277
Neuroscience Letters, 128 (1991) 277-280
© 1991 ElsevierScientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100380N NSL 07896
Myelin maintenance by Schwann cells in the absence of axons J o h n W. H e a t h 1, G r a h a m e J. K i d d 1'*, B r u c e D. T r a p p 2 a n d P e t e r R. D u n k l e y 1 The Neuroscience Group, Faculty of Medicine, University of Newcastle, New South Wales (Australia) and 2Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (U.S.A.)
(Received 18 March 1991; Accepted 18 April 1991) Key words: Schwann cell regulation; Myelin maintenance; Wallerian degeneration
Formation and maintenance of myelin sheaths in the peripheral nervous system are regulated by unknown molecular interactions that are thought to depend upon physical contact between Schwann cells and axons. However, recent studies describing axons surrounded by two concentric myelin internodes in the superior cervical ganglion (SCG) of normal rodents have demonstrated that the outer myelin internodes are maintained without physical contact with the axon. To determine whether the centrally enclosed axon has atrophic effect in maintaining these remote outer internodes, we have produced axonal degeneration by surgical or chemical means. The results indicate that maintenance of myelin internodes totally displaced from axonal contact depends neither upon the presence of the axon nor on diffusible axonal factors. A further implication of these studies is that myelin breakdown during Wallerian degeneration is regulated by a positive signal which originates in degenerating nerves, rather than solely by loss of axonal trophic substances.
Development of the m a m m a l i a n nervous system depends upon membrane interactions between a variety of cells and their processes. Axonal regulation of Schwanrt cell phenotype is well known and represents one of the most stereotyped and extensive cellular interactions in the nervous system. Whether a Schwann cell myelinates a single axon (myelinated fibres) or ensheathes multiple axons (unmyelinated fibres) is determined by the axons with which it is associated [1, 25, 26]. Axons also stimulate Schwann cell division, mediate basal lamina formation around the Schwann cell, and regulate the a m o u n t of myelin Schwann cells produce [2, 19]. These axonal influences leading to myelin formation are thought to require physical contact between the axolemma and Schwann cell plasma membranes, but how these influences are mediated at the molecular level is poorly understood. Axonal degeneration in a peripheral nerve results in a sequence of events that includes disruption of contact between the axolemma and the Schwann cell plasma membrane [6], myelin sheath disruption [21, 24], invasion of activated macrophages I16, 21], decreased myelin
*Present address: Department of Neurology, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21205, U.S.A. Correspondence: J. Heath, Department of Neurology, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21205, U.S.A. Fax: (1) 61-49~82928.
protein gene expression [17, 18, 23], Schwann cell expression of nerve growth factor receptor [22] and Schwann cell division [4, 14, 20]. These observations have led to the d o g m a that, like myelin sheath formation, myelin maintenance depends upon continuous physical contact between axon and Schwann cell, and that loss o f an axonal signal results in dedifferentiation o f Schwann cells to a non-myelinating state [7-9, 14]. However, because it has been difficult to separate the effects of disrupted axonal contact from the cascade of cellular events which accompany Wallerian degeneration, this hypothesis has never been tested directly. 'Double myelination' represents the outcome o f complete displacement of a myelinating Schwann cell from axonal contact due to invasion of another Schwann cell at a node or heminode [11]. The invading (now 'inner') cell typically invests the axon closely and myelin formation by this cell gives rise to the double ensheathment [11]. Characteristically the displaced (now 'outer') myelin internode remains intact [10]. Further, once displaced the outer internode undergoes infolding, a progression of events which includes the compaction of juxtaposed Schwann cell plasma membranes to form new myelin lamellae [10]. Immunocytochemical studies using antibodies against myelin-associated glycoprotein have indicated that ordered synthesis and translocation of this molecule continues during this process [5]. In addition, radio-labelled lipids and amino acids are incorporated into the outer myelin sheath (Heath and Gould, unpub-
