Simultaneous intrastriatal and intranigral grafting (double grafts) in the rat model of Parkinson's disease

Brain Research Reviews 32 Ž2000. 328–339 www.elsevier.comrlocaterbres

Short review

Simultaneous intrastriatal and intranigral grafting ždouble grafts / in the rat model of Parkinson’s disease I. Mendez a

a,b,)

, K.A. Baker a , M. Hong

b

Neural Transplantation Laboratory, Departments of Anatomy and Neurobiology, Room 12H1, Sir Charles Tupper Medical Building, Dalhousie UniÕersity, Halifax, NoÕa Scotia, Canada, B3H 4H7 b Surgery, DiÕision of Neurosurgery, Dalhousie UniÕersity, Halifax, NoÕa Scotia, Canada, B3H 4H7

Abstract Experimental and clinical studies of neural transplantation in Parkinson’s disease have focused on the placement of fetal dopaminergic grafts not in their ontogenic site Žsubstantia nigra. but in the main nigral target area Žstriatum.. The reason for this is the apparent inability of intranigral nigral grafts to extend axons for long distances reinnervating the ipsilateral striatum. This review presents previous work by our laboratory wI. Mendez, M. Hong, Reconstruction of the striato-nigro-striatal circuitry by simultaneous double dopaminergic grafts: a tracer study using fluorogold and horseradish peroxidase, Brain Res. 778 Ž1997. 194–205; I. Mendez, D. Sadi, M. Hong., Reconstruction of the nigrostriatal pathway by simultaneous intrastriatal and intranigral dopaminergic transplants, J. Neurosci. 16 Ž1996. 7216–7227x using a new transplantation strategy aimed at restoring dopaminergic innervation of the nigra and striatum by simultaneous dopaminergic transplants placed in the substantia nigra and ipsilateral striatum Ždouble grafts. in the 6-hydroxydopamine lesioned adult rat brain. These double grafts achieve not only greater striatal reinnervation than the standard intrastriatal grafts but also produce a faster and more complete behavioural recovery six weeks after transplantation. Injection of the retrograde tracer fluorogold into the striatum and nigra resulted in fluorescent labeled cells within the intranigral graft and the intrastriatal graft and surrounding striatum, respectively suggesting that these double grafts promote at least partial reconstruction of the nigrostriatal dopaminergic pathway. This double graft strategy may have potential implications in clinical neural transplantation for Parkinson’s disease. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Intrastriatal grafting; Intranigral grafting; Parkinson’s disease

Contents 1. Introduction .

.......................................................................

329

2. Materials and methods . . . . . . . . . . 2.1. Experimental design . . . . . . . . . 2.2. Animals and 6-OHDA lesions . . . . 2.3. Microtransplantation . . . . . . . . . 2.4. Post-transplant behavioral assesment 2.5. Immunohistochemistry . . . . . . . 2.6. Fluorogold injections . . . . . . . . 2.7. WGA-HRP injections . . . . . . . . 2.8. Statistics . . . . . . . . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

330 330 330 330 330 330 330 331 331

3. Results . . . . . . . . . . . . . 3.1. 6-OHDA lesions . . . . . 3.2. Single transplants . . . . 3.3. Double transplants . . . . 3.4. Intrastriatal FG injections

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

331 331 332 332 332

)

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

Corresponding author. Fax: q1-902-473-3343; e-mail: [email protected]

0165-0173r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 1 7 3 Ž 9 9 . 0 0 0 9 1 - 0

I. Mendez et al.r Brain Research ReÕiews 32 (2000) 328–339 3.5. Intranigral fluorogold and WGA-HRP injections . 3.6. Rotational behaviour . . . . . . . . . . . . . . .

................................................. .................................................

4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Enhancement of striatal reinnervation . . . . . . . . . 4.2. Enhancement of functional recovery . . . . . . . . . 4.3. Double grafts: a new transplantation strategy for PD?

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

334 334 336 336

.....................................................................

337

..........................................................................

337

1. Introduction Evidence for the growth and survival of fetal dopaminergic grafts and their reversal of biochemical and locomotor deficits in animal models of Parkinson’s disease ŽPD. is well documented. However, restoration of the nigrostriatal pathway has not yet been achieved by the use of current grafting strategies. To date, the main transplant strategy has been to place nigral grafts not in their ontogenic site Žsubstantia nigra. but in their target area Žstriatum.. However, restoration of the nigrostriatal circuitry with dopaminergic neurons and their dendrites in the substantia nigra ŽSN. and terminals in the striatum may be essential for more complete alleviation of the variety of symptoms in PD w56x. Previously, it has been demonstrated that dopamine is released from dendrites of pars compacta neurons in the pars reticulata w7,8x. This dendritic release of dopamine has been shown to be important in modulating g-aminobutyric acid ŽGABA. release by the descending striatonigral pathway w58x. The main reason for the ectopic placement of dopaminergic grafts in the striatum is the apparent inability of fetal nigral grafts placed in the nigra to grow axons for long distances capable of reaching their striatal target. Possible factors contributing to this failure may in part result from a lack of endogenous cues directing axonal growth or the presence of inhibitory factors in myelinated pathways of the adult central nervous system ŽCNS. w5,54,59–61x. Although restoration of efferent nigrostriatal connections has been demonstrated by ectopic placement of nigral grafts in the striatum w9,19,41x, afferent striatonigral connections are lacking w11x. This lack of graft innervation by the host striatum may result in a failure by the host to regulate graft activity. Conversely, in transplant strategies where the graft has been placed in the ontogenic site such as intrastriatal striatal transplants in the Huntington’s model, afferent connections are abundant w75,76,80x. In an attempt to enhance axonal growth from the SN to the striatum, several strategies have been attempted. Several studies have incorporated ‘‘bridging’’ techniques in an effort to reinnervate the striatal target by intranigral dopaminergic grafts. Fetal striatal tissue cell suspensions and ibotenic and kainic acid injections have been made

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

333 334

. . . .

