Brain Advance Access originally published online on March 14, 2006
Brain 2006 129(5):1319-1329; doi:10.1093/brain/awl057
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Remyelination of dorsal column axons by endogenous Schwann cells restores the normal pattern of Nav1.6 and Kv1.2 at nodes of Ranvier
1 Department of Neurology and Center for Neuroscience and Regeneration Research, Yale School of Medicine, New Haven, 2 Rehabilitation Research Center, VA Connecticut Healthcare System, West Haven, CT, USA and 3 Department of Clinical Neuroscience, King's College London, Guy's Campus, London, UK
Correspondence to: Joel A. Black, PhD Neuroscience Research (127A), VA Connecticut Healthcare System, 950 Campbell Avenue, West Haven, CT 06518, USA E-mail joel.black{at}yale.edu
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Summary |
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Demyelination of CNS axons occurs in a number of pathological conditions, including multiple sclerosis and contusion-type spinal cord injury. The demyelination can be repaired by remyelination in both humans and rodents, and even within the CNS remyelination can be achieved by endogenous and/or exogenous Schwann cells, the myelinating cells of the PNS. Remyelinated axons can often conduct impulses securely, but the organization of ion channels at long-term remyelinated nodes is not known. In the present study, the expression of voltage-gated sodium (Nav) and potassium (Kv) channels along central axons remyelinated by endogenous Schwann cells has been studied in lesions induced more than 1 year previously by the intraspinal injection of ethidium bromide (EB). The expression of the channels at long-term nodes formed by Schwann cell remyelination has been compared with that present in nascent nodes formed in the adult at 18 and 23 days post-EB injection. Immunohistochemical studies revealed that long-term nodes formed by Schwann cell remyelination exhibit a clustering of Nav1.6 sodium channels within the nodal membrane, with the Shaker-type potassium channel Kv1.2 segregated within the juxtaparanodal region, similar to the arrangement at normal mature CNS nodes. Nav1.2 was not detected at nodes formed by Schwann cells at any stage of their development. Moreover, Nav1.6, but not Nav1.2, was clustered at nascent nodes formed by remyelinating Schwann cells 18 and 23 days following EB injection. These observations show that endogenous Schwann cells can establish and maintain nodes of Ranvier on central axons for over one year, and that the nodes exhibit an apparently normal distribution of sodium and potassium channels, with Nav1.6 the predominant subtype of sodium channel present at such nodes at all stages of their development.
Key Words: demyelination; remyelination; Schwann cell; sodium channel; spinal cord
Abbreviations: Caspr = contactin-associated protein; EB = ethidium bromide; MOG = myelin oligodendrocyte glycoprotein; Nav = voltage-gated sodium; Kv = voltage-gated potassium
Received September 20, 2005. Revised December 14, 2005. Accepted February 10, 2006.
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Introduction |
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Demyelination occurs in several CNS disorders, including multiple sclerosis, viral infection and contusion-type spinal cord injury. Demyelination can be an important cause of neurological deficits because demyelination either delays or blocks impulse conduction (McDonald and Sears, 1970
; Smith et al., 1979; Blight, 1983; Rodriquez et al., 1987; Waxman, 1992). Demyelinated axons can be repaired by remyelination in both humans (Prineas et al., 1993; Chang et al., 2002) and animals. Indeed, in some experimental rodent models of demyelination repair can be effectively complete, achieved either by endogenous Schwann cells (Blakemore, 1975; Felts and Smith, 1992; Gilson and Blakemore, 2002) or cells of the oligodendrocyte lineage (Keirstead and Blakemore, 1999). Moreover, remyelination has also been achieved by the transplantation of a variety of exogenous myelin-producing cells into experimentally demyelinated lesions (Baron-Van Evercooren et al., 1992; Chari and Blakemore, 2002; Baron-Van Evercooren and Blakemore, 2004; Pluchino et al., 2004; Mackay-Sim, 2005). Remyelination by both endogenous and exogenous myelinating cells has been demonstrated to restore conduction that is sufficiently secure that few conduction deficits can be detected (Felts and Smith, 1992; Honmou et al., 1996; Akiyama et al., 2002), but how this is achieved in terms of channel organization is not known.
