Differential responses of spinal axons to transection: influence of the NG2 proteoglycan

Experimental Neurology 192 (2005) 299 – 309 www.elsevier.com/locate/yexnr

Differential responses of spinal axons to transection: influence of the NG2 proteoglycan Romulo de Castro Jr.*, Rokhsareh Tajrishi, Jennifer Claros, William B. Stallcup Developmental Neurobiology Program, The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA Received 25 March 2004; revised 29 September 2004; accepted 9 November 2004

Abstract Spinal cord transections were performed in wild type and NG2 proteoglycan null mice in order to study penetration of regenerating axons into the scar that forms in response to this type of injury. Aside from the presence or absence of NG2, the features of the transection scar did not differ between the two genotypes. In both cases, the rostral and caudal spinal cord stumps were separated by collagenous connective tissue that was continuous with the spinal cord meninges. In wild type mice, oligodendrocyte progenitors, macrophages, and microvascular pericytes contributed to up-regulation of NG2 expression in and around the scar. Substantial amounts of non-cell associated NG2 were also observed in the scar. The abilities of two classes of spinal axons to penetrate the transection scar were examined. Serotonergic efferents and calcitonin gene-related peptide-positive sensory afferents both were observed within the lesion, with calcitonin gene-related peptide-positive axons exhibiting a greater capability to penetrate deeply into the scar tissue. These observations demonstrate inherent differences in the abilities of distinct types of neurons to penetrate the scar. Significantly, growth of serotonergic axons into the transection scar was observed twice as frequently in wild type mice as in NG2 knockout mice, suggesting a stimulatory role for the proteoglycan in regeneration of these fibers. These findings run counter to in vitro evidence implicating NG2 as an inhibitor of nerve regeneration. This work therefore emphasizes the importance of including in vivo models in evaluating the responses of specific types of neurons to spinal cord injury. D 2004 Elsevier Inc. All rights reserved. Keywords: Spinal cord injury; Transection; Scar; Nerve regeneration; NG2 proteoglycan; Serotonin; Calcitonin gene-related peptide

Introduction Recovery from various types of nerve injuries represents an important clinical problem that has been extremely resistant to effective treatment. In particular, lesions to the central nervous system (CNS) pose a serious obstacle to recovery of function. Compared to axons in the peripheral nervous system (PNS), axons in the CNS have a very limited ability to regenerate and recover function following injury. This discrepancy has been attributed in large part to the formation in the lesioned CNS of a glial scar that provides a physical and/or molecular barrier to axon regeneration (Bunge et al., 1997; Fawcett and Asher,

* Corresponding author. Fax: +1 858 646 3197. E-mail address: [email protected] (R. de Castro). 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2004.11.027

1999; Fitch and Silver, 1997). Compounding the problem are the varied types of neurons that must regenerate to allow functional recovery. Axonal projections from different neurons may well differ in their response to elements of the glial scar. Moreover, each class is likely to have its own specific requirements for survival, growth stimulation, and guidance (Novikova et al., 2002; Pataky et al., 2000; Tetzlaff et al., 1994). Although present at the correct place and time during normal development, many of these cues may no longer be present at the time of injury. Due to its complex variety of neuronal cell types, the brain appears to offer an overwhelmingly complex system for effective repair. Even the spinal cord contains enough different types of axons to pose a severe challenge. In addition to several types of descending efferent fibers and ascending afferent tracts, there are intraspinal neurons that need to be regenerated. Advances in our understanding of

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the regeneration process will require careful analysis of the response of individual classes of axons to injury, as well as to putative stimulatory and inhibitory factors in the environment of the lesion. Glial scars in the CNS are thought to contain several types of molecules that are inhibitory to axon re-growth. Myelin-associated glycoprotein (Filbin, 1995; McKerracher et al., 1994), Nogo (Chen et al., 2000; Fouad et al., 2001; GrandPre et al., 2000; Woolf, 2003), and semaphorins (De Winter et al., 2002; Pasterkamp and Verhaagen, 2001) have all been proposed as molecules that may act as inhibitors of axon regeneration in the lesioned CNS. Chondroitin sulfate proteoglycans (CSPGs) have also been widely discussed as a class of molecules that establish boundaries in the CNS (Brittis et al., 1992; Snow et al., 1990, 1991; Yamaguchi, 2000) and act as inhibitors of axon regeneration (Bradbury et al., 2002; Davies et al., 1997; Fitch and Silver, 1997; Lemons et al., 1999; McKeon et al., 1999; Moon et al., 2001). Among the CSPGs, the NG2 proteoglycan is a prominent candidate for inhibition of axon regeneration, based on its reported ability to inhibit neurite outgrowth in vitro (Chen et al., 2002a,b; Dou and Levine, 1994, 1997; Fidler et al., 1999; Ughrin et al., 2003) and its dramatic in vivo up-regulation at sites of neural injury (Jones et al., 2002, 2003; Keirstead et al., 1998; Levine, 1994; McTigue et al., 2001; Rezajooi et al., 2004; Watanabe et al., 2002; Zhang et al., 2001). Although this combination of properties offers tantalizing circumstantial evidence in favor of NG2 as an inhibitor of nerve regeneration, no in vivo studies have been published in support of the hypothesis. In this report, we have compared the ability of two classes of axons [calcitonin gene-related protein (CGRP)positive and serotonergic] to penetrate the transection scar that forms in response to severing the mouse spinal cord. In addition, we have examined the potential effect of the NG2 proteoglycan on regeneration of these axons by performing parallel transection studies in wild type and NG2 null mice. The results demonstrate that the respective axon types differ in their abilities (1) to penetrate the transection scar and (2) to respond to the presence of NG2 in the environment. Significantly, we could not demonstrate improved axon regeneration in the NG2 null mouse, and thus were unable to provide support for the hypothesis that NG2 is inhibitory to nerve re-growth in the injured spinal cord.

