Effect of Fetal Striatal and Astrocyte Transplants into Unilateral Excitotoxin-Lesioned Striatum

(C)Frcund Publishing House Ltd., 1993

Effect of Fetal Striatal and Astrocyte Transplants into UnilateralExcitotoxin-Lesioned Striatum

Sunny Y. Lu1, Sarah K. Pixley2, Dwaine F. Emerich1., Michael N. Lehman2 and Andrew B. Norman1,2,3

Division ofNeuroscience, Departments of1psychiatry, 2Anatomy, and 3physiologyUniversity of Cincinnati College ofMedicine, Cincinnati, OH 45267, USA

*Present address: CytoTherapeutics, Inc., Four Richmond Square, Providence, R102906

ABSTRACT

Studies have suggested that neurotrophicmechanisms may underlie transplant-inducedfunctional recovery. Astrocytes have beenreported to be a source of neurotrophic factors.The present study examined the possible role ofcultured astrocytes in promoting recovery ofapomorphine-induced rotation behavior in ratswith unilateral kainic acid (KA) lesions of thestriatum. Five weeks after the lesions, one groupof rats received fetal striatal tissue (E17)transplants, another group received transplantsof cultured astrocyte suspension, and theremaining rats received sham transplants andserved as controls. Apomorphine-inducedrotation behavior was tested 4 weeks after theKA lesions, and 5 and 10 weeks following thetransplantation. The KA-induced rotationbehavior was reduced by the striatal transplantsbut not by the cultured astrocyte transplants 5and 10 weeks following the transplantation.Histochemicai analysis indicated that the striataltransplants had survived and grown andcontained neurons and glia with similarmorphology to those in the host brain.Immunocytochemical analysis of the astrocytetransplant sites revealed heavy glial fibrillaryacidic protein and OX-42 staining in thetransplant areas, suggesting that thetransplanted astrocytes may have survived in

Reprint address:Andrew B. NormanDivision of NeuroscienceDepartment of PsychiatryUniversity of Cincinnati College of MedicineCincinnati, OH 45267-0559, USA

the host brain. Although fetal striataltransplants can ameliorate apomorphine-induced rotation behavior, transplants ofastrocytes alone may not be sufficient to reversethe functional deficits produced by KA lesions.

KEY WORDS

neural transplants, glia, apomorphine,behavior, dopamine receptors, kainic acid

rotation

INTRODUCTION

It has been demonstrated that neural transplantscan promote functional recovery of lesion-inducedbehavioral deficits in animal models of Parkinson’sdisease, Huntington’s disease, and Alzheimer’sdisease /4,11,16,17,27,33/. Despite the clinicalpotential of neural transplants, the mechanisms bywhich transplanted tissue exerts its beneficial ffectsare largely unknown. Recent studies have indicatedthat transplanted astrocytes may play an importantrole in transplant-induced recovery. Kesslak et al./18/reported that transplants of cultured fetal ratcortical astrocytes were able to reverse th’ebehavioral deficits induced by lesions of the frontalcortex. In addition, Kliot et al. /20/indicated thattransplants of millipore membranes coated withembryonic astrocytes facilitated the regeneration ofcrashed dorsal root fibers into the spinal cord.

Glial cells have been reported to synthesize andsecrete neurotrophic and neurotropicfactors, toincrease neuronal survival and to facilitate axonalgrowth in vivo and in vitro /21,22,32,37/.Furthermore, glial cells are important to maintain

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homeostasis of the neuronal environment andremove neurotoxic materials such as glutamate/6/.Glial cells within transplanted striatal tissues mayplay a positive role in the ability of transplants toexert their beneficial effects. However, it is not dearwhether transplants of astrocytes alone aresufficient to reverse striatal excitotoxic lesion-induced deficits. The present study furtherinvestigated the possible role of transplantedastrocytes in promoting recovery of lesion-inducedbehavioral deficits in rats with excitotoxin lesions ofthe striatum.

MATERIALS AND METHODS

Subjects

Three groups of adult male rats (Sprague-Dawley, 200 g, n--28) received unilateral kainic acid(5 nmol) lesions of striatum. The coordinates wereAP=0.3 mm, ML--2.6 mm from bregma andDV--5.6 mm from dura/31/. Five weeks after thelesions, one group of rats (n---9) received fetal ratstriatal tissue transplants (El7), another group(n--l l) received transplants of a suspension ofastrocytes cultured from newborn (0-3 days) ratstriatum. The remaining group (n--8) received sham(equivalent volume of saline) transplants and servedas the control group.

