276

th'am Research, 502 (199l) 276-280 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0(X~6-8993/91/$03.5(l A I)ONIS 00068993911708~4

BRES 17088

Effects of aging on quinolinic acid lesions in rat striatum Stephen E Finn, Bradley T. Hyman, Elsdon Storey, Joanne M. Miller and M. Flint Beal Neurochemistry Laboratory, Neurology Service, Massachusetts General Hospital and Harvard Medical School. Boston, MA 02114 (U.S.A.) (Accepted 4 June 1991) Key words: Aging; Uric acid; N-Methyt-D-aspartatc; Kynurenic acid

Several neurologic illnesses in which excitotoxic mechanisms may play a role increase in prevalence with age. In the present study wc examined the susceptibility of rats to quinolinic acid striatal lesions at 1, 4 and 20 months of age, and susceptibility to N-methyl-o-aspartate (NMDA) at 1 and 4 months of age. The extent of the lesions was quantitated with measurements of substancc P-like immunoreactivity (SPLI) and y-aminobutyric acid (GABA). The lesions in the 4- and 20-month-old age groups showed significantly smaller depletions of SPLI and GABA than those in 1-month-old animals. Neuropeptide Y-like immunoreactivity (NPYLI) and somatostatin-like immunoreactivity (SLI) were unchanged in the lesioned striata. NMDA lesions were also attenuated in 4-month- and 12-month-old animals as compared with l-month-old animals. Uric acid concentrations showed marked dose-dependent increases in the lesioned striatum, and to a lesser extent in the overlying cerebral cortex, in all 3 age groups. There were no changes of SLI, NPYLI or SPLI with aging in the cerebral cortex or hippocampus. Kynurenine and kynurenic acid concentrations showed significant increases with aging in frontal cortex. The present results show a reduced susceptibility of animals to striatal quinolinic acid and NMDA lesions with normal aging. The delayed onset of several neurodegenerative illnesses is therefore unlikely to be due to an increasing susceptibility to excitotoxin lesions with aging. INTRODUCTION

d u c e d by striatal q u i n o l i n i c acid lesions in 1-, 4- and 20m o n t h - o l d animals. We also e x a m i n e d N M D A lesions in

E x c i t a t o r y a m i n o acids h a v e b e e n i m p l i c a t e d in the neuronal

degeneration

which occurs in c e r e b r a l isch-

1-, 4- and 12-month-old animals. In a d d i t i o n we e x a m i n e d the effects of aging on n e u r o c h e m i c a l m a r k e r s in

e m i a , h y p o g l y c e m i a , e p i l e p s y , H u n t i n g t o n ' s disease and A l z h e i m e r ' s disease 15. H u n t i n g t o n ' s disease ( H D ) typi-

o v e r l y i n g c e r e b r a l c o r t e x and h i p p o c a m p u s .

cally exhibits an o n s e t in m i d d l e age, while A l z h e i m e r ' s

MATERIALS AND METHODS

disease s h o w s an i n c r e a s i n g p r e v a l e n c e with i n c r e a s i n g

One-month-, 4-month- and 20-month-old male Sprague-Dawley rats were obtained from Zivic-Miller. Quinolinic acid and NMDA were obtained from Sigma (St. Louis, MO). Animals were anesthetized with pentobarbital 50 mg/kg i.p. Quinolinic acid (240 nmol) or NMDA (200 nmol) were dissolved in I /A of phosphatebuffered saline (pH 7.4) and were injected into the left striatum at the coordinates 8.4 mm anterior to the interaural line, 2.6 mm lateral to the sagittal suture, and 4.5 mm ventral to the dura 4. Injections were made with a 10 ~1 Hamilton syringe fitted with a 30gauge blunt-tipped needle. All injections were made over 1 min, and the needle was left in place for a further 2 min before being slowly withdrawn. Ten animals of each age group were lesioned with each excitotoxin, however there were 9 and 8 survivors, respectively, in the 4and 20-month-old quinolinic acid groups. Animals were sacrificed at 1 week post-lesion by decapitation, their brains promptly removed, and the anterior striatum, overlying cortex and hippocampus were dissected from 2-mm-thick slices as previously described 4. Care was taken to exclude the globus pallidus. Samples of cerebral cortex for evaluation of transmitter changes associated with aging were taken from the unlesioned hemisphere. The dissected tissue was placed in I ml of chilled 0.1 N HCI. Tissue samples were subsequently sonicated, extracted and assayed for SLI, NPYLI, SPLI and y-aminobutyric acid (GABA) as previously described 1'5;6'j7. Kynurenic acid concentrations were measured by HPLC with fluorometric detection 33. Cateeholamines and related compounds were

