- Email: [email protected]
The effects of combining chondroitinase ABC and NEP1-40 on the corticospinal axon growth in organotypic co-cultures
Neuroscience Letters 476 (2010) 14–17
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
The effects of combining chondroitinase ABC and NEP1-40 on the corticospinal axon growth in organotypic co-cultures Toshio Nakamae ∗ , Nobuhiro Tanaka, Kazuyoshi Nakanishi, Naosuke Kamei, Hirofumi Sasaki, Takahiko Hamasaki, Kiyotaka Yamada, Risako Yamamoto, Bunichiro Izumi, Mitsuo Ochi Department of Orthopaedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan
a r t i c l e
i n f o
Article history: Received 18 February 2010 Received in revised form 15 March 2010 Accepted 18 March 2010 Keywords: Chondroitinase ABC NEP1-40 Axon regeneration Spinal cord injury Organotypic co-culture
a b s t r a c t The area surrounding the injured spinal cord is a non-permissive milieu for axonal growth due to the inhibitory factors, especially chondroitin sulfate proteoglycan (CSPG) and Nogo. Recent studies have reported that chondroitinase ABC (ChABC) or Nogo-66(1-40) antagonist peptide (NEP1-40) promote axonal growth after spinal cord injury. But no study has addressed the effects on spinal cord injury of combining ChABC and NEP1-40. Previously, we described an organotypic co-culture system using the brain cortex and spinal cord from neonatal rats. In this study, we examined whether the combination of ChABC and NEP1-40 creates an action that promotes corticospinal axon growth in organotypic co-cultures. Organotypic co-cultures of brain and spinal cord were prepared from rats, and ChABC or NEP1-40 was delivered to them. To examine the effects of this combination these two drugs were applied together. We counted the number of labeled axons with DiI and assessed the immunoreactivity of CSPG and Nogo. Axonal growth was enhanced by infusing ChABC or NEP1-40 compared with that in the control group, whereas synergistic effects of combined administration of ChABC and NEP1-40 on axonal growth were not observed. There is a possibility that ChABC and NEP1-40 affect the same intracellular pathways and have no synergistic influence on axonal growth. © 2010 Elsevier Ireland Ltd. All rights reserved.
Spinal cord injury (SCI) in adult mammals results in sensory and motor deficits. Recent studies have demonstrated significant functional and histological recovery of injured spinal cord in animal models. However, there is a non-permissive milieu for repair surrounding the injured spinal cord [7,11,12]. After a central nerve system (CNS) injury, several axonal growth inhibitory factors, including the chondroitin sulfate proteoglycan (CSPG) and Nogo, are upregulated around the injured spinal cord. CSPG is originally the extracellular matrix contained within a glial scar. Nogo is one of the myelin-associated proteins and is thought to regulate the abnormal axonal growth in the embryogenic stage. Recent studies have reported that digestion and blocking of these factors induce promotion of axonal growth and functional recovery [1,3,5,6]. For example, chondroitinase ABC (ChABC) induces axonal regeneration and functional recovery by digesting the glycosaminoglycan (GAG) side chain of CSPG [1,3] and Nogo-66(1-40) antagonist peptide (NEP1-40) promotes axonal growth following SCI due to blocking Nogo-66/NgR interaction [5,6]. But it is not enough to assess the effects of axonal growth quantitatively, and the therapeutic effects
∗ Corresponding author. Tel.: +81 82 257 5233; fax: +81 82 257 5234. E-mail address: [email protected] (T. Nakamae). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.03.049
are not well known. Furthermore, there is no study that addresses the effect of combining ChABC and NEP1-40 on spinal cord injury. Previously we demonstrated how an organotypic co-culture system using the brain cortex and spinal cord from neonatal rats helps to assess the corticospinal tract’s (CST) axonal growth quantitatively and facilitates the analysis of factors that regulate axonal growth [10,15,16]. The purpose of this study was to examine whether the combination of these two extrinsic approaches, ChABC and NEP1-40 application, facilitate axonal growth from the brain cortex to the spinal cord using an organotypic co-culture system. Our research methods were reviewed and approved by the ethical committee of Hiroshima University. An organotypic co-culture of brain cortex and spinal cord was prepared as reported previously [16]. Brains and thoracic spinal cords were collected from Sprague–Dawley (SD) rats on postnatal day 7 (P7). The brains were sectioned using a Vibratome (Dosaka EM, Kyoto, Japan). The region of the sensorimotor cortex was dissected from the coronal sections, and thoracic spinal cord was bisected in the sagittal plane using a razor blade. The dissected cortex and spinal cord were placed on membranes (Millicell-CM; Millipore, Billerica, MA, USA), in 1 ml of serum-based medium (50% basal medium Eagle with Earle’s Salts (BME; Sigma, St. Louis, MD, USA), 25% heat inactivated horse serum (Gibco, Grand Island, NY, USA) 25% Earle’s Balanced Salt Solution
T. Nakamae et al. / Neuroscience Letters 476 (2010) 14–17
15
Fig. 1. Axons projecting from the brain cortex to the spinal cord. A photomicrograph of an organotypic co-culture (a). Minimal axons are seen in the control group (b). Axonal growth is promoted by the administration of ChABC (c) or NEP1-40 (d) or both ChABC and NEP1-40 (e). (b–e) High magnification of the box pointed by arrow head in a photomicrograph (a). (a) Bar = 200 m, (b–e) bar = 40 m.
