Cellular and extracellular components at nodes of Ranvier in rat white matter

Rat CNS nodes of Ranvier were investigated by electron microscopy and immunohistochemistry. Nodes along thin callosal axons possess tiny node gaps containing few or no astrocytic processes. Nodes along thick spinal axons exhibit spatious node gaps containing relatively few irregularly arranged astrocytic processes. Antibodies against HNK-1, chondroitin sulfate, tenascin or NSP-4 do not label small nodes but stain large nodes. We conclude that rat CNS fibers do not exhibit a strict relation between nodal complexity and fiber size comparable to that found in rat PNS fibers.     

Features associated with paranodal demyelination at a specialized site in the non-pathological nervous system

Experimental demyelination in the CNS and PNS have been shown in some cases to exhibit a paranodal distribution. The electric organ of the gymnotid Sternarchus is composed of specialized axons which generate external electric fields. The structure of the nodes of Ranvier changes characteristically along the course of these specialized non-pathological axons. The nodes of Ranvier in two locations along the fibers are markedly enlarged. At the enlarged nodes, but not at normal nodes from the same fibers, the paranodal myelin exhibits morphological features associated with paranodal demyelination. These features include termination of the innermost myelin lamellae at distances of up to 200 μm from the nodal gap. The results indicate that these morphological findings are not necessarily associated with pathological demyelination, and suggest that remodeling of the myelin sheath, including programmed demyelination, may play a role in the development of certain specialized neural systems.     

Ultrastructural and cytochemical observations in a case of dominantly inherited hypertrophic (Charcot-Marie-Tooth) neuropathy

Ultrastructural and cytochemical studies were carried out on the sural nerve of a 6 1/2 year old girl with dominantly inherited hypertrophic (Charcot-Marie-Tooth) neuropathy. Electron microscopy revealed a paucity of myelinated fibers, with inappropriately thin myelin sheaths and onion-bulb formations associated with those fibers that were myelinated. In some cases the nodal axolemma was folded so as to form irregular excrescences. At other nodes, the non-myelinated gap was enlarged. Following staining with ferric ion and ferrocyanide, dense precipitates were observed on the cytoplasmic surface of the axolemma at some nodes of Ranvier, as in normal peripheral axons. At other nodes, staining was attenuated or absent. The latter result is similar to our findings in the dy/dy dystrophic mouse. These results are consistent with the hypothesis that, in dominantly inherited hypertrophic neuropathy, there are abnormalities of structure of the axolemma, in addition to an abnormality of the myelin sheath.     

Cation binding at the node of Ranvier in biopsied peripheral nerves of patients with Charcot-Marie-Tooth disease type 1A and hereditary neuropathy with liability to pressure palsies

The node of Ranvier in myelinated fibers is known to have an affinity to bind cations. Demyelination and remyelination due to abnormal expression of a myelin protein may affect cation binding or vice versa under pathological conditions. To study the cation binding at the node of Ranvier in inherited demyelinating neuropathies associated with over- and under-expression of the peripheral myelin protein 22 (PMP-22), the reaction with ferric ion and ferrocyanide was used to visualize the cation binding sites in biopsied nerves of four patiens with Charcot-Marie-Tooth disease type 1A (CMT1A) and two patients with hereditary neuropathy with liability to pressure palsies (HNPP), and the results were compared with those of four patients having acquired neuropathies with normal PMP-22 expression. In CMT1A, nodal widening or paranodal demyelination was associated with dense precipitates focally on both sides of the widened node. Although fainter precipitates were present at the node between remyelinated internodes, the percentage of nodes exhibiting the reaction product between normal and remyelinated internodes was not statistically different from that between normal internodes in CMT1A. In acquired neuropathies, on the other hand, the difference was significant between the two (P < 0.05), with reduction between normal and remyelinated internodes. At the nodes neighboring demylinated internodes, the percentage of nodes exhibiting the reaction product was reduced significantly in both CMT1A and acquired neuropathies, but to a lesser degree in CMT1A. Precipitates were clearly seen at the nodes neighboring a tomaculum in HNPP. The results suggest that preserved cation binding at the node may allow nerves to keep the electrical excitability in CMT1A and HNPP where myelin remodeling takes place at high frequency. Received: 6 June 1995 / Revised: 28 September 1995 / Revised, accepted: 29 November 1995     

