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Keywords:

  • axon;
  • bone morphogenetic proteins;
  • neuron;
  • regeneration;
  • spinal cord

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor-β superfamily. BMPs regulate several crucial aspects of embryonic development and organogenesis. The reemergence of BMPs in the injured adult CNS suggests their involvement in the pathogenesis of the lesion. Here, we demonstrate that BMPs are potent inhibitors of axonal regeneration in the adult spinal cord. The expression of BMP-2/4 is elevated in oligodendrocytes and astrocytes around the injury site following spinal cord contusion. Intrathecal administration of noggin – a soluble BMP antagonist—leads to enhanced locomotor activity and reveals significant regrowth of the corticospinal tract after spinal cord contusion. Thus, BMPs play a role in inhibiting axonal regeneration and limiting functional recovery following injury to the CNS.

Abbreviations used
BBB

Basso-Beattie-Bresnahan

BDA

biotin dextran amine

BMPs

bone morphogenetic proteins

GFAP

glial fibrillary acidic protein

PBS

phosphate-buffered saline

It is well known that in the adult mammalian central nervous system (CNS), injured axons exhibit very limited regenerative ability. Because of the lack of appropriate axonal regeneration, traumatic damage to the adult brain and spinal cord frequently causes permanent neuronal deficits. After a CNS injury, various neurite outgrowth inhibitors are present around the damaged site and are considered to be, at least in part, responsible for the lesion-induced poor axonal regeneration and functional deficits. Among these inhibitors, the myelin-associated glycoprotein Nogo and the oligodendrocyte-myelin glycoprotein are well-characterized (Yiu and He 2006). In fact, the inactivation or blocking of these inhibitors, receptors, and intracellular signals leads to an enhancement in functional recovery as well as axonal growth; however, there remains some controversy regarding this finding (Yiu and He 2006). In addition, recent publications have proposed roles for other proteins, including ephrin-B3, Semaphorin 4D (Sema 4D), chondroitin sulfate proteoglycans (CSPGs), and repulsive guidance molecule, in inhibiting axonal regeneration following CNS injury (Yiu and He 2006). These proteins are well known for their roles in generating the neuronal network during the developmental stages (Harel and Strittmatter 2006). Therefore, cues that play roles in generating the nervous system may act as inhibiting molecules for the axons in the adult nervous system.

Bone morphogenetic proteins (BMPs) are members of a unique subfamily of the transforming growth factor-β superfamily. Bone morphogenetic proteins have a diverse array of functions, including cell fate decision, proliferation, and survival (Miyazono et al. 2005). Bone morphogenetic proteins are particularly involved in the generation of the CNS during development. It is important to note that the adult rat spinal cord responds to injury with a dramatic increase in the expression of BMP-2 and BMP-7 (Setoguchi et al. 2001, 2004). Although BMPs have been reported to inhibit neurogenesis and alternatively promote gliogenesis, the inhibition of BMPs by the transplantation of neural stem cells engineered to express noggin – an endogenous antagonist of BMP action – into the injured spinal cord does not antagonize terminal astroglial differentiation in the grafted stem cells (Enzmann et al. 2005). Inconsistent with this observation, it was reported that the differentiation of neural precursor cells into neurons and oligodendrocytes was promoted when noggin-expressing neural precursor cells were transplanted after SCI (Setoguchi et al. 2004). As these studies focus on the differentiation of transplanted precursor cells, the effect of endogenous BMPs on the pathogenesis of SCI remains a significant question to be addressed. In this study, we demonstrate that the local administration of noggin following SCI significantly facilitates improvement in locomotor activity and axonal regrowth.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Surgical procedure

