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Analysis of axon-sheath relations during early wallerian degeneration
BRAIN RESEARCH
199
ANALYSIS OF A X O N - S H E A T H RELATIONS D U R I N G E A R L Y WALLERIAN DEGENERATION
R. L. F R I E D E AND A. J. M A R T I N E Z
Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106 (U.S.A.) (Accepted October 20th, 1969)
INTRODUCTION
A rapid increase in the volume of axoplasm in swollen fibers is accompanied by an extension of the myelin sheath due to slippage of the myelin lamellae; the direction of slippage is 'outward', resulting in a wider sheath having fewer turns of lamellae6,12. The present investigation attempts to clarify the changes in the myelin sheath that develop upon an acute reduction in the volume of axoplasm as it occurs during the initial phase of WaUerian degeneration. 'Inward' slippage of the myelin lamellae around the shrinking axis cylinder resulted in contraction of the sheath with increased thickness relative to the size of the axis cylinder. MATERIAL AND METHODS
Twenty-one adult rats of Sprague-Dawley strain were sacrificed l, 2, 3, 4, 6, 8 and 10 days after bilateral transection of the sciatic nerves. Treatment of the specimens was the same as for the preceding reports6,12. Briefly, it consisted of fixation in situ with glutaraldehyde, postosmication, and embedding in Maraglas. Measurements were made in electron micrographs, or in light microscopic photographs of 1 # m sections, magnification 2000. The method of measuring nerve fibers in 1 #m sections had to be modified because fibers undergoing Wallerian degeneration are rarely straight and cylindrical. It is difficult, therefore, to cut sections precisely perpendicular to the fiber axis after the 3rd day and undesirable variation results when axon circumference is compared with sheath thickness. This variation can be reduced by measuring both the inner and outer circumferences of the myelin sheath; these values allow calculation of the ratio of the caliber of the fiber and its axis cylinder even if the fiber is cut slightly oblique. This method does not eliminate the error in the determination of absolute fiber size; however, the latter has little influence on the interpretation of the present data. Brain Research, 19 (1970) 199-212
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R. L. FRIEDE AND A. J. MARTINEZ
RESULTS
Definitions The term axis cylinder, as used in this text, defines the space inside the myelin sheath, regardless of whether or not it contains axoplasm. Collapse of the sheath signifies a marked reduction in the volume of the axis cylinder without change in its circumference. Contraction of the sheath defines a reduction in the volume of the axis cylinder because of reduction in its circumference. Collapse and contraction may concur.
Changes in nerve fibers undergoing Wallerian degeneration, in 1 Izm sections All samples were taken at least 2 m m distal to the transection, excluding the distal stumps containing reactive axon swellings. Cross-sections of the nerves showed no definite changes on the 1st day after transection. By the 2nd day, the majority of the fibers were shrunken or collapsed and the variation in the volume of axoplasm was greater than in normal fiber populations. The collapse of fibers advanced through the 3rd and 4th day, resulting in many excessively angulated, bean-shaped, and irregular fiber forms. Irregularities in sheath structure were also evident by the 4th day and increased thereafter; they consisted of local splitting or inhomogeneity of the sheath, or of two concentric rings of myelin. The two rings were of approximately equal thickness for the majority of fibers, as shown by the histogram in Fig. 1. By the 6th and 81h days, m a n y of the smaller fibers had relatively small or ill-defined axis cylinders; large 'myelin forms' without discernible axis cylinders also became apparent and subsequently increased in frequency. Thinly myelinated fibers,
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SHEATHS WITH TWO CONCENTRIC RINGS OF MYELIN : THICKNESS OF THE RINGS
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Fig. 1. Histograms of the thickness of the inner and outer ring of myelin for cross-sections of fibers showing two separate, concentric rings of myelin sheath. The two rings are usually of equal thickness, supporting the view that they represent pouches of the sheath protruding into the axis cylinder.
Brain Research, 19 (1970) 199-212
AXON-SHEATHRELATIONSDURING WALLERIANDEGENERATION
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TABLE I CHANGES IN THE DENSITY OF T H I N L Y MYELINATED FIBERS*
Day after transection 1
2 3 4 6 8 10
Number of fibers counted
Percent of thin fibers
192 243 287 195 393 311 370
28 24.6 8.7 5.9 5.3 3.8 3.3
* For the convenience of counting, thinly myelinated fibers were identified as fibers of 0.7 pm or less sheath thickness.
which constituted approximately 28 ~ of the fiber population in normal nerves (Table I), were greatly diminished after the 3rd or 4th day. By the 10th day, fibers with a discernible large axis cylinder were a minority, as most fibers showed an indistinct or absent axis cylinder; irregularities in sheath structure abounded. The features were essentially alike in longitudinal sections. Serial reconstructions from 1 /zm sections demonstrated excessive folding and pouching of the sheath, the pouches often protruding into the axis cylinder (Fig. 2). Measurements o f fibers in 1 # m sections
The proportions of sheath and axis cylinder were determined from measurements of the inner and outer circumferences of the sheaths (Material and Methods). In normal sciatic nerves the ratio for the circumferences of the fiber and its axis cylinder was 1.0-0.7 for all fibers regardless of their caliber. The value agrees with the most widely quoted ratios, and with measurements of electron micrographs 7. A change in the slope of the regression curve was noticed by the 2nd day; it increased consistently with each successive phase of Wallerian degeneration (Fig. 3 and Table II). Accordingly, there were progressive changes in the proportions of axis cylinder and sheath that could not be explained by the collapse of the fiber and that were consistent with sheath contraction. Representative axon-sheath ratios (Fig. 3) illustrate the relative increases in sheath thickness for 3 fibers of different calibers. All correlation coefficients were above 0.92 and statistically highly significant. Sheath contraction, therefore, did not affect the scatter of the fiber scores. The change in the slopes of the regression curves was associated with a change in intercepts (Table II), which could not be explained by assuming that all sheaths contract by the same percentage regardless of fiber caliber. The curves imply that the sheaths of thin fibers contracted more than those of thick fibers. On the 2nd day, the intercept for zero circumference of the axis cylinder (encircled in Fig. 3) was larger than the circumference of normal non-myelinated fibers (Table II); in other words, Brain Research, 19 (1970) 199-212
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R . L . FRIEDE AND A. J. MARTINEZ
Fig. 2. Collapse of the axis cylinder with contortion of the sheath and protrusion of pouches into the axonal space is shown by reconstruction of a fiber from serial 1/~m sections. 1,000 ×.
the regression curves did not allow for the existence of non-myelinated fibers at and after this day. By the 6th day, the intercepts had moved well into the range of thin myelinated fibers, suggesting that thin myelinated fibers disappear earlier than thick myelinated fibers. Counts of thin and thick fibers (Table I) verified this conclusion. The intercepts for zero circumference of the axis cylinder corresponded to finite values for the circumference of the fiber, indicating the existence of a highly unusual fiber form having a thick myelin sheath but no axis cylinder. These characteristics corresponded precisely to the features of the so-called 'myelin forms' that have little or no space for the axis cylinder (Fig. 5). Accordingly, 'myelin forms' should be redefined as extreme or terminal stages of sheath contraction. It should be emphasized that the features of 'myelin forms' were predicted by the computer. No
Brain Research, 19 (1970) 199-212
AXON-SHEATH
RELATIONSDURING WALLERIANDEGENERATION
203
TABLE II STATISTICAL REGRESSION ANALYSES OF THE DATA IN FIGS.
