Myelin phagocytosis by macrophages and nonmacrophages during Wallerian degeneration.

The literature concerning Schwann cells (SCs) and macrophages in myelin phagocytosis during Wallerian degeneration is reviewed. SCs carry out the first step in the removal of myelin by segmenting myelin and then incorporating the degraded myelin. The recruited macrophages then join in the myelin-phagocytosis event, appearing to make full use of their original phagocyte abilities until the end of myelin clearance. The molecular mechanisms of the two cells underlying myelin phagocytosis are thought to be different; myelin phagocytosis by SCs being lectin-mediated, i.e., opsonin-independent, whereas that of macrophages is mainly opsonin-dependent. It is important to note that SCs and macrophages cooperatively accomplish myelin phagocytosis.

Morphological basis of nitric oxide production and its correlation with the polysialylated precursor cells in the dentate gyrus of the adult guinea pig hippocampus.

Neurogenesis in the hippocampus persist throughout life and precursors of neurons reside in the granule cell layer of the dentate gyrus. Until now, the role of nitric oxide (NO) in the phenomenon has been unclear. By using specific antibodies and a confocal laser scanning microscope, the localization of NO synthase (NOS) was examined in the dentate gyrus of the adult guinea pig in relation with the neuronal precursor marker highly polysialylated neural cell adhesion molecule (PSA-N-CAM). Observation of single immunolabeled sections has revealed that both the PSA-N-CAM- and most NOS-positive cells were localized in the granule cell layer of the dentate gyrus. The former were small in size and showed a punctate, clustered immunoreaction with an irregular cellular margin, whereas the latter showed somewhat diverse cellular profiles. Some NOS-positive neurons had elliptical-like morphology with elongated dendrites, whereas others were small, irregularly shaped and mostly lacking dendritic spines. Double immunolabeling has revealed that NOS-immunoreactivity intermingled, as well as colocalized, with that of PSA-N-CAM, particulary in the granule cell layer. The doubly stained cells were morphologically indistinguishable from PSA-N-CAM single positive cells. These results not only suggest the role of NO production in adult hippocampal neurogenesis, but also indicate that some PSA-N-CAM-expressing neuronal precursors produce NO.

Age differences in morphological patterns of axonal sprouting and multiple innervation of neuromuscular junctions during muscle reinnervation following nerve crush injury.

During the first 4-20 weeks after sciatic nerve crushing injury regrowing axons return to the neuromuscular junction and its reformation is in progress. During this time period age differences in patterns of axonal reinnervation from Wistar rats, with special reference to multiple axonal innervation and sprouting, was morphologically investigated using a neuronal marker (protein gene product 9.5). In young (4 months old) and aged (24 months old) animals, terminal outgrowth at the junction consisted of offshoots extending out from the junctional zone (extraterminal sprouts), and an extraterminal sprout extending to an adjacent endplate (endplate-to-endplate connections). Endplate-to-endplate connections and a nodal sprout served as partners of multiple axonal innervation. Large and complex junctions were formed by multiple innervation and elaboration of terminal branching. The most obvious changes in aged animals were as follows. (1) There were consistently more frequent numbers of extraterminal sprouting, endplate-to-endplate connections, and multiple innervation. The rates of process extension in extraterminal sprouting, however, displayed a significant drop at 4 and 8 weeks post-crush. (2) Late in reinnervation (12, 20 weeks), persistent aberrant changes in axonal reinnervation were more frequently observed, such as clumping of poorly organized nerve bundles, aggregates of multiple extensions, and poorly developed endplate-to-endplate connections, along with disorderly development of nerve terminals. Thus, age affects the reinnervating and sprouting capabilities of axons giving rise to persistent compensatory (though impaired) growth, extension, and branching in the formation of motor pathways during muscle reinnervation and endplate regeneration. The spatiotemporal relationship of these axonal changes to that of the postsynaptic receptor region is discussed.

Molecular cloning of two guinea pig liver gap junction proteins, connexin32 and connexin26.