Acknowledgements . References

329

along a path from the intranigral graft to the striatum w13,81,82x. In those studies, the investigators noted TH-immunoreactive axons extending along the ‘‘bridge’’, reinnervating the ipsilateral striatum. The observation that glutamate and kainate induce the up-regulation of glial cell line-derived neurotrophic factor ŽGDNF. w23,24x and that GDNF is expressed in developing striatal tissue w68x, led Wang and colleagues w71x to evaluate the efficacy of GDNF bridges in promoting striatal reinnervation by intranigral dopaminergic nigral grafts. Injections of GDNF along a pathway from the SN to the ipsilateral striatum promoted the long-distance axonal outgrowth from intranigral dopaminergic grafts w71x and dopamine release w70x to and in the ipsilateral striatum, respectively. Implantation of Schwann cells overexpressing GDNF as ‘‘bridges’’ from SN to striatum have also been shown to promote axonal outgrowth w78x. Xenografts have also been tried in an attempt to escape the inhibitory factors that may prevent axonal growth in myelinated tracts in the adult central nervous system ŽCNS.. Long distance axonal growth along the nigrostriatal tract in lesioned rats was observed by implantation of human ventral mesencephalic and forebrain tissue along this pathway w73,74x. Transplantation in the developing CNS, an environment that is presumably more conducive to axonal growth, is another strategy that has been employed to investigate the ability of intranigral dopaminergic grafts to reinnervate the striatum. Fetal dopaminergic grafts have been placed into the nigra of bilaterally lesioned neonatal pups at postnatal day 3 w45x. TH-immunoreactive fibers were observed growing from the graft along the nigrostriatal pathway reinnervating the ipsilateral striatum. This striatal reinnervation correlated well with the behavioural recovery observed in the transplanted pups w47x. These observations suggest that reconstruction of the nigrostriatal pathway and restoration of dopamine to both the SN and striatum may be necessary for optimal graft function in animal models of PD. The double graft strategy may also have relevance in clinical neural transplantation. In the past decade, over 100 patients with PD have received intrastriatal fetal nigral grafts in several centres worldwide with encouraging results w14,17,18,25– 28,30,31,33–37,52,65,72,77x. Although the clinical results

330

I. Mendez et al.r Brain Research ReÕiews 32 (2000) 328–339

thus far are promising, clinical improvement has been variable and transplanted patients have not been cured with some remaining severely disabled. It is clear that before neural transplantation can be used as a routine therapeutic procedure in the treatment of PD, a number of issues need to be resolved w40,49x. A crucial question yet to be answered is, what are the optimal targetŽs. for transplantation? Determination of the optimal targetŽs. is essential in order to maximize the clinical efficacy of neural transplantation in PD. In this review, we will discuss the anatomical reconstruction of the nigrostriatal pathway and enhanced functional recovery by dopaminergic implants placed simultaneously in the striatum and SN of 6-hydroxydopamine Ž6-OHDA.-lesioned adult rats.

2. Materials and methods 2.1. Experimental design A total of 42 female Wistar rats Ž200–225 g. were used in these experiments. Sixteen rats received simultaneous intranigral and intrastriatal transplants of 800,000 fetal ventral mesencephalic ŽFVM. cells Ždouble grafts., receiving 400,000 cells in each of the SN and striatum. In another group, 4 animals received intranigral FVM implants and intrastriatal transplants of non-dopaminergic Žcortical. cells. Eight rats were randomly divided into two groups and received transplants of 800,000 FVM cells in the striatum Ž n s 4. or SN Ž n s 4. only. Ten rats had unilateral 6-OHDA lesions only and 4 rats served as normal controls. 2.2. Animals and 6-OHDA lesions Rats received two stereotaxic injections of 6-OHDA into the right ascending nigrostriatal dopaminergic pathway under pentobarbital anaesthesia. Following a two week recovery period, animals were challenged with amphetamine Ž5 mgrkg i.p.. and their rotational scores collected over a 90-min period. Only animals exhibiting a mean ipsilateral rotational score of 9 or more full body turnsrmin were included in the studies. 2.3. Microtransplantation The microtransplantation procedure previously described by Nikkhah and colleagues w48x was used. Briefly, fetuses Žembryonic day 14. were removed from pregnant Wistar rats anaesthetized with pentobarbital. The ventral mesencephalons were dissected out and single cell suspensions prepared and stereotactically injected into the brains of 6-OHDA lesioned animals. The tissue was dissected in DMEM and incubated in 0.1% trypsinr0.05% DNAserDMEM at 378C for 20 min and rinsed 4 times in

0.05% DNAserDMEM. Incubated tissue was then mechanically dissociated until a milky, homogenous single cell suspension was achieved. Cell suspensions with a final cell concentration of approximately 200,000 cellsrml were used with a cell viability ) 98% as determined by the trypan blue dye exclusion method. A total of 800,000 cells were implanted per rat. In animals implanted with double grafts, the striatum received 400,000 cells and the nigra also received 400,000 cells. Single-grafted animals received a total of 800,000 cells in either the striatum or SN. Implantation of the dopaminergic cell suspensions occurred at the following coordinates for the striatum; anteroposterior ŽAP. s q1.3, q0.6 and q0.3; mediolateral ŽML. s y2.1, y2.9 and y3.7; DV s y5.5 and y4.3 and for the SN at AP s y4.8, y5.0 and y5.3; ML s y2.0, y2.3 and y2.6; DV s y8.4 and y8.2, y8.3 and y8.1 and y8.2 and y8.0. All AP and ML coordinates are in mm from Bregma and the surface of the brain for DV coordinates; toothbar at y3.3. 2.4. Post-transplant behaÕioural assessment Functional recovery was assessed by amphetamine-induced rotational behaviour using a computerised-video activity monitor system ŽVideomex, Columbus Instruments, Columbus, OH.. Behavioural testing was carried out every 3 weeks following transplantation. 2.5. Immunohistochemistry Six to eight weeks after transplantation, rats received an overdose of pentobarbital and were perfused transcardially with 4% paraformaldehyde. The fixative was followed by a solution of 10% glucose in phosphate buffer. The brain was cryoprotected by placement in a solution of buffered 30% sucrose overnight. Coronal and sagittal sections were cut on a freezing microtome and collected serially in phosphate buffer. Sections were processed for TH immunohistochemistry using a primary rabbit-anti-TH antiserum Ž1:500 Pel Freez, Rogers, AR.. Visualization of the TH-immunoreactivity was carried out with the ABC-kit and 3,3X-diaminobenzidine. 2.6. Fluorogold injections For retrograde tracing with fluorogold, six animals with double grafts Ž n s 6., three animals with 6-OHDA lesions only Ž n s 3., and two normal controls Žno lesion or grafts. Ž n s 2. received stereotactic injections of the retrograde neuronal tracer fluorogold ŽFG. Ž2% in 0.9% saline, Fluorochrome, Denver, CO. 6 weeks following grafting. FG was injected into the striatum Ž0.4 ml per injection. using a microcapillary cannula Ž40–50 mm tip diameter. at the following coordinates: AP s 0.5 and y0.5; ML s y2.0 and y3.5; DV s y6.0 and y4.0; toothbar at y3.3. Three animals with double grafts received stereotactic

I. Mendez et al.r Brain Research ReÕiews 32 (2000) 328–339

331

Fig. 1. A representative parasagittal section through a rat brain of an animal with both intrastriatal and intranigral grafts Ždouble grafts.. Many TH-immunoreactive cells can be observed within both the intrastriatal and intranigral grafts. A dense halo of TH-immunoreactivity is seen surrounding the intrastriatal graft with lighter immunostaining being observed throughout the entire striatal area. Scale bar: 500 mm.

injections Ž0.1 ml per injection. of FG into the SN at the following coordinates: AP s y5.0; ML s y2.3; DV s y8.2; toothbar at y3.3. Ten days after the injections, animals were anaesthetized, perfused, and their brains sectioned in the same manner as described for immunohistochemistry Žsee above.. Both coronal and sagittal sections

were mounted and analyzed for FG labelling using a fluorescence microscope equipped with a filter for 365 nm excitation. Adjacent sections were stained for TH as described above. In sections stained for TH, the total number of fluorescent neurons and TH-immunopositive neurons were

Fig. 2. A schematic representation of a parasagittal section through the rat brain demonstrating the sites of injection of FG and HRP into the intrastriatal and intranigral grafts of double-grafted animals.