Materials and methods Animal surgery and postoperative care All procedures were performed in compliance with the NIH Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Burnham Institute’s Animal Care and Use Committee. C57Bl/6 male mice were obtained from homozygous matings in our colonies of NG2 wild type and knockout mice (Grako et al., 1999). The

animals were 3–4 months old at the time of the initial surgery. At the conclusion of each experiment, genotypes were re-examined by Southern blotting and/or by immunohistochemistry to confirm the presence or absence of NG2. For our studies, we used a complete spinal cord transection model in order to avoid possible ambiguities in the interpretation of results. Following complete interruption of all nerve tracts in the spinal cord, the observation of any intact axons must be due to nerve regeneration rather than to the presence of nerves that were not severed by the original surgery. Prior to surgery, animals were anesthetized by intraperitoneal administration of 125 mg/kg ketamine plus 7 mg/ kg xylazine. Depth of anesthesia was assured by confirming the lack of response to a noxious foot pinch. The animal’s back was shaved and aseptically scrubbed with chlorhexidine. A longitudinal incision was made along the dorsal midline to expose the spinal column, and a laminectomy was performed at the T9–T10 vertebral level. The exposed spinal cord was then severed at this site by several transverse passes of a #11 scalpel to ensure complete transection (in this case, the stumps visibly retract, leaving a space at the lesion site). The wound was closed with sterile metal clips. An analgesic (buprenorphine, 0.01–0.05 mg/kg sc) was administered as needed. Immediately after wound closure, a solution of dextrose (0.01%) and antibiotic (enrofloxacin, 0.50%) in normal saline was administered subcutaneously (0.33 ml/g body weight). Animals initially recovered from surgery in a warm environment and were later returned to their original quarters. Food and water were provided at the floor of the cage for easy access. Dextrose and antibiotics were administered and bladders were expressed 2–3 times daily until the animals resumed eating and drinking normally and urinary incontinence disappeared (usually within 1–2 weeks). Urine and feces were inspected for signs of infection, and the animals were checked periodically for weight loss. Complications, such as dehydration, hypothermia, wound infection, autophagia, etc., were treated as recommended by the Burnham Institute veterinarian. Tissue collection, processing, and histology At designated time points, animals were re-anesthetized (as above) and transcardially perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (in PBS). Spinal cord segments containing the lesion site were removed, post-fixed overnight, and immersed in 30% sucrose prior to embedding in OCT compound, freezing, and sagittal sectioning (20-Am serial sections were collected). Some sections were stained by the Masson–Trichrome procedure, while others were used for immunohistochemical analyses with the following antibodies: rat monoclonal antibody against mouse macrophage marker (F4/80, 1:50, Biosource International), rat monoclonal antibody against mouse CD31 (Pharmingen, La Jolla, CA), rabbit and guinea

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Fig. 1. Masson–Trichrome staining of spinal cord at 8 weeks post-transection. Blue staining indicates collagenous material. Red and dark red areas are nervous tissue. Arrows point to the meningeal lining of the spinal cord and to the scar tissue between stumps. (A) Compressed scar, NG2 null mouse. (B) Extended scar, NG2 null mouse. (C) Compressed scar, wild type mouse. (D) Extended scar, wild type mouse. Scale bar in A = 50 Am.

pig antibodies against NG2 (4 Ag/ml, Ozerdem et al., 2001, 2002), rabbit antibody against platelet-derived growth factor alpha receptor (PDGF aR, 8 Ag/ml; Nishiyama et al., 1996a,b), rat monoclonal antibody against glial fibrillary acidic protein (GFAP, 2.5 Ag/ml, Zymed Laboratories, Inc.), rabbit antibody against calcitonin gene-related peptide (CGRP, 1:200, Peninsula Laboratories, Inc.), and rabbit antibody against serotonin (5-hydroxytryptamine or 5-HT, 1:1000, Oncogene Research Products). Double staining was performed for the following pairs of antigens: NG2 + F4/80, NG2 + CD31, NG2 + GFAP, NG2 + PDGF aR, PDGF aR + GFAP, CGRP + GFAP, 5-HT + GFAP. Secondary antibodies conjugated to the fluorescent dyes Alexa 488 (Green) or Alexa 594 (Red) were obtained from Molecular Probes (Eugene, OR) and used at a 1:500 dilution. Analysis of axon growth into the transection scar To determine the boundaries of the transection scar, sagittal sections were obtained at several levels (lateral to

medial) through the injured spinal cord. Masson–Trichrome staining was performed to discriminate between collagenous material (blue) and nervous tissue (red). PDGF aR + GFAP double staining was also performed to visualize the meninges enclosing the nerve stumps (PDGF aR) and astrocytic processes (GFAP) bordering the scar area. Fluorescence microscopy was used to examine sections that were double stained for CGRP and GFAP. CGRPpositive axons within the scar (i.e., the area bordered by GFAP-positive astrocytic processes) were identified in representative sections from the medial and lateral regions of the injured spinal cord. Similarly, for quantification of serotonergic axon sprouting, we used sections that were double stained for 5-HT and GFAP to identify 5-HT-positive axons within the scar. For this analysis, we examined every second section from a complete serial sectioning of the injured spinal cord sample (20-Am sections). Because 5-HTpositive axons run in and out of the plane of sectioning,

Fig. 2. GFAP and PDGF aR expression in transected spinal cord. Double immunofluorescence labeling for GFAP (red) and PDGF aR (green) was performed on longitudinal spinal cord specimens 8 weeks after transection. (A) Compressed scar, wild type mouse. (B) Extended scar, NG2 null mouse. Arrows indicate PDGF aR-positive meningeal lining. Arrowheads identify GFAP-positive processes within the scar. Asterisks denote cavities filled with scar tissue. Scale bars = 50 Am.

