Astrocytes in rat fetal cerebral cortical homografts following implantation into adult rat spinal cord

62

Bram Re~eu~, a 409 (198"j 6 . - "~

BRE 12499

Astrocytes in rat fetal cerebral cortical homografts following implantation into adult rat spinal cord J a m e s R C o n n o r 1'2 a n d J e r a l d J B e r n s t e m l-~ I Laboratory of Central Nervous System Regeneratton, Veterans Admmtstratton Med:¢ al Center, Washmgton D( ( I; ~ A ) and Departments of 2phvstologv and ~Neurosurgery, George Washmgton Unn'ersttv, ~chool o[ Medtcme Washington, D( (U ~ A ) (Accepted 2 September 1986) Key words Homograft, Astrocyte Implantation. Spinal cord injury, lmmunohlstochemlstrx

The present experiment examines astrocytes m fetal cerebral cortical homografts to adult rat spinal cords Fhe purpose of this stud', ~s to determine ff astrocytes are structurally orgamzed within the graft Also, the presence or absence of astroghos~s may be an md*cator of the metabohc status of the graft Embryomc cerebral cortex was taken at 14 days gestatlonal age and transplanted into adult sp,nal cord at the level of the s~xth thorac,c vertebra The homografts were examined at the hght and electron m~croscop~c levels from 7 days postlmplantatmn (PI) to 6 months PI wlth ghal fibrlllary acidic protein antiserum which is a specxfic lmmunohlstohemlcal marker for astrocytes At 7 days PI, ~mmunoreactlve astrocytes were present only at the periphery of the graft and appeared to be associated w,th blood vessels By 30 days PI, normal protoplasmic astrocytes were present throughout the graft No hypertroph,ed astrocvtes arc present at 30 days PI, but the numerical densaty of astrocytes ~s greater than that of normal cerebral cortical gray matter Fibrous astrocytes are present m the periphery of the ~mplant and many of these astrocytes extended their processes between the host and the graft Occasional ghal scarring is observed between the gray matter of the host and graft but generally no ghal scar occurred m the interface between the graft and the host gray matter By 45 clays PI, hypertrophied astrocytes can be seen m the graft, but are confined In th~s age group to penvascular regions Ghosis progressed w~thm the graft throughout the duration of the study, predominating w,thm the graft by 5 months PI The results of this study demonstrate that astrocytlc processes can be shared by the host and graft and that the appearance of hypertrophied astrocytes possibly indicating a decrease m the funcuonal capacity m the graft does not occur until after 30 days PI

ical c o n t a c t which m u s t p r e c e d e f u n c t i o n a l integra-

INTRODUCTION

tion In r e c e n t years the l i t e r a t u r e on the use of trans-

G i v e n t h e s e c o n s i d e r a t i o n s , this study was de-

signed to follow the d e v e l o p m e n t , and d e s c r i b e the

plants as a possible c u r e for n e u r o l o g i c a l d | s o r d e r s

m o r p h o l o g i c a l characteristics of astrocytes in fetal

has b u r g e o n e d

T h e s e studies with rare e x c e p t i o n

c e r e b r a l cortical transplants to adult rat splnat c o r d

h a v e f o c u s e d on the survival o f n e u r o n s , n e u r o p e p ttdes and n e u r o t r a n s m l t t e r s 9 12 K n o w l e d g e o f astro-

using ghal f i l a m e n t acidic p r o t e i n ( G F A P ) a n t i s e r u m

cytes tn transplants of fetal c e n t r a l n e r v o u s system

ghal f i l a m e n t s within astrocytes ll i~

which is a specific i m m u n o h l s t o c h e m l c a l m a r k e r for

could p r e s e n t t m p o r t a n t r e f o r m a t i o n for d e t e r m m m g the structural graft

and f u n c t i o n a l o r g a n i z a t i o n of the

MATERIALS AND METHODS

A l s o , the p r e s e n c e o r a b s e n c e of astroghosls

m a y be an i n d i c a t o r of the m e t a b o l i c status (vtabthty)

