Expression of interleukin-6 receptor α in normal and injured rat sciatic nerve

Neuroscience 152 (2008) 601– 608

EXPRESSION OF INTERLEUKIN-6 RECEPTOR ␣ IN NORMAL AND INJURED RAT SCIATIC NERVE R. LARA-RAMÍREZ,1 E. SEGURA-ANAYA, A. MARTÍNEZ-GÓMEZ AND M. A. R. DENT*

kin-11 (IL-11), all of which use the common receptor subunit gp130 for signal transduction. IL-6 acts by binding to the interleukin-6 receptor alpha (IL6-R␣), a process that triggers the association of this complex with the ubiquitous signal-transducing molecule gp130. Following formation and activation of the IL-6 receptor complex, IL-6 activates the Janus kinases/signal transducers and activators of transcription (JAK/STAT) and the mitogen-activated protein kinase (MAPK) signaling pathways (Heinrich et al., 1995). The IL6-R␣ also exists in a soluble form found in serum with agonistic effects, produced either by shedding or by alternative splicing (Peters et al., 1998). It has been shown that IL-6 and IL6-R␣ mRNAs are expressed in vitro by immortalized Schwann cells or primary culture Schwann cells (Bolin et al., 1995; Grothe et al., 2000). In peripheral nerves, IL-6 was detected by RTPCR and Southern blot (Bourde et al., 1996; Kurek et al., 1996). Expression of both IL-6 and IL6-R␣ in the sciatic nerve was subsequently confirmed by in situ hybridization in Schwann cells and vascular endothelial cells (Grothe et al., 2000). Also, IL-6 was detected during sciatic nerve degeneration in vitro and in vivo (Reichert et al., 1996) and work on other peripheral nerves has shown that the IL-6 signal affects peripheral nerve regeneration. Genetic deletion of IL-6 retards the speed of regeneration of the facial nerve in 15% (Galiano et al., 2001), and the regeneration of axotomized hypoglossus nerve is delayed when IL6-R␣ is inhibited with specific antibodies (Hirota et al., 1996). In contrast, IL-6 deficient mice do not show a significant difference in the regeneration of injured sciatic nerve (Inserra et al., 2000), probably because other cytokines, like LIF, replace IL-6 function in this process (Kurek et al., 1996). After nerve injury by crush or transection, IL-6 mRNA is upregulated in the first few hours and dramatically decreases soon after (Bolin et al., 1995; Bourde et al., 1996; Grothe et al., 2000; Ito et al., 1998; Kurek et al., 1996). IL6-R␣ mRNA also increases having a peak between 2 and 7 days after injury and decreases gradually over 28 days (Ito et al., 1998). In vitro, Schwann cells secrete IL-6 that induces LIF expression, which in turn induces macrophage chemoattractant protein-1 (MCP-1), suggesting that IL-6 plays a role in regulating macrophage infiltration during injury (Tofaris et al., 2002). Thus, although there are many studies on cytokine induction after peripheral nerve injury, the localization of IL6-R␣ in the normal sciatic nerve and during Wallerian degeneration has not been described. To investigate this, we have determined the protein expression of IL6-R␣ in normal and injured rat sciatic nerve.

Laboratorio de Neurociencias, Facultad de Medicina, Universidad Autónoma del Estado de México, Apartado Postal 428, Toluca, Edo. de México, México CP 50000

