Axon-myelin sheath relations of oligodendrocyte unit phenotypes in the adult rat anterior medullary velum

Axon-oligodendrocyte relations of Rip-immunolabelled and dye-injected oligodendrocyte units are characterised in the adult rat anterior medullary velum (AMV). Each oligodendrocyte unit comprised the oligodendrocyte cell body, processes and the internodal myelin segments they support. Oligodendrocyte units corresponded to classically described type I/II or type III/IV unit phenotypes which respectively myelinated discrete populations of small and large diameter axons, delineated by a myelinated fire diameter of 2-4 microns (diameter of the axon plus its myelin sheath). Within units, mean fibre diameter was directly related to mean internodal length and inversely related to the number of myelin sheaths in the unit. The relationship between fibre diameter and internodal length was retained in units which myelinated axons of different diameters, indicating that axon diameter was an important determinant of the longitudinal dimensions of myelin sheaths. We also show that type III/IV units maintained a far greater volume of myelin than type I/II units. It was concluded that type I/II and III/IV oligodendrocytes represent two functionally and morphologically distinct phenotypes whose distribution densities were determined by the diameter and spatial dispersion of axons.     

Differential expression of the L- and S-isoforms of myelin associated glycoprotein (MAG) in oligodendrocyte unit phenotypes in the adult rat anterior medullary velum

We have previously demonstrated differences in the expression of carbonic anhydrase II (CAII) in oligodendrocyte units myelinating small and large diameter fibres in the anterior medullary velum (AMV) of the adult rat (each unit comprises the cell body, processes and myelin sheaths). Others have indicated that myelin composition may also vary with respect to myelin basic protein (MBP) and proteolipid protein (PLP), and the small (S)- and large (L)-isoforms of myelin associated glycoprotein (MAG). In this study, we have determined the expression of myelin proteins in oligodendrocyte unit phenotypes I–IV, which myelinate fibres ranging in diameter from 0.3–12 m diameter in the AMV, by using double immunolabelling for Rip, which labels entire units, and MBP, PLP, myelin oligodendrocyte glycoprotein (MOG), L-MAG and S-MAG. We show differences in the expression of L- and S-MAG in units which myelinate different diameter fibres: (1) type I/II units myelinating small diameter fibres had a L-MAG+/S-MAG–/CAII+ phenotype; (2) type II/III units myelinating different diameter fibres had a L-MAG+/S-MAG+/CAII+ phenotype; (3) type III/IV units myelinated large diameter fibres had a L-MAG+/S-MAG+/CAII– phenotype. All units, irrespective of fibre diameter, expressed Rip, MBP, PLP and MOG. The results indicate that type I–IV units may be variants of a single oligodendrocyte population and that phenotypic differences are determined by the diameter of fibres within the unit. The possible significance of metabolic and biochemical differences between oligodendrocytes myelinating small and large diameter axons are discussed with reference to the pathology of demyelination.     

The relationship between developing oligodendrocyte units and maturing axons during myelinogenesis in the anterior medullary velum of neonatal rats

Myelinogenesis was investigated in whole-mounted anterior medullary vela from rats aged postnatal day (P) 10–12, using double immunofluorescence labelling with Rip and anti-neurofilament 200 (NF200) antibodies, to identify oligodendrocytes and axons, respectively. A number of discrete phases of maturation of oligodendrocyte units were recognised. (1) Promyelinating oligodendrocytes co-expressed Rip and Myelin basic Protein and formed axonal associations, prior to ensheathment. (2) Transitional oligodendrocytes contained both ensheathing and non-ensheating processes. (3) Myelinating oligodendrocytes were established after a period of remodelling (in which non-ensheathing processes were lost), appearing as oligodendrocyte unit morphological phenotypes with a definitive number of incipient myelin sheaths. (4) Maturation of myelinating oligodendrocytes was defined as the establishment of internodal sheath lengths and the redistrubution of myelin basic protein from the cell somata and radial processes into the myelin sheaths only. Myelination was probably related to the maturational state of the axons, since it was initiated when the latter had attained a critical diameter of between 0.2 and 0.4 m, coincident with the expression of NF200. Oligodendrocyte differentiation and myelination of the AMV were asynchronous and multifocal, and at P10: (1) axons which were destined to be of the largest calibre in the adult AMV were already myelinated by early developing oligodendrocytes, whilst those which were destined to be the smallest calibre in the adult were unmyelinated, but ultimately became ensheathed by late developing oligoendrocytes; (2) axons were sequentially ensheathed by early developing myelinating oligodendrocytes and late developing promyelinating oligodendrocytes; (3) all axons were small calibre; (4) oligodendrocyte units exhibited polymorphism. Thus, the development of oligodendrocyte morphological phenotypes was not related solely to either the physical dimension of axon calibre at the time of ensheathment, nor oligodendrocyte birth dates.     