332

I. Mendez et al.r Brain Research ReÕiews 32 (2000) 328–339

counted. An approximation of graft cell number was calculated according to Abercrombie’s formula w1x and the percentage of neurons that were both fluorescent and TH-immunoreactive was estimated. 2.7. WGA-HRP injections For anterograde wheat germ agglutinin–horse radish peroxidase ŽWGA-HRP. tracing, four animals with double

grafts Ž n s 4., three animals with 6-OHDA lesions only Ž n s 3., and two normal controls Žno lesion or grafts. Ž n s 2. received stereotactic injections of the anterograde neuronal tracer horse radish peroxidase ŽHRP. Ž2.5% in 0.9% saline. 6 weeks, post-transplantation. HRP was injected into the SN Ž0.1 ml. using a microcapillary cannula Ž40–50 mm tip diameter. at the following coordinates: AP s y5.0; ML s y2.3; DV s y8.2; toothbar at y3.3. Two days after the injections, the animals were anesthetized, perfused with 1.0% paraformaldehyde containing

Fig. 3. Fluorescence photomicrographs of a parasagittal section of the SN in animals with FG injections into the ipsilateral intrastriatal graft. ŽA. FG labelled cells within the substantia nigra pars compacta and pars reticulata in a non-6-OHDA lesioned animal. ŽB. A paucity of fluorescence was observed in the contralateral nigral region of the same rat. ŽC. FG labelled cells in an animal following a 6-OHDA lesion. ŽD. Fluorescent cells in an intranigral graft. ŽE. High power view of retrogradely labelled cells in the nigral region in the lesion-only rat seen in ŽC.. ŽF. High-power view of the retrogradely labelled cells within the intranigral graft. Scale bar: A, B, C, D, 100 mm; E, F, 50 mm.

I. Mendez et al.r Brain Research ReÕiews 32 (2000) 328–339

1.25% glutaraldehyde in 0.1 M phosphate buffer, and their brains sectioned in a similar manner to that described for immunohistochemistry Žsee above.. Sections were processed for HRP histochemistry using the TMB method. Briefly, sections were prewashed in distilled water for 10 min and then placed in a pre-reaction solution 0.0005% TMB, 0.1% sodium nitroferricyanide acetate buffer for 20 min prior to reaction with hydrogen peroxide. Following this reaction, sections were rinsed with acetate buffer, mounted on gelatin-coated slides and coverslipped with fluoromount. Adjacent sections were stained for TH as described above.

333

3.3. Double transplants 3.3.1. TH immunohistochemistry Clusters of a large number of TH-immunoreactive neurons were found in both the intranigral and intrastriatal grafts ŽFig. 1.. As in the single-grafted animals, a dense network of TH-immunoreactive fibers was observed within the graft and extended into the host brain in all directions

2.8. Statistics Statistical analysis for between-group differences was assessed at P - 0.05 using two-way ANOVA followed by Tukey’s post-hoc test.

3. Results 3.1. 6-OHDA lesions Stereotactic injection of 6-OHDA in the ascending nigrostriatal dopaminergic pathway produced a complete obliteration of TH-immunoreactive cells in the ipsilateral SN and disappearance of TH-immunoreactive fibers in the ipsilateral medial forebrain bundle ŽMFB. and striatum. 3.2. Single transplants 3.2.1. Intranigral grafts Intranigral grafts were localized within the region of the ventral mesencephalon. Clusters of TH-immunoreactive cells were observed on the periphery of the graft area with fewer cells being seen in the centre of the graft. Within the area of the graft, a dense network of TH-immunoreactive fibers was seen and few processes were observed to extend beyond the graft-host interface for short distances in all directions, but no fibers were traced to the ipsilateral striatum. 3.2.2. Intrastriatal grafts Intrastriatal grafts formed dense clusters of TH-immunoreactive perikarya and fibers with the clusters of TH-immunoreactive cells being primarily observed on the graft periphery, with the center of the graft containing only a few scattered cell bodies. A dense network of TH-immunopositive fibers was also observed within the intrastriatal graft itself with numerous fibers penetrating the graft–host interface in all directions forming a halo of dense TH-immunoreactivity around the graft. Those fibers could be traced for up to 2 mm beyond the host–graft border.

Fig. 4. ŽA. Fluorescence photomicrograph of a parasagittal section of a double grafted rat SN demonstrating the site of FG injection. ŽB. and ŽC. High-power view of FG labelled cells within the ipsilateral striatum and intrastriatal graft of the same animal as ŽA.. Scale bar: A, 125 mm; B, 75 mm; C, 25 mm.

334

I. Mendez et al.r Brain Research ReÕiews 32 (2000) 328–339

with the most substantial fiber outgrowth occurring from the intranigral graft in the rostral direction. Those fibers projected rostrally along the MFB medially and the internal capsule laterally and could be traced from the intranigral graft to the ipsilateral striatum and in some cases, into the intrastriatal graft. A dense network of fibers was also found within the intrastriatal grafts with TH-immunoreactive processes extending in all directions within the host striatum. The extent of striatal reinnervation in rats receiving double grafts was much greater than in animals with intrastriatal grafts only. In some animals with double grafts, TH-immunoreactivity was present throughout the entire striatum, which was not observed in animals with intrastriatal grafts only. 3.4. Intrastriatal FG injections Following injection of FG into the striatum ŽFig. 2., diffuse labeling throughout the striatum and intrastriatal graft was observed and many retrogradely labeled cells could be seen within the ipsilateral intranigral graft. The fluorescent-labeled cells were randomly distributed in the body of the transplant, which differed from labeled cells seen in the pars reticulata of lesion only animals. In normal control animals Ž n s 2. without either a lesion or transplant, numerous densely packed, labeled cells were seen in the ipsilateral pars compacta ŽFig. 3.. Fluorescent cells were more loosely arranged within pars reticulata. Approx-

imately 11.5 " 4.9% of the TH-immunopositive cells were found to also be fluorescent and 75.3 " 26.5% of fluorescent cells were also TH-immunopositive. 3.5. Intranigral fluorogold and WGA-HRP injections Injection of FG into the substantia nigra resulted in diffuse labeling of the intranigral transplant and numerous cells in the ipsilateral striatum. Fluorescent-labeled cells were seen throughout the striatum and globus pallidus including a few labeled cells in the body of the intrastriatal graft ŽFig. 4.. Some of the fluorescent cells seen in the body of the intrastriatal graft were also immunoreactive for TH. Injections of HRP were confined to the intranigral graft. HRP-filled fibers could be traced from the intranigral graft to the ipsilateral striatum where a dense pattern of HRP reaction product was observed. HRP labeling was concentrated primarily around the intrastriatal graft and to a lesser extent within the graft ŽFig. 5.. In rats with 6-OHDA lesions only, no HRP labeling in the striatum was observed. 3.6. Rotational behaÕiour Animals were tested for rotational behaviour every 3 weeks following grafting. Compensation of rotational behaviour was observed 3 weeks after transplantation in