Abstract—Interleukin-6 (IL-6) is a pleiotropic cytokine synthesized by many different cells after appropriate stimulation. IL-6 binds first to the interleukin-6 receptor alpha (IL6-R␣) and then this complex binds to the signal-transducing gp130 receptor, forming a functional hexameric receptor complex. We observed by Western blot analysis with anti-IL6-R␣ two bands of ⬃80 kDa and ⬃110 kDa in the rat sciatic nerve, cerebral cortex, spleen, pancreas and liver, corresponding to the mature glycosylated form and possibly to the dimer of the non-glycosylated precursor protein. By immunohistochemistry, high levels of IL6-R␣ expression are observed in nonmyelinating Schwann cells. In myelinating Schwann cells IL6-R␣ is present as discrete dots in the perinuclear region, in distinct membrane domains of the Schwann cell sheath and at the nodes of Ranvier, suggesting that IL6-R␣ is clustered both on the axonal side of the node and within the Schwann cells. After sciatic nerve crush injury IL6-R␣ is upregulated in denervated Schwann cells between the myelin ovoids during the period of Schwann cell proliferation. The expression of IL6-R␣ continues during the period of remyelination, suggesting that IL6-R␣ might be involved in both Schwann cell proliferation and remyelination of the rat sciatic nerve. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: nodes of Ranvier, cytokine, Wallerian degeneration, Schwann cells, myelin, crush injury.

Interleukin-6 (IL-6) is a pleiotropic cytokine with a wide range of biological activities. It can induce proliferation, differentiation or maturation depending on the target cell and cellular context (Simpson et al., 1997). In diabetesrelated neuropathy IL-6 shows a potential neuroprotective action (Andriambeloson et al., 2006). In the nervous system, IL-6 promotes neuron survival, growth and differentiation, neurotransmitter metabolism, and is involved in the formation of dendrites (Gadient and Otten, 1995; Grothe et al., 2000; Heinrich et al., 1995). IL-6 is functionally and structurally similar to other cytokines such as leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM), cardiotrophin-1 (CT-1) and interleu1

Present address: Department of Zoology, University of Oxford, The Tinbergen Building, South Parks Road, Oxford OX1 3PS, UK. *Corresponding author. Tel: ⫹52-722-217-3552x222; fax: ⫹52-722217-4142 (request fax tone). E-mail address: [email protected] (M. A. R. Dent). Abbreviations: GFAP, glial fibrillary acidic protein; IL-6, interleukin-6; IL6-R␣, interleukin-6 receptor alpha; LIF, leukemia inhibitory factor; MBP, myelin basic protein; NF200, neurofilament 200 kDa.

0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.01.014

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EXPERIMENTAL PROCEDURES Teased nerve preparations All procedures involving animals were carried out in accordance with the Institutional Guide for Ethical Animal Experimentation (National Autonomous University of Mexico), minimizing the number of animals used and their suffering. Male Wistar rats (250 – 350 g) were used in all experiments. Rats were killed by ether or CO2 overexposure. Crushed sciatic nerves, sympathetic trunk and sciatic nerve from normal adult rats were excised and desheathed. The nerves were split into manageable strands and then teased gently onto poly-L-lysine-coated microscope slides in a drop of PBS using 23-gauge needles as previously described (Jessen and Mirsky, 1984). The teased nerves were allowed to air-dry for at least 1 h before immunostaining.

Surgery Rats were anesthetized with CO2 and anhydrous ether during the surgery. Anesthesia was confirmed and monitored by the absence of pain reflex. The surgery was performed using aseptic conditions. Briefly, the left leg muscles were separated at the mid-thigh level to uncover the sciatic nerve, which was then crushed. The sciatic nerves were crushed with fine forceps #7 (Dumont, Switzerland) for 30 s and rats were allowed to recover for 5 h, 3, 9, 18, 21 and 30 days after crush. The undamaged contralateral nerves and normal sciatic nerves were used as controls. Proximal and distal parts of the injured nerves were teased separately.

