AR and RS designed the experiments and carried them out with AL; AR, RS, and MS wrote the paper; YS helped with in vitro experiments. The gene array project was conducted by JJ-H, NA, and GR.
The authors declare that they have no competing interests.
Chondroitin sulfate proteoglycan (CSPG) is a major component of the glial scar. It is considered to be a major obstacle for central nervous system (CNS) recovery after injury, especially in light of its well-known activity in limiting axonal growth. Therefore, its degradation has become a key therapeutic goal in the field of CNS regeneration. Yet, the abundant de novo synthesis of CSPG in response to CNS injury is puzzling. This apparent dichotomy led us to hypothesize that CSPG plays a beneficial role in the repair process, which might have been previously overlooked because of nonoptimal regulation of its levels. This hypothesis is tested in the present study.
We inflicted spinal cord injury in adult mice and examined the effects of CSPG on the recovery process. We used xyloside to inhibit CSPG formation at different time points after the injury and analyzed the phenotype acquired by the microglia/macrophages in the lesion site. To distinguish between the resident microglia and infiltrating monocytes, we used chimeric mice whose bone marrow-derived myeloid cells expressed GFP. We found that CSPG plays a key role during the acute recovery stage after spinal cord injury in mice. Inhibition of CSPG synthesis immediately after injury impaired functional motor recovery and increased tissue loss. Using the chimeric mice we found that the immediate inhibition of CSPG production caused a dramatic effect on the spatial organization of the infiltrating myeloid cells around the lesion site, decreased insulin-like growth factor 1 (IGF-1) production by microglia/macrophages, and increased tumor necrosis factor alpha (TNF-α) levels. In contrast, delayed inhibition, allowing CSPG synthesis during the first 2 d following injury, with subsequent inhibition, improved recovery. Using in vitro studies, we showed that CSPG directly activated microglia/macrophages via the CD44 receptor and modulated neurotrophic factor secretion by these cells.
Our results show that CSPG plays a pivotal role in the repair of injured spinal cord and in the recovery of motor function during the acute phase after the injury; CSPG spatially and temporally controls activity of infiltrating blood-borne monocytes and resident microglia. The distinction made in this study between the beneficial role of CSPG during the acute stage and its deleterious effect at later stages emphasizes the need to retain the endogenous potential of this molecule in repair by controlling its levels at different stages of post-injury repair.
Michal Schwartz and colleagues describe the role of chondroitin sulfate proteoglycan in the repair of injured tissue and in the recovery of motor function during the acute phase after spinal cord injury.
Every year, spinal cord injuries paralyze about 10,000 people in the United States. The spinal cord, which contains bundles of nervous system cells called neurons, is the communication superhighway between the brain and the body. Messages from the brain travel down the spinal cord to control movement, breathing, and other bodily functions; messages from the skin and other sensory organs travel up the spinal cord to keep the brain informed about the body. All these messages are transmitted along axons, long extensions on the neurons. The spinal cord is protected by the bones of the spine but if these are displaced or broken, the axons can be compressed or cut, which interrupts the information flow. Damage near the top of the spinal cord paralyzes the arms and legs (tetraplegia); damage lower down paralyzes the legs only (paraplegia). Spinal cord injuries also cause other medical problems, including the loss of bowel and bladder control. Currently there is no effective treatment for spinal cord injuries. Treatment with drugs to reduce inflammation has, at best, only modest effects. Moreover, because damaged axons rarely regrow, most spinal cord injuries are permanent.
One barrier to recovery after a spinal cord injury seems to be an inappropriate immune response to the injury. After an injury, microglia (immune system cells that live in the nervous system), and macrophages (blood-borne immune system cells that infiltrate the injury) become activated. Microglia/macrophage activation can be either beneficial (the cells make IGF-1, a protein that stimulates axon growth) or destructive (the cells make TNF-α, a protein that kills neurons), so studies of microglia/macrophage activation might suggest ways to treat spinal cord injuries. Another possible barrier to recovery is “chondroitin sulfate proteoglycan” (CSPG). This is a major component of the scar tissue (the “glial scar”) that forms around spinal cord injuries. CSPG limits axon regrowth, so attempts have been made to improve spinal cord repair by removing CSPG. But if CSPG prevents spinal cord repair, why is so much of it made immediately after an injury? In this study, the researchers investigate this paradox by asking whether CSPG made in the right place and in the right amount might have a beneficial role in spinal cord repair that has been overlooked.
