asymmetry of the node of ranvier - journal of cell...

J. Cell Set. 3, 341-356 (1968) 3 4 I

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ASYMMETRY OF THE NODE OF RANVIER

P. L. WILLIAMS AND R. KASHEF*Department of Anatomy, Guy's Hospital Medical School, London, S.E. 1

SUMMARY

The nodal constrictions of Ranvier in normal limb nerves of mammals are bounded byasymmetric paranodal bulbs, the proximal bulb (nearer the cell body) being larger in alldimensions. This investigation was undertaken to determine whether the asymmetry resultsfrom partial damming of proximo-distally flowing axoplasm at the nodal constriction, orwhether other features of local growth patterns are more relevant.

The degree of asymmetry was estimated on teased, osmicated fibres from normal immatureand mature limb nerves to skin and muscle and on ventral nerve roots. Estimates were alsomade on the central processes of dorsal root ganglionic cells with their contrasting direction offlow, after alteration of growth patterns in regenerates of crushed immature and mature nerves,and in the recurrent laryngeal nerve which pursues an exceptional course in relation to sur-rounding tissues. A polarization of asymmetry with larger proximal bulbs was found in un-complicated limb nerves and after simultaneous regeneration and limb growth followingcrushing of immature nerves. Mixed populations (that is, with no preferred direction of asym-metry and often symmetrical bulbs) were found in dorsal and ventral nerve roots and the apicalrecurved segment of the recurrent laryngeal, whilst the mature regenerates closely approachedthe symmetrical condition. The descending and ascending limbs of the recurrent laryngealnerve showed a reversal of polarization with respect to the cell body, but similar with respect tothe cephalo-caudal body axis. It was concluded that damming of directionally flowing axo-plasm was not causally related to the formation of asymmetric bulbs. The overall interstitialgrowth pattern of limb nerve elongation as revealed by internodal distance studies contrastswith the differential growth and maturation gradients shown by the various limb segmentsthrough which the nerve passes. These differences result in a relative movement between themyelinating and elongating Schwann cell and the surrounding limb tissues. The movementsare considerable and unidirectional throughout most of the limb, the Schwann cells becomingrelatively further removed from the limb apex, whilst the movements minimize at the limb rootand apex. It is suggested that the altered mechanical conditions operating at the 'advancing'and 'trailing' ends of the cell are related to the dimensional differences of the paranodalapparatus comprising the bulb at each end of the cell, and secondarily result in asymmetricnodes of Ranvier.

INTRODUCTION

The first recognition and account of the periodic (nodal) constrictions shown bymammalian peripheral myelinated nerve fibres (Ranvier, 1871, 1873, z^7^) includeda description of bulbous dilatations of fibre contour in the immediate paranodalregions. Each bulb presented a longitudinal grooving in surface view which cor-responded to an underlying regular folding of the myelin sheath which was seen intransverse section. In one illustration, the two bulbs adjacent to a single node wereshown as asymmetric structures, all dimensions shown (including myelin sheath

• Present address: Department of Anatomy, School of Medicine, University of Iran,Teheran, Evin, Iran.

22 Cell Scl. 3

342 P. L. Williams and R. Kashef

thickness) of one bulb being less than those of its neighbour. Further dimensionalstudies of this asymmetry awaited an analysis by Lubinska & Lukaszewska (1956), whoexamined teased fibres from fixed peripheral nerve trunks. In this situation theyaffirmed the presence of asymmetric bulbs which, further, showed an overall polaritywith the bulb of larger dimensions sited proximal to the node, i.e. nearer to the cellbody of origin of the axonal process. This appearance they compared with the dilata-tion and 'nodal damming' of some fibres proximal to an experimental constrictionwhich had been demonstrated by Weiss & Hiscoe (1948) and used in support of ahypothesis of proximo-distal flow of cytoplasm occurring in neurites. Apparentsimilarities between the two sets of observations led Lubinska & Lukaszewska tosuggest that partial damming of distally flowing axoplasm might occur in normal nervesat the mid-nodal constriction and result in a proximal bulb of larger dimensions.Later investigations by Lubinska (1958), however, concerning the morphology ofnodes between normal internodal segments and adjoining, short, intercalated seg-ments led her to modify this view and to consider that the causation of normal asym-metry must await further researches.