Secure conduction in normal myelinated axons is facilitated by a precise organization of cellular processes and ion channels at nodes of Ranvier. This organization includes the segregation into discrete domains of voltage-gated sodium channels (Nav), septate-like axo-glial junctions containing contactin and contactin-associated protein (Caspr; also known as paranodin) and Shaker-type potassium channels (Kv1) (for reviews, see Rasband and Trimmer, 2001a; Girault and Peles, 2002; Bhat, 2003). Nav channels are concentrated at high density within the nodal axolemma, and the Nav1.6 isoform predominates at mature nodes of Ranvier (Caldwell et al., 2000), although Nav1.2 is accumulated at immature nodes in some CNS tracts (Boiko et al., 2001; Kaplan et al., 2001; Jenkins and Bennett, 2002; Rios et al., 2003). Recently, it was demonstrated that Nav1.2 immunolabelling reappears at nodes in adult mouse optic nerve in the model of inflammatory demyelination, experimental allergic encephalomyelitis (EAE), suggesting that remyelinated nodes formed by oligodendrocytes may recapitulate the developmental clustering of Nav1.2 at immature nodes (Craner et al., 2003). However, whether the appearance of this nodal Nav1.2 in EAE is a temporary phenomenon, or a permanent switch, is not known.
It is well established that many demyelinated axons are acutely remyelinated by Schwann cells in spinal cord demyelinating lesions induced by injection of ethidium bromide (EB) into the dorsal columns (see Blakemore, 2005). Current evidence demonstrates that Schwann cell myelination of CNS axons can persist for extensive periods of time (>1 year; Felts and Smith, 1992, 1996), although the molecular architecture of the Schwann cell remyelinated nodes is unknown. In the present study, we have examined the expression of Nav channel isoforms Nav1.2 and Nav1.6 at the nodes of spinal cord axons remyelinated by Schwann cells, and of Kv1.2 channels at the juxtaparanodal regions of such axons. The results demonstrate that axons remyelinated for at least 1 year by Schwann cells exhibit mature nodal characteristics, with accumulation of Nav1.6, but not Nav1.2, at nodes and localization of Kv1.2 within juxtaparanodal regions. These results demonstrate that long-term Schwann cell myelination of demyelinated CNS axons is stable for periods >1 year in terms of nodal ion channel organization, and provide a molecular correlate for previous studies demonstrating near-normal conduction properties of these axons.
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Lesion induction
Focal demyelinating lesions in the spinal cord dorsal column were induced as described previously (Felts et al., 1997
). Briefly, 16 adult male Sprague–Dawley rats (Harlan, Bicester, UK) were anaesthetized with halothane, a quarter laminectomy was performed at the T12 lamina and EB (2 x 0.5 µl, 0.5 mg/ml in saline) injected unilaterally into the dorsal column using a drawn glass micropipette held in a micromanipulator. The wound was closed and the rats allowed to survive for 18, 23 or 378–587 days. The number of lesions analysed is specified below (see Quantitative analysis).
Tissue processing
Rats were deeply anaesthetized and perfused through the left ventricle with 4% paraformaldehyde in 0.14 M Sorensen's phosphate buffer, pH 7.4. The spinal cords were removed and immersion fixed for a total fixation time of 20–25 min, blocked into 3–5 mm segments, cryoprotected in 30% sucrose in phosphate-buffered saline (PBS) and frozen in optimal cutting temperature (OCT) compound (Tissue-Tek, Torrance, CA). In some instances, 0.5 mm blocks were obtained between adjacent 3–5 mm segments and processed for plastic sections, in order to identify Schwann cell remyelinated regions.
