Guidelines for cadaver dissection in education and research of clinical medicine (The Japan Surgical Society and The Japanese Association of Anatomists).

This article translates the guidelines for cadaver surgical training (CST) published in 2012 by Japan Surgical Society (JSS) and Japanese Association of Anatomists from Japanese to English. These guidelines are based on Japanese laws and enable the usage of donated cadavers for CST and clinical research. The following are the conditions to implement the activities outlined in the guidelines. The aim is to improve medicine and to contribute to social welfare. Activities should only be carried out at medical or dental universities under the centralized control by the department of anatomy under the regulation of Japanese law. Upon the usage of cadavers, registered donors must provide a written informed-consent for their body to be used for CST and other activities of clinical medicine. Commercial use of cadavers and profit-based CST is strongly prohibited. Moreover, all the cadaver-related activities except for the commercial-based ones require the approval of the University's Institutional Review Board (IRB) before implementation. The expert committee organized at each university for the implementation of CST should summarize the implementation of the program and report the details of the training program, operating costs, and conflicts of interest to the CST Promotion Committee of JSS.

Evaluation of serum phosphorylated neurofilament subunit NF-H as a prognostic biomarker in dogs with thoracolumbar intervertebral disc herniation.

OBJECTIVE: To investigate whether pNF-H is a prognostic biomarker of spinal cord injury (SCI) in paraplegic dogs with thoracolumbar intervertebral disc herniation (IVDH). STUDY DESIGN: Prospective, case-control clinical study ANIMALS: Dogs (n = 60) with SCI from IVDH and 6 healthy dogs. METHODS: Serum from 60 thoracolumbar IVDH dogs (Grade 4: 22 dogs; Grade 5: 38 dogs) collected 1-3 days after injury, and 6 control dogs, was analyzed using enzyme-linked immunosorbent assay (ELISA) against a phosphorylated form of the high-molecular-weight neurofilament subunit NF-H (pNF-H). Serum pNF-H levels were compared between different IVDH grades and their prognostic value was investigated. RESULTS: pNF-H levels were significantly greater in Grade 5 than Grade 4 dogs. There were significant differences in pNF-H levels between dogs that regained voluntarily ambulation and those that did not. All 8 dogs that had high pNF-H levels 1-3 days after injury did not regain the ability to walk after surgery. CONCLUSIONS: Serum pNF-H levels might be a biomarker for predicting prognosis of canine SCI.

Safety of autologous bone marrow stromal cell transplantation in dogs with acute spinal cord injury.

OBJECTIVE: To assess the feasibility and safety of transplantation of autologous bone marrow stromal cell (BMSC) in dogs with acute spinal cord injury (SCI). STUDY DESIGN: An open-label single-arm trial. ANIMALS: Dogs (n = 7) with severe SCI from T6 to L5, caused by vertebral fracture and luxation. METHODS: Decompressive and stabilization surgery was performed on dogs with severe SCI caused by vertebral fracture and luxation. Autologous BMSCs were obtained from each dog's femur, cultured, and then injected into the lesion in the acute stage. Adverse events and motor and sensory function were observed for >1 year after SCI. RESULTS: Follow-up was 29-62 months after SCI. No complications (eg, infection, neuropathic pain, worsening of neurologic function) were observed. Two dogs walked without support, but none of the 7 dogs had any change in sensory function. CONCLUSIONS: Autologous BMSC transplantation is feasible and safe in dogs with acute SCI. Further studies are needed to determine the efficacy of this therapy.

Guidelines for cadaver dissection in education and research of clinical medicine (The Japan Surgical Society and The Japanese Association of Anatomists).

This article translates the guidelines for cadaver surgical training (CST) published in 2012 by Japan Surgical Society (JSS) and Japanese Association of Anatomists from Japanese to English. These guidelines are based on Japanese laws and enable the usage of donated cadavers for CST and clinical research. The following are the conditions to implement the activities outlined in the guidelines. The aim is to improve medicine and to contribute to social welfare. Activities should only be carried out at medical or dental universities under the centralized control by the department of anatomy under the regulation of Japanese law. Upon the usage of cadavers, registered donors must provide a written informed-consent for their body to be used for CST and other activities of clinical medicine. Commercial use of cadavers and profit-based CST is strongly prohibited. Moreover, all the cadaver-related activities except for the commercial-based ones require the approval of the University's Institutional Review Board (IRB) before implementation. The expert committee organized at each university for the implementation of CST should summarize the implementation of the program and report the details of the training program, operating costs, and conflicts of interest to the CST Promotion Committee of JSS.

