Paranodal Axolemma Research Articles

Morphological Progression of Myelin Abnormalities in IgM-Monoclonal Gammopathy of Undetermined Significance Anti-Myelin-Associated Glycoprotein Neuropathy

To characterize the morphological progression of neuropathy associated with immunoglobulin M-monoclonal gammopathy of undetermined significance with anti-myelin-associated glycoprotein antibody, we assessed histopathologic features of sural nerve specimens from 15 patients, emphasizing widely spaced myelin (WSM), demyelination, and tomaculous changes. The frequency of WSM correlated with that of demyelination and tomaculous appearance in teased-fiber preparations. In longitudinal sections at nodes of Ranvier and paranodal regions, the spaces between terminal myelin loops, particularly those adjacent to the node of Ranvier, were widened, indicating an early change before demyelination, and there was concomitant swelling of terminal myelin loops. Some conspicuously swollen terminal myelin loops were detached from the paranodal axolemma, thereby widening the nodes of Ranvier. Tomacula coexisted frequently with redundant myelin loops and WSM, particularly in the outermost layer of myelin sheaths, suggesting that loosening of the outer layers contributes to their formation. By immunofluorescence microscopy, immunoglobulin M and myelin-associated glycoprotein were colocalized in paranodal regions and Schmidt-Lanterman incisures. Confocal analysis revealed colocalization of immunoglobulin M and complement product C3d corresponding to the area of WSM. Thus, morphological changes in terminal myelin loops, formation of WSM at paranodes, and subsequent dissociation from paranodal axolemma (which may be associated with activation of the complement pathway) likely contribute to demyelination in this condition. Loosening of compact myelin seems to contribute to tomacula formation.

Contactin orchestrates assembly of the septate-like junctions at the paranode in myelinated peripheral nerve.

Rapid nerve impulse conduction depends on specialized membrane domains in myelinated nerve, the node of Ranvier, the paranode, and the myelinated internodal region. We report that GPI-linked contactin enables the formation of the paranodal septate-like axo-glial junctions in myelinated peripheral nerve. Contactin clusters at the paranodal axolemma during Schwann cell myelination. Ablation of contactin in mutant mice disrupts junctional attachment at the paranode and reduces nerve conduction velocity 3-fold. The mutation impedes intracellular transport and surface expression of Caspr and leaves NF155 on apposing paranodal myelin disengaged. The contactin mutation does not affect sodium channel clustering at the nodes of Ranvier but alters the location of the Shaker-type Kv1.1 and Kv1.2 potassium channels. Thus, contactin is a crucial part in the machinery that controls junctional attachment at the paranode and ultimately the physiology of myelinated nerve.

Insulin receptor in rat peripheral nerve: its localization and alternatively spliced isoforms.

Diabetic neuropathy accompanies both Type 1 and Type 2 diabetes, although it shows in both humans and animal models distinct differences between the two types of diabetes. Progressive paranodal degenerations occurring in Type 1, but not in Type 2, diabetes is believed to account for the more severe functional deficits in Type 1 diabetic rats. This suggests that factors other than hyperglycemia, such as insulin deficiency, may play a pathogenetic role. In this study, we investigated the immunolocalization of the insulin receptor (IR) and the expression of its two alternatively spliced isoforms in adult rat peripheral nerve. Adult male Wistar rats 6-8 months of age were examined. Both light and ultrastructural immunohistochemistry was employed for localization of IR. The antibody was a mouse monoclonal antibody raised against the beta-subunit of human IR. Reverse transcription polymerase chain reaction (RT-PCR) was used to identify the two IR isoforms in peripheral nerve and seven other organs. Localization of the mRNA message was assessed by in situ hybridization. IR was localized to paranodal terminal Schwann cell loops and microvilli and to the paranodal axolemma. Furthermore, IR immunoreactivity was also present in Schmidt-Lantermann incisures. Endoneurial vessels showed IR localization on plasma membranes and in endocytotic vesicles of endothelial cells and pericytes. A high intensity of immunostained IR was found in close proximity to interendothelial tight junctions. Peripheral nerve showed, like the brain, predominantly the high affinity IR lacking exon 11. The mRNA message was localized to Schwann cells, endothelial cells and pericytes. Peripheral nerve expresses predominantly the high affinity IR, which is localized to strategic structures associated with the blood-nerve barrier and the paranodal ion-channel barrier.