Immunocytochemistry
Tissue from unoperated (control) and lesioned rats was processed for immunocytochemistry as described previously (Black et al., 2004). Briefly, 10 µm longitudinal cryosections of the dorsal columns were cut and mounted on Fisher Superfrost Plus glass slides, and the sections were processed for immunofluorescent detection of Nav1.2, Nav1.6, Kv1.2, Caspr, P0, myelin oligodendrocyte glycoprotein (MOG), ezrin and neurofilament. Primary antibodies utilized were polyclonal Nav1.2 (1 : 100; Alomone, Jersusalem), monoclonal Nav1.2 (1 : 100; Upstate Biotechnology, Charlottesville, VA), polyclonal PN4 (Nav1.6) (1 : 100, Sigma), polyclonal Kv1.2 (1 : 100, Alomone), polyclonal Caspr (1 : 500, Rasband et al., 1999a; a generous gift of Dr M. Rasband, University of Connecticut), monoclonal Caspr IgG and IgM (1 : 300, a generous gift of Dr M. Rasband), monoclonal P0 (1 : 1000, Sasaki et al., 2004; a gift from Dr J. J. Archelos, University of Graz, Austria), goat polyclonal MOG (1 : 15, R & D Systems, Minneapolis, MN), monoclonal ezrin (1 : 50, Abcam, Cambridge, MA), and chicken polyclonal NF-H (1 : 5000, Encor, Alachua, FL). Secondary antibodies used were goat anti-rabbit IgG-Cy3 (1 : 2000, Amersham, Piscataway, NJ), goat or donkey anti-mouse IgG- or IgM-Alexa Fluor 488 or Alexa Fluor 546 (1 : 1000, Molecular Probes, Eugene, OR), donkey anti-goat IgG-Alexa Fluor 546 (1 : 1000, Molecular Probes) and goat anti-chicken Alexa Fluor 633 (1 : 1000, Molecular Probes).
Quantitative analysis
For analysis of control (unlesioned) and lesioned spinal cords, multiple images of longitudinal sections of the dorsal columns were acquired with a NIKON Eclipse E600 confocal microscope. Images were obtained from 4 control and 4 lesioned rats at 378–587 days post-EB, 5 lesioned rats at 23 days post-EB and 3 lesioned rats at 18 days post-EB. The number of nodes analysed for each condition is given in Table 1. Quantitative results for nodes labelled with Nav1.2, Nav1.6 and Kv1.2 are expressed as mean percentage ± standard deviation.
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Nodal Nav channel expression in control dorsal columns and characteristics of the EB lesion
Study of control adult rat spinal cord dorsal columns confirmed the observations of Caldwell et al. (Caldwell et al., 2000
; Schaller and Caldwell, 2000) that identified Nav1.6 as the predominant sodium channel isoform at mature, normal nodes of Ranvier (Supplementary Fig. 1). In addition, Nav1.2 was not observed at nodes in control dorsal columns.
In common with previous studies (Felts and Smith, 1992; Blakemore et al., 1995), the repaired EB-injected spinal cord lesion was found to consist of a central core of central axons remyelinated by Schwann cells, surrounded by a shell composed of axons remyelinated by oligodendrocytes. The core and shell were distinct, so that in transverse plastic sections taken through the spinal cord at either end of the block examined immunohistochemically in longitudinal section, the border between the two areas was clear (data not shown). Furthermore, the shell of oligodendrocyte repair completely encased the region remyelinated by Schwann cells, so that all the axons bordering this region were remyelinated by oligodendrocytes.
Nodal Nav channel expression in 378–587 days post-EB spinal cord lesions
Within the core of Schwann cell remyelination, the nodes formed by Schwann cells were unambiguously identified with two distinct markers of Schwann cell myelination: P0 and ezrin. P0 is a major component of PNS myelin and is not normally expressed in the CNS (for review, see Eichberg, 2002; Supplementary Fig. 2A and B). Likewise, ezrin, a member of the ezrin-radixin-moesin (ERM) family of proteins that have been shown to accumulate within microvillar processes of myelinating Schwann cells at PNS nodes (Melendez-Vasquez et al., 2001; Scherer et al., 2001) is localized at nodes within normal sciatic nerve but not within dorsal columns (Supplementary Fig. 2C and D).
A representative low-magnification montage of a dorsal column >1 year following EB injection and immunolabelled for P0/MOG, a component of CNS but not PNS myelin (see Johns and Bernard, 1999), and ezrin is shown in Fig. 1. The region of P0 immunofluorescence corresponding to remyelination by endogenous Schwann cells is clearly distinguishable within the dorsal columns, and it is encased by MOG immunolabelling (Fig. 1A). Similarly, ezrin immunolabelling of lesioned dorsal column demonstrates the localized presence of ezrin-positive processes, consistent with Schwann cell remyelination, surrounding remyelinated nodes within the lesion; ezrin was not observed at nodes along adjacent axons myelinated by oligodendrocytes (Fig. 1B). Double labelling experiments with ezrin and P0 antibodies demonstrated that the perinodal ezrin labelling is bounded by P0-positive Schwann cell myelin (Fig. 1B inset).