Correlation Significance Slope coefficient
3 AND
6
Intercept (units)
Calculateddiameter of fibers for zero axon circumference (#m)
0.18" 0.03 1.19 1.22 1.26 2.70 4.09 2.97
0.34 0.06 2.2 2.3 2.4 5.1 7.7 5.6
A. Measurements in 1 #m sections (Fig. 3)
Normal 1 day 2 days 3 days 4 days 6 days 8 days 10 days
0.96 0.93 0.92 0.94 0.98 0.96 0.95 0.99
< 0.01 < 0.01 -< 0.01 < 0.01 < 0.01 < 0.01 < 0.01 <~0.01
0.69 0.73 0.79 0.81 0.86 0.94 0.98 0.99
B. Measurements in electron micrographs (Fig. 6)
2 days 4 days 8 days
0.95 0.96 0.97
< 0.01 < 0.01 < 0.01
0.72 0.84 0.88
4.16"* 12.80 19.80
3.9 12.0 18.6
* Scales in Fig. 3 are converted to #m; one unit equals 5.9 #m. ** One unit equals 2.95 #m.
measurements of myelin forms were entered into the statistical analysis; the prediction of their features - - thick sheaths lacking axis cylinders - - was based entirely on the regression analysis of fibers that still had sizeable axis cylinders. Histochemical observations in 1 tzm sections
A histochemical technique for the demonstration of NAD-diaphorase activity in individual mitochondria in plastic-embedded 1 /zm thick sections6, tz was applied to the present material. By the 1st day, the axoplasmic density of mitochondria in myelinated fibers appeared only slightly diminished. Some of the fibers showed small focal accumulations of mitochondria in the axoplasm adjacent to the nodes of Ranvier by the 1st and 2nd day (Fig. 4), probably identical to the transient accumulations of mitochondria observed with the electron microscope by Webster23; they were not seen at later phases. Axoplasmic mitochondria were markedly diminished or absent on the 2nd and 3rd day. A few axoplasmic mitochondria with enzyme activity could be detected by careful scrutiny of the fibers up to the 8th day, but the vast majority had disappeared after the 3rd day. Swollen axons near the cut surface of the distal nerve stump showed dense accumulations of mitochondria similar to those in the proximal stumps, although densities appeared not to be as high. The Schwann cell cytoplasm showed relatively little changes in mitochondrial density. Pools of cytoplasm with densely crowded mitochondria were attached to the sheaths during the first 4 days (Fig. 4), giving the impression that these changes were due to redistribution of cytoplasm along the internodes. At later phases, there was a Brain Research~ 19 (1970) 199-212
204
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Fig. 3. Wallerian degeneration is accompanied by a progressive change in the slope of the relation between the inner and outer circumferences of the myelin sheath. Determination of these parameters eliminates error due to slightly oblique cutting of the fiber (see Material and Methods). Statistical regression analyses of the data are given in Table I. The changes in the ratio of axon diameter to fiber diameter are shown for fibers of 25, 60 and 85 /~m circumference for each set. A progressive thickening of the sheath in relation to the axis cylinder is evident for the thin fibers. The open circles for the 2nd and 8th days are measurements of the outer circumference of 'myelin forms'; these were not entered into the computer analysis. Curves for days 4 and 10 (Table I) are not shown.
gne vacuolization and reduced mitochondrial density of the cytoplasm associated with collapsed fibers, with fibers with irregularities of sheath structure, or with myelin Brain Research~ 19 (1970) 199-212
AXON-SHEATH RELATIONSDURING WALLERIAN DEGENERATION
205
Fig. 4. Demonstration of mitochondria by the reaction for NAD-diaphorase in 1 /~m thick plasticembedded sections. Top : Mitochondria are either greatly diminished, or have disappeared, from the axoplasm 3 days after cutting the nerve. High mitochondrial density is found in the Schwann cell cytoplasm. Middle: Transient accumulations of mitochondria in the proximity of the nodes of Ranvier are found on the 1st (shown) and on the 2nd day, probably identical to those described by Webster 23. Bottom: By the 4th day, many of the Schwann cells show less than normal mitochondrial density, apparently due to a fine vacuolization of their cytoplasm. Early 'Btingner's bands' show moderate mitochondrial density. All 1,000 x.
forms. The m i t o c h o n d r i a l density in the cytoplasm of BiJngner's bands, was either equal to or less t h a n that in n o r m a l S c h w a n n cells; Biingner's b a n d s were n u m e r o u s by the 10th day (Fig. 4).
Fine structure of fibers Electron microscopic studies were done only for rats killed 2, 4, 6 a n d 8 days after transection of their nerves; the observations are reported only briefly, as numerous p u b l i c a t i o n s are available on the subject (see Discussion). Disintegration of
Brain Research, 19 (1970) 199-212
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R . L . FRIEDE AND A. J. MARTINEZ
neurofilaments and microtubules was prevalent 2 and 4 days after transection, and ftoccular material was distributed irregularly throughout the axis cylinder. Mitochondria and smooth endoplasmic reticulum were sparse or absent; the persistent mitochondria showed retrogressive changes such as swelling and vacuolization. No changes were found in the myelin sheaths. The Schwann cell cytoplasm appeared unchanged by the 2nd day but elongated mesaxons and leaflets of myelin were occasionally found by the 4th day. The axoplasm of non-myelinated fibers showed the same changes as described above. A reduced number of non-myelinated fibers per Schwann cell was noticed by the 2nd day, with m a n y of the profiles appearing smaller than normal; invaginated surface membranes of Schwann cells suggestive o f ' e m p t i e d ' mesaxons were also seen and prevailed by the 4th day. Axis cylinders were 'empty' or contained floccular or membranous debris by the 6th and 8th days, when changes in the myelin sheath and in the Schwann cell cytoplasm became more prominent. The latter showed separation into numerous islands underneath the intact basement membrane, each bound by a continuous
Fig. 5. Electron micrograph 6 days after transection shows a 'myelin form' without discernible axis cylinder, encompassed by Schwann cell cytoplasm. The latter shows separation of several cytoplasmic islands encompassed by a common basement membrane. 28,800 x. Brain Research, 19 (1970) 199-212
AXON-SHEATH RELATIONS DURING WALLERIAN DEGENERATION
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plasma membrane (Fig. 5). The cytoplasm in these islands resembled that in the main pool of Schwann cell cytoplasm in showing filaments, microtubules, ribosomes, smooth and granular endoplasmic reticulum and mitochondria. Convoluted aggregates of myelin leaflets were found in some fibers. Many myelin sheaths were without distinct changes in structure, although they appeared more angulated than normal and relatively thick as compared with their axis cylinder. Myelin forms generally showed variation in interperiod width and irregular foldings and contortions of the lamellae. Most were encompassed by a generous pool of cytoplasm showing extensive separation into 'islands'. Non-myelinated fibers could not be positively identified, as the attribution of Bfingner's bands to myelinated or non-myelinated became a matter of conjecture if no remnants of myelin were present.