age. T h e r e is, h o w e v e r , little i n f o r m a t i o n a v a i l a b l e a b o u t a g e - d e p e n d e n t susceptibility to e x c i t o t o x i n lesions. R e c e n t studies o f c u l t u r e d n e u r o n s h a v e s h o w n that a r e d u c t i o n in i n t r a c e l l u l a r e n e r g y levels e n h a n c e s glutam a t e toxicity 28. In c u l t u r e d c e r e b e l l a r n e u r o n s a reduction in g l u c o s e , e x c l u s i o n o f o x y g e n , or a d d i t i o n of inhibitors o f o x i d a t i v e p h o s p h o r y l a t i o n o r o f the s o d i u m / p o t a s s i u m p u m p m a r k e d l y e n h a n c e b o t h g l u t a m a t e and N - m e t h y l - D - a s p a r t a t e ( N M D A ) toxicity 14"28. A r e d u c t i o n in m i t o c h o n d r i a l o x i d a t i v e m e t a b o l i s m a c c o m p a n i e s n o r mal aging 35, and m i g h t t h e r e f o r e be e x p e c t e d to i n c r e a s e susceptibility to e x c i t o t o x i n lesions. O n the o t h e r h a n d , r e d u c t i o n s in n u m b e r s of N M D A r e c e p t o r s 3°, increases in k y n u r e n i c acid c o n c e n t r a t i o n s 27 and r e d u c t i o n s in striatal e x c i t a t o r y s y n a p t i c i n p u t all o c c u r with n o r m a l aging ~3. E a c h of t h e s e factors m i g h t be e x p e c t e d to r e d u c e susceptibility to striatal e x c i t o t o x i n lesions with increasing age. In t h e p r e s e n t study we e x a m i n e d t h e s e issues by c o m p a r i n g the e x t e n t o f n e u r o n a l d e g e n e r a t i o n in-

Correspondence: M.E Beal, Neurology Research 4, Massachusetts General Hospital, Boston, MA 02114. U.S.A.

277 measured by H P L C with 16 channel electrochemical detection 25. Protein m e a s u r e m e n t s were made on the sonicate using a fluorometric assay m. Neurochemical m e a s u r e m e n t s in the striatum were compared with the unlesioned (control) side and expressed as percentagcs of control. Wc have prcviously found that the right (control) values do not differ from saline-injected controls 3. Comparisons were made using non-paired Student's t-test (two-tailed) or one way analysis of variance. Results are expressed as the mean -+ S.E.M. Three animals with N M D A lesions were examined histologically in each of the 1- and 4-month-old groups. At l0 days, animals were deeply anesthetized with pentobarbital and perfused with ice-cold saline, followed by phosphate-buffered 4% paraformaldehyde, pH 7.3. Brains were post-fixed for 24 h and then transferred to 30% glycerol/0.1 M Tris-buffered saline, pH 7.4 cryoprotectant solution. Brains were sectioned at 50 micron intervals using a sledge microtome and stained for Nissl substance (thionin), and glial fibrillary acid protein (GFAP) immunoreactivity as previously described3.

TABLE I

Effects of aging on neuropeptides in cerebral cortex and hippocampus All values are pmol/mg protein. There were no significant differenccs by A N O V A .