Immunohistochemistry Teased nerves were fixed in 4% w/v paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PF) for 20 min and then washed three times in PBS for 5 min. Non-specific binding sites were blocked with 10% non-fat skimmed milk powder in PBS (blocking solution) overnight at 4 °C, prior to incubation with anti-IL6-R␣ antibody [1:100] (Genzyme Diag) in PBS, 0.1% Tween 20 (Sigma) and 10 ␮l of blocking solution at 4 °C overnight in a humid chamber. After washing in PBS, the teased nerves were incubated with biotinylated anti-rabbit IgG antibody (1:500) (Vector Laboratory) in PBS, 0.1% Tween 20, and 10 ␮l of blocking solution at 4 °C for 1 h, and then incubated in ABC kit (Vector Elite reagent) for 30 min. After additional washes, peroxidase was developed with 3,3=-diaminobenzidine (Sigma) for 10 min. To counterstain the nuclei with Thionin, the slides were incubated for 5 min in 1.3% w/v Thionin (Sigma), dehydrated and mounted in Permount medium (Fisher Chemical). For fluorescence immunostaining, after the second antibody the slides were incubated with fluoresceinavidin D (1:500) (Vector Laboratory) for 30 min at 4 °C, washed and mounted with Vectashield (Vector Laboratories). For double immunohistochemistry, teased nerves were incubated with mouse anti-glial fibrillary acidic protein (GFAP) conjugated with Cy3 (1:1500) (Sigma) overnight at 4 °C. For myelin basic protein (MBP) and neurofilament 200 kDa (NF200) immunostaining, teased nerves were fixed with methanol for 10 min and blocked for 1 h with 10% milk in PBS (0.2% Tween-20, 0.5% Triton-100) at 4 °C. Mouse monoclonal anti-MBP antibody (Calbiochem) was added (1:1000) in PBS with 5% milk (0.02% Tween20, 0.05% Triton-100) and mouse monoclonal anti-NF200 antibody clone NE14 (Boehringer Mannheim) was added (1:3000) in PBS, 0.1% Tween 20 and 10 ␮l of blocking solution at 4 °C overnight in a humid chamber. The samples were washed with PBS and incubated with anti-mouse Alexa-Red (1:2000) (Molecular Probes) for 1 h at 4 °C. Some preparations were also stained with DAPI/Hoescht for 5 min. The samples were washed with PBS and mounted with Vectashield. Controls were performed by omission of the primary antibody. Specimens were examined by epifluorescence on an Olympus BX60 microscope, followed by image manipulation with Image-Pro Plus 4.5.1 or by confocal microscopy

on an Olympus FluoViewTM FV1000 (1.3b Viewer) microscope. The 3D deconvolution and 3D reconstruction of the images were done using the Image-Pro Plus software.

Immunoblot analysis Sciatic nerves, cerebral cortex, spleen, pancreas and liver tissue were homogenized directly in SDS solubilization buffer (2% w/v SDS, 2 mM EDTA, 2 mM EGTA, 5 mM Tris–Cl, 1 mM PMSF pH 7), and protease inhibitors (0.5 mg/ml each of antipain, pepstatin, amastatin, leupeptin, bestatin, trypsin inhibitor and 3 U/ml of aprotinin). The protein concentration was estimated and 10 ␮g/␮l of total protein was denatured for 5 min at 95 °C in non-reducing Laemmli electrophoresis sample buffer. The samples and 10 ␮l of Benchmark Prestained Protein Ladder (Invitrogen) were subjected to 12% SDS-PAGE at 50 mA for 2 h. Proteins were transferred onto nitrocellulose membrane (Amersham) in ice-cold transfer buffer (25 mM Tris–Cl, 192 mM glycine, 0.2% w/v SDS, 20% v/v methanol) at 35 mA for 16 –17 h, and the membrane was then stained with Ponceau Red to confirm the success of the transfer. The membrane was blocked with 10% milk in TBST (10 mM Tris–Cl, 150 mM NaCl, 0.05% Tween) for 3 h, then incubated with anti-IL6-R␣ (1:3000) in TBST, 5% milk for 1 h, and further washed three times in TBST for 5 min. The blot was incubated with biotinylated anti-rabbit antibody (1:3000) in TBST, 5% milk for 1 h, followed by ABC kit (Vector Laboratories) for 30 min. The signal was developed with ECL reagent (Amersham) and captured on a HyperfilmTM ECL (Amersham).