278
Fig. I. Response of completely displaced outer internodes to surgically (a,d) and chemically induced (b,c) axonal degeneration, a: initial response to treatment involveddegeneration of the axon (Ax) and disruption of the inner sheath (M~,arrowheads). In contrast, the outer sheath (Mo) remained intact. Ni, nucleus of inner Schwann cell. b: eventually, the outer sheath (Mo) enclosed the inner Schwann cell (Si), redundant basal lamina (arrows) and numerous collagen fibrils (*) but no axon or myelin. Inset shows the intact lamellar structure of this outer sheath, c: at later time-points, the inner Schwann cells (nuclei N 1, N2) formed bands of Biingner within the outer sheath (Mo). Inset shows the integrity of this outer sheath, d: where axonal regeneration was encouraged, axonal sprouts (A') invaded the bands of Biingner within the outer sheath (Mo) to form typical unmyelinated fibres. Arrows, mesaxons; *, collagen. Inset shows the outer sheath (Mo) which enclosed these fibres (arrowhead). Treatments: (a,d) 1 day and 5 days post-surgery, respectively;(b,c) guanethidine sulphate (50 mg/kg/day i.p.) for 3 weeks and 6 weeks, respectively. Bars (a)= 2/tm, (b)= 1 ~m, (c)= 2 /~m, (d) = 0.5/~m. Insets (b,c) = 0.25/zm; (d) = 2/tm.
lished data). The outer S c h w a n n cell c o n t i n u e s to m a i n tain its sheath in the absence of a x o n a l contact, suggesting either that the outer cell n o longer requires axonal influences for o n g o i n g myelin m a i n t e n a n c e or that diffusible a x o n a l factors are operating. T o determine whether survival of the outer sheath is d e p e n d e n t o n diffusible
axonal influences we have produced degeneration o f superior cervical ganglion (SCG) postganglionic a x o n s in rats a n d mice by t r a n s g a n g l i o n i c nerve transection, a n d by chemical s y m p a t h e c t o m y using chronic treatm e n t with g u a n e t h i d i n e [3, 12]. In the S C G the axons involved in d o u b l e m y e l i n a t i o n
279
arise from postganglionic sympathetic nerve cell bodies located inferiorly in the ganglion, and they exit the ganglion via the external carotid nerve. Thus, ganglionic transection 1 mm inferior to the origin of the external carotid nerve produced the desired Wallerian degeneration of these axons. Analysis was performed 1 mm superior (i.e. distal) to the lesion plane to avoid non-specific traumatic effects. Rats were anaesthetised with a single i.p. injection of chloral hydrate (0.36 mg/100 g b.wt., dissolved in 0.9% saline). Mice were pretreated with diazepam (Valium 10, Roche; 0.5 mg/100 g b.wt.) injected i.p. After a 5 min interval they were lightly anaesthetised with Innovar-Vet solution i.p. (fentonyl-droperidol, Janssen, Australia) at a rate of 0.15 ml/100 g body weight of a 1:5 dilution in saline. Surgical anaesthesia was then induced and maintained by intravenous injection of the diluted Innovar-Vet solution in 0.1 ml increments via a tail vein cannula. Animals were sacrificed 1 day-16 weeks post-operatively. Chronic treatment of rats with guanethidine (50 mg/kg/day, i.p.) produces degeneration of the entire adrenergic neuron [3, 12]; animals were sacrificed after treatment for 4 days to 6 weeks. Tissues were fixed, embedded and individual profiles pursued through serial sections as previously described [10-12]. Overall, our findings using guanethidine and surgery were similar. Axonal degeneration occurred within 1-5 days after surgery and within 10 days after guanethidine administration [12] (Figs. 1a and 2). Degeneration of the inner myelin sheath followed in a typical Wallerian pattern of myelin splitting and ovoid formation. About 75 % of outer sheaths also degenerated in the same manner at this time (not shown). EM analysis of initial outer sheath degeneration in serial sections indicated that these internodes represented early stages of displacement and still retained axonal contact [11].