Axo-glial interactions regulate the localization of axonal paranodal proteins.

Mice incapable of synthesizing the abundant galactolipids of myelin exhibit disrupted paranodal axo-glial interactions in the central and peripheral nervous systems. Using these mutants, we have analyzed the role that axo-glial interactions play in the establishment of axonal protein distribution in the region of the node of Ranvier. Whereas the clustering of the nodal proteins, sodium channels, ankyrin(G), and neurofascin was only slightly affected, the distribution of potassium channels and paranodin, proteins that are normally concentrated in the regions juxtaposed to the node, was dramatically altered. The potassium channels, which are normally concentrated in the paranode/juxtaparanode, were not restricted to this region but were detected throughout the internode in the galactolipid-defi- cient mice. Paranodin/contactin-associated protein (Caspr), a paranodal protein that is a potential neuronal mediator of axon-myelin binding, was not concentrated in the paranodal regions but was diffusely distributed along the internodal regions. Collectively, these findings suggest that the myelin galactolipids are essential for the proper formation of axo-glial interactions and demonstrate that a disruption in these interactions results in profound abnormalities in the molecular organization of the paranodal axolemma.

Imbalances in N-CAM, SAM and polysialic acid may underlie the paranodal ion channel barrier defect in diabetic neuropathy

Breakdown of protective tissue barrier systems characterizes the chronic diabetic complications affecting the retina, and peripheral and central nerve tracts. The progressive damages to the blood–retina-, blood–nerve-, and paranodal ion channel barriers have pathophysiological consequences for the relentless progression of these complications. The continuing damage to the paranodal ion channel barrier in the spontaneously diabetic BB/W rat is associated with an increasingly irreversible nerve conduction defect, due to impaired nodal Na + currents associated with displacement of nodal Na + channels across the damaged paranodal barrier. The structural substrate for the mechanical barrier of the paranode is provided by electron-dense junctional complexes made up by a moiety of neural cell adhesive–(N-CAM), neural–glial adhesive (Ng-CAM), substrate adhesive molecules (SAMs) and polysialic acid (PSA). To further explore the mechanisms underlying the protective barrier defect in diabetic neuropathy we examined the expression and immunolocalization of these molecules in peripheral nerve. In 6-month diabetic BB/W rats, direct and indirect ELISAs revealed significantly up-regulated N-CAM ( P<0.05), tenascin (Ng-CAM) ( P<0.001) and N-cadherin (A-CAM) ( P<0.03). On the other hand, SAMs showed little change, except for PSA which showed a significantly ( P<0.03) decreased concentration in the diabetic nerve. Immunocytochemical identification of these molecules revealed no visually detectable differences between diabetic and control rats. In conclusion, these data suggest that imbalances between highly interactive molecules responsible for the adhesiveness between terminal Schwann cell loops and the paranodal axolemma may underlie the critical paranodal barrier defect in diabetic neuropathy.

