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Outcome Following Intrastriatal Fetal Mesencephalic Grafts for Parkinson's Patients Is Directly Related to the Volume of Grafted Tissue
EXPERIMENTAL NEUROLOGY ARTICLE NO.
146, 536–545 (1997)
EN976577
Outcome Following Intrastriatal Fetal Mesencephalic Grafts for Parkinson’s Patients Is Directly Related to the Volume of Grafted Tissue Oleg V. Kopyov,* Deane ‘‘Skip’’ Jacques,* Abraham Lieberman,† Christopher M. Duma,* and Robert L. Rogers* *Neurosciences Institute, Good Samaritan Hospital, Los Angeles, California 90017; and †Barrows Neurological Institute, Phoenix, Arizona
The effect of varying the volume of grafted fetal mesencephalic tissue was studied in patients with idiopathic Parkinson’s disease in a single-blinded study. Evaluations were performed according to the Core Assessment Program for Intracerebral Transplantation and videotaped both prior to transplantation and in 3-month intervals after transplantation. One group, low-volume grafts (six subjects; mean age, 57.2 years), received ventral mesencephalon grafts from one to two donors with an approximate volume up to 20 mm3, while the second group, high-volume grafts (seven subjects; mean age, 59.5 years), received ventral mesencephalon grafts from three or more donors with an approximate volume of 24 mm3. Both groups of patients demonstrated significant improvement over presurgical baseline scores on all major parameters. The high-volume group had significantly greater improvements on all the UPDRS scores and also better performance on a variety of motor performance tasks over that seen among low-volume patients. These results indicate that variations of fetal graft volume do have an impact on clinical outcome. r 1997 Academic Press
INTRODUCTION
A recent development in clinical neuroscience has been the possibility of fetal grafting for Parkinson’s disease. The implantation of fetal tissue provides the patient with dopaminergic neurons that connect with the host tissue and replace deteriorating adult neurons (4, 7, 22, 28, 32). Studies in animals with chemically induced parkinsonism have consistently demonstrated improvement after intracerebral grafts of ventral mesencephalic tissue (1, 8, 9, 11, 27, 33; for review see 2, 3). The results of neurotransplantation in humans with idiopathic Parkinson’s disease (PD) have not been so consistent; however, some studies report improvement in the major PD symptoms, while others have been less successful (12, 13, 15, 18, 21, 24, 26, 29, 31; for review see 20, 25). This variability among results of clinical testing of fetal transplantation for PD raises the question of which graft parameters may determine outcome. 0014-4886/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
Investigation of the factors which influence fetal transplantation outcome has been conducted in animal models (23, 30, 34). Intrastriatal grafting of nigral tissue only partially restores local levels of dopamine in rats (30, 34). When fetal grafts were placed into functionally discrete areas of the striatum, behavioral changes associated with that particular region were observed, suggesting a local effect of the graft (23). These studies provide evidence that such factors as the distribution and volume of transplanted fetal tissue may exert a profound effect on the degree of functional recovery which can be obtained. While there are several factors which can influence functional outcome in human patients as well (for review see 20, 25), the issue of tissue volume is a central one for continuing clinical trials. Previous studies have provided all subjects with approximately the same volume of fetal tissue, and only two of these studies have included more than seven subjects (15, 24). The present study investigated the effect of varying the volume of grafted tissue, with other variables maintained constant, for 13 patients. All patients received the same surgical procedure and were kept blind as to which experimental condition (high or low tissue volume) they were assigned. Although all subjects exhibited functional improvement, the highvolume patients demonstrated greater improvement than low-volume patients on UPDRS scores, ability to perform activities of daily living, and timed Care Assessment Program for Intracerebral Transplantation (CAPIT) assessment of dexterity for the upper extremities. An essential component of transplantation research has been the utilization of controls on which to base comparisons and rule out placebo effects. In clinical trials, however, this approach poses ethical questions regarding performance of neurosurgery on a patient without any prospect of improving the patients’ condition. In the present paper the experimental question was posed such that one independent variable was manipulated to varying degrees, allowing all subjects to receive viable transplant surgery under wellcontrolled conditions.
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MATERIALS AND METHODS
Patient Selection Only PD patients diagnosed according to the CAPIT protocol (5), including a good response to levodopa, participated in the study. Selection criteria for admittance to the study included a fluctuating response to levodopa (carbidopa), Hoehn–Yahr Stage III or less during the ‘‘on’’ state and stage III or more during the ‘‘off’’ state, clinically meaningful disability in activities of daily living (ADL) during off state, and a stable dose of L-dopa for a least 6 months were required. Exclusion criteria were dementia, a previous intracranial procedure (e.g., pallidotomy or thalamotomy), or clinically meaningful psychiatric, medical, or neoplastic disease. Patients were screened serologically prior to entry for viral and other antibodies. Patients were not included if there was evidence of infection with syphilis, HIV, HTLV, or hepatitis. Subjects and Protocol Thirteen subjects met the selection criteria and were entered into the study. Six subjects were randomly assigned to receive a volume of fetal tissue up to about 20 mm3 (1 or 2 donors) and the other 7 patients received the larger volume of fetal grafts over 24 mm3 (3 or more donors). Six patients, ranging in age from 46 to 73 years with a mean age of 57.2 6 9.5, received the lower volume (low-volume). The higher volume group (highvolume) ranged in age from 52 to 67 years with a mean of 59.5 years 6 7.2. Table 1 summarizes the demographics and presurgical L-dopa intake and Hoehn–Yahr scores for both experimental groups. The assessment protocol required an initial baseline evaluation period for a minimum of 3 months. During this time the patient was stabilized on medications and evaluated for baseline levels of functioning and performance. Most patients were followed for 1 year before TABLE 1 Patient Demographics Number GestaPD donors tional Age duration implanted age (years) Sex (years) (mean) (weeks) Low-volume group
52.7
2F 4M
6SD 68.17 Range (46–73) High-volume group 59.65 2F 5M 6SD 66.70 Range (52–67)
13.3 64.3 (9–20) 10.3 62.5 (8–1)
2.0
Hoehn & Yahr ‘‘on’’
7.7
2.0
60.88 (7–9)
60.58 (1–3)
3.1
7.3
2.1
60.34 (3–4)
61.3 (6–10)
60.42 (1.5–3.0)
60.0
537
