J. Cell Set. 13, 46i-477 (i973) 461Printed in Great Britain

SOME ASPECTS OF REMYELINATION AFTER

DEMYELINATION PRODUCED BY THE

INTRANEURAL INJECTION OF

LYSOPHOSPHATIDYL CHOLINE

SUSAN M. HALLDepartment of Anatomy, Guy's Hospital Medical School, London, SEi 9RT, U.K.

SUMMARYThe morphology of remyelination following demyelination induced by the intraneural

injection of lysophosphatidyl choline, LPC, has been examined in the mouse sciatic nerve, atperiods up to 240 days post-injection. It was found that, in many fibres, the process resembledprimary myelinogenesis. There was a moderate Schwann cell proliferation; those Schwanncells not involved in remyelination remained closely associated with the remyelinating Schwanncell/axon unit, within a common basal lamina tube. Numerous small axons, considered to besprouts from the remyelinating axon, were observed lying in contact with the ' supernumerarySchwann cells'.

In a small population of fibres, however, atypical morphological features were consistentlyseen: (i) multiple mesaxons, indicating probable remyelination by tunication; (ii) paranodalreorganization in the junctional zone; (iii) the formation of internodal ' pseudonodes', whichsubsequently underwent transition into incisures of Schmidt-Lanterman.

These structures are discussed in terms of the re-establishment of the Schwann cell/axonrelationship.

INTRODUCTION

The intraneural injection of lysophosphatidyl choline, LPC, 10 mg/ml, produces awell defined, dose-dependent, demyelinating lesion (Hall & Gregson, 1971). This paperdescribes the morphological aspects of remyelination in mature, previously-myelinatedfibres, following the intraneural injection of LPC, over a period up to 240 dayspost-injection.

MATERIAL AND METHODS

Adult mice were used throughout the experiment.Injection of the sciatic nerve was performed as described previously (Hall & Gregson, 1971).

The concentration of the standard demyelinating dose of LPC was 10 mg/ml; the volumeinjected was approximately 2 x io"4 ml.

Preparation of the nerve bundle for light microscopy

In 10 mice, at intervals from 25 to 65 days after injection of LPC, the sciatic nerve from apoint immediately distal to the sciatic notch to the lower end of the femur, was carefullyremoved and laid on a card under slight tension. The specimen was placed in 3 % glutaraldehydein Millonig's phosphate buffer, pH 7-3, for 2 h at 4 °C, washed in phosphate buffer and post-fixed in 1 % OsO4 in phosphate buffer pH 7-3, for 2 h at 4 °C. When the nerve was in washing

462 S. M. Hall

fluid, the bundle was examined with oblique incident light, the remyelinating zone identi-fied and this segment of the bundle was removed for postfixation and teasing. Teasing of fibreswas carried out in 33 % glycerine under a dissecting microscope using mounted needles: thepreparations were mounted in 33 % glycerine.

Preparation of the nerve bundle for electron microscopy

Thirty fibre bundles previously injected with LPC 10 mg/ml were examined electron micro-scopically at intervals from 15 to 240 days post-injection. Specimens were fixed in 3 % glutar-aldehyde and postfixed in 1 % OsO4 as described above. After osmication they were dehydratedin an ascending series of ethanols, carried through propylene oxide, and infiltrated and em-bedded in TAAB resin. Ultrathin sections were cut on an LKB ultramicrotome, and examinedin RCA EMU 3 and RCA EMU 4 electron microscopes.

RESULTS

In the following account the term 'normal' will be applied to the apparently unin-volved internodes proximal and distal to the lesion, and ' junctional zone' to the regionin which these internodes are continuous with the remyelinating internodes.

Light-microscope findings

Fibres were examined 25-65 days after injection of 10 mg/ml LPC. In osmicated,teased fibre bundles, the extent of the lesion was initially clearly defined as a pale-staining area interposed between strongly osmiophilic regions proximally and distally.Most of the internodes within this area exhibited morphological characteristics similarto those of newly myelinating fibres: thin myelin sheaths, simple paranodal contoursgradually increasing in complexity with time, and closed Schmidt-Lanterman inci-sures. The remyelinating internodes displayed a wide range of internodal lengths,from 100 /im to lengths that fell within the normal range for the diameter of the fibre.

