There is increasing evidence that bone marrow stromal cells (BMSC) have the potential to migrate into the injured neural tissue and to differentiate into the CNS cells, indicating the possibility of autograft transplantation therapy. The present study was aimed to clarify whether the mouse BMSC can migrate into the lesion and differentiate into the CNS cells when transplanted into the mice subjected to focal cerebral infarct or spinal cord injury. The BMSC were harvested from mice and characterized by flow cytometry. Then, the BMSC were labeled by bis-benzimide, a nuclear fluorescence dye, over 24 h, and were stereotactically transplanted into the brain or spinal cord of the mice. The cultured BMSC expressed low levels of CD45 and high levels of CD90 and Sca-1 on flow cytometry. A large number of grafted cells survived in the normal brain 4 weeks after transplantation, many of which were located close to the transplanted sites. They expressed the neuronal marker including NeuN, MAP2, and doublecortin on fluorescent immunohistochemistry. However, when the BMSC were transplanted into the ipsilateral striatum of the mice subjected to middle cerebral artery occlusion, many of the grafted cells migrated into the corpus callosum and injured cortex, and also expressed the neuronal markers 4 weeks after transplantation. In particular, NeuN was very useful to validate the differentiation of the grafted cells, because the marker was expressed in the nuclei and was overlapped with bis-benzimide. Similar results were obtained in the mice subjected to spinal cord injury. However, many of the transplanted BMSC expressed GFAP, an astrocytic protein, in injured spinal cord. The present results indicate that the mouse BMSC can migrate into the CNS lesion and differentiate into the neurons or astrocytes, and that bis-benzimide is a simple and useful marker to label the donor cells and to evaluate their migration and differentiation in the host neural tissues over a long period.
A surprising shortage of information surrounds the mechanism by which bone marrow stromal cells (BMSC) restore lost neurologic functions when transplanted into the damaged central nervous system. To clarify the issue, the BMSC were cocultured with the neurons using two paradigms: the cell-mixing coculture technique and three-dimensional coculture technique. The green fluorescent protein (GFP)-expressing BMSC were cocultured with the PKH-26-labelled neurons, using cell mixing coculture technique. GFP-positive, PKH-26-negative cells morphologically simulated the neurons and significantly increased the expression of MAP-2, Tuj-1, nestin, and GFAP. GFP/nestin-positive, PKH-26-negative cells increased from 13.6% +/- 6.7% to 32.1% +/- 15.5% over 7 days of coculture. They further enhanced Tuj-1 expression when cocultured with neurons exposed to 100 microM of glutamate for 10 min. About 20-30% of GFP-positive cells became positive for PKH-26 through coculture with the neurons, but the doubly positive cells did not increase when cocultured with glutamate-exposed neurons. Alternatively, the BMSC significantly ameliorated glutamate-induced neuronal damage when cocultured with the three-dimensional coculture technique. The protective effect was more prominent when coculture was started prior to glutamate exposure than when coculture was started just after glutamate exposure. ELISA analysis revealed that the BMSC physiologically produce NGF, BDNF, SDF-1alpha, HGF, TGFbeta-1, and IGF-1 and significantly enhanced the production of NGF and BDNF when cocultured with glutamate-exposed neurons. These findings strongly suggest that the BMSC may protect and repair the damaged neurons through multiple mechanisms, including transdifferentiation, cell fusion, and production of growth factors.
Despite recent developments in innovative treatment strategies, stroke remains one of the leading causes of death and disability worldwide. Stem cell therapy is currently attracting much attention due to its potential for exerting significant therapeutic effects on stroke patients. Various types of cells, including bone marrow mononuclear cells, bone marrow/adipose-derived stem/stromal cells, umbilical cord blood cells, neural stem cells, and olfactory ensheathing cells have enhanced neurological outcomes in animal stroke models. These stem cells have also been tested via clinical trials involving stroke patients. In this article, the authors review potential molecular mechanisms underlying neural recovery associated with stem cell treatment, as well as recent advances in stem cell therapy, with particular reference to clinical trials and future prospects for such therapy in treating stroke.
Recent basic experiments have strongly suggested that cell transplantation therapy may promote functional recovery in patients with spinal cord injury (SCI). However, a safe and efficient transplantation technique still remains undetermined. This study, therefore, was aimed to clarify whether fibrin matrix could be a useful scaffold in bone marrow stromal cell (BMSC) transplantation for the injured spinal cord. To clarify the issue, three-dimensional structure of fibrin matrix was assessed and the green fluorescent protein (GFP)-expressing BMSC were cultured in fibrin matrix. The rats were subjected to spinal cord hemisection at T8 level, and the vehicle, BMSC or BMSC-fibrin matrix construct was implanted into the cavity. Neurologic function was serially evaluated. Using immunohistochemistry, we evaluated the survival, migration and differentiation of the transplanted cells at 4 weeks after transplantation. In the initial in vitro study, the BMSC could survive in fibrin matrix for 2 weeks. The animals treated with the BMSC-fibrin matrix construct showed significantly more pronounced recovery of neurologic function than vehicle- or BMSC-treated animals. Fibrin scaffold markedly improved the survival and migration of the transplanted cells. There was no significant difference in the percentage of cells doubly positive for GFP and microtubule-associated protein 2 between the animals treated with BMSC-fibrin matrix construct and those treated with BMSC, but a certain subpopulation of GFP-positive cells morphologically simulated the neurons in the animals treated with BMSC-fibrin matrix construct. These findings strongly suggest that fibrin matrix may be one of the promising candidates for a potential, minimally invasive scaffold for injured spinal cord, and that such strategy of tissue engineering could be a hopeful option in regeneration therapy for patients with SCI.
