Katsuhiko Maruichi

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.

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.

Graded model of diffuse axonal injury for studying head injury‐induced cognitive dysfunction in rats

Diffuse axonal injury (DAI) plays a major role in the development of cognitive dysfunction, emotional difficulties and behavioral disturbances in patients following closed head injury, even when they have no definite abnormalities on conventional MRI. This study aimed to develop a highly controlled and reproducible model for DAI that simulates post‐traumatic cognitive dysfunction in humans. Sprague‐Dawley (SD) rats were subjected to impact acceleration head injury, using a pneumatic impact targeted to a steel disc centered onto their skull. The severity of injury was graded as three levels by adjusting the driving pressure at 60, 70 or 80 pounds per square inch. In vivo MRI was obtained 2 days post‐injury. Cognitive function was evaluated using the Morris water maze at 1 and 2 weeks post‐injury. HE staining and immunohistochemistry were performed to assess neuronal and axonal damages after 2 weeks. MRI demonstrated that this model induced no gross structural modification in the brain. The degree and duration of cognitive dysfunction were dependent on the force of impact. Histological analysis revealed the force‐dependent damage of the neurons and microtubule‐associated protein 2‐positive axons in the neocortex. Hippocampal damage was much less pronounced and was not linked to cognitive dysfunction. This is the first report that precisely evaluates the threshold of impact energy to lead to neocortical damage and cognitive dysfunction in rodents. This model would be suitable for clarifying the complex mechanisms of post‐traumatic brain damage and testing novel therapeutic approaches against post‐traumatic cognitive dysfunction due to diffuse axonal damage.

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.

Transplanted bone marrow stromal cells improves cognitive dysfunction due to diffuse axonal injury in rats

Diffuse axonal injury (DAI) often leads to persistent cognitive dysfunction in spite of the lack of gross lesions on MRI. Therefore, this study was aimed to evaluate whether transplanted bone marrow stromal cells (BMSC) can improve DAI‐induced cognitive dysfunction or not. The rats were subjected to impact acceleration head injury, using a pneumatic high‐velocity impactor. The BMSC were harvested from the mice and were cultured. The BMSC (4.0 × 105 cells) or vehicle were stereotactically transplanted into the right striatum at 10 days post‐injury. Cognitive function analysis was repeated at 1, 2, and 4 weeks post‐injury, using the Morris water maze test. Histological analysis was performed at 2, 8 and 20 weeks post‐injury, using double fluorescence immunohistochemistry. Transplanted BMSC were widely distributed in the injured brain and gradually acquired the phenotypes of neurons and astrocytes over 20 weeks. In addition, they significantly improved DAI‐induced cognitive dysfunction as early as 2 weeks post‐injury, although their processes of neuronal differentiation were not completed at this time point. The findings suggest that the engrafted BMSC may exhibit this early beneficial effect on cognitive function by producing neuroprotective or neurotrophic factors. In conclusion, direct transplantation of BMSC may serve as a novel therapeutic strategy to enhance the recovery from DAI‐induced cognitive impairment.

Transplanted bone marrow stromal cells protect neurovascular units and ameliorate brain damage in stroke-prone spontaneously hypertensive rats