An orderly development of paranodal axoglial junctions and bracelets of Nageotte in the rat sural nerve

The present study was designed to assess the normal development of the paranodal apparatus with particular emphasis on axoglial junctions (AGJs) which constitute the paranodal barrier system. The sural nerve was examined in 10- and 31-day-old rats. During the early phase of myelination AGJ attachment of terminal myelin loops to the axolemma proceeded from the node to the internode. The frequency of terminal loops with AGJ attachment increased with fiber growth. As myelination advanced internodal-most loops became almost 100% attached to the axolemma by AGJs, whereas at the same time an increasing number of nodal-most loops were unattached, suggesting a lack of AGJ formation at this site. The formation of bracelets of Nageotte increased with the progressive addition of myelin loops. They formed most frequently at the juxtanodal interface between unattached and attached loops, probably reflecting crowding of terminal loops along the unchanged length of the paranodal axolemma. The findings suggest a complex but orderly age- and fiber size-dependent maturation process of the paranode and its structural barrier system. The present data will serve as a basis for the evaluation of this anatomical region in regenerating and remyelinating fibers in various neuropathies.

An orderly development of paranodal axoglial junctions and bracelets of Nageotte in the rat sural nerve

The present study was designed to assess the normal development of the paranodal apparatus with particular emphasis on axoglial junctions (AGJs) which constitute the paranodal barrier system. The sural nerve was examined in 10- and 31-day-old rats. During the early phase of myelination AGJ attachment of terminal myelin loops to the axolemma proceeded from the node to the internode. The frequency of terminal loops with AGJ attachment increased with fiber growth. As myelination advanced internodal-most loops became almost 100% attached to the axolemma by AGJs, whereas at the same time an increasing number of nodal-most loops were unattached, suggesting a lack of AGJ formation at this site. The formation of bracelets of Nageotte increased with the progressive addition of myelin loops. They formed most frequently at the juxtanodal interface between unattached and attached loops, probably reflecting crowding of terminal loops along the unchanged length of the paranodal axolemma. The findings suggest a complex but orderly age- and fiber size-dependent maturation process of the paranode and its structural barrier system. The present data will serve as a basis for the evaluation of this anatomical region in regenerating and remyelinating fibers in various neuropathies.

Developmental changes at the node and paranode in human sural nerves: morphometric and fine-structural evaluation

Developmental alterations of paranodal fiber segments have not been investigated systematically in human nerve fibers at the light- and electron-microscopic level. We have therefore analyzed developmental changes in the fine structure of the paranode in 43 human sural nerves during the axonal growth period up to 5 years of age, and during the subsequent myelin development up to 20 years and thereafter. The nodal, internodal, and paranodal axon diameters reach their adult values at 4-5 years of age. The ratio between internodal and paranodal axon diameters remains constant at 1.8-2.0. Despite a considerable increase in myelin sheath thickness, the length of the paranodal myelin sheath attachment zone at the axon does not increase correspondingly, because of attenuation, separation from the axolemma, and piling up of myelin loops in the paranode. Separation of variable numbers of terminal myelin loops from the underlying axolemma results in the formation of bracelets of Nageotte, whereas the transverse bands of these loops disappear. The adaptation of the paranodal myelin sheath to axonal expansion during development probably occurs by uneven gliding of the paranodal myelin loops simultaneously with internodal slippage of myelin lamellae. Since mechanically stabilizing structures (tight junctions and desmosomes between adjacent paranodal myelin processes; transverse bands between myelin loops and paranodal axolemma) are unevenly arranged, especially during rapid axonal growth, paranodal axonal growth with simultaneous adaptation of the myelin sheath is probably discontinuous with time.

The influence of nodal constriction on conduction velocity in myelinated nerve fibers

Myelinated nerve fibers exhibit a complex anatomy in the nodal region which includes a marked nodal-paranodal constriction and an intricate paranodal structure where the myelin sheath is separated from the axon by a narrow periaxonal space. In this study, a recently developed computational model of the mammalian myelinated nerve fiber based on electron microscopic data was employed to examine the effect of the nodal-paranodal axonal radius and periaxonal space width on the conduction of action potentials. These findings indicate that the nodal-paranodal constriction promotes higher conduction velocities by minimizing the component of the nodal capacity contributed by the paranodal axolemma. Model prediction of optimal nodal-paranodal radii is correlated with radii determined in experimental anatomical studies.