RESULTS Immunoblot analysis of IL6-R␣ in different tissues Western blotting with anti-IL6-R␣ of whole sciatic nerve, cerebral cortex, spleen, pancreas and liver revealed two bands of ⬃80 kDa and ⬃110 kDa (Fig. 1). The ⬃80 kDa band corresponds to the glycosylated monomer of the mature form of IL6-R␣, while the ⬃110-kDa band might correspond to a non-glycosylated dimer of IL6-R␣. All of the tissues we studied expressed IL6-R␣, but differences in the levels of expression were observed (Fig. 1). Immunolocalization of IL6-R␣ in the sciatic nerve IL6-R␣ expression was examined by immunohistochemistry in teased preparations of normal adult rat sciatic

Fig. 1. Immunoblot analysis of IL6-R␣ in different tissues. Anti-IL6-R␣ antiserum recognizes two bands of ⬃80 kDa and ⬃110 kDa in sciatic nerve (SN), cerebral cortex (C), spleen (S), pancreas (P) and liver (L).

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Fig. 2. IL6-R␣ is present in both myelinating and non-myelinating Schwann cells of the adult rat sciatic nerve. Confocal images show that IL6-R␣ is strongly expressed in non-myelinating Schwann cells (arrowheads in A, D), which were double-labeled with mAbs against GFAP (B), and co-distributed with GFAP (C). However, IL6-R␣ does not co-localize with MBP in the sciatic nerve (D, G), or in the sympathetic trunk (E). In myelinating Schwann cells, perinuclear staining of IL6-R␣ (arrow in F) is present in preparations counterstained with DAPI. IL6-R␣ is expressed at the nodes of Ranvier (arrows in D, G) when double-labeled against MBP. IL6-R␣ is also present in the internodal Schwann cell membrane (arrowheads in G, H). Double-immunostaining of IL6-R␣ and NF200 (H) shows co-distribution at the nodes of Ranvier (arrows in H), elsewhere, it does not co-localize with NF200. (I, J) 3D deconvolution of the double-labeling of IL6-R␣ and NF200. On a cross-section at the middle of the internode (J) at the level of the line in (I) IL6-R␣ is localized in the abaxonal Schwann cell membrane (arrow in J) and in the middle of the nerve fiber the axon is stained with NF200. Scale bars⫽10 ␮m (A–C, F, G, J); 25 ␮m (D); 50 ␮m (E); 20 ␮m (H, I).

nerve (Fig. 2), which were double labeled with mAbs against GFAP and MBP to identify non-myelinating and myelinating Schwann cells, respectively. Laser confocal microscopy shows that IL6-R␣ is strongly present in non-myelinating Schwann cells as discrete dots (Fig. 2A and D). When we double-labeled against GFAP (Fig. 2B), IL6-R␣ co-distributed with GFAP (Fig. 2C). To examine this further, we teased the cervical sympathetic trunk where more than 90% of the axons are unmyelinated (Aguayo et al., 1976), and double-labeled against IL6-R␣ and MBP. In these preparations, IL6-R␣ is present in non-myelinating Schwann cells but absent from those cells expressing MBP (Fig. 2E). In most myelinating Schwann cells of the sciatic nerve a perinuclear dot staining of IL6-R␣ is present (Fig. 2F). IL6-R␣ is also present at the nodes of Ranvier and on the abaxonal side of the Schwann cell membrane close to

the node when we double-stained against MBP (Fig. 2D and G). When we double labeled against IL6-R␣ and NF200 to identify the axons, IL6-R␣ only co-distributed with NF200 at the nodes of Ranvier (Fig. 2H). The results suggest that IL6-R␣ is present both on the axonal side of the node and within the Schwann cells. There is also a dotted expression of IL6-R␣ on the abaxonal side of the Schwann cell membrane near the node and along the internode (Fig. 2G–J). To examine this further, we performed 3D deconvolution of the double labeling of IL6-R␣ and NF200 of the nerves (Fig. 2I). On a cross-section of one of the internodes of this preparation (Fig. 2J), we observed the presence of IL6-R␣ on the periphery of the fiber corresponding to the abaxonal side of the Schwann cell membrane, and in the middle the axon is stained with NF200 (Fig. 2J), but not with IL6-R␣. These results confirm that in the internodes IL6-R␣ is not present in the axons.

