Combined treatment with chondroitinase ABC and treadmill rehabilitation for chronic severe spinal cord injury in adult rats

Accepted Manuscript Title: Combined treatment with chondroitinase ABC and treadmill rehabilitation for chronic severe spinal cord injury in adult rats Author: Munehisa Shinozaki Akio Iwanami Kanehiro Fujiyoshi Shouichi Tashiro Kazuya Kitamura Shinsuke Shibata Hiroshi Fujita Masaya Nakamura Hideyuki Okano PII: DOI: Reference:

S0168-0102(16)30108-0 http://dx.doi.org/doi:10.1016/j.neures.2016.07.005 NSR 3967

To appear in:

Neuroscience Research

Received date: Revised date: Accepted date:

18-5-2016 20-7-2016 26-7-2016

Please cite this article as: Shinozaki, Munehisa, Iwanami, Akio, Fujiyoshi, Kanehiro, Tashiro, Shouichi, Kitamura, Kazuya, Shibata, Shinsuke, Fujita, Hiroshi, Nakamura, Masaya, Okano, Hideyuki, Combined treatment with chondroitinase ABC and treadmill rehabilitation for chronic severe spinal cord injury in adult rats.Neuroscience Research http://dx.doi.org/10.1016/j.neures.2016.07.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highlights C-ABC and rehabilitation ameliorate severe and chronic spinal cord injury.

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Graphical abstract

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Combined treatment with chondroitinase ABC and treadmill rehabilitation for chronic severe spinal cord injury in adult rats

Munehisa Shinozaki1, Akio Iwanami2, Kanehiro Fujiyoshi2, Shouichi Tashiro3, Kazuya Kitamura2, Shinsuke Shibata1, Hiroshi Fujita4, Masaya Nakamura2, and Hideyuki Okano1

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Department of Physiology, Keio University School of Medicine, Shinanomachi 35,

Shinjuku-ku, Tokyo, Japan, 1608582 2

Department of Orthopedic Surgery, Keio University School of Medicine,

Shinanomachi 35, Shinjuku-ku, Tokyo, Japan, 1608582 3

Department of Rehabilitation, Keio University School of Medicine, Shinanomachi 35,

Shinjuku-ku, Tokyo, Japan, 1608582 4

Department of Glycoresearch, Central Research Laboratories, Seikagaku Corporation,

1253, Tateno 3-chome, Higashiyamato-shi, Tokyo, Japan, 2070021

Corresponding authors Masaya Nakamura Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Tel: +81-3-5363-3812, Fax: +81-3-3353-6597 E-mail: [email protected]

Hideyuki Okano

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Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Tel: +81-3-5363-3747, Fax: +81-3-3357-5445 E-mail: [email protected]

Key words: chondroitin sulfate proteoglycan, glial scar, neural plasticity, axonal regeneration

Abbreviations SCI: spinal cord injury CSPG: Chondroitin sulfate proteoglycan C-ABC: Chondroitinase-ABC BBB: Basso, Beattie, and Bresnahan HPLC: High-performance liquid chromatography PBS: Phosphate-buffered saline H&E: Hematoxylin & eosin LFB: Luxol fast blue PBST: PBS/0.5% Tween-20 RT: Room temperature GAP-43: growth-associated protein 43 5-HT: 5-hydroxytryptamine CST: Corticospinal tract WGA: Wheat germ agglutinin PNN: Perineuronal net

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Funding: This work was supported by the Research Center Network for the Realization of Regenerative Medicine from the Japan Science and Technology Agency (JST) and Japan Agency for Medical Research and Development (A-MED) to M.N. and H.O.

