developmental and pathological changes at the node and paranode in human sural nerves

MICROSCOPY RESEARCH AND TECHNIQUE 34:422-435 (1996)

Developmental and Pathological Changes at the Node and -

Paranode in Human Sural Nerves J. MICHAEL SCHRODER Department of Neuropathology, Medical Faculty of the Rheinisch-Westfalische Technische Hochschule, 52074 Aachen, Germany

KEY WORDS Node of Ranvier, Development, Sural nerve, Axon, Myelin sheath, Paranodal junction, Human

ABSTRACT The nodes and paranodes of peripheral nerve fibers are complex structures that are especially prone to artificial and pathological changes which have to be distinguished from normal developmental changes. Alterations during normal development are mainly caused by an increase in axonal diameter and myelin sheath thickness. The nodal, and paranodal axon diameters in human sural nerves reach their adult values a t 3-5 years of age, simultaneously to the internodal diameter. The ratio between internodal and paranodal axon diameters remain relatively constant, with an average value of 1.8 to 2.0 (range: 1.6 to 2.5). Despite a considerable increase of the number of myelin lamellae, the length of the paranodal myelin sheath attachment zone at the axon does not increase correspondingly, because of (1) attenuation of the terminal myelin loops, (2) separation of some of these from the axolemma, and their piling up in the paranode. Separation of variable numbers of terminal myelin loops from the underlying axolemma results in the formation of the spines on the “double bracelet epineux” of Nageotte, while 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. Artificial changes are caused by insufficient fixation or mechanical stress during excision and further handling (cutting, dedydrating, embedding) of nerves whereas pathological changes may be induced by a multitude of causes. An attempt to classify these changes is presented in Table 2. o 1996 Wiley-Liss, Inc.

INTRODUCTION Developmental changes of peripheral nerve fibers in

man are documented in detailed morphometric studies, especially those concerning the increase in the length and thickness of internodal fiber segments (Behse, 1990; Friede and Beuche, 1985; Gutrecht and Dyck, 1970; Jacobs and Love, 1985; Origuchi, 1981; Schroder et al., 1978, 1988). Axonal diameters of sural nerve fibers were found to increase until the age of about 5 years, showing their most rapid growth during the first months of life. In contrast, there is an asynchronous thickening of myelin sheaths which continues up to the age of about 14-20 years (Schroder et al., 1978, 1988). The process of myelination ends asymptotically similar to other developmental processes and is therefore difficult to determine; the final myelin thickness is probably reached during the third decade. Later on, it may still increase on the account of axonal thickness but this indicates already pathological myelin thickening due to axonal atrophy and secondary, concentric “slippage” of the myelin lamella spiral which could be due either to mechanical or more complex biochemical rearrangement of the bilipid layers of the myelin sheath with all its attachment devices.

Concerning the nodes of Ranvier (Ranvier, 1871/72), some ultrastructural data on normal developing peripheral and central nerve fibers of animals (Geren Uzman and Nogueira-Graf, 1957; Robertson, 1962; Berthold and Skoglund, 1965, 1967, 1968; Berthold, 1968; Wax-

man and Forster, 1980; Wiley-Livingston and Ellisman, 1980; Tao-Cheng and Rosenbluth, 1980a,b; Hildebrand and Waxman, 1984) and man (Gamble, 1966; Hilde- brand and Skoglund, 1967; Dunn, 1970) are available. However, there are only a few morphometrical measurements concerning developmental changes of nodal and paranodal segments in cats (Berthold, 1968, 1974, 19781, and only one systematic study in man (Bertram and Schroder, 1993). Measurements at adult feline nodes (Berthold, 1978) have revealed that the ratio of internodal to nodal axon diameters is almost the same in large as in small fibers. This has also been established for mature and immature fibers in man (Bertram and Schroder, 1993). Axon-caliber increases a t the paranode cause complex rearrangements of the paranodal apparatus of the myelin sheath during additional growth of myelin lamellae. These developmental changes have been studied in a series of sural nerves selected from a large biopsy and autopsy file on which the present study is based. Knowledge of the developmental changes is essential for differentiating normal from pathological and artificial changes (see Table 2).

MATERIALS AND METHODS The nodes of Ranvier in a series of 43 human sural

nerves from 5 preterm and 3 mature neonates, 26 in-

Received February 13, 1995; accepted in revised form March 20, 1995. Address reprint requests to Dr. J.M. Schroder, Institut fur Neuropathologie

der RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany.

