Toshiya Osanai

Near-infrared fluorescence labeling allows noninvasive tracking of bone marrow stromal cells transplanted into rat infarct brain.

BACKGROUND:Noninvasive imaging techniques would be needed to validate the therapeutic benefits of cell transplantation therapy for central nervous system disorders. OBJECTIVE:To evaluate whether near-infrared (NIR)-emitting fluorescence tracer, quantum dots, would be useful to noninvasively visualize the bone marrow stromal cells (BMSC) transplanted into the infarct brain in living animals. METHODS:Rat BMSCs were labeled with QD800. In vitro and in vivo conditions to visualize NIR fluorescence were precisely optimized. The QD800-labeled BMSCs were stereotactically transplanted into the ipsilateral striatum of the rats subjected to permanent middle cerebral artery occlusion 7 days after the insult. Using the NIR fluorescence imaging technique, the behaviors of BMSCs were serially visualized during the 8 weeks after transplantation. RESULTS:NIR fluorescence imaging could noninvasively detect the NIR fluorescence emitted from the transplanted BMSCs engrafted in the peri-infarct neocortex through the scalp up to 8 weeks after transplantation. The intensity gradually increased and reached the peak at 4 weeks. The results were supported by the findings on ex vivo NIR fluorescence imaging and histological analysis. CONCLUSION:NIR fluorescence imaging is valuable in monitoring the behaviors of donor cells in the rodent brain. The results would allow new opportunities to develop noninvasive NIR fluorescence imaging as a modality to track the BMSCs transplanted into the brain.

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.

Noninvasive transplantation of bone marrow stromal cells for ischemic stroke: preliminary study with a thermoreversible gelation polymer hydrogel.

OBJECTIVERecent studies have indicated that bone marrow stromal cells (BMSCs) have the potential to improve neurological function when transplanted into animal models of cerebral infarct. However, it is still undetermined how the BMSCs should be transplanted to obtain the most efficient therapeutic benefits safely. The aim of this study was to assess whether a thermoreversible gelation polymer (TGP) hydrogel acts as a noninvasive, valuable scaffold in BMSC transplantation for infarct brain. METHODSThe mice were subjected to permanent middle cerebral artery occlusion. Vehicle, BMSC suspension, or the BMSC-TGP construct was transplanted onto the ipsilateral intact neocortex at 7 days after the insult. Neurological symptoms were assessed throughout the experiments. The fate of the transplanted BMSC was examined 8 weeks after transplantation with immunohistochemistry. RESULTSTGP hydrogel completely disappeared and provoked no inflammation in the host brain. Many transplanted cells were widely engrafted in the ipsilateral cerebrum, including the dorsal neocortex adjacent to the cerebral infarct in the BMSC-TGP construct—treated mice. Their number was significantly larger than in the BMSC-treated mice. The majority were positive for both NeuN and MAP2 and morphologically simulated the neurons. CONCLUSIONThe findings suggest that surgical transplantation of tissue-engineered BMSCs onto the intact neocortex enhances the engraftment of donor cells around the cerebral infarct. These data may be useful in developing a noninvasive but efficient paradigm in neural tissue engineering. TGP hydrogel can be a promising candidate for valuable scaffolds in BMSC transplantation for central nervous system disorders because of its unique biochemical properties.

Therapeutic effects of intra-arterial delivery of bone marrow stromal cells in traumatic brain injury of rats--in vivo cell tracking study by near-infrared fluorescence imaging.

BACKGROUND: A noninvasive and effective route of cell delivery should be established to yield maximal therapeutic effects for central nervous system (CNS) disorders. OBJECTIVE: To elucidate whether intra-arterial delivery of bone marrow stromal cells (BMSCs) significantly promotes functional recovery in traumatic brain injury (TBI) in rats. METHODS: Rat BMSCs were transplanted through the ipsilateral internal carotid artery 7 days after the onset of cortical freezing injury. The BMSCs were labeled with fluorescent dye, and in vivo optical imaging was employed to monitor the behaviors of cells for 4 weeks after transplantation. Motor function was assessed for 4 weeks, and the transplanted BMSCs were examined using immunohistochemistry. RESULTS: In vivo optical imaging and histologic analysis clearly demonstrated that the intra-arterially injected BMSCs were engrafted during the first pass without systemic circulation, and the transplanted BMSCs started to migrate from the cerebral capillary bed to the injured CNS tissue within 3 hours. Intra-arterial BMSC transplantation significantly promoted functional recovery after cortical freezing injury. A subgroup of BMSCs expressed the phenotypes of neurons, astrocytes, and endothelial cells around the injured neocortex 4 weeks after transplantation. CONCLUSION: Intra-arterial transplantation may be a valuable option for prompt, noninvasive delivery of BMSCs to the injured CNS tissue, enhancing functional recovery after TBI. In vivo optical imaging may provide important information on the intracerebral behaviors of donor cells by noninvasive, serial visualization.

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.

Bone marrow stromal cells and bone marrow-derived mononuclear cells: Which are suitable as cell source of transplantation for mice infarct brain?