receiving the implants. All evaluation sessions pre- and posttransplant were videotaped and the tapes were rated, with the exception of rigidity scores, by a neurologist blind to the group assignment of the patient. Each patient underwent MR imaging, neurological evaluation, and cognitive assessment during the baseline period. Patients were evaluated in both on and off conditions. Off periods were defined as being off levodopa for 12 h (18 h if subject was taking long-acting form of L-dopa) and at least 1 h after rising. Table 2 lists the battery of tests that were administered at each evaluation session along with a brief description of the test. Postsurgical evaluation was performed approximately 6 months following the operation. We are continuing to follow these patients at 3-month intervals to assess the long-term effects of the procedure. In each specific case, the decision to use two donors (low-volume group) or three or more donors (highvolume group) was made based on the availability of fetal tissue at the time of surgery. Neither the gynecologist, neurosurgeon, evaluator, nor the patient had prior knowledge of the availability of tissue on the day of implantation, except that at least two suitable fetal cadavers had to be available. Statistically, this was treated as a blind randomization. Surgical Procedure The surgical procedure was identical for both patient groups with four tracts stereotactically implanted into the posterior two-thirds of the putamen of each hemisphere. The fetal tissue was implanted through a superior entrance point. Placement of the grafts into the posterior section of the putamen was based on previous positron emission tomography studies which indicated that this area is most depleted among PD patients (19). A modified Backlund needle was inserted into the most inferior point of the target with the tip of the needle 4 mm lower than the actual Z value of the intended placement of the graft. The needle was opened briefly to check for bleeding. A modified catheter (Target Therapeutics; 0.04 in. i.d.) was connected to a tuberculin syringe and filled with autologous plasma to avoid injection of the storage medium into the patient’s brain tissue. Prior to the surgery a piece of fetal tissue was slowly aspirated into the free end of the catheter so as to conform to the inner shape of the catheter with as little damage to the fetal tissue as possible producing a cylindrical shaped graft about 5 to 8 mm in length. In-house testing in rodents demonstrated a better survival rate of grafted tissue with this method as opposed to cutting the tissue into smaller pieces in order to fit into the catheter. The catheter was then placed into the transplantation needle about 4 mm above the tip. The window was opened and the needle was withdrawn for 4 mm while the catheter remained
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TABLE 2 Assessment Measures Assessment measure (mg) UPDRS
Conditions a
L-Dopa
ADL Dyskinesia
On Off On Off Intensity
Duration AIM Hours off/day Single-dose L-dopa test Stand–walk–sit Pronation–supination
Finger dexterity
Foot tapping
Hoen–Yahr
Effect wears off On Off On, right On, left Off, right Off, left On, right On, left Off, right Off, left On, right On, left Off, right Off, left On Off
Self-rating a
Description of procedure Optimal levels adjusted for maximal effects Standardized scale for rating mentation, behavior, mood, and motor performance Rating scale for behaviors associated with normal daily activities, e.g., dressing, hygiene, handwriting, etc. 0, absent; 1, minimal; 2, patient aware but does not interfere with motor acts; 3, impaired movement but still functional; 4, intense interference; 5, violent disruption of motor activities. 0, absent; 1, only during motor tasks; 2, 25–50%; 3, 51–75%; 4, 76–99%; 5, 100% of waking hours Patient’s self-assessment of amount of involuntary movement Hourly assessment of presence of dyskinesia Measures the time and intensity of a single dose of L-dopa by repeating timed tests after patient is off medication for at least 12 h Timed test of patient starting in sitting position, rising, walking 7 m, and then returning to original sitting position Time required to perform 20 cycles of alternating tapping movements of the palm and dorsum of the hand on the knee
Time required for tapping the thumb with the forefinger and then with each finger in rapid succession for 10 repetitions
Number of successive foot tapping movements performed in 60 s
Rates the extent of motor involvement from 0.0, no signs of PD, to 5, bedridden Self-assessment scale of patient’s condition
On–Off L-dopa; right–left side of body.
stationary leaving the tip of the catheter at the intended target and significantly reducing the risk of even minor bleeding. Both catheter and needle were simultaneously withdrawn with concurrent reciprocal injection of the tissue into the path left by the needle. Six minutes were allowed to pass so that intracerebral pressure and/or mild swelling would not force the transplant to follow the needle as it was retracted. The same procedure was repeated for each tract. Fetal Tissue Preparation Fetal tissue was obtained from physicians in the local area using the guidelines recommended by the National Institutes of Health. Only after an elective abortion was performed was the donor approached, and informed consent was subsequently obtained from the donor. The patient and the tissue donor were unknown to each other and no monetary or other inducements were offered to the patient, gynecologist, or clinic. A maternal blood sample was obtained and the following serologic tests were performed: HIV; hepatitis A, B, and
C; HTLVI; and VDRL. Fetal tissue from donors with a history of genital herpes, cancer, asthma, lupus, rheumatoid arthritis, allergies, vasculitis of autoimmune origin, or drug abuse was excluded. Gestation of the fetal cadaver was determined according to crown-to-rump length measured by ultrasound and ranged from 6 to 9 weeks (16). Surgery was performed within 48 h of the abortion. The intact cranium was transported to the hospital in a general purpose serum-free medium at 14°C (Ultraculture, Whittaker Bioproducts, plus 5 mmol of L-glutamine and 10 µg/ml of kanamycin). The rostral half of the ventral mesencephalon was dissected and a tissue fragment of approximately 2 3 4 3 2 mm obtained under sterile conditions which was then further dissected into smaller sections. Dissections were carried out in the same serum-free culture and then put through 10 serial baths of the culture medium with antibiotic before final storage at 14°C. Areas of the fetal brain tissue adjacent to the dissected ventral mesencephalon were treated similarly and
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VOLUME OF FETAL GRAFTS FOR PARKINSON’S AFFECTS OUTCOME
fixed for electron and light microscopy and immunocytochemistry for retrospective analysis of viability and functional specificity. Immediately before transplantation, cell viability was assessed by trypan blue staining. Minimum acceptance for transplantation was 80% viable cells. Perioperative Management Doses of levodopa were initially adjusted as necessary to provide optimal treatment of symptoms (6). Patients had to remain at a stable dose of L-dopa for at least 3 months prior to surgery. Following surgery, antiparkinsonian medications were reinstated at their preoperative dose and efforts were made to maintain this dose throughout the study. However, postsurgical levels of levodopa were adjusted as indicated by changes
TABLE 3 Statistical Comparisons of the Effects of the Amount of Fetal Tissue Transplanted Test
Condition
L-dopa
UPDRS Hoen–Yahr ADL Dyskinesia
On Off On Off On Off Intensity Duration
AIM Hours off/day Single-dose L-dopa test Effect Wear-off Stand–walk–sit On Off Pronation–supination On, right On, left Off, right Off, left Finger dexterity On, right On, left Off, right Off, left Foot tapping On, right On, left Off, right Off, left Self-rating
Main (time)
Main (fetus)
0.0001 N.S. 0.0004 N.S. 0.0003 N.S. 0.008 N.S. 0.0001 0.04 0.003 0.006 0.001 N.S. 0.01 0.009 0.04 0.01 0.0009 0.005 0.004 0.0001 N.S. 0.0002 0.04 0.0001 N.S. N.S. 0.0007 0.01 0.04 0.0001 0.02 N.S. 0.01 N.S. 0.01 0.0002 N.S. 0.03 N.S. 0.05 0.001 N.S. 0.006 N.S. 0.03 N.S. 0.02 0.001 0.005 N.S. 0.004 0.01 0.002 0.005
Inter- Pairaction wise N.S. 0.01 0.01 0.04 0.05 0.001 0.04 N.S. N.S. 0.05 0.004 N.S. 0.05 N.S. 0.03 0.01 0.01 0.04 0.03 0.02 0.01 0.008 0.003 N.S. N.S. N.S. N.S. 0.01
N.S. * * N.S. * * * * * * * * * N.S. * * * * N.S. * * * * N.S. N.S. N.S. N.S. *
Note. Main (time), main effect contrasting presurgical baseline to 6-month posttransplant follow-up. Main (fetus), main effect due to amount of tissue transplanted. Interaction, tests the relative relationships between the two experimental groups changing following transplant (For example, if both groups were the same before surgery but diverged significantly at follow-up this would produce a significant interaction; see Fig. 2). Pairwise comparison, uses Scheffe test to directly compare the two groups at the follow-up assessment (* 5 P , 0.05; N.S. 5 not significant).