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Fig. 6. Measurements made in electron micrographs at three phases of Wallerian degeneration show changes comparable to those shown in Fig. 3. Only fibers with a solid sheath (i.e. without splitting of the myelin leaflets) were measured. One unit corresponds to 2.95 ym. Statistical regression analyses of the data are given in Table I.
Brian Research, 19 (1970) 199-212
15
208
R . L. F R I E D E A N D A. J. M A R T I N E Z
Measurements in electron micrographs
Measurements in electron micrographs were made with the same technique as those in 1 # m sections to facilitate comparison of the sets of data. Care was taken to measure only fibers with solid myelin sheaths, eliminating sources of errors such as a thickening of the sheath due to splitting of its myelin lamellae, or due to the presence of narrow clefts of Schmidt-Lantermann which may have been unnoticed in 1 # m sections. The curves were extremely similar to those obtained from 1 ,urn sections (Fig. 6) validating the use of the latter, and adding evidence to the concept of sheath contraction. The interperiod width of myelin lamellae in contracted sheaths was also measured and was found to be approximately 160 A - - the same as in normal fibers - - except for sites where splitting of myelin leaflets had occurred. DISCUSSION
The measurements presented in this report show that early Wallerian degeneration is characterized by a progressive change in the relation between the caliber of the axis cylinder and the thickness of the myelin sheath, indicating sheath contraction. The extreme end-stage of sheath contraction are the 'myelin forms' having numerous turns of myelin lamellae around a minimal or absent axis cylinder. The increase in sheath thickness cannot be explained by the formation of new myelin, as biochemical studies on Wallerian degeneration agree in showing no change in lipid composition of the nerves during the initial 8 days 9. Increase in sheath thickness, also, cannot be explained by the collapse of the axis cylinder, although contraction coexists with collapse. Our data, therefore, evidence contraction of the sheath by inward slippage of the myelin lamellae, producing a sheath of increasing thickness around a shrinking axis cylinder. This process is comparable in nature, but opposite in direction, to the expansion of sheaths of swollen axons. It appears to be more marked in thin myelinated fibers than in thick myelinated fibers, in contrast with sheath expansion, which was found to affect fibers of all calibers by the same degree. Wallerian degeneration may be divided into two phases. (1) The first is characterized by a readjustment or contraction of the sheath around the shrinking axis cylinder, without significant myelin degeneration. Even the appearance of the conspicuous 'myelin forms' during this period signifies excessive contraction rather than myelin breakdown. Sheath contraction lasts for about 8-10 days and is followed by (2) the second phase of Wallerian degeneration, characterized by sheath disintegration. This phase is characterized by biochemical degradation of the myelin 9 and by loss of its histochemical staining properties 15. This concept of Wallerian degeneration reconciles some of the apparent contradictions between the morphological and biochemical data. Another time-honored concept on Wallerian degeneration is questioned by our data. It is generally accepted that thick fibers degenerate earlier than thin fibers. Our data, in contrast, show that the thinly myelinated fibers disappear much faster than the thick ones. The traditional dictum of the early disappearance of the thick Brain Research, 19 (1970) 199-212
AXON-SHEATH RELATIONS DURING WALLERIAN DEGENERATION
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fibers is probably a misinterpretation of the progressive shrinkage of the axis cylinder. If all fibers change successively into the next lower classes, one would have the impression that the thick fibers disappear first, at least if the caliber of the axis cylinder only is taken into account. Coexistent with contraction of the sheath, there is also collapse of the sheath, evident by the appearance of angulated and contorted fiber profiles. One striking manifestation of collapse are pouches of sheath which appear as two concentric myelin rings in cross-sections. That these profiles, indeed, represent pouches protruding deeply into the axis cylinder is demonstrated by histograms showing that the two rings have equal thickness for most of the fibers (Fig. 1). If the concentric rings were intussusceptions of sheath, a substantial number of fibers would show three rings, which was not the case. If they were gaping clefts of Schmidt-Lantermann, cross-sections would show any conceivable ratio of the thickness of the inner and outer rings of myelin. Our observations on the fine structure of nerve fibers undergoing Wallerian degeneration agree with numerous previous electron microscopic investigations20,22, except for the timing of the degeneration of non-myelinated fibers and for the probably interrelated problem in identifying the cytoplasmic islands underneath the basement membrane of fibers or inside the collapsed Schwann tubes. These 'islands' have been described as survival, or very slow degeneration, respectively, of nonmyelinated fibersS,S,ll,lS; as pseudopodes of Schwann cells13,~4; or as Btingner's bands3, 21. During the later phases of Wallerian degeneration, when regeneration commences, identification of these structures is further complicated by the fact that regenerating axons may actually coexist with Schwann cellprocesses inside the Schwann tubesl4,1s,2L Our electron microscopic observations of early disappearance of non-myelinated fibers agree with the changes predicted by the regression curves in Figs. 3 and 6, both indicating that non-myelinated axons disappear very fast, the majority by the 2nd or 3rd day, leaving behind only 'empty' Schwann cells with extensively folded and indented surface membranes. The same rapid disappearance of non-myelinated fibers was found in spinal nerves and roots13,14,16 and in the iris following ganglionectomy17. Previous reports on prolonged survival, or exceptionally slow degeneration of non-myelinated fibers 5,8,~,19 do not illustrate the characteristic features of bundles of non-myelinated fibers encompassed by a common Schwann cell envelope. The illustrations in these reports are rather similar to those called Btingner's bands, or regenerating axons, respectively, by other authors. Hence, the claims concerning the exceptionally slow degeneration of non-myelinated fibers should be dismissed, based on doubtful identification. Nathaniel and PeasO3,14 describe both regenerating axons and pseudopodes of Schwann cells underneath the basement membrane of fibers during early Wallerian degeneration (4 days after crush); some of the profiles, called regenerating axons, are indistinguishable from the structures described as Biingner's bands by Thomas 21 and by B15mcke and Niedorf3. Furthermore, regenerating axons were described 2 cm below the division 7 days after transection of the sciatic nervO, which is incompatible Brain Research, 19 (1970) 199-212
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R. L. FRIEDE AND A. J. MARTINEZ