PartlyDisplaced
Completely Displaced Internode
J ~'~.,m~,~ Band of BOngner
Fig. 2. Summary of results. Upon axonal degeneration, myelin internodes making contact with the axon also degenerate, but completely displaced outer internodes remain intact for at least 16 weeks (vertical scale exaggerated).
In contrast, where the outer Schwann cell completely lacked direct contact with the central axon, the outer sheath remained intact during the phase of degeneration and phagocytosis of the axon and inner sheath (Fig. la). These events continued until there was no evidence remaining of the central axon or inner myelin sheath. At this stage, the surviving outer cell and myelin sheath enclosed the inner Schwann cell and its processes, basal lamina and a substantial number of collagen fibrils (Fig. lb). Bands of Biingner also developed within the confines of outer surviving cells. As a result, multiple inner Schwann cells were located within surviving myelin internodes at some levels of section (Fig. lc). Macrophages were also frequently present but did not attack the outer sheath (not shown). Surviving outer sheaths remained intact for up to 16 weeks after surgical lesion and 6 weeks after guanethidine treatment, the longest periods examined in this study. Axonal regeneration within surviving outer sheaths was not observed following guanethidine and rarely observed in experiments where the lower pole of the SCG was surgically resected. The organelle content of sprouts and their characteristic pattern of Schwann cell ensheathment including formation of mesaxons (Fig. 1d) distinguished them from profiles in which only the inner Schwann cells and their processes remained within surviving sheaths (Fig. lb,c). In other experiments where axonal sprouting was encouraged by sparing the inferior pole of the lesioned ganglion (1 day to 16 weeks survival), axonal sprouts were often present and showed a propensity to associate with Schwann cells of the inner bands of Biingner. Thus, we are confident that regenerating axons were not a source of trophic factors for outer sheath maintenance. Myelin maintenance by Schwann cells in the SCG therefore does not depend upon membrane-membrane contact between axolemma and Schwann cell periaxonal membranes, nor upon diffusible axonal factors. In addition, recent studies by Perry and colleagues [13, 15] indicate that the initiation of rapid axonal and myelin sheath breakdown during Wallerian degeneration depends on the presence of macrophages in the endoneurial space. Current concepts about Schwann cell interaction and the sequence of the events which follow axonal transection therefore require re-evaluation. Based on the results of the present study and those of Perry's laboratory [13, 15], we propose that Wallerian degeneration involves the following sequence of events. A positive signal originating from degenerating axons induces myelin sheath breakdown. This signal is potentiated by infiltrating macrophages and is transmitted to myelinating Schwann cells that are in physical contact with a degenerating axon.
280 W e t h a n k I n g r i d R u d o l p h for s e c r e t a r i a l assistance. T h i s w o r k w a s s u p p o r t e d by g r a n t s f r o m the N a t i o n a l M u l t i p l e Sclerosis S o c i e t y o f A u s t r a l i a ( J . W . H . , G . J . K . ) , the R a m a c i o t t i F o u n d a t i o n ( J . W . H . ) , a n d t h e N a t i o n a l I n s t i t u t e s o f H e a l t h ( B . D . T . ) . B . D . T . is a H a r r y W e a v e r N e u r o s c i e n c e S c h o l a r o f the N a t i o n a l M u l t i p l e Sclerosis Society of America. 1 Aguayo, A.J., Epps, J., Charron, L. and Bray, G.M., Multipotentiality of Schwann cells in cross anastomosed and grafted myelinated and unmyelinated nerves: quantitative microscopy and autoradiography, Brain Res., 104 (1976) 1-20. 