Abstract: A 6-year-old, neutered male Saint Bernard dog was presented with a 1-month history of ataxia, hypermetria and head tilt. High-field magnetic resonance imaging revealed a mass in the cerebellar vermis. During necropsy examination, a cream-coloured irregular area was observed in the cerebellar white matter. Microscopically, the mass comprised a diffuse neoplastic proliferation of spindle cells with oval pleomorphic nuclei in the white and grey matter of the cerebellum and pons and in the subpial area. Neoplastic infiltration was not found in the cerebrum. Immunohistochemistry revealed that the neoplastic cells were positive for vimentin and partially positive for glial fibrillary acidic protein. Based on these findings, the neoplastic lesion was diagnosed as gliomatosis cerebelli, without involvement of the cerebrum.
Abstract: ABSTRACT: BACKGROUND: Microglia/macrophages and T lymphocytes (T-cells) accumulate around motor and primary sensory neurons that are regenerating axons but there is little or no microglial activation or T-cell accumulation around axotomised intrinsic CNS neurons, which do not normally regenerate axons. We aimed to establish whether there was an inflammatory response around the perikarya of CNS neurons that were induced to regenerate axons through a peripheral nerve graft. RESULTS: When neurons of the thalamic reticular nucleus (TRN) and red nucleus were induced to regenerate axons along peripheral nerve grafts, a marked microglial response was found around their cell bodies, including the partial enwrapping of some regenerating neurons. T-cells were found amongst regenerating TRN neurons but not rubrospinal neurons. Axotomy alone or insertion of freeze-killed nerve grafts did not induce a similar perineuronal inflammation. Nerve grafts in the corticospinal tracts did not induce axonal regeneration or a microglial or T-cell response in the motor cortex. CONCLUSIONS: These results strengthen the evidence that perineuronal microglial accumulation (but not T-cell accumulation) is involved in axonal regeneration by intrinsic CNS and other neurons.
Abstract: Keap1 is proposed to be a sensor protein of electrophilic compounds and a transducer of the signal from electrophilic compounds for transcriptional activation. Thus, the use of keap1 gene-knockout (KO) mice is a straightforward approach in order to clarify the molecular background for the use of electrophilies as neuroprotective compounds. In the present report, we investigated the question as to how the deletion of the keap1 gene affects the activities of Nrf2 and survival of immature cortical neurons. In cortical cultures prepared from wild-type (WT) mice, Keap1 was expressed in the neurons, and Nrf2 protein was retained in their cytoplasm; whereas Nrf2 was translocated into the nuclei of neurons and phase 2 enzymes were constitutively activated in the cortical cultures from KO mice. Consistent with these results, cortical neurons from KO mice showed increased resistance to oxidative stress induced by high concentrations of glutamate and rotenone. These results suggest that the absence of Keap1 constitutively activates Nrf2, which then induces the phase 2 enzymes in neurons and induces increased resistance of cortical neurons to oxidative stress. This report is the first report to show that Keap1 is a key regulator of cell defense mechanisms of CNS neurons against oxidative stress.
Abstract: TAP-like (TAPL; ABCB9) is a half-type ATP-binding cassette (ABC) transporter that localizes in lysosome and putatively
conveys peptides from cytosol to lysosome. However, the physiological role of this transporter remains to be elucidated.
Comparison of genome databases reveals that TAPL is conserved in various species from a simple model organism,
Caenorhabditis elegans, to mammals. C. elegans possesses homologous TAPL genes: haf-4 and haf-9. In this study, we
examined the tissue-specific expression of these two genes and analyzed the phenotypes of the loss-of-function mutants
for haf-4 and haf-9 to elucidate the in vivo function of these genes. Both HAF-4 and HAF-9 tagged with green fluorescent
protein (GFP) were mainly localized on the membrane of nonacidic but lysosome-associated membrane protein homologue
(LMP-1)-positive intestinal granules from larval to adult stage. The mutants for haf-4 and haf-9 exhibited granular
defects in late larval and young adult intestinal cells, associated with decreased brood size, prolonged defecation cycle,
and slow growth. The intestinal granular phenotype was rescued by the overexpression of the GFP-tagged wild-type
protein, but not by the ATP-unbound form of HAF-4. These results demonstrate that two ABC transporters, HAF-4 and
HAF-9, are related to intestinal granular formation and some other physiological aspects.
Abstract: Pulmonary surfactant is synthesized and secreted by pulmonary alveolar type II epithelial cells (type II cells). It passes through the alveolar lining fluid and adsorbs to the air-liquid interface. The process from secretion to adsorption is not yet entirely understood. To acquire a detailed understanding of this process, we used multiple observations of type II cells isolated from rat lungs under electron microscopy (EM) and confocal laser scanning microscopy (CLSM). Transmission EM observation demonstrated a loosening process of the intracellular lamellar bodies from the inside to the outside of the cell. Scanning EM observation revealed bubble-like protrusions from the cell surface, and differential interference contrast microscopy illustrated the protrusions expanding with time. CLSM observation with FM 1-43, a fluorescent membrane probe, revealed that the bubble-like protrusions were composed of phospholipids. Thus, we have demonstrated that isolated rat type II cells protrude intracellular lamellar bodies by forming bubble-like structures, possibly enabling them to adsorb to the air-liquid interface directly. These observations suggest a new mechanism for surfactant secretion from type II cells.
Abstract: We report a cervical intraspinal cyst in a dog that was initially tetraparetic but spontaneously recovered completely. MRI revealed a well-demarcated intraspinal cyst located dorsally to a degenerated intervertebral disc. The location of the cyst and its signal features on MRI resembled those of discal cysts previously reported in humans. It has been reported in dogs that clinical signs of a intraspinal cyst are similar to those of intervertebral disc herniation and both conditions require surgical intervention. Unexpectedly, our case showed rapid spontaneous recovery and the follow-up MRI revealed complete resolution of the intraspinal cyst and spinal cord compression. Spontaneous recovery of degenerative intraspinal cyst may occur in dogs, similar to rare human cases as reported previously.
Abstract: By using high-resolution, conventional, and neuromelanin-sensitive magnetic resonance imaging techniques, we reviewed the normal anatomy of the nuclei consisting of monoamine neurons such as dopaminergic, noradrenergic, and serotoninergic neurons and noted the changes in these nuclei that occur in some degenerative and psychiatric disorders. Multimodal MR images can directly or indirectly help in identifying the substantia nigra, locus ceruleus, and raphe nuclei that contain monoamine neurons. Neuromelanin-sensitive magnetic resonance imaging can detect signal alterations in the substantia nigra pars compacta and/or locus ceruleus that occur in Parkinson's disease and psychiatric disorders such as depression and schizophrenia. This technique seems to be promising for the noninvasive evaluation of the pathological or functional changes in the monoamine system that occur in degenerative and psychiatric disorders.
Abstract: In contrast to compact myelin, the series of paranodal loops located in the outermost lateral region of myelin is non-compact; the intracellular space is filled by a continuous channel of cytoplasm, the extracellular surfaces between neighboring loops keep a definite distance, but the loop membranes have junctional specializations. Although the proteins that form compact myelin have been well studied, the protein components of paranodal loop membranes are not fully understood. This report describes the biochemical characterization and expression of Opalin as a novel membrane protein in paranodal loops. Mouse Opalin is composed of a short N-terminal extracellular domain (amino acid residues 1-30), a transmembrane domain (residues 31-53), and a long C-terminal intracellular domain (residues 54-143). Opalin is enriched in myelin of the central nervous system, but not that of the peripheral nervous system of mice. Enzymatic deglycosylation showed that myelin Opalin contained N- and O-glycans, and that the O-glycans, at least, had negatively charged sialic acids. We identified two N-glycan sites at Asn-6 and Asn-12 and an O-glycan site at Thr-14 in the extracellular domain. Site-directed mutations at the glycan sites impaired the cell surface localization of Opalin. In addition to the somata and processes of oligodendrocytes, Opalin immunoreactivity was observed in myelinated axons in a spiral fashion, and was concentrated in the paranodal loop region. Immunogold electron microscopy demonstrated that Opalin was localized at particular sites in the paranodal loop membrane. These results suggest a role for highly sialylglycosylated Opalin in an intermembranous function of the myelin paranodal loops in the central nervous system.