[Draft of guidelines for human body dissection for clinical anatomy education and research and commentary].

This article analyses the Draft of Guidelines for Human Body Dissection for Clinical Anatomy Education and Research drawn by the Study Group for Future Training Systems of Surgical Skills and Procedures established by the Fiscal Year 2010 research program of the Ministry of Health, Labor and Welfare. The purpose of the Draft of Guidelines is: First, to lay out the required basic guidelines for human cadaver usage to allow medical and dental faculty to conduct clinical education and research in accordance with existing regulations. Second, the guidelines are expected to give physicians a regulatory framework to carry out cadaver training in accordance with the current legal framework. This article explains the Draft of Guidelines in detail, outlines the future of cadaver training, and describes issues which must still be solved.

[Draft of Guidelines for Human Body Dissection for Clinical Anatomy Education and Research and commentary].

This article analyses the Draft of Guidelines for Human Body Dissection for Clinical Anatomy Education and Research drawn by the Study Group for Future Training Systems of Surgical Skills and Procedures established by the Fiscal Year 2010 research program of the Ministry of Health, Labor and Welfare. The purpose of the Draft of Guidelines is: First, to lay out the required basic guidelines for human cadaver usage to allow medical and dental faculty to conduct clinical education and research in accordance with existing regulations. Second, the guidelines are expected to give physicians a regulatory framework to carry out cadaver training in accordance with the current legal framework. This article explains the Draft of Guidelines in detail, outlines the future of cadaver training, and describes issues which must still be solved.

Nectin: an adhesion molecule involved in formation of synapses.

The nectin-afadin system is a novel cell-cell adhesion system that organizes adherens junctions cooperatively with the cadherin-catenin system in epithelial cells. Nectin is an immunoglobulin-like adhesion molecule, and afadin is an actin filament-binding protein that connects nectin to the actin cytoskeleton. Nectin has four isoforms (-1, -2, -3, and -4). Each nectin forms a homo-cis-dimer followed by formation of a homo-trans-dimer, but nectin-3 furthermore forms a hetero-trans-dimer with nectin-1 or -2, and the formation of each hetero-trans-dimer is stronger than that of each homo-trans-dimer. We show here that at the synapses between the mossy fiber terminals and dendrites of pyramidal cells in the CA3 area of adult mouse hippocampus, the nectin-afadin system colocalizes with the cadherin-catenin system, and nectin-1 and -3 asymmetrically localize at the pre- and postsynaptic sides of puncta adherentia junctions, respectively. During development, nectin-1 and -3 asymmetrically localize not only at puncta adherentia junctions but also at synaptic junctions. Inhibition of the nectin-based adhesion by an inhibitor of nectin-1 in cultured rat hippocampal neurons results in a decrease in synapse size and a concomitant increase in synapse number. These results indicate an important role of the nectin-afadin system in the formation of synapses.

Structures of filum terminale and characteristics of ependymal cells of its central canal in rats.

The filum terminale (FT) is a potential source of ependymal cells for transplantation. The present study was performed to clarify the characteristics of ependymal cells of the central canal (CC) of the FT in rats. The FT was a thin strand continuous with the conus medullaris (CM), a caudal end of the main spinal cord, situated at the L3-4 level in adult rats. The border between the CM and FT was not visible, but could be defined as the site where the strand was as thin as its more caudal segment. While the CM contained an appreciable amount of white and grey matter associated with the CC at its center, the FT had no or only a negligible amount of such spinal cord parenchymal tissue. The FT was tracked ca. 4 cm from the site defined above to the level of S4-5 in adult rats. The rostral part of the FT (FTI) included within the cauda equina is exposed to cerebrospinal fluid, whereas the more caudal part (FTE) was surrounded by a dense layer of connective tissue. Almost all ependymal cells were immunostained for Sox2, Sox9, FoxJ1, and CD133, generally recognized immunochemical markers for ependymal cells of the CC in the spinal cord. Ependymal cells of the CC of FT exhibited almost the same structural and immunohistochemical characteristics as those of the CC of the main spinal cord. Ependymal cells of FTI covered by a thin layer of connective tissue are considered appropriate for transplantation.