All surgical intervention and subsequent care and treatment of all animals used in this study were in strict accordance with the guidelines of the Special Committee on Animal Welfare of Chiba University. Anesthetized (2% halothane) female Sprague-Dawley rats (180–220 gm) underwent a laminectomy at the T9/T10 vertebral level, thereby exposing the spinal cord. We contused the spinal cords by using the Infinite Horizon Spinal Cord Impactor (Precision Systems and Instrumentation, Lexington, KY, USA). The impact force was set at 200 kdyn24. Immediately after SCI, the rats were fitted with an osmotic minipump for a period of 2 weeks (volume, 200 μL; rate of infusion, 0.5 μL/h; Alzet 2002; Durect Corp., Cupertino, CA, USA). These pumps were filled with human Fc (18 animals, 17.8 μg/kg/day over 2 weeks; Sigma–Aldrich) or recombinant mouse noggin (11 animals, 17.8 μg/kg/day over 2 weeks; R&D Systems, Minneapolis, MN, USA). The minipump was placed under the skin on the animal’s back, and a silastic tube connected to the outlet of the minipump was placed under the dura of the spinal cord at the site of the contusive injury with the tip lying immediately rostral to the injury site. The tube was sutured to the spinous process just caudal to the laminectomy site to anchor it in place. Afterward, the muscle and skin layers were sutured. The bladder was expressed by manual abdominal pressure at least twice a day until bladder function was restored. Sham-operated rats (three animals) underwent a laminectomy at the T9/10 vertebral level, and the minipump was placed without contusive spinal cord injury. Rats in which the silastic tubes slipped out from the dura were excluded from the statistical analysis.

Behavioral testing

Behavioral recovery was assessed in an open field environment for 10 weeks after the injury by using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale (Basso et al. 1995). Quantification was performed in a blinded manner by two observers. The difference in the judged counts among the observers was within 1 point on the BBB scoring scale.

Anterograde labeling of the corticospinal tract

Eight weeks after injury, descending corticospinal tract (CST) fibers were labeled with biotin dextran amine (BDA; 10% in saline, total 4 μL per cortex, MW 10 000; Molecular Probes, Eugene, OR, USA) that was injected bilaterally (2 mm posterior to the bregma, 2 mm lateral to the bregma, and at a depth of 1.5 mm) under anesthesia. For each injection (four points per cortex, 1 μL for each points), 0.25 μL BDA was delivered for a 30 s period via glass capillaries with internal diameters of 15–20 μm attached to a microliter syringe (Hamilton, Reno, NV, USA). The rats were killed by perfusion with phosphate-buffered saline (PBS) followed by fixation in 4% paraformaldehyde 14 days after the BDA injection. The animals’ spinal cords were dissected and post-fixed overnight in the same fixative. Cryopreservation was undertaken in 30% sucrose in PBS. The portion of the spinal cord located 5 mm rostral and 5 mm caudal to the lesion site (total length, 10 mm) was embedded in Tissue-Tek OCT. These blocks were sectioned (50 μm) in the sagittal or transverse plane, retaining each section. In both the cases, the transverse sections were also obtained from the spinal cord located more than 5 mm rostral and caudal to the injury site. These sections were washed three times in PBS for 1 h, and incubated in the reducing agent NaBH4 (10 mg/mL). They were then permeabilized with PBS containing 0.3% Triton X-100 followed by incubation for 2 h with Alexa Fluor 488-conjugated streptavidin (1 : 400; Invitrogen, Carlsbad, CA, USA) in PBS with 0.15% bovine serum albumin.