Age

Somatostatin

Neuropeptide Y Substance P

Cerebral cortex 1 month 4 months 20 m o n t h s

0.73 -+ 0.05 0.65 -+ 0.03 0.61 -+ 0.04

0.91 -+ 0.l)8 0.96 +- 0.ll7 0.90 +- 0.08

0.042 +_ 0.003 0.034 +- 11.002 0.033 -+ 0.1)03

Hippocampus lmonth 4 months 20 m o n t h s

1.36 -+ 0.12 1.39 -+ 0.15 1.22 -+ 0.08

2.18 -+ 0.13 2.07 + 1/.13 1.90 + 0.07

0.094 -+ 0.008 0.085 + 0.007 0.064 + 0.005

RESULTS

The quinolinic acid lesions resulted in a significant (P < 0.001) 57% depletion of SPLI in the 1-month-old animals; whereas in the 4- and 20-month-old animals the depletion, although significant (P < 0.05), was only 2 0 25% (Fig. l). Similarly, significant (P < 0.01) G A B A depletions of 47% were only seen in the 1-month-old animals, whereas the depletions in the 4- and 20-month-old animals were not significant. SLI and NPYLI were unaffected by any of the lesions. Dopamine concentrations, used as a marker of striatal afferents, were unaffected,

]

Sornatostatln-like Irnrnunoreactivity

]

Neuropepttde Y-like Imrnunoreactivity

]

Substance P-like lrnmunoreactivi~

]

GAFtA

150

although 3,4-dihydroxyphenylacetic acid ( D O P A C ) concentrations were significantly increased in all groups (data not shown). Uric acid concentrations were the most sensitive neurochemical markers of the lesions. In the 1-month-old animals, they were significantly increased from 3.2 + 0.2 ng/mg protein on the unlesioned side to 21.7 -+ 5.1 on the lesioned side (P < 0.001). In the 4and 20-month-old groups, respectively, they increased from 3.2 ± 0.2 to 11.2 -+ 2.3 ng/mg protein (P < 0.01), and 4.3 -+ 0.3 to 19.8 -+ 5.2 ng/mg protein (P < 0.01). Xanthine concentrations showed no significant changes. The overlying cerebral cortex showed smaller significant (P < 0.01) increases in uric acid from 4.6 ± 0.5 to 10.4 -+ 2.1, 5.0 ± 0.4 to 10.6 -+ 1.6 and 5.3 ± 0.7 to 15.7 ± 3.3 ng/mg protein in the 3 age groups, respectively. There were no significant changes in SLI, NPYLI or SPLI in either cerebral cortex or hippocampus amongst the various age groups (Table I). There were, however,

SubmIonceP-Irke Irnrnunoeeoctivib] v--4 0 ¢--

100

"2.7 . . . . . . . . . . . . . .

•

C O

*

~,

I

50

1 Month

4 Months

20 Months

Fig. 1. Effects of quinolinic acid (240 nmol) striatal lesions on SPLI and G A B A in 1-, 4- and 20-month-old animals. SLI and NPYLI showed no significant changes. The SPLI and G A B A depletions were significantly greater in the l-month-old animals. *P < 0.05, **P < 0.0l.

100 ......................................................................................

50

i

1 Month

4 Months

12 Months

Fig. 2. Effects of N M D A (200 nmol) striatal lesions on SPLI and G A B A in 1-, 4- and 12-month-old animals. SPLI and G A B A reductions wcrc greater in the 1-month-old animals. *P < 0.05, **P < 0.01.

278 sions in 1-, 4- a n d 1 2 - m o n t h - o l d animals. T h e e x t e n t of !iii~ilia!!~¸!¸

ill

; ! )iiiii?i~

the SPLI a n d G A B A d e p l e t i o n was again greater in the 1 - m o n t h - o l d a n i m a l s t h a n in the 4- or 1 2 - m o n t h - o l d animals (Fig, 2). T h e SPLI a n d G A B A were d e p l e t e d ap-

!:i

proximately

50%

in

l-month-old

animals,

35%

in

4 - m o n t h - o l d a n i m a l s , a n d 25% in 1 2 - m o n t h - o l d a n i m a l s , Histologic e x a m i n a t i o n with G F A P staining s h o w e d that N M D A lesions in the 4 - m o n t h - o l d g r o u p were characterized by a m o r e i n t e n s e local r e s p o n s e , as c o m p a r e d with a larger, m o r e diffuse lesion seen in the 1 - m o n t h old g r o u p (Fig. 3). In 1 - m o n t h - o l d a n i m a l s , uric acid c o n c e n t r a t i o n s s h o w e d d o s e - d e p e n d e n t significant ( P < 0.01) increases with i n c r e a s i n g doses of N M D A . A dose of 12.5 n m o l resulted in an increase fl'om 6.5 ± 0.7 to 16.4 ± 1.7 n g / m g p r o t e i n , a dose of 50 n m o l from 5.2 -+0.5 to 19.3 ± 4.8 ng/mg p r o t e i n a n d 100 n m o l from 6.3 ± 0.6 to 25.8 ± 4.2 n g / m g p r o t e i n (n = 8 in each group). In saline controls there was n o significant difference bet w e e n the 2 striata (5.8 ± 0.4 versus 7.7 ± 0.8 ng/mg protein). DISCUSSION i~i~!iiii!i!i,