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Fig. 3. NG2 expression in the spinal cord 1 week after transection. Immunolabeling for NG2 was compared in wild type and NG2 null spinal cords 1 week after the injury. (A) Wild type cord at the site of injury. (B) Wild type cord away from injury site. (C) NG2 null cord at the site of injury. (D) NG2 null cord away from injury site. Dashed lines in A and C mark the boundary between the lesion site and the uninjured spinal cord. Arrows in A and B identify NG2-positive oligodendrocyte progenitors. Scale bar in A = 50 Am.

the entire length of an axon is never visible in any given section. It is therefore not possible to determine with confidence whether visible axon segments belong to the same or different fibers. Instead of attempting to count the number of labeled fibers per section, we instead quantified

the percentage of sections containing labeled fibers in order to provide an estimate of the extent of 5-HT axonal sprouting. This value was determined by dividing the number of sections that contained 5-HT-positive axons within the scar by the total number of sections examined.

Fig. 4. Identity of cells expressing NG2 in the transected spinal cord. Several double labeling schemes were used to identify cell types that express NG2 2 weeks after spinal cord transection. (A–C) NG2 (green) + GFAP (red) immunostaining. (A) NG2 only; (B) GFAP only; (C) merged. Asterisks (B) denote GFAP ( ) NG2 (+) connective tissue making up the scar. Note GFAP-positive projections into the scar tissue from rostral and caudal stumps. Rostral lies to the right, caudal to the left. Scale bar in A = 50 Am. (D) NG2 (green) + CD31 (red) immunostaining of section neighboring the one in A–C; inset in C shows approximate location of photo. Arrows indicate pericyte investment of newly-formed blood vessels within the scar tissue. Scale bar = 50 Am. (E) NG2 (green) + F4/80 (red) immunostaining of section neighboring the one in A–C and different from D; inset in C shows approximate location of photo. Arrows point to NG2 (+) F4/80 ( ) oligodendrocyte progenitor cells. Arrowheads point to NG2 (+) F4/80 (+) macrophages [other macrophages are NG2 ( )]. Scale bar = 50 Am. (F) NG2 (red) + PDGF aR (green) immunostaining at a site distant (inset in A) from injury. Co-labeling for PDGF aR confirms these NG2-positive cells as oligodendrocyte progenitors. Scale bar = 50 Am.

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Results Characteristics of the transection scar Immediately after spinal cord transection, the cord stumps retract, leaving a space separating the rostral and caudal elements. In a series of events somewhat unique to the mouse, this space is initially filled by a blood clot, which is replaced over a period of weeks by a bridge of collagenous connective tissue (Fujiki et al., 1996; Inman and Steward, 2003; Ma et al., 2001; Zhang et al., 1996). One of our original goals was to compare the size and extent of this bridging tissue in wild type and NG2 knockout mice as a means of determining the possible importance of the NG2 proteoglycan in formation of the transection scar. However, the variability of the bridging tissue did not allow a rigorous comparison of this type. We were only able to conclude that transection scars exhibit two general types of morphology, as shown in Fig. 1. Panels A (knockout) and C (wild type) are illustrative of cases in which the rostral and caudal stumps of the spinal cord remain in close proximity. In these cases, the transection scar is a compact structure whose bgrainQ runs perpendicular to that of the spinal cord. Panels B (knockout) and D (wild type) are representative of cases in which the cord stumps have undergone more extensive retraction. In these cases, the scar is an extended, constricted structure whose bgrainQ runs parallel to that of the cord. The figure indicates that the presence or absence of NG2 does not appear to be a factor in determining scar morphology, since both compressed and extended scars were observed in each of the two genotypes (NG2+/+ and NG2 / ). Mechanical forces encountered during surgery and subsequent recovery would seem to be a more likely factor in determining the extent of stump retraction and therefore the morphological characteristics of the bridging tissue. Despite their different morphologies, the two types of scars exhibit similarities in composition. The Masson– Trichrome staining of wild type and NG2 null specimens (Fig. 1) shows that in both compressed and extended scars, the bridging tissue is continuous with the meningeal lining of the uninjured spinal cord. Like the meninges, the bridging tissue is immunoreactive for PDGF aR in both scar morphologies (shown in Fig. 2A for a compressed scar in the wild type mouse and Fig. 2B for an extended scar in the NG2 null mouse). This structure completely separates the rostral and caudal stumps of the spinal cord, which are characterized by the presence of dense networks of GFAPpositive processes (Figs. 2A, B). Cavities within the GFAPrich regions also contain PDGF aR-positive connective tissue (asterisks in Fig. 2A). In turn, GFAP-positive processes extend into PDGF aR-positive areas in both types of scars (arrowheads in Fig. 2B). GFAP and PDGF aR labeling of compressed scars in null mice and extended scars in wild type mice were indistinguishable from the

