Neuroscience, Vol. 3. pp. 1241-1250. Pergamon Press
Ltd.1978.Printedin
Great Britain.
A SCANNING ELECTRON MICROSCOPIC STUDY OF PERIPHERAL NERVE DEGENERATION AND REGENERATION Department
M. R. GERSHENBAUMand F. J. ROI~EN of Anatomy, Rutgers Medical School-CMDNJ, Piscataway, NJ 08854, U.S.A.
Abstract-The left sciatic nerves of male Holtzman rats were exposed and crushed with a hemostat for 3 s at a point midway between the inferior gluteal nerve and the bifurcation into the tibia1 and common peroneal nerves. Normal myelinated axons appear in the scanning electron microscope as relatively smooth cylindrical structures with interweaving strands of collagen fibers coursing over their length. Nodes of Ranvier are seen as constrictions along the myelinated fibers. After crushing, the nerve fibers swell, and interruptions appear in the nodal and internodal regions causing a beaded morphology. The myelin sheaths fragment into numerous spherical bodies as the degenerative process progresses. Some remnants of myelin debris are found within the nerves 60 days following the lesion A few regenerating myelinated axons are observed within the connective tissue fibers and myelin debris distal to the crushed regions approx 15 days after placement of the lesion. By day 30 the nerves are relatively normal in appearance. The scanning electron micrographs provide a three-dimensional picture of the dynamic processes which occur during nerve degeneration and regeneration. These findings are correlated with previous studies and can serve as a basis for future scanning electron microscopic studies in this area of research.
of a peripheral nerve, there are profound morphological alterations in the nerve fibers and connective tissues involved in the lesion. Electron microscopy has enabled investigators to analyze the ultrastructural events associated with Wallerian degeneration and peripheral nerve regeneration (OHMI, 1961; O’DALY & IMAEDA, 1967; BALLIN &
AFTER injury or severance
hemostat were covered with adhesive tape to limit damage to the nerve during the crush. Scanning electron microscopy
Two rats were killed at each 3day interval from 3 days to 30 days after placement of the lesion; an additional two animals were killed at day 60. To prepare the sciatic nerves for SEM observations, the nerves were exposed and THOMAS,1969; CRAVIOTO,1969; SINGER& STEINBERG, fixed in situ for 10 min with 3% glutaraldehyde (Electron 1972; CALABRE-ITA,MUNGER & GRAHAM, 1973; HUDMicroscopy Sciences, Fort Washington, Pa.) buffered with 0.05 M cacodylate [mixed 1: 1 with Pucks Saline G (Grand SON & KLINE, 1975; GHABRIEL& ALLT, 1977). These Island Biological Co., Grand Island, NY)] before being studies provide a good representation of the overall changes which occur during the degeneration and re- removed and fixed overnight at 4°C. (The segments of scigeneration of myelinated axons, but it is often ex- atic nerves removed for SEM observations were approximately 15 mm long and stretched from a point just below tremely difficult to interpret the three-dimensional the level of the inferior gluteal nerve to the bifurcation process from the two-dimensional images obtained into the tibia1 and common peroneal nerves. The nerves with thin sections. In order to aid in the interprewere not excised more distally because of the difficulty tation of the ultrastructural changes seen with the encountered in the removal of the connective tissue covertransmission electron microscope we employed scanings from the smaller branches.) The nerves were rinsed ning electron microscopy (SEM) to study the alterin saline and post-fixed in 1% osmium tetroxide (Electron Microscopy Sciences, Fort Washington, Pa.) buffered with ations in surface morphology which occur during 0.2 M cacodylate at 4°C for 12 hr. After post-fixation the Wallerian degeneration and subsequent regeneration of the nerve fibers. This study can further serve to epineurium and perineurium were carefuHy removed under a dissecting microscope. The exposed bundles of axons facilitate the use of SEM in future studies of nerve were teased apart and dehydrated in a graded series of degeneration and regeneration. acetone solutions. After two changes of 100% acetone, the nerve fibers were dried in a critical point drying apparatus (Sorvall) and mounted on specimen studs with silver conEXPERIMENTAL PROCEDURES ducting paint. The fibers were then coated with gold in Surgical technique a Denton vacuum evaporator and examined with a JEOL The left sciatic nerves of 25 male Holtzman rats JSM-U3 scanning electron microscope operated at 25 kV. (200-240g) under ether anesthesia were exposed and For these observations we examined three regions of the crushed with a hemostat for 3 s at a point midway between continuous nerve segment: (a) near the site of the crush, the inferior gluteal nerve and the bifurcation into the tibia1 (b) approximately 4-6 mm proximal to the crush site and and common peroneal nerves. The serrated jaws of the (c) approximately 5-8 mm distal to the lesion. It should be noted that some slight variations in the time course Abbreviation: SEM, scanning electron microscopy. of the degenerative and regenerative processes were 1241
1242
ivl K.
GLRSH~ NHAGM
observed between nerve segments within each specific time period. Micrographs depicting the most representative regions were therefore chosen to illustrate best the sequence of events which occur during Wallerian degencration and subsequent nerve regeneration.