0 1996 WILEY-LISS, INC.

ALTERATIONS AT THE NODE OF RANVIER 423

fants, and 9 adolescents or adults were investigated by light and electron microscopy (Bertram and Schroder, 1993). The material was obtained in 27 cases by biopsy and, because of the low frequency of nerve biopsies par- ticularly in the perinatal age groups, in 16 cases by autopsy. Since the number of infantile cases suitable for this study was still small, some cases with neuro- muscular disorders affecting solely the motor system and a few cases with borderline or sparse changes were also included, whereas otherwise, cases with no known diseases of the peripheral nervous system were selected. In addition, electron micrographs were obtained from a non-selected series of 536 sural nerves studied for diagnostic purposes by electron microscopy (out of a larger, light microscopically investigated series of 3,757 nerve specimens) to illustrate representative pathological alterations.

The nerve specimens were prepared using standard methods (Schrder et al., 1978). They were fEed in 0.1 M phosphate-buffered 3.9% glutaraldehyde, cut into pieces approximately 2 mm in length, postfixed in a 0.1 M phosphate-buffered solution of 2% osmium tetroxide, dehydrated, and embedded in epoxy resin. Semithin sections about 1 pm in thickness were cut with glass knives, and were stained with Toluidine blue or para- phenylendiamine. Ultrathin sections were cut using a diamond knife, mounted on copper grids, and con- trasted with uranyl acetate and lead citrate.

For ultrastructural investigations, a Philips 400T electron microscope (Eindhoven, The Netherlands) was used. Nodes of Ranvier with symmetrical appearance cut a t the maximal diameter of the axon were evaluated on electron micrographs of well-oriented longitudinal sections.

RESULTS Developmental Changes

Light microscopic measurements of nodal, paranodal, and internodal axon segments of the five largest myelinated nerve fibers in each nerve reveal an almost proportionate caliber increase at all three sites during the initial 3-5 years of age (Table 1). As shown in Figure 2, there is a rapid growth of paranodal diameters [D(P)I before and during the first year of age, fol- lowed by a period of less rapid growth, until adult values of about 3.5-4 pm are reached at the age of 2-3 years. The developmental curve of the axonal diameter at the paranode and at the internode [D(I)l can be characterized by an exponential function according to the formula y = a[l-exp(-h.x)l+ b [y = D(P), D(I).in p.m, x = (age-4) in months]. The non-linear regression analysis using this model yields high rnl values of 0.94 for paranodal and 0.89 for internodal calibers, indicating that the regression lines presented in Figure 2 significantly describe the change of axonal calibers with age (for y = D(P): a = 2.45 pm, b = 1.29 pm, X = -0.102 months-’; for y = D(1): a = 4.33 pm, b = 2.61 km, A = 0.119 months-’; P = 0.05). There is a trend toward a slight reduction of internodal diameters after the age of about 200 months, but linear regression analysis does not establish a significant decrease at the period studied between 200 and 600 months (r = -0.44 and a

= -0.0015 with P = 0.32 for H,: a # 0) (Bertram and Schroder, 1993).

The ratio of internodal to paranodal diameters, which varies considerably within the perinatal age group, tends to decrease slightly with age. However, this trend is not statistically significant (P = 0.13) within the age interval investigated.

A linear relationship between internodal and paranodal diameters can also be observed when comparing fibers of different sizes in different age groups. The parameters of the regression lines [D(P) = a.D(I) + bl do not show any significant change with age so that the relationship of paranodal to internodal diameters appears to be independent of the fiber caliber group and of age (for statistical details see Bertram and Schroder, 1993).

The results of electron microscopic-measurements are listed in Table 1. Despite a considerable increase of myelin sheath thickness (or number of lamellae) during development, the length of the myelin attachment zone does not elongate correspondingly, when fibers of the same caliber group are compared (Fig. 3). The myelin attachment zone of fibers of the smaller caliber group, however, tends to be shorter in comparison with those of larger fibers of the same nerve. The largest attachment zone that we observed measured approximately 7.8 pm.

The length of the nodal axon segment shows no clear developmental changes, with slightly lower values in fibers of smaller caliber (Table 1). The bulging of the axon at the node shows marked asymmetry and variations in different caliber groups, and lacks any obvious change with age. The height of the nodal gap also varies considerably and shows no clear increase after 2 months of age.