with the normal speed of fusion of the stumps of transected nerves. Hence, the need for more precise criteria for the identification of these profiles is evident. Regenerating axons show an orderly arrangement of neurofilaments and microtubules at all phases of regeneration, since these extend into the immediate proximity of the clusters of vesicles found in the tips of growth cones 4. At late phases of regeneration, the formation of a mesaxon and the deposition of myelin readily identify a regenerating axon. In contrast, the 'islands', or 'pseudopodes', of Schwann cell cytoplasm are characterized by the following features: (a) They appear en m a s s e during the initial phase of Wallerian degeneration and are found at sites where the occurrence of regenerating axons is unlikely. (b) They are initially located between the basement membrane and the cytoplasmic envelope of myelinated fibers 21. Later, after degeneration of the fibers, they occur in bundles inside the collapsed Schwann tubes. (c) The cytoplasm first lacks filamentous structures, but formation of filaments may occur at later phases of Wallerian degeneration2,L (d) There is a complete lack of growth cones during their formation. By applying these criteria, we identified the majority of profiles in our material as 'islands', or processes, of Schwann cell cytoplasm. The mechanism producing separation of Schwann cell processes is not entirely clear. They have been considered finger-like proliferations or pseudopodes10,13,14. It is more likely that the early appearance of such profiles between the sheath and the basement membrane of myelinated fibers indicates damage by rapid slipping of the sheath, as has been proposed for swollen fibers in the preceding reports6,12. The coaxial movement of myelin leaflets evidently meets less resistance than the spinning of larger pools of cytoplasm around the fiber, regardless of whether the direction of slippage is inwards or outwards. Hence, the cytoplasmic strands or islands are likely the product of disruption of the Schwann cell envelope by rapid slippage of the sheath. Their first appearance in swollen fibers and in fibers undergoing Wallerian degeneration coincides, in each case, with the period of sheath adjustment. The timing of these periods differs in the two situations. Finally, we like to point out that there is a tendency, in the electron microscopic literature, to consider every change in the fine structure of the myelin sheath as indicative of a primary alteration of myelin. The present data show that changes in the sheath may develop in consequence to primary alterations in the volume of axoplasm. Thus, definition of the mechanism producing alteration in the fine structure of the sheath requires a careful quantitative analysis of all aspects of fiber structure. SUMMARY
Measurements of axon-sheath relations of fiber populations undergoing early Wallerian degeneration showed contraction of the sheath around the shrinking axis cylinder concurrent with a collapse of the axis cylinder. Collapse was associated with folding and pouching of the sheath. Contraction was produced by inward slippage of the myelin leaflets, producing a progressively thicker sheath around a shrinking axis cylinder. The so-called 'myelin forms' represent the end-stages of Brain Research,
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AXON-SHEATH RELATIONS DURING WALLERIANDEGENERATION
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sheath contraction, characterized by a relatively thick sheath a r o u n d a m i n i m a l or n o n - e x i s t e n t axis cylinder. Accordingly, W a l l e r i a n degeneration consists of two phases; the first is characterized by sheath a d a p t a t i o n (contraction) a n d the second by sheath disintegration. Sheath c o n t r a c t i o n proceeds faster in the thinly m y e l i n a t e d fibers than in the thick ones. The axons of n o n - m y e l i n a t e d fibers disappear first. These observations were c o m p a r e d with the r e d i s t r i b u t i o n a n d the eventual loss of axonal mitochondria. ACKNOWLEDGEMENTS This investigation was supported by U.S. Public Health G r a n t NB 06239 a n d Special Fellowship NB 19903 from the N a t i o n a l Institute of Neurological Diseases a n d Strokes.
REFERENCES 1 BARTON, A. A., An electron microscope study of degeneration and regeneration of nerve, Brain,
85 (1962) 799-808. 2 BLtJMCKE,S., Elektronenoptische Untersuchungen an Schwann'schen Zellen w/ihrend der Waller'schen Degeneration peripherer Nerven, Verh. Dtsch. Ges. Path., 6 (1965) 346-349. 3 BL~JMCKE, S., AND NIEDORF, H. R., Electron microscope studies of Schwann cells during the Wallerian degeneration with special reference to the cytoplasmic filaments, Acta neuropath. (Berl.), 6 (1966) 46-60. 4 DEL CERRO, P., AND SNIDER, R. S., Studies on the developing cerebellum. Ultrastructure of the growth cones, J. comp. Neurol., 133 (1968) 341-362. 5 FISHER, E. R., AND TURANO, A., Schwann cells in Wallerian degeneration, Arch. Path., 75 (1963)
517-527. 6 FRIEDE, R. L., AND MARTINEZ, A. J., Analysis of the process of sheath expansion in swollen nerve fibers, Brain Research, 19 (1970) 165-182. 7 FRIEDE, R. L., AND SAMORAJSKI, T., Relation between the number of myelin lamellae and axon circumference in fibers of vagus and sciatic nerves of mice, J. comp. NeuroL, 130 (1967) 223-232.
8 HONJIN, R., NAKAMURA,T., AND ]MURA, M., Electron microscopy of peripheral nerve fibers. III. On the axoplasmic changes during Wallerian degeneration, Okafimas Folia anat. jap., 33 (1959) 131-156. 9 JOHNSON, A. C., MCNABB, A. R., AND ROSSITER,R. J., Chemistry of Wallerian degeneration, Arch. Neurol. Psychiat. (Chic.), 64 (1950) 105-121. 10 LAMPERT, P. W., AND SCHOCHET, S. S., JR., Demyelination and remyelination in lead neuropathy, J. Neuropath. exp. Neurol., 27 0968) 527-545. 11 LEE, J., Electron microscopy of Wallerian degeneration, J. comp. NeuroL, 120 (1963) 65-79. 12 MARTINEZ, A. J., AND FRIEDE, R. L., Accumulation of axoplasmic organelles in swollen nerve fibers, Brain Research, 19 (1970) 183-198. 13 NATHANIEL,E. J. H., AND PEASE, D. C., Degenerative changes in rat dorsal roots during Wallerian degeneration, J. Ultrastruct. Res., 9 (1963) 511-532. 14 NATHANIEL, E. J. H., AND PEASE, C., Regenerative changes in rat dorsal roots following Wallerian degeneration, J. Ultrastruct. Res., 9 (1963) 533-549. 15 NOBACK, C. R., AND MONTAGNA, W., Histochemical studies of the myelin sheath and its fragmentation products during Wallerian (secondary) degeneration, J. comp. NeuroL, 97 (1952) 211-232. 16 OHraI, S., Electron microscopic study on Wallerian degeneration of the peripheral nerve, Z. Zellforsch., 54 (1961) 39-67. 17 ROTH, C. D., AND RICHARDSON, K. C., Electron microscopical studies on axonal degeneration in the rat iris following ganglionectomy, Amer. J. Anat., 124 (1969) 341-360. Brain Research, 19 (1970) 199-212
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18 SATINSKY,D., PEPE, F. A., AND LIU, C. N., The neurilemma cell in peripheral nerve degeneration and regeneration, Exp. Neurol., 9 (1964) 441-451. 19 TAXI, J., t~tude au microscope 61ectronique de la dSgSndrescence wallerienne des fibres nerveuses amySliniques, C. R. Acad. Sci. (Paris'), 248 (1959)2796-2798. 20 TERRY, R. D., AND HARKIN, J. C., Wallerian degeneration and regeneration of peripheral nerves. In S. A. KOREY (Ed.), The Biology of Myelin, Harper (Hoeber), New York, 1959, pp. 303-320. 21 THOMAS, P. K., Changes in the endoneurial sheaths of peripheral myelinated nerve fibres during Wallerian degeneration, J. Anat. (Lond.), 98 (1964) 175-182. 22 VIAL, J. D., The early changes in axoplasm during Wallerian degeneration, J. biophys, bioehem. Cytol., 4 (1958) 551-556. 23 WEBSTER, DEF. H., Transient, focal accumulation of axonal mitocbondria during the early stages of Wallerian degeneration, J. Cell Biol., 12 (1962) 361-383.