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There is also IL6-R␣ expression in the vascular endothelial cells (data not shown). Expression of IL6-R␣ during Wallerian degeneration To investigate IL6-R␣ expression during nerve injury the sciatic nerve was crushed and the distal and proximal regions, and contralateral nerves were studied. The distal regions were teased and at 5 h after crush the same pattern of expression of IL6-R␣ as in the normal sciatic

nerve was observed (data not shown). At 3 days, the formation of ovoids of degenerated myelin sheaths stained with MBP, and expression of IL6-R␣ is observed in denervated Schwann cells between the myelin ovoids (Fig. 3A and B), mainly in areas close to the nucleus (Fig. 3C). IL6-R␣ staining is also observed in denervated non-myelinated Schwann cells that have started to proliferate (Fig. 3C). When double labeled against IL6-R␣ (Fig. 3D) and GFAP (Fig. 3E), IL6-R␣ co-distributed with GFAP (Fig. 3F).

Fig. 3. Expression of IL6-R␣ in the distal region of the sciatic nerve at 3 and 9 days after crush. At 3 days after crush, teased sciatic nerve preparations were double-labeled with anti-IL6-R␣ and mAbs against MBP (A, B). IL6-R␣ is present between the myelin ovoids that express MBP. In DAB preparations counterstained with Thionin (which stains the nuclei) IL6-R␣ is expressed in the denervated areas close to the nucleus (arrowhead in C) and in denervated non-myelinating Schwann cells (arrow in C) that have started to proliferate. Double-staining with IL6-R␣ (D) and GFAP (E) shows that IL6-R␣ codistributed with GFAP (F). At 9 days after crush, double-labeling with anti-IL6-R␣ and mAbs against MBP (G, H) shows expression of IL6-R␣ in denervated Schwann cells. There are some MBP⫹ ovoids present and few fibers (arrowhead in H) have a thin coat of MBP expression along the Schwann cell (arrow in H). Double-labeling with anti-IL6-R␣ and mAbs against GFAP (I), shows that all the fibers visualized with IL6-R␣ express GFAP. When double-labeled with IL6-R␣ and NF200 (J), IL6-R␣ does not co-distribute with NF200. Scale bars⫽10 ␮m (A–C, H–J); 20 ␮m (D–G).

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At 9 days all denervated Schwann cells express IL6-R␣, and there are few MBP⫹ myelin ovoids (Fig. 3G and H). There are also a few IL6-R␣⫹ cells with a thin coat of MBP expression along the Schwann cells (Fig. 3H), suggesting that remyelination has already taken place in some fibers. At this stage all denervated Schwann cells stained for IL6-R␣ express GFAP (Fig. 3I). When the fibers were double labeled against IL6-R␣ and NF200 to identify the axons (Fig. 3J), IL6-R␣ did not co-localize with NF200, suggesting that IL6-R␣ is present only in the denervated Schwann cell membrane, but is not in the axolemma. At 18 days after crush, double-staining against IL6-R␣ and MBP

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shows three different types of fibers expressing only IL6-R␣ or having few MBP⫹ myelin ovoids (Fig. 4A and B), fibers IL6-R change to Greek alpha⫹ expressing a thin coat of MBP along the Schwann cell in which remyelination is taking place (arrow in Fig. 4A), and fibers already remyelinated that are forming (arrowhead in Fig. 4B) or have formed (arrows in Fig. 4B and C) nodes of Ranvier. The latter cells express IL6-R␣ in the abaxonal side of the Schwann cell membrane and at the nodes of Ranvier. When we double-labeled with IL6-R␣ (Fig. 4D and G) and GFAP (Fig. 4E and H) at 18 and 21 days after injury, respectively, IL6-R␣ co-distributed with GFAP only in a few