Tissue preparation and immunohistochemistry

For immunohistochemistry, fresh frozen tissues were obtained from an uninjured spinal cord and from spinal cords at 1, 3, and 7 days after injury. After induction of deep anesthesia with diethyl ether, the rats were decapitated and the spinal cords were dissected out, embedded in Tissue-Tek OCT, and immediately frozen on dry ice at −80°C. A series of sagittal sections were cut at 16 μm on a cryostat and mounted on aminopropyltriethoxysilane (APS) coating Superfrost-Plus slides (Matsunami, Osaka, Japan). The sections were fixed in 4% paraformaldehyde for 1 h at 25°C, washed three times with PBS, and blocked in PBS containing 5% bovine serum albumin and 0.1% Triton X-100 for 1 h at 25°C. The sections were incubated with primary antibodies overnight at 4°C and washed three times with PBS, followed by incubation with fluorescein-conjugated secondary antibodies (1 : 1000; Invitrogen) for 1 h at 25°C. The anti-TuJ1 (1 : 500), monoclonal anti-MOSP (1 : 200; Chemicon, Temecula, CA, USA), monoclonal anti-glial fibrillary acidic protein (anti-GFAP) (1 : 400; Sigma–Aldrich), monoclonal anti-BMP-2/4 (1 : 50; R&D systems), polyclonal anti-BMP-RIa antibody (Abgent, San Diego, CA, USA), polyclonal anti-BMP-RII antibody (1 : 50; Abgent), monoclonal anti-CD11b antibody (BD Biosciences, San Jose, CA, USA), monoclonal anti-CSPG antibody (Sigma–Aldrich), or polyclonal anti-phospho smad1/5/8 antibody (Chemicon Int, Inc.) was used as the primary antibody. Samples were examined under a fluorescence microscope.

To assess the glial scar formation after SCI, the 10-mm-long sagittal sections of the injured spinal cords obtained from 5 Fc-treated control rats and six noggin-treated rats (as described above) were immunostained. The monoclonal anti-GFAP antibody was used as the primary antibody, and the glial scar formation was quantitatively analyzed using Scion Image software. Digital photomicroscopic images were converted into gray scale and collected using identical intensity settings with a threshold of 1-234/255 for GFAP-positive areas and a threshold of 1-248/255 for the whole area of the sagittal section. The area of GFAP-positive cells was then calculated as a percentage of the total area of each section. We calculated the estimated volume of the injured spinal cord by the same method.

Quantification

The rats that did not exhibit BDA uptake, as estimated in the axial sections 10 mm rostral from the site of the lesion, were excluded from the quantification. In total, we examined and compared the regenerative responses of 5 Fc-treated control rats and 6 noggin-treated rats after SCI. The total pixel number for the BDA-positive fibers in the axial section 10 mm rostral from the lesion site was assessed. To reconstruct serial parasagittal sections completely, all the serial 50-μm-thick sections were evaluated. All the serial sagittal sections were reconstructed into axial images, and the pixel number for the BDA-labeled fibers in the gray matter was counted using Scion Image software (Scion Corporation, Frederick, MD, USA) from 4 mm above to 4 mm below the lesion center. The pixel number of BDA-labeled fibers was calculated as a percentage of the fibers observed 4 mm above the lesion.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Enhanced expression of BMP-RII and BMP-2 after SCI

To assess whether BMPs play a role in inhibiting axonal regeneration after SCI, we performed immunohistochemistry to investigate the distribution pattern of BMP-RII, receptor for BMPs, in the adult rat spinal cord. Fresh frozen sections of the spinal cords were obtained at 1, 3, and 7 days after SCI as well as from sham-operated control rats. Double immunostaining was performed using anti-TuJ1/anti-BMP-RII antibodies (Fig. 1a), anti-GFAP/anti-BMP-2/4 antibodies (Fig. 1b), or anti-myelin/oligodendrocyte-specific protein (MOSP)/anti-BMP-2/4 antibodies (Fig. 1c). Although we observed minimal signals for BMP-RII in the sham-operated control spinal cords (Fig. 1a), the induction of BMP-RII in the TuJ1-positive neurons was observed in the gray matter of the spinal cord at 7 day following SCI (Fig. 1a). The BMP-RII signals appeared in the neurons as early as 1 day after SCI (data not shown). The noggin treatment did not change the expression of BMP-RII after SCI (Fig. 1d). We further determined the distribution pattern of BMP-RIa, another receptor for BMPs. Similar to the findings in BMP-RII, the induction of BMP-RIa in the TuJ1-positive neurons was observed in the gray matter of the spinal cord at 7 day following SCI (Fig. 1e).