Effects of Intrathecal Injection of the Conditioned Medium from Bone Marrow Stromal Cells on Spinal Cord Injury in Rats.

Bone marrow stromal cells (BMSCs) have been studied for the treatment of spinal cord injury (SCI). In previous studies, we showed that the transplantation of BMSCs, even though they disappeared from the host spinal cord within 1-3 weeks after transplantation, improved locomotor behaviors and promoted axonal regeneration. This result led to the hypothesis that BMSCs might release some neurotrophic factors effective for the treatment of SCI. The present study examined this by injecting the conditioned medium (CM) of BMSCs to treat SCI in rats. The spinal cord was contusion-injured, followed immediately by continuous injection for 2 weeks of the CM of BMSCs through the cerebrospinal fluid via the 4th ventricle using an Alzet osmotic pump. Locomotor behaviors evaluated by the Basso-Beattie-Bresnahan score were markedly improved in the CM-injection group, compared with the control group, at 1 to 4 weeks post-injection. The contusion-injured site of the spinal cord was identified as an astrocyte-devoid area, which contained no astrocytes but was filled with collagen matrices and empty cavities of various sizes. Collagen matrices contained type I collagen and laminin. Numerous axons extended through the collagen matrices of the astrocyte-devoid area. Axons were surrounded by Schwann cells, exhibiting the same morphological characteristics as peripheral nerve fibers. The density of axons extending through the astrocyte-devoid area was higher in the CM-injection group, compared with the control group. CM injection had beneficial effects on locomotor improvements and tissue repair, including axonal regeneration, meaning that the BMSC-CM stimulated the intrinsic ability of the spinal cord to regenerate. Activation of the intrinsic ability of the spinal cord to regenerate by the injection of neurotrophic factors such as BMSC-CM is considered to be a safe and preferable method for the clinical treatment of SCI.

Specific induction of neuronal cells from bone marrow stromal cells and application for autologous transplantation.

Bone marrow stromal cells (MSCs) have the capability under specific conditions of differentiating into various cell types such as osteocytes, chondrocytes, and adipocytes. Here we demonstrate a highly efficient and specific induction of cells with neuronal characteristics, without glial differentiation, from both rat and human MSCs using gene transfection with Notch intracellular domain (NICD) and subsequent treatment with bFGF, forskolin, and ciliary neurotrophic factor. MSCs expressed markers related to neural stem cells after transfection with NICD, and subsequent trophic factor administration induced neuronal cells. Some of them showed voltage-gated fast sodium and delayed rectifier potassium currents and action potentials compatible with characteristics of functional neurons. Further treatment of the induced neuronal cells with glial cell line-derived neurotrophic factor (GDNF) increased the proportion of tyrosine hydroxylase-positive and dopamine-producing cells. Transplantation of these GDNF-treated cells showed improvement in apomorphine-induced rotational behavior and adjusting step and paw-reaching tests following intrastriatal implantation in a 6-hydroxy dopamine rat model of Parkinson disease. This study shows that a population of neuronal cells can be specifically generated from MSCs and that induced cells may allow for a neuroreconstructive approach.

Neuroprotective effect of bone marrow-derived mononuclear cells promoting functional recovery from spinal cord injury.