Abstract: BACKGROUND: We investigated alterations in the substantia nigra pars compacta (SNc) and locus ceruleus (LC) in schizophrenic and depressive patients by using a neuromelanin-sensitive magnetic resonance imaging (MRI) technique that enables direct visualization of these nuclei and examined whether this technique could distinguish between these disorders and healthy subjects. METHODS: Using a neuromelanin-sensitive T1-weighted MRI technique, we examined 20 schizophrenia patients, 18 depressive patients, and 34 healthy control subjects. The signal intensities of the areas corresponding to the SNc and LC were measured, and the contrast ratios (CR) to the adjacent white matter were calculated. RESULTS: The CR of the SNc was significantly higher in schizophrenic patients (22.6 +/- 5.6) than in depressive patients (19.2 +/- 4.7) and healthy control subjects (19.6 +/- 3.8), whereas the CR of the LC in depressive patients (7.7 +/- 2.4) was significantly lower than that in healthy control subjects (11.0 +/- 3.9) and schizophrenic patients (10.0 +/- 3.1). Further, the difference in the CR between the SNc and LC was significantly greater in schizophrenic patients (12.6 +/- 6.7) than in control subjects (8.6 +/- 4.1). CONCLUSIONS: Neuromelanin-sensitive MRI enables visualization of alterations in the SNc and LC that are observed in schizophrenia and depression.
Abstract: We evaluated the preservation of ultra-structure and immunoreactivity in cryosections of central nervous system tissue mounted with and stored in a sucrose-gelatin solution for one month at -20 degrees C or -80 degrees C. The ultra-structure of synaptic structure in these sections was well preserved and comparable to that of freshly cut cryosections. Quantitative analysis of mitochondrial ultra-structure demonstrated gradually lower degrees of preservation in sections stored at -20 degrees C and -80 degrees C compared with that in freshly cut sections. We observed distinct metabotropic glutamate receptor 1 (mGluR1)-immunogold labelling at peri-synaptic sites in freshly cut sections and also in those stored at -20 degrees C and -80 degrees C. Quantitative analysis of mGluR1 immunoreactivity revealed that the total number of immunogold particles per synapse and the number of non-specifically bound particles were similar under all three conditions. However, the percentage of gold particles bound to a specific synaptic region was greatest in freshly cut sections (79.0%) and progressively lower in sections stored at -20 degrees C (76.1%), in which sections were not frozen, and in sections stored at -80 degrees C (68.0%). These data indicate that ultra-thin cryosections may be conveniently stored in a sucrose-gelatin solution at -20 degrees C for cryoultramicrotomy-immunolabelling.
Abstract: Affinity-purified Shiga toxin (Stx) 2 given intraperitoneally to mice caused weight loss and hind-limb paralysis followed by death. Globotriaosylceramide (Gb(3)), the receptor for Stx2, was localized to neurons of the central nervous system (CNS) of normal mice. Gb3 was not found in astrocytes or endothelial cells of the CNS. In human cadaver CNS, we found Gb(3) in neurons and endothelial cells. Mouse Gb(3) localization was confirmed by immunoelectron microscopy. In Stx2-exposed mice, anti-Stx2-gold immunoreaction was positive in neurons. During paralysis, after Stx2 injection, multiple glial nuclei were observed surrounding motoneurons by electron microscopy. Also revealed was a lamellipodia-like process physically inhibiting the synaptic connection of motoneurons. Ca2+ imaging of cerebral astrocytic end-feet in Stx2-treated mouse brains suggested that the toxin increased neurotransmitter release from neurons. In this article, we propose that the neuron is a primary target of Stx2, affecting neuronal function and leading to paralysis.
Abstract: Galactocerebroside and sulfatide are two major glycolipids in myelin; however, their independent functions are not fully understood. The absence of these glycolipids causes disruption of paranodal junctions, which separate voltage-gated Na(+) and Shaker-type K(+) channels in the node and juxtaparanode, respectively. In contrast to glial cells in the central nervous system (CNS), myelinating Schwann cells in the peripheral nervous system (PNS) possess characteristic structures, including microvilli and Schmidt-Lanterman incisures, in addition to paranodal loops. All of these regions are involved in axo-glial interactions. In the present study, we examined cerebroside sulfotransferase-deficient mice to determine whether sulfatide is essential for axo-glial interactions in these PNS regions. Interestingly, marked axonal protrusions were observed in some of the nodal segments, which often contained abnormally enlarged vesicles, like degenerated mitochondria. Moreover, many transversely cut ends of microvilli surrounded the mutant nodes, suggesting that alignments of the microvilli were disordered. The mutant PNS showed mild elongation of nodal Na(+) channel clusters. Even though Caspr and NF155 were completely absent in half of the paranodes, short clusters of these molecules remained in the rest of the paranodal regions. Ultrastructural analysis indicated the presence of transverse bands in some paranodal regions and detachment of the outermost several loops. Furthermore, the numbers of incisures were remarkably increased in the mutant internode. Therefore, these results indicate that sulfatide may play an important role in the PNS, especially in the regions where myelin-axon interactions occur.
Abstract: Axonal regeneration after crush injury of the sciatic nerve has been intensely studied for the elucidation of molecular and cellular mechanisms. Neurite extension factor1 (Nrsn1) is a unique membranous protein that has a microtubule-binding domain and is specifically expressed in neurons. Our studies have shown that Nrsn1 is localized particularly in actively extending neurites, thus playing a role in membrane transport to the growing distal ends of extending neurites. To elucidate the possible role of Nrsn1 during peripheral axonal regeneration, we examined the expression of Nrsn1 mRNA by in situ hybridization and Nrsn1 localization by immunocytochemistry, using a mouse model. The results revealed that during the early phase of axonal regeneration of motor nerves, Nrsn1 mRNA is upregulated in the injured motor neuron. Nrsn1 is localized in the cell bodies of motor neurons and at the growing distal ends of regenerating axons. These results indicate that Nrsn1 plays an active role in axonal regeneration as well as in embryonic development.
Abstract: Electrophilic neurite outgrowth-promoting prostaglandin (NEPP) compounds protect neurons from oxidative insults. At least part of the neuroprotective action of NEPPs lies in induction of hemeoxygenase-1 (HO-1), which, along with other phase II enzymes, serve as a defense system against oxidative stress. Here, we found that, by using fluorescent tags and immunoprecipitation assays, NEPPs are taken up preferentially into neurons and bind in a thiol-dependent manner to Keap1, a negative regulator of the transcription factor Nrf2. By binding to Keap1, NEPPs prevent Keap1-mediated inactivation of Nrf2 and, thus, enhance Nrf2 translocation into the nucleus of cultured neuronal cells. In turn, Nrf2 binds to antioxidant/electrophile-responsive elements of the HO-1 promoter to induce HO-1 expression. Consistent with this notion, NEPP induction of an HO-1 reporter construct is prevented if the antioxidant-responsive elements are mutated. We show that NEPPs are neuroprotective both in vitro from glutamate-related excitotoxicity and in vivo in a model of cerebral ischemia/reperfusion injury (stroke). Our results suggest that NEPPs prevent excitotoxicity by activating the Keap1/Nrf2/HO-1 pathway. Because NEPPs accumulate preferentially in neurons, they may provide a category of neuroprotective compounds, distinct from other electrophilic compounds such as tert-butylhydroquinone, which activates the antioxidant-responsive element in astrocytes. NEPPs thus represent a therapeutic approach for stroke and neurodegenerative disorders.