Nodes of Ranvier and myelin sheath dimensions along exceptionally thin myelinated vertebrate PNS axons

Summary The trigeminal alveolar branch in the lower jaw of the cichlidTilapia mariae was examined by light and electron microscopy on single and serial sections, and by light microscopy on teased fibre preparations. The principal purpose was to find out if the exceptionally thin myelinated axons (d < 1 m) present in this nerve possess true nodes of Ranvier, and to determine the dimensions of their myelin sheaths. This necessitated analysis of the whole size range of myelinated fibres, with respect to nodal and internodal morphology. The results show that the exceptionally thin myelinated fibres exhibit primitive nodal regions, with patches of axolemmal undercoating, and few Schwann cell processes in the node gap. This contrasts with the more complex nodal organization seen in larger trigeminal alveolar branch fibres. For the whole population of myelinated fibres the number of myelin lamellae increases rectilinearly with axon diameter, and sheath length increases with fibre diameter according to a logarithmic expression. The myelin sheaths of the exceptionally thin trigeminal alveolar branch fibres are composed of 10–20 lamellae, and extend 35–50 m along the axon. These results show that the structural complexity of nodal regions in the trigeminal alveolar branch decreases with decreasing fibre size, that the exceptionally thin myelinated trigeminal alveolar branch fibres possess primitive nodes and that they have very short myelin sheaths. Our crude theoretical calculations suggest that these fibres might be capable of saltatory conduction.     

Three-dimensional morphology of astrocytes and oligodendrocytes in the intact mouse optic nerve

Summary The three-dimensional morphology of astrocytes and oligodendrocytes was analysed in the isolated intact mature mouse optic nerve, by correlating laser scanning confocal microscopy and camera lucida drawings of single cells, dye-filled with lysinated rhodamine dextran or horseradish peroxidase, respectively. These techniques enabled the entire process field of single dye-filled cells to be visualized in all planes and resolved the fine details of glial morphology. Morphometric analysis showed that the processes of all astrocytes had branches ending at the pial surface, on blood vessels, and freely in the nerve; branches ending in the nerve were described to end at nodes of Ranvier in the accompanying paper. Astrocytes were classified into a single morphological population in which each cell subserved multiple functions. The results of this study do not support the contention that astrocytes can be subdivided into two morphological and functional subtypes, namely type-1 and type-2, which have processes ending either at the glia limitans or at nodes, respectively. Three-dimensional analysis of oligodendrocyte units, defined as the oligodendrocyte, its processes and the axons it ensheaths, showed the provision of single myelin segments for an average of 19 nearby axons (range 12–35) with a mean internodal length of 138 m (range 50–350 m). Mouse optic nerve oligodendrocytes were a homogeneous population and were markedly similar to those in the rat optic nerve. The results of our analysis of oligodendrocyte morphology are consistent with the view that the number and internodal length of myelin sheaths supported by a single oligodendrocyte are related to the diameter of the ensheathed axons.     

Nodal axon diameter correlates linearly with internodal axon diameter in spinal roots of the cat

Nodal and internodal axon diameters of individual myelinated nerve fibres were measured electron microscopically in fibre samples from serially sectioned L₇ ventral and dorsal spinal root of young adult cats. Axon cross-sectional area at the node of Ranvier in axons more than 4 μm in diameter was reduced to less than 20% of its internodal value. Internodal and nodal axon diameters showed a rectilinear distribution and linear regression analysis gave coefficients of correlation between 0.93 and 0.99. An individual nodal diameter value could be fitted to an internodal axon diameter value in a 95% prediction interval of ± 1.06–2.41 μm.     

