Search

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.

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.

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.

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.

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.

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.

Purpose This study was conducted to assess the safety and feasibility of intrathecal transplantation of autologous bone marrow-derived mononuclear cells for the treatment of patients with spinal cord injury. Methods Ten patients were included in the study. Approximately 120 ml of bone marrow aspirate was obtained from bilateral iliac bone of patients with spinal cord injury. Isolation of mononuclear cells was performed using Ficoll density-gradient centrifugation. Bone marrow mononuclear cells were transplanted into cerebrospinal fluid by lumbar puncture. Functional tests were performed prior to the cell transplantation and six months after cell transplantation. The patients were carefully observed for up to six months. Results In 5 patients with AIS A prior to cell transplantation, 1 patient converted to AIS B six months after cell transplantation. In 5 patients with AIS B, 1 patient converted to AIS D and 2 patients to AIS C. MRI did not show any complication. Two patients showed slight anemia after aspiration of bone-marrow cells, which returned to normal level within a several weeks. Conclusion The results of this study suggest that this method may be safe and feasible.

Purpose To determine whether intrathecal administration of cultured autologous bone marrow stromal cells (BMSCs) is safe and feasible for treatment of subacute spinal injury. Methods Five patients with complete tetraplegia due to cervical spinal injury on admission were included. A small amount of bone marrow was obtained during surgery for spinal fusion. BMSCs were cultured, reaching 107-108 cells. The properties and functional efficacy of the BMSCs were verified with surface marker analysis and a neurite extension test. BMSCs were administered by lumbar puncture. The patients were closely observed for 6 months, and the Committee on Effectiveness and Safety of Clinical Treatment (CESCT) evaluated safety. Results No adverse responses were observed in biochemical and radiographic examinations. The CESCT did not recognize any harmful effects of the transplantation, and concluded it was safe for treatment. The patients were further followed up for 1 to 4 years with no adverse responses. The recovery of American Spinal Injury Association Impairment Scale (AIS) B and C patients at transplantation was rapid and remarkable, but gradual or limited in AIS A patients. Conclusion This study demonstrated that intrathecal administration of cultured autologous BMSCs is safe and feasible for treatment of spinal cord injury.

Choroid plexus (CP) epithelial cells (CPECs) produce cerebrospinal fluid (CSF) to provide the CNS with a specialized microenvironment. Our previous study showed that the conditioned medium of cultured CPECs enhanced the survival and neurite extension of hippocampal neurons. The present study examined the ability of cultured CPECs to protect against ischemic brain injury when transplanted into the CSF. Rats were subjected to a transient occlusion of the middle cerebral artery, followed by an injection of cultured CPECs into the fourth ventricle. The injection markedly reduced neurological deficits and infarction volume within 24 h. Other beneficial effects were (1) a reduction in number of apoptotic and inflammatory cells, (2) an up-regulation of the mRNA expression of an anti-apoptotic effecter, cAMP-response element binding protein, and (3) a down-regulation of the production of pro-inflammatory factors such as interleukin-1 beta and inducible nitric oxide synthase. The injected CPECs were located within the ventricles and on the brain's surface, not in the ischemic foci, suggesting that they exert their effects by releasing diffusible neuroprotective factors into the CSF. The transplantation of CPECs via CSF is a potential new strategy for protecting against ischemic brain injury.

It is said that bone marrow stromal cells (BMSCs) are able to differentiate into different kinds of cells, including Schwann cells, under relevant conditions (Dezawa et al., Eur J Neurosci 2001;14:1771-1776). In the previous paper, we demonstrated that chitosan gel sponge is one of the effective scaffolds for regenerating axons of the rat sciatic nerve (Ishikawa et al., J Biomed Mater Res A 2007;83:33-40). In the present study, we examined whether BMSC-derived Schwann cells with chitosan gel sponges were one of the effective scaffolds for peripheral nerve regeneration in rats. BMSC-derived cells with Schwann cell characteristics were labeled by green fluorescent protein using a retrovirus. An 8-mm gap was made by removing a nerve segment from the rat peripheral nerve, and chitosan gel sponges containing BMSC-derived Schwann cells were grafted, sandwiching the proximal and distal stumps of the transected nerve. Rats were sacrificed at 7, 14, and 28 days, and 2 and 4 months after transplantation. Immunohistochemistry demonstrated that regenerating axons were found near transplanted Schwann cells 7 days after surgery and extended into the host distal nerve segment at 14 days after surgery. Electron microscopy showed that transplanted Schwann cells formed myelin sheaths on regenerating axons 1 month after transplantation. The mean diameter of myelinated fibers was increased from 2.58 mum at 2 months to 2.84 mum at 4 months postsurgery. This study indicates that chitosan gel sponges containing BMSC-derived Schwann cells have strong potentiality as a graft that can be used for peripheral nerve regeneration.