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examples shown in Fig. 2 (data not shown). These histochemical similarities reinforce the notion that differences in scar morphology have a mechanical rather than biochemical origin. Expression of NG2 after spinal cord transection In wild type mice with spinal cord lesions, we observed up-regulation of NG2 expression at the injury site within a few days after the injury. NG2 expression in the lesion site is maximal at 2 weeks and decreases in intensity by 6 weeks (data not shown). At 1 week post-transection, the pattern of NG2 staining at the injury site is quite different from that seen in the uninjured portion of the cord at a distance from the lesion (Figs. 3A, B). Away from the lesion, NG2 expression is seen on cell bodies and processes (Fig. 3B). Within and around the lesion, the NG2 staining pattern is more complex. Near the lesion site, cellular staining can still be observed, but staining within the lesion is more amorphous in nature (Fig. 3A). As expected, in the NG2 null mouse, labeling for NG2 is absent from both injured and uninjured areas of the spinal cord (Figs. 3C, D). The relationship between NG2 labeling and the GFAPpositive borders of the transected spinal cord are shown in Figs. 4A–C. The amorphous pattern of heavy NG2 staining at the injury site (indicated by asterisks in Fig. 4B) suggests the presence of significant quantities of non-cell associated proteoglycan, as observed in the injured rat spinal cord (Jones et al., 2002). As also described for the rat model of spinal cord injury (Jones et al., 2002), macrophages (Fig. 4E) and oligodendrocyte progenitors (Fig. 4F) both contribute to the expression of NG2 in the mouse model. In addition, NG2 is expressed by perivascular cells associated with blood vessels growing into the scar tissue (Fig. 4D). All three of these diverse cells types were also found at the injury site in NG2 null mice, albeit without cell surface NG2 (data not shown). At 6 weeks post-injury, high levels of cellassociated NG2 were still observed at the lesion sites of wild type mice, but the level of non-cellular NG2 was much decreased (data not shown). This may be due to degradation of the proteoglycan by proteinases such as MMP-9 (Larsen et al., 2003). Enhanced scar penetration by serotonergic efferents in the presence of NG2 In the mouse spinal cord, the primary sources of serotonergic projections are the raphe nuclei in the brainstem. Previous studies have found that serotonin immunoreactivity virtually disappears from the caudal spinal cord after transection (in rabbits: Carlsson et al., 1963; in rats: Lu et al., 2002) or compression (in mice; Inman and Steward, 2003). Our immunohistochemical analyses confirm these observations at 8 weeks post-transection in both wild type and NG2 knockout mice. Fig. 5B illustrates the presence of serotonergic fibers interspersed among GFAP-positive processes in the

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wild type spinal cord stump rostral to the transection scar. Caudal to the scar, no serotonergic fibers are evident (Fig. 5A). In both genotypes (NG2+/+ and / ) of animals, we observed serotonergic fibers sprouting into the scar beyond GFAP-positive areas of the rostral stump (arrows in Fig. 5C). Distances covered by these sprouts into the scar were less than 50 Am, never approaching the midline of the lesion. As a means of quantifying penetration of serotonergic fibers into the scar, we serially sectioned the injured spinal cord and determined the percentage of sections in which fibers penetrated into the scar (see Materials and methods). Because of their different morphologies/geometries, results

from compressed and extended scars were initially compiled separately. Table 1 shows that 58% of sections from compressed scars in wild type mice contained serotonergic fibers, while only 31.5% of the corresponding sections from NG2 null mice contained such fibers. Similar results were seen in extended scars (62% in wild types versus 30% in knockouts). Thus, serotonergic axon sprouting was roughly twice as common within transection scars in wild type mice than in corresponding scars in NG2 knockouts (t test for the entire data set, P = 0.002, n = 3 animals of each genotype), suggesting that the presence of NG2 in the environment is conducive to the sprouting of these axons.

Fig. 5. Serotonergic fibers sprout into the scar. (A–C) 5-HT (green) + GFAP (red) staining of NG2 (+/+) spinal cord at 8 weeks post-transection. (A) Caudal to injury site. (B) Rostral to injury site. 5-HT fibers persist within GFAP (+) areas of the rostral stump whereas virtually all 5-HT staining has disappeared from caudal areas. (C) Higher magnification rostral to injury site (different section from A and B). Stacked confocal images show 5-HT (+) fibers (arrows) growing beyond GFAP (+) areas. Scale bar in C = 50 Am.

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Table 1 5-HT and CGRP axon growth into the scar Animal ID#

NG2 phenotype

Scar morphology

5-HT (+)/total sections examined

5-HT (+) %

1019 1020 1076 1062 1069 1070

wt wt wt ko ko ko

comp comp ext comp comp ext

28/46 22/40 29/47 14/38 10/38 14/46

61 55 62 37 26 30

Ave % 60*

31*

CGRP (+)/total sections examined

CGRP (+) %

4/4 4/4 4/4 2/4 2/4 4/4

100 100 100 50 50 100

Comp = compressed, ext = extended. (+) Axons located within the connective tissue scar (delineated with GFAP staining). * For 5-HT data set, P = 0.002 (wt vs. ko).

Scar penetration by CGRP-positive primary sensory afferents In transected spinal cords, CGRP-positive fibers were observed both rostral and caudal to the injury site, most likely emanating from intact afferents above and below the

lesion. More interestingly, CGRP-positive fibers were also observed within the transection scar (Fig. 6C, NG2 null specimen; Fig. 6D, wild type specimen), suggestive of sprouting into this connective tissue from rostral and caudal afferents. Previous studies have documented sprouting from neighboring afferents presumably to innervate spinal neu-

Fig. 6. CGRP fibers penetrate deeply within the scar. (A and B) CGRP immunostaining of NG2 ( / ) spinal cord at 8 weeks post-transection shows highly arborized and varicose fibers (arrows) rostral (A) and caudal (B) to the injury site. Scale bar in A = 50 Am. (C) Merged CGRP (green) + GFAP (red) immunostaining of NG2 ( / ) spinal cord at 8 weeks post-transection; inset shows the location of the image with respect to the rostral stump. CGRP (+) fibers found near the midline of the scar (arrows) follow the grain, are linear, and lack varicosities. (D) Merged CGRP + GFAP immunostaining of NG2 (+/+) spinal cord at 8 weeks post-transection. Arrows point to CGRP fibers emanating from the caudal stump, following the grain of the scar tissue.

