Brain Research, 550 (1991) 231-238 © 1991 Elsevier Science Pubhshers B V 0006-8993/91/$03 50 A DONIS 000689939116626T

231

BRES 16626

Sham transplantation protects against 6-hydroxydopamine-induced dopaminergic toxicity in rats" behavioral and morphological evidence S e r g e P r z e d b o r s k l 1'2, M a r c L e v l v l e r 3, V l a d l m l r K o s t l c 1 , V e r n l c e J a c k s o n - L e w i s 1 , A l f r e d

D o l h s o n 1, D o n M Gash 4, Stanley F a h n 1 and Jean Lud C a d e t 1 1Laboratory of Prechmcal Neuroscwnces, Columbia Umversay, College of Physicians and Surgeons, New York, New York 10032, (U S A ), Departments of 2Neurology and of 3Neurosurgery, H6pital Erasme, Umverstt~ Llbre de Bruxelles, B-1070 Brussels (Belgmm) and 4Department of Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 (U S A ) (Accepted 18 December 1990)

Key words Parkmson's disease, 6-Hydroxydopamme, Stnatum, Rotation behavior, Transplantatmn, Protection, Receptor binding autora&ography, [3H]SCH 23390, [3H]Mazmdol

Admmlstratmn of the neurotoxm 6-hydroxydopamme (6-OHDA) to rat brain causes biochemical and neuroanatomlcal changes to the nlgrostnatal dopammerglc pathway similar to those observed m Parkmson's disease (PD) Although the cause of PD is unknown, It has been hypothesized that the neurodegeneratlve changes seen m PD might result from exposure to a neurotoxm Therefore, strategies for hmmng neurotoxm-mduced dopammerglc damages, hke those caused by 6-OHDA, may be of both chmcal and basic interest Accordingly, we tested the ability of both fetal neural (stnatum) and fetal non-neural (hver) tissue implants to protect the rat stnatum against the toxic effects of a subsequent mtrastnatal mlectmn of 6-OHDA Non-grafted rats (lesmn only) showed amphetamine-reduced rotational behavmr and a decrease in stnatal [3H]mazmdol-labeled dopamme uptake sites after 6-OHDA rejection In contrast, the animals grafted with stnatum or hver showed no behavmral or bmchemlcal changes Interestingly, sham-transplanted control ammais were also protected against the 6-OHDA-mdueed toxicity These results suggest that the resistance of the dopammerglc system against 6-OHDA neurotoxloty observed m grafted and sham-transplanted ammals is hkely to be related to the surgical procedure itself This observatmn points to a possible role for surgery-related events in the clinical improvement described in PD patients who underwent intracerebral transplantation INTRODUCTION Parklnson's disease ( P D ) is a c o m m o n degenerative d i s o r d e r of the central nervous system 36 characterized p r l m a n l y by a progressive loss of dopamlnerglc cells in the substantla nigra pars c o m p a c t a (SNc) 2'36 T h e observatmn of a significant decrease in the stnatal d o p a m i n e ( D A ) levels associated with a loss of dopamlnergic neurons led to the introduction of L - D O P A in the t r e a t m e n t of this disorder 36 H o w e v e r , long-term t h e r a p y of P D patients with D A r e p l a c e m e n t is associated with a n u m b e r of m o t o r and psychiatric comphcatlons 1836 Thus, recent efforts have been centered on developing new therapeutical strategies capable of arresting or i m p e d i n g the progress of the disease 19'27"36 40 A l t h o u g h the exact etiopathogenesis of PD remains u n k n o w n , the possibihty of a n e u r o t o x i n - m e d i a t e d mechanism for the neuronal d e g e n e r a t i o n in P D has been p r o p o s e d H'36 T h e hypothesis of neurotoxic damage was strengthened by the finding that a simple organic molecule, 1 - m e t h y l - 4 - p h e n y l - l , 2 , 3 , 6 - t e t r a h y d r o p y n d i n e (MPTP),