T h e process for r e m o v i n g fetal cortices for implan-

neurons

tation and the t r a n s p l a n t a t i o n p r o c e d u r e is identical

a n d / o r n e u r o n a l p r o c e s s e s m a y r e q m r e bridges of

to that r e p o r t e d p r e v i o u s l y ~-s T i m e d - p r e g n a n t Spra-

ghal fibers u p o n which t h e y can m i g r a t e 26 b e t w e e n

g u e - D a w l e y rats f r o m ZlvlC L a b o r a t o r i e s w e r e anes-

the h o m o g r a f t and host m o r d e r to establish t h e phys-

t h e t i z e d with C h l o r o p e n t (180 m g / k g ) on successive

of the

graft

Correspondence

Furthermore.

transplanted

J J Bernstem. CNS Research (151Q). Veterans Admmtstratlon Medical Center Washington, 1)( 20422 U b A

0006-8993/87/$03 50 © 1987 Elsevier Science Pubhshers B ¥ (Biomedical Dwtslon)

63 days and two pups were r e m o v e d by Ceasarlan secnon trom the same uterine horn on embryonic days 14 ( E l 4 ) and 15 ( E l 5 ) The contralateral uterine horn was lett undisturbed so that the pups delivered atter a normal gestanon period could be used as developmental controls The tetus was placed in a cold complete T } r o d e - R m g e r s solunon and the cerebral cortex was dissected lree and transierred to a second dish of cold Ringers solunon A 0 5 x 1 0-ram piece of tetal cerebral cortex was aspirated into a 30-gauge needle attached to a 50-/tl Hamilton synnge The aspirated e m b r v o m c cortex was pressure-rejected subdurallv on the lett side between the dorsal horn and mldllne at the level o| the sixth thoracic ~ertebra (Ta) o! a host adult (300 g) S p r a g u e - D a ~ l e ~ , male rat

sections were incubated m rabbit serum rather than In G F A P anhserum Following the lncubahon m the primary anttbody overmght, the sections were treated with an antl-rabb~t IgG traction p r e p a r e d m goat (1 20, Miles Lab) and incubated in p e r o x l d a s e antlperoxldase (1 30) prior to t r e a t m e n t with a 3,3'dlammobenzldlne solution [or 30 mm The brown precipitate at the reaction site was mtensihed by osm~cat~on The sections were m o u n t e d on gelatincoated shdes d e h y d r a t e d and c o v e r q l p p e d Some sechons were stained with Cress1 ~lolet prior to dehydrahon to md m ldentll-lcatlon ot the gralt F o r electron mlcroscop'~, the sections were deh'~drated and then fiat e m b e d d e d m resin tot c o m b i n e d hght and electron microscopic examination

(prewous b anesthetized) Following the transplantation G e l t o a m was placed over the exposed spinal cord and the wound closed All the host ammals rec e l l e d a 0 1 ml injection of Longlcfl (Fort D o d g e Laboratories) Host rats (4 m each group) were allowed to survive for 7 14, 30, 45 days or 2, 3, 4, 5, or 6 months p o s n m p l a n t a n o n (PI) Pups taken from the same m o t h e r that had provided the embryos for the transplant were used as normal developmental controls and were s a c n h c e d at either 8, 20, 23, or 311 days postnatally (3 per group) To collect the tissue, the rats were anesthetized with Chloropent and peflused through the ascending aorta with 0 9cc sahne followed by 4c'~, paraformaldehyde m II 1 M phosphate-buffered sahne (PBS) If the tissue were to be used ior electron microscopic e x a m l n a n o n , the animal was perfused with a 4-step procedure 22 (l) 0 9cc saline, (n) 4ec p a r a f o r m a l d e hyde and 0 2ce glutaraldeh~de, (hi) 0 f M cacod~late bufter, (~x) 4ec p a r a f o r m a l d e h v d e The spinal cord ~sas exposed and the region containing the graft remos ed and sectioned either horizontally or long~tudlnalh at 5 0 u m on a Lancer Vlbratome The brains ot the control pups ~ e r e r e m o v e d and cut coronal b at 5{]/tm Sections ~ e r e rinsed m 0 1 M PBS and p r e t r e a t e d m NaIO~ ((I 111 M), N a B H a (1(I mg/ml) 5~f D M S O and goat serum ( 1 31)) Each step m the p r e t r e a t m e n t procedure was separated b~ a series of 3 PBS nn~es (5 mm each) The tissue was incubated overmght m a 4 o( cold r o o m m G F A P a n n s e r u m (1 1000) The G F A P antiserum had been generated m rabb~t (supphed b~ Lawrence F Eng, Palo Alto, C A ) so control