TABLE 4 Comparisons of the Two Groups at Follow-Up Low-volume
High-volume
Test
Mean
SD
Mean
SD
UPDRS–on UPDRS–off Hoehn–Yahr–on Hoehn–Yahr–off ADL–on ADL–off L-Dopa Dyskinesia–intensity Dyskinesia–duration Self-rating scale PS–on, right PS–on, left PS–off, right PS–off, left FD–on, right FD–on, left FD–off, right FD–off, left SWS–on SWS–off FT–on, right FT–on, left FT–off, right FT–off, left Dopa effect Dopa wear Hours off AIM
20.6 51.0 1.2 2.5 17.0 23.3 716.6 2.6 2.0 236.3 32.1 26.8 18.2 13.0 46.3 41.5 28.4 23.6 10.6 20.4 42.0 43.4 25.2 21.2 65.0 128.0 8.7 27.3
17.9 27.2 0.8 0.5 5.5 9.6 377.7 1.2 0.6 55.1 8.3 9.1 7.1 4.0 8.2 6.8 1.1 9.8 3.2 5.5 12.1 11.3 15.8 10.0 15.0 21.6 4.3 15.5
14.2 33.7 1.2 1.9 9.0 19.2 321.4 1.5 1.0 23.5 34.4 32.4 23.5 23.7 46.8 43.7 31.8 29.0 9.3 14.3 43.2 38.8 29.5 27.8 35.0 160.0 3.0 10.5
6.7 10.8 0.3 0.1 3.2 4.6 195.4 0.7 0.5 32.1 4.3 6.0 3.9 5.1 7.9 3.4 8.1 5.3 1.2 4.6 8.4 11.0 5.2 7.5 6.4 33.1 1.4 8.7
in the patients symptoms. Even though the CAPIT committee recommended keeping L-dopa doses at presurgical levels, we were frequently unable to do this because it produced severe dyskinesias in a majority of our patients, especially in the first 3 to 4 weeks. This result correlates with earlier reports from other laboratories (26). Patients were placed on cyclosporin A starting with a loading dose of 10 mg/kg and gradually decreased to a dose of 3 mg/kg over a period of 6 to 8 weeks and discontinued after 18 months. Renal function was monitored routinely. Prophylactic pre- and postoperative antibiotics were administered. Care was taken to avoid drugs which are known to be teratogenic or embryocidal. MRI was performed on the first postoperative day. Statistical Analysis Analysis strategy was to first perform a two-factor analysis of variance (ANOVA) with the two main effects being treatment (pre- versus postsurgical levels) and volume of transplanted tissue. The main effect of the treatment was handled as a repeated factor. Significant main effects due to tissue volume or a significant interaction of tissue volume by treatment were always
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FIG. 1. Comparison of the percentage change in motor UPDRS scores per amount of transplanted fetal tissue from presurgical baseline levels contrasting low-volume and high-volume transplant groups both on and off L-dopa.
followed by pairwise comparisons of the two experimental groups using the Scheffe method to contrast the high- and low-volume groups during the follow-up assessment. Raw data scores were always used in the ANOVAs, although graphs are presented as percentage of change from baseline in order to emphasize the magnitude of treatment effects over intrasubject variability. RESULTS
All patients demonstrated significant improvements when compared to pretreatment baseline. On many of the assessment measures, a significant effect of graft volume was also observed, as the scores of the highvolume group were significantly greater than the scores of the low-volume group. The statistical comparisons for all measures are summarized in Table 3, while group means and standard deviations of the treatment groups during follow-up assessment are presented in Table 4. Comparisons of the levels of L-dopa administration indicated a significant main effect due to fetal transplantation, but did not reveal any difference attributable to the volume of fetal tissue grafted. Although efforts were made to maintain patients at their presurgical levels of L-dopa, in many cases this resulted in adverse symptoms and the dosage had to be adjusted downward. There was one surgical complication which resulted in an asymptomatic unilateral intraputamenal hemorrhage. Two other patients had transitory mild disorientation in space and time which resolved within 3 to 4 weeks postoperatively. All patients showed significant improvement on the degree of involvement of parkinsonism, as measured by
the Unified Parkinson’s Disease Rating Scale (UPDRS). Scores of the high-volume group improved by 59.6% while on L-dopa and 48.2% while off L-dopa, while the low-volume group improved by only 19.9% while on L-dopa and 13.3% while off L-dopa (Fig. 1). While UPDRS scores improved for all patients, patients with high-volume grafts demonstrated significantly greater improvement than patients receiving low-volume grafts (Fig. 1). This difference was observed in ANOVA analysis as a significant main effect due to treatment and a significant interaction between the two main effects. The Scheffe comparisons indicated a significant difference between the UPDRS scores of low-volume and high-volume groups after the treatment regardless of whether or not the patients were on L-dopa. When patients were classified into groups according to their percentage of improvement on the UPDRS, within the low-volume group half or more of the subjects had less than 25% improvement in UPDRS scores. In the highvolume group all of the patients had scores improved more than 25% while on medication, and six of seven patients had greater than 25% improvement when off L-dopa (Table 5). TABLE 5 Categorization of Amount of Improvement on UPDRS Degree of improvement Group Low-volume High-volume
Medication status
0–25%
26–50%
51–75%
.76%
On L-dopa Off L-dopa On L-dopa Off L-dopa
3 4 0 1
1 1 3 4
2 1 2 1
0 0 2 1
VOLUME OF FETAL GRAFTS FOR PARKINSON’S AFFECTS OUTCOME
541
FIG. 2. Comparison between percentage change in high- and low-volume groups on Hoehn–Yahr and ADL scores per amount of transplanted fetal tissue.
