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Bilateral fetal nigral transplantation into the postcommissural putamen in Parkinson's disease.

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Bilateral Fetal Nigral Transplantation
into the Postcommissural Putamen
in Padunson’s Disease
Thomas B. Freeman,”? C. Warren Olanow, MD,”“ Robert A. Hauser, MD,$ G. Michael Nauert, M D , t t
Donald A. Smith, MD,” Cesario V. Borlongan, MD,” Paul R. Sanberg, MD,”tB Douglas A. Holt, MD,n
Jeffrey H. Kordower, PhD,$$ Francois J. G. Vingerhoets, MD,§§ Barry J. Snow, MD,@
Donald Calne, MD,@ and Lisa L. Gauger, BSc$
We performed fetal nigral tansplantations in 4 Parkinson’s disease (PD)patients. Solid grafts were bilaterally implanted
into the postcommissural putamen using 3 to 4 donors per side aged 6%to 9 weeks postconception. Transplant deposits
were separated by no more than 5 mm in three dimensions. Cyclosporine was employed for a total of 6 months.
Patients were evaluated at baseline and at 1, 3, and 6 months postoperatively. Striatal 18-fluorodopa uptake was
assessed by positron emission tomography at baseline and at 6 months postoperatively. The procedure was well
tolerated in all patients. One patient had a clinically asymptomatic superficial cortical hemorrhage along the needle
tract and a second had transient postoperative confusion and hallucinations. All patients experienced clinically meaningful benefit. Significant improvement ( p < 0.05) was detected in total UPDRS score during the “off” state, SchwabEngland disability score during the “off” state, percent “off” time, and percent “on” time with dyskinesia. Increased
striatal fluorodopa uptake was observed bilaterally in each patient, with mean increases of 53% on the right ( p =
0.01) and 33% on the left ( p = 0.08). Our study demonstrated clear and consistent improvement in clinical features
and striatal fluorodopa uptake following fetal tissue transplantation in patients with advanced PD whose condition
was not improved preoperatively by drug manipulation. These preliminary results are encouraging and support further
studies to evaluate grafting strategies as a therapy for PD.
Freeman TB, Olanow, CW, Hauser RA, Nauert GM, Smith DA, Borlongan CV, Sanberg PR, Holt DA,
Kordower JH, Vingerhoets FJG, Snow BJ, Calne D, Gauger LL..Bilateral fetal nigral transplantation
into the postcommissural putamen in Parkinson’s disease. Ann Neurol 1995;38:379-388
Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is associated with loss of neurons
in the substantia nigra pars compacta and a decline in
striatal dopamine. Current therapy is based primarily
on a dopamine replacement strategy using the dopamine precursor levodopa. However, chronic levodopa
therapy is associated with adverse effects and increasing disability. Therefore, there has been a search for
alternative therapies that can reverse functional disability in patients with advanced P D who cannot be satisfactorily controlled with existing medications. Neural
grafting is a rational consideration as a therapy for P D
because (i) P D is associated with a relatively wellcharacterized and specific dopamine neuronal degeneration, (ii) dopamine replacement therapy provides dramatic clinical benefit, (iii) dopamine neurons subserve
a modulatory function and under physiological conditions provide tonic stimulation of striatal dopamine receptors, and (iv) there is a well-defined target area for
transplantation. Several studies demonstrated the capacity of intrastriatal grafts of embryonic dopaminergic
neurons to survive, produce dopamine, form connections with host neurons, and provide long-lasting amelioration of motor dysfunction in animal models of parkinsonism (reviewed in [l}).These studies led to the
initial clinical trials of fetal nigral transplantation as a
treatment for PD 12-71,However, the observed benefits have been inconsistent and for the most part modest. This may relate to the specific transplant variables
employed such as target site, number of donors, donor
age, and distribution of implanted tissue within the target region. We performed fetal nigral transplantation
From the Departments of ‘Surgery, ?Pharmacology and Experimental Therapeutics, :Neurology, $Psychiatry, and ‘Medicine, University of South Florida, Tampa; ““Department of Neurology, Mount
Sinai Medical Center, New York, NY; -?twoman’s Center, Tampa,
FL; $$Department of Neuroscience, Rush Presbyterian Medical
Center, Chicago, IL; and @Division of Neurology, University of
British Columbia, University Hospital, Vancouver, British Columbia, Canada.
Received Feb 21, 1995, and in revised form Apr 18. Accepted for
publication May 4,1995.