Internodal microvilli of Schwann cells of myelinated fibres in lizard spinal roots project onto unmyelinated axons

Summary Tufts of microvilli originating from the internodal cytoplasm of Schwann cells associated with myelinated axons in apparently normal lizard spinal roots have been studied under the electron microscope by means of both single and serial sections. More than one tuft of internodal microvilli may arise from a single Schwann cell. Sometimes mitochondria and more frequently an organelle resembling a multivesicular body with a clear matrix can be found in the Schwann cell cytoplasm underlying a tuft of internodal microvilli. The dimensions (length: 0.4–1.0 m; diameter: 40–70 nm) and structure of internodal microvilli of the Schwann cell are very similar to those of nodal microvilli of the same cell. Each tuft of internodal microvilli projects towards an adjacent unmyelinated axon which at this site is partly devoid of its own Schwann cell sheath. Thus a single Schwann cell may be related to a myelinated axon and an unmyelinated axon at the same time. Patches of a dense axolemmal undercoating (which could be portions of the cytoskeleton) are present in the unmyelinated axon in close spatial correlation with internodal microvilli. The factors which could induce the formation of internodal microvilli as well as the possible role (or roles) of these microvilli are briefly discussed.     

Characteristic variations of relative myelin sheath thickness in 11 nerves of the rat

Summary Computer-assisted measurements of relative myelin sheath thickness (the g ratio) were made in 11 peripheral nerves of the rat. The scatter diagrams showed nerve-specific variations in the distribution of relative myelin sheath thickness. Myelinated fibers of less than 3.5 m axon diameter had relatively thin myelin sheaths, particularly in the splanchnic, vagus and glossopharyngeal nerves. The oculomoter nerve had two fiber populations clearly set apart in terms of relative myelin sheath thickness. Thickly myelinated fibers were found in facial and hypoglossal nerves. No single functional modality was evident for the thinly myelinated fibersThis study was supported by grant 609 from the Deutsche Forschungsgemeinschaft     

Occurrence of long non-myelinated axonal segments intercalated in myelinated, presumably sensory axons: electron microscopic observations in the dog atrial endocardium

Summary Electron microscopy of serial sections revealed the occurrence of long non-myelinated segments in myelinated, presumably sensory axons running in the left atrial endocardium of normal adult dogs. Four such non-myelinated segments were analysed in three myelinated axons. They varied from 20 to 150 m in length, and differed from nodes of Ranvier in being invested by Schwarm cells in the manner of unmyelinated nerve fibres. Short non-myelinated portions (20–25 m long) were associated with a single Schwann cell, whereas the longest such segment (150 gmm) had five. The non-myelinated axonal segments were non-varicose and similar in diameter (1.2–3.0 m) to adjacent myelinated segments, which had myelin sheaths 6–25 lamellae thick. The cytoplasm of the non-myelinated axonal segments contained numerous neurofilaments and microtubules, some mitochondria and smooth endoplasmic reticulum. The short non-myelinated segments were enclosed by perineurium, whereas the long non-myelinated segment was devoid of perineurium at its mid-portion; instead fibroblast-like cells made a loose boundary around the axon at this level. The significance of these non-myelinated segments was discussed with special emphasis on the question of whether they result from focal degeneration of the myelin sheath (demyelination) or are generally present in the preterminal regions of some axons.     

Myelination and remyelination in the regenerating visual system of the goldfish

Summary The remyelination of regenerated optic axons was investigated in goldfish following either optic nerve crush or ouabain retinal intoxication. Axons grown after nerve crushing acquire thinner myelin sheaths than axons originating from reconstituted ganglion cells. If axons of reconstituted ganglion cells are crushed and allowed to regenerate, the subsequent myelination is weaker than that of control axons not interrupted by crushing, but stronger than that of axons of preexisting retinal ganglion cells.The present results suggest that a neuron is capable of inducing a normally developed myelin sheath when its axon contacts an oligodendrocyte the first time, whereas a neuron whose axon contacts an oligodendrocyte the second time is not capable of forming a normal myelin sheath in the adult animal. The present results also support the notion that the oligodendrocyte requires a neuronal signal for myelin sheath formation.Supported by the Deutsche Forschungsgemeinschaft (Wo 215/5)     