Abstract: PURPOSE: To investigate age-related changes in the locus ceruleus (LC) in healthy subjects using neuromelanin magnetic resonance (MR) imaging at 3 Tesla. METHODS: We examined 64 healthy volunteers (aged 23 to 80 years) using neuromelanin-sensitive T1-weighted images and measured the contrast of areas of high signal intensity corresponding to the LC. RESULTS: A pair of punctate areas of high signal intensity that represented neuromelanin within the noradrenergic neurons of the LC was easily recognized in all subjects. The contrast ratio of the LC to the adjacent pontine tegmentum increased to the age of 40 to 59 years and gradually and significantly decreased in elderly subjects. This correlates well with pathologically proven age-related changes in neuromelanin content within the LC. CONCLUSION: Age-related variance should be considered when determining the existence of abnormalities in the LC.
Abstract: We carried out an investigation to identify neuromelanin-containing noradrenergic and dopaminergic neurons in the locus ceruleus and substantia nigra pars compacta of healthy volunteers and patients with Parkinson's disease using a newly developed magnetic resonance imaging technique that can demonstrate neuromelanin-related contrast. The high-resolution neuromelanin images obtained by a 3-T scanner revealed high signal areas in the brain stem and these corresponded well with the location of the locus ceruleus and substantia nigra pars compacta in gross specimens. In Parkinson's disease patients, the signal intensity in the locus ceruleus and substantia nigra pars compacta was greatly reduced, suggesting depletion of neuromelanin-containing neurons. We conclude that neuromelanin magnetic resonance imaging can be used for direct visualization of the locus ceruleus and substantia nigra pars compacta, and may help in detecting pathological changes in Parkinson's disease and related disorders.
Abstract: We examined each step of the protocol for ultracryotomy for central nervous system tissue in order to define and overcome some of the methodological difficulties. The following three steps emerged as critical for the method's success: (1) pretreatment of grids to render them hydrophilic immediately before use; (2) careful collection of ultrathin cryosections during ultracryotomy; (3) removal of the appropriate amount of excess poly(vinyl alcohol)-uranyl acetate (PVA-UA) prior to drying after staining with PVA-UA. By taking account of the three critical steps described above, we succeeded in obtaining ultrathin cryosections, including serial sections, with excellent preservation of ultrastructure, as well as semithin cryosections which are useful for evaluating the quality of the samples and for selecting areas of interest for ultrastructural analysis. Cytoplasmic organelles in neurons and glial cells, and the fine structure of synapses and myelinated fibers were well preserved. The localization of gold particles after immunostaining for astrocytic glutamate transporter (GLAST), metabotropic glutamate receptor 1 (mGluR1) and neurofilament protein was consistent with previous reports and ultrastructure was well-preserved in all cases. These findings should be helpful to researchers wishing to carry out ultrastructural and immunogold analyses of cryosections of nervous tissue.
Abstract: Low temperature scanning electron microscopy of frozen-fractured specimens under cryo-protecting, non-dehydrating, and non-etching "wet" conditions, that is, direct cryo-SEM, was followed by transmission electron microscopy (TEM) with the same neural tissue specimens. In comparison to replica TEM, direct cryo-SEM can obtain images with a smooth gradation of contrast. The major advantage of direct cryo-SEM combined with TEM was that time was saved in SEM preparation. It had a high potentiality at a wide-range survey of multi-dimensional specimen structures with less-artifacts. Because the specimens were prepared as quickly as possible under "wet" conditions, the target structures could be examined under lower through higher magnifications. In the present study, neuronal and glial elements, such as plasma membranes and cell organelles that include the synaptic vesicles, were localized on the fractured surface. In subsequent TEM examination, it was confirmed that the underlying internal structures could be further characterized from cytological as well as molecular biological aspects. In addition, direct cryo-SEM distinctively demonstrated small intra-membrane particles (ca. 10 nm in diameter). However, due to electron lucency, they could not be confirmed in the re-processed TEM specimens. Applying the present protocol, stereological and internal architectural examinations of the neural tissues have been simultaneously conducted at ultra-fine levels.
Abstract: Mice aged 1, 4 or 8 weeks were inoculated with haemagglutinating encephalomyelitis virus (HEV), strain 67N, by the intracerebral (i.c.), intranasal (i.n.), intraperitoneal (i.p.), subcutaneous (s.c.), intravenous (i.v.) or oral route, with different doses. In 1-week-old mice, mortality and mean time to death were mostly the same regardless of the inoculation route, except for the oral route, which appeared to be the least effective. The virus killed 4-week-old mice readily by all routes of inoculation except the oral, and 8-week-old mice by i.c., i.n. or s.c. inoculation. In descending order of efficacy, the routes of HEV infection were: i.c., i.n., s.c., i.p., i.v. and oral. To follow the spread of HEV from peripheral nerves to the central nervous system (CNS), the virus was inoculated subcutaneously into the right hind leg of 4-week-old mice. The virus was first detected in the spinal cord on day 2, and in the brain on day 3. The brain titres became higher than those of the spinal cord, reaching a maximum of 10(7)PFU/0.2 g when the animals were showing CNS signs. Viral antigen was first detected immunohistochemically in the lumbar spinal cord and the dorsal root ganglion ipsilateral to the inoculated leg; it was detected later in the pyramidal cells of the hippocampus and cerebral cortex, and in the Purkinje cells of the cerebellum but not in the ependymal cells, choroid plexus cells or other glial cells. The infected neurons showed no cytopathological changes.
Abstract: We investigated hippocampal substructure in the rat, cat, dog, and human by means of magnetic resonance imaging to elucidate phylogenetic differences in longitudinal organization. Multidirectional high-resolution images obtained with a 3 T scanner revealed that the dorsal part of the hippocampus was well developed in the rat, cat, and dog brain, and was homologous to the hippocampal tail, a poorly-developed posterior part, in the human. We conclude that the dorsal hippocampus of laboratory animals corresponds to the hippocampal tail in the human brain, which is considered to be hypoplastic and of less importance clinically than more anterior regions. These data may help in understanding phylogenetic, and in correlating results from animal experiments with clinical findings on the functions and pathologies of the human hippocampus.
Abstract: Basic studies were carried out to apply frozen allogeneic nerve grafts in dogs after wide-ranging defects of the brachial plexus due to surgical resection of tumor. In this study, morphological variations in branching patterns of the brachial plexus were examined in ten beagle dogs, to evaluate whether the brachial plexus might represent a useful source of allogeneic nerve grafts. Spatial relationships between the axillary lymph node, which had the possibility of carcinomatous metastasis, and the musculocutaneous (MC) nerve, which was important for the function of the forelimbs, were also investigated. In all ten cases examined, the brachial plexus received ventral roots from the fifth cervical nerve to the first thoracic nerve. No significant variation in the branching pattern was found in any nerve except the phrenic, MC and dorsal thoracic nerves. Four communicating branches were observed and had some morphological variations which might be negligible for nerve grafting. Considering previous physiological and anatomical reports, the most important nerve to be reunited in graft operations for functional recovery is the radial nerve. The MC nerve and median or ulnar nerve should also be considered as possibilities for reuniting. Distances between the axillary lymph nodes and the MC nerve ranged from 11.2 mm to 21 mm (mean +/- SD: 16.1 +/- 2.3 mm). In conclusion, it was suggested that morphological variations in the brachial plexus were technically acceptable to apply allogeneic nerve grafts at least in beagle dogs.