Some authors (Causey & Palmer, 1952, 1953; Causey, i960) remained unimpressedby the degree or significance of the nodal asymmetry, but others (Williams & Kashef,1961; Williams & Landon, 1963; Kashef, 1966) have confirmed its presence in manysites in both fixed and fresh sections and teased preparations and recently in vivo usingtrans-illumination and incident illumination techniques (Williams & Wendell-Smith,unpublished observations). Further, Williams & Landon (1963) pointed out that theparanodal bulb was not to be regarded as a simple dilatation of the axon and its sheaths,but comprised a complex series of alterations of form and content of both axon andsatellite cell. This they termed the ' paranodal apparatus' and suggested that it formedpart of a highly ordered two-cell interchange system at the node (Landon & Williams,

The present investigation was undertaken further to examine the relative signifi-cance of proximo-distal flow patterns or features of local growth patterns as causativefactors in the genesis of nodal asymmetry. To this end, asymmetry was analysedquantitatively in normal peripheral nerves and ventral roots, in situations wheredivergence of flow direction is to be expected, that is, in the central and peripheralprocesses of dorsal root ganglionic cells, in situations with altered growth patternsduring regeneration after crush lesions of young and mature nerves and finally wherea nerve follows an exceptional course during growth, for example, the recurrentlaryngeal nerve.

MATERIAL AND METHODS

Specimens of mature normal nerve were obtained from 6 adult laboratory stockrabbits. Twelve specimens of each of the following were used: the nerve to the medialhead of the gastrocnemius muscle (N.G.M.) from the distal half of the thigh, the suralcutaneous nerve in the thigh and leg, lumbar dorsal nerve roots central to the ganglion,and lumbar ventral nerve roots. Two neonatal rabbits provided 4 normal immature

Asymmetry of node of Ranvier 343

sciatic nerves and their branches. Four mature rhesus monkeys provided 8 furtherspecimens of the N.G.M. and sural nerves and 4 specimens of the left cervical vagusand its recurrent laryngeal branch. The latter were divided into descending, recurved(related to the aortic arch) and ascending parts, before processing.

In 4 adult rabbits and 4 neonatal rabbits, the left sciatic nerve was exposed underNembutal anaesthesia and using smooth-tipped forceps (1 mm wide), the tibial andsural nerves were firmly crushed for 10 sec in the upper thigh, and the wound re-sutured. Nerve regeneration (and limb growth in the neonate) were allowed to pro-ceed for 6 months before removal and processing of segments of the N.G.M., sural nerveand tibial nerve from the distal half of the thigh.

In each case, 2-cm lengths of nerve were exposed under Nembutal anaesthesia, afine perineurial ligature was introduced on one side of the nerve to indicate theproximal end of the specimen and the specimen plus ligature removed and affixed toa card frame under slight tension to restore the pre-removal length of the specimen.Fixation in 10 % formal-saline and post-osmication in 1 % osmium tetroxide solutionwas followed by storage in 66% glycerol as described by Vizoso & Young (1948).Subsequently each specimen was teased on a slide in a pool of 66 % glycerol underthe dissecting microscope and individual fibres isolated throughout part of their length(usually 3-6 internodal segments), whilst remaining attached at one end to the markerligature. Measurements were made with a Leitz microscope fitted with a sensitivescrew ocular micrometer at a magnification of x 300 and were corrected to the nearest0-5 ji. Sources and magnitudes of the error in such techniques have been reviewed indetail by Wendell-Smith & Williams (1959), Williams & Wendell-Smith (i960) andKashef (1966). Estimates of the mean internodal diameter (i.e. the total fibre diameterincluding the axon and its sheath of compact myelin) were made from 5 measurementsat equidistant points along the internode, together with estimates of the maximumtransverse diameter of the neighbouring paranodal bulb profiles. The data within eachspecimen group were pooled, each group including measurements on 300-500 nodes,i.e. 600-1000 paranodal bulbs. In some cases plots were made of the mean percentageincrease in fibre diameter at the paranode in the various fibre diameter groups. In allcases the degree of asymmetry was studied by plotting the maximum diameter of theproximal bulb against the maximum diameter of its adjacent distal bulb and the dis-tribution of points examined in relation to the 'line of symmetry' drawn at 45° to theco-ordinates and passing through the origin.