Address correspondence to D r OIanow, Department of Neurology,
Mount Sinai Medical Center, One Gustave L. Levy Place, Box 1137,
New York, NY 1o029,
Copyright 0 1995 by the American Neurological Association
379
in PD patients using a protocol designed to maximize
the likelihood of graft survival and dopamine reinnervation of the target site based o n existing information. Solid fetal nigral grafts were bilaterally implanted
into the posterior (postcommissural) putamen using 3
to 4 donors per side aged 6Il2 t o 9 weeks postconcepd o n (PC). Fetal deposits were separated by n o more
than 5 m m in three dimensions. We present the clinical
and positron emission tomography (PET) results at 6
months in the first 4 patients w h o have undergone fetal
nigral transplantation according t o this protocol.
Materials and Methods
Patient Selestion
Patients with PD were selected from the Movement Disorder Center at the University of South Florida. PD was diagnosed according to the core assessment program for intracerebral transplantation (CAPIT) protocol [60] and included
resting tremor, rigidity, bradykinesia, and a good response
to levodopa therapy. Entry criteria included stable dose of
levodopaicarbidopa for a minimu.m of 3 months prior to entry into the study, Hoehn-Yahr stage of 111 or less during
the “on” state, clinically meaningful disability during the “off’
state, and predictable motor fluctuations. Exclusion criteria
included dementia; a previous intracranial procedure; a clinically significant medical, neoplasti’c,or infectious disease; and
a clinically significant laboratory abnormality. Patients were
screened serologically prior to entry into the study for the
following: human immunodeficictncy virus type 1 (HIV-1;
antibody [Ab], antigen [Aglj, HIV-2 (Ab), human T-cell
lymphotropic virus type 1 (HTLV-I; Ab), hepatitis A virus
(HAV; IgM), hepatitis B ( H B :surface Ag, H b core Ab),
hepatitis C virus (HCV; Ab), cytomegalovirus (CMV; IgM,
IgG), toxoplasma (IgG, IgM), syphilis (rapid plasma reagent
[RPR]), and herpes simplex virus (HSV; IgG). Patients were
not included in the study if there was serological evidence
of infection with syphilis, HIV, HTLV, or hepatitis. Patients
who were CMV or toxoplasma negative were also excluded
to eliminate the risk of transplanting these common contagious agents to a naive recipient.
Stiidy Design
Patients who met entry criteria and signed informed consent
were entered into the study. Solid grafts of fetal human mesencephalon derived from donor embryos aged 6‘/2to 9 weeks
PC were implanted bilaterally into the postcommissural putamen in staged procedures separated by approximately 4
weeks. Implanted tissue was derived from 3 to 4 embryos
per side and placed so that deposits were distributed at
approximately 5-mm intervals throughout the threedimensional configuration of the postcommissural putamen.
Cyclosporine (CsA) was employed for approximately 6
months. Patients were evaluated at baseline and at 1, 3, and
6 months postoperatively. Each evaluation included UPDRS,
Hoehn-Yahr, and Schwab-England assessments during both
“on” and “off” states in accordance with the CAPIT protocol.
Timed motor tests (supination-pronation,stand-walk-sit, step
seconds, Purdue Peg-Board, finger tapping, and hand-arm
movements between two points) were also performed during
“on” and “off’ states. Evaluations during “off’ states were
performed in the early morning, approximately 12 hours
380 Annals of Neurology
after the previous dose of medication (“practically defined
off”). “On” scores were determined at the time of peak response to the morning dose of levodopa. Percent “on” time
with and without dyskinesia was calculated based on a selfassessment calendar. All evaluations were performed by the
same investigator and a stanilartlized video examination was
obtained at the time of each evaluation. Striatal 18fiuoroclopa (FD) uptake on PET was assessed preoperatively
and 6 months postoperatively.
Tissue was obtained from women undergoing elective abortion in accordance with federal, state, and local laws; National
Institutes of Health ( N I H jguidelines; and the Uniform Anatomical Gift Act as adapted by the Stare of Florida. liiformed
consent for the use of the cadaver fetus was sought only
after the patient had signed surgical consent for abortion. N o
monetary or other inducement was provided to the patient,
abortionist, o r abortion clinic. Donation of embryonic tissue
was made without restriction as to individual recipient or
whether the tissue would be employed in human trials. A
low-pressure aspiration abortion technique was employed using sterile technique [XI. There was no alteration in the indicarion, timing, or methodology of the abortion procedure.
Each donor was screened using maternal blood for HIV-1
(Ab, Agj. HIV-2 (Ab), HTLV-I (Ab), HAV (IgM), hepatitis
B virus, ( H B surface Ag, H B core Abj, HCV (Ab), CMV
(IgM), toxoplasma (IgM), syphilis (RPRj, and HSV (IgIcI).
