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Co-grafted embryonic striatum increases the survival of grafted embryonic dopamine neurons

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THE JOURNAL OF COMPARATIVE NEUROLOGY 399:530–540 (1998)
Co-Grafted Embryonic Striatum
Increases the Survival of Grafted
Embryonic Dopamine Neurons
CARYL E. SORTWELL,1* TIMOTHY J. COLLIER,2 AND JOHN R. SLADEK, JR.1
of Neuroscience, Chicago Medical School, North Chicago, Illinois 60064
2Department of Neurological Sciences, Rush Presbyterian St. Luke’s Medical School,
Chicago, Illinois 60612
1Department
ABSTRACT
To enhance the current therapeutic benefit of dopamine (DA) neuron grafts in Parkinson’s
disease, strategies must be developed that increase both DA neuron survival and fiber
outgrowth into the denervated striatum. Previous work in our laboratory has demonstrated
that dopaminergic neurons grow to greater size when co-grafted with striatal cell suspensions
and display extensive tyrosine hydroxylase-positive (TH1) projections, but no conclusion
could be reached concerning enhancement of survival of grafted DA neurons. The aim of the
present study was to characterize further the potential trophic effects of striatal co-grafts on
grafted mesencephalic DA neuron survival. Unilaterally lesioned male Fischer 344 rats were
grafted with either a suspension of mesencephalic cells or with both mesencephalic and
striatal cell suspensions. Co-grafts were either mixed together or placed separately into the
striatum. Lesioned rats receiving no graft served as controls. Rotational behavior was
assessed following amphetamine challenge at 2 weeks prior to grafting and at 4 and 8 weeks
following grafting. Only rats receiving co-grafts of nigral and striatal suspensions separated
by a distance of 1 mm showed significant behavioral recovery from baseline rotational
asymmetry. Both mixed and separate striatal co-grafts were associated with a doubling of DA
neuron survival compared with solo mesencephalic grafts. In the mixed co-graft experiment,
DA neurite branching appeared enhanced and TH-rich patches were observed, whereas with
co-grafts that were separated, TH1 innervation of the intervening host striatum was
increased significantly. These results provide the first evidence suggesting that nigral-striatal
co-grafts, particularly those placed separately and in proximity to each other, increase both
DA neuron survival and neurite extension from the mesencephalic component of the grafts.
J. Comp. Neurol. 399:530–540, 1998. r 1998 Wiley-Liss, Inc.
Indexing terms: transplant; substantia nigra; Parkinson’s disease; trophic striatum; dopamine
The abnormal motor function that is symptomatic of
Parkinson’s disease (PD) is not usually apparent until
dopamine (DA) levels in the striatum have dropped to less
than 20% of normal (Hornykiewicz, 1988). Attempts to
increase DA levels through the transplantation of pharmacologically relevant cells has provided a novel therapeutic
strategy for PD. Implants of substantia nigra DA cells to
the caudate nucleus in monkeys significantly alleviate
specific behavioral deficits following 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) treatment (Taylor et
al., 1990, 1991). However, human clinical studies with
fetal tissue transplants have not resulted in similar success. With few exceptions, patients have experienced either no improvement or modest improvement (Ahlskog,
1993; Olanow et al., 1996). In the most recent case study, in
which all grafted patients experienced significant clinical
r 1998 WILEY-LISS, INC.
benefit (Freeman et al., 1995), tissue from six to eight
donors was transplanted to each recipient. For the transplant approach to become a significant therapeutic option,
strategies must be developed to enhance the survival of
grafted DA neurons.
The striatum provides a target for DA-containing innervation from the substantia nigra during development of
the nigrostriatal pathway. Multiple influences, including
Grant sponsor: NIH; Grant numbers: F32 NS09646–01, RSA K05-MH
00643, and AG10851.
*Correspondence to: Caryl E. Sortwell, Ph.D., Department of Neurological Sciences, Rush-Presbyterian-St. Luke’s Medical Center, Tech 2000,
Suite 200, 2242 W. Harrison Street, Chicago, IL 60612.
E-mail: csortwel@rush.rpslmc.edu
Received 14 April 1997; Revised 21 May 1998; Accepted 25 May 1998
STRIATAL CO-GRAFTS AND DOPAMINE NEURON SURVIVAL
extracellular matrix components and trophic factors, contribute to the establishment of this circuitry. There is
abundant evidence in in vitro systems that striatal target
tissue exerts both tropic and trophic effects on developing
mesencephalic DA neurons (Prochiantz et al., 1979; Daguet
et al., 1980; Hemmendinger et al., 1981; Hoffman et al.,
1983; Tomozawa and Appel, 1986; Dal Toso et al., 1988;
Niijima et al., 1990; Dong et al., 1993; Takeshima et al.;
1994, Carvey et al., 1996). Attempts have been made to
extend these effects observed in culture to the mammalian
brain. DA neurons survived and innervated their embryonic striatal targets when co-grafted to the anterior chamber of the eye of adult rats (Olson et al., 1979). Greater host
reinnervation density was produced by intrastriatal grafts
of mixed embryonic mesencephalic and striatal cell suspensions than by mesencephalic grafts alone (Brundin et al.,
1986). Moreover, embryonic striatal grafts were reinnervated preferentially over host striatum (DeBeaurepaire
and Freed, 1987). Significantly larger DA cell bodies and
extensive TH-positive (TH1) projections were seen when
both mixed and separate striatal-nigral co-grafts were
studied, respectively (Yurek et al., 1990). Embryonic striatal tissue is effective in promoting TH-positive fiber outgrowth from mesencephalic grafts placed homotopically in
the substantia nigra (Dunnett et al., 1989). A strong
correlation exists between co-graft-enhanced TH1 fiber
outgrowth and more rapid or enhanced functional recovery
from denervation (Brundin et al., 1986; Dunnett et al.,
1989; Yurek et al., 1990). More recently, mixed nigralstriatal grafts exhibited a more robust and long-lasting
turning response in a bilaterally lesioned model (Costantini et al., 1994). In nonhuman primates, dopaminergic
neurites displayed preferential growth from a separate
nigral graft to the accompanying striatal graft (Sladek et
al., 1993).
