Shinozaki, Munehisa



School of Medicine, Department of Orthopaedic Surgery (Shinanomachi)


Project Senior Assistant Professor (Non-tenured)/Project Assistant Professor (Non-tenured)/Project Lecturer (Non-tenured)


Research Areas 【 Display / hide

  • Life Science / Clinical pharmacy

  • Life Science / Physiology

Research Keywords 【 Display / hide

  • regeneration


Papers 【 Display / hide

  • A non-invasive system to monitor in vivo neural graft activity after spinal cord injury

    Ago K., Nagoshi N., Imaizumi K., Kitagawa T., Kawai M., Kajikawa K., Shibata R., Kamata Y., Kojima K., Shinozaki M., Kondo T., Iwano S., Miyawaki A., Ohtsuka M., Bito H., Kobayashi K., Shibata S., Shindo T., Kohyama J., Matsumoto M., Nakamura M., Okano H.

    Communications Biology (Communications Biology)  5 ( 1 )  2022.12

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    Expectations for neural stem/progenitor cell (NS/PC) transplantation as a treatment for spinal cord injury (SCI) are increasing. However, whether and how grafted cells are incorporated into the host neural circuit and contribute to motor function recovery remain unknown. The aim of this project was to establish a novel non-invasive in vivo imaging system to visualize the activity of neural grafts by which we can simultaneously demonstrate the circuit-level integration between the graft and host and the contribution of graft neuronal activity to host behaviour. We introduced Akaluc, a newly engineered luciferase, under the control of enhanced synaptic activity-responsive element (E-SARE), a potent neuronal activity-dependent synthetic promoter, into NS/PCs and engrafted the cells into SCI model mice. Through the use of this system, we found that the activity of grafted cells was integrated with host behaviour and driven by host neural circuit inputs. This non-invasive system is expected to help elucidate the therapeutic mechanism of cell transplantation treatment for SCI.

  • biPACT: A method for three-dimensional visualization of mouse spinal cord circuits of long segments with high resolution

    Nakanishi K., Shinozaki M., Nagoshi N., Nakamura M., Okano H.

    Journal of Neuroscience Methods (Journal of Neuroscience Methods)  379 2022.09

    ISSN  01650270

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    Background: The spatial complexity of neuronal circuits in the central nervous system is a hurdle in understanding and treating brain and spinal cord injury (SCI). Although several methods have recently been developed to render the spinal cord transparent and label specific neural circuits, three-dimensional visualization of long segments of spinal cord with high resolution remains challenging for SCI researchers. New Method: We present a method that combines tissue staining of neuronal tracts traced with biotinylated dextran amine (BDA) and a modified passive clarity clearing protocol to describe individual fibers in long segments of mouse spinal cord. Results: Corticospinal tract was traced with BDA with a mouse model of thoracic spinal cord injury. The spinal cord was stained and cleared in two weeks with four solutions: staining solution, hydrogel solution, clearing solution, and observation solution. The samples were observed with a light-sheet microscope, and three-dimensional reconstruction was performed with ImageJ software. High resolution-images comparable with tissue sections were obtained continuously and circumferentially. By tiling, it was possible to obtain high-resolution images of long segments of the spinal cord. The tissue could be easily re-stained in case of fading. Comparison with existing methods: The present method does not require special equipment such as vacuum devices, can label specific circuits without genetic technology, and re-staining rounds can be easily implemented. Conclusions: By using simple neural staining and clearing methods, it was possible to acquire a wide range of high-resolution three-dimensional images of the spinal cord.

  • Long Preservation of AAV-Transduced Fluorescence by a Modified Organic Solvent-Based Clearing Method

    Lu T., Shinozaki M., Nagoshi N., Nakamura M., Okano H.

