The Effect of Sertoli Cells on Xenotransplantation and Allotransplantation of Ventral Mesencephalic Tissue in a Rat Model of Parkinson's Disease

Abstract

Intra-striatal transplantation of fetal ventral mesencephalic (VM) tissue has a therapeutic effect on patients with Parkinson's disease (PD). Sertoli cells (SCs) possess immune-modulatory properties that benefit transplantation. We hypothesized that co-graft of SCs with VM tissue can attenuate rejection. Hemi-parkinsonian rats were generated by injecting 6-hydroxydopamine into the right medial forebrain bundle of Sprague Dawley (SD) rats. The rats were then intrastriatally transplanted with VM tissue from rats or pigs (rVM or pVM), with/without a co-graft of SCs (rVM+SCs or pVM+SCs). Recovery of dopaminergic function and survival of the grafts were evaluated using the apomorphine-induced rotation test and small animal-positron emission tomography (PET) coupled with [¹⁸F] DOPA or [¹⁸F] FE-PE2I, respectively. Immunohistochemistry (IHC) examination was used to determine the survival of the grafted dopaminergic neurons in the striatum and to investigate immune-modulatory effects of SCs. The results showed that the rVM+SCs and pVM+SCs groups had significantly improved drug-induced rotational behavior compared with the VM alone groups. PET revealed a significant increase in specific uptake ratios (SURs) of [¹⁸F] DOPA and [¹⁸F] FE-PE2I in the grafted striatum of the rVM+SCs and pVM+SCs groups as compared to that of the rVM and pVM groups. SC and VM tissue co-graft led to better dopaminergic (DA) cell survival. The co-grafted groups exhibited lower populations of T-cells and activated microglia compared to the groups without SCs. Our results suggest that co-graft of SCs benefit both xeno- and allo-transplantation of VM tissue in a PD rat model. Use of SCs enhanced the survival of the grafted dopaminergic neurons and improved functional recovery. The enhancement may in part be attributable to the immune-modulatory properties of SCs. In addition, [¹⁸F]DOPA and [¹⁸F]FE-PE2I coupled with PET may provide a feasible method for in vivo evaluation of the functional integrity of the grafted DA cell in parkinsonian rats.

Keywords: Parkinson’s disease; positron emission tomography; sertoli cell; transplantation; ventral mesencephalic tissue.

Figure 1

Experimental flowchart. [¹⁸F]-DOPA and [¹⁸F] FE-PE2I PET scans were performed at three time points (once before and twice after a unilateral 6-hydroxydopamine (6-OHDA) lesion was made to medial forebrain bundle of the animals). Behavior test refers to the apomorphine-induced rotation test. Transplantation was performed two weeks after the 6-OHDA lesion was made. At six weeks after the lesion, the rats were sacrificed for immunohistochemistry (IHC) studies.

Figure 2

Isolation of Sertoli cells (SCs). IHC staining was used to identify SCs isolated from the testis. Staining included (a) nuclear red staining (biomarker of nucleus) and (b) immunostaining for FSHr (biomarker of SCs). (c) SCs were identified as double-labeled cells. (d) Flow cytometry showed different fluorescence intensity in M1 (cell suspension only stained with florescent secondary antibody) and M2 (cell suspension stained with FSHr primary antibody and florescent secondary antibody). The SCs (M2) exhibited a tremendously shifted peak as compared to the control (M1). (e) The purity of the SCs was calculated by flow cytometry.

Figure 3

The apomorphine-induced rotational behavior test was used to evaluate dopaminergic (DA) function of hemiparkinsonian rats that received different neural grafts. The test was performed before and after transplantation. * p < 0.05; *** p < 0.001.

Figure 4

PET images of ¹⁸F-DOPA uptake distribution in rat brains. (a) Coronal (upper panel) and horizontal (lower panel) sections of each group were acquired 50–80 min after injection of ¹⁸F-DOPA. Left, middle, and right columns of each group represent the ¹⁸F-DOPA uptake before the 6-OHDA lesion, after the 6-OHDA lesion, and after transplantation with different grafting materials, respectively. (b) Specific uptake ratios (SURs) of ¹⁸F-DOPA of the grafted striatum at different time points and under different grafting conditions (* p < 0.05).

