Masaru Kawabuchi

Differential response of macrophage subpopulations to myelin degradation in the injured rat sciatic nerve.

Molecular mechanisms of myelin removal by macrophages were explored by examining the immunophenotypes of macrophages following injury of rat sciatic nerve, using a combined method of immunohistochemistry and confocal laser microscopy. In the crush injury model, the involvement in myelin clearance of a cytoplasmic antigen specific for monocytes/macrophages, ED1, was evident. The obvious recruitment of ED1-immunoreactive (-ir) cells was detected first at the crush injury site and then in the distal stump within which Wallerian degeneration had occurred. Double labelling revealed that the ED1-ir cells, except for monocyte-like round cells, always phagocytosed myelin basic protein-ir myelin debris. On the other hand, the expression of ED2, a surface antigen specific for resident macrophages, was significantly different; ED2-ir cells also increased while myelin removal was progressing from day 3 to day 7, but only some of the cells were engaged in myelin phagocytosis. The poor capacity of myelin phagocytosis by ED2-ir cells was supported by the transection model, in which the proximal stump was ligated to suppress regeneration. ED2 may be involved in events other than myelin removal, providing a local environment conducive to axonal regeneration. Our findings thus seem to suggest that ED1 is one of the most reliable markers for cells carrying out myelin phagocytosis, whereas ED2 may participate in entirely different functions. The expression of complement receptor type 3, OX42, was similar to that of ED1 in terms of the swift recruitment of immunopositive cells, their distribution with close association to myelin debris and their high phagocytotic capacity. This supports previously reported in vitro evidence that myelin phagocytosis by macrophages may be complement-mediated.

SPECIFIC LOCALIZATION OF GAP JUNCTION PROTEIN, CONNEXIN45, IN THE DEEP MUSCULAR PLEXUS OF DOG AND RAT SMALL INTESTINE

Abstract Cellular networks of pacemaker activity in intestinal movements are still a matter of debate. Because gap-junctional intercellular communication in the intestinal wall may provide important clues for understanding regulatory mechanisms of intestinal movements, we have attempted to clarify the distribution patterns of three types of gap junction proteins. Using antibodies for connexin40, connexin43, connexin45, smooth muscle actin, and vimentin, immunocytochemical observations were made with the confocal laser scanning microscope on cryosections of fresh-frozen small intestine and colon of the dog and rat. Connexin 45 was localized along the deep muscular plexus of the small intestine in both dog and rat. Double labeling studies revealed that connexin45 overlapped with vimentin –, but not actin-positive areas, indicating the fibroblast-like nature of the cells, rather than their being smooth muscle-like. Connexin43 immunoreactivity appeared along the smooth muscle cell surface in the outer circular layer of the small intestine of both animals. Connexin 40 immunoreactivity was not observed in the muscle layer other than in the wall of large blood vessels. It is suggested that connexin45-expressing cells along the deep muscular plexus of dog and rat small intestine are likely to act as a constituent of a pacemaker system, which may include a conductive system, by forming a cellular network operating via specific types of gap junctions.

Involvement of PITPnm, a Mammalian Homologue of Drosophila rdgB, in Phosphoinositide Synthesis on Golgi Membranes

Phosphatidylinositol transfer protein (PITP) is involved in phospholipase C-mediated signaling and membrane trafficking. We previously reported cloning and characterization of a gene encoding for membrane-bound PITP, named PITPnm, that is a mammalian homologue of the Drosophila retinal degeneration B (rdgB) gene (Aikawa, Y., Hara, H., and Watanabe, T. (1997)Biochem. Biophys. Res. Commun. 236, 559–564). Here we report the subcellular localization of PITPnm protein and provide evidence for its involvement in phosphatidylinositol 4-phosphate (PtdIns 4-P) synthesis. PITPnm is an integral membrane protein that largely localized in close association with membranes of Golgi vacuoles and the endoplasmic reticulum (ER). The amino terminus region of PITPnm was exposed to cytoplasmic side. Interaction with various phosphoinositides was observed in the amino terminus region spanning from 196 amino acids to 257 amino acids of PITPnm. At the amino terminus regions of 1–372 amino acids, PITPnm formed a complex with type III PtdIns 4-kinase. The transmembrane and carboxyl-terminal portions (residues 418–1242) functioned to retain the PITPnm in the Golgi vacuole. These results suggest that PITPnm plays a role in phosphoinositide synthesis on the Golgi vacuoles and possibly in the PtdIns signaling pathway in mammalian cells.

