NG2 and phosphacan are present in the astroglial scar after human traumatic spinal cord injury
© Buss et al; licensee BioMed Central Ltd. 2009
Received: 26 November 2008
Accepted: 15 July 2009
Published: 15 July 2009
A major class of axon growth-repulsive molecules associated with CNS scar tissue is the family of chondroitin sulphate proteoglycans (CSPGs). Experimental spinal cord injury (SCI) has demonstrated rapid re-expression of CSPGs at and around the lesion site. The pharmacological digestion of CSPGs in such lesion models results in substantially enhanced axonal regeneration and a significant functional recovery. The potential therapeutic relevance of interfering with CSPG expression or function following experimental injuries seems clear, however, the spatio-temporal pattern of expression of individual members of the CSPG family following human spinal cord injury is only poorly defined. In the present correlative investigation, the expression pattern of CSPG family members NG2, neurocan, versican and phosphacan was studied in the human spinal cord.
An immunohistochemical investigation in post mortem samples of control and lesioned human spinal cords was performed. All patients with traumatic SCI had been clinically diagnosed as having "complete" injuries and presented lesions of the maceration type.
In sections from control spinal cord, NG2 immunoreactivity was restricted to stellate-shaped cells corresponding to oligodendrocyte precursor cells. The distribution patterns of phosphacan, neurocan and versican in control human spinal cord parenchyma were similar, with a fine reticular pattern being observed in white matter (but also located in gray matter for phosphacan). Neurocan staining was also associated with blood vessel walls. Furthermore, phosphacan, neurocan and versican were present in the myelin sheaths of ventral and dorsal nerve roots axons. After human SCI, NG2 and phosphacan were both detected in the evolving astroglial scar. Neurocan and versican were detected exclusively in the lesion epicentre, being associated with infiltrating Schwann cells in the myelin sheaths of invading peripheral nerve fibres from lesioned dorsal roots.
NG2 and phosphacan were both present in the evolving astroglial scar and, therefore, might play an important role in the blockade of successful CNS regeneration. Neurocan and versican, however, were located at the lesion epicentre, associated with Schwann cell myelin on regenerating peripheral nerve fibres, a distribution that was unlikely to contribute to failed CNS axon regeneration. The present data points to the importance of such correlative investigations for demonstrating the clinical relevance of experimental data.
The loss of function following human traumatic spinal cord injury (SCI) is often permanent and results in a serious limitation to the patients' quality of life. Despite considerable progress in recent years, the underlying mechanisms responsible for the failure of axonal regeneration after SCI remain only partially understood.
At the spinal cord lesion site, the initial damage to the parenchyma is followed by a complex cascade of secondary events including breakdown of the blood brain barrier (BBB), infiltration of blood-derived inflammatory cells, oedema, excitotoxicity and ischemia. This early phase of secondary parenchymal damage is followed by the removal of tissue debris, resulting in fluid filled cystic cavitation and the deposition of extracellular matrix (ECM) proteins at the lesion epicentre . The surrounding scar is largely composed of astrocytes and a dense, irregular network of their processes. Traumatic injuries which include damage to spinal nerve roots and the meninges also induce fibroblast, meningeal cell and Schwann cell invasion into the lesion site [2–4]. All these cell populations contribute to the production of a dense ECM that presents itself as a molecular barrier to axonal regeneration [1, 3, 5].
A major class of growth-inhibitory molecules associated with CNS scar tissue is the family of chondroitin sulphate proteoglycans (CSPGs). CSPGs are highly sulphated molecules that consist primarily of one of six possible core proteins, each of which is coated, to varying extents with glycosaminoglycan (GAG) side chains. The CSPGs are expressed throughout the developing CNS, where their axon growth-repulsive properties are believed to play an important role in determining nerve fibre pattern formation. In adulthood, expression levels are generally much lower. The use of broad specificity antibodies in a number of experimental lesion paradigms in adult animals, including cortical injury, nigro-striatal axotomy and SCI, has demonstrated the elevated expression of CSPGs [6, 7]. Pharmacological interventions involving the enzymatic degradation of CSPG-GAG side chains have demonstrated a substantially enhanced axonal regeneration and functional recovery following spinal cord injury .