The general ultrastructural organization of nodes of Ranvier in nerve fibers of the perinatal age group cor- responds to that of adult nerve fibers. This implies chains of desmosome-like structures, tight junctions between adjacent terminations of myelin lamellae, and the spiral axoglial junctional complex, characterized by the so-called transverse bands.

However, certain features in the perinatal age group are not seen in adult nodes of Ranvier. As shown in Figure la, transverse bands of single terminal loops are missing at various sites, such as at the center of the termination zone or at terminations of the innermost lamellae, without removal of the corresponding loops from the axon. The transverse bands of the adjacent loops, however, appear to be well developed. Some- times, deficient transverse bands have been noted at several neighboring loops (Fig. la). The lamellae of terminal loops with missing transverse bands also may lack paranodal desmosome-like structures, although they are prominent in other myelin loops of the same paranode (Fig. la).

At later stages in large nerve fibers, long opposing rows of terminal myelin loops are separated from the axolemma, thereby piling up into the surrounding myelin (Figs. la, 5, 6a). This phenomenon is the ultrastructural correlate of the spines on Nageotte’s spiny double bracelets (“double bracelet 6pineux”). The number and size of these structures are significantly higher

424 J.M. SCHRODER

TABLE 1. Results of electron microscopic measurements at nodes of Ranuier in fibers of different sizes and at different ages in well oriented longitudinal sectioms'

N A C L(P) D(P) D(M) L UN) D(N) NB NG LP/DM LP/L 4

6

10

32

39

-2

0

2

62

204

1

1

m

m

1

1

d s

8

1

1

slm

slm

1

1

8

Nm 8

8

3.3- 3.85 4.52- 5.39 3.4- 3.86 3.17- 4.6 3.88- 4.35 4.5- 5.55 3.35- 3.78 2.54- 3.57 4.6- 5.39 4.28- 4.76 2.86- 3.65 3.73- 3.97 3.49- 4.6 3.89- 4.76 2.54- 3.57 4.13- 5.9 3.75- 5.9 4.2- 5.5

1.3- 1.9 1.43- 2.14 1.37

1.58

2.05 2.7 2.14- 2.06 1.15- 1.61 0.71- 0.87 2.14- 3.5 2.38

1.27- 1.51 1.27

3.85- 4.36 4.04- 5.55 1.47- 2.18 2.78- 3.69 1.51- 1.9 1.19- 1.55

0.25

0.24

0.2

0.17

0.39

0.32

0.24

0.16

0.47

0.47

0.22

0.24

1.11

1.03

0.2

2.1

0.46

0.36

23

20

15

13

28

24

17

15

40

39

20

19

91

84

16

122

38

29

0.93- 1.37 1.35- 1.47 1.37- 1.43 0.95

0.81- 1.37 1.58- 2.14 0.75- 0.93 1.98

0.95- 1.19 0.71- 1.1 1.59- 1.67 1.11- 127 1.19- 1.47 0.95- 1.03 1.35- 1.59 1.19- 1.35 1.35- 1.51 0.79- 0.99

1.43 - 1.68 1.47 - 1.74 1.26- 1.83 1.59- 2.1 2.05- 1.68 2.06- 1.98 1.15- 124 0.79- 0.87 2.06- 226 2.22- 2.5 1.27- 1.63 1.19- 1.78 3.69- 3.89 3.49- 3.73 1.51- 1.59 2.58- 2.78 1.43- 1.75 1.15- 1.23

0.12- 022 0.08- 0.16 0.23

0.16- 024 0.06- 022 0.0- 0.04 0.0- 0.09 0.0- 0.04 0.0- 028 0.12- 022 0.08- 028 1.16- 0.59 0.0- 02 0.16- 0.24 0.0- 0.14 0.0- 02 0.14- 022 0.0- 0.08

0.19- 028 0.16- 0.32 0.17- 0.37 0.24- 0.6 0.06- 028 0.2- 0.36 0.12- 0.43 0.08- 0.16 0.48- 0.79 0.55- 0.79 0.16- 0.4 0.55- 1.11 0.48- 1.15 0.36- 0.99 0.36- 0.71 0.46- 0.95 0.51- 0.67 0.32- 0.48