Abstract: Poliovirus (PV), when injected intramuscularly into the calf, is incorporated into the sciatic nerve and causes an initial paralysis of the inoculated limb in transgenic mice carrying the human PV receptor (hPVR/CD155) gene. Here, we demonstrated by using an immunoelectron microscope that PV particles exist on vesicle structures in nerve terminals of neuromuscular junctions. We also demonstrated in glutathione S-transferase pull-down experiments that the dynein light chain, Tctex-1, interacts directly with the cytoplasmic domain of hPVR. In the axons of differentiated rat PC12 cells transfected with expression vectors for hPVRs, vesicles composed of PV and hPVR alpha, as well as a mutant hPVR alpha (hPVRM alpha) that had a reduced ability to bind Tctex-1, colocalized with Tctex-1. However, vesicles containing PV, dextran, and hPVR alpha had only retrograde motion, while those containing PV, dextran, and hPVRM alpha had anterograde or retrograde motion. Topical application of the antimicrotubule agent vinblastine to the sciatic nerve reduced the amount of virus transported from the calf to the spinal cord. These results suggest that direct efficient interaction between the cytoplasmic domain and Tctex-1 is essential for the efficient retrograde transport of PV-containing vesicles along microtubules in vivo.
Abstract: Introduction of clinical high-field MRI has raised interest in the use of clinical imaging--the efficacy of which has not yet been fully established--in daily practice. A high signal-to-noise ratio and profound susceptibility effects can improve the spatial resolution and image contrast of clinical imaging, whereas the heating effects of the radio frequency tend to prolong acquisition time. As well, inhomogeneities in the static or local magnetic fields can have a negative effect on image quality. The T(1) prolongation may affect T(1) contrast yet improve the enhancement effect of gadolinium chelate and the inflow effect of MR angiography. High-contrast imaging, such as the short inversion-time inversion recovery technique, can provide excellent intra- and extracerebral contrast comparable to microscopic or macroscopic specimens. High-field systems can also be applied to microscopic imaging. High-field MRI is expected to have an increased clinical impact in the near future. Technological advances tailored to high-field systems, as well as the accumulation of scientific evidence, will be necessary to establish its predominance over conventional MRI.
Abstract: The postsynaptic site of the excitatory synapse, which is composed of the postsynaptic density (PSD) attached to the postsynaptic membrane, is a center for synaptic plasticity. To reveal the molecular organization and functional regulation of the postsynaptic site, we cloned a 70 kDa protein that is concentrated in PSDs using a monoclonal antibody against the PSD. This protein, named PSD-Zip70, is highly homologous to the human FEZ1/LZTS1 gene product. PSD-Zip70 contains an N-myristoylation consensus sequence, a polybasic cluster in the N-terminal region and four leucine-zipper motifs in the C-terminal region. Light and electron microscopy showed that this protein was localized to the dendritic spines, especially in the PSD and the postsynaptic membrane. Fractionation of the synaptic plasma membrane demonstrated that PSD-Zip70 was localized to the PSD and the dendritic raft. In Madin-Darby canine kidney (MDCK) cells, exogenous PSD-Zip70 was targeted to the apical plasma membrane of microvilli, and its N-myristoylation was necessary for this targeting. In hippocampal neurons, N-myristoylation was also required for the membrane localization and the C-terminal region was critically involved in the synaptic targeting. These results suggest that PSD-Zip70 may be involved in the dynamic properties of the structure and function of the postsynaptic site.
Abstract: BACKGROUND AND PURPOSE: A reduction in the area of the substantia nigra (SN) has been shown in patients with Parkinson disease. The substantia nigra is anteroinferolateral to the red nucleus, and it is important to precisely locate its true anatomic location to accurately measure SN area. Our purpose was to determine the exact location of the substantia nigra by correlating imaging and anatomic findings. We also attempted to quantitate SN area in patients with Parkinson disease compared with that in healthy control subjects on the basis of proton density-weighted spin-echo (SE) and fast short inversion time inversion-recovery (STIR) MR imaging findings. METHODS: In four healthy volunteers, dual-echo SE and fast STIR MR images were obtained in three orthogonal planes and an oblique coronal plane. These images were correlated with anatomic specimens to determine the location of the SN. The area of the SN was also measured on oblique coronal fast STIR images obtained at a plane perpendicular to the SN in 22 patients with Parkinson disease and in 22 age- and sex-matched healthy volunteers. RESULTS: The true anatomic location of the SN, anteroinferolateral to the red nucleus, was accurately identified, not on T2-weighted images, but on proton density-weighted SE images and fast STIR images as an area of hyperintense gray matter. The hypointense area seen on T2-weighted images corresponded to the anterosuperior aspect of the SN and to the adjacent crus cerebri. No statistically significant differences were noted in the size of the SN when the oblique coronal images of patients with Parkinson disease were compared with those of the control groups. CONCLUSION: The SN is located mainly beneath the red nucleus. Its location cannot be determined on the basis of T2-weighted imaging results but rather on the basis of proton density-weighted SE or fast STIR findings. SN volume loss is not found in Parkinson disease, and this finding is compatible with that of recent pathology reports in the literature.
Abstract: Corneas of tadpole, mouse, rat, guinea pig, rabbit, cat, cattle, and human were examined by TEM and SEM in a comparative study. The differences between species were noted mainly by using TEM. Bowman's layer showed a tendency to be well developed in higher mammals. Tadpoles lack a Bowman's layer, lower mammals have a thin Bowman's layer, and higher mammals have a thick Bowman's layer. The boundary between the substantia propria and Descemet's membrane was distinct in higher mammals. On the other hand, there are no differences in thickness of the collagen fibrils that constitute Bowman's layer and those of the substantia propria. NaOH digestion was utilized for SEM preparation. SEM imaging revealed a textured appearance of the epithelial side of Bowman's layer. In Descemet's membrane, fibrous long spacing (FLS) fiber-like structures, which are arranged in parallel to the endothelium, were observed by both TEM and SEM. To our knowledge, this is the first report of SEM observations of FLS fiber-like structures on the endothelial surface of Descemet's membrane. SEM at a plane normal to the plane of the cornea showed that Descemet's membrane has a piled laminar structure. Descemet's membrane is closely associated with the collagen layer of the substantia propria. Collagen fibrils invading from the substantia propria into Descemet's membrane were observed with both TEM and SEM.
Abstract: Poliovirus receptor (hPVR/CD155) is a cell surface glycoprotein that belongs to the immunoglobulin superfamily but its natural function remains unknown. Two membrane-bound isoforms, hPVRalpha and hPVRdelta, are known to date, and they differ only in the amino acid sequence of their cytoplasmic domains. To gain an insight into the possible function of the cytoplasmic domains, we examined the localization of introduced hPVRalpha and hPVRdelta in polarized epithelial cells deficient of native hPVRs. Basolateral sorting of hPVRalpha was observed in Madine-Darby canine kidney cells expressing mu1B, but not in LLC-PK1 porcine kidney cells deficient in mu1B. Distribution of hPVRdelta, however, occurred both on the apical and basolateral plasma membranes of these two cell lines. Basolateral sorting of hPVRalpha was also seen in LLC-PK1 cells that expressed an intact exogenous mu1B, but not in the cells that expressed a mutant mu1B lacking binding ability to tyrosine-containing signals. These results indicate that mu1B is involved in the distribution of hPVRalpha to the basolateral membrane. Comparative distribution analysis of hPVRalpha using a series of mutants with truncations and substitutions in the cytoplasmic tail demonstrated that determinant for the basolateral sorting resided in the tyrosine-containing motif of the cytoplasmic tail. Furthermore, yeast two hybrid analysis strongly suggested that the tyrosine motif directly interacted with mu1B protein. Thus, basolateral sorting of hPVRalpha appears to involve the interaction with mu1B through a tyrosine motif existing in the cytoplasmic domain.
