Shunsuke Yano

Bone marrow stromal cells protect and repair damaged neurons through multiple mechanisms

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 μM 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‐1α, HGF, TGFβ‐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.

Role of SDF-1/CXCR4 system in survival and migration of bone marrow stromal cells after transplantation into mice cerebral infarct

Recent studies have indicated that bone marrow stromal cells (BMSC) have the potential to improve neurological function when transplanted into animal models of cerebral infarction. However, it is still obscure how the transplanted BMSC restore the lost neurological function. In this study, therefore, we aimed to elucidate the role of stromal cell-derived factor-1 (SDF-1) and its specific receptor, CXCR4, in BMSC transplantation into the brain subjected to cerebral infarction. The BMSC were harvested from the wild type (WT) and CXCR4-knockout (CXCR4-KO) mice and were cultured. The mice were subjected to permanent middle cerebral artery occlusion. The WT or CXCR4-KO BMSC was injected into the ipsilateral striatum 7 days after the insult. Motor function of the animals was serially evaluated, using a rotarod treadmill. Using fluorescence immunohistochemistry, we evaluated the distribution and phenotype of the transplanted cells 4 weeks after transplantation. Recovery of motor function in the WT BMSC-transplanted mice was more pronounced than in the CXCR4-KO-transplanted mice and the vehicle-treated ones. SDF-1 was extensively expressed in peri-infarct area. In the WT BMSC-transplanted mice, the transplanted cells were extensively distributed in the ipsilateral hemisphere, and many of them migrated towards the peri-infarct area and expressed the proteins specific for neurons and astrocytes, although these findings were not observed in the CXCR4-KO-transplanted mice. The results suggest that the SDF-1/CXCR4 system may play a critical role in the survival, proliferation and migration of the transplanted BMSC and contribute to recovery of neurological function.

Do bone marrow stromal cells proliferate after transplantation into mice cerebral infarct? : A double labeling study

The present study was aimed to clarify the proliferation capacity of the bone marrow stromal cells (BMSC) transplanted into the brain. The BMSC were harvested from green fluorescence protein (GFP)-transgenic mice, grown to the confluency and passed three times. They were labeled by co-culture with Ferucarbotran, a superparamagnetic iron oxide (SPIO) agent. The proportions of the SPIO-positive cells were evaluated from P3 to P7, using Turnbull blue staining. The GFP-BMSC labeled by Ferucarbotran were transplanted into the ipsilateral striatum of the mice brain subjected to permanent focal ischemia at 7 days after the insult. The distribution and differentiation of GFP- and SPIO-positive cells in the brain were studied 3 months after transplantation, using immunohistochemistry and Turnbull blue staining. As the results, the proportions of the SPIO-positive cells gradually decreased from 93.6% at P3 to 6.5% at P7. Fluorescence immunohistochemistry revealed that the GFP-positive cells were widely distributed around infarct and partially expressed MAP2 and NeuN 3 months after transplantation. However, only a smaller number of SPIO-positive cells could be detected on Turnbull blue staining. The ratio of the SPIO- to GFP-positive cells was approximately 2.7%. The results strongly suggest that the BMSC repeat proliferation many times, migrate into the lesion, and partially express the neuronal phenotype in the host brain during 3 months after transplantation. The double labeling technique would be valuable to prove the proliferation of the transplanted cells in the host tissue because GFP gene and SPIO nanoparticles have different inheritance characteristics.

Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: A novel material for CNS tissue engineering

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.

The effects of neuronal induction on gene expression profile in bone marrow stromal cells (BMSC)—a preliminary study using microarray analysis