can induce selective lesions of d o p a m m e r g i c cells in the SNc in several animal species and h u m a n subjects and causes a clinical s y n d r o m e indistinguishable from P D 34'49 Earlier, U n g e r s t e d t s2 had d e m o n s t r a t e d that another c o m p o u n d , 6 - h y d r o x y d o p a m i n e ( 6 - O H D A ) , was selectively toxic to catecholaminergic n e u r o n s T h e specificity of that c o m p o u n d for d o p a m i n e r g l c neurons is e n h a n c e d by selectively blocking its u p t a k e into n o r a d r e n e r g i c neurons 8 These observations stimulated the idea that PD might result from single or multiple exposures to either exogenous or e n d o g e n o u s toxic agent(s) 11 19,5o, perhaps chemically r e l a t e d to the a b o v e - m e n t i o n e d c o m p o u n d s A l t h o u g h the relevance of drug-reduced animal m o d e l s of PD is d e b a t a b l e 34, it is r e a s o n a b l e to predict that the elucidation of m o l e c u l a r m e c h a m s m s that could prevent or i m p e d e n e u r o t o x l n - l n d u c e d d a m a g e s to the D A system might help to b e t t e r u n d e r s t a n d the etlopathogenesis of P D A similar a r g u m e n t can be m a d e m the case of animal m o d e l s of o t h e r n e u r o d e g e n e r a t l v e disorders Thus, it was shown that the c o n c o m i t a n t transplantation of n e o n a t a l striatal cells could p r e v e n t the toxic

Correspondence S Przedborskl, Black Bldg, RM ~ 307, 630 West, 168th Street, Columbia Umverslty, College of Physicians & Surgeons, New York, N Y 10032, U S A

232 e f f e c t s o t m t r a s t r l a t a l m j e c t t o n o f t h e exc~totoxlns kam~c acid a n d q u m o h m c acid ( Q A ) 51 W e h a v e s u b s e q u e n t l y r e p o r t e d t h a t t h e m t r a s t r m t a l t r a n s p l a n t a t i o n o f fetal s t r l a t u m p r i o r to t h e mject~on o f Q A p r o t e c t s t h e a n i m a l s agamst the strtatal rons ~

d e l e t e r i o u s e f f e c t s o f th~s e x c l t o t o x m o n

acetylchohne- and

enkephahn-contalnlng neu-

In t h e p r e s e n t s t u d y , w e t e s t e d t h e specificity o f

t h e s e o b s e r v a t i o n s by a s s e s s m g t h e a b t h t y o f fetal grafts to p r o t e c t rat s t n a t a l D A effects of OHDA

a

subsequent

t e r m i n a l s a g a m s t t h e toxtc mtrastrtatal

rejection

of

6-

Hereto, we report both behavtoral and morpho-

logical e v i d e n c e f o r t h e r e s i s t a n c e o f t h e n t g r o s t r t a t a l D A system to 6 - O H D A

m rats w h t c h h a v e u n d e r g o n e ttssue

g r a f t i n g o r s h a m g r a f t i n g 12 d a y s p r i o r t o m t r a s t r t a t a l injection of the neurotoxln