Experimental investigations into the role of individual CSPG family members have revealed distinct expression patterns and functions in the traumatically injured CNS. NG2 as an integral membrane proteoglycan shares no homology to other proteins and is present on the surface of oligodendrocyte precursors in vitro  and in the developing and adult rodent CNS [10, 11]. Correlative investigations in human post mortem tissue have demonstrated a similar oligodendrocyte precursor distribution in the adult human CNS . NG2 has been reported to undergo a strong up-regulation at the lesion site after experimental traumatic SCI, being detected on oligodendrocyte precursors and invading macrophages [13, 14]. Furthermore, treatment with antibodies specifically neutralizing the NG2 proteoglycan has revealed significantly increased axon regeneration following experimental SCI .
Neurocan belongs to the lecticans, a sub-division of the CSPG family that is characterized by a hyaluron-binding domain and a C-type lectin domain, both of which enable these molecules to interact with ECM proteins such as tenascin-R . Neurocan is a nervous system-specific CSPG  and both in vitro and in vivo studies point to an axon growth-repulsive role [18, 19]. Its presence in regions of active fibre growth during development suggests that neurocan delineates boundaries of neuronal outgrowth and that it may be important for neuronal pattern formation [20, 21]. In various CNS lesion paradigms, including SCI, both transient , and long term  re-expressions of neurocan by astrocytes have been reported.
A second member of the lectican sub-family is versican. This CSPG is also developmentally regulated, being expressed in white matter tracts around the time of myelination . This distribution of versican in the white matter has also been described by others, and an axon growth-inhibitory role suggested . Furthermore, in vivo investigations have demonstrated an early up-regulation of this CSPG following experimental lesions, the distribution of which was compatible with a regeneration-blocking effect [22, 26]. Cells of the oligodendrocytic lineage were reported to be responsible for the post-traumatic up-regulation of versican following spinal lesions.
Phosphacan is an alternatively spliced variant of the receptor-type protein tyrosine phosphatase. It does not belong to the lectican sub-family of CSPGs but is, nevertheless, able to bind to ECM proteins such as N-CAM and tenascin . In the CNS, its expression peaks during development and in the adult the distribution is more restricted. In vitro studies have demonstrated a growth-inhibitory effect of phosphacan on several neuronal populations [28, 29]. In vivo experiments, following brain and spinal cord injury, have demonstrated an initial decrease in phosphacan concentration at the lesion site [22, 23]. However, a marked increase in the expression of phosphacan has been observed in long-term astroglial scars .
In contrast to the detailed functional and spatio-temporal information that has been obtained using small laboratory animals, there is relatively little correlative data on the expression of CSGPs following traumatic human spinal cord injury. An immunohistochemical investigation in traumatically injured post mortem human spinal cord has demonstrated an up-regulation of CSPGs at the lesion site in human traumatic SCI, but this was spatially correlated with the presence of migrating Schwann cells rather than reactive astrocytes . We have therefore performed a more detailed immunohistochemical investigation on the expression pattern of distinct members of the CSPG family in samples of post mortem human spinal cord, taken from patients who died at a range of survival times following severe macerating SCI.