These studies illustrate the predictive validity of in vitro
experiments. Striatal cells serve as a chemotactic attractant and enhance neurite outgrowth from mesencephalic DA neurons both in vitro and in vivo. However,
enhanced survival of mesencephalic neurons has been
reported only in culture. The present experiment provides
the first direct evidence that co-grafted striatal cells
influence the directionality and density of DA neurite
extension and enhance mesencephalic DA neuron survival. To investigate whether target-derived regulation of
DA neuron survival and neurite extension is contact
dependent or is mediated by diffusible substances, both a
mixed and separate co-graft condition were included. This
research has been presented previously in abstract form
(Sortwell et al., 1996, 1997).
MATERIALS AND METHODS
Lesions and behavioral assessment
Thirty-five male Fischer 344 rats (200–225 g) were
used in this study. Protocols were approved by the Institutional Animal Care and Use Committee of the Chicago
Medical School. Stereotaxic injections of 6-hydroxydopamine (6-OHDA) were made unilaterally into the nigrostriatal pathway of anesthetized rats (30 mg/kg, pentobarbital).
Each rat received a total of 18 µg of 6-OHDA (in 0.9%
NaCl/0.2% ascorbic acid) in two injections over a period of
3 minutes each, one in the vicinity of the medial forebrain
bundle (AP 24.3, ML 11.2, DV 27.5) and the other in the
pars compacta of the substantia nigra (AP 24.8, ML 11.5,
531
DV 27.5). Baseline measures of amphetamine-induced
rotational behavior were obtained at 2 weeks following the
6-OHDA lesion. Lesioned animals were given an injection
of 5.0 mg/kg (i.p.) amphetamine and placed in cylindrical
bowls in which rotational behavior was quantified by a
MacIntosh rotometer program starting 15 minutes after
injection, for a period of 70 minutes. The rotation quantitation computer program and cylinders were designed by
Dennis Levin and generously provided by Dr. Barry Hoffer.
Animals meeting the criterion of six ipsilateral turns per
minute or greater were grafted 2 weeks later. Rats were
tested for rotational asymmetry following amphetamine
challenge at 4 and 8 weeks after grafting. Completeness of
the lesion was verified histologically at the conclusion of
the experiment.
Dissection and transplantation procedures
Timed pregnant female Fischer 344 rats were injected
with 5-bromo-28-deoxyuridine (BrdU) at 24 and 12 hours
(50 mg in 50% EtOH/kg, i.p.) prior to tissue dissection to
label dividing cells and allow for graft identification.
Mesencephalic and striatal brain regions were dissected
by using sterile techniques from embryonic day (E15)
fetuses and separately pooled in oxygenated, cold, sterile
calcium-magnesium-free buffer, pH 7.1 (CMF). Care was
taken to dissect out only the lateral ganglionic eminence
(LGE), the primordial anlagen that develops into the
striatum (Pakzaban et al., 1993; Deacon et al., 1994). Cell
suspensions of embryonic mesencephalic or striatal tissue
were then prepared through a series of CMF rinses,
incubated in 0.125% trypsin (Gibco, Gaithersburg, MD),
rinsed in CMF again, and triturated in 0.004% DNase to
disperse the cells into solution. This cell suspension method
generates a primary culture containing approximately
3–5% tyrosine hydroxylase-positive (TH1) neurons (unpublished observations). Trypan blue was added to a sample of
cell suspension and viewed in a hemocytometer to assess
cell viability and to determine cell counts. Cell suspensions
of greater than 95% viability were diluted for transplantation to a concentration of 60,000 cells/µl in CMF. Cells were
loaded into a 10-µl syringe with a 25-gauge needle and
attached to a stereotaxic needle holder for implantation.
Following behavioral screening, lesioned rats were
grafted with either (A) mesencephalic cell suspensions
alone (i.e., solo mesencephalic graft), (B) mixed striatal
and mesencephalic cell suspensions (i.e., mixed co-graft),
or (C) separate striatal and mesencephalic cell suspensions (i.e., separate co-graft). Mesencephalic cell suspensions alone (group A) and mixed co-grafts (group B) were
transplanted into the dorsomedial region of the striatum
(AP 10.7, ML 12.0, DV 26.5). In the separate co-graft
group (group C), mesencephalic cells were transplanted to
the above coordinates with the striatal suspension transplanted at a 1-mm distance lateral in the same coronal
plane (AP 10.7, ML 13.0, DV 26.5). In all transplant
groups, equal numbers of the same mesencephalic suspension were grafted to ensure initial grafting of an equivalent number of DA neurons. Approximately 180,000 cells
in a 3-µl volume were injected into each implantation site.