RESULTS In order to view the individual myelinated fibers with the SEM, connective tissue elements are pulled or teased away from the nerves. In Fig. lA, these connective tissue elements can easily be identified. The epineurium appears as a thick fibrous network surrounding the entire nerve while the perineurium surrounds only the individual fasicles of the nerve. In these preparations (Fig. 18) normal myelinated axons are seen as relatively smooth cylindrical structures which interweave with strands of collagen and elastic fibers that course over their length. The axons vary in diameter and are closely packed within the nerve bundles. Nodes of Ranvier are occasionally observed and appear as annular constrictions along the myelinated fibers (Fig. 1B and C). Details of the nodal regions are obscured by the overlying connective tissue sheath (sheath of Plenk and Laidlaw) and basal lamina. Small, branching fibrous structures are found running longitudinally along the myelinated axons for various distances (Fig. lB, C and D). These frequently cross to adjacent axons where they often merge with other similar fibers (Fig. 1C and D). These fibers are continuous across the nodes (Fig. 1C) and may be intimately associated with the connective tissue sheath covering the axons (Fig. IB, C and D). Such structures are difficult to identify positively with the SEM but may represent: (a) the unmyelinated component of the sciatic nerve, (h) dense aggregations of collagen fibers, or (c) a combination of both. Proximal
region
No significant changes from normal are observed in any of the regions proximal to the lesion for all time periods studied after crushing the sciatic nerves. These proximal regions look essentially the same as the normal nerve shown in Fig. 1B. Therefore, to avoid redundancy, only the reactive crush and distal segments are described and illustrated, even though the proximal regions were studied. Crush and distal
regions
Three days following placement of the lesion, gross morphological alterations are observed in the crush and distal regions. The myelin sheaths of nerve fibers in the vicinity of the crush (Fig. 1E) are swollen with numerous cytoplasmic bulges occurring along the length of the degenerating axons. Schwann cells appear to be retracting from presumptive nodes of Ranvier and several breaks or constrictions in the internodal regions of the nerve fiber can be found. At this stage, it is often very diffiThree days post-lesion.
and F J. RON K
cult to dilTerentiatc between node. anti II:~W:II~I~~I~ internodal breaks due to the outer connectl\~‘ ti~ue coverings. In the nerve segment distal tcr I!I< lls\~q,:~ (Fig. IF) the nerve fibers appear m ;+ ~mewha~ dtf?& ent stage of degeneration than those in :IIV :.I~I*II region. The myelinated axons arc dissoc~.~tcd jnt;> elliptical segments of varying size. The gaps separat mg these segments are greater than lhosr found Ilcar the region of the crush. Occasionally. those cliip~cl~& appear to be joined only by remnants of the ctmn~~:tive tissue sheath. :Vinr duys post-lesion. In the area of the crush (Fig. 2A) many degenerating fibers begin to fragment into large spherical globules which are orient4 in a linear fashion along the major axis of Ihc IWVC. The globules are covered by strands of collagen and other debris. In the distal region of the nerves (Fig. 2B), fragmentation of the myetin sheath doe5 not appear as extensive as that found near the lesion. Globules of myclin are smaller and seem to hc enclosed by the connective tissue sheath. Very often fibers are found which exhibit a shrunken and Jiatorted morphology. In both the crush and disral regions (Fig. ZA and B), the amount of connecti\r tissue covering the damaged fibers seems to have mcreased while the axons have lost the smooth appearance observed in normal and proximal ncrvc tegments. Fifteen duys post-lrsion. At this stage, the large globules found in the crush region (Fig. 2c‘) arc apparently fragmented into smaller pieces. In some areas fragments of the main spheroids are torn Amy during preparation of the nerves for SEM, leaving behind depressions on the fiber surfaces (Fig. 2Ct. Beneath the degenerative fragments. nerve fibers drc observed which in many respects appear to be normal in surface morphology (Fig. 2C). These most probably represent regenerating fibers that have grow-n into the region of the crush. In the distal segment (Fig. ?D), the globules of myelin are not fragmented as extensively as those found near the lesion. These myetin globules are similar to those seen in the crush region 9 days post-lesion (Fig. 2A). Remnants of axons are seen coursing through the connective tissue fibers. Few regenerating fibers can be identified in this region. Twenty days post-lesion. In the area of the crush (Fig. 3A), globules are further fragmented into smaller spherical pieces. These pieces appear scattered with other bits of debris over the surfaces of regenerating fibers. By day 20, regenerating fibers are also observed in the distal segment of the nerves (Fig. 38). Myelin fragments found in this region are generally larger than those found near the crush site. Thirty days post-lesion. By this time, many normal looking regenerated axons can be observed spanning the region of the crush (Fig. 3C). Remnants of my&n debris are still found in this area after 30 days. The segment distal to the crush (Fig. 3D) appears IO he very similar to the crush region.
Nerve degeneration and regeneration in scanning EM
1243
Discussion
distal. The reason for this remains unclear. Further fragmentation of the spheroids and ovoids into Wallerian degeneration in the peripheral nervous smaller pieces takes place as the degenerative process system has been studied extensively with light and continues. Small globules break off of the larger myetransmission electron microscopy but little has been lin pieces and are often expelled into the endoneurial done to correlate these findings with the threedimenspace (Fig. 2C) as reported by previous investigators sional view obtainable with scanning electron micro@‘DALY & IMAEDA,1967; WILLIAMS& HALL, 1971b; scopy. Some earlier SEM observations on normal and CALAEXRETTA et al., 1973). By 30 days few globules degenerating air dried, teased nerve preparations were can be found in the endoneurial space. In several made by SPENCER& LIEBERMAN (1971). In their study cases the myelin debris has been reported to be phathe air-drying produced an artifactual ‘pattern of pitgocytized by invading macrophages (TERRY& HARlike depressions’ not seen in our specimens and in KIN, 1959; OHMI, 1961; O’DALY & IMAEDA,1967; general distorted the entire surface of the nerve fibers. WILLIAMS& HALL, 1971b), a fact disputed by those The significance of critical point drying in the prepwho contend that the Schwann cells are responsible aration of biological material for SEM has been well for myelin digestion (NATHANIEL& PEASE, 1963; documented (PORTER,KELLEY& AND~EWS,1972). As SATINSKY, PEPE& LIU, 1964; CRAVIOTO,1969; SINGER described previously, normal myelinated axons & STEINBERG, 1972). We have not, however, identified appear as relatively smooth cylindrical structures with these macrophages in our preparations. connective tissue fibers coursing over the surface Regenerating fibers, as shown by our transmission (Fig. 1B). They are often associated with longitudinal electron microscopic studies (unpublished observafibrous bands (Fig. lB, C and D) which seem to be tions), can be observed growing between the myelin similar in appearance to the three-dimensional reconstructions of unmyelinated fibers made by AGUAYO debris in crush segments by 15 days post-lesion (Fig. 2C). By 20 days they are found in the regions & BRAY (1974), but could be bundles of collagen fibers or a combination of the two. These bands are distal to the crush (Fig. 3B). Although most nerve fibers appear to be relatively normal in the medial continuous across the nodes of Ranvier and appear and distal portions of the nerve bundles after 30 days, to be $mly attached to the endoneurial sheath (Fig. 1C). We also occasionally observed the ‘fluting’ there are still fragments of myelin debris in the in the paranodal regions described by HESS& YOUNG endoneurial space at this time. Such debris persists even after 60 days post-lesion. (1952). In which direction does Wallerian degeneration proChanges after nerve crush
ceed?