13.2- 14.5 18.83- 22.46 17.0- 19.3 18.56- 25.6 9.95- 15.4 14.06- 17.43 14.0- 15.75 16.39- 23.03 9.79- 11.47 8.92- 16.54 13.0- 14.59 15.54- 16.54 3.14- 4.14 3.78- 4.62 12.8- 18.03 2.0- 2.95 7.76- 12.8 11.67- 15.2

0.14- 0.1 7 0.23- 027 0.23- 026 0.24- 0.34 0.14- 0.1 7 0.19- 023 0.2- 022 0.17- 0.24 0.12- 0.14 0.11- 021 0.14- 0.18 0.2- 021 0.04- 0.05 0.05- 0.06 0.16- 022 0.03- 0.05 0.09- 0.16 0.15- 0.19 . .. . ~.

'All measurements are presented in pm. Despite considerable increase of myelin sheath thickness and of the number of lamellae during development, the length of the myelin sheath attachment zone did not elongate correspondingly. The length of the nodal axon segment did not show any clear changes, with slightly lower values in fibers of the smaller caliber group. The bulging of the axons at the node showed marked variations in different caliber groups but remained constant, lacking obvious increaea with age. N = case number; A = age in months, C = fiber caliber group (1 = large, m = medium, s = small); UP) = length of myelin sheath attachment zone; D(P) = paranodal diameter of axon; D(M) = myelin sheath thickness; L = number of lamellae; UN) = nodal length; D(N) = nodal diameter; NB = maximal nodal axonal bulging (both sides); NG = height of nodal gap.

in the large nodes of adults than in those of young individuals.

Pathological Changes The most common changes of the node and paranode

are listed in Table 2. One of the most frequent changes is swelling of terminal myelin loops. This change, however, may also be artificial (Fig. la). Fine vesicular disintegration (Fig. lb) of paranodal myelin loops is considered a reliable pathological change whereas formation of membranous whorls (multilamellated, myeloid bodies) again may be real or artificial (see Fig. 6a). Various forms of separation of the loops from the axolemma may indicate incipient paranodal or segmental demyelination; however, developmental changes and piling up of terminal lamellae into the compact myelin sheath have to be distinguished. When Schwann cell processes are seen between the axon and terminal myelin loops (Fig. lb) this is considered a real, not an artificial change.

Another severe pathological change consists of an incorporation of granular material into the intraperiod

line (the former extracellular space) between terminal myelin loops separated from the axolemma at the paranode (Fig. 6a,b). A similar change may be seen at Schmidt-Lanterman clefts in inflammatory neuropathies (Schroder and Himmelmann, 1992).

Following remyelination, different alterations of myelin attachment to the axolemma may be seen. There may be variable distances between the attachment zones of adjacent terminal myelin loops (Figs. 5a, 8a- el. This change is designated as a "pseudonode" when Schwann cell cytoplasm is incorporated between the otherwise compacted myelin lamellae as it is seen in Schmidt-Lanterman clefts (for references see Hirano, 1984). One of the most important and common changes at nodes consists of an asymmetry of paranodes due to remyelination of a segmentally demyelinated internode adjacent to a normal one (Fig. 712). This is a relatively, not an absolutely, reliable sign of segmental or paranodal demyelination and remyelination and nearly always of high diagnostic significance. On very rare conditions, the transitional zone of axonal degen- eration and regeneration may be met a t a node of Ranvier resulting in a similar situation of a contact


Fig. 1. a: Five-month-old infant succumbing from glomerulone- phritis. Several terminal myelin loops are separated from the axolemma (arrowheads), a t one site showing desmosomes (big arrow). Some loops are artificially swollen (s). There are no transverse bands at the terminal loops adjacent to the node (small arrows). b Cockayne syndrome in a 5-year-old girl. The innermost myelin lamella sepa- rates 6 other terminal loops from the axon, indicated by asterisks between the arrowheads. Another group of lamellae is elevated adjacent to a fine vesicular type of focal degradation (V). Other terminal loops are piled up into the myelin sheath in a regular fashion. There is a cleft-like structure including some glycogen granules overlaying

a single mitochondrion in the abaxonal cytoplasm of the Schwann cell. A small membranous cytoplasmic body is seen in the axoplasm (which may have been induced by prolonged fixation in glutaraldehyde with phosphate buffer or in buffer alone). c: Normal sural nerve of a 17-year-old boy with necrotizing myopathy of unknown cause. Double rows of terminal loops are seen at several sites separated from the axolemma thereby symmetrically piling up into the surrounding myelin (ultrastructural correlate of the spines on the “double bracelet Bpineux” of Nageotte). Artificial warping of myelin lamellae presumably caused by minor mechanical squeezing is indicated by an arrowhead. a x , x 40,000.

