Schwann Cell Processes Guide Regeneration of Peripheral Axons

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Young-Jin Son and Wesley J. Thompson come immunoreactive (Astrow et al., 1994). There is a Center for Developmental Biology similar up-regulation of immunoreactivity in Schwann cells and Department of Zoology of the nerve following axonal degeneration. University of Texas Motor axons commonly reinnervate muscle fibers by Austin, Texas 78712 growing down the old endoneurial Schwann cell tubes leading to each endplate. Some axons continue to grow beyond their endplates (Tello, 1907; Ramon y Cajal, 1928; Summary Gutmann and Young, 1944; Letinsky et al., 1976; Gorio et al., 1983), forming so-called "escaped" fibers. In some Terminal Schwann cells overlying the neuromuscular cases these escaped fibers grow to reinnervate nearby junction sprout elaborate processes upon muscle de- endplates. Muscle fibers reinnervated by an escaped fiber nervation. We show here that motor axons use these from an adjacent endplate as well as by an axon regenerat- processes as guides/substrates during regeneration; ing down the old Schwann cell tube become polyneuro- in so doing, they escape the confines of endplates and nally innervated (Rich and Lichtman, 1989). We wondered grow between endplates to generate polyneuronal in- whether the processes extended by terminal Schwann nervation. We also show that Schwann cells in the cells play a role in the generation of escaped fibers and nerve provide similar guidance. Axons extend from the polyneuronal reinnervation. Using immunocytochemistry, cut end of a nerve in association with Schwann cell we found that motor axons use processes of terminal processes and appear to navigate along them. The pro- Schwann cells as guides during reinnervation. This guid- cesses extend from axotomized nerves at the same ance is not unique to terminal Schwann cells but extends rate and in the same manner as they do from axon- to Schwann cells of the nerve. Upon severing a nerve, containing nerves. The rate of process extension limits Schwann cells elaborate processes from the cut end that the rate at which axons regenerate. Thus, Schwann act as guides for extension of peripheral axons. Moreover, cell processes lead and guide peripheral regeneration. the rate of axon regeneration from these cut ends is limited by the rate of Schwann cell extension. Thus, Schwann Introduction cells act as leaders during regeneration.

Schwann cells play a crucial role in regeneration of periph- Results eral axons, as regeneration is deficient in their absence (Hall, 1986). Axons that successfully regenerate in a nerve Upon Denervation, Terminal Schwann Cells lacking living Schwann cells are accompanied by Elaborate Processes That Grow Schwann cells from the intact portion of the nerve, but the to Interconnect Endplates rate of regeneration is reduced (Anderson et al., 1983). The rat neuromuscular junction can be identified easily in How Schwann cells promote regeneration is less clear. muscle whole-mounts double labeled with MAb 4E2 and These cells have been proposed to form a cellular bridge an antibody to the 200 kDa neurofilament protein. The spanning the gap between ends of severed nerves (Aber- anti-neurofilament antibody labels the long, unbranched crombie and Johnson, 1942), to secrete tropic and trophic preterminal axon and the larger terminal branches in the agents for neurons (Politis et al., 1982; Kuffler, 1986; Heu- junction (Figure 1B). As documented extensively else- mann et al., 1987), and to generate a basal lamina support- where (Astrow et al., 1992, 1994), labeling with MAb 4E2 ive of nerve growth (Ide et al., 1983). Although Schwann is postsynaptic, marking in particular the edges of the syn- cells clearly form a preferred substrate for axonal regener- aptic gutters and Z discs in the vicinity of the endplate ation in vitro (Failon, 1985; Ard et al., 1987; Bixby et al., (Figure 1A). There is no detectable labeling of Schwann 1988; Hopkins and Bunge, 1991), controversy still exists cells at the junction, which however can be labeled using as to whether Schwann cells act as leaders or followers an antibody to a general Schwann cell marker, the protein during regeneration in vivo (Keynes, 1987; Reynolds and S-100 (Reynolds and Woolf, 1992; Astrow et al., 1994). Woolf, 1993). Use of anti-S-100 shows that terminal Schwann cells ex- Recently, Reynolds and Woolf (1992) showed that termi- tend short processes which cover the nerve terminal, but nal Schwann cells overlying the neuromuscular junction these processes are confined to the endplate. Following sprout elaborate processes following muscle denervation. denervation, immunoreactivity for 4E2 disappears post- During another study, we generated a monoclonal anti- synaptically (Astrow et al., 1994), but appears in myelinat- body (MAb 4E2) that marks terminal Schwann cells and ing and nonmyelinating Schwann cells of the nerve and their processes following muscle denervation. The epitope in the terminal Schwann cells and their processes (Figure recognized by this antibody is normally present postsynap- 1C) (Astrow et al., 1994). tically at the neuromuscular junction (Astrow et al., 1992). To generate muscles in which we could examine the However, following denervation, this postsynaptic immu- relationship between Schwann cell processes and regen- noreactivity disappears, and terminal Schwann cells be- erating axons, soleus was denervated by crushing its Neuron 126

