Influence of cell preparation and target location on the behavioral recovery after striatal transplantation of fetal dopaminergic neurons in a primate model of Parkinson's disease

Abstract

Surgeries involving transplantation of fetal dopamine (DA) neurons into the caudate-putamen of patients with Parkinson's disease (PD) have been performed in various clinical trials to examine a potential restoration of motor function. The absence of studies in non-human primates to define the best transplantation protocols have lead to the use of a broad variety of techniques that potentially could have a major impact on the clinical outcome. The effects of using different cell and tissue preparation, and surgical targets, remain unknown. For this purpose, 20 St. Kitts African Green Monkeys (AFG) rendered parkinsonian by i.m. injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were balanced into 4 groups and unilaterally grafted in the (a) caudate or (b) putamen with fetal ventral mesencephalic (VM) tissue as (c) solid pieces or as a (d) cell suspension. By 9 months post-transplantation all animals showed significant and similar behavioral improvement as determined by a UPDRS based PD scale. Postmortem analyses showed that VM transplants survived in all animals. They were located in both surgical target sites, producing a broad DA reinnervation of the targeted nuclei that could also extend to the non-grafted nucleus on the ipsilateral side. Although no differences between groups were found in survival of DA neurons or degree of DA reinnervation, there was a significant correlation between striatal reinnervation and behavioral recovery only in animals transplanted in the putamen surgical target. Additionally, there was in general a stronger glial reaction to solid grafts than to cell suspensions. These studies provide data for the optimal time course, cell preparation and surgical targets for systematic examinations of both potential benefits and side effects of dopamine neuron cell transplantation in primate models of PD.

Figure 1

Experimental design. Twenty animals were treated with MPTP (an average of 3.1 mg/kg for each monkey; described in text). According to baseline severity in the last month prior to transplantation, animals were randomly allocated into 4 groups to receive small solid pieces (less than 1 mm × 1 mm × 1 mm) or cell suspensions from monkey fetal VM, either in the caudate or the putamen targets. One week after transplantation parkinsonism and normal behavior were evaluated 5 days/week for 9 months. After these 9 months animals were perfused for histological evaluation.

Figure 2

A. Parkinsonism was evaluated several times each week. The mean scores during each month from baseline, two months after MPTP treatment, and each of 10 months after transplantation until sacrifice are shown. An analysis of variance, including all 20 monkeys, showed that there were significant improvements in the final 2 months, compared with earlier periods after transplantation, although the parkscores did not return fully to baseline by that time (effect of treatment month [F=56.45, df=12, 2709, p=0.0001]). Significant differences (p<0.05) from a Newman Keuls post hoc analysis are indicated by letters: Months with the same letter are not different from each other. B. To illustrate the effect of parkinsonism severity, the mean parkscore and mean of normal daily activities included in “healthy behavior” during the last month before transplantation (M02) were compared with the mean scores during the last month of the study (T10). For this analysis animals with a parkscore <5 (“very mild”) were analyzed separately. Analysis by severity groups showed a dramatic improvement of parkscore in severe animals, and a quantitatively smaller improvement in moderate, mild and very mild animals, although the percentage improvements were very similar at 40 to 60% in all four groups. Similarly, “healthy behavior” improved significantly in severe and moderate animals, but not in the mild and very mild animals, which had more normal healthy behavior scores before transplantation.

Figure 3

A regression analysis on the “park score” (black line) and the “healthy behavior” (red line) summary factors were calculated and the predicted slopes plotted for the baseline (Transplant Day ‘TDAY’) before −60, after MPTP (TDAY 0 to −60) and after grafting (0 to 270) were plotted for “all animals” and for the subset of the most “severe animals” with the 95% confidence interval of the predicted slope. Baselines did not show significant slopes All monkeys analyzed together, showed a positive slope in “park score” prior to grafting (slope +0.407, p<0.0005), and a significant improvement for the period after grafting (slope −0.046, p<0.0001). In the total group, the “healthy behaviors” showed no significant differences in the slope before grafting or after, whereas the severe animals (which had a lower pre-grafting level of “healthy behaviors”) showed a significant increase in these behaviors after grafting (slope +0.0235, p<0.0001). Slopes and significance levels were determined by linear regressions (GLM, Statistical Analysis System).

Figure 4

TH immunostaining showed surviving grafts in the 20 transplanted animals. Macroscopically, cell suspension grafts showed a typical organization, with aggregates of TH positive cells in the periphery, and a reticular area with small TH positive cells, in the centre of the graft. There was no significant difference in the number of surviving TH+ cells in the 4 transplantation groups (Two way ANOVA and Newman-Keuls post-hoc test). Scale bar 1mm.

Figure 5

Representative low power photomicrographs of TH immunostained coronal sections from an animal per group. Sections were color-coded and optical density of TH innervation was quantified (scale 0-254), using NIH image 1.61 software. In all the transplantation groups, VM grafts provided a significant increase of dopaminergic innervation in the Cd or Put target, and also in the other striatal nucleus (Put or Cd respectively), compared with the non-grafted contralateral striatum (Table 1B). There were no significant differences between transplantation groups. Scale bar 5 mm.

Figure 6

Representative photomicrographs from the 4 transplantation groups, of a GFAP immunostaining showing the astroglial reaction around the grafts. Solid grafts both at Cd (B) and Put (C) locations elicit a significantly larger astroglial reaction of fibrous hypertrophic astrocytes compared with cell suspension grafts at the same locations (A-B). * p<0.05, unpaired t test. Scale bar 250 μm.

Figure 7

Representative photomicrographs from the 4 transplantation groups, of the graft-host border stained with CD68 to analyze activated microglia. Stereological quantification did not show differences in the microglial reaction between graft locations or between tissue preparations. p >0.05 unpaired t test. Scale bar 60 μm.