426

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9-

J.M. SCHRODER

-

-

Age in months Fig. 2. Light microscopic measurements of internodal [D(I)I and paranodal [D(P)I diameters of the

largest sural nerve fibers in relation to age. In both axonal segments, an almost parallel caliber increase is seen with advancing age, the non-linear regression analysis indicating exponential caliber growth during the life interval investigated, according to the formula y = a~[l-exp(h.x)l + b. (Reproduced from Bertram and Schrtider, 1993, with permission of Springer-Verlag GmbH & Co. KG.)

between a thick normal and a thinly remyelinated internode.

Other Pathological Changes Further pathological aspects a t the node of Ranvier

listed in Table 2 but not illustrated in the present article, especially axoglial dysjunction in human diabetic nerves, are presented by Giannini and Dyck (see article this issue). Loosening of the myelin sheaths a t the paranode as it is seen in dysproteinemic neuropathies is discussed by J. Jacobs (see article this issue). Changes of the axon-Schwann cell network indicating processes of degradation of axonal components by the adaxonal cytoplasma of the Schwann cell at the tran- sition zone between the paranode and the internode are described in detail by Gatzinsky (see article this issue).

Artificial Changes at the Node of Ranvier Artifacts at the paranode are easily identified in au-

tolytic, swollen nerves from autopsy cases if the corpses were not cooled or autopsied shortly after death. More difficult is the interpretation of membranous whorls at the site of the paranodes where they occur as fre-

quently as at Schmidt-Lanterman incisures. There is no doubt that membranous whorls (or “myeloid bodies” or “membranous cytoplasmic bodies,” MCBs) can occur as a truly pathological phenomenon, e.g., in chloro- quine neuromyopathy (Schroder and Himmelmann, 1992). The high lipid content of the Schwann cell cytoplasm at paranodes and Schmidt-Lanterman clefts is obviously prone to physicochemical precipitation of proteolipids although this is seen in many cells or even extracellularly at a large number of conditions.

DISCUSSION Knowledge of normal developmental changes is im-

portant for distinguishing pathological and artificial alterations at nodes of Ranvier in peripheral human sural and other nerves. The study of Bertram and Schrii- der (1993) on the development of internodal, paranodal, and nodal axon segments in large sural nerve fibers in man has revealed a simultaneous and proportionate caliber increase that is most rapid during the prenatal and the first postnatal months. Adult values of axon diameters are reached by the age of about 3-5 years, confirming earlier measurements on internodal axon


I

2 0 4

I

5 -

4 -

3 -

2 -

1 -

0

2 0 4

0 ' I I I I I I

0 20 40 60 80 100 120 140

Number of myelin lamellae

Fig. 3. Relationship between the length of the paranodal myelin sheath attachment zone [L(P)I and the number of myelin lamellae (L) of the myelin sheath in corresponding fibers (compare Table 1). The bars indicate the range between the lowest and the highest value measured on either side of a single node. The numbers labelling the burs indicate the age of the studied individuals in months. In contrast

segments (Schroder et al., 1978, 1988). The regression line for the measurements of the axon caliber at the paranode is non-linear and shows an exponential increase with age which is of high statistical significance during the period investigated (Fig. 2). A similar, significant exponential increase of internodal diameters has also been established, although a slight, statistically non-significant reduction is visible after the age of about 200 months.

The ratio of internodal to paranodal calibers remains constant during development, except for a slight but statistically non-significant decrease. True paranodal caliber growth is apparent from our measurements as- suming that the largest fibers found in immature nerves are also the largest subsequently. There are convincing arguments for this hypothesis: (1) Gutrecht and Dyck (19701, who have evaluated fiber diameters in infantile human sural nerves, have noted a bimodal distribution of the calibers, the second peak moving toward higher values with increasing age. (2) Adult nerves show a positive correlation between internodal

to considerable addition of lamellae during development, the length of the paranode does not elongate correspondingly when fibers of the same caliber group are compared. The paranodes of the small fibers within one sural nerve, however, tend to be shorter than those of the respective large fibers.

length and fiber diameter; the number of established internodes generally does not increase during development (Boycott, 1903; Vizoso and Young, 19481, large and small fibers being subject to the same growth rate in total length. Since the absolute number of internodes of large fibers is lower than that of smaller fibers, myelination in large fibers must thus have started earlier. According to Duncan (1934), and Peters and Muir (19591, myelination usually begins when axons have reached a caliber of about 1 pm, so that those fibers that are the first to be myelinated correspond to the largest fibers in mature nerves.

