JP2006327942A - Axon-regenerating agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an axon-regenerating agent capable of regenerating central nerve such as spinal cord, etc., even in the presence of a substance inhibiting the regeneration of the axon. <P>SOLUTION: This axon-regenerating agent is provided by using an Rho kinase inhibitor as a main component. Preferably, the Rho kinase inhibitor is Y-27632 or fasudil. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an axonal regeneration agent capable of regenerating a central nerve such as a spinal cord.

  If the central nervous system such as the spinal cord is damaged due to injury or cerebrovascular disorder caused by a traffic accident or the like, the nerve function is lost and cannot be regenerated. In contrast to the regeneration of peripheral nerves. Damage to the central nerve often results in partial or complete paralysis because the central nerve cannot be regenerated when damaged. Therefore, regenerating damaged central nerves is an important issue in the medical field.

  On the other hand, there is a report (see Non-Patent Document 1) that an adult central nerve axon regenerates through a peripheral nerve graft, and the main cause of the adult central nerve not regenerating is the locality surrounding nerve cells. It is suggested that it is in an environment.

  So far, three proteins derived from myelin have been discovered as substances that inhibit axonal regeneration in the central nervous system of adults (see Non-Patent Document 2). They are Nogo, myelin-binding glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (OMgp), all functioning through p75 (see Non-Patent Documents 3 to 4).

  One of the key intracellular substances in elucidating the signal transduction mechanism downstream of p75 is RhoA, a small GTPase. Activation of RhoA inactivates the actin skeletal system, resulting in inhibition of axonal extension and collapse of the growth cone. That is, when Nogo, MAG, and OMgp activate RhoA via p75, it is considered that the extension of neuronal processes is suppressed (see Non-Patent Documents 3 to 4). The activation of RhoA by MAG and Nogo via p75 is guided by the dissociation of RhoA and Rho GDI by p75 (see Non-Patent Document 5). In addition to RhoA, its effector, Rho kinase, is thought to play an important role in controlling axonal extension, but it has not yet been elucidated how it controls axonal extension.

  It has been reported that axonal extension requires dynamic rearrangement of the cytoskeleton (see Non-Patent Document 6), and in the growth cone at the distal end of the axon, cytoskeleton protein microtubules and actin are reported. Report that it is involved in axonal extension (see Non-Patent Document 7), and that axonal extension requires the interaction between actin and microtubules (Non-Patent Document 7- 10). Therefore, it is considered that dynamic actin promotes axonal extension by promoting microtubule transport and polymerization (see Non-Patent Document 11). Thus, microtubules are considered important in controlling axonal extension.

  By the way, CRMP-2 is one of the important factors that control the movement of microtubules in axonal extension, and CRMP-2 has been reported to bind to tubulin and promote microtubule formation (Non-Patent Document). 13). In this report, overexpression of CRMP-2 promotes axonal extension, and a mutant lacking microtubule polymerization activity is reported to suppress axonal extension. From the above, it is suggested that a complex of CRMP-2 and tubulin gathers at the distal end of the axon, regulates microtubule polymerization, and is involved in axonal extension.