Postnatal development of Schwann cells at neuromuscular junctions, with special reference to synapse elimination

The neuromuscular junctions (NMJs) of postnatal rat soleus muscles were examined by immunohistochemical staining for S100, a marker of Schwann cells (SCs), and for protein gene product 9.5, a neuronal marker, to elucidate the involvement of SCs in synapse elimination. The morphological maturation of S100-immunoreactive terminal SCs at NMJs proceeded with the gradual increase in their number. The number of terminal SCs per NMJ was one or two at postnatal day (P) 7, reaching the adult number at P28, when it became three or four. Confocal laser scanning microscopic analysis of multi-innervated NMJs, whose number decreased between P7 and P14, revealed a change in the ratio between terminal SCs and axons with age. At P7, the ratio between axons and terminal SCs per NMJ was ≥2:1, which was exactly the reverse of that in adults, while at P14 this had changed to 2:2. A structural change appeared to occur at the same time at the preterminal region, this being prior to the establishment of a 1:1 relationship between axon and SC sheath which was detected at P14, with the ≥2:1 relationship seeming to occur at P7. Thus, synapse elimination seems to proceed, at least for one week, with the gradual loss of axons which are at different stages of maturation with respect to their spatial relationship with SCs. From our results it seems unlikely that SCs play an active role in selecting a single axon to survive.

Neostigmine Myopathy Is a Calcium Ion-Mediated Myopathy Initially Affecting the Motor End-plate

Morphological techniques were used to determine the acute and chronic effects of neostigmine on rat muscles. Transient calcium deposits, eliminated by prior treatment of sections with ethyleneglycol bis (aminoethylether) tetracetate (EGTA), were independent of fiber type and found at sites corresponding to neostigmine-induced focal lesions. The dimension and number of focal lesions and calcium deposits gradually decreased with chronic drug treatment. Size, shape, and density of the calcium deposits varied. Alterations in the motor nerve terminal, synaptic space, and junctional fold persisted even when banding patterns at the motor end plates were intact. Characteristic intermediate findings consisted of rod bodies and ribosomal clusters. Such clusters were frequently mingled with clumped sarcoplasmic reticulums, T-systems, or mitochondria. Despite continued administration of neostigmine, focal myopathic changes, other than in the synaptic region of the end plates, were reversible.

HSP27 is markedly induced in Schwann cell columns and associated regenerating axons

It is well known that regenerating axons enter Schwann cell (SC) columns, within which they grow to reinnervate the appropriate targets. The current study detected a marked induction of a 27‐kDa heat shock protein (HSP27) in the SC columns of crush‐injured rat sciatic nerves. Immunohistochemical studies showed the first appearance of strong HSP27‐immunoreactive linear structures in the proximal stump near an injury site 7 h after an operation. The HSP27‐immunoreactive linear structures crossed the injury site to the distal stump 2 days after the operation. They then extended in a more proximal and more distal direction and were found to have propagated through the entire length of the nerve 1 week after the operation. This pattern of expression was maintained until 3 weeks after the operation. Double‐immunofluorescent labeling and confocal laser microscopy confirmed that the linear structures consisted of SC columns and associated multiple axons. The HSP27‐immunoreactive SC columns expressed glial fibrillary acidic protein, but not S‐100 protein. Electron microscopy and immunoelectron microscopy demonstrated that reactive Schwann cells (SCs) and the associated axons with an outgrowing profile exhibited a strong immunoreactivity to HSP27, with the former containing a greater number of bundles of intermediate filaments. It is suggested that HSP27 may play an essential role in axonal outgrowth, especially by contributing to cytoskeletal dynamics in SCs. GLIA 42:1–11, 2003.

The spatiotemporal relationship among schwann cells, axons and postsynaptic acetylcholine receptor regions during muscle reinnervation in aged rats

To morphologically define the aging‐related features during muscle reinnervation the spatiotemporal relationships among the major components of the neuromuscular junctions (NMJs) were investigated. A total of 64 rats, 30 adults (4 months old) and 34 aged adults (24 months old), were used. Between 1 and 12 weeks after sciatic nerve‐crushing injury, cryosections of skeletal muscle were single or double labeled for S100, a marker of Schwann cells (SCs), for protein gene product 9.5, a neuronal marker, and for α‐bungarotoxin (α‐BT), a marker of the acetylcholine receptor site (AChR site), and then observed by confocal laser microscopy. The most obvious age changes were noted: (1) the regenerating SCs and axons were delayed in their arrival at the NMJ, (2) the dimensions of terminal SCs and AChR sites displayed a drastic and long‐lasting drop (for terminal SCs, during 1–8 weeks; for AChR sites, during 1–12 weeks); (3) the degree of spatial overlap between AChR sites and terminal SCs was markedly low until 8 weeks post‐crush; (4) damage and poor formation in the SCs, terminal axons and AChR sites, together with poor process extension from the terminal SC or terminal axon, were pronounced; (5) persistent aberrant changes, such as multiple innervation and terminal axon sprouting, together with poorly formed collateral innervation, nerve bundles, and NMJs, more frequently occurred in the later reinnervation period. Thus, with aging, regeneration is impaired during the period in which regenerating SC strands and axons extend into NMJs and the subsequent establishment of nerve‐muscle contact is in progress. A complex set of morphological abnormalities between or among the TSCs, terminal axons, and AChR sites may be important in slowing of regeneration and reinnervation in aged motor endplates. Anat Rec 264:183–202, 2001.