Neural cell transplantation, a new therapeutic strategy for replacing injured neural components and obtaining functional recovery, has shown beneficial effects in animal models. Use of this strategy in human patients, however, requires that a number of serious issues be addressed, including ethics, immunorejection, and the therapeutic time window within which the procedure will be effective. Bone marrow-derived mononuclear cells (BM-MNC) are attractive for transplantation because they can be used as an autograft, can be easily collected within a short time period, and do not have to be cultured. In a rat model of spinal cord injury (SCI), we transplanted BM-MNC at 1 h after SCI at Th 8-9 by injecting them into the cerebrospinal fluid (CSF), and investigated the effect of this on neurologic function. In the acute stage of injury, we found a neuroprotective antiapoptotic effect, with an elevated concentration of hepatocyte growth factor in CSF. At 1 week after transplantation, the Basso-Beattie-Bresnahan locomotor score had increased significantly over its base-line value. In the chronic stage of injury, we observed suppressed cavity formation and functional improvement. We conclude that transplantation of BM-MNC after SCI has a remarkable neuroprotective effect in the acute stage of injury, suppressing cavity formation, and contributing to functional recovery. Our results suggest that transplantation of BM-MNC via the CSF is a potentially effective means of enhancing functional recovery after SCI in humans.

Effects of Multiple Injection of Bone Marrow Mononuclear Cells on Spinal Cord Injury of Rats.

The effects of multiple injection of bone marrow mononuclear cells (BMNCs) on spinal cord injury (SCI) were compared with those of single injection in rats. BMNCs separated by density-gradient centrifugation from a bone marrow perfusate were injected three times (once weekly) through the cerebrospinal fluid (CSF) via the fourth ventricle, and the locomotor improvement and tissue recovery, including axonal regeneration, were compared with those of single injection. While the single-injection group showed a steep elevation of the Basso-Beattie-Bresnahan (BBB) score 1 week after transplantation, the multiple-injection group maintained a similar steep elevation for 2 weeks after transplantation, and the BBB scores of the multiple-injection group remained thereafter at a level approximately 2-3 points higher than those of the single-injection group until the end of the experiment. There were significant differences between the single- and multiple-injection groups at 3, 4, and 8 weeks after transplantation. The difference in BBB scores at 8 weeks after transplantation suggested that there was a marked difference in the quality of locomotor behaviors between the single-and multiple-injection groups at this stage. An extensive outgrowth of regenerating axons through the astrocyte-devoid areas and a marked reduction of cavity formation were found in both the single- and multiple-injection groups. There were, however, no significant differences in the density of regenerating axons or volumes of cavities between the single- and multiple-injection groups. These results showed that although tissue recoveries were similar between single and multiple injection, the multiple injection of BMNCs was more beneficial for locomotor improvement than single injection for the treatment of SCI. Considering the technically simple and low-cost procedures for the preparation and injection of BMNCs, multiple injection of BMNCs by lumbar puncture has an advantage over single injection on clinical application.

Possibility of using nerve segments dissected from human cadavers for grafting: preliminary report.

An intercostal nerve obtained from a human cadaver 6 h post-mortem was transplanted into the rat sciatic nerve and nerve regeneration was observed 4 and 8 weeks after surgery. Sciatic nerves from deceased rats up to 2 days post-mortem were also transplanted for comparison. Good nerve regeneration was observed through the human cadaver-derived graft to the distal segment at the medial plantal nerve 8 weeks after surgery. The results of the present study indicate the possibility that nerves from human cadavers can be used for nerve grafting in clinical applications.

Increase in bFGF-responsive neural progenitor population following contusion injury of the adult rodent spinal cord.

The number of neural progenitor cells, especially nestin+ cells or BrdU-uptake cells is sparse in the normal adult rodent spinal cord. However, in the present study, we show that after spinal cord injury (SCI), many ordinarily quiescent cells were activated to become nestin+ and undergo mitosis (BrdU+) in the ependymal layer as well as in the parenchyma of the spinal cord. Nestin+ cells and BrdU+ cells were in most cases immunohistochemically GFAP+, some of which displayed radial glial cell morphology and partly participated in the border formation of the lesion. The culturing of injured rat spinal cord tissues generated more neurospheres earlier than did the culturing of intact tissues, and these neurosphere cells were multipotent and bFGF-responsive. Immunohistochemical analysis showed that there existed many bFGF+ cells after SCI, the number of which were almost 15 times greater than that in an intact spinal cord. Increased bFGF production after SCI might activate quiescent progenitor cells, and thus initiate their cell proliferation. Finally, SCI to the nestin-promoter green fluorescent protein (GFP) transgenic mice showed broad proliferation of progenitor cells that were induced in the injured spinal cord. The culturing of injured spinal cord tissues from these transgenic mice provides direct evidence that neurospheres can be generated by SCI-activated nestin+ cells. Thus, the activation of bFGF-responsive progenitor cells and the concomitant increase in the population of bFGF+ cells following SCI might be beneficial for spinal cord repair if these progenitor cells are properly manipulated.