Electron microscopic nerve fiber caliber analysis in the nerve branch to the venter rostralis of the M. digastricus in mice

Nerve fiber counts and caliber analysis were performed with an electron microscope on 5 nerve branches to the venter rostralis of m. digastricus (VRD) from 3 mice. Branches contained an average of 42.4 (79.4%) myelinated fibers and 11.0 (20.6%) unmyelinated fibers. 68.4% of the sheaths of the myelinated fibers in the nerve branches were 6.5-8.5 microns (peak 7.0-7.5 microns) in major diameter and 76.4% were 5.0-7.5 microns (peak 6.0-6.5 microns) in minor diameter, 68.8% of the axons of the myelinated fibers were 4.5-6.5 microns (peak 5.5-6.5 microns) in major diameter and 76% were 3.5-5.5 microns (peak 4.0-5.0 microns) in minor diameter. The frequency distribution of the diameters showed that the nerve branch to VRD consisted of extra large myelinated fibers as in the r. digastricus to the venter caudalis of m. digastricus (VCD). Average ratios of the areas of the myelin sheaths to those of axons of the myelinated fiber transverse sections in the various axon area classes of the nerve branches were 0.7-2.9, and those ratios tended to decrease in the larger axon classes.     

Regeneration and remyelination of Xenopus tadpole optic nerve fibres following transection or crush

Optic nerves of stage 54-56 Xenopus laevis tadpoles were either transected or crushed, and subsequent Wallerian degeneration, regeneration, and remyelination were examined. After 4 days, normal myelinated fibres were no longer present in the distal stump, and only a few unmyelinated fibres remained. After 10-13 days, the distal nerve consisted mainly of a core of reactive astrocytes with enlarged processes and scattered oligodendrocytes which persisted throughout the degenerative period. Regenerating axons traversed the site of the lesion and extended into the distal stump within 13-15 days. As regeneration progressed, astrocytic processes extended radially from the optic nerve's central cellular core and formed longitudinal compartments for regenerating axons. Between 15-19 days, a few regenerating fibres were remyelinated and by 35 days, more axons were surrounded either by thin collars of oligodendrocyte cytoplasm or by 1-3 spiral turns of myelin membrane. By 95 days, the number of myelinated fibres had increased to about 50% of those present in control nerves. Their myelin sheaths were normal in appearance and thickness relative to their respective axon diameters. The largest axons were surrounded by compact sheaths with 4-9 lamellae.     

Macromolecular structure of axon membrane and action potential conduction in myelin deficient and myelin deficient heterozygote rat optic nerves

Summary The macromolecular structure of the axon membrane in optic nerves from 25-day-old male littermate control and myelin deficient (md) rats and 16-month-old md heterozygotic rats was examined with quantitative freeze-fracture electron microscopy.The axon membrane of control optic nerves displayed an asymmetrical partitioning of intramembranous particles (IMPs); P-fracture faces of myelinated internodal axon membrane were more particulate than those of pre-myelinated axons (1600 1100 m^–2, respectively), while relatively few IMPs (150 m^–2) were present on external faces (E-faces) of internodal or pre-myelinated axon membrane. Amyelinated axons of md optic nerves also exhibited an asymmetrical partitioning of IMPs; protoplasmic membrane face (P-face) IMP densities, taken as a group, exhibited a wide range (600–2300 m^–2) and, in most regions, E-faces displayed a relatively low IMP density (175 m^–2). Axons of > 0.4 m diameter exhibited significantly greater mean P-face IMP density than axons < 0.4 m diameter. Aggregations of E-face IMPs (350 m^–2) were occasionally observed along amyelinated axon membrane from md optic nerves.Optic nerves from md heterozygote rats exhibit myelin mosaicism, permitting examination of myelinated and amyelinated axon membrane along the same tract. The axon membrane exhibits different ultrastructure in these two domains. Myelinated internodal axon membrane from md heterozygote optic nerves exhibits similar P- and E-face IMP densities to those of control internodal axolemma (1800 and 140 m^–2, respectively). Amyelinated axons in the heterozygote exhibit a membrane structure similar to amyelinated axons in md optic nerve. P-face IMP density of large diameter (> 0.4 m) amyelinated axons from md heterozygote optic nerves is significantly greater than that of small calibre (< 0.4 m) axons. In most regions, amyelinated axon membrane exhibits a relatively low E-face IMP density (200 m^–2); however, focal aggregations (400 m^–2) of E-face particles are present.Electrophysiological recordings demonstrate that amyelinated axons in md optic nerves support the conduction of action potentials. Compound action potentials in md optic nerves exhibit a monophasic configuration, even at 20-days postnatal, similar to that of pre-myelinated optic nerve of 7-day-old normal rats. Moreover, conduction velocities in the amyelinated 20-day-old md optic nerve are similar to those displayed by pre-myelinated axons from 7-day-old optic nerves. These results are consistent with persistence of action potential conduction in md axons, despite the absence of myelination in the optic nerves of the md mutant.     

