggests the p r e s e n c e of a common m e c h a n i s m u n d e r l y i n g the d e l a y e d n e u r o n a l death after b r a i n ischemia (i0) and neuron damage i n d u c e d by the kainoids. Since it seems reasonable to assume that A C R O acts p r i m a r i l y on kainate r e c e p t o r s a c c o r d i n g to p h a r m a c o l o g i c a l studies in vitro (9,15,18), the m a r k e d regional d i f f e r e n c e in n e u r o n a l e x c i t a t i o n and damage b e t w e e n kainate and A C R O would be e x p l a i n e d by a s s u m i n g d i f f e r e n t subtypes of kainate receptors for A C R O and kainate. A C R O causes a significant d e p o l a r i z a t i o n in the spinal dorsal root fibers of the n e w b o r n rat slightly more e f f e c t i v e l y than k a i n a t e (16). Since dorsal root C-fibers of immature rats are e f f e c t i v e l y d e p o l a r i z e d by kainate and domoate but weakly by N M D A or A M P A (1,3), A C R O p o s s e s s e s a c o n s i d e r a b l y h i g h a f f i n i t y for k a i n a t e receptors. A C R O is, however, c o n s i d e r a b l y less potent t h a n kainate in i n h i b i t i n g [3H]kainate b i n d i n g to the rat spinal cord s y n a p t i c m e m b r a n e s (2). Above e v i d e n c e suggests that there is a k a i n a t e r e c e p t o r subtype in the rat spinal cord r e s p o n d i n g p r e f e r e n t i a l l y to A C R O (tentatively called "ACRO receptor"). A human d i s e a s e analogous to the p a r a p a r e t i c rats i n d u c e d by A C R O is 'stiffman syndrome', a p r o g r e s s i v e n e u r o l o g i c a l d i s e a s e c h a r a c t e r i z e d by slowly a s c e n d i n g r i g i d o - s p a s t i c i t y with p a i n f u l
PL-96
Selective Neuron Damage by Acromelic Acid
Vol. 49, No. 14, 1991
spasms of limb a n d axial m u s c u l a t u r e (6). The salient n e u r o p a t h o l o g i c a l c h a n g e of s t i f f m a n s y n d r o m e is the m a r k e d loss of spinal i n t e r n e u r o n s a n d e x t e n s i v e g l i o s i s s p a r i n g b o t h large m o t o n e u r o n s in the v e n t r a l h o r n s a n d the c o r t i c o s p i n a l t r a c t (8,20). Since e x c i t a t o r y a m i n o acids are i m p l i c a t e d as a p a t h o g e n e s i s of seveal n e u r o l o g i c a l d i s e a s e s a f f e c t i n g the spinal c o r d i n c l u d i n g a m y o t r o p h i c l a t e r a l s c l e r o s i s (ALS), G u a m A L S - p a r k i n s o n - d e m e n t i a c o m p l e x a n d n e u r o l a t h y r i s m (13), the close s i m i l a r i t y of c l i n i c o p a t h o l o g i c a l p i c t u r e d e s e r v e s s c r u t i n y of the m e c h a n i s m u n d e r l y i n g n e u r o n d a m a g e i n d u c e d by ACRO. Acknowledaements We t h a n k to Prof. H a r u h i s a S h i r a h a m a for the g e n e r o u s gift of A C R O a n d t o Mrs K u m i k o M i u r a for e x c e l l e n t t e c h n i c a l a s s i s t a n c e . This study was p a r t i a l l y s u p p o r t e d by a G r a n t - i n - A i d for S c i e n t i f i c R e s e a r c h a n d a G r a n t - i n - A i d for S c i e n t i f i c R e s e a r c h on P r i o r i t y A r e a s f r o m the M i n i s t r y of Education, S c i e n c e a n d C u l t u r e of Japan. References i. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12.
13. 14. 15. 16.
17. 18.
19. 20.
S.G. A G R A W A L A N D R.H.EVANS, Br. J. Pharmac. 87 3 4 5 - 3 5 5 (1986). H. A I Z A W A AND S. KWAK, Neurosci. Res., in p r e s s (1991). A.W. B R O W N AND J . B . B R I E R L E Y , J. Neurol. Sci. 16 59-84 (1972). R.H. EVANS, S.J.EVANS, P.C.POOK, AND D . C . S U N T E R , Br. J. Pharmac. 91 531-537 (1987). S. G E L F A N AND I.M.TARLOV, J. Physiol. 146 594-617 (1959). E.E. GORDON, D . M . J A N U S Z K O , AND L.KAUFMAN, Am. J. Med. 42 5 8 2 - 5 9 9 (1967). S. HADZOVIC, Iugoslav. Physiol. P h a r m a c o l . A c t a ~ 97-100 (1970). D.A. HOWELL, A.J.LEES, AND P . J . T O G H I L L , J. Neurol. N e u r o s u r g . P s y c h i a t . 42 773-785 (1979). M. I S H I D A AND H. SHINOZAKI, B r a i n Res. 474 3 8 6 - 3 8 9 (1988). T. KIRINO, B r a i n Res. 239 57-69 (1982). K. KONNO, K. HASHIMOTO, Y. OHFUNE, H. S H I R A H A M A A N D T. M A T S U M O T O , J. Am. Chem. Soc. ii0 4 8 0 7 - 4 8 1 5 (1988). S. KWAK, H. AIZAWA, M. ISHIDA, Y. G O T O H AND H. SHINOZAKI, A m i n o Acids: Chemistrv. B i o l o a v a n d M e d i c i n e . G . L u b e c a n d G . A . R o s e n t h a l (eds), 318-326, ESCOM, L e i d e n (1990). J.W. OLNEY, Ann. Rev. Pharmacol. Toxicol. 30 47-71 (1990). J.E. SCHWOB, T.FULLER, J.L.PRICE, AND J.W.OLNEY, N e u r o s c i e n c e 991-1014 (1980). H. SHINOZAKI, P r o g r e s s N e u r o b i o l . 30 3 9 9 - 4 3 5 (1988). H. SHINOZAKI, E x c i t a t o r v A m i n o A c i d Receptors~ P. K r o g s g a a r d L a r s e n a n d J.J. H a n s e n (eds), in press, Ellis Howard, C h i c h e s t e r (1991) H. SHINOZAKI, I S H I D A M., G O T O H Y. AND K W A K S., B r a i n Res. 503 3 3 0 - 3 3 3 (1989). H. SHINOZAKI, I S H I D A M., G O T O H Y. AND K W A K S, A m i n o Acids: C h e m i s t r y . B i o l o g y a n d Medicine. G . L u b e c a n d G . A . R o s e n t h a l (eds), 281-293, ESCOM, L e i d e n (1990). G.R. STEWART, C.F. ZORUMSKI, M.T. PRICE, AND J.W. OLNEY, Exp. Neurol. ii0 127-138 (1990). A.M. WHITELY, M.SWASH, AND H.URICH, B r a i n 99 27-42 (1976).