This study was aimed to assess whether bone marrow stromal cells (BMSC) could ameliorate brain damage when transplanted into the brain of stroke‐prone spontaneously hypertensive rats (SHR‐SP). The BMSC or vehicle was stereotactically engrafted into the striatum of male SHR‐SP at 8 weeks of age. Daily loading with 0.5% NaCl‐containing water was started from 9 weeks. MRIs and histological analysis were performed at 11 and 12 weeks, respectively. Wistar‐Kyoto rats were employed as the control. As a result, T2‐weighted images demonstrated neither cerebral infarct nor intracerebral hemorrhage, but identified abnormal dilatation of the lateral ventricles in SHR‐SP. HE staining demonstrated selective neuronal injury in their neocortices. Double fluorescence immunohistochemistry revealed that they had a decreased density of the collagen IV‐positive microvessels and a decreased number of the microvessels with normal integrity between basement membrane and astrocyte end‐feet. BMSC transplantation significantly ameliorated the ventricular dilatation and the breakdown of neurovascular integrity. These findings strongly suggest that long‐lasting hypertension may primarily damage neurovascular integrity and neurons, leading to tissue atrophy and ventricular dilatation prior to the occurrence of cerebral stroke. The BMSC may ameliorate these damaging processes when directly transplanted into the brain, opening the possibility of prophylactic medicine to prevent microvascular and parenchymal‐damaging processes in hypertensive patients at higher risk for cerebral stroke.

Beneficial Effects of Bone Marrow Stromal Cell Transplantation on Axonal Regeneration in Injured Spinal Cord

Recent 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 transplanted BMSCs restore the function of the dorsal corticospinal tract (dCST) in the injured spinal cord. Rats were subjected to incomplete SCI, using a pneumatic impact device. Then BMSC suspension or vehicle was transplanted into the rostral site of the SCI at 7 days after the injury. Fluoro-ruby (FR; Molecular Probes), a fluorescent axonal tracer, was injected into the dorsal funiculus of the rostral site of the SCI 63 days after the injury. BMSC transplantation significantly enhanced functional recovery of the hind limbs. The number of FR-labeled fibers in the dCST at the caudal site of the SCI was significantly higher in the BMSC-transplanted animals than in the vehicle-transplanted animals. Some of the engrafted BMSCs were positive for FR, neuronal nuclear antigen (NeuN). and microtubule-associated protein 2 (MAP2) in the gray matter. The findings suggest that the transplanted BMSCs acquire neural cell phenotypes around the injury site and contribute to rebuilding neural circuits, including those in the CST. promoting functional recovery of the hind limbs.

Thermoreversible Gelation Polymer (TGP) Hydrogel as a Degradable Scaffold for Bone Marrow Stromal Cell Transplantation

Recent studies have indicated that bone marrow stromal cells (BMSCs) have the potential to improve neurologic function when transplanted into animal cerebral infarct models. However, it is still undetermined how the BMSCs should be transplanted to safely obtain the most efficient therapeutic benefits. Therefore, this study was carried out aiming to assess whether a thermoreversible gelation polymer (TGP) hydrogel would act as a noninvasive. valuable scaffold in BMSC transplantation for brain infarct. Mice were subjected to permanent middle cerebral artery occlusion. Vehicle, BMSC suspension, or a BMSC-TGP construct was transplanted onto the ipsilateral intact neocortex at 7 days after the insult. The TGP hydrogel completely disappeared and provoked no inflammation in the host brain. In the BMSC-TGP construct-treated mice, many transplanted cells were widely engrafted in the ipsilateral cerebrum, including the dorsal neocortex adjacent to the cerebral infarct. The number of these transplanted cells was significantly larger than that in the BMSC-treated mice; the majority of the cells were positive for both neuronal nuclear antigen (NeuN) and microtubule-associated protein 2 (MAP2) and morphologically simulated neurons. These findings suggest that the surgical transplantation of tissue-engineered BMSCs onto the intact neocortex enhances the engraftment of donor cells around a cerebral infarct. TGP hydrogel, because of its unique biochemical properties, could be a promising candidate as a valuable scaffold in BMSC transplantation for central nervous system disorders.

Noninvasive Optical Tracking of Bone Marrow Stromal Cells Transplanted into Rat Cerebral Infarct

Background and Purpose It is quite important to track donor cells transplanted into the central nervous system (CNS) in order to evaluate the therapeutic effects of cell transplantation therapy. Therefore, in this preliminary study we aimed to evaluate whether fluorescent quantum dots (QDs) would be useful in noninvasive cell tracking in cerebral infarcts of rats.