A comparison of internodal and paranodal axon calibers in fibers of different sizes shows a significantly linear relationship in all six cases analyzed (Bertram and Schroder, 1993). The fact that the regression analysis does not show any significant change with age indicates a constant relationship between paranodal and internodal diameters, independent of both fiber size and age.

In nodes of Ranvier of developing sural nerves, we

428 J.M. SCHRODER

Fig. 4. a-c: Neuropathy with liability to pressure palsy in a 63-year-old man. Abnormal hemi-node with a demyelinated internode on the left side and separation of terminal myelin loops from the axon by uncompacted Schwann cell processes (asterisk) on the right, shown in different planes of section. It is not clear whether this change is still reversible or indicates incipient demyelination. a, x 13,000; b, X 42,000; c, ~50 ,000 .

have repeatedly observed the absence of transverse bands at isolated or a t several adjacent terminal loops of individual paranodes not removed from the axolemma. This has only been detected in young individuals with no known neurologic disorder, and not in normal adults, indicating that this is indeed a normal developmental phenomenon.

Deficient transverse bands have frequently been observed at the innermost terminal loops, as noted also by Berthold (1968) in developing feline nerves. Since myelination progresses by the spiral movement of the

inner Schwann cell “tongue” around the axon (Speidel, 1964; Dunn, 1970; Tao-Cheng and Rosenbluth, 1980a,b, 1982; Sims et al., 1988; Schroder et al., 1988), the absence of transverse bands at this site probably indicates delayed synthesis following spiral growth of the innermost process during myelination. This assump- tion is consistent with the observation that transverse bands are missing at the initial stages of myelination (Tao-Cheng and Rosenbluth, 1982) and at the innermost loops in nerves with ongoing remyelination (Allt, 1969). Since many transverse bands are nevertheless

Fig. 5. a,b Myotonic dystrophy with peripheral neuropathy in a 40-year-old female. A large, inner myelin loop projecting into the axoplasm is labelled by an M, an outpouching of the axolemma into the paranode by an A. The latter is filled with normal and abnormal organelles such as mitochondria, free or membrane-bound glycogen, and multilamellated (“myeloid) bodies. The nodal axoplasm, shown at higher magnification in b, contains numerous vesicles, tubules,

neurofilaments, mitochondria, and multilamellated (“dense”) bodies presumably of lysosomal origin. There is no axon-Schwann cell network. On the lower left side, the terminal myelin loops are discontinuously connected to the axolemma with some lamellae terminating more distant to the node (arrowhead). Only very few mitochondria are apparent in the abaxonal cytoplasm of the Schwann cell. a, x 9,000; b, X 21,000.

Fig. 6. 4 b Adrenomyeloneuropathy (clinical diagnosis without typical cytoplasmic inclusions) in a 59-year-old male. There is axo- plasmic outpouching at two sites (arrows) and “extracellular” deposition of a granular material between elevated terminal myelin loops at two other sites (arrowheads). The latter are shown at higher magnification in b. The axon shows on the right side of the node (presumably

proximal site) numerous vesicular or tubular profiles whereas on the left much fewer organelles, predominantly dense bodies, are apparent. A multilamellated (“myeloid”) body is seen among the terminal loops in the lower left part of the parancde. Only very few mitochondria are included in the nodal cytoplasm of the Schwann cell. a, x 11,600; b, X 62,000.

