Kazuho Hirata

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.

A monoamine in the gustatory cell of the frog's taste organ

SummaryFluorescence histochemistry reveals that in the frogs taste organ a yellow fluorescence is regularly observed at the most basal region of the sensory epithelium. The fluorescence has a strong intensity, but it fades rapidly upon the UV-irradiation. The peak of the emission spectrum is at 520 mμ. Following reserpine treatment the yellow fluorescence is markedly reduced, but not depleted completely. From these characteristics the monoamine fluorescence is regarded as representing 5-HT (serotonin).The ultrastructural study on sensory epithelia shows that the terminal portions of gustatory cell processes are localized at the basal region. These portions are filled with dense cored vesicles (700–1000 Å in diameter) and frequently opposed with nerve fibers penetrating into the epithelium. The gustatory cell processes are also interposed between the terminal portions or nerve fibers. The cytoplasm of the gustatory cell process is characterized by many mitochondria, fine filaments and glycogen particles, but contains few cored vesicles. The distribution of terminal portions of gustatory cell processes seems to correspond fairly well to that of the monoamine fluorescence observed discontinuously along the basal lamina. Accordingly it is concluded that the fluorigenic monoamine is localized in the cored vesicles of the gustatory cell.

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.

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.

An improved method for avulsion of lumbar nerve roots as an experimental model of nitric oxide-mediated neuronal degeneration

A root avulsion lesion on the spinal nerve of adult animals is a useful technique to make a model for axotomy-induced motoneuronal degeneration, which is thought to be mediated by nitric oxide (NO). Here, we show a simplified version of extravertebral avulsion in the young adult rat. The L4 nerve always runs under the transverse process of the L5 vertebra, which is located just rostral to the delineation of the iliac crest. We used the iliac crest as a clue for the identification of the L4 nerve during surgery, including before skin incision. In almost all animals the L4 nerve was successfully avulsed at the exit point from the spinal cord. This experimental result was similar to that shown in the previous literature; the number of either Nissl-stained or ChAT-immunoreactive (-ir) motoneurons (MN) gradually decreased, while NOS immunoreactivity was induced in the MN after avulsion. Furthermore, a combined method of confocal laser scanning microscopy and double fluorescent procedures carried out in this model suggested the existence of cellular interaction between NOS-ir MN and OX42-ir or ED1-ir microglia. It is concluded that this simple and fast method of spinal root avulsion is very useful for making a reproducible model of NO-mediated MN cell death, with which the mechanism of neuronal cell death, including neuron-glia interaction, can be further explored.

Effect of nitric oxide synthase inhibition on Schistosoma japonicum egg‐induced granuloma formation in the mouse liver

Nitric oxide (NO) plays diverse roles in a variety of pathological processes. We investigated the role of NO in Schistosoma japonicum egg‐induced granuloma formation in a mouse hepatic model. Immunohistological analysis revealed that there is the most intense and extensive inducible nitric oxide (iNOS) expression 2 weeks after egg implantation, and thereafter it decreased considerably with time. Treatment with nitric oxide synthase inhibitors, NIL (lN6‐ (iminoethyl)‐lysine) or Nω‐nitro‐l‐arginine methyl ester (l‐NAME), resulted in two different types of unusual granulomas at 2 weeks. One type showed suppressed fibrosis, while another showed foreign body‐type multinuclear cell formation which frequently appeared particularly when 50 µg/ml NIL was given. At 3 weeks following treatment, fibrotic granulomas with scanty peripheral cellularity was obvious. However, there were no apparent changes after this period (at 4 weeks). Cytokine analysis in NIL‐treated mice showed a significant increase of IL‐4 and IL‐13 production at 2 weeks. These findings indicated that nitric oxide contributes to granuloma development during the early stages, probably through the regulation of Th2 cytokine production.

Heme oxygenase1 (HSP-32) is induced in myelin-phagocytosing Schwann cells of injured sciatic nerves in the rat.

Schwann cells participate in myelin phagocytosis in the early stage of Wallerian degeneration, prior to the recruitment of macrophages. This is the first report that Schwann cells induce heme oxygenase‐1 (HO‐1), a 32‐kDa heat shock protein, only when they have transformed into myelin‐phagocytosing cells from myelinating cells (days 2–3) immediately after crush injury of rat sciatic nerves. Double immunofluorescent labelling for HO‐1 and transferrin receptors revealed that HO‐1‐immunoreactive Schwann cells also expressed transferrin receptors suggesting activation of iron metabolism. The transient induction of HO‐1 in Schwann cells may contribute to the adaptive function in an altered environment when the cells have lost contact with axons, and may play a crucial role in the ensuing regeneration.

Immunohistochemical studies on neuron-specific enolase in developing rat vallate papillae

SummaryThe detailed morphology of the nerve fibers and the taste bud cells in developing vallate papillae of the rat tongue was investigated utilizing the immunoperoxidase technique to detect neuron-specific enolase (NSE). For convenience of description, five stages of development were defined: Stage 1, the fifteenth and the sixteenth embryonic day (E15–E16): NSE like immunoreactive (NSE-) nerve fibers, with some random arborization, appeared around the median lingual sulcus at the base of the tongue; Stage 2 (E16–E17): NSE-nerve fibers invading the central core of newly formed vallate papilla and underlying the apical epithelium of the papilla; Stage 3 (E18–E21): round-shaped undifferentiated NSE-taste bud cells appearing in the apical epithelium; Stage 4, the first day of postnatal age (P1): NSE-taste bud cells migrated to the side epithelium, lining the gutter beneath which the nerve plexus formed during E18–E21, and extended cytoplasmic process toward the surface and/or the basal lamina; Stage 5 (P3–P5): NSE-nerve fibers and spindle-shaped NSE-taste bud cells with a typical figure of taste bud cells appeared in newly formed taste buds in the side epithelium, lining the gutter. The sequential topographic development of nerve preceding NSE-taste bud cells in precise morphological locations, suggests that the ingress of precursor NSE-taste bud cells and their subsequent differentiation are contingent upon initial neural derived ontologic signals.