Two principal types of distribution were found. First, a mixed type where the pointsare evenly distributed about the line of symmetry and which approaches more or lessclosely a symmetrical distribution where the linear regression line of the distributionis almost coincident with the line of symmetry. Secondly, the type where the greatmajority of points and the linear regression line are approximately parallel to the lineof symmetry but widely removed above (or below) it and where the population is saidto show a polarization of asymmetry. These two types of regression are illustrated inFig. 1. To limit the rather bulky amount of graphic illustration involved in the in-vestigation, the present account is confined to 4 typical regression lines and 6 of themore significant scatter diagrams, each containing 80-120 randomly selected points.

344 P- L- Williams and R. Kashef

The remaining general results are tabulated in Tables i and 2 and summarizeddiagrammatically in Figs. 9-13.

RESULTS

Neonatal rabbits

In the normal sciatic nerve of neonatal rabbits 60% of the myelinated fibresmeasured showed no enlargement in the paranodal region. The remaining 40 %showed slight paranodal enlargement and the proximal and distal bulbs were sym-metrical within the limits of the measuring technique.

30 r

25

20

9 15

73 10

Line ofsymmetry

Sural cutaneous nerv<polarized nodes

6-month regenerateof crushed mature tibial nerve

—symmetrical nodes

10 15 20Distal bulb maximum diameter (ji)

25 30

Fig. 1. Graph showing typical regression lines for proximal paranodal bulb maximumdiameter against distal bulb maximum diameter in the normal mature sural cutaneousnerve and a 6-month regenerate after crushing a mature tibial nerve. The line for thesural nerve corresponds to the scatter diagram of Fig. 4; n = 95; regression co-efficient = 0-950; standard error of estimate of proximal diameter on distal diameter= 1-043 /*• This line is typical of a nodal population with a polarization of asym-metry. The line for the crushed, regenerate tibial nerve corresponds to scatter diagramFig. 7; n = 117; regression coefficient = 0-966; standard error of estimate of proximalon distal diameter = 1 -045 fi. This line is typical of a nodal population approachingthe symmetrical condition.


Normal mature limb nerves (Table i; Figs. 1-4, 9, 10, 16, 17)

In both rabbit and rhesus monkey both the N.G.M. and the sural nerve possessedwell-formed paranodal bulbs, and as the quantitative findings in the two were similarthe graphs reproduced here are limited to the rabbit.

Table 1

Nerve

N.G.M., normal adultSural nerve, normal adultLumbar dorsal roots,normal adult

Lumbar ventral roots,normal adult

Crushed mature N.G.M.and tibial nerve, 6-monthregenerate

Crushed mature suralnerve, 6-monthregenerate

Crushed neonatal N.G.M.,6-month regenerate

Crushed neonatal tibial

No. ofnodes

measured

4 2 03683 2 0

3 H

446

4 0 2

386

1 1 2

Paranodeswhere P > D

(%)

758346

44

28

31

76

7 1

Paranodeswhere P -

(%)

171319

13

37

36

18

18

= DParanodes

where P < D(%)

8

435

43

35

33

6

1 1

nerve, 6-monthregenerate

Crushed neonatalperoneal nerve, 6-monthregenerate

Crushed neonatalsural nerve, 6-monthregenerate

320

480

70

73

17

1 2 15

Abbreviations: P, proximal bulb maximum diameter; D, distal bulb maximum diameter.

Figure 2 illustrates the relation of paranodal diameter to internodal diameter. Insmall-diameter fibres the proximal bulb is some 50 % wider than the internodereducing to some 23 % in large fibres. In contrast, the distal bulb ranges from 27 %wider in small fibres to 15 % in large fibres. Further details of this relationship are notimmediately relevant here, but in a further survey to be published it will be shownthat these general findings hold for other mammalian groups and some birds andamphibia.

Figures 3 and 4 show the degree of bulbar asymmetry and its polarization in theN.G.M. and sural nerve of the rabbit. In no case is 100% polarization found; never-theless there is a marked directional preponderance; for example, in 368 nodesmeasured in the sural nerve, 83 % had larger proximal bulbs, only 4 % larger distalbulbs and 13 % were equal within the limits of the measuring technique.


Dorsal and ventral nerve roots (Table i; Figs. 5, 6, 9, 10)

In both cases a mixed population is seen; for example, in the ventral root group of314 nodes measured, 44% had larger proximal bulbs, 43 % larger distal bulbs and13 % were equal.