The researchers bruised a small section of the spinal cord of mice to cause hind limb paralysis, and then monitored the recovery of movement in these animals. They also examined the injured tissue microscopically, looked for microglia and infiltrating macrophages at the injury site, and measured the production of IGF-1 and TNF-α by these cells. Inhibition of CSPG synthesis immediately after injury impaired the functional recovery of the mice and increased tissue loss at the injury site. It also altered the spatial organization of infiltrating macrophages at the injury site, reduced IGF-1 production by these microglia/macrophages, and increased TNF-α levels. In contrast, when CSPG synthesis was not inhibited until two days after the injury, the mice recovered well from spinal cord injury. Furthermore, the interaction of CSPG with a cell-surface protein called CD44 activated microglia/macrophages growing in dishes and increased their production of IGF-1 but not of molecules that kill neurons.
These findings suggest that, immediately after a spinal cord injury, CSPG is needed for the repair of injured neurons and the recovery of movement, but that later on the presence of CSPG hinders repair. The findings also indicate that CSPG has these effects, at least in part, because it regulates the activity and localization of microglia and macrophages at the injury site and thus modulates local immune responses to the damage. Results obtained from experiments done in animals do not always accurately reflect the situation in people, so these findings need to be confirmed in patients with spinal cord injuries. However, they suggest that the effect of CSPG on spinal cord repair is not an inappropriate response to the injury, as is widely believed. Consequently, careful manipulation of CSPG levels might improve outcomes for people with spinal cord injuries.
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The poor recovery of the central nervous system (CNS) following an injury is generally attributed to the accumulation of compounds that mediate self-perpetuating degeneration, the presence of growth inhibitors [
Accumulated data suggest, however, that a tightly regulated and timely immune response is needed for recovery [
Two mouse strains were used in the experiments, inbred adult wild-type C57Bl/6J mice (supplied by the Animal Breeding Center of The Weizmann Institute of Science) and CX3CR1GFP/+ mice (a generous gift of Stefan Jung at the Weizmann Institute). The CX3CR1GFP/+ mice are heterozygotic mice with green fluorescent protein (GFP) inserted in the CX3CR1 locus in one allele, while a normal allele enables the continued expression of CX3CR1 [
The experiments described in
Paraffin-embedded spinal cord sections were prepared from the lesion site 14 d after injury.
(A and B) Sections were immunolabeled for IGF-1 (A) or BDNF (B) (scale bar 500 μm).
(C) Sections were labeled with IB-4 (green), to identify microglia/macrophages and IGF-1 (red; left panels) or BDNF (yellow; right panels) (scale bars, 10 μm; arrows indicate double-labeled cells).
(D) Labeling by CS-56 (blue), a marker of CSPG and IGF-1 (red, upper panels) colocalized at the margins of the lesion site. BDNF expression (yellow, lower panels) is not specifically colocalized with CS-56 immunoreactivity (blue, all panels; scale bar, 100 μm). High-power images of the boxed area in the left panels are shown on the right (scale bar, 20 μm).
(E and F) Quantitative analysis of IGF-1 (E) and BDNF (F) immunoreactivity, at the epicenter and the margins of the lesion calibrated to either intensity per square millimeter. Total intensity in the examined region was normalized to the size of the area (left graphs, arbitrary units, Student
(G) Sections from GFP-chimeric mice labeled for GFP (blood-derived macrophages; green) in non-injured and injured mice (scale bar, 100 μm).
(H and I) Lesion site in chimeric injured mice, labeled for CS-56 (blue) (H) and GFP (green) or IB-4 (red) (I) and GFP (green; scale bars 10 μm).
(J) High magnification of cells from the marginal area of the lesion, indicating that both GFP-positive (green) and GFP-negative cells express IGF-1 (red; scale bar, 20 μm). Arrows indicate blood-derived macrophages (GFP-positive) cells expressing IGF-1. In all panels, boundaries between the epicenter and the margins are marked by dashed line.
After spinal cord injury, mice were injected IP with xyloside (1.2 mg/mouse/d, for 6 d), a pharmacological inhibitor of CSPG synthesis.
(A) CS-56 staining of spinal cord sections for the presence of CSPG (scale bar, 250 μm).
