Axonal regeneration through the fibrous scar in lesioned goldfish spinal cord

Neuroscience 284 (2015) 134–152

AXONAL REGENERATION THROUGH THE FIBROUS SCAR IN LESIONED GOLDFISH SPINAL CORD A. TAKEDA, Y. ATOBE, T. KADOTA, R. C. GORIS AND K. FUNAKOSHI *

INTRODUCTION Traumatic injury to the mammalian central nervous system (CNS) results in scar tissue. The scar that forms in response to an injury that opens the meninges contains fibrous scar tissue in the core and glial scar tissue in the surrounding parenchyma (Silver and Miller, 2004). Glial scars are characterized by astrocyte hypertrophy, which is a consequence of the overexpression of intermediate filament proteins, such as glial fibrillary acidic protein (GFAP). The fibrous scar, on the other hand, is formed of a dense collagen meshwork and fibroblasts. The scar is thought to produce various extracellular matrix (ECM) molecules that inhibit axonal regrowth in the CNS (reviewed by Fawcett and Asher, 1999; Condic and Lemons, 2002; Grimpe and Silver, 2002; Yiu and He, 2006; Galtrey and Fawcett, 2007; Kawano et al., 2012). Among them, chondroitin sulfate proteoglycans (CSPGs), which inhibit axonal growth in vitro (Snow et al., 1990; Dou and Levine, 1994; Friedlander et al., 1994), are upregulated by astrocytes and oligodendrocyte precursor cells after injury and are speculated to be a major inhibitory factor in glial scar tissue (McKeon et al., 1995, 1999; Fitch and Silver, 1997; Haas et al., 1999; Asher et al., 2000, 2002; Jones et al., 2002, 2003a; Tang et al., 2003). NG2, a major CSPG that inhibits axonal growth in vitro and in vivo, increases at the lesion site after CNS injury in mammals (Dou and Levine, 1994; Levine, 1994; Fidler et al., 1999; Chen et al., 2002; Ughrin et al., 2003; Tan et al., 2006). The scar also contains ECM molecules with permissive roles for axonal regeneration, such as laminin (reviewed by Condic and Lemons, 2002). In vitro findings suggest that laminin stimulates the axonal outgrowth of sensory ganglion cells, PC12 cells, developing CNS neurons, and injured neurons (Baron-van Evercooren et al., 1982; Manthorpe et al., 1983; Rogers et al., 1983; Liesi et al., 1984a; Letourneau et al., 1988; Liesi and Silver, 1988; Frise´n et al., 1995). Laminin is strongly upregulated after injury to the CNS, as well as after injury to the peripheral nervous system (Liesi et al., 1984b; Sosale et al., 1986; Doyu et al., 1993; Risling et al., 1993; Agius and Cochard, 1998; Wallquist et al., 2002). Furthermore, laminin is expressed on the surface of cells that ensheath the axons sprouting into the scar, suggesting a role for laminin in axonal growth in the injured spinal cord (Frise´n et al., 1995). Previous studies raised the possibility of successful axonal regeneration through the spinal injury site when permissive signals exceed inhibitory signals in the scar (Jones et al., 2003b; Silver and Miller, 2004; Lu et al., 2007). The

Department of Neuroanatomy, Yokohama City University School of Medicine, Yokohama, Kanagawa 236-0004, Japan

Abstract—Spontaneous nerve regeneration beyond the scar frequently occurs in fish after spinal cord lesions, in contrast to mammals. Here we examined the spatiotemporal relationship between the fibrous scar and axonal regeneration in the goldfish. Within 1 week after hemisection of the spinal cord, the open wound was closed by a fibrous scar that was demarcated from the surrounding nervous tissue by the glia limitans, which was immunoreactive for laminin. Within 1 week after hemisection, regenerating axons entered the fibrous scar, and were surrounded by laminin-coated tubular structures continuous with the glia limitans. Regenerating axons that initially entered the fibrous scar were usually accompanied by glial processes. Within 2–3 weeks after hemisection, the tubular structures became enlarged, and the regenerating axons increased in number, fasciculating in the tubules. Glial processes immunoreactive for glial fibrillary acid protein and 5-hydroxytryptamine neurons then entered the tubular structures to associate with the regenerating axons. The tubular structures developed further, creating tunnels that penetrated the fibrous scar, through which the regenerating axons passed. At 6–12 weeks after hemisection, the fibrous scar was smaller and the enlarged tunnels contained many glial processes and several axons. The findings of present study demonstrated that, following spinal lesions in goldfish, regenerating axons enter and pass the scar tissue. The regenerating axons first enter the fibrous scar with glial elements and then grow through laminincoated tubular structures within the fibrous scar. Invasion by glial processes and neuronal elements into the tubular structures reduces the fibrous scar area and allows for more regenerating axons to pass beyond the fibrous scar. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: teleost, spinal injury, fibrous scar, laminin, serotonin, regrowth. *Corresponding author. Address: Department of Neuroanatomy, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. Tel: +81-45-787-2569; fax: +81-45-782-7251. E-mail address: [email protected] (K. Funakoshi). Abbreviations: 5-HT, 5-hydroxytryptamine; AMCA, aminomethylcoumarin; BSA, bovine serum albumin; CNS, central nervous system; CSPGs, chondroitin sulfate proteoglycans; ECM, extracellular matrix; GFAP, glial fibrillary acidic protein; NDS, normal donkey serum; Nflm, nucleus of the medial longitudinal fasciculus; PB, phosphate buffer; PBS, phosphate-buffered saline; PFA, paraformaldehyde. http://dx.doi.org/10.1016/j.neuroscience.2014.09.066 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 134

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downregulation of inhibitory factors such as CSPGs in the spinal lesion model leads to the growth of regenerating axons beyond the lesion site (Bradbury et al., 2002; Yick et al., 2003; Caggiano et al., 2005; Huang et al., 2006; Cafferty et al., 2007). In contrast to mammals, spontaneous axonal regeneration beyond the injury site in the CNS is reported in anamniotes and reptiles. The fish showed excellent recovery of motor activity after spinal injury (Koppanyi, 1955). In the goldfish Carassius auratus, the majority of descending spinal projections from several brain nuclei regenerate beyond the lesion site within 4–12 weeks (Sharma et al., 1993). In goldfish, the midbrain–spinal pathway, which is critical for locomotion in fish, also regenerates beyond the lesion site to re-innervate the appropriately innervated target spinal motoneurons and interneurons (Takeda et al., 2007). In goldfish, spinal injury produces a scar containing fibrous tissue, but regenerating axons grow past the scar (Bernstein, 1964; Bernstein and Bernstein, 1967). The bridges formed by ependymoglial cells seem to permit the passage of regenerating axons through the scar tissue (Bernstein and Bernstein, 1969). The mechanisms by which regenerating axons penetrate the fibrous scar, however, have not yet been established. Furthermore, scar organization and interaction between glial cells and the ECM that contribute to axonal regeneration have not been examined using histochemical methods. In the present study, therefore, we used combined immunohistochemistry and tracer methods, and electron microscopy to show that regenerated axons enter the fibrous scar with accompanying glial processes, and grow within the scar tissue through the laminin-coated tubular structures. We also showed that the fibrous scar was gradually reduced to allow more regenerating axons and glial processes to penetrate the scar. Little is known about whether the molecules expressed in the scar function as inhibitory factors in goldfish. The present study showed the expression of a CSPG molecule, NG2, in the lesion site and its relationship with the regenerating axons. We previously reported that newly generated neurons migrate to the lesion site after traumatic injury in goldfish, and some of the neurons persist for a long time and differentiate to express 5-hydroxytryptamine (5-HT; Takeda et al., 2008). 5-HT stimulates axonal growth, probably by inducing glia to produce neurotrophins (Lauder, 1993). The present study also showed that 5-HT neurons were associated with the regenerating axons in the fibrous scar.