However, there was a small population of fibres, both in the junctional zoneand in the remyelinating region, comprising 5-10% of the total number of fibresexamined, in which the following atypical features were consistently observed.(a) Attenuation of the normal paranodal myelin sheath in the junctional zone, some-times accompanied by swelling of the bulb. In some cases, the whole thickness of thesheath did not round off into a typical paranodal bulb; some part of it continued, muchthinned, along the fibre for a further 20-30 /im, finally forming a new paranodal bulbapposing that of the succeeding remyelinating internode. The point at which the thinmyelin was continuous with the thicker sheath, at what was judged to be the originalsite of the paranodal termination on the basis of the presence of characteristic myelinprofiles, was usually indicated by a typical Schmidt-Lanterman incisure, and/orextensive myelin infolding. Careful through-focusing at this point often revealed anosmiophilic band, possibly the remnant of the curved border of the original paranode:this structure was clearly distinguishable from the loosely wound spiral apparatus ofGolgi-Rezzonico commonly found at the level of the Schmidt-Lanterman incisure inosmicated fibres (Fig. 1 A-E). (b) Short, apparently normally-myelinated internodes incontinuity with remyelinating internodes proximally and distally. The paranodalbulbs of the former generally showed considerable myelin infolding and occasionalsmall osmiophilic globules within the Schwann cytoplasm of the paranode (Fig. 1 F).

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(c) Irregularities of sheath thickness along the remyelinating internode, particularlynear the Schwann nucleus (Fig. 1 G). (d) 'Node-like' discontinuities in the remyelinat-ing sheath, which were difficult to categorize on the basis of their position along theinternode; e.g. they were frequently observed in perinuclear regions, and were neverassociated with myelin profiles.

Electron-microscope findings

After the intraneural injection of 10 mg/ml LPC, the myelin sheath underwent acharacteristic progressive vesicular breakdown, while the axon and Schwann cellapparently remained undamaged (Hall & Gregson, 1971). Remyelination began beforemyelin debris had been completely removed from the Schwann cells: by 20 days manyaxons were surrounded by 2-6 lamellae of compacting and compacted myelin. In mostfibres, remyelination was indistinguishable from myelination during ontogeny. Therewas a population of fibres, however, representing about 10% of the total number offibres in the bundle, in which this was not the case. The salient morphological featuresof the altered Schwann cell/axon relationship found in these fibres, in approximatetemporal sequence, will now be described.

During the first 2 weeks post-injection, large single axons, up to 8 /tm in diameter,were observed, associated with numerous slender microtubule-containing cytoplasmicprocesses, the cellular complex being surrounded by a loose, often serpentine, basallamina envelope. Occasionally, these processes were of markedly different electrondensity, suggesting either that more than one Schwann cell was represented, or thatsome of the processes were derived from another cell type, e.g. a macrophage, whichhad entered the basal lamina tube (Figs. 2, 3).

Initially, the cytoplasmic fingers made intermittent contact with the axonal circum-ference, hence stretches of axon were separated from the endoneurial space only bySchwann cell basal lamina (Fig. 2). The area of axonal surface covered by cytoplasmprogressively increased until the axon was completely invested by overlapping tonguesof Schwann cell cytoplasm: an arrangement which, when seen in transverse section,produced a system of multiple mesaxons (Figs. 4, 5). The contrasting electron densityof some of the cytoplasmic processes emphasized the continued presence of more thanone Schwann cell per axon at any point along the length of the remyelinating fibre. Itwas not possible to determine the relative contribution to the compacting sheath beingmade by a particular cell, except in fortunate sections passing through the cell body.

Remyelination was well established in all affected fibres by 20 days post-injection;the main criterion for recognizing remyelinating fibres at this and subsequent stagesbeing the presence of an inappropriately thin sheath for the diameter of the axon. Asthe sheath became compacted, and the number of lamellae encircling the axonincreased, fewer multiple mesaxons were observed, until, by the time the sheath con-sisted of 8 lamellae, they had almost all disappeared, leaving only definitive externaland internal mesaxons.