(B) Quantitative analysis of CSPG intensity in the diameter of 1 mm around the epicenter (arbitrary units; Student
(C) Staining for IGF-1 (red; scale bar, 250 μm).
(D) Quantitative analysis of IGF-1 immunoreactivity per square millimeter, at the site (arbitrary units; Student
(E) Staining for BDNF (yellow; scale bar, 250 μm).
(F) Quantitative analysis of BDNF immunoreactivity at the site (arbitrary units; nonsignificant according to Student
(G) Representative photomicrographs of CS-56-labeled spinal cord sections from mice treated with xyloside (scale bar, 250 μm). The boxed area in the left panel is magnified on the right and shows labeling for IGF-1 (red) and CS-56 (CSPG; white; scale bar, 20 μm).
(H) Scheme showing experimental time scale. Bone marrow chimeras were generated by reconstitution of irradiated C57BL/6J mice with CX3CR1GFP/+ bone marrow. After 2 mo, chimeric mice were subjected to spinal cord injury and injected IP with xyloside.
(I) Staining for GFP (green; blood-borne macrophages) and IB-4 (red; microglia/macrophages) at the injury site in control (left) and in xyloside-treated (right) animals (scale bar, 100 μm; **
(A) Staining for myelin by Luxol (blue) and to nuclei by Nissel (pink) (scale bar, 500 μm).
(B) Quantitative analysis of the size of the site of injury as a function of the xyloside dosages, determined by Luxol and Nissel staining (ANOVA, F[3,17] = 37.5,
(C) Immunohistochemistry of xyloside-treated spinal cords, using anti-GFAP (green) antibodies and IB-4 labeling (red) (scale bar, 250 μm).
(D) Mean locomotor score (BMS) of individual mice on day 36 after spinal cord injury with and without xyloside treatment (immediately after injury 0.8 mg/mouse/d; Student
(A) Schematic representation of the experimental time scale. Mice were subjected to contusive spinal cord injury and were treated with xyloside (0.8 mg/mouse/d for 6 d) at different time points after the injury. Locomotion was recorded, and is given by the mean locomotor scores (BMS) for each group.
(B) Xyloside application started 2 d after the injury (repeated measures ANOVA, F[1,15] = 7.426 [between groups],
(C) Xyloside application started immediately after the injury (two factor repeated measures ANOVA, F[1,14] = 15.481 [between groups],
(D) Xyloside application started 7 d after the injury (repeated measures ANOVA, F[1,15] = 0.093 [between groups],
(E) Fold change in functional recovery on day 30 after the injury between each treatment compared to their matched untreated control (ANOVA, F[2,27] = 16.64,
(F) BDA tracing of the corticospinal tract, caudal to the lesion site in a xyloside-treated mouse; photomicrographs show cornal sections excised from mice treated with xyloside on day 0 (left) or 2 (right) after the injury (scale bars, 250 μm). Insert shows the site from which the images were taken.
(G) Quantitative analysis of BDA labeling. To quantify labeled fibers caudal to the lesion, we calculated the labeling caudal to the lesion site relative to the amount of BDA rostal to the lesion for each animal (mean ± SD; Student
(H) Immunohistochemistry of xyloside-treated spinal cords, using anti-GFAP (green) antibodies and IB-4 labeling (red; scale bars, 250 μm).
(I) Quantitative analysis of the size of the injury site as a function of the treatment, determined by Luxol and Nissel staining (ANOVA, F[3,26] = 43.03,
(J) CS-56 staining of spinal cord sections, excised from mice 14 d after the injury, for the presence of CSPG (scale bar, 250 μm). The dashed line demarcates the lesion site, defined based on GFAP labeling.
(K) Quantification of CSPG intensity (ANOVA, F[3,12] = 7.619,
(L) Western blot analysis of CSPG levels in the control group (PBS) and in the groups receiving xyloside treatment on day 0 or day 2 after the injury (excised 7 d after the injury). β-actin was used as a control for protein levels. Fold decrease in CSPG levels, relative to PBS control, are shown (
ANOVA in (I) and (K) followed by the Fisher test for differences among groups (significant at the 5% level). Asterisks (G, I, and K) denote statistically significant differences between the indicated groups, or compared to the relevant control: *
Spinal cords were excised (14 d after the lesion) from animals that were subjected to spinal cord injury and treated with either PBS or with xyloside administered on day 2 or day 7 following the insult.