Total page: 37 pages Figure: 7 figures

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Abstract There are more than 50 times the number of chronic-phase spinal cord injury (SCI) patients than there are acute patients, and over half of all SCI patients are severely disabled. However, research focusing on chronic severe contusional SCI remains very rare. Here, we evaluated whether chondroitinase ABC (C-ABC), a degradative enzyme directed against chondroitin sulfate proteoglycans (CSPGs), and treadmill rehabilitation could exert synergistic therapeutic actions against chronic severe contusional SCI. First, we induced severe contusional SCI in adult rats, and administered C-ABC intrathecally at 6 weeks post-injury for a period of one week. Next, we performed treadmill rehabilitation from weeks 6 to 14 after SCI, for a total period of eight weeks. The initiation of treadmill rehabilitation triggered slight recovery between weeks 6 and 9, whereas C-ABC administration stimulated a third phase of recovery between weeks 12 and 14. Histologically, the C-ABC-treated rats showed an increase in the transverse residual tissue area and the extent of neuronal fiber regeneration at a site caudal to the lesion epicenter, and regrowth of putatively regenerating serotonergic fibers was significantly increased at the epicenter. We suggest that, when combined with intensive rehabilitation, C-ABC may play a beneficial role, even in severe and chronic SCI.

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Significance Statement Approximately one-half of admitted spinal-cord-injury patients suffer from severe motor symptoms and are non-ambulatory in the chronic phase, but most studies of spinal cord injury focus on mild-to-moderate contusion injury in the acute-to-subacute phase. The present study utilized a combination of chondroitinase ABC and rehabilitation in severe and chronic contusional spinal cord injury in rats and revealed the effectiveness, and limits, of this combined regimen.

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Introduction Although it had long been thought that the central nervous system is incapable of repair after injury, recent studies on spinal cord injury (SCI) indicate the possibility of tissue regrowth and axonal regeneration. Some clinical trials are now showing the efficacy of various therapeutic approaches against loss of function following incomplete SCI. Approximately one-half of admitted SCI patients suffer from severe symptoms and are non-ambulatory, and most patients progress into the chronic phase. Therefore, a principal goal of SCI research is to develop methods to treat such serious and chronically injured cases and to restore function. Severe contusional injury is more difficult to treat, while most SCI studies focus on mild-to-moderate contusion injury, and relatively few reports address the management of severe contusional SCI (Iwanami et al., 2005; Yokota et al., 2015) . In addition, the treatment of chronic injury is more difficult than that of acute or subacute injury, due to variation in pathological conditions in the acute, subacute, and chronic injury phases of SCI. Karimi-Abdolrezaee et al. (2006) revealed a reduction in the survival of the transplanted cells in the chronic phase. Furthermore, it has been shown that an insufficient effect of chronic transplantation compared with subacute transplantation was possibly due to phase-dependent changes in the intraspinal environment, such as an increased infiltration of anti-inflammatory M2 macrophages in the sub-acute phase (Kumamaru et al., 2013; Nishimura et al., 2013). Only a few reports have shown favorable outcomes in chronic contusional injury (Zurita and Vaquero, 2006). The mechanisms underlying the difficulties of chronic-phase treatment have been discussed extensively. Secretion of chondroitin sulfate proteoglycans (CSPGs) at

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the lesion site is one of the major characteristics of chronic SCI. CSPGs are potent inhibitors of axonal regeneration and have been shown to inhibit neural outgrowth in vitro. NG2 is the key inhibitory CSPG that is expressed after SCI and its expression persists during the chronic phase (Jones et al., 2002; Niederost et al., 1999). Chondroitinase-ABC (C-ABC) is a CSPG-digesting enzyme that has been studied for decades for its potential in improving axonal regeneration and motor function after SCI. Bradbury et al. described effective motor recovery with chondroitinase treatment after SCI (Bradbury et al., 2002); this report was followed by a study of the combinational effect of chondroitinase with transplantation (Ikegami et al., 2005; Karimi-Abdolrezaee et al., 2012). Effects on the sensory (Massey et al., 2006) and respiratory systems (Alilain et al., 2011) have also been reported. However, few reports to date have focused on the treatment of chronic contusional SCI, and to our knowledge, neither C-ABC stand-alone therapy nor C-ABC in combination with an existing therapy has been tested in chronic and severe contusional SCI. The present study evaluated C-ABC in rat models of severe and chronic contusion SCI. Due to the lack of an effective drug to treat chronic SCI, such patients receive rehabilitation therapy; therefore, we combined C-ABC treatment with intensive treadmill rehabilitation. First, we induced severe contusion SCI in adult rats, followed by intrathecal application of C-ABC at six weeks post-injury, for a period of one week. Treadmill training was then performed in weeks 6 to 14 post SCI. The combinatorial approach enhanced axonal regrowth, thereby promoting functional recovery in SCI model rats. Our findings suggest the potential efficacy of C-ABC administration when combined with physical rehabilitation, even in severe and chronic contusional SCI cases.