Fig 1 The tran,,planted homograft (t) ~ in the center o f the ml-

crograph Se~en da~s PI lrnmunorcacme astroc'~tes stained with GFAP antlsermn are tound onl; m the periphery ol the grMt and arc trequentl'y associated wlth blood xe~,sels(arrow) 81Ix

64 RESULTS

7 days PI The ~mmunoreactwe astrocytes m the homograft are confined to the periphery of the implant at th~s Ume period No ~mmunoreactwe astrocyuc somata are present m the graft, and astrocyUc processes are only found along blood vessels which are entering the graft (F~g 1) No reaction product ~s seen m the cen-

within the central p o r u o n s ol the gratt Irl thl~ t~1 group, although there are some ,ueas ot densely, packed cells within the center ot lhe gr,dt u hlch art. not penetrated by t m m u n o r e a c t l ~ ghal hber~ Astrocyt~c ~omata and many gllal libels ot noltladl ~l/c are present m the outer 1/3 ot the gratt ()ccas~onall~ hypertrophied astrocvtes (enlarged stmla and broad

tral porUon of the graft The region of the graft ~s not

thick processes) are seen outside ~t the gralt w~thm the host but the graft ~s not demarcated from the host by ~mmunoreactwe ghal tflamc tv~

encapsulated with ~mmunoreactwe ghal processes and very httle ghosls is present m the host

30 daw PI

14 days P1 Very thin, free astrocyt~c processes are v~s~ble

B) 30 days PI, the entire homogratt is tilled with astrocytes, and all blood vessels have ghal endteet on them The astrocytes m the center of the gratt arc

t1¢

B Fig 2 Immunoreacnve astrocytes m a 30-day-old graft The density of astrocytes and astrocytlc processes m the graft ~s far greater than the astrocyt~c denstty m the normal cerebral cortex in young adult rats Most of the GFAP-posmve cells appear to be morphologically normal protoplasmic astrocytes 40×

Fig 3 Astrocvtes m the normal 2~-da~-old adult rat cerebral cortex from layers V and VI following Hnmunoreat~Uonwith GFAP antiserum The density of the astroc~tes in th~s figure ~ much less compared to the density of aslrocvtes m F~g 2 but the o'~erall morphological appearance ol the astrocvtes in th~ two figures ~ss~mflar 80×

65 structurally normal protoplasmic astrocytes with a stellate appearance Although the stellate astrocytes in the tuner 2/3 of the graft are structurally normal, there is a clear hyperplastic response (Fig 2) compared to the normal density of astrocytes found in the gra~, matter of the cerebral cortex at a comparable developmental age, or e~en in the adult (Fig 3) In the penpher~ of the graft, hbrous astrocytes are ahgned either parallel or perpendicularly to the graft/ host lntertace The perpendicularly oriented ghal tibets pass through a cell sparse region in the periphery of the implant (Fig 4) similar to caudate fibers 1 in the normal cerebral cortex (Fig 5) These ghal fibers e~ther enter the host/gratt interface to apparently loin the ghal processes m the interface or continue