Double myelination of axons in the sympathetic nervous system

Summary Relationships between axons and Schwann cells in myelinated fibres of the superior cervical (sympathetic) ganglion have been examined in normal adult rats. In cross-sections through the ganglion up to 4 % of myelinated fibres were focally encircled by an additional myelinating Schwann cell, forming regions termed double myelination. In these regions and elsewhere in the ganglion, the structure of the inner fibre (axon and myelinating Schwann cell) conformed to the relationships expected on the basis of numerous previous investigations on normal peripheral nerve. However, the outer Schwann cell and myelin sheath, which formed an annulus around the inner fibre, was remarkable in that it apparently made no direct contact either with the centrally enclosed axon or with any neighbouring axon, yet appeared largely if not completely intact. In addition, the increasing frequency of double myelination in older animals and the rarity of myelin degeneration in the same ganglia indicate that the outer Schwann cell, and in particular its myelin sheath, persist for some period in an isolated form. Double myelination was not located in non-sympathetic peripheral nerve samples from the same animals. Double myelination may result from the displacement of one myelin internode by the interposition of another Schwann cell rendering the original Schwann cell redundant. There was no involvement of haematogenous cells as occurs in some demyelinating conditions. While some parallels may be found with previous studies, this would appear to be the first report of apparent survival of myelin in a Schwann cell not making, as far as could be determined in the present study, at least partial direct axonal contact. These observations on sympathetic nerve may provide a new perspective on axon-Schwann cell signalling.     

Interactions between Schwann cells and CNS axons following a delay in the normal formation of central myelin

Summary Irradiation of the rat spinal cord during the first postnatal week results in a profound reduction of oligodendrocyte myelin formation in the dorsal funiculi (DF). Despite this absence of myelin, however, axons in the irradiated region in the DF increase in diameter and approximate the size distribution seen in the control spinal cord. By 25 days of age Schwann cells are present in the irradiated DF where they undergo cell division and myelinate the axons. During the early stages of this myelin formation, these intraspinal Schwann cells exhibit a relationship to axons that is somewhat different from that seen in the normal developing peripheral nervous system (PNS). For example, within a given region, intraspinal Schwann cells myelinate axons of large diameter prior to ensheathing bundles of small diameter axons. Additionally, during myelination a Schwann cell will surround a single axon with multiple processes which appear to compete for contact with the axolemma. On axons of larger diameter, the elaboration of these processes is so excessive that it is often difficult to trace them back to the parent Schwann cell. Later, when a single process establishes several spirals about an axon, additional processes are no longer elaborated, and the extra processes disappear as myelin formation advances to the stage of compact lamellae. Thereafter, the myelin sheath continues to form in a normal manner. Excess processes have been observed during myelinogenesis in the normal developing PNS, but their frequency in that environment is much less than in the irradiated cord. These observations support the hypothesis that the signal(s) to initiate myelin formation are expressed on the axolemmal surface and are controlled by the neuron. In addition, these observations suggest that the delay in myelination results in an affinity or tropism between axons and Schwann cells which exceeds the level existing at the normal time of myelin formation.     