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ANALYSIS OF A X O N - S H E A T H RELATIONS D U R I N G E A R L Y WALLERIAN DEGENERATION
R. L. F R I E D E AND A. J. M A R T I N E Z
Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106 (U.S.A.) (Accepted October 20th, 1969)
INTRODUCTION
A rapid increase in the volume of axoplasm in swollen fibers is accompanied by an extension of the myelin sheath due to slippage of the myelin lamellae; the direction of slippage is 'outward', resulting in a wider sheath having fewer turns of lamellae6,12. The present investigation attempts to clarify the changes in the myelin sheath that develop upon an acute reduction in the volume of axoplasm as it occurs during the initial phase of WaUerian degeneration. 'Inward' slippage of the myelin lamellae around the shrinking axis cylinder resulted in contraction of the sheath with increased thickness relative to the size of the axis cylinder. MATERIAL AND METHODS
Twenty-one adult rats of Sprague-Dawley strain were sacrificed l, 2, 3, 4, 6, 8 and 10 days after bilateral transection of the sciatic nerves. Treatment of the specimens was the same as for the preceding reports6,12. Briefly, it consisted of fixation in situ with glutaraldehyde, postosmication, and embedding in Maraglas. Measurements were made in electron micrographs, or in light microscopic photographs of 1 # m sections, magnification 2000. The method of measuring nerve fibers in 1 #m sections had to be modified because fibers undergoing Wallerian degeneration are rarely straight and cylindrical. It is difficult, therefore, to cut sections precisely perpendicular to the fiber axis after the 3rd day and undesirable variation results when axon circumference is compared with sheath thickness. This variation can be reduced by measuring both the inner and outer circumferences of the myelin sheath; these values allow calculation of the ratio of the caliber of the fiber and its axis cylinder even if the fiber is cut slightly oblique. This method does not eliminate the error in the determination of absolute fiber size; however, the latter has little influence on the interpretation of the present data. Brain Research, 19 (1970) 199-212
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R. L. FRIEDE AND A. J. MARTINEZ
RESULTS
Definitions The term axis cylinder, as used in this text, defines the space inside the myelin sheath, regardless of whether or not it contains axoplasm. Collapse of the sheath signifies a marked reduction in the volume of the axis cylinder without change in its circumference. Contraction of the sheath defines a reduction in the volume of the axis cylinder because of reduction in its circumference. Collapse and contraction may concur.
Changes in nerve fibers undergoing Wallerian degeneration, in 1 Izm sections All samples were taken at least 2 m m distal to the transection, excluding the distal stumps containing reactive axon swellings. Cross-sections of the nerves showed no definite changes on the 1st day after transection. By the 2nd day, the majority of the fibers were shrunken or collapsed and the variation in the volume of axoplasm was greater than in normal fiber populations. The collapse of fibers advanced through the 3rd and 4th day, resulting in many excessively angulated, bean-shaped, and irregular fiber forms. Irregularities in sheath structure were also evident by the 4th day and increased thereafter; they consisted of local splitting or inhomogeneity of the sheath, or of two concentric rings of myelin. The two rings were of approximately equal thickness for the majority of fibers, as shown by the histogram in Fig. 1. By the 6th and 81h days, m a n y of the smaller fibers had relatively small or ill-defined axis cylinders; large 'myelin forms' without discernible axis cylinders also became apparent and subsequently increased in frequency. Thinly myelinated fibers,
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TABLE I CHANGES IN THE DENSITY OF T H I N L Y MYELINATED FIBERS*
Day after transection 1
2 3 4 6 8 10
Number of fibers counted
Percent of thin fibers
192 243 287 195 393 311 370
28 24.6 8.7 5.9 5.3 3.8 3.3
* For the convenience of counting, thinly myelinated fibers were identified as fibers of 0.7 pm or less sheath thickness.
which constituted approximately 28 ~ of the fiber population in normal nerves (Table I), were greatly diminished after the 3rd or 4th day. By the 10th day, fibers with a discernible large axis cylinder were a minority, as most fibers showed an indistinct or absent axis cylinder; irregularities in sheath structure abounded. The features were essentially alike in longitudinal sections. Serial reconstructions from 1 /zm sections demonstrated excessive folding and pouching of the sheath, the pouches often protruding into the axis cylinder (Fig. 2). Measurements o f fibers in 1 # m sections
The proportions of sheath and axis cylinder were determined from measurements of the inner and outer circumferences of the sheaths (Material and Methods). In normal sciatic nerves the ratio for the circumferences of the fiber and its axis cylinder was 1.0-0.7 for all fibers regardless of their caliber. The value agrees with the most widely quoted ratios, and with measurements of electron micrographs 7. A change in the slope of the regression curve was noticed by the 2nd day; it increased consistently with each successive phase of Wallerian degeneration (Fig. 3 and Table II). Accordingly, there were progressive changes in the proportions of axis cylinder and sheath that could not be explained by the collapse of the fiber and that were consistent with sheath contraction. Representative axon-sheath ratios (Fig. 3) illustrate the relative increases in sheath thickness for 3 fibers of different calibers. All correlation coefficients were above 0.92 and statistically highly significant. Sheath contraction, therefore, did not affect the scatter of the fiber scores. The change in the slopes of the regression curves was associated with a change in intercepts (Table II), which could not be explained by assuming that all sheaths contract by the same percentage regardless of fiber caliber. The curves imply that the sheaths of thin fibers contracted more than those of thick fibers. On the 2nd day, the intercept for zero circumference of the axis cylinder (encircled in Fig. 3) was larger than the circumference of normal non-myelinated fibers (Table II); in other words, Brain Research, 19 (1970) 199-212
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R . L . FRIEDE AND A. J. MARTINEZ
Fig. 2. Collapse of the axis cylinder with contortion of the sheath and protrusion of pouches into the axonal space is shown by reconstruction of a fiber from serial 1/~m sections. 1,000 ×.
the regression curves did not allow for the existence of non-myelinated fibers at and after this day. By the 6th day, the intercepts had moved well into the range of thin myelinated fibers, suggesting that thin myelinated fibers disappear earlier than thick myelinated fibers. Counts of thin and thick fibers (Table I) verified this conclusion. The intercepts for zero circumference of the axis cylinder corresponded to finite values for the circumference of the fiber, indicating the existence of a highly unusual fiber form having a thick myelin sheath but no axis cylinder. These characteristics corresponded precisely to the features of the so-called 'myelin forms' that have little or no space for the axis cylinder (Fig. 5). Accordingly, 'myelin forms' should be redefined as extreme or terminal stages of sheath contraction. It should be emphasized that the features of 'myelin forms' were predicted by the computer. No
Brain Research, 19 (1970) 199-212
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RELATIONSDURING WALLERIANDEGENERATION
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TABLE II STATISTICAL REGRESSION ANALYSES OF THE DATA IN FIGS.