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Materials and Methods Animals Adult female Sprague-Dawley rats (n = 61, weight = 200–220 g, CLEA Japan, Inc., Tokyo, Japan) were used in this study. The animals were housed doubly in standard plastic cages (26 x 42 cm) under the conditions of a 12-h light/dark cycle with ad libitum access to food and water. General activity, the urine condition, and the absence of symptoms of allodynia were checked twice daily after SCI. Hardwood sawdust bedding was replaced by soft paper bedding for several days. Antibiotics (ampicillin, Meiji Seika Pharma, Tokyo, Japan) were injected for 1 week after SCI and for three days after other surgeries. All surgical interventions and animal care procedures were performed in accordance with the Laboratory Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals (National Institute of Health, Bethesda, MD, USA) and the Guidelines and Policies for Animal Surgery provided by the Animal Study Committee of the Central Institute for Experimental Animals of Keio University (Tokyo, Japan). The study protocol was approved by the Ethics Committee of Keio University (Approval No. 12082).

Contusion SCI model Animals were anesthetized with a mixture of ketamine plus xylazine (30 + 3 mg/kg, administered intraperitoneally). A laminectomy was performed at thoracic vertebral level T10 as previously described (Kitamura et al., 2007; Mukaino et al., 2010; Zhang et al., 2014). An impact force of 250 kdyn was delivered to the spinal cord with

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an IH impactor (Precision Systems and Instrumentation, Fairfax Station, VA, USA) to induce a severe contusion injury. This force was chosen because pilot studies revealed that milder (200 kdyn) injuries allowed the animals to walk via their hindlimbs over time, thus failing to recreate the recovery course of severe SCI (data not shown). The muscles and skin were closed in layers, and the animals were placed into a temperature-controlled chamber until thermoregulation was re-established. Manual voiding of the bladder was performed twice daily until reflex bladder emptying was re-established. Two rats died during the surgery because of respiratory failure. All animals showed complete paraplegia after SCI. General activity was reduced in all animals on the second day but recovered by the third day. Residual urine had reduced by 1 week. The condition of the skin, appetite, and the color of the urine showed no abnormality throughout the procedure. Severe contusional injury of the thoracic spinal cord induced by an IH impactor is relatively unstable, as previously reported (Nessler et al., 2006; Shinozaki et al., 2013). Twelve rats were excluded from the present study because they showed mild impairment after SCI (over 5 in the Basso, Beattie, and Bresnahan locomotor rating scale six weeks after SCI), which did not match our experimental design.

C-ABC treatment and rehabilitation Six weeks after contusion SCI at T10, animals were anesthetized with a mixture of ketamine plus xylazine, and an additional laminectomy was performed at T12. A silicon tube (Alzet rat IT catheter, 7741) (DURECT Corp., Cupertino, CA, USA) was attached to an osmotic minipump and inserted into the subarachnoid space. The

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osmotic minipump (volume = 200 l, rate = 1 l/h, time course = 7-day delivery; Alzet pump model 200, DURECT Corp.) was filled with C-ABC solution (40 U/200 l) (Seikagaku Corp., Tokyo, Japan) for the C-ABC group, or inactivated C-ABC (same dose, but pretreated at 60°C for 24 h) for the vehicle control and no-treatment control groups, as previously reported (Karimi-Abdolrezaee et al., 2012). Treadmill rehabilitation was initiated in both the C-ABC group and the vehicle control group at 6 weeks after SCI. Quadrupedal training was performed for 30 min/day for 5 days/week until 14 weeks post-injury (Fig. 1) with a Rodent Robot 3000 robotic device (Robomedica Inc., Irvine, CA, USA), as previously reported (Shah et al., 2013; Ward et al., 2014). In brief, the rats were progressively trained to step for as long as they could at the fastest speed, e.g., starting the treadmill at 10 cm/s and gradually increasing the speed to 20 cm/s. During training, the weight support was adjusted to 60–80% of the body weight to facilitate voluntary stepping for each rat. The arms of the device for the support of steps were not used, and all steps were performed actively, and not passively.