Regenerates of crushed mature nerves (Table 1, Figs. 1, 7, 11, 18, 19)

In sural, N.G.M. and tibial nerves, regenerated for 6 months after a crush lesion, amixed population is found. The points are closely and evenly distributed about theline of symmetry and the distributions are termed symmetrical.

60 r-

50

40

30

IQ

20

10

Proximal bulbs

Distal bulbs

0 2 4 6 8 10Total fibre diameter (mean internodal diameter) (ft)

12

Fig. 2. Graph showing the % increase in fibre diameter at the paranode compared withthe internode in the N.G.M. D = maximum diameter of paranodal bulb, d = meaninternodal diameter (i.e. the mean of 5 measurements at equidistant points alonginternode). Each point represents the mean of 50 estimates within a particular fibrediameter grouping. The lines are calculated regression lines. For proximal bulbs,regression coefficient = —0-301; standard error of % on fibre diameter = 0-73%.For distal bulbs, regression coefficient = —0-632; standard error of % on fibrediameter = 0-83 %.

Regenerates of crushed immature nerves (Table 1; Figs. 8, 12)

After crushing of the neonatal nerve followed by regeneration of the nerve and limbgrowth for 6 months, the N.G.M. sural, tibial and peroneal nerves all develop a well-marked proximo-distal polarization.


Table 2

Part of recurrentlaryngeal nerve

No. ofnodes

measured

Paranodeswhere

P > D (%)

Paranodeswhere

Paranodeswhere

DescendingRecurvedAscending

34O300386

8838

74018

52268

Abbreviations: P, proximal bulb maximum diameter; D, distal bulb maximum diameter.

30 r

25

20

15

•a 10

10 15 20 25 30

Distal bulb maximum diameter (ji)

Fig. 3. Scatter diagram showing the relation of proximal bulb maximum diameter todistal bulb maximum diameter in the nerve to the medial head of the gastrocnemiusmuscle in the rabbit. n = 84. There is a well-marked polarization of asymmetry.

Cervical vagus and its recurrent laryngeal branch (Table 2; Figs. 13, 15)

The left cervical vagus and the descending part of its recurrent branch showedmainly larger proximal bulbs. The recurved part showed a mixed population withmany symmetrical bulbs whilst the ascending part showed an interesting reversal ofpolarity with mainly larger distal bulbs.


DISCUSSION

The observations recorded here on normal limb nerves to skin and muscle, all ofwhich have relatively direct courses in fascial planes, passing from the limb plexusperipherally towards the limb apex to reach their destination, are in full accord with thequantitative findings of Lubinska & Lukaszewska (1956). The majority of nodesexhibit an asymmetry polarized with the larger bulb nearer the root of the limb (and

30 1-

25

20

15

I 10

2

10 15 20Distal bulb maximum diameter (/*)

25 30

Fig. 4. Scatter diagram showing the relation of proximal bulb maximum diameter todistal bulb maximum diameter in the sural cutaneous nerve of the rabbit, n — 95.See also the linear regression line in Fig. 1. There is a well-marked polarization ofasymmetry.

in this instance nearer the cell body of origin). As pointed out by these authors, theasymmetry is sufficiently obvious in most cases to enable an observer to establish theproximo-distal orientation of an otherwise unidentifiable, isolated stretch of nervefibre. These general findings are consistent with a hypothesis that proximo-distal flowis causally related to nodal asymmetry.

Lubinska (1964) has reviewed the general evidence for flow in neurites. Whilstquantitative aspects of the translocation and lability of the various cytoplasmic sub-units and molecular species involved remain relatively unexplored, as do the mechan-isms of translocation, evidence exists for a net outward flow along neurites away fromthe cell body. Such flows are most marked during growth and regeneration and mini-


mize as maturity is approached. Remarkable supporting evidence for the existenceand direction of flow, at least in excised, avascular explants of ganglia and the proximalparts of their associated neurites under the conditions of tissue culture, has recentlybeen offered by Weiss, Taylor & Pillai (1962), using time-lapse cinematography, andin invertebrate neurons using micro-injection techniques by Kerkut & Walker (1962).