Figure 5

PET images of [¹⁸F] FE-PE2I uptake distribution in rat brains. (a) Coronal (upper panel) and horizontal (lower panel) sections of each group were acquired 20–40 min after injection of [¹⁸F]FE-PE2I. Left, middle, and right columns of each group represent [¹⁸F]FE-PE2I uptake before the 6-OHDA lesion, after the 6-OHDA lesion, and after transplantation with different grafting materials, respectively. (b) SURs of [¹⁸F] FE-PE2I in the grafted striatum at different time points and under different grafting conditions (* p < 0.05).

Figure 6

Photomicrographs of tyrosine hydroxylase immunoreactive (TH-ir) cells, and quantification results in hemiparkinsonian rat striatum at four weeks following transplantation. TH-ir cell bodies and fibers in the grafted side (right side of the coronal brain sections) of the rat ventral mesencephalic (rVM), rVM+SCs, pig ventral mesencephalic (pVM), and pVM+SCs groups. Greater densities of TH-ir cell bodies and fibers were noted in the rVM+SCs and pVM+SCs groups as compared to the rVM and pVM groups. Quantification data were consistent with the IHC results. (a) Sham group. (b) SC group. (c) rVM group. (d) rVM+SCs group. (e) pVM group. (f) pVM+SCs group. (g) Quantification of TH-ir cell density. (h) Optical density (OD) ratio in grafted striatum (* p < 0.05; ** p < 0.01).

Figure 7

SCs and DA cells co-existed in the grafted striatum of the rVM+SCs and pVM+SCs groups four weeks after transplantation. Brain sections were stained with a dopaminergic neuron marker (TH) and SCs marker (FSHr). (a) rVM+SCs group. (b) pVM+SCs group. (c) Quantification data of FSHr-ir cell numbers. Scale bar = 20 μm.

Figure 8

Photomicrographs of DAT-ir and quantification results at four weeks after transplantation. DAT-ir cell bodies in the grafted sides of the rVM, rVM+SCs, pVM, and pVM+SCs groups. The densities of DAT-ir cell bodies in the rVM+SCs and pVM+SCs groups were higher than those of the rVM and pVM groups. Quantification data were consistent with the IHC results. (a) Sham group. (b) SC group. (c) rVM group. (d) rVM+SCs group. (e) pVM group. (f) pVM+SCs group. (g) Quantification of DAT-ir cell density. (h) OD ratio in grafted striatum (* p < 0.05; *** p < 0.01). Scale bar = 100 μm.

Figure 9

Immunofluorescence staining with anti-Iba1 and anti-OX6 antibody and quantification results at four weeks after transplantation. A severe immune response in grafted striatum was noted in the rVM group and pVM group. Co-grafted groups showed lower microglia cell numbers compared with the rVM and pVM groups. Quantification results showed that Iba1-ir and OX6-ir were obviously increased in the rVM and pVM groups. The OX-6+/Iba1+ ratio was lower in the rVM+SCs and pVM+SCs groups than in the rVM and pVM groups. (a) Sham group. (b) SCs group. (c) rVM group. (d) rVM+SCs group. (e) pVM group. (f) pVM+SCs group. (g) Quantification of Iba1-ir and OX-6-ir cell numbers. (h) OX-6+/Iba1+ ratio. Scale bar = 20 μm.

Figure 10

Grafted striatum and contralateral striatum stained with anti-CD3 antibody. Photomicrographs of grafted striatum: (a) Sham group, (b) SCs group, (c) rVM group, (d) rVM+SCs group, (e) pVM group and (f) pVM+SCs group. Photomicrographs of contralateral striatum: (g) Sham group, (h) SCs group, (i) rVM group, (j) rVM+SCs group, (k) pVM group and (l) pVM+SCs group. The number of CD3-ir cells in the rVM group and pVM group were greater than in the rVM+SCs group and pVM+SCs. (m) Quantification of CD3-positive cells in striatal grafts at four weeks after transplantation. The number of CD3-ir cells were increased in the grafted striatum of the rVM, pVM, and pVM+SCs groups at four weeks after transplantation (** p < 0.01, the grafted side compared to each contralateral side). The number of CD3-ir cell in the grafted striatum of the rVM+SCs and pVM+SCs groups was markedly less than in the rVM group and pVM group (* p < 0.05; ** p < 0.01). Scale bar = 50 μm.

Figure 11

Pearson’s correlation analysis of ¹⁸F-DOPA and [¹⁸F]FE-PE2I in the grafted striatum of the rVM, rVM+SCs, pVM, and pVM+SCs groups. In all groups, Pearson’s R square was greater than 0.9, indicating a high correlation between the SUR of ¹⁸F-DOPA and [¹⁸F]FE-PE2I.