(A) Quantitative analysis of IGF-1 immunoreactivity per square millimeter at the site (ANOVA, F[2,11] = 22.78,
(B) Quantitative analysis of IGF-1 protein concentration determined by ELISA of spinal cord tissue (ANOVA, F[3,12] = 32.138,
(C) Experimental time scale. CX3CR1GFP/+ > wild type bone marrow chimeras were generated by reconstitution of irradiated C57BL/6J mice with CX3CR1GFP/+ bone marrow. Chimeric mice were subjected to spinal cord injury and injected IP with xyloside starting from day 2 after the injury.
(D) Staining for GFP (green; blood-borne macrophages) and IB-4 (red; microglia/macrophages) at the injury site in control (PBS) and in delayed (day 2) xyloside-treatment group (scale bar, 250 μm).
ANOVA followed by the Fisher test for differences between groups; significant differences at the 5% level are denoted by asterisks. All the groups in (B) were significantly different from uninjured control.
(A) IB-4 labeling (green) and Hoechst (nuclear; blue) of microglia cultured on PDL or on CSPG for 48 h showing morphological changes in the CSPG-cultured microglia (scale bar, 10 μm).
(B) BrdU incorporation showing increased proliferation induced by microglia cultured on CSPG relative to PDL-cultured microglia (scale bar, 100 μm).
(C) Quantitative analysis of the proportion of BrdU-incorporating cells in the total population of IB-4+ cells (Student
(D) Semi-quantitative PCR analyses of IGF-1 and BDNF expression by microglia cultured for 12 h on PDL or on CSPG. Values represent relative amounts of amplified mRNA normalized against β-actin in the same sample, and are represented as fold induction relative to control microglia cultured on PDL.
(E) IGF-1 (red) and BDNF (green) expression in microglia cultured for 18 h on PDL or on CSPG (scale bar, 20 μm). Hoechst labeling is blue.
(F) Quantitative analysis of IGF-1 immunoreactivity in microglia cultured for 12, 18, and 48 h on CSPG (ANOVA, F[2,6] = 185.2,
(G) Semi-quantitative PCR analyses of IRS-1 expression by microglia cultured on PDL and on CSPG.
(H) Semi-quantitative PCR analyses of MMP-2 and MMP-9 mRNA in microglia cultured on PDL and on CSPG for various time periods. Values represent relative amounts of amplified mRNA normalized against β-actin in the same sample, and are represented as fold change in microglia cultured on CSPG relative to PDL at the same time point (C, CSPG; P, PDL).
(I) Nitric oxide levels in the culture media of microglia cultured for 48 h on PDL or CSPG or in the presence of LPS (50 ng/ml) (ANOVA, F[2,9] = 114.9,
(J) Semi-quantitative PCR analysis of TNF-α expression indicating that TNF-α was not increased in CSPG-activated microglia, but was significantly increased upon activation of microglia by LPS (12 h). Values represent relative amounts of amplified mRNA normalized against β-actin in the same sample, and are represented as fold induction relative to control microglia cultured on PDL.
(K–M) Cells were cultured on PDL and CSPG, 24 h prior to their stimulation with LPS for an additional 24 h. Quantitative analysis of TNF-α production (K) or cell-body size (L) in microglia activated by increasing doses of LPS (M), representative photos; TNF-α (red) and IB-4 (green) labeling of microglia (scale bar, 20 μm). Two-way ANOVA was used for statistical analysis in (K) (F[7,17] = 34.3,
(A) Anti-phospho-ERK1/2 labeling (green) indicating increased phosphorylation of ERK1/2 in microglia (IB-4; red) cultured on CSPG. In this culture, addition of CD44-neutralizing antibodies to the medium resulted in decreased ERK1/2 phosphorylation (scale bar, 20 μm). Arrows indicate microglia labeled with pERK1/2.