EXPERIMENTAL PROCEDURES Materials Goldfish, C. auratus (n = 87; body length 10–12 cm, body weight 30–70 g), were obtained commercially (Nomoto Fish Farm Co. Ltd., Yokohama, Japan) and maintained in an aquarium at 25–28 °C. All procedures were performed according to the standards established by the NIH Guide for the Care and Use of Laboratory Animals and the protocols were approved by the Yokohama City University Committee for Animal

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Research. All efforts were made to minimize the number of animals used and their suffering. Hemisection of the spinal cord For better quantitative evaluation of the regenerating axons and behavioral activities, the injury was made at the upper spinal level. Full transection at the upper spinal level frequently produces permanent separation of the spinal cord. Lateral hemisection is a useful model for the sequential observation of the injured tissue because of its efficient repair of the injury. The fish (n = 80) were deeply anesthetized with 0.02% tricaine methanesulfonate (MS-222, Sigma–Aldrich Chemical Co., St. Louis, MO, USA) in water, and placed on ice. An incision of the dorsal skin was made at the level just caudal to the cranium, the muscles were retracted, and the post-temporal bone and vertebrae were exposed. The rostral segments of the spinal cord were exposed after removing the bones, and a frontal hemisection of the left side of the spinal cord was performed 500 lm caudal to the first spinal nerve. The hemisection was performed by inserting the blades of small scissors at a right angle to the spinal surface along the posterior median septum. After the wound was sutured and sealed with aerosol plastic dressing (Yoshitomi-Seiyaku, Osaka, Japan), the fish were allowed to recover. The five remaining fish (control group) did not undergo the hemisection procedure. Tracer injection Five control fish and lesioned fish (RDA group: n = 35) with survival periods of 0 d (n = 5), 1 week (n = 5), 2 weeks (n = 5), 3 weeks (n = 5), 4 weeks (n = 5), 6 weeks (n = 5), and 12 weeks (n = 5) after hemisection were anesthetized with 0.02% MS-222 in water, and placed on ice. An incision was made in the appropriate region of the dorsal skin, and the rostral segments of the spinal cord were exposed. The spinal cord was cut at the level of the second spinal nerve on the same side as the first incision. The level of the second incision was more caudal than that of the first incision, and the distance between the first and second incisions was 5 mm. A piece of paper soaked with 10mg/ml tetramethylrhodamine dextran amine (RDA; 1%; 0.5 ll; molecular weight = 3000; Invitrogen, Carlsbad, CA, USA) in phosphate-buffered saline (PBS; 0.1 M; pH 7.4) was inserted into the lesion. The survival time after tracer injection in the RDA group was 3 days. The remaining 45 lesioned fish (non-RDA group) and 1 control fish did not undergo the tracer injection. The lesioned fishes were survived 3 d (n = 5), 1 week (n = 6), 2 weeks (n = 5), 3 weeks (n = 9), 4 weeks (n = 5), 6 weeks (n = 10), and 12 weeks (n = 5) after hemisection. Motor activity measurements The motor activity of each fish was quantified by measuring the duration (in seconds) of upright posture per minute. Observations were made immediately

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before killing the non-RDA group with survival periods of 3 d (n = 5), 1 week (n = 5), 2 weeks (n = 5), 3 weeks (n = 5), 4 weeks (n = 5), 6 weeks (n = 5), and 12 weeks (n = 5) after hemisection. One-minute measurements were obtained three times for each fish. Mean duration was calculated and compared using the Mann–Whitney U-test with Bonferroni correction. To verify the role of regenerating axons through the lesion site in the improvement of motor activity, the spinal cord of an additional non-RDA group with a survival period of 6 weeks after hemisection (n = 5) was re-hemisectioned at the same level. Three days after the re-hemisection, motor activity measurements were calculated in the same manner. Mean duration of upright posture in the re-hemisection group and that in the non-RDA group at 6 weeks after hemisection was compared using the Mann–Whitney U-test. Tissue preparation After the indicated survival period, the fish (Total: n = 83, Control group: n = 6, RDA group: n = 35, non-RDA group: n = 42) were re-anesthetized and perfused transcardially with saline containing 1% heparin, followed by 0.1 M phosphate buffer (PB, pH 7.4) containing 4% paraformaldehyde (PFA). The spinal cord including the hemisectioned level was removed immediately, postfixed in 0.1 M PB containing 4% PFA for 5–6 h at 4 °C, and left overnight in 0.1 M PB containing 25% sucrose at 4 °C for cryoprotection. The brains of the control group (n = 3) and the RDA-group (n = 6) were also removed and postfixed in the same manner. They were used for observation of retrogradely labeled neurons in the brain. The spinal cord and brain were embedded in TissueTek OCT compound (Sakura, Tokyo, Japan), and frozen in liquid nitrogen. The frozen specimens were cut serially into 20-lm-thick horizontal sections, and thawmounted on gelatin-coated slides in a cryostat (Moriyasu-Konetsu, Osaka, Japan), equipped with a microtome (Microm, Walldorf, Germany). Sections were arranged in five series comprising every fifth section. All the sections were dried for 1 h at room temperature, postfixed in 0.1 M PB containing 4% PFA for 30 min, and rinsed in 0.1 M PBS (pH 7.4) containing 0.3% Triton X-100 (PBST, pH 7.4) for 10–20 min. Triple fluorescent immunohistochemistry The sections of the spinal cord were used for fluorescent immunohistochemistry, as shown in Table 1. A series of sections of the RDA group (n = 35) was incubated in a moist chamber overnight at 4 °C with a mixture of mouse monoclonal antibody against GFAP (50 lg/ml, clone G-A-5, Sigma–Aldrich), rat polyclonal antibody against 5-HT (1:10, AbD Serotec, Oxfordshire, UK), and rabbit polyclonal antibody against laminin (20 lg/ml, Sigma–Aldrich) diluted with 1% normal donkey serum (NDS), 0.2% bovine serum albumin (BSA), and 0.1% NaN3 in 0.1 M PBST. After several rinses with 0.1 M PBST, the sections were at room temperature incubated for 3 h with a mixture of secondary antibodies, i.e., Cy5conjugated donkey anti-mouse IgG (10 lg/ml; Jackson