Most Schwann cells within the affected region appeared undamaged by LPCtreatment (Hall & Gregson, 1971). A recent quantitative study has demonstrated amoderate proliferation of Schwann cells during remyelination after LPC injection,

464 S. M. Hall

and a concomitant halving of the internodal length in the majority of remyelinatingfibres, although in 10 % of the fibre population the remyelinated internodal length waswithin the normal range for the fibre diameter (Gregson & Hall, 1973). From com-parisons of I-/4IT1 resin sections with montages prepared from low-power electronmicrographs of adjacent thin sections, it was apparent that approximately 60 % of theendoneurial cells were Schwann cells which were not involved in remyelinating thedenuded axons. These, supernumerary Schwann cells (SSC), presumably arose bymitotic division from the original population of Schwann cells. Mitotic figures inSchwann cells, which are not features of normal mature sciatic nerve, were seenrelatively frequently during the early stages of remyelination.

The SSC were always observed close to a remyelinating Schwann cell/axon unit,forming a characteristic cellular complex, bounded by the basal lamina covering theexternal surface of the SSC (Fig. 4). The space between the basal lamina covering theouter surface of the remyelinating Schwann cell and that covering the inner surface ofthe SSC usually contained collagen fibrils, oriented parallel with the long axis of thefibre. The collagen observed in this situation, and the endoneurial collagen surroundingthe outer aspect of the SSC had an average diameter of 30 run, somewhat less than thediameter reported for endoneurial collagen in normal fibres (Thomas & Jones, 1967):a similar finding has recently been described in the proximal stump of a divided nerve(Morris, Hudson & Weddell, 1972). During the early stages of remyelination, cellscontaining pale-staining lipid globules and other forms of myelin debris were some-times observed within this space.

A characteristic feature of the SSC was their association with small axonal profiles,0-2-0-5 /im in diameter, which were considered to be non-myelinated sprouts from theremyelinating axons. The relationship between these sprouts and their 'parent'Schwann cell was unlike that existing between axon and Schwann cell in a bundle ofmature, unmyelinated fibres, in that the sprouts frequently exhibited only tangentialcontact with Schwann cytoplasm (Fig. 4). Neither SSC nor axonal sprouts extendedinto the fibre bundle beyond the level of the mid-internodal point of the first normally-myelinated internodes proximal and distal to the lesion. As remyelination continued,many of the sprouts displayed morphological changes consistent with their incipientdegeneration, e.g. disintegration of the axolemma and axoplasmic organelles and theconcomitant appearance of small axoplasmic vacuoles.

Representative fields were viewed at later stages of remyelination, up to 240 dayspost-injection. The most striking feature of the fibre bundles sampled was the pro-gressive disappearance of SSC and sprouts such that, by 240 days, many remyelinatedaxons were surrounded by empty basal lamina tubes, or by double or single fragmentsof basal lamina, or were not associated with any cellular remnants.

Although a survey of transverse sections is informative in determining the nature ofthe re-established Schwann cell/axon relationship, it was felt that the possible ultra-structural correlates of the atypical internodes observed in teased fibres were morelikely to be detected in longitudinal sections. The areas which were selected for par-ticular study were the nodes of Ranvier, and internodal myelin, which was examinedfor evidence of thinning and node-like discontinuities.

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The node of Ranvier

The typical remyelinating pattern of the node will be briefly described. In the junc-tional zone, the demyelinating paranode, filled with myelin debris, and limitedexternally by fine Schwann cell processes and Schwann cell basal lamina, contrastedsharply with the paranode of the adjacent normal internode. By 35 days, in the major-ity of fibres, the organization of the remyelinating paranode/node region resembled thatof its immature counterpart: there was little or no axonal constriction at the midnodalpoint, and only a slight indication of swelling of the paranodal bulbs, with consequentlyminimal deviation from the overall cylindrical profile of the internode. In the junc-tional zone it was easy to distinguish between the paranodal regions of the normal andremyelinating internodes: in the former, many of the small dense terminal loops failedto reach the axolemma, becoming arranged in the familiar herring-bone pattern, whilein the latter, the less numerous loops were paler, filled with abundant Schwann cellcytoplasm, and all loops reached the axolemma in a longitudinal series (Fig. 6).

Irregular, finger-like processes of Schwann cell cytoplasm were often observedprojecting into the endoneurial space within the loose basal lamina envelope aroundthe node. Frequent examples of the processes of one Schwann cell 'over-riding' thoseof an adjacent Schwann cell were seen in the junctional zone and in the remyelinatingregion. Similar findings have been reported in remyelination after experimentalallergic neuritis (Ballin & Thomas, 1969) and after segmental demyelination caused bydiphtheria toxin (Allt, 1969).