(B) Quantitative analysis of pERK1/2-positive, IB-4-positive cells. Data are from one of at least three independent experiments in replicate cultures (P, PDL; C, CSPG; ANOVA, F[3,17] = 158.7,
(A) Mean locomotor score (BMS) for each group during the 60-d recovery period (repeated measures ANOVA, F[1,31] = 15.47 [between groups],
(B–D) BMS scores of individual mice on day 60 after spinal cord injury (Student
(E) Quantitative analysis of the IB-4-labeled area, indicative of the lesion site (Student
(F) Quantitative analysis of BDA labeling. Sections that contained more than two labeled fibers caudal to the lesion site were counted and presented in percentage (Student
(G) IGF-1 immunoreactivity (red) in microglia treated with CSPG-DS in the presence or absence of LPS (scale bar, 20 μm).
(H) Quantitative analysis of IGF-1 immunoreactivity in the CSPG-DS-treated microglia, with (gray) and without LPS (black) (ANOVA, F[5,19] = 10.63,
(I) BDNF immnoreactivity (green) and Hoechst labeling (blue) in microglia treated with CSPG-DS (scale bar, 10 μm).
(J) Quantitative analysis of BDNF immunoreactivity in the CSPG-DS-treated microglia (ANOVA, F[2,9] = 23.16,
(K) BDNF labeling (yellow) of microglia in the lesion site (scale bar, 20 μm). The image depicts the marginal area of the lesion.
(L) IGF-1 staining (red) of microglia in the lesion (scale 20 μm). The image depicts the epicenter of the lesion. The dashed line demarcates the lesion site. All data in (G)–(J) are from one of at least three independent experiments with replicate cultures.
*
The spinal cords of anesthetized mice were exposed by laminectomy at T12, and a force of 200 kdyn was placed for 1 s on the cord by using the Infinite Horizon spinal impactor (Precision Systems, Lexington, Kentucky), a device shown to inflict a well-calibrated injury of the spinal cord. The animals were maintained on twice daily bladder expression. Functional recovery from spinal cord contusion in mice was determined by hind limb locomotor performance. Recovery was scored by the Basso mouse scale (BMS), an open-field locomotor rating scale [
Wild-type C57Bl/6J mice were killed and their spinal cords were removed 14 d after spinal cord injury. A section of 1 mm2 from the lesion area or from a noninjured area 1 cm distally to the injury site (repeated with six mice per group) was excised. The excised tissue was homogenized in PBS. Similarly, in an independent experiment, we excised the lesion area in PBS or xyloside-treated (0.8 mg/mouse for 6 d, immediately, 2 or 7 d after the injury) mice (four mice per group). Two freeze-thaw cycles were performed to break the cell membranes, and the homogenates were centrifuged for 5 min at 5,000
C57BL/6J-CX3CR1GFP/+ chimeric mice were generated by lethal whole body irradiation (950 rad, with shielding of the brain) of C57BL/6J mice followed by reconstitution with 4 × 106 bone marrow cells isolated from the CX3CR1GFP/+ mice (harvested from the femora and tibiae, by flushing the bones with Dulbecco PBS under aseptic conditions, and then collected and washed by centrifugation for 10 min at 1,000 rpm at 4 °C), in which GFP is expressed under the promoter of the chemokine receptor, CX3CR1. In the chimeric mice formed following bone marrow reconstitution, the bone marrow-derived cells (blood-borne monocytes) originating from the CX3CR1GFP/+ mice express GFP; however, the resident microglia are GFP-negative. This system thus allows the resident microglia and the infiltrating macrophages to be distinguished. The mice were grafted with bone marrow cells 2 mo before the spinal cord injury.
Xyloside (4-methylumbelliferyl-β-D-xylopyranoside; Sigma-Aldrich) was injected intraperitoneally (IP) every day starting either immediately after the injury, 2 d later, or 7 d after the injury. The mice were injected twice daily for 5 d. The mice were analyzed for functional recovery with the BMS, cytokine expression by ELISA, and CSPG expression by Western blotting (using wild-type C57Bl/6J mice) and histology (using wild-type and chimeric mice). For functional analysis, mice were examined up to 60 d after injury. For histological analysis mice were killed 14 d after the injury. Several dosages of xyloside, adapted from another experimental system [
CSPG-DS (6-sulfated disaccharides; Sigma-Aldrich), were administered to mice by repeated intravenous injections (5 μg dissolved in PBS) on days 1, 4, 7, 10, and 13 after injury.