Intercellular coupling of hepatocytes through gap junctions facilitates exchange of small metabolites or ions, and contributes to maintenance of tissue homeostasis. As protein constituents of the liver gap junction channels, connexin32 (Cx32) and Cx26 have been identified. By use of rat cDNA probes, we cloned cDNAs for guinea pig homologs of Cx32 and Cx26, and compared their amino acid sequences with those of other species. The deduced primary structure of guinea pig Cx32 was 283 amino acids long and contained 98% identical amino acids to the rat and human Cx32. Only six amino acid exchanges were detected between the guinea pig and rat Cx32. On the contrary, the deduced amino acid sequence of guinea pig Cx26 (226 amino acids long) was 91 and 89% identical to the rat and human Cx26, respectively. Twenty-one amino acid exchanges were found between the guinea pig and rat, and the divergence was mostly located in cytoplasmic domains of Cx26. These results suggest that Cx26 shows structural diversity between species, while Cx32 is highly conserved.

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.

Age-related NADPH-diaphorase positive bodies in the lumbosacral spinal cord of aged rats.

In the course of a morphological investigation of age-related changes in the rat spinal cord, using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, we found abundant NADPH-d positive bodies, which were characteristically expressed in the aged lumbosacral spinal cord. Together with a normally stained fiber network and a few neurons, the dense, spheroidal NADPH-d positive bodies occurred in portions of the sacral dorsal spinal cords, such as the dorsal commissural nucleus, intermediolateral nuclei, and superficial dorsal horn, and were scattered throughout the dorsal white column. These NADPH-d positive bodies were occasionally observed in a fibrous structure. Two morphologically distinctive subsets of NADPH-d positive bodies were noted in the spinal cord of rats aged 8 to 36 months: 1) highly-dense spheroidal shapes with sharp edges; 2) moderately-dense spheroidal or multiangular shapes with a central "core" and a peripheral "halo". The quantitative analysis, particularly the stereological measurement, confirmed a gradual increase in the incidence and size of NADPH-d positive bodies with increasing age. With nNOS immunohistochemistry, no corresponding structures to NADPH-d positive bodies were detected in aged rats; thus NADPH-d activity is not always specific to the NO-containing neural structures. The major distribution of the NADPH-d positive bodies in the aged lumbosacral spinal cord indicates some anomalous changes in the neurite, which might account for a disturbance in the aging pathway of the autonomic and sensory nerve in the pelvic visceral organs.

The occurrence of nitric oxide synthase-containing axonal baskets surrounding large neurons in rat dorsal root ganglia after sciatic nerve ligation.

To clarify the possible role of nitric oxide (NO) induced in primary sensory neurons after peripheral axotomy, NO synthase (NOS) immunohistochemistry was carried out on rat L5 dorsal root ganglia after sciatic nerve ligation. The results were compared with the expression of 27-kDa heat shock protein (HSP27), a neuroprotective molecule. In intact animals, NOS-immunoreactive neurons represented about 2% of all dorsal root ganglion (DRG) neurons, whereas HSP27-immunoreactive neurons comprised about 14%. After sciatic nerve ligation, both neurons increased, in number and immunoreactivity, reaching a maximum at 2 weeks, when NOS- and HSP27-immunoreactive neurons represented about 33 and 66%, respectively. NOS-immunoreactive neurons then remained unchanged until 7 weeks although HSP27-immunoreactive neurons showed a slight decline. The increased NOS-immunoreactive neurons were preferentially small (100-500 microm(2)) and coexpressed with HSP27 (about 87%). On the other hand, in the proximal stump of sciatic nerves, numerous NOS-immunoreactive fibers with a regenerative profile appeared transiently (2-4 weeks). At higher magnification, an axonal sprout from the NOS-immunoreactive small DRG neurons was found to form a basket-like structure (or basket) mostly around the cell body of NOS-negative large neurons. Retrograde labeling with a fluorescent tracer showed that both neurons sent peripheral axon collaterals to the sciatic nerve. The appearance of this unique structure was most prominent after depletion of the NOS-immunoreactive regenerating fibers in the sciatic nerve (at 7-9 weeks). The findings suggest that NO might be involved in not only axonal regeneration but also the rewiring of two classes of DRG neurons after peripheral nerve injury.

Expression of nitric oxide synthase and 27-kD heat shock protein in motor neurons of ventral root-avulsed rats.