Fig 4 Ghal fibers in the periphery of the 30-day-old graft are perpendicular to the ghal scar (white arrows) which separates the graft gra~ matter (counterstained with Cresyl violet) from the host white matter The perpendicular astrocytic processes pass through a cell-sparse area m the outermost regions of the graft 78x

through the interface to extend between the host and graft The density of perpendicularly oriented ghal fibers is always much greater in areas where a ghal scar is present in the host/graft interface A gllai scar (dense band of ghal fibers encapsulating the graft) is generally but not always present where the graft is contiguous to host white matter However, where the host gray matter and the graft are contiguous, they are generally not separated by lmmunoreactlve ghal processes

45 days PI In this PI survival group gllosJs as determined by enlarged astrocytlc somata, with broad, thickened (hypertrophied) processes are seen for the first time

Fig 5 In the normal adult rat cerebral cortex, ghal fibers which are l m m u n o r e a c n v e with G F A P antiserum extend from the plal surface through the cell-sparse molecular layer These ~strocvtic processes are similar to the a r r a n g e m e n t of ghal fibers in the penpher,¢ of the cerebral corucal graft (Fig 4) A n astroCVtlC soma ~s visible in the upper regions of the molecular layer (short dark arrow) A stellate-shaped, protoplasmic astrocvte Is seen in laver II (clear arrow) 146×

66 wlthm the graft

The hypertrophied astrocytes are

usually in association with the vasculature (Fig 6) but are otherwise not confined to a region (l c center vs periphery) of the graft Throughout the gratt man) structurally normal protoplasmic astrocytes ,ire still present At the periphery ot the graft, fibrous astrocytes with an orientation parallel to the interface between the host and gratt are still present, part,cularl~ where the graft is contiguous with the gra) matter o! the host The astrocytic hbers with an orientation perpendicular to the interface are found only intrequentl) and the cell sparse region through which they had

growing towards host gray tn,ittet m t h e , entral horr, aStl ocvtlc processes extend or br ~d~~ a ~ oss t l~c m l~ race lblg 7) Man) regton~ e',p¢,Jall\ ,It the' mid,lace between the host g],lx mdlt¢~ md e~alt ,,i~ ,,ltll not separated by gl,al scarrme

2 - 0 months PI Bx 2 months PI, hypertrophied astrocytes are present throughout the implant and ar~ no longer tmuted to a per,vascular association (F~g ~1 Some thin ,istrocyhc processes are still present but by ~ months P1 non-hypertrophied astroc,~tlC plocesses are r a r d v

the host and the graft, particularly where the gralt is

seen By 5 months PI and persisting throughout the last month of this study, all lmmunoreactlve astrocytes In the graft possess enlarged somata, thick,

Fig 6 By 45 days PI ghosls IS present in the graft, but is conhned to the penvascular regions Large (hypertrophied) astrocync processes and an increase m GFAP reaction product are seen associated with blood vessels (*) In the graft whde other astrocytes in the graft are increased m number, but do not contam hypertrophied processes (compare to Fig 2) 73 x

Fig 7 Astrocytic processes (arrow) can be found which traverse the interface between the graft (t) and the host (h), partlcularlv where the graft is contiguous ~ath the gray matter ol the host spinal cord This mlcrograph was taken at 45 days PI but these 'bndgmg' astrocytes can be found between 45 days and 6 months following the implantation 152x

passed is no longer present In the interface between

67 broad prlmar), secondar) and occaslonall) ternary branches No structurally normal protoplasmic astrocytes are seen in the graft Although ghosls predominates within the gratt there are still regions at the host/gratt interface where a ghal encapsulation does not separate the host and the graft The data on the

DISCUSSION

Fig 8 By 2 months PI astroghosls is ewdent throughout the graft Astroc,ctes with enlarged somata (arrows) which are filled with GFAP reacnon product are frequentl~ lound Man,~ of the h~pertrophmd astrocytes also have enlarged pnmar~ processes By this t~me period, the astroghosls is distributed throughout the graft 73x