Fig. 5. A representative coronal section of an intrastriatal graft in an animal with double grafts. ŽA. Section immunostained for TH. A dense halo of TH-immunoreactivity is noted surrounding the graft. ŽB. Darkfield photomicrograph labelled for HRP reaction product of the intrastriatal graft following injection of HRP into the ipsilateral intranigral graft. HRP labelling is concentrated outside of the graft with less labelling being observed in the graft itself. Scale bar: 150 mm.

I. Mendez et al.r Brain Research ReÕiews 32 (2000) 328–339

335

4.1. Enhancement of striatal reinnerÕation

Fig. 6. Amphetamine Ž5 mgrkg, i.p..-induced rotational behaviour in rats following 6-OHDA lesioning of the ascending nigrostriatal pathway Žstipled bars. and 6 weeks following implantation of dopaminergic grafts into the striatum Ž ns 4. or nigra Ž ns 4. or into both nigra and striatum Ždouble grafts. Ž ns6.. Each bar represents mean"S.E.M. number of rotations made in 60 min; U p) 0.05 compared with pre-grafting; UU p) 0.05 compared with intrastriatal and intranigral graft groups.

animals with double grafts only. This compensation was significant Ž P - 0.05. when compared with pre-transplantation values and rats with single grafts. Animals with double grafts had complete restoration of amphetamine-induced rotational behaviour 6 weeks after grafting. Animals with intrastriatal grafts alone also improved their rotational scores, however did not achieve the level of compensation observed in rats with double grafts. In contrast, animals with intranigral grafts only did not improve their rotational scores. The differences in the rotational scores between the rats with double grafts and either intrastriatal or intranigral grafts were statistically significant ŽFig. 6..

4. Discussion The ability of long axonal outgrowth along myelinated pathways in animal models of neural transplantation has been observed when ‘‘bridging’’ strategies w13,73,74, 78,81,82x are used or when transplants are placed within the immature CNS w15,38,67x. Long distance TH-immunoreactive axonal outgrowth from intranigral dopaminergic grafts to the ipsilateral striatum has also been demonstrated in neonatal rats w45,47x. This review discusses previous work by our laboratory providing evidence of reconstruction of the dopaminergic nigrostriatal pathway in the adult CNS by simultaneous intrastriatal and intranigral dopaminergic grafts w42,43x. This work suggests that double grafts may encourage axonal outgrowth and guidance in the adult host brain.

Numerous studies have demonstrated reinnervation of the dopamine-depleted striatum by ectopic placement of fetal nigral grafts within the striatum w3,4,12,41x. Demonstration of graft integration into the ventral mesencephalon has been demonstrated by homotopically placed nigral grafts w46,50x, but no evidence of axonal outgrowth along the nigrostriatal pathway was observed w46x. In our studies, animals with intranigral dopaminergic grafts alone did not demonstrate long distance axonal growth along the nigrostriatal pathway thus confirming those previous observations. Several strategies have been employed in an attempt to reconstruct the nigrostriatal pathway in the adult CNS by using ‘‘bridging’’ techniques w13,72,73,78,81,82x. Our studies have shown that fetal nigral transplants placed simultaneously in the adult striatum and ventral mesencephalon have the capacity to reconstruct at least partially, the nigrostriatal pathway ŽFig. 7.. It appears that the intrastriatal graft provides cues to promote the guidance of axons from the intranigral transplant along the internal capsule to the striatum and vice versa. Currently we are also investigating the extent of dopaminergic reinnervation of the pallidal segments and subthalamic nucleus in double-grafted animals, with these structures normally receiving dopaminergic innervation w20,21,29,32,51x. Fetal nigral neurons appear to have the ability for long-distance axonal growth w22,63x and reconstruction of the nigrostriatal pathway when implanted into a permissive environment such as the neonatal rat w45,47x. It is possible that the intrastriatal nigral transplant has a trophic effect on the intranigral graft promoting the growth and guidance of TH-immunoreactive axons. Previous studies have demonstrated that fetal striatal tissue can stimulate the outgrowth of dopaminergic fibers in vitro w10,53x. This notion is supported by a recent study in which increased dopaminergic neurite outgrowth was observed when co-grafts of fetal striatal and ventral mesencephalic tissue were placed within close proximity to one another in the host striatum w64x. It is possible that neurotrophic factors such as GDNF, which is expressed in the developing striatum w68x may be involved in the increased TH-immunoreactive fiber outgrowth observed with fetal striatal co-grafts. Many results have been compiled to support the notion that GDNF enhances fiber outgrowth from dopaminergic grafts w2,20,57,62,71x. It has similarly been demonstrated that FVM grafts express brain-derived neurotrophic factor ŽBDNF. mRNA in oculo w69x. In two recent studies, BDNF was observed to enhance the fiber outgrowth of dopaminergic FVM cells in vivo and in vitro w66,79x, suggesting a neurotrophin-dependant mechanism by which intrastriatal nigral grafts enhance striatal reinnervation. Regardless of the mechanism involved, it appears that simultaneous homotopic and ectopic placement of developing fetal nigral cell suspensions provides an environment

336

I. Mendez et al.r Brain Research ReÕiews 32 (2000) 328–339

Fig. 7. A schematic representation of a parasagittal section through the rat brain illustrating the hypothetical reconstruction of the nigrostriatal pathway and enhancement of striatal reinnervation by double grafts.