In the case of the mixed co-graft, approximately 360,000
cells (180,000 mesencephalon/180,000 striatum) in a 6-µl
volume were injected. Transplant surgeries were conducted in alternating order by group to prevent differences
in DA neuron viability caused by lengthy postdissection
532
C.E. SORTWELL ET AL.
intervals (Brundin et al., 1985). A control group of lesioned
rats with no grafts was also included.
Immunohistochemical analysis
Histological evaluation of surviving, grafted DA neurons
and patterns of neurite outgrowth was performed immediately following the completion of behavioral assessment
(9 weeks). Rats were deeply anesthetized (50 mg/kg,
pentobarbital, i.p.) and perfused intracardially with 4%
paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Each
brain was removed, postfixed for 24 hours, and immersed
in 30% sucrose in phosphate buffer. Fixed brains were
sliced on a freezing microtome (40 µm) and free-floating
brain sections were immunostained by using antisera
directed against TH (Eugene Tech, Ridgefield Park, NJ;
rabbit anti-bovine TH 1:10,000, overnight at 25°C). Sections for immunostaining of bromodeoxyuridine (BrdU)labeled nuclei were exposed to 0.1 N HCl for 30 minutes
prior to primary incubation (Boehringer Manheim, Indianapolis, IN; mouse anti-BrdU 1:500, overnight at 25°C).
Triton X-100 was added to the phosphate buffer (0.3 % for
TH, 0.8% for BrdU) during incubations and rinses to
permeabilize cell and nuclear membranes. The Vector ABC
detection kit employing horseradish peroxidase staining
was used following primary incubation. Manual counts
were made at 2003 magnification in every third 40-µmthick section of TH1 neurons identified within grafts of
each graft recipient. The sum of these counts was then
adjusted according to the method of Abercrombie (1946).
These cell counts were collected to provide information
concerning relative survival of grafted TH1 neurons among
experimental groups. Rats with grafts improperly placed
in the ventral region of the striatum were only included in
the morphological analysis and not included in the behavioral analysis since variations in graft placement have
been demonstrated to influence rotational recovery (Dunnet et al., 1981, 1983; Björklund et al., 1980).
Dopaminergic innervation density
Quantification of the density of TH1 striatal reinnervation from grafted mesencephalon was performed by using
Mocha computer imaging software (Jandel Scientific, San
Rafael, CA). Fields of 200 3 300 µm (at 1003 magnification) immediately lateral to the graft site in all three graft
conditions were measured in the three sections per animal
determined to possess the highest number of surviving DA
neurons. These values were compared with striatal densities 2 mm lateral to graft sites, as well within unlesioned
striatum, lesioned nongrafted striatum, and TH-rich
patches of mixed striatal-nigral co-grafts. Optical density
within the corpus callosum was measured to provide a
control for nonspecific background staining. All measurements were taken by using the same light intensity and
threshold settings to exclude areas of zero density, i.e.,
blood vessels.
Statistical analysis
Statistical analysis was performed by using the SuperANOVA statistics package. ANOVA for repeated measures
was used to analyze the data obtained from rotational
assessment and ANOVA was used to analyze the data from
manual counts of DA neurons and TH1 innervation
densities. The Fisher’s Protected LSD post hoc test was
used to determine significant differences.
Fig. 1. Effect of striatal co-grafts on rotational asymmetry following amphetamine challenge. Groups of animals included: 6-OHDAlesioned rats that received no graft (Lesion Only, n 5 5), mesencephalic solo grafts (Solo Graft, n 5 8), mixed striatal-nigral co-grafts
(Mixed Co-graft, n 5 6), and separate striatal co-grafts (Sep Co-graft,
n 5 6). The average number of ipsilateral rotations per minute
were recorded over a 70-minute interval. Values represent the mean 6
SEM for each group at 2 weeks following 6-OHDA lesion (Baseline)
and 4 weeks and 8 weeks following transplantation. Significant
differences from baseline rotational scores are denoted by asterisks
(P # 0.05).
RESULTS
Rotational asymmetry
Twenty-nine of the 35 6-OHDA-lesioned rats exhibited
average ipsilateral turning rates of 6 turns per minute or
greater after amphetamine challenge. These rats subsequently were assigned to either a transplant or control
group. There were no significant differences in baseline
rotational scores between the different transplant groups
(F [3, 20] 5 0.199; P $ 0.05). At 4 and 8 weeks following
grafting only the separately co-grafted rats (n 5 6) displayed average ipsilateral rotation rates that were significantly recovered compared with baseline (P # 0.05). In
contrast, solo grafted rats (n 5 8) did not display a
significant reduction in amphetamine-induced rotations at
8 weeks following transplantation (P $ 0.05). Similarly,
mixed striatal co-grafted rats (n 5 6) did not display
significant reductions in rotational asymmetry (P $ 0.05).