Behavioral assessment Open field hindlimb locomotor activity was evaluated using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale (Basso et al., 1995). Briefly, this involved placing the animal in a circular open field and evaluating the movement of both hindlimbs for individual joint movements, as well as paw posture, weight support, forelimb/hindlimb coordination, paw angle, and overall trunk stability. Scores were calculated according to the 0–21-point BBB scale for each hindlimb and averaged to give the animal an overall score (James et al., 2011; Kaneko et al., 2006). Locomotor activity was evaluated on days 1 and 7 post-injury and weekly thereafter for 14 weeks.

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The results were quantified in a blinded manner by two observers. Tactile hyperalgesia was assessed with harmless mechanical stimuli of the hind paw using a von Frey monofilament (Semmes-Weinstein Anesthesiometer; Stoelting, IL, USA). The up-down paradigm was used to calculate the threshold (Chaplan et al., 1994; Tashiro et al., 2015).

High-performance liquid chromatography (HPLC) assay Rats in the C-ABC (n = 5) and vehicle control (n = 6) groups were euthanized at the end of the infusion period (7 weeks after SCI) under deep anesthesia. The injured spinal cord tissues (10 mm in length) were removed and immediately frozen in liquid nitrogen and stored at −80°C until use. Glycosaminoglycans in the spinal cord were analyzed as previously described (Shinmei et al., 1992). Each specimen was digested with actinase E, and the digest was centrifuged. The supernatant was treated with C-ABC and chondroitinase AC-II. The sample was subjected to ultrafiltration, and the unsaturated

disaccharides

in

(2-acetamide-2-deoxy-3-O-(b-d-gluco-4-enepyranosyl

the uronic

filtrate acid)-d-galactose,

2-acetamide-2-deoxy-3-O-(b-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose, and

2-acetamide-2-deoxy-3-O-(b-d-gluco-4-

enepyranosyluronic

acid)-6-O-sulfo-d-galactose) derived from chondroitin sulfate were analyzed by HPLC. The disaccharides in each sample were eluted with a gradient of 0–100 mm sodium sulfate, and the effluent was monitored using a fluorimeter. The amount of each unsaturated disaccharide was calculated from the peak area, and the content of the disaccharide isomers per unit weight of spinal cord was investigated.

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Histological examination Animals were deeply anesthetized by intraperitoneal administration of ketamine plus xylazine and intracardially perfused with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). Tissue samples were immersed in 10% sucrose in PBS at 4°C for 24 h, placed in 30% sucrose in PBS for 48 h, and embedded in optimal cutting temperature compound. The embedded tissue was immediately frozen in liquid nitrogen and cut into 20-m sagittal or axial sections (Nori et al., 2011; Yasuda et al., 2011). The sections were histologically evaluated by hematoxylin & eosin (H&E) staining, Luxol fast blue (LFB) staining, and immunohistochemistry. Immunostaining for CSPGs was performed as previously reported (Ikegami et al., 2005). Briefly, after blocking with Blocking-One reagent (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature (RT), the sections were incubated in PBS/0.5% Tween-20 (PBST)-azide containing monoclonal mouse CS56 antibody against chondroitin sulfate (1:200) (Sigma Chemical Co., St. Louis, MO, USA) overnight at RT. After four 5-min washes with PBS, the sections were incubated in PBST-azide containing the appropriate Alexa Fluor-conjugated secondary antibody (1:1000) (Invitrogen, Carlsbad, CA, USA) for 1 h at RT. After three 5-min washes with PBS, the sections were coverslipped with Fluoromount mounting medium (Diagnostic BioSystems, Pleasanton, CA, USA), and the images were acquired on a Zeiss LSM 700 upright confocal microscope (Carl Zeiss, Oberkochen, Germany) at the same settings and in single sessions. Immunostaining for the axonal marker RT-97/neurofilament 200, the distal axon/growth cone marker growth-associated protein 43 (GAP-43), and the neurotransmitter 5-hydroxytryptamine (5-HT)/serotonin was performed on sagittal