Three-dimensional fine structure of cytoskeletal-membrane interactions at nodes of Ranvier

Cytoskeleton-membrane-extracellular matrix interactions at the node of Ranvier were examined in both central and peripheral axons by combining three different methods for tissue preparation with three different electron microscopic techniques for imaging supramolecular structure. Conventional and three-dimensional high voltage electron microscopy of thin and semithick sections of tissues stained en bloc with ferric chloride revealed the presence of transcellular structures across the nodal gap traversing the paranodal glial-axonal junction. These structures penetrate both axonal and glial membranes and are further traced to the cortical axoplasm. This observation was verified by an examination of similar regions in rapidly-frozen freeze-substituted fresh axons. The filamentous nature of these structures, their focal attachment to the external true surface of the nodal and paranodal axolemma and their association with membrane particles were visualized in deep etch rotary-shadow replicas. At the node, both extracellular gap-crossing filaments and membrane-cytoskeletal linkers in the nodal axoplasm are joined to one of the prominent membrane particles of the nodal axolemma. At the paranodal axo-glial junction, the anchoring site of these membrane-cytoskeleton linkers are found on the linear arrays of 16 nm particles. Thus, cytoplasmic filaments and extracellular filaments or bridge structures are involved in the membrane-cytoskeletal interaction at the node and paranode. Some of these membrane particles are known to play a role in ionic conductances known to occur at this site. An additional role in cell adhesion or maintenance of the membrane specialization of this functionally important site of axolemma is now indicated.

Segmental demyelination in Peripheral Nerves of Old Cats

This study of the fine structure of sciatic nerve branches in normal old cats provides evidence indicating that segmental demyelination may account, in part, for the significant decrease with age in the mean axonal conduction velocity in these hindlimb nerves. Fibers of different diameters exhibited focal abnormalities of their myelin sheath. Lipid-like droplets and granulo-vacuolar debris were present in distended portions of the inner adaxonal rim and in the outer cytoplasmic compartment of the Schwann cell. These inclusions extended into the cytoplasm of the paranodal myelin loops and clefts of Schmidt-Lantermann. There also occurred disruption of the axoglial junctions and separation of the myelin loops from the paranodal axolemma which widens the nodes of Ranvier. Complete disruption of one or more contiguous segments of the myelin sheath was produced by interlamellar splitting and ballooning along the major dense and intraperiod lines. Axonal degeneration occurred less frequently and was not present in all hindlimb nerves.

A distributed-parameter model of the myelinated nerve fiber

This paper presents a new model for the characterization of electrical activity in the nodal, paranodal and internodal regions of isolated amphibian and mammalian myelinated nerve fibers. It differs from previous models in the following ways: (1) in its ability to incorporate detailed anatomical and electrophysiological data; (2) in its approach to the myelinated nerve fiber as a multi-axial cable; and (3) in the numerical algorithm used to obtain distributed model equation solutions for potential and current. The morphometric properties are taken from detailed electron microscopic anatomical studies (Berthold & Rydmark, 1983a, Experientia 39, 964-976). The internodal axolemma is characterized as an excitable membrane and model-generated nodal and internodal membrane action potentials are presented. A system of describing equations for the equivalent network model is derived, based on the application of Kirchoff's Current Law, which take the form of multiple cross-coupled parabolic partial differential equations. An implicit numerical integration method is developed and the numerical solution implemented on a parallel processor. Non-uniform spatial step sizes are used, enabling detailed representation of the nodal region while minimizing the number of total segments necessary to represent the overall fiber. Conduction velocities of 20.2 m sec-1 at 20 degrees C for a 15 microns diameter amphibian fiber and 57.6 m sec-1 at 37 degrees C for a 17.5 microns diameter mammalian fiber are achieved, which agrees qualitatively with published experimental data at similar temperatures (Huxley & Stämpfli, 1949, J. Physiol., Lond. 108, 315-339; Rasminsky, 1973, Arch, Neurol. 28, 287-292). The simulation results demonstrate the ability of this model to produce detailed representations of the transaxonal, transmyelin and transfiber potentials and currents, as well as the longitudinal extra-axonal, periaxonal and intra-axonal currents. Also indicated is the potential contribution of the paranodal axolemma to nodal activity as well as the presence of significant longitudinal currents in the periaxonal space adjacent to the node of Ranvier.