Correlation Significance Slope coefficient
3 AND
6
Intercept (units)
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0.18" 0.03 1.19 1.22 1.26 2.70 4.09 2.97
0.34 0.06 2.2 2.3 2.4 5.1 7.7 5.6
A. Measurements in 1 #m sections (Fig. 3)
Normal 1 day 2 days 3 days 4 days 6 days 8 days 10 days
0.96 0.93 0.92 0.94 0.98 0.96 0.95 0.99
< 0.01 < 0.01 -< 0.01 < 0.01 < 0.01 < 0.01 < 0.01 <~0.01
0.69 0.73 0.79 0.81 0.86 0.94 0.98 0.99
B. Measurements in electron micrographs (Fig. 6)
2 days 4 days 8 days
0.95 0.96 0.97
< 0.01 < 0.01 < 0.01
0.72 0.84 0.88
4.16"* 12.80 19.80
3.9 12.0 18.6
* Scales in Fig. 3 are converted to #m; one unit equals 5.9 #m. ** One unit equals 2.95 #m.
measurements of myelin forms were entered into the statistical analysis; the prediction of their features - - thick sheaths lacking axis cylinders - - was based entirely on the regression analysis of fibers that still had sizeable axis cylinders. Histochemical observations in 1 tzm sections
A histochemical technique for the demonstration of NAD-diaphorase activity in individual mitochondria in plastic-embedded 1 /zm thick sections6, tz was applied to the present material. By the 1st day, the axoplasmic density of mitochondria in myelinated fibers appeared only slightly diminished. Some of the fibers showed small focal accumulations of mitochondria in the axoplasm adjacent to the nodes of Ranvier by the 1st and 2nd day (Fig. 4), probably identical to the transient accumulations of mitochondria observed with the electron microscope by Webster23; they were not seen at later phases. Axoplasmic mitochondria were markedly diminished or absent on the 2nd and 3rd day. A few axoplasmic mitochondria with enzyme activity could be detected by careful scrutiny of the fibers up to the 8th day, but the vast majority had disappeared after the 3rd day. Swollen axons near the cut surface of the distal nerve stump showed dense accumulations of mitochondria similar to those in the proximal stumps, although densities appeared not to be as high. The Schwann cell cytoplasm showed relatively little changes in mitochondrial density. Pools of cytoplasm with densely crowded mitochondria were attached to the sheaths during the first 4 days (Fig. 4), giving the impression that these changes were due to redistribution of cytoplasm along the internodes. At later phases, there was a Brain Research~ 19 (1970) 199-212
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Fig. 3. Wallerian degeneration is accompanied by a progressive change in the slope of the relation between the inner and outer circumferences of the myelin sheath. Determination of these parameters eliminates error due to slightly oblique cutting of the fiber (see Material and Methods). Statistical regression analyses of the data are given in Table I. The changes in the ratio of axon diameter to fiber diameter are shown for fibers of 25, 60 and 85 /~m circumference for each set. A progressive thickening of the sheath in relation to the axis cylinder is evident for the thin fibers. The open circles for the 2nd and 8th days are measurements of the outer circumference of 'myelin forms'; these were not entered into the computer analysis. Curves for days 4 and 10 (Table I) are not shown.
gne vacuolization and reduced mitochondrial density of the cytoplasm associated with collapsed fibers, with fibers with irregularities of sheath structure, or with myelin Brain Research~ 19 (1970) 199-212
AXON-SHEATH RELATIONSDURING WALLERIAN DEGENERATION
205
Fig. 4. Demonstration of mitochondria by the reaction for NAD-diaphorase in 1 /~m thick plasticembedded sections. Top : Mitochondria are either greatly diminished, or have disappeared, from the axoplasm 3 days after cutting the nerve. High mitochondrial density is found in the Schwann cell cytoplasm. Middle: Transient accumulations of mitochondria in the proximity of the nodes of Ranvier are found on the 1st (shown) and on the 2nd day, probably identical to those described by Webster 23. Bottom: By the 4th day, many of the Schwann cells show less than normal mitochondrial density, apparently due to a fine vacuolization of their cytoplasm. Early 'Btingner's bands' show moderate mitochondrial density. All 1,000 x.
forms. The m i t o c h o n d r i a l density in the cytoplasm of BiJngner's bands, was either equal to or less t h a n that in n o r m a l S c h w a n n cells; Biingner's b a n d s were n u m e r o u s by the 10th day (Fig. 4).
Fine structure of fibers Electron microscopic studies were done only for rats killed 2, 4, 6 a n d 8 days after transection of their nerves; the observations are reported only briefly, as numerous p u b l i c a t i o n s are available on the subject (see Discussion). Disintegration of
Brain Research, 19 (1970) 199-212
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R . L . FRIEDE AND A. J. MARTINEZ
neurofilaments and microtubules was prevalent 2 and 4 days after transection, and ftoccular material was distributed irregularly throughout the axis cylinder. Mitochondria and smooth endoplasmic reticulum were sparse or absent; the persistent mitochondria showed retrogressive changes such as swelling and vacuolization. No changes were found in the myelin sheaths. The Schwann cell cytoplasm appeared unchanged by the 2nd day but elongated mesaxons and leaflets of myelin were occasionally found by the 4th day. The axoplasm of non-myelinated fibers showed the same changes as described above. A reduced number of non-myelinated fibers per Schwann cell was noticed by the 2nd day, with m a n y of the profiles appearing smaller than normal; invaginated surface membranes of Schwann cells suggestive o f ' e m p t i e d ' mesaxons were also seen and prevailed by the 4th day. Axis cylinders were 'empty' or contained floccular or membranous debris by the 6th and 8th days, when changes in the myelin sheath and in the Schwann cell cytoplasm became more prominent. The latter showed separation into numerous islands underneath the intact basement membrane, each bound by a continuous
Fig. 5. Electron micrograph 6 days after transection shows a 'myelin form' without discernible axis cylinder, encompassed by Schwann cell cytoplasm. The latter shows separation of several cytoplasmic islands encompassed by a common basement membrane. 28,800 x. Brain Research, 19 (1970) 199-212
AXON-SHEATH RELATIONS DURING WALLERIAN DEGENERATION
207
plasma membrane (Fig. 5). The cytoplasm in these islands resembled that in the main pool of Schwann cell cytoplasm in showing filaments, microtubules, ribosomes, smooth and granular endoplasmic reticulum and mitochondria. Convoluted aggregates of myelin leaflets were found in some fibers. Many myelin sheaths were without distinct changes in structure, although they appeared more angulated than normal and relatively thick as compared with their axis cylinder. Myelin forms generally showed variation in interperiod width and irregular foldings and contortions of the lamellae. Most were encompassed by a generous pool of cytoplasm showing extensive separation into 'islands'. Non-myelinated fibers could not be positively identified, as the attribution of Bfingner's bands to myelinated or non-myelinated became a matter of conjecture if no remnants of myelin were present.