Peroxidase activity at CNS nodes of Ranvier and in initial axon segments of lumbosacral alpha-motoneurons after intramuscular administration of horseradish peroxidase

The occurrence of peroxidase activity in central (CNS) and peripheral nervous system (PNS) parts of alpha-motor axons was studied by light and electron microscopy in adult cats after injection of horseradish peroxidase (HRP) into the medial gastrocnemius muscle. The intrafunicular parts of the axons were virtually free of HRP-positive bodies except at a few nodes of Ranvier. Most of these nodes were weakly HRP-positive and contained, irrespective of a survival time between 25 and 48 h, only a few HRP-positive bodies randomly scattered in the nodal axoplasm. In contrast to this and as described elsewhere (J. Neurocytol., 15 [1986] 253-260), the nodal regions of alpha-motor axons at the level of the ventral root showed strong and characteristic accumulations of HRP-activity. The initial axon segments and adjoining axonal parts contained many HRP-positive bodies. We conclude that the CNS and the PNS parts of an alpha-motor axon differ with regard to the way nodal regions interact with retrogradely transported HRP. Possible mechanisms behind this difference are discussed.     

Lysosomal activity in developing cat alpha-motor axons under normal conditions and during retrograde axonal transport of horseradish peroxidase.

The occurrence of acid phosphatase (AcPase)-positive bodies, i.e., lysosomes, in lumbosacral alpha-motor axons of kittens, 0-16 weeks of age, was analyzed by light and electron cytochemical methods under normal conditions and after intramuscular injection of horseradish peroxidase (HRP). Axonal lysosomes were rare early postnatally. In 3-week-old animals, a few AcPase-positive bodies appeared in the axoplasm at some nodes of Ranvier in the peripheral nervous system (PNS) and internodally in the intrafunicular motor axon parts within the central nervous system (CNS). From 6 weeks postnatally, a nodal concentration of AcPase-positive bodies was also noted in the CNS. The number of AcPase-positive bodies continued to increase gradually in the course of neuronal maturation. In 16-week-old animals, axonal AcPase activity was still at considerably lower levels than at adult stages. At all ages, acid hydrolase-containing organelles were most commonly found at ventral root nodes. After injection of HRP in the medial gastrocnemius muscle, accumulations of AcPase-positive bodies were seen in the axoplasm at some PNS nodes of the HRP-injected sides of kittens aged 8, 12, and 16 weeks. Incubation for demonstration of both HRP and AcPase activity showed that some organelles at HRP-transporting nodes contained both types of reaction product. The nodal AcPase activity in the intrafunicular, CNS parts of alpha-motor axons of the HRP-exposed sides did not differ from that of the contralateral, uninjected sides. In view of our previous observations in alpha-motor neurons of adult cats in which a lysosome-mediated degradation of axonally transported materials may take place at PNS nodes of Ranvier, the present study illuminates possible differences in the ability to interfere with axonal transport between developing and mature neurons. The infrequent presence of lysosomes in developing alpha-motor axons and the implied disability of their nodal regions to interfere with axonally transported constituents in a way similar to that seen in adult animals may be of significance in that trophic and chemical signals can pass unhindered between the periphery and perikaryon. However, this could also have negative consequences for the vulnerable immature neuron in that various materials retrieved at the axon terminals outside the CNS are permitted a more-or-less free access to the perikaryon.     