The central process of a dorsal root ganglionic cell is therefore a site where any netflow is expected to proceed from the cell body towards the spinal cord and thereafter

30

25

20

15

XI

10

80,

5 10 15 20Distal bulb maximum diameter

25 30

Fig. 5. Scatter diagram showing the relation of proximal bulb maximum diameterto distal bulb maximum diameter in the ventral lumbar nerve roots of the rabbit.n = 91. There is a mixed population.

within the cord towards the synaptic terminals. If the direction of flow is an importantfactor in the genesis of asymmetric paranodal bulbs, a reversal of polarity is to beexpected at the point of bifurcation into central and peripheral processes; that is, onthe central process the larger bulbs would be sited away from the cord (or nearer thecell body). In fact, a mixed population is found, some with larger proximal bulbs, somewith larger distal bulbs and yet others symmetrical. To explain such findings on thebasis of flow patterns it is necessary to postulate opposite directions of flow in neigh-bouring fibres or in adjacent segments of the same fibre, or transient alteration ofdirection in the roots, whilst preserving a constant direction in the peripheral parts ofthe fibre. The possibility of such variations seems remote and these findings thereforedo not support the hypothesis of flow as a causative factor of nodal asymmetry.

35© P. L. Williams and R. Kashef

Further doubts are raised by the finding of a similar mixed population in ventral nerveroots which transforms to an almost uniform proximo-distal polarity outside theintervertebral foramen.

A factor common to both dorsal and ventral nerve roots but contrasting sharplywith limb nerves concerns the tissue environment of each during maturation. Thenerve root fibres mature surrounded by a fluid-filled subarachnoid space, from whichthey are separated by tenuous connective tissue sheaths which 'attach' to other

30

25

20

-aE

15

•ao

5 10 15 20Distal bulb maximum diameter (ji)

25 30

Fig. 6- Scatter diagram showing the relation of proximal bulb maximum diameter todistal bulb maximum diameter in the dorsal lumbar nerve roots of the rabbit, n — 86.There is a mixed population.

structures only at the pial surface of the spinal cord and at the intervertebral foramen.Under these conditions the growing nerve root will be subjected to a uniform set ofmechanical forces at all points along its suspended length. Further, as it develops ina fluid pool, there is no possibility of relative movements occurring between the nerveroot and surrounding closely packed tissues. The limb nerves, however, pass to theirdestinations in planes between other closely packed tissues and it is instructive toexamine the growth patterns of these tissues in the various limb segments comparedwith what is known of nerve growth in the limbs. Vizoso & Young (1948), reviewingthe evidences of Boycott (1904) and Takahashi (1908) and the results of their ownresearches, concluded that the length of the internodal segments during growth in-


creases with the length of the whole nerve and that the number of internodal segmentsin a nerve remains constant after the onset of myelination and throughout develop-ment. Increases in internodal length were found to be greater in regions with greaterlongitudinal growth, as shown, for example, by a comparison of calf nerves and facialnerves (Vizoso, 1950) and a comparison of lateral-line nerves and branchial nerves(Thomas & Young, 1949). However, the correspondence between increase in inter-nodal length and increase in length of the part in which the nerve lies was much less

30 r

25

20

15

J3

•a

I10

10 15 20Distal bulb maximum diameter (ji)

25 30

Fig. 7. Scatter diagram showing the relation of proximal bulb maximum diameter todistal bulb maximum diameter in a 6-month regenerate after a crush lesion of amature tibial nerve in the rabbit, n = 117. There is a mixed, approaching symmetricaldistribution. See also linear regression line in Fig. 1.

close when a single segment of the limb was considered (for example, the ulnar nerveand forearm (Vizoso, 1950)) than when internodal length and total length of the partsin which the whole nerve lies is considered (Thomas & Young 1949). It would seemtherefore that, considered as a whole, the growth in length of a nerve is approximatelyinterstitial, with a regular increase in internodal length more closely related to thetotal length increase of the various subsections of the body through which it passesfrom origin to termination. The discrepancy found by Vizoso (1950) may well lie inthe differential growth patterns of the various limb segments which change with thestage of development. The early limb-bud mesoderm shows a well-known proximo-distal gradient with the proximal mesoderm developing at a greater rate and reaching


a more advanced maturational stage sooner than the distal mesoderm (Tschumi, 1957;Milaire, 1965). Postnatally, however (including most of the period of myelination), aless well known reversal of this gradient occurs, the evidence for which has beenreviewed by Tanner (1962). For example, in the human leg at 2 years of age the foothas already reached 52 % of its adult length, the calf 42 % and the thigh only 37 %.Consideration of an approximately interstitial growth of a nerve, with a regular in-crease of internodal length related to its total increase in length, growing through a

