Therapeutic effects of intranigral transplantation of mesenchymal stem cells in rat models of Parkinson's disease.

Stem cell transplantation is a promising tool for the treatment of neurodegenerative disorders, including Parkinson's disease (PD); however, the therapeutic routes and mechanisms of mechanical approaches to stem cell transplantation must be explored. This study tests the therapeutic effect of transplantation of rat bone marrow mesenchymal stem cells (MSCs) into the substantia nigra (SN) of the PD rat. 5-Bromo-2-deoxyuridine-labeled rat MSCs were transplanted into the SN of the 6-hydroxydopamine-injected side of PD rat brains. The behavioral changes in PD rats were examined before and 4 and 8 weeks after MSC transplantation. The expression of tyrosine hydroxylase (TH) in the SN and the striatum and the survival and differentiation of MSCs were assessed by immunohistochemical and double immunofluorescence techniques. Abnormal behavior of PD rats was significantly improved by the administration of bone marrow MSCs, and the number of TH-positive cells in the SN and the optical density of TH-positive fibers in the striatum were markedly increased. Transplanted MSCs can survive and migrate in the brain and differentiate into nestin-, neuron-specific enolase-, and GFAP-positive cells. Our findings suggest that transplantation of rat bone marrow MSCs into the SN of PD rats may provide therapeutic effects. © 2016 Wiley Periodicals, Inc.

ES cell-derived renewable and functional midbrain dopaminergic progenitors.

During early development, midbrain dopaminergic (mDA) neuronal progenitors (NPs) arise from the ventral mesencephalic area by the combined actions of secreted factors and their downstream transcription factors. These mDA NPs proliferate, migrate to their final destinations, and develop into mature mDA neurons in the substantia nigra and the ventral tegmental area. Here, we show that such authentic mDA NPs can be efficiently isolated from differentiated ES cells (ESCs) using a FACS method combining two markers, Otx2 and Corin. Purified Otx2(+)Corin(+) cells coexpressed other mDA NP markers, including FoxA2, Lmx1b, and Glast. Using optimized culture conditions, these mDA NPs continuously proliferated up to 4 wk with almost 1,000-fold expansion without significant changes in their phenotype. Furthermore, upon differentiation, Otx2(+)Corin(+) cells efficiently generated mDA neurons, as evidenced by coexpression of mDA neuronal markers (e.g., TH, Pitx3, Nurr1, and Lmx1b) and physiological functions (e.g., efficient DA secretion and uptake). Notably, these mDA NPs differentiated into a relatively homogenous DA population with few serotonergic neurons. When transplanted into PD model animals, aphakia mice, and 6-OHDA-lesioned rats, mDA NPs differentiated into mDA neurons in vivo and generated well-integrated DA grafts, resulting in significant improvement in motor dysfunctions without tumor formation. Furthermore, grafted Otx2(+)Corin(+) cells exhibited significant migratory function in the host striatum, reaching >3.3 mm length in the entire striatum. We propose that functional and expandable mDA NPs can be efficiently isolated by this unique strategy and will serve as useful tools in regenerative medicine, bioassay, and drug screening.

Functional dopaminergic neurons derived from human chorionic mesenchymal stem cells ameliorate striatal atrophy and improve behavioral deficits in Parkinsonian rat model.

Human chorionic mesenchymal stem cells (HCMSCs) have been recognized as a desirable choice for cell therapy in neurological disorders such as Parkinson's disease (PD). Due to invaluable features of HCMSCs including their immunomodulatory and immunosuppressive properties, easily accessible and less differentiated compared to other types of MSCs, HCMSCs provide a great hope for regenerative medicine. Thus, the purpose of this study was to determine the in vitro and in vivo efficacy of HCMSCs-derived dopaminergic (DA) neuron-like cells with regard to PD. Initially, HCMSCs were isolated and underwent a 2-week DA differentiation, followed by in vitro assessments, using quantitative real-time polymerase chain reaction, immunocytochemistry, patch clamp recording, and high-performance liquid chromatography. In addition, the effects of implanted HCMSCs-derived DA neuron-like cells on the motor coordination along with stereological alterations in the striatum of rat models of PD were investigated. Our results showed that under neuronal induction, HCMSCs revealed neuron-like morphology, and expressed neuronal and DA-specific genes, together with DA release. Furthermore, transplantation of HCMSCs-derived DA neurons into the striatum of rat models of PD, augmented performance. Besides, it prevented reduction of striatal volume, dendritic length, and the total number of neurons, coupled with a diminished level of cleaved caspase-3. Altogether, these findings suggest that HCMSCs could be considered as an attractive strategy for cell-based therapies in PD.