When the degree of motor dysfunction was assessed with the Hoehn–Yahr scale, scores were reduced an equivalent amount for all patients while either on or off medication (Fig. 2). There was no effect of groups. An assessment of the patients’ abilities to perform normal daily tasks (ADL) showed the same general pattern as the UPDRS scores described above. Reductions in ADL scores among the high-volume subjects averaged 49.1% and 37.0% (On and Off L-dopa, respectively), while the low-volume group had reductions of 3.3% and 23.1% (Fig. 2). All patients improved in their ADL abilities following fetal transplantation, but a significantly greater improvement was observed among patients in the highvolume group than in the low-volume group (Fig. 2). In timed performance tests for movement of the upper and lower extremities, improvement was assessed according to outlines established by the CAPIT committee (5). Tests of upper extremity performance indicated significant improvement of all patients over baseline performance levels, on both sides of the body while either on or off L-dopa medication (Fig. 3). The patients receiving the high volume of transplanted tissue showed significantly larger improvements than those who had the low-volume grafts. Improvements in upper extremity performance among the high-volume group ranged from 52.9% (finger dexterity; off L-dopa; right)
to 20.6% (finger dexterity; on L-dopa; left), while the low-volume transplant group tended to show slight increases and some decreases in performance levels (Fig. 4). Timed performance tests involving the lower extremities (foot tapping and stand–walk–sit) showed the same patterns as described above for upper extremity tests, but failed to show statistical significance. Although the magnitude of improvements on these tests were similar and in some instances even greater than the upper extremity tests, higher within-subject variances likely prevented the means from being outside the 95% confidence interval. Tests of the patients’ and families’ own perception and accounting of their improvements included a selfrating scale (SELF) and a log of the amount of involuntary movements (AIM). The patients were not aware of the amount of tissue grafted. Patients in the highvolume group reported improvements of 143% for SELF and 33% for AIM, while the low-volume group reported 40% for SELF and 27.3% for AIM. Differences on both self-assessment scales were statistically significant. DISCUSSION
The present study investigated fetal mesencephalic grafting for PD, involving a larger number of subjects
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FIG. 3. Comparison between percentage change in high- and low-volume groups on timed performance tasks involving the upper extremities per amount of transplanted fetal tissue.
than most previous studies, as well as the role of varying tissue volume in clinical outcome. All patients demonstrated a significant improvement in performance and function, regardless of volume group, when compared to their stabilized baseline presurgical values. Patient requirements for L-dopa were substantially reduced from baseline levels about equally in both experimental groups. A significant difference was observed between the grafting groups as well, so that patients receiving higher volumes of transplanted mesencephalic fetal tissue had significantly greater improvements in functioning and larger reductions of Parkinsonian motor symptoms than patients receiving the lower volume grafts. The unexpectedly large difference between lowvolume and high-volume groups probably requires additional information (e.g., regarding the growth potential variability of the tissue from different donors) to be definitely interpreted. One of the most controversial subjects in clinical neurotransplantation is the use, or lack thereof, of control subjects for comparing the effects of the procedure. The importance of a control group is clear from our knowledge of placebo effects as well as from results of previous studies which have demonstrated that nonspecific mechanisms such as lesions produced by
the surgical intervention or neurotrophic influences of the graft may play a role in graft effects (10, 14, 17). The only complete double-blind placebo control for this procedure would require a sham stereotactic neurosurgery identical to that used for the implantation of fetal tissue, which involves substantial risk with no promise of benefit to the patient. Not a single published report on fetal tissue transplantation for the treatment of PD has applied the rigorous controls now considered standard for the demonstration of the clinical efficacy of new drug treatments. The strategy that we have employed to create a control group in the present study was to have all subjects receive stereotactically implanted mesencephalic fetal tissue while varying the amount of tissue grafted. This approach directly addressed the role of fetal graft volume and clearly demonstrated that a higher volume of tissue resulted in significantly improved outcomes. However, this approach also provided a controlled experiment, since both groups of subjects were treated identically with the exception of the volume of tissue (number of donors) transplanted. The observed differences cannot, therefore, be explained by placebo effects. The only plausible explanations for the significant differences between the two groups after the surgery would have to be attributed to either signifi-
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543
FIG. 4. Comparison between percentage change in high- and low-volume groups on timed performance tasks involving the lower extremities per amount of transplanted fetal tissue.
cant increases among the high-volume group or by decrements among the low-volume group. Since even the low-volume group in this case also demonstrated significant improvements over their presurgical levels, the group differences could not be attributed to declines in the low-volume group. One often-discussed aspect of fetal transplantation among PD patients is the variability of the effects of the procedure (20, 25). Although most patients treated with grafts have shown some improvement, the degree of change has varied from barely noticeable to dramatic improvements in the patient’s lifestyle. Investigations of variables which produce optimal results and the identification of predisposing variables which affect the efficacy of the procedure will likely provide much important information that can be used to refine and optimize the procedure. Many questions remain concerning fetal neurotransplantation. The optimal amount of tissue grafted, the distribution of the grafts, and the preparation of the tissue, as well as the effects of various patient predisposing factors including age and premorbid level of PD, are all poorly understood issues which could be tested by contrasting their relative effects between ‘‘treatment’’ groups. While placebo-controlled clinical trials are both necessary and desirable, alternative comparative experimental methods can be utilized in circumstances which
present ethical difficulties. Given the substantial cost of clinical trials coupled with risk to patients of sham neurosurgery, investigations focusing on specific variables of interest while giving all experimental groups viable treatment can represent a means of obtaining valuable information to advance the field and ultimately lead to development of an optimal procedure. ACKNOWLEDGMENTS The authors thank Good Samaritan Hospital and its Board of Trustees for their generous financial and moral support, Dr. Kaaren Sipes Eagle for assistance with this project, and Steve Jones for organizing this project. The protocol and consent form for this study were approved by the Institutional Review Board of Good Samaritan Hospital. All patients read and signed the consent form before entering the study.
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Kordower, J. H., T. B. Freeman, B. J. Snow, F. Vingerhoets, E. J. Mufson, P. R. Sanberg, R. A. Hauser, D. A. Smith, G. M. Nauert, D. P. Perl, and C. W. Olanow. 1995. Neuropathological evidence of graft survival and striatal reinnervation after the transplantation of fetal mesencephalic tissue in a patient with Parkinson’s disease. N. Engl. J. Med. 332: 1118–1124. Leenders, K. L., E. P. Salmon, P. Tyrrell, D. Perani, D. J. Brooks, H. Sager, T. Jones, C. D. Marsden, and S. J. Frackowiak. 1990. The nigrostriatal dopaminergic system assessed in vivo by positron emission tomography in healthy volunteer subjects and patients with Parkinson’s Disease. Arch. Neurol. 47: 1290– 1298. Lindvall, O. 1994. Neural transplantation in Parkinson’s disease. In Functional Neural Transplantation (S. B. Dunnett and A. Bjorklund, Eds.), pp. 103–137. Raven Press, New York. Lindvall, O., P. Brundin, H. Widner, S. Rehncrona, B. Gustavii, R. Frackowiak, K. L. Leenders, G. Sawle, J. C. Rothwell, C. D. Marsden, and A. Bjorklund. 1990. Grafts of fetal dopamine neurons survive and improve motor function in Parkinson’s disease. Science 247: 574–577. Mahalik, T. J., T. E. Finger, I. Stromberg, and L. Olson. 1985. Substantia nigra transplants into denervated striatum of the rat: ultrastructure of graft and host interconnections. J. Comp. Neurol. 240: 60–70. Mandel, R., P. Brundin, and A. Bjorklund. 1990. The importance of graft placement and task complexity for transplant-induced recovery of simple and complex sensorimotor deficits in dopamine denervated rats. Eur. J. Neurosci. 2: 888–894. Molina, H., R. Quinones, L. Alvarez, et al. 1991. Transplantation of human fetal mesencephalic tissue in caudate nucleus as treatment for Parkinson’s disease: The Cuban experience. In Intracerebral Transplantation in Movement Disorders: Experimental Basis and Clinical Experiences (O. Lindvall, A. Bjorklund, and H. Widner, Eds.), pp. 99–110. Elsevier, Amsterdam. Olanow, C. W., J. H. Kordower, and T. B. Freeman. 1996. Fetal nigral transplantation as a therapy for Parkinson’s disease. Trends Neurosci. 19: 102–109. Peschanski, M., G. Defer, J. P. N’Guyen, F. Ricolfi, J. C. Monfort, P. Remy, C. Geny, Y. Samson, P. Hantraye, and R. Jeny, et al. 1994. 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: 487–499. Rioux, L., D. P. Gaudin, L. K. Bui, L. Gregoire, T. DiPaolo, and P. J. Bedard. 1991. Correlation of functional recovery after a 6-hydroxydopamine lesion with survival of grafted fetal neurons and release of dopamine in the striatum of the rat. Neuroscience 40: 123–131. Schmidt, R. H., M. Ingvar, O. Lindvall, U. Stenevi, and A. Bjorklund. 1982. Functional activity of substantia nigra grafts reinnervating the striatum: neurotransmitter metabolism and [ 14C]2-deoxy-D-glucose autoradiography. J. Neurochem. 38: 737– 748. Spencer, D. D., 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, E. L. Kier, R. Makuch, P. B. Hoffer, and D. E. Redmond. 1992. Unilateral transplantation of human fetal mesencephalic tissue into the caudate nucleus of patients with Parkinson’s disease. N. Engl. J. Med. 327: 1541– 1548. Strecker, R. E., T. Sharp, P. Brundin, T. Zetterstrom, U. Ungerstedt, and A. Bjorklund. 1987. Autoregulation of dopamine release and metabolism by intrastriatal nigral grafts as revealed by intracerebral dialysis. Neuroscience 22: 169–178.