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rons that have lost their input after spinal cord injury (Christensen and Hulsebosch, 1997; Krenz and Weaver, 1998, Krenz et al., 1999). Here, we report that such sprouting extends to the non-neural scar tissue. Compared to serotonergic fibers, these CGRP-positive axons projected for longer distances within the scar, and were frequently observed at the midline of the lesion more than 500 Am from the scar boundary. Mature CGRP fibers within normal nervous tissue (Figs. 6A and B) are highly arborized and exhibit numerous varicosities characteristic of synapse formation (Merighi et al., 1989, 1991). In contrast, fibers in the scar follow the bgrainQ of the scar, are linear, and seem to lack varicosities, consistent with their nascent ontogeny (Figs. 6C and D). CGRP-positive fibers were observed in transection scars in both wild type and NG2 knockout animals. With the limited number of specimens remaining after the analysis of serotonergic sprouting, we were able to show in wild type animals that 8 out of 8 sections from compressed scars and 4 out of 4 sections from extended scars contained CGRPpositive fibers (Table 1). In NG2 null animals, CGRPpositive fibers were present in 4 of 8 sections in compressed scars and 4 of 4 sections in extended scars. While the small number of specimens does not permit rigorous statistical comparison of the results, the data do not support the idea that NG2 might be inhibitory to regeneration of these axons. If anything, CGRP-positive fibers would appear to be similar to serotonergic fibers, exhibiting more frequent scar penetration in the presence of NG2.

Discussion It has been previously reported that axons are able to grow into the scar that forms after severing the mouse spinal cord (Zhang et al., 1996). A key result of our studies is the demonstration that distinct classes of axons differ in their ability to penetrate this scar tissue. Serotonergic and CGRP-containing axons both exhibit the ability to grow into the transection scar, albeit with different degrees of effectiveness. Although differing in several specific details with earlier reports, our results are in general agreement with the concept that spinal axons can behave differently following injury (Inman and Steward, 2003). In light of the fact that neurons and their axons express cell adhesion molecules and cell surface receptors for chemoattractants and extracellular matrix components in a cell type-specific manner, the differences in their abilities to penetrate the spinal cord transection scar should probably not be surprising. Serotonergic fibers were previously reported to be associated with astrocytic processes penetrating the margins of the transection scar (Inman and Steward, 2003). Our results suggest that these neurons can penetrate somewhat more deeply into the lesion, clearly beyond the boundary marked by GFAP-positive astrocytes. These fibers therefore

have at least a limited ability to grow beyond the rostral spinal cord stump into the connective tissue of the lesion itself. CGRP-positive axons projected even more deeply into the transection scar, often reaching the midline of the lesion. These observations highlight differences in the ability of various classes of axons to sprout into the scar that forms after spinal cord transection. Our results with transection lesions contrast somewhat with a previous study on compression injuries (Inman and Steward, 2003). These workers found that CGRP-positive processes were not among the species of sensory axons capable of penetrating a compression lesion. These types of differences in the histological outcomes of compression versus transection injuries to the mouse spinal cord have been noted previously (Jacob et al., 2003), possibly stemming from biochemical and morphological differences between the two types of scars. One of our goals was to assess the possible contribution of the NG2 proteoglycan in making the transection scar inhibitory to axon regeneration. This has been done by comparing scar formation and axon regeneration in wild type and NG2 knockout mice. In wild type mice, the lesion site is filled with NG2 immunoreactive material, reaching a maximum at 2 weeks post-transection. Levels of diffusely distributed NG2, probably representing proteoglycan that has been proteolytically shed from cell surfaces (Jones et al., 2002), decline over the ensuing weeks. In contrast, NG2 continues to be expressed on the surfaces of oligodendrocyte progenitors, macrophages, and microvascular pericytes in and around the lesion. These three cell types are also associated with the lesions in NG2 null mice, albeit without the proteoglycan on their surfaces. The proteoglycan therefore does not appear to influence the participation of these cells types in the response to injury. The presence or absence of NG2 also appears to have little effect on the general morphological characteristics of the transection scar. Compressed and extended scars occurred in both genotypes. Also, in both wild type and NG2 null mice, the lesion was bordered by GFAP-positive astrocytes and was filled by connective tissue that appeared to be continuous with (and possibly derived from) the PDGF aR-positive meninges. On the basis of previous reports of the ability of NG2 to inhibit neurite outgrowth (Chen et al., 2002a,b; Dou and Levine, 1994, 1999; Fidler et al., 1999; Ughrin et al., 2003), we envisioned that axon regeneration into the transection scar might be improved in the NG2 null mouse. However, this was not the case. CGRP-positive fibers grew deeply into transection scars in both wild type and knockout mice. Most intriguing was the significant decrease in serotonergic fiber penetration into transection scars in the NG2 null mouse, implying that NG2 could play a facilitatory role in the regeneration of these fibers. Although nerve regeneration has not been previously studied in the NG2 null mouse, in vivo nerve injury studies in wild type mice have already provided some indications that NG2 is not necessarily inhibitory to nerve growth.