In a small proportion of fibres, however, morphological features were observedwhich differed from those described above: deviations from standard nodal morpho-logy were seen both within the remyelinating area, and in the junctional zone, wherenot only the remyelinating paranode, but also the paranode of the otherwise normalinternode were involved.

At the remyelinating paranode, a staggered arrangement of terminal loops wassometimes observed, in which small groups of 5 or 6 lamellae peeled off from the mainsheath, the intervals between adjacent groups being 2-6 /tm (Fig. 7). At the pointwhere the lamellae became separated from the sheath the major dense line was splitto enclose a loop of Schwann cell cytoplasm which reached the axolemma. These loopswere indistinguishable in their mode of formation and morphology from those asso-ciated with the conventional paranode. This pattern often involved as many as 5 suc-cessive groups of lamellae; in this way it was possible for a sheath in which 12 lamellaewere present at the mid-internodal point, to be reduced to only 3 or 4 lamellae at theparanode. Less frequently, similar structures were observed at considerable distancesfrom the paranode.

Alterations in paranodal organization were seen in some otherwise apparentlynormally-myelinated internodes within the junctional zone, so that at least twostructures which possessed paranodal characteristics were present at the same end of asingle internode. The main criteria for identifying the site of the original paranodewere (a) the presence of terminal loops exhibiting a typical mature configuration andrelationship with the axolemma; and (b) the presence of isolated spheres of compact

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myelin and out-pushings of the sheath into surrounding Schwann cell cytoplasm -both indicative of changes in overall sheath contour associated with the crenations of amature paranode (Fig. 8). A variable number of the outermost myelin lamellae peeledoff from this site, and continued as an extension of the sheath, investing the adjacentaxon with a thin layer of compact myelin (Fig. 8), often consisting of no more than8—io lamellae, finally forming a new paranodal termination which was apposed by aremyelinating paranode.

In many cases this staggered arrangement of groups of cytoplasmic loops resembledthat described at the remyelinating paranode (see above). In other fibres, however, thetransition between the bulk of the sheath and the aberrant external lamellae was morecomplex: the major dense line of the latter split to enclose Schwann cytoplasm, butwas reconstituted at the axonal side of the cytoplasmic inclusion to continue, with itsassociated intraperiod line, as a lamella of compact myelin. Thus cytoplasm which,presumably, would have been incorporated in characteristic terminal loops appearedinstead in a typical Schmidt-Lanterman incisure (Figs. 8, 9). In several of these'incisures', desmosome-like thickenings and microtubules were present, as they are inthe normal incisure (Hall & Williams, 1970).

Examination of semi-serial longitudinal sections of selected teased single fibresrevealed a further type of transition from thick to thin myelin, in which the sheathexhibited an area of lamellar disorganization where some lamellae were continuousbetween the 2 regions, while others ended blindly in loops of Schwann cytoplasm:a similar pattern has been described in demyelinating central fibres (Harrison,McDonald, Ochoa & Ohlrich, 1972). This arrangement was often associated withextensive ballooning of the whole thickness of the sheath, such as may be observed inFig. 1 A.

In some paranodes in the junctional zone, the terminal loops were separated fromthe axolemma by finger-like processes of Schwann cell cytoplasm, continuous with thatexternal to the sheath and in the nodal gap (Fig. 10). Occasionally, the paranode of thenormally-myelinated internode appeared to end blindly as a loop of compact myelinenclosing the paranodal terminal loops of cytoplasm. The relationship between theSchwann cell of this internode and that of the adjacent remyelinating paranode acrossthe nodal gap appeared normal (Fig. 11).

Changes along the remyelinating internode

A consistent finding, in about 10% of the fibre population, was a node-like structureoccurring within individual Schwann cells, so that the sheath appeared to be dis-continuous within the cell. These structures, hereafter referred to as 'pseudonodes',closely resembled nodes of Ranvier in some aspects of their basic organization, e.g.2 groups of apposing Schwann cytoplasm-filled loops, separated in this case, not bygap substance, but by Schwann cytoplasm continuous with that in the loops; trans-verse thickenings of the outer aspect of the axolemma; and increased density of theaxoplasm immediately subjacent to the associated axolemma (Elfvin, 1968). TheSchwann cytoplasm within the loops was abundant, as it is in the immature node,frequently extending as much as 0-5-1 /tm from the axolemmal surface (Figs. 12, 13).