conducive to the long-distance growth of TH-immunoreactive axons. 4.2. Enhancement of functional recoÕery It is clear from our experiments that animals receiving double grafts had a more complete restoration of motor symmetry than single-grafted rats, although there was also a significant but not complete reduction in rotational asymmetry in animals with intrastriatal grafts only. Contralateral rotations were observed in double grafted animals, an effect that was not encountered in rats with intrastriatal grafts only. In animals receiving an intranigral graft a reduction in rotational behaviour was not observed. This lack of rotational compensation in intranigrally grafted animals most likely relates to the failure of the intranigral graft to also reinnervate the striatum since the degree of striatal reinnervation correlates with the extent of functional recovery w55x. In our studies we have demonstrated a more extensive reinnervation of the striatum in double grafted animals. Although extensive reinnervation of the striatum may relate to the enhanced behavioural recovery observed, the intranigral graft itself may have also contributed to the increased behavioural recovery. We are currently exploring the role of the intranigral graft in behavioural recovery by destroying the intranigral graft after restoration of rotational symmetry in double-grafted animals. Dopaminergic reinnervation of the nigra by the intranigral graft may be important for the beneficial functional effects in the double graft strategy. It is known that dopamine is released from pars compacta dendrites in the pars reticulata w7,8x. Nigral dopamine has been observed to be important in enhancing pars reticulata neuronal activity by inhibiting striatonigral GABA release through D1 dopamine receptors located on the descending striatonigral axon terminals w39,58x. However, double grafts may en-

hance functional recovery also by reestablishing the dopaminergic innervation of other basal ganglia structures, such as the subthalamic nucleus ŽSTN.. The STN receives nigral dopaminergic efferents w21,29,51x and dopamine appears primarily to reduce STN neuronal activity w6x. In a recent study, intrastriatal dopaminergic grafts reversed increases in cytochrome oxidase activity in the entopeduncular nucleus and SNr but not the STN w44x, suggesting that intrastriatal dopaminergic grafts are unable to normalize STN activity. Thus, reinnervation of the STN may be important for enhancing functional recovery as unilateral lesions of the nigrosubthalamic dopaminergic pathway result in ipsiversive turning upon amphetamine challenge w16x. We hypothesize that double grafts promote enhancement of functional recovery of the striatum, nigra and other basal ganglia structures normally receiving dopaminergic innervation, in particular the STN. The extent of reinnervation of the STN by intranigral dopaminergic grafts is currently being investigated by our laboratory. 4.3. Double grafts: a new transplantation strategy for PD? Many variables undoubtedly influence the efficacy of neural transplantation for PD w40x. A crucial variable still to be resolved is the optimal placement of the graft in the basal ganglia. To date, the main transplant strategy has been to place nigral grafts in the striatum. However, restoration of the dopaminergic innervation of the SN and STN in addition to the striatum may be essential for a more complete alleviation of motor symptoms in PD. These studies suggest that a double graft strategy may accomplish a more complete reinnervation of the basal ganglia dopaminergic circuitry resulting in increased functional recovery. These observations may have potential implications in optimizing the efficacy of clinical neural transplantation in Parkinsonian patients.

I. Mendez et al.r Brain Research ReÕiews 32 (2000) 328–339

Acknowledgements The authors wish to thank Mr. Damaso Sadi for his superior technical assistance and all the students that helped make these studies possible. w18x

References w1x M. Abercrombie, Estimation of nuclear population from microtome sections, Anat. Rec. 94 Ž1946. 239–247. w2x C. Apostolides, E. Sandford, M. Hong, I. Mendez, Glial cell line-derived neurotrophic factor improves intrastriatal graft survival of stored dopaminergic cells, Neuroscience 83 Ž1998. 363–372. w3x A. Bjorklund, R.H. Schmidt, U. Stenevi, Functional reinnervation of ¨ the neostriatum in the adult rat by use of intraparenchymal grafting from the substantia nigra, Cell Tissue Res. 212 Ž1980. 39–45. w4x J.P. Bolam, T.F. Freund, A. Bjorklund, S.B. Dunnett, A.D. Smith, ¨ Synaptic input and local output of dopaminergic neurons in grafts that functionally reinnervate the host neostriatum, Exp. Brain Res. 68 Ž1987. 131–146. w5x D.S. Cadelli, M.E. Schwab, Myelin-associated inhibitors of neurite outgrowth and their role in CNS regeneration, Ann. N.Y. Acad. Sci. 633 Ž1991. 234–240. w6x G.A. Campbell, M.J. Eckardt, F.F. Weight, Dopaminergic mechanisms in subthalamic nucleus of rat: analysis using horseradish peroxidase and microiontophoresis, Brain Res. 333 Ž1985. 261–270. w7x A. Cheramy, V. Leviel, J. Glowinski, Dendritic release of dopamine in the substantia nigra, Nature 289 Ž1981. 537–542. w8x A. Cheramy, A. Nieoullon, J. Glowinski, In vivo evidence for a dendritic release of dopamine in cat substantia nigra, Appl. Neurophysiol. 42 Ž1979. 57–59. w9x D.J. Clarke, P. Brundin, R.E. Strecker, O.G. Nilsson, A. Bjorklund, ¨ Human fetal dopamine neurons grafted in a rat model of Parkinson’s disease: ultrastructural evidence for synapse formation using tyrosine hydroxylase immunocytochemistry, Exp. Brain Res. 73 Ž1988. 115–126. w10x S. Denis-Donini, J. Glowinski, A. Prochiantz, Specific influence of striatal target neurons on the in vitro outgrowth of mesencephalic dopaminergic neurites: a morphological qualitative study, Neuroscience 3 Ž1983. 2292–2299. w11x G. Doucet, Y. Murata, P. Brundin, O. Bosler, N. Mons, M. Geffard, C.C. Ouimet, A. Bjorklund, Host afferents into intrastriatal trans¨ plants of fetal ventral mesencephalon, Exp. Neurol. 106 Ž1989. 1–19. w12x S.B. Dunnett, A. Bjorklund, Mechanisms of function of neural grafts ¨ in the adult mammalian brain, J. Exp. Biol. 132 Ž1987. 265–289. w13x S.B. Dunnett, D.C. Roger, S.J. Richards, Nigrostriatal reconstruction after 6-OHDA lesions in rats: combination of dopamine-rich nigral grafts and nigrostriatal ‘‘bridge’’ grafts, Exp. Brain Res. 75 Ž1989. 523–535. w14x A. Fine, P. Gaudet, R. Holness, D. King, J. Fisk, A. Lang, B. Snow, L. Heffernan, R. Poirier, R. Redman, T. Hosokawa, C. Leopold, M. Mackay-Lyons, H. Robertson, F. Fingerhut, D. Calne, Fetal dopaminergic neural transplantation for treatment of Parkinson’s disease: outcome after one year, Can. J. Neurol. Sci. 21 ŽS2. Ž1994. S27. w15x M.K. Floeter, E.J. Jones, Connections made by transplants to the cerebral cortex of rat brains damaged in utero, J. Neurosci. 4 Ž1984. 141–150. w16x G. Flores, J. Valencia, M.G. Rosales, A. Sierra, J. Aceves, Appearance of EMG activity and motor asymmetry after unilateral lesions of the dopaminergic innervation to the subthalamic nucleus in the rat, Neurosci. Lett. 162 Ž1993. 153–156. w17x C.R. Freed, R.E. Breeze, N.L. Rosenberg, S.A. Schneck, E. Kriek,