ImmunoResearch Laboratories, West Grove, PA, USA), Cy2-conjugated donkey anti-rat IgG (10 lg/ml; Jackson ImmunoResearch Laboratories), and aminomethylcoumarin (AMCA)-conjugated donkey anti-rabbit IgG (10 lg/ml; Jackson ImmunoResearch Laboratories), diluted with 1% NDS, 0.2% BSA, and 0.1% NaN3 in 0.1 M PBST. The specificity of mouse monoclonal antibody against GFAP (clone G-A-5) in the goldfish spinal cord was confirmed previously (Alunni et al., 2005). A series of sections of the non-RDA group (n = 35) was incubated with a mixture of mouse monoclonal antibody against GFAP (50 lg/ml), rabbit polyclonal antibody against fish collagen-1 (20 lg/ml, Abcam, Cambridge, UK), and goat polyclonal antibody against fibroblast growth factor-1 (FGF-1; 4 lg/ml, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The sections were then incubated with a mixture of Cy3-conjugated donkey anti-mouse IgG (10 lg/ml; Jackson ImmunoResearch Laboratories), Cy5-conjugated donkey anti-goat IgG, and Cy2-conjugated donkey anti-rabbit IgG or AMCA-conjugated donkey anti-rabbit IgG. Double fluorescent immunohistochemistry A series of sections of the RDA group (n = 35) was incubated with a mixture of rabbit polyclonal antiserum cocktail to neurofilament (NF; 1:150, NA 1297, Affinity Research Products, Exeter, UK) and mouse monoclonal antibody against GFAP (50 lg/ml). The sections were then incubated with a mixture of Cy5-conjugated donkey anti-mouse IgG, and Cy2-conjugated donkey anti-rabbit IgG. Another series of sections of the RDA group was incubated with a mixture of rabbit polyclonal antibody against laminin (20 lg/ml), and mouse monoclonal antibody against GFAP (50 lg/ml). The sections were then incubated with a mixture of Cy5-conjugated donkey anti-mouse IgG, and Cy2-conjugated donkey anti-rabbit IgG or AMCA-conjugated donkey anti-rabbit IgG. Another series of sections of the RDA group was incubated with a mixture of rabbit polyclonal antibody against NG2 (10 lg/ml, Chemicon, Temecula, CA, USA), and mouse monoclonal antibody against GFAP (50 lg/ml). The sections were then incubated with a mixture of Cy5-conjugated donkey anti-mouse IgG, and Cy2-conjugated donkey anti-rabbit IgG. A series of sections of the non-RDA group (n = 35) was incubated with a mixture of mouse monoclonal antibody against GFAP (50 lg/ml), and rabbit polyclonal antibody against fish collagen-1 (20 lg/ml). The sections were then incubated with a mixture of Cy3conjugated donkey anti-mouse IgG, and Cy2-conjugated donkey anti-rabbit IgG or AMCA-conjugated donkey anti-rabbit IgG. Another series of sections of the nonRDA group was incubated with a mixture of mouse monoclonal antibody against a neuronal marker, acetylated tubulin (1:100, Clone 6–11B-1, Sigma– Aldrich), and rabbit polyclonal antibody against laminin (20 lg/ml, Sigma–Aldrich). The sections were then incubated with a mixture of Cy2-conjugated donkey antimouse IgG or Cy3-conjugated donkey anti-mouse IgG, and Cy2-conjugated donkey anti-rabbit IgG or Cy5conjugated donkey anti-rabbit IgG. Another series of

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A. Takeda et al. / Neuroscience 284 (2015) 134–152 Table 1. Summary of the experiments 1. Immunohistochemical and tract-tracing study Group Hemisection

RDA group

non-RDA group

Control

n = 35

n = 35

Survival time

RDA injection

Immunostaining

3 d, 1 w, 2 w, 3 w, 4 w, 6 w, 12 w

+

Glial fibrillary acidic protein, laminin Glial fibrillary acidic protein, Glial fibrillary acidic protein, Glial fibrillary acidic protein, Glial fibrillary acidic protein, growth factor-1 Glial fibrillary acidic protein, Acetylated tubulin, Laminin Glial fibrillary acidic protein, Acetylated tubulin, Nestin Glial fibrillary acidic protein Collagen-1 5-Hydroxytryptamine Laminin

3 d, 1 w, 2 w, 3 w, 4 w, 6 w, 12 w

n=1

1w

n=5 n=5 n=1

6 w (re-sectioning)

5-Hydroxytryptamine, Neurofilaments Laminin NG2 Collagen-1, fibroblast Collagen-1 Vimentin

+ Glial fibrillary acidic protein Collagen-1 5-Hydroxytryptamine laminin

2. Electron microscopic study Group Hemisection Control

n=4 n=1

Survival time 3w

sections of the non-RDA group was incubated with a mixture of mouse monoclonal antibody against vimentin (Clone V9; 0.5 mg/ml, Dako), and rabbit polyclonal antibody against GFAP (15 lg/ml, Dako). The sections were then incubated with a mixture of Cy2-conjugated donkey anti-mouse IgG, and Cy5-conjugated donkey anti-rabbit IgG. Another series of sections of the nonRDA group was incubated with a mixture of rabbit polyclonal antibody against zebrafish nestin (1:50, Anaspec Inc., Fremont, CA, USA), and mouse monoclonal antibody against acetylated tubulin (1:100). The sections were then incubated with a mixture of Cy3-conjugated donkey anti-mouse IgG, and Cy2conjugated donkey anti-rabbit IgG. Single fluorescent immunohistochemistry Sections of the control group (n = 1) and the non-RDA group 1 week after hemisection (n = 1) was incubated with rabbit polyclonal antibody against fish collagen-1 (20 lg/ml), rabbit polyclonal antibody against laminin (20 lg/ml), mouse monoclonal antibody against GFAP (50 lg/ml), or rat polyclonal antibody against 5-HT (1:10), respectively. The sections were then incubated with Cy2-conjugated donkey anti-rabbit IgG, Cy5conjugated donkey anti-mouse IgG, or Cy5-conjugated donkey anti-rat IgG. Observation and imaging All the sections were examined with an epifluorescence microscope (Leica DMR; Leica, Wetzlar, Germany)

equipped with excitation filters. Images obtained with a CCD camera (Leica DC 20; Leica) were digitally read into a computer through DC Viewer Software (Leica). Some sections were also examined with confocal laser scanning microscopy (Zeiss LSM 510; Carl-Zeiss, Jena, Germany) to construct 3D images. Contrast and brightness were adjusted with Adobe Photoshop Software (Adobe, San Jose, CA, USA). Quantitative analysis of fibrous scar area For quantitative analysis of the fibrous scar area, a series of sections of the RDA group that were immunostained with anti-GFAP antibody were used. The fibrous scar area was defined as an area including small cavities as well as fibrous tissue. Therefore, the area negative for GFAP immunoreactivity in each section was measured as the fibrous scar area with ImageJ software. The sum total of area for each fish at 3 d, 1 week, 2 weeks, 6 weeks, and 12 weeks after hemisection was calculated. The fibrous scar area was compared among the different time points using the Mann–Whitney U-test. Quantitative analysis of number of nerve fibers penetrating the scar A series of sections of the RDA group (n = 35) and control group (n = 5) were used for a quantitative analysis of regenerating fibers. The number of nerve fibers that grew beyond the level of the fibrous scar in each section was calculated by counting RDA-labeled fibers on the hemisection side at a level 0.5 mm rostral

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to the hemisection level, i.e., 5.5 mm rostral to the tracer injection site. The sum total of nerve fibers for fish of the control group, and each fish at 3 d, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, and 12 weeks after hemisection was calculated. The number of nerve fibers penetrating the scar was compared among the different time points using the Mann–Whitney U-test. Quantitative analysis of number of retrogradely labeled neurons In the control group (n = 3), and the RDA-group 6 weeks (n = 3) after hemisection, retrogradely labeled neurons were counted in sequential sections of the brain and the spinal cord rostral to the hemisection level. Transmission electron microscopy For the electron microscopy study, control fish (n = 1) and lesioned fish (n = 4) with survival periods of 3 weeks after hemisection were deeply anesthetized with 0.04% MS222 in water, and placed on ice. The fishes were then perfused transcardially with saline containing 1% heparin, followed by 0.1 M PB (pH 7.4) containing 2% PFA and glutaraldehyde (GA). Spinal cord specimens were removed immediately and postfixed overnight in the same fixative. After dehydration with a graded ethanol series, the sections were embedded in a mixture of Epon and Araldite. Ultrathin sections were cut using an Ultratome (LKB: Leica), counterstained with 4% uranyl acetate and 1% lead citrate, and examined under an electron microscope (H-7500; Hitachi, Tokyo, Japan).