MATERIALS AND METHODS Ammals Adult female Long-Evans rats weighing 200-220 g (Charles River) at the begmmng of the experiment were used The ammals were housed 5 per cage m a temperature-controlled room with a 12-h hght-dark cycle and were given free access to food and water ad hbltum Prior to all experiments, rats were randomly d m d e d into 4 groups strlatum-transplanted, hver-transplanted, sham-transplanted and lesion only Surgery The ammals were anesthetized with chloral hydrate (400 mg/kg i p ) and were placed m a stereotaxlc frame with the mouth bar set at - 3 3 mm The skin was opened and two burr holes were made with a small drill on the right skull at the following coordinates bregma A/P 0 0 mm, LAT - 2 0 mm and A/P 1 6 mm, LAT - 2 0 mm For donor tissue, pregnant Long-Evans rats were sacrificed by decapitation and their fetuses (aged 15-17 days of gestaUon) were rapidly removed and put on ice For the stnatum-tran~planted rats, fetal stnata were d~ssected out under a m~croscope and were maintained in sterile, ice-cold calcmm-free, magnesmm-free buffer (CMF 0 15 M NaCI. 0 008 M Na2HPO4, 0 0027 M KCI, 0 0015 M KHPO4, 0 026 M NaHCO 3, pH 7 4, sterilized by autoclave) containing 0 1% glucose, 50 ~g/ml gentamycln, 2 5 /xg/ml funglzone)38, until transplantation (less than 2 h) 47 Fetal strlata were minced m small pmces using mlcrodlssection scissors and aspirated mto a sterile 18-gauge needle wMch was attached to the stereotaxic apparatus The needle was lowered into the host brain and a bulk of 1-2 mm 3 (~2/~1) of tissue was inserted per site into the strmtum over a period of 30 s The needle was left in place for an addmonal 2 mln 22 A first bulk of fetal stnatum (graft No 1) was implanted into the stnatum at the following coordinates bregma A/P 0 0 mm, LAT - - 2 0 mm, D/V -5 5 mmn, and a second bulk (graft No 2) at bregma A/P 1 6 mm, L A T - 2 0 mm, D/V -5 5 mm according to the Atlas of Paxlnos and Watson~ For the hvertransplanted rats, small blocks of fetal liver were dissected out, prepared and implanted into the strtatum of the host using the same technique, volume and coordinates For sham-transplanted rats, ammals underwent the same surgical procedure but 2/~1 of CMF was injected instead of fetal tissue At the end of the surgical procedure, the skin was closed and the rats were returned to their cages Lesion Twelve days after surgery, the animals were again anesthetized and placed m the stereotaxlc frame as described above A 2 5-ktg//~l solution of 6-OHDA was prepared with ascorbtc acid (0 2 mg/ml) added to prevent 6-OHDA auto-oxidation Th~s solution was kept

on ice in a hght-proof tube until used A partial umlatcral lesion ~t the strlatal dopamlnerglc svstem on the right side ~as induced by injecting 2/d of 6-OHDA at I ul/mtn using a 5.1 Hamilton syrmgL The dosage and the rate o1 injection were based on our prc*~ous study using mtrastrlatal injection of 6-OHDA" ~'' qhe rejection ol 6-OHDA was made at a site eqmdlstant and ~) 5 mm lateral to the two grafts b r e g m a A / P 0 8 m m , L A T - 2 5 m m D / V - ~ 5 m m For lesion only rats. non-grafted animals received onl~¢ the mtrastrmtal injection o! 6-OHDA at the same coordmatts Rotational beha~ tor All animals were tested for rotational behavior Induced by both 0 5 mg/kg apomorphme (s c ) and 3 mg/kg amphetamine 0 P )~ ~0at the beginning of the experiment (basehne), 8 days after transplantahon (posttransplant), and 15 and 21 days after lntrastnatal rejection of 6-OHDA (postleston) These doses o! apomorphme and amphetamine have been used in our laboratory 7~ ~o and caused consistent circling behavior in rats with prior lesion ot the mgrostnatal DA pathway Rats were allowed a two-day rest period between each apomorphme and amphetamine challenge Rotational behavior was measured in mechamcal rotometers52 The number of net rotations (clockwise-counterclockwise) was recorded at Intervals of 10 mln for 60 rain Tissue preparation Four days after the last behavioral test (1 e 25 days postlesion), rats were sacrificed by decapitation The brains were rapidly removed, rinsed in ice-cold saline, frozen by slow immersion in lsopentane cooled on dry ice and then kept at -80 °C until sectioned Coronal sections (20/~m) were cut m a cryostat at -20 °C. thaw-mounted onto gelatin-coated glass slides (2 sections per shde), dried at 4 °C under negatwe pressure for 2 h and then kept in a sealed shde box at -20 °C until use (at least 72 h) A total of 20 rats were used for the autoradlographlc studies (5 per group) Receptor bmdmg assay~ DA D 1 receptors were labeled with [3H]SCH 23390 (71 3 Cl/mmol, NEN, MA) according to the method described by Dawson et al 14 with minor modificat~ons 7 The sections were premcubated for 10 mln at 25 °C in Tns-HCI (pH 7 4) containing 120 mM NaCI 5 mM KCI 2 mM CaCI2, 1 mM MgCI 2 before incubation (25 °C, 60 mm) in the same buffer with 1 25 nM of [3H]SCH 23390 After incubation, the sections were washed 2 x 5 mm m ice-cold buffer, dipped m ice-cold distilled water and rapidly dried under a stream of cold air Non-specific binding was defined as that occurring in the presence of 1 ktM of unlabeled R(+)-SCH 23390 (RBI, MA) and accounted for less than 10% of the total binding In order to better quantify the effects of 6-OHDA on DA terminals, we labeled DA uptake sites with [3H]mazmdol (15 0 C1/mmol, NEN, MA) according to the methods described by Javitch et al 2~ The sections were premeubated (4 °C, 15 mm) in 50 mM Trls-HCI (pH 7 9) containing 120 mM NaCI, 5 mM KCI They were then incubated (4 °C, 60 mm) in 50 mM Tns-HCt (pH 7 9) containing 300 mM NaCI, 5 mM KCI, 300 nM desmethyhmlpramme (DMI) with 15 nM [3H]mazmdol DMI was used to block binding to noreplnephrlne uptake sitesz8 After incubation, the sections were washed 2 × 3 mm in ice-cold buffer, dipped in ice-cold distilled water and rapidly dried under a stream of cold air Non-specific binding was defined as that occumng m the presence of 30/xM of benztroplne and accounted for 20% of the total bmdmg Dried labeled sections were placed in hght-proof X-ray cassettes along with trmum plashc standards ([3H]Mtcro-scales, Amersham) and were then apposed to trmum sensmve film (Hyperfilm, Amersham) for 10 days at 25 °C for [3H]SCH 23390 and 20 days at 4 °C for [~H]mazmdol The films were then developed with D-19 developer (Kodak) and fixed with Kodak Rapid Fix The optical densities of the developed autoradlographJc films were quantified using a RAS-3000 computerized ~mage analysis system (Amersham) Optical densmes were converted to fmol of radlohgand bound pcr nag of tissue using a standard curve ([~H]mtcro-