Spinal cord injury often results in devastating dysfunction and disability. When a spinal cord is injured, various symptoms are presented depending on the segments of the damage and the degree. If cervical spinal damage is severe, tetraplegia results. If damage occurs at levels higher than C4, diaphragmatic movement will be impaired, and the patient has to live being connected with the ventilator on the bed. Patients will suffer from acute hyperesthesia or severe chronic pain, urinary and rectal dysfunction, and autonomic dystonia as well as motor and sensory deficits. In Japan, there are more than 100,000 victims suffering from spinal injury, and a new 5,000 to 6,000 patients are added every year. In the Unites States, about 250,000 to 400,000 people are living with spinal cord injury, and there are about 11,000 to 13,000 new injuries every year. The number of incidence is increasing. The majority of them result from motor vehicle or sports injuries, violence, or falls. An injured central nervous system never regenerates. This has long been thought as a medical common sense terms. Therefore, the principal object for the treatment of spinal injury was mainly purposed how to minimize the progression of secondary injuries and maintain the remnant function of the spine. For the purpose of preventing secondary spinal cord injury, spine stabilization for the fracture or dislocation and rehabilitation were the main strategy in the treatment. There has been no successful treatment for the severe spinal cord injury to recover the function satisfactorily. However, if spinal cord damage is functionally improved even at the minimum, it will affect not only the physical, mental, and economic status of patients and their families, but also the medical resources of society. Recently, regenerative treatments with stem cells are in the limelight. However, there are some serious problems such as ethical ones to be solved for the study with stem cells. We reported significant recovery of motor function in rats with experimental spinal cord injury treated by transplanting bone marrow stromal cells (BMSCs) in the cerebrospinal fluid (CSF). Based on that study, we aimed at the clinical application of this treatment, and actually planned a clinical trial of spinal cord injury treatment by transplanting patient’s autologous BMSCs into CSF in the acute phase after spinal cord injury, at Kansai Medical University Hospital. We have developed a detailed protocol for the clinical trial. The medical ethics committees of the institutions have approved the protocol officially. This clinical trial aims to treat a damaged spinal cord by a novel method of injecting BMSCs into CSF through the lumbar puncture, and assess the safety and efficacy of the procedure. Although we have experienced only a single case, a committee that monitors the data to assess the efficacy and safety of the trial with members independent of this study team has evaluated the safety of the trial in this case, approved to continue the study, and agreed to submit a report of the first case. In addition, Japan Spinal Cord Foundation strongly requested to disclose the course of the first case. Therefore, we would like to publish the report of the first case to enhance research work on the new strategy for the difficult treatment of spinal cord injury. Submitted for publication July 6, 2007. Accepted for publication September 18, 2007. Copyright © 2008 by Lippincott Williams & Wilkins From the Emergency and Critical Care Center (F.S., T.N., M.I., Y.M., A.H., Y.M.), Kansai Medical University, Osaka, Japan; Department of Plastic and Reconstructive Surgery (Y.S.), Kitano Hospital, Tazuke Research Institute, Osaka, Japan; Department of Clinical Trial Design and Management (R.O., M.F.), Translational Research Center, Kyoto University Hospital, Kyoto, Japan; Department of Occupational Therapy (C.I.), Faculty of Nursing and Rehabilitation, Aino University, Osaka, Japan. This work was supported, in part, by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science, Japan. Presented in part at the 8th Asian Pacific Conference on Disaster Medicine, November 22, 2006, Tokyo, Japan; at the Shanghai International Conference on Trauma and Emergency Medicine, May 13, 2006, Shanghai, China; and as a poster at the 66th Annual Meeting of the American Association for the Surgery of Trauma, September 27–29, 2007, Las Vegas, Nevada. Address for reprints: Toshio Nakatani, MD, Emergency and Critical Care Center, Kansai Medical University, Osaka, Japan; email: nakatant@ takii.kmu.ac.jp.

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.

The subventricular zone (SVZ) of the lateral ventricle of the mammalian forebrain is the major site in which neural progenitor cells (NPC) persist in the adult brain. The NPC are located beneath ventricular ependymal cells and have the capacity to self-renew and continuously produce neurons and glial cells. We have shown previously that neurospheres can be obtained from the brain of deceased adult rats and that neurosphere cells survive after transplantation into the spinal cord. In the present study, we investigated whether fresh NPC from living adult rats can survive and be integrated into host tissues after transplantation into the adult rat spinal cord of the same strain. We used rats expressing transgenic green fluorescent protein (GFP) as a donor to identify the transplanted NPCs. The SVZ tissues were obtained from the striatal wall of the lateral ventricle of adult GFP-rats and were grafted into lesions of the spinal cord at the cervical level. Two to 3 weeks after grafting, NPC migrated through the host tissue 0.5-1 mm away from the implantation site, and were integrated into the white matter of the host spinal cord. Surviving NPC exhibited immunohistochemical phenotypes of astrocytes (glial fibrillary acidic protein), but not for neurons (alpha-tubulin III) or oligodendrocytes (Rip; Hybridoma Bank, Iowa City, IA, USA). Thus, NPC from the SVZ of adult rats can survive and differentiate into at least astrocytes, which can then be integrated into host tissue after transplantation into spinal cord lesions in the adult rat.

Bone marrow stromal cells (MSCs) have great potential as therapeutic agents. We report a method for inducing skeletal muscle lineage cells from human and rat general adherent MSCs with an efficiency of 89%. Induced cells differentiated into muscle fibers upon transplantation into degenerated muscles of rats and mdx-nude mice. The induced population contained Pax7-positive cells that contributed to subsequent regeneration of muscle upon repetitive damage without additional transplantation of cells. These MSCs represent a more ready supply of myogenic cells than do the rare myogenic stem cells normally found in muscle and bone marrow.

We have developed a nerve regeneration material consisting of alginate gel crosslinked with covalent bonds. in the first part of this study, we attempted to analyze nerve regeneration through alginate gel in the early stages within 2 weeks. in the second part, we tried to regenerate cat peripheral nerve by using alginate tubular or non-tubular nerve regeneration devices, and compared their efficacies. Four days after surgery, regenerating axons grew without Schwann cell investment through the partially degraded alginate gel, being in direct contact with the alginate without a basal lamina covering. One to 2 weeks after surgery, regenerating axons were surrounded by common Schwann cells, forming small bundles, with some axons at the periphery being partly in direct contact with alginate. At the distal stump, numerous Schwann cells had migrated into the alginate 8-14 days after surgery. Remarkable restorations of the 50-mm gap in cat sciatic nerve were obtained after a long term by using tubular or non-tubular nerve regeneration material consisting mainly of alginate gel. However, there was no significant difference between both groups at electrophysiological and morphological evaluation. Although, nowadays, nerve regeneration materials being marketed mostly have a tubular structure, our results suggest that the tubular structure is not indispensable for peripheral nerve regeneration.

Many neurodegenerative diseases are attributed to the degeneration of neurons with subsequent functional loss. Cell transplantation is a strategy with potential for treating such diseases, and many kinds of cells are considered candidates for transplantation therapy. Bone marrow stromal cells (MSCs) have great potential as therapeutic agents, as they are easy to isolate and expand from patients without serious ethical and technical problems. The authors have found a method for the highly efficient, exclusive and specific induction of functional postmitotic neuronal cells from both rat and human MSCs. Gene transfer of Notch intracellular domain (NICD) followed by the administration of certain trophic factors induced mature neurons expressing neuronal markers, some of which showed action potentials. Induced neurons were transplanted to animal models of neurodegenerative disorders, including Parkinson's disease and ischaemic brain injury, resulting in the successful integration of transplanted cells and improvement in function of the transplanted animals. This review summarises the respective potentials, benefits and drawbacks of MSC-derived neurons, and discusses the possibility of their clinical application in neurodegenerative diseases.