Cytochemical localization of K+-NPPASE in the heminode of Ranvier in the rat optic nerve.

The localization of ouabain sensitive, K+-dependent p-nitrophenylphosphatase (K+-NPPase), a component of the transport adenosine triphosphatase (Na+, K+-ATPase) system, was ultracytochemically investigated in the heminode of Ranvier in the rat optic nerve. K+-NPPase activities were noted on the nodal and paranodal axolemma, and on the paranodal myelin lateral loops. In the nodal region, the axolemma, which lies above the dense cytoplasmic undercoating, showed remarkably strong K+-NPPase activity.

Role of sorbitol accumulation and myo-inositol depletion in paranodal swelling of large myelinated nerve fibers in the insulin-deficient spontaneously diabetic bio-breeding rat. Reversal by insulin replacement, an aldose reductase inhibitor, and myo-inositol.

Axo-glial dysjunction refers to the disruption of important junctional complexes that anchor terminal loops of myelin to the paranodal axolemma in diabetic human and animal peripheral nerve. Neither axo-glial dysjunction nor the preceeding acute localized paranodal swelling has been specifically attributed to discrete metabolic consequences of insulin deficiency or hyperglycemia. Two metabolic sequelae of hyperglycemia in diabetic nerve, sorbitol accumulation via aldose reductase, and (Na,K)-ATPase deficiency related to myo-inositol depletion, were explored as possible underlying causes of acute paranodal swelling in the spontaneously diabetic bio-breeding rat. 3 wk of insulin replacement, or therapy with an aldose reductase inhibitor or myo-inositol completely reversed paranodal swelling in sural nerve fibers after 3 wk of untreated insulin deficiency. These observations suggest that insulin deficiency and hyperglycemia cause reversible paranodal swelling, and ultimately poorly reversible axo-glial dysjunction, via the myo-inositol-related (Na,K)-ATPase defect rather than by the osmotic effects of sorbitol accumulation within nerve fibers.

Nodal and paranodal structural changes in frog optic nerve during early Wallerian degeneration.

Ultrastructural changes in the nodal and paranodal regions of myelinated nerve fibres of frog optic nerves were studied during early stages of Wallerian degeneration. The earliest changes seen include retraction of paranodal loops of myelin from the axolemma and disconnection of paranodal myelin loops from myelin lamellae. These paranodal changes are asymmetric around the node and may be more advanced on either the proximal or distal side. Axoplasmic changes, including segregation of microtubules from neurofilaments, disorientation of microtubules and accumulation of abnormal organelles at nodes, appear shortly. In some axons the 'undercoating' along the widened nodal surfaces becomes patchy, and blebs appear in the nodal axolemma. In freeze-fracture replicas a mixture of particle clusters and particle-free areas appears in both E- and P-faces of the nodal axolemma. Blebs remain particle free. Initially, E-face particles remain segregated to the node and are present only at much lower concentrations in the demyelinated paranodal axolemma, suggesting that they are not freely mobile at this stage. Nodal E-face particles begin to decrease on day 5 associated with an increase in particles at the adjacent demyelinated paranode, and by day 11 the particle distribution is uniformly low over the entire extent of the nodal and demyelinated paranodal axolemma. If nodal E-face particles represent sodium channels, as has been proposed, the sequence of changes in Wallerian degeneration would be compatible with a gradual redistribution of nodal sodium channels into the demyelinated paranode.