Patients who served as the control group
Cause of death
PM fixation time
Patients who died after traumatic injury to the spinal cord
PM fixation time
Transverse sections (5 μm thick) were collected onto poly-L-lysine-coated slides and allowed to dry. Sections were de-waxed in xylene, rehydrated and unless otherwise stated, were microwaved in 10 mM citrate buffer (pH 6) for 3 × 3 minutes. Sections for neurocan, versican and phosphacan immunohistochemistry were not microwaved; instead they were incubated in 1 U/ml chondroitinase ABC for 2 hours at 37°C. Sections for NG2 were neither microwaved nor treated with chondroitinase ABC. Non-specific binding was blocked by incubation in 0.1 M PBS containing 3% normal goat serum and 0.5% Triton X-100 for 30 minutes. Next, sections were incubated in the primary antibody, overnight at room temperature. The primary antibodies used were: monoclonal mouse anti-human NG2 (clone B5, undiluted cell culture supernatant from ATCC cultures and antibody 9.2.27, diluted 1:50; gift from Prof. R. Reisfeld, Scripps Research Institute), monoclonal mouse anti-neurocan (Chemicon, diluted 1:100), polyclonal rabbit anti-versican (Acris antibodies, diluted 1:1.000), monoclonal mouse anti-phosphacan (Chemicon, diluted 1:500), polyclonal rabbit anti-GFAP (DAKO, diluted 1:2.500), polyclonal rabbit anti-myelin basic protein (MBP) (Chemicon, diluted 1:1.000) and polyclonal rabbit anti-neurofilament (NF, Sigma-Aldrich, diluted 1:2.000).
Following extensive rinsing steps in 0.1 M PBS, sections were incubated in biotinylated horse anti-mouse or anti-rabbit antibody (Vector Laboratories, diluted 1:500) for 1 hour at room temperature. As described earlier, incubation with the biotinylated secondary antibody was followed by the Vector ABC system and a subsequent incubation in diaminobenzidine for visualization of the reaction product. For negative controls the primary antibody was omitted.
For double immunofluorescence, sections were de-waxed in xylene and rehydrated. Treatment with chondroitinase ABC (1 U/ml) for 2 hours at 37°C (for neurocan, versican and phosphacan immunohistochemistry) was followed by blockade of non-specific binding by incubation in 3% normal goat serum and 0.5% triton X-100 in 0.1 M PBS for 30 minutes and subsequent incubation overnight at room temperature in a combination of the following primary antibodies: monoclonal mouse anti-NG2 (clone B5, cell culture supernatant undiluted and 9.2.27, diluted 1:10), monoclonal mouse anti-neurocan (Chemicon, diluted 1:20), polyclonal rabbit anti-versican (diluted 1:20), monoclonal mouse anti-phosphacan (diluted 1:50), monoclonal mouse anti-neurofilament (NF, Sigma, Clone52, diluted 1:100), polyclonal rabbit anti-NF (diluted 1:1,000), polyclonal rabbit anti-GFAP (diluted 1:1.000), polyclonal rabbit anti-MBP (Chemicon, diluted 1:500) and polyclonal rabbit anti-laminin (Sigma, diluted 1:50). After the subsequent incubation with Alexa 594 (red-fluorescence)-conjugated goat anti-mouse and Alexa 488 (green fluorescence)-conjugated goat anti-rabbit secondary antibodies (Molecular Probes, diluted 1:500) for 3 hours at room temperature, slides were cover-slipped in Moviol. For negative controls, the primary antibodies were omitted.
Amount of immunopositive cells at different survival times after human SCI
Distribution of NG2, neurocan, versican and phosphacan in the normal adult human spinal cord
Morphological appearance of the lesion site
The intermediate zone included the extremities of the lesion site and their interface with the adjacent damaged, but non-degenerating, CNS parenchyma. At 24 days after injury, the remaining astrocytes appeared activated and produced a dense, irregular scar. At longer survival times, the intermediate zone showed a clear demarcation between the Schwann cell area and the astroglial scar (Fig. 4C). In most cases, cystic cavitation could also be seen in between the 2 areas (Fig. 4D).
Early survival times (2 to 11 days post insult)
In the less severely affected areas at the border of the lesion epicentre, the density of astrocytic cells and their network of processes was dramatically decreased compared to control cases (not shown). In accordance with the observations at the lesion core, immunohistochemistry for neurocan, versican and phosphacan revealed no immunoreactive structures in the CNS parenchyma. At the PNS-CNS interface, the immunopositive PNS myelin staining for all 3 CSPGs, stopped abruptly (Fig 5B). NG2 staining was found in some round to oval-shaped cells in the intermediate zone. Furthermore, some stellate-shaped cells were NG2 immunopositive, however their numbers were initially reduced by 2–8 days post injury in comparison to control cases but had returned to near normal levels by 10–11 days after SCI (Table 3 and Fig. 5C–D).