In addition, fetal tissue immediately adjacent to the mesencephalon was cultured for aerobic and anaerobic bacteria,
yeast, HSV, anci CMV. Donors were excluded if there was
evidence of infection with HIV-I or -2, HTLV-I, syphilis,
or hepatitis B or C. In addition fetal tissue was not employed
if the mother had a history of prostitution, injection drug
abuse, more than 10 transfusions i n the preceding year, evidence of active vaginal herpes, a temperature higher than
lOO.j”F, o r a white blood cell (WBC) count higher than
15,000imni‘.
Donor age was staged according t o the atlas of O’Rahilly
and Muller [‘,I for donors younger than PC week 8 and for
older donors by a combination of foot length, heel length,
and greatest length [ l o , 111. The mesencephalon was dissected and stored in “hibernation medium” at 8°C for up to 2
days. Immediately before transplantation, the mesencephalon
was further dissected into ,/,-mmi pieces. The final tissue
dissection was performed in chilled Hank’s balanced salt solution (HBSS, Gibco).
LoizlizatioTi
of
Turget
Pariencs were placed in a standard CRW magnetic resonance
imaging (MRI)-compatible stereotactic frame using local anesthesia. The putamen was visualized on a high-field-strength
MRI ( 1.5 T) using a fast spin-echo sequence (TR 3200iTE
17imatrix 256 x 256ifield of view 3 cm). Axial images were
obtained using contiguous 3-mm sections extending from below the putamen to above the caudate in a plane such that a
perpendicular line passing through the mid putamen would
intersect the coronal suture. Coronal sections were obtained
in 3-mm intervals in a plane perpendicular to the axial cuts
beginning 3 cm anterior to the coronal suture and progressing caudally through the putamen. Target sites for im-
Vol 38 No 3 September 1995
plantation were based on a determination of the “zero point”
in the putamen defined as the point midway between its
rostra1 and caudal aspects on the lowest axial section. This
point was the initial target site for tansplancation and permitted identification of all other target sites based on a single
stereotactic measurement.
SzIrgicul Procediwe
After target sites had been determined, patients were transferred to the operating theater and sedated with fentanyl and
Propofol. The airway was protected with a laryngeal mask
airway. A grid array in the shape of the putamen, with holes
at 5-mm intervals, was placed onto the stereotactic frame.
The grid array was aligned so that its axial plane was parallel
to the plane of the axial MRI and its longitudinal axis parallel
to the axis of the midline of the brain. While the patient was
under local anesthesia, a burr hole was placed to accommodate the site of entry of the stereotactic transplant needle.
Initially, a cortical entry point 3 cm lateral to midline was
employed. After the first operation on the third patient (the
fifth procedure), a cortical entry point 1.5 cm from the midline was utilized so that the superficial needle tract remained
entirely within the superior frontal gyrus. The transplant needle consisted of an outer cannula with a diameter of 1.5 mm
and an inner cannula with a diameter of 1.2 mm tapering to
0.9 mm (the size of a 20 gauge needle). Tissue was microdissected into l/i-mmipieces and aspirated into the stereotactic
needle using a 100-p.1 Hamilton syringe in a volume of approximately 16 to 20 p.1 of HBSS. The transplant needle was
then placed into the “zero point” on the grid array and directed to the “zero point” of the putamen. Each needle tract
contained tissue from one half of a mesencephalon ( = one
substantia nigra). Within each needle tract, four deposits in
a volume of approximately 4 to 5 p.1 were implanted at approximately 5-mm intervals. Injections were made at a rate
of 2 p,1/30 sec with an interval of 1 minute between deposits.
Following the last deposit in each tract, 3 p.1 of HBSS was
injected, and the needle was left in place for 2 minutes to
avoid graft withdrawal. Subsequent needle trajectories utilized the same burr hole and cortical entry point by angling
the grid array in the sagittal and coronal planes. A total of
six to eight needle tracts per side were made.
Periuperatiaie Management
Immunosuppression with CsA, 6 mgikglday, was initiated 2
weeks before the first transplant procedure, reduced to 2 mg/
kgiday 2 weeks after the second procedure, and discontinued
after 6 months. Renal function was monitored on a routine
basis. Antibiotic treatment with piperacillin ( 3 g intravenously every 6 hours), ceftazidime (Fortaz) (2 mg intravenously every 8 hours and fluconazole (200 mg intravenously/
orally every morning) was initiated immediately before surgery and continued for S days or for a full course of appropriate treatment if cultures were positive. Vancomycin (1 gm
intravenously every 12 hours) was used in place of piperacillin if patients were allergic to penicillin. MRI was performed
on the first postoperative day. Following surgery, antiparkinsonian medications were reinstituted at their preoperative
dose and efforts were made to maintain this dose throughout
the study except in the case of toxicity or an inadequate
clinical response.