S. David, A.D. J. et al. Aguayo, Science 214, 931-3 (Nov 20, 1981). L. McKerracher, M.M. J. et al. Winton, Neuron 36, 345-8 (Oct 24, 2002). T.A. Yamashita, H .; Higuchi, M .; Tohyama, J Cell Biol 157, 565-70 (May 13, 2002). K. C. Wang, J. et al. A. Kim, R.A. Sivasankaran, R.A. Segal, Z .; He, Nature 420, 74-8 (Nov 7, 2002). T.A. Yamashita, M .; Tohyama, Nat Neurosci 6, 461-7 (May, 2003). P. W. Baas, L.M. Luo, Neuron 32, 981-4 (Dec 20, 2001). P. R. Gordon-Weeks, Bioessays 13, 235-9, (May, 1991). P. R. Gordon-Weeks, J Neurocytol 22, 717-25 (Sep, 1993). C. H. Lin, P.M. Forscher, J Cell Biol 121, 1369-83 (Jun, 1993). J. et al. F. Challacombe, D.C. M.M. Snow, P.M. C. Letourneau, J Cell Sci 109, 2031-40 (Aug, 1996). F. Bradke, C.I. G. Dotti, Science 283, 1931-4 (Mar 19, 1999). Y. Fukata, T .; J. et al. Itoh, T .; Kimura, C.I. Manager, T.M. Nishimura, T .; Shiromizu, H. et al. Watanabe, N.A. Inagaki, A.A. Iwamatsu, H .; Hotani, K .; Kaibuchi, Nat Cell Biol 4, 583-91 (Aug, 2002). N. Inagaki, K .; Chihara, N.A. Arimura, C.I. Manager, Y.M. Kawano, N .; Matsuo, T .; Nishimura, M .; Amano, K.M. Kaibuchi, Nat Neurosci 4, 781-2 (Aug, 2001). P. Doherty, M.M. Fruns, P.M. Seaton, G.C. Dickson, C.D. H. Barton, T.W. A. Sears, F.A. S. Walsh, Nature 343, 464-6 (Feb 1, 1990) Z. Li, C.I. D. Aizenman, H.M. T.A. Cline, Neuron 33, 741-50 (Feb 28, 2002). M.M. Amano, M.M. Ito, K.K. Kimura, Y .; Fukata, K .; Chihara, T .; Nakano, Y. et al. Matsuura, K .; Kaibuchi, J Biol Chem 271, 20246-9 (Aug 23, 1996).

  However, the above report on CRMP-2 is a report on the formation of CRMP-2 microtubules, but the relationship with substances that inhibit axonal regeneration such as MAG and Nogo has not been considered, and axonal regeneration is inhibited. It has not been considered to enable axonal regeneration even in the presence of substances.

  In view of the above problems, an object of the present invention is to provide an axonal regeneration agent capable of regenerating central nerves such as the spinal cord even in the presence of a substance that inhibits axonal regeneration.

  As a result of intensive studies on the above problems, the present inventors have found that MAG and Nogo phosphorylate CRMP-2 in cerebellar granule cells of young rats, and CRMP-2 non-phosphorylated form in cerebellar granule cells. It has also been discovered that the process of inhibiting the extension of protrusions by MAG or Nogo is lost when. As a result of further intensive studies, it was confirmed that this phosphorylation was Rho kinase-dependent, and it was conceived that inhibition of axonal regeneration could be suppressed by using a Rho kinase inhibitor. The present invention has been reached.

  That is, the present invention provides an axonal regeneration agent mainly composed of a Rho kinase inhibitor. In this case, the Rho kinase inhibitor is preferably Y-27632 or fasudil, as will be described in the experimental examples described later.

  As described above, the present invention can provide an axonal regeneration agent that can regenerate central nerves such as spinal cord even in the presence of a substance that inhibits axonal regeneration.

  One of the forms of the axonal regeneration agent according to the present invention is mainly composed of a Rho kinase inhibitor. Here, a Rho kinase inhibitor refers to a substance that lowers the activity of Rho kinase, and Rho kinase refers to a protein kinase activated by RhoA. Examples of Rho kinase inhibitors are not particularly limited, but Y-27632 and fasudil are preferred. This axonal regeneration agent is more effective for the affected area.

  Examples of the administration method of the axonal regeneration agent according to this embodiment include intravenous administration. In the case of intravenous injection, 30 mg can be administered 2 to 3 times a day.

  The axonal regeneration agent according to this embodiment can be obtained from Calbiochem in the case of Y-27632 and from Asahi Kasei in the case of fasudil.

  By doing in this way, the axonal regeneration agent which can reproduce | regenerate central nerves, such as a spinal cord, can be provided even in presence of the substance which inhibits axonal regeneration.

In the following, experiments conducted for the purpose of verifying the configuration and effects of the present invention will be described in detail.