Lesioned-only rats (n 5 5) showed a trend toward increased ipsilateral rotation rates at 8 weeks; however, this
increase did not achieve significance (P $ 0.05). These
behavioral results are illustrated in Figure 1.
5-Bromo-28-deoxyuridine labeling
BrdU labeling of the mesencephalic and striatal suspensions allowed for clearer visualization of cells in the
grafted regions and proved to be useful in distinguishing
grafted striatal tissue from host striatal tissue. Within all
graft types, BrdU-labeled nuclei of different staining intensities were apparent (Fig. 2).
STRIATAL CO-GRAFTS AND DOPAMINE NEURON SURVIVAL
533
Fig. 2. 5-Bromo-28-deoxyuridine (BrdU) labeling of grafted embryonic mesencephalic and striatal cells. A: Solo mesencephalic graft (ng).
B: Mixed striatal-nigral co-graft (mg). C: Separate nigral (ng) and
striatal (sg) co-graft. D: At higher magnification, individual BrdU-
labeled nuclei of different staining intensities are apparent (arrows) in
the striatal portion of the co-graft. H, host striatum. Scale bars 5 200
µm in C (applies to A–C), 50 µm in D.
Mesencephalic graft morphology
dense TH-immunoreactive patches that often appeared to
form robust pericellular arrays (Fig. 4B). At higher magnifications, DA neurons within the co-grafts appeared to
possess numerous neuritic branches (Fig. 5B).
Separate striatal co-grafts (n 5 7) appeared longer in the
dorsal-ventral axis than their solo counterparts. The mesencephalic portion of the separate co-grafts spanned at
least 360 µm in the rostral-caudal axis. The striatal
portion of the co-graft appeared as an unstained area
approximately 800–1,000 µms lateral to the mesencephalic portion and was only faintly distinguishable from
the host striatum. DA neurons within the mesencephalic
portion appeared to be present in clusters throughout the
graft (Figs. 3C,F, 4C). In a few co-grafts TH-immunoreactive patches were present in the striatal portion of the
co-graft (Fig. 3C,F). A dense halo of TH1 innervation was
observed interposed between the mesencephalic and striatal portion of the separate co-grafts (Fig. 3C,F). This
innervation into the surrounding and intervening host
striatum appeared to be denser and also to innervate a
larger host area than that seen in either the solo or mixed
striatal co-grafted rats. At higher magnifications, TH1
neurites could be observed extending toward the regions of
the striatal graft (Fig. 4C). In general, grafted DA neurons
in the separate co-grafts were similar in individual morphology to the DA neurons within the solo and mixed
striatal co-grafts (Fig. 5C).
Unilateral injection of 6-OHDA produced a near total
absence of TH immunoreactivity within the lesioned host
striatum. All grafted rats (n 5 24) contained visible grafts
with numerous TH1 neurons. In solo mesencephalic
grafted rats (n 5 9), coronal sections containing TH1
neurons spanned at least 240 µm in the rostral-caudal axis
and were visualized typically as thin tracts of DA neurons
surrounded by a distinct halo of TH1 innervation in the
host striatum (Figs. 3A,D, 4A). Individual TH1 neurites
could be traced for a limited distance into the surrounding
host parenchyma (Fig. 4A). At higher magnifications, TH1
neurons within the solo mesencephalic grafts appeared
unipolar or bipolar, possessing a few apical dendrites
and/or axonal branches (Fig. 5A).
Mixed striatal co-grafts (n 5 8) were typically droplet
shaped and occupied a larger portion of the lesioned
striatum than the solo grafts. These grafts were found to
span at least 320 µm in the rostral-caudal axis. TH1
neurons were found to be scattered throughout the graft in
clusters, rather than the usual location around the graft
periphery (Figs. 3B,E, 4B). While a halo of TH1 innervation was apparent in the mixed co-grafts, the majority of
TH1 fibers were visible within the co-graft rather than in
the host striatum (Fig. 4B). The most remarkable component of the mixed striatal co-grafts was the presence of
534
C.E. SORTWELL ET AL.
Fig. 3. Low-power views of solo mesencephalic grafts (A,D), mixed
striatal-nigral co-grafts (B,E) and separate striatal-nigral co-grafts
(C,F) in the denervated rat striatum labeled with antisera against
tyrosine hydroxylase (TH). A,D: Coronal sections are shown through
solo mesencephalic grafts containing a total of 640 and 222 dopamine
(DA) neurons, respectively. B,E: Examples of mixed nigral-striatal
co-grafts are depicted that possessed totals of 2,514 and 1,521 DA
neurons. Dense TH-immunoreactive patches are apparent within the
mixed co-graft (asterisks). C,F: Two separate nigral-striatal co-grafts
are shown. These two grafts contain totals of 1,143 and 1,050 DA
neurons, respectively, in the nigral portions of the separate co-graft.
Dense TH-immunoreactive patches are visible within areas in the
striatal portion of the separate co-graft (asterisks, sg) and robust
TH-positive innervation of the intervening host striatum is also
apparent (arrows). Scale bar 5 300 µm.
Survival of dopamine neurons
neurons initially transplanted. Separate striatal co-grafts
(n 5 7) contained an average of 953 TH1 neurons,
representing an approximate survival rate of 11–18%.