Reversible parkinsonism due to chronic bilateral subdural hematomas.

Subdural hematoma is a rare cause of secondary parkinsonism. We report a 65-year-old woman with reversible parkinsonism due to bilateral chronic subdural hematomas. Symmetrical parkinsonism evolved acutely 45 days after a trivial head injury. Mild pyramidal signs were also present on her left side. MRI revealed bilateral chronic subdural hematomas. The patient's parkinsonism was completely abolished one month after successful neurosurgical evacuation of the hematomas.

Recovery of chronic parkinsonian monkeys by autotransplants of carotid body cell aggregates into putamen.

We have studied the effect of unilateral autografts of carotid body cell aggregates into the putamen of MPTP-treated monkeys with chronic parkinsonism. Two to four weeks after transplantation, the monkeys initiated a progressive recovery of mobility with reduction of tremor and bradykinesia and restoration of fine motor abilities on the contralateral side. Apomorphine injections induced rotations toward the side of the transplant. Functional recovery was accompanied by the survival of tyrosine hydroxylase-positive (TH-positive) grafted glomus cells. A high density of TH-immunoreactive fibers was seen reinnervating broad regions of the ipsilateral putamen and caudate nucleus. The nongrafted, contralateral striatum remained deafferented. Intrastriatal autografting of carotid body tissue is a feasible technique with beneficial effects on parkinsonian monkeys; thus, this therapeutic approach could also be applied to treat patients with Parkinson's disease.

Cellular and functional recovery of Parkinsonian rats after intrastriatal transplantation of carotid body cell aggregates.

We have tested the suitability of chromaffin-like carotid body glomus cells for dopamine cell replacement in Parkinsonian rats. Intrastriatal grafting of cell aggregates resulted in almost optimal abolishment of motor asymmetries and deficits of sensorimotor orientation. Recovery of transplanted animals was apparent 10 days after surgery and progressed throughout the 3 months of the study. The behavioral effects were correlated with the long survival of glomus cells in the host brain. In host tissue, glomus cells were organized into glomerulus-like structures and retained the ability to secrete dopamine. Several weeks after transplantation, dopaminergic fibers emerged from the graft, reinnervating the striatal gray matter. The special durability of grafted glomus cells in the conditions of brain parenchyma could be related to their sensitivity to hypoxia, which is known to induce cell growth, excitability, and dopamine synthesis. This work should stimulate research on the clinical applicability of carotid body autotransplants in Parkinson's disease.

The functional impact of the intrastriatal dopamine neuron grafts in parkinsonian rats is reduced with advancing disease.

Clinical trials involving intrastriatal transplants of human embryonic mesencephalic tissue have provided proof-of-principle that nigral dopamine (DA) neurons can survive and functionally integrate into the host neural circuitry. However, the degree of graft-induced symptomatic relief differs significantly between the patients. This variability has led to investigations aimed at identifying factors that could affect the clinical outcome. The extent and pattern of dopaminergic denervation in the brain may be one of the major determinants of the functional outcome after intrastriatal DA cell grafts. Here, we report that in animals subjected to an intrastriatal 6-hydroxydopamine lesion of the striatal dopaminergic afferent, the integrity of the host dopaminergic innervation outside the areas innervated by the graft is critical for optimal function of DA neurons placed in the striatum. Established graft-induced functional recovery, as assessed in the stepping and cylinder tests, was compromised in animals in which the dopaminergic lesion was extended to include also the medial and ventral striatum as well as the cortical and limbic DA projections. Poor clinical outcome after transplantation may, thus, at least in part, be caused by dopaminergic denervation in areas outside the graft-innervated territories, and similarly beneficial effects initially observed in patients may regress if the degeneration of the host extrastriatal DA projection systems proceeds with advancing disease. This would have two implications: first, patients with advanced disease involving the ventral striatum and/or nonstriatal DA projections would be unlikely to respond well to intrastriatal DA grafts and, second, to retain the full benefit of the grafts, progression of the disease should be avoided by, for example, combining cell therapy with a neuroprotective approach.

Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats.

In vitro expansion of central nervous system (CNS) precursors might overcome the limited availability of dopaminergic neurons in transplantation for Parkinson's disease, but generating dopaminergic neurons from in vitro dividing precursors has proven difficult. Here a three-dimensional cell differentiation system was used to convert precursor cells derived from E12 rat ventral mesencephalon into dopaminergic neurons. We demonstrate that CNS precursor cell populations expanded in vitro can efficiently differentiate into dopaminergic neurons, survive intrastriatal transplantation and induce functional recovery in hemiparkinsonian rats. The numerical expansion of primary CNS precursor cells is a new approach that could improve both the ethical and the technical outlook for the use of human fetal tissue in clinical transplantation.

The Impact of Ghrelin on the Survival and Efficacy of Dopaminergic Fetal Grafts in the 6-OHDA-Lesioned Rat.

Ghrelin is a peptide produced in the gut with a wide range of physiological functions. Recent studies have suggested it may have potential as a neuroprotective agent in models of Parkinson's disease, reducing the impact of toxic challenges on the survival of nigral dopaminergic neurons. The presence of the ghrelin receptor (GHSR1a) on the dopaminergic neurons of the substantia nigra raises the possibility that a potential application for this property of ghrelin may be as an adjunctive neuroprotective agent to enhance and support the survival and integration of dopaminergic cells transplanted into the striatum. Thus far, inconsistent outcomes in clinical trials for fetal cell transplantation have been linked to low rates of cell survival which we hypothesize could be ameliorated by the presence of ghrelin. To explore this, we confirmed the expression of the GHSR1a and related enzymes on e14 ventral mesencephalon. To determine a functional effect, five groups of female Sprague-Dawley rats received a unilateral 6-OHDA lesion to the medial forebrain bundle and four received an intrastriatal graft of e14 ventral mesencephalic cells. Grafted rats received saline; acyl-ghrelin (10 µg/kg); acyl-ghrelin (50 µg/kg) or the ghrelin agonist JMV-2894 (160 µg/kg) i.p. for 8 weeks. An effect of ghrelin at low dose on hippocampal neurogenesis indicated blood-brain barrier penetrance and attainment of biologically relevant levels but neither acyl-ghrelin nor JMV-2894 improved graft survival or efficacy.

Behavioral and neurochemical responses derived from dopaminergic intrastriatal grafts in hemiparkinsonian rats engaged in a novel motor task.

BACKGROUND: Putative treatments derived from in vivo stem cell transplant-derived dopamine (DA) in hemiparkinsonian rats have been assessed via DA-agonist-induced rotations involving imbalanced intra-hemispheric striatal DA receptor stimulation. However, such tests obscure the natural responses of grafts to sensory stimuli, and drug-induced plasticity can modify the circuit being tested. Thus, we propose an alternative testing strategy using a novel water tank swimming apparatus. NEW METHOD: Microdialysis was used to compare striatal DA levels when rats were: (1) in a rest-phase within a bowl-shaped apparatus, or (2) in an active forced-swim phase within a specially-equipped water tank. Resting-phase DA release levels were compared with active-phase levels obtained while rats were required to swim in the water-tank task. Behavioral variables such as asymmetric circling while swimming (rotations), front-limb strokes, and front-limb reaches were captured by a camera for analysis. RESULTS AND COMPARISON WITH EXISTING METHODS: Transplanted cells had a very modest effect on percentage of contralateral front-limb strokes, but did not reduce lesion-induced rotational asymmetry in the swim task. Neither striatal DA levels, nor their breakdown products, were significantly different between transplanted and sham-transplanted groups. Our new behavioral test eliminates the need for pharmacological stimulation, enabling simultaneous assessment of DA released in resting and active phases to explore graft control. CONCLUSIONS: Our new method allows for accurate assessments of stem cell therapy for PD as an alternative to "rotation" tests. Use of natural motivations to engage in sensory-driven motor tasks provides more accurate insights into ongoing graft-derived behavioral support.