P o i iflnii Emix.)Zon Tonzog~wphj
PET was performed at the University of British Columbia
on an ECAT 953-3 1B camera according t o a standard protocol [ 121. Briefly, medication was withheld overnight and the
patient was given 200 mg of carbidopa orally 1 hour before
scanning. The patient was positioned in the scanner using
gantry-mounted lasers to align the orbitomeatdl line with the
detector rings. Head movement was restrained by an individuallv molded thermoplastic face mask that was used for subsequent scans. A transmission scan was performed to correct
for attcnuation of radioactivity. FD (3-5 mCi) was injected
intravenously and 12 sequential scans were made at 10minute intervals. Serial arterial blood samples were obtained
to define peak level and elimination time course of blood
raclioactivity. The peripheral metabolism of FD to 3-0methylfluorodopa was determined using an alumina extraction method 1131. Regions of interest (ROls) were placed
on an integral image generated from the last 60 minutes
of emission data. Four circular ROIs measuring 8.8 mm in
diameter were positioned manually along the long axis o f
each striatum so that one covered the head of the caudate
and three covered the putamen. Three background circular
ROIs were placed on the occipitoparietal cortex on each side,
taking care t o avoid the ventricles. Similar sets of ROIs were
placed o n the five contiguous slices where the striatum was
best seen. The scans were analyzed to determine the striatal
FD uptake rate constant ( K , )using the method of Patlak and
colleagues [ 141. The K, for each ROI in each slice was averaged to determine the mean K , for the right and left caudate
and putamen. T o determine the significance o f changes between scans in individual subjects, we used the data generated in separate studies of the reprociucibility of FD PET
in normal and P D patients scanned with a similar protocol
[ i s , 161.
Stutistical AnaIyriJ
For each variable, a repeated-measures analysis of variance
was performed using PROC GLM in the SAS statistical software package. To test for the presence of an overall time
effect on each variable, a univariate adjusted F test for withinsubject effects was used. This test was followed up by testing
each of the means at 1, 3, and 6 months postoperatively for
a difference from the mean at the preoperative baseline visit
by using an I; test derived from the repeated-measures
model.
Results
Four patients underwent bilateral transplant procedures according to the above-described protocol and
have been followed for 6 months. Patient data at the
time of entry are summarized in Table 1. Surgery was
well tolerated and patients were discharged from the
hospital within 48 to 72 hours. All cultures of fetal
tissue (n = 29) were negative and antibiotics were
discontinued after 5 days. MRI was performed the day
following surgery. No evidence of blood was detected
within any of the transplant sites. MRI studies with
gadolinium were performed after the first four procedures and did not demonstrate areas of enhancement
Freeman et al: Fetal Transplantation
381
Table 1. Baseltne Charactertsttcs
Age Duration UPDRS
Patient
(onloffl
No
Sex (yr) (yr)
HoehnSchwab- Yahr
% “off” % “on”
England score
time
wldyskinesia score
(onlofo
Selegiline
Levodopal
Hydrochloride
(Sinemet) Pergolide (Deprenyl)
(mglday) (mglday) (mglday)
48
30
30.5
28
1,700
800
700
800
~
1
2
3
4
M
M
F
M
UPDRS
=
59
39
61
50
8
12
22
15
17/78
13170
27.5168.5
221104.5
19.5
30
69.5
57
1.513
113
214
213
7.5
10
5
5
0
0.75
1.5
1.5
unified Parkinson’s disease rating scale.
suggestive of a breakdown in the blood-brain barrier
(BBB).
A comparison of clinical scores at baseline and at 6
months following the transplant procedure is shown
in Table 2. Significant improvement ( p < 0.05) was
detected in total UPDRS score during the “off’ state,
Schwab-England disability score during the “off’ state,
percent “off” time, and percent “on” time with dyskinesia. Percent “off’ time was reduced from a mean of
34.1% at baseline to 12.1% at the time of the final
visit. Dyskinesia was markedly reduced in all patients
and disappeared in 2. These blenefits were observed in
each patient and generally be,gan between postoperative months 1 and 3 (Fig. 1). Painful “off’ period dystonia, present at baseline in Patients 1, 2, and 4, virtually
disappeared by the +month postoperative visit. Gait
was improved during “off’ periods in 3 patients and a
marked reduction in falling was detected in 1. The
number and duration of freezing episodes, noted preoperatively in Patients 2 and 4.,were reduced following
surgery. No significant benefit was detected in any of
the timed motor functions. Each patient was believed
to be substantially improved on global assessment performed by both patient and physician. The daily levodopa dose was reduced at 3 months in Patient 1 from
6 to 5 tablets of Sinemet CR 50/200 and from 5 to 4
tablets of Sinemet 25/100 because of mild confusion.