image

Figure 1.  Induction of BMP receptors after SCI. (a) BMP-R II expression is induced in the neurons in the gray matter 7 days after SCI. Fixed tissues were obtained from the spinal cords of sham-operated rats (Sham) or rats with SCI (SCI). The sections were double-stained with the anti-TuJ1 and anti-BMP-RII antibodies. BMP-RII is expressed in the neuronal cell bodies in the gray matter rostral to the lesion site at 7 day following SCI, whereas few signals were observed in the sham-operated controls. Scale bar: 10 μm. (b) Double-labeling with anti-BMP-RII and anti-GFAP antibodies shows no colocalization in the epicenter area 7 days after SCI (SCI). SCI + Noggin; SCI with the noggin treatment. Scale bar: 100 μm. (c) Double-labeling with anti-BMP-2/4 and anti-MOSP antibodies shows no colocalization in the epicenter area 7 days after SCI (SCI). SCI + Noggin; SCI with the noggin treatment. Scale bar: 100 μm. (d) Induction of BMP-R II is observed 7 days after SCI (SCI). The noggin treatment did not change the expression of BMP-R II (SCI + Noggin). Scale bar: 100 μm. (e) BMP-RIa expression is induced in the neurons in the gray matter 7 days after SCI. Fixed tissues were obtained from the spinal cords of sham-operated rats (Sham) or rats with SCI (SCI). The sections were double-stained with the anti-TuJ1 and anti-BMP-RIa antibodies. BMP-RIa is expressed in the neuronal cell bodies in the gray matter rostral to the lesion site at 7 day following SCI, whereas few signals were observed in the sham-operated controls. SCI + Noggin; SCI with the noggin treatment. Scale bar: 100 μm.

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The immunoreactivity for BMP-2/4 was very weak in the sham-operated spinal cords (Fig. 2a–c) and was strongly enhanced in MOSP-positive oligodendrocytes (Fig. 2a) and GFAP-positive astrocytes (Fig. 2b), but not in Tuj1-positive neurons (Fig. 2c) around the injury site at 7 day after SCI. The noggin treatment did not change the expression of BMP-2/4 after SCI (Fig. 2d). The immunoreactivity for BMP-2/4 was observed in CD11b-positive microglia/macrophages (Fig. 2d), which were accumulated around the damaged tissues after SCI. The BMP-2/4 signal was observed within 1 day after SCI (data not shown). The noggin treatment did not change the expression of BMP-2/4 after SCI (Fig. 2e). Thus, BMP-RII and BMP-RIa induced in the neurons, whereas the level of BMP-2/4 increased in oligodendrocytes as well as in reactive astrocytes and microglia/macrophages around the injury site after SCI. These findings are consistent with the previous observations showing up-regulation of BMP-2 and BMP-7 following SCI (Setoguchi et al. 2001, 2004), and therefore, increase the prospects of the inhibition of axonal regrowth by BMPs after SCI.

image

Figure 2.  Induction of BMP-2/4 after SCI. (a) Double-labeling with anti-BMP-2/4 and anti-MOSP antibodies shows colocalization in the epicenter area 7 days after SCI. Faint signals for BMP-2/4 were observed in the sham-operated control spinal cords. Scale bar: 10 μm. (b) Double-labeling with anti-BMP-2/4 and anti-GFAP antibodies shows co-localization in the epicenter area 7 days after SCI. Faint signals for BMP-2/4 were observed in the sham-operated control spinal cords. Scale bar: 10 μm. (c) Double-labeling with anti-BMP-2/4 and anti- TuJ1 antibodies shows no co-localization in the epicenter area 7 days after SCI (SCI). SCI + Noggin; SCI with the noggin treatment. Scale bar: 100 μm. (d) Double-labeling with anti-BMP-2/4 and anti-CD11b antibodies shows co-localization in the epicenter area 7 days after SCI (SCI). Faint signals for BMP-2/4 as well as CD11b were observed in the sham-operated control spinal cords. SCI + Noggin; SCI with the noggin treatment. Scale bar: 100 μm. (e) Induction of BMP-2/4 is observed 7 days after SCI (SCI). The noggin treatment did not change the expression of BMP-R II (SCI + Noggin). Scale bar: 100 μm.