Transplantation of bone marrow stromal cells containing the neurturin gene in rat model of Parkinson's disease.

The experiment was to evaluate the therapeutic benefit of transplanted bone marrow stromal cells (BMSCs) transfected with a kind of neurotrophic factor gene, neurturin (NTN) gene, in treating the rat model of Parkinson's disease (PD). The 6-OHDA-lesioned rats were assigned to one of three groups, those receiving BMSCs transfected with NTN gene, those receiving untransfected BMSCs containing a void plasmid and those receiving phosphate buffer solution (PBS). Treatments were injected into the right striatum (6-OHDA-lesioned side). One to six months post-transplantation, apomorphine-induced rotational behavior was observed. One month after transplantation, green fluorescent protein (GFP)/NTN, GFP/glial fibrillary acidic protein (GFAP), GFP/neuron specific enolase (NSE) and GFP/tyrosine hydroxylase (TH) fluorescence determinations of brain sections were carried out. One to six months after transplantation, brain sections containing striatum and substantia nigra were stained for TH. In situ hybridization and Western blots were used to determine NTN mRNA and protein concentration, respectively, in affected brain regions. High performance liquid chromatography (HPLC) was used to measure the dopamine (DA) content in the lesioned striatum 1 and 3 month(s) post-transplantation. The results were shown that: in the first 3 months after transplantation, the number of rotations was lower in NTN-transplant group than the void vector group, and during 1-6 months post-transplantation, the number of rotations was lower in both transplant groups than that in the PBS group (P<0.05). One month after transplantation, we detected GFP/NTN-, GFP/GFAP- and GFP/NSE-labeled cells in the transplantation area of the NTN-transplanted group, but no obvious GFP/TH labeled cells were found. Quantitative analysis of TH-positive cells 1 to 6 months after transplantation indicated that there were no significant differences between groups in survival rates of TH-positive neurons in the lesioned substantia nigra (P>0.05). In situ hybridization and Western blot identified NTN mRNA and protein expression in the transplantation area of the NTN-transplanted group. After transplantation of NTN-expressing cells, DA content in the lesioned striatum was significantly higher in the transgenic group than that in the void vector group or the PBS group (P<0.05). The overall therapeutic effects of the NTN-transplanted group were superior to those of the void plasmid group and the PBS group. The mechanisms by which transgenic therapy treats PD might involve functional enhancement of residual dopaminergic neurons by NTN, which significantly reduces the number of rotations in animals, but not increase the numbers of existing dopaminergic neurons.

Liver transplantation in a patient with rapid onset parkinsonism-dementia complex induced by manganism secondary to liver failure.

Bilateral and symmetric globus-pallidus hyperintensities are observed on T1-weighted MRI in most of the patients with chronic liver failure, due to manganese accumulation. We report a 53-year-old man, with rapid onset parkinsonism-dementia complex associated with accumulation of manganese in the brain, secondary to liver failure. A brain MRI was performed and a high signal on T1-weighted images was seen on globus-pallidus, as well as on T2-weighted images on the hemispheric white-matter. He was referred to a liver-transplantation. The patient passed away on the seventh postoperative day. Our findings support the concept of the toxic effects of manganese on the globus-pallidus. The treatment of this form of parkinsonism is controversial and liver-transplantation should not be considered as first line treatment but as an alternative one.

KA-bridged transplantation of mesencephalic tissue and olfactory ensheathing cells in a Parkinsonian rat model.