Axo-glial relations in the retina-optic nerve junction of the adult rat: electron-microscopic observations

Summary The retina-optic nerve junction (ROJ) was examined by electron microscopy in adult rats, with particular emphasis on the unmyelinated-myelinated nerve fibre transition. Both single sections and serial sections were used. The non-retinal part of the ROJ is covered by an extensively folded glia limitans, facing the choroidea, sciera and pia mater. The blood vessels within the ROJ follow a transverse course and are surrounded by unusually wide perivascular spaces with a glia limitans-like outer delimitation. The endothelial cells exhibit numerous pinocytotic vesicles on their abluminal aspect. In the unmyelinated part of the ROJ the axons are embedded in an extensive meshwork of fibrous astrocytic processes. Some unmyelinated axons exhibit patches of axolemmal undercoating with externally associated astrocytic processes. Typical oligodendrocytes are not found, but a few small dark glial cells of unknown identity can be observed. Atypical ensheathment and myelination of axons at this level by ectopic Schwann cells occurred in one case. In the transition segment of the ROJ a pattern similar to that along dysmyelinated axons is observed, including aberrant axo-glial contacts, unusually thin and short myelin sheaths, intercalated unmyelinated segments, distorted myelin termination regions, bizarre paranodes and myelin termination regions without associated nodally differentiated axolemma. Neither sheath length nor number of myelin lamellae is related to axon diameter in the transition region. Axon diameter tends to be somewhat larger at myelinated than unmyelinated levels of the same axon. We suggest that the unusual axo-glial relations in this region are due to a deficient proliferation and differentiation of oligodendroglial cells, and that the pattern of glial ensheathment in the ROJ might be a consequence of the locally deficient blood-brain barrier.     

Degeneration of myelinated sympathetic nerve fibres following treatment with guanethidine

Summary The specificity and characteristics of the degeneration of myelinated axons after chronic guanethidine treatment have been investigated in sympathetic and non-sympathetic nerves. Adult male Sprague-Dawley rats aged approximately 43 weeks were treated with guanethidine sulphate (50 mg per kg body weight per day) for between ten days and six weeks. Tissues were examined by qualitative and quantitative light and electron microscopy. In the superior cervical (sympathetic) ganglion (SCG), guanethidine treatment produced a 78% decrease (P = 0.009) in the mean number of myelinated fibres at a standard level of section, compared to the contralateral control ganglion which was removed surgically prior to drug treatment. This reduction in the treated SCG was apparent after 10 days, though complete degeneration of nerve cell bodies was not widespread at this stage. Degeneration of unmyelinated axons was extensive. Degenerating myelinated fibres were consistently small in diameter (up to 3m). In individual myelinated fibres the earliest signs of degeneration involved disruption of axonal organelles, particularly the cytoskeleton, and focal widening of the periaxonal space. Myelin breakdown followed these events; degeneration of myelin still associated with a structurally intact axon was not observed. Myelin breakdown appeared to take place initially within the Schwann cell, at least to the stage of loosened membranes. However, infiltrating cells were also involved in myelin phagocytosis. At all stages of treatment some small diameter myelinated fibres remained intact, and there was no evidence of degeneration of the larger diameter fibres (up to 15 m) which are consistently present in small numbers in the SCG. In the cervical sympathetic trunk, which carries preganglionic axons to the SCG and the vagus and sciatic nerves, degeneration only of unmyelinated axons was detected. These results indicate that guanethidine does not exert a primary degenerative influence on myelin or myelinating Schwann cells and that the myelin degeneration observed in the SCG is a secondary result of the previously documented selectively destructive effect of guanethidine on postganglionic sympathetic neurons. Surviving, small diameter myelinated fibres in the SCG could be either preganglionic or processes of resistant postganglionic neurons, while the larger diameter fibres are likely to be somatic. While the cervical sympathetic trunk, vagus and sciatic nerves all contain postganglionic sympathetic fibres it appears that few of these are myelinated, at least at the levels sampled in this study.     