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Most pseudonodes were asymmetrical, and consequently, there were correspondingdifferences in the thickness of the internodal myelin sheaths on either side of thesediscontinuities.

As the remyelinating sheath increased in thickness, lamellar continuity was estab-lished between the 2 sides of the pseudonode. However, the major dense line in thisregion remained split, so that Schwann cytoplasm was incorporated into the otherwisecompact sheath in a manner similar to that described above for the reorganized para-node in the junctional zone, i.e. in a typical Schmidt-Lanterman incisure (Figs. 14,15). Folding and disruption of the sheath at the point of transition between pseudo-node and incisure were commonly observed. By 40 days post-injection, all pseudo-nodes had undergone this transformation. So, too, had the isolated groups of cytoplas-mic loops which peeled off unidirectionally from the sheath, particularly near theremyelinating paranode: it was not unusual at this stage, therefore, to find Schmidt-Lanterman incisures in which the innermost lamellae terminated at the axolemma innode-like cytoplasmic loops. By 240 days post injection no trace of incisures withmodified inner lamellae could be found.

DISCUSSION

The morphological response of the majority of fibres in a bundle following the ad-ministration of LPC 10 mg/ml is essentially similar to that reported after other types ofexperimentally-induced segmental demyelination (e.g. Allt, 1969; Ballin & Thomas,1969; Raine, Wisniewski & Prineas, 1969; Gregson & Hall, 1973). As with remye-lination following the induction of experimental allergic encephalitis and experimentalallergic neuritis in the peripheral nervous system, the mechanism of remyelinationreported above does not exactly resemble primary myelinogenesis, in that a number ofaxons exhibit a more complex relationship with their Schwann cell than simple spirali-zation will allow, and appear to be initially remyelinated by tunication (Raine et al.1969; Allt, 1972).

While there was very little evidence of axonal degeneration, a quantitative study hasdemonstrated a reduction in the number of large diameter axons during the firstmonth post-injection (Gregson & Hall, 1973). As remyelination progressed, theirnumber gradually increased, although the pre-injection numbers of large fibres werenot regained (S. Hall, unpublished observations). There was a corresponding reduc-tion in the numbers of small axonal profiles associated with the supernumerarySchwann cells with time: since these small axons did not extend into the fibre bundledistal to the lesion, they presumably failed to effect any peripheral contact, and hencedegenerated. It is considered that these axonal profiles are the non-myelinated sproutsof an originally-myelinated axon, rather than bundles of normally unmyelinatedaxons which had been incorporated within the common basal lamina tube of aremyelinating unit. Small axonal profiles were frequently seen amongst myelin debriswithin the basal lamina tubes of demyelinating internodes 3 days after injection ofLPS (S. Hall, unpublished observations) and in the peripheral cytoplasm of re-myelinating Schwann cells, and subsequently in the space between the latter and

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supernumerary Schwann cells (Fig. 4). It is suggested that sprouts may run alongthe surface of the remyelinating Schwann cell, before becoming associated with thebasal lamina envelope of a supernumerary Schwann cell, within which they travel inparallel with the parent remyelinating axon.

The stimulus for this sprouting is not known. It is unlikely, however, to be theresult of direct axonal attack by LPC, since there were no overt signs of axonal damage.It is possible that sprouting is elicited by the loss of the normal contact between theaxon and its Schwann cell.

A striking and persistent reduction in axonal diameter following primary demye-lination in the peripheral nervous system has been reported by Raine et al. (1969).After the intraneural injection of LPC, there is a partially reversible reduction in thenumber of large-diameter fibres within the bundle at the level of the lesion (Gregson &Hall, 1973). It does not necessarily follow that LPC selectively attacks fibres within aparticular diameter range: although the action of LPC is dose-dependent, the demye-lination it produces occurs irrespective of calibre. Axonal narrowing may be a responseto loss of myelin and/or the 'normal' Schwann cell/axon relationship. It could alsobe related to sprouting, if this activity involves a significant local redistribution ofaxoplasm.

Nodes of Ranvier

It is interesting that LPC-induced demyelination typically involves whole inter-nodes (Gregson & Hall, 1973). The majority of nodes in the junctional zone comprisedapposing normally-myelinated and remyelinating paranodes: if any of the formersustained damage as a consequence of the administration of LPC, this must be pre-sumed to have been minimal, and of a type that was repaired without recognizableultrastructural alteration.