Schwann cell remyelination in experimental herpes simplex encephalitis at the trigeminal root entry zone

Inoculation of mice on the cornea with herpes simplex virus, type I, results in demyelination of central nervous system (CNS) axons at the trigeminal root entry zone. This study examined the process of remyelination in this area. Between eight and 15 days after corneal infection, increasing numbers of Schwann cells appeared on the CNS side of the trigeminal root entry zone, where they encircled the demyelinated CNS axons. Remyelination of CNS axons by Schwann cells began between 12 and 15 days and increased during the following weeks. Remodeling of remyelinated internodes continued during the nine weeks of observation. No infectious virus could be cultured 15 days after infection, although latent virus was recovered from the dorsal root ganglia at this time. The disruption of astrocytes on the CNS side of the trigeminal root entry zone during the early stages of infection and the proximity of Schwann cells to the CNS trigeminal root entry zone appear to be important factors affecting CNS remyelination.     

Sodium channels in normal and regenerated feline ventral spinal roots

Regenerated and remyelinated nerve fibers have shorter internodes and thus more nodes than normal mature fibers. This requires either a decrease in the number of sodium channels per node or an increase in the number of channels per fiber or both. The purpose of this investigation was to determine what happens to sodium channel number, as estimated by 3H-saxitonin (STX) binding, in regenerated fibers and to relate this to nodal number. Five adult cats underwent cryoaxotomy of ventral root levels L5, L6, L7, and S1 on the left side. After regeneration for 16-45 weeks, binding parameters were determined. On the right (control) side, binding was consistent with that in unoperated animals (b = 1.3, Bmax = 10.2 +/- 0.4 fmol/mg wet, Kd = 0.6 +/- 0.1 nM). However, the regenerated nerves showed a 3.5-fold increase in maximal binding (b = 1.3, Bmax = 36.1 +/- 0.5, Kd = 0.45 +/- 0.4). Computer-aided histologic analysis of the regenerated roots revealed a decrease in fiber size; a significant decrease in internodal length for fibers in a given size class; and a 1.35-fold increase in total fibers per root. These factors account for a 2.36-fold increase in nodes per milligram (wet). The number of STX binding sites per regenerated node was calculated to be 1.95 X 10(6) (1.31, 3.07, 95% confidence limits), whereas it was 1.26 X 10(6) (0.78, 2.02) for the control roots. The difference was not significant (p greater than 0.05). It is concluded that, in regeneration, the increase in nodal number is accompanied by an increase in sodium channels, so that the number of channels per node is normal or slightly increased. There is a marked increase in channels per fiber and an even greater increase in channels per anterior horn cell. The implications of these data for nodal reorganization in remyelination are discussed.     

Caspr reveals an aggregation of nodes and flanking node free zones at the rat trigeminal sensory root and dorsal root entry zones

The sensory root entry zone demarcates the transition from the peripheral nervous system (PNS) to the central nervous system (CNS). In this study, we describe the organization of nodes of Ranvier at the trigeminal sensory and dorsal root entry zones of the rat. Caspr immunoreactivity (IR) was used to identify the paranodal region of nodes of Ranvier, while L-MAG-IR was used to identify CNS oligodendrocytes. Immunofluorescence confocal microscopy revealed a dense aggregation of nodes precisely at the PNS to CNS transition with prominent node-depleted zones on either side, while L-MAG-IR was confined to ensheathing fibers on the central side of nodes located in this dense band and identified these as transitional nodes. Morphometric analysis of the PNS and CNS sides of the trigeminal and the PNS side of the dorsal root entry zones confirmed the presence of virtually node-free domains flanking the transitional zone. Further, the reappearance of nodes on the far side of the node-free zones strongly correlated with nodal diameter, with small nodes reappearing first. These findings suggest that the PNS/CNS transition may represent the initial site of myelination of the primary afferent axon within this area.     