The results of this study demonstrate (1) tetal cerebral cortical homografts to adult rat spinal cord estabhsh a suftlclent metabohc state (as determined by lack of ghosls) to promote cellular growth and differentlatlon for at least 30 days after implantation, (n) graft and host share astrocytlc processes creating a potential pathway for neuronal migration, and (in) no ghal scar which completely encapsulated the gratt could be round at any age Pl A dense ghal band generally separates host white matter from the graft, but even th~s finding is not consistent ~z The absence ot dstrogllOSlS m the graft for at least the first 30 days after implantation ~s suthclent t~me to permit cell growth and differentiation 2 4 i~ In our previous studies using vasoactlve intestinal polypeptide (VIP) I~ and somatostatm (SOM) ~ neuronal and especially dendritic development did not progress beyond that seen at 45 days PI Thus, the first indication of astroghosls observed in the present study corresponds to the apparent termination of development of peptidergic neurons and their dendritic trees It is not clear whether the increased numerical density of astrocytes in the 30-day-old graft represents a hyperplasla of astrocytes or an increased packing density Based on the density of cells In the Cresyl vlolet-counterstalned graft, it IS likely that the increase in astroghal lmmunoreactlvlty compared to the normal cerebral cortex is due to increased packing density This finding of an increase in astrocytic processes initially observed in the graft at 30 days PI IS a consistent finding in cerebral cortical homografts having been reported in cortical-cortical grafts 7, cerebellar cortex-cerebral cortex grafts 7, lntracerebral hlppocampal transplants 29, and lntraocular cerebral cortex grafts s 16 The latter study showed that astrocytic hyperplasla may be delayed by thyrotdectomy and thus can be influenced by the thyroid hormone

absence of ghal scarring between the host and graft must be viewed with caution as electron microscopic examination of the intermediate zone between the host and graft revealed that not all bands of ghal filaments reacted with G F A P antiserum (Fig 9) even though they were m the same plane of section with reactive filaments

No astrocytic patterns were d~sccrnlble within the Implant Specglcally, no radial ghal fibers were observed from the center of the graft to the periphery tor neurons to use as guides for m~gratmg as m normal development 2a The absence of ghal orgamzatlon may be responsible for the lack of orientation among VIP-posltlVe bipolar neurons in cerebral cortical grafts to spinal cord I~ and lack of laminar organization in cortical-cortical grafts 14 1Q In a recent study, laml-

68

F~g 9 An electron mlcrograph of the mterface area between the host and graft shown at the hght m~croscoplc lexel m F~g 7 Ill regions where gray matter of the graft and host are contiguous, Immunoreactwe ghal fibers frequently do not encapsulate tht. gratt Follov~lng lmmunoreaetmn with GFAP antiserum the region shown m F~g 7 was sectmned for electron m~croscop~ Etectr,,n mlcroscop~c exam matron of the interface regmn revealed the presence of both ~mmunoreaet~ve(clear arrowheads) and non-~mm~Hu~react~veIblat.k arrows) ghal filament bundles in astrocytes (* indicates astrocvtlc nucleus) 6370x

nar organization of transplants could be achieved by usmg sheets of cerebral cortical tissue placed into 'shallow depressions' m the host (newborn) cerebral cortex 1° In the present study, a cell-sparse zone was observed m the periphery of the graft in the 30-daysPI survwal group Thts cell-sparse zone resembled the molecular layer of the cerebral cortex and included astroghal fibers which perpendtcularly traversed the region In the normal adult cerebral cortex, astrocytlc fibers oriented perpendicularly to the pml surface traverse the molecular layer These astrocytic processes in the normal cerebral cortex were descrtbed by A n d n e z e n 1 and referred to as 'caudate fl-

bres Whether the cell-sparse regmn m the gratt is actually a molecular zone and doe, represent some torm o4 laminar organization wIthin the gralt is unclear However, as the graft continued to grow, the cell-sparse zone was not observed m the graft after 45 days P1 Many of the perpendicularly oriented astrocytic fibers however remained and extended through the intermediate zone between the host and graft The sharmg ot astroghal processc, by the host and gra)t was evident at the earhest time period (7 days) investigated Initially. the astrocytlc processe, were associated with the vasculature which penetrated the