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sections according to the avidin-biotin-peroxidase method (Vector Laboratories, Burlingame, CA, USA). Briefly, sections were incubated with 0.3% hydrogen peroxide in absolute methanol for 30 min at RT. After blocking with Blocking-One reagent for 1 h at RT, the sections were incubated with PBST-azide containing monoclonal mouse anti-neurofilament 200 antibody, clone RT-97 (1:200) (Millipore, Billerica, MA, USA), monoclonal mouse anti-GAP-43 antibody (1:200) (Chemicon, Carlsbad, CA, USA), and polyclonal goat anti-5-HT antibody (1:200) (ImmunoStar, Hudson, WI, USA). The sections were then incubated with biotinylated secondary donkey anti-mouse or goat IgG (1:1000) (Jackson ImmunoResearch, West Grove, PA, USA) at RT, followed by avidin-biotin complex (1:500) (Vector Laboratories). Lastly, the sections were stained by using diaminobenzidine and mounted onto slides for visualization. Images were obtained via fluorescence microscopy under a BZ-9000 fluorescence microscope (Keyence Corp., Osaka, Japan).

Anterograde and trans-synaptic labeling of the corticospinal tract Descending corticospinal tract (CST) fibers and their trans-synaptic connections to anterior horn cells (Abematsu et al., 2010; Broadwell and Balin, 1985; Fabian and Coulter, 1985) were evaluated at 14 weeks after SCI. CST fibers were labeled with Alexa Fluor 488-conjugated wheat germ agglutinin (WGA) (1.0% in saline, 4.0 l/cortex; Invitrogen) injected under deep anesthesia into the left and right motor cortices (coordinates = 2 mm posterior and lateral to the bregma; depth = 1.5 mm). For each injection, WGA (2 l) was delivered over a 60-s period via a 10-m inner diameter

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glass capillary attached to a microliter syringe (Hamilton Co., Reno, NV, USA). Four days after WGA injection, animals were intracardially perfused with 4% paraformaldehyde in 0.1 M PBS. Tissue samples were immersed in 10% sucrose in PBS at 4°C for 24 h, placed in 30% sucrose in PBS for 48 h, and embedded in optimal cutting temperature compound. The embedded tissue was immediately frozen in liquid nitrogen and cut into 20-m sagittal sections. Images were obtained under a BZ-9000 fluorescence microscope.

Quantitative analysis of stained tissue sections To quantify the spinal cord tissue/axonal population of interest in H&E/LFB-stained and immunostained sections, BZ-HIC software (Keyence Corp., Osaka, Japan) was employed with the threshold values maintained at a constant level for all BZ-HIC analyses. CS56-stained-CSPG-positive areas were quantified by scanning midsagittal sections, with subsequent transverse tiling from 2 mm rostral to 2 mm caudal to the lesion epicenter. The lesion area was quantified by capturing H&E/LFB-stained images in axial sections at the epicenter and 4.0 mm rostral and caudal to the epicenter at 100 × magnification. RT-97-positive areas were quantified by scanning the sagittal sections, with transverse tiling at the lesion epicenter and 2 mm rostral and caudal to the epicenter at 400 × magnification. GAP-43- and 5-HT-positive areas were quantified by scanning the sagittal sections, with transverse tiling from 2 mm rostral to 2 mm caudal to the epicenter at 400 × magnification. WGA-positive areas were quantified by scanning midsagittal sections, with transverse tiling from 6 mm rostral to 6 mm caudal to the epicenter.