Figure 1. Following Muscle Denervation, Terminal Schwann Cells Gain Immunoreactivity for MAb 4E2 and Grow Processes That interconnect Endplates Muscle fibers from normal muscle (A and B) and from muscle 3 days after the second of two nerve crushes (C and D) were double labeled with MAb 4E2 (A and C) and anti-neurofilament (B and D). At innervated endplates, immunoreactivity for MAb 4E2 marks the synaptic gutters containing the nerve terminal processes and the Z discs in the vicinity of the endplate. No labeling is present in the terminal Schwann cells. Following denervation, Schwann cells become immunoreactive and extend processes between endplates (C). Four endplates, labeled 1-4, can be identified by the accumulations of cell bodies of Schwann cells and by an old Schwann cell tube (arrows) leading to each. Processes interconnecting endplates 1 and 4 and endplates 3 and 4 have been marked with a '~/." A process also interconnects endplates 1 and 2. Regenerating motor axons have arrived in the muscle, but in this region only one endplate (#1) has been reinnervated, in this case by an axon growing along the old Schwann cell tube (C and D). Bars, 10 ~m (B), 20 ~m (D).

nerve just outside the muscle and then recrushing the same site 3 days later. Reinnervation of muscle (as assayed by muscle contractions to stimulation of the nerve) began about 3 days following the second nerve crush. Thus, this denervation regimen allowed a period of about 5 days for terminal Schwann cells to grow before the begin- ning of reinnervation. Muscles were examined 2-20 days after the second nerve crush, but most observations were made 3-5 days after the second crush, during the early stages of reinnervation. Because of the varying distances motor neurons must grow to reinnervate different parts of this large muscle, endplates at various stages of reinnervation were present in individual muscles at these times. Endplates in these muscles were identified by the Schwann cell bodies that remain clustered at these sites, at least for short periods following denervation (Figure 1C). Figure 2. Regenerating Axons Grow beyond Endplates by Following Each endplate could also be identified by the presence of Schwann Cell Processes a MAb 4E2-1abeled Schwann cell tube that was previously Muscle examined 3 days after the second crush. occupied by the preterminal axon (Figure 1C). (A) Labeling of the Schwann cells with MAb 4E2. Within 5 days after denervation, many of the terminal (B) Labeling of the regenerating axon by anti-neurofilament. The axon Schwann cell processes had grown to contact adjacent (arrow) has grown down the old Schwann cell tube, reinnervated the endplates (Figure 1C). Often these inter-endplate connec- endplate, and then grown sprouts onto the Schwann cell processes extended from this endplate. The tip of one nerve sprout is marked tions were fasciculations of several Schwann cell pro- with a "v." Note that the Schwann cell processes extend further than cesses. These interconnections formed in the absence of the nerve sprouts, and some Schwann cell processes are unoccupied any reinnervation. by nerve sprouts. Bar, 10 I~m. Axon Guidance by Schwann Cells 127

Figure 3. Polyneuronal Innervation Is Generated by Growth of Axons along Schwann Cell Processes Linking Adjacent Endplates Muscle examined 3 days after second nerve crush by labeling with MAb 4E2 (A, C, and E) and anti-neurofilament antibody (B, D, and F). In each pair of pictures, MAb 4E2-1abeled Schwann cell processes have extended between two endplates, labeled 1 and 2. Endplate 1 has been reinnervated by an axon (left arrow in [B], [D], and [F]) regenerating down an old Schwann cell tube. This axon has extended a sprout along the Schwann cell processes (asterisks) that has reinnervated (B and D) or is approaching (F) endplate 2. In (A) and (B), endplate 2 has also been reinnervated by an axon (right arrow in [B]) regenerating down an old Schwann cell tube; thus, this endplate has become polyneuronally reinnervated. In (C) and (D), an axon (right arrow in [C]) has approached endplate 2 by growing down an old Schwann cell tube. In (F) the tip of the sprout between the endplates is marked with a "v." Bar, 10 ~.m.