Three days following the nerve crush, extensive swelling and retraction of the Schwann cells from the nodal regions can be seen. This has been reported to be one of the earliest morphological changes to occur after crushing (RAM~NY CAJAL, 1928; BALLIN & THOMAS, 1969; WILLIAMS& HALL, 1971a,b). Simultaneously we have seen breaks and constrictions occurring in the internodal regions of these fibers causing the formation of primary ellipsoids of greatly varying size. In the distal area the ellipsoids are more numerous than those found near the crush and tend to be qeparated by larger gaps, giving the impression that the distal region is in a different stage of the degenerative process than the crush site. These distal ellipsoids often appear to be joined solely by remnants of endoneurial connective tissue. The endoneurial tubes and basement membranes have been shown by THOMAS(1964) to undergo extensive collapse and folding during Wallerian degeneration, while the continuity of these structures appears to be maintained during the degenerative process providing tubes in which the Schwann cells may proliferate. By 9 days post-lesion the primary myelin ellipsoids fragment into spheroids and ovdids which are often oriented in a linear fashion along the longitudinal axis of the nerve bundles (Fig. 2A and B). The orientation of these myelin remnants is usually in better alignment in the regions closer to the crush site than those more
The direction in which Wallerian degeneration proceeds has long been controversial. While some researchers have reported a centrifugal degeneration of the nerve fibers (ROSENBLUETH & DEL Pozo, 1943; CAUSEY& PALMER,1953; JOSEPH,1973) others found no difference along the length of the fiber or believe that the wave of degeneration passes centripetally (ERLANGER8~ SCHOEPFLE,1946; DONAT & WrsNIEWSKI,1973). LUBINSKA(1977) has shown that in the early stages of Wallerian degeneration, ovoid (ellipsoid) formation in the cut phrenic nerve begins near the lesion and progressively spreads in a distal direction at rates of 46250mm/day depending on fiber diameter. In this study we first examined the lesioned nerves shortly after the formation of ellipsoids had occurred. It was apparent that the ellipsoidal segmentation was more prominent distally than near the region of the crush. This is probably due to the trauma created by the lesioning process, but differences in the spread of Wallerian degeneration between cut and crushed nerves should not be excluded. At a later stage, a reversal in the rate of myelin breakdown seems to occur. The fragmentation and digestion of the myelin ellipsoids appears to proceed more rapidly in the region of the crush than in the distal area, thus giving the appearance of a shift in tlie direction of the degenerative process. Since the spread of degeneration down the fiber is so rapid
1244
M. R.
GERSHENBACJMand
F.
J. ROISFN
FIG. 1. Normal sciatic nerves. (A) A cross-section of the rat sciatic nerve as observed by scanning electron microscopy. The epineurium (EPI) consists of a thick fibrous meshwork which surrounds the entire nerve while the perineurium (PERI) surrounds only the nerve fascicles (65 x ), (B) These structures are relatively smooth with strands of interweaving connective tissue fibers coursing over their length. The node of Ranvier (arrow) appears as a constriction on the myelinated fiber. Note the fibrous longitudinal bands (arrowheads) (1600x). (C) The fibrous bands appear to be tightly applied to the endoneurial sheath as they cross the node of Ranvier, yet loosely applied to the nerve fiber below it. Branches can be seen coming off of the main band (arrowheads) (4500x). (D) The band of fibers can be seen crossing from one axon to another where it becomes tightly attached to the endoneurial sheath. It appears to merge with a smaller band (arrowheads) (2100 x ). Three days post-lesion. (E) Crush: Cytoplasmic bulges and swellings appear in the vicinity of the crush after 3 days. Interruptions along the length of the nerve fibers are seen (arrowheads). It is often difficult to distinguish between nodes of Ranvier and incomplete intemodal breaks in this micrograph (1500 x ). (F) Distal: The nerve fibers are separated into ellipsoids of various sizes. Often these ellipsoids appear to be joined only by remnants of the endoneurial connective tissue (arrowheads) (1500 x). FIG. 2. Nine days post-lesion. (A) Crush: The degenerating fibers become fragmented into large spherical globules which are oriented in a linear fashion along the long axis of the nerve. Note the increase of connective tissue fibers (1500 x ). (B) Distal: Globules of myelin debris in the distal segment are in general smaller than those found near the crush. These globules appear to be enclosed by the connective tissue sheath (1500 x ). Fifteen days post-lesion. (C) Crush: The larger globules are fragmented into many smaller pieces. Occasionally some of these pieces are torn away during sample preparation leaving behind depressions on the surface of the fibers (arrowheads). Nerve fibers which appeared normal in surface morphology can be found adjacent to these degenerating myelin fragments (arrows) (1500 x ). (D) Distal. The spherical globules do not appear to be as extensively fragmented in this region as compared to those in the crush segment. Note the abundance of collagen fibers (1500 x 1. FIG. 3. Twenty days post-lesion. (A) Crush: Many nerve fibers appear to have a normal surface morphology. There is still an abundance of connective tissue fibers and degenerative debris (1500 x ). (B) Distal: Some regenerating nerve fibers are found in the distal segment at this time (arrowhead). There is more debris and connective tissue in this region than in the crush segment (1500 x ). Thirty days post-lesion. (C) Crush: Although most of the myelinated axons appear to be normal, there is still evidence of myelin debris (1500 x ). (D) Distal: Most fibers are normal in appearance but there are still accumulations of myelin fragments and connective tissue fibers (1500 x I.
FIG. 3.