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In initial experiments, we examined the clustering of Nav1.2 and Nav1.6 at nodes formed by Schwann cell remyelination, identified using the paranodal marker Caspr (Menegoz et al., 1997
; Peles et al., 1997) in sections that were serial to sections labelled for P0. In these sections of dorsal columns >1 year post-EB injection, Nav1.2 was not detectable at any nodes (Fig. 2A), while virtually all Caspr-delimited nodes exhibited Nav1.6 immunolabelling (Fig. 2B). Neither Nav1.2 nor Nav1.6 immunolabelling was observed in paranodal, juxtaparanodal or internodal regions. Subsequently, we performed triple-immunofluorescent labelling for Nav1.2 or Nav1.6, Caspr and P0 to identify the Caspr-demarcated nodes as unambiguously formed by remyelinating Schwann cells (Fig. 2C and D). Consistent with the double-label experiments, Nav1.2 was not detected at the Caspr-delimited nodes bounded by P0-positive Schwann cell myelin (Fig. 2C), while most of these nodes exhibited Nav1.6 clustering.
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The percentage of nodes formed by Schwann cells that exhibited clusters of Nav1.2 and Nav1.6 is shown in Fig. 2E. The nodes were identified by Caspr labelling, either in sections serial to sections immunolabelled with P0, or by double labelling with a combination of Caspr and P0. Both methods of identifying nodes formed by Schwann cells gave similar results. Virtually all (95–100%) nodes in the long-term lesions displayed Nav1.6 immunofluorescence, and 0% exhibited Nav1.2 labelling, similar to the pattern at nodes in normal control spinal cord.
At the boundaries of Schwann cell and oligodendrocyte remyelinated regions, occasional nodes were observed that were formed on one side by a Schwann cell and on the opposite side by an oligodendrocyte (Fig. 3). At these hybrid nodes, there was invariably a focal cluster of Nav1.6, and not Nav1.2. These observations provide the first demonstration that Schwann cell–oligodendrocyte hybrid nodes accumulate the sodium channel isoform (Nav1.6) present at normal mature nodes (Caldwell et al., 2000).
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In order to verify the results obtained using P0 immunolabelling as a marker of spinal cord axons remyelinated by Schwann cells, we also reacted lesioned dorsal column sections with ezrin antibody. Nav1.6 immunolabelling was present at 90% of nodes (Fig. 4E and G) identified by ezrin labelling of Schwann cell microvillar processes, while Nav1.2 immunofluorescence was not detected at any such nodes (Fig. 4B and G). These data are consistent with the results obtained with P0 immunolabelling and demonstrate that, as at normal spinal cord nodes, Nav1.6 is the predominant sodium channel isoform at long-term nodes remyelinated by Schwann cells within the CNS.
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It was not possible in the cryosections to distinguish with certainty nodes formed by oligodendrocyte remyelination from adjacent normal tissue. However, given that the region remyelinated by Schwann cells was routinely encased by a border remyelinated by oligodendrocytes, and given that the nodes surrounding Schwann cell regions exhibited foci of Nav1.6 bordered by Kv1.2 (data not shown), it is likely that the nodes formed by oligodendrocyte remyelination achieved a normal adult channel configuration, like the nodes formed by Schwann cells.
Kv1.2 at juxtaparanodes within 378–587 days post-EB spinal cord lesions
In addition to the spatial segregation of sodium channels at normal nodes, Shaker-type potassium channels Kv1 are clustered within the juxtaparanodal region (for review, see Rasband, 2004). In order to examine the localization of Kv1 channels in dorsal column axons remyelinated by Schwann cells >1 year post-EB injection, we examined the distribution of Kv1.2 (Rasband and Trimmer, 2001b) in control and lesioned spinal cords. The immunocytochemical localization of Kv1.2 channels in lesioned spinal cord was carried out using a protocol similar to that utilized for Nav channels, with Schwann cell remyelinated regions identified by P0 and ezrin labelling. In control spinal cord axons, Kv1.2 accumulated in juxtaparanodal regions of most, but not all, axons, although Kv1.2 was also observed in the paranodal areas of some axons, in agreement with previous descriptions (Rasband and Trimmer, 2001b) (e.g. Fig. 5A). The distribution of Kv1.2 was similar, irrespective of whether nodes remyelinated by Schwann cells were identified by Caspr labelling in sections serial to sections labelled for P0 (Fig. 5B), by combined Caspr and P0 immunolabelling (Fig. 5C) or by ezrin immunoreactivity (Fig. 5D). Kv1.2 was expressed in the juxtaparanodal regions of a high percentage (
70–90%) of nodes formed by Schwann cell remyelination (Fig. 5E); at some Caspr-demarcated nodes, incursion of Kv1.2 channels into paranodal regions was observed (Fig. 5B).
