an electron microscope study

J. Anat. (1987), 150, pp. 277-286 With 5 figures Printed in Great Britain

277

The development of Schmidt-Lanterman incisures: an electron microscope study J. R. SMALL*, M. N. GHABRIEL* t AND G. ALLT* * Reta Lila Weston Institute of Neurological Studies, Middlesex Hospital Medical School, t Department of Anatomy, Charing Cross and Westminster Medical School, London W6 8RF and t Department of Anatomy and Biology as Applied to Medicine, Middlesex Hospital Medical School, London WiN 8AA

(Accepted 25 March 1986) INTRODUCTION

The mode of formation of Schmidt-Lanterman incisures during development of the myelinated nerve fibre has not previously been systematically investigated. While such a hiatus in our extensive knowledge of the myelin sheath may appear surprising, it is quite representative of our very incomplete understanding of the biology of Schmidt-Lanterman incisures (for review see Ghabriel & Allt, 1981). Several studies have been concerned with the morphology of Schmidt-Lanterman incisures in the normal myelinated nerve fibre (Cajal, 1928; Hiscoe, 1947; Robertson, 1958; Blakemore, 1969; Hall & Williams, 1970; Schnapp & Mugnaini, 1975), in Wallerian degeneration (Cajal, 1928; Webster, 1965; Williams & Hall, 1971 a, b; Ghabriel & Allt, 1979a, b) and subsequent regeneration (Hiscoe, 1947; Cooper & Kidman, 1984), in axon-sparing demyelination (Webster, 1964; Hall & Gregson, 1971; Wisniewski & Raine, 1971) and remyelination (Ghabriel & Allt, 1980a, b; Cooper & Kidman, 1984). However in normal development incisures have been investigated mainly in respect of their quantitative relationship with fibre size (Hiscoe, 1947). In contrast to the moderate progress over the last two decades in our knowledge of the morphology of incisures, their functional importance has been the subject of little experimental investigation and hence speculative roles are numerous (Ghabriel & Allt, 1981). If, as has been proposed, incisures are essential for the maintenance of myelin, then they are likely to be present during the earliest stages of myelination. We have therefore determined the stage at which incisures make their first appearance and the mode of their subsequent maturation. MATERIALS AND METHODS

Twelve Sprague-Dawley immature male rats weighing between 9 and 40 g were used in this study. Two rats were killed at each of the following time intervals post partum: 5, 8, 10, 12, 15 and 21 days. Animals were anaesthetised by inhalation of a mixture of oxygen and nitrous oxide, administered at rates of 300 ml and one litre per minute respectively. The sural nerves were exposed in both popliteal fossae and fixed in situ for 10 minutes with a freshly prepared phosphate-buffered mixture of paraformaldehyde (4 %) and glutaraldehyde (0-5 %) (- 1470 m mol, pH 7 4). The nerves were excised and primary fixation was continued in cold fixative (4 C) for 2 hours, followed by washing in phosphate buffer and secondary fixation for 1 hour

278

J. R. SMALL, M. N. GHABRIEL AND G. ALLT

Table 1. Number and type of incisures examined at each time interval in transverse and longitudinal sections Time interval

5d

8d

Sectoral Subcircumferential Circumferential Total

30 -

28 1 3 32

30

15 d

21 d

Total

Transverse sections 5 35 31 5 6 6 3 7 13 18 54 39

4 2 10 16

133 20 36 189

10 d

12 d

Longitudinal sections Asymmetric M o I C Symmetric M

o

C Total

1 2 1 -

-

1 2 6 1 -

1 8 14 1 -

-

2

6 8 5 1

4

2 1

'60

-

4

2

8 1 3 20 23 27 15 27 4 10 M, middle; 0, outer or abaxonal; I, inner or adaxonal; C, complete incisures. For full explanation see Results.