Regenerating Axons Extend beyond Endplates Polyneuronal Innervation Is a Consequence by Following Schwann Cell Processes of Schwann Cell Guidance For most muscle fibers, reinnervation, assayed by immu- In our examination of muscles 3 days after the second nostaining with antibodies to neurofilament and MAb 4E2, nerve crush, we encountered many examples in which an began with the regeneration of axons down the old axon had not only reinnervated an endplate through an Schwann cell tubes leading to endplates (Figure 1D). How- old Schwann cell tube but also had grown beyond this ever, these axons did not stop their growth upon arrival endplate to reinnervate an adjacent endplate (Figures 3A- at the endplate. Rather, they continued to grow onto the 3D). In each of these cases, the axons were clearly associ- processes extended by the Schwann cells from the end- ated with Schwann cell processes. Once again, deviations plate (Figures 2A and 2B). In all cases in which axons in the course of Schwann cells were matched precisely extended beyond the endplate, they were associated with by deviations in the course of axons. Often, endplates Schwann cell processes. Deviations in the course of the receiving an axon from an adjacent endplate were also Schwann cell processes were matched closely by the course reinnervated by axons regenerating down their old of the axons. Examples were found in which the Schwann Schwann cell tubes, and thus were polyneuronally inner- cell processes were much longer than the axons with vated (Figures 3A and 3B). which they were associated, and in these cases, the axons We encountered instances in which two endplates were appeared to be navigating along the Schwann cells (Fig- interconnected by Schwann cell processes, one of the ure 2). endplates was reinnervated, and an axonal process was Neuron 128

Figure 4. The Consequences of Schwann Cell Guidance during Muscle Reinnervation Persist after MAb 4E2 Imm unoreactivity Is Lost from Schwann Cells Muscles at later stages of reinnervation were double labeled with MAb 4E2 (A and C) and anti-neurofilament (B and D) or with anti-S-100 (E) and anti-neurofilament (F). (A and B) Three endplates in a muscle 10 days after the second nerve crush. An axon has reinnervated endplate 2 by growing down a Schwann cell tube and sprouted to innervate the adjacent endplates. MAb 4E2 labeling associated with the sprouts has almost disappeared and is replaced by postsynaptic labeling at the reinnervated endplates. (C and D) A pair of endplates 20 days after the second nerve crush. Both endplates have been reinnervated by axons (arrows) growing down Schwann cell tubes, but endplate 1 also sends a sprout to reinnervate endplate 2. MAb 4E2 immunoreactivity is now entirely postsynaptic. (E and F) Inter-endplate nerve sprouts are still associated with Schwann cell processes. Two junctions identified by the accumulations of Schwann cell bodies are labeled 1 and 2 in (E). Each junction has been reinnervated by an axon (arrows) growing down its old Schwann cell tube. Additional reinnervation is by reciprocal sprouts between these two endplates. Schwann cells labeled with anti-S-100 are associated with each nerve process. Bars, 10 p.m (A-D), 15 ~m (E and F).