ALTERATIONS AT THE NODE OF RANVIER 43 1

TABLE 2. Classification of developmental, pathological, and artificial changes at the no& of Ranvier

Developmental changes Separation of single or multiple terminal myelin loops

from the axolemma associated with Loss of transverse bands Loss of tight junctions between neighbouring terminal

Loss of desmosomes loops

Attenuation of terminal myelin loops Piling up of opposing terminal myelin lamellae

(“ear-of-barley” pattern) Pathological changes

Finally leading to paranodal or segmental demyelination Swelling

Microvacuolation of terminal myelin loops (Fig. lb) Vesicular swelling (may be artificial)

Precipitation of multilamellar “myeloid membranous bodies (“whorls”) (may be artificial) (Figs. 6a, 7c)

Detachment of terminal myelin loops from the axon (“axo-glial dysjunction”) (may be developmental) (Fig. lb)

Incomplete with or without intervening macrophages Complete with or without subsequent paranodal or

segmental demyelination Undercoating of individual terminal myelin loops by

Schwann cell processes (Fig. lb) Loosening of myelin lamellae (enlargement of the

Deposition of granular substances between terminal

Invasion by mononuclear cells (macrophages,

Axonal outpouching into the paranode (Figs. 5a,b, 6a) Abnormal cytoplasmic inclusions Nodal deposition of antibodies

Attenuation of paranodes (Fig. 7c) Discontinuous attachment of terminal myelin loops

along the axon (Fig. 5a; 8) Formation of pseudonodes Non-compaction of myelin loops

intraperiod line)

myelin loops (extracellular material at the site of the intrapericd line) (Fig. 6)

lymphocytes)

Following remyelination

Artificial changes Swelling of myelin loops [can be real, non-artificial (Fig.

Warping or other mechanic deformations of myelin

Precipitation of multilamellar, myeloid bodies (can be real,

la)]

lamellae (Fig. lc)

non-artificial) (Figs. 6a; 7c)

well differentiated at inner loops in developing nerves, an intermittent course of myelination may be assumed.

On the other hand, the absence of transverse bands of individual terminal loops has also been detected at various other sites of the attachment zone of the myelin sheath in the present material and in feline nerves (Berthold, 1968). Comparable nodes of large nerve fibers in infants (Fig. la) and adults (Figs. 6,7) show an increasing number and size of the double-rowed piles of terminal loops separated from the axolemma; these are known to be the ultrastructural correlate of the “spines” on the “double bracelet 6pineux” of Nageotte (1911). Ultrastructurally, the symmetrically piled up myelin terminals may show an “ear-of-barley” appearance (Thomas et al., 1993). The absence of transverse bands, especially adjacent to previously separated loops, appears to represent early stages of this piling up and requires secondary separation of terminal loops that had previously been attached to the axolemma.

The physiological significance of the piled-up myelin lamellae, although they are regularly observed in large nerve fibers, has not been discussed in the literature. As illustrated in Figure 3, the length of the paranodal myelin sheath attachment zone does not increase pro- portionately despite considerable apposition of lamellae during development. This limitation in the length of the attachment zone results from (1) attenuation of the terminal loops, and (2) separation of numerous lamellae from the axon. Teleologically, this limitation in length of the paranode may be of advantage because there is caliber constriction at this axon segment (Ber- tram and Schriider 1993). Such a constriction may, at least in part, result from the transverse bands helically surrounding the axon and attaching the myelin sheath to the axon. It may be concluded from the investigations of Cooper and Smith (1974) or Stanley et al. (1980) that a longer constricted axon segment impedes axonal transport or causes lower conduction velocity.

Dissolution of transverse bands at individual loops not yet removed from the axolemma has also been described previously in pathological conditions as a fea- ture of incipient paranodal demyelination following, e.g., cyanide-intoxication (Hirano and Zimmerman, 19711, injection of trypsin (Yu and Bunge, 1975), appli- cation of serum from Guillain-BarrB patients (Hirano et al., 19711, experimental allergic neuritis (Allt, 19751, or chronic endoneurial ischemia (Sladky et al., 1991) and diabetic neuropathy (for references see Gi- annini and Dyck article this issue).

The developmental expansion of the constricted paranodal axon segment implies complex adaptational re-arrangements of paranodal myelin lamellae. One possibility for adaptation would be the simultaneous increase in the circumference of the axolemma and of the terminal myelin loops. This would be consistent with the observed maintenance of desmosomes and tight junctions during development. However, if the geometry of a whole fiber segment including the con- tinuity of the paranodal with the corresponding internodal parts of lamellae is considered, the adaptation of the paranodal myelin sheath to axonal expansion can be more convincingly explained by the gliding of the terminal loops simultaneously with internodal slippage of myelin lamellae (Bertram and Schroder, 1993). Slippage of myelin lamellae as a mechanism of adjustment of the myelin sheath to axonal expansion is accepted for the internodal (Friede and Martinez, 1970; Friede and Miyagishi, 1971) and also discussed for the paranodal parts of lamellae (Jones and Cavanagh, 1983), although a more complex rearrangement of lipid molecules along the bilipid membrane may be involved (Yoshikawa et al., 1990).