Similarly, mixed striatal co-grafts (n 5 8) contained an
average of 954 surviving DA neurons, a survival rate of
Mesencephalic grafts transplanted alone (n 5 9) displayed an average of 481 surviving DA neurons. This value
represents an approximate survival rate of 5–9% of the DA
STRIATAL CO-GRAFTS AND DOPAMINE NEURON SURVIVAL
535
than lesioned, nongrafted striatum. As suggested by qualitative examination, the intervening striatum between
separately co-grafted striatum and mesencephalon contained a significantly denser innervation of TH1 fibers
than the striatum immediately adjacent to both the solo
and the mixed co-graft groups (P # 0.01). This increased
density between separate striatal-nigral co-grafts reflected a 50% restoration of dopaminergic innervation
compared with unlesioned striatum. When TH fiber innervation in fields further lateral (2 mm) to the graft sites was
examined, no significant differences were noted when
compared with control measurements in lesioned, nongrafted striatum. Lastly, measurements taken within TH1
fiber-rich patches of mixed co-grafts revealed a marked
increase in fiber density, reaching a level 40% greater than
unlesioned striatum. Figure 7 illustrates the differences in
striatal dopaminergic reinnervation from the three graft
types.
DISCUSSION
Fig. 4. Effects of co-grafted striatal target tissue on the directionality and length of grafted mesencephalic dopamine (DA) neurites. A: In
this example of a solo mesencephalic graft, tyrosine hydroxylase
(TH)-immunoreactive neurites (arrows) can be seen extending only a
limited distance into the host striatum. B: In mixed nigral-striatal
co-grafts, whereas a halo of TH immunoreactivity is apparent along
the border of the co-graft (open arrows), much denser TH innervation
(asterisks) remains within the boundary of the graft (host-graft border
demarcated by black on white arrows). C: Long individual DA neurites
(black arrowheads) are readily visible extending from the nigral
portion to the striatal portion of this separate co-graft. sg, striatal
graft. Scale bars 5 100 µm in A (applies to A and B), 50 µm in C.
11–18%, although the number of surviving DA neurons
was more variable than in the separately co-grafted condition. The increased DA neuron survival in the separate
and mixed striatal co-graft groups was significant when
compared with survival rates of the solo mesencephalic
grafts (P # 0.05). Figure 6 illustrates the striatal co-graft
effects on DA neuron viability.
Extent of TH1 reinnervation
of the host striatum
Dopaminergic reinnervation of the host striatum immediately adjacent to all graft types was significantly denser
Previous evidence suggests that striatal co-grafts may
be used to stimulate neurite extension of grafted mesencephalic DA neurons and enhance recovery of lesion-induced
rotational asymmetry. We now have demonstrated that
co-grafted striatal cells also exhibit a trophic effect on
grafted DA neurons reflected by increased DA neuron
survival. Separate striatal co-grafts and mixed striatal
co-grafts displayed a significant twofold increase in DA
neuron survival when compared with solo mesencephalic
grafts. The present findings also confirm earlier reports in
which striatal co-grafts placed at a short distance from
mesencephalic grafts appear to attract dopaminergic neurites (Yurek et al., 1990), significantly enhancing innervation of the intervening lesioned host striatum in the
process. These morphological effects of striatal co-grafts
appear to influence behavioral parameters directly. Separate co-grafts produced a significant reduction in rotational asymmetry at both 4 and 8 weeks after transplantation. Neither solo grafts nor mixed striatal co-grafts
displayed significant behavioral improvement at these
time intervals.
Cell survival of DA neurons is enhanced when mesencephalic neurons are co-cultured with striatal cells (Hoffman
et al., 1983; Dong et al., 1993), intact or denervated striatal
extracts (Dal Toso et al., 1988; Niijima et al., 1990; Carvey
et al., 1996), or striatal oligodendrocyte-type-2 astrocyte
(O-2A) progenitor cells (Takeshima et al., 1994). Although
this effect has been observed in vitro, it had never been
documented to occur in the co-graft situation prior to our
initial reports (Sortwell et al., 1996, 1997). One reason for
this discrepancy could be the inclusion of nontarget tissue
in the striatal portion of the co-graft in previous studies. In
those co-graft studies (Olson et al., 1979; Brundin et al.,
1986; DeBeaurepaire and Freed, 1987; Dunnett et al.,
1989; Yurek et al., 1990; Sladek et al., 1993), a distinction
might not have been made between the dissection of the
lateral ganglionic eminence (LGE) to the exclusion of the
medial ganglionic eminence (MGE). The MGE develops
into the globus pallidus (Pakzaban et al., 1993; Deacon et
al., 1994), an inappropriate target for developing nigral
neurons. Inclusion of the MGE in the striatal portion of the
co-graft may have diluted the effects of LGE-derived
trophic factors, particularly in cell suspension grafts where
the number of co-grafted cells remains constant. Our
present results illustrating the trophic effect of striatal
536
Fig. 5. High-power photographs depicting the individual cellular
morphology of dopamine (DA) neurons within solo mesencephalic
grafts (A), mixed striatal-nigral co-grafts (B), and separate striatalnigral co-grafts (C). A: Tyrosine hydroxylase (TH)-positive neurons
(arrows) in this solo mesencephalic graft possess few apical dendrites
and/axonal branches. B: DA neurons within this mixed nigral-striatal
Fig. 6. Striatal co-grafts increase the survival of grafted mesencephalic dopamine (DA) neurons. Groups of animals included: mesencephalic solo grafts (Solo Graft, n 5 9), mixed striatal-nigral co-grafts
(Mixed Co-graft, n 5 8), and separate striatal co-grafts (Sep Co-graft,
n 5 7). Tyrosine hydroxylase (TH)-positive neurons were counted in
every third 40-µm section and the sum of these counts corrected
according to the method of Abercrombie (1946). Values represent the
mean 6 SEM for each group at 9 weeks following transplantation.