Abstract: The molecular mechanisms involved in preventing regenerating dorsal root axons from entering the spinal cord at the dorsal root entry zone (DREZ) are obscure. We used immunohistochemistry, in situ hybridization, and electron microscopy to study axonal regeneration after dorsal rhizotomy in adult rats and its relationship to cellular changes and the distribution of putative growth inhibitory molecules in this region. Astrocyte processes, ending as bulb-shaped expansions, grew up to 700 microm into the basal lamina tubes of injured roots, where regenerating axons were also present. Some of these axons approached or reached the DREZ but grew no further; others turned back toward the ganglion, suggesting the presence of repulsive cues in or near the DREZ. Tenascin-C mRNA and protein and CSPG stub immunoreactivity were strongly upregulated in the roots after rhizotomy, but were only weakly expressed in the DREZ. Tenascin-R immunoreactivity was confined to CNS tissue, and unaffected by rhizotomy. Large, rounded GFAP-negative, NG2-immunoreactive cells, a few of which were OX42 positive, were found in the DREZ following rhizotomy. Astrocyte processes projecting into the roots were tenascin-R and NG2 negative. Hence, only NG2-expressing cells and tenascin-R were appropriately situated to inhibit regeneration through the DREZ.
Abstract: Haemagglutinating encephalomyelitis virus, strain 67N, was used to inoculate 1-, 2-, 4- and 8-week-old rats by the intracerebral (i.c.), intranasal (i.n.), intraperitoneal (i.p.), subcutaneous (s.c.), intravenous (i.v.) and oral routes with graded doses. The routes of infection, in descending order of efficacy, were: i.ci.ns.ci.pi.v. and oral. Rats aged 1 and 2 weeks were generally similar in terms of mortality and mean time to death, regardless of inoculation route, except for the oral route, which had little effect. In comparison with the 1- and 2-week-old rats, the 4-week-old rats were less susceptible to the virus by all routes. Eight-week-old rats inoculated by the i.ci.n. or s.c. routes died, but all those inoculated by other routes survived. To follow the spread of virus in the central nervous system, 4-week-old rats inoculated by the i.c. route were examined. The virus was first detected in the brain on day 1 and in the spinal cord on day 2. The viral titres in both tissues reached a plateau of 10(7) plaque-forming units (PFU)/0.2 g by day 4, at which time clinical signs had developed. By immunohistochemical analysis, virus-specific antigen was found first in the pyramidal cells of the hippocampus and cerebral cortex, and later in the large-sized neurons of the pons and spinal cord. Still later (day 4) immunolabelling was found in Purkinje cells of the cerebellum, but not in the ependymal cells, choroid plexus or other glial cells. Copyright Harcourt Publishers Ltd.
Abstract: Microglia may contribute to cell death in neurodegenerative diseases. We studied the activation of microglia in affected regions of Huntington disease (HD) brain by localizing thymosin beta-4 (Tbeta4), which is increased in reactive microglia. Activated microglia appeared in the neostriatum, cortex, and globus pallidus and the adjoining white matter of the HD brain, but not in control brain. In the striatum and cortex, reactive microglia occurred in all grades of pathology, accumulated with increasing grade, and grew in density in relation to degree of neuronal loss. The predominant morphology of activated microglia differed in the striatum and cortex. Processes of reactive microglia were conspicuous in low-grade HD, suggesting an early microglia response to changes in neuropil and axons and in the grade 2 and grade 3 cortex, were aligned with the apical dendrites of pyramidal neurons. Some reactive microglia contacted pyramidal neurons with huntingtin-positive nuclear inclusions. The early and proximate association of activated microglia with degenerating neurons in the HD brain implicates a role for activated microglia in HD pathogenesis.
Abstract: Neuregulins (NRGs), a large group of structurally related signaling proteins, are likely to have important roles in the development, maintenance and repair of the nervous system and other selected tissues. We have demonstrated, by using the major form of NRG cloned from the mouse cerebellum that both the soluble form and the membrane anchored form of NRG may serve different functions in synaptogenesis. The soluble form of NRG was produced by proteolytic cleavage of the membrane anchored form of NRG. The proteolytic cleavage was promoted by protein kinase activation. The cleaved form of NRG trans-synaptically regulated the expression of the NMDA (N-methyl-D-aspartate) receptor subunit NR2C as neurally-derived factors, whereas the membrane anchored form of NRG showed a homophilic binding activity between NRGbeta1s. In adult mice the membrane anchored form of NRG was concentrated in neuro-terminals of both granule cells and pontocerebellar mossy fibers. The fact that NRG can be functionally viewed as cell recognition molecules as well as neurotrophic agents suggests new possibilities for the important class of molecules.
Abstract: The Mahoney strain of poliovirus type 1 (OM) is generally unable to cause paralysis in mice. We isolated a mouse-adapted mutant, PV1/OM-SA (SA), from the spinal cord of a mouse that had been intracerebrally inoculated with OM. SA showed mouse neurovirulence only with intraspinal inoculation, and the infected mice developed a flaccid paralysis, which was indistinguishable from that observed in poliovirus-sensitive transgenic mice inoculated with OM. SA antigens were detected in neurons of the spinal cords of the infected mice. Nucleotide (nt) sequence analysis revealed 9 nt changes on the SA genome, resulting in three amino acid (a.a.) substitutions, i.e., one each in the capsid proteins VP4 and VP1 and in the noncapsid protein 2C. To identify the key mutation site(s) for the mouse neurovirulence, virus recombinants between OM and SA were constructed by using infectious cDNA clones of these two viruses and tested for their mouse neurovirulence after inoculation via an intraspinal route. The results indicated that a mutation at nt 928 (replacement of A with G), resulting in a substitution of Met for Ile at a.a. 62 within VP4, was responsible for conferring the mouse neurovirulence phenotype of the mutant SA. The mutation in VP4 may render the virus accessible to a molecule that acts as a virus receptor and is located on the surfaces of neurons of the mouse spinal cord. This molecule appears not to be expressed in the mouse brain.
Abstract: Axonal and glial reactions to traumatic injury were compared between the caudal and rostral border of the lesion after freeze-injury to the C3 dorsal funiculus by attaching a liquid nitrogen-cooled copper probe to the dorsum of the rat spinal cord. The axonal and glial changes were examined up to 60 days postoperative by light and electron microscopy and immunohistochemistry for neurofilaments. Regenerative axonal changes and the appearance of numerous undifferentiated cells were found at the caudal border 7 days after cryoinjury. In contrast, such axonal and cellular reactions were scarce at the rostral border. Undifferentiated cells clearly manifested their phenotypes by differentiating into oligodendrocytes or astrocytes 11 days postinjury. The results indicated that glial cell reactions occurred in association with regenerative axonal changes at the proximal stump of the injured nerve fibers, suggesting that regenerating and demyelinated naked axons could be responsible for the appearance of the immature glial cells.
Abstract: Swine hemagglutinating encephalomyelitis virus (HEV) strain 67N was inoculated into the sciatic nerve or the right leg crural muscle of rats. In both cases, the virus was isolated first from the caudal half of the spinal cord on day 2 after inoculation, and from the rostral half of the spinal cord and the brain on day 3. The virus titers in the brain reached a maximum when the infected rats developed CNS symptoms on day 5. Using confocal laser scanning microscope, fluorescent positive cells were first found in the lumbar dorsal root ganglion (DRG) and spinal cord ipsilateral of the inoculated leg on day 3. Antigen positive neurons were found bilaterally in the lumbar DRG and spinal cord on day 4. On day 5 specific fluorescence was observed in the neurons of the cerebral cortex, hippocampus, brainstem and Purkinje cells in the cerebellum.