233 small dtfferences m behavtor were observed between strlatum-, hver- and sham-transplanted antmals, none of these reached slgmficance On the other hand, apomorphme administration d~d not cause stgnlficant changes m the number of net rotations in any of the 4 groups of rats at either 15 or 21 days postleston compared to the posttransplant act]vtty (Fzg 1B)

scales) Regmnal distribution of binding for both [3H]SCH 23390 and [3H]mazmdol was quantified m 8 different coronal planes corresponding A/P from bregma 2 2 mm (No 1), 1 7 mm (No 2), 12ram(No 3),07ram(No 4),02mm(No 5),-03ram(No 6), - 0 8 mm (No 7 ) , - 1 3 r a m ( N o 8) from lesion o n l y ( n = 5), stnatum-transplanted (n -- 5), hver-transplanted (n = 5) and sham-transplanted (n = 5) rats The regions of interest were defined according to Paxmos and Watson 41 and included olfactory tubercule, nucleus accumbens, caudate-putamen complex Each of these regions were outhned with a hand-hold cursor The radioligand binding assays were carried out on adjacent sections of the same brain region from each animal For each coronal plane, readings from at least 4 separate sections were obtained and averaged for total and non-specific binding

[3H]Mazmdol-labeled DA uptake sites In lesion only antmals, a marked decrease m [3H]mazmdol binding, that ranged from -62 to - 8 9 % , was observed through strlatal planes Nos 1-7 on the lesioned side compared to the contralateral non-lesioned szde

Htstology After autoradlographic studies, representative sections were stained for Cresyl violet Additional sections, which have not been used for the receptor binding assays, were stained for Cresyl violet, hematoxdm and eosln as well as lmmunostamed for the ghal fibriilary acidic protein (GFAP) The rabb]t antI-GFAP was a gift of Dr J Goldman (Department of Neuropathology, Columbia Umversity, NY) and Its charactenstlcs 0 e sensmvity and specificity) have been reported elsewhere z3

A.