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Noggin treatment enhances functional improvement after SCI

Noggin is a 32-kDa glycoprotein secreted by the Spemann organizer of Xenopus embryos. Noggin antagonizes the action of BMPs by blocking binding to the receptors; further, it induces the formation of neural tissue and dorsalizes the ventral mesoderm. Further, noggin binds directly to BMP-2 and BMP-4 with high affinity and to BMP-7 with low affinity; however, it does not bind to activin or TGF-β (Yanagita 2005). Therefore, noggin is an excellent tool to estimate the specific involvement of BMPs in the pathogenesis of SCI. Prior to in vivo assessment, we investigated whether noggin could neutralize the effect of rhBMP-2 in vitro. The results show that the noggin treatment completely abolished the effect of rhBMP-2 on the neurite growth of the CGNs in vitro (data not shown).

We then decided to assess the effect of noggin in vivo. We employed a rat spinal contusion model since it is relevant to clinical conditions. The spinal cords at the thoracic level (T9 and T10) were contused by an impactor force of 200 kdyn (Cao et al. 2005). Either noggin or a human Fc acting as a control was delivered via osmotic minipumps with intrathecal catheters close to the thoracic injury site. The sham-operated rats underwent laminectomy and catheter placement without contusion. The locomotor performance of the animals was monitored over a period of 10 weeks after SCI. The sham-operated rats achieved full scores according to the BBB locomotor rating scale (Basso et al. 1995) within 3 weeks (Fig. 3a), although we observed mild worsening of the locomotor activity during the first 2 weeks. This may be due to the compression of the spinal cord by the intrathecal catheters. All the injured rats became almost completely paraplegic on the first day after the injury (Fig. 3a) and then gradually displayed partial recovery of locomotor behavior as assessed by the BBB scores. There was no difference in the BBB scores of the noggin-treated rats and the Fc-treated control rats up to 4 weeks after the SCI. The locomotor performance thereafter showed increased improvement in the rats treated with noggin as compared to those treated with the control Fc. On an average, rats treated with the control Fc attained a BBB score of 9.5, whereas those treated with noggin achieved a significantly better BBB score of 13.0 at 10 weeks after the surgery. Thus, the noggin treatment improved locomotor recovery following spinal cord contusion in rats.

image

Figure 3.  Noggin treatment promotes locomotor recovery after SCI. (a) The BBB score was determined at the indicated times after thoracic contusion in the control Fc-treated (control Fc), noggin-treated (Noggin), and sham-operated (sham) rats. Data are represented as the mean ± SEM of 18, 11, and 3 rats for the Fc-treated control, noggin-treated, and sham-operated rats, respectively. The average score of the noggin-treated group is statistically different from that of the control Fc group. *, p < 0.05 (Student’s t-test) compared with the control. (b) The volume of the spared spinal cord after SCI. There was no significant difference between the volumes of the control Fc-treated and noggin-treated rat spinal cords. Data are represented as the mean ± SEM of 18 Fc-treated control rats and 11 noggin-treated rats.

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Noggin treatment induces the sprouting of corticospinal tract axons after SCI

Initially, we examined whether the noggin treatment reduced or increased the lesion volume. There was no significant difference in the total volume of the spinal cord between the control and noggin-treated groups (Fig. 3b), suggesting that the noggin treatment did not increase or decrease the extent of destruction of the tissues after SCI.

