Takao Yasuhara

Transplantation of Human Neural Stem Cells Exerts Neuroprotection in a Rat Model of Parkinson's Disease

Neural stem cells (NSCs) possess high potencies of self-renewal and neuronal differentiation. We explored here whether transplantation of human NSCs cloned by v-myc gene transfer, HB1.F3 cells, is a feasible therapeutic option for Parkinsons disease. In vivo, green fluorescent protein-labeled HB1.F3 cells (200,000 viable cells in 3 μl of PBS) when stereotaxically transplanted (same-day lesion-transplant paradigm) into the 6-hydroxydopamine-lesioned striatum of rats significantly ameliorated parkinsonian behavioral symptoms compared with controls (vehicle, single bolus, or continuous minipump infusion of trophic factor, or killed cell grafts). Such graft-derived functional effects were accompanied by preservation of tyrosine hydroxylase (TH) immunoreactivity along the nigrostriatal pathway. Grafted HB1.F3 cells survived in the lesioned brain with some labeled with neuronal marker mitogen-activated protein 2 and decorated with synaptophysin-positive terminals. Furthermore, endogenous neurogenesis was activated in the subventricular zone of transplanted rats. To further explore the neuroprotective mechanisms underlying HB1.F3 cell transplantation, we performed cell culture studies and found that a modest number of HB1.F3 cells were TH and dopamine and cAMP-regulated phosphoprotein 32 positive, although most cells were nestin positive, suggesting a mixed population of mature and immature cells. Administration of the HB1.F3 supernatant to human derived dopaminergic SH-SY5Y cells and fetal rat ventral mesencephalic dopaminergic neurons protected against 6-hydroxydopamine neurotoxicity by suppressing apoptosis through Bcl-2 upregulation, which was blocked by anti-stem cell factor antibody alone, the phosphatidylinositol 3-kinase/Akt inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one] alone, or a combination of both. These results suggest that HB1.F3 cell transplantation exerts neuroprotective effects against dopaminergic depletion in vitro and in vivo because of trophic factor secretion and neuronal differentiation.

Exercise exerts neuroprotective effects on Parkinson's disease model of rats

Recent studies demonstrate that rehabilitation ameliorates physical and cognitive impairments of patients with stroke, spinal cord injury, and other neurological diseases and that rehabilitation also has potencies to modulate brain plasticity. Here we examined the effects of compulsive exercise on Parkinsons disease model of rats. Before 6-hydroxydopamine (6-OHDA, 20 microg) lesion into the right striatum of female SD rats, bromodeoxyuridine (BrdU) was injected to label the proliferating cells. Subsequently, at 24 h after the lesion, the rats were forced to run on the treadmill (5 days/week, 30 min/day, 11 m/min). As behavioral evaluations, cylinder test was performed at 1, 2, 3, and 4 weeks and amphetamine-induced rotational test was performed at 2 and 4 weeks with consequent euthanasia for immunohistochemical investigations. The exercise group showed better behavioral recovery in cylinder test and significant decrease in the number of amphetamine-induced rotations, compared to the non-exercise group. Correspondingly, significant preservation of tyrosine hydroxylase (TH)-positive fibers in the striatum and TH-positive neurons in the substantia nigra pars compacta (SNc) was demonstrated, compared to the non-exercise group. Additionally, the number of migrated BrdU- and Doublecortin-positive cells toward the lesioned striatum was increased in the exercise group. Furthermore, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor increased in the striatum by exercise. The results suggest that exercise exerts neuroprotective effects or enhances the neuronal differentiation in Parkinsons disease model of rats with subsequent improvement in deteriorated motor function.