Significant differences from the solo mesencephalic graft transplant
group are denoted by asterisks (P # 0.05).
co-grafts have been confirmed by a very recent manuscript
published during this manuscript’s review process. Mixed
nigral-LGE co-grafts were also demonstrated to possess an
increased survival of DA neurons (Costantini and SnyderKeller, 1997).
C.E. SORTWELL ET AL.
co-graft (arrows) are often obscured by dense neuritic branching and
fiber ingrowth into TH-immunoreactive patches (asterisk). C: Neuritic
branching of DA neurons (arrows) within the nigral portion of this
separate nigral-striatal co-graft appears similar to the branching of
DA neurons in the solo mesencephalic grafts depicted in A. Scale bar 5
100 µm.
Our behavioral and viability results appear to disagree
with the first of two studies conducted by Costantini et al.
(1994). In this study, mixed striatal co-grafts did produce
an augmented turning response in a bilaterally lesioned
model without significant effects on DA neuron viability. In
fact, a trend toward decreased survival of DA neurons in
the mixed striatal-nigral grafts was reported. Inconsistencies between their results and ours may be due to methodological differences: different embryonic age of the transplanted tissue and a different behavioral model. For
example, the bilateral 6-OHDA model employed in their
study allows for a larger range with which to measure the
efficacy of the transplants. It is possible that the behavioral effects of mixed striatal co-grafts are too small to be
detected in the unilateral 6-OHDA model. The most striking contrast between our experimental design and their
earlier design is the extreme difference in the number of
cells implanted. In their 1994 study a total of 500,000–
1,000,000 cells were implanted compared with our 180,000–
360,000 cells. Perhaps a larger graft volume is a suboptimal grafting condition that offsets any trophic effects of the
striatal co-graft through increases in reactive gliosis and
by limiting the access of grafted neurons to nutrients from
host blood vessels. Nikkhah et al. (1994) found that the
microtransplantation of an equal amount of mesencephalic cells in smaller multiple deposits enhances grafted
DA neuron viability almost 300% and an experiment in
which the vascularization of grafts was enhanced also
reported increases in graft volume (Finger and Dunnett,
1989). The most compelling evidence that the larger graft
volume used in the Costantini et al. (1994) study may have
proved detrimental to DA neuron survival is made available by results of their second, more recent study. When
STRIATAL CO-GRAFTS AND DOPAMINE NEURON SURVIVAL
Fig. 7. Separate striatal co-grafts increase the dopaminergic reinnervation of the adjacent host striatum. Fields (1003) within the host
striatum immediately lateral to the transplant site were measured for
density of TH1 reinnervation. Groups include sections from: mesencephalic solo grafts (Solo Graft, n 5 5), mixed striatal-nigral co-grafts
(Mixed Co-graft, n 5 5), separate striatal co-grafts (Sep Co-graft, n 5
5), lesioned striatum only (Lesioned, n 5 6), and unlesioned striatum
(Unlesioned, n 5 6). Values represent the mean 6 SEM for each group
at 9 weeks following transplantation. All transplant groups and the
unlesioned striatum possessed significantly denser TH1 striatal
innervation than lesioned striatum. Significant increases in TH1
striatal density compared with both the solo mesencephalic graft
transplant group and the mixed co-graft group are denoted by
asterisks (P # 0.01).
grafted mesencephalic cell number was decreased (from
500,000 to 280,000) DA neuron survival in the mixed
striatal co-graft condition increased from three-to-fourfold compared with mesencephalon grafted alone (Costantini et al., 1997).
Dense TH-immunoreactive patches in mixed striatal
co-grafts were observed in the present study, confirming
previous observations (Jaeger, 1986; Costantini et al.,
1994). Both a contact-dependent phenomena as well as
diffusible substances have been implicated in the enhancement of mesencephalic DA neuron survival, whereas the
effects on nerve terminal differentiation are attributed to
direct contact with striatal target neurons (Dong et al.,
1993). These in vitro findings parallel what we observed in
our present study. Separate co-grafts, in which diffusible
communication is maintained, but direct contact between
striatal and mesencephalic cells is abolished, produced
increased DA survival. However, mixed co-grafts, in which
cellular contact between the two tissue types occurs, both
exhibited increased DA neuron survival and contained
zones of intensely TH immunoreactive patches within the
graft.