The pathology of Parkinson's disease (PD) results mainly from nigrostriatal pathway damage. Unfortunately, commonly used PD therapies do not repair the disconnected circuitry. It has been reported that using kainic acid (KA, an excitatory amino acid) in bridging transplantation may be useful to generate an artificial tract and reconstruct the nigrostriatal pathway in 6-hydroxydopamine (6-OHDA) lesioned rats. In this study, we used KA bridging and a co-graft of rat olfactory ensheathing cells (OECs) and rat E14 embryonic ventral mesencephalic (VM) tissue to restore the nigrostriatal pathway of the PD model rats. The methamphetamine-induced rotational behaviour, 4-[18 F]-ADAM (a selectively serotonin transporter radioligand)/micro-PET imaging, and immunohistochemistry were used to assess the effects of the transplantation. At 9 weeks post-grafting in PD model rats, the results showed that the PD rats undergoing VM tissue and OECs co-grafts (VM-OECs) exhibited better motor recovery compared to the rats receiving VM tissue transplantation only. The striatal uptake of 4-[18 F]-ADAM and tyrosine hydroxylase immunoreactivity (TH-ir) of the grafted area in the VM-OECs group were also more improved than those of the VM alone group. These results suggested that OECs may enhance the survival of the grafted VM tissue and facilitate the recovery of motor function after VM transplantation. Moreover, OECs possibly promote the elongation of dopaminergic and serotonergic axon in the bridging graft. Copyright © 2015 John Wiley & Sons, Ltd.

The role of positron emission tomography in the assessment of human neurotransplantation.

Positron emission tomography (PET) using tracers of the dopaminergic system has been used to measure striatal function in a small number of parkinsonian patients undergoing neurotransplantation procedures. Some postoperative scans have shown an unequivocal increase in presynaptic dopaminergic function at the graft site, providing evidence of graft survival independent of clinical assessment. Combined PET and magnetic resonance imaging (MRI) images provide the facility to explore the relationship between graft placement, survival and clinical efficacy.

Nerve growth factor increases survival of dopaminergic graft, rescue nigral dopaminergic neurons and restores functional deficits in rat model of Parkinson's disease.

In the present study, an attempt has been made to explore the neuroprotective and neurorescue effects of nerve growth factor (NGF) on grafted cells and on host nigral dopaminergic neurons, respectively. NGF was co-transplanted with fetal ventral mesencephalic cells (VMC) in the striatum of 6-hydroxydopamine (6-OHDA) lesioned rat model of Parkinson's disease (PD). In the other groups fetal VMC and NGF were transplanted alone. Twelve weeks post-transplantation, a significant restoration was observed in D-amphetamine induced rotations (stereotypy), spontaneous locomotor activity, striatal and nigral dopamine (DA) and 3,4-dihydroxy-phenyl acetic acid (DOPAC) levels in co-transplanted rats as compared to VMC alone transplanted rats. Higher number of surviving tyrosine hydroxylase immunoreactive (TH-ir) neurons and significantly increased fiber outgrowth from graft was evident in co-transplanted rats as compared to VMC alone transplanted rats. Further, a significant increase was also observed in substantia nigra TH-ir neurons count in co-transplanted rats, exhibiting a potential neuroprotective and neurorescue effects of NGF on nigrostriatal dopaminergic neurons. The results suggest that NGF at the time of transplantation exhibits neuroprotective effect on transplanted VMC as well as neurorescue effect on remaining host nigral dopaminergic neurons, leading to better functional restoration.

Reversible parkinsonism induced by hypercalcemia and primary hyperparathyroidism.

We describe a patient who had the recent onset of both primary hyperparathyroidism and parkinsonism. Surgical removal of a parathyroid adenoma was followed by spontaneous resolution of the parkinsonism. Hypoparathyroidism and other disorders of calcium metabolism are recognized causes of secondary parkinsonism, while hyperparathyroidism is not. We review the relevant literature, which supports the hypothesis that hypercalcemia may have induced cytotoxic changes in the basal ganglia of this patient.

Xenogeneic adrenal medulla graft rejection rather than survival leads to increased rat striatal tyrosine hydroxylase immunoreactivity.