(EBSS; Sigma), 1 mM l-glutamine and 0.5% d-glucose) in a 6-well tissue culture plate. The cortex and the spinal cord were incubated for 1 day, on the second day, the spinal cord pieces were aligned next to the white matter of the cortex (Fig. 1a). The co-cultures were incubated in a humidified atmosphere with 5% CO2 at 37 ◦ C. The medium was replaced every 3 days. The co-cultures were incubated for up to 14 days. Drugs were administered into the co-cultures just after the cortical tissue and the spinal cord tissue came into contact with one another. They were dropped onto the junction of the brain cortex and spinal cord in a quantity of 2 l every 3 days during incubation. To degrade CSPG, ChABC (Seikagaku, Tokyo, Japan) was delivered to the co-culture at a concentration of 1 U/ml (ChABC group). To block Nogo-66/NgR interaction, NEP1-40 (Sigma) was applied at a concentration of 0.4 mg/ml (NEP group). To examine the effects of the combination, these two drugs were dropped together onto the co-cultures (combined group). For the control, administration of only medium was performed (control group). The next stage was for the co-culture tissues to be fixed onto the membranes and incubated overnight at 4 ◦ C in 4% paraformaldehyde followed by immunostaining with mouse monoclonal antibodies against CSPG (1:500, Chemicon), and rabbit polyclonal antibodies against Nogo (1:100, Santa Cruz, CA, USA). We assessed the degradation of chondroitin sulfate CS-GAG with mouse monoclonal antibodies against 2B6 (1:500, Seikagaku). 2B6 is recognized an epitope on the CSPG core proteins but not the intact CS-GAG. On the following day, the tissues were exposed for 1 h to Alexa Fluor 488-conjugated goat anti-rabbit antibodies (1:400, Molecular Probes, Eugene, OR, USA) and Alexa Fluor 568conjugated goat anti-mouse antibodies (1:400, Molecular Probes) at room temperature, and observed under a confocal laser microscope (Carl Zeiss, Oberkochen, Germany). Axon projections from the cortex to the spinal cord were labeled by anterograde tracing with DiI. The co-cultures were fixed for 5 days in 4% paraformaldehyde at 4 ◦ C. Small crystals of DiI were placed on the center of the cortex, and the co-cultures were incubated for another 14 days in 0.1 M phosphate buffer in a humidified atmosphere at 37 ◦ C. The co-cultures were mounted on glass slides and covered with Vectashield (Vector, Burlingame, CA, USA) and glass coverslips. To analyze the axonal growth, we counted the number of labeled axons passing through a reference line running
parallel to the junction between the brain cortex and spinal cord 500, 1000 and 1500 m from the junction toward the spinal cord. We evaluated the axonal growth using images that were taken at a magnification of 200×, at different focal planes. All fibers crossing the reference line were counted, and the counts were expressed as the average number of axons per culture. Results were expressed as mean ± standard errors (S.E.). The statistical significance of differences in parameters was assessed by the Kruskal–Wallis test with a post hoc test using the Scheffe procedure. For all data collection, the researchers were blinded to the group identities. In the ChABC group, the immunostaining of CSPG decreased around the junction of brain cortex and spinal cord compared with the control group (Fig. 2a and b), whereas with 2B6 expression increased (Fig. 2c and d). On the other hand, immunoreactivity of Nogo-A decreased by applying NEP1-40 (Fig. 2e and f). In the control group, rare labeled axons were seen (Fig. 1b). The average number of axons that extended 500 m past the junction was 0.6 ± 0.3 (n = 12 cultures/group), whereas in the ChABC group and in the NEP group, axonal growth was enhanced in comparison to the control group (Fig. 1c and d). In the ChABC group and in the NEP group, the average number of axons that extended 500 m past the junction was 3.9 ± 0.4 (n = 12 cultures/group) and 5.1 ± 1.3 (n = 8 cultures/group), respectively. The average number of axons in the ChABC group and in the NEP group was statistically significantly higher than that in the control group. In the combined group (Fig. 1e), the average number of axons that extended 500 m past the junction was 4.9 ± 1.0 (n = 12 cultures/group). This was statistically significantly higher than that in the control group, but this was not significantly higher than that in the ChABC group or in the NEP group. In the ChABC group, NEP group and combined group, the average number of axons that extended 1000 or 1500 m past the junction was not statistically significantly higher than that in the control group (Fig. 3). After spinal cord injury, axonal growth of adult mammalians is limited in part by the presence of growth inhibitory molecules in the glia scar and CNS myelin. CSPGs and three myelin-associated proteins, namely Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), are thought to be the main molecular obstacles to axonal growth [2,4,7,11]. CSPGs are
16
T. Nakamae et al. / Neuroscience Letters 476 (2010) 14–17
Fig. 2. Immunological staining of CSPG, 2B6 and Nogo. In the control group, immunoreactivity of CSPG (red) increases (a), whereas in the ChABC group (b), CSPG expression decreases compared with the control group. Immunoreactivity of 2B6 (red) increases in the ChABC group (d) compared with the control group (c). In the control group, immunoreactivity of Nogo-A (green) increases (e). Compared with the control group, Nogo-A expression decreases in the NEP1-40 group (f). (a, b, e, f) Bar = 20 m, (c, d) bar = 100 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
upregulated via astrocytes and oligodendrocytes in the injury site after SCI to limit axonal regeneration [13,17–19]. CSPGs are extracellular matrix molecules and they belong to a family of molecules characterized by a protein core to which large sulfated GAG chains are attached. It has been reported that ChABC liberates CS-GAGs from CSPG core proteins to render a more permissive substrate for axonal outgrowth [1,3]. On the other hands, a 66-residue domain of Nogo (Nogo-66) is expressed on the surface of oligodendrocytes and can inhibit axonal outgrowth through an axonal Nogo-66 receptor (NgR). The Nogo-66(1-40) antagonist peptide (NEP1-40) blocks Nogo-66 or CNS myelin inhibition of axonal outgrowth in vitro [6]. However, no study has addressed the combined blockade of CSPG and Nogo inhibitor after SCI. In this study, we have examined the effects of the combined therapies of ChABC and NEP1-40 using an organotypic co-culture system with brain cortex and spinal cord from neonatal rats [10,15,16]. The advantage of this co-culture system is to assess CST axonal growth quantitatively and to facilitate the analysis of factors that regulate axonal growth. Oishi et al. reported that a major advantage of the co-cultures is that they allow easy delivery of
agents at known concentrations, and quantitative assessment of axon growth, and so the cultures will be useful for screening potential growth promoting substances and for combinatorial strategies [16]. They have also demonstrated that the failure of axonal growth is due to the development of a non-permissive tissue substrate with an organotypic co-culture, and that the most prominent difference between the P3 co-cultures and the P7 co-cultures is the amount of gliosis [16]. Glia scars not only present a physical barrier, but also produce molecules such as CSPG, which hamper axonal regeneration. Previously, we demonstrated that immunoreactivity of CSPG was observed around the junction of the brain cortex and spinal cord in the P3 co-culture group, and that immunoreactivity of CSPG increased in the P7 co-culture group [15]. In the present study, we demonstrated that the administration of ChABC caused CSPG expression to decrease and 2B6 expression to increase and furthermore, axonal growth from the brain cortex to the spinal cord was promoted by the administration of ChABC. Additionally, administration of NEP1-40 reduced the expression of Nogo and induced axonal growth significantly in an organotypic co-culture. This result
T. Nakamae et al. / Neuroscience Letters 476 (2010) 14–17
17
treatment for spinal cord injury through the appropriate combination of blocking agents of axonal growth inhibitory factors. Acknowledgement The authors thank Prof. Norio Sakai (Department of Molecular and Pharmacological Neuroscience, Hiroshima University) for the use of the instruments. References
Fig. 3. The quantitative assessment of axonal growth from the cortex to the spinal cord. The average number of axons in the ChABC group and that in the NEP1-40 group are statistically significantly higher than that in the control group at a distance of 500 m from the junction. The average number of axons in the combination group is statistically significantly higher than that in the control group, but this is not significantly higher than that in the ChABC group or in the NEP group (*p < 0.05 Scheffe).