30 r

25

20

E3

. | 15

a.a

1 1 0a

25 300 5 10 15 20

Distal bulb maximum diameter (ji)

Fig. 8. Scatter diagram showing the relation of proximal bulb maximum diameter todistal bulb maximum diameter in a 6-month regenerate after a crush lesion of a neonataltibial nerve in the rabbit, n — 100. There is a well-marked polarization of asymmetry.

number of Umb segments with varying maturation patterns, leads to interesting con-clusions. Figure 14 illustrates such a system based upon figures for the human upperlimb (Simmons, 1944; Tanner, 1962). Figure 14A and B show the proportions of thelimb divided into hand, forearm and upper arm at maturity and at two years of agerespectively, drawn to the same scale. Figure 14 C and D represent the mature and the2-year limb proportions drawn to the same length, to facilitate a comparison of relativenodal positions. In each case 9 equidistant nodes are represented. Thus, during growththe total number of nodes has remained constant and internodal length has changedin proportion to total limb length, whilst the relative proportions of the Umb segmentshave changed. Comparison of the nodal positions in the two cases shows that whilstthe position of the terminal nodes remains unchanged, the remaining nodes have


moved relative to the limb tissues in a direction towards the limb root. For example,node X is sited well into the forearm in the immature limb but is placed in the distalupper arm in the mature limb. Such movements are substantial and would probablybe even greater if accurate growth studies were available to encompass the wholeperiod of myelination. The preceding analysis is based upon figures for human limb

Nerve to muscle Ventral root

0 Jc -4CPolarized bulbs Mixed bulbs

10

•'

Cutaneous nerve

=C.

Dorsal root

Polarized bulbs Mixed bulbs

Crush of normal mature nerve Crush

Regeneration •

Remyehnation with short internodes

i dt tSymmetrical bulbs

12 Crush of immature nerve CrushRegeneration, remyelination, l i m b f T r ^ Tgrowth, elongating internodes

Polarizedbulbs

13 Recurrent laryngeal nerve

Recurvedpart

Descending part

Polarization reversed withrespect to cell body

TAscending part

Figs. 9-13. Diagrams summarizing the predominant form of nodal asymmetry foundin each of the experimental situations examined in this paper.

Fig. 9. The mixed directions of asymmetry and the symmetrical bulbs typical ofventral nerve roots and the polarized asymmetry found in limb nerves to muscle.

Fig. 10. The mixed population characteristic of dorsal nerve roots and the polarizedasymmetry of peripheral cutaneous nerves.

Fig. 11. After crush of a mature nerve, axonal regrowth is followed by remyelinationwith typical short internodal segments. Here, limb growth, Schwann-cell elongationand relative movement are absent and the node population exhibits symmetrical bulbs.

Fig. 12. After crush of an immature nerve and axonal regrowth, remyelination occurswith elongating internodal segments and limb growth. Here a well-defined polariza-tion of nodal asymmetry develops.

Fig. 13 illustrates the reversal of the direction of polarization with reference to theposition of the cell body to be found in the descending and ascending parts of therecurrent laryngeal nerve.

354 P- L. Williams and R. Kashef

growth which are more complete than for any other animal. However, Vizoso &Young's figures concerning constancy of node number are based upon the sciatic nerveof the rabbit, whilst the skeletal growth figures of Lowrance (1953) show that similardifferential growth patterns exist in the rabbit's leg. Whilst formal proof of suchdirectional relative movements is lacking, the available growth figures are most sug-gestive. The alternative involves a precise correlation of internodal length of each partof the nerve with the particular growth pattern of each region of the body throughwhich it passes. This would result in substantial alterations in the internodal length/diameter relationship at a number of points along the same nerve trunk within a limb,

Upper arm Forearm HandA 1 t:::::::::::::::::::::::::: T""""'^^M^^^^M Adult

• « • » » • » « mx 9 equidistant nodes of Ranvier

B I IS- ~^^^^M 2 years i 3 In. t

C I MMmil^^Mmimi^mxtms^^^^^^^^^^^M Adultx» » » • « • » > •

D I immmsmmvm^^m^W^^WKK^^KKKnKM^i i years

Fig. 14. Diagram illustrating the different proportions of the length of the upper arm,forearm and hand in the human upper limb at 2 years of age and in the mature state.A and C represent the proportions in the mature limb and B at 2 years of age. D repre-sents the proportions at 2 years scaled up to the length of C. In each case 9 equi-distant nodes are represented, i.e. internodal distance has increased with total limblength. Terminal nodes are unchanged in position but the remaining nodes have movedrelative to the limb tissues towards the root of the limb (e.g. compare the positions ofnode x in each case).