Double myelination of axons in the sympathetic nervous system of the mouse. I. Ultrastructural features and distribution

Summary This study has examined the structural features and distribution of doubly myelinated axons in normal adult and aged mice. Investigation focused on the superior cervical ganglion (SCG) and paravertebral sympathetic ganglia, which were extensively serial-sectioned for light and electron microscopy. In the SCG, the principal features of doubly myelinated regions were that an apparently normal myelinated axon was enclosed for part of its length by an additional (outer) myelinating Schwann cell. The separate nature of the inner and outer Schwann cells was emphasized by the consistent presence of individual nuclei in each, and by the presence of endoneurial space, often containing collagen fibrils, between the inner and outer cells. In some cases more than a single outer Schwann cell was present, arranged serially along the inner myelinated fibre. While double myelination forms through a mechanism involving displacement of an original myelinating Schwann cell by an interposed Schwann cell (see companion paper), we here provide evidence that in some instances the outer Schwann cell fails to retain any direct axonal contact, either with the axon centrally enclosed within the configuration or with any neighbouring axon. In contrast to the rat, delicate cytoplasmic processes often extended from the lateral extremes of outer Schwann cells. However, again no evidence for axonal contact was found, and similar processes also extended from the paranodal region of some singly myelinated non-displaced Schwann cells. Without exception the outer myelin sheath remained structurally intact, and characteristically underwent a series of conformational changes (progressive infolding of the paranodes and new areas of myelin compaction) which infer a continuing capacity of the outer Schwann cell to translocate myelin-specific components in a co-ordinated manner. A basal lamina was always present on the abaxonal plasma membrane of the outer cell, but not on the adaxonal surface except in areas involved in infolding, thus retaining the polarity which existed at the time of displacement from the axon. At single cross-sectional levels through the SCG, up to approximately 4% of myelinated axons were involved in double myelination. Double myelination was not detected in the sciatic nerve or in the paravertebral ganglia, thus indicating a predilection for the SCG as a site of development of these configurations. Though not challenging the role of the axon in initiating the formation of myelin, these data indicate that in this tissue myelin maintenance does not require direct contact between axonal and Schwann cell plasma membranes.     

Ultrastructural and immunohistochemical analysis of axonal regrowth and myelination in membranes which form over lesion sites in the rat visual system

Summary Glial-connective tissue membranes which form bridges over lesion cavities in the brachial and pretectal region of the rat visual system contain regenerated myelinated and unmyelinated axons. The lesions were made between 10 and 16 days postnatal—a time at which neonatal regeneration would not be expected. A detailed ultrastructural study of these membrane bridges has been undertaken in order to describe the cellular and extracellular conditions that are associated with the regeneration, myelination and continued survival of identified retinal and other axons.The lesion-induced membrane bridges possessed a limiting surface of fibroblasts and were composed of glial cells, macrophages, endothelial cells, pericytes and collagen. There was some variability in the ultrastructural appearance of the glial cells; the majority of criteria indicate that they were astrocytes. These astrocytes formed glia limitans-like surfaces beneath the fibroblasts. They contained numerous filaments and extended fine, electron-dense cytoplasmic processes, often arranged into lamellated stacks. Basal lamina was present on the outer surfaces of the astrocytes. Astrocytic processes isolated clusters of myelinated and unmyelinated axons in lacunae which may have served as conduits for axonal elongation. This suggests a role for these astrocytes in the regeneration and maintenance process which appears to recapitulate events which occur during normal development. Interestingly, regrowing retinal axons were never found adjacent to astrocytic surfaces possessing a basal lamina.We did not detect evidence of Schwann cell invasion into the lesion. By ultrastructural criteria the myelin ensheathment which occurred on the larger axons in the membrane bridge was of central rather than peripheral type. The cytoplasmic domain external to the sheath was limited to a small tongue; no basal lamina invested the fibre; and the periodicity of the myelin was equivalent to that of other CNS structures. Similarly, the CNS character of the myelin was demonstrated by intense immunostaining of myelin sheaths for myelin basic protein and phospholipid protein and lack of staining for the PNS component P_o. The oligodendrocytes responsible for this myelination may either have extended cytoplasmic processes from the adjacent neuropil, or may have differentiated from precursor cells within the membrane bridge.