w19x

w20x

w21x

w22x

w23x

w24x

w25x

w26x

w27x

w28x

w29x

w30x

w31x w32x

337

J.X. Qi, T. Lone, Y.B. Zhang, J.A. Snyder, T.H. Wells, L.O. Ramig, L. Thomson, J.C. Mazziotta, S.C. Huang, S.T. Grafton, D. Brooks, G. Sawle, G. Schroter, A.A. Ansari, Survival of implanted fetal dopamine cells and improvement 12 to 46 months after transplantation for Parkinson’s disease, New Engl. J. Med. 327 Ž1992. 1549– 1555. T.B. Freeman, C.W. Olanow, R.A. Hauser, G.M. Nauert, D.A. Smith, C.V. Borlongan, P.R. Sanberg, D.A. Holt, J.H. Kordower, F.J. Vingerhoets, B.J. Snow, D. Calne, L.L. Ganger, Bilateral fetal nigral transplantation into the postcommissural putamen in Parkinson’s disease, Ann. Neurol. 38 Ž1995. 379–388. T.F. Freund, J.P. Bolam, A. Bjorklund, U. Stenevi, S.B. Dunnett, ¨ J.F. Powell, A.D. Smith, Efferent synaptic connections of grafted dopaminergic neurons reinnervating the host neostriatum: a tyrosine hydroxylase immunocytochemical study, J. Neurosci. 5 Ž1985. 603– 616. A.C. Granholm, J.L. Mott, K. Bowenkamp, S. Eken, S. Henry, B.J. Hoffer, P.A. Lapchak, M.R. Palmer, C. van Horne, G.A. Gerhardt, Glial cell line-derived neurotrophic factor improves survival of ventral mesencephalic grafts to the 6-hydroxydopamine lesioned striatum, Exp. Brain Res. 116 Ž1997. 29–38. O.K. Hassani, C. Franc¸ois, J. Yelnik, J. Feger, Evidence for a ´ dopaminergic innervation of the subthalamic nucleus in the rat, Brain Res. 749 Ž1997. 88–94. J.P. Herman, D.N. Abrous, M.M. Le, Anatomical and behavioural comparison of unilateral dopamine-rich grafts implanted into the striatum of neonatal and adult rats, Neuroscience 40 Ž1991. 465–475. A. Ho, A.C. Gore, C.S. Weickert, M. Blum, Glutamate regulation of GDNF gene expression in the striatum and primary striatal astrocytes, NeuroReport 6 Ž1995. 1454–1458. C. Humpel, B. Hoffer, I. Stromberg, S. Bektesh, F. Collins, L. ¨ Olson, Neurons of the hippocampal formation express glial cell line-derived neurotrophic factor messenger RNA in response to kainate-induced excitation, Neuroscience 59 Ž1994. 791–795. O.V. Kopyov, D.S. Jacques, A. Lieberman, C.M. Duma, R.L. Rogers, Outcome following intrastriatal fetal mesencephalic grafts for Parkinson’s patients is directly related to the volume of grafted tissue, Exp. Neurol. 146 Ž1997. 536–545. J.H. Kordower, T.B. Freeman, E.Y. Chen, E.J. Mufson, P.R. Sanberg, R.A. Hauser, B. Snow, C.W. Olanow, Fetal nigral grafts survive and mediate clinical benefit in a patient with Parkinson’s disease, Mov. Disord. 13 Ž1998. 383–393. J.H. Kordower, T.B. Freeman, B.J. Snow, F.J. Vingerhoets, E.J. Mufson, P.R. Sanberg, R.A. Hauser, D.A. Smith, G.M. Nauert, D.P. Perl, C.W. Olanow, Neuropathological evidence of graft survival and striatal reinnervation after the transplantation of fetal mesencephalic tissue in a patient with Parkinson’s disease, New Engl. J. Med. 332 Ž1995. 1118–1124. J.H. Kordower, J.M. Rosenstein, T.J. Collier, M.A. Burke, E.Y. Chen, J.M. Li, L. Martel, A.E. Levey, E.J. Mufson, T.B. Freeman, C.W. Olanow, Functional fetal nigral grafts in a patient with Parkinson’s disease: chemoanatomic, ultrastructural, and metabolic studies, J. Comp. Neurol. 370 Ž1996. 203–230. B. Lavoie, Y. Smith, A. Parent, Dopaminergic innervation of the basal ganglia in the squirrel monkey as revealed by tyrosine hydroxylase immunohistochemistry, J. Comp. Neurol. 289 Ž1989. 36–52. M. Levivier, S. Dethy, F. Rodesch, M. Peschanski, A. Vandesteene, P. David, D. Wikler, S. Goldman, T. Claes, F. Biver, C. Liesnard, M. Goldman, J. Hildebrand, J. Brotchi, Intracerebral transplantation of fetal ventral mesencephalon for patients with advanced Parkinson’s disease. Methodology and 6-month to 1-year follow-up in 3 patients, Stereotact. Funct. Neurosurg. 69 Ž1997. 99–111. O. Lindvall, Update on fetal transplantation: the Swedish experience, Mov. Disord. 13 Žsuppl1. Ž1998. 83–87. O. Lindvall, A. Bjorklund, Dopaminergic innervation of the globus ¨ pallidus by collaterals from the nigrostriatal pathway, Brain Res. 172 Ž1979. 169–173.