46 106

in phosphate-buffered 1 % osmium tetroxide (pH 7 4). The osmicated nerves were divided into 2-5 mm segments which were dehydrated, embedded in Araldite and orientated for both transverse and longitudinal sectioning. Semithin (1 ,um) sections were stained with toluidine blue and ultrathin sections with uranyl acetate and lead citrate. A total of 295 incisures was exanined by electron microscopy. RESULTS

On the basis of their appearance in transverse section, incisures were classified in relation to their circumferential extent. Using this criterion three groups (Figs. 1-4) were identified: (a) sectoral incisures, involving less than a half of the circumference of the fibres (Figs. 1C, 3 B), (b) subcircumferential incisures, involving a half or more of the fibre circumference (Figs. 2B, 4A), (c) circumferential incisures, involving the whole circiumference of the fibre (Figs. 2D, 4C, D). The number of incisures examined at each time interval is shown in Table 1. In transverse sections, 189 incisures were examined in total and were identified according to their circumferential extent as belonging to one of the above three groups. It was apparent that there was a marked reduction in the number of sectoral incisures in relation to the total with increasing time intervals. Thus sectoral incisures accounted for all incisures at 5 days, most (28 out of 32) at 8 days but only a minority (4 out of 16) at 21 days. Conversely the number of circumferential incisures increased proportionally at later time intervals. Thus by 21 days most of the examined incisures were circumferential. A few subcircumferential incisures were identified at most time intervals. Sectoral incisures showed not only a progressive reduction in their relative number with increasing time but also a change in their morphology. The majority of sectoral incisures at 5 and 8 days, and to a lesser extent at 12 days occupied the whole myelin

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Fig. 2 (A-D). Diagrams of myelinating nerve fibres containing nine (A-B) and fourteen (C-D) myelin lamellae. (A) and (C) are three dimensional appearances of nerve fibres showing developing incisures seen through the Schwann cell surface. The incisure in (A) shows an early diagonal spread in relation to the long axis of the fibre (differential movements between consecutive myelin lamellae) and a circumferential spread around the fibre. A cross section of the fibre is shown in (B) and passes through three cytoplasmic compartments of the incisure. In (C) the incisure shows a complete spiral and a marked diagonal inclination. A cross section at the middle of the cone (arrow) is shown in (D) and displays two turns of the cytoplasmic spiral. Abbreviations as in Figure 1.

sheath radially (Figs. 1 C, 3 B) at the same transverse plane. By contrast at later time intervals the majority of sectoral incisures were present, in a single transverse section, only in the outer or inner lamellae. Semi-serial sectioning confirmed that this change in appearance in transverse section was due to a progressive diagonal spread of the incisuie (Fig. 1 A). Employing their appearance in longitudinal sections, incisures were classified according to their symmetry on both sides of the axon into two groups (Figs. 1 A,

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Fig. 4 (A-D). Transverse sections of nerve fibres 10 days postnatally showing incisures at various stages of circumferential spread, (A) subcircumferential (between arrows), (B-D) circumferential incisures. (B) and (C) are serial sections of the same incisure. (A) x 28000; (B) x 26000; (C) x 26000; (D) x 29000.

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Fig. 5 (A-E). Longitudinal sections of developing fibres at 12 days (A, B) and 10 days (C-E) postnatally showing incisures between arrows: asymmetric inner (A), symmetric complete (B), asymmetric outer (C), symmetric outer (D) and symmetric inner (E). (A) x 24000; (B) x 22000; (C) x 26000; (D) x 28000; (E) x 26000.

283

284

J. R. SMALL, M. N. GHABRIEL AND G. ALLT a progressive and sharp increase in the proportion of symmetric incisures to the extent that all incisures examined at 5 days were asymmetric and at 21 days all were symmetric. In addition there was a gradual increase in the proportion of complete symmetric incisures. Furthermore incomplete incisures were more common in the inner lamellae (adaxonally) at earlier time intervals and among the outer lamellae (abaxonally) at later time intervals (Table 1). Qualitatively, most incisures at later stages contained cytoplasm sufficiently electron-dense to obscure any contained organelles (Figs. 4D, 5B). At earlier stages of maturation the incisural cytoplasm, with a reduced electron density, could be seen to contain organelles including microtubules, filaments, ribosomes (Fig. 3 B) and occasional desmosome-like bands. Mitochondria were not observed. The adjacent external Schwann cell cytoplasm and plasma membrane usually contained coated vesicles and coated caveolae. Both qualitatively and quantitatively the most substantial changes in incisural morphology occurred between 8 and 12 days while incisures at 21 days were almost indistinguishable from those of adult rat sural nerve and nerves were therefore not examined at later time intervals. DISCUSSION