2 Bunge, R.P., The cell of Schwann. In A.K. Asbury, G.M. McKhann and W.I. McDonald (Eds.), Diseases of the Nervous System. Clinical Neurobiology, Heinemann Medical, London, 1986, pp. 153-162. 3 Burnstock, G., Evans, B., Gannon, B.J., Heath, J.W. and James, V., A new method of destroying adrenergic nerves in adult animals using guanethidine, Br. J. Pharmacol., 43 ( 1971) 295-30 l. 4 Clemence, A., Mirsky, R. and Jessen, K.R., Non-myelin-forming Schwann cells proliferate rapidly during Wallerian degeneration in the rat sciatic nerve, J. Neurocytol., 18 (1989) 185-192. 5 Heath, J.W. and Trapp, B.D., Changing immunocytochemieal localisation of myelin-associated glycoprotein in myelin internodes displaced from axonal contact, Proc. Aust. Neurosci. Soc., 1 (1990) 84. 6 Hirano, A. and Dembitzer, H.M., The periaxonal space in an experimental model of neuropathy: the mutant Syrian hamster with hindleg paralysis, J. Neurocytol., 10 (1981) 261-269. 7 Jessen; K.R., Mirsky, R. and Morgan, L., Myelinated, but not unmyelinated axons, reversibly down-regulate N-CAM in Schwann cells, J. Neurocytol., 16 (1987) 681~588. 8 Jessen, K.R., Morgan, L. and Mirsky, R., Axonal signals regulate the differentiation of non-myelin-forming Schwann cells: an immunohistoehemical study of galactocerebroside in transected and regenerating nerves, J. Neurosci., 7 (1987) 3362-3369. 9 Jessen, K.R., Morgan, L., Stewart, H.J.S. and Mirsky, R., Three markers of adult non-myelin-forming cells, 217c (Ran-l), A5E3 and GFAP: development and regulation by neuron-Schwann cell interactions, Development, 109 (1990) 91-103. 10 Kidd, G.J. and Heath, J.W., Double myelination of axons in the sympathetic nervous system of the mouse. I. Ultrastructural features and distribution, J. Neurocytol., 17 (1988) 245 261. 11 Kidd, G.J. and Heath, J.W., Double myelination of axons in the sympathetic nervous system of the mouse. II. Mechanisms of Formation, J. Neurocytol., 17 (1988) 263 276. 12 Kidd, G.J., Heath, J.W. and Dunkley, P.R., Degeneration ofmyelinated sympathetic nerve fibres following treatment with guanethidine, J. Neurocytol., 15 (1986) 561 572.
13 Lunn, E.R., Perry, V.H., Brown, M.C., Rosen, H. and Gordon, S., Absence of Wallerian degeneration does not hinder regeneration in peripheral nerve, Eur. J. Neurosci., 1 (1989) 27-33. 14 Oaklander, A.L. and Spencer, P.S., Cold blockade of axonal transport activates premitotic activity of Schwann cells and Wallerian degeneration, J. Neurochem., 50 (I 988) 490-496. 15 Perry, V.H., Brown, M.C., Lunn, E.R., Tree, P. and Gordon, S., Evidence that very slow Wallerian degeneration in C57BL/OIa mice is an intrinsic property of the peripheral nerve, Eur. J. Neurosci., 2 (1990) 802-808. 16 Perry, V.H. and Gordon, S., Macrophages and microglia in the nervous system, Trends Neurol. Sci., l I (1988) 273-277. 17 Poduslo, J.F., Dyck, P.J. and Berg, C.T., Regulation of myelination: Schwann cell transition from a myelin-maintaining state to a quiescent state after permanent nerve transection, J. Neurochem., 44 (1985) 388-400. 18 Politis, M.J., Sternberger, N., Ederle, K. and Spencer, P.S., Studies on the control of myelinogenesis. IV. Neuronal induction of Schwann cell protein synthesis during nerve fibre regeneration, J. Neurosci., 2 (1982) 1252-1266. 19 Raine, C.S., Morphology of myelin and myelination. In P. Morell (Ed.), Myelin, 2nd edn., Plenum, New York, 1984, pp. 1--50. 20 Spencer, P.S., Politis, M.J., Pellegrino, R.G. and Weinberg, H.J., Control of Schwann cell behaviour during nerve degeneration and regeneration. In A. Gorio, H. Millesi and S. Mingrino (Eds.), Posttraumatic Peripheral Nerve Regeneration: Experimental Basis and Clinical Implications, Raven, New York, 198 I, pp. 411-426. 21 Stoll, G., Griffin, J.W., Li, C.Y. and Trapp, B.D., Wallerian degeneration in the peripheral nervous system: participation of both Schwann cells and macrophages in myelin degradation, J. Neurocytol., 18 (1989) 671 683. 