•i~~(i~i~!!~!#~

Fig. 3. GFAP staining of representative NMDA lesions at 1 month (top) and 4 months (bottom) of age. The NMDA lesions in the 4-month-old animals produced smaller and more sharply circumscribed lesions than the larger, more diffuse lesions seen in the 1-month-old animals.

significant increases in b o t h k y n u r e n i n e a n d k y n u r e n i c acid in frontal cortex with aging, as well as increases in t r y p t o p h a n in the h i p p o c a m p u s . T h e r e was a t r e n d towards i n c r e a s e d t r y p t o p h a n in the cerebral cortex, b u t it was n o t significant. A follow-up e x p e r i m e n t e x a m i n e d N M D A striatal le-

Excitatory a m i n o acids m a y play a role in n e u r o n a l d e a t h in a variety of n e u r o l o g i c illnesses j5. T h e most c o m p e l l i n g e v i d e n c e has l i n k e d N M D A receptors to n e u ronal d e g e n e r a t i o n in b o t h ischemia a n d hypoglycemia. In e x p e r i m e n t a l m o d e l s of these c o n d i t i o n s n e u r o n a l d a m a g e can be b l o c k e d with N M D A antagonists. T h e r e is also e v i d e n c e that H D m a y be m e d i a t e d by a n N M D A i n d u c e d excitotoxic process. T h e r e is p r e f e r e n t i a l loss of N M D A receptors in H D striatum, a n d a n i m a l m o d e l s with N M D A agonists closely m i m i c the n e u r o c h e m i c a l features of HD 3'4"36. A l z h e i m e r ' s disease m a y also involve excitotoxic m e c h a n i s m s ~. H D characteristically has its o n s e t in m i d d l e age, w h e r e a s the i n c i d e n c e of A l z h e i m e r ' s disease increases progressively with increas-

TABLE ll Effects of aging on monoamines and related compounds in cerebral cortex and hippocampus All values are ng/mg protein except kynurenic acid which is pg/mg protein. N.D., not determined. Age

N o r e p i n e p h r i n Guanosine e

Tyrosine

Dopamine

Tryptophan

Kynurenine

Kynurenicacid

Cerebral cortex 1 month 0.83 ± 0.10 4 months 0.85 -+ 0.08 20 months 1.06 ± 0.06

41.1 ± 2.9 36.6 ± 2.2 41.3 ± 2.4

26.5 ± 2.0 24.4 ± 1,8 26.2 +- 2.1

2.26 ± 0.57 1.85 ± 0.49 2.89 ± 0.68

14.6 + 0.9 15.7 ± 1.2 16.5 -+ 1.6

0,31 -± 0.01 0.53 ~: 0,27 0.54 ± 0.11"

7.57 ± I).55 9:78 ± 0,99* 12.5 -+ 1.62"

Hippocampus 1 month 4 months 20 months

55.5 -+ 2.0 73.0 + 2.9 73.3 ± 6.7

25.2 -+ 2.1 29.2 +- 1.4 33.0 ± 1.9

I).30 + 0.05 0,25 + 0.02 I).25 ± 0.03

12.4 -+ 1.7 19.5 + 0.6* 20.0 ± 11.9"

0.21 + 0.05 0,23 ± 0.01 0.24 ± 0.02

N.D.

1.11 +- 0.06 1.07 -+ 0.07 1.42 -+ 0.1l

P < 0.05 as compared with 1-month group.

N.D. N.D.