In Patient 4, the daily dose of levodopa was changed
from 200 mg four times a day administered as Sinemet
CR to 150 mg four times a day administered as regular
Sinemet. The dose and timing of levodopa and other
antiparkinsonian drugs were otherwise not changed
during the course of this study.
The results of FD PET in individual patients at baseline and at 6 months are shown in Table 3 and illustrated in Figure 2. Bilateral iincreases in puraminal Ki
were observed in each patient. Mean putaminal K, increased by 53% on the right ( p = 0.012, t test, twotailed) and by 33% on the left ( p = 0.08). There were
smaller but nonsignificant increases in the caudate K,’s.
Patient 1 experienced confusion and hallucinations
beginning 3 weeks after the :second transplant procedure, possibly due to subclinical seizure activity. Men-
382
85150
90165
90140
85150
Annals of Neurology V~ol 38
Table 2.Cornpartson of Mean ( & standard error of mean)
BaJeline and &Month Scores Following Fetal Ntgral
Trampla n tat t v n
UPDRS “on”
ADL
Motor
Total
UPDRS “off’
ADL
Motor
Total
% “Off’ time
‘%
“On”
, time with
Baseline
6 Months
p Value
7.3 t 1.1
12.6 t 2.6
19.9 t 3.1
5.9 & 1.6
10.1 +- 2.4
16.0 ? 3.7
NS
NS
NS
32.6
47.6
80.3
34.1
44.0
t 1.4
t 7.4
?
8.3
t 4.7
t 11.6
20.1 lr 1.2 0.05
37.9 & 10.1 NS
58.0 2 10.5 0.05
12.1 5 3.7
0.05
3.8 & 2.4
0.05
dyskinesia
Hoehn-Yahr
1.6 t 0.2
1.4
5
0.2
NS
3.3 -+ 0.3
2.8
&
0.3
NS
87.5 t 1.4
87.5
?
2.5
NS
51.3 t 5.2
72.5
i
1.4
0.05
score “on”
Hoehn-Yahr
score “off”
Schwab-England
score “on”
Schwab-England
score “off’
UPDRS = unified Parkinson’s disease rating scale; ADL = activities
of daily living; NS = not significant.
tal status improved following introduction of carbamazepine and reduction of levodopa dose. Patient 3
had an asymptomatic superficial cortical hemorrhage
detected on routine postoperative MRI. No other adverse events were encountered. CsA was well tolerated
in all patients and was not associated with any alteration
in measures of renal function.
Discussion
We report significant improvement in clinical function
and striatal FD uptake 6 months following fetal nigral
transplantation in 4 patients with advanced PD. Significant improvement was observed in total UPDRS
score during “off’ periods, percent “on” time, percent
“on” time without dyskinesia, and functional disability
during “off’ state. Gait was improved in 3, painful “off
period” dystonia resolved in 3, and freezing episodes
disappeared in 2. Increased striatal FD uptake was de-
No 3 September 1995
120
100
8
u)
B
3
80
60
40
20
0
Baseline
1
3
6
3
6
3
6
Months
A
B&eIine
1
Months
B
80
60
40
20
n
Baseline
1
Months
C
Fig. 1 . Score of each patient at baseline and postoperative month
I , 3 , and 6 for (A) (unified Parkinson’s disease rating scale:
UPDRS) “off;” (B)percent “ o f l time, and (C) percent “on” time
with dyskinesia.
tected in grafted regions in all patients. These benefits
were attained with minimal alterations in levodopa dosage and without introduction of any other antiparkinsonian medication.
It is difficult to compare our results with other results reported in the literature because of differences
in patient selection, transplantation technique, and
method of evaluation. Nonetheless, our results appear
to compare favorably to those reported 6 months following transplantation in the peer-reviewed literature
[ 2 , 4 , 6, 7 ) including patients with l-methyl-4phenyl- 1,2,3,6-tetrahydropyridine (MPTP)-induced
parkinsonism [17). All of our patients experienced
clinically relevant improvement that persisted throughout the 6-month observation period. This time course
of recovery is similar to that observed in other studies
C4). Increased striatal FD uptake on PET at this time
point, as we detected in each patient, has only been
reported for 2 other patients [7, 181. Interestingly,
each underwent transplantation with multiple donors.