22 Taniuchi, M., Clark, H.B. and Johnson, E.M., Induction of nerve growth factor receptor in Schwann cells after axotomy, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 4094-4098. 23 Trapp, B.D., Hauer, P. and Lemke, G., Axonal regulation of myelin protein mRNA levels in actively myelinating Schwann cells, J. Neurosci., 8 (1988) 3515 3521. 24 Vital, C. and Vallat, J.-M., Ultrastructural Study of the Human Diseased Peripheral Nerve, 2nd edn., Elsevier, New York, 1987, 290 pp. 25 Weinberg, H.J. and Spencer, P.S., Studies on the control of myelinogenesis. I. Myelination of regenerating axons after entry into a foreign unmyelinated nerve, J. Neurocytol., 4 (1975) 395-418. 26 Weinberg, H.J. and Spencer, P.S., Studies on the control of myelinogenesis. II. Evidence for neuronal regulation of myelin production, Brain Res., 113 (1976) 363-378.
Neuroscience Letters, 128 (1991) 277-280
© 1991 ElsevierScientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100380N NSL 07896
Myelin maintenance by Schwann cells in the absence of axons J o h n W. H e a t h 1, G r a h a m e J. K i d d 1'*, B r u c e D. T r a p p 2 a n d P e t e r R. D u n k l e y 1 The Neuroscience Group, Faculty of Medicine, University of Newcastle, New South Wales (Australia) and 2Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (U.S.A.)
(Received 18 March 1991; Accepted 18 April 1991) Key words: Schwann cell regulation; Myelin maintenance; Wallerian degeneration
Formation and maintenance of myelin sheaths in the peripheral nervous system are regulated by unknown molecular interactions that are thought to depend upon physical contact between Schwann cells and axons. However, recent studies describing axons surrounded by two concentric myelin internodes in the superior cervical ganglion (SCG) of normal rodents have demonstrated that the outer myelin internodes are maintained without physical contact with the axon. To determine whether the centrally enclosed axon has atrophic effect in maintaining these remote outer internodes, we have produced axonal degeneration by surgical or chemical means. The results indicate that maintenance of myelin internodes totally displaced from axonal contact depends neither upon the presence of the axon nor on diffusible axonal factors. A further implication of these studies is that myelin breakdown during Wallerian degeneration is regulated by a positive signal which originates in degenerating nerves, rather than solely by loss of axonal trophic substances.
Development of the m a m m a l i a n nervous system depends upon membrane interactions between a variety of cells and their processes. Axonal regulation of Schwanrt cell phenotype is well known and represents one of the most stereotyped and extensive cellular interactions in the nervous system. Whether a Schwann cell myelinates a single axon (myelinated fibres) or ensheathes multiple axons (unmyelinated fibres) is determined by the axons with which it is associated [1, 25, 26]. Axons also stimulate Schwann cell division, mediate basal lamina formation around the Schwann cell, and regulate the a m o u n t of myelin Schwann cells produce [2, 19]. These axonal influences leading to myelin formation are thought to require physical contact between the axolemma and Schwann cell plasma membranes, but how these influences are mediated at the molecular level is poorly understood. Axonal degeneration in a peripheral nerve results in a sequence of events that includes disruption of contact between the axolemma and the Schwann cell plasma membrane [6], myelin sheath disruption [21, 24], invasion of activated macrophages I16, 21], decreased myelin
*Present address: Department of Neurology, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21205, U.S.A. Correspondence: J. Heath, Department of Neurology, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21205, U.S.A. Fax: (1) 61-49~82928.