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Despite the fact that dramatic up-regulation of NG2 expression is always seen in nerve injury models (Jones et al., 2002; Keirstead et al., 1998; Levine, 1994; McTigue et al., 2001; Watanabe et al., 2002; Zhang et al., 2001), the proteoglycan appears to have little effect on the growth patterns of some types of axons. Regenerating sciatic nerves readily grow through an NG2-rich environment without evidence of inhibition (Rezajooi et al., 2004). In the injured spinal cord, some classes of axons, including CGRPpositive fibers and NGF-responsive projections, preferentially associate with NG2-rich substrata within the lesion (Jones et al., 2003). This is consistent with our observation that serotonergic axons are more abundant in the transection scars of wild type mice than NG2 null mice. It has been suggested that the ability of axons to penetrate a particular environment reflects the balance between stimulatory and repulsive cues (Jones et al., 2003). Thus, regions that are rich in NG2 may also be relatively richer in L1 and laminin, positive cues for axon growth that could overcome any inhibitory effects of NG2. This idea may be compatible with observed differences in the ability of mixed substrata (NG2 + L1, NG2 + laminin) to support neurite outgrowth from different types of neurons in vitro (Dou and Levine, 1994; Schneider et al., 2001). One caveat to our conclusions regards the possible existence of differences between wild type and NG2 null mice prior to spinal cord injury. If the wild type and knockout animals were to have pre-existing discrepancies in the number and/or properties of 5-HT or CGRP tract axons, then post-injury responses could be the result of those differences. Without extensive characterization of the two lines of mice, we cannot rule out this possibility. However, it seems unlikely due to the fact that NG2 is not expressed by neurons, but instead is found on oligodendrocyte progenitors (Nishiyama et al., 1996a,b). Since 5-HT and CGRP tracts are unmyelinated, there is little reason to suppose that their development would be affected by the absence of NG2. In summary, we suggest that specific classes of axons differ in their ability to regenerate into the scar that forms in response to spinal cord transection. Although in wild type mice NG2 expression is greatly increased in and around the scar, our studies comparing wild type and NG2 null mice do not provide evidence to support an inhibitory role for NG2 in the regeneration of two specific classes of spinal axons. Neither do our data absolutely rule out an inhibitory function for NG2 in the regeneration of other axon types. The take-home lesson from these studies is that the response of distinct neuronal cell types to injuries and putative inhibitory molecules (NG2 in this case) needs to be examined on a case-by-case basis using both in vitro and in vivo models. While in vitro models are limited by their failure to mimic faithfully all characteristics of the normal environment, in vivo models also suffer from their inherent complexity and variability. A combination of strategies is therefore warranted in order to maximize the chance of drawing valid conclusions.

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Acknowledgments This work was supported by NIH grant PO1 HD25938. We are very grateful to Robin Newlin and Jennifer Freund for technical assistance with histology and imaging, and to Jun-ichi Fukushi and Adriana Charbono for help with animal surgery and rehabilitation.

References Bradbury, E.J., Moon, L.D., Popat, R.J., King, V.R., Bennett, G.S., Patel, P.N., Fawcett, J.W., McMahon, S.B., 2002. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416 (6881), 636 – 640 (Apr. 11). Brittis, P.A., Canning, D.R., Silver, J., 1992. Chondroitin sulfate as a regulator of neuronal patterning in the retina. Science 255 (5045), 733 – 736 (Feb. 7). Bunge, R.P., Puckett, W.R., Hiester, E.D., 1997. Observations on the pathology of several types of human spinal cord injury, with emphasis on the astrocyte response to penetrating injuries. Adv. Neurol. 72, 305 – 315. Carlsson, A., Magnusson, T., Rosengren, E., 1963. 5-hydroxytryptamine of the spinal cord normally and after transection. Experientia 19, 359 (July 15). Chen, M.S., Huber, A.B., van der Haar, M.E., Frank, M., Schnell, L., Spillmann, A.A., Christ, F., Schwab, M.E., 2000. Nogo-A is a myelinassociated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature 403 (6768), 434 – 439 (Jan 27). Chen, Z.J., Negra, M., Levine, A., Ughrin, Y., Levine, J.M., 2002a. Oligodendrocyte precursor cells: reactive cells that inhibit axon growth and regeneration. J. Neurocytol. 31 (6–7), 481 – 495 (Jul.–Aug.). Chen, Z.J., Ughrin, Y., Levine, J.M., 2002b. Inhibition of axon growth by oligodendrocyte precursor cells. Mol. Cell. Neurosci. 20 (1), 125 – 139 (May). Christensen, M.D., Hulsebosch, C.E., 1997. Spinal cord injury and antiNGF treatment results in changes in CGRP density and distribution in the dorsal horn in the rat. Exp. Neurol. 147 (2), 463 – 475 (Oct.). Davies, S.J., Fitch, M.T., Memberg, S.P., Hall, A.K., Raisman, G., Silver, J., 1997. Regeneration of adult axons in white matter tracts of the central nervous system. Nature 390 (6661), 680 – 683 (Dec. 18–25). De Winter, F., Holtmaat, A.J., Verhaagen, J., 2002. Neuropilin and class 3 semaphorins in nervous system regeneration. Adv. Exp. Med. Biol. 515, 115 – 139. Dou, C.L., Levine, J.M., 1994. Inhibition of neurite growth by the NG2 chondroitin sulfate proteoglycan. J. Neurosci. 14 (12), 7616 – 7628 (Dec.). Dou, C.L., Levine, J.M., 1997. Identification of a neuronal cell surface receptor for a growth inhibitory chondroitin sulfate proteoglycan (NG2). J. Neurochem. 68 (3), 1021 – 1030 (Mar.). Fawcett, J.W., Asher, R.A., 1999. The glial scar and central nervous system repair. Brain Res. Bull. 49 (6), 377 – 391 (Aug.). Fidler, P.S., Schuette, K., Asher, R.A., Dobbertin, A., Thornton, S.R., Calle-Patino, Y., Muir, E., Levine, J.M., Geller, H.M., Rogers, J.H., Faissner, A., Fawcett, J.W., 1999. Comparing astrocytic cell lines that are inhibitory or permissive for axon growth: the major axoninhibitory proteoglycan is NG2. J. Neurosci. 19 (20), 8778 – 8788 (Oct. 15). Filbin, M.T., 1995. Myelin-associated glycoprotein: a role in myelination and in the inhibition of axonal regeneration? Curr. Opin. Neurobiol. 5 (5), 588 – 595 (Oct.). Fitch, M.T., Silver, J., 1997. Glial cell extracellular matrix: boundaries for axon growth in development and regeneration. Cell Tissue Res. 290 (2), 379 – 384 (Nov.). Fouad, K., Dietz, V., Schwab, M.E., 2001. Improving axonal growth and