RESULTS Improvement of motor activity after spinal cord lesion Immediately after transection of the spinal cord on the left side, the bodies of the fish curved to the right, and the fish lay on their right side on the bottom of the tank (Fig. 1A). They could not perform rhythmic and sequential locomotive movements, but sporadic movements of their fins and trunk muscles were observed on the intact side of the body. The body curvature gradually decreased, and the fish regained the ability to assume a normal upright posture and swim straight for short distances within 3 weeks after hemisection (Fig. 1B). Motor activity was significantly improved at 4 weeks after hemisection compared to that at 1 week after hemisection (Fig. 1C). Motor activity was significantly decreased in the re-hemisected group (Fig. 1D). Fibrous scar formation within 1 week after spinal cord lesion Hemisection of the spinal cord produced an open wound at the lesion site. A fibrous scar that was immunoreactive for fish collagen-1 and laminin formed at the lesion site within 1 week after hemisection (Fig. 2A–D). The fibrous scar was distinguished from the surrounding nervous tissue, which contained many GFAP-immunoreactive glial processes. In the surrounding nervous tissue, no hypertrophic change of

the glial processes was observed, whether near or far from the lesion site (Fig. 2E, F). The fibrous scar contained a few GFAP-positive glial processes, and was thus recognized as a GFAP-negative zone under a fluorescence microscope (Fig. 3A–F). Between the fibrous scar and the surrounding nervous tissue, the glia limitans that was immunoreactive for laminin, was partially present (Fig. 3D). The glia limitans appeared continuous with the meninges (Fig. 3D). In the fibrous scar, there were many laminin-coated tubular structures (Fig. 3G). Most of the strongly lamininimmunoreactive tubules appeared to be blood vessels, but some laminin-immunoreactive tubules were continuous with the glia limitans bordering the fibrous scar and the nervous tissue. Many of these tubular structures contained GFAP-immunoreactive glial processes (Fig. 3H–J). Thus, the tubular structures appeared to be nervous tissue protruding into the fibrous scar. 5-HT-positive neurons were present in the fibrous scar, as well as the surrounding nervous tissue (Fig. 2G, H). The fibrous scar reached its maximum size 7 d after the hemisection, and gradually decreased within 3 weeks after hemisection. The fibrous scar area was significantly reduced at 6 and 12 weeks after the hemisection compared with that 1 week after hemisection (Fig. 4). Axonal intrusion into the fibrous scar within 1 week after spinal cord lesion The tract tracing study using RDA in combination with multiple immunohistochemistry showed that regenerating axons entered the fibrous scar within 1 week after the hemisection. Although some of the regenerating axons were associated with GFAP-positive fibers, many labeled axons were not in close proximity to the GFAP fibers (Fig. 5A–D). Intrusion of glial processes into the fibrous scar was usually preceded by that of neuronal elements. Furthermore, double labeling of acetylated tubulin and laminin showed that many acetylated tubulin-immunoreactive regenerating axons in the fibrous scar were not associated with the laminincoated tubular structures. Although some regenerating axons were partly covered by the laminin-coated tubular structures, the tip of axons was not in close proximity to the laminin-coated tubules (Fig. 5E–G). Development of tubular structures in the fibrous scar and axonal regeneration 2–3 weeks after spinal cord lesion Two to three weeks after the spinal hemisection, the large fibrous scar containing collagen-1 and FGF-1 immunoreactive materials was still present in the lesion site (Fig. 6A–D). The number of laminin-coated tubular structures appeared higher than that at 1 week after hemisection. Some tubules had a large diameter, and contained many GFAP-immunoreactive glial processes. Some of these tubules developed to create tunnels that penetrated the fibrous scar (Fig. 6E–G). Thin tubules that contained a few GFAP-immunoreactive glial processes were also present (Fig. 6H–K). GFAP-immunoreactive

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Fig. 1. Motor activity of goldfish recovers within 1–3 months after spinal hemisection. A: Three days after hemisection of the spinal cord on the left side, the body of the fish curved to the right, and the fish lay on its right side on the bottom of the tank. B: Four weeks after hemisection, the fish regained its normal upright posture and swam straight for short distances. C: The motor activity of fish at 3 d, 1 week, 2 weeks, 3 weeks, 4 weeks, and 12 weeks after spinal hemisection was evaluated by measuring the duration of upright posture per minute. Motor activity 4 weeks after hemisection was significantly recovered compared to that 1 week after hemisection. Motor activity 12 weeks after hemisection was significantly greater than that 4 weeks after hemisection. Values are means ± SD. Significant differences are indicated by ⁄ (P < 0.05) and ⁄⁄ (P < 0.01). D: The motor activity of fish at 6 weeks after spinal hemisection was compared with that at 3 d after spinal re-hemisection following the 6-weeks survival period after the first hemisection. Motor activity of re-hemisected fish was significantly reduced compared with that 6 weeks after the initial hemisection. Values are means ± SD. Significant differences are indicated by ⁄ (P < 0.05) and ⁄⁄ (P < 0.01).

fibers were present exclusively within the laminin-coated tubular structures in the fibrous scar. Within 2–3 weeks after the hemisection, although some regenerating axon tips were not in close proximity to the laminin-coated tubular structures tubules (Fig. 7A–D), the majority of regenerating axons in the fibrous scar were observed inside the laminin-coated tubules (Fig. 7E–H). 5-HT immunoreactive neurons migrated into the tubules, lined the inner walls of the tubules. Development of tunnels containing glial processes and regenerating axons 6–12 weeks after the spinal lesion Six weeks after hemisection, the glia limitans bordering the fibrous scar and the surrounding nervous tissue was developed (Fig. 8A). The laminin-coated tubular structures in the fibrous scar also developed and increased in diameter. Some significantly enlarged tubules containing many GFAP-positive glial processes, created tunnels that penetrated the fibrous scar

(Fig. 8B). Many thin tubules coated with laminin also invaginated from the glia limitans into the fibrous scar (Fig. 8E, J). The number of 5-HT neurons lining the tubular walls had also significantly increased (Fig. 8D, I, N). Six weeks after hemisection, many RDA-labeled axons passed the fibrous scar through large laminincoated tunnels (Fig. 8C, G, L). These nerve bundles were closely associated with 5-HT neurons, as well as to GFAP-immunoreactive glial processes (Fig. 8H, M). Some RDA-labeled axons unaccompanied by glial processes were also observed 6 weeks after the hemisection. No RDA-labeled axons entered the fibrous scar by routes other than the tubules at this stage. Twelve weeks after hemisection, the diameter of the tunnels in the fibrous scar was further increased (Fig. 9A). Further invasion of GFAP-immunoreactive glial processes into the tunnels promoted the replacement of fibrous tissue by nervous tissue, and the glial processes that filled the tunnels occupied most of the lesion site (Fig. 9B). This resulted in a significant reduction of the fibrous scar area (Fig. 4). Twelve

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the fibrous scar (Fig. 10A, B). RDA-labeled axons did not enter the NG-2 positive fibrous tissue (Fig. 10C). Six weeks after hemisection, NG2 was still expressed in both the fibrous scar and the glial tissue, but immunoreactivity appeared to be significantly lower (Fig. 10D, E). In this stage, there were some RDAlabeled axons penetrating the NG2-positive zone in the fibrous tissue (Fig. 10F). Vimentin and nestin expression in the scar tissue Vimentin immunoreactivity, a marker for ependymoglial cells, was observed in the ependymal layer around the central canal, but not in the fibrous scar or the surrounding glial tissue after hemisection (Fig. 11A–D). Nestin immunoreactivity was observed in the cells scattered in the parenchyma surrounding the lesion site. They did not migrate into the lesion site or the fibrous scar tissue at 1 week after hemisection. No nestinimmunoreactive cells were observed at 2 weeks after hemisection or thereafter (Fig. 11E, F). Number of RDA-labeled axons beyond the hemisection level

Fig. 2. The fibrous scar formed at the lesion site showed immunohistochemical profiles very different from those in the intact fish. A, B: Immunoreactivity for fish collagen-1 was observed in the meninges (arrow) at normal (A), and in the fibrous scar at the lesion site as well as the meninges (arrow) 1 week after hemisection (B). C, D: Immunoreactivity for laminin was observed in the meninges (arrow) at normal (C), and in the fibrous scar at the lesion site as well as the meninges (arrow) 1 week after hemisection (D). E, F: GFAP-immunoreactive fibers were observed in the entire parenchyma at normal (E), but were lacking at the fibrous scar 1 week after hemisection (F). G, H: 5-HT Immunoreactive neurons (arrow) were scattered at normal (G), but accumulated at the lesion site (arrows in H). Scissor marks: hemisection site. Dashed line: midline. Scale bar = 50 lm in H (applies also to A–G).