Moderate to late survival times (24 days to 30 years post insult)
At 24 days after trauma, Schwann cell migration was detected within the damaged spinal parenchyma in the vicinity of spinal nerve roots. These cells infiltrated up to 900 μm into the spinal cord tissue with a decreasing density towards the more central regions. Furthermore, outside the CNS parenchyma, root-like structures of various sizes could be found around the original nerve roots . These structures, with an often more complex and convoluted appearance in comparison to nerve roots in control tissue demonstrated scattered immunoreactivity for neurocan, versican and phosphacan. At the 24 days survival time, only occasional axons in the neuromas were surrounded by neurocan, versican or phosphacan immunopositive myelin rings (Fig. 5E). NG2 was similar to that seen at 10 and 11 day survival times, with round to oval cells scattered around the lesion core (not shown). No immunoreactivity could be found in the root-like structures.
At 24 days after SCI, the intermediate zone was devoid of infiltrating Schwann cells. Instead, the first signs of astrocytic scar formation were visible . In these regions of gray and white matter, a densely packed mass or network of diffusely stained GFAP-positive processes could be seen (Fig. 5F). NG2 and phosphacan immunohistochemistry revealed a staining pattern very similar to GFAP with a dense, irregular pattern of processes without identifiable cell bodies (Fig. 5G–H). At survival times of 4 months and longer after SCI, the territory of the densely packed GFAP-positive astroglial scar was clearly distinguishable and distinct from that of the Schwann cell dominated lesion core. At these survival times, NG2 staining revealed a dense, mostly diffuse staining pattern in the astrocytic scar. No clearly identifiable immunopositive cell bodies could be found in this area. Phosphacan immunohistochemistry also revealed a similar distribution to that of GFAP-positive astrocytes, also without clearly identifiable cell bodies (Fig. 6G–I). Double immunofluorescence confirmed the nearly identical distribution patterns of both NG2 and phosphacan with that of the reactive GFAP-positive astroglia in this area (Fig. 3H–I). In the astroglial scar, occasional, single thin nerve fibres could be found within the CSPG-rich ECM (Fig. 3J). At no survival time could neurocan- or versican-immunopositive structures be found in the intermediate zone of human SCI (not shown).
Investigations using experimental animals have demonstrated a central role for CSPGs in the lack of regeneration after SCI. Most members of this proteoglycan family have demonstrated up-regulation after spinal injury, being prominent in the astroglial component of the scar [13, 14, 22]. Furthermore, the application of chondroitinase ABC (an enzyme capable of degrading GAG side chains) after a spinal cord contusion injury has resulted in increased nerve fibre outgrowth associated with enhanced functional recovery . The application of NG2-specific antibodies following transection injuries to the rat spinal cord has led to a significant improvement in the regeneration of lesioned dorsal column axons . Despite the clear identification of the re-expression of numerous CSPGs in experimental animal models of traumatic injury and the demonstration, both in vitro and in vivo, of the axon-growth repulsive effect of these molecules, there have been relative few correlative investigations demonstrating the expression of such functionally important molecules in post mortem human nervous tissue following injury. In one such publication, a broad specificity CSPG antibody (CS-56, Sigma) was used to demonstrate that the lesion induced distribution of this proteoglycan family was not associated with the evolving astroglial scar. It was, however, associated with migrating Schwann cells and regenerating peripheral nerve fibres within the lesion epicentre. Such a distribution did not point to a central role for CSPGs in the growth-inhibitory milieu of the glial scar after human SCI .