Expression of amyloid precursor protein‐like molecule in astroglial cells of the subventricular zone and rostral migratory stream of the adult rat forebrain

In adult mammals, new neurons in the subventricular zone (SVZ) of the lateral ventricle (LV) migrate tangentially through the rostral migratory stream (RMS) to the olfactory bulb (OB), where they mature into local interneurons. Using a monoclonal antibody for the β‐amyloid precursor protein (APP) (mAb 22C11), which is specific for the amino‐terminal region of the secreted form of APP and recognizes all APP isoforms and APP‐related proteins, immunoreactivity was detected in specific subpopulations of cells in the SVZ and RMS of the adult rat forebrain. In the SVZ, APP‐like immunoreactivity was detected in the ependymal cells lining the LV and some of the subependymal cells. The latter were regarded as astrocytes, because they were positive for the glial markers, S‐100 protein (S‐100) and glial fibrillary acidic protein (GFAP). APP‐like immunoreactive astrocytes exhibited strong labelling of the perinuclear cytoplasm and often possessed a long, fine process similar to that found with radial glia. The process extended to an APP‐like immunoreactive meshwork in the RMS that consisted of cytoplasmic processes of astrocytes forming ‘glial tubes’. Double‐immunofluorescent labelling with a highly polysialylated neural cell adhesion molecule (PSA‐NCAM) confirmed that the APP‐like immunoreactive astrocytes in the SVZ and meshwork in the RMS made close contact with PSA‐NCAM‐immunopositive neuroblasts, suggesting an interaction between APP‐containing cells and neuroblasts. This region of the adult brain is a useful in vivo model to investigate the role of APP in neurogenesis.

Age affects reciprocal cellular interactions in neuromuscular synapses following peripheral nerve injury.

Studies of the influence of age on regeneration and reinnervation in the peripheral nervous system (PNS) and neuromuscular junction (NMJ) are reviewed, with a particular focus on aged and denervated skeletal muscles. The morphological and functional features of incomplete regeneration and reinnervation are compared between adult and aged animals. In addition, some possible mechanisms of the age-related defects will be discussed. Increased fragmentation or damage in individual components of the NMJ (terminal Schwann cells (TSCs), axon terminals and acetylcholine receptor sites occurs during muscle reinnervation following PNS injury in the aged animals. The capacity to produce ultraterminal sprouting or multiple innervation secondary to PNS injury is maintained, but not the capacity to eliminate such anomalous axonal profiles. The frequency and accuracy of reoccupation of the synaptic sites by TSCs and axon terminals are impaired. Thus, despite the capability of extending neural processes, the rate at which regenerating nerve fibers grow, mature and precisely appose the postsynaptic muscle fiber is impaired, resulting in the failure of re-establishment of the normal single motor innervation in the NMJ. A complex set of cellular interactions in the NMJ are known to participate in the neurotrophism and neurotrophism to support growth of the regenerating and sprouting axons and their pathfinding to direct the target muscle fiber. Besides the capability of α-motoneurons, signaling originating from the TSCs and muscle may be impaired during aging.

Three-dimensional structures of c-Kit-positive cellular networks in the guinea pig small intestine and colon

Cryosections and whole-mount preparations of the guinea pig small intestine and colon were single or double immunolabeled using the anti-c-Kit and protein gene product 9.5 antibodies. Immunolabeled specimens were observed under a confocal laser scanning microscope. The main findings of the present study are: (1) the distribution and profiles of three-dimensional structures of c-Kit-positive cellular networks in the small intestine and colon, and (2) the anatomical relations of c-Kit-positive cells to the enteric nerves in the layers. In the small intestine, c-Kit-positive cellular networks were observed at levels of the deep muscular plexus and myenteric plexus. The c-Kit-positive cellular networks ran along or overlay the nerve fibers at the deep muscular plexus, while they showed the reticular structures intermingled with the nerve elements at the myenteric plexus. In the colon, c-Kit-positive cellular networks were observed at levels of the submuscular plexus and myenteric plexus, and were further identified within the circular and longitudinal muscle layers as well as in the subserosal layer. In the circular muscle layer, c-Kit-positive cells surrounded the associated nerve fibers and extended several long processes toward the adjacent c-Kit-positive cells. The c-Kit-positive cellular networks within the longitudinal muscle layer as well as in the subserosal layer were not associated with the nerve fibers. In the layers of the intestinal wall with c-Kit-positive cells, the cellular networks of the interstitial cells were identified in ultrastructure. The characteristic profiles of c-Kit-positive cellular networks provide a morphological basis upon which to investigate the mechanisms regulating intestinal movement.