The integrity of the descending CST was then assessed by injecting biotinylated dextran amine (BDA) into the bilateral sensory-motor cortices. For a group of animals (five control rats and six noggin-treated rats), blocks extending 5 mm rostral and 5 mm caudal to the center of the injury were sectioned in the sagittal plane (Fig. 4m and n). Rostral and caudal segments of 10 mm each were sectioned in the axial plane (Fig. 4a–l). Representative axial sections of Fc-treated control rats (Fig. 4a–c) and the noggin-treated rats (Fig. 4g–i) taken at 10 mm above the lesion are shown. Quantitative analysis of BDA-labeled fibers was performed by counting the number of these fibers (Fig. 4o). The total number of labeled fibers observed 10 mm above the lesion was approximately equal in the Fc-treated control rats (n = 5) and the noggin-treated rats (n = 6), indicating that the extent of BDA uptake was the same in both the groups. Almost no fibers were observed 10 mm caudal to the lesion site in the ventral part of the dorsal or dorsolateral column in the noggin-treated rats (Fig. 4j, k) as well as in the control rats (Fig. 4d, e). This demonstrated that no dorsal or dorsolateral CST axons were spared since axons that are spared and that extend into the caudal segments should be present in their normal locations. However, the sprouting axons extended through the gray matter to a greater extent than in the white matter in rats treated with noggin (Fig. 4j and l) but not in the corresponding regions of the Fc-treated control rats (Fig. 4f). The longitudinal sections across the lesion site revealed lesser retraction of the dorsal CST bundles from the lesion epicenter in rats treated with noggin (Fig. 4n) than in those from the lesion epicenter in the control rats (Fig. 4m). We then reconstructed serial parasagittal sections of injured spinal cords and estimated the number of labeled fibers. The number of BDA-labeled fibers in the spinal cord was counted from 4 mm above to 4 mm below the lesion epicenter and was calculated as a percentage of the fibers observed 4 mm above the lesion (Fig. 4p). In the control rats, the percentage of fibers observed was low, whereas the number of labeled fibers observed rostral (1 and 2 mm) and caudal (2 mm) to the lesion site of the spinal cord in rats treated with noggin was significantly higher. Since we observed no labeled fibers in the normal locations of the dorsal CST caudal to the lesion site in any of the injured rats (Fig. 4d, j), the increase in the number of labeled fibers after the noggin treatment was not resulting from an increased survival of the dorsal CST axons. Thus, these results demonstrate that noggin treatment promoted significant fiber growth from the intact ventral CST or the injured dorsal CST after SCI. In addition, this increased growth of the CST occurred at the proximal as well as the distal segments of the CST.

image

Figure 4.  Noggin promotes the sprouting of CST axons after SCI. (a–l) Representative axial sections of BDA-labeled CST fibers. Anterograde-labeled CST fibers in control Fc-treated (a and d) or noggin-treated (g and j) spinal cords 10 weeks after SCI; 10 mm rostral (a and g) and 10 mm caudal (d and j) from the lesion epicenter. Arrows in (j) indicate sprouting labeled CST fibers. Scale bars: 1 mm. Higher magnification views of the boxed regions in (a), (d), (g), and (j) are shown to the right (b, c, e, f, h, i, k, and l). (m and n) Anterograde-labeled CST fibers in control Fc-treated (m) and noggin-treated (n) spinal cords 10 weeks after injury. The rostral site is indicated to the left, and the epicenter areas of the lesion are indicated by asterisks. Bundles of the descending CST (indicated by arrows) are closer to the epicenter in the noggin-treated rats (n) than in the control Fc-treated rats (m). Scale bars: 1 mm. (o) The number of labeled corticospinal axons 10 mm rostral to the lesioned site in rats treated with the control Fc (n = 5) and noggin (n = 6). No significant difference is observed between these groups. (p) Serial parasagittal sections were reconstructed and evaluated. The number of BDA-labeled fibers in the gray matter was counted from 4 mm above to 4 mm below the lesion epicenter and calculated as a percentage of the fibers observed 4 mm above the lesion. Collateral sprouting as indicated by the BDA-labeled fibers in the gray matter increased in the rats treated with noggin (also see l) but not in the corresponding regions of the control Fc-treated rats (also see f). The x-axis indicates specific locations along the rostro-caudal axis of the spinal cord. The y-axis indicates the ratio of the number of BDA-labeled fibers at the indicated site to those 4 mm rostral to the lesion site. *, p < 0.05 compared with the control (Student’s t-test). Error bars indicate SEM.