Stattstwal analysts One-way analysis of variance (ANOVA) was used to assess the effects of the grafts on the unilateral lntrastnatal 6-OHDA-mduced lesmn, on the amphetamine- and apomorphine-mduced rotations and on the concentrations of radlohgands bound in the different strlatal planes In all cases, Scheffe F-test was used for posthoc comparison of the means The null hypothesis was relected at the 0 05 level All value~ are expressed as means + standard error of the means (S E M )

Post-Transplant 15 Days Post.Lesion 21 Days Post-Lesion

300-

i ¢n

200-

E *~

100"

~k /t.

Z

-100

RESULTS

Lesion Only

Rotattonal behavtor When tested at 8 days posttransplantatmn, none of the 3 grafted groups (stnatum, hver or sham) displayed any change m amphetamine- or apomorphme-mduced rotation behavior m companson to basehne (data not shown) 6-OHDA-mduced strtatal lesions resulted in robust net amphetamme-mduced rotations lpstlateral to the lesioned side m the non-grafted animals (lesmn only) at 15 days (+260 4 + 52 8 turns/h) and at 21 days (+286 6 + 34 1 turns/h) postlesion (Fig 1A) In contrast, no slgmficant change in the number of net rotations was observed in strlatum-transplanted animals at etther 15 days (-2 5 + 7 8 turns/h) or at 21 days (-6 0 + 8 2) postleslon compared to the posttransplant actlvtty (Ftg 1A) There was a small increase m net rotations lpstlateral to the lesioned side m the hver-transplanted (15 days = +35 4 + 18 9 turns/h and 21 days = +35 2 + 25 4 turns/h) and the sham animals (15 days = + 15 0 + 18 2 turns/h and 21 days = + 112 + 104 turns/h) compared to the posttransplant activity (F~g 1A) Rotational actwmes recorded m grafted animals (smatum, hver and sham) were all significantly lower (P < 0 001) than those observed m the lesion only animals (Fig 1A) Although

[] • •

Strlatum

Liver

Sham

6O

B.

[]

40

3

Post.transplant 15 days Post-lesion 21 days Post-Leston

20

2.

T TITI

Z -20

-40

Lesion Only

Strlatum

Liver

Sham

F]g 1 Amphetamine- (A) and apomorphine-lnduced (B) rotaUonal behaviors m rats tested 8 days posttransplant or postsham surgery and 15 and 21 days post 6-OHDA-mduced lesion Ammals were implanted with either fetal stnatal tissue (stnatum), fetal hver tissue (liver) or were injected with a slmdar volume of calcmm-free, magnesmm-free buffer (sham) A fourth group of rats had only mtrastnatal rejection of 6-OHDA and no graft (lesion only) Bars represent mean number of net turns per h (clockwise-counterclockwise) corrected for baseline for 5 animals per group and the error bars represent the S E M Positive values represent net rotatmns lpsllateral to the lesioned side and negative values contralateral to the lesioned side Statistical differences between the means were tested by a one-way ANOVA followed by a Scheffe F-test * = slgmficantly different from controls (lesion only) at P < 0 001

234 (Figs 2 and 3) Topographically, slgmflcant changes m b m d m g e x t e n d e d from 1 4 mm anterior to 1 6 mm posterior to the site of the 6 - O H D A rejection In contrast, only a small reductmn m blndmg ( - 3 0 % ) was seen m the most posterior strmtal plane (No 8) which is located about 2 1 mm posterior to the site ot mlectmn (Fig 2) In a d d • o n , a mean 20% reduction m [~H]m a z m d o l b m d m g at the level of the nucleus accumbens and olfactory tubercule was found (F~g 3) In grafted ammals, no slgmficant decreases m [~H]mazlndol b m d m g were found on the lesmned side c o m p a r e d to the contralateral non-lesioned side in any of the s t n a t a l planes studied (Figs 2 and 3) In addition, the b m d m g of [3H]mazlndol on the lesmned side was stgmficantly higher m grafted ammals (strmtum, hver, sham) c o m p a r e d to non-grafted ammals (leston only) in all s t n a t a l planes except for plane No 8 (Fig 2) Furthermore, there were no significant differences m the m a g m t u d e of bmdlng changes between stnatum-transp l a n t e d , hver-transplanted or sham ammals and no slgmficant &fference m [3H]mazlndol bmdlng was observed on the contralateral non-lesioned side among the 4 different groups