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Statistical analysis All quantifiable data are reported as the mean ± the standard error of the mean. The results of the histological examinations and the infrared sensor system among three groups were analyzed using one-way ANOVA followed by post hoc pairwise multiple comparisons using the Tukey-Kramer method. The results of the BBB scores among three groups were analyzed using ANOVA followed by post hoc pairwise multiple comparisons using a nonparametric test. For histological examinations of CSPG, an unpaired two-tailed Student’s t-test was used for single comparisons between the C-ABC and vehicle control groups. Correlations between the functional BBB scores and HE/LFB positive areas were examined using Spearman tests. In each case, * P < 0.05 and ** P < 0.01 were considered statistically significant.

Results Behavioral performance after severe SCI Immediately after contusion SCI, all animals showed complete paraplegia (BBB score = 0), which was followed by slight recovery until the chronic phase. The no-treatment control group reached a behavioral plateau (BBB score = 3.3 ± 0.12) and showed no increase in functional recovery at 5 weeks after SCI and thereafter (Fig. 2A). By contrast, the C-ABC and vehicle control with rehabilitation groups exhibited a second recovery phase after the initiation of treadmill training. Notably, the C-ABC group demonstrated a third recovery phase at 12–14 weeks after SCI, and showed significant differences in final BBB scores compared with the no-treatment control group (F(2, 23)=3.81, n=24, P=0.039).

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We evaluated the natural course of sensory sensitivity and the effect of the C-ABC and/or the rehabilitation on allodynia with the von Frey hair test. There was no consistent tendency to exhibit hypersensitivity, and the standard deviation was large in each group (Fig. 2B). Hence, there was no difference in hypersensitivity during the observed period among the three groups.

C-ABC-mediated digestion of CSPGs To determine the efficacy of C-ABC toward the digestion of CSPGs within the injured spinal cord, two analyses were performed. First, HPLC was used to measure CSPG levels just after the cessation of C-ABC treatment (i.e., 7 weeks after SCI; Fig. 1), and demonstrated a significantly lower CSPG content in the C-ABC group relative to the vehicle control group (t(9)=2.27, n=11, P=0.048, Fig. 3A). Next, immunostaining for CSPGs was conducted in midsagittal sections to evaluate the long-term effect of enzymatic digestion. Consistent with the HPLC results, a pronounced CS56-stained, CSPG-positive area was observed at the lesion epicenter in the vehicle control with rehabilitation group at 14 weeks post-SCI, whereas the CSPG-positive area was diminished in size in the C-ABC group (t(5)=6.85, n=7, P=0.0010, Fig. 3B, C). Contusion SCI caused destructive changes at the lesion site, including neuronal/tissue loss and spinal cord atrophy in H&E/LFB-stained sections (Fig. 3D). Cystic cavity formation was also observed rostral to the lesion epicenter. Quantitative analysis revealed a significant difference in the transverse area of the caudal injured spinal cord (+4 mm) in the C-ABC group (n=12, HE-rostral; F(11, 2)=0.03, P=0.97, HE-epicenter; F(11, 2)=6.9, P=0.015, HE-caudal; F(11, 2)=6.66, P=0.017, LFB-rostral; F(11, 2)=0.23, P=0.80, LFB-epicenter; F(11, 2)=10.2, P=0.0049, LFB-caudal; F(11,

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2)=12.0, P=0.0029, Fig. 3E and F). Scatterplots between the BBB score and the residual area showed a significant correlation in those areas (n=12, HE-rostral; r=-0.4, P=0.21, HE-epicenter; r=0.18, =0.57, HE-caudal; r=0.71, P=0.0094, LFB-rostral; r=0.02, P=0.95, LFB-epicenter; r=0.76, P=0.0038, LFB-caudal; r=0.45, P=0.14, Fig. 4A - 4F). Hence, C-ABC administration can effectively reduce CSPG content and safeguard against long-term atrophic changes in the distal spinal cord.