69 host, but m the older grafts, a clear and consistent vasculature relationship could not be estabhshed The developmental pattern of G F A P lmmunoreachvlty beginning m the periphery and then extending along the vasculature to the central gray m a t t e r ~s consistent with the reports of normal d e v e l o p m e n t of astrocytes using G F A P anhserum 6 It has been hypothesized that m normal develo p m e n t , astrocytes provide a grading path upon which axons migrate p a m c u l a r l y m estabhshmg the corpus callosum 26, but this wewpolnt has been challenged by showing that astrocytes m the developing corpus callosum have no preferentml orientation 27 F u r t h e r m o r e , growing axons in the developing spinal cord actuall) precede the arrival ot astrocytes -'s Consequently, it cannot be concluded from the data m the present study that the purpose of the bridging aslrocytes is to provMe a path for axons to enter and leave the graft, although such a role for astroc)tes has been postulated for substantm mgra grafts is What is clear Is that astrocytes d e m o n s t r a t e morphological connechons between the host and graft and if they are necessary for neuronal mlgratmn, they are present The observation that astrocytic processes may form a bridge across the Intermediate zone between the host and the graft rather than isolate the graft or the host from the other ~s consistent with observaUons lrom other types of central nervous system transplants including those from substantm mgra Is hlppocampus es and spinal cord > Not only astrocync processes, but astrocytlc somata have been reported to enter the host from the transplant 21 These studms and the present study suggest that astrocytes do not form a barrier to the potential neuronal hbers and somata that may pass between host and graft Further support of this norton can be found m our previous observations that following l m p l a n t a h o n of fetal cerebral cortex into spinal cord, VIP and SOM neurons (which could have only arisen from the implant) can be lound adlacent to anterior m o t o r horn cells-" ~ In addition, serotonln and substance P-containing nerve fibers enter the graft from the host ~e These cons~deratums and the ~mmunmoh~stochem~cal data from

the present m v e s n g a n o n indicate that a ghal scar does not form a barrier whmh encapsulates the transplanted hssue Consequently, at least morphological lntegrahon between the host and graft is possible and the potential for functional integration exists The observation that not all ghal hlaments m the host/graft interface immunoreact with G F A P antiserum is of academic interest, but probably not ol functional slgmficance because the ghal hbers m the mterrace apparently do not serve as a barrier to host/ graft interaction A number of explanations for the lack ot lmmunoreactlvlty of these hlaments exist, among them are dflferentlal hxatum, lack of anUbod) penetration or perhaps &flerent maturatlonal states ol the gllal filament bundles or even different types ol hlaments Finally, there is a posslbfllt) that the onset of astrogllosls in the graft represents a specific onset of astroc~nc dysfunction rather than a reflection of a change m metabolic status ot the graft Although th~s question is dffhcult to address dlrectlx ~ e examined astrocytes m the graft tot the presence ot Rosenthal fibers -'s ~hlch may be assocmted ~ l t h an abnormallt,¢ m the degradation or p r o d u c h o n o[{ ghal hlaments IThe Rosenthal fibers do not stare with G F A P antiserum > thus the c~rtoplasm of the l m m u n o r e a c h v e astrocytes m the present stud) would have had Islets of non-~mmunoreachvltv a fmdmg which was not observed In addition ultrastructural e x a m m a h o n also faded to reveal the presence ot Rosenthal fibers A t the electron rnlcroscoplc lexel, these hbers a p p e a r as an electron-dense mass always assocmted ~ l t h ghal filaments The failure to obserxe Rosenthal fibers in the present study is taken as indication that the astrogllosls ls secondary m response to d~mmlshlng wablhty of the graft tot reasons that are not knov~n at present A(KNOWLEDGEMENTS The authors are grateful to Dr Lawrence Eng ( V A , Palo Alto C A ) for generousl~ supplying the G F A P antiserum This work was s u p p o r t e d by the Veterans A d m i n i s t r a t i o n

70

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23 24

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