5'-nucleotidase localization in the brains and spinal cords of adult normal and dysmyelinating mutant (shiverer) mice

Immunocytochemical staining with the antibody against mouse liver 5'-nucleotidase revealed 5'-nucleotidase antigenicity in myelinated fibers in the brains and in myelinated fibers and some interfascicular oligodendroglia in the spinal cords of normal adult mice. Although the 5'-nucleotidase specific activity in adult shiverer mouse CNS tissue homogenates had been shown to be normal, immunocytochemical staining with anti-mouse-5'-nucleotidase could be demonstrated in CNS tissue sections from only 2 out of 10 of the mutant animals. In tissue from these animals the staining, which was relatively faint, was localized specifically to cell-bodies, usually arranged in rows, and to material oriented parallel to nerve fibers. This pattern of immunostaining with anti-5'-nucleotidase resembled the immunostaining with anti-carbonic anhydrase but not with anti-glial-fibrillary-acidic-protein. This suggested that the rows of cells were oligodendrocytes, not astrocytes, and that the material parallel to nerve fibers might consist of oligodendrocyte processes wrapped loosely around axons. The antibody against rat 5'-nucleotidase, as distinguished from mouse, immunostained only the blood vessels in the shiverer mouse CNS, a finding similar to a previous observation in the normal mouse CNS. From these findings it was inferred that the primary loci of 5'-nucleotidase in the shiverer mouse CNS were interfascicular oligodendrocytes, their processes, and blood vessels, and in the normal mouse CNS, the myelin in some tracts, the blood vessels, and some interfascicular oligodendrocytes.     

Specific staining of the axon membrane at nodes of Ranvier with ferric ion and ferrocyanide

Ferric ion and ferrocyanide were used as stains for light and electron microscopy of peripheral nerves. In rat sciatic nerves, it was found that ferric ion preferentially binds to the cytoplasmic surface of the axon membrane at nodes of Ranvier but not at internodal regions. In myelinated axons in the electric organ of the gymnotid fish, Sternarchus albifrons, the small excitable nodes are similarly stained, but the larger inexcitable nodes are not stained by ferric ion. Staining of the inner surface of the nodal membrane appears to be related to a structural specialization of this membrane, rather than accessibility to stain. Our data thus show a chemical differentiation of the inner surface of the axon membrane between nodes and internodes in normal peripheral nerve fibers and between the inner surface of the axon membrane at active nodes, inactive nodes, and the internodes in the Sternarchus electrocyte axons.     

Basic fibroblast growth factor immunoreactivity in the peripheral motor system of the rat

We have used immunohistochemistry and electron microscopy to investigate the distribution of basic fibroblast growth factor (bFGF) in the peripheral motor system of the adult rat. In the lumbar segments of the spinal cord, bFGF immunoreactivity (bFGF-I) was seen in motor neurons and glial cells but not in axons. The neuronal immunostaining was seen as two or three intensely fluorescent spots in the nuclei with weak and more diffuse staining of the perinuclear cytoplasm. In the sciatic nerve, bFGF-I was seen in Schwann cells with strong intensity at the nodes of Ranvier. Axonal immunostaining could not be detected. At the electron microscopic level, the intense nodal immunostaining of Schwann cells was confirmed and was found to be localized to the Schwann cell membrane at the nodal gap. The intensity of staining decreased with distance from the node. In the soleus and gastrocnemius muscles, bFGF-I was seen at the motor endplates in sites corresponding to the motor nerve terminals in addition to faint staining within the muscle fibers. Electron microscopy showed that bFGF-I was localized within the nerve terminals. Histochemical localization of bFGF in the peripheral motor system is compatible with the functions ascribed for this protein in this system.     

Nodal and paranodal structure during Wallerian degeneration in frog spinal nerve

The nodal and paranodal regions of myelinated peripheral nerve fibers in frogs were examined at sequential times (1-24 days) during Wallerian degeneration. In the region up to 3 mm distal to the transection, paranodal demyelination and axoplasmic degeneration became apparent on day 4 and progressed to involve most of the nodes by day 8. The E-fracture face of the axolemma showed a patchy distribution of nodal particles and some paranodal demyelination on days 4 and 6. On day 8, nodal particles were evenly distributed at low concentration and the adjacent demyelinated paranodal regions showed a corresponding increase in particle density, suggesting redistribution of the nodal particles. The sequence of changes seen in comparable to that in Wallerian degeneration of central nervous system (CNS) fibers but progressed more rapidly in the peripheral nervous system (PNS). In addition a higher proportion of PNS fibers shows pathological changes at corresponding time periods.     