found somewhere along the Schwann cell processes link- persist, and.they may ultimately participate in the myelina- ing the two endplates (Figures 3E and 3F). Images like tion of these processes (Rich and Lichtman, 1989). these suggest that the neural connections running between endplates were generated by axons navigating Schwann Cells and Axons Extend from the Cut End along pathways pre-established by the Schwann cells. of a Nerve At survival times longer than 5 days following the second We wondered whether guidance of regenerating axons nerve crush, many cases of axons extending between end- by Schwann cell processes was a phenomenon restricted plates could still be found. However, it was often difficult to terminal Schwann cells. Accordingly, we examined how to discern the Schwann cell processes associated with axons and Schwann cells grew from the cut end of a for- these axons (Figures 4A and 4B). At these longer survival eign nerve transplanted onto the surface of the soleus times, MAb 4E2 immunoreactivity in the Schwann cells muscle. Previous examination of such transplants has had declined, and postsynaptic immunoreactivity had re- shown that axons, in the companyof Schwann cells, reach turned, as expected from a previous study (Astrow et al., the surface of the muscle but do not form synapses with 1994). At the longest survival times we examined (20 muscle fibers unless the muscle is denervated (cf. Korneli- days), MAb 4E2 staining was entirely postsynaptic (Fig- ussen and Sommerschild, 1976). ures 4C and 4D). However, in these cases Schwann cells Double labeling transplants with anti-neurofilament anti- were still associated with the inter-endplate connections, body and MAb 4E2 revealed that Schwann cells and axons because in other muscles labeling with anti-S-100 re- extended together from the cut end of the nerve and grew vealed their presence (Figures 4E and 4F). Thus, Schwann over the surface of the muscle (Figure 5A). Schwann cells cells that presu m ably formed the su bstrate for axon growth emerging from the cut end of the nerve formed chiefly Axon Guidance by Schwann Cells 129

.7 transplanted nerve depends on the presence and/or growth of axons, we implanted nerves that had been resected at a more proximal site 4 days earlier. Thus, these transplants contained no axons. Schwann cells grew out onto the muscle in the same fashion as when axons were present (Figure 5C), except the mat of Schwann cell processes tended to be less dense. To compare the rates of extension of the growth front across the muscle with and without axons, we transplanted a previously resected, ax- onless nerve in the right hindlimb and an axon-containing nerve in the left hindlimb in each of six rats. After 7 days, the distance from the cut end of the transplant to the most distal nerve or Schwann cell outgrowth on the muscle was measured. Axons and Schwann cells in the left hindlimb or Schwann cells alone in the right hindlimb had extended 1.0-1.5 mm. Thus, in both cases the rate of extension was approximately 0.2 mm/day.

Schwann Cell Extension Limits the Rate of Axonal Regeneration The rate of Schwann cell or Schwann cell plus axon extension from the transplants is substantially less than the re- ported regeneration of axons through a nerve (approximately 3 mm/day; Jacobson and Guth, 1965), in which the axons grow in basal lamina tubes containing Schwann Figure 5. Schwann Cell Processes Extend from the Cut End of a cells and their processes. To see whether Schwann cell Nerve to Form Pathwaysfor RegeneratingAxons extension might limit the rate of axon regeneration, we The cut, distal end of a foreign nerve was transplantedto the surface performed nerve transplants but delayed axonal regenera- of soleus. tion by crushing the nerve 1.3 cm proximal to the end of (A) Double labelingwith MAb 4E2 (green)and anti-neurofilamentanti- the transplant and then recrushing the same site 3 days body (red) 7 days after transplantationshows that Schwann cell and axonal processes have grownout over the musclesurface. Axons are later. In three rats examined 4 days after the second crush, associated with Schwann cells and their processes, although some regenerating axons were found near the original tip of the Schwann cell processesare unoccupied by axons. transplant, but the Schwann cell processes that had grown (B) Higher magnificationview of muscle like that in (A) showing the out over the surface of the muscle (for a distance of approx- navigation of a regeneratingaxon along Schwann cells. (C) Schwann cell processesextend from the cut end of a nerve that imately 1.5 mm) were devoid of regenerating axons. In was previously resected and thus contained no axons. A single three companion animals examined 6 days after the sec- Schwann cell and its bipolar processesare indicatedwith the "v" sym- ond crush, regenerating axons had grown to the tips of the bols. The cell body of this Schwann cell is seen underneaththe right Schwann cell processes. Thus, during the 2 days between "v." The absenceof anti-neurofilamentlabeling (red) confirms that no axons were present. This result shows that Schwann cell extension these two