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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.

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.

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.

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.

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

The present study, using confocal laser scanning microscopy and immunoelectron microscopy, examined the intracellular localization of tyrosine-phosphorylated proteins in cultured mouse dorsal root ganglion neurons with special reference to their growth cones. The growth cone is the specialized structure formed at the growing tip of the axon; characteristically highly motile with filopodia on the surface, it is responsible for the extension and guidance of the neurites to the appropriate targets during nerve regeneration. It has been suggested that protein-tyrosine phosphorylation plays an important role in the intracellular signal transduction that regulates the extension and motility of growth cones. By fluorescence immunocyto-chemistry, phosphotyrosine immunoreactivity was found in the growth cones and neurites. Some of the filopodia exhibited strong immunoreactivity at their tips. By immunoelectron microscopy, a large number of immunogold particles (gold particles conjugated to the secondary antibody) were seen to be distributed in the cytoplasm and some were observed on the plasma membrane in the growth cones, whereas in the neurites the density of immunogold particles was the same in the axoplasm as on the plasma membranes. These findings suggest that in the growth cones phosphotyrosines might mainly be involved in intracellular signaling for maintaining their high motility whereas in the neurites they might mostly be associated with the receptor proteins at the plasma membrane for adhesion as well as for growth of neurites. Thus, tyrosine phosphorylation might contribute to different functions for growth cones and neurites.

The present study defines the cellular composition of chemosensory corpuscles (taste buds) present in the mucosa overlying the arytenoid cartilages of the larynx in serial sections studied by electron microscopy. Three cell types can be defined in such chemosensory corpuscles. Basal cells are relatively undifferentiated in terms of cytologic characteristics and have been identified presumptively as the stem cells for differentiation of the other two cell types. Sustentacular or supporting cells are characterized by the presence of apical electron-opaque granules that apparently are extruded into the lumen of the pore, and are, thus, the source of the electron-opaque extracellular material present between the microvilli protruding from the apex of the cells into the chemosensory corpuscle pore. The chemosensory cells are characterized by the presence of synaptic specializations with appended nerve fibers at the base of the cell, the presence of specific cytoplasmic secretory granules, as well as numerous clusters of typical synaptic vesicles, and apical microvilli that extend into the pore of the corpuscle. Other cell profiles encountered in chemosensory corpuscles are cells in various stages of degeneration or transition, to one of the other cell types, from undifferentiated basal cells. Two types of synaptic specializations have been identified as being associated with chemosensory cells. The first is characterized by an increased electron-opacity of the respective plasma membranes of nerve fiber and chemosensory cell. In this situation numerous synaptic vesicles usually are present in the cytoplasm of the chemosensory cell subjacent to the membrane densities. A second type of synaptic association is characterized by the presence of subsurface cisterns of agranular endoplasmic reticulum in the cytoplasm of the chemosensory cell, and numerous synaptic vesicles often are present in the axoplasm of the subjacent neurite, suggesting a reciprocal synapse. These findings are interpreted to indicate that only one cell type is specialized for transducing chemical signals into neural activity and that this cell is modulated by activity of the nervous system.

Mechanoreceptive sensory corpuscles (murine Meissner corpuscles) in the toe pad skin of mice, consisting of axon terminals and lamellar cells, were studied following freeze-substitution in order to clarify the plasma membrane relationships between axon terminals and lamellar cells. Tissue preservation of corpuscles was excellent when the corpuscle was located within 10 microns from the contact surface with the precooled metal block. The axolemmata appeared more electron-opaque than did plasma membranes of lamellar cells. The inner leaflet of the unit membranes was thicker than the outer leaflet in the axolemma, and the contour of cell plasma membranes was relatively smooth and straight. Characteristic focal or regional approximations of plasma membranes were noted between the axon and abutting lamellae. Such membrane appositions resembled gap junctions, although no gap junctions were found between the axon and lamellae in chemically fixed materials. Similar gap junction-like close appositions of plasma membranes also were found between neighboring lamellae. These approximations occurred more frequently than typical gap junctions seen in chemically fixed materials. These findings indicate that there may be a relationship of the plasma membranes in the axon terminals and in abutting lamellae as well as between neighboring lamellae that have not been identified as yet in conventional chemically fixed material. Another striking finding was that basal laminae on lamellar cells exhibited the same electron opacity as the surrounding connective tissue matrix and thus the two are indistinguishable from one another. Furthermore, the lamina lucida was not evident, and basal lamina material was directly contiguous with the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

The sequential changes of the morphology and the histochemically demonstrated, nonspecific cholinesterase (ChE) activity of the denervated digital corpuscle (Meissner corpuscle) of the mouse were observed by light and electron microscopy for 6 months after transection of the sciatic nerve. A fragment of the sciatic nerve, approximately 1 cm long, was removed from the distal stump to prevent reinnervation. Within one day following nerve division, the axon terminals began to manifest degenerative changes: the axoplasm became electron-opaque and some mitochondria became disorganized. The lamellar cell processes (lamellar) also exhibited atrophic changes: they became thinner and more electron opaque. The lamellar cell body became smaller due to a decreased amount of cytoplasm and contained few organelles except for some free ribosomes. These changes advance with time. Around 4-6 months after denervation, the corpuscle was small, consisting of a few thin, shrunken, atrophic lamellar cells. The ChE activity persisted and could be demonstrated in the interlamellar spaces, but its intensity decreased with time so that the corpuscle was hardly identifiable by light microscope histochemistry around 4-6 months after denervation. During this time, the precipitates of the reaction product were scattered as small particles throughout the interlamellar spaces. The enzyme activity in the cisternae of the rough endoplasmic reticulum and the nuclear envelope of the lamellar cell body was no longer demonstrable 5 days or less after denervation. These findings indicate that the lamellar cell is dependent on the axon terminal to maintain its normal morphology and the function of ChE synthesis.