[3H]SCH 23390 labeled DA D~ receptors Because rotational b e h a v m r m d u c e d by a m p h e t a m m e can be modtfied by the status of the d o p a m m e r g l c postsynapt~c structures 43 and because intrastrtatal inJection of 6 - O H D A might have induced non-specific tissue necros~s 7 wtthln that structure, we also m e a s u r e d [3H]SCH 23390-labeled striatal D A D l receptors ~ D A D~ receptors have been localized to strtatai neurons by both fluorescent hgands 4 and m SltU h y b n d l z a t m n ~5 Similar concentrations of [3H]SCH 23390 b m d m g were o b s e r v e d on the lesmned side and the contralateral non-lestoned side in all stnatal planes for each animal T h e r e were no slgmficant differences m [3H]SCH 23390 b m d l n g a m o n g the different groups of animals and none of the a u t o r a d m g r a m s g e n e r a t e d & s p l a y e d areas of low b m d m g that mtght c o r r e s p o n d to a loss of strtatal neuronal elements due to tissue necrosis

Htstology N e e d l e tracts were ldenufied easily in sections stained w~th Cresyl w o l e t o r h e m a t o x y h n and eosln (not shown) T h e tracts traversed the overlaymg cerebral cortex and corpus callosum to reach the s t n a t u m The tracts, mcludmg the region containing the graft, were surr o u n d e d by a thin GFAP-pOSltlVe cuff and a few G F A P - p o s i t t v e reactive astrocytes were tdentlfied wtthin the VlCtmty The grafts (fetal s t n a t u m or hver) a p p e a r e d as a healthy bulk o f tissue at the tip of the cannula tract without evidence of necros~s In several cases, a m o d e r -

o =_.X -

*

*

*

100' 80"

60' 40-

20 :

~

o

22

17

1

iil 0

0

0

[] []

Lesion Only Strlatum

Liver []

08

Sham

13

Coronal strlatal plane from B r e g m a ( m m )

Fig 2 Effect ot undateral mtrastrmtal injection of 6-OHDA on [~H]mazmdol-labeled dopamme uptake sites m grafted, shamgrafted and non-grafted rats Ammals were implanted with either fetal strlatal tissue (strmtum) fetal hver tissue (hver) or were injected with a similar volume of calcium-free, magnesmm-free buffer (sham) A fourth group of rats had only mtrastnatal rejection of 6-OHDA and no graft (lesion only) [~H]Mazlndol bmdmg was performed as described m the Mater•Is and Methods Bars represent the mean ratio ol [~H]mazmdol bmdmg (lesioned/ non-lesmned × 100) and the error bars represent the S E M for 5 animals per group Statistical ddferences between the means were tested by a one-way ANOVA followed by a Scheffe F-test * = slgmficantly different from controls (lesmn only) at P < 0 01, * = slgmflcantlv different from controls (lesion only) at P < 0 001 ate m f l a m m a t o r y reaction could be o b s e r v e d with scattered spots of hemostderln p~gments w~thln the cannula tract and m the vlcmlty ot the graft H e m a t o x y h n and eosm stamlng showed that the n o r m a l t~ssue orgamzatlon of both the cerebral cortex and strlatum was well preserved, no areas of h e m o r r h a g e or necroms were Identified H o w e v e r , a mild g e n e r a h z e d • c r e a s e m strmtal GFAP-posltlVe l m m u n o r e a c t w l t y was o b s e r v e d on the stde of the lesmn c o m p a r e d to the contralaterai non-lestoned side A t higher magntficatlon, this increase m G F A P - l m m u n o r e a c t l v l t y was identified as a mild ghal reactmn characterized by an m c r e a s e d n u m b e r of G F A P positive thick tortuous fibers and a few heavdy labeled cell bodies (not shown) DISCUSSION In the present study, we c o n f r m that a partial unilateral lesmn of the strmtal D A system can be reduced by the &rect rejection of 6 - O H D A into the strmtum 7 ~0 29 30 All of the rats which received only the m t r a s t n a tal rejection of 6 - O H D A (lesion only) displayed a robust rotational behavior ipstlateral to the lesion after the adminlstratmn of a m p h e t a m m e Since m the present study, only a hmtted destruction of the s t n a t a l d o p a m i n ergtc system was o b s e r v e d (rangmg from 62 to 89%), we did not expect the d e v e l o p m e n t of postsynaptlc dopammerglc r e c e p t o r supersensmvlty on the stde of the lesion and consequently no significant a p o m o r p h m e -

