J. Anat. (1967), 101, 1, pp. 45-55 45With 8 figuresPrinted in Great Britain

The cellular response to nerve injury2. Regeneration of the perineurium after nerve section

P. K. THOMAS AND D. G. JONESInstitute of Neurology, Queen Square, and Department of Anatomy,

University College, London

INTRODUCTION

When a peripheral nerve is divided, the cut ends normally retract from each other.Restoration of the continuity of the nerve depends upon outgrowths from both cutends. The outgrowth from the distal stump, which is at least as extensive as theneuroma that grows out from the proximal stump (Young, Holmes & Sanders, 1940),has been investigated by electron microscopy in a previous study (Thomas, 1966). Itwas found to consist of fibrous connective tissue containing fibroblasts and otherconnective tissue cells within which were embedded columns of Schwann cellscontinuous with the Schwann cell columns (bands of Bungner) of the distal stump.From light microscope observations, Denny-Brown (1946) described a vigorous con-tribution of cells to the outgrowth from the distal stump arising from the perineuriumas well as from the endoneurium. Normal perineurial cells display a characteristicmorphology in electron microscope preparations, but Thomas (1966) was unable torecognize such cells in the outgrowth.When regeneration occurs across the gap in the nerve, groups of Schwann cells

and associated axons become surrounded by perineurial cells so that the junctionalregion comes to consist of numerous small fascicles (Masson, 1932; Nageotte, 1932).The origin of these perineurial cells has not yet been established and has been investi-gated in the present study.

MATERIAL AND METHODS

The investigation was performed on the sural nerve of eight mature albino rats.Under pentobarbitone sodium (Nembutal) and ether anaethesia, the nerve wasdivided with scissors in the portion of its course where it overlies the upper part ofthe lateral belly of the gastrocnemius muscle and a small segment removed from oneend so that the stumps lay separated by a gap of about 0-5 cm. The animals werethen allowed to survive for periods of 2-8 weeks before biopsy. At biopsy, againunder Nembutal and ether anaesthesia, the distal part of the bridge of tissue that hadgrown across between the two cut ends was removed and fixed at 4 'C by immersionfor 3 h in 1% osmium tetroxide in mammalian Ringer solution, buffered to pH 7.4with veronal-acetate. Following dehydration in graded concentrations of ethanol,some specimens were stained for 4 h by immersion in 1 % phosphotungstic acid inabsolute ethanol. The specimens were then embedded in Araldite, and sections cutwith a Porter-Blum microtome. The sections were collected on carbon-coated gridsand examined with a Siemens Elmiskop I. The sections from the material not

P. K. THOMAS AND D. G. JONES

treated with phosphotungstic acid were stained on the grids with lead citrate for5-10 min according to the method of Venable & Coggeshall (1965), except that a 1 u%aqueous solution was employed, sometimes preceded by staining with 40 aqueousuranyl acetate for 5 min.

RESULTS

Normal perineurial structureThe structure of the perineurium has been examined with the electron microscope

by a number of previous investigators (Rohlich & Knoop, 1961; Thomas, 1963;Shanthaveerappa, Hope & Bourne, 1963; Gamble, 1964; Gamble & Eames, 1964;Waggener, Bunn & Beggs, 1965), but certain further observations on normal materialrelevant to the present study have been made.The perineurium of the rat sural nerve trunk usually consists of four to five

lamellae composed of flattened cells possessing basement membrane (basementlamina; Fawcett, 1963) on both inner and outer aspects (Fig. 1). The endoplasmicreticulum and Golgi complex are usually sparse, but the cells contain numerouspinocytotic vesicles (Fig. 2). As reported previously (Thomas, 1963), adjacent cellsare closely related to one another, either by extensive overlapping of their contiguousmargins or by the interdigitation of cell processes (Fig. 2). The gap separating thesurface membrane of cells in contact with one another is variable, but may be aslittle as - 90 A (Fig. 2). Such 'tight junctions' may show a quintuple-layered arrange-ment, the gap between the two adjacent cell membranes displaying a central denseline bordered by two zones of lesser density (Fig. 3), although this is not alwaysevident. They correspond to zonulae occludentes as defined by Farquhar & Palade(1963). The perineurial cells also possess numerous hemidesmosomes (Kelly, 1966).These are of relatively simple form, thickenings of the cell membrane on surfacescovered by basement membrane being associated with an increased density in theunderlying cytoplasm (Fig. 4). Tonofibrils are not usually visible. External to suchregions there is generally a narrow dense layer separated from the surface membraneby a lighter zone - 40 A in thickness (Fig. 4). These appearances are most obvious inthe material stained with phosphotungstic acid, but are also apparent after stainingwith uranyl acetate and lead citrate. They are of similar type to those described inthe basal layer of the corneal epithelium (Whitear, 1960) and human epidermis(Odland, 1964), but are often of considerably greater width.The perineurial lamellae are separated by gaps that contain collagen fibrils orien-