Benefits in our patients were obtained using a protocol that was designed to optimize donor age, number
of donors, distribution of cells, and target site. We used
cells derived from donors aged 6% to 9 weeks PC.
The optimal donor age for graft survival following
transplantation is thought to be from the time dopaminergic cells first appear in the ventricular zone to
when they differentiate and extend neuritic processes
[ 191. Once neuritic processes are formed, cells are less
likely to survive transplantation, possibly because they
may be axotomized during dissection and preparation.
Dopamine neurons first appear in the ventricular zone
at 5% to 6% weeks PC [20, 21). Neuritic process are
first identified at PC week 8 and reach the striatum at
PC week 9. These observations suggest that the ideal
donor age for grafting human embryonic mesencephalic dopamine cells is between 5% and 9 weeks PC.
Our previous study on human to rodent nigral xenografts confirmed that optimal survival is obtained with
embryos aged 5 Y 2 to 8 weeks PC for suspension grafts
and 6‘/2 to 9 weeks PC for solid grafts [22). We employed solid grafts and hence used fetal donors aged
6% to 9 weeks PC. Only three other groups C2, 5, 71
have consistently utilized donor tissue from this donor
age “window.”
We employed fetal nigral tissue derived from 3 to 4
donors per side. The precise number of donors required to provide functional benefit in patients with
PD is unknown and can only be estimated from animal
experiments. The smallest number of transplanted dopaminergic neurons demonstrated to provide significant behavioral improvement is 120 in the rodent [231
and 2,000 in the marmoset 1241. The human striatum
is substantially larger and it is likely that a greater number of neurons will be required to achieve meaningful
clinical benefit. It has been estimated that in humans
60,000 dopamine neurons project to the putamen and
that up to 20,000 dopamine neurons from a single
human fetus survive transplantation into immunosuppressed rodents [25). It may thus be necessary to transplant mesencephalic tissue from 3 donors into each
putamen to restore complete dopaminergic activity.
However, full dopaminergic innervation may not be
required to reverse the symptoms associated with striatal dopamine deficiency as clinical features of PD are
not seen prior to a 60 to 80% reduction in nigral neu-
Freeman et al: Fetal Transplantation
383
Table 3 . 18-Fluorodnpa Uptake (ml . min-’ . cc-’1 Before and 6 Months after Nigral Grafting
~
Patient No.
Putamen
1
2
3
4
Mean ( ? SEM)
Caudate nucleus
1
2
3
4
*
Mean ( SEM)
bp
< 0.05.
< 0.01.
‘p
=
d p
R Pregraft
R Postgraft
L Pregraft
L Postgraft
0.007 1
0.0069
0.0068
0.0075
0.0071
0.0105a
0.0098a
0.0126b
0.0104”
0.0108 rfr 0.0006a
0.0087
0.0052
0.0109
0.0076
0.0081
0.0012
0.01 1la
0.0 10Yb
0.0122
0.0089
0.0108 ? 0.0007‘
0.0143
0.0135
0.0177
0.0121
0.0144
0.0144
0.0107
0.0198
0.0129
0.0145
0.0019
0.0143
0.0136
0.0223
0.0129
0.0158
0.0130
0.0143
0.0153
0.0144
0.0143
2
0.0002
* 0.0005
?
0.0012 (NS)
0.08.
NS = not significant compared to baseline; SEM = standard error of mean.
Fig. 2. Preoperattve (top row) and postoperative (bottom row)
juorodopa positron emission tomograpbj scans in the 4 Parkinson’s disease patients who underwent bilateral transplantation.
Images have been normalized t o background activity in each indioidual and scaled to each other. Note that striatal juorodopa
uptake in the putamen is increased bilaterally in each patient.
384
Annals of Neurology
Vol 38 No 3 September 1995
C
?
?
0.0022 (NS)
rons and striatal dopamine (26}. On the other hand, it
is not known what percent of embryonic cells survive
transplantation into the human striatum. To maximize
the likelihood of obtaining a detectable clinical benefit,
we employed a minimum of 3 donors per side in our
protocol. Only one other group has bilaterally implanted 3 or more donors per side 117). Interestingly,
while these patients had MPTP-induced parkinsonism
and not PD, the pattern and magnitude of benefit in
this group most closely resemble those observed in our
patients.
We distributed tissue within the target region so that
deposits were separated by no more than 5 mm in
three dimensions. Even if the correct number of donor
cells are transplanted, improper distribution may result
in a suboptimal clinical response. We estimate, based
on our experimental observations in rodents, that human fetal nigral neurons implanted into the striatum
extend processes for approximately 2.5 mm. Further,
dopamine diffusion from implanted dopaminergic neurons is highly limited 127, 281. This implies that donor
cells should be distributed throughout the putamen at
intervals no greater than 5 mm in all three dimensions.