Bone marrow stromal cells (BMSC) have been anticipated as a donor for cell type for transplantation therapy in various neurological disorders. However, their neurogenic capacity still remains undetermined. In this study, we aimed to clarify whether in vitro chemical treatment promotes their neuronal differentiation on the level of gene expression. Mice BMSC were cultured with medium supplemented with DMSO, retinoic acid, and basic fibroblast growth factor, and their morphology and expression of neuronal markers were evaluated. Subsequently, using microarray and RT-PCR techniques, the treatment-induced changes in the gene expression profile were analyzed. After exposure to the medium, the BMSC simulated a neuron-like appearance and increased their immunoreactivity for nestin and Tuj-1. Microarray analysis revealed that the BMSC per se express the multilineage cellular genes, including those associated with the neuron. Chemical treatment significantly decreased the expression of genes related to mesenchymal cells and increased the expression of 5 neuron-associated genes. Microarray and RT-PCR analyses also demonstrated that the BMSC express the genes for several growth factors including NGF-beta and BDNF, indicating their therapeutic role in protecting the injured central nervous system. The present results suggest that at least a certain subpopulation of the BMSC have the potential to alter their gene expression profile in response to the surrounding environment and may possibly protect the host tissue by secreting neuroprotective factors.

Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy.

Objective. Recent reports have indicated that bone marrow stromal cells (BMSCs) have the potential to improve neurological function when transplanted into models of central nervous system (CNS) disorders, including traumatic spinal cord injury. In this study, the authors aimed to clarify the underlying mechanism through which BMSCs supported CNS regeneration in the spinal cord. Methods. The authors topically applied mouse BMSCs expressing green fluorescence protein (0.4-4 × 104 cells) on the organotypic spinal cord slice culture prepared from 6-day-old rat pups (n = 17). They were co-cultured for 3 weeks after the slice culture started, and the behavior of the applied BMSCs was serially observed using a fluorescence bioimaging technique. The authors completed a histological analysis at the end of the co-cultures and evaluated the profiles of the cultured BMSCs using microarray and immunocytochemistry techniques. Results. The fluorescence bioimaging showed that the BMSCs survived and made a cluster on the slice during the experiments. They also induced a morphological change in the slice within 48 hours of co-culture. Immunohistochemistry analysis showed that the BMSCs promoted a marked neurite extension toward their cluster and some of the BMSCs expressed Tuj-1, an early neuronal marker. Analysis by microarray and immunocytochemistry revealed that BMSCs highly expressed the matrix metalloproteinases (MMPs), stromal cell—derived factor-1, and its specific receptor CXCR4. Conclusions . These findings suggest that the donor BMSCs can support CNS regeneration due to their acquisition of a suitable environment for differentiation and promotion of neurite extension via MMPs and chemokines.

Spinal subdural hematoma: a sequela of a ruptured intracranial aneurysm?

BACKGROUND A case of spinal subdural hematoma (SSDH) following subarachnoid hemorrhage (SAH) because of a ruptured internal carotid aneurysm is described. Such a case has never been reported. CASE DESCRIPTION A 52-year-old woman underwent a craniotomy for a ruptured internal carotid aneurysm. A computed tomography scan showed that SAH existed predominantly in the posterior fossa and subdural hematoma beneath the cerebellar tentorium. Intrathecal administration of urokinase, IV administration of fasudil hydrochloride, and continuous cerebrospinal fluid (CSF) evacuation via cisternal drainage were performed as prophylactic treatments for vasospasm. On the sixth postoperative day, the patient complained of severe lower back and buttock pain. Magnetic resonance imaging showed a subdural hematoma in the lumbosacral region. Although the mass effect was extensive, the patient showed no neurologic symptoms other than the sciatica. She was treated conservatively. The hematoma dissolved gradually and had diminished completely 15 weeks later. Her pain gradually subsided, and she was discharged 7 weeks later without any neurologic deficit. CONCLUSION Although the exact mechanism of SSDH in this case is unclear, we speculate that this SSDH was a hematoma that migrated from the intracranial subdural space. Low CSF pressure because of continuous drainage and intrathecal thrombolytic therapy may have played an important role in the migration of the hematoma through the spinal canal. It is important to recognize the SSDH as a possible complication of the SAH accompanied with intracranial subdural hematoma.