The results of this study can be interpreted on the assumption that there are two distinct mechanisms of incisural development which begin at different stages in myelinogenesis. Thus the variety of incisural appearances described can be related to the formation of either primary incisures or secondary incisures. Primary incisures are found from the onset of myelination and always extend across the whole thickness of the myelin sheath (i.e. complete), they are represented in transverse section by sectoral incisures and in longitudinal section by 'asymmetric' incisures. The interpretation of these appearances is that from incipient myelination onwards there is a failure of both compaction of myelin and extrusion of Schwann cell cytoplasm over radial segments of the myelin sheath. During maturation the primary incisures spread circumferentially around the myelin sheath, becoming first 'subcircumferential' and later 'circumferential', as viewed in transverse section and symmetric' in longitudinal section. By contrast, secondary incisures by definition begin their formation during later stages of myelination in regions of compact myelin, initially occupying only the inner or outer lamellae and therefore appear incomplete radially. On their initial appearance they predominantly involve the entire circumference of the sheath and thus appear in transverse section as 'circumferential' and in longitudinal section as 'symmetric'. Less commonly secondary incisures may appear in a sector of the sheath, but still in regions of compact myelin, and thus appear 'sectoral' in transverse section and 'asymmetric' in longitudinal section. Maturation of secondary incisures is accompanied by radial and circumferential spread to occupy all the layers of the myelin sheath. The spread of primary and secondary incisures involves the separation of previously compact myelin lamellae and the insertion of Schwann cell cytoplasm. Both primary and secondary incisures thus become differentiated into mature incisures characteristic of the adult peripheral nerve, at which stage they become indistinguishable from each other. While the present study is the first systematic investigation of incisural formation during normal development, a mechanism has been suggested previously, involving

Development of Schmidt-Lanterman incisures

285

the transformation of redundant myelin folds (Glimstedt & Wohlfart, 1960; Friede & Samorajski, 1969). We have found no evidence to support this hypothesis and furthermore have commonly identified distinct stages in the maturation of incisures (e.g. sectoral, asymmetric incisures) without any associated redundant myelin folds. Conversely, redundant myelin folds, which were not present in all fibres, were frequently seen not to be associated with identifiable incisures. In remyelination after segmental demyelination a mechanism of incisural formation from internodal myelin terminal loops has been proposed (Hall, 1973). In the present study of incisural formation during ontogeny only very occasionally did we observe internodal myelin terminal loops in relation to developing incisures. The mechanism of incisural formation described in the present study, both for primary and secondary incisures, assumes a degree of plasticity of myelin sheath structure. Circumferential and radial spread of incisures presupposes a separation of myelin major dense lines to incorporate a spiral of incisural cytoplasm and a dilatation of the intraperiod line gaps to form the characteristic spiral of extracellular space. In addition the elaboration of a tight junctional system typical of the differentiated Schmidt-Lanterman incisure (Ghabriel & Allt, 1981) must accompany incisural spread. SUMMARY

The development of Schmidt-Lanterman incisures was investigated in the rat sural nerve during an active phase of postnatal myelination (5-21 days postpartum). Two distinct populations of incisures were recognised and the following nomenclature for their developmental stages is proposed. Primary incisures which appear ab initio in myelination and always extend across the whole radial thickness of the myelin sheath but initially around only part of its circumference. Consequently they appear in transverse section as sectoral incisures (occupying less than half the circumference) and in longitudinal section as asymmetric incisures (involving one side only of the myelin sheath). Secondary incisures appear later, in regions of a compact myelin sheath, initially traversing only part of its radial thickness but commonly occupying its whole circumference. Thus they usually appear in transverse section as circumferential incisures and in longitudinal section as symmetric incisures (involving both sides of the myelin sheath). Less commonly secondary incisures may form in a sector of the myelin sheath but still in regions of compact myelin and thus appear asymmetric in longitudinal section and sectoral in transverse section. Secondary incisures appear mainly adaxonally in the earlier stages examined and mainly abaxonally in the later stages. The maturation of primary and secondary incisures into the radially and circumferentially complete incisure characteristic of the mature myelinated nerve fibre is described. The above mechanisms of incisural formation are contrasted with mechanisms previously suggested to occur during normal development and remyelination and related to the plasticity and ultrastructure of the myelin sheath. We are grateful to Lynn Mackenzie, John Syrett, John Bowles and Derek Couturier for technical assistance and to Jackie Walker for typing the manuscript.

286


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