Wild-type C57Bl/6J mice that were followed for functional recovery were anesthetized 60 d after the injury, and injected bilaterally using a stereotaxic frame, with the high-resolution anterograde tracer BDA (10,000 MW lysine-fixable biotin dextran amine; Molecular Probes, Eugene, OR; 1.2 μl of 10% wt/vol BDA solution in 0.01 M PB). BDA was injected into both motor cortices. A 30-gauge Hamilton needle was lowered through the cortex (1 mm) and four injections of 0.3 μl per site were administered within a perimeter defined coronally by bregma 1.7 mm to −0.7 mm, and sagittally 0.5–1.5 mm from the sagittal suture, bilaterally. Injections were performed over 3–5 min and the needle was slowly removed. The mice were killed and perfused 14 d after BDA injection. For histological assessment of the BDA tracing, spinal cords, and brains were dissected, and sections (30 μm) of the thoracic spinal cord comprising the lesion site were cut either horizontally (floating) or longitudinally, and stained for BDA by the use of either nickel-enhanced diaminobenzidine protocol [
Mice subjected to spinal cord injury were killed 14 d later, their spinal cords were prepared for histology and analyzed as described before [
Myelin integrity was qualitatively examined on paraffin-embedded sections that were stained with Luxol fast blue for myelin and Nissel for the nuclei and the thin cytoplasmic layer around them. With the aid of Image-Pro (Media Cybernetics) the results were analyzed by determination of the density or by measurement of the lesion area by an observer who was blinded to the treatment received by the mice. For tissue cultured cell phosphorylated ERK labeling, mouse anti phosphoERK (Santa Cruz) was used. For analysis, 500–1,000 cells were sampled for each marker.
For microscopic analysis a Nikon florescent microscope was used (Nikon E800). Intensity of staining was calibrated using Image-Pro Plus software by an observer blind to the identity of the slides. To calibrate intensity of labeling, an average of sampled background areas was subtracted from the total intensity counted. To demonstrate intensity per square millimeter, the total intensity in the examined region was normalized to the size of the area. To determine cell numbers, we counted the cells using manual tagging within the Image-Pro Plus software. To determine intensity per cell, total intensity in the examined region was normalized to the number of cells. For each assessment, four or five animals per group were examined, sections from three different depths were examined, and at least 1,000 cells per group were included. The margins and the lesion size were defined by the area demarcated by GFAP immunolabeling or Luxol/Nissel staining (the lesion was identified as the area that was not labeled for myelin by Luxol) and quantified by Image-Pro Plus software.
Wild-type C57Bl/6J mice were killed 7 d after the spinal cord injury, and sections of the spinal cord (5 mm around the lesion site) were removed. The sections were shock-frozen in liquid nitrogen and homogenized in 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris (pH 8.0) containing proteinase inhibitor cocktail (Sigma), 1 mM leupeptin, and 1 mM pepstatin. The homogenates were incubated for 1 h and centrifuged at 10,000
To prepare cultures of microglia, a distinct group of neonatal (P0–P1) C57Bl/6J mice was used. The mice were killed and their brains were stripped of their meninges and minced with scissors under a dissecting microscope (Zeiss, Stemi DV4, Germany) in Leibovitz-15 medium (Biological Industries, Beit Ha-Emek, Israel). After trypsinization (0.5% trypsin, 10 min, in 5% CO2 at 37 °C), the tissue was triturated. The cell suspension was washed in culture medium (DMEM supplemented with 10% fetal calf serum [Sigma-Aldrich], 1 mM l-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin). The mixed brain glial cells were cultured in 5% CO2 at 37 °C in 75-cm2 Falcon tissue culture flasks (BD Biosciences) that had been coated with poly-d-lysine (PDL) (10 μg/ml; Sigma-Aldrich) in borate buffer (15.45 g boric acid [Merck] dissolved in 500 ml of sterile water [pH 8]) for 1 h, then rinsed thoroughly with sterile, glass-distilled water. The medium was changed after 24 h in culture, and every second day thereafter, for a total culture time of 10–14 d. Microglia were shaken off the primary mixed brain glial cell cultures (150 rpm, 6 h, 37 °C) with maximum yields between day 10 and 14, and seeded (105 cells/ml) on coverslips coated with PDL or CSPG (Sigma-Aldrich) for the indicated time periods. Cells were grown in culture medium for microglia (RPMI-1640 medium [Sigma-Aldrich] supplemented with 10% fetal calf serum, 1 mM l-glutamine, 1 mM sodium pyruvate, 50 μM β-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin). Since CSPG was used in the present study as a matrix component rather than as a soluble compound, its effect was monitored as a function of time, rather than as a function of dose. Cell proliferation was visualized by staining with 5-bromo-deoxyuridine (BrdU, 2.5 μM; Sigma-Aldrich). Lipopolysaccharide (LPS; Sigma-Aldrich) was added to the culture medium after the cells were cultured on CSPG or treated with CSPG-DS. For neutralization assays, anti-mouse CD44 (BD Pharmingen) neutralizing antibody was used. CSPG-DS was added to PDL-cultured microglia at the indicated concentrations.