protein gene expression [17, 18, 23], Schwann cell expression of nerve growth factor receptor [22] and Schwann cell division [4, 14, 20]. These observations have led to the d o g m a that, like myelin sheath formation, myelin maintenance depends upon continuous physical contact between axon and Schwann cell, and that loss o f an axonal signal results in dedifferentiation o f Schwann cells to a non-myelinating state [7-9, 14]. However, because it has been difficult to separate the effects of disrupted axonal contact from the cascade of cellular events which accompany Wallerian degeneration, this hypothesis has never been tested directly. 'Double myelination' represents the outcome o f complete displacement of a myelinating Schwann cell from axonal contact due to invasion of another Schwann cell at a node or heminode [11]. The invading (now 'inner') cell typically invests the axon closely and myelin formation by this cell gives rise to the double ensheathment [11]. Characteristically the displaced (now 'outer') myelin internode remains intact [10]. Further, once displaced the outer internode undergoes infolding, a progression of events which includes the compaction of juxtaposed Schwann cell plasma membranes to form new myelin lamellae [10]. Immunocytochemical studies using antibodies against myelin-associated glycoprotein have indicated that ordered synthesis and translocation of this molecule continues during this process [5]. In addition, radio-labelled lipids and amino acids are incorporated into the outer myelin sheath (Heath and Gould, unpub-
278
Fig. I. Response of completely displaced outer internodes to surgically (a,d) and chemically induced (b,c) axonal degeneration, a: initial response to treatment involveddegeneration of the axon (Ax) and disruption of the inner sheath (M~,arrowheads). In contrast, the outer sheath (Mo) remained intact. Ni, nucleus of inner Schwann cell. b: eventually, the outer sheath (Mo) enclosed the inner Schwann cell (Si), redundant basal lamina (arrows) and numerous collagen fibrils (*) but no axon or myelin. Inset shows the intact lamellar structure of this outer sheath, c: at later time-points, the inner Schwann cells (nuclei N 1, N2) formed bands of Biingner within the outer sheath (Mo). Inset shows the integrity of this outer sheath, d: where axonal regeneration was encouraged, axonal sprouts (A') invaded the bands of Biingner within the outer sheath (Mo) to form typical unmyelinated fibres. Arrows, mesaxons; *, collagen. Inset shows the outer sheath (Mo) which enclosed these fibres (arrowhead). Treatments: (a,d) 1 day and 5 days post-surgery, respectively;(b,c) guanethidine sulphate (50 mg/kg/day i.p.) for 3 weeks and 6 weeks, respectively. Bars (a)= 2/tm, (b)= 1 ~m, (c)= 2 /~m, (d) = 0.5/~m. Insets (b,c) = 0.25/zm; (d) = 2/tm.