TRANSPLANTED BONE MARROW STROMAL CELLS PROMOTE AXONAL REGENERATION AND IMPROVE MOTOR FUNCTION IN A RAT SPINAL CORD INJURY MODEL

OBJECTIVERecent studies have indicated that bone marrow stromal cells (BMSCs) have the potential to improve neurological function when transplanted into animal models of spinal cord injury (SCI). However, it is still unclear how the transplanted BMSCs promote functional recovery after SCI. In this study, therefore, we evaluated how the transplanted BMSCs restore the function of the dorsal corticospinal tracts in the injured spinal cord. METHODSThe rats were subjected to incomplete SCI by means of a pneumatic impact device. BMSC or vehicle transplantation into the rostral site of SCI was performed at 7 days after injury. Neurological symptoms were assessed throughout the experiments. Fluoro-Ruby was injected into the dorsal funiculus of the rostral site of SCI at 63 days after injury. The fate of the transplanted BMSCs was examined using immunohistochemistry. RESULTSBMSC transplantation significantly enhanced functional recovery of the hind limbs. The number of Fluoro-Ruby–labeled fibers of the dorsal corticospinal tracts at the caudal site of SCI was significantly higher in the BMSC-transplanted animals than in the vehicle-transplanted animals. Some of the engrafted BMSCs were positive for Fluoro-Ruby, NeuN, and MAP2 in the gray matter, suggesting that they acquired neuronal phenotypes and built synaptic connection with the hosts neural circuits. Others in the white matter morphologically simulated the astrocytes and were also positive for glial fibrillary acidic protein. CONCLUSIONThe findings suggest that the transplanted BMSCs acquire neural cell phenotypes around the injury site and contribute to rebuild the neural circuits, including the corticospinal tract, promoting functional recovery of the hind limbs.

Synergistic effects of bone marrow stromal cells and a Rho kinase (ROCK) inhibitor, Fasudil on axon regeneration in rat spinal cord injury

Transplanted bone marrow stromal cells (BMSC) promote functional recovery after spinal cord injury (SCI) through multiple mechanisms. A Rho kinase inhibitor, Fasudil also enhances axonal regeneration. This study was aimed to evaluate whether combination therapy of BMSC transplantation and Fasudil further enhances axonal regeneration and functional recovery in rats subjected to SCI. Fasudil or vehicle was injected for 2 weeks. BMSC or vehicle transplantation into the rostral site of SCI was performed at 7 days after injury. Neurological symptoms were assessed throughout the experiments. Fluoro‐Ruby was injected into the dorsal funiculus of the rostral site of SCI at 63 days after injury. The fate of the transplanted BMSC was examined using immunohistochemistry. BMSC transplantation significantly increased the number of Fluoro‐Ruby ‐labeled fibers of the dorsal corticospinal tracts at the caudal site of SCI, enhancing functional recovery of the hind limbs. Some of the engrafted BMSC were positive for Fluoro‐Ruby, neuronal specific nuclear protein and microtubule‐associated protein‐2, suggesting that they acquired neuronal phenotypes and built synaptic connection with the hosts neural circuits. Fasudil treatment also improved axonal continuity, but did not promote functional recovery. Combination therapy dramatically increased the number of Fluoro‐Ruby‐labeled fibers of the dorsal corticospinal tracts at the caudal site of SCI, but did not further boost the therapeutic effects on locomotor function by BMSC transplantation. The findings suggest that BMSC transplantation and Fasudil provide synergistic effects on axon regeneration after SCI, although further studies would be necessary to further enhance functional recovery.

A case of pediatric thoracic SCIWORA following minor trauma.

Abstract.Case report: A case of spinal cord injury without radiological abnormality (SCIWORA) at the thoracic level is reported. A 14-year-old girl fell backwards from a low chair and hit her back on the floor. It left her bent forward markedly. After taking a nap, she found herself unable to walk. Neurological examination revealed flaccid paraparesis, hypalgesia below the L-1 level, and bladder and bowel dysfunction. MR imaging revealed marked edema in the thoracic spinal cord. Results and conclusion: The patient was treated conservatively and showed gradual improvement in her symptoms, finally becoming independently ambulant. The spinal cord edema was less pronounced on the follow-up MR imaging. The clinical course and findings of MR imaging in this case demonstrated mid-thoracic SCIWORA caused by hyperflexion of the thoracic spine.