In some fibres, however, there were changes within the paranodal region of other-wise normally-myelinated internodes. Since the manner in which the main body of thesheath terminated at the axolemma satisfied the morphological criteria for identifyinga mature paranode, this would support the view (a) that much of the paranoderemained relatively undamaged in its original site, and (b) by implication, that thethin sheath which peeled off from the outermost aspect of the main sheath and extendedalong the adjacent axon was newly synthesized by the Schwann cell. The alternativeview, that most of the original paranode retracts intact to a new site, leaving behind athinly-myelinated sheath and a correspondingly attenuated paranode, is thoughtimprobable on the following grounds: (a) the immature form of this paranodal'remnant'; (b) the absence of detached terminal loops along the axolemma, a pheno-menon which has recently been reported in demyelinating central nodes (Harrisonet al. 1972); (c) the staggered termination of the myelin lamellae in some cases, sug-gesting the progressive outgrowth of Schwann processes along the axon; (d) the lack ofexcessive infolding and disruption of the main body of the sheath beyond the immediateparanodal region. Complex infolding of the sheath was always observed in the occa-sional instances of normally-myelinated internodes 'intercalated' between remyelinat-

Remyelination 469

ing internodes (Fig. 1 F), where it is probable that retraction of paranodal myelin hasoccurred.

A noticeable feature of remyelination in most reports in the literature is the way inwhich Schwann cells rapidly establish contact with all but the shortest lengths ofdenuded axons (e.g. Thomas & Lascelles, 1966). In the present study, it is clear that apopulation of Schwann cells within the junctional zone are stimulated to initiate andmaintain the myelination of a length of axon beyond their original territorial bound-aries (as defined by a paranodal termination). This behaviour implies the priormobilization and extension of exploratory processes of Schwann cytoplasm from theseparanodes into the demyelinating region. The examples of Schwann cell processesdissecting terminal loops away from the paranodal axolemma, and ' over-riding' theparanodes of remyelinating Schwann cells, are consistent with such activity; moreover,they indicate that it is a relatively widespread phenomenon, and not confined to thosefibres in which paranodal reorganization occurs.

It is reasonable to assume that in fibres such as that in Fig. 8, processes growing outfrom the paranodal cytoplasm in the early stages of remyelination are able to extendaround, and subsequently to myelinate, the adjacent axon, because their progress isunimpeded by the presence of a neighbouring Schwann cell. Furthermore, it is likelythat they are only halted when the remaining available, i.e. demyelinated, axon hasbeen invested either by the Schwann cell by which it was originally myelinated, or bya migrating supernumerary Schwann cell. In the majority of cases, however, theseprocesses are obviously displaced by a competing Schwann cell before they contact theaxon, since they take no part in remyelination, and remain within redundant folds ofbasal lamina surrounding the node.

Why do mature Schwann cells exhibit such atypical behaviour? The production ofperipheral cytoplasmic processes could be a localized repair mechanism, triggered, ina few cases, by the direct action of LPC on paranodal myelin, producing limitedparanodal demyelination; it may reflect the effects of an altered relationship with anadjoining Schwann cell in which total demyelination has occurred; or it may be aresponse to the presence of a denuded axonal surface. Allt (1969) has demonstratedrepair of minimally damaged paranodes in cases where widening of the nodal gap isless than 15 /tm: fibres such as those in Figs. 8 and 9 demonstrate the capacity of theSchwann cell to effect even more complex 'repair'.

Changes along the remyelinating internode

The difficulties in extrapolating from individual micrographs in attempting toanalyse 3-dimensional organization in vivo are well known. The various groups ofcytoplasm-filled loops abutting on to the axolemma of the remyelinating internode maybe expressions of one way in which the Schwann cell/axon relationship is re-establishedafter demyelination. Bearing in mind the population of fibres in which, in transversesection, the axons were found to be surrounded by multiple processes of Schwanncytoplasm, it is probable that these structures are examples, in longitudinal section, oftunication by regularly-overlapping processes, all of which are derived from the sameSchwann cell.