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Widner, H., J. Tetrud, S. Rehncrona, B. Snow, P. Brundin, B. Gustavii, A. Bjorklund, O. Lindvall, and J. W. Langston. 1992. Bilateral fetal mesencephalic grafting in two patients with parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). N. Engl. J. Med. 327: 1556–1563. Wuerthele, S. M., W. J. Freed, L. Olson, J. Morihisa, L. Spoor, R. J. Wyatt, and B. J. Hoffer. 1981. Effect of dopamine agonists and antagonists on the electrical activity of substantia nigra neurons transplanted into the lateral ventricle of the rat. Exp. Brain Res. 44: 1–10.
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146, 536–545 (1997)
EN976577
Outcome Following Intrastriatal Fetal Mesencephalic Grafts for Parkinson’s Patients Is Directly Related to the Volume of Grafted Tissue Oleg V. Kopyov,* Deane ‘‘Skip’’ Jacques,* Abraham Lieberman,† Christopher M. Duma,* and Robert L. Rogers* *Neurosciences Institute, Good Samaritan Hospital, Los Angeles, California 90017; and †Barrows Neurological Institute, Phoenix, Arizona
The effect of varying the volume of grafted fetal mesencephalic tissue was studied in patients with idiopathic Parkinson’s disease in a single-blinded study. Evaluations were performed according to the Core Assessment Program for Intracerebral Transplantation and videotaped both prior to transplantation and in 3-month intervals after transplantation. One group, low-volume grafts (six subjects; mean age, 57.2 years), received ventral mesencephalon grafts from one to two donors with an approximate volume up to 20 mm3, while the second group, high-volume grafts (seven subjects; mean age, 59.5 years), received ventral mesencephalon grafts from three or more donors with an approximate volume of 24 mm3. Both groups of patients demonstrated significant improvement over presurgical baseline scores on all major parameters. The high-volume group had significantly greater improvements on all the UPDRS scores and also better performance on a variety of motor performance tasks over that seen among low-volume patients. These results indicate that variations of fetal graft volume do have an impact on clinical outcome. r 1997 Academic Press
INTRODUCTION
A recent development in clinical neuroscience has been the possibility of fetal grafting for Parkinson’s disease. The implantation of fetal tissue provides the patient with dopaminergic neurons that connect with the host tissue and replace deteriorating adult neurons (4, 7, 22, 28, 32). Studies in animals with chemically induced parkinsonism have consistently demonstrated improvement after intracerebral grafts of ventral mesencephalic tissue (1, 8, 9, 11, 27, 33; for review see 2, 3). The results of neurotransplantation in humans with idiopathic Parkinson’s disease (PD) have not been so consistent; however, some studies report improvement in the major PD symptoms, while others have been less successful (12, 13, 15, 18, 21, 24, 26, 29, 31; for review see 20, 25). This variability among results of clinical testing of fetal transplantation for PD raises the question of which graft parameters may determine outcome. 0014-4886/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
Investigation of the factors which influence fetal transplantation outcome has been conducted in animal models (23, 30, 34). Intrastriatal grafting of nigral tissue only partially restores local levels of dopamine in rats (30, 34). When fetal grafts were placed into functionally discrete areas of the striatum, behavioral changes associated with that particular region were observed, suggesting a local effect of the graft (23). These studies provide evidence that such factors as the distribution and volume of transplanted fetal tissue may exert a profound effect on the degree of functional recovery which can be obtained. While there are several factors which can influence functional outcome in human patients as well (for review see 20, 25), the issue of tissue volume is a central one for continuing clinical trials. Previous studies have provided all subjects with approximately the same volume of fetal tissue, and only two of these studies have included more than seven subjects (15, 24). The present study investigated the effect of varying the volume of grafted tissue, with other variables maintained constant, for 13 patients. All patients received the same surgical procedure and were kept blind as to which experimental condition (high or low tissue volume) they were assigned. Although all subjects exhibited functional improvement, the highvolume patients demonstrated greater improvement than low-volume patients on UPDRS scores, ability to perform activities of daily living, and timed Care Assessment Program for Intracerebral Transplantation (CAPIT) assessment of dexterity for the upper extremities. An essential component of transplantation research has been the utilization of controls on which to base comparisons and rule out placebo effects. In clinical trials, however, this approach poses ethical questions regarding performance of neurosurgery on a patient without any prospect of improving the patients’ condition. In the present paper the experimental question was posed such that one independent variable was manipulated to varying degrees, allowing all subjects to receive viable transplant surgery under wellcontrolled conditions.