338

I. Mendez et al.r Brain Research ReÕiews 32 (2000) 328–339

w33x O. Lindvall, P. Brundin, H. Widner, S. Rehncrona, B. Gustavii, R. Frackowiak, K.L. Leenders, G. Sawle, J.C. Rothwell, C.D. Marsden, A. Bjorklund, Grafts of fetal dopamine neurons survive and improve ¨ motor function in Parkinson’s disease, Science 247 Ž1990. 574–577. ˚ w34x O. Lindvall, S. Rehncrona, P. Brundin, B. Gustavii, B. Astedt, H. Widner, T. Lindholm, A. Bjorklund, K.L. Leenders, J.C. Rothwell, ¨ R. Frackowiak, C.D. Marsden, B. Johnels, G. Steg, R. Freedman, ˚ Seiger, M. Bygdeman, I. Stromberg, B.J. Hoffer, A. L. Olson, ¨ Human fetal dopamine neurons grafted into striatum in two patients with severe Parkinson’s disease. A detailed account of methodology and a 6-month follow-up, Arch. Neurol. 46 Ž1989. 615–631. w35x O. Lindvall, G. Sawle, H. Widner, J.C. Rothwell, A. Bjorklund, D. ¨ Brooks, P. Brundin, R. Frackowiak, C.D. Marsden, P. Odin, S. Rehncrona, Evidence for long-term survival and function of dopaminergic grafts in progressive Parkinson’s disease, Ann. Neurol. 35 Ž1994. 172–180. w36x O. Lindvall, H. Widner, S. Rehncrona, P. Brundin, P. Odin, B. Gustavii, R. Frackowiak, K.L. Leenders, G. Sawle, J.C. Rothwell, A. Bjorklund, C.D. Marsden, Transplantation of fetal dopamine ¨ neurons in Parkinson’s disease: one-year clinical and and neurophysiological observations in two patients with putaminal implants, Ann. Neurol. 31 Ž1992. 155–165. w37x Clinica Puerta de Hierro Neural Transplantation Group, J.J. LopezLozano, G. Bravo, B. Brera, I. Millan, ´ J. Dargallo, J. Salmean, ´ J. Urıa, ´ J. Insausti, Long-term improvement in patients with severe Parkinson’s disease after implantation of fetal ventral mesencephalic tissue in a cavity of the caudate nucleus: 5-year follow up in 10 patients, J. Neurosurg. 86 Ž1997. 931–942. w38x R.D. Lund, J.D. Radel, M.H. Hankin, H. Klassen, P.J. Coffey, J.N.P. Rawlins, Developmental and functional integrations of retinal transplants with host rat brains. In: A. Bjorklund, A.J. Aguayo, D. ¨ Ottoson ŽEds.., Brain Repair, MacMillan, London, 1990, pp. 327– 340. w39x L.P. Martin, B.L. Waszczak, D1 agonist-induced excitation of substantia nigra pars reticulata neurons: mediation by D1 dopamine receptors of striatonigral terminals via a pertussis toxin-sensitive coupling pathway, J. Neurosci. 14 Ž1994. 4494–4506. w40x V. Mehta, J. Spears, I. Mendez, Neural transplantation in Parkinson’s disease, Can. J. Neurol. Sci. 24 Ž1997. 292–301. w41x I. Mendez, K. Elisevich, B. Flumerfelt, Dopaminergic innervation of substance P-containing striatal neurons by fetal nigral grafts: an ultrastructural double-labeling immunocytochemical study, J. Comp. Neurol. 308 Ž1991. 66–78. w42x I. Mendez, M. Hong, Reconstruction of the striato-nigro-striatal circuitry by simultaneous double dopaminergic grafts: a tracer study using fluorogold and horseradish peroxidase, Brain Res. 778 Ž1997. 194–205. w43x I. Mendez, D. Sadi, M. Hong, Reconstruction of the nigrostriatal pathway by simultaneous intrastriatal and intranigral dopaminergic transplants, J. Neurosci. 16 Ž1996. 7216–7227. w44x N. Nakao, M. Ogura, K. Nakai, T. Itakura, Intrastriatal mesencephalic grafts affect neuronal activity in basal ganglia nuclei and their target structures in a rat model of Parkinson’s disease, J. Neurosci. 18 Ž1998. 1806–1817. w45x G. Nikkhah, M.G. Cunningham, M.A. Cenci, R. McKay, A. Bjorklund, Dopaminergic transplants into the substantia nigra of ¨ neonatal rats with bilateral 6-OHDA lesions. I. Evidence for anatomical reconstruction of the nigrostriatal pathway, J. Neurosci. 15 Ž1995. 3548–3561. w46x G. Nikkhah, M.G. Cunningham, A. Jodicke, U. Knappe, A. ¨ Bjorklund, Improved graft survival and striatal reinnervation by ¨ microtransplantation of fetal nigral cell suspensions in the rat Parkinson model, Brain Res. 633 Ž1994. 133–143. w47x G. Nikkhah, M.G. Cunningham, R. McKay, A. Bjorklund, ¨ Dopaminergic transplants into the substantia nigra of neonatal rats with bilateral 6-OHDA lesions. II. Transplant-induced behavioural recovery, J. Neurosci. 15 Ž1995. 3562–3570.

w48x G. Nikkhah, M. Olsson, J. Eberhard, C. Bentlage, M.G. Cunningham, A. Bjorklund, A microtransplantation approach for cell suspen¨ sion grafting in the rat Parkinson model: a detailed account of the methodology, Neuroscience 63 Ž1994. 57–72. w49x C.W. Olanow, J.H. Kordower, T.B. Freeman et al., Fetal nigral transplantation as a therapy for Parkinson’s disease, Trends Neurosci. 19 Ž1996. 102–109. w50x M. Olsson, G. Nikkhah, C. Bentlage, A. Bjorklund, Forelimb akine¨ sia in the rat Parkinson model: differential effects of dopamine agonists and nigral transplants as assessed by a new stepping test, J. Neurosci. 15 Ž1995. 3863–3875. w51x A. Parent, Y. Smith, Differential dopaminergic innervation of the two pallidal segments in the squirrel monkey Ž Saimiri sciureus ., Brain Res. 426 Ž1987. 397–400. w52x M. Peschanski, G. Defer, J.P. N’Guyen, F. Ricolfi, J.C. Monfort, P. Remy, C. Geny, Y. Samson, P. Hantraye, R. Jeny, A. Gaston, Y. Keravel, J.D. Degos, P. Cesaro, Bilateral motor improvement and ´ alteration of L-dopa effect in two patients with Parkinson’s disease following intrastriatal transplantation of foetal ventral mesencephalon, Brain 117 Ž1994. 487–499. w53x A. Prochiantz, U. DiPorzio, A. Kato, B. Berger, J. Glowinski, In vitro maturation of mesencephalic dopaminergic neurons from mouse embryos is enhanced in the presence of their striatal target cells, Proc. Natl. Acad. Sci. U.S.A. 76 Ž1979. 5387–5391. w54x O. Raineteau, W.J. Z’Graggen, M. Thallmair, M.E. Schwab, Sprouting and regeneration after pyramidotomy and blockade of the myelin-associated neurite growth inhibitors NI35r250 in adult rats, Eur. J. Neurosci. 11 Ž1999. 1486–1490. w55x L. Rioux, D.P. Gaudin, L.K. Bui, L. Gregoire, T. DiPaolo, P.J. ´ Bedard, Correlation of functional recovery after a 6-hydroxydopa´ mine lesion with survival of grafted fetal neurons and release of dopamine in the striatum of the rat, Neuroscience 40 Ž1991. 123–131. w56x H.A. Robertson, Synergistic interactions of D1- and D2-selective dopamine agonists in animal models for Parkinson’s disease: sites of action and implications for the pathogenesis of dyskinesias, Can. J. Neurol. Sci. 19 Ž1992. 147–152. w57x C. Rosenblad, A. Martinez-Serrano, A. Bjorklund, Glial cell line-de¨ rived neurotrophic factor increases survival, growth and function of intrastriatal fetal nigral dopaminergic grafts, Neuroscience 75 Ž1996. 979–985. w58x A. Ruffieux, W. Schultz, Dopaminergic activation of reticulata neurones in the substantia nigra, Nature 285 Ž1980. 240–241. w59x L. Schnell, M.E. Schwab, Axonal regeneration in the rat spinal cord produced by an antibody against myelin-associated neurite growth inhibitors, Nature 343 Ž1990. 269–272. w60x L. Schnell, M.E. Schwab, Sprouting and regeneration of lesioned corticospinal tract fibers in the adult rat spinal cord, Eur. J. Neurosci. 5 Ž1993. 1156–1171. w61x M.E. Schwab, Myelin-associated inhibitors of neurite growth and regeneration in the CNS, Trends Neurosci. 13 Ž1990. 452–456. w62x S.R. Sinclair, C.N. Svendsen, E.M. Torres, D. Martin, J.W. Fawcett, S.B. Dunnett, GDNF enhances dopaminergic cell survival and fibre outgrowth in embryonic nigral grafts, NeuroReport 7 Ž1996. 2547– 2552. w63x A.M. Snyder-Keller, R.K. Carder, R.D. Lund, Development of dopamine innervation and turning behaviour in dopamine-depleted infant rats receiving unilateral nigral transplants, Neuroscience 30 Ž1989. 779–794. w64x C.E. Sortwell, T.J. Collier, J.R. Sladek Jr., Co-grafted embryonic striatum increases the survival of grafted embryonic dopamine neurons, J. Comp. Neurol. 399 Ž1998. 530–540. w65x D.D. Spencer, R.J. Robbins, F. Naftolin, K.L. Marek, T. Vollmer, C. Leranth, R.H. Roth, L.H. Price, A. Gjedde, B.S. Bunney, K.J. Sass, J.D. Elsworth, L. Kier, R. Makuch, P.B. Hoffer, D.E. Redmond Jr., Unilateral transplantation of human fetal mesencephalic tissue into the caudate nucleus of patients with Parkinson’s disease, New Engl. J. Med. 327 Ž1992. 1541–1548.