Abstract: Sciatic nerves were excised from 3 beagle dogs about 5 h after their sacrifice, treated three times by freezing and thawing, and stored in physiological saline for 3 months at -20 degrees C until used. Nerve segments 5 cm in length prepared from these stored nerves were transplanted to the common peroneal nerve in the right hindlimb of beagle dogs. Sixteen beagle dogs in total were used, in four treatment groups of two pairs each studied at 1 and 3 months. Five-hundred microliters basic fibroblast growth factor (bFGF) of two different concentrations (10 micrograms/300 microliters and 100 micrograms/300 microliters) which were impregnated in 0.5 ml gelatin hydrogels was applied around the sutured allografts. Autografting was also done in 4 beagle dogs, with no bFGF application. One month after the grafting, no regenerating nerves extended beyond the middle of the transplant in any of the allografts, except in the autografts in which a number of regenerated (myelinated) axons were present. Three months after the grafting, an abundance of myelinated axons was found at the middle of the graft: the numbers of axons per 10(4) micron 2 were 22.6 in the autografts and 10.6, 10.4 and 19.2 in the allografts treated with no bFGF, low-dose bFGF, and high-dose bFGF, respectively. Regenerating axons extended into the host nerve: the numbers of myelinated axons at the level 1.5 cm distal to the distal suture were 35.7, 0.9, 3.8, and 12.1 per 10(4) micron 2 in the above respective order. Although it was inferior in quality to the autograft, peripheral nerve regeneration was extensive in the distal nerve using freeze-thawed and bFGF-treated allografts at 3 months. Electromyography showed that the peroneus longus muscle responded to the electrical stimuli given at the site proximal to the transplant in all four groups. These data indicate that a 5-cm acellular nerve segment containing Schwann cell basal laminae can be used successfully as an allograft without any immunosuppressants and that exogenously applied bFGF can improve nerve regeneration by enhancing the growth of regenerating axons.
Abstract: Excimer laser (KrF excimer laser, 248 nm wavelength) was used to damage cellular components in the dorsal funiculus at the lumbar level (L2) of the rat spinal cord. An open lesion was not found at the irradiation site on the spinal cord. However, the cytological examination revealed that cellular components were damaged to the depth of 200-500 microm from the pial surface. The characteristic feature was that at the border of the lesion, many axons remained naked but intact after their myelin sheaths had been completely disintegrated. Such naked axons were subsequently remyelinated by mature or immature glial cells. Mature oligodendrocytes, while retaining their cytoplasmic processes connected with the myelin sheaths of unaffected axons, extended new cytoplasmic processes on nearby naked axons and made new myelin sheaths around them. In contrast, 7 days after the irradiation, numerous immature glial cells appeared in association with naked axons, and some of them were differentiated into oligodendrocytes forming thin myelin sheaths on naked axons. These findings suggest that demyelinated axons can cause the proliferation and probably dedifferentiation of the oligodendrocyte lineage. The use of lasers provides a unique experimental model of demyelination and remyelination in the central nervous system of adult mammals.
Abstract: Rat pheochromocytoma PC12 cells die when cultured in serum-free medium. Neurotrophic factors can rescue PC12 cells from cell death, and induce neuronal differentiation. To further investigate the relationship among cell death, survival, and differentiation, the bcl-2 cDNA, which is known to prevent apoptosis in various types of cells, was transfected into PC12 cells. Six monoclonal bcl-2-transfected cell lines were isolated and confirmed to express mRNA and protein product of bcl-2. The wild-type and bcl-2-transfected PC12 cells were kept to adhere to collagen-coated dishes at the initiation of serum-free experiments to avoid cellular damage due to detachment of the cells by trituration. Even under the conditions, the control PC12 cells mostly died within 24 h, when cultured in serum-free medium, whereas those expressing Bcl-2 survived even for 7 days in serum-free medium. Moreover, outgrowth of long processes in the bcl-2-transfected cells was only observed under the condition to keep the cells attached to the dishes in serum-free medium without any additive neurotrophic or growth factors. Neurofilament medium protein, which is a neuron-specific cytoskeletal component, was also expressed in the differentiated cells, suggesting that the long processes in bcl-2-transfected PC12 cells are neurites. However, neuronal differentiation of PC12 cells expressing Bcl-2 was not observed when cultured in serum-containing medium. Accordingly, survival of PC12 cells expressing Bcl-2 under the condition which cells usually die may be accompanied with neuronal differentiation.
Abstract: To demonstrate three-dimensional architecture of regenerating axons growing through basal lamina tubes in cryoinjured nerve graft, backscattered electron (BSE) imaging in a scanning electron microscope (SEM) was used to visualize immunostained axons. Regenerating axons immunostained with an antibody against the 200 kD neurofilament protein (RT97) were clearly visualized in BSE images as bright components pursuing an irregular, often spiral course within the basal lamina tubes, and commonly branching within the tubes. The morphology of these structures corresponded closely to that of putative regenerating axons in SEM preparations following application of the potassium hydroxide-collagenase digestion method. The present approach, however, is a considerable improvement on the latter, providing three-dimensional information together with the identification of regenerating axons.
Abstract: We are interested to know whether expression of a lineage-specific growth factor receptor is deterministic to lineage commitment during hematopoiesis. For this purpose, we introduced the human c-fms gene into the multipotential stem cell clone LyD9 and two myeloid progenitor clones, L-GM3 and L-G3, cells that differentiate in response to granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte (G)-CSF, respectively. Although LyD9 cells have differentiation potential to become macrophages, c-fms transfectants of LyD9 and L-GM3 cells did not differentiate in response to human macrophage (M)-CSF. However, c-fms transfectants of L-G3 cells differentiated to neutrophils in response to human M-CSF. These results indicate that the M-CSF receptor requires a specific signal transduction pathway to exert its differentiational and proliferative effects. Furthermore, the M-CSF receptor can convey a granulocyte-type differentiation signal possibly by cooperating with the G-CSF receptor signal transduction pathway. The c-fms-transfected LyD9 cells as well as the original LyD9 cells differentiated predominantly into GM-CSF- and G-CSF-responsive cells by coculturing with PA6 and ST2 stromal cells, respectively. The results indicate that differentiation lineage is not affected by premature expression of the M-CSF receptor. Instead, the stromal cell used for coculture apparently controls lineage-selective differentiation of the multi-potential stem cell line.
Abstract: To study differences in the distribution of proteoglycans and their relationships to collagen fibrils in the cornea and sclera, bovine cornea and sclera were examined histochemically using the ruthenium red (RR) staining method. RR-positive granules (30 nm in diameter) were present in association with fine filamentous materials (3 nm in diameter and 30-100 nm in length) in the interfascicular spaces of collagen bundles in both the corneal stroma and sclera. The amount of these materials was smaller in the sclera than in the cornea. The characteristic band-like arrangement of RR-positive granules connected by filamentous materials at intervals of 80-100 nm was found only in the cornea. In enzyme digestion experiments, tissue sections were treated by chondroitinase ABC, AC, and keratanase before RR staining. The RR-positive granules and their associated filamentous materials were darkly stained after chondroitinase AC or keratanase digestion, but displayed markedly lighter staining after chondroitinase ABC digestion. These results indicate that RR-positive granules and filamentous materials contain dermatan sulfate proteoglycan.
Abstract: With the use of ulnar nerves of cynomolgus monkeys, the present study examined whether basal laminae of Schwann cells can serve as conduits for regenerating axons in nerve allografts from non-human primates. A segment of ulnar nerve was transected distal to the elbow joint one week before grafting. In Group A, a distal segment of the transected nerve was transplanted, after freezing and thawing, into the ulnar nerve of another monkey, at a level that corresponded to that from which the graft was taken. In Group B (the control group), the segment of nerve was grafted in the same manner but without cryotreatment. Two weeks, five weeks, eight weeks, and five months after grafting, the graft and the host nerve were examined with light and electron microscopy. Within two weeks after grafting in Group A, after degradation of the cellular components of the Schwann cells, the basal laminae of the Schwann cells were intact in the form of tubes. Within five weeks, many regenerating axons grew out into these basal lamina tubes in the three-centimeter-long grafts and extended into the host nerve. As seen at the wrist (seven centimeters from the distal suture) five months after grafting, the axons exhibited fully mature myelination both in the graft and in the host nerve. In contrast, in Group B, in which the Schwann cells had not been disrupted by cryotreatment, cellular components and connective-tissue matrices, including basal laminae, had been degraded and had been replaced by invading cells, which filled the endoneurial spaces of the graft. Five months after grafting, axonal growth had been arrested in the graft one centimeter distal to the proximal suture. The beneficial effect in Group A appears to have been the result of the retention and preservation of intact basal laminae of Schwann cells after rapid removal of killed Schwann cells and myelin debris. Killing of Schwann cells by freezing before grafting may abolish the immune response to the Schwann cells in allografts and lead to fragmentation and disruption of myelin, which facilitates the rapid removal of myelin by macrophages.