Transplantation surgery

1. Striatal transplants

The techniques for tissue preparation andtransplantation have been previously described indetail/3,7/. Briefly, fetal striatal tissue was obtainedfrom commercially-purchased timed-pregnantfemale Sprague-Dawley rats (Zivic Miller). Undersodium pentobarbital (40 mg/kg i.p.) anesthesia, alaparotomy was performed in order to expose theuterus, and embryos were then extractedindividually. After the removal of the cranium andoverlying integument, the fetal brain was removedand submerged in sterile lactated Ringer’s solution.Under a dissecting microscope, the cortex waspeeled laterally and the half-moon-shaped fetalstriatal tissue was removed. The dissected fetalstriatum was then aspirated into a glass capillary

needle (25 5/8 G) connected to a Hamilton syringe(50 1) and undissociated cells were thenstereotaxically delivered into the lesioned hoststriatum. Tissue was injected at the rate of one ttlper min, the needle was left in place for twoadditional min and then the needle was raised 0.8mm. The procedure was repeated until 4 lxl wasdelivered (approximately 1-1.5 mm3 of fetal striataltissue). The coordinates were AP---0.3 mm, ML--3.0mm from bregma and the initial DV coordinate was6.0 mm from dura/31/.

2. Cultured astrocyte transplants

Astrocytes were prepared by a modification ofthe procedure of McCarthy and de Vellis /24/ andMorrison and de Vellis/25/. Briefly, rat striatumwas dissected away from the brain tissue of thenewborn rat pups, meninges were removed and thetissue was minced with fine scissors. The tissue wastreated for 30 min with 0.025% trypsin and 0.02%EDTA (trypsin/EDTA) and then dissociated bypassage through a plastic 10 ml pipet. The resultingcell suspension was plated into 75 cm2 cultureflasks (Falcon) at a density of 2x107 cells per flask.The culture medium (changed every 2-3 days) wasHam’ s F-10/Dulbecco’ s modified Eagle’s medium,1:1 (vol/vol), with 1.2 g/1 NaHCO2, 15 mM HEPESand 10% (vol/vol) fetal calf serum (FCS). After 10-14 days in culture, the flasks were capped tightlyand shaken overnight at 37C on a rotary shaker.The medium was removed and replaced with freshmedium and the astrocytes were maintained asbefore until use. After approximately 3 weeks theastrocytes were removed from the dish by treatingwith trypsin/EDTA, then medium with 10% FCS toinactivate the trypsin. Cells were then rinsed twicein Hank’s balanced salt solution (HBSS) (Ca2+ andMg2+ free) by centrifugation (7 min at 1000 g),viable cell counts were determined by trypan bluetreatment and a hemocytometer and the cells wereresuspended at 3.2x105 viable cells/4 Ixl HBSS. Thesame multiple injection transplant procedure wasused as described for the fetal striatal tissue. Thecell suspension was stereotaxically injected into thelesioned striatum at the coordinates AP--0.3 mm,ML---3.0 mm from bregma and the initial DVcoordinate was 6.0 mm from dura/31/.

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Behavioral testing

Behavioral testing was performed 4 weeks afterthe KA lesion, and repeated at 5 and 10 weeksfollowing transplantation.

Animals were individually placed in an open fieldenvironment consisting of a Plexiglas box(dimensions: 40 x 40 cm) and left to habituate for20-30 min. Animals were then injected withapomorphine (1 mg/kg s.c., Sigma Chemical Co.)dissolved in normal saline containing 0.07%ascorbate. Sensitization of apomorphine-inducedrotation behavior occurs in unilateral KA lesionedrats /28/. Therefore, rats received two priminginjections of apomorphine (1 mg/kg s.c.) androtations were measured following the thirdinjection. A three day period separated eachinjection. Rotations were continuously countedvisually in five min periods until all rotationbehavior ceased. Rotations were defined ascomplete 360 turns and were reported as the netdifference between the two directions.

Statistical analysis of rotational behavior usedtwo-way analysis of variance (ANOVA).Preplanned comparisons were used to compare thetransplant groups with the KA lesion group.

Immunocytochemistry

Glial fibrillary acidic protein (GFAP) and OX-42immunocytochemistry was used to visualizeastrocytes and microglia respectively.

The rats for these studies were intracardiallyperfused with isotonic saline for 1-2 min, followedby 30 min of ice-cold 4% paraformaldehyde. Brainswere post-fixed 1.5-2 h in 4% paraformaldehydeand left in 20% sucrose overnight. The brains weresectioned (30 lxm) on a freezing microtome intocold phosphate buffered saline (PBS). Alternatesections were processed for GFAP and OX-42immunocytochemistry and the other sections werestained with cresyl violet.