308

R. de Castro Jr. et al. / Experimental Neurology 192 (2005) 299–309

functional recovery after experimental spinal cord injury by neutralizing myelin associated inhibitors. Brain Res. Brain Res. Rev. 36 (2–3), 204 – 212 (Oct.). Fujiki, M., Zhang, Z., Guth, L., Steward, O., 1996. Genetic influences on cellular reactions to spinal cord injury: activation of macrophages/ microglia and astrocytes is delayed in mice carrying a mutation (WldS) that causes delayed Wallerian degeneration. J. Comp. Neurol. 371 (3), 469 – 484 (Jul. 29). Grako, K.A., Ochiya, T., Barritt, D., Nishiyama, A., Stallcup, W.B., 1999. PDGF (alpha)-receptor is unresponsive to PDGF-AA in aortic smooth muscle cells from the NG2 knockout mouse. J. Cell Sci. 112 (Pt. 6), 905 – 915 (Mar.). GrandPre, T., Nakamura, F., Vartanian, T., Strittmatter, S.M., 2000. Identification of the Nogo inhibitor of axon regeneration as a reticulon protein. Nature 403 (6768), 439 – 444 (Jan. 27). Inman, D.M., Steward, O., 2003. Ascending sensory, but not other longtract axons, regenerate into the connective tissue matrix that forms at the site of a spinal cord injury in mice. J. Comp. Neurol. 462 (4), 431 – 449 (Aug. 4). Jacob, J.E., Gris, P., Fehlings, M.G., Weaver, L.C., Brown, A., 2003. Autonomic dysreflexia after spinal cord transection or compression in 129 Sv, C57BL, and Wallerian degeneration slow mutant mice. Exp. Neurol. 183 (1), 136 – 146 (Sep.). Jones, L.L., Yamaguchi, Y., Stallcup, W.B., Tuszynski, M.H., 2002. NG2 is a major chondroitin sulfate proteoglycan produced after spinal cord injury and is expressed by macrophages and oligodendrocyte progenitors. J. Neurosci. 22 (7), 2792 – 2803 (Apr. 1). Jones, L.L., Sajed, D., Tuszynski, M.H., 2003. Axonal regeneration through regions of chondroitin sulfate proteoglycan deposition after spinal cord injury: a balance of permissiveness and inhibition. J. Neurosci. 23 (28), 9276 – 9288 (Oct. 15). Keirstead, H.S., Levine, J.M., Blakemore, W.F., 1998. Response of the oligodendrocyte progenitor cell population (defined by NG2 labelling) to demyelination of the adult spinal cord. Glia 22 (2), 161 – 170 (Feb.). Krenz, N.R., Weaver, L.C., 1998. Sprouting of primary afferent fibers after spinal cord transection in the rat. Neuroscience 85 (2), 443 – 458 (Jul.). Krenz, N.R., Meakin, S.O., Krassioukov, A.V., Weaver, L.C., 1999. Neutralizing intraspinal nerve growth factor blocks autonomic dysreflexia caused by spinal cord injury. J. Neurosci. 19 (17), 7405 – 7414 (Sep. 1). Larsen, P.H., Wells, J.E., Stallcup, W.B., Opdenakker, G., Yong, V.W., 2003. Matrix metalloproteinase-9 facilitates remyelination in part by processing the inhibitory NG2 proteoglycan. J. Neurosci. 23 (35), 11127 – 11135 (Dec. 3). Lemons, M.L., Howland, D.R., Anderson, D.K., 1999. Chondroitin sulfate proteoglycan immunoreactivity increases following spinal cord injury and transplantation. Exp. Neurol. 160 (1), 51 – 65 (Nov.). Levine, J.M., 1994. Increased expression of the NG2 chondroitin-sulfate proteoglycan after brain injury. J. Neurosci. 14 (8), 4716 – 4730 (Aug.). Lu, J., Feron, F., Mackay-Sim, A., Waite, P.M., 2002. Olfactory ensheathing cells promote locomotor recovery after delayed transplantation into transected spinal cord. Brain 125 (Pt. 1), 14 – 21 (Jan.). Ma, M., Basso, D.M., Walters, P., Stokes, B.T., Jakeman, L.B., 2001. Behavioral and histological outcomes following graded spinal cord contusion injury in the C57Bl/6 mouse. Exp. Neurol. 169 (2), 239 – 254 (Jun.). McKeon, R.J., Jurynec, M.J., Buck, C.R., 1999. The chondroitin sulfate proteoglycans neurocan and phosphacan are expressed by reactive astrocytes in the chronic CNS glial scar. J. Neurosci. 19 (24), 10778 – 10788 (Dec. 15). McKerracher, L., David, S., Jackson, D.L., Kottis, V., Dunn, R.J., Braun, P.E., 1994. Identification of myelin-associated glycoprotein as a major myelin-derived inhibitor of neurite growth. Neuron 13 (4), 805 – 811 (Oct.). McTigue, D.M., Wei, P., Stokes, B.T., 2001. Proliferation of NG2-positive