Neurogenesis has been described in limited regions of the adult mammalian brain. In this study, we showed that the ependymal layer of the 3rd ventricle is a neurogenic region in the adult rat brain. DiI labeling of the 3rd ventricle revealed that neural progenitor cells were derived from cells at the ependymal layer of the adult 3rd ventricle. The mitosis of these progenitor cells at the ependymal layer was promoted by bFGF administration. Combination of BrdU administration, nestin/GFAP immunohistochemistry, and labeling by GFP-recombinant adenoviral infection (vGFP) indicated that at least some tanycytes might be neural progenitor cells in the ependymal layer of the 3rd ventricle. Tracing by vGFP indicated that neural progenitor cells may have migrated from the 3rd ventricle to the hypothalamic parenchyma, where they were integrated into neural networks by forming synapses. In addition, some BrdU(+) neurons had immunoreactivity for orexin A in the hypothalamus. These results indicate that neural progenitor cells exist in the ependymal layer of the adult rat 3rd ventricle and that they may differentiate into neurons functioning in the hypothalamus.

We previously demonstrated that choroid plexus epithelial (modified ependymal) cells (CPECs) differentiated into astrocytes after grafting into the spinal cord. In the present study, we examined whether CPECs from rats at postnatal 1 day (P1), 7 day (P7), and 8 weeks (P8W) can function as neural progenitor cells that give rise to neurons and glial cells. Cell spheres were produced in cultures of whole tissue of the choroid plexus from the fourth ventricle of rats at each postnatal period. beta-tubulin class III (Tuj-1), glial fibrillary acid protein (GFAP)-, and O4-positive cells differentiated from cell spheres in the differentiation medium. We produced a monoclonal antibody 3E6 specifically labeling microvilli of CPECs. Using this monoclonal antibody, CPECs were isolated from the choroid plexus of P8W rats by cell sorter (FACS). Immunocytochemistry confirmed that there was no contamination from fibroblasts, endothelial cells, macrophages, or Schwann cells in the FACS-isolated 3E6-labeled cells. Cell spheres formed in the cultures of these 3E6-labeled CPECs. After expansion, these cell spheres gave rise to Tuj-1- (5%), GFAP- (45%), and O4-positive cells (0.16%). The remaining cells (45%) were unlabeled neural or glial markers. Some CPECs of the P8W rat were immunohistochemically stained with lineage-associated markers of Musashi-1 and epidermal growth factor-receptor (EGF-R). In addition, infusion of EGF or fibroblast growth factor-2 (FGF2) into the ventricle increased the number of bromodeoxyuridine (BrdU)-positive cells among CPECs from 0.03% (untreated) to 1.14% (38-fold, EGF) and 1.03% (35-fold, FGF2), respectively. These findings indicate that neural progenitor cells exist among CPECs in the rat.

Choroid plexus ependymal cells (CPECs) were known to promote axonal growth when choroid plexus is grafted into the adult rat spinal cord. The present study was carried out to examine whether CPECs promote axonal outgrowth from neurons derived from the CNS in vitro. Hippocampal neurons were cocultured on CPEC monolayers. After 24 h, neurite extension was evaluated using various parameters in comparison with cultures grown on poly-L-lysine (PLL)-coated plates and cocultures grown on astrocyte monolayers. The primary neurite length and total neurite length were longest in the cocultures with CPECs. The number of primary neurites and the number of branches were larger in the cultures with CPECs than in the cultures on PLL-coated plates, but almost the same as in the cocultures with astrocytes. Next, we examined whether the neurite extension-promoting effect occurring within 24 h is due primarily to contact with the CPECs or to factors secreted by CPECs into the culture medium. The CPEC monolayers were killed by ethanol fixation, and neurons cultured on them. The neurons extended long neurites with elaborate branching, as in the case of cocultures grown on living CPECs. On the other hand, CPEC-conditioned medium exhibited less promoting effect on neurite outgrowth from hippocampal neurons. These results indicate that CPECs have a capacity to promote neurite outgrowth from CNS neurons in vitro , and that surface plasma membrane-bound components of CPECs strongly contribute to the enhancement of neurite outgrowth in the present coculture system.

The effects of bone marrow stromal cells (BMSCs) on the repair of injured spinal cord and on the behavioral improvement were studied in the rat. The spinal cord was injured by contusion using a weight-drop at the level of T8-9, and the BMSCs from the bone marrow of the same strain were infused into the cerebrospinal fluid (CSF) through the 4th ventricle. BMSCs were conveyed through the CSF to the spinal cord, where most BMSCs attached to the spinal surface although a few invaded the lesion. The BBB score was higher, and the cavity volume was smaller in the rats with transplantation than in the control rats. Transplanted cells gradually decreased in number and disappeared from the spinal cord 3 weeks after injection. The medium supplemented by CSF (250 microl in 3 ml medium) harvested from the rats in which BMSCs had been injected 2 days previously promoted the neurosphere cells to adhere to the culture dish and to spread into the periphery. These results suggest that BMSCs can exert effects by producing some trophic factors into the CSF or by contacting with host spinal tissues on the reduction of cavities and on the improvement of behavioral function in the rat. Considering that BMSCs can be used for autologous transplantation, and that the CSF infusion of transplants imposes a minimal burden on patients, the results of the present study are important and promising for the clinical use of BMSCs in spinal cord injury treatment.

Freeze-dried alginate sponge cross-linked with covalent bonds has been demonstrated to enhance nerve regeneration in peripheral nerves and spinal cords. The present study examined, at early stages after surgery, the outgrowth of regenerating axons and reactions of astrocytes at the stump of transected spinal cord in young rats. Two segments (Th7-8) were resected, and alginate was implanted in the lesion. As controls, collagen gel was implanted in place of alginate or the lesion was left without implantation. Two and 4 weeks after surgery, nerve outgrowth and astrocyte reactions were examined. Many regenerating axons, some of which were accompanied by astrocytic processes, were found to extend from the stump into the alginate-implanted lesion. In the all nonimplanted animals, large cystic cavities were formed at both interfaces with no definite axonal outgrowth into the lesion. In collagen-implanted animals, cavity formation was found in some rats, and regenerating axons once formed at the stumps did not extend further into the lesion. Astrocytic processes extending into alginate-implanted lesion had no basal laminae, whereas those found in control experiments were covered by basal laminae. These findings suggest that alginate contributed to reducing the barrier composed of connective tissues and reactive astrocytic processes, and served as a scaffold for the outgrowth of regenerating axons and elongation of astrocytic processes.