    International Journal of Molecular Sciences (International Journal of Molecular Sciences)  23 ( 17 )  2022.09

    ISSN  16616596

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    The development of tissue clearing technologies allows 3D imaging of whole tissues and organs, especially in studies of the central nervous system innervated throughout the body. Although the three-dimensional imaging of solvent-cleared organs (3DISCO) method provides a powerful clearing capacity and high transparency, the rapid quenching of endogenous fluorescence and peroxide removal process decreases its practicability. This study provides a modified method named tDISCO to solve these limitations. The tDISCO protocol can preserve AAV-transduced endogenous EGFP fluorescence for months and achieve high transparency in a fast and simple clearing process. In addition to the brain, tDISCO was applied to other organs and even hard bone tissue. tDISCO also enabled us to visualize the long projection neurons and axons with high resolution. This method provides a fast and simple clearing protocol for 3D visualization of the AAV- transduced long projection neurons throughout the brain and spinal cord.

  • 3D imaging of supraspinal inputs to the thoracic and lumbar spinal cord mapped by retrograde tracing and light-sheet microscopy

    Lu T., Shinozaki M., Nagoshi N., Nakamura M., Okano H.

    Journal of Neurochemistry (Journal of Neurochemistry)  162 ( 4 ) 352 - 370 2022.08

    ISSN  00223042

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    The supraspinal inputs play a major role in tuning the hindlimb locomotion function. While most research on spinal cord injury (SCI) with rodents is based on thoracic segments, the difference in connectivity of the supraspinal centers to the thoracic and lumbar cord is still unknown. Here, we combined retrograde tracing and 3D imaging to map the connectivity of supraspinal neurons projecting to thoracic (T9-vertebral) and lumbar (T13-vertebral) spinal levels in adult female mice. We dissected the difference in connections of corticospinal neurons (CSNs), rubrospinal neurons, and reticulospinal neurons projecting to thoracic and lumbar cords. The ratio of double-labeled neurons is higher in T13-vertebral projection CSNs and parvocellular part of the red nucleus (RPC) than in T9-vertebral projection. Using the Cre-DIO system, we precisely targeted CSNs projecting to T9-vertebral or T13-vertebral. We found that abundant axon branches communicated with the red nucleus and reticular formation and distributed from cervical gray matter to the lumbar cord. Their collateral branches showed a distinct innervation pattern in thoracic and lumbar gray matters and a similar distribution pattern in the cervical spinal cord. These results revealed the difference in connectivity between the thoracic and lumbar projection supraspinal centers and clarified the collateralization of thoracic/lumbar projection CSNs throughout the brain and spinal cord. This study highlights brain-spinal cord neural networks and the complexity of the axon terminals of spinal projection CSNs, which could contribute to the development of targeted therapeutic strategies connecting CST fibers and hindlimb function recovery. (Figure presented.) Cover Image for this issue:

  • Treadmill Training for Common Marmoset to Strengthen Corticospinal Connections After Thoracic Contusion Spinal Cord Injury

    Kondo T., Saito R., Sato Y., Sato K., Uchida A., Yoshino-Saito K., Shinozaki M., Tashiro S., Nagoshi N., Nakamura M., Ushiba J., Okano H.

    Frontiers in Cellular Neuroscience (Frontiers in Cellular Neuroscience)  16 2022.04

    ISSN  16625102

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    Spinal cord injury (SCI) leads to locomotor dysfunction. Locomotor rehabilitation promotes the recovery of stepping ability in lower mammals, but it has limited efficacy in humans with a severe SCI. To explain this discrepancy between different species, a nonhuman primate rehabilitation model with a severe SCI would be useful. In this study, we developed a rehabilitation model of paraplegia caused by a severe traumatic SCI in a nonhuman primate, common marmoset (Callithrix jacchus). The locomotor rating scale for marmosets was developed to accurately assess the recovery of locomotor functions in marmosets. All animals showed flaccid paralysis of the hindlimb after a thoracic contusive SCI, but the trained group showed significant locomotor recovery. Kinematic analysis revealed significantly improved hindlimb stepping patterns in trained marmosets. Furthermore, intracortical microstimulation (ICMS) of the motor cortex evoked the hindlimb muscles in the trained group, suggesting the reconnection between supraspinal input and the lumbosacral network. Because rehabilitation may be combined with regenerative interventions such as medicine or cell therapy, this primate model can be used as a preclinical test of therapies that can be used in human clinical trials.

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