Marrow stromal cells: implications in health and disease in the nervous system.

Chronic degenerative diseases and traumatic injuries are responsible for a decline in neuronal function, which often limit life span. While solid organ transplantation such as liver and kidney has been already applied for thousands of patients, great limitation exists in case of nervous system. Cell transplantation is one of the strategies with potential for treatment of such neural disorders, and many kinds of cells including embryonic stem cells and neural stem cells have been considered as candidates for transplantation therapy. Bone marrow stromal cells (MSCs) have great potential as therapeutic agents, since they are easy to isolate and can be expanded from patients without serious ethical and technical problems. We found a method for the highly efficient and specific induction of functional neurons and Schwann cells from both rat and human MSCs. Induced neurons and Schwann cells were transplanted in animal models of Parkinson's disease, stroke, peripheral nerve injury, and spinal cord injury resulting in the successful integration of transplanted cells and improvement in behavior of transplanted animals. Here we focus on the respective potentials of MSC-derived cells and discuss the possibility of clinical application in neurodegenerative and neurotraumatic diseases.

Points regarding cell transplantation for the treatment of spinal cord injury.

Transplantation of somatic cells, including bone marrow stromal cells (BMSCs), bone marrow mononuclear cells (BMNCs), and choroid plexus epithelial cells (CPECs), enhances the outgrowth of regenerating axons and promotes locomotor improvements. They are not integrated into the host spinal cord, but disappear within 2-3 weeks after transplantation. Regenerating axons extend at the spinal cord lesion through the astrocyte-devoid area that is filled with connective tissue matrices. Regenerating axons have characteristics of peripheral nerves: they are associated with Schwann cells, and embedded in connective tissue matrices. It has been suggested that neurotrophic factors secreted from BMSCs and CPECs promote "intrinsic" ability of the spinal cord to regenerate. Transplanted Schwann cells survive long-term, and are integrated into the host spinal cord, serving as an effective scaffold for the outgrowth of regenerating axons in the spinal cord. The disadvantage that axons are blocked to extend through the glial scar at the border of the lesion is overcome. Schwann cells have been approved for clinical applications. Neural stem/progenitor cells (NSPCs) survive long-term, proliferate, and differentiate into glial cells and/or neurons after transplantation. No method is available at present to manipulate and control the behaviors of NPSCs to allow them to appropriately integrate into the host spinal cord. NPSP transplantation is not necessarily effective for locomotor improvement.

Cell transplantation for the treatment of spinal cord injury - bone marrow stromal cells and choroid plexus epithelial cells.

Transplantation of bone marrow stromal cells (BMSCs) enhanced the outgrowth of regenerating axons and promoted locomotor improvements of rats with spinal cord injury (SCI). BMSCs did not survive long-term, disappearing from the spinal cord within 2-3 weeks after transplantation. Astrocyte-devoid areas, in which no astrocytes or oligodendrocytes were found, formed at the epicenter of the lesion. It was remarkable that numerous regenerating axons extended through such astrocyte-devoid areas. Regenerating axons were associated with Schwann cells embedded in extracellular matrices. Transplantation of choroid plexus epithelial cells (CPECs) also enhanced axonal regeneration and locomotor improvements in rats with SCI. Although CPECs disappeared from the spinal cord shortly after transplantation, an extensive outgrowth of regenerating axons occurred through astrocyte-devoid areas, as in the case of BMSC transplantation. These findings suggest that BMSCs and CPECs secret neurotrophic factors that promote tissue repair of the spinal cord, including axonal regeneration and reduced cavity formation. This means that transplantation of BMSCs and CPECs promotes "intrinsic" ability of the spinal cord to regenerate. The treatment to stimulate the intrinsic regeneration ability of the spinal cord is the safest method of clinical application for SCI. It should be emphasized that the generally anticipated long-term survival, proliferation and differentiation of transplanted cells are not necessarily desirable from the clinical point of view of safety.