sets of observations, axons had regenerated is independentof regeneratingaxons. Bars, 20 ~m (A), 10 p.m (B), 40 over Schwann cell processes (now approximately 2.0 mm ~m (C). in length) that had been established on the muscle over the course of 9 days. This result shows that axons regenerate over the surface of the muscle more rapidly in the bipolar processes that usually fasciculated into bundles. presence of pre-established Schwann cell processes (a Axons were always associated with Schwann cells along minimum of 2.0 mm in 2 days, or 1.0 mm/day) than they their entire neurofilament-labeled length. Most commonly, do if the Schwann cells must accompany them in their the tips of the Schwann cells and axons furthermost from growth (0.2 mm/day; see above). the cut end of the nerve were coextensive. However, in most cases not all the Schwann cells were occupied by Discussion axons, and occasionally the Schwann cell tips were in advance of the neurofilament labeling. In these cases, We believe our results show that Schwann cells function axons were found in the apparent act of navigating along as leaders rather than followers during regeneration. Ter- the Schwann cell processes (Figure 5B). By observing im- minal Schwann cells and Schwann cells of the nerve, in plants at different times, it is clear that the Schwann cells the complete absence of axons, can establish processes and axons slowly advance across the surface of the that later serve as substrates for axonal growth. When muscle. axons and Schwann cells extend together from the cut end of a nerve, they proceed no faster than the Schwann Schwann Cell Extension from the Cut End of cells proceed alone. On the other hand, axons are capable a Nerve Occurs in the Absence of Axons of more rapid extension if a Schwann cell pathway is pre- To determine whether Schwann cell extension from the pared for them. While these conclusions come from obser- Neuron 130

vations of nerve growth from endplates and on the surface tiguous fibers by the same motor neuron (e.g., Figures 4A of muscles, they likely also apply to the growth that occurs and 4B). That the degree of fiber type grouping depends when axons regenerate across the gap between the ends upon the severity of the nerve lesion is again likely a conse- of a severed nerve. Considering the results of others (Ide quence of the time the fibers remain denervated and the et al., 1983; Hall, 1986), axons regenerating within the extent of Schwann cell interconnections formed among basal lamina tubes of nerves may also grow preferentially endplates. upon Schwann cells and their processes. Several studies of muscle cross-reinnervation have re- The number of observations made in this study of axon ported that, when a foreign nerve is transplanted to the tips extending along Schwann cell processes (e.g., those proximal end of the soleus muscle and the innervation of processes extended between endplates) is small, despite soleus itself is then destroyed, the foreign nerve begins the fact that we believe Schwann cells lead axons during to make ectopic synapses with the muscle fibers. If axons regeneration. We believe the small number is explained of the foreign nerve approach the endplate zone of soleus, by the difference in speed with which axons and Schwann then these axons avoid making ectopic synapses in a cells extend. Since axons extend rapidly if Schwann cell zone, the so-called "refractory zone," within about 1 mm pathways are available, the likelihood of finding an axon of the old endplates (L~mo et al., 1988). Instead, these at some intermediate point along a short Schwann cell axons make synaptic contact at the old endplates. We process is probably small. believe that the elaboration of processes by terminal Schwann cells plays a role in the generation of this refrac- Schwann Cells Explain Several Phenomena tory zone. As shown by Reynolds and Woolf (1992), these of Muscle Reinnervation processes can be extended for a considerable distance Our observations offer explanation for the "escaped" fibers from the endplate. A foreign axon encountering one of the observed by neuroanatomists studying muscle reinnerva- Schwann cell processes extended from the denervated tion early in this century (Tello, 1907; Ramon y Cajal, endplates would likely follow this process to the endplate 1928). From our study, escaped fibers represent the zone and make a synapse at this preferred site for synapse growth of axons onto the processes of Schwann cells ex- formation rather than on the muscle surface surrounding tended from denervated endplates. In this regard it is inter- the endplate. esting to note that Letinsky et al. (1976), in their electron Several investigators have observe d effects of so-called microscopic study of reinnervation of frog muscle, de- "conditioning lesions." In some of these experiments, it scribed escaped fibers that were wrapped by Schwann has been observed that regeneration following the second cells; they also serially reconstructed an example in which of two temporarily separated nerve crushes given at the a regenerating axon appears to be following a Schwann same site on a peripheral nerve is