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VOLUME OF FETAL GRAFTS FOR PARKINSON’S AFFECTS OUTCOME
MATERIALS AND METHODS
Patient Selection Only PD patients diagnosed according to the CAPIT protocol (5), including a good response to levodopa, participated in the study. Selection criteria for admittance to the study included a fluctuating response to levodopa (carbidopa), Hoehn–Yahr Stage III or less during the ‘‘on’’ state and stage III or more during the ‘‘off’’ state, clinically meaningful disability in activities of daily living (ADL) during off state, and a stable dose of L-dopa for a least 6 months were required. Exclusion criteria were dementia, a previous intracranial procedure (e.g., pallidotomy or thalamotomy), or clinically meaningful psychiatric, medical, or neoplastic disease. Patients were screened serologically prior to entry for viral and other antibodies. Patients were not included if there was evidence of infection with syphilis, HIV, HTLV, or hepatitis. Subjects and Protocol Thirteen subjects met the selection criteria and were entered into the study. Six subjects were randomly assigned to receive a volume of fetal tissue up to about 20 mm3 (1 or 2 donors) and the other 7 patients received the larger volume of fetal grafts over 24 mm3 (3 or more donors). Six patients, ranging in age from 46 to 73 years with a mean age of 57.2 6 9.5, received the lower volume (low-volume). The higher volume group (highvolume) ranged in age from 52 to 67 years with a mean of 59.5 years 6 7.2. Table 1 summarizes the demographics and presurgical L-dopa intake and Hoehn–Yahr scores for both experimental groups. The assessment protocol required an initial baseline evaluation period for a minimum of 3 months. During this time the patient was stabilized on medications and evaluated for baseline levels of functioning and performance. Most patients were followed for 1 year before TABLE 1 Patient Demographics Number GestaPD donors tional Age duration implanted age (years) Sex (years) (mean) (weeks) Low-volume group
52.7
2F 4M
6SD 68.17 Range (46–73) High-volume group 59.65 2F 5M 6SD 66.70 Range (52–67)
13.3 64.3 (9–20) 10.3 62.5 (8–1)
2.0
Hoehn & Yahr ‘‘on’’
7.7
2.0
60.88 (7–9)
60.58 (1–3)
3.1
7.3
2.1
60.34 (3–4)
61.3 (6–10)
60.42 (1.5–3.0)
60.0
537
receiving the implants. All evaluation sessions pre- and posttransplant were videotaped and the tapes were rated, with the exception of rigidity scores, by a neurologist blind to the group assignment of the patient. Each patient underwent MR imaging, neurological evaluation, and cognitive assessment during the baseline period. Patients were evaluated in both on and off conditions. Off periods were defined as being off levodopa for 12 h (18 h if subject was taking long-acting form of L-dopa) and at least 1 h after rising. Table 2 lists the battery of tests that were administered at each evaluation session along with a brief description of the test. Postsurgical evaluation was performed approximately 6 months following the operation. We are continuing to follow these patients at 3-month intervals to assess the long-term effects of the procedure. In each specific case, the decision to use two donors (low-volume group) or three or more donors (highvolume group) was made based on the availability of fetal tissue at the time of surgery. Neither the gynecologist, neurosurgeon, evaluator, nor the patient had prior knowledge of the availability of tissue on the day of implantation, except that at least two suitable fetal cadavers had to be available. Statistically, this was treated as a blind randomization. Surgical Procedure The surgical procedure was identical for both patient groups with four tracts stereotactically implanted into the posterior two-thirds of the putamen of each hemisphere. The fetal tissue was implanted through a superior entrance point. Placement of the grafts into the posterior section of the putamen was based on previous positron emission tomography studies which indicated that this area is most depleted among PD patients (19). A modified Backlund needle was inserted into the most inferior point of the target with the tip of the needle 4 mm lower than the actual Z value of the intended placement of the graft. The needle was opened briefly to check for bleeding. A modified catheter (Target Therapeutics; 0.04 in. i.d.) was connected to a tuberculin syringe and filled with autologous plasma to avoid injection of the storage medium into the patient’s brain tissue. Prior to the surgery a piece of fetal tissue was slowly aspirated into the free end of the catheter so as to conform to the inner shape of the catheter with as little damage to the fetal tissue as possible producing a cylindrical shaped graft about 5 to 8 mm in length. In-house testing in rodents demonstrated a better survival rate of grafted tissue with this method as opposed to cutting the tissue into smaller pieces in order to fit into the catheter. The catheter was then placed into the transplantation needle about 4 mm above the tip. The window was opened and the needle was withdrawn for 4 mm while the catheter remained
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TABLE 2 Assessment Measures Assessment measure (mg) UPDRS
Conditions a
L-Dopa
ADL Dyskinesia
On Off On Off Intensity
Duration AIM Hours off/day Single-dose L-dopa test Stand–walk–sit Pronation–supination
Finger dexterity
Foot tapping
Hoen–Yahr
Effect wears off On Off On, right On, left Off, right Off, left On, right On, left Off, right Off, left On, right On, left Off, right Off, left On Off
Self-rating a
Description of procedure Optimal levels adjusted for maximal effects Standardized scale for rating mentation, behavior, mood, and motor performance Rating scale for behaviors associated with normal daily activities, e.g., dressing, hygiene, handwriting, etc. 0, absent; 1, minimal; 2, patient aware but does not interfere with motor acts; 3, impaired movement but still functional; 4, intense interference; 5, violent disruption of motor activities. 0, absent; 1, only during motor tasks; 2, 25–50%; 3, 51–75%; 4, 76–99%; 5, 100% of waking hours Patient’s self-assessment of amount of involuntary movement Hourly assessment of presence of dyskinesia Measures the time and intensity of a single dose of L-dopa by repeating timed tests after patient is off medication for at least 12 h Timed test of patient starting in sitting position, rising, walking 7 m, and then returning to original sitting position Time required to perform 20 cycles of alternating tapping movements of the palm and dorsum of the hand on the knee
Time required for tapping the thumb with the forefinger and then with each finger in rapid succession for 10 repetitions
Number of successive foot tapping movements performed in 60 s
Rates the extent of motor involvement from 0.0, no signs of PD, to 5, bedridden Self-assessment scale of patient’s condition
On–Off L-dopa; right–left side of body.