Differences in intramembranous particle distribution in the paranodal axolemma are not associated with functional differences of dorsal and ventral roots

Freeze-fracture methods were used to study intramembranous particles, which are believed to represent ectopic sodium channels in the paranodal axolemma, and their possible association with differences in action potential electrogenesis of maturing rat dorsal and ventral root fibers. Our results indicate that there is no significant association between paranodal axon membrane structure and the functional difference of dorsal and ventral root axons.

Filipin-sterol complexes at nodes of Ranvier.

Using the filipin-sterol technique, regional heterogeneity in the axonal and Schwann cell plasma membranes was investigated at the node of Ranvier and paranodes. Filipin-sterol complexes were abundant at the nodal axolemma but infrequent throughout the paranodal axolemma. The paranodal Schwann cell plasma membrane was rich in complexes which extended over the nodal Schwann cell microvilli. There were no regional differences in filipin labelling of the nodal-paranodal Schwann cell plasma membrane in relation to features such as paranodal cytoplasmic columns or mesaxonal furrows. However, the paranodes of adjacent Schwann cells were sometimes markedly different from each other in the amount of filipin labelling. The extent to which filipin labelling is indicative of cholesterol membrane content is discussed and the findings are related to current concepts of distribution, mobility and interaction of protein and lipid in biomembranes, with particular reference to the nodal axolemma.

Dependence of axolemmal differentiation on contact with glial cells in chronically demyelinated lesions of cat spinal cord

Chronically demyelinated lesions of cat dorsal columns were created by focal injection of the glial toxin ethidium bromide. Freeze-fracture studies show that the center of the lesion, which is devoid of glial cells and processes, contains axons having neither node-like nor paranodal-type membrane specializations. Near the margin of the lesion,, however, where axons are in contact with glial cells, the axolemma sometimes displays focal accumulations of E- and P-face particles resembling those at nodes of Ranvier. In cases where the adjacent cell could be identified, it had the characteristics of an astrocyte. Linear indentations of the axolemma displaying a paracrystalline pattern like that of the paranodal axolemma also occur in the marginal region. Here, the adjacent cell had the characteristics of an oligodendrocyte. These specializations may be closely associated with each other or spatially separate. Normal nodal and paranodal specializations were absent throughout the lesion at all time periods examined. These findings support the view that both the formation and the maintenance of nodal and paranodal axon membrane specialization require contact with glial cells.

Demyelination-induced plasticity in the axon membrane: An ultrastructural cytochemical study of lead neuropathy in the rat

We examined the distribution of ferric ion-ferrocyanide stain (a marker for excitable regions of myelinated fibers) in the lead-induced demyelinating neuropathy of the rat. By electron microscopy, we found that paranodal degeneration resulted in spreading of the reaction product from nodal to internodal axolemma. During repair, nodal-like stained areas formed at the contact zones between preremyelinating Schwann cells. These data suggest that the location and extent of excitable axonal regions are influenced by axoglial relationships. Additionally, some fibers displayed staining at paranodal axolemma adjacent to demyelinated segments, suggesting it might be an alternative site for impulse generation in demyelinated fibers.

Rapid alterations of the axon membrane in antibody-mediated demyelination.

Alterations of nodal and paranodal axolemma of the rat sciatic nerve were investigated in antigalactocerebroside serum-induced demyelination. A ferric ion-ferrocyanide (FeFCN) stain that appears to stain the regions with a high sodium channel density in nerve fibers was applied. When acute conduction block was initiated 20 to 180 minutes after the antiserum injection, myelin terminal loops began to be detached from the paranodal axolemma and reaction product of FeFCN stain originally localized at the nodes decreased in density and extended to the paranodal axolemma. By the time that complete conduction block was established, 5 hours after the injection, FeFCN stain was barely detectable around the nodal area. The loss of staining was associated with detachment and vesiculovacuolar degeneration of the paranodal myelin. This rapid deterioration and disappearance of normal cytochemical characteristics of the axolemma in the presence of only modest paranodal demyelination could be a morphological correlate of the loss of excitability of the axon membrane.