235

Fig 3 Representauve color-codedtransformed autoradlograms showmg [3H]mazmdol(left column) and [3H]SCH23390 (right column) binding m coronal sections through the stnatum from non-grafted (A-C) and grafted (B-D) rats subsequently lesioned with an mtrastnatal mlectton of 6-OHDA, CPu, caudate-putamen nucleus, NAc, nucleus accumbens, OTu, olfactory tubercule The rank order of binding is dark-red > hght-red > yellow > green > blue > purple > white

reduced contralateral rotahons Dopamme receptor supersensmwty on the lesioned s~de is found only after lesions destroying more than 90% of the stnatal dopammerglc afferents 9'25 In addmon, the absence of significant apomorphme-mduced rotational behawor ~psdateral to the les~on md~cates that the postsynapt~c D A structures have not been altered by the mtrastnatal mject~on of 6 - O H D A 43 Morphologically, the type of lesion used in the present study mduces about an 80% reduction in [3H]mazlndol-labeled D A uptake sites which extends about 1 4 mm both anterior and postenor from the s~te of lnlecuon However, contrary to the observed extent of the stnatal lesion m the antero-postenor d~mens~on, the D A mnervatlon of the nucleus accumbens and olfactory tubercule was moderately affected Furthermore, h~stological examination showed a preservation of the normal stnatal Ussue orgamzat~on and the absence of necros~s around the site of reJection Slmdarly, [3H]SCH 23390labeled D 1 receptor generated autoradlograms did not reveal any defect which would correspond to the loss of stnatal neurons 3 7 T h i s ewdence of preservatton of strlatal structures is m contrast to the situation observed

after mtrastnatal mlect~on of exc~totoxms, which produces degeneration of neurons m the region of the rejected site, while spanng axons of passage and nerve termmals 39,46 In contrast, the mtrastnatal mlect~on of 6 - O H D A to grafted rats did not reduce either the behavioral or the morphological changes observed m non-grafted rats (lesion only) For instance, grafted rats did not show s~gmficant amphetamine-induced rotational behavior at either 2 or 3 weeks after the 6 - O H D A lesion nor did they exhibit a significant reduction m [3H]mazmdol binding In addmon, we did not find any slgmficant difference m the protectwe effect assocmted w~th transplantation between the rats grafted w~th fetal stnatal t~ssue and rats grafted w~th fetal hver tissue Although we have only tested these two types of fetal t~ssue, th~s finding indicates that the observed protection of stnatal D A system against 6 - O H D A toxicity might not be mediated by (a) grafted tissue-specific mechamsm(s) Furthermore, complete protection was also found in sham-grafted rats, th~s result supports the poss~bd~ty that the observed resistance to 6 - O H D A tOXlOty is hkely related to a host-medmted