and such evidence is lacking. Any deviation from such a correspondence must neces-sarily result in relative movement, which could well confer a polarity on the elongatingand myelinating cell in the sense of possessing 'advancing' and 'trailing' ends inrelation to surrounding tissues.

To examine further the role of flow and the presence or absence of axial elongation,the asymmetry of nodes in regenerates' after crush lesions of mature and immaturenerves was assessed. In both cases proximo-distal flow was pronounced, whereaselongation and relative movement was confined to regeneration in the immatureanimal, where limb growth continues.

In the crushed mature nerve, remyelination occurs largely after the young neuriteshave approached their end organs. Np further limb elongation occurs and myelinatedfibres of all diameters mature with a common short internodal length (Vizoso &Young, 1948). Here, elongation and relative movement are absent and the nodes ofRanvier develop well-formed paranodal bulbs which are virtually symmetrical. Theimmature regenerates, however, after substantially normal limb growth, developasymmetric nodes which are polarized with the larger bulbs nearer the root of the


limb. Evidently, regeneration itself does not preclude the development of asymmetry.However, simple elongation of the Schwann cell in a relatively homogeneous environ-ment (the fluid-filled subarachnoid space) does not result in a polarization of asym-metric nodes.

These observations on regenerates again do not support the flow hypothesis asrelated to nodal asymmetry, but could be explained in terms of relative movement ofthe developing Schwann cell.

Finally, it seemed important to examine the nodes of different regions of the samenerve which are reversed in their directional relationship to surrounding tissues. Sucha situation obtains in the descending, recurved and ascending parts of the recurrentlaryngeal nerve. Here the descending and ascending parts show a marked polarizationof asymmetry, whereas the recurved part shows a mixed population. In the descend-ing and ascending parts the paranodal bulbs nearer the head are larger. Thus, thedirection of polarization in the two parts is the same relative to the cranio-caudalbody axis (and growth gradients) but is reversed in the two parts relative to the cellbody of origin and the direction of axoplasmic flow.

Taken together, these findings allow a firm rejection of the hypothesis of partialdamming of a proximo-distally flowing column of axoplasm by the mid-nodal con-striction as causally related to nodal asymmetry. They are consistent with the viewthat elongation and relative movement of the developing Schwann cell are responsiblefor the polarization. Williams & Landon (1963) showed that the larger (i.e. 'trailing')paranodal bulb shows deeper and more extensive grooving of the myelin sheath, agreater accumulation of organelle-bearing Schwann-cell cytoplasm and greater flutingof the subjacent axon. It is suggested therefore that different mechanical conditionsoperate at the ' advancing' and ' trailing' ends of the cell during maturation and resultin dimensional differences of the folded paranodal myelin sheath and associatedfeatures of the paranodal apparatus. On this view, the asymmetry of the node ofRanvier is a secondary phenomenon, merely reflecting a primary polarization of eachSchwann cell. Final proof of this must await more critical growth studies of both nerveand limb, more direct estimates of the relative movements involved and a fullerunderstanding of the forces operating on cells undergoing mutual translation.

REFERENCES

BOYCOTT, A. C. (1904). On the number of nodes of Ranvier in different stages of growth ofnerve fibres in the frog. jf. Physiol, Lond. 30, 370-380.

CAUSEY, G. (i960). The Cell of Scltwaim. Edinburgh and London: Livingstone.CAUSEY, G. & PALMEH, E. (1952). Early changes in degenerating mammalian nerves. Proc. R.

Soc. B 139, 597-609.CAUSEY, G. & PALMER, E. (1953). The centrifugal spread of structural changes at the nodes in

degenerating mammalian nerves. .7. Anat. 87, 185-191.KASHEF, R. (1966). The Node of Ranvier—a Comparative and Dimensional Study. Ph.D. Thesis,

University of London.KERKUT, G. A. & WALKER, R. J. (1962). Marking individual nerve cells through electrophoresis

of ferrocyanide from a microelectrode. Stain Technol. 37, 217-219.LANDON, D. N. & WILLIAMS, P. L. (1963). Ultrastructure of the node of Ranvier. Nature,

Lond. 199, 575-577-


LOWHANCE, E. Q. (1953). Roentgenographic record of skeletal growth in relation to age andbody weight of the rabbit; caJcaneus and tibia. Growth 17, 183-189.