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Sodium channel clustering in nascent remyelinated spinal cord nodes
Our initial studies examined sodium channel expression at nodes formed on dorsal column axons by Schwann cell remyelination at >1 year following EB demyelination and demonstrated that Nav1.6, but not Nav1.2, was detected at these nodes. To determine whether Nav1.2 clusters might be present at newly formed remyelinated dorsal column nodes, similar to that described in nascent nodes in developing optic nerve (Boiko et al., 2001
; Rios et al., 2003), we examined dorsal column lesions at 18 and 23 days following EB injection. Previous studies have demonstrated that extensive demyelination occurs within the first week following EB injection and that by 14–21 days post-injection large numbers of demyelinated axons are present, as well as signs of early Schwann cell remyelination (Felts and Smith, 1992, 1996). Thus, at these two early post-injection times substantial numbers of nascent nodes formed by remyelinating Schwann cell are present within the lesion. Nodes formed by remyelinating Schwann cells in the 18-day post-injection dorsal columns were identified using Caspr/P0 and ezrin labelling, and in the 23-day post-injection dorsal columns by Caspr immunolabelling in sections serial to sections labelled for P0 and ezrin (Fig. 6), similar to that described for lesions >1 year post-injection. At 18 days post-injection (Fig. 6A and C), 0 out of 51 Caspr/P0-defined nodes displayed Nav1.2 clustering, and 0 out of 33 ezrin-identified nodes exhibited Nav1.2 accumulation. Similarly, at 23 days post-injection (Fig. 6E and G) 0 out of 24 Caspr-delimited nodes exhibited detectable Nav1.2 labelling, and 1 out of 76 ezrin-defined nodes displayed Nav1.2 clustering. Thus, during a period of remyelination when nascent nodes are actively being formed, only 1 out of 184 nodes exhibited detectable Nav1.2 clustering.
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In contrast, at 18 days post-injection (Fig. 6B and D) 14 out of 22 Caspr/P0-defined nodes displayed Nav1.6 clustering, and 10 out of 15 ezrin-identified nodes exhibited Nav1.6 accumulation. In addition, at 23 days post-injection (Fig. 6F and H) 26 out of 26 Caspr-demarcated nodes displayed Nav1.6 clustering and 84 out of 87 ezrin-identified nodes exhibited Nav1.6 accumulation. These data indicate that clustering of Nav1.6 was present at
75% of nascent nodes at 18 days post-injection, and nearly 100% of nodes at 23 days post-injection. Consistent with the clustering of Nav1.6, but not Nav1.2, at nodes newly formed by remyelination, binary heminodes (indicating nascent nodal formation; Rios et al., 2003) were observed in the early remyelinating lesion, and these developing nodes invariably displayed clusters of Nav1.6 (e.g. Fig. 6I and J). These observations strongly suggest that Nav1.6 is the predominant sodium channel subtype present at newly established nodes formed by remyelinating Schwann cells in EB-lesioned dorsal columns. |
Discussion |
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The ability of experimentally demyelinated CNS axons to be remyelinated by a variety of exogenous and endogenous myelin-forming cells, including oligodendrocyte precursor cells, olfactory ensheathing cells and Schwann cells, has been well established (Franklin, 2002
; Kocsis et al., 2004; Pluchino et al., 2004; Lubetzki et al., 2005; but see Lachapelle et al., 2005), although, in most instances, only a relatively short period of remyelination (3–6 weeks) has been examined. It has also been demonstrated that remyelination within previously demyelinated regions is accompanied by restoration of near-normal conduction properties (Smith et al., 1979, 1981; Felts and Smith, 1992; Honmou et al., 1996; Akiyama et al., 2002). Notably, in some long-term remyelinated lesions (>1 year) the improvement in conduction properties persists (Felts and Smith, 1992). The molecular mechanisms supporting this functional recovery of CNS axons have not been explored previously.