There are few studies that denote whether bone marrow stromal cells (BMSC) and bone marrow‐derived mononuclear cells (MNC) show the same therapeutic effects, when directly transplanted into the infarct brain. This study therefore aimed to compare their biological properties and behaviors in the infarct brain. Mouse BMSC were harvested and cultured. Mouse MNC were obtained through centrifugation techniques. Their cell markers were analyzed with FACS analysis. The MNC (106 cells; n = 10) or BMSC (2 × 105 cells; n = 10) were stereotactically transplanted into the ipsilateral striatum of the mice subjected to permanent middle cerebral artery occlusion at 7 days after the insult. Their survival, migration, and differentiation in the infarct brain were precisely analyzed using immunohistochemistry 4 weeks after transplantation. The MNC were positive for CD34, CD45, CD90, but were negative for Sca‐1. The BMSC were positive for CD90 and Sca‐1. The transplanted BMSC, but not MNC, extensively migrated into the peri‐infarct area. Approximately 20% of the transplanted BMSC expressed a neuronal marker, NeuN in the infarct brain, although only 1.4% of the transplanted MNC expressed NeuN. These findings strongly suggest that there are large, biological differences between MNC and BMSC as cell sources of regenerative medicine for ischemic stroke.

Moyamoya disease presenting with subarachnoid hemorrhage localized over the frontal cortex: case report

BACKGROUND In moyamoya disease, intracranial bleeding is known to occur because of the rupture of saccular aneurysms in the circle of Willis or because of the rupture of dilated, fragile moyamoya vessels. The former causes subarachnoid hemorrhage (SAH), and the latter causes intracerebral or intraventricular hemorrhage. CASE DESCRIPTION In this report, we describe the case of a 34-year-old woman with moyamoya disease who suddenly developed headache and jacksonian seizure. Plain computed tomographic scans on admission revealed SAH localized over the left frontal cortex. The patient was diagnosed with moyamoya disease on cerebral angiography. However, no aneurysm was found on cerebral angiography. Positron emission tomography showed the reduction of CBF and its reactivity to acetazolamide and the elevation of CBV in the left hemisphere. She underwent STA to MCA anastomosis and indirect synangiosis. Intraoperative observations revealed that the pial arterioles were markedly dilated on the brain surface. The CBF in the left hemisphere significantly improved after surgery. The patient has experienced no further episode of cerebral ischemia or intracranial bleeding. CONCLUSIONS Subarachnoid hemorrhage of unknown cause is quite rare in moyamoya disease. Based on the findings in the present case, the dilated collateral arteries on the brain surface may rupture and cause SAH over the cerebral cortex, which is the third cause of intracranial bleeding in patients with persistent cerebral ischemia due to moyamoya disease.

Impact of ageing on biological features of bone marrow stromal cells (BMSC) in cell transplantation therapy for CNS disorders: Functional enhancement by granulocyte-colony stimulating factor (G-CSF)

This study was designed to clarify the effects of donor age on biological features of bone marrow stromal cells (BMSC), one of the candidates for cell transplantation therapy for CNS disorders, because many aged patients might require such therapy. This study was also aimed to test whether ex vivo treatments with granulocyte‐colony stimulating factor (G‐CSF) could modify biological properties of BMSC from aged donors and enhance its therapeutic effects in an animal model of traumatic brain injury. The BMSC were harvested from young (6‐week‐old) and aged (100‐week‐old) rats. The ageing significantly increased the senescence‐associated β‐galactosidase (SA‐β‐gal) activity of the cultured BMSC, and decreased their proliferative capacity and production of nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF). As the next step, the rats were subjected to brain freezing injury by applying liquid nitrogen onto the neocortex through the thinned skull. The 6‐week BMSC, 100‐week BMSC, G‐CSF‐treated 100‐week BMSC or vehicle were stereotactically injected into the ipsilateral striatum at 7 days post‐injury. Transplantation of the 6‐week BMSC, but not 100‐week BMSC, significantly improved locomotor function. However, treatment of the 100‐week BMSC with 0.1 µmol of G‐CSF significantly improved their proliferation activity and growth factor production, and recovered therapeutic effects in the injured brain. In conclusion, donor age may largely determine biological aspects of BMSC. G‐CSF may contribute to improve the outcome of BMSC transplantation therapy for CNS disorders in aged patients.

Increased Blood Viscosity in Ischemic Stroke Patients with Small Artery Occlusion Measured by an Electromagnetic Spinning Sphere Viscometer

BACKGROUND AND PURPOSE High blood viscosity causes blood stagnation and subsequent pathological thrombotic events, resulting in the development of ischemic stroke. We hypothesize that the contribution of blood viscosity may differ among ischemic stroke subtypes based on specific pathological conditions. We tried to verify this hypothesis by measuring blood viscosity in acute ischemic stroke patients using a newly developed electromagnetic spinning sphere (EMS) viscometer. METHODS Measurements in acute ischemic stroke patients were performed 4 times during admission and data were compared with those obtained from 100 healthy outpatient volunteers. RESULTS We enrolled 92 patients (cardioembolism: 25, large artery atherosclerosis: 42, and small artery occlusion [SAO]: 25) in this study. Comparisons of blood viscosity between the ischemic stroke subgroups and control group revealed that blood viscosity at the date of admission was significantly higher in the SAO group (5.37 ± 1.11 mPa⋅s) than in the control group (4.66 ± .72 mPa⋅s) (P < .01). Among all subtype groups showing a reduction in blood viscosity after 2 weeks, the SAO group showed the highest and most significant reduction, indicating that SAO patients had the most concentrated blood at the onset. CONCLUSIONS Blood viscosity was significantly increased in the SAO group at the date of admission, which indicated the contribution of dehydration to the onset of ischemic stroke. The importance of dehydration needs to be emphasized more in the pathogenesis of SAO. The clinical application of the EMS viscometer is promising for understanding and differentiating the pathogenesis of ischemic stroke.

Utility of noncontrast-enhanced time-resolved four-dimensional MR angiography with a vessel-selective technique for intracranial arteriovenous malformations

To evaluate the utility of a vessel‐selective four‐dimensional (4D) magnetic resonance angiography (MRA) technique for the evaluation of intracranial arteriovenous malformations (AVMs).