(A) Materials and methods (1) Cell culture Dorsal root ganglion cells were obtained by combining dorsal root ganglia from 6-10 day old rats in 0.025% trypsin solution, pulverizing them at 37 ° C. Incubated for 5 minutes. Also, cerebellar granule cells were obtained by combining cerebellums from rats in 5 ml of 0.025% trypsin solution, pulverizing, and incubating at 37 ° C. for 10 minutes.

  Next, 10% FCS-containing DMEM medium was added, and the cells were collected by centrifugation at 1000 rpm. The cells were plated in Sato medium (Non-patent Document 15) on a chamber slide coated with poly-L-lysine. For growth assays, plated cells were incubated for 24 hours, fixed with 4% paraformaldehyde, and monoclonal antibodies (TuJ1, Covance Research Products, Inc., for the purpose of recognizing neuron-specific β-tubulin III protein. (US) immunostained. Then, the length of the longest process (axon) or the total neurite length of β-tubulin III positive neurons was measured. In order to evaluate axonal regeneration, MAG (25 μg / ml) and Nogo were added to the medium after the plate. Cerebellar granule cells were transfected with a non-phosphorylated CRMP-2 plasmid with a myc tag or a CRMP-2 deletion mutant plasmid. Replated 24 hours after transfection and further incubated for 24 hours. In order to confirm the transfected cells, the cells were stained with an anti-myc antibody (manufactured by Sigma).

(2) Western blot After washing a 3.5 cm dish in which cells were cultured with PBS three times, a lysis buffer (1% NP-40 (trade name, nonionic surfactant), 0.1% SDS, 50 Cells with 10 mM Tris-HCl [pH 7.4], 2 mM EDTA, 150 mM NaCl, 1 mM sodium orthovanadate, 1 mM sodium fluoride, protease inhibitor (Roche Biochemicals) for 10 minutes at 4 ° C. Was dissolved. Thereafter, the mixture was centrifuged for 15 minutes, and the lysate was fixed at a protein concentration using a protein assay kit (manufactured by BioRad), then added with a sample buffer, boiled for 5 minutes and then subjected to SDS-PAGE. Blot analysis was performed. As the antibody, polyclonal anti-phosphorylated CRMP-2 antibody (manufactured by Kaikai Lab.), Polyclonal anti-CRMP-2 antibody (manufactured by Kaikai Lab.), Polyclonal anti-phosphorylated cofilin antibody (manufactured by Promega), polyclonal anti-cofilin antibody (Promega), polyclonal anti-phosphorylated MLC antibody (Asahi Kasei), and polyclonal anti-MLC antibody (Asahi Kasei) were used.

(3) Immunostaining To exclude monomeric tubulin, 0.1 M phosphate buffer (PB), 10 mM EGTA, 2 mM MgCl2 (pH 6.9), 4% paraformaldehyde, 0.2% Triton Nerve cells were fixed with X-100 (trade name) for 30 minutes. EB1 was fixed with methanol at −20 ° C. for 5 minutes. Other staining was performed with 4% paraformaldehyde and 0.1M PB. The fixed nerve cells were washed with PBS, blocked with 5% calf serum, 0.1% Triton X-100, 0.1% albumin, and reacted with the primary antibody. Anti-β tubulin III antibody was used for microtubule staining. In order to evaluate the distribution of APC and EB1, double staining was performed using TuJ1 or anti-nerve fiber antibody simultaneously with polyclonal anti-APC antibody (manufactured by Promega) and polyclonal anti-EB1 antibody (manufactured by Promega). Next, the secondary antibody was reacted and observed with a fluorescence microscope. To quantify fluorescence, the exposure time and camera settings were fixed.