C-ABC treatment combined with treadmill exercise promotes regeneration of neuronal axons Immunostaining for RT-97 was conducted in sagittal sections of the injured spinal cord to evaluate existing axon fibers at 14 weeks after SCI. In the vehicle and the no-treatment groups, the RT-97-positive area caudal to the lesion epicenter (+2 mm) was smaller than that of the epicenter and the corresponding rostral (−2 mm) site (Fig. 5A). However, the RT-97-positive area caudal to the epicenter was significantly increased in the C-ABC group compared with the vehicle control and no-treatment groups (F(10, 2)=15, n=11, P=0.0021, Fig. 5B). Immunostaining for GAP-43 and 5-HT was also performed to examine axonal regeneration within the sagittal sections at 14 weeks post-SCI (Fig. 6). GAP-43 is a marker of actively extending fibers. Some regenerated GAP-43-postive fibers were found at the lesion epicenter and rostral to the lesion, but few were found caudally (Fig. 6A). The size of the overall GAP-43-positive area was moderately increased in the C-ABC group relative to the vehicle control and the no-treatment groups (F(10, 2)=30, n=11, P=0.00019, Fig. 6B).

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Serotonergic raphespinal tract axons are important descending fibers in terms of motor functional recovery (Kaneko et al., 2006; Saruhashi et al., 1996). In the vehicle control and no-treatment groups, the serotonin (5-HT)-positive area decreased as the rostral proximity to the lesion epicenter increased, and the fibers were scarcely discerned at regions caudal to the lesion site (Fig. 6C). Contrarily, the 5-HT-positive area in the C-ABC group did not differ markedly with the rostral distance, and some 5-HT-positive fibers were even found caudal to the lesion epicenter (n=11, rostral-2.0 mm; F(9, 2)=0.032, P=0.97, rostral-1.6 mm; F(9, 2)=6.2, P=0.028, rostral-1.2 mm; F(9, 2)=1.0, P=0.42, rostral-0.8 mm; F(9, 2)=6.5, P=0.025, rostral-0.4 mm; F(9, 2)=7.8, P=0.017, epicenter; F(9, 2)=7.0, P=0.02, caudal-0.4 mm; F(9, 2)=1.9, P=0.22, caudal-0.8 mm; F(9, 2)=1.7, P=0.26, caudal-1.2 mm; F(9, 2)=4.4, P=0.059, caudal-1.6 mm; F(9, 2)=2.5, P=0.15, caudal-2.0 mm; F(9, 2)=5.0, P=0.045, Fig. 6D). Finally, WGA-mediated trans-synaptic neuronal fiber tracing was performed in midsagittal sections to evaluate CST fiber regeneration from the motor cortex. All groups revealed CST fibers extending from the rostral side of the spinal cord toward the lesion epicenter, but the axons gradually decreased short of the epicenter (Fig. 7A). Quantitative analysis of the WGA-labeled CST fibers showed that the fibers reached more closely to the epicenter in the C-ABC group than in the vehicle control and no-treatment groups, and the fibers nearly contacted the cystic cavity (n=12, rostral-6 mm; F(11, 2)=0.91, P=0.44, rostral-5.5 mm; F(11, 2)=1.1, P=0.37, rostral-5.0 mm; F(11, 2)=9.3, P=0.0064, rostral-4.5 mm; F(11, 2)=9.3, P=0.0065, rostral-4.0 mm; F(11, 2)=11.2, P=0.00036, rostral-3.5 mm; F(11, 2)=4.4, P=0.047, rostral-3.0 mm; F(11, 2)=8.2, P=0.0093, rostral-2.5 mm; F(11, 2)=5.0, P=0.034, rostral-2.0 mm; F(11, 2)=5.0, P=0.034, rostral-1.5 mm; F(11, 2)=1.1, P=0.37, rostral-1.0 mm; F(11, 2)=1.1, P=0.37,

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rostral-0.5 mm; F(11, 2)=0.69, P=0.53, epicenter; F(11, 2)=0.94, P=0.43, Fig. 7B). In the caudal area, however, no WGA-labeled fibers were apparent in any group. Neurons that synapsed with the CST were additionally explored via WGA tracing, but no stained cells were observed caudal to the lesion site.