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Noggin treatment does not affect the glial scar formation after SCI

BMPs are known to promote the astrocytic differentiation of embryonic and adult neural stem cells. Since reactive astrocytes are sources of axonal growth inhibitors such as CSPG, the inhibition of astrocytic differentiation by noggin may result in increased axonal growth after SCI. Thus, we quantitatively analyzed reactive astrogliosis in injured rat spinal cords. The sagittal sections of the injured spinal cords obtained from the control and the noggin-treated rats were immunostained using the anti-GFAP antibody to detect the reactive astrocytes. We observed intensive immunoreactivity for GFAP around the lesion site following SCI both in the control rats (Fig. 5a and c) and the noggin-treated rats (Fig. 5b and d). The total areas immunoreactive for GFAP around the lesion epicenter were measured to estimate the extent of astrogliosis at 10 weeks after SCI. Results demonstrate that the extent of astrogliosis was not significantly different between the groups (Fig. 5e). We further examined expression of chondroitin sulfate proteoglycans (CSPGs), potent inhibitors of axon regeneration (Yiu and He 2006). However, expression of CSPG was not significantly changed by the Noggin treatment following SCI (Fig. 5f). In addition, we observed no significant increase in the CD11b-positive cells by the noggin treatment after SCI (data not shown). Finally, we assessed whether the noggin treatment inhibited the signaling pathway downstream of BMPs. We performed immunohistochemistry and found that the Noggin treatment effectively attenuated the phosphorylation of Smad1/5/8 (Fig. 5g). Thus, the noggin effectively suppressed the effect of BMP-2/4. These findings do not support the hypothesis that the noggin treatment decreased the number of reactive astrocytes around the lesion site, thus allowing the axons to elongate beyond the glial scar.

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Figure 5.  Glial scar formation after SCI. (a–d) Representative photomicrographs of control Fc-treated (a and c) and noggin-treated rats (b and d). Sagittal sections of injured spinal cords were immunostained with the anti-GFAP antibody (green). (c and d) Higher magnification views of (a) and (b), respectively. Scale bars: (a and b) 200 μm, (c and d) 50 μm. (e) Quantification of the glial scar formation. The sagittal sections obtained from five control-Fc and six noggin-treated rats were analyzed using Scion Image software. The area of positive-GFAP immunoreactivity was calculated as a percentage of the total area of each section. Data are represented as the mean ± SEM of three independent experiments. No significant difference was observed between the two groups. (f) CSPG expression is induced 7 days after SCI (control SCI). Fixed tissues were obtained from the spinal cords of sham-operated rats (Sham), rats with SCI (control SCI), or rats with SCI and the noggin treatment (Noggin). The sections were double-stained with the anti-GFAP and anti-CSPG antibodies. The induction of CSPG is still observed after SCI with the noggin treatment. (g) Phosphorylation of smad1/5/8 was observed in the neurons after SCI, whereas the noggin treatment attenuated it. Double-labeling with anti-phospho smad1/5/8 and anti-TuJ1 antibodies shows colocalization in the epicenter area 7 days after SCI (control SCI). Faint signals for phospho smad1/5/8 were observed in the sham-operated control spinal cords (Sham) as well as the noggin-treated SCI rats spinal cords (Noggin) Scale bar: 100 μm.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In this study, we observed that the expression of BMP-2 was induced in reactive astrocytes and oligodendrocytes around the injury site after SCI. It is evident that CNS myelin exerts multiple layers of inhibitory influences in vivo as well as in vitro (Yiu and He 2006). In addition to myelin, another important source of inhibition is the glial scar, which forms after CNS injury. Many astrocytes in the injured area often become hypertrophic, adopt a reactive phenotype, and release CSPG (McKeon et al. 1991). After injury, CSPG expression is rapidly up-regulated by reactive astrocytes, forming an inhibitory gradient that is highest at the center of the lesion. Intrathecal administration of chondroitinase ABC, an enzyme that removes glycosaminoglycan (GAG) chains from the protein core, following SCI promoted both the regeneration of various axon tracts as well as functional recovery (Bradbury et al. 2002). Therefore, in combination with these findings, our data suggest that multiple inhibitors expressed in the reactive astrocytes as well as oligodendrocytes may form a barrier to inhibit axonal sprouting and functional recovery.