Root avulsion of adult spinal nerves causes the subacute cell loss of motor neurons. To explore the mechanisms of the elimination of motor neurons, we investigated the expression of two molecules--neuronal nitric oxide synthase (nNOS) as a cytotoxity marker and a 27-kD heat shock protein (HSP27) as a cytoprotection marker--in rat spinal motor neurons after ventral root avulsion, using immunofluorescent labeling technique for confocal laser microscopy. A drastic cell loss of motor neurons occurred during the first week following the avulsion, and the surviving motor neurons fell to approximately 60% of the control value at one week. Subsequent cell loss proceeded slowly, as the surviving motor neurons decreased to 35% at nine weeks. HSP27 immunohistochemistry showed that normal spinal motor neurons consisted of two types of motor neurons: HSP27-negative small motor neurons (< 500 micrometer2 ) (about 30%), and HSP27-positive large motor neurons (> 500 micrometer2) (about 70%). At one week, all of the HSP27-negative small motor neurons had died and only HSP27-positive large motor neurons survived. This event was followed by the induction of nNOS in the surviving large motor neurons, which showed a significant upregulation of HSP27. HSP27-negative small motor neurons were thus found to be more vulnerable to avulsion than HSP27-positive large motor neurons, suggesting that HSP27 may have protected the avulsed motor neurons from cell death. In addition, NO was involved in the gradual cell death of large motor neurons. The persistent upregulation of HSP27 and its colocalization with nNOS in surviving motor neurons may imply a keen competition in motor neuron survival between cytotoxic and cytoprotective systems.

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.

The spatiotemporal characterization of endplate reoccupation, with special reference to the superposition patterns of the presynaptic elements and the postsynaptic receptor regions during muscle reinnervation.

In this study, an immunohistochemical investigation was carried out to define spatiotemporal characteristics of superposition patterns of the presynaptic elements and the postsynaptic acetylcholine receptor (AChR) sites during the period of endplate regeneration after sciatic nerve crush. The extent of close correspondence of terminal Schwann cell (TSC)-, or axon terminal-, apposing AChR sites was quantitated with three-dimensional images of neuromuscular junctions (NMJs) taken under confocal laser-scanning microscopy. After 3-weeks post-crush (wpc), reoccupation of regenerating TSCs and later arriving axon terminals proceeded within the scope of previously denervated AChR plaques. During this period, the areas of presynaptic elements and the areas of postsynaptic elements were highly correlated. TSCs rapidly reoccupied a greater part of the postsynaptic receptors. In contrast, there was a slower increase of the contact areas of AChR sites overlapped by the axon terminals. Reoccupation by the presynaptic elements at 20 wpc was almost completed in a majority of NMJs, but some anomalous changes still continued to occur in a small proportion of the NMJs (20-30%). Our results suggest that: (a) with gradual increase of the contact areas between presynaptic and postsynaptic elements, imperfect reinnervation and regeneration, due to spatial mismatching or unbalanced growth between presynaptic and postsynaptic elements, result in sporadic remodeling; (b) the difference in superposition patterns between TSCs and axon terminals depends on the ability of making alignment to the endplate gutters in regenerating NMJs; and (c) a complex set of anatomical relationships among the three endplate components affects the process of endplate reoccupation synthetically.

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 beta-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.

N-myc downstream-regulated gene 1 expression in injured sciatic nerves.

N-myc downstream-regulated gene 1 (NDRG1)/RTP/Drg1/Cap43/rit42/TDD5/Ndr1 is expressed ubiquitously and has been proposed to play a role in growth arrest and cell differentiation. A recent study showed that mutation of this gene is responsible for hereditary motor and sensory neuropathy-Lom. However, the role of this gene in the peripheral nervous system is not fully understood. In our study, rabbit polyclonal antibodies were raised against this gene product and were used to examine changes in its expression over the time course of Wallerian degeneration and ensuing regeneration after crush injury of mouse sciatic nerves. Fluorescent immunohistochemistry showed that NDRG1 was expressed over the intact nerve fibers. Double labeling with a Schwann cell (SC) marker, S-100 protein (S-100), revealed that NDRG1 was localized in the cytoplasm of S-100-positive Schwann cells (SCs). NDRG1 expression was maintained in the early stage of myelin degradation but was then markedly depleted at the end stage of myelin degradation when frequent occurrence of BrdU-labeled SCs was observed (at 7-9 days). The depletion of NDRG1 at this time point was also confirmed by Western blotting analysis. NDRG1 expression finally recovered at the stage of remyelination, with immunoreactivity stronger than that in intact nerves. These findings suggest that NDRG1 may play an important role in the terminal differentiation of SCs during nerve regeneration.