stationary leaving the tip of the catheter at the intended target and significantly reducing the risk of even minor bleeding. Both catheter and needle were simultaneously withdrawn with concurrent reciprocal injection of the tissue into the path left by the needle. Six minutes were allowed to pass so that intracerebral pressure and/or mild swelling would not force the transplant to follow the needle as it was retracted. The same procedure was repeated for each tract. Fetal Tissue Preparation Fetal tissue was obtained from physicians in the local area using the guidelines recommended by the National Institutes of Health. Only after an elective abortion was performed was the donor approached, and informed consent was subsequently obtained from the donor. The patient and the tissue donor were unknown to each other and no monetary or other inducements were offered to the patient, gynecologist, or clinic. A maternal blood sample was obtained and the following serologic tests were performed: HIV; hepatitis A, B, and
C; HTLVI; and VDRL. Fetal tissue from donors with a history of genital herpes, cancer, asthma, lupus, rheumatoid arthritis, allergies, vasculitis of autoimmune origin, or drug abuse was excluded. Gestation of the fetal cadaver was determined according to crown-to-rump length measured by ultrasound and ranged from 6 to 9 weeks (16). Surgery was performed within 48 h of the abortion. The intact cranium was transported to the hospital in a general purpose serum-free medium at 14°C (Ultraculture, Whittaker Bioproducts, plus 5 mmol of L-glutamine and 10 µg/ml of kanamycin). The rostral half of the ventral mesencephalon was dissected and a tissue fragment of approximately 2 3 4 3 2 mm obtained under sterile conditions which was then further dissected into smaller sections. Dissections were carried out in the same serum-free culture and then put through 10 serial baths of the culture medium with antibiotic before final storage at 14°C. Areas of the fetal brain tissue adjacent to the dissected ventral mesencephalon were treated similarly and
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VOLUME OF FETAL GRAFTS FOR PARKINSON’S AFFECTS OUTCOME
fixed for electron and light microscopy and immunocytochemistry for retrospective analysis of viability and functional specificity. Immediately before transplantation, cell viability was assessed by trypan blue staining. Minimum acceptance for transplantation was 80% viable cells. Perioperative Management Doses of levodopa were initially adjusted as necessary to provide optimal treatment of symptoms (6). Patients had to remain at a stable dose of L-dopa for at least 3 months prior to surgery. Following surgery, antiparkinsonian medications were reinstated at their preoperative dose and efforts were made to maintain this dose throughout the study. However, postsurgical levels of levodopa were adjusted as indicated by changes
TABLE 3 Statistical Comparisons of the Effects of the Amount of Fetal Tissue Transplanted Test
Condition
L-dopa
UPDRS Hoen–Yahr ADL Dyskinesia
On Off On Off On Off Intensity Duration
AIM Hours off/day Single-dose L-dopa test Effect Wear-off Stand–walk–sit On Off Pronation–supination On, right On, left Off, right Off, left Finger dexterity On, right On, left Off, right Off, left Foot tapping On, right On, left Off, right Off, left Self-rating
Main (time)
Main (fetus)
0.0001 N.S. 0.0004 N.S. 0.0003 N.S. 0.008 N.S. 0.0001 0.04 0.003 0.006 0.001 N.S. 0.01 0.009 0.04 0.01 0.0009 0.005 0.004 0.0001 N.S. 0.0002 0.04 0.0001 N.S. N.S. 0.0007 0.01 0.04 0.0001 0.02 N.S. 0.01 N.S. 0.01 0.0002 N.S. 0.03 N.S. 0.05 0.001 N.S. 0.006 N.S. 0.03 N.S. 0.02 0.001 0.005 N.S. 0.004 0.01 0.002 0.005
Inter- Pairaction wise N.S. 0.01 0.01 0.04 0.05 0.001 0.04 N.S. N.S. 0.05 0.004 N.S. 0.05 N.S. 0.03 0.01 0.01 0.04 0.03 0.02 0.01 0.008 0.003 N.S. N.S. N.S. N.S. 0.01
N.S. * * N.S. * * * * * * * * * N.S. * * * * N.S. * * * * N.S. N.S. N.S. N.S. *
Note. Main (time), main effect contrasting presurgical baseline to 6-month posttransplant follow-up. Main (fetus), main effect due to amount of tissue transplanted. Interaction, tests the relative relationships between the two experimental groups changing following transplant (For example, if both groups were the same before surgery but diverged significantly at follow-up this would produce a significant interaction; see Fig. 2). Pairwise comparison, uses Scheffe test to directly compare the two groups at the follow-up assessment (* 5 P , 0.05; N.S. 5 not significant).
TABLE 4 Comparisons of the Two Groups at Follow-Up Low-volume
High-volume
Test
Mean
SD
Mean
SD
UPDRS–on UPDRS–off Hoehn–Yahr–on Hoehn–Yahr–off ADL–on ADL–off L-Dopa Dyskinesia–intensity Dyskinesia–duration Self-rating scale PS–on, right PS–on, left PS–off, right PS–off, left FD–on, right FD–on, left FD–off, right FD–off, left SWS–on SWS–off FT–on, right FT–on, left FT–off, right FT–off, left Dopa effect Dopa wear Hours off AIM
20.6 51.0 1.2 2.5 17.0 23.3 716.6 2.6 2.0 236.3 32.1 26.8 18.2 13.0 46.3 41.5 28.4 23.6 10.6 20.4 42.0 43.4 25.2 21.2 65.0 128.0 8.7 27.3
17.9 27.2 0.8 0.5 5.5 9.6 377.7 1.2 0.6 55.1 8.3 9.1 7.1 4.0 8.2 6.8 1.1 9.8 3.2 5.5 12.1 11.3 15.8 10.0 15.0 21.6 4.3 15.5
14.2 33.7 1.2 1.9 9.0 19.2 321.4 1.5 1.0 23.5 34.4 32.4 23.5 23.7 46.8 43.7 31.8 29.0 9.3 14.3 43.2 38.8 29.5 27.8 35.0 160.0 3.0 10.5
6.7 10.8 0.3 0.1 3.2 4.6 195.4 0.7 0.5 32.1 4.3 6.0 3.9 5.1 7.9 3.4 8.1 5.3 1.2 4.6 8.4 11.0 5.2 7.5 6.4 33.1 1.4 8.7
in the patients symptoms. Even though the CAPIT committee recommended keeping L-dopa doses at presurgical levels, we were frequently unable to do this because it produced severe dyskinesias in a majority of our patients, especially in the first 3 to 4 weeks. This result correlates with earlier reports from other laboratories (26). Patients were placed on cyclosporin A starting with a loading dose of 10 mg/kg and gradually decreased to a dose of 3 mg/kg over a period of 6 to 8 weeks and discontinued after 18 months. Renal function was monitored routinely. Prophylactic pre- and postoperative antibiotics were administered. Care was taken to avoid drugs which are known to be teratogenic or embryocidal. MRI was performed on the first postoperative day. Statistical Analysis Analysis strategy was to first perform a two-factor analysis of variance (ANOVA) with the two main effects being treatment (pre- versus postsurgical levels) and volume of transplanted tissue. The main effect of the treatment was handled as a repeated factor. Significant main effects due to tissue volume or a significant interaction of tissue volume by treatment were always
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FIG. 1. Comparison of the percentage change in motor UPDRS scores per amount of transplanted fetal tissue from presurgical baseline levels contrasting low-volume and high-volume transplant groups both on and off L-dopa.
followed by pairwise comparisons of the two experimental groups using the Scheffe method to contrast the high- and low-volume groups during the follow-up assessment. Raw data scores were always used in the ANOVAs, although graphs are presented as percentage of change from baseline in order to emphasize the magnitude of treatment effects over intrasubject variability. RESULTS
All patients demonstrated significant improvements when compared to pretreatment baseline. On many of the assessment measures, a significant effect of graft volume was also observed, as the scores of the highvolume group were significantly greater than the scores of the low-volume group. The statistical comparisons for all measures are summarized in Table 3, while group means and standard deviations of the treatment groups during follow-up assessment are presented in Table 4. Comparisons of the levels of L-dopa administration indicated a significant main effect due to fetal transplantation, but did not reveal any difference attributable to the volume of fetal tissue grafted. Although efforts were made to maintain patients at their presurgical levels of L-dopa, in many cases this resulted in adverse symptoms and the dosage had to be adjusted downward. There was one surgical complication which resulted in an asymptomatic unilateral intraputamenal hemorrhage. Two other patients had transitory mild disorientation in space and time which resolved within 3 to 4 weeks postoperatively. All patients showed significant improvement on the degree of involvement of parkinsonism, as measured by
the Unified Parkinson’s Disease Rating Scale (UPDRS). Scores of the high-volume group improved by 59.6% while on L-dopa and 48.2% while off L-dopa, while the low-volume group improved by only 19.9% while on L-dopa and 13.3% while off L-dopa (Fig. 1). While UPDRS scores improved for all patients, patients with high-volume grafts demonstrated significantly greater improvement than patients receiving low-volume grafts (Fig. 1). This difference was observed in ANOVA analysis as a significant main effect due to treatment and a significant interaction between the two main effects. The Scheffe comparisons indicated a significant difference between the UPDRS scores of low-volume and high-volume groups after the treatment regardless of whether or not the patients were on L-dopa. When patients were classified into groups according to their percentage of improvement on the UPDRS, within the low-volume group half or more of the subjects had less than 25% improvement in UPDRS scores. In the highvolume group all of the patients had scores improved more than 25% while on medication, and six of seven patients had greater than 25% improvement when off L-dopa (Table 5). TABLE 5 Categorization of Amount of Improvement on UPDRS Degree of improvement Group Low-volume High-volume
Medication status
0–25%
26–50%
51–75%
.76%
On L-dopa Off L-dopa On L-dopa Off L-dopa
3 4 0 1
1 1 3 4
2 1 2 1
0 0 2 1
VOLUME OF FETAL GRAFTS FOR PARKINSON’S AFFECTS OUTCOME
541
FIG. 2. Comparison between percentage change in high- and low-volume groups on Hoehn–Yahr and ADL scores per amount of transplanted fetal tissue.