weeks after hemisection, the enlarged tunnels provided pathways for a number of long descending and ascending tracts to penetrate the scar, and a bundle of RDA-labeled axons ran along the glial processes in the broad hole-like structures in the fibrous scar (Fig. 9C). Many 5-HT neurons were present close to the lamininpositive tunnel wall, but the majority of 5-HT neurons was observed inside the tunnels and did not contact the tunnel wall. Some RDA-labeled axons were in close proximity to 5-HT neurons (Fig. 9D). Expression of CSPGs in the scar tissue NG2-immunoreactive deposits were not observed within 1 to 2 weeks after the hemisection. They were observed 3 weeks after the hemisection in the fibrous scar. NG2 immunoreactivity was also observed in the surrounding nervous tissue, but significantly lower levels than that in

In intact fish, nearly 600 RDA-labeled fibers were observed at the level 5.5 mm rostral to the RDA injection (Fig. 12). From 3 d to 2 weeks after hemisection, few RDA-labeled fibers were observed at the level 0.5 mm rostral to hemisection. The number of RDA-labeled fibers was significantly reduced compared to that in intact fish. No RDA-labeled fibers penetrating through the fibrous scar were observed. Three weeks after the hemisection, the number of RDA-labeled fibers just rostral to the lesion site had significantly increased compared to that 2 weeks after hemisection. Four weeks after hemisection, the number of RDA-labeled fibers rostral to the lesion site increased to more than 500, which was significantly greater than that 3 weeks after hemisection. There were no significant differences between the number of RDAlabeled fibers 4 weeks after hemisection, and that 6 or 12 weeks after hemisection. Retrogradely labeled neurons in the brainstem Many RDA-labeled neuronal perikarya were observed in the diencephalon and brainstem of the control group and the RDA group 6 weeks after hemisection. In the diencephalon, labeled neurons were observed in the nucleus ventromedialis (NVMD; Fig. 13A, F). In the mesencephalon, labeled neurons were observed in the nucleus of the medial longitudinal fasciculus (Nflm) on both sides and in the red nucleus on the side contralateral to the injection (Fig. 13B, F). In the rhombencephalon, labeled neurons were observed in the nucleus reticularis (NR: Fig. 13C, F), the octaval nucleus (NO; Fig. 13D, F), as well as in the facial lobe (FL; Fig. 13E, F). Most of the labeled fibers were observed in the medial longitudinal fasciculus, the bulbospinal tract, the vestibulospinal tract, and the secondary gustatory tract. No RDA-labeled neuronal

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Fig. 3. At 1 week after hemisection, a fibrous scar was bordered from surrounding GFAP-immunoreactive glial tissue by the laminin-positive glial limitans. Some GFAP-immunoreactive glial processes entered the fibrous scar through the laminin-coated tubular structures. A–C: The fibrous scar was filled with collagen-1 immunoreactive matrix (A), and considered to be the GFAP-negative zone in the lesion site, although some GFAPimmunoreactive glial processes enter the fibrous scar (B). A merged image of A and B is shown in C. D–F: The fibrous scar was immunoreactive for laminin (D). Laminin was also expressed on the glia limitans (arrows in D) that bordered the fibrous scar and surrounding nervous tissue immunoreactive for GFAP (E). The glia limitans was continuous with the meninges (black arrowheads in D). Blood vessels (white arrowheads in D) were also immunoreactive for laminin. A merged image of D and E is shown in F. G-I: The laminin-coated tubular structures in the fibrous scar (arrows in G) were continuous with the glia limitans. Glial processes immunoreactive for GFAP (arrowheads in H) entered the tubular structures. A merged image of G and H is shown in I. J: The GFAP immunoreactive glial processes within the laminin-coated tubular structures were also shown as a 3D image. Scissor marks: hemisection site. Dashed line: midline. Scale bars = 50 lm in C (applies also to A and B), 100 lm in F (applies also to D and E), and 20 lm in I (applies also to G and H) and J.

perikarya, on the other hand, were observed 3 days after hemisection. No RDA-labeled neuronal perikarya were observed in the spinal cord both in intact fishes and the RDA group. Ultrastructure of the regenerating axons in the lesion site Three weeks after spinal hemisection, the fibrous scar in the lesion site was demarcated from the surrounding nervous tissue by glial limitans. Electron microscopy observation of the glial limitans revealed a basement membrane between the glial process and the fibrous tissue. Breaking through the glia limitans, regenerating axons protruded into the fibrous tissue from the surrounding nervous tissue. These axons were usually in close contact with glial processes containing small bundles of glial filaments (Fig. 14A). The glial processes

sometimes had a digitate structure, the surface of which was coated by the basement membrane (Fig. 14A). Direct contacts between the regenerating axons and the basement membrane were not observed. Three weeks after spinal hemisection, fasciculi and bundles of regenerating axons were observed among the fibroblasts and collagen fibers at the lesion center. The nerve bundles were wrapped by glial processes, whose surface was coated by the basement membrane (Fig. 14B). In the lesion center 3 weeks after the spinal hemisection, tubular structures ensheathing the regenerating axons, dendrites, glial processes, glial cell bodies, and phagocytes were observed among the fibrous tissue (Fig. 14C, D). The peripheral wall of the tubular structures was formed by glial processes, and the basement membrane coated the surface of the glial processes (Fig. 14E). Desmosome-like structures form

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Fig. 4. The fibrous scar area measured as a GFAP-negative zone. The fibrous scar area reached its maximum size 1 week after hemisection. The fibrous scar area 6 or 12 weeks after hemisection was significantly reduced compared to that 2 weeks after hemisection. Values are means ± SD. Significant differences are indicated by ⁄ (P < 0.05) and ⁄⁄ (P < 0.01).

Fig. 5. At 1 week after hemisection, some regenerating axons entered the fibrous scar. A–D: RDA-labeled regenerating axons (arrow in A), also labeled with neurofilament immunoreactivity (arrow in B) entered the fibrous scar. A merged image of A and B is shown in C. Regenerating axons in the fibrous scar were not associated with the GFAP-immunoreactive glial processes (arrowhead in D). E–G: Regenerating axons labeled with acetylated tubulin (arrows in E) entered the fibrous scar. Although regenerating axons were partly covered by the laminin-coated tubular structures (arrowhead in F), the axon tips were not associated with the laminin-coated tubular structures. A merged image of E and F is shown in G. Scissor mark: hemisection site. Scale bars = 20 lm in D (applies also to A–C) and G (applies also to E and F).

the contacts between glial processes and neighboring glial process (Fig. 14D). The spatiotemporal change of the tissue organization in the lesion site is summarized in Fig. 15.