I. Mendez et al.r Brain Research ReÕiews 32 (2000) 328–339 w66x C. Spenger, C. Hyman, L. Studer, M. Egli, L. Evtouchenko, C. Jackson, A. Dahl-Jorgenson, R.M. Lindsay, R.W. Seiler, Effects of BDNF on dopaminergic, serotonergic and GABAergic neurons in cultures of human fetal ventral mesencephalon, Exp. Neurol. 133 Ž1995. 50–63. w67x B.B. Stanfield, D.D.M. O’Leary, Fetal occipital cortical neurones transplanted to the rostral cortex can extend and maintain a pyramidal tract axon, Nature 313 Ž1985. 135–137. w68x I. Stromberg, L. Bjorklund, M. Johansson, A. Tomac, F. Collins, L. ¨ ¨ Olson, B. Hoffer, C. Humpel, Glial cell line-derived neurotrophic factor is expressed in developing but not adult striatum and stimulates developing dopamine neurons in vivo, Exp. Neurol. 124 Ž1993. 401–412. w69x I. Stromberg, C. Humpel, Expression of BDNF and trkB mRNAs in ¨ comparison to dopamine D1 and D2 receptor mRNAs and tyrosine hydroxylase-immunoreactivity in nigrostriatal in oculo co-grafts, Brain Res., Dev. Brain Res. 84 Ž1995. 215–224. w70x F.I. Tang, L.T. Tien, F.C. Zhou, B.J. Hoffer, Y. Wang, Intranigral ventral mesencephalic grafts and nigrostriatal injections of glial cell line-derived neurotrophic factor restore dopamine release in the striatum of 6-hydroxydopamine-lesioned rats, Exp. Brain Res. 119 Ž1998. 287–296. w71x Y. Wang, L.T. Tien, P.A. Lapchak, B.J. Hoffer, GDNF triggers fiber outgrowth of fetal ventral mesencephalic grafts from nigra to striatum in 6-OHDA lesioned rats, Cell Tissue Res. 286 Ž1996. 223–233. w72x G.K. Wenning, P. Odin, P. Morrish, S. Rehncrona, H. Widner, P. Brundin, J.C. Rothwell, R. Brown, B. Gustavii, P. Hagell, M. Jahanshahi, G. Sawle, A. Bjorklund, D.J. Brooks, C.D. Marsden, ¨ N.P. Quinn, O. Lindvall, Short- and long-term survival and function of unilateral intrastriatal dopaminergic grafts in Parkinson’s disease, Ann. Neurol. 42 Ž1997. 95–107. w73x K. Wictorin, P. Brundin, B. Gustavii, O. Lindvall, A. Bjorklund, ¨ Reformation of long axon pathways in adult rat central nervous system by human forebrain neuroblasts, Nature 347 Ž1990. 556–558. w74x K. Wictorin, P. Brundin, H. Sauer, O. Lindvall, A. Bjorklund, Long ¨ distance directed axonal growth from human dopaminergic mesen-

w75x

w76x

w77x

w78x

w79x

w80x

w81x

w82x

339

cephalic neuroblasts implanted along the nigrostriatal pathway in 6-hydroxydopamine lesioned adult rats, J. Comp. Neurol. 323 Ž1992. 475–494. K. Wictorin, C.C. Ouimet, A. Bjorklund, Intrinsic organization and ¨ connectivity of intrastriatal striatal transplants in rats as revealed by DARPP-32 immunohistochemistry: specificity of connections with the lesioned host brain, Eur. J. Neurosci. 1 Ž1989. 690–701. K. Wictorin, R.B. Simberley, O. Isacson, L.W. Swanson, A. Bjorklund, Connectivity of striatal grafts implanted into the ibotenic ¨ acid-lesioned striatum. III. Efferent projecting graft neurons and their relation to host afferents within the grafts, Neuroscience 30 Ž1989. 313–330. H. Widner, J. Tetrud, S. Rehncrona, B. Snow, P. Brundin, B. Gustavii, A. Bjorklund, O. Lindvall, J.W. Langston, Bilateral fetal ¨ mesencephalic grafting in two patients with Parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, New Engl. J. Med. 327 Ž1992. 1556–1563. M.J. Wilby, S.R. Sinclair, E.M. Muir, R. Zietlow, K.H. Adcock, P. Horellou, J.H. Rogers, S.B. Dunnett, J.W. Fawcett, A glial cell line-derived neurotrophic factor-secreting clone of the Schwann cell line SCTM41 enhances survival and fiber outgrowth from embryonic nigral neurons grafted to the striatum and lesioned substantia nigra, J. Neurosci. 19 Ž1999. 2301–2312. D.M. Yurek, W. Lu, S. Hipkens, S.J. Wiegand, BDNF enhances functional reinnervation of the striatum by grafted fetal dopamine neurons, Exp. Neurol. 137 Ž1996. 105–118. F.C. Zhou, N. Buchwald, Connectivities of the striatal grafts in adult rat brain: a rich afference but scant striatonigral efference, Brain Res. 504 Ž1989. 15–30. F.C. Zhou, Y.H. Chiang, Excitochemical-induced bridging directs axonal growth of transplanted neurons to distal target, Cell Transplant. 4 Ž1995. 103–112. F.C. Zhou, Y.H. Chiang, Y. Wang, Constructing a new nigrostriatal pathway in the Parkinsonian model with bridged neural transplantation in substantia nigra, J. Neurosci. 16 Ž1996. 6965–6974.