236 response to the surgical p r o c e d u r e itself, regardless of the i m p l a n t e d tissue Various m e c h a m s m s m~ght underly this protective effect O n e posstbihty is that the surgical grafting p r o c e d u r e by itself might have sttmulated a local productlon of trophlc factors which protected the host's strtatum against the neurotoxln This hypothesis is s u p p o r t e d by previous findings showing that mechanical or chemical injury to adult rat brain ellotS the accumulation of n e u r o n o t r o p h t c acttv~ty m the les~oned area 2° 37 and that nerve growth factor reduces the toxicity of 6 - O H D A in rats ~6 In addition, it has been shown recently that e p i d e r m a l growth factor partially attenuates the loss of tyrosine hydroxylase lmmunoreactlvity which occurs after unilateral transection of the mgrostnatal p a t h w a y 5s Since the m a j o r i t y of neuronotroph~c growth factors are of n o n - n e u r o n a l o n g m s, the hypothesized factors might be derived from glial cells, a view which Is consistent with our findings of increased strlatal ghosls on the side of the surgery F u r t h e r support for ghal-derived growth factors is provided by studies which had r e p o r t e d p r o m o t i o n of neuronal recovery in the brain I ~2 and neuronal survival in cultures 24 by ghal cells Macrophages, which might have accumulated in the area of postsurglcal i n f l a m m a t o r y reaction or e n t e r e d the t~ssue through the surgery-induced dtsruptlon of the b l o o d brain barrier 44 4~ are a n o t h e r possible source of neuronotrophlc factor production 6 Alternatively, 6 - O H D A reduced deleterious effects are thought to be medmted by the production of oxygen-free radicals such as the superoxlde anion and hydrogen peroxide J2 i~ ~J which are toxic to cells el Partial protection agamst the effects of 6 - O H D A has been provtded by different scavengers of oxygen-based free radicals l° 12 It is thus possible that the cellular reactions occurring m the brain after the grafting p r o c e d u r e might have led to an increase in scavenging activity responstble for the resistance to the toxin This argument is m accord with the recent demonstration that nerve growth factor t r e a t m e n t , whtch Increases the activity of catalase, provides protection against the toxic

effects of hydrogen p e r o x i d e 26 Nevertheless, postoperattve brain e d e m a might have increased the fluid content of the grafted strtatum, which might have decreased the concentratton of the toxm and in turn its toxtcltV Fmall~ although the stability ot 6 - O H D A in VlVO has been recently d o c u m e n t e d 17, auto-oxidation ot 6 - O H D A mtght have been sttmulated by local factors related to the graftmg surgtcal p r o c e d u r e O u r present data do not identify the specific underlying protective mechantsms agamst 6 - O H D A - i n d u c e d lnjurtes to the nigrostrlatal d o p a m l n e r g l c system Nevertheless, when taken t o g e t h e r wtth our p r e w o u s demonstration that v e n t n c u l a r grafts of fat ttssue assoctated with the lntraventrlcular infusion of nerve growth factor is effective in reducing 6 - O H D A - l n d u c e d parkmsonism in rat 42, these data support the concept that the effects of transplantation could occur through processes other than a d~rect restoration or protection m e d t a t e d by the grafted tissues themselves These findings are of interest in connection with studies that have used grafts to treat parkinsonlan s y m p t o m s in h u m a n and e x p e r i m e n t a l animals27 48 Thus far, the identity ot the molecular mechanisms underlying the t h e r a p e u t i c effects of neuronal grafting has r e m a i n e d controversial ~ 54 The present data suggest that transplantation might act through a protective mechanism which prevents further degeneration of dopamlnergtc neurons, in accordance with the neurotoxlc hypothesis of PD 1~ 19 36 This argument raises the possibility of using transplantation 'early during the course of the illness because a smaller n u m b e r of cells would be available for 'rescue' after a long duration of PD F u r t h e r characterizatton of these protecttve tactors mtght open avenues for possible preventive intervention in n e u r o d e g e n e r a t i v e disorders Acknowledgements Serge PrzedborskJ Is Research Assistant of the National Fund for Soentlfic Research (Beigmm) and ts also supported by the Parklnson s disease Foundation (New York, U S A ) Vladlmlr Kostlc ts supported by a Fulbnght Scholarship We are very grateful to Prol J Brotchl (Dept of Neurosurgery U L B , Brussels) for his support to this project

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