Sept2 is a member of the septin family of GTPases. Septins form filaments in a GTP-form dependent manner, and are involved in cytokinesis from yeast to mammals; however, some mammalian septins, including Sept2, are expressed in the brain, a tissue in which almost all the cells are postmitotic. Recently, some functions of mammalian septin other than cytokinesis such as vesicle transport have been reported. However, mammalian septin's physiological functions are still unclear. The present study revealed that Sept2 co-localizes with the astrocyte glutamate transporter GLAST in the Bergmann glial processes facing axons and synapses. Biochemical analyses demonstrated that Sept2 bound directly to the carboxy-terminal region of GLAST in a GDP-form dependent manner. Expression of constitutive GDP-form Sept2 mutant reduced the glutamate uptake activity of GLAST via internalization of GLAST from cell surface. Thus Sept2 may regulate GLAST-mediated glutamate uptake by astrocytes, which is important for appropriate transmitter signalling in the cerebellum.

We have developed an alginate gel crosslinked with covalent bonds for regeneration of dermis, nerve, and bone. Recently, a novel matrix (H/A gel) which consists of heparin and alginate covalently crosslinked with ethylenediamine, was designed. It can stabilize and release biologically active basic fibroblast growth factor (bFGF) for 1 month, which is one of the heparin-binding growth factors. In the present report, we examined the effect of this novel H/A gel on nerve regeneration in the rat sciatic nerve. In this study, regenerated axons in H/A gel with bFGF grew faster than in ordinary alginate gel with bFGF in the early stage. Myelinated fibers showed a tendency to increase in diameter toward the normal size in the later stage. Nerve bundles in the implantation exhibited minimal fibrosis and good vascularization. H/A gel with bFGF exhibited better-developed vascularization than ordinary alginate gel with bFGF. These findings suggested that H/A gel with bFGF could serve not only as an efficient cellular scaffold, but also as a stabilizing matrix for bFGF for peripheral nerve regeneration. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 661–668, 2004

A biodegradable copolymer of poly L-lactic acid and epsilon-caprolactone (PLAC) was manufactured into a tube, in which a denatured skeletal muscle segment was placed longitudinally. This model tube was implanted as a guide to promote nerve regeneration across a 5 cm gap in the rabbit sciatic nerve. Five months after implantation, good nerve regeneration was found throughout the graft and in the distal host nerve. The population (29.6/16 x 10(2) microm(2)) of regenerated nerves in the graft was higher than that of the contralateral normal sciatic nerve (18.0/16 x 10(2) microm(2)). Regenerated nerve fibers extended to the distal host nerve. The number of myelinated fibers was 13.7/16 x 10(2) microm(2) at a level 1.5 cm from the distal suture. The diameters (below 2 microm) of most regenerated myelinated (nerves in the graft and in the distal host nerve were much smaller than those (6-8 microm) of normal nerves. Electrophysiological evaluation showed that the hindlimb muscle (gastrocnemius) was innervated by motor nerves in all animals 5 months after implantation. These results indicate that the PLAC tube with a denatured muscle segment inside provided good conditions for nerve fiber regrowth. The PLAC tube is thought to protect the denatured muscle segment from rapid dissociation in the host tissue.

Recent studies have shown that catenins play a pivotal role in neuronal signalling during vertebrate development. In order to study the significance of beta-catenin in the developing mouse retina, the localization of beta-catenin was examined by immunohistochemistry from embryonic day (E) 12 to adult mice. Immunoreactivity for beta-catenin was found in ganglion cells of the retina at E12, and extended to the inner and outer plexiform layer as well as the ganglion-cell layer with the strongest immunolabelling from E16 through to postnatal day (P) 5. The immunoreactivity of ganglion cells was distributed on the cell surface. Thereafter, the immunoreactivity gradually decreased, being limited to the inner plexiform layer and ganglion-cell layer, including the nerve-fiber layer in P10. By P16, the weak immunoreactivity was detected in the inner plexiform layer and ganglion-cell layer, and almost disappeared in the adult retina. No distinct immunoreactivity was found in the retinal pigment epithelium. The reverse transcription polymerase chain reaction showed that beta-catenin messenger ribonuclic acid was detected at E12, E16, P1 and P16, and thereafter markedly decreased, being weakest in the adult. These findings show that beta-catenin is expressed during development at the sites of synaptic connections of inner and outer plexiform layers, and on the ganglion cells and their fibers in the retina, suggesting that beta-catenin might play an important role in the synapse formation and ganglion-cell development during the morphogenesis of the retina.

Our previous study revealed that alginate gel cross-linked with covalent bonds promoted peripheral nerve regeneration in the cat and rat. The present study analyzed nerve regeneration through alginate gel in the early stages within 2 weeks and the late stages up to 21 months after implantation. Four days after surgery, regenerating axons grew without Schwann cell investment through the partially degraded alginate gel, being in direct contact with the alginate without a basal lamina covering. Numerous mast cells infiltrated into the alginate. One to 2 weeks after surgery, regenerating axons were surrounded by common Schwann cells to form small bundles, with some axons at the periphery being partly in direct contact with alginate. At the distal stump, numerous Schwann cells had migrated into the alginate 8-14 days after surgery. They had no basal laminae. The diameter of regenerated myelinated fibers was small (approximately 1 micro m) at 8 weeks, but increased in diameter, having a distribution pattern similar to that of normal nerve 21 months after surgery. Much better nerve regeneration was found in alginate gel-, than collagen sponge-, and fibrin glue-implanted distal stump 12 months after surgery. These results indicate that alginate gel has good biocompatibility for regenerating axon outgrowth and Schwann cell migration, and that regenerated fibers can have a diameter as thick as that of normal fibers in the long term. Alginate gel is a promising material for use as an implant for peripheral nerve regeneration.

Here we report a novel method of supplying cultured neurosphere cells to the injured spinal cord, by injection of cells into the cerebrospinal fluid (CSF) through the fourth ventricle or cisterna magna. Hippocampus-derived neurosphere cells, isolated from a transgenic rat fetus expressing green fluorescent protein, were transplanted into the CSF of a rat with spinal cord injury. It was found that injected cells were extensively transported by CSF within the subarachnoidal space, and survived as clusters on the pial surface of the spinal cord. The most notable finding was that a large number of injected cells migrated into the lesion site and integrated into the injured spinal cord tissues.

Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling. We previously found that a membrane-anchored glycoprotein, RECK, negatively regulates MMP-9 and inhibits tumor invasion and metastasis. Here we show that RECK regulates two other MMPs, MMP-2 and MT1-MMP, known to be involved in cancer progression, that mice lacking a functional RECK gene die around E10.5 with defects in collagen fibrils, the basal lamina, and vascular development, and that this phenotype is partially suppressed by MMP-2 null mutation. Also, vascular sprouting is dramatically suppressed in tumors derived from RECK-expressing fibrosarcoma cells grown in nude mice. These results support a role for RECK in the regulation of MMP-2 in vivo and implicate RECK downregulation in tumor angiogenesis.