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Fig. 6. Measurements made in electron micrographs at three phases of Wallerian degeneration show changes comparable to those shown in Fig. 3. Only fibers with a solid sheath (i.e. without splitting of the myelin leaflets) were measured. One unit corresponds to 2.95 ym. Statistical regression analyses of the data are given in Table I.
Brian Research, 19 (1970) 199-212
15
208
R . L. F R I E D E A N D A. J. M A R T I N E Z
Measurements in electron micrographs
Measurements in electron micrographs were made with the same technique as those in 1 # m sections to facilitate comparison of the sets of data. Care was taken to measure only fibers with solid myelin sheaths, eliminating sources of errors such as a thickening of the sheath due to splitting of its myelin lamellae, or due to the presence of narrow clefts of Schmidt-Lantermann which may have been unnoticed in 1 # m sections. The curves were extremely similar to those obtained from 1 ,urn sections (Fig. 6) validating the use of the latter, and adding evidence to the concept of sheath contraction. The interperiod width of myelin lamellae in contracted sheaths was also measured and was found to be approximately 160 A - - the same as in normal fibers - - except for sites where splitting of myelin leaflets had occurred. DISCUSSION
The measurements presented in this report show that early Wallerian degeneration is characterized by a progressive change in the relation between the caliber of the axis cylinder and the thickness of the myelin sheath, indicating sheath contraction. The extreme end-stage of sheath contraction are the 'myelin forms' having numerous turns of myelin lamellae around a minimal or absent axis cylinder. The increase in sheath thickness cannot be explained by the formation of new myelin, as biochemical studies on Wallerian degeneration agree in showing no change in lipid composition of the nerves during the initial 8 days 9. Increase in sheath thickness, also, cannot be explained by the collapse of the axis cylinder, although contraction coexists with collapse. Our data, therefore, evidence contraction of the sheath by inward slippage of the myelin lamellae, producing a sheath of increasing thickness around a shrinking axis cylinder. This process is comparable in nature, but opposite in direction, to the expansion of sheaths of swollen axons. It appears to be more marked in thin myelinated fibers than in thick myelinated fibers, in contrast with sheath expansion, which was found to affect fibers of all calibers by the same degree. Wallerian degeneration may be divided into two phases. (1) The first is characterized by a readjustment or contraction of the sheath around the shrinking axis cylinder, without significant myelin degeneration. Even the appearance of the conspicuous 'myelin forms' during this period signifies excessive contraction rather than myelin breakdown. Sheath contraction lasts for about 8-10 days and is followed by (2) the second phase of Wallerian degeneration, characterized by sheath disintegration. This phase is characterized by biochemical degradation of the myelin 9 and by loss of its histochemical staining properties 15. This concept of Wallerian degeneration reconciles some of the apparent contradictions between the morphological and biochemical data. Another time-honored concept on Wallerian degeneration is questioned by our data. It is generally accepted that thick fibers degenerate earlier than thin fibers. Our data, in contrast, show that the thinly myelinated fibers disappear much faster than the thick ones. The traditional dictum of the early disappearance of the thick Brain Research, 19 (1970) 199-212
AXON-SHEATH RELATIONS DURING WALLERIAN DEGENERATION
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fibers is probably a misinterpretation of the progressive shrinkage of the axis cylinder. If all fibers change successively into the next lower classes, one would have the impression that the thick fibers disappear first, at least if the caliber of the axis cylinder only is taken into account. Coexistent with contraction of the sheath, there is also collapse of the sheath, evident by the appearance of angulated and contorted fiber profiles. One striking manifestation of collapse are pouches of sheath which appear as two concentric myelin rings in cross-sections. That these profiles, indeed, represent pouches protruding deeply into the axis cylinder is demonstrated by histograms showing that the two rings have equal thickness for most of the fibers (Fig. 1). If the concentric rings were intussusceptions of sheath, a substantial number of fibers would show three rings, which was not the case. If they were gaping clefts of Schmidt-Lantermann, cross-sections would show any conceivable ratio of the thickness of the inner and outer rings of myelin. Our observations on the fine structure of nerve fibers undergoing Wallerian degeneration agree with numerous previous electron microscopic investigations20,22, except for the timing of the degeneration of non-myelinated fibers and for the probably interrelated problem in identifying the cytoplasmic islands underneath the basement membrane of fibers or inside the collapsed Schwann tubes. These 'islands' have been described as survival, or very slow degeneration, respectively, of nonmyelinated fibersS,S,ll,lS; as pseudopodes of Schwann cells13,~4; or as Btingner's bands3, 21. During the later phases of Wallerian degeneration, when regeneration commences, identification of these structures is further complicated by the fact that regenerating axons may actually coexist with Schwann cellprocesses inside the Schwann tubesl4,1s,2L Our electron microscopic observations of early disappearance of non-myelinated fibers agree with the changes predicted by the regression curves in Figs. 3 and 6, both indicating that non-myelinated axons disappear very fast, the majority by the 2nd or 3rd day, leaving behind only 'empty' Schwann cells with extensively folded and indented surface membranes. The same rapid disappearance of non-myelinated fibers was found in spinal nerves and roots13,14,16 and in the iris following ganglionectomy17. Previous reports on prolonged survival, or exceptionally slow degeneration of non-myelinated fibers 5,8,~,19 do not illustrate the characteristic features of bundles of non-myelinated fibers encompassed by a common Schwann cell envelope. The illustrations in these reports are rather similar to those called Btingner's bands, or regenerating axons, respectively, by other authors. Hence, the claims concerning the exceptionally slow degeneration of non-myelinated fibers should be dismissed, based on doubtful identification. Nathaniel and PeasO3,14 describe both regenerating axons and pseudopodes of Schwann cells underneath the basement membrane of fibers during early Wallerian degeneration (4 days after crush); some of the profiles, called regenerating axons, are indistinguishable from the structures described as Biingner's bands by Thomas 21 and by B15mcke and Niedorf3. Furthermore, regenerating axons were described 2 cm below the division 7 days after transection of the sciatic nervO, which is incompatible Brain Research, 19 (1970) 199-212
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R. L. FRIEDE AND A. J. MARTINEZ