tated both longitudinally and obliquely to the axis of the nerve trunk (Fig. 1),together with elastic fibres (Fig. 4) which are often closely associated with thebasement membrane of the perineurial cells. The collagen fibrils are of comparablediameter to those of the endoneurium, averaging - 515 A, and are of smallerdiameter than those of the epineurium, where they average -800 A. The changebetween collagen of perineurial and epineurial type takes place abruptly at the outer-most perineurial lamella (Fig. 1). The gaps also contain occasional cells with elon-gated processes directed circumferentially around the fascicle, but differing fromthose of the perineurial lamellae in that they lack basement membrane and thattheir processes do not come into close contact with the processes of other cells

46

The cellular response to nerve injury. 2

Fig. I. Transverse section through perineurium (pn.) of rat sural nerve. ep.,FEpineurium; en.,endoneurium. Phosphotungstic acid stain._+ ~~~~~ris,,.w_~

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Fig. 2. Junctions between perineurial cells. The gap between the adjacent cell membranes isreduced to 90 A at regions indicated by arrows. p.v., Pinocytotic vesicles. Uranyl acetate and

lead citrate stain.

47

P. K. THOMAS AND D. G. JONES

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Fig. 4. Hemidesmosomes (hd.) at surface of perineurial cells. b.m., Basement membrane; c.f.,collagen fibrils; ef, elastic fibres. Phosphotungstic acid stain.

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The cellular response to nerve injury. 2(Fig. 5). It seems likely that they are fibroblasts, although they usually do not showan extensive endoplasmic reticulum. Fibroblasts of similar appearance are oftenseen immediately internal or external to the perineurium.~~~~~~~~~and myelnate axns The wer seatdby, irreula bude fcllgnfbs

w~~TC;hicwer not clevarl *organze int shah surndg th Scwn cells.In

also preenThatspossessedtoelrongate processesshwhingchlprasmifed thea irough th tissueadecrledn indiidal Schwaentoprnerac ellso groupnfcll. Fg).Pop gTheydcidsplayedth

possssigarelaiveyRvlumnertous cyopls with an xtesiv enoplsmireiclumOthciernsimilarecellsnshowedaunctioncomplsseintheinvestmentwthebasementsmembrane.rTe proctiesssoSadjacentscells whereofencnassctianed onnmecintedbocasdeionaltigh jutons. Tight junctionsahavedbyreenladudesribedobetwen fibroblasts

Inthe sptecimenotaknenatlhsonger intervals after operation,thmegroupsoflSchwann

celshdthcrced appeardancl osmhacllfscils beingru surone bycells thg)Te ipate whermobvioulygclfaueofprnuilte(Figls.7,8)bTeseg cellsdposssebasement membraneanhadesinalessielvoluminous cytoplasmwtandapol developed endoplasmicrtclmThetirulatera exthensionslfrequenlyshontied mutinopletpinocytoetiveice bandshowedthmemidaesmosoesprossesse by nomlprnuilclsadjacentcellswereofeinctatndoncedbcoselyassocaltedhwinthione. antedslyigmliTightjunctions.hvendscie ewe Everybat

4n Anssteculture101Devis & James (1964).

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49

P. K. THOMAS AND D. G. JONES

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Fig. 6. Transverse section through junctional zone between cut ends of nerve 3 weeks after section.Groups of axons (ax.) and Schwann cells (Sc.) are separated by cells with elongated processes

resembling fibroblasts (fb.). Uranyl acetate and lead citrate stain.

50

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The cellular response to nerve injury. 2

Fig. 7. Transverse section through junctional zone between cut ends of nerve 8 weeks aftersection. Groups of myelinated and unmyelinated axons (ax.) and Schwann cells (Sc.) are sepa-rated by perineurial cells (p.c.). The central bundle also contains a fibroblast (fb.) and an eosino-phil leucocyte (e.l.). Phosphotungstic acid stain.

4-2

51

P. K. THOMAS AND D. G. JONES

gradation was observed between such cells and the cells of fibroblastic appearanceseen at the earlier stages of regeneration. Fibroblasts were also present within thefascicles at later stages, together with scattered leucocytes and macrophages (Figs.7, 8).

It was not possible to make a precise correlation between the appearances foundand the time interval after operation, the stage of regeneration, as judged by thedegree of myelinization and the organization of the perineurium, being somewhatvariable. Moreover, the appearances tended to vary from place to place in theregenerating tissue. The subdivision into fascicles by cells resembling fibroblastsrather than by cells of obvious perineurial appearance was not seen later than threeweeks after operation.