Hippocampus-derived neurospheres were prepared from transgenic rat fetuses expressing green fluorescent protein (GFP), and transplanted into an alginate-filled lesion of young rat spinal cord. One, two and four weeks after transplantation, a large number of grafted cells survived, many of which expressed immunoreactivity for glial fibrillary acidic protein, and a few expressed immunoreactivity for beta-tubulin III. The grafted cells closely attached to the host tissue including astrocytes at the border of the lesion. It was notable that numerous GFP-positive cells had migrated within host spinal cord tissue up to 2 mm away from the implanted site 4 weeks postoperation. These results demonstrate that rat fetal hippocampus-derived neurosphere cells could survive, differentiate, extensively migrate, and integrate well into the host spinal cord tissue.

Muscle segments excised from rabbit biceps femoris muscles were treated with detergent sodium dodecyl sulphate to denature cellular constituents, and each was autografted in a 5 cm gap of the sciatic nerve in the same rabbit. Axonal regrowth through the grafts and reinnervation into the host sciatic nerves and muscles were studied morphologically, and electrophysiologically, 4 months after grafting. Regenerating axons accompanied by Schwann cells extended through basal lamina tubes of the grafts into the distal host nerves. Reinnervation of the tibialis anterior muscles by motor nerves was confirmed by recovery of the compound muscle action potentials (CMAP) and the reinnervation of the muscle spindles was demonstrated by electron microscopy. These findings indicated that the basal lamina tubes of denatured muscles were effective scaffolds through which the regenerating nerve fibers grew across as large a gap as 5 cm.

The high-affinity IgE receptor (FcepsilonRI) on mast cells and basophils consists of a ligand-binding alpha-chain and two kinds of signaling chains, a beta-chain and disulfide-linked homodimeric gamma-chains. Crosslinking by multivalent antigen results in the aggregation of the bound IgE/alpha-chain complexes at the cell surface, triggering cell activation, and subsequent internalization through coated pits. However, the precise topographical alterations of the signaling beta- and gamma-chains during stimulation remain unclarified despite their importance in ligand binding/signaling coupling. Here we describe the dynamics of FcepsilonRI subunit distribution in rat basophilic leukemia cells during stimulation as revealed by immunofluorescence and immunogold electron microscopy. Immunolocalization of beta- and gamma-chains was homogeneously distributed on the cell surfaces before stimulation, while crosslinking with multivalent antigen, which elicited optimal degranulation, caused a distinct aggregation of these signaling chains on the cell membrane. Moreover, only gamma- but not beta-chains were aggregated during the stimulation that evoked suboptimal secretion. These findings suggest that high-affinity IgE receptor beta- and gamma-chains do not co-aggregate but for the most part form homogenous aggregates of beta-chains or gamma-chains after crosslinking.

The epithelial cells of the choroid plexus are a continuation of the ventricular ependymal cells and are regarded as modified ependymal cells. The present study was carried out to determine the influence of choroid plexus ependymal cells (CPECs) on axonal growth in vitro. Choroid plexuses were dissected from the fourth ventricle of postnatal day-1–10 mice, mechanically dissociated, and plated in fibronectin-coated culture dishes. CPECs had spread into monolayers with few endothelial cells in 3-week cultures. Some macrophages were scattered on the monolayer of CPECs. Dorsal root ganglia (DRG) were excised from mouse fetuses of 14-day gestation, dissociated with trypsin and cocultured on the CPEC monolayers. For comparison, dissociated DRG neurons were cocultured on astrocyte monolayers or cultured on laminin-coated plates. After 4.5 h culturing, the cultures were fixed and immunohistochemically double-stained for neurites and CPECs using antibodies against β-tubulin III and S-100 β, respectively. It was demonstrated that neurons extended many long neurites with elaborate branching on the surface of S-100-stained CPECs. In contrast, DRG neurons cultured on the astrocytes and on the laminin-coated plates had much shorter primary neurites with fewer branches than those cultured on the CPECs. The total length of neurites including primary neurites and their branches, of a single DRG neuron was 285 ± 14, 395 ± 15 and 565 ± 12 μm on the laminin-coated plates, on astrocytes and on CPECs, respectively. Scanning electron microscopy revealed extension of neurites with well-developed growth cones on the ependymal cells. These results suggest that CPECs have a great capacity to promote neurite outgrowth from DRG neurons in vitro.

Synaptosomal-associated protein 25 has been regarded as one of the target-associated soluble N-ethylmaleimide-sensitive fusion attachment protein receptors essential for exocytosis of vesicles in synapses. We have previously reported that cleavage of syntaxin, which is another target-associated soluble N-ethylmaleimide-sensitive fusion attachment protein receptor, with botulinum neurotoxin C1 resulted in inhibition of neurite extension and morphological changes including growth cone collapse and large vacuole formation. As an attempt to explore the mechanism of growth cone extension, we examined the ultrastructural localization of synaptosomal-associated protein 25 in growth cones with or without treatment of botulinum neurotoxin A, which cleaves synaptosomal-associated protein 25. In dorsal root ganglion neurons, light microscopy demonstrated synaptosomal-associated protein 25 immunoreactivity throughout the neurons, including the cell bodies, neurites and growth cones. Using electron microscopy, gold signals immunoreactive for synaptosomal-associated protein 25 were identified diffusely in the cytoplasm of the growth cones. In contrast, in PC-12 cells, a large number of gold signals were localized on the plasma membranes. High levels of signal were also found in the cytoplasm in the central region of the growth cones. We also confirmed that botulinum neurotoxin A treatment reduced neurite extension by about 50%. However, both in dorsal root ganglion neurons and in PC-12 cells we found no differences in the ultrastructure nor in the localization of synaptosomal-associated protein 25 between growth cones with and without toxin treatment. These results indicate that cleavage of synaptosomal-associated protein 25 inhibits growth cone extension in a manner different than that of syntaxin cleavage. The results of this study suggest the possibility that synaptosomal-associated protein 25 is involved in growth cone extension through a process independent of vesicle fusion.