faster than that observed cell process extended from the endplate. That the inci- after only a single crush (cf. Brown and Hopkins, 1981). dence of escaped fibers should depend upon the duration In agreement with conclusions reached by Sj6berg and of denervation (Gutmann and Young, 1944) is likely a con- Kanje (1990), our results suggest that Schwann cells could sequence of the time required for growth of Schwann cell explain the effects of such conditioning lesions: the first processes. damage to the nerve leads to preparation of a Schwann Our observations also explain the polyneuronal innerva- cell path distal to the lesion, and consequently, axons re- tion seen in adult muscles upon reinnervation. It is clear generate more quickly following a subsequent lesion. that some polyneuronal innervation is created by the growth of axons along Schwann cell processes linking ad- Other Roles for Schwann Cells during Regeneration jacent endplates. The increased incidence of such poly- and Development neuronal reinnervation following repeated nerve crush We believe our results raise additional questions about (Rich and Lichtman, 1989) can also be explained. Re- roles Schwann cells might play in axon growth and regres- peated nerve crush delays reinnervation so that Schwann sion during development and reinnervation. cell processes have more time to extend between end- As was clear from the original study of Reynolds and plates and so that more pathways for the generation of Woolf (1992), Schwann cell processes extended from de- polyneuronal reinnervation are created. nervated endplates regress following reinnervation. Just Reinnervation of adult muscles can change the distribu- as it is unclear what it is that signals terminal Schwann tion of fibers contained in single motor units: the fibers in cells to extend processes, it is also unclear what it is about single units, rather than being intermixed with those of reinnervation that causes these cells to withdraw their pro- other units as they normally are, occur in large, contiguous cesses. A more careful examination of the withdrawal of groups. Under the influence of the motor neuron rein- Schwann cell processes and the escaped fibers associ- nervating them, fibers in these single motor units become ated with them following reinnervation might give hints converted to the same fiber type. This reinnervation pat- about the mechanism. Such information is relevant to nor- tern and fiber type conversion results in so-called "fiber mal development and the course of reinnervation. At least type groups," a condition diagnostic of certain neurologic some of the polyneuronal innervation resulting from rein- lesions (Kugelberg et al., 1970). This pattern of reinnerva- nervation of adult muscles is transient (cf. Rich and Licht- tion can be explained by the Schwann cell processes ex- man, 1989), as is most of the polyneuronal innervation tended between fibers during their denervation. This established on developing muscle fibers (Brown et al., Schwann cell guidance results in the reinnervation of con- 1976). The loss of this polyneuronal innervation is associ- Axon Guidance by Schwann Cells 131

ated with the displacement of nerve terminals from muscle a rabbit polyclonal anti-S-100A (Z628, DAKO, Carpinteria, CA) diluted fibers and the withdrawal of the preterminal axon back 1:400; and a mouse monoclonal anti-NF200 kDa (RPN1103, Amer- sham, Arlington Heights, IL) diluted 1:50. Secondary antibodies in- into the intramuscular nerve (Korneliussen and Jansen, cluded the following: fluorescein-conjugated sheep F(ab')2 fragment 1976; Riley, 1981). Since Schwann cells promote the ex- anti-mouse (absorbed against rat serum proteins; F-2266, Sigma, St. tension of axons, they might also be involved in determin- Louis, MO) diluted 1:200; rhodamine-conjugated goat F(ab')2fragment ing their retraction. It will be interesting to determine what anti-rabbit (#55671, Cappel, Durham, NC) diluted 1:400; fluorescein- role, if any, Schwann cells play in axon retraction. conjugated rabbit anti-mouse IgG2b (#610-4242, Rockland, Gilberts- ville, PA) diluted 1:200; and rhodamine-conjugated goat anti-mouse If Schwann cells guide axons during regeneration, could IgG~ (#1070-03, Southern Biotechnology, Birmingham, AL) diluted they also provide guidance during development similar to 1:200. Double labeling with 4E2 and anti-S-100 employed anti-mouse that provided by glial cells in developing insects (Bastiani and anti-rabbit second antibodies conjugated to different fluoro- and Goodman, 1986)? While it seems unlikely that chromes. Because of their different isotypes (4E2 is an IgG2b; anti- neurofilament is an IgG~), double labeling with the two mouse mono- Schwann cells could provide the kind of specific guidance clonals employed type-specific anti-mouse antisera conjugated to necessary, for example, to ensure that motor neurons different fluorochromes. reach the correct muscles (Landmesser, 1980), it is possi- The procedures used for immunostaining are essentially those de- ble that Schwann cells provide pathways that are then scribed previously (Astrow et al, 1994). Whole mounts of muscles