Recent studies have indicated that bone marrow stromal cells (BMSC) may improve neurological function when transplanted into an animal model of CNS disorders, including cerebral infarct. However, there are few studies that evaluate the therapeutic benefits of intracerebral and intravenous BMSC transplantation for cerebral infarct. This study was aimed to clarify the favorable route of cell delivery for cerebral infarct in rats. The rats were subjected to permanent middle cerebral artery occlusion. The BMSC were labeled with near infrared (NIR)-emitting quantum dots and were transplanted stereotactically (1 × 10⁶ cells) or intravenously (3 × 10⁶ cells) at 7 days after the insult. Using in vivo NIR fluorescence imaging technique, the behaviors of BMSC were serially visualized during 4 weeks after transplantation. Motor function was also assessed. Immunohistochemistry was performed to evaluate the fate of the engrafted BMSC. Intracerebral, but not intravenous, transplantation of BMSC significantly enhanced functional recovery. In vivo NIR fluorescence imaging could clearly visualize their migration toward the cerebral infarct during 4 weeks after transplantation in the intracerebral group, but not in the intravenous, group. The BMSC were widely distributed in the ischemic brain and some of them expressed neural cell markers in the intracerebral group, but not in the intravenous group. These findings strongly suggest that intravenous administration of BMSC has limited effectiveness at clinically relevant timing and intracerebral administration should be chosen for patients with ischemic stroke, although further studies would be warranted to establish the treatment protocol.
M oyamoya disease (MMD) is characterized by the presence of net-like collateral vessels at the brain base that are caused by progressive major cerebral artery occlusion. 1Executive function/attention and working memory, primarily mediated by the lateral prefrontal region, are impaired, suggesting that lateral prefrontal ischemia is responsible for neurocognitive dysfunction.2,3 A recent investigation revealed the association of neurocognitive dysfunction with reduced cerebral blood flow.3 Nevertheless, not all patients with neurocognitive dysfunction had cerebral infarction on conventional MRI. Thus, ischemia-induced subtle microstructural alterations, which are beyond the detectability of conventional MRI, underlie neurocognitive dysfunction in MMD.Subtle gray matter changes, not shown on conventional MRI, are successfully detected in many diseases, such as mild cognitive impairment and schizophrenia, through voxel-byvoxel comparison of gray matter density on 3-dimensional (3D) MRI.4,5 Diffusion tensor imaging (DTI) is reportedly highly sensitive to microstructural alterations in diffusion characteristics of white matter. [6][7][8][9] To the best of our knowledge, no reports have evaluated gray matter changes in MMD using 3D MRI. There are only few reports on DTI assessments of MMD white matter integrity.6-8 Nevertheless, these reports used a specified region-of-interest approach and evaluated only 2 major DTI indices, such as fractional anisotropy (FA) and mean diffusivity (MD). Voxel-based analysis of white matter can provide detailed topographical characteristics of white matter integrity, and tractography can show the integrity of the major white matter tracts that run in anatomic regions. Furthermore, additional information for characterizing chronic ischemia-induced white matter damage can be extracted by incorporating other major DTI indices, such as axial diffusivity (AD) and radial diffusivity (RD).Here, we investigated the brain's microstructure across different regions in adult MMD by a voxel-based analysis of gray and white matter and tractography, and evaluated the relationship of these microstructural alterations with hemodynamic compromise and neurocognitive dysfunction.Background and Purpose-The mechanisms underlying frontal lobe dysfunction in moyamoya disease (MMD) are unknown. We aimed to determine whether chronic ischemia induces subtle microstructural brain changes in adult MMD and evaluated the association of changes with neuropsychological performance. Methods-MRI, including 3-dimensional T1-weighted imaging and diffusion tensor imaging, was performed in 23 adult patients with MMD and 23 age-matched controls and gray matter density and major diffusion tensor imaging indices were compared between them; any alterations in the patients were tested for associations with age, ischemic symptoms, hemodynamic compromise, and neuropsychological performance. Results-Decrease in gray matter density, associated with hemodynamic compromise (P<0.05), was observed in the posterior cingulate cortex of pa...
Intracerebral injection of the vasoconstrictor peptide, endothelin-1 (ET-1), has been used as a method to induce focal ischemia in rats. The relative technical simplicity of this model makes it attractive for use in mice. However, the effect of ET-1 on mouse brains has not been firmly established. In this study, we determined the ability of ET-1 to induce focal cerebral ischemia in four different mouse strains (CD1, C57/BL6, NOD/SCID, and FVB). In contrast to rats, intracerebral injection of ET-1 did not produce a lesion in any mouse strain tested. A combination of ET-1 injection with either CCA occlusion or N(G)-nitro-l-arginine methyl ester (l-NAME) injection produced only a small infarct and its size was strain-dependent. A triple combination of CCA occlusion with co-injection of ET-1 and l-NAME produced a lesion in all mouse strains tested, and this resulted in a significant motor deficit. However, lesion size was still relatively small and strain-dependent. This study shows that ET-1 has a much less potent effect for producing an infarct in mice than rats.
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