In contrast to previous reports denying the occurrence of axonal regeneration of the dorsal column (DC) projections, here we demonstrate for the first time that marked regeneration occurs spontaneously after transection in infant rats. Transection was made sharply so as to produce edema-free lesions without subsequent formation of either scars or cysts. Transganglionic labeling of axons revealed that regenerated axons ascended in the normal tract in a manner similar to normal projections as a tightly-packed fasciculus and terminated densely in the nucleus gracilis. The present study indicates that failure of regeneration of DC axons is due to neither intrinsic deficiency of regrowth potential nor globally-inhospitable axonal environment but rather the local conditions of the lesion site.

In the peripheral nerve, regenerating axonal sprouts usually emanate at nodes of Ranvier, and extend as growth cones along the inner surface of Schwann cells and/or through Schwann cell columns in the distal nerve segment. In order to elucidate the significance of Ca(2+)-independent protein kinase C in nerve regeneration, localizations of delta, epsilon and zeta subtypes were examined immunocytochemically in sprouts and growth cones of regenerating axons, as well as in normal intact nerves in the rat sciatic nerve. In normal nerves, intense immunoreactivities of delta, epsilon and zeta subtypes were present in axons of both myelinated and unmyelinated fibres. Subcellularly, the distribution of these subtypes in the axoplasm was patchy, and discontinuous in the axolemma and subaxolemmal peripheral zones of myelinated nerves. Some thin myelinated axons showed no immunoreactivity for epsilon subtype. Schwann cells of both myelinated and unmyelinated fibres had moderate immunoreactivities for each subtype. In areas of nerve regeneration, axonal sprouts at nodes of Ranvier, and growth cones extending along Schwann cell basal laminae, had intense immunoreactivities for delta, epsilon and zeta subtypes which are distributed diffusely throughout the axoplasm, and on the entire axolemma. In the sprouts, immunoreactivity for epsilon subtype was strong on the axolemma, but weak or almost absent in the axoplasm. These data, together with those of our previous study, indicate that Ca(2+)-independent protein kinase C subtypes (delta, epsilon and zeta) have basically the same distribution patterns as those of Ca(2+)-dependent subtypes in sprouts and growth cones of regenerating axons, as well as in normal intact axons; albeit epsilon subtype is somewhat different in distribution and intensity from delta and zeta subtypes. It is suggested that Ca(2+)-independent subtypes are involved in maintaining growth cone activities along with the Ca(2+)-dependent subtypes.

Caveolae are small plasmalemmal invaginations which are assumed to play various physiological functions. In the present study, distribution of two caveolae-specific proteins, the plasmalemmal Ca(2+)-pump and caveolin, was examined in the corneal epithelium in the normal state and after artificial wounding.A central epithelial ablation was made in the mouse cornea by a razor blade. After various intervals, the corneas were excised, fixed, and rapidly frozen. The specimens were subjected to immunofluorescence microscopy and immunoelectron microscopy, using antibodies against the plasmalemmal Ca(2+)-pump or caveolin.In the normal corneal epithelium, both plasmalemmal Ca(2+)-pump and caveolin were observed along the cell surface by immunofluorescence microscopy, and were localized to caveolae by immunogold electron microscopy. In the regenerating epithelium, 12-18 h after injury, plasmalemmal Ca(2+)-pump was seen as many dots in the cytoplasm by immunofluorescence microscopy; in contrast, caveolin persisted along the cell surface. Immunoelectron microscopy revealed that the labeling for the plasmalemmal Ca(2+)-pump was located around membranous structures in the cytoplasm and was scarce along the plasma membrane, while caveolin remained in caveolae. The Ca(2+)-pump regained normal distribution when the wound was closed. By quantitation in electron micrographs, the number of caveolae per unit plasma membrane length was found to be decreased in the wounded corneal epithelium.The present results indicate that caveolae undergo compositional modification during the wound healing process of the corneal epithelium. Considering putative caveolar functions, the phenomenon may be related to possible fluctuations of the intracellular Ca(2+)-concentration in the regenerating epithelium.

Schwann cell basal lamina tubes serve as attractive conduits for regeneration of peripheral nerve axons. In the present study, by using basal lamina tubes prepared by in situ freeze-treatment of rat saphenous nerve, the effects of exogenously applied basic fibroblast growth factor (bFGF) on peripheral nerve regeneration was examined 2 and 5 days after bFGF administration. Regenerating axons were observed by light and electron microscopy using PGP9.5-immunohistochemistry for specific staining of axons. In addition, the localizations of bFGF and its receptor (FGF receptor-1) were examined by immunohistochemistry using anti-bFGF antibody and anti-FGF receptor-1 antibody, respectively. Regenerating axons extended further in the bFGF-administered segment than in the bFGF-untreated control segment. Electron microscopy showed that regenerating axons grew out unaccompanied by Schwann cells. Findings concerning angiogenesis and Schwann cell migration were very similar between the bFGF treated and control nerve segment. bFGF-immunoreactivity was not detected in the control nerve segment. In contrast, bFGF-immunoreactivity was detected on the basal lamina tubes as well as on the plasmalemma of regenerating axons facing the basal lamina in the bFGF treated nerve segment up to 5 days after administration, suggesting that exogenous bFGF can be retained in the basal lamina for several days after administration. FGF receptor was detected on the plasma membrane of regenerating axons where they abutted the basal lamina. These results indicate that bFGF could promote the extension of early regenerating axons by directly influencing the axons, but not via Schwann cells or angiogenesis.

Peripheral nerve regeneration comprises the formation of axonal sprouts, their outgrowth as regenerating axons and the reinnervation of original targets. This review focuses on the morphological features of axonal sprouts at the node of Ranvier and their subsequent outgrowth guided by Schwann cells or by Schwann cell basal laminae. Adhesion molecules such as N-CAM, L1 and N-cadherin are involved in the axon-to-axon and axon-to-Schwann cell attachment, and it is suggested that integrins such as alpha 1 beta 1 and alpha 6 beta 1 mediate the attachment between axons and Schwann cell basal laminae. The presence of synaptic vesicle-associated proteins such as synaptophysin, synaptotagmin and synapsin I in the growth cones of regenerating axons indicates the possibility that exocytotic fusion of vesicles with the surface axolemma supplies the membranous components for the extension of regenerating axons. Almost all the subtypes of protein kinase C have been localized in growth cones both in vivo and in vitro. Protein kinase C and GAP-43 are implicated to be involved in at least some part of the adhesion of growth cones to the substrate and their growth activity. The significance of tyrosine kinase in growth cones is emphasized. Tyrosine kinase plays an important role in intracellular signal transduction of the growth of regenerating axons mediated by both nerve trophic factors and adhesion molecules. Growth factors such as NGF, BDNF, CNTF and bFGF are also discussed mainly in terms of the influence of Schwann cells on regenerating axons.