(4) Rho assay The Rho assay was performed as previously described using the GST-Rho binding region (Non-Patent Document 14). That is, the tissue was fixed with 4% paraformaldehyde and 0.1 M PB (pH 7.4) for 1 hour at room temperature. After washing with PBS, blocking was performed for 1 hour with a blocking buffer obtained by adding 5% albumin and 0.1% Triton X-100 to PBS, and a blocking buffer containing 15 ug / ml GST-Rho binding region was applied at 4 ° C. Reacted overnight. After washing with PBS, it was again fixed with 2% paraformaldehyde and 0.1 MPB for 10 minutes at room temperature, and washed with PBS. In addition to a monoclonal anti-GST antibody (manufactured by Santa Cruz) as a primary antibody, double staining was performed using an anti-nerve fiber antibody to evaluate the distribution of Rho. Cultured nerve cells were fixed with 4% paraformaldehyde, and similarly stained with monoclonal anti-GST antibody and anti-nerve fiber antibody. The secondary antibody was reacted and then observed with a fluorescence microscope.

(5) Image analysis and statistics Each distribution was quantified using anti-APC antibody, anti-EB1 antibody, and anti-β tubulin III antibody. The growth cone or the middle of the axon was selected and the fluorescence density was measured. At the same time, the background fluorescence density was also measured and subtracted to obtain the final average fluorescence density. All data were expressed as mean ± standard error and unpaired Student's t-test was performed.

(6) Surgical procedure Rats were anesthetized with 2% halothane. Female Wistar rats (250-300 grams) were subjected to laminectomy at thoracic spinal level 9-10 to expose the spinal cord. The spinal cord was completely injured with a scalpel, and a 10 mm to 15 mm spinal cord was removed around the damaged site. 1 mg / ml Y-27632 (manufactured by Calbiochem) dissolved in PBS was soaked in a small gel by 10 ul and placed at the site of spinal cord injury.

(7) Tissue specimens that had not been damaged and tissues that had been damaged for 2 hours were obtained as fresh frozen tissues. Rats were deeply anesthetized and the spinal cord was removed by cutting the neck. The tissue was embedded in Tissue Tek and frozen at −80 ° C. using dry ice. The section was cut into a sagittal shape with a thickness of 20 μm using a cryostat. Sections were fixed with 4% paraformaldehyde and 0.1 M PB (pH 7.4) for 1 hour at room temperature, washed with PBS, and room temperature using PBS containing 5% albumin and 0.1% Triton X-100. For 1 hour. To exclude monomeric tubulin, tissues were fixed with 0.1 MPB, 10 mM EGTA, 2 mM MgCl2 (pH 6.9), 4% paraformaldehyde, 0.2% Triton X-100 for 1 hour.

(B) Results First, the results of Western blotting on cerebellar granule cells of 7-day-old young rats are shown in FIGS. 1 (A) to (C).

  FIG. 1 (A) shows the results when the cultured cerebellar granule cells are not stimulated with Nogo and when stimulated for 10 minutes at the concentrations (10, 30, 100, 300 nM) shown in the upper part of the figure. . The lysate was Western blotted with anti-phosphorylated CRMP-2 antibody (p-CRMP2), anti-phosphorylated cofilin antibody (p-cofilin), and anti-phosphorylated MLC antibody (p-MLC). The second row shows all CRMP-2.

  According to this figure, it was confirmed that a large amount of CRMP-2 was expressed in these neurons (second stage), and that phosphorylated CRMP-2 was also confirmed. It was also confirmed that CRMP-2 phosphorylation had a dose dependency on Nogo.

  FIG. 1 (B) shows the results of adding Rho-kinase inhibitors Y-27632 (Y) and fasudil (HA) to cultured cerebellar granule cells. Here, it was confirmed that the addition of Y-27632 (Y) and fasudil (HA) reduced the phosphorylation of CRMP-2 by Nogo. That is, it was confirmed that the phosphorylation of CRMP-2 has Rho kinase dependency.

  FIG. 1C shows the results when the cultured cerebellar granule cells were not stimulated with MAG and were stimulated for a predetermined time (10 minutes, 30 minutes, 60 minutes, 25 ug / ml each). It was confirmed that CRMP-2 was phosphorylated by MAG, while the phosphorylation level of cofilin was not changed. MAG phosphorylated CRMP-2 within 10 minutes, and MAG and Nogo were found to phosphorylate CRMP-2 in young cerebellar granule cells with Rho kinase dependency.