DISCUSSION Improvement of motor activity and increases in the number of regenerating fibers across the lesion site The present retrograde tracing study showed that spinally descending pathways from several nuclei in the

diencephalon, mesencephalon, and rhombencephalon regenerated after spinal hemisection. The present results are consistent with the previous report by Sharma et al. (1993), which used a complete transection model. In the goldfish, the midbrain region including the Nflm is a locomotion center that controls the bilateral and unilateral rhythmic movement of the trunk and tail (Kobayashi et al., 2009). The medial longitudinal fasciculus is the major descending pathway for spinal projections from the midbrain locomotion center (Uematsu and Todo, 1997). The behavioral observations in the present study could indicate that the medial longitudinal fasciculus on

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Fig. 6. At 2 week after hemisection, area of the fibrous scar was reduced. GFAP-immunoreactive glial processes enter the fibrous scar exclusively through the laminin-coated tubular structures. A–D: The large fibrous scar containing collagen-1 immunoreactive materials was still present in the lesion site (A). GFAP immunoreactive glial processes (arrow in B) enter the fibrous scar (arrowheads in A). A merged image of A and B is shown in C. FGF-1 immunoreactive materials were also present in the fibrous scar (arrow in D). E–G: GFAP-immunoreactive glial processes (arrow in F) protruded into the fibrous scar. They were exclusively present within the laminin-coated tubular structures (asterisks in E). A merged image of E and F is shown in G. H–J: GFAP-immunoreactive glial processes (arrows in I) were exclusively present within the laminin-coated tubular structures (asterisks in H). A merged image of H and I is shown in J. K: The GFAP immunoreactive glial processes (arrow) within the laminin-coated tubular structure (asterisk) were also shown as a 3D image. Scissor marks: hemisection site. Dashed line: midline. Scale bars = 50 lm in D (applies also to A–C), 100 lm in G (applies also to E and F), and 20 lm in J (applies also to H and I) and K.

the lesion side was damaged, because the medial longitudinal fasciculus below the medullary level is responsible for ipsilateral movement (Uematsu and Todo, 1997). Although the present study suggests that the increase in the number of regenerating axons penetrating the lesion site correlates with the recovery of swimming ability, this did not necessarily indicate that motor activity recovered to normal levels, because the present study did not evaluate the precise motor activities. A previous study showed that C-starts primarily conducted by Mauthner cells are functionally impaired to some extent (Zottoli and Freemer, 2003). In addition, some spinally projecting brain nuclei do not regenerate across the lesion site after spinal transection (Sharma et al., 1993), or project inappropriately to the peripheral nerve roots, as demonstrated in the crush injury model of the goldfish (Bentley and Zottoli, 1993). These previous observations are consistent with the present finding that the number of RDA-labeled regenerating axons did not reach the levels observed in intact fish. Recovery of swimming activity might be primarily induced by the regeneration of descending projections from the midbrain locomotion center. Regeneration of projections from the Nflm to motor neurons and interneurons in the spinal cord has been demonstrated in the goldfish hemisection model (Takeda et al., 2007).

The present study also showed that re-sectioning of the lesion site in recovered fish significantly impaired motor activity. This observation provides strong evidence against the possibility that recovery after spinal hemisection might be due to collateral sprouting of the intact descending tracts on the contralateral side. A previous study provided evidence for neurogenesis after spinal injury in the goldfish (Takeda et al., 2008). Here, however, we could not evaluate the significance of de novo neural circuits for functional recovery after goldfish spinal injury. Organization of the scar Axonal regeneration after spinal lesions in the goldfish was first reported almost 90 years ago (see Bernstein, 1964). Previous histological studies showed that scar tissue forms at lesion sites after spinal transection, and regenerating axons pass through the scar (Bernstein, 1964; Bernstein and Bernstein, 1967). Scar tissue in the goldfish is reported to contain fibrous tissue at the core and glial tissue in the surrounding parenchyma (Bernstein, 1964; Bernstein and Bernstein, 1967). The present study confirmed that the fibrous scar contains collagen-1. These findings suggest that the scar formed

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Fig. 7. At 2 weeks after hemisection, regenerating axons entering the fibrous scar were covered by the laminin-coated tubular structures. A–C: Although the acetylated tubulin-immunoreactive regenerating axons (arrow in A) were partly covered by the laminin-coated tubular structures (asterisk in B), regenerating axon tips were not associated with the laminin-coated tubular structures. A merged image of A and B is shown in C. D: The acetylated tubulin-immunoreactive axons (arrow) and the laminin-coated tubular structure (asterisk) were shown as a 3D image. E–G: A fascicle of acetylated tubulin-immunoreactive regenerating axons (arrow in E) was exclusively present within the laminin-coated tubular structures (asterisk in F). A merge of E and F was shown in G. H: The acetylated tubulin-immunoreactive axons (arrow) within the laminin-coated tubular structure (asterisk) were shown as a 3D image. A cross section of the laminin-coated tubular structure (asterisk) and the acetylated tubulinimmunoreactive axon (arrow) at the indicated site (dashed line) were also shown in an inset. Scissor marks: hemisection site. Scale bars = 10 lm in D, and 20 lm in C (applies also to A and B), G (applies also to E and F) and H.

after spinal hemisection in goldfish is somewhat similar to that observed in mammals, and that these conditions appear to be distinct from those in the zebrafish and amphibians in which the formation of scar tissue has not been reported. It is unclear, however, whether these models lack the fibrous scar in fact, because thorough characterization of the lesion site with chemical markers has not been performed. Further histochemical investigation in these and other species might be necessary to determine if the axonal regenerating found in the present study is the model specific to the goldfish or more universal one across the vertebrates. The origin of the ECM molecules in the fibrous scar is not clear. In mammals, the meningeal cells that migrate to the lesion site make an important contribution to the formation of the fibrous scar (Shearer and Fawcett, 2001; Kawano et al., 2012). The meningeal cells in goldfish might also migrate to the lesion site and proliferate to contribute to the formation of the fibrous scar, because the glia limitans that demarcated the fibrous scar from the surrounding nervous tissue was continuous with the meninges. The tissue surrounding the fibrous scar contains many GFAP-expressing glial processes. This glial tissue in goldfish, however, differs in some aspects from the glial scar that forms in mammals. First, the glial fibers in goldfish do not appear hypertrophic and there is little increase in GFAP expression. These findings suggest that the glial reaction to injury is quite different from that in mammals. The difference in glial reactivity might be due to the nature of the glial cells. In the normal spinal cord of teleosts, stellate-shaped astrocytes are only rarely observed in specific regions of the brainstem

(Ka´lma´n, 1998). GFAP expression in the teleost spinal cord is observed in the ependymal plexus, a dense network of glial processes that covers the gray matter around the central canal, in the ependymoglial cells within the ependymal layer and their radial fibers, and in the perivascular glia within the white matter (carp, Cyprinus carpio; Ka´lma´n, 1998; rainbow trout, Oncorhynchus mykiss; Alunni et al., 2005; and mullet, Chelon labrosus; Arochena et al., 2004). Even after injury, typical ‘‘astrocyte’’-like cells were not observed in the present study. Organization of the tubular structure The present immunofluorescence study suggested that almost all of the regenerating axons were wrapped by the laminin-coated tubular, and that the enlargement of laminin-coated tubular structures is relevant to the axonal regrowth beyond the fibrous scar in goldfish (Fig. 15). The present electron microscopy observations demonstrating the basement membrane between the glial processes and the surrounding fibrous tissue in the scar, suggests that the laminin-coated tubular structures observed in the immunofluorescence study might correspond to the glial processes accompanied by the basement membrane. Furthermore, the desmosome-like contacts with neighboring glial processes suggest that these processes are derived from astrocytes. Tight junctions or desmosomes were observed in the astrocytes in the optic nerve of the goldfish (Wolburg and Ka¨stner, 1984). Tubular structures ensheathing the sprouting axons are also found in the fibrous scar after spinal injury of the rat (Frise´n et al., 1995). In the rat, the sheath is

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Fig. 8. At 6 weeks after hemisection, many regenerating axons and GFAP-immunoreactive glial processes enter the fibrous scar through the laminin-coated tubular structures. A–D: Fibrous scar tissue was demarcated from the nervous tissue by the glia limitans, which was strongly immunoreactive for laminin (arrowheads in A). Laminin was also strongly expressed in the large tubes (asterisks in A) continuous with the glia limitans. GFAP-immunoreactive glial processes (arrows in B) filled the tubes, which penetrated the fibrous scar to create tunnels. Many RDAlabeled axons (arrows in C) entered the tube. 5-HT neurons (arrowheads in D) fibers migrated into the tube, and lined the tube wall. E–N: Higher magnification images of a repaired lesion site. Laminin was expressed in the wall of the tubular structures (asterisks in E and J) that protruded into the fibrous scar. GFAP-immunoreactive glial processes (arrows in F and K) entered the tubular structures. RDA-labeled axons (arrows in G and L) entered the tube in the fibrous scar. Merged images of F and G, and K and L are shown in H and M, respectively. 5-HT neurons (arrowheads in I and N) migrated into the tube, and lined the tube wall. Scissor marks: hemisection site. Dashed line: midline. Dotted line: border of the fibrous tissue. Scale bars: 50 lm in D (applies also to A–C), 20 lm in I (applies also to E–H) and N (applies also to J–M).

formed predominantly by astrocytes, and the cell surface is immunoreactive for laminin. Therefore, formation of the laminin-coated tubular structures by glial cells might be a common process for guiding the regenerating axons in the fibrous scar both in mammals and goldfish after spinal hemisection. Unlike goldfish, however, the tubules in mammals do not form tunnels, through which regenerating axons penetrate the scar. Axonal regeneration through the scar The present study demonstrated the axonal regeneration of the descending spinal tracts through the fibrous scar with retrograde tract tracing using RDA-3000. Regeneration of the descending spinal tracts through the lesion site was previously observed in an anterograde tract tracing study in the goldfish after spinal hemisection (Takeda et al., 2007). As RDA-3000 is mainly transported retrogradely, labeled fibers in the fibrous scar after RDA injection might be regenerated descending spinal tracts which had originated in the nuclei in the diencephalon and brainstem. The present study, on the other hand, does not exclude the possibility that RDA was incorporated by the ascending spinal tracts and transported anterogradely. Some labeled fibers entering the fibrous scar from the caudal border might be ascending spinal tracts.

Significant roles of the glial processes for axonal regrowth A glial contribution to axonal regeneration through the scar has been suggested in the goldfish by the previous studies. Ependymoglial cells proliferating from the central canal after a spinal lesion migrate to the lesion site to constitute the scar tissue, and the processes of these cells then form a bridge as the supporting matrix for regenerating axons (Bernstein and Bernstein, 1969). Another study, on the other hand, showed that some regenerating axons grow without direct association with glial cells, and GFAP-positive glial processes trail behind the growing axons, suggesting that the outgrowth of regenerating axons does not depend on a direct association with ependymoglial cells (Nona and Stafford, 1995). The present immunofluorescence study showed that intrusion of the GFAP-positive glial processes into the fibrous scar was frequently preceded by the entry of regenerating axons into the fibrous scar (Fig. 15 stage 2). Furthermore, some regenerating axons entering the fibrous scar were wrapped by the thin tubular structures, which contained no GFAP-immunoreactive glial processes. The findings of the electron microscopy study, however, confirmed that axons protruding into the fibrous scar were usually in close contact with the glial

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Fig. 9. At 12 weeks after hemisection, laminin-coated tubular structures were enlarged to make tunnel, through which many regenerating axons penetrated the scar. Laminin was still expressed in the fibrous tissue, which contained a small cavity (demarcated by line in A). GFAP-immunoreactive glial processes (arrow in B) filled the large tunnel (asterisk in A). The fibrous scar area (demarcated by dotted line in B) was significantly reduced. A bundle of RDA-labeled axons (arrow in C) entered the large tunnel and penetrated the lesion site. Many 5-HT neurons (arrowheads in D) migrated into the large tunnel. Scissor mark: hemisection site. Dashed line: midline. Dotted line: border of the fibrous tissue. Scale bars = 50 lm in D (applies also to A–C).

processes. These glial processes might have been negative for GFAP-immunoreactivity because they did not contain well-developed filaments in their cytoplasm. Close contact between the glial processes and the regenerating axons was also observed deep in the lesion site at an early stage, suggesting that the glial processes might be associated with axonal elongation within the fibrous scar. Glial processes, on the other hand, did not precede the regenerating axons.

Therefore, it is unclear whether the glial processes play a significant role in guiding the regenerating axons. The growth cones of regenerating retinal axons in the goldfish are also in contact with astrocytes after optic nerve transection (Wolburg and Ka¨stner, 1984; Strobel and Stuermer, 1994). It is unclear, however, whether the glial processes are also associated with axonal regeneration in the late stage, because glial processes wrapped around bundles of several axons did not appear to be in direct contact with all of the axons in the large bundles. The contribution of glial processes to axonal regeneration after spinal injury is suggested in many other species. In the rat as mentioned above, astrocyte processes constitute tubular structures, through which regenerating axons to enter the scar (Frise´n et al., 1995). In the turtle Trachemys dorbigni (Rehermann et al., 2009), the sea lamprey Petromyzon marinus (Lurie et al., 1994), the newt Notophthalmus viridescens (Zukor et al., 2011) and the zebrafish (Goldshmit et al., 2012), all of which allow regenerating axons to pass the lesion site, regenerating axons appear to be guided by glial processes in the lesion site. A recent study in the newt suggested that the processes of ependymoglial cells extending longitudinally into the lesion site might guide the regenerating axons to pass the lesion site (Zukor et al., 2011). Migration of glial cells in the fibrous scar Migration of ependymoglial cells or ependymal cells plays a significant role in axonal regeneration after tail amputation with spinal injury in the black ghost knife fish (Apteronotus albifrons) and urodele amphibians (Egar and Singer, 1972; Nordlander and Singer, 1978; Anderson and Waxman, 1981; Anderson et al., 1984). In these animals, regenerated axons grow in close contact with processes of ependymal cells that proliferate in response to injury (reviewed by Chernoff et al., 2003). In

Fig. 10. NG2 was expressed in the lesion site. A–C: At 3 weeks after hemisection, the lesion site near the midline contained GFAP-immunoreactive glial processes, as shown in A. NG2 was expressed in the fibrous scar (arrowheads in B), as well as in the glial tissue surrounding the fibrous scar. An RDA-labeled fiber (black arrow in C) ran through the area filled with GFAP-immunoreactive glial processes near the midline. Another RDAlabeled axon (white arrow in C) appeared to turn in front of the fibrous scar. D–F: At 6 weeks after hemisection, the fibrous tissue was almost completely replaced by nervous tissue containing many GFAP-immunoreactive glial processes, as shown in D. NG2 (arrowheads in E) was expressed in the fibrous tissue and in the nervous tissue adjacent to the fibrous tissue. An RDA-labeled axon (arrow in F) penetrated the NG2-rich area in the fibrous scar. Scissor mark: hemisection site. Dashes line: midline. Dotted line: border of the fibrous tissue. Scale bars = 50 lm in C (applies also to A and B) and F (applies also to D and E).

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the glial cells in the fibrous scar originate from the ependymoglial cells. Alternatively, it is possible that cells other than the ependymoglial cells, such as meningeal cells or Schwann cells, migrate into the fibrous scar to come in close contact with the regenerating axons, and play a role in the formation of the tubular structures in the fibrous scar. Role of laminin in axonal regeneration beyond the scar Laminin is expressed on the basal lamina of vascular endothelial cells, and the pia mater in the intact CNS (Falk et al., 1999; Yong et al., 2012). After the spinal hemisection, laminin on the tubular structures might be formed by interactions between the glial elements inside the tubules and the fibrous tissue outside the tubular walls, in the same way as the glial limitans formed at the border between the fibrous scar tissue and surrounding nervous tissue. Because laminin is a potent stimulatory factor for axonal outgrowth, laminin on the tubules or tunnels might have a role in the axonal regrowth beyond the fibrous scar in the goldfish. Our electron microscopy study in goldfish, however, did not confirm direct contact between the regeneration axons and the basement membrane associated with the glial processes. The role of 5-HT neurons in axonal regeneration