Fig. 4. Expression of IL6-R␣ in the distal region of the sciatic nerve at 18, 21 and 30 days after crush. At 18 days after injury, teased sciatic nerve preparations were double-labeled with anti-IL6-R␣ and mAbs against MBP (A–C). All denervated Schwann cells express IL6-R␣, some have few MBP⫹ ovoids (arrowhead in A), some show a thin coat of MBP labeling along the Schwann cell (arrow in A) and some are forming (arrowhead in B) or have formed (arrows in B, C) nodes of Ranvier. When the fibers were double-labeled with anti-IL6-R␣ (D, G) and GFAP (E, H) at 18 and 21 days after injury, respectively, IL6-R␣ co-distributed with GFAP only in a few fibers (arrow in F, I), but the majority of the fibers only expressed IL6-R␣. At 30 days after crush, the same expression pattern as the normal nerve is observed when double-labeled with IL6-R␣ and MBP (J, K). Scale bars⫽10 ␮m (A–C, G–I, K); 20 ␮m (D–F, J).

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cells (Fig. 4F and I), but the majority of the Schwann cells that express IL6-R␣ did not express GFAP (Fig. 4F and I). At 30 days after injury, the expression pattern of IL6-R␣ is the same as the normal nerve (Fig. 4J and K), suggesting that the sciatic nerve has already regenerated. The proximal region and the contralateral nerves showed no changes in the IL6-R␣ expression pattern at all times examined after crush compared with the normal sciatic nerve (data not shown).

DISCUSSION The cDNA of the human IL6-R␣ encodes a protein of 468 amino acids. The mature form of IL6-R␣ is a glycosylated 80 kDa form of the predicted 50 kDa precursor protein (Hirata et al., 1989; Varghese et al., 2002). By Western blot analysis the rabbit antiserum against the IL6-R␣ recognizes two bands, one of ⬃80 kDa corresponding to the mature glycosylated molecule and another one of ⬃110 kDa, which might represent a dimer of the non-glycosylated precursor protein of 50 kDa. Both bands were observed in the sciatic nerve as well as in cerebral cortex, spleen, pancreas and liver. This confirms that IL6-R␣ is expressed at the protein level in the sciatic nerve, as well as in the other tissues we studied. The results show that IL6-R␣ is constitutively expressed in the normal adult sciatic nerve. The receptor is expressed at high levels in non-myelinating Schwann cells. It is also present in myelinating Schwann cells, in distinct membrane domains of the internodal cytoplasm, at the nodes of Ranvier, and in the Schwann cell membrane close to the node, possibly in the microvilli, which are found in this region (Thanos et al., 1998). These results raise the possibility that IL6-R␣ signaling may play a role in the maintenance of the fine molecular architecture of the relationship between Schwann cells and axons in the rat sciatic nerve, however, the details of the signaling pathway remain to be investigated. It has been reported that Schwann cells de-differentiate when axonal contact is lost, then they re-enter the cell cycle and adopt a phenotype similar to that of immature Schwann cells (Jessen and Mirsky, 2004). The expression of cytokines during Wallerian degeneration plays an important role in regulating the degradation of myelin and the conversion of the Schwann cells from the myelinating phenotype to the denervated Schwann cell. This change in phenotype in the distal stump involves downregulation of myelin-associated genes and upregulation of several regenerating associated genes (Fenrich and Gordon, 2004). For example, the cytokines LIF and IL-6 are upregulated in denervated Schwann cells (Fenrich and Gordon, 2004). IL-6 mRNA shows a rapid and robust increase after nerve injury (Bolin et al., 1995; Bourde et al., 1996; Grothe et al., 2000; Ito et al., 1998; Kurek et al., 1996). Also, the levels of IL6-R␣ mRNA increase after a crush injury of the sciatic nerve, with a peak value between 2 and 3 days, followed by a gradual decrease until reaching almost normal levels at 28 days (Ito et al., 1998). At the protein level we observed that IL6-R␣ is upregulated during Wallerian degeneration and is down-regulated during the period of regener-