Fig. 8. Transverse section through junctional zone between cut ends of nerve 8 weeks aftersection. Groups of Schwann cells (Sc.) with myelinated and unmyelinated axons (ax.) are sepa-rated by processes of perineurial cells (p.c.). Phosphotungstic acid stain.

DISCUSSION

It is customary to employ the term perineurium to describe the lamellated sheathsurrounding the individual fascicles of a peripheral nerve, although there has beensome discussion as to its exact definition. Shanthaveerappa & Bourne (1962, 1964)suggested that the cellular laminae of the perineurium should be referred to as theperineurial epithelium, and Lehman (1957, 1959) had earlier employed the termneurothelium for the same structure. These authors described the perineurium asbeing a condensation of the surrounding collagenous connective tissue immediately

52

The cellular response to nerve injury. 2external to the cellular layer. The term epineurium was reserved for the moreperipheral collagen binding the fascicles together. A similar view was also taken byRohlich & Weiss (1955) and Clara & Ozer (1959). The present electron microscopeobservations suggest that it is more satisfactory to consider that the boundarybetween perineurium and epineurium is at the outermost perineurial lamella, as thecollagen changes in type at this point. This demarcation was adopted by Rohlich& Knoop (1960), Thomas (1963) and Waggener, Bunn & Beggs (1965). The composi-tion of the perineurium revealed by the present study corresponds closely to thedescription given by Denny-Brown (1946), who described it as being made up oflamellae of flattened mesothelial cells, separated by layers of collagen, together withoccasional fibroblasts.

Physiological studies have shown that the connective tissue sheath of peripheralnerves acts as a diffusion barrier (Feng & Gerard, 1930; Feng & Liu, 1949; Rashbass& Rushton, 1949; Crescitelli, 1951; Causey & Palmer, 1953; Shanes, 1954) althoughthis had been denied by Lorente de N6 (1950). It is now clear that the perineurium isthe layer responsible for this effect (Lehman, 1953, 1957; Krnjevic, 1954; Rohlich &Weiss, 1955), this conclusion being supported by electron microscope observationson its morphological features (Thomas, 1963) and on the diffusion of ferritin(Waggener, et al., 1965). It has been accepted that the perineurium plays an importantrole in isolating the environment of the nerve fibres from the surrounding tissues(Denny-Brown, 1946; Shanthaveerappa & Bourne, 1962, 1963), although the precisesignificance of this has not yet been established, and it probably has importantmechanical properties (Sunderland & Bradley, 1952; Sunderland, 1965). The presentinvestigation has confirmed earlier light and electron microscope studies (Masson,1932; Nageotte, 1932; Denny-Brown, 1946; Terry & Harkin, 1957) that the peri-neurium is reconstituted around nerve fibres regenerating across gaps in nervetrunks, giving rise to multiple small fascicles.The developmental origin of the perineurium has been disputed. The generally

held view has been that it is a specialized mesodermal tissue (Denny-Brown, 1946).Masson (1942), on the other hand, believed that both the perineurium and theendoneurial fibroblasts were of neural crest origin. It has been claimed that themeninges are derived from the neural crest (Harvey & Burr, 1926; Harvey, Burr &van Campenhout, 1933). If the perineurium is an extension of the leptomeninges, asoriginally suggested by Key & Retzius (1876) and more recently by Shanthaveerappa& Bourne (1962), Benke & Rohlich (1964) and Poldcek (1966), this might indicate asimilar origin for the two tissues. It was for this reason that Shanthaveerappa &Bourne referred to the perineurial cells as an epithelium. However, the experimentsof Harvey & Burr were not confirmed by Flexner (1929) and have not been generallyaccepted. Gamble & Breathnach (1965), in developing human peripheral nerve,found that the perineurium was derived from cells morphologically resemblingfibroblasts. This is also true of the present observations on regenerating nerve.Although not of course conclusive, both these observations suggest that the perineu-rium is a mesodermal tissue, the lamellar cells being modified fibroblasts. Theacquisition of basement membrane during this process is of interest.

53

54 P. K. THOMAS AND D. G. JONES

SUMMARY

An electron microscope study has been made on the regeneration of the perineuriumof the rat sural nerve following removal of a short segment of the nerve. Groups ofSchwann cells and associated axons that regenerate across the gap become surroundedby cells having the morphological features of fibroblasts and these gradually assume

the appearance of typical perineurial cells. The region of the gap in the nerve, afterregeneration, therefore comes to consist of multiple small fascicles, each bounded byperineurium.

We wish to thank Miss J. Slatford for technical assistance and Professor W. H.McMenemy for laboratory facilities at Maida Vale Hospital. A personal grant toone of us (P. K. T.) from the National Fund for Research into Poliomyelitis andother Crippling Diseases is gratefully acknowledged. We also wish to thank ProfessorJ. Z. Young, F.R.S., and Dr E. G. Gray for helpful discussion.

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