47O S. M. Hall

The fact that the most complex arrangement, the pseudonode, was frequentlyobserved in a perinuclear position is interesting since it has been shown that, in somedeveloping fibres, the earliest envelopment of the axon is by perinuclear processes,providing the axon with a system of separate mesaxons (Webster, 1971). The simplestinterpretation of the development of the pseudonode is that it is formed by the pre-cocious investment of a segment of denuded axon by 2 relatively long Schwann cellprocesses of approximately equal length and width, derived from the same Schwanncell. As these processes are wrapped around the axon, the latter is progressivelycovered by a more continuous area of the Schwann cell, until, at the point where theprocesses originate from their cell body, the myelin lamellae become continuous acrossthe 'pseudonodal gap'.

It is unlikely that such structures are formed as a specific response of Schwann cellsremyelinating after treatment with LPC, since they have also been observed inremyelination following cyanide intoxication (S. Hall, unpublished observations), andas an unusual feature of developing nerve (Geren Uzman & Nogueira-Graf, 1957),although in the latter description their subsequent transition into Schmidt-Lantermanincisures does not occur.

An interesting feature of the pseudonodes and some of the reorganized paranodes isthe transition from nodal to incisural characteristics: such a transformation emphasizesthe similarity in ultrastructural organization of these specialized regions of the normalmyelin sheath. The significance of the inclusion of Schwann cytoplasm within thesheath after the lamellae have become continuous at the pseudonode is uncertain, asindeed it is in the Schmidt-Lanterman incisure in the normal fibre: it may be thatthese cytoplasmic strips provide relatively large interfaces for the addition of newmembrane material during remyelination. There was evidence of lamellar disruptionat the transition between pseudonode and incisure, suggesting that this is a labileregion of the sheath. By 60 days post-injection, pseudonodes were no longer observed.It seems likely, therefore, that the cytoplasmic processes are withdrawn as remye-lination proceeds, and/or are incorporated into a remodelled sheath in some way.

The presence of regular transverse thickenings of the axolemma and the increaseddensity of the underlying axoplasm have been cited as morphological specializationspeculiar to the nodal region in normal fibres (Elfvin, 1968). Their association withmany of the loops terminating along the remyelinating internode belies such specificity;it may be that they represent specialized contact sites between the Schwann cell andaxon. It is unlikely that they mark the site of former paranodes, since careful examina-tion of long stretches of axolemma covering axons prior to the start of remyelination,has failed to reveal them.

Two main points have emerged from the present study: (a) the chronic response tothe intraneural injection of LPC 10 mg/ml is one of remyelination as opposed toregeneration, substantiating the claim the LPC produces a primary demyelination(Hall & Gregson, 1971); and (b) certain atypical features have been consistentlyobserved in a small population of fibres in the remyelinating region and junetionalzone which are presumably related to the re-establishment of the Schwann cell/axonrelationship.

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The author was supported by a grant from the Medical Research Council.

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MORRIS, J. H., HUDSON, A. R. & WEDDELL, G. (1972). A study of degeneration and regenerationin the divided rat sciatic nerve based on electron microscopy. II. The development of the'regenerating unit ' . Z. Zellforsch. mikrosk. Anat. 124, 103-130.

RAINE, C. S., WISNIEWSKI, H. & PRINEAS, J. (1969). An ultra-structural study of experimentaldemyelination and remyelination. II. Chronic experimental allergic encephalomyelitis in theperipheral nervous system. Lab. Invest. 21, 316—327.

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(Received 23 January 1973)

472 S. M. Hall

B eftFig. i A—G. Teased, osmicated fibres, 50 days post-injection, K6hler illumination.A-E are of the junctional zone, showing the thinning and ballooning of the myelin sheathof normally-myelinated internodes in their paranodal regions. Complex myelin foldingis present in c and D. F illustrates an unusually short normally-myelinated internode,'intercalated' between 2 remyelinating internodes. As in c and D, the paranodalmyelin profiles are complex. G is of a length of remyelinating internode: carefulthrough-focusing reveals alterations in thickness of the myelin sheath along theinternode. Scale lines indicate 10 /im in A-E and G and 50 /im in F.

Figs. 2-15 are electron micrographs of fibres in the junctional zone and remyelinatingarea, 15-45 days post-injection. Lack of space precludes a more comprehensive presen-tation of material, and micrographs of fibres from 50-240 days have not been included.As has been pointed out in the text, the changes that occurred during this time wereessentially a gradual loss of supernumerary Schwann cells, axon sprouts and pseudo-nodes, and a gradual increase in thickness of the sheath.Figs. 2-5. Transverse sections of fibres in remyelinating area, 20 days post-injectionof LPC.