When the degree of motor dysfunction was assessed with the Hoehn–Yahr scale, scores were reduced an equivalent amount for all patients while either on or off medication (Fig. 2). There was no effect of groups. An assessment of the patients’ abilities to perform normal daily tasks (ADL) showed the same general pattern as the UPDRS scores described above. Reductions in ADL scores among the high-volume subjects averaged 49.1% and 37.0% (On and Off L-dopa, respectively), while the low-volume group had reductions of 3.3% and 23.1% (Fig. 2). All patients improved in their ADL abilities following fetal transplantation, but a significantly greater improvement was observed among patients in the highvolume group than in the low-volume group (Fig. 2). In timed performance tests for movement of the upper and lower extremities, improvement was assessed according to outlines established by the CAPIT committee (5). Tests of upper extremity performance indicated significant improvement of all patients over baseline performance levels, on both sides of the body while either on or off L-dopa medication (Fig. 3). The patients receiving the high volume of transplanted tissue showed significantly larger improvements than those who had the low-volume grafts. Improvements in upper extremity performance among the high-volume group ranged from 52.9% (finger dexterity; off L-dopa; right)
to 20.6% (finger dexterity; on L-dopa; left), while the low-volume transplant group tended to show slight increases and some decreases in performance levels (Fig. 4). Timed performance tests involving the lower extremities (foot tapping and stand–walk–sit) showed the same patterns as described above for upper extremity tests, but failed to show statistical significance. Although the magnitude of improvements on these tests were similar and in some instances even greater than the upper extremity tests, higher within-subject variances likely prevented the means from being outside the 95% confidence interval. Tests of the patients’ and families’ own perception and accounting of their improvements included a selfrating scale (SELF) and a log of the amount of involuntary movements (AIM). The patients were not aware of the amount of tissue grafted. Patients in the highvolume group reported improvements of 143% for SELF and 33% for AIM, while the low-volume group reported 40% for SELF and 27.3% for AIM. Differences on both self-assessment scales were statistically significant. DISCUSSION
The present study investigated fetal mesencephalic grafting for PD, involving a larger number of subjects
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FIG. 3. Comparison between percentage change in high- and low-volume groups on timed performance tasks involving the upper extremities per amount of transplanted fetal tissue.
than most previous studies, as well as the role of varying tissue volume in clinical outcome. All patients demonstrated a significant improvement in performance and function, regardless of volume group, when compared to their stabilized baseline presurgical values. Patient requirements for L-dopa were substantially reduced from baseline levels about equally in both experimental groups. A significant difference was observed between the grafting groups as well, so that patients receiving higher volumes of transplanted mesencephalic fetal tissue had significantly greater improvements in functioning and larger reductions of Parkinsonian motor symptoms than patients receiving the lower volume grafts. The unexpectedly large difference between lowvolume and high-volume groups probably requires additional information (e.g., regarding the growth potential variability of the tissue from different donors) to be definitely interpreted. One of the most controversial subjects in clinical neurotransplantation is the use, or lack thereof, of control subjects for comparing the effects of the procedure. The importance of a control group is clear from our knowledge of placebo effects as well as from results of previous studies which have demonstrated that nonspecific mechanisms such as lesions produced by
the surgical intervention or neurotrophic influences of the graft may play a role in graft effects (10, 14, 17). The only complete double-blind placebo control for this procedure would require a sham stereotactic neurosurgery identical to that used for the implantation of fetal tissue, which involves substantial risk with no promise of benefit to the patient. Not a single published report on fetal tissue transplantation for the treatment of PD has applied the rigorous controls now considered standard for the demonstration of the clinical efficacy of new drug treatments. The strategy that we have employed to create a control group in the present study was to have all subjects receive stereotactically implanted mesencephalic fetal tissue while varying the amount of tissue grafted. This approach directly addressed the role of fetal graft volume and clearly demonstrated that a higher volume of tissue resulted in significantly improved outcomes. However, this approach also provided a controlled experiment, since both groups of subjects were treated identically with the exception of the volume of tissue (number of donors) transplanted. The observed differences cannot, therefore, be explained by placebo effects. The only plausible explanations for the significant differences between the two groups after the surgery would have to be attributed to either signifi-
VOLUME OF FETAL GRAFTS FOR PARKINSON’S AFFECTS OUTCOME
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FIG. 4. Comparison between percentage change in high- and low-volume groups on timed performance tasks involving the lower extremities per amount of transplanted fetal tissue.
cant increases among the high-volume group or by decrements among the low-volume group. Since even the low-volume group in this case also demonstrated significant improvements over their presurgical levels, the group differences could not be attributed to declines in the low-volume group. One often-discussed aspect of fetal transplantation among PD patients is the variability of the effects of the procedure (20, 25). Although most patients treated with grafts have shown some improvement, the degree of change has varied from barely noticeable to dramatic improvements in the patient’s lifestyle. Investigations of variables which produce optimal results and the identification of predisposing variables which affect the efficacy of the procedure will likely provide much important information that can be used to refine and optimize the procedure. Many questions remain concerning fetal neurotransplantation. The optimal amount of tissue grafted, the distribution of the grafts, and the preparation of the tissue, as well as the effects of various patient predisposing factors including age and premorbid level of PD, are all poorly understood issues which could be tested by contrasting their relative effects between ‘‘treatment’’ groups. While placebo-controlled clinical trials are both necessary and desirable, alternative comparative experimental methods can be utilized in circumstances which
present ethical difficulties. Given the substantial cost of clinical trials coupled with risk to patients of sham neurosurgery, investigations focusing on specific variables of interest while giving all experimental groups viable treatment can represent a means of obtaining valuable information to advance the field and ultimately lead to development of an optimal procedure. ACKNOWLEDGMENTS The authors thank Good Samaritan Hospital and its Board of Trustees for their generous financial and moral support, Dr. Kaaren Sipes Eagle for assistance with this project, and Steve Jones for organizing this project. The protocol and consent form for this study were approved by the Institutional Review Board of Good Samaritan Hospital. All patients read and signed the consent form before entering the study.
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