Nerve degeneration and regeneration in scanning EM (LUBINSKA,
1977), a fairly uniform process over the entire length of a small nerve should be expected, unless other factors are involved. One ‘such factor may be related to the fact that the proliferation of non-neuronal cells in vitro is directly dependent upon the presence of neurons (WOOD & BUNGE, 1975; MCCARTHY& PARTLOW, 1976; WOOD, 1976). SinOe the neurons are capable of exerting some types of influence on the Schwann cells, it is possible that the rate of myelin breakdown and digestion by the
1249
Schwann cells may be enhanced by contact with the regenerating nerve fibers. This could explain the apparent shift in the direction of Wallerian degeneration in crushed nerves, as well as the numerous conflicting reports.
Acknowledgements-This work was supported by a research grant, NS 11299, from the National Institutes of Health, and a grant from the Kroc Foundation.
REFERENCES AGUAYOA. J. & BRAY G. M. (1975) Experimental pathology of unmyelinated nerve fibers. In Peripheral Neuropothy (eds DYCX P. J., THOMASP. K. & LAMBERT E. H.).Vol. 1, pp. 363-390. Saunders, Philadelphia. BALLIN R. H. M. & THOMASP. K. (1969) Changes at the node of Ranvier during Wallerian degeneration: an electron microscope study. Acta Neuropath. (Be&) 14, 237-249. CALAEIRETTA A. M., MUNGER B. L. L GRAHAMW. P. (1973) The ultrastructure of degenerating rat sciatic nerves. J. Surg. Res. 14, 465-471. CAUSEYG. & PALMERE. (1953) The centrifugal spread of structural change at the nodes in degenerating mammalian nerves. J. Anut. 87, 185-191. CRAVIOTOH. (1969) Walierian degeneration: ultrastructural and histochemical studies. Bull. Los An&es Neural. Sot. 34, 233-253. DONATJ. R. & WISNIEWSKI H. M. (1973) The spatio-temporal pattern of Wallerian degeneration in mammalian peripheral nerves. Brain Res. 53, 41-53. ERLANGERJ. & SCHOEPFLE G. M. (1946) A study of nerve degeneration and regeneration. Am. J. Physiof. 147, 550-581. GHABRIELM. N. & ALLT G. (1977) Regeneration of the node of Ranvier; a light and electron microscope study. Acta Neuropath. (Be&) 37, 153-163. HF..%A. & YOUNG J. Z. (1952) The nodes of Ranvier. Proc. R. Sac. B 140, 301-320. HUDSONA. R. & KLINE D. G. (1975) Progression of partial experimental injury to peripheral nerve-2. Light and electron microscopic studies. J. Neurosurg. 42, 15-22. JOSEPHB. S. (1973) Somatofugal events in Wallerian degeneration: a conceptual overview. Brain Res. 59, 1-18. LUBINSKAL. (1977) Early course of Wallerian degeneration in myelinated fibres of the rat phrenic nerve. Brain Res. 130, 47-63.
MCCARTHVK. D. & PARTLOWL. M. (1976) Neuronal stimulation of c3H]thymidine incorporation by primary cultures of highly purified non-neuronal cells. Brain Res. 114, 41.5-426. NATHANIELE. J. H. & PEASED. C. (1963) Degenerative changes in rat dorsal roots during Wallerian degeneration. J. W~tr~rruct. Res. 9, 511-532. G’DALY J. A. & IMAEDAT. (1967) Electron microscopic study of Wallerian degeneration in cutaneous nerves caused by mechanical injury. Lab. Invest. 17, 744-766. 0t-1~1 S. (1961) Electron microscopic study on Wallerian degeneration of peripheral nerve. 2. Zellforsch. mikrosk, Anat. 34, 3967. PORTERK. R., KELLEYD. & ANDREWSP. M. (1972) The preparation of cultured cells and soft tissues for scanning electron microscopy. In Proc. Fifth Ann. Stereoscan Colloquium, pp. l-19. Kent Cambridge Scientific Company, Morton Grove, Illinois. RAM~N Y., CAJAL S. (1928) Degeneration and Regeneration of the Nervous System, Vol. I. Oxford University Press, London. ROSENBLUETH A. & DEL Fozo E. C. (1943) The centrifugal course of Wallerian degeneration. Am. J. Physiof. 139, 247-254. SATINSKY D., PEPE F.
A. & LIU C. N. (1964) The neurilemma cell in peripheral nerve degeneration and regeneration. Expl Neurol. 9, 441-451. SINGERM. & STEINBERG M. C. (1972) Wallerian degeneration: a reevaluation based on transected and colchicine-poisoned nerves in the amphibian, Triturus. Am. J. Anat. 133, 51-84. SPENCERP. S. & LIEBERMAN A. R. (1971) Scanning electron microscopy of isolated ~ri~heral nerve fibres, normal surface structure and alterations proximal to neuromas, 2. Zelfforsch mikrosk. Anat. 119, 534-551. TERRYR. D. & HARKIN J. C. (1959) Wallerian degeneration and regeneration of peripheral nerves. In The Biology ofbfyelin (ed. KOREYS. R.), pp. 303-320. Hoeber-Harper, New York. THOMASP. K. (1964) Changes in the endoneurial sheaths of peripheral myelinated nerve fibres during Wallerian degeneration. J. Anar. 98, 175-182. WILLIAMSP. L. & HALL S. M. (1971u) Prolonged in eiuo observations of normal peripheral nerve f&es and their acute reactions to crush and deliberate trauma. J. Anat. 108, 397-408. WILLIAMSP. L. & HALL S, M. (19716) Chronic Wallerian degeneration-an in uivo and ultrastructural study. J. Anat. 109, 487-503.
1250
M. R. GERSHENBAUMand F. J. ROISEN
WOOD P. M. (1976) Separation of functional Schwann cells and neurons from normal peripheral Res.115,361-375. WOOD P. M. & BUNGE R. P. (1975) Evidence that sensory axons are mitogenic for Schwann 256, 662-664.