Nitric oxide release was assayed according to the method of Griess [
These procedures were performed as previously described [
Data were analyzed using the Student
After CNS injury, microglia/macrophages become activated and accumulate at the lesion site. We first examined neurotrophic factor expression by these cells. We inflicted a well-calibrated contusive injury at the thoracic segments of the mice spinal cords (T-12), which resulted in paralysis of the hind limbs. To delineate the lesion site in spinal cord sections, we used GFAP labeling. We found that microglia/macrophages located at the lesion site expressed high levels of IGF-1 in addition to BDNF (
The observed localized expression of IGF-1 encouraged us to examine its association with CSPG, shown to be expressed mainly at the margins of the lesion. Immunohistochemical analysis showed that microglia/macrophages that were spatially associated with CSPG expressed IGF-1 in abundance (
The increased expression of IGF-1 by the microglia/macrophages that were spatially associated with CSPG could reflect a direct interaction of these cells with CSPG. Alternatively, it could be an outcome of additional differences between the margins and the epicenter of the lesion, such as the origin of the microglia/macrophages; a preferential presence of either microglia or blood-borne monocytes [
To examine whether CSPG directly affects the microglia and the blood-borne monocytes found in its proximity, we used xyloside, a pharmacological inhibitor of CSPG biosynthesis. Xyloside inhibits the biosynthesis of CSPG, and has been previously used to study the role of CSPG in axonal growth both in vivo and in vitro [
To address the relevance of CSPG production to the recovery from injury, we first assessed the size of the lesion site by immunohistochemical analysis. Staining for myelin with Luxol fast blue, and for cell nuclei with Nissel, revealed a dose-dependent increase in the size of the lesion site in correlation with the increase in the xyloside dosages (
To further evaluate the relevance of CSPG production to functional recovery, we performed an additional experiment applying xyloside immediately after injury, and assessed the functional recovery using the Basso Mouse Scale, BMS. This scale evaluates locomotion in an open field, where a score of 0 indicates complete paralysis and a score of 9 indicates normal function. Treatment with xyloside immediately after injury significantly reduced the spontaneous recovery, resulting in lower motor function of the hind limbs (
The observed beneficial role of CSPG and the well-documented inhibitory effect of CSPG on axonal growth [
Based on these findings, we wished to examine whether the delayed (day 2) xyloside treatment, compared to the immediate treatment, differentially affected the microglia/macrophages and their distribution at the lesion site. We found that the inhibition of CSPG synthesis during the subacute phase (delayed [day 2] treatment), resulted in increased IGF-1 immunolabeling (
As described above, CSPG participated in the spatial organization of the microglia/macrophages at the lesion site; in the absence of CSPG (in mice receiving xyloside treatment immediately after the injury), the compartmentalized organization of the site was disrupted and macrophages also invaded the epicenter of the lesion (
To gain insight into the underlying mechanism of CSPG effects on microglia/macrophages, we employed in vitro assays using primary cultures of mouse microglia. Microglia cultured on an inert substrate, PDL, were used as a basal reference. Cultured microglia at rest do not show the classical ramified morphology of microglia in vivo [
Examination of the direct effect of CSPG on microglial expression of BDNF and IGF-1 in vitro, in line with our results in vivo, revealed an increase in the mRNA of IGF-1 but not of BDNF (
IGF-1 is recognized as a key factor in neuronal survival [
MMPs are endogenous proteolytic enzymes that can degrade CSPG [
Examination of the microglia cultured on CSPG revealed no detectable increase in nitric oxide (measured in terms of nitrate levels in the cultured media;
CD44 is a well-characterized receptor of CSPG [
Some of the most important lines of evidence supporting a negative effect of CSPG on CNS repair come from studies in which degradation of CSPG by ChABC, even if it is administered immediately after an insult [
In the case of CSPG, its degradation by ChABC results in the formation of a 6-sulfated disaccharide (CSPG-DS) [