The Ca(2+)-dependent intercellular adhesion molecule cadherin is known to be linked to the cytoskeleton by the protein catenin, an association of which appears to be important for the cell-adhesion function of cadherin. Catenin consists of three subtypes-alpha, beta, and gamma. In our previous study, N-cadherin was shown to be localized on the plasmalemma of normal and regenerating chick peripheral nerve. Thus, as alpha N-catenin is a subtype of alpha-catenin (which is specifically associated with N-cadherin), we investigated the immunolocalization of alpha N-catenin in normal and regenerating chick sciatic nerve. In normal nerve, unmyelinated axons exhibited either intense or weak alpha N-catenin immunoreactivity throughout the axoplasm, whereas myelinated axons were completely immunonegative. Regenerating axons, including those derived from parent myelinated axons, showed alpha N-catenin immunoreactivity of variable intensities in growth cones and axon shafts. Schwann cells were invariably devoid of immunoreactivity. Thus alpha N-catenin is not necessarily bound to the surface plasmalemma, but is distributed throughout the cytoplasm, suggesting that most alpha N-catenin molecules are dissociated from N-cadherin.

Localization of synaptotagmin, a Ca2+-binding protein associated with synaptic vesicles, in regenerating axons was investigated by immunocytochemistry in the injured rat sciatic nerves. The early regenerating axonal sprouts emanating from nodes of Ranvier exhibited synaptotagmin immunoreactivity on vesicles, vacuoles and surface plasma membranes. In the well-developed regenerating sprouts extending through the space between Schwann cell basal lamina and myelin sheath of the parent axon, the growing tips, i.e., typical growth cones, exhibited an intense immunoreaction on vesicles, vacuoles and surface plasma membranes, while the stem regions where the sprouts were continuous with the parent axon exhibited almost no immunoreaction on any organelles including plasma membranes. These findings suggest that synaptotagmin-immunoreactive vesicles and vacuoles might be utilized for the supply of membrane components to the surface plasma membrane in the growth cone. Synaptotagmin which is known to regulate membrane fusion of synaptic vesicles with presynaptic plasma membranes in a Ca2+-dependent manner in synapses may participate in the regulation of the membrane fusion between the vesicles and plasma membranes in growth cones.

Objective—To compare methods for harvesting canine bone marrow stromal cells (BMSCs) and determine the biological properties of canine BMSCs at successive passages in vitro. Sample—BMSCs collected from the femurs of 9 Beagles. Procedures—A fibroblast assay was performed to compare 2 methods for harvesting BMSCs: the aspiration and perfusion method. Flow cytometric analysis was performed to evaluate the cell surface markers. Changes in proliferative activity were analyzed by examining radioactivity of hydrogen 3-thymidine. Cell senescence was studied via senescence-associated β-galactosidase staining, and differentiation properties (osteogenesis and adipogenesis) were estimated in association with passage. Results—The aspiration method yielded significantly more fibroblasts than the perfusion method. The cells harvested by both methods gave positive results for CD44 and CD90 and negative results for CD34 and CD45. After induction, the cells had osteogenic and adipogenic phenotypes. The biological properties of BMSCs harvested by the aspiration method were estimated in association with passage. With increasing number of passages, the proliferative activity was reduced and the proportion of cells with senescence-associated β-galactosidase staining was increased. The capacity of differentiation was reduced at passage 3. Conclusions and Clinical Relevance—The aspiration method was superior for collection of BMSCs. In early passages, canine BMSCs had the proliferative activity and potential of osteogenic and adipogenic differentiation, but this decreased with increased number of passages. Consideration of passage will be important to the success of any strategy that seeks to regenerate tissue though the use of BMSCs.

OBJECTIVES Severe intervertebral disc herniation causes complete paraplegia and loss of pain sensation in canines. The prognosis is poor, even when decompression surgery is performed immediately after onset. Studies suggest that bone marrow-derived mononuclear cells will regenerate the injured spinal cord and restore neurologic function. This study was conducted to assess the clinical efficacy of bone marrow-derived mononuclear cell autotransplanting in severe cases of canine intervertebral disc herniation. MATERIALS AND METHODS Eighty-two dogs (miniature dachshunds) with severe thoracolumbar intervertebral disc herniation were used. All had intervertebral disc herniation accompanied by paraplegia and loss of pain perception. In 36 dogs, bone marrow-derived mononuclear cells were autotransplanted to the lesioned spinal cord immediately after decompression surgery. Bone marrow was collected from the proximal humerus and subjected to density gradient centrifugation to isolate the bone marrow-derived mononuclear cells. The remaining 46 dogs (receiving surgical treatment only) were assigned as controls. Therapeutic efficacy was compared based on the rate of ambulatory recovery. RESULTS Ambulatory recovery was observed in 88.9% and 56.5% of animals in the bone marrow-derived mononuclear cells and control groups, and a significant difference was found. No complications were found in bone marrow-derived mononuclear cells group. CONCLUSIONS Bone marrow-derived mononuclear cell transplanting revealed a significant increase in the recovery rate and, as has been reported in rats and humans, bone marrow-derived mononuclear cell autotransplanting shows efficacy in canines as well.

The localization of protein kinase C (PKC) α, β and γ subspecies in sensory axon terminals of muscle spindles in the plantar lumbrical muscles of rat was investigated by light and electron microscopic immunocytochemistry using monoclonal and polyclonal antibodies. Immunoreactivity for these subspecies was detected specifically in sensory axon terminals which wound spirally around the intrafusal muscle fibres of the muscle spindle. Immunostaining was found to be stronger with polyclonal than with monoclonal antibodies. By electron microscopy, immunoreactivity for α, β and γ subspecies was almost diffusely distributed in the cytoplasm of the axon terminal, and the overall pattern of distribution of immunoreactivity was similar for all three subspecies. In the cases of a and β subspecies, some intensely immunostained regions were found in the cytoplasm, but no definite subcellular structures corresponding to such regions could be identified. Considering that PKC plays a crucial role in the regulation of ion channels, it is suggested that PKC might be involved in the control of mechanoelectric transduction in sensory axon terminals.