(Accepted
25 July
1978)
nerve
tlrqirt
cellh. SU?:N~
&~~rr,
i IW
Ltd.1978.Printedin
Great Britain.
A SCANNING ELECTRON MICROSCOPIC STUDY OF PERIPHERAL NERVE DEGENERATION AND REGENERATION Department
M. R. GERSHENBAUMand F. J. ROI~EN of Anatomy, Rutgers Medical School-CMDNJ, Piscataway, NJ 08854, U.S.A.
Abstract-The left sciatic nerves of male Holtzman rats were exposed and crushed with a hemostat for 3 s at a point midway between the inferior gluteal nerve and the bifurcation into the tibia1 and common peroneal nerves. Normal myelinated axons appear in the scanning electron microscope as relatively smooth cylindrical structures with interweaving strands of collagen fibers coursing over their length. Nodes of Ranvier are seen as constrictions along the myelinated fibers. After crushing, the nerve fibers swell, and interruptions appear in the nodal and internodal regions causing a beaded morphology. The myelin sheaths fragment into numerous spherical bodies as the degenerative process progresses. Some remnants of myelin debris are found within the nerves 60 days following the lesion A few regenerating myelinated axons are observed within the connective tissue fibers and myelin debris distal to the crushed regions approx 15 days after placement of the lesion. By day 30 the nerves are relatively normal in appearance. The scanning electron micrographs provide a three-dimensional picture of the dynamic processes which occur during nerve degeneration and regeneration. These findings are correlated with previous studies and can serve as a basis for future scanning electron microscopic studies in this area of research.
of a peripheral nerve, there are profound morphological alterations in the nerve fibers and connective tissues involved in the lesion. Electron microscopy has enabled investigators to analyze the ultrastructural events associated with Wallerian degeneration and peripheral nerve regeneration (OHMI, 1961; O’DALY & IMAEDA, 1967; BALLIN &
AFTER injury or severance
hemostat were covered with adhesive tape to limit damage to the nerve during the crush. Scanning electron microscopy
Two rats were killed at each 3day interval from 3 days to 30 days after placement of the lesion; an additional two animals were killed at day 60. To prepare the sciatic nerves for SEM observations, the nerves were exposed and THOMAS,1969; CRAVIOTO,1969; SINGER& STEINBERG, fixed in situ for 10 min with 3% glutaraldehyde (Electron 1972; CALABRE-ITA,MUNGER & GRAHAM, 1973; HUDMicroscopy Sciences, Fort Washington, Pa.) buffered with 0.05 M cacodylate [mixed 1: 1 with Pucks Saline G (Grand SON & KLINE, 1975; GHABRIEL& ALLT, 1977). These Island Biological Co., Grand Island, NY)] before being studies provide a good representation of the overall changes which occur during the degeneration and re- removed and fixed overnight at 4°C. (The segments of scigeneration of myelinated axons, but it is often ex- atic nerves removed for SEM observations were approximately 15 mm long and stretched from a point just below tremely difficult to interpret the three-dimensional the level of the inferior gluteal nerve to the bifurcation process from the two-dimensional images obtained into the tibia1 and common peroneal nerves. The nerves with thin sections. In order to aid in the interprewere not excised more distally because of the difficulty tation of the ultrastructural changes seen with the encountered in the removal of the connective tissue covertransmission electron microscope we employed scanings from the smaller branches.) The nerves were rinsed ning electron microscopy (SEM) to study the alterin saline and post-fixed in 1% osmium tetroxide (Electron Microscopy Sciences, Fort Washington, Pa.) buffered with ations in surface morphology which occur during 0.2 M cacodylate at 4°C for 12 hr. After post-fixation the Wallerian degeneration and subsequent regeneration of the nerve fibers. This study can further serve to epineurium and perineurium were carefuHy removed under a dissecting microscope. The exposed bundles of axons facilitate the use of SEM in future studies of nerve were teased apart and dehydrated in a graded series of degeneration and regeneration. acetone solutions. After two changes of 100% acetone, the nerve fibers were dried in a critical point drying apparatus (Sorvall) and mounted on specimen studs with silver conEXPERIMENTAL PROCEDURES ducting paint. The fibers were then coated with gold in Surgical technique a Denton vacuum evaporator and examined with a JEOL The left sciatic nerves of 25 male Holtzman rats JSM-U3 scanning electron microscope operated at 25 kV. (200-240g) under ether anesthesia were exposed and For these observations we examined three regions of the crushed with a hemostat for 3 s at a point midway between continuous nerve segment: (a) near the site of the crush, the inferior gluteal nerve and the bifurcation into the tibia1 (b) approximately 4-6 mm proximal to the crush site and and common peroneal nerves. The serrated jaws of the (c) approximately 5-8 mm distal to the lesion. It should be noted that some slight variations in the time course Abbreviation: SEM, scanning electron microscopy. of the degenerative and regenerative processes were 1241
1242
ivl K.
GLRSH~ NHAGM
observed between nerve segments within each specific time period. Micrographs depicting the most representative regions were therefore chosen to illustrate best the sequence of events which occur during Wallerian degencration and subsequent nerve regeneration.