LUBINSKA, L. (1958). Short intemodes 'intercalated' in nerve fibres. Acta Biol. exp., Vars. 18,117-136.

LUBINSKA, L. (1964). Axoplasmic streaming in regenerating and normal nerve fibres. InProgress in Brain Research, vol. 13; pp. 1-71. Amsterdam: Elsevier.

LUBINSKA, L. & LUKASZEWSKA, I. (1956). Shape of myelinated nerve fibres and proximo-distalflow of axoplasm. Acta Biol. exp., Vars. 17, 115-133.

MILAIRE, J. (1965). Aspects of limb morphogenesis in mammals. In Organogenesis (ed. R. L.De Haan & H. Ursprung), pp. 283-300. New York, Chicago, San Francisco, Toronto,London: Holt, Rinehart & Winston.

RANVIER, M. L. (1871). Contributions a l'histologie et a la physiologie des nerfs peripheriques.C. r. hebd. Sianc. Acad. Set., Paris 73, 1168-1171.

RANVIER, M. L. (1875). Sur les elements conjonctifs de la moelle epiniere. C. r. hebd. Se'anc.Acad. Set., Paris 77, 1299-1302.

RANVIER, M. L. (1873). Lefons sur l'histologie du systbne nerveux. Paris: Savy.SIMMONS, K. (1944). The Brush Foundation study of child growth and development. II.

Physical growth and development. Monogr. Soc. Res. Child. Dev. 9, no. 1.TAKAHASHI, K. (1908). Some conditions which determine the length of internodes found on

the nerve fibres of the leopard frog, Rana pipiens. J. comp. Neurol. 18, 167-197.TANNER, J. M. (1962). Growth at Adolescence. Oxford: Blackwell.THOMAS, P. K. & YOUNG, J. Z. (1949). Internodal length in nerves of fishes. J. Anat. 83,

336-350.TSCHUMI, P. A. (1957). Growth of hind-limb bud of Xenopus laevis and its dependence upon

the epidermis. J. Anat. 91, 149-171.Vizoso, A. D. (1950). The relationship between internodal length and growth in human nerves.

J. Anat. 84, 342-353-Vizoso, A. D. & YOUNG, J. Z. (1948). Internodal length and fibre diameter in developing and

regenerating nerves. ,7. Anat. 82, no-134.WEISS, P. & HISCOE, H. B. (1948). Experiments on the mechanism of nerve growth. J. exp.

Zool. 107, 3I5-395-WEISS, P., TAYLOR, A. C. & PILLAJ, P. A. (1962). The nerve fibre as a system in continuous

flow: microcinematographic and electronmicroscopic demonstrations. Science, N. Y. 136,33O.

WENDELL-SMITH, C. P. & WILLIAMS, P. L. (1959). The use of teased preparations and frozensections in quantitative studies of mammalian peripheral nerve. Q. Jl microsc. Sci. 100,499-508.

WILLIAMS, P. L. & KASHEF, R. (1961). Asymmetry of the node of Ranvier—an experimentalstudy. J. Anat. 95, 610.

WILLIAMS, P. L. & LANDON, D. N. (1963). Paranodal apparatus of peripheral myelinated nervefibres of mammals. Nature, Lond. 198, 670-673.

WILLIAMS, P. L. & WENDELL-SMITH, C. P. (i960). The use of fixed and stained sections inquantitative studies of peripheral nerve. Q. Jl ntiscrosc. Sci. 101, 43-54.

(Received 19 September 1967)

Fig. 15. A group of nodes with mixed directions of asymmetry from the recurved partof the recurrent laryngeal nerve of the rhesus monkey.Fig. 16. A typical asymmetric node from the polarized population of the N.G.M.of the rabbit.Fig. 17. A typical asymmetric node from the polarized population of the suralcutaneous nerve of the rabbit.Figs. 18 and 19. Typical symmetrical nodes from a 6-month regenerate followinga crush lesion of the mature tibial nerve of the rabbit.

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