Abstract: A predenervated sciatic nerve segment, which had been treated by repeated freezing and thawing to kill Schwann cells, was grafted to the original sciatic nerve in the rat. Three to five days later, the graft was chemically fixed and treated by KOH-collagenase digestion, a treatment which selectively removes almost all non-cellular elements including the collagen fibrils and basal laminae from the tissue, thus making it possible to observe regenerating axons by scanning electron microscopy. Debris of degraded Schwann cells and myelin sheaths remained in the form of "columns", and several thick (2-3 microns in diameter) and thin (less than 1 micron in diameter) axons ran singly or in bundles on such "cell debris columns." Thick axons have an almost straight contour, while there were various swellings at intervals along the thin axons. In most cases, the growing tips of regenerating axons were swollen as growth cones ranging from 2 microns to 5 microns in diameter. Growth cones exhibited fusiform to polygonal variations in structure and had only a few filopodial processes on the surface.
Abstract: To examine whether the 3-4-cm-long allogeneic basal lamina tubes of Schwann cells serve as conduits for regenerating axons in rabbits, allogeneic saphenous nerve, which had been predenervated and pretreated by freezing, were transplanted from Japanese White rabbits (JW) to New Zealand White rabbits (NW). Animals were killed 1, 2, 6, 8, and 14 weeks after transplantation, and the cytology at the mid-portion of the grafts was examined by electron microscopy. The distal portion of the host saphenous nerves was also examined 14 weeks after grafting. Myelin sheath debris was phagocytosed by macrophages, while the basal lamina of Schwann cells were left intact in the form of tubes. Regenerating axons were first found in such basal lamina tubes 2 weeks after grafting, and gradually increased in number. Host Schwann cells accompanied the regenerating axons behind their growing tips, separating them into individual fibers and forming thin myelin sheaths on thick axons by 6 weeks after grafting. Regenerating nerves were divided into small compartments by new perineurial cells. Newly formed blood vessels were situated outside the compartment 8 weeks after grafting. The percentage of myelinated fibers in the regenerating nerves was roughly 10% at 8 weeks and 30% at 14 weeks after grafting. The diameter of the regenerating axons, both myelinated and unmyelinated, was less than that of normal axons at all the stages examined. Numerous regenerating axons, some of which were fully myelinated, were found at the site 10 mm distal to the distal end of the graft 14 weeks after grafting.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: The role of basal laminae as conduits for regenerating axons in an allogeneic graft was examined by transplanting a 3 cm long segment of the sciatic nerve from the Brown Norway to the Fischer 344 strain of rat. These strains are not histocompatible with each other. In order to compare the nerve regeneration in variously treated grafts, three different types of graft were employed: non-treated (NT), predenervated (PD), and predenervated plus freeze-treated (PDC) grafts. The cytology of nerve regeneration through these grafts was examined by electron microscopy at four, seven, 14, 30 and 60 days after grafting. In the PDC graft, in which Schwann cells were dead on grafting, basal laminae were well preserved in the form of tubes after Schwann cells and myelin sheaths had been removed at seven days after grafting. Regenerating axons accompanied by immature host Schwann cells grew out through such basal lamina tubes in the same fashion as observed in our previous studies. By day 14, axons extended as far as the middle of the graft. In the proximal part they were separated into individual fibres and even thinly myelinated by Schwann cells. On the other hand, in the NT and PD grafts in which Schwann cells were alive on grafting, most Schwann cells and myelin sheaths appeared to undergo autolytic degeneration by day 14, while Schwann cell basal laminae were left almost intact in the form of tubes. A few regenerating axons were seen associated with Schwann cells in the proximal portion by day seven. It is probable that host Schwann cells moved into the graft after donor cells had been degraded. Schwann cell basal laminae tended to be damaged at the site of extensive lymphoid cell infiltration. By day 30, regenerating axons had arrived at the distal end of the graft in all three types of graft: in the PDC graft thick axons were fully myelinated, whereas in the PD graft they were only occasionally myelinated and in the NT graft most axons were still surrounded by common Schwann cells. By 60 days after grafting, regenerating axons were well myelinated in the host nerve as observed 1 cm distal to the apposition site in all the three types of graft. These findings show that Schwann cell basal laminae can serve as pathways (most efficiently in the PDC graft) for regenerating axons in a 3 cm long allograft in the rat.
Abstract: In an attempt to identify regenerating axons in the central nervous system, a partial transection of the dorsal funiculus in the rat spinal cord was carried out with a pair of microdissection scissors, and a nylon thread loop was inserted into the lesion to demarcate the severed tissue. Nerve regeneration through the demarcated lesion was observed 4-20 days after the operation by light and electron microscopy. In the early stage, many naked axons appeared from the caudal part of the lesion, and some of these further extended into the demarcated space. They contained an accumulation of mitochondria, smooth-surfaced endoplasmic reticulum and vesicles in the axoplasm; this axoplasmic feature indicated that they were regenerating axons. They gradually increased in number, and took highly irregular courses exhibiting various fluctuations in diameter throughout their lengths. Immature Schwann cells as well as glial cells including oligodendrocytes and astrocytes appeared in close association with these regenerating axons. Oligodendrocytes eventually formed thin myelin sheaths. On the other hand, naked axons were present deflecting outside the thread loop; they showed no axoplasmic characteristics as described above. These axons could be regarded as uninjured ones merely undergoing demyelination due to the surgery. Thus, regenerating axons were clearly distinguished from merely demyelinated ones, and some of them were shown to grow through the traumatic lesion in the dorsal funiculus of the rat spinal cord.
Abstract: The distribution of carbonic anhydrase (CA) activity was studied by electron microscopic histochemistry in rat Pacinian corpuscles, Meissner corpuscles and Merkel cell-neurite complexes using the cobalt bicarbonate method. The distribution of CA activity in these axon terminals was compared to the activity in sciatic nerve axons. An intense enzymatic CA activity was demonstrated in axon terminals of both Pacinian and Meissner corpuscles, while a weak activity was found within the axoplasm of terminals abutting Merkel cells. Some large- and medium-sized axons in sciatic nerves exhibited an intense activity. These findings indicate that large- or medium-diameter sensory axons innervating corpuscular endings have an intense CA activity extending from their somata to their sensory terminals. Axons to Merkel-neurite complexes differ in CA activity from those innervating Meissner and Pacinian corpuscular endings.
Abstract: Cryo-treated nerves whose Schwann cells had been killed by repeated freezing and thawing were xenogenically grafted into sciatic nerves from rats (Wistar, as donor) to mice (ddy strain, as recipient) to examine whether Schwann cell basal lamina tubes of cryo-treated xenogeneic grafts were effective conduits for regenerating axons. For comparison and evaluation of the effectiveness of this technique, experiments using grafts without the cryo-treatment were carried out. Cells in cryo-treated xenografts degraded into cell debris immediately after grafting and then were phagocytized by macrophages. After the cellular components had been removed from the graft, Schwann cell basal laminae remained intact in situ, serving as conduits for the regenerating axons. The process of nerve regeneration was almost the same as that observed in cryo-treated auto- and allografts, except that the regeneration was slightly delayed in the xenogeneic graft. In contrast, an extensive cell infiltration occurred in the non-treated grafts. It appeared that the donors Schwann cells in the graft deteriorated due to immunological reactions and were finally eliminated by macrophages, leaving their basal laminae undamaged in situ. The initiation of nerve regeneration including perineurial sheath formation in non-treated grafts was, therefore, significantly delayed, but once begun, it proceeded in the same manner as in the cryo-treated grafts. These findings strongly indicate that Schwann cell basal laminae can serve as effective pathways for regenerating axons even in the xenograft. Moreover, cryo-treated xenogeneic grafts are more desirable than non-treated ones, since dead Schwann cells in the former can be removed in the early period (4-14 days) from the graft without causing any immunological reaction, thus resulting in the facilitation of nerve regeneration.