  Rho kinase is known to phosphorylate various substances, for example, directly phosphorylating and activating myosin light chain (MLC) (Non-patent Document 16). In this neuron, Nogo is MLC. Was not phosphorylated (FIG. 1 (A)). Furthermore, LIM kinase activated by Rho kinase (in mammals, LIM kinase phosphorylates cofilin and thereby actin is depolymerized. Especially in Drosophila, cofilin is essential for axon growth). In cerebellar granule cells, neither Nogo nor MAG changed the phosphorylation level of cofilin. From the above, it was suggested that CRMP-2 plays an important role in the suppression of axonal extension via MAG and Nogo.

  FIG. 2 (A) shows cerebellar granule cells (WT) transfected with CRMP-2 with a non-phosphorylated myc tag and cerebellar granule cells transfected with a mutant not expressing CRMP-2 ( Fig. 5 shows the results of immunostaining when ΔC) was cultured and re-plated for 24 hours, and stimulated with Nogo or not stimulated, and further cultured for 24 hours. In the figure, a photo shows a cell expressing GFP, b shows a cell expressing GFP treated with Nogo, and c shows a non-phosphorylated CRMP-2. The photograph of d shows the cell which treated the cell which expressed non-phosphorylated CRMP-2 with Nogo, and the photograph of e shows the cell which CRMP-2 is not expressing, respectively. The cerebellar granule cells of young rats used are those 7 days after birth.

  FIG. 2 (B) shows a neurite outgrowth assay of cerebellar granule cells. In the figure, “GFP” expresses cells expressing GFP, and “WT” expresses non-phosphorylated CRMP-2. “ΔC” indicates a cell in which CRMP-2 is not expressed, “GFP + Nogo” indicates a case in which Nogo is added to a cell expressing GFP at 100 nM, and “WT + Nogo” indicates that 100 NM is added to WT. The case where Nogo is added is shown. The data shows the mean value ± standard error.

  As a result, it was confirmed that protrusion growth was suppressed by Nogo stimulation, but when non-phosphorylated CRMP-2 with a myc tag was transfected into cerebellar granule cells and this CRMP-2 was overexpressed, It was confirmed that it prominently promotes protrusion extension. Further, Nogo was added to these cells, but there was no inhibition of protrusion extension. Cerebellar granule cells transfected with a mutant not expressing CRMP-2 lacked microtubule polymerization activity, but showed similar results as above. Therefore, it was confirmed that inactivation of CRMP-2 is related to inhibiting the effects of MAG and Nogo.

  FIG. 3 (A) shows the result of immunostaining in the case where dorsal root ganglion cells of young rats are not stimulated with MAG (control, Y-27632) or fixed after stimulation with MAG (MAG, MAG + Y27632). Indicates. Here, at the same time as fixing the cells, the monomeric tubulin is removed to stain only the polymerized microtubules so that the microtubules polymerized by the neurites can be directly observed. FIG. 3 (B) quantifies the relative microtubule density (comparison with the control) in each state. * Indicates statistical significance (p <0.05 in Student's t test). Moreover, although it is substantially the same as the experiment similar to FIG. 3 (A) and (B), the result of the experiment conducted about the total amount of tubulin without removing monomeric tubulin is shown in FIG. ).

  According to the results shown in FIGS. 3 (A) and 3 (B), it was confirmed that polymerized microtubules were distributed along the axon when not stimulated with MAG. The density of polymerized microtubules in the axon has decreased. However, even when treated with MAG, in the presence of Y-27632, it was confirmed that the density of the polymerized microtubules was restored to the control level. However, it was confirmed that Y-27632 alone does not show any effect. In addition, it has confirmed that the level of all tubulin did not show the significant change by MAG or Y-27632 (FIG. 3 (C), (D)). Therefore, MAG could be thought to inhibit CRMP-2 activity and control to reduce microtubule levels downstream of Rho kinase. In addition, the same result was able to be obtained also when processing with Nogo instead of MAG about the above (illustration omitted).

  From the results so far, it is considered that axon growth involves the control of axon microtubules. Presumably, CRMP-2 is inactivated by MAG or Nogo, resulting in inhibition of microtubule polymerization.

  By the way, various molecules are involved in the control of microtubules, and APC was focused on and examined as another substance that may control the polymerization of microtubules in axons.

  APC binds directly to the positive end of the microtubule either directly through the base region or indirectly through the EB1 binding region. Localization of APC and EB1 at the tip of the axon is related to axon growth by NGF. Based on the above, it was thought that these molecules may be involved in the axon extension inhibitory action of myelin-derived factors. Since it is small in cerebellar granule cells and inappropriate for this assay, the localization of APC and EB1 in neurites was visualized by immunostaining using dorsal root ganglion cells of young rats. FIG. 4 (A) shows the results of double staining performed on APC and β-tubulin, FIG. 4 (B) shows the relative average fluorescence density of APC in the growth cone, FIG. 4 (C) shows nerve fibers, The result of double staining of EB1 is shown in FIG. 4D, and the relative average density of EB1 in the growth cone is shown. In addition, the data of FIG. 4 (B) and (D) show an average value +/- standard error.

  According to the result shown in FIG. 4 (A), it was confirmed that APC was strongly stained in the cell body, neurite, and axon distal end (arrow head in FIG. 4 (A)). When treated with MAG, the same APC localization was observed at the tip of the neurite (FIGS. 4A and 4B). It was also confirmed that EB1 was stained at the tip of the neurite (FIG. 4C, arrowhead). However, this localization was not changed by MAG treatment (FIGS. 4C and D). Therefore, it was confirmed that APC and EB1 are not involved in suppression of protrusion extension via MAG.

  Next, to evaluate how RhoA plays a role in damaged axons, the activity of Rho in axons after spinal cord injury was examined. Here, dorsal root ganglion cells that were not stimulated with MAG or stimulated for 30 minutes (25 μg / ml) were fixed, and after reacting with the GST-Rho binding region, activated RhoA was stained with an anti-GST antibody and visualized. . A photograph of the results is shown in FIG. 5 (A), and FIG. 5 (B) shows the relative mean fluorescence density of activated RhoA in the growth cone (data is mean ± standard error). Further, in FIG. 5 (A), the result of staining activated RhoA is shown in the upper stage, and the result of staining nerve fibers as nerve markers is shown in the lower stage.

  According to FIGS. 5 (A) and 5 (B), neurites are hardly stained in cells not stimulated with MAG (RhoA activity is weak), whereas in cells stimulated with MAG, both growth cones and axons have RhoA. The remarkable activity was confirmed. Since this phenomenon is observed in growing axons and growth cones, it is considered that the polymerization of microtubules is reduced by the activation of RhoA and Rho kinase. Note that in FIG. 5B, * indicates statistically significant (p <0.05 in Student's t test).

  Then, this time, the spinal cord of the rat was damaged, and after 2 hours, the tissue around the damaged site was taken out and stained in the same manner as above, and the result is shown in FIG. FIG. 5C shows the sagittal stage of the injured spinal cord, and enlarges the white matter on the mouth side by about 15 mm from the damaged site. After spinal cord injury, RhoA was activated in nerve fiber positive axons (right figure, SCI 2h), whereas it was confirmed that the control was not (left figure, control). Thus, RhoA was found to be very active around the damaged axon.

  The results thus far show that MAG and Nogo phosphorylate and inactivate CRMP-2, thereby reducing microtubule polymerization in nerve cells, and in agreement with this, RhoA is present after spinal cord injury. It was also shown to be activated.

  Therefore, after spinal cord injury, the level of polymerized tubulin was investigated, and it was verified whether what was observed in vitro was also observed in vivo. Fresh frozen sections were prepared 2 hours after spinal cord injury and stained with Tuj1 antibody. The result is shown in FIG. FIG. 6 (A) shows the case where the total amount of tubulin was used without removing the monomeric β-tubulin III, and FIG. 6 (B) shows the polymerization after removing the monomeric β-tubulin III. Only microtubules are stained.

  As a result, in FIG. 6 (A), β tubulin III was present in the white matter at the site adjacent to the damaged site, and the β tubulin level did not change 2 hours after spinal cord injury (SCI 2h). Even if Y-27632 was allowed to act on the damaged site, the staining pattern was not affected (Y-27632).

  On the other hand, in FIG. 6B, it can be confirmed that the polymerized microtubules are distributed along the white matter axon (control), and the microtubules polymerized at a site adjacent to the damaged site at 2 hours after the spinal cord injury. Levels were significantly reduced and observed on the oral and caudal sides of the damaged site (SCI), but when a gel soaked with Y-27632 was placed in the damaged site, the decrease in polymerized microtubules was reduced. It was confirmed that the control level completely returned to the control level (Y-27632 + SCI). That is, it is considered that the decrease in polymerized microtubules occurred because RhoA and Rho kinase were activated by spinal cord injury. CRMP-2 was also double-stained using Tuj1 antibody and confirmed to be expressed in white matter axons and gray matter cell bodies. These results suggest that CRMP-2 inactivation may play an important role in the depolymerization of microtubules after spinal cord injury.

(A) The cultured cerebellar granule cells were not stimulated with Nogo or stimulated at a predetermined concentration for 10 minutes, and the lysate was Western blotted with anti-phosphorylated CRMP-2 antibody, anti-phosphorylated cofilin antibody, and anti-phosphorylated MLC antibody. . (B) The figure which shows the result of the Western blot in the case where Y-27632 (Y) and fasudil (HA) which are Rho kinase inhibitors are added with respect to the cultured cerebellar granule cell. (C) Western blotting results when cultured cerebellar granule cells are not stimulated with MAG or stimulated for the indicated time. (A) 24 cerebellar granule cells (WT) transfected with CRMP-2 with a non-phosphorylated myc tag or cerebellar granule cells (ΔC) transfected with a mutant not expressing CRMP-2. The figure which shows the result of the immuno-staining at the time of carrying out culture | cultivation for a time, replate, and stimulating or not stimulating with Nogo, and also culturing for 24 hours. (B) The figure which shows the axon growth assay result of a cerebellar granule cell. (A) The figure showing the result of immunostaining when dorsal root ganglion cells of young rats were not stimulated with MAG, or were stimulated and fixed for 30 minutes, and monomeric tubulin was removed. (B) The figure which shows the result of having quantified the relative microtubule density (comparison with control) in each state. (C) The figure which shows the result of the immunostaining when dorsal root ganglion cells of young rats are not stimulated with MAG or fixed after stimulation for 30 minutes. (D) The figure which shows the result of having quantified the relative microtubule density (comparison with control) in each state. (A) The figure which shows the result of the double dyeing | staining of (beta) -tubulin and APC. (B) The figure which shows the relative average fluorescence density of APC in a growth cone. (C) The figure which shows the result of the double dyeing | staining of a nerve fiber and EB1. (D) The figure which shows the relative average fluorescence density of EB1 in a growth cone. (A) The figure showing the result of staining the activated RhoA and nerve fibers after stimulation of dorsal root ganglion cells with MAG or after stimulation for 30 minutes (25 μg / ml). (B) The relative mean fluorescence density of activated RhoA in the growth cone. (C) A diagram showing a sagittal cut of the damaged spinal cord. (A) The figure which dye | stained (beta) tubulin III specific to a neuron using Tuj1 antibody. (B) The figure which shows the result at the time of dyeing | staining only the microtubule which removed the monomer tubulin at the time of tissue fixation. The figure which shows the result of having performed double dyeing | staining using the anti- CRMP-2 antibody (upper figure) and Tuj1 antibody (lower figure) with respect to the sagittal section.

Claims (2)

  An axonal regenerative agent mainly composed of a Rho kinase inhibitor.   The axonal regeneration agent according to claim 1, wherein the Rho kinase inhibitor is Y-27632 or Fasudil.