cells and altered oligodendrocyte numbers in the contused rat spinal cord. J. Neurosci. 21 (10), 3392 – 3400 (May 15). Merighi, A., Polak, J.M., Fumagalli, G., Theodosis, D.T., 1989. Ultrastructural localization of neuropeptides and GABA in rat dorsal horn: a comparison of different immunogold labeling techniques. J. Histochem. Cytochem. 37 (4), 529 – 540 (Apr.). Merighi, A., Polak, J.M., Theodosis, D.T., 1991. Ultrastructural visualization of glutamate and aspartate immunoreactivities in the rat dorsal horn, with special reference to the co-localization of glutamate, substance P and calcitonin-gene related peptide. Neuroscience 40 (1), 67 – 80. Moon, L.D., Asher, R.A., Rhodes, K.E., Fawcett, J.W., 2001. Regeneration of CNS axons back to their target following treatment of adult rat brain with chondroitinase ABC. Nat. Neurosci. 4 (5), 465 – 466 (May). Nishiyama, A., Lin, X.H., Giese, N., Heldin, C.H., Stallcup, W.B., 1996. Co-localization of NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells in the developing rat brain. J. Neurosci. Res. 43 (3), 299 – 314 (Feb. 1). Nishiyama, A., Lin, X.H., Giese, N., Heldin, C.H., Stallcup, W.B., 1996. Interaction between NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells is required for optimal response to PDGF. J. Neurosci. Res. 43 (3), 315 – 330 (Feb. 1). Novikova, L.N., Novikov, L.N., Kellerth, J.O., 2002. Differential effects of neurotrophins on neuronal survival and axonal regeneration after spinal cord injury in adult rats. J. Comp. Neurol. 452 (3), 255 – 263 (Oct. 21). Ozerdem, U., Grako, K.A., Dahlin-Huppe, K., Monosov, E., Stallcup, W.B., 2001. NG2 proteoglycan is expressed exclusively by mural cells during vascular morphogenesis. Dev. Dyn. 222 (2), 218 – 227 (Oct.). Ozerdem, U., Monosov, E., Stallcup, W.B., 2002. NG2 proteoglycan expression by pericytes in pathological microvasculature. Microvasc. Res. 63 (1), 129 – 134 (Jan.). Pasterkamp, R.J., Verhaagen, J., 2001. Emerging roles for semaphorins in neural regeneration. Brain Res. Brain Res. Rev. 35 (1), 36 – 54. (Mar.). Pataky, D.M., Borisoff, J.F., Fernandes, K.J., Tetzlaff, W., Steeves, J.D., 2000. Fibroblast growth factor treatment produces differential effects on survival and neurite outgrowth from identified bulbospinal neurons in vitro. Exp. Neurol. 163 (2), 357 – 372 (Jun.). Rezajooi, K., Pavlides, M., Winterbottom, J., Stallcup, W.B., Hamlyn, P.J., Liebermann, A.R., Anderson, P.N., 2004. NG2 proteoglycan expression in the peripheral nervous system: upregulation following injury and comparison with CNS lesions. Mol. Cell. Neurosci. 25 (4), 572 – 584. Schneider, S., Bosse, F., D’Urso, D., Muller, H., Sereda, M.W., Nave, K., Niehaus, A., Kempf, T., Schnolzer, M., Trotter, J., 2001. The AN2 protein is a novel marker for the Schwann cell lineage expressed by immature and nonmyelinating Schwann cells. J. Neurosci. 21 (3), 920 – 933 (Feb. 1). Snow, D.M., Lemmon, V., Carrino, D.A., Caplan, A.I., Silver, J., 1990. Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro. Exp. Neurol. 109 (1), 111 – 130 (Jul.). Snow, D.M., Watanabe, M., Letourneau, P.C., Silver, J., 1991. A chondroitin sulfate proteoglycan may influence the direction of retinal ganglion cell outgrowth. Development 113 (4), 1473 – 1485 (Dec.). Tetzlaff, W., Kobayashi, N.R., Giehl, K.M., Tsui, B.J., Cassar, S.L., Bedard, A.M., 1994. Response of rubrospinal and corticospinal neurons to injury and neurotrophins. Prog. Brain Res. 103, 271 – 286. Ughrin, Y.M., Chen, Z.J., Levine, J.M., 2003. Multiple regions of the NG2 proteoglycan inhibit neurite growth and induce growth cone collapse. J. Neurosci. 23 (1), 175 – 186 (Jan. 1). Watanabe, M., Toyama, Y., Nishiyama, A., 2002. Differentiation of proliferated NG2-positive glial progenitor cells in a remyelinating lesion. J. Neurosci. Res. 69 (6), 826 – 836 (Sep. 15). Woolf, C.J., 2003. No Nogo: now where to go? Neuron 38 (2), 153 – 156. (Apr. 24). Yamaguchi, Y., 2000. Lecticans: organizers of the brain extracellular matrix. Cell. Mol. Life Sci. 57 (2), 276 – 289 (Feb.).

R. de Castro Jr. et al. / Experimental Neurology 192 (2005) 299–309 Zhang, Z., Fujiki, M., Guth, L., Steward, O., 1996. Genetic influences on cellular reactions to spinal cord injury: a wound-healing response present in normal mice is impaired in mice carrying a mutation (WldS) that causes delayed Wallerian degeneration. J. Comp. Neurol. 371 (3), 485 – 495 (Jul. 29).

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Zhang, Y., Tohyama, K., Winterbottom, J.K., Haque, N.S., Schachner, M., Lieberman, A.R., Anderson, P.N., 2001. Correlation between putative inhibitory molecules at the dorsal root entry zone and failure of dorsal root axonal regeneration. Mol. Cell. Neurosci. 17 (3), 444 – 459 (Mar.).