Discussion There are more than 50 times more chronic SCI patients than acute patients in the United States. More than half of all SCI patients are severely disabled and non-ambulatory (National Spinal Cord InjuryStatistical, 2014; Zorner et al., 2010); contusive trauma is the most common cause of SCI (Anderson et al., 2005). Nevertheless, studies of “chronic” and “severe” contusional SCI are rare (Du et al., 2015; Granger et al., 2012; Granger et al., 2013; Hall et al., 2010; Munoz-Quiles et al., 2009; Woerly et al., 2001; Zurita and Vaquero, 2006), and only a few reports have evaluated effectiveness in locomotor recovery (Granger et al., 2012; Munoz-Quiles et al., 2009; Woerly et al., 2001; Zurita and Vaquero, 2006). In the present study, we treated chronic severe contusional SCI model rats with treadmill rehabilitation only or in combination with C-ABC. Rats in the no-treatment control group showed little functional motor recovery at 14 weeks after SCI, whereas rehabilitation alone resulted in a slight recovery of hindlimb function. Combinatorial C-ABC/rehabilitation therapy delivered an improved functional outcome compared to rehabilitation alone, with significantly increased locomotor scores. The histology of the spinal cord demonstrated a large cavity at the lesion epicenter in vehicle control and no-treatment rats, as opposed to a significant increase in the residual tissue and regenerated RT-97-, GAP-43-, and 5-HT-positive fibers in the C-ABC group. WGA-labeled CST fibers also extended more

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proximal to the lesion epicenter in the C-ABC group. This study demonstrates for the first time the effectiveness of the combination of C-ABC and rehabilitation in animal models of severe and chronic contusional SCI.

Durable effect of C-ABC on CSPG content in the injured spinal cord The effect of C-ABC on CSPG content in the injured spinal cord was assessed at 7 weeks post-SCI via HPLC and at 14 weeks post-SCI via immunohistochemistry analysis. We found a significant decrease in CSPG levels in the C-ABC vs. vehicle control group, indicative of the long-term impact of enzymatic proteoglycan digestion. Previous work showed that CSPG mRNA expression increases during the subacute phase of SCI, and decreases thereafter (Nishimura et al., 2013). One-time administration of C-ABC in the chronic phase of SCI may thus provide sustained benefits regarding CSPG reduction. Whereas C-ABC is bacteria-derived enzyme, an endogenous human enzyme, ADAMTS-4, reportedly also digests proteoglycans (Cua et al., 2013). CSPGs are widely distributed after contusion SCI and form a large mass at the lesion epicenter, and the effect of an endogenous enzyme secreted from marginal residual tissue is likely somewhat limited (Tauchi et al., 2012). Particularly after severe SCI, only a small amount of residual tissue remains, and thus cannot secrete sufficient amounts of endogenous proteoglycan-degrading enzyme to digest large quantities of CSPGs generated after contusion. Under such conditions, intervention with the application of exogenous C-ABC seems inevitable for optimal proteoglycan removal and functional

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recovery.

C-ABC administration combined with treadmill exercise promotes axonal regeneration after severe SCI In addition to the substantial axonal degeneration that occurs just after SCI at the lesion epicenter, subsequent neuronal degeneration generally occurs in the rostral and caudal spinal cord. Because Wallerian degeneration of ascending fibers and retrograde degeneration of descending fibers gradually occur in regions rostral to the injury site, the defect area of neuronal fiber damage typically extends beyond the lesion epicenter. In the present study, however, regenerating fibers rostral to the lesion site were observed in all three groups, with the C-ABC group showing pronounced regeneration of 5-HT-positive serotonergic and GAP-43-positive fibers. C-ABC promotes new branching and/or axonal extension through the digestion of CSPGs, which are otherwise deposited around the dystrophic endballs of neuronal fibers (Davies et al., 1999; Tom et al., 2004). Previously, digestion of CSPGs by C-ABC (Karimi-Abdolrezaee et al., 2012) and knockdown of xylosyltransferase (Grimpe and Silver, 2004) promoted axonal regeneration and branching in animal models, resulting in improved functional recovery after SCI. The facilitation of axonal regeneration and branching via CSPG digestion has also been demonstrated in animal models of transection of the nigrostriatal tract and hemisection of the cervical cord (Massey et al., 2006; Moon et al., 2001). With respect to intracellular signal transduction, the transmembrane protein tyrosine phