Address correspondence and reprint requests to Toshihide Yamashita, Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: firstname.lastname@example.org
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.
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
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 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.
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.
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).
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.
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.
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.
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.
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.
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.