Nav and Kv channels are targeted to specific domains in long-term remyelinated nodes
In the present study, the distribution of sodium (Nav) and potassium (Kv) channels at mature (>1 year) nodes of Ranvier formed on dorsal column axons by remyelinating Schwann cells was found to be similar to that observed at normal nodes. As at normal nodes (Girault and Peles, 2002; Salzer, 2002; Bhat, 2003; Poliak and Peles, 2003), the populations of channels were sharply delimited, with Nav channels clustered at the nodal axolemma and Kv channels at the juxtaparanode. No Nav1.2 or Nav1.6 channels were detected beneath the myelin sheath in remyelinated internodes, in accordance with the observation that sodium channels are present only in low density beneath the myelin sheath in normal axons (Waxman and Ritchie, 1993). Of the seven sodium channel isoforms expressed within nervous tissue (Goldin et al., 2000), Nav1.6 has been demonstrated to be the predominant isoform at mature normal nodes in both the PNS and CNS (Caldwell et al., 2000), and such channels were also found to be the dominant isoform at mature remyelinated nodes in this study.
Unlike sodium channels, Kv1 potassium channels are generally not observed in the nodal axolemma (Wang et al., 1993; Mi et al., 1995). Instead, Shaker-type Kv1 channels are aggregated within juxtaparanodal domains, and their segregation to this region is dependent on intact paranodal axo-glial junctions (Rasband et al., 1999b, 2004). In the absence of intact paranodal junctions, Kv1 channels can encroach into paranodal and even nodal domains, leading to abnormalities in conduction parameters (Couetzee et al., 1996; Dupree et al., 1998; Bhat et al., 2001; Boyle et al., 2001). These abnormalities in distribution are absent in mature nodes formed by remyelinating Schwann cells.
The results presented here clearly demonstrate that, in spinal cord dorsal column axons, long-term remyelination (>1 year) by endogenous Schwann cells is associated with mature nodal characteristics, that is, clusters of Nav1.6 at nodes, Caspr-rich paranodal junctions and aggregations of Kv1.2 in juxtaparanodal regions. These observations are consistent with previous observations that Schwann cell myelin is stable for at least 24 weeks following EB-induced spinal cord demyelination, and is not supplanted by oligodendrocyte myelin (Gilson and Blakemore, 2002). The mature molecular organization of the axon membrane remyelinated by Schwann cells is in agreement with electrophysiological studies that demonstrate near-normal conduction properties in the chronically remyelinated spinal cord (Felts and Smith, 1992), although the conduction properties in themselves do not allow an accurate prediction of the composition of ion channels at the nodes of Ranvier. Transplantation of exogenous Schwann cells into demyelinated dorsal columns is also associated with the restoration of normal conduction properties (Honmou et al., 1996).