Fig. 11. Cells immunoreactive for vimentin or nestin were not observed in the fibrous scar. A–D: Double fluorescent images of the lesion site 3 weeks after hemisection. Vimentin immunoreactivity (arrows in A and C) was observed in the ependymal layer reacting strongly with GFAP antiserum (B), but not in the laminin-immunoreactive fibrous scar tissue (D). E and F: Double fluorescent images of the lesion site 1 week after hemisection. Many nestin-immunoreactive cells (arrow in E) were observed in nervous tissue surrounding the lesion site, but not in the lesion site. Acetylated tubulin-immunoreactive nerve fibers (arrow in F) entered the lesion site, but they were not associated with nestin cells. Scissor marks: hemisection site. Dashed line: midline. Scale bars = 50 lm in B, D, F (applies also to A, C, and E).

the case of spinal transection at the rostral levels in urodeles, the proliferated ependymal cells appeared to transform into mesenchymal cells expressing vimentin to replace the gap, and then re-epithelialize to create an epithelial tube, through which regenerating axons grow (O’Hara et al., 1992; Chernoff et al., 2003). A recent study in zebrafish also raised the possibility that glial cells proliferating from the central canal migrate to the lesion site, and make bridges along which the regenerating axons pass the lesion site. Glial cells in the lesion site first express nestin and vimentin, but not always GFAP (Goldshmit et al., 2012). Immunofluorescence studies using antibodies against glial markers such as vimentin and nestin, however, did not confirm the migration of ependymoglial cells into the lesion site in goldfish. Therefore, it is not clear whether

A recent study showed that serotonergic fibers spontaneously regenerate into and cross beyond the scar more powerfully than other types of fibers in the case of spinal hemisection with a wire knife in the rat (Schiwy et al., 2009). Furthermore, serotonergic fibers enter the lesion site at the earliest stage of regeneration in the turtle, which also exhibits spontaneous axonal regeneration beyond the lesion (Rehermann et al., 2009). These findings suggest that serotonergic fibers have regenerative ability in the scar environment, and that 5-HT secreted in the scar has a role in the regeneration process. In goldfish, spinal injury activates neurogenesis and some newborn neurons expressing 5-HT persist for a long time in the lesion site (Takeda et al. (2008)). The 5-HT neurons might have a role as a growth-promoting or growth-regulating factor in guiding regenerating axons through the lesion site. In support of this idea, the number of 5-HT neurons inside the tunnels increased in parallel with the increase of the regenerating axons passing through the tunnels. It is possible that 5-HT fine-tunes axon guidance by inducing local collapse responses in regenerating axons, as suggested in the snail Lymnaea stagnalis (Koert et al., 2001). Do CSPGs play an inhibitory role in axonal regeneration in the scar? In mammals, NG2 molecules in the scar have long been assumed to be a major inhibitory factor for axonal regeneration (Dou and Levine, 1994; Fidler et al., 1999; Chen et al., 2002; Ughrin et al., 2003; Tan et al., 2006). NG2-expressing cells in mammals are assumed to

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Fig. 12. The number of RDA-labeled fibers 5.5 mm rostral to the injection level (0.5 mm rostral to the hemisection level) in intact fish, and fish at 3 d, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, and 12 weeks after hemisection. Values are means ± SD. Significant differences are indicated by ⁄ (P < 0.05).

Fig. 13. Many RDA-labeled neuronal perikarya were observed in the diencephalon and brainstem after RDA injection into the left spinal cord. A: Neurons in the NVMD (arrows) were labeled at 6 weeks after hemisection. B: Neurons in the Nflm (arrows) on the both sides and red nucleus (arrowhead) on the right side were labeled at 6 weeks after hemisection. C: Neurons in the NR (arrows) were labeled at 6 weeks after hemisection. D: Neurons in the NO (arrows) were labeled at 6 weeks after hemisection. E: Neurons in the FL (arrows) were labeled at 6 weeks after hemisection. F: The number of ipsilaterally labeled neuronal perikarya in the nuclei of intact fish, and fish at 6 weeks after hemisection. Values are means ± SD. Dashed line: midline. Scale bars = 100 lm in A–E.

represent oligodendrocyte precursor cells with multipotent stem cell-like characteristics, whereas NG2 is also expressed by macrophages and meningeal cells (Jones

et al., 2002; Tan et al., 2005; Rhodes et al., 2006; Trotter et al., 2010). The present study did not provide evidence of an inhibitory role of NG2 for axonal growth,

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Fig. 14. Ultrastructure of the lesion site 3 weeks after the spinal hemisection. A: A regenerating axon (a) breaking through the glia limitans, protruding into the fibrous tissue containing fibroblasts (fb) and collagen fibers (asterisk), from the nervous tissue. The axon (a) was associated with the glial process (gp), which contain glial filaments (arrow). The surface of the glial process was coated by the basement membrane (arrowheads). B: In the fibrous scar, a bundle of regenerating axons (a) was present among the fibrous tissue containing fibroblasts (fb) and collagen fibers (asterisk). The glial process (gp) coated by basement membrane (arrow) wrapped the axon bundle. C: A tubular structure ensheathing the regenerating axons (a), dendrites (dr), glial processes (gp), glial cell bodies (gc), and phagocytes (pc) containing lysosomes (white arrows) was observed among the fibrous scar containing fibroblasts (fb) and collagen fibers (asterisk). D: Higher magnification of the inset of C. A glial process (gp) containing glial filaments (arrows) were in close contact with axons (a) and dendrite (dr). Desmosome-like structure (white arrowhead) was present between the glial process and neighboring glial process. The glial process forming the tubular wall was coated by the basement membrane (arrowhead). A phagocyte (pc) contained lysosomes (white arrows). E: Higher magnification of the inset of C. The peripheral wall of the tubular structures was formed by glial processes (gp), which were coated by the basement membrane (arrowhead). dr: dendrite. fb: fibroblast. Asterisk: collagen fibers. Scale bars = 1 lm in A, B, D, E, and 2 lm in C.

because many regenerating axons entered the fibrous scar to penetrate the tubular structures before NG2 was expressed in the fibrous scar. Furthermore, the present findings indicated that many RDA-labeled axons penetrated the NG2-positive area, suggesting that the inhibitory role of NG2 is not critical for axonal regeneration in goldfish. Some other studies, on the other hand, suggest that NG2-expressing cells provide a favorable substrate for growing axons in the developing cerebral cortex

(Bergles et al., 2000; Lin et al., 2005; Yang et al., 2006). Moreover, these NG2-expressing cells might make a permissive bridge for regenerating sensory axons to pass through a region of inhibitory proteoglycans in the injured spinal cord (Busch et al., 2010). The present study in goldfish revealed no morphologically close relationship between NG2-positive structures and regenerating axons in the fibrous scar. Therefore, the growth-promoting role of NG2 or NG2-expressing cells in goldfish remains unclear.

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Fig. 15. Schematic representation of the axonal regeneration process following spinal injury in goldfish is shown. Stage 1: The lesion site separated by the tissue injury is filled with the fibrous scar, which is continuous with the meninges. Small cavities are formed in the center of the fibrous tissue. The fibrous tissue and the surrounding nervous tissue are demarcated by a laminin-positive basement membrane (glia limitans). Stage 2: Regenerating axons enter the fibrous scar, and were surrounded by laminin-coated tubular structures that were continuous with the glia limitans. The regenerating axons were accompanied with glial processes, coated by basement membrane expressing laminin. Stage 3: The laminin-coated tubules develop to create tunnels that penetrate the fibrous scar. The regenerating axons pass the fibrous scar through the tunnels. The regenerating axons are closely associated with GFAP-positive glial processes, as well as with 5-HT neurons migrating into the tubules. Stage 4: As the diameter of the tunnels is further increased, the glial processes filling the tunnels occupy most of the lesion site, and thus the fibrous scar area is significantly reduced. The number of regenerating axons passing the fibrous scar is significantly increased.

Acknowledgements—We are grateful to Mrs. M. Kobayashi, Mr. T. Koga, and Mr. H. Inoue for their expert technical aid. This study was supported in part by Grant-in-aid #20500312 and #23500414 from the Japan Society for Promotion of Science.

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(Accepted 17 September 2014) (Available online 5 October 2014)