279 ing age. In the present study we therefore examined the effects of aging on excitotoxin lesions at several different time points in rats. Equivalent injections of quinolinic acid were made in the striatum in 1-, 4- and 20-month-old rats. The extent of the lesions was determined by measuring substance P and G A B A concentrations. The 1-month-old animals showed significantly greater depletions of both substance P and G A B A than the two older age groups, which were equivalent in the extent of the reductions. N M D A striatal lesions were also attenuated both neurochemically and histologically in 4- and 12-month-old animals as compared with 1-month-old animals. The older animals therefore were less susceptible to striatal excitotoxin lesions than younger animals. Previous studies of susceptibility to excitotoxin lesions have been largely confined to neonatal animals. Campochiaro and Coyle J2 showed that susceptibility kainic acid striatal toxicity is first elicitable 7 days after birth, and then increases progressively to adult levels by 21 days after birth. The increase in susceptibility parallels the development of glutamatergic innervation of the striaturn. Quinolinic acid also showed no toxicity in 7-dayold rat pups, however ibotenic acid is toxic at this age~8' 31 McDonald et al. showed that N M D A lesions in 7-day-old rats were 21-fold larger than those seen in adult (3-month-old) rats 2~'. In rat hippocampus, N M D A receptors peak at day 7 followed by a decrease to adult levels by postnatal day 1314"34 In older animals there are a variety of processes which could affect susceptibility to excitotoxin lesions. In two strains of mice reductions in N M D A receptors have been shown with aging 3°. N M D A receptors also decrease with aging in rat striatum and cerebral cortex 23'32. Electrophysiologic studies have shown that N M D A receptor antagonists block visual responses of cortical neurons more effectively in kittens than in adult cats 36. These electrophysiologic changes are accompanied by a reduction in the number of N M D A receptors after 4 weeks of age 9. Another potential mechanism is age-related increases in kynurenic acid, an endogenous antagonist of excitatory amino acids 27. A further protective mechanism which may occur with aging is a reduction in cortico-striatal glutamatergic input. Striatal excitotoxin lesions with a variety of excitatory amino acid receptor agonists are dependent on intact cortical innervation. With aging, glutamate release from slices of frontal cortex is unchanged in response to potassium, however glutamate content is reduced 17% 16. Electrophysiologic studies have shown decreased striatal neuronal excitability in aged (24-month-old) rats as compared with younger age

groups 13. Similar findings were reported in neocortex 2. There is also a reduction in the number of dendritic spines on striatal neurons 2°. In cats there is a decrease in synaptic density in the caudate nucleus in both middle-aged and aged animals, suggesting a decrease in the number of excitatory synaptic inputs 24. The present results indicate that factors mitigating N M D A excitotoxin lesions occur with normal aging in rat striatum. We found an increase in kynurenic acid content with normal aging, confirming a previous report 27. Whether this increase with aging exerts a neuroprotective effect remains to be determined, It is likely that both reduced numbers of N M D A receptors and reduced excitatory corticostriatal synaptic input play a role. The present study shows that marked dose-dependent increases in uric acid concentrations occur at 7 days after a striatal excitotoxin lesion. Marked increases also occur with 7 day cortical excitotoxin lesions; however concentrations are normal at 3 and 6 months after striatal lesions 3. The cause of these increases is unclear. Excitotoxin lesions by depleting intracellular ATP 8 may change the N A D H / N A D + ratio, which can regulate xanthine oxidase activity 21. Increased uric acid is observed in ischemic brain tissue for 48 h after middle cerebral artery occlusion 22. Increased uric acid could also arise from degradation of adenosine, which can be released by excitatory amino acids 2~. There are few studies of neuropeptide changes which occur with normal aging. In the present study there were no significant changes in SLI, N P Y L I or SPLI concentrations with normal aging in either overlying cerebral cortex or hippocampus. A previous study also failed to show changes in somatostatin or substance P in cerebral cortex with normal aging 1~. In aged primates we have observed decreases in somatostatin and neuropeptide Y in cerebral cortex, but only in the 31- to 34-year-old age group which also exhibits both cognitive impairment and frequent senile plaques 7. The present results argue against an age-dependent increase in susceptibility to N M D A excitotoxin lesions being responsible for the delayed onset of HD. Whether the increasing prevalence of Alzheimer's disease with advancing age relates to changes in susceptibility to excitotoxin lesions remains unanswered, since it is possible that a n o n - N M D A excitotoxin mechanism may be involved.

Acknowledgements. The secretarial assistance of Sharon Melanson is gratefully acknowledged. Supported by NIA Grant IP05AG(15134 and NINCDS 16367.

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