Only two other groups addressed the issue of tissue
distribution. Freed and coworkers 12) separated needle
tracts by 4 mm in the anteroposterior but not the mediolateral plane of the putamen. Spencer and colleagues 161 separated deposits by 4 mm but only transplanted tissue into the caudate nucleus.
We implanted fetal tissue into the postcommissural
putamen. Based on our desire to optimize the concentration of implanted nigral cells, we chose to limit
placement of grafts into one somatotopically defined
region. In rodent and primate experiments, functional
recovery following dopamine implantation is sitespecific (24, 29, 30). Embryologically, differentiation
of the postcommissural putamen differs from that of
the anterior putamen, which is more closely linked spatially and temporally to the caudate nucleus 1317. In
PD there are several reasons for selecting the postcommissural putamen as the primary site for neural grafting. Both autopsy and PET studies in PD demonstrated
greater dopamine depletion within the posterior putamen than the anterior putamen/caudate nucleus (26,
321, and degeneration of the substantia nigra preferentially occurs in regions that project to the posterior
putamen 133). In primates, the postcommissural putamen receives input from the precentral motor fields
[34, 351 and microstimulation studies within the posterior putamen evoke discrete movements of contralateral body parts (361. In contrast, the caudate nucleus
and anterior putamen are less related to primary motor
circuitry and receive input primarily from the prefrontal cortex and frontal eyefields [35, 371. Thus the postcommissural putamen appears to be distinct from the
anterior putamen/caudate and a rational target site for
fetal nigral grafting in PD. No other group has exctusively targeted the postcommissural putamen or implanted such a high concentration of fetal nigral tissue
into this specific region. It is possible that implantation
into the caudate will provide additional benefits and
that best results will be obtained with transplantation
into both the putamen and caudate.
We transplanted tissue bilaterally into the postcommissural putamen. Following unilateral transplantation
into the putamen, improvement primarily occurred on
the contralateral side in both rodents [30) and PD patients [2, 31.Further, PET demonstrated that following
unilateral transplantation, FD uptake is increased on
the transplanted side but declines on the unoperated
side ( 5 , 18). These observations suggest that better
results may be obtained with bilateral grafts. Only two
other groups have transplanted bilaterally 12, 171 and
only the former involved PD patients.
We employed immunosuppression with CsA. The
central nervous system (CNS) is a relatively immunologically privileged site and the need for immunosuppression in neural transplantation is not established.
Fetal allografts in rodents and nonhuman primates have
been observed to survive for extended periods of time
without immunosuppression [38, 397. Transplantation
of fetal tissues into the CNS of nonhuman primates
does not induce a detectable immunological response
or donor-specific sensitization ( 4 0 ) and clinical improvement has been reported in some patients who
received fetal grafts without immunosuppression (23.
However, there is concern that the surgical trauma or
the graft itself could disrupt the BBB arid permit the
immune system access to graft antigens within the
brain (411. CsA has been shown to improve survival
of xenografts in rodent models 123, 4 2 ) and there are
examples of allogenic neural graft rejection in immunologically disparate rodents 1431. This is particularly relevant in our protocol where tissue from 6 to 8 immunologically unrelated donors is transplanted into a
single individual. Further, lack of graft rejection in rodents and primates does not ensure similar results in
humans. CsA was not employed by the group reporting the highest proportion of clinical failures {44}
and there are as yet no autopsy-proved examples of
robust graft survival following fetal nigral transplantation in nonimmunosuppressed patients. As failure to
employ immunosuppression might preclude optimal
graft survival and clinical response, we elected to use
immunosuppression.
The mechanism responsible for the clinical benefit
observed in our patients cannot be established based
on this open clinical trial. In animal models, behavioral
improvement following fetal nigral transplantation is
thought to relate primarily to survival of grafted neurons, neuritic outgrowth with synaptic connectivity,
and graft-derived dopamine production [451. How-
Freeman et al: Fetal Transplantation 385
ever, several additional factors could contribute to a
beneficial clinical response following nigral transplantation. These include a placebo effect, increased dopamine availability due to a breakdown in the BBB, immunosuppressants, and host-derived sprouting due to
surgical trauma or a trophic effect.