Adrenal medulla has often been used as a donor tissue for transplantation into damaged central nervous system, with functional effects ranging from very good to nonexistent. The grafts have often been associated with morphological evidence of stimulated recipient dopaminergic fiber plasticity. The interpretation of these results has been difficult due to variable but mostly poor graft survival. The present study combines two experiments which evaluated the effects of intrastriatal xenogeneic adrenal medullary cell suspension grafts on rat recipients. First, bovine adrenal medulla cell suspension grafts of various compositions were tested for their functional and morphologic effects on immunosuppressed hemiparkinsonian rats. In the second experiment, graft rejection was allowed to occur in half of the rats in order to determine a possible contribution of the inflammatory/immune response to increased dopaminergic fiber plasticity of the recipient. At 28 days, grafts of all cell types survived well in immunosuppressed rats, but none of the grafted cell types was associated with either an amelioration of amphetamine-induced rotation or an increase in striatal tyrosine hydroxylase immunoreactivity around the graft site. The latter phenomenon was observed only in the nonimmunosuppressed rats with rejected grafts. Our findings strongly support the role of inflammatory/immune response to grafting in stimulating dopaminergic fiber plasticity and in the appearance of sprouting.

Evidence for plasticity of the dopaminergic system in parkinsonism.

A series of compensatory mechanisms within the dopaminergic system have been shown to maintain clinical function in the presence of dopamine loss. Experimental evidence for increased presynaptic dopamine turnover owing to increased dopamine synthesis, release, and reduced reuptake exists. Direct evidence that these mechanisms maintain extracellular dopamine levels is provided by intracerebral microdialysis techniques. Postsynaptic denervation supersensitivity clearly occurs with D2 dopamine receptors, although this is less evident with D1 receptors. Similarly, mechanisms of plasticity have been shown to be relevant in human postmortem and Positron Emission Tomographic studies of patients with Parkinson's disease. However, although presynaptic increases in dopamine turnover are well documented, postsynaptic D1 and D2 receptor changes have been more difficult to establish, mainly because of methodological difficulties. D2, but not D1, receptor increases have been documented in drug naive Parkinsonian patients with PET techniques. In transplantation of adrenal gland to striatum in animal models and patients with Parkinsonism where clinical improvement occurs, plasticity of host response may be as important as plasticity of the graft. Although some elements of the compensatory mechanism of dopamine plasticity may be deleterious, such as dyskinesias owing to dopamine receptor supersensitivity, the overall effect of delay and minimization of the clinical expression of disease is advantageous. An even greater understanding of the mechanisms involved may assist in developing future therapeutic strategies.

Co-grafted embryonic striatum increases the survival of grafted embryonic dopamine neurons.

To enhance the current therapeutic benefit of dopamine (DA) neuron grafts in Parkinson's disease, strategies must be developed that increase both DA neuron survival and fiber outgrowth into the denervated striatum. Previous work in our laboratory has demonstrated that dopaminergic neurons grow to greater size when co-grafted with striatal cell suspensions and display extensive tyrosine hydroxylase-positive (TH+) projections, but no conclusion could be reached concerning enhancement of survival of grafted DA neurons. The aim of the present study was to characterize further the potential trophic effects of striatal co-grafts on grafted mesencephalic DA neuron survival. Unilaterally lesioned male Fischer 344 rats were grafted with either a suspension of mesencephalic cells or with both mesencephalic and striatal cell suspensions. Co-grafts were either mixed together or placed separately into the striatum. Lesioned rats receiving no graft served as controls. Rotational behavior was assessed following amphetamine challenge at 2 weeks prior to grafting and at 4 and 8 weeks following grafting. Only rats receiving co-grafts of nigral and striatal suspensions separated by a distance of 1 mm showed significant behavioral recovery from baseline rotational asymmetry. Both mixed and separate striatal co-grafts were associated with a doubling of DA neuron survival compared with solo mesencephalic grafts. In the mixed co-graft experiment, DA neurite branching appeared enhanced and TH-rich patches were observed, whereas with co-grafts that were separated, TH+ innervation of the intervening host striatum was increased significantly. These results provide the first evidence suggesting that nigral-striatal co-grafts, particularly those placed separately and in proximity to each other, increase both DA neuron survival and neurite extension from the mesencephalic component of the grafts.