Neuroprotective effects of vascular endothelial growth factor (VEGF) upon dopaminergic neurons in a rat model of Parkinson's disease

Vascular endothelial growth factor (VEGF) has previously been shown to display neuroprotective effects following ischemia, suggesting that VEGF may potentially be applied as a neuroprotective agent for the treatment of other neurological diseases. In this study, we investigated the neuroprotective capacity of VEGF in a model of Parkinsons disease. VEGF was found to be neuroprotective against cell death of primary E14 murine ventral mesencephalic neurons induced by 6‐hydroxydopamine (6‐OHDA) treatment in vitro. Further, rats receiving a continuous infusion of VEGF into the striatum via encapsulated hVEGF‐secreting cells (baby hamster kidney‐VEGF) displayed a significant decrease in amphetamine‐induced rotational behavior and a significant preservation of tyrosine hydroxylase‐positive neurons and fibers compared with control animals. VEGF likely functions via direct mechanisms by signaling through the neuropilin receptor expressed upon dopaminergic neurons in response to 6‐OHDA treatment. Further, VEGF is likely to promote neuroprotection indirectly by activating the proliferation of glia and by promoting angiogenesis. Our results support a potential neuroprotective role for VEGF in the treatment of Parkinsons disease.

Intravenous grafts recapitulate the neurorestoration afforded by intracerebrally delivered multipotent adult progenitor cells in neonatal hypoxic-ischemic rats

Once hypoxic-ischemic (HI) injury ensues in the human neonate at birth, the resulting brain damage lasts throughout the individuals lifetime, as no ameliorative treatments are currently available. We have recently shown that intracerebral transplantation of multipotent adult progenitor cells (MAPCs) results in behavioral improvement and reduction in ischemic cell loss in neonatal rat HI-injury model. In an attempt to advance this cellular therapy to the clinic, we explored the more practical and less invasive intravenous administration of MAPCs. Seven-day-old Sprague-Dawley rats were initially subjected to unilateral HI injury, then 7 days later received intracerebral or intravenous injections of allogeneic rat MAPCs. On post-transplantation days 7 and 14, the animals that received MAPCs via the intracerebral or intravenous route exhibited improved motor and neurologic scores compared with those that received vehicle infusion alone. Immunohistochemical evaluations at day 14 after transplantation revealed that both intracerebrally and intravenously transplanted MAPCs were detected in the ischemic hippocampal area. The degree of hippocampal cell preservation was almost the same in the two treatment groups and greater than that in the vehicle group. These results show that intravenous delivery of MAPCs is a feasible and efficacious cell therapy with potential for clinical use.

Intravenous administration of mesenchymal stem cells exerts therapeutic effects on parkinsonian model of rats: Focusing on neuroprotective effects of stromal cell-derived factor-1α

BackgroundMesenchymal stem cells (MSCs) are pluripotent stem cells derived from bone marrow with secretory functions of various neurotrophic factors. Stromal cell-derived factor-1α (SDF-1α) is also reported as one of chemokines released from MSCs. In this research, the therapeutic effects of MSCs through SDF-1α were explored. 6-hydroxydopamine (6-OHDA, 20 μg) was injected into the right striatum of female SD rats with subsequent administration of GFP-labeled MSCs, fibroblasts, (i.v., 1 × 107 cells, respectively) or PBS at 2 hours after 6-OHDA injection. All rats were evaluated behaviorally with cylinder test and amphetamine-induced rotation test for 1 month with consequent euthanasia for immunohistochemical evaluations. Additionally, to explore the underlying mechanisms, neuroprotective effects of SDF-1α were explored using 6-OHDA-exposed PC12 cells by using dopamine (DA) assay and TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining.ResultsRats receiving MSC transplantation significantly ameliorated behaviorally both in cylinder test and amphetamine-induced rotation test compared with the control groups. Correspondingly, rats with MSCs displayed significant preservation in the density of tyrosine hydroxylase (TH)-positive fibers in the striatum and the number of TH-positive neurons in the substantia nigra pars compacta (SNc) compared to that of control rats. In the in vitro study, SDF-1α treatment increased DA release and suppressed cell death induced by 6-OHDA administration compared with the control groups.ConclusionsConsequently, MSC transplantation might exert neuroprotection on 6-OHDA-exposed dopaminergic neurons at least partly through anti-apoptotic effects of SDF-1α. The results demonstrate the potentials of intravenous MSC administration for clinical applications, although further explorations are required.