Long-term remyelination fails to reconstitute normal thickness of central myelin sheaths

The relationship between the thickness of the myelin sheath and the square area of the axon was measured in both short-term and long-term remyelinated axons following demyelination in the central nervous system, and compared with that of controls. The normal linear relationship between thickness of myelin sheath and axon size was not present in either group of remyelinated axons even though there was a trend, not statistically significant, in the long-term remyelinated animals to approach that of normal animals.     

Olfactory ensheathing cells exhibit unique migratory, phagocytic, and myelinating properties in the X-irradiated spinal cord not shared by Schwann cells

Although several studies have shown that Schwann cells (SCs) and olfactory ensheathing cells (OECs) interact differently with central nervous system (CNS) cells in vitro, all classes of adult myelin-forming cells show poor survival and migration after transplantation into normal CNS. X-irradiation of the spinal cord, however, selectively facilitates migration of oligodendrocyte progenitor cells (OPCs), but not SCs, revealing differences in in vivo migratory capabilities that are not apparent in intact tissue. To compare the in vivo migratory properties of OECs and SCs and evaluate the potential of migrating cells to participate in subsequent repair, we first transplanted freshly isolated GFP-expressing adult rat olfactory bulb-derived OECs and SCs into normal and X-irradiated spinal cords. Both OECs and SCs showed limited survival and migration in normal spinal cord at 3 weeks. However, OECs, unlike SCs, migrated extensively in both grey and white matter of the X-irradiated spinal cord, and exhibited a phagocytic phenotype with OX-42 staining on their processes. If a X-irradiated and OEC transplanted spinal cord was then subjected to a focal demyelinating lesion 3 weeks after transplantation, OECs moved into the delayed demyelinated lesion and remyelinated host axons with a peripheral-like pattern of myelin. These results revealed a clear difference between the migratory properties of OECs and SCs in the X-irradiated spinal cord and demonstrated that engrafted OECs can participate in repair of subsequent lesions.     

Early onset of degenerative changes at nodes of Ranvier in alpha-motor axons of Cntf null (-/-) mutant mice

The nodes of Ranvier are sites of specific interaction between Schwann cells and axons. Besides their crucial role in transmission of action potentials, the nodes of Ranvier and in particular the paranodal axon-Schwann cell networks (ASNs) are thought to function as local centers in large motor axons for removal, degradation, and disposal of organelles. In order to test whether ciliary neurotrophic factor (CNTF), which is present at high levels in the Schwann cell cytoplasm, is involved in the maintenance of these structures, we have examined lumbar ventral root nerve fibers of alpha-motor neurons by electron microscopy in 3- and 9-month-old Cntf null ((-/-)) mutant mice. Nerve fibers and nodes of Ranvier in 3-month-old Cntf(-/-) mutants appeared morphologically normal, except that ASNs were more voluminous in the mutants than in wild-type control animals at this age. In 9-month-old Cntf(-/-) animals, morphological changes, such as reduction in nerve fiber and axon diameter, myelin sheath disruption, and loss of ASNs at nodes of Ranvier, were observed. These findings suggest that endogenous CNTF, in addition to its role in promoting motor neuron survival and regeneration, is needed for long-term maintenance of alpha-motor nerve fibers. The premature loss of paranodal ASNs in animals lacking CNTF, which seems to be a defect related to a disturbed interaction in the nodal region between the axon and its myelinating Schwann cells, could impede the maintenance of a normal milieu in the motor axon, preceding more general neuronal damage.     

Conduction properties of central nerve fibers remyelinated by Schwann cells.