Fig. 2. Segment of a demyelinated axon, now almost surrounded by cellularprocesses of varying electron density. A small stretch of axolemma (short arrow)remains covered only by basal lamina (long arrow). Note microtubules in paler cyto-plasmic process, x31800.

Fig. 3. The axon (ax) is now completely surrounded by the processes of 2 differentcells: the dark-staining process (arrow) is itself enclosed by paler cytoplasmic processescontinuous with the more extensive mass of Schwann cytoplasm in the lower half ofthe micrograph. Note the difference in the diameter of the collagen (c) at the top right(within basal lamina tube), and top left (outside basal lamina tube), of the micrograph,x 10600.

Fig. 4. Typical remyelinating cellular complex. The thin processes of the super-numerary Schwann cell (iO are associated with numerous small axonal sprouts (a").Some of these sprouts (short arrows) lie free in the space between the SSC and theremyelinating Schwann cell (s) and one (long arrow) lies in a furrow in the remye-linating Schwann cell. Three mesaxons may be observed within the remyelinatingSchwann cell: note asymmetry in the circumferential distribution of the myelin lamel-lae. The entire complex is surrounded by a continuous serpentine basal lamina (A).Collagen (c) is present in the endoneurial space, and between s' and s. x 21200.

Fig. 5. As in Fig. 4, triple mesaxons are present around the axon. The cellularprocesses contain numerous mitochondria, suggesting that this section has been madenear a remyelinating paranode — compare with Fig. 7. x 21200.

Rpmvelination

474 s- M. Hall

Figs. 6—i i are longitudinal sections of nodes of Ranvier in the junctional zone andremyelinating area.

Fig. 6. Typical node of Ranvier in the junctional zone. Compare the herring-bonepattern of the terminal loops in the upper paranode with the immature pattern in thelower paranode, and differences in thickness between myelin sheath (m) on either sideof node. X7950.

Fig. 7. Remyelinating node, in which groups of cytoplasm-filled loops can be seenpeeling off from the compact sheath of both paranodes (arrows). The arrangement inthe lower paranode, where the 2 groups are almost continuous, was seen in severalnormally-myelinated paranodes in the junctional zone. Note numerous mitochondriaand dense bodies in the loops. In later examples of similar nodes where the cytoplasm-filled loops were present, the latter had transformed into Schmidt-Lantermanincisures in the outer layers of the (relatively thicker) sheath, x 10600.

Fig. 8. Paranodal reorganization of normally-myelinated internodes in the junctionalzone. Note occasional redundant folds of basal lamina, x 5000.

Fig. 9. Paranodal reorganization of normally-myelinated internodes in the junctionalzone. The number of lamellae in the compact sheath are reduced by approximately50 % by this transformation from paranode to incisure. x 21 200.

Fig. 10. Normally-myelinated paranode, in which the terminal loops of paranodalcytoplasm are separated from the axolemma by a narrow process of Schwann cytoplasm(arrow), x 33000.

Fig. 11. Paranodal region of a normally-myelinated internode: the terminal loopsof the paranode have been enclosed in a blind loop of myelin, and the whole issurrounded by Schwann cytoplasm, x 10600.

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476 S. M. Hall

Figs. 12, 13. Longitudinal sections of typical pseudonodes. The mode of formation andappearance of these loops of cytoplasm are indistinguishable from that of the remyeli-nating paranodes in Figs. 6 and 7. In Fig. 13, which is a higher magnification of part ofFig. 12, note the transverse thickenings of the axolemma (arrow). Fig. 12, x 12000;Fig. 13, x 48000.

Figs. 14, 15. These micrographs illustrate the transformation from pseudonode intoSchmidt-Lanterman incisure in remyelinating internodes 45 days post-injection. Notethe increase in density of the axoplasm underlying the loops in Fig. 14 (arrow). In viewof the overall good preservation of myelin and axoplasmic organelles, the disorganiza-tion of the loops (#) in Fig. 14, suggests that this is not a fixation artifact, but indicativeof the lability of the entire region, possibly because this is an area in which remodellingof the sheath is occurring. Some disorganization is present at the point of transition inFig. 15. In Fig. 15, note the microtubules in the pseudonodal cytoplasm and in the'incisural' cytoplasm. Fig. 14, x 18550; Fig. 15, x 50000.

Remyelination

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