The following primary antibodies were used:GFAP (Dako, 1:5,000) and OX-42 (SeroTEC,1:5,000). The duration of incubation was 24 h withthe primary antibody in PBS containing 0.1% TritonX-100 and 1% normal goat serum for GFAPstaining, and 1% normal horse serum for OX-42

staining. After a rinse in PBS, sections wereincubated for 1 h in biotinylated secondaryantibodies (goat anti-rabbit IgG for GFAP, andhorse anti-mouse IgG for OX-42). After a rinse inPBS, sections were incubated 1 h in avidin-biotin-HRP conjugate (Vetor Laboratories). A five minincubation in 3,3’-diaminobenzidine tetrachloride(DAB) and hydrogen peroxide was done tocomplete the reaction.

Receptor autoradiography

Under deep pentobarbital anesthesia, the ratswere intracardially perfused with ice-cold saline.Brains were removed and immediately buried inpowdered dry ice, then frozen at -70C until use.Brains were mounted in a cryostat at -20C and20 tm thick sections were cut. Alternate brainsections were thaw mounted on gelatinizedmicroscope slides, and four sets of slides wereobtained. One set was stained with cresyl violet andthe remainder of the sets were used to determineautoradiography of D1 and D2 dopamine andmuscarinic cholinergic receptors, respectively.

For receptor autoradiography, the slide-mountedsections were dried at 4C in a sealed containerwith desiccant overnight. For D and D2 dopaminereceptors, the slides were immersed in a bufferedsolution containing either [3H]SCH23390 or[3H]spiperone, respectively. The buffer consisted of50 mM Tris-HC1 (pH=7.7, 25C) containing 125mM NaC1 and 5 mM MgC12. Ketanserin (40 nM)was also added to prevent binding of[3H]SCH23390 /15/ and [3H]spiperone to 5-HT2receptors. The final concentration of[3H]SCH23390 or [3H]spiperone was 1 nM or 0.5nM, respectively. Some of the slides were incubatedwith the radioligand and 1 lxM (+)butaclamol fordetermination of non-specific binding. For thelabeling of muscarinic cholinergic receptors, theslides were immersed in Tris-HC1 (pH--7.7, 25C)containing 130 mM NaC1 and 0.5 nM [3H]QNB forone hour. Some slides were incubated with theradioligand and 1 tM atropine for determination ofnon-specific binding.

Slides were incubated in the appropriate solutionfor one hour, rinsed twice in ice-cold buffer without

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radiolabel for five min each, rapidly dipped intodistilled water and then dried rapidly using cold airfrom a blow dryer. When the sections were fullydried, the slides were placed in an X-ray cassetteand in a photographic dark room, the slides werejuxtaposed to a sheet of LKB ultrofilm and the X-ray cassettes sealed. The X-ray cassettes werestored at 2C for 10-16 days for the [3H]SCH23390and [3H]QNB labeled sections, 25 to 35 days forthe [3H]spiperone labeled sections.

RESULTS

Rotational behavior

There was no spontaneous rotation behaviorfollowing the placement of the rats into the openfield. Four weeks after the KA lesion, the ratsdisplayed rotation ipsilateral to the lesion followingthe injection of apomorphine, consistent with alesion in the more posterior aspect of the striatum/29/. As shown in Table 1, no significant differencein the number of rotations was found between thelesion and transplant groups [F(2,25)--0.72, p>0.5].Five weeks after the transplantation the number ofrotations in the striatal transplant group wasmarginally reduced compared to the sham transplantgroup although the difference failed to reachsignificance (p=0.056). The astrocyte transplantgroup did not differ from the sham transplant group.Ten weeks after the transplantation, the striataltransplant group showed a significant reduction inthe number of rotations compared to the sham

transplant group (p<0.002). There was nodifference between the astrocyte transplant groupand the sham transplant group.

Histochemistry

As shown in Fig. 1A, Nissl staining revealed thatthe striatal transplants had survived and grown inthe lesioned host striatum and contained manyneurons of similar morphology to those in the hostbrain. Glial cells were more dense surrounding thetransplants (Fig. 1A). Transplant survival and asimilar morphological appearance of transplant cellswas observed in all of the animals.

The KA lesioned striatum is often characterizedby depletion of neurons with dense but evenlydistributed glial cells. In all of the animals, in areasof striatum where the astrocytes had beentransplanted, a greater density of glial cells wasobserved compared to the KA lesioned striatum andto the striatum in sham transplanted rats.

Immunocytochemical staining

A high density of GFAP stained astrocytes orOX-42 stained microglia was observed in the areascontaining striatal and astrocyte transplantscompared to that in the host. There was also anincrease in the density of GFAP and OX-42 stainingin the KA lesioned striatum relative to theunlesioned striatum. However, a higher density ofGFAP and OX-42 staining were often observed inastrocyte transplant areas (Fig. 2).