RESULTS In order to view the individual myelinated fibers with the SEM, connective tissue elements are pulled or teased away from the nerves. In Fig. lA, these connective tissue elements can easily be identified. The epineurium appears as a thick fibrous network surrounding the entire nerve while the perineurium surrounds only the individual fasicles of the nerve. In these preparations (Fig. 18) normal myelinated axons are seen as relatively smooth cylindrical structures which interweave with strands of collagen and elastic fibers that course over their length. The axons vary in diameter and are closely packed within the nerve bundles. Nodes of Ranvier are occasionally observed and appear as annular constrictions along the myelinated fibers (Fig. 1B and C). Details of the nodal regions are obscured by the overlying connective tissue sheath (sheath of Plenk and Laidlaw) and basal lamina. Small, branching fibrous structures are found running longitudinally along the myelinated axons for various distances (Fig. lB, C and D). These frequently cross to adjacent axons where they often merge with other similar fibers (Fig. 1C and D). These fibers are continuous across the nodes (Fig. 1C) and may be intimately associated with the connective tissue sheath covering the axons (Fig. IB, C and D). Such structures are difficult to identify positively with the SEM but may represent: (a) the unmyelinated component of the sciatic nerve, (h) dense aggregations of collagen fibers, or (c) a combination of both. Proximal
region
No significant changes from normal are observed in any of the regions proximal to the lesion for all time periods studied after crushing the sciatic nerves. These proximal regions look essentially the same as the normal nerve shown in Fig. 1B. Therefore, to avoid redundancy, only the reactive crush and distal segments are described and illustrated, even though the proximal regions were studied. Crush and distal
regions
Three days following placement of the lesion, gross morphological alterations are observed in the crush and distal regions. The myelin sheaths of nerve fibers in the vicinity of the crush (Fig. 1E) are swollen with numerous cytoplasmic bulges occurring along the length of the degenerating axons. Schwann cells appear to be retracting from presumptive nodes of Ranvier and several breaks or constrictions in the internodal regions of the nerve fiber can be found. At this stage, it is often very diffiThree days post-lesion.
and F J. RON K
cult to dilTerentiatc between node. anti II:~W:II~I~~I~ internodal breaks due to the outer connectl\~‘ ti~ue coverings. In the nerve segment distal tcr I!I< lls\~q,:~ (Fig. IF) the nerve fibers appear m ;+ ~mewha~ dtf?& ent stage of degeneration than those in :IIV :.I~I*II region. The myelinated axons arc dissoc~.~tcd jnt;> elliptical segments of varying size. The gaps separat mg these segments are greater than lhosr found Ilcar the region of the crush. Occasionally. those cliip~cl~& appear to be joined only by remnants of the ctmn~~:tive tissue sheath. :Vinr duys post-lesion. In the area of the crush (Fig. 2A) many degenerating fibers begin to fragment into large spherical globules which are orient4 in a linear fashion along the major axis of Ihc IWVC. The globules are covered by strands of collagen and other debris. In the distal region of the nerves (Fig. 2B), fragmentation of the myetin sheath doe5 not appear as extensive as that found near the lesion. Globules of myclin are smaller and seem to hc enclosed by the connective tissue sheath. Very often fibers are found which exhibit a shrunken and Jiatorted morphology. In both the crush and disral regions (Fig. ZA and B), the amount of connecti\r tissue covering the damaged fibers seems to have mcreased while the axons have lost the smooth appearance observed in normal and proximal ncrvc tegments. Fifteen duys post-lrsion. At this stage, the large globules found in the crush region (Fig. 2c‘) arc apparently fragmented into smaller pieces. In some areas fragments of the main spheroids are torn Amy during preparation of the nerves for SEM, leaving behind depressions on the fiber surfaces (Fig. 2Ct. Beneath the degenerative fragments. nerve fibers drc observed which in many respects appear to be normal in surface morphology (Fig. 2C). These most probably represent regenerating fibers that have grow-n into the region of the crush. In the distal segment (Fig. ?D), the globules of myelin are not fragmented as extensively as those found near the lesion. These myetin globules are similar to those seen in the crush region 9 days post-lesion (Fig. 2A). Remnants of axons are seen coursing through the connective tissue fibers. Few regenerating fibers can be identified in this region. Twenty days post-lesion. In the area of the crush (Fig. 3A), globules are further fragmented into smaller spherical pieces. These pieces appear scattered with other bits of debris over the surfaces of regenerating fibers. By day 20, regenerating fibers are also observed in the distal segment of the nerves (Fig. 38). Myelin fragments found in this region are generally larger than those found near the crush site. Thirty days post-lesion. By this time, many normal looking regenerated axons can be observed spanning the region of the crush (Fig. 3C). Remnants of my&n debris are still found in this area after 30 days. The segment distal to the crush (Fig. 3D) appears IO he very similar to the crush region.
Nerve degeneration and regeneration in scanning EM
1243
Discussion
distal. The reason for this remains unclear. Further fragmentation of the spheroids and ovoids into Wallerian degeneration in the peripheral nervous smaller pieces takes place as the degenerative process system has been studied extensively with light and continues. Small globules break off of the larger myetransmission electron microscopy but little has been lin pieces and are often expelled into the endoneurial done to correlate these findings with the threedimenspace (Fig. 2C) as reported by previous investigators sional view obtainable with scanning electron micro@‘DALY & IMAEDA,1967; WILLIAMS& HALL, 1971b; scopy. Some earlier SEM observations on normal and CALAEXRETTA et al., 1973). By 30 days few globules degenerating air dried, teased nerve preparations were can be found in the endoneurial space. In several made by SPENCER& LIEBERMAN (1971). In their study cases the myelin debris has been reported to be phathe air-drying produced an artifactual ‘pattern of pitgocytized by invading macrophages (TERRY& HARlike depressions’ not seen in our specimens and in KIN, 1959; OHMI, 1961; O’DALY & IMAEDA,1967; general distorted the entire surface of the nerve fibers. WILLIAMS& HALL, 1971b), a fact disputed by those The significance of critical point drying in the prepwho contend that the Schwann cells are responsible aration of biological material for SEM has been well for myelin digestion (NATHANIEL& PEASE, 1963; documented (PORTER,KELLEY& AND~EWS,1972). As SATINSKY, PEPE& LIU, 1964; CRAVIOTO,1969; SINGER described previously, normal myelinated axons & STEINBERG, 1972). We have not, however, identified appear as relatively smooth cylindrical structures with these macrophages in our preparations. connective tissue fibers coursing over the surface Regenerating fibers, as shown by our transmission (Fig. 1B). They are often associated with longitudinal electron microscopic studies (unpublished observafibrous bands (Fig. lB, C and D) which seem to be tions), can be observed growing between the myelin similar in appearance to the three-dimensional reconstructions of unmyelinated fibers made by AGUAYO debris in crush segments by 15 days post-lesion (Fig. 2C). By 20 days they are found in the regions & BRAY (1974), but could be bundles of collagen fibers or a combination of the two. These bands are distal to the crush (Fig. 3B). Although most nerve fibers appear to be relatively normal in the medial continuous across the nodes of Ranvier and appear and distal portions of the nerve bundles after 30 days, to be $mly attached to the endoneurial sheath (Fig. 1C). We also occasionally observed the ‘fluting’ there are still fragments of myelin debris in the in the paranodal regions described by HESS& YOUNG endoneurial space at this time. Such debris persists even after 60 days post-lesion. (1952). In which direction does Wallerian degeneration proChanges after nerve crush
ceed?