Mannitol facilitates neurotrophic factor up‐regulation and behavioural recovery in neonatal hypoxic‐ischaemic rats with human umbilical cord blood grafts

We recently demonstrated that blood–brain barrier permeabilization using mannitol enhances the therapeutic efficacy of systemically administered human umbilical cord blood (HUCB) by facilitating the entry of neurotrophic factors from the periphery into the adult stroke brain. Here, we examined whether the same blood–brain barrier manipulation approach increases the therapeutic effects of intravenously delivered HUCB in a neonatal hypoxic‐ischaemic (HI) injury model. Seven‐day‐old Sprague–Dawley rats were subjected to unilateral HI injury and then at day 7 after the insult, animals intravenously received vehicle alone, mannitol alone, HUCB cells (15k mononuclear fraction) alone or a combination of mannitol and HUCB cells. Behavioural tests at post‐transplantation days 7 and 14 showed that HI animals that received HUCB cells alone or when combined with mannitol were significantly less impaired in motor asymmetry and motor coordination compared with those that received vehicle alone or mannitol alone. Brain tissues from a separate animal cohort from the four treatment conditions were processed for enzyme‐linked immunosorbent assay at day 3 post‐transplantation, and revealed elevated levels of GDNF, NGF and BDNF in those that received HUCB cells alone or when combined with mannitol compared with those that received vehicle or mannitol alone, with the combined HUCB cells and mannitol exhibiting the most robust neurotropic factor up‐regulation. Histological assays revealed only sporadic detection of HUCB cells, suggesting that the trophic factor–mediated mechanism, rather than cell replacement per se, principally contributed to the behavioural improvement. These findings extend the utility of blood–brain barrier permeabilization in facilitating cell therapy for treating neonatal HI injury.

Therapeutic targets and limits of minocycline neuroprotection in experimental ischemic stroke

BackgroundMinocycline, a second-generation tetracycline with anti-inflammatory and anti-apoptotic properties, has been shown to promote therapeutic benefits in experimental stroke. However, equally compelling evidence demonstrates that the drug exerts variable and even detrimental effects in many neurological disease models. Assessment of the mechanism underlying minocycline neuroprotection should clarify the drugs clinical value in acute stroke setting.ResultsHere, we demonstrate that minocycline attenuates both in vitro (oxygen glucose deprivation) and in vivo (middle cerebral artery occlusion) experimentally induced ischemic deficits by direct inhibition of apoptotic-like neuronal cell death involving the anti-apoptotic Bcl-2/cytochrome c pathway. Such anti-apoptotic effect of minocycline is seen in neurons, but not apparent in astrocytes. Our data further indicate that the neuroprotection is dose-dependent, in that only low dose minocycline inhibits neuronal cell death cascades at the acute stroke phase, whereas the high dose exacerbates the ischemic injury.ConclusionThe present study advises our community to proceed with caution to use the minimally invasive intravenous delivery of low dose minocycline in order to afford neuroprotection that is safe for stroke.

Neurorescue effects of VEGF on a rat model of Parkinson's disease

Vascular endothelial growth factor (VEGF) has been shown to display neuroprotective effects on dopaminergic (DA) neurons. Here, we investigated the neurorescue effects of VEGF on 6-hydroxydopamine (6-OHDA)-treated DA neurons in vitro and in vivo. Initially, we examined in vitro whether 1, 10, or 100 ng/ml of VEGF administration at 2 or 4 h after 6-OHDA treatment rescued DA neurons derived from E14 murine ventral mesencephalon. The earlier treatment of VEGF suppressed 6-OHDA-induced loss of DA neurons more than the delayed treatment. Next, we examined whether the continuous infusion of VEGF had neurorescue effects in a rat model of Parkinsons disease. We established a human VEGF secreting cell line (BHK-VEGF) and encapsulated the cells into hollow fibers. The encapsulated cells were unilaterally transplanted into the striatum of adult rats at 1 or 2 weeks after 6-OHDA lesions, and animals subsequently underwent behavioral and immunohistochemical evaluations. Compared to lesioned rats that received BHK-Control capsules, lesioned rats transplanted with BHK-VEGF capsules showed a significant reduction in the number of amphetamine-induced rotations, a significant preservation of TH-positive neurons in the substantia nigra pars compacta, and a remarkable glial proliferation in the striatum, with the earlier transplantation exerting much more benefits than the delayed transplantation. Parallel studies revealed that the observed in vitro and in vivo neurorescue effects were likely mediated by VEGFs angiogenic and glial proliferative effects, as well as its direct effects on the neurons. Our results suggest that VEGF is a highly potent neurorescue molecule for Parkinsons disease therapy.