Whereas it was not surprising that the separate striatal
co-grafts displayed significant behavioral recovery given
that there is a reported correlation between DA neuron
survival and behavioral recovery (Rioux et al., 1991), what
was unexpected was the lack of behavioral improvement in
the mixed co-graft group. This may be related to a prefer-
537
ence of the grafted DA neurons in the mixed condition to
innervate the co-grafted striatal cells rather than the host
striatum (DeBeaurepaire and Freed, 1987). In fact, TH1
innervation within patches of mixed striatal co-grafts was
extremely dense, exceeding the fiber density of the intact
host striatum by 40%. However, enhanced reinnervation of
the host striatum does not occur with the mixed co-grafts
when compared with solo grafts. Thus, the separate striatal co-graft approach seems optimal to enhance both DA
neuron survival and reinnervation of the host striatum.
There is abundant evidence in vitro that striatal target
tissue exerts tropic effects on the directionality and length
of mesencephalic DA neurites. In the presence of striatal
cells, mesencephalic cells show stimulated development
(Prochiantz et al., 1979) and enhanced DA uptake, synthesis, and release (Prochiantz et al., 1979; Daguet et al.,
1980). Additionally, mesencephalic neuronal processes are
lengthened by co-culturing with striatal cells and soluble
striatal extracts (Hemmendinger et al., 1981; Tomozawa
and Appel, 1986). The degree of dopaminergic innervation
to the denervated striatum and subsequent integration
into the host striatal circuit also is considered crucial to
establishing graft efficacy (Björklund et al., 1987; Björklund, 1992). Since the diffusion of DA is a restricted
phenomenon (Sendelbeck and Urquhart, 1985), tonic diffusion from grafted DA neurons can only replenish a limited
area of the striatum. A common finding in rodents is that
DA neuron fiber outgrowth is limited to the immediate
vicinity of the graft. In investigations in rodents where DA
neuron fiber density and length is reported, the greatest
lengths that fibers are seen penetrating is only 1–2 mm
into the host caudate nucleus (Freed et al., 1980; Freed,
1983; Stromberg et al., 1985; Mahalik et al., 1985). Furthermore, the average DA terminal density in the graftreinnervated striatum for a graft containing 1,000 DA
neurons has been estimated to be around 40% of normal
(Doucet et al., 1990; Manier et al., 1991). In the present
study, the separate striatal co-grafts produced significantly enhanced TH1 neurite outgrowth into the surrounding striatum, particularly in host striatal regions interposed between the mesencephalic and striatal grafts,
when compared with solo or mixed co-graft neurite outgrowth. This separate co-graft-induced increase in reinnervation density reached an average level of 50% of intact
striatum. These results confirm a tropic effect of the
separate striatal co-grafts on both the directionality and
density of TH1 innervation, as suggested previously (Yurek
et al., 1990).
The only co-grafted cells used in the present study were
striatal cells; therefore whether or not other co-grafted
target and nontarget cells can exert trophic effects is a
valid question. Data generated in previous studies suggest
that, compared with other target and nontarget tissue,
striatal target tissue exerts the maximal effect on both
tropic and trophic parameters in mesencephalic grafts and
cultures. For example, maximal DA neuron axonal maturation and upregulation of DA transporter mRNA occurs
when mesencephalon is co-cultured with striatum, with
significantly smaller effects elicited by target cortical cells,
and no effect exerted by nontarget rostral tectum (Hemmendinger et al., 1981; Perrone-Capano et al., 1996).
Similar effects were reported on DA neuron survival where
again striatal co-culturing induced maximal effects on DA
neuron survival when compared with the lesser effects or
538
nonexistent effects of target cortex and nontarget cerebellum, hippocampus, olfactory bulb, and liver (Tomazawa
and Appel, 1986; Dong et al., 1993). In rats, only nigral
tissue and cells co-grafted with striatum and not other
tissues including cerebellum, cortex, and spinal cord were
effective in promoting extensive fiber outgrowth and islands of intense catecholamine fluorescence (Brundin et
al., 1986; Jaeger, 1986; Dunnett et al., 1989). These many
experiments provide evidence to support the concept that
the effect observed in the present experiment on DA
neuron survival and fiber outgrowth is specific for the
co-grafted striatal target cells.
Separate and mixed striatal co-grafts had equivalent
effects on enhancing mesencephalic DA neuron survival.
Therefore it is likely that the striatal co-graft serves as a
source of diffusible neurotrophic substances that are not
provided by the adult host striatum, or are provided in
increased amounts by embryonic striatum. Several known
growth factors, such as basic fibroblast growth factor
(bFGF; Knusel et al., 1990), epidermal growth factor (EGF;
Casper et al., 1991), insulin-like growth factor 1 (IGF-1;
Knusel et al., 1990), brain-derived neurotrophic factor
(BDNF; Beck et al., 1993), neurotrophin-3 (Hyman et al.,
1993), neurotrophin-4/5 (Hynes et al., 1993), and glial cell
line-derived growth factor (GDNF; Lin et al., 1993) have
been demonstrated to promote survival of DA neurons to
varying degrees, acting either directly or indirectly. Several known trophic factors, such as BDNF (Spenger et al.,
1995), neurotrophin-3 and neurotrophin-4/5 (Hyman et
al., 1994), and GDNF (Lin et al., 1993) have been demonstrated to promote survival of DA neurons in vitro to
varying degrees. However, to date, only GDNF has been
localized specifically to striatal target areas during development (Schaar et al., 1993; Stromberg et al., 1993;
Choi-Lundberg and Bohn, 1995). In the adult striatum,
high to moderate expression of GDNF, platelet-derived
growth factor (PDGF), and transforming growth factor
(TGF) has been detected (Sasahara et al., 1991; Seroogy et
al., 1991; Unsicker et al., 1991; Yeh et al., 1991; Springer et
al., 1994; Blum and Weickert, 1995; Trupp et al., 1997).