Three days following the nerve crush, extensive swelling and retraction of the Schwann cells from the nodal regions can be seen. This has been reported to be one of the earliest morphological changes to occur after crushing (RAM~NY CAJAL, 1928; BALLIN & THOMAS, 1969; WILLIAMS& HALL, 1971a,b). Simultaneously we have seen breaks and constrictions occurring in the internodal regions of these fibers causing the formation of primary ellipsoids of greatly varying size. In the distal area the ellipsoids are more numerous than those found near the crush and tend to be qeparated by larger gaps, giving the impression that the distal region is in a different stage of the degenerative process than the crush site. These distal ellipsoids often appear to be joined solely by remnants of endoneurial connective tissue. The endoneurial tubes and basement membranes have been shown by THOMAS(1964) to undergo extensive collapse and folding during Wallerian degeneration, while the continuity of these structures appears to be maintained during the degenerative process providing tubes in which the Schwann cells may proliferate. By 9 days post-lesion the primary myelin ellipsoids fragment into spheroids and ovdids which are often oriented in a linear fashion along the longitudinal axis of the nerve bundles (Fig. 2A and B). The orientation of these myelin remnants is usually in better alignment in the regions closer to the crush site than those more
The direction in which Wallerian degeneration proceeds has long been controversial. While some researchers have reported a centrifugal degeneration of the nerve fibers (ROSENBLUETH & DEL Pozo, 1943; CAUSEY& PALMER,1953; JOSEPH,1973) others found no difference along the length of the fiber or believe that the wave of degeneration passes centripetally (ERLANGER8~ SCHOEPFLE,1946; DONAT & WrsNIEWSKI,1973). LUBINSKA(1977) has shown that in the early stages of Wallerian degeneration, ovoid (ellipsoid) formation in the cut phrenic nerve begins near the lesion and progressively spreads in a distal direction at rates of 46250mm/day depending on fiber diameter. In this study we first examined the lesioned nerves shortly after the formation of ellipsoids had occurred. It was apparent that the ellipsoidal segmentation was more prominent distally than near the region of the crush. This is probably due to the trauma created by the lesioning process, but differences in the spread of Wallerian degeneration between cut and crushed nerves should not be excluded. At a later stage, a reversal in the rate of myelin breakdown seems to occur. The fragmentation and digestion of the myelin ellipsoids appears to proceed more rapidly in the region of the crush than in the distal area, thus giving the appearance of a shift in tlie direction of the degenerative process. Since the spread of degeneration down the fiber is so rapid
1244
M. R.
GERSHENBACJMand
F.
J. ROISFN
FIG. 1. Normal sciatic nerves. (A) A cross-section of the rat sciatic nerve as observed by scanning electron microscopy. The epineurium (EPI) consists of a thick fibrous meshwork which surrounds the entire nerve while the perineurium (PERI) surrounds only the nerve fascicles (65 x ), (B) These structures are relatively smooth with strands of interweaving connective tissue fibers coursing over their length. The node of Ranvier (arrow) appears as a constriction on the myelinated fiber. Note the fibrous longitudinal bands (arrowheads) (1600x). (C) The fibrous bands appear to be tightly applied to the endoneurial sheath as they cross the node of Ranvier, yet loosely applied to the nerve fiber below it. Branches can be seen coming off of the main band (arrowheads) (4500x). (D) The band of fibers can be seen crossing from one axon to another where it becomes tightly attached to the endoneurial sheath. It appears to merge with a smaller band (arrowheads) (2100 x ). Three days post-lesion. (E) Crush: Cytoplasmic bulges and swellings appear in the vicinity of the crush after 3 days. Interruptions along the length of the nerve fibers are seen (arrowheads). It is often difficult to distinguish between nodes of Ranvier and incomplete intemodal breaks in this micrograph (1500 x ). (F) Distal: The nerve fibers are separated into ellipsoids of various sizes. Often these ellipsoids appear to be joined only by remnants of the endoneurial connective tissue (arrowheads) (1500 x). FIG. 2. Nine days post-lesion. (A) Crush: The degenerating fibers become fragmented into large spherical globules which are oriented in a linear fashion along the long axis of the nerve. Note the increase of connective tissue fibers (1500 x ). (B) Distal: Globules of myelin debris in the distal segment are in general smaller than those found near the crush. These globules appear to be enclosed by the connective tissue sheath (1500 x ). Fifteen days post-lesion. (C) Crush: The larger globules are fragmented into many smaller pieces. Occasionally some of these pieces are torn away during sample preparation leaving behind depressions on the surface of the fibers (arrowheads). Nerve fibers which appeared normal in surface morphology can be found adjacent to these degenerating myelin fragments (arrows) (1500 x ). (D) Distal. The spherical globules do not appear to be as extensively fragmented in this region as compared to those in the crush segment. Note the abundance of collagen fibers (1500 x 1. FIG. 3. Twenty days post-lesion. (A) Crush: Many nerve fibers appear to have a normal surface morphology. There is still an abundance of connective tissue fibers and degenerative debris (1500 x ). (B) Distal: Some regenerating nerve fibers are found in the distal segment at this time (arrowhead). There is more debris and connective tissue in this region than in the crush segment (1500 x ). Thirty days post-lesion. (C) Crush: Although most of the myelinated axons appear to be normal, there is still evidence of myelin debris (1500 x ). (D) Distal: Most fibers are normal in appearance but there are still accumulations of myelin fragments and connective tissue fibers (1500 x I.
FIG. 3.