In the present investigation, the expression pattern of 4 individual members of the CSPG family was studied in patients who died at various survival times after SCI. In contrast to the previous studies, which used a general immunohistochemical marker for CSPG, the present investigation demonstrated that NG2 and phosphacan were indeed present in the lesion-induced astroglial scar, supporting the notion that these molecules may contribute to the lack of axonal regeneration following human SCI. This data strongly suggests that monoclonal CS-56 antibody may not be capable of detecting, with sufficient sensitivity, all members of this protein family in wax embedded sections.
In sections from control spinal cord, NG2 immunoreactivity was restricted to stellate-shaped cells corresponding to oligodendrocyte precursor cells. This pattern was identical to a previous study in unlesioned human CNS using several antibodies including the 9.2.27 antibody . The distribution patterns of phosphacan, neurocan and versican in control human spinal cord parenchyma were similar, with a fine reticular pattern being observed in white matter (but also located in gray matter for phosphacan). Neurocan staining was also associated with blood vessel walls. This distribution pattern is identical to a previous study in human brain tissue . Although experimental studies have demonstrated an interaction of CSPGs with proteins involved in nerve fibre organisation  and phosphacan has been shown to interact with neuronal cell adhesion molecules , the exact function of these proteins in the normal CNS remains unknown. In contrast to the present investigation, showing the presence of phosphacan, neurocan and versican in the myelin sheaths of control and regenerating ventral and dorsal nerve roots axons, others have reported a distribution pattern associated with nerve root endoneurium . However, the co-localisation of certain CSPGs with myelin is not without precedent. Others have clearly demonstrated the association of brevican and V2 versican with a myelin fraction extracted from porcine spinal cord. Furthermore, much of the axon growth inhibitory capacity was attributed to these CSPGs [4, 25].
After SCI, the distribution pattern of all four CSPGs changed significantly. At survival times of 2 to 8 days after injury, no NG2 immunopositive structures were detectable at the lesion epicentre. However, by 10–24 days, NG2-positive cells could, once again, be identified at the lesion core as well as in the intermediate zone. At the lesion epicentre, double immunofluorescence of such NG2-positive cells revealed that they were macrophages. In the intermediate zone, both round to oval shaped macrophages and stellate-shaped oligodendrocyte precursor cells were NG2 immunopositive. Although others have reported NG2-expression by Schwann cells, it is unlikely that such cells contributed to the NG2 signal observed in the present investigation, since an earlier report from our group using the same human SCI cases was unable to find any evidence of Schwann cell migration in this particular region with an anti-NGFr antibody . An experimental spinal cord contusion injury model has demonstrated NG2-positive non-myelinating Schwann cells, oligodendrocyte precursor cells and macrophages at the lesion site . NG2 was reported to be largely present in the ECM at the lesion core, which contained numerous migrating fibroblasts and Schwann cells. The NG2-positive lamellae were closely associated with P0-myelinated axons. In accordance with these results, the present human SCI cases also demonstrated NG2-positive lamellae in the lesion epicentre. Regenerating nerve fibres, originating from injured spinal nerve roots  were embedded within the NG2-positive ECM. It is possible that these lamellae might have played a supporting role in the growth of PNS axons from lesioned nerve roots.
The post lesion survival times of the cases used in the present investigation indicate that the deposition of an NG2-positive matrix took place between 24 days and 4 months after human SCI. The spatial distribution correlates with the animal data , however the timing of NG2-expression in traumatically injured human spinal cord is substantially delayed [13, 35]. In the present investigation, only macrophages located at the lesion epicentre were found to be immunoreactive and, thus, may have been responsible for NG2 deposition. As described by others, migrating Schwann cells or oligodendrocyte precursor cells (OPCs) may also have contributed to the CSPG-rich ECM . However, the present investigation was not able to give any direct evidence to support this notion. It is possible that sub-optimal antigen preservation or antigen retrieval may have been responsible for an incomplete representation of NG2 distribution. The inevitable delays that occur before human post mortem tissues can undergo fixation certainly contribute to this problem. NG2 was, nonetheless, clearly detectable in macrophages and in the ECM of both the Schwann cell-dominated area as well as in the astroglial scar being devoid of Schwann cells.