ation, reaching normal levels at 30 days after crush injury, suggesting that the sciatic nerve has already regenerated. Several cytokines, including IL-6 and transforming growth factor-␤ (TGF-␤) are secreted by both macrophages and denervated Schwann cells, and are released into the distal nerve stump after nerve injury (Fenrich and Gordon, 2004). It has been shown that IL-6 secreted by Schwann cells plays a role after nerve injury by regulating macrophage infiltration (Tofaris et al., 2002). Also, the recruited macrophages release cytokines after nerve injury, such as IL-1 and IL-6 (Fu and Gordon, 1997), and macrophage invasion is temporally correlated with Schwann cell mitosis (Jiménez et al., 2005). IL-6 signal has also been involved in cell proliferation. For example, the IL6-R/IL6 chimera protein induces proliferation in different cell lines (Haggiag et al., 2001; Zvibel et al., 2004). Thus, it is possible that during Wallerian degeneration, the IL-6 secreted by both Schwann cells and macrophages upregulates IL6-R␣ in Schwann cells, which might be involved in Schwann cell proliferation. It has been reported that demyelination starts 1 or 2 days after injury and the myelin sheath is fragmented into “ovoids” or “ellipsoids” (Koeppen, 2004). We observed that at 3 days after nerve crush IL6-R␣ is expressed in denervated myelinating Schwann cells, mainly in areas close to the nucleus, but it is not present in the degenerated myelin that is MBP⫹. Also, denervated non-myelinating Schwann cells express high levels of IL6-R␣ and have started to proliferate, in agreement with the fact that non-myelinating Schwann cells proliferate earlier than myelinating Schwann cells (Clemence et al., 1989). Remyelination starts when the dividing Schwann cells spread along the regenerating axon and initiate spiraling of the membrane forming a thin coat of myelin (Bunge et al., 1986, 1990; Garbay et al., 2000). Then, the myelin lamellae increase in number in proportion to the axon size (Hildebrand et al., 1994), with a further thickening of the myelin sheath and the formation of nodes of Ranvier and myelin incisures. At 9 days after injury we observed very few cells with a thin coat of myelin that are MBP⫹, suggesting that remyelination has already started in some fibers. This is in agreement with previous work showing that remyelination initiates 1–2 weeks after nerve crush (Bisby, 1995; Nagarajan et al., 2002). However, at 18 days there are cells with a thin coat of myelin and cells already myelinated forming nodes of Ranvier, showing that not all Schwann cells are at the same stage of regeneration. IL6-R␣ has also been implicated in myelination. It has been shown that the chimeric protein IL6R/IL6 which triggers the gp130 receptor system is involved in myelination. It induces MBP and P0 mRNAs and proteins in dorsal root ganglia (DRG) cultures from 14-day-old mouse embryos (Haggiag et al., 1999), and in vivo it increases fourfold the number of myelinated nerve fibers after nerve injury (Haggiag et al., 2001). In newborn mice, conditional inactivation of gp130 causes loss of myelin sheaths (Betz et al., 1998). Also, when the melanoma cell line B16/10.9 is treated with the IL6R/IL6 chimera protein, it undergoes morphological transdifferentiation from a melanocytic to a glial phenotype characterized by

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the transcriptional activity of both P0 and MBP promoters and accumulation of myelin gene products (Slutsky et al., 2003). IL6-R␣ and GFAP are expressed in all denervated Schwann cells at 3 and 9 days after injury, but at 18 and 21 days after lesion GFAP is downregulated in those Schwann cells that form myelin and is only expressed in non-myelinating Schwann cells, as it has been reported previously (Jessen et al., 1990). However, IL6-R␣ is present in both myelinating and non-myelinating Schwann cells. Thus, it is possible that IL6-R␣ is involved in the remyelination process, since IL6-R␣ continues to be expressed during this period in myelinating Schwann cells. In conclusion, it is possible that after nerve crush IL6-R␣ might be important in both Schwann cell proliferation and remyelination. Acknowledgments—We thank Prof. R. Mirsky and Dr. Bob Amess for critically reading and correcting the manuscript, Biol. Juan Carlos Rivera-Mulia for assistance with the figures and the deconvolution system, MVZ Claudia Rivera-Cerecedo for providing us with the rats used in this work. This work was supported by Conacyt-México, grant 33540-N and UAEMex grant 1977/2004 to M. A. R. Dent.

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(Accepted 9 January 2008) (Available online 16 January 2008)