Nav channels at early Schwann cell remyelinated nodes
Previous studies in sciatic nerve (Boiko et al., 2001) and optic nerve (Boiko et al., 2001; Kaplan et al., 2001; Jenkins and Bennett, 2002; Rios et al., 2003) have demonstrated that Nav1.2 is distributed along premyelinated axons and is transiently present at immature nodes in these tracts. There is subsequently a transition from Nav1.2 to Nav1.6 at these nodes; in the optic nerve, the transition occurs over several weeks (Boiko et al., 2001; Rios et al., 2003), whereas in the sciatic nerve Nav1.2 is replaced by Nav1.6 over a briefer period of 2–3 days (Boiko et al., 2001). In the present study in rat spinal cord, we detected Nav1.6 and not Nav1.2 as the predominant sodium channel at nascent remyelinating nodes. These results are consistent with the report by Schafer et al., (2006) that, in the lysolecithin-induced model of PNS demyelination/remyelination, Nav1.6, but not Nav1.2, is detected at newly formed nodes. In contrast, Dupree et al., (2005) have reported that Nav1.2 is clustered in nascent nodes in corpus callosum axons following cuprizone-induced demyelination. It is possible that Nav1.2 clustering at nodes after remyelination is tract-specific; consistent with this speculation, previous demonstrations of Nav1.2 at remyelinated nodes have been reported in studies of optic nerve (Craner et al., 2003; Rasband et al., 2003) and corpus callosum (Dupree et al., 2005), that is, for axons with neurons of origin (retinal ganglion cells; cortical neurons) that express Nav1.2 (Westenbroek et al., 1989; Fjell et al., 1997). In contrast, the neurons (dorsal root ganglion) projecting axons to the dorsal columns and sciatic nerve do not express Nav1.2 in the adult (Felts et al., 1997). We also cannot entirely exclude the possibility that a very rapid transition from Nav1.2 to Nav1.6 occurred at the earliest formed nodes, so that there was only a very transient period of Nav1.2 expression at immature nodes that we were unable to detect. Such a transition would be more similar to that in the developing PNS than the CNS, and this could correspond with remyelination by the peripheral myelinating cell. However, several lines of evidence argue that, at best, any period of nodal clustering of Nav1.2 is very brief: (i) we detected only 1 out of 184 nascent nodes with detectable Nav1.2 clustering at 18 and 23 days following EB injection, a time when active remyelination is occurring (Felts and Smith, 1996) and it would be anticipated that Nav1.2 would be most detectable, as demonstrated in developing optic nerve (e.g. Fig. 2A inset); (ii) Schwann cell remyelination of lysolecithin-induced demyelination in the rat sciatic nerve is not accompanied by clustering of Nav1.2 at remyelinating nodes (Schafer et al., 2006); and (iii) Nav1.6, and not Nav1.2, was invariably clustered at binary nodes, which are indicative of a very early stage of node development (Vanick et al., 1996; Rasband et al., 1999a; Boiko et al., 2001). It is most likely, therefore, that Nav1.6 is the predominant sodium channel at nascent, as well as mature, Schwann cell remyelinated nodes in the lesioned spinal cord.
Clinical implications
Although repair by remyelination ultimately fails in many axons in multiple sclerosis, axons can undergo remyelination by both oligodendrocytes and Schwann cells in this disease (Ghatak et al., 1973; Prineas and Connell, 1979; Itoyama et al., 1985). Remyelination by Schwann cells, although less common, may offer the advantage that the internodes of peripheral-type myelin might be less vulnerable to immune attack than internodes formed by oligodendrocytes (Prineas et al., 1984; Hughes, 1985; Lucchinetti et al., 2000). This potential advantage is accompanied by the observation from the present study that the new nodes formed by remyelinating Schwann cells stably express a mature configuration of voltage-gated ion channels. Furthermore, central axons repaired by Schwann cells are able to function almost as well as normal (Felts and Smith, 1992), and Schwannian repair of CNS axons can be promoted by transplantation therapies (e.g. Blakemore, 1977; Baron-Van Evercooren et al., 1992; Avellana-Adalid et al., 1998; Brierley et al., 2001; Kohama et al., 2001; Lankford et al., 2002). In this regard, it has recently been demonstrated that autologous Schwann cells transplanted into demyelinated macaque spinal cord can effect robust remyelination, and these cells transplanted into demyelinated mouse spinal cord promote functional and anatomical repair (Bachelin et al., 2005). Enhanced functional recovery has been reported when the macaque Schwann cells were transfected with vectors to overexpress brain-derived neurotrophic factor (BDNF) or neurotrophin 3 (NT-3) and transplanted into demyelinated mouse spinal cord (Girard et al., 2005). Collectively, these observations encourage a view that Schwann cells are tractable candidates for cell replacement therapies aimed at restoration of function following central demyelination.
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Supplementary material |
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Supplementary data are available at Brain Online.
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Acknowledgements |
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The authors thank Hannah Morgan for excellent technical support. This work was supported in part by grants from the Medical Research Service and Rehabilitation Service, Department of Veterans Affairs and the National Multiple Sclerosis Society (S.G.W.: RG1912), and by the Multiple Sclerosis Society of Great Britain and Northern Ireland (K.J.S.). The authors also thank the Nancy Davis Foundation and Destination Cure for support. The Center for Neuroscience and Regeneration Research is a collaboration of the Paralyzed Veterans of America and the United Spinal Association with Yale University.
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