reflects the contribution of Nogo-66 to axonal growth inhibition after SCI. However, the combined treatment of these two drugs did not improve compared to the results when ChABC or NEP1-40 was used separately. One might assume that ChABC affected Nogo and NEP1-40 affected CSPG. But Mingorance et al. reported that NEP140 treatment did not modify CSPG up-regulation and conversely, ChABC treatment did not affect Nogo-A or NgR expression level [14]. There might be a possibility that the amount of axonal regeneration in this culture system is maximal after either treatment; hence there could be no additive effect. But there is another study, which used the same organotypic co-cultures of P7 rats, showing the superior axon growth after transplantation of neural progenitor cells compared with the axon growth in the present study [10]. This data indicates that there is not saturation effect after treatments of ChABC and/or NEP1-40 in present study. We believed that axon growth could be enhanced if a non-permissive milieu for repair changed to the permissive state. Recently, glial inhibitors and intracellular signaling mechanisms have been studied [8,9,14]. The signaling components that are common to both CSPG and myelin inhibition are the activation of RhoA and the rise in intracellular calcium. Whilst the signals downstream of RhoA that lead to the action cytoskeleton are well characterized, the relationship between components upstream of RhoA and the role of calcium influx are still ambiguous. In this study, ChABC alone may reduce inhibitory intracellular pathways to an inactivated level at which the incubation with NEP1-40 has no additive effect. We therefore speculate that the signaling of CSPG and Nogo converges in the inhibitory intracellular pathways after SCI. Further studies are necessary in order to confirm a sufficiently effective
[1] A.W. Barrit, M. Davies, F. Marchand, R. Hartley, J. Grist, P. Yip, S.B. McMahon, E.J. Bradbury, Chondroitinase ABC promotes sprouting of intact and injured spinal systems after spinal cord injury, J. Neurosci. 26 (2006) 10856–10867. [2] P. Bovolenta, F. Wandosell, M. Nieto-Sampedro, Characterization of a neurite outgrowth inhibitor express after CNS injury, Eur. J. Neurosci. 5 (1993) 454– 465. [3] E.J. Bradbury, L.D. Moon, R.J. Popat, V.R. King, G.S. Bennett, P.N. Patel, J.W. Jawcett, S.B. McMahon, Chondroitinase ABC promotes functional recovery after spinal cord injury, Nature 416 (2002) 636–640. [4] P. Caroni, M.E. Schwab, Antibody against myelin-associated inhibitor of neurite growth neutralizes nonpermissive substrate properties of CNS white matter, Neuron 1 (1988) 85–96. [5] M. Domeniconi, M.T. Filbin, Overcoming inhibitors in myelin to promote axonal regeneration, J. Neurol. Sci. 233 (2005) 43–47. [6] T. GrandPre, S. Li, S.M. Strittmatter, Nogo-66 receptor antagonist peptide promotes axonal regeneration, Nature 417 (2002) 547–551. [7] N.Y. Harel, S.M. Strittmatter, Can regenerating axons recapitulate developmental guidance during recovery from spinal cord injury? Nat. Rev. Neurosci. 7 (2006) 603–616. [8] Y. Hasegawa, M. Fujitani, K. Hata, M. Tohyama, S. Yamagishi, Y. Yamashita, Promotion of axon regeneration by myelin-associated glycoprotein and Nogo through divergent signals downstream of Gi/G, Neuroscience 24 (2004) 6826–6832. [9] Z. He, V. Koprivica, The Nogo signaling pathway for regeneration block, Rev. Neurosci. 27 (2004) 341–368. [10] N. Kamei, Y. Oishi, N. Tanaka, O. Ishida, Y. Fujiwara, M. Ochi, Neural progenitor cells promote corticospinal axon growth in organotypic co-cultures, Neuroreport 15 (2004) 2579–2583. [11] B.P. Liu, W.B. Cafferty, S.O. Budel, S.M. Strittmatter, Extracellular regulators of axonal growth in the adult central nervous system, Philos. Trans. R. Soc. Lond. B Biol. Sci. 361 (2006) 1593–1610. [12] A.W. McGee, S.M. Strittmatter, The Nogo-66 receptor: focusing myelin inhibition of axon regeneration, Trends Neurosci. 26 (2003) 193–198. [13] R.J. MaKeon, R.C. Schreiber, J.S. Rudge, J. Silver, Reduction of neurite outgrowth in a model of glia scaring following CNS injury is correlated with the expression of inhibitory molecules on reactive astrocytes, J. Neurosci. 11 (1991) 3398–3411. [14] A. Mingorance, M. Sole, V. Muneton, A. Martinez, M. Nieto-Sampedro, E. Soriano, J.A. del Rio, Regeneration of lesioned entorhino-hippocampal axons in vitro by combined degradation of inhibitory proteoglycans and blockade of Nogo-66/NgR signaling, FASEB J. 20 (3) (2006) 491–493. [15] T. Nakamae, N. Tanaka, K. Nakanishi, N. Kamei, H. Sasaki, T. Hamasaki, K. Yamada, R. Yamamoto, Y. Mochizuki, M. Ochi, Chondroitinase ABC promotes corticospinal axon growth in organotypic cocultures, Spinal Cord 47 (2) (2009) 161–165. [16] Y. Oishi, J. Baratta, R.T. Robertson, O. Steward, Assessment of factors regulating axon growth between the cortex and spinal cord in organotypic co-cultures: effect of age and neurotrophic factors, J. Neurotrauma 21 (2004) 339–356. [17] A. Sandvig, M. Berry, L.B. Barrett, A. Butt, A. Logan, Myelin- , reactive glia- , and scar-derived CNS axon growth inhibitors: expression, receptor signaling, and correlation, Glia 46 (2004) 225–251. [18] X. Tang, J.E. Davies, S.J. Davies, Change in distribution cell associations, and protein expression levels of NG2, neurocan, phosphacan, brevican, versican V2, and tenascin-C during acute to chronic maturation of spinal cord scar tissue, J. Neurosci. 71 (2003) 427–444. [19] M.J. Winton, C.I. Dubreuil, D. Lasko, N. Leclerc, L. McKerracher, Characterization of new cell permeable C3-like proteins that inactivate Rho and stimulate neurite outgrowth on inhibitory substrates, J. Biol. Chem. 277 (2002) 32820–32829.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
The effects of combining chondroitinase ABC and NEP1-40 on the corticospinal axon growth in organotypic co-cultures Toshio Nakamae ∗ , Nobuhiro Tanaka, Kazuyoshi Nakanishi, Naosuke Kamei, Hirofumi Sasaki, Takahiko Hamasaki, Kiyotaka Yamada, Risako Yamamoto, Bunichiro Izumi, Mitsuo Ochi Department of Orthopaedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan
a r t i c l e
i n f o
Article history: Received 18 February 2010 Received in revised form 15 March 2010 Accepted 18 March 2010 Keywords: Chondroitinase ABC NEP1-40 Axon regeneration Spinal cord injury Organotypic co-culture
a b s t r a c t The area surrounding the injured spinal cord is a non-permissive milieu for axonal growth due to the inhibitory factors, especially chondroitin sulfate proteoglycan (CSPG) and Nogo. Recent studies have reported that chondroitinase ABC (ChABC) or Nogo-66(1-40) antagonist peptide (NEP1-40) promote axonal growth after spinal cord injury. But no study has addressed the effects on spinal cord injury of combining ChABC and NEP1-40. Previously, we described an organotypic co-culture system using the brain cortex and spinal cord from neonatal rats. In this study, we examined whether the combination of ChABC and NEP1-40 creates an action that promotes corticospinal axon growth in organotypic co-cultures. Organotypic co-cultures of brain and spinal cord were prepared from rats, and ChABC or NEP1-40 was delivered to them. To examine the effects of this combination these two drugs were applied together. We counted the number of labeled axons with DiI and assessed the immunoreactivity of CSPG and Nogo. Axonal growth was enhanced by infusing ChABC or NEP1-40 compared with that in the control group, whereas synergistic effects of combined administration of ChABC and NEP1-40 on axonal growth were not observed. There is a possibility that ChABC and NEP1-40 affect the same intracellular pathways and have no synergistic influence on axonal growth. © 2010 Elsevier Ireland Ltd. All rights reserved.
Spinal cord injury (SCI) in adult mammals results in sensory and motor deficits. Recent studies have demonstrated significant functional and histological recovery of injured spinal cord in animal models. However, there is a non-permissive milieu for repair surrounding the injured spinal cord [7,11,12]. After a central nerve system (CNS) injury, several axonal growth inhibitory factors, including the chondroitin sulfate proteoglycan (CSPG) and Nogo, are upregulated around the injured spinal cord. CSPG is originally the extracellular matrix contained within a glial scar. Nogo is one of the myelin-associated proteins and is thought to regulate the abnormal axonal growth in the embryogenic stage. Recent studies have reported that digestion and blocking of these factors induce promotion of axonal growth and functional recovery [1,3,5,6]. For example, chondroitinase ABC (ChABC) induces axonal regeneration and functional recovery by digesting the glycosaminoglycan (GAG) side chain of CSPG [1,3] and Nogo-66(1-40) antagonist peptide (NEP1-40) promotes axonal growth following SCI due to blocking Nogo-66/NgR interaction [5,6]. But it is not enough to assess the effects of axonal growth quantitatively, and the therapeutic effects
∗ Corresponding author. Tel.: +81 82 257 5233; fax: +81 82 257 5234. E-mail address: [email protected] (T. Nakamae). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.03.049
are not well known. Furthermore, there is no study that addresses the effect of combining ChABC and NEP1-40 on spinal cord injury. Previously we demonstrated how an organotypic co-culture system using the brain cortex and spinal cord from neonatal rats helps to assess the corticospinal tract’s (CST) axonal growth quantitatively and facilitates the analysis of factors that regulate axonal growth [10,15,16]. The purpose of this study was to examine whether the combination of these two extrinsic approaches, ChABC and NEP1-40 application, facilitate axonal growth from the brain cortex to the spinal cord using an organotypic co-culture system. Our research methods were reviewed and approved by the ethical committee of Hiroshima University. An organotypic co-culture of brain cortex and spinal cord was prepared as reported previously [16]. Brains and thoracic spinal cords were collected from Sprague–Dawley (SD) rats on postnatal day 7 (P7). The brains were sectioned using a Vibratome (Dosaka EM, Kyoto, Japan). The region of the sensorimotor cortex was dissected from the coronal sections, and thoracic spinal cord was bisected in the sagittal plane using a razor blade. The dissected cortex and spinal cord were placed on membranes (Millicell-CM; Millipore, Billerica, MA, USA), in 1 ml of serum-based medium (50% basal medium Eagle with Earle’s Salts (BME; Sigma, St. Louis, MD, USA), 25% heat inactivated horse serum (Gibco, Grand Island, NY, USA) 25% Earle’s Balanced Salt Solution
T. Nakamae et al. / Neuroscience Letters 476 (2010) 14–17
15
Fig. 1. Axons projecting from the brain cortex to the spinal cord. A photomicrograph of an organotypic co-culture (a). Minimal axons are seen in the control group (b). Axonal growth is promoted by the administration of ChABC (c) or NEP1-40 (d) or both ChABC and NEP1-40 (e). (b–e) High magnification of the box pointed by arrow head in a photomicrograph (a). (a) Bar = 200 m, (b–e) bar = 40 m.
(EBSS; Sigma), 1 mM l-glutamine and 0.5% d-glucose) in a 6-well tissue culture plate. The cortex and the spinal cord were incubated for 1 day, on the second day, the spinal cord pieces were aligned next to the white matter of the cortex (Fig. 1a). The co-cultures were incubated in a humidified atmosphere with 5% CO2 at 37 ◦ C. The medium was replaced every 3 days. The co-cultures were incubated for up to 14 days. Drugs were administered into the co-cultures just after the cortical tissue and the spinal cord tissue came into contact with one another. They were dropped onto the junction of the brain cortex and spinal cord in a quantity of 2 l every 3 days during incubation. To degrade CSPG, ChABC (Seikagaku, Tokyo, Japan) was delivered to the co-culture at a concentration of 1 U/ml (ChABC group). To block Nogo-66/NgR interaction, NEP1-40 (Sigma) was applied at a concentration of 0.4 mg/ml (NEP group). To examine the effects of the combination, these two drugs were dropped together onto the co-cultures (combined group). For the control, administration of only medium was performed (control group). The next stage was for the co-culture tissues to be fixed onto the membranes and incubated overnight at 4 ◦ C in 4% paraformaldehyde followed by immunostaining with mouse monoclonal antibodies against CSPG (1:500, Chemicon), and rabbit polyclonal antibodies against Nogo (1:100, Santa Cruz, CA, USA). We assessed the degradation of chondroitin sulfate CS-GAG with mouse monoclonal antibodies against 2B6 (1:500, Seikagaku). 2B6 is recognized an epitope on the CSPG core proteins but not the intact CS-GAG. On the following day, the tissues were exposed for 1 h to Alexa Fluor 488-conjugated goat anti-rabbit antibodies (1:400, Molecular Probes, Eugene, OR, USA) and Alexa Fluor 568conjugated goat anti-mouse antibodies (1:400, Molecular Probes) at room temperature, and observed under a confocal laser microscope (Carl Zeiss, Oberkochen, Germany). Axon projections from the cortex to the spinal cord were labeled by anterograde tracing with DiI. The co-cultures were fixed for 5 days in 4% paraformaldehyde at 4 ◦ C. Small crystals of DiI were placed on the center of the cortex, and the co-cultures were incubated for another 14 days in 0.1 M phosphate buffer in a humidified atmosphere at 37 ◦ C. The co-cultures were mounted on glass slides and covered with Vectashield (Vector, Burlingame, CA, USA) and glass coverslips. To analyze the axonal growth, we counted the number of labeled axons passing through a reference line running
parallel to the junction between the brain cortex and spinal cord 500, 1000 and 1500 m from the junction toward the spinal cord. We evaluated the axonal growth using images that were taken at a magnification of 200×, at different focal planes. All fibers crossing the reference line were counted, and the counts were expressed as the average number of axons per culture. Results were expressed as mean ± standard errors (S.E.). The statistical significance of differences in parameters was assessed by the Kruskal–Wallis test with a post hoc test using the Scheffe procedure. For all data collection, the researchers were blinded to the group identities. In the ChABC group, the immunostaining of CSPG decreased around the junction of brain cortex and spinal cord compared with the control group (Fig. 2a and b), whereas with 2B6 expression increased (Fig. 2c and d). On the other hand, immunoreactivity of Nogo-A decreased by applying NEP1-40 (Fig. 2e and f). In the control group, rare labeled axons were seen (Fig. 1b). The average number of axons that extended 500 m past the junction was 0.6 ± 0.3 (n = 12 cultures/group), whereas in the ChABC group and in the NEP group, axonal growth was enhanced in comparison to the control group (Fig. 1c and d). In the ChABC group and in the NEP group, the average number of axons that extended 500 m past the junction was 3.9 ± 0.4 (n = 12 cultures/group) and 5.1 ± 1.3 (n = 8 cultures/group), respectively. The average number of axons in the ChABC group and in the NEP group was statistically significantly higher than that in the control group. In the combined group (Fig. 1e), the average number of axons that extended 500 m past the junction was 4.9 ± 1.0 (n = 12 cultures/group). This was statistically significantly higher than that in the control group, but this was not significantly higher than that in the ChABC group or in the NEP group. In the ChABC group, NEP group and combined group, the average number of axons that extended 1000 or 1500 m past the junction was not statistically significantly higher than that in the control group (Fig. 3). After spinal cord injury, axonal growth of adult mammalians is limited in part by the presence of growth inhibitory molecules in the glia scar and CNS myelin. CSPGs and three myelin-associated proteins, namely Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), are thought to be the main molecular obstacles to axonal growth [2,4,7,11]. CSPGs are