Extremely low levels of BDNF are detected in the adult
striatum with immunocytochemistry; however mRNA levels indicate that BDNF is not present in adult striatum
unless seizures are induced (Schmidt-Kastner et al., 1996)
or dopaminergic stimulation occurs (Okazawa et al., 1992).
This suggests that in the adult, levels of BDNF are not
constant and that production of BDNF may increase to
play a role in a lesioned or injury-induced state. It also is
possible that undefined striatal-derived trophic factors
may be released by the striatal co-graft, such as those
involved in the trophic effects of striatal extracts (Dal Toso
et al., 1988) and striatal O-2A progenitor cells (Takeshima
et al., 1994).
Since multiple trophic factors can influence nigral DA
neurons, striatal co-grafts may provide grafted DA neurons with a continuous source of endogenous factors at
their appropriate developmental timepoints. Studies of
infusion of a variety of growth factors in conjunction
with neural grafts reveal enhanced graft viability both in
oculo and in the denervated striatum (Giacobini et al.,
1990, 1991; Steinbusch et al., 1990; Stromberg et al., 1993;
Takayama et al.,1995; Rosenblad et al., 1996; Wang et al.,
1996). As it may prove difficult to infuse appropriately the
identical cascade of trophic factors that are provided by the
C.E. SORTWELL ET AL.
striatal co-graft, the co-graft approach may prove comparable or even superior in exerting trophic/tropic effects.
The mechanism by which co-cultured and co-grafted
striatal cells enhance the survival of DA neurons may be
by supplying trophic factors that protect DA neurons from
programmed cell death, i.e., apoptosis. These factors may
be unavailable in the adult host striatum. The ‘‘neurotrophic factor hypothesis’’ suggests that survival of developing neurons depends on the secretion of specific factors
from target cells that the neurons innervate. These trophic
signals appear to exert their supportive effect by suppressing an intrinsic cell ‘‘suicide’’ program (Raff et al., 1993),
i.e., an apoptotic program that engages when such signals
are absent. In cultures of sympathetic neurons, nerve
growth factor (NGF) deprivation induces apoptosis,
whereas NGF-exposed neurons are protected (Deckwerth
and Johnson, 1993). Exposure of mesencephalic cultures to
GDNF reduces the rate of apoptotic cell death (Clarkson et
al., 1995). In the nigrostriatal system, lesions of the
striatum during development result in apoptosis in the
substantia nigra (Macaya et al., 1994). Until recently, it
was assumed that cell death in mesencephalic grafts
primarily was necrotic, i.e., the result of inadequate vascularization or trauma to the graft during tissue dissection or
transplantation. However, apoptosis has been suggested to
be a normal part of graft development, particularly at
early times following grafting (Mahalik et al., 1994;
Sortwell et al., 1997). These findings suggest that grafts
may be under the guidance of the same genetic mechanisms that function in naturally developing neural systems and may offer insights into how striatal co-grafts
enhance survival of grafted mesencephalic neurons through
the inhibition of apoptosis.
The development of strategies that increase grafted DA
neuron survival and promote innervation of the host
striatum are critical to the success of neural transplantation for PD. Currently, the survival of DA neurons in
human grafts is estimated to be about 5–10% (Olanow et
al., 1996) and although neurite extension from an average
graft can extend through a large cross-sectional area
within the caudate, the density of this reinnervation
approximates only 40% of that seen in the intact striatum
(Doucet et al., 1990; Manier et al., 1991). These shortcomings suggest that the use of multiple donors and multiple
placement sites may be necessary for enhanced and sustained therapeutic benefits. The use of multiple donors
may be impractical and lead to other problems, such as
immunological rejection. An alternate strategy is to increase the survival and neurite outgrowth of grafted
DA neurons. Taken collectively, our data suggest that
1) embryonic striatal co-grafts exert a trophic effect on DA
neurons by significantly augmenting cell survival, 2) striatal co-grafts placed separately at short distances also exert
a tropic effect that significantly enhances dopaminergic
innervation of the lesioned striatum, and 3) enhanced
neurite outgrowth and augmented DA neuron survival
may participate in enhanced restoration of neural circuitry necessary for behavioral recovery from parkinsonism.
ACKNOWLEDGMENTS
We are grateful for the excellent technical advice and
assistance of Barbara Blanchard and Brian Daley. This
work was supported by NIH grants F32 NS09646–01
STRIATAL CO-GRAFTS AND DOPAMINE NEURON SURVIVAL
(to C.E.S.), RSA K05-MH 00643(to D.E.R.), and AG10851
(T.J.C.).
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increase, survival, dopamine, embryonic, striatum, neurons, grafted
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