Demyelination of central axons arises from a number of conditions, including multiple sclerosis and spinal cord compression. The demyelination disrupts conduction and leads directly to the production of symptoms. Repair of the demyelination by peripheral myelinating cells could potentially relieve the symptoms, but the conduction properties of central axons remyelinated by Schwann cells have yet to be studied in detail. This paper examined the conduction properties of such axons. Large focal demyelinating and remyelinating lesions were induced in the dorsal columns of rats by the intraspinal injection of ethidium bromide. Recordings of compound action potentials conducted through these lesions were then made at various recovery times. Thus the changing conduction properties of the affected fibers could be correlated with the different stages of lesion development. During the early stages of demyelination there was widespread conduction block, with no evidence of appreciable conduction occurring with prolonged latency or refractory period of transmission (RPT). However, with the onset of remyelination by Schwann cells, conduction was restored in many axons, and most, if not all, of the affected axons eventually showed successful conduction through the lesion. Initially the conduction was characterized by very prolonged latency, long RPT, and an inability to conduct fast trains of impulses. These deficits became less prominent as remyelination progressed. In chronically remyelinated axons the RPT was restored to within normal limits, although some deficit in both conduction velocity and the ability to conduct trains of impulses persisted. Since these deficits were not severe we conclude that remyelination of central demyelinated axons by Schwann cells should be effective in promoting the restoration of normal function.     

Aberrant remyelination of axons after heat injury in the dorsal funiculus of rat spinal cord

Summary We studied the course of demyelination and subsequent remyelination of nerve fibers after heat injury in the dorsal funiculus of the rat spinal cord. Four weeks after heat treatment, we observed, in addition to normally remyelinated axons, a few aberrantly remyelinated axons which had both CNS-and PNS-type myelin sheaths: the CNS-type myelin sheaths were always situated inside the PNS-type sheaths. This finding indicates that in some conditions Schwann cells can form myelin sheaths around those formed by oligodendrocytes.     

Immunosuppression promotes CNS remyelination in chronic virus-induced demyelinating disease.

Immunosuppression using cyclophosphamide or anti-T cell monoclonal antibodies (mAbs) directed at CD4 or CD8 promoted remyelination of CNS axons in the spinal cords of mice infected chronically with Theiler's virus. Treatment with a mAb directed at class II major histocompatibility gene products did not increase the extent of CNS remyelination. Following immunosuppressive treatment, quantitative morphometry revealed a five- to sevenfold increase in new myelin synthesis. Proliferating nervous system cells were identified at the edges of remyelinated lesions by their incorporation of [3H]thymidine. CNS remyelination occurred in mice depleted of selected subsets of T lymphocytes despite the local persistence of viral antigen. These findings indicate that CNS remyelination occurs as a normal consequence of primary myelin injury, but factors associated with immune T cells somehow impair remyelination. Interference with the function of immune T cells enhances CNS remyelination by oligodendrocytes. Similar depletion of immune T cells may allow for enhanced remyelination in the CNS of patients with chronic multiple sclerosis.     

Disruption of nodal architecture in skin biopsies of patients with demyelinating neuropathies

Cutaneous nerves represent the most distal part of the sensory nervous system. We took advantage of the good discernibility of longitudinal myelinated fibers in skin biopsies to analyze the distribution of nodal and paranodal proteins in neuropathies and to assess nodal disorganization as a diagnostic marker of demyelinating neuropathy (NP). We analyzed myelinated nerve fibers in skin biopsies from the finger and the proximal leg of 52 prospectively recruited patients with different peripheral neuropathies and 17 controls. We performed immunohistochemical double labeling with anti‐MBP, anti‐PGP9.5, anti‐caspr, anti‐pan‐neurofascin, and anti‐pan‐sodium‐channel. Three potential features of demyelinating NP could be established: elongated nodes of Ranvier and dispersion of contactin‐associated protein (caspr) staining were found more often in demyelinating than in axonal neuropathies (p < 0.05) and were not detectable in normal controls. Broadening of neurofascin staining was detectable more often in demyelinating neuropathies compared with normal controls (p < 0.05). Our data suggest that pathological changes of nodal architecture can be visualized in skin biopsies and that the detection of elongated nodes of Ranvier and alterations in the distribution of paranodal proteins may be useful in the diagnostic assessment of peripheral NP.