Transplantation of bone marrow-derived stem cells: a promising therapy for stroke.

Stroke remains a major cause of death in the US and around the world. Over the last decade, stem cell therapy has been introduced as an experimental treatment for stroke. Transplantation of stem cells or progenitors into the injured site to replace the nonfunctional cells, and enhancement of proliferation or differentiation of endogenous stem or progenitor cells stand as the two major cell-based strategies. Potential sources of stem/progenitor cells for stroke include fetal neural stem cells, embryonic stem cells, neuroteratocarcinoma cells, umbilical cord blood-derived nonhematopoietic stem cells, and bone marrow-derived stem cells. The goal of this article is to provide an update on the preclinical use of bone marrow-derived stem cells with major emphasis on mesenchymal stem cells (MSCs) and multipotent adult progenitor cells (MAPCs) because they are currently most widely applied in experimental stroke studies and are now being phased into early clinical trials. The phenotypic features of MSCs and MAPCs, as well as their application in stroke, are described.

Behavioral and histological characterization of intrahippocampal grafts of human bone marrow-derived multipotent progenitor cells in neonatal rats with hypoxic-ischemic injury.

Children born with hypoxic-ischemic (HI) brain injury account for a significant number of live births wherein no clinical treatment is available. Limited clinical trials of stem cell therapy have been initiated in a number of neurological disorders, but the preclinical evidence of a cell-based therapy for neonatal HI injury remains in its infancy. One major postulated mechanism underlying therapeutic benefits of stem cell therapy involves stimulation of endogenous neurogenesis via transplantation of exogenous stem cells. To this end, transplantation has targeted neurogenic sites, such as the hippocampus, for brain protection and repair. The hippocampus has been shown to secrete growth factors, especially during the postnatal period, suggesting that this brain region presents as highly conducive microenvironment for cell survival. Based on its neurogenic and neurotrophic factor-secreting features, the hippocampus stands as an appealing target for stem cell therapy. Here, we investigated the efficacy of intrahippocampal transplantation of multipotent progenitor cells (MPCs), which are pluripotent progenitor cells with the ability to differentiate into a neuronal lineage. Seven-day-old Sprague-Dawley rats were initially subjected to unilateral HI injury, which involved permanent ligation of the right common carotid artery and subsequent exposure to hypoxic environment. At day 7 after HI injury, animals received stereotaxic hippocampal injections of vehicle or cryopreserved MPCs (thawed just prior to transplantation) derived either from Sprague-Dawley rats (syngeneic) or Fisher rats (allogeneic). All animals were treated with daily immunosuppression throughout the survival period. Behavioral tests were conducted on posttransplantation days 7 and 14 using the elevated body swing test and the rotarod to reveal general and coordinated motor functions. MPC transplanted animals exhibited reduced motor asymmetry and longer time spent on the rotarod than those that received the vehicle infusion. Both syngeneic and allogeneic MPC transplanted injured animals did not significantly differ in their behavioral improvements at both test periods. Immunohistochemical evaluations of graft survival after behavioral testing at day 14 posttransplantation revealed that syngeneic and allogeneic transplanted MPCs survived in the hippocampal region. These results demonstrate for the first time that transplantation of MPCs ameliorated motor deficits associated with HI injury. In view of comparable behavioral recovery produced by syngeneic and allogeneic MPC grafts, allogeneic transplantation poses as a feasible and efficacious cell replacement strategy with direct clinical application. An equally major finding is the observation lending support to the hippocampus as an excellent target brain region for stem cell therapy in treating HI injury.