Gliding of terminal loops in relation to the axolemma and to each other may be impeded by mechanically stabilizing junctions, which therefore would have to be dissolved. Since most of the transverse bands, tight junctions, and some desmosomes are well preserved during the age-period studied, a discontinuous course of development of myelin with relatively short and therefore barely detectable growth phases may be assumed. Indeed, there is some evidence for an intermittent course of myelination and therefore presumably

Fig. 7. The same case as in Figure 6. Type I node (according to the classification of Phillips et al., 1972) with terminal myelin loops a p proaching the axon at a low angle (a) and type II node (b) with myelin loops approaching the axon at a steep angle; both types of nodes are considered normal. Cytoplasm of the Schwann cell is incorporated into the outer parts of the paranodal myelin sheath (arrows) containing an increased number of glycogen particles. A minor axon-Schwann cell network is seen in the lower part of the nerve fiber in a (arrowheads).

Tubular and vesicular components of the axons are ac-cumulated above the nodes in a and b, presumably indicating the proximal sites in respect to the perikaryon. Glycogen-like granules are more frequent in the distal paranodal portion of the axon. The nodal segment of the axon is unusually dense in a. c: Fkmyelinated internode with thin myelin on the right, opposite to a presumably pre-existing thick myelin sheath on the left. There are multilamellated (“myelinoid”) figures in the axon and in the myelin sheath. a, x 8,000; b, x 12,000; c, x 11,000.

Fig. 8. a-e: Peripheral neuronopathy in a 38-year-old male with inclusion body myositis. Terminal myelin loops are discontinuously attached to the axon at various sites (arrowheads) some of which are shown at higher magnification in b-e. Otherwise the myelin sheath appears to be quite normal although the Schmidt-Lanterman incisures are somewhat irregularly distributed. a, x 13,000; b, 48,500; c,d, x 40,000; e, x 42,000.

434 J.M. SCHRODER

for fiber development in general. Analysis of transverse sections of developing myelin sheaths has revealed, that the external "tongue" process and the in- ternal mesaxon are not positioned randomly. Instead, they show a definite tendency to be located in the same quadrant of the sheath (Bertram and Schroder, 1993). This suggests that myelin development is intermittent with additional turns being formed by brief cyclic bursts, rather than by continuous growth activity. Cy- clic growth appears to be characteristic for nerve fiber development, including axonal expansion. Our observations are compatible with previous findings in developing central nerve fibers (Peters, 1964). Transverse band dissolvement a t several neighboring loops possi- bly represents an initial or final stage of growth and adaption.

By experimental destruction of transverse bands, changes of the electrical potential caused by rapid so- dium currents can be abolished (Mueller-Mohnssen et al., 19741, presumably because of liberation of paranodal potassium channels (Chiu and Ritchie, 1980, 1981; Binah and Palti, 1981; Smith et al., 1982). The functional importance of the integrity of the paranode has also been established by studies showing that even minor lesions of the paranodal myelin are effective in slowing impulse conduction (Koles and Rasminsky, 1972; Rasminsky and Sears, 1972). Furthermore, a complete conduction block can be caused not only by advanced paranodal demyelination (Ochoa et al., 1973) but also by slight paranodal changes (Shahani and Sumner, 19811, presumably because of shunts in the transverse-band-limited paranodal spiral pathway, thereby lowering its impedance. Conduction block has also been noted in association with nodal and paranodal deposits of anti-GM1 antibodies in a human sural nerve (Santoro et al., 1990). However, the temporary paranodal changes during normal development as described in the present study do not appear to be severe enough to cause significant functional deficits or to contribute essentially to functional immaturity of developing nerve fibers.

No systematic classification of ultrastructural pathological changes at the node or paranode is available in the literature attributing the various changes to certain etiological conditions. An attempt to list these changes in a systematical order (Table 2) must therefore be preliminary and awaits further differentiation and explanation. However, there is no doubt that the number of changes at the node of Ranvier may be large, but it is certainly limited, whereas the number of different causes for these changes appears to be indefi- nite.

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