It is unlikely that benefits observed following nigral
grafting in P D are solely due to a placebo effect as
benefits are not detected immediately, a similar pattern
of improvement has been observed in other nigral
transplant studies {Z,4, 17}, and benefits are associated
with an increase in striatal FD uptake, which increases
over time 151. Lindvall and coauthors { 5 ] also argued
that a placebo response is unlikely because improvement primarily occurs on the contralateral side following a unilateral transplant procedure. There is no evidence to suggest a long-lasting breakdown of the BBB.
Experimental data suggest thai: the BBB closes rapidly
following neural transplantation 1461. Contrastenhanced MRI in our patients as well as in other studies [ 5 ] showed no evidence of BBB breakdown following transplantation. Carbidopa was not detected in the
ventricular cerebrospinal fluid of adrenal transplant patients following levodopa/carbidopa administration
[47). Finally, clinical benefit following transplantation
was most pronounced during “off’ episodes, when circulating levels of levodopa are presumably at their lowest. The possibility that CsA might influence the signs
and symptoms of P D and confound interpretation of
the transplant effect is also considered. CsA increases
spontaneous and amphetamine-induced locomotor behavior in Sprague-Dawley rats [48). There is also some
evidence suggesting that inflammation or autoimmunity may contribute to the pathogenesis of PD [49,
50). Controlled studies to more definitively determine
the effect of CsA in PD are required.
It is possible that the graft or the lesion associated
with the transplant procedure could induce sprouting
of residual host fibers. Implants of adrenal medulla into
mice 1511, monkeys [ 5 2 ] , and PD patients [ 5 3 ] can
induce a tyrosine hydroxylase-immunoreactive (THIR) sprouting response from residual host ventral mesencephalic dopaminergic neurons despite failure of adrenal cells to survive. Nonetlheless, it is unlikely that
sprouting due to the surgical trauma or a trophic effect
is responsible for the clinical. improvement observed
in the present study. All published observations of
TH-IR sprouting occurred in the caudate nucleus.
There have been no reports of sprouting in the posterior putamen, the target of transplantation in this project. N o behavioral improvemlents have been noted in
primate models of parkinsonism following sham surgery with injection of saline solution [54] or implantation of nonnigral tissue [ 5 5 ] . Increased striatal FD uptake observed on PET could be due to host-derived
sprouting rather than survival of grafted neurons.
However, striatal FD uptake is not increased following
adrenal transplantation in monkeys {56} or in PD patients [57} despite greater striatal trauma. Finally, we
now have autopsy confirmation of robust survival of
implanted fetal nigral neurons without evidence of
host-derived sprouting in a PD patient 18 months following a fetal nigral transplantation procedure [SS].
Based on the above, it is likely that the functional recovery observed in our patients is due, at least in part,
to survival of grafted nigral neurons.
The mechanism responsible for the dramatic reduction in percent “off’ time and dyskinesia in our patients
is unknown. Change in levodopa dose does not readily
account for these benefits as improvement was observed in 2 patients in whom the dose of levodopa was
not changed and prior to change of dose in the other
2. Further, preoperative dose manipulation did not
provide simultaneous benefit in these parameters in
any patient. A graft-related increase in dopamine neurons and terminals could permit more physiological
storage of dopamine and stimulation of dopamine receptors, thereby accounting for an increase in “on”
time coupled with a reduction in levodopa-induced
dyskinesia C59).
In summary, we demonstrated clinical benefit and
increased striatal FD uptake on PET 6 months following fetal nigral transplantation in patients with advanced PD. As progressive improvement beyond 6
months has been reported 151, it is possible our patients will experience further benefit and long-term follow-up is essential. The quality of improvement we
observed may relate to the specific transplant variables
employed, including restricted donor age window,
multiple donors, bilateral transplantation, and wide dispersion of grafts throughout the three-dimensional
configuration of the postcommissural putamen. Further studies to define the transplant variables associated
with the optimal clinical response are required. In addition, the need for immunosuppression in neural transplantation and any effect CsA may have on the signs
and symptoms of P D must be determined. The results
of our uncontrolled clinical trial are encouraging. However, fetal transplantation must still be considered an
experimental procedure and ultimately, studies that
more precisely define the contribution of the lesion
and placebo effect will be necessary before its value as
a treatment for PD can be established.
This work was supported by a grant from the National Parkinson
Foundation.
We would like to thank Sandoz Pharmaceuticals for kindly providing
cyclosporine, and T. Malapira, Ligia Gomes, MD, John Sinnott, 111,
MD, Carlos Martinez, MD, Ray Widen, PhD, Kevin Clifford, Diana
Walters, and Eric Cosman, PhD, for their assistance.
The UBCiTRIUMF PET group assisted with scanning.
386 Annals of Neurology Vol 38 No 3 September 1995
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