The adult rat spinal cord responds to injury with a dramatic increase in the expression of BMP-2 and BMP-7 (Setoguchi et al. 2001, 2004). Although BMPs have been reported to inhibit neurogenesis and alternatively promote gliogenesis, the inhibition of BMPs by the transplantation of neural stem cells engineered to express noggin – an endogenous antagonist of BMP action – into the injured spinal cord does not antagonize terminal astroglial differentiation in the grafted stem cells (Enzmann et al. 2005). It was also reported that the differentiation of neural precursor cells into neurons and oligodendrocytes was promoted when noggin-expressing neural precursor cells were transplanted after SCI (Setoguchi et al. 2004). As these studies focus only on the differentiation of transplanted precursor cells, the effect of endogenous BMPs on the pathogenesis of SCI remains a significant question to be addressed. Thus, our present study suggesting that BMPs play a role in inhibiting axon regeneration in vivo provides new roles of BMPs in pathological conditions.

We observed no significant change in the reactive astrogliosis or lesion volume, as measured by the total volume of the spinal cord, in the control SCI rats and noggin-treated SCI rats. These observations were surprising as a previous report demonstrated that the transplantation of the neural precursor cells engineered to express noggin increased the lesion volume after SCI (Enzmann et al. 2005). Although the reason for these conflicting results is yet to be elucidated, it is possible that the amount of noggin used in this study had a minimal effect on astrogliosis and tissue destruction. We chose the two-week window for infusing noggin, as many other protocols including Rho-kinase inhibitors and the antibody to repulsive guidance molecule employed the same time window for the treatment of SCI (Kubo et al. 2007; Yamashita et al. 2007). However, it is possible that the additional noggin treatment over a longer time period has additional effects on the locomotor recovery and/or gliosis. In addition, it is noted that our study did not assess myelination of the oligodendrocytes after SCI. These issues should be examined in the future.

We recently reported that BMP-2 inhibits the neurite outgrowth of postnatal cerebellar neurons in vitro (Matsuura et al. 2007). Although receptor-regulated Smad proteins are activated by BMP-2, this signal transduction is not necessary for the inhibitory effect of BMP-2. Interestingly, BMP-2 activates LIM-kinase 1 in the neurons, and the dominant negative form of LIM-kinase 1 abolishes the effect of BMP-2. Thus, LIM-kinase might play a role in inhibiting axon regeneration after SCI.

In summary, our results demonstrate that BMPs are important inhibitory molecules for successful neuroregeneration in vivo and suggest that their manipulation would be useful for the treatment of human spinal injuries.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This work was supported by a Research Grant from the National Institute of Biomedical Innovation (05–12), Grant-in-Aid for Young Scientists (S) from JSPS, and by the Research Grant (18A-8) for Nervous and Mental Disorders from the Ministry of Health, Labour and Welfare.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References