In the present material, no phosphacan-positive structures could be seen at the lesion epicentre or in the intermediate zone at early survival times of up to 11 days after SCI. Twenty-four days after injury, the evolving astroglial scar in the intermediate zone contained phosphacan with a dense network of irregularly distributed fibres. This distribution, which corresponded to that of astroglial GFAP was seen in all cases with survival times longer than 24 days after SCI. This temporal and spatial expression pattern suggests that reactive astrocytes may have been responsible for the increased phosphacan expression following human SCI. Such a distribution following experimental spinal cord injury has already been suggested by others [22, 23]. However, since no clearly identifiable phosphacan-positive cell population could be found, the astroglial origin of this proteoglycan following human SCI must remain speculative. Together with NG2, phosphacan may represent an important growth-inhibitory molecule that is associated with the developing astroglial scar and thus, may be of functional importance for the inhibitory glial environment following human traumatic SCI.
Apart from being associated with the astrocytic scar, phosphacan was also present in the lesion epicentre with a distribution pattern that corresponded to neurocan and versican. This lesion area contained migrating fibroblasts, meningeal cells and migrating Schwann cells and was strictly separated from the surrounding astrocytic scar . All three CSPGs were associated with the myelin sheaths of nerve fibres that were mostly arranged in root-like structures and were derived from regenerating spinal nerve roots . Furthermore, no nerve fibres were detected traversing the interface between the lesion epicentre and the astroglial scar. Therefore, the distribution pattern of neurocan and versican at the lesion epicentre in the present post mortem human material is not able to support the notion that either molecule contributes to the failure of CNS axon regeneration after human SCI. However, in the absence of definitive proof of the peripheral source of all nerve fibres within the lesion core, it cannot be excluded that some Schwann cells and their myelin sheaths including the CSPGs were associated with re-growing CNS axons. The function of CSPGs in the myelin sheaths surrounding PNS nerve fibres at the lesion core is currently not known.
The involvement of four members of the CSPG family has been demonstrated in the post-traumatic events after human SCI. This investigation comprised of 15 patients who died at a range of different survival times after trauma. Due to the difficulties in obtaining post mortem human tissue specimens, only one case could be studied at each survival time. Although cases with similar survival times demonstrated similar immunohistochemical staining patterns, the present results need to be interpreted with caution, and further studies with more human cases per survival time would be of significant value.
The present data extends our investigations into the cellular and molecular composition of the lesion site of severe macerating human SCI [31, 36, 37]. The distribution pattern of individual members of the CSPG family varies significantly after human SCI. NG2 and phosphacan were both present in the evolving astroglial scar and, therefore, might have played an important role in the blockade of successful CNS regeneration. Neurocan and versican, however, were located at the lesion epicentre, associated with Schwann cell myelin on regenerating peripheral nerve fibres, a distribution that was unlikely to contribute to failed CNS axon regeneration. This interpretation certainly needs to take into account the limited number of time points studied after human SCI. It is possible that transient up-regulation of various CSPGs may have been missed due to a lack of appropriate specimens at these time points. The present data emphasises the importance and potential usefulness of comparing data obtained with experimental animals with that obtained from human post mortem tissues. This is of particular relevance for the identification of possible key functional molecules that are believed to play a major role in the failure of axonal regeneration following traumatic human spinal cord injury.
Blood brain barrier
Central nervous system
Chondroitin sulphate proteoglycan
Glial fibrillary acidic protein
Myelin Basic Protein
Oligodendrocyte Precursor Cell
Peripheral nervous system
Spinal cord injury.
The authors thank S. Lecouturier for excellent technical assistance and Prof. B. Stallcup for technical discussion. This work was supported by a grant from the Deutsche Stiftung Querschnittslähmung (DSQ).
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