Nerve degeneration and regeneration in scanning EM (LUBINSKA,
1977), a fairly uniform process over the entire length of a small nerve should be expected, unless other factors are involved. One ‘such factor may be related to the fact that the proliferation of non-neuronal cells in vitro is directly dependent upon the presence of neurons (WOOD & BUNGE, 1975; MCCARTHY& PARTLOW, 1976; WOOD, 1976). SinOe the neurons are capable of exerting some types of influence on the Schwann cells, it is possible that the rate of myelin breakdown and digestion by the
1249
Schwann cells may be enhanced by contact with the regenerating nerve fibers. This could explain the apparent shift in the direction of Wallerian degeneration in crushed nerves, as well as the numerous conflicting reports.
Acknowledgements-This work was supported by a research grant, NS 11299, from the National Institutes of Health, and a grant from the Kroc Foundation.
REFERENCES AGUAYOA. J. & BRAY G. M. (1975) Experimental pathology of unmyelinated nerve fibers. In Peripheral Neuropothy (eds DYCX P. J., THOMASP. K. & LAMBERT E. H.).Vol. 1, pp. 363-390. Saunders, Philadelphia. BALLIN R. H. M. & THOMASP. K. (1969) Changes at the node of Ranvier during Wallerian degeneration: an electron microscope study. Acta Neuropath. (Be&) 14, 237-249. CALAEIRETTA A. M., MUNGER B. L. L GRAHAMW. P. (1973) The ultrastructure of degenerating rat sciatic nerves. J. Surg. Res. 14, 465-471. CAUSEYG. & PALMERE. (1953) The centrifugal spread of structural change at the nodes in degenerating mammalian nerves. J. Anut. 87, 185-191. CRAVIOTOH. (1969) Walierian degeneration: ultrastructural and histochemical studies. Bull. Los An&es Neural. Sot. 34, 233-253. DONATJ. R. & WISNIEWSKI H. M. (1973) The spatio-temporal pattern of Wallerian degeneration in mammalian peripheral nerves. Brain Res. 53, 41-53. ERLANGERJ. & SCHOEPFLE G. M. (1946) A study of nerve degeneration and regeneration. Am. J. Physiof. 147, 550-581. GHABRIELM. N. & ALLT G. (1977) Regeneration of the node of Ranvier; a light and electron microscope study. Acta Neuropath. (Be&) 37, 153-163. HF..%A. & YOUNG J. Z. (1952) The nodes of Ranvier. Proc. R. Sac. B 140, 301-320. HUDSONA. R. & KLINE D. G. (1975) Progression of partial experimental injury to peripheral nerve-2. Light and electron microscopic studies. J. Neurosurg. 42, 15-22. JOSEPHB. S. (1973) Somatofugal events in Wallerian degeneration: a conceptual overview. Brain Res. 59, 1-18. LUBINSKAL. (1977) Early course of Wallerian degeneration in myelinated fibres of the rat phrenic nerve. Brain Res. 130, 47-63.
MCCARTHVK. D. & PARTLOWL. M. (1976) Neuronal stimulation of c3H]thymidine incorporation by primary cultures of highly purified non-neuronal cells. Brain Res. 114, 41.5-426. NATHANIELE. J. H. & PEASED. C. (1963) Degenerative changes in rat dorsal roots during Wallerian degeneration. J. W~tr~rruct. Res. 9, 511-532. G’DALY J. A. & IMAEDAT. (1967) Electron microscopic study of Wallerian degeneration in cutaneous nerves caused by mechanical injury. Lab. Invest. 17, 744-766. 0t-1~1 S. (1961) Electron microscopic study on Wallerian degeneration of peripheral nerve. 2. Zellforsch. mikrosk, Anat. 34, 3967. PORTERK. R., KELLEYD. & ANDREWSP. M. (1972) The preparation of cultured cells and soft tissues for scanning electron microscopy. In Proc. Fifth Ann. Stereoscan Colloquium, pp. l-19. Kent Cambridge Scientific Company, Morton Grove, Illinois. RAM~N Y., CAJAL S. (1928) Degeneration and Regeneration of the Nervous System, Vol. I. Oxford University Press, London. ROSENBLUETH A. & DEL Fozo E. C. (1943) The centrifugal course of Wallerian degeneration. Am. J. Physiof. 139, 247-254. SATINSKY D., PEPE F.
A. & LIU C. N. (1964) The neurilemma cell in peripheral nerve degeneration and regeneration. Expl Neurol. 9, 441-451. SINGERM. & STEINBERG M. C. (1972) Wallerian degeneration: a reevaluation based on transected and colchicine-poisoned nerves in the amphibian, Triturus. Am. J. Anat. 133, 51-84. SPENCERP. S. & LIEBERMAN A. R. (1971) Scanning electron microscopy of isolated ~ri~heral nerve fibres, normal surface structure and alterations proximal to neuromas, 2. Zelfforsch mikrosk. Anat. 119, 534-551. TERRYR. D. & HARKIN J. C. (1959) Wallerian degeneration and regeneration of peripheral nerves. In The Biology ofbfyelin (ed. KOREYS. R.), pp. 303-320. Hoeber-Harper, New York. THOMASP. K. (1964) Changes in the endoneurial sheaths of peripheral myelinated nerve fibres during Wallerian degeneration. J. Anar. 98, 175-182. WILLIAMSP. L. & HALL S. M. (1971u) Prolonged in eiuo observations of normal peripheral nerve f&es and their acute reactions to crush and deliberate trauma. J. Anat. 108, 397-408. WILLIAMSP. L. & HALL S, M. (19716) Chronic Wallerian degeneration-an in uivo and ultrastructural study. J. Anat. 109, 487-503.
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M. R. GERSHENBAUMand F. J. ROISEN
WOOD P. M. (1976) Separation of functional Schwann cells and neurons from normal peripheral Res.115,361-375. WOOD P. M. & BUNGE R. P. (1975) Evidence that sensory axons are mitogenic for Schwann 256, 662-664.
(Accepted
25 July
1978)
nerve
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cellh. SU?:N~
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