Abstract: The purpose of this study was to examine whether the basal laminae of Schwann cells in allografts could survive immunological rejection and serve as a conduit for regenerating nerves, as in the case of autogenic nerve grafts. Allografts of nerves were carried out using sciatic nerves of mice after the grafts had been repetitively frozen to kill their Schwann cells. Two mouse strains, C57BL/6N and C3H/HeN, were used, as they are known to differ in major histocompatibility complex. The mid-portion of the grafted nerve segments was examined by electron microscopy. In addition, the toe pad skin and lumbrical muscles were examined for determining whether regenerating nerves reinnervate sensory end organs and motor endplates. The process of nerve regeneration in the allograft was the same as that seen in the autograft. Cells in the graft disintegrated into cell debris and were phagocytized by macrophages, whereas the basal laminae of Schwann cells were not removed by macrophages, remaining in the form of tubes or scaffolds. Regenerating nerve fibers grew out through such basal lamina scaffolds, keeping in contact with the inner surface. Digital sensory corpuscles and motor endplates of the operated side were well reinnervated. The results indicate that the basal laminae of Schwann cells of the allograft may survive and serve as a conduit for regenerating axons in the same way as in the case of an autograft.
Abstract: The presence of macrophages in the outer bulb region of mouse, monkey and human Pacinian corpuscles was demonstrated by light and electron microscopy. In the normal, nontreated, Pacinian corpuscles, a few particular cells were located in the spaces between lamellae of the outer bulb. These cells contained numerous vesicles and vacuoles, and various cytoplasmic processes. When horseradish peroxidase (HRP) was injected locally or systemically, many HRP-positive cells, which were considered to be similar to the particular cells described above, were found in the outer bulb region of the corpuscles. Electron microscopy revealed that these cells contained HRP in vesicles and vacuoles, suggesting that they were macrophages vigorously taking up exogenous HRP. Macrophages in the Pacinian corpuscles are considered to work as scavengers to keep the inner environment of the corpuscles clear and constant with regard to its macromolecular content.
Abstract: Basal laminae were separated from Schwann cells of mouse sciatic nerves by sonification, and the distributions of lectin-binding sites were demonstrated by electron microscopy using ferritin-conjugated lectins. Only three out of the 11 lectins examined were bound to the basal laminae of Schwann cells: they were Ricinus communis agglutinin-I (RCA-I), Canavalia ensiformis agglutinin (ConA) and Triticum vulgaris agglutinin (wheat germ agglutinin, WGA). It was notable that WGA was bound more densely to the cellular side than to the interstitial side, whereas in the case of RCA-I and ConA there were no differences in the binding density on the two sides of the basal lamina. These results indicate that there are sugar residues such as beta-D-galactose, alpha-D-mannose, alpha-D-glucose and beta(1-4) linked N-acetyl-D-glucosamine in the Schwann cell basal laminae. The first three sugar residues are almost equally densely distributed on the cellular and interstitial sides of the basal laminae, whereas beta(1-4) linked N-acetyl-D-glucosamine is more densely distributed on the cellular than on the interstitial side. This result suggests that the basal lamina has a polarity in chemical composition between the cellular and interstitial sides. These findings are discussed in the context of the preferential attachment of regenerating axons to the cellular side of the Schwann cell basal laminae.
Abstract: The localization of laminin and fibronectin was examined on the basal laminae of Schwann cells. Basal laminae from sciatic nerves were isolated by sonication, and the localization of laminin and fibronectin on such isolated basal laminae was studied by immunoferritin histochemistry. Laminin was localized mainly on the cellular side (i.e. the side originally facing the Schwann cell plasma membrane) of the basal laminae. On the other hand, fibronectin was found to be present as aggregates only on the interstitial side (i.e., the side originally facing the endoneurial connective tissue) of the basal laminae. Thus, the locations of laminin and fibronectin were distinctly different. It is presumed that laminin might be involved in the attachment of axons and Schwann cells to the basal laminae, while fibronectin mediates the adhesion of the basal laminae to connective tissue elements, including the collagen fibrils. These findings are discussed from a standpoint of nerve regeneration through the basal laminae scaffolds of Schwann cells.
Abstract: Nerve segments approximately 7 mm long were excised from the predegenerated sciatic nerves of mice, and treated 5 times by repetitive freezing and thawing to kill the Schwann cells. Such treated nerve segments were grafted into the original places so as to be in contact with the proximal stumps. The animals were sacrificed 1, 2, 3, 5, 7 and 10 days after the grafting. The grafts were examined by electron microscopy in the middle part of the graft, i.e. 3-4 mm distal to the proximal end and/or near the proximal and distal ends of the graft. In other instances, the predegenerated nerve segments were minced with a razor blade after repetitive freezing and thawing. Such minced nerves were placed in contact with the proximal stumps of the same nerves. The animals were sacrificed 10 days after the grafting. Within 1-2 days after grafting, the dead Schwann cells had disintegrated into fragments. They were then gradually phagocytosed by macrophages. The basal laminae of Schwann cells, which were not attacked by macrophages, remained as empty tubes (basal lamina scaffolds). In the grafts we examined, no Schwann cells survived the freezing and thawing process. The regenerating axons always grew out through such basal lamina scaffolds, being in contact with the inner surface of the basal lamina (i.e. the side originally facing the Schwann cell plasma membrane). No axons were found outside of the scaffolds. One to two days after grafting, the regenerating axons were not associated with Schwann cells, but after 5-7 days they were accompanied by Schwann cells which were presumed to be migrating along axons from the proximal stumps. Ten days after grafting, proliferating Schwann cells observed in the middle part of the grafts had begun to sort out axons. In the grafts of minced nerves, the fragmented basal laminae of the Schwann cells re-arranged themselves into thicker strands or small aggregations of basal laminae. The regenerating axons, without exception, attached to one side of such modified basal laminae. Collagen fibrils were in contact with the other side, indicating that these modified basal laminae had the same polarity in terms of cell attachment as seen in the ordinary basal laminae of the scaffolds.(ABSTRACT TRUNCATED AT 400 WORDS)
Abstract: Basal laminae (BL) were separated from Schwann cells of rat sciatic nerves by means of weak sonication, and the anionic sites of the BL were demonstrated by using cationized ferritin (CF) or ruthenium red (RR). CF particles were deposited in clusters at intervals of 100-150 nm on the interstitial side of the BL facing the connective tissue, while the cellular side facing the Schwann cell plasmalemma showed only an occasional deposition of CF particles. RR-positive sites were found only on the interstitial side with a pattern of distribution comparable to that of CF-binding sites. These results indicate that the patterns of anionic site distribution are different between the inner and outer surfaces of the Schwann cell BL.
Abstract: To distinguish axons from Schwann cell processes in the denervated (Büngner's bands) and reinnervated peripheral nerves, the nearest-neighbor distance of intermediate filaments (NND) was measured in axons and Schwann cells from denervated and subsequent regenerating peripheral nerves. It was revealed that the NND was much larger in regenerating axons (41.9 +/- 14.1 nm) than in Schwann cell processes (23.1 +/- 7.1 nm in regeneration and 19.7 +/- 5.8 nm in denervation). In addition, the NND was also measured in the normal adult and developing peripheral nerves, and it became clear that in all cases the NND in axons (29.0-41.9 nm) was larger than in Schwann cells (19.7-23.1 nm). Thus, it can be generally considered that the NND is larger in axons than in Schwann cells. This fact can be used for the distinction between axons and Schwann cell processes, when the latter have a profile similar to that of the former as in Büngner's bands and in the regenerating nerves.