NTAK/neuregulin-2 secreted by astrocytes promotes survival and neurite outgrowth of neurons via ErbB3.

NTAK (neural- and thymus-derived activator for ErbB kinases), also known as neuregulin-2 (NRG2), is a member of the epidermal growth factor (EGF) family, which binds directly to ErbB3 and ErbB4, and transactivates ErbB2. NTAK/NRG2 is structurally homologous to NRG1. The biological function of NTAK/NRG2 still remains unknown, especially in the nervous system, whereas NRG1 is known to be essential for nervous system function. In the present study, we examined the functions of NTAK/NRG2 secreted from astrocytes to neurons. NTAK/NRG2 was expressed in both neurons and astrocytes, as evidenced by immunohistochemical staining and RT-PCR methods. The conditioned medium (CM) from astrocytes promoted survival and neurite outgrowth of neurons. The CM stimulated phosphorylation of ErbB3 in neurons. When phosphorylation of ErbB3 was blocked by AZD8931, an ErbB3 inhibitor, neuronal survival and neurite outgrowth were reduced. Conversely, canertinib, an ErbB4 inhibitor, did not affect survival or neurite outgrowth of neurons. Survival and neurite outgrowth of neurons were lower in CM of NTAK/NRG2-knockdown astrocytes than in the CM of control astrocytes, whereas the CM of NRG1-knockdown astrocytes had little effect on survival and neurite outgrowth. The present study demonstrated that NTAK/NRG2 secreted from astrocytes bound to ErbB3 on neurons, and promoted neuronal survival and neurite extension in vitro.

Transplantation of choroid plexus epithelial cells into contusion-injured spinal cord of rats.

PURPOSE: The effect of the transplantation of choroid plexus epithelial cells (CPECs) on locomotor improvement and tissue repair including axonal extension in spinal cord lesions was examined in rats with spinal cord injury (SCI). METHODS: CPECs were cultured from the choroid plexus of green fluorescent protein (GFP)-transgenic rats, and transplanted directly into the contusion-injured spinal cord lesions of rats of the same strain. Locomotor behaviors were evaluated based on BBB scores every week after transplantation until 4 weeks after transplantation. Histological and immunohistochemical examinations were performed at 2 days, and every week until 5 weeks after transplantation. RESULTS: Locomotor behaviors evaluated by the BBB score were significantly improved in cell-transplanted rats. Numerous axons grew, with occasional interactions with CPECs, through the astrocyte-devoid areas. These axons exhibited structural characteristics of peripheral nerves. GAP-43-positive axons were found at the border of the lesion 2 days after transplantation. Cavity formation was more reduced in cell-transplanted than control spinal cords. CPECs were found within the spinal cord lesion, and sometimes in association with astrocytes at the border of the lesion until 2 weeks after transplantation. CONCLUSION: The transplantation of CPECs enhanced locomotor improvement and tissue recovery, including axonal regeneration, in rats with SCI.

Tlx, an orphan nuclear receptor, regulates cell numbers and astrocyte development in the developing retina.

Tlx belongs to a class of orphan nuclear receptors that underlies many aspects of neural development in the CNS. However, the fundamental roles played by Tlx in the control of eye developmental programs remain elusive. By using Tlx knock-out (KO) mice, we show here that Tlx is expressed by retinal progenitor cells in the neuroblastic layer during the period of retinal layer formation, and it is critical for controlling the generation of appropriate numbers of retinal progenies through the activities of cell cycle-related molecules, cyclin D1 and p27Kip1. Tlx expression is restricted to Müller cells in the mature retina and appears to control their proper development. Furthermore, we show that Tlx is expressed by immature astrocytes that migrate from the optic nerve onto the inner surface of the retina and is required for their generation and maturation, as assessed by honeycomb network formation and expression of R-cadherin, a critical component for vasculogenesis. The impaired astrocyte network formation on the inner retinal surface is accompanied by the loss of vasculogenesis in Tlx KO retinas. Our studies thus indicate that Tlx underlies a fundamental developmental program of retinal organization and controls the generation of the proper numbers of retinal progenies and development of glial cells during the protracted period of retinogenesis.