16
T. Nakamae et al. / Neuroscience Letters 476 (2010) 14–17
Fig. 2. Immunological staining of CSPG, 2B6 and Nogo. In the control group, immunoreactivity of CSPG (red) increases (a), whereas in the ChABC group (b), CSPG expression decreases compared with the control group. Immunoreactivity of 2B6 (red) increases in the ChABC group (d) compared with the control group (c). In the control group, immunoreactivity of Nogo-A (green) increases (e). Compared with the control group, Nogo-A expression decreases in the NEP1-40 group (f). (a, b, e, f) Bar = 20 m, (c, d) bar = 100 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
upregulated via astrocytes and oligodendrocytes in the injury site after SCI to limit axonal regeneration [13,17–19]. CSPGs are extracellular matrix molecules and they belong to a family of molecules characterized by a protein core to which large sulfated GAG chains are attached. It has been reported that ChABC liberates CS-GAGs from CSPG core proteins to render a more permissive substrate for axonal outgrowth [1,3]. On the other hands, a 66-residue domain of Nogo (Nogo-66) is expressed on the surface of oligodendrocytes and can inhibit axonal outgrowth through an axonal Nogo-66 receptor (NgR). The Nogo-66(1-40) antagonist peptide (NEP1-40) blocks Nogo-66 or CNS myelin inhibition of axonal outgrowth in vitro [6]. However, no study has addressed the combined blockade of CSPG and Nogo inhibitor after SCI. In this study, we have examined the effects of the combined therapies of ChABC and NEP1-40 using an organotypic co-culture system with brain cortex and spinal cord from neonatal rats [10,15,16]. The advantage of this co-culture system is to assess CST axonal growth quantitatively and to facilitate the analysis of factors that regulate axonal growth. Oishi et al. reported that a major advantage of the co-cultures is that they allow easy delivery of
agents at known concentrations, and quantitative assessment of axon growth, and so the cultures will be useful for screening potential growth promoting substances and for combinatorial strategies [16]. They have also demonstrated that the failure of axonal growth is due to the development of a non-permissive tissue substrate with an organotypic co-culture, and that the most prominent difference between the P3 co-cultures and the P7 co-cultures is the amount of gliosis [16]. Glia scars not only present a physical barrier, but also produce molecules such as CSPG, which hamper axonal regeneration. Previously, we demonstrated that immunoreactivity of CSPG was observed around the junction of the brain cortex and spinal cord in the P3 co-culture group, and that immunoreactivity of CSPG increased in the P7 co-culture group [15]. In the present study, we demonstrated that the administration of ChABC caused CSPG expression to decrease and 2B6 expression to increase and furthermore, axonal growth from the brain cortex to the spinal cord was promoted by the administration of ChABC. Additionally, administration of NEP1-40 reduced the expression of Nogo and induced axonal growth significantly in an organotypic co-culture. This result
T. Nakamae et al. / Neuroscience Letters 476 (2010) 14–17
17
treatment for spinal cord injury through the appropriate combination of blocking agents of axonal growth inhibitory factors. Acknowledgement The authors thank Prof. Norio Sakai (Department of Molecular and Pharmacological Neuroscience, Hiroshima University) for the use of the instruments. References
Fig. 3. The quantitative assessment of axonal growth from the cortex to the spinal cord. The average number of axons in the ChABC group and that in the NEP1-40 group are statistically significantly higher than that in the control group at a distance of 500 m from the junction. The average number of axons in the combination group is statistically significantly higher than that in the control group, but this is not significantly higher than that in the ChABC group or in the NEP group (*p < 0.05 Scheffe).