pro- selected by the projecting neurons. There have been sev- cessed for S-100 and NF200 were fixed for 7-10 min in 1% phosphate- buffered paraformaldehyde, rinsed in rat Ringer's solution for 30 min, eral investigations of this possibility (reviewed in Keynes, permeabilized by incubation in absolute MeOH for 10 min at -20°C, 1987) with conflicting results (cf. Noakes and Bennett, rinsed for 30 min in phosphate-buffered saline (PBS), blocked by 0.2% 1987; Dahm and Landmesser, 1988; Carpenter and Holly- bovine serum albumin in PBS for 30 rain, and incubated for 1 hr or day, 1992). One problem seems to be determining whether overnight with a mixture of both antibodies at room temperature. Mus- cles that were processed for MAb 4E2 and NF200 were fixed and axons or Schwann cells lead during the establishment of permeabilized by cold MeOH alone. Binding of primary antibodies was peripheral nerves. While some effort seems to have been visualized following a 1 hr incubation in a mixture of the appropriate directed at ascertaining that the neuronal markers used fluorochrome-conjugated secondary antibodies. All antibodies were in these studies stain the furthest extent of the neuronal diluted with PBS containing Triton X-100 (0.3%), bovine serum albu- growth cones, there is perhaps less certainty that the glial min (0.2%), and NaAzide (0.1%). Following immunostaining, connec- tive tissue was cleared from the surfaces of the muscle, and a layer markers stain the furthest extent of glial growth cones/pro- of fibers 5-6 muscle fibers thick was peeled from each side of the cesses. A further difficulty is that of distinguishing between muscle, mounted in fluorescence mounting medium (FITC-Guard, axon and Schwann cell processes in electron micrographs Testog, Chicago, IL), and examined using a Nikon microscope (Payer, 1979). On the other hand, it is also possible that equipped for epifluorescence. Images were acquired using appro, Schwann cells play an entirely different role in develop- priate rhodamine or fluorescein filter sets and an integrating CCD camera connected to a Macintosh computer equipped with a frame ment than that which they appear to play in regeneration. grabber and running NIH Image software. Montages of images Lastly, a number of experiments have demonstrated the achieved at different focal planes were constructed using Adobe Pho- ability of grafts of peripheral nerves or Schwann cells to toshop. In some cases image contrast was enhanced. Images were promote regeneration by axons of the CNS (Bray et al., cropped and labeled using Adobe Photoshop and Canvas software and printed using a Sony color printer. For color photographs, the 1987). Experiments suggest that living Schwann cells are images acquired with the fluorescein and rhodamine filter sets were required for this growth promotion (Smith and Stevenson, recolored green and red, respectively, using Adobe Photoshop and 1988) and that the regeneration, at least in vitro, occurs then combined. along cordons of Schwann cells (Hopkins and Bunge, 1991). Our results suggest that the ability of Schwann cells Acknowledgments to promote CNS regeneration may be limited bythe extent We thank S. Astrow, R. Balice-Gordon, J. Larimer, M. A. Rankin, J. to which Schwann processes from the implants can pene- Trachtenberg, and H. Zakon for their critique of earlier versions of this trate the CNS and provide pathways for axon extension. manuscript. This work was supported by grants from the NIH (NS 20480) and NSF (BNS-9009682). Experimental Procedures The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby Animals and Surgery marked "advertisement" in accordance with 18 USC Section 1734 Soleus muscles of adult Wistar rats were denervated aseptically under solely to indicate this fact. ether anesthesia by crushing the soleus nerve with #5 forceps approximately 2 mm away from the muscle. The same site was recrushed Received July 26, 1994; revised September 16, 1994. again 3 days later. At varying times after the second crush, the animals were euthanized with ether, and the soleus muscle was removed for References immunolabeling. For nerve transplantation, the superficial branch of the peroneal Abercrombie, M., and Johnson, M. L (1942). The outwandering of nerve was cut and deflected to the surface of the soleus muscle in cells in tissue cultures of nerves undergoing Wallerian degeneration. ether-anesthetized rats. In some cases this transplantation was per- J. Exp. Biol. 19, 266-283. formed 4 days following resection (under ether anesthesia) of a more Anderson, P. N., Mitchell, J., Mayor, D., and Stauber, V. V. (1983). proximal piece of the peroneal nerve. After varying survival times, An ultrastructural study of the early stages of axonal regeneration the animals were euthanized with ether, and the soleus muscle was through rat nerve grafts. Neuropathol. Appl. Neurobiol. 9, 455-466. removed for immunolabeling. Ard, M. D., Bunge, R. P., and Bunge, M. B. (1987). Comparison of the Schwann cell surface and Schwann cell extracellular matrix as Whole-Mount Immunolabeling promoters of neurite growth. J. 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