lished data). The outer S c h w a n n cell c o n t i n u e s to m a i n tain its sheath in the absence of a x o n a l contact, suggesting either that the outer cell n o longer requires axonal influences for o n g o i n g myelin m a i n t e n a n c e or that diffusible a x o n a l factors are operating. T o determine whether survival of the outer sheath is d e p e n d e n t o n diffusible
axonal influences we have produced degeneration o f superior cervical ganglion (SCG) postganglionic a x o n s in rats a n d mice by t r a n s g a n g l i o n i c nerve transection, a n d by chemical s y m p a t h e c t o m y using chronic treatm e n t with g u a n e t h i d i n e [3, 12]. In the S C G the axons involved in d o u b l e m y e l i n a t i o n
279
arise from postganglionic sympathetic nerve cell bodies located inferiorly in the ganglion, and they exit the ganglion via the external carotid nerve. Thus, ganglionic transection 1 mm inferior to the origin of the external carotid nerve produced the desired Wallerian degeneration of these axons. Analysis was performed 1 mm superior (i.e. distal) to the lesion plane to avoid non-specific traumatic effects. Rats were anaesthetised with a single i.p. injection of chloral hydrate (0.36 mg/100 g b.wt., dissolved in 0.9% saline). Mice were pretreated with diazepam (Valium 10, Roche; 0.5 mg/100 g b.wt.) injected i.p. After a 5 min interval they were lightly anaesthetised with Innovar-Vet solution i.p. (fentonyl-droperidol, Janssen, Australia) at a rate of 0.15 ml/100 g body weight of a 1:5 dilution in saline. Surgical anaesthesia was then induced and maintained by intravenous injection of the diluted Innovar-Vet solution in 0.1 ml increments via a tail vein cannula. Animals were sacrificed 1 day-16 weeks post-operatively. Chronic treatment of rats with guanethidine (50 mg/kg/day, i.p.) produces degeneration of the entire adrenergic neuron [3, 12]; animals were sacrificed after treatment for 4 days to 6 weeks. Tissues were fixed, embedded and individual profiles pursued through serial sections as previously described [10-12]. Overall, our findings using guanethidine and surgery were similar. Axonal degeneration occurred within 1-5 days after surgery and within 10 days after guanethidine administration [12] (Figs. 1a and 2). Degeneration of the inner myelin sheath followed in a typical Wallerian pattern of myelin splitting and ovoid formation. About 75 % of outer sheaths also degenerated in the same manner at this time (not shown). EM analysis of initial outer sheath degeneration in serial sections indicated that these internodes represented early stages of displacement and still retained axonal contact [11].
PartlyDisplaced
Completely Displaced Internode
J ~'~.,m~,~ Band of BOngner
Fig. 2. Summary of results. Upon axonal degeneration, myelin internodes making contact with the axon also degenerate, but completely displaced outer internodes remain intact for at least 16 weeks (vertical scale exaggerated).
In contrast, where the outer Schwann cell completely lacked direct contact with the central axon, the outer sheath remained intact during the phase of degeneration and phagocytosis of the axon and inner sheath (Fig. la). These events continued until there was no evidence remaining of the central axon or inner myelin sheath. At this stage, the surviving outer cell and myelin sheath enclosed the inner Schwann cell and its processes, basal lamina and a substantial number of collagen fibrils (Fig. lb). Bands of Biingner also developed within the confines of outer surviving cells. As a result, multiple inner Schwann cells were located within surviving myelin internodes at some levels of section (Fig. lc). Macrophages were also frequently present but did not attack the outer sheath (not shown). Surviving outer sheaths remained intact for up to 16 weeks after surgical lesion and 6 weeks after guanethidine treatment, the longest periods examined in this study. Axonal regeneration within surviving outer sheaths was not observed following guanethidine and rarely observed in experiments where the lower pole of the SCG was surgically resected. The organelle content of sprouts and their characteristic pattern of Schwann cell ensheathment including formation of mesaxons (Fig. 1d) distinguished them from profiles in which only the inner Schwann cells and their processes remained within surviving sheaths (Fig. lb,c). In other experiments where axonal sprouting was encouraged by sparing the inferior pole of the lesioned ganglion (1 day to 16 weeks survival), axonal sprouts were often present and showed a propensity to associate with Schwann cells of the inner bands of Biingner. Thus, we are confident that regenerating axons were not a source of trophic factors for outer sheath maintenance. Myelin maintenance by Schwann cells in the SCG therefore does not depend upon membrane-membrane contact between axolemma and Schwann cell periaxonal membranes, nor upon diffusible axonal factors. In addition, recent studies by Perry and colleagues [13, 15] indicate that the initiation of rapid axonal and myelin sheath breakdown during Wallerian degeneration depends on the presence of macrophages in the endoneurial space. Current concepts about Schwann cell interaction and the sequence of the events which follow axonal transection therefore require re-evaluation. Based on the results of the present study and those of Perry's laboratory [13, 15], we propose that Wallerian degeneration involves the following sequence of events. A positive signal originating from degenerating axons induces myelin sheath breakdown. This signal is potentiated by infiltrating macrophages and is transmitted to myelinating Schwann cells that are in physical contact with a degenerating axon.
