In vivo imaging and evaluation of therapeutic effect of the neural stem cell treatment in 6-OHDA induced mouse model of Parkinsons disease

Abstract

Transplantation of neural stem cells (NSCs) has been proposed and shown efficacy in animal models of Parkinsons disease (PD) as a disease course modifying treatment. There are two major therapeutic mechanisms of neural stem cell transplantation, which are replacement of degenerated dopaminergic neuron and alteration of host environment by paracrine effect. However, it is not clear that which one is the major therapeutic mechanism in the 6-hydroxydopamine (6-OHDA) induced mouse model of PD, because survival of transplanted stem cell has not been quantified. Exosome has been emerged as an active component of the paracrine effect in stem cell therapy, yet has not been evaluated in PD model. Thus, the purpose of the study was to monitor and quantify the survival of transplanted neural stem cells stably expressing the enhanced luciferase gene, and to evaluate therapeutic effect of neural stem cell (NSC) and exosome-mimetic nanovesicle (NV) derived from NSC in 6-OHDA induced mouse model of PD. The mouse model of PD was induced by unilateral injection of 6-OHDA in right striatum. The behavioral tests, and positron emission tomography (PET) imaging with [18F]N-(3-fluoropropyl)-2-beta-carbomethoxy-3-beta-(4-iodophenyl)nortropane ([18F]FP-CIT) were conducted. Human fetal-derived immortal neural stem cell line, HB1.F3, is transduced with an enhanced firefly luciferase retroviral vector (F3-effLuc cells). F3-effLuc cells or F3-effLuc derived exosome-mimetic NV (F3-NV) were injected into the right striatum of mouse model of PD. Dead F3-effLuc cells, human foreskin fibroblasts transduced with effLuc (HFF-effLuc cells) and PBS are used as controls. In vivo bioluminescence imaging for the cells and fluorescence imaging for F3-NV were repeated until the signal remained. Two and four weeks after transplantation, behavioral tests were repeated. Mice were sacrificed 1 hour after injection of apomorphine at 4 weeks after transplantation. Immunostain for tyrosine hydroxylase (TH), dopamine transporter (DAT) and cFOS were done at the level of caudateputamen. Also, gene expression microarray was conducted using lesioned striatum after treatment. Quantitative real time polymerase chain reaction (RT-PCR) was done to confirm the expression of the target genes selected from microarray. All 6-OHDA-injected mice showed side biased motor impairment and markedly decreased [18F]FP-CIT uptake in the right striatum. After transplantation of F3-effLuc, HFF-effLuc cells in PD mice, bioluminescence signals were visualized on the right side of the brain. Quantified bioluminescence intensity of the transplanted F3-effLuc and HFF-effLuc cells gradually decreased until it was undetectable by 10 days. And there were no bioluminescence signal in dead F3-effLuc cell treated or PBS treated mice. Injected Cy7 labeled F3-NVs were visualized by in vivo fluorescence imaging at 3 hours after injection, but not visualized at 24 hours after injection. Four weeks after transplantation, there was no significant difference in [18F]FP-CIT imaging, TH and DAT stain of PD models regardless of types of treatment (F3-effLuc, F3-NV, HFF-effLuc and PBS). At four weeks after transplantation, remained F3-effLuc cells were found by microscopic examination, however, the cells showed negative TH stain. The results indicate that there was no evidence of dopaminergic neuron replacement by transplanted F3-effLuc cells. However, side biased motor impairment was attenuated by F3-effLuc cell transplantation at 4 weeks after the treatment but not by HFF-effLuc, or PBS. F3-NV treated mice showed a tendency of improvement of side biased behavior at 2 weeks after injection, but no significant improvement at 4 weeks after the treatment. Striatal cFOS expression which is an indicator of supersensitivity was lower in F3-effLuc treated striatum than F3-NV, HFF-effLuc or PBS treated one. Microarray and host cell specific quantitative RT-PCR revealed up regulation of genes related with immune response, regulation of apoptosis, and neurogenesis in F3-effLuc cell treated striatum compare to HFF-effLuc or PBS treated one. Survival of transplanted F3-effLuc cell was successfully monitored by in vivo bioluminescence imaging. Side biased motor impairment and striatal supersensitivity of PD mouse was improved by F3-effLuc transplantation. F3-NV showed tendency of behavioral improvement initially however the change was not sustained until 4 weeks after treatment. There was no evidence of direct replacement of dopaminergic neuron by transplanted F3-effLuc cell. However, genes related to immune response, regulation of apoptosis, neurogenesis were up regulated in host striatum after F3-effLuc treatment. Thus, F3-effLuc induced host gene alteration might be the mechanism of behavioral improvement.