TABLE 1Effect of astrocyte and striatal transplants on apomorphine-induced rotation behavior in rats with unilateral kainic acid

lesions of the striatum

Total number of rotation..$

Lesion (n--8)Lesion and astrocyte transplant (n=11)Lesion and striatal transplant (n=9)

Pretransplant 5 weeks post 10 weeks post_428

_63 658

_72 721 __. 104

371 __. 46 685 *__ 92 651 +/- 71463 __. 63 424

___89 361 +/- 49*

Rotation behavior in response to apomorphine (1 mg/kg s.c.) was measured at four weeks following unilateral KA lesion(pretransplant) and at five and at 10 weeks posttransplant. Values represent the mean +/- SEM number of ipsilateral rotations fromthe number of animals shown in parentheses. Significantly different from lesion values *p<0.002, ANOVA.

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Fig. 1: Receptor autoradiograms from the striatal transplant in lesioned host striatum. The transplant (14 weeks post-transplantation) indicated by the arrows, can be seen in the cresyl violet-stained section (A). The distribution of[3H]SCH23390 binding to D dopamine receptors (B), [3H]spiperone binding to D2 dopamine receptors (C) and[3H]QNB binding to muscarinic receptors (D). Note the high degree of correspondence between the high-density patchesof dopamine D1 and D2 and muscarinic receptors. T=transplant.

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Receptor autoradiography

Autoradiography for striatal transplants revealedthat dopamine D1 (Fig. 1B) and D2 (Fig. 1C)receptors and muscarinic cholinergic receptors (Fig.1D) were present in the transplants with a patchydistribution. However, these receptors were absentin the astrocyte transplants (data not shown).

DISCUSSION

Neurotrophic mechanisms have been suggestedto play an important role in transplant-inducedfunctional recovery /1,5,18,19/. Since glial cellshave been reported to synthesize and secrete trophicand tropic factors which improve neuronal survivaland axon regeneration following lesions/10,26,32,34,37/, one possible source ofneurotrophic factors is transplanted glial cells. Inaddition, glial cells have important functions inmaintaining ionic and pH balance, and absorbendogenous excitotoxins.

Our results indicated that striatal but notcultured astrocyte transplants ameliorated therotational behavior induced by apomorphine in ratswith unilateral KA lesions. This result is consistentwith the finding of Kesslak et al. /19/ thattransplants of fetal hippocampal tissue into KAlesioned hippocampus facilitated the behavioralrecovery measured in an alternation task, whereasastrocyte transplants did not. A previous study byKesslak et al. /18/ reported that transplants ofcultured astrocytes were effective in acceleratingthe rate of spontaneous behavioral recovery afterfrontal cortex ablation. The lack of effect oftransplants of purified astrocytes in promoting KA-induced deficits may be due to insufficient amountsof trophic factors released from the transplants.However, most importantly, the depletion ofneurons and circuitry by KA produces a long-termfunctional deficit, which cannot be reversed byastrocytes or trophic factors alone. Repletion ofneurons and reconstruction of neural circuitry byneural tissue transplants may be essential forfunctional recovery.

Whether the presence of D and D2 dopamine ormuscarinic receptor binding sites in the striatal

transplant contributes to the behavioral recoveryobserved in the rats from the striatal transplantgroup is uncertain. Norman et al. /30/reported asimilar behavioral recovery produced by striataltransplants in which few receptors were found. Thelack of D, D2 and muscarinic receptors in theastrocyte transplanted area indicated that thedepletion of intrinsic striatal neurons due to KAlesion is not affected by astrocyte transplants. Thislack of neuronal elements may be partiallyresponsible for the poor effect of astrocytetransplants in ameliorating KA-induced deficits.

Recent studies have indicated that there aredifferences in the ability of immature and matureastrocytes to facilitate plastic changes in adult brain.Immature astrocytes can synthesize trophic factorsto support neuronal survival, produce a permissiveenvironment for neurite extension and reduce scarformation /10,35/. In contrast, mature astrocytesproduce a non-permissive environment for axongrowth and increase scar formation /36/. Matureastrocytes may not promote functional recovery. Inthe present studies, it is possible that the astrocyteswhich were grown from newborn striatum and werein culture for approximately three weeks may havedeveloped characteristics of mature astrocytes.

Cultured astrocytes have been reported tosurvive and migrate following transplantation/2,13,14/. In the present study, histochemical andimmunocytochemical staining indicated the presenceof a high density of glial cells in the areas containingastrocyte transplants and suggested the survival ofthe transplanted astrocytes. However, it is difficultto distinguish the transplanted astrocytes from anyhost reactive astrocytes using the presenttechniques, since either migration of transplantedastrocytes out of the transplant site and/ormigration of host reactive astrocytes into thetransplant site has been reported/8,9,38/. Labelingof astrocytes prior to transplantation is necessary todetermine the survival and migration of transplantedastrocytes.

ACKNOWLEDGEMENTS

This study was supported by MH45253 fromADAMHA. We are grateful to Dr. Paul R. Sanberg

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for helpful discussion. We would like to thank MelFariello for manuscript preparation.

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