reflects the contribution of Nogo-66 to axonal growth inhibition after SCI. However, the combined treatment of these two drugs did not improve compared to the results when ChABC or NEP1-40 was used separately. One might assume that ChABC affected Nogo and NEP1-40 affected CSPG. But Mingorance et al. reported that NEP140 treatment did not modify CSPG up-regulation and conversely, ChABC treatment did not affect Nogo-A or NgR expression level [14]. There might be a possibility that the amount of axonal regeneration in this culture system is maximal after either treatment; hence there could be no additive effect. But there is another study, which used the same organotypic co-cultures of P7 rats, showing the superior axon growth after transplantation of neural progenitor cells compared with the axon growth in the present study [10]. This data indicates that there is not saturation effect after treatments of ChABC and/or NEP1-40 in present study. We believed that axon growth could be enhanced if a non-permissive milieu for repair changed to the permissive state. Recently, glial inhibitors and intracellular signaling mechanisms have been studied [8,9,14]. The signaling components that are common to both CSPG and myelin inhibition are the activation of RhoA and the rise in intracellular calcium. Whilst the signals downstream of RhoA that lead to the action cytoskeleton are well characterized, the relationship between components upstream of RhoA and the role of calcium influx are still ambiguous. In this study, ChABC alone may reduce inhibitory intracellular pathways to an inactivated level at which the incubation with NEP1-40 has no additive effect. We therefore speculate that the signaling of CSPG and Nogo converges in the inhibitory intracellular pathways after SCI. Further studies are necessary in order to confirm a sufficiently effective
[1] A.W. Barrit, M. Davies, F. Marchand, R. Hartley, J. Grist, P. Yip, S.B. McMahon, E.J. Bradbury, Chondroitinase ABC promotes sprouting of intact and injured spinal systems after spinal cord injury, J. Neurosci. 26 (2006) 10856–10867. [2] P. Bovolenta, F. Wandosell, M. Nieto-Sampedro, Characterization of a neurite outgrowth inhibitor express after CNS injury, Eur. J. Neurosci. 5 (1993) 454– 465. [3] E.J. Bradbury, L.D. Moon, R.J. Popat, V.R. King, G.S. Bennett, P.N. Patel, J.W. Jawcett, S.B. McMahon, Chondroitinase ABC promotes functional recovery after spinal cord injury, Nature 416 (2002) 636–640. [4] P. Caroni, M.E. Schwab, Antibody against myelin-associated inhibitor of neurite growth neutralizes nonpermissive substrate properties of CNS white matter, Neuron 1 (1988) 85–96. [5] M. Domeniconi, M.T. Filbin, Overcoming inhibitors in myelin to promote axonal regeneration, J. Neurol. Sci. 233 (2005) 43–47. [6] T. GrandPre, S. Li, S.M. Strittmatter, Nogo-66 receptor antagonist peptide promotes axonal regeneration, Nature 417 (2002) 547–551. [7] N.Y. Harel, S.M. Strittmatter, Can regenerating axons recapitulate developmental guidance during recovery from spinal cord injury? Nat. Rev. Neurosci. 7 (2006) 603–616. [8] Y. Hasegawa, M. Fujitani, K. Hata, M. Tohyama, S. Yamagishi, Y. Yamashita, Promotion of axon regeneration by myelin-associated glycoprotein and Nogo through divergent signals downstream of Gi/G, Neuroscience 24 (2004) 6826–6832. [9] Z. He, V. Koprivica, The Nogo signaling pathway for regeneration block, Rev. Neurosci. 27 (2004) 341–368. [10] N. Kamei, Y. Oishi, N. Tanaka, O. Ishida, Y. Fujiwara, M. Ochi, Neural progenitor cells promote corticospinal axon growth in organotypic co-cultures, Neuroreport 15 (2004) 2579–2583. [11] B.P. Liu, W.B. Cafferty, S.O. Budel, S.M. Strittmatter, Extracellular regulators of axonal growth in the adult central nervous system, Philos. Trans. R. Soc. Lond. B Biol. Sci. 361 (2006) 1593–1610. [12] A.W. McGee, S.M. Strittmatter, The Nogo-66 receptor: focusing myelin inhibition of axon regeneration, Trends Neurosci. 26 (2003) 193–198. [13] R.J. MaKeon, R.C. Schreiber, J.S. Rudge, J. Silver, Reduction of neurite outgrowth in a model of glia scaring following CNS injury is correlated with the expression of inhibitory molecules on reactive astrocytes, J. Neurosci. 11 (1991) 3398–3411. [14] A. Mingorance, M. Sole, V. Muneton, A. Martinez, M. Nieto-Sampedro, E. Soriano, J.A. del Rio, Regeneration of lesioned entorhino-hippocampal axons in vitro by combined degradation of inhibitory proteoglycans and blockade of Nogo-66/NgR signaling, FASEB J. 20 (3) (2006) 491–493. [15] T. Nakamae, N. Tanaka, K. Nakanishi, N. Kamei, H. Sasaki, T. Hamasaki, K. Yamada, R. Yamamoto, Y. Mochizuki, M. Ochi, Chondroitinase ABC promotes corticospinal axon growth in organotypic cocultures, Spinal Cord 47 (2) (2009) 161–165. [16] Y. Oishi, J. Baratta, R.T. Robertson, O. Steward, Assessment of factors regulating axon growth between the cortex and spinal cord in organotypic co-cultures: effect of age and neurotrophic factors, J. Neurotrauma 21 (2004) 339–356. [17] A. Sandvig, M. Berry, L.B. Barrett, A. Butt, A. Logan, Myelin- , reactive glia- , and scar-derived CNS axon growth inhibitors: expression, receptor signaling, and correlation, Glia 46 (2004) 225–251. [18] X. Tang, J.E. Davies, S.J. Davies, Change in distribution cell associations, and protein expression levels of NG2, neurocan, phosphacan, brevican, versican V2, and tenascin-C during acute to chronic maturation of spinal cord scar tissue, J. Neurosci. 71 (2003) 427–444. [19] M.J. Winton, C.I. Dubreuil, D. Lasko, N. Leclerc, L. McKerracher, Characterization of new cell permeable C3-like proteins that inactivate Rho and stimulate neurite outgrowth on inhibitory substrates, J. Biol. Chem. 277 (2002) 32820–32829.