Immunohistochemical analysis of the lesion site using anti-CSPG antibody revealed that treatment with CSPG-DS had no effect on CSPG levels, indicating that the beneficial effect of CSPG-DS treatment was not caused indirectly by inhibition of CSPG expression (
Staining for GFAP and IB-4 indicated significant tissue preservation in the CSPG-DS–treated mice relative to controls treated with PBS (
We also examined the potential effects of CSPG-DS on microglia in vitro. Incubation of microglia on PDL in the presence of CSPG-DS resulted, as with the intact CSPG, in a dose-dependent increase in IGF-1 levels (
To further study the effect of CSPG-DS in vivo, we examined the injured spinal cords treated with CSPG-DS or PBS for IGF-1 and BDNF (
The results of this study suggest that CSPG, an extracellular component of the glial scar, exerts a beneficial effect on CNS recovery from injury, in part by inducing IGF-1 and MMP expression by microglia/macrophages and attenuating TNF-α levels. This microglial modulation was mediated, at least in part, by the CD44 receptor. Our data further suggest that, following injury to the CNS, CSPG plays a beneficial role in its recovery that can be achieved only by careful regulation of its presence: blockage of CSPG production immediately after spinal cord injury decreased spontaneous recovery, whereas restriction of CSPG biosynthesis to the acute phase improved recovery.
The intensive secretion of CSPG reported after a CNS injury [
The contribution of blood-borne monocytes to the recovery is still a subject of debate [
The direct role of CSPG in controlling microglial and macrophage behavior after spinal cord injury was demonstrated here by blockage of CSPG biosynthesis, rather than inducing its degradation of existing CSPG. Degradation, unlike blockage of biosynthesis, can result in the formation of potentially new CSPG-derived active compounds such as CSPG-DS. Blockage of biosynthesis immediately after the injury resulted in reduced expression of IGF-1 by microglia/macrophages, loss of cellular compartmentalization in the lesion site, and decreased functional recovery relative to untreated controls. In contrast, when CSPG biosynthesis was allowed to take place during the first 2 d and was then inhibited, this treatment resulted in enhanced recovery, preservation of the site organization and of neurotrophic factor levels. Moreover, since both treatments (immediate and delayed to day 2) resulted in overall decrease in CSPG levels, it is likely that the improved recovery observed in the delayed treatment (day 2) should be attributed mostly to the presence of CSPG in the acute phase after injury.
Thus, these results indicate that the effect of CSPG after spinal cord injury is not an all-or-none phenomenon; it is a function of timing and level. Accordingly, our observations might explain some of the results described in the literature, in which various conditions of enzymatic degradation led to differing extents of recovery. For example, moderate (rather than intense) application of CSPG-degrading enzymes was reportedly more effective than intensive degradation [
The observation that CSPG induces MMP expression by microglia in vitro, in light of the need for temporally regulated degradation of CSPG in the process of repair, might point to a potential feedback regulation of CSPG by the same microglia/macrophages as those activated by it. This point, however, requires further investigation.
To conclude, our study does not argue against the beneficial effect of CSPG degradation, but rather suggests that the timing and the extent of degradation should be carefully selected according to the changing requirements of the ongoing dynamic repair process [
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We thank Dr. Stefan Jung for providing us with CX3CR1GFP/+ mice, Hillary Voet for assistance with the statistical analysis, Shelley Schwarzbaum for editing the manuscript, and Yaniv Ziv, Gil Lewitus, Ayal Ronen, and Rinat Levi for their constructive comments. MS holds the Maurice and Ilse Katz Professorial Chair in Neuroimmunology.
biotinylated dextran amine
brain-derived neurotrophic factor
Basso mouse scale
5-bromo-deoxyuridine
chondroitinase ABC
central nervous system
chondroitin sulfate proteoglycan
glial fibrillary acid protein
green fluorescent protein
insulin-like growth factor 1
intraperitoneal(ly)
insulin receptor substrate 1
lipopolysaccharide
matrix metalloproteinase
poly-
standard deviation
tumor necrosis factor alpha