280 W e t h a n k I n g r i d R u d o l p h for s e c r e t a r i a l assistance. T h i s w o r k w a s s u p p o r t e d by g r a n t s f r o m the N a t i o n a l M u l t i p l e Sclerosis S o c i e t y o f A u s t r a l i a ( J . W . H . , G . J . K . ) , the R a m a c i o t t i F o u n d a t i o n ( J . W . H . ) , a n d t h e N a t i o n a l I n s t i t u t e s o f H e a l t h ( B . D . T . ) . B . D . T . is a H a r r y W e a v e r N e u r o s c i e n c e S c h o l a r o f the N a t i o n a l M u l t i p l e Sclerosis Society of America. 1 Aguayo, A.J., Epps, J., Charron, L. and Bray, G.M., Multipotentiality of Schwann cells in cross anastomosed and grafted myelinated and unmyelinated nerves: quantitative microscopy and autoradiography, Brain Res., 104 (1976) 1-20. 2 Bunge, R.P., The cell of Schwann. In A.K. Asbury, G.M. McKhann and W.I. McDonald (Eds.), Diseases of the Nervous System. Clinical Neurobiology, Heinemann Medical, London, 1986, pp. 153-162. 3 Burnstock, G., Evans, B., Gannon, B.J., Heath, J.W. and James, V., A new method of destroying adrenergic nerves in adult animals using guanethidine, Br. J. Pharmacol., 43 ( 1971) 295-30 l. 4 Clemence, A., Mirsky, R. and Jessen, K.R., Non-myelin-forming Schwann cells proliferate rapidly during Wallerian degeneration in the rat sciatic nerve, J. Neurocytol., 18 (1989) 185-192. 5 Heath, J.W. and Trapp, B.D., Changing immunocytochemieal localisation of myelin-associated glycoprotein in myelin internodes displaced from axonal contact, Proc. Aust. Neurosci. Soc., 1 (1990) 84. 6 Hirano, A. and Dembitzer, H.M., The periaxonal space in an experimental model of neuropathy: the mutant Syrian hamster with hindleg paralysis, J. Neurocytol., 10 (1981) 261-269. 7 Jessen; K.R., Mirsky, R. and Morgan, L., Myelinated, but not unmyelinated axons, reversibly down-regulate N-CAM in Schwann cells, J. Neurocytol., 16 (1987) 681~588. 8 Jessen, K.R., Morgan, L. and Mirsky, R., Axonal signals regulate the differentiation of non-myelin-forming Schwann cells: an immunohistoehemical study of galactocerebroside in transected and regenerating nerves, J. Neurosci., 7 (1987) 3362-3369. 9 Jessen, K.R., Morgan, L., Stewart, H.J.S. and Mirsky, R., Three markers of adult non-myelin-forming cells, 217c (Ran-l), A5E3 and GFAP: development and regulation by neuron-Schwann cell interactions, Development, 109 (1990) 91-103. 10 Kidd, G.J. and Heath, J.W., Double myelination of axons in the sympathetic nervous system of the mouse. I. Ultrastructural features and distribution, J. Neurocytol., 17 (1988) 245 261. 11 Kidd, G.J. and Heath, J.W., Double myelination of axons in the sympathetic nervous system of the mouse. II. Mechanisms of Formation, J. Neurocytol., 17 (1988) 263 276. 12 Kidd, G.J., Heath, J.W. and Dunkley, P.R., Degeneration ofmyelinated sympathetic nerve fibres following treatment with guanethidine, J. Neurocytol., 15 (1986) 561 572.
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