Hydrosyringomyelia was produced experimentally by the injection of kaolin into the cisterna magna of the rabbit, and the ultrastructural changes of the spinal cord surrounding the syrinx were investigated 2, 4, and 6 weeks after injection by transmission electron microscopy. The ependyma at the ventral part of the central canal was flat and stretched, whereas, in the dorsal part, it was split, and the syrinx extended through the dorsal median plane in most animals. Extracellular edema was found in the subependymal white matter and in and around the posterior median septum. Many nerve fibers surrounding the syrinx were in varying stages of axonal degeneration. Myelin sheaths were split, thinned, and completely lost in many nerve fibers. In some fibers, the axons were totally lost, leaving the myelin sheaths as empty tubes. Astrocytic processes containing a large number of glial filaments covered the nerve fibers adjacent to the syrinx and partially replaced the edematous area. The perivascular spaces were enlarged, especially near the syrinx and in the dorsal white matter. Oligodendrocytes remained undamaged, and the remyelination by oligodendrocytic processes was seen on some denuded axons. Sometimes, this further remyelination was abortive, especially where the edema was severe. The ultrastructural changes of the neural tissue and their sequences were identical, in most respects, to those of hydrocephalus and noncommunicating syringomyelia. The oligodendrocytic remyelination with ongoing demyelination found in this model has many similarities to those in experimental hydrocephalus.

Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein which is implicated in neurotransmitter release. Rabphilin-3A is expressed mainly in brain, but its subcellular localization remains to be clarified. Immunohistochemical analysis has revealed that Rabphilin-3A is most abundant in the synaptic area of the rat cerebellum, retina, and neuromuscular junction. Ultrastructural analysis of the neuromuscular junction using the immunogold method indicates that Rabphilin-3A is localized on the synaptic vesicle. Subcellular fractionation analysis of rat brain has shown that Rabphilin-3A is most highly concentrated in the purified synaptic vesicle fraction. These results indicate that Rabphilin-3A is localized on the synaptic vesicle in the presynapse.

Objective—To evaluate effects of transplantation of bone marrow stromal cells (BMSCs) into the CSF for the treatment of chronic spinal cord injury in dogs that had not responded by 1 month after decompressive surgery. Animals—23 dogs. Procedures—Dogs with paraplegia and loss of nociception in the pelvic limbs for at least 1 month after decompressive surgery were assigned to transplantation or control groups. Dogs in the transplantation group received BMSCs injected into the CSF 1 to 3 months after decompressive surgery. Dogs in the control group did not receive additional treatments. Improvements in gait, proprioceptive positioning, and nociception were evaluated by use of the Texas Spinal Cord Injury Scale for ≥ 6 months after BMSC transplantation. Results—6 of 10 dogs in the transplantation group regained the ability to walk, whereas only 2 of 13 dogs in the control group regained the ability to walk. Scores for the Texas Spinal Cord Injury Scale in the transplantation group were significantly higher than scores in the control group at the endpoint of the study (6 months after BMSC transplantation or after decompressive surgery for the transplantation and control groups, respectively). Only 1 dog (transplantation group) recovered nociception. All dogs from both groups had fecal and urinary incontinence. No complications were observed in relation to BMSC transplantation. Conclusions and Clinical Relevance—Injection of BMSCs into the CSF caused no complications and could have beneficial effects on pelvic limb locomotion in dogs with chronic spinal cord injuries.

Following crush injury of rat sciatic nerve, strong synaptophysin immunoreactivity was demonstrated in the regenerating sprouts that emerged from the proximal nodes of Ranvier and in their growth cones that extended through the space between Schwann cell basal lamina and myelin sheath of the parent axon. These findings suggest that synaptophysin is involved in the growth regulation of regenerating sprouts.

Ultrastructural localization of protein kinase C (PKC) beta-subspecies in neuromuscular junctions of the rat lumbrical muscle was investigated by the immunoperoxidase and immunofluorescence methods. By light microscopy, PKC beta-like immunoreactivity (PKC beta-LIR) was found in the axon terminal expansions as well as in the preterminal axons. By confocal laser scanning microscopy, the staining for PKC beta-like immunoreactivity was more intense in the presynaptic regions just in contact with the acetylcholine receptor stained by FITC-alpha-bungarotoxin. By electron microscopy, PKC beta-like immunoreactivity was distributed non-uniformly in the terminal expansions. In the terminal expansions, PKC beta-like immunoreactivity was accumulated in the presynaptic regions in contact with the post-synaptic folds. This accumulation was approximately 0.1-0.2 microns in diameter, which comprised a part of the presynaptic plasma membrane and a group of synaptic vesicles adjacent to it. Weak immunoreactivity was also found diffusely in the axoplasmic matrix. The discrete presynaptic accumulation of PKC beta-subspecies may represent the strategical localization specialized for the effective regulation of neurotransmitter release.

Sprouting from crush-injured rat sciatic nerves was studied by electron microscopy to clarify how regenerating sprouts are generated at the node of Ranvier and extended distally in the injured nerves. Three hours after injury, sprouting had already begun in a few nodes: part of the dense submembranous undercoat had disappeared from the nodal axolemma, and the axolemma was slightly evaginated where the undercoat was avoided. The sprout contained clear vesicles of about 50 nm in diameter in an amorphous axoplasm. In addition, vacuoles measuring 100-200 nm in diameter and multivesicular bodies were commonly found within or near these early sprouts. Six to 12 h after injury, nodes of Ranvier with sprouts increased in number in the region 1-1.5 mm proximal to the lesion. Sprouts grew toward the overlying Schwann cell basal lamina, and extended through the space between the basal lamina and Schwann cell plasmalemma (or myelin sheath). In the stem region continuous with the parent axon, regenerating sprouts displayed prominent neurofilaments, which were randomly arranged at 1 day post-injury, but oriented longitudinally by 5 days post-injury.

Immunocytochemical demonstration of protein kinase C (PKC) subspecies (α, β, γ) was carried out in Pacinian corpuscles of rat hind feet using monoclonal or polyclonal antibodies against each of these subspecies. The inner core cells and lamellae and the Schwann cell cytoplasm of the nerve fiber innervating the corpuscle were strongly positive for PKC α-immunoreactivity (IR). In contrast, the axon terminal and the outer core did not display any positive α-IR. Very weak PKC β-IR was detected in the ultraterminal region of the axon terminal, while the trunk region showed no immunoreactivity. Very faint PKC β-IR was found also in the lamellar cells located at the periphery of the inner core and the endoneurial fibroblasts in the intermediate layer. PKC γ-IR was not detected in any part of the corpuscle. The strong PKC α-IR in the inner core and the presence of absence of PKC α-, β-, and γ-IR in the axon terminal are discussed from the point of view of the functional aspects of each part.