with the normal speed of fusion of the stumps of transected nerves. Hence, the need for more precise criteria for the identification of these profiles is evident. Regenerating axons show an orderly arrangement of neurofilaments and microtubules at all phases of regeneration, since these extend into the immediate proximity of the clusters of vesicles found in the tips of growth cones 4. At late phases of regeneration, the formation of a mesaxon and the deposition of myelin readily identify a regenerating axon. In contrast, the 'islands', or 'pseudopodes', of Schwann cell cytoplasm are characterized by the following features: (a) They appear en m a s s e during the initial phase of Wallerian degeneration and are found at sites where the occurrence of regenerating axons is unlikely. (b) They are initially located between the basement membrane and the cytoplasmic envelope of myelinated fibers 21. Later, after degeneration of the fibers, they occur in bundles inside the collapsed Schwann tubes. (c) The cytoplasm first lacks filamentous structures, but formation of filaments may occur at later phases of Wallerian degeneration2,L (d) There is a complete lack of growth cones during their formation. By applying these criteria, we identified the majority of profiles in our material as 'islands', or processes, of Schwann cell cytoplasm. The mechanism producing separation of Schwann cell processes is not entirely clear. They have been considered finger-like proliferations or pseudopodes10,13,14. It is more likely that the early appearance of such profiles between the sheath and the basement membrane of myelinated fibers indicates damage by rapid slipping of the sheath, as has been proposed for swollen fibers in the preceding reports6,12. The coaxial movement of myelin leaflets evidently meets less resistance than the spinning of larger pools of cytoplasm around the fiber, regardless of whether the direction of slippage is inwards or outwards. Hence, the cytoplasmic strands or islands are likely the product of disruption of the Schwann cell envelope by rapid slippage of the sheath. Their first appearance in swollen fibers and in fibers undergoing Wallerian degeneration coincides, in each case, with the period of sheath adjustment. The timing of these periods differs in the two situations. Finally, we like to point out that there is a tendency, in the electron microscopic literature, to consider every change in the fine structure of the myelin sheath as indicative of a primary alteration of myelin. The present data show that changes in the sheath may develop in consequence to primary alterations in the volume of axoplasm. Thus, definition of the mechanism producing alteration in the fine structure of the sheath requires a careful quantitative analysis of all aspects of fiber structure. SUMMARY
Measurements of axon-sheath relations of fiber populations undergoing early Wallerian degeneration showed contraction of the sheath around the shrinking axis cylinder concurrent with a collapse of the axis cylinder. Collapse was associated with folding and pouching of the sheath. Contraction was produced by inward slippage of the myelin leaflets, producing a progressively thicker sheath around a shrinking axis cylinder. The so-called 'myelin forms' represent the end-stages of Brain Research,
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AXON-SHEATH RELATIONS DURING WALLERIANDEGENERATION
211
sheath contraction, characterized by a relatively thick sheath a r o u n d a m i n i m a l or n o n - e x i s t e n t axis cylinder. Accordingly, W a l l e r i a n degeneration consists of two phases; the first is characterized by sheath a d a p t a t i o n (contraction) a n d the second by sheath disintegration. Sheath c o n t r a c t i o n proceeds faster in the thinly m y e l i n a t e d fibers than in the thick ones. The axons of n o n - m y e l i n a t e d fibers disappear first. These observations were c o m p a r e d with the r e d i s t r i b u t i o n a n d the eventual loss of axonal mitochondria. ACKNOWLEDGEMENTS This investigation was supported by U.S. Public Health G r a n t NB 06239 a n d Special Fellowship NB 19903 from the N a t i o n a l Institute of Neurological Diseases a n d Strokes.
REFERENCES 1 BARTON, A. A., An electron microscope study of degeneration and regeneration of nerve, Brain,
85 (1962) 799-808. 2 BLtJMCKE,S., Elektronenoptische Untersuchungen an Schwann'schen Zellen w/ihrend der Waller'schen Degeneration peripherer Nerven, Verh. Dtsch. Ges. Path., 6 (1965) 346-349. 3 BL~JMCKE, S., AND NIEDORF, H. R., Electron microscope studies of Schwann cells during the Wallerian degeneration with special reference to the cytoplasmic filaments, Acta neuropath. (Berl.), 6 (1966) 46-60. 4 DEL CERRO, P., AND SNIDER, R. S., Studies on the developing cerebellum. Ultrastructure of the growth cones, J. comp. Neurol., 133 (1968) 341-362. 5 FISHER, E. R., AND TURANO, A., Schwann cells in Wallerian degeneration, Arch. Path., 75 (1963)
517-527. 6 FRIEDE, R. L., AND MARTINEZ, A. J., Analysis of the process of sheath expansion in swollen nerve fibers, Brain Research, 19 (1970) 165-182. 7 FRIEDE, R. L., AND SAMORAJSKI, T., Relation between the number of myelin lamellae and axon circumference in fibers of vagus and sciatic nerves of mice, J. comp. NeuroL, 130 (1967) 223-232.
8 HONJIN, R., NAKAMURA,T., AND ]MURA, M., Electron microscopy of peripheral nerve fibers. III. On the axoplasmic changes during Wallerian degeneration, Okafimas Folia anat. jap., 33 (1959) 131-156. 9 JOHNSON, A. C., MCNABB, A. R., AND ROSSITER,R. J., Chemistry of Wallerian degeneration, Arch. Neurol. Psychiat. (Chic.), 64 (1950) 105-121. 10 LAMPERT, P. W., AND SCHOCHET, S. S., JR., Demyelination and remyelination in lead neuropathy, J. Neuropath. exp. Neurol., 27 0968) 527-545. 11 LEE, J., Electron microscopy of Wallerian degeneration, J. comp. NeuroL, 120 (1963) 65-79. 12 MARTINEZ, A. J., AND FRIEDE, R. L., Accumulation of axoplasmic organelles in swollen nerve fibers, Brain Research, 19 (1970) 183-198. 13 NATHANIEL,E. J. H., AND PEASE, D. C., Degenerative changes in rat dorsal roots during Wallerian degeneration, J. Ultrastruct. Res., 9 (1963) 511-532. 14 NATHANIEL, E. J. H., AND PEASE, C., Regenerative changes in rat dorsal roots following Wallerian degeneration, J. Ultrastruct. Res., 9 (1963) 533-549. 15 NOBACK, C. R., AND MONTAGNA, W., Histochemical studies of the myelin sheath and its fragmentation products during Wallerian (secondary) degeneration, J. comp. NeuroL, 97 (1952) 211-232. 16 OHraI, S., Electron microscopic study on Wallerian degeneration of the peripheral nerve, Z. Zellforsch., 54 (1961) 39-67. 17 ROTH, C. D., AND RICHARDSON, K. C., Electron microscopical studies on axonal degeneration in the rat iris following ganglionectomy, Amer. J. Anat., 124 (1969) 341-360. Brain Research, 19 (1970) 199-212
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18 SATINSKY,D., PEPE, F. A., AND LIU, C. N., The neurilemma cell in peripheral nerve degeneration and regeneration, Exp. Neurol., 9 (1964) 441-451. 19 TAXI, J., t~tude au microscope 61ectronique de la dSgSndrescence wallerienne des fibres nerveuses amySliniques, C. R. Acad. Sci. (Paris'), 248 (1959)2796-2798. 20 TERRY, R. D., AND HARKIN, J. C., Wallerian degeneration and regeneration of peripheral nerves. In S. A. KOREY (Ed.), The Biology of Myelin, Harper (Hoeber), New York, 1959, pp. 303-320. 21 THOMAS, P. K., Changes in the endoneurial sheaths of peripheral myelinated nerve fibres during Wallerian degeneration, J. Anat. (Lond.), 98 (1964) 175-182. 22 VIAL, J. D., The early changes in axoplasm during Wallerian degeneration, J. biophys, bioehem. Cytol., 4 (1958) 551-556. 23 WEBSTER, DEF. H., Transient, focal accumulation of axonal mitocbondria during the early stages of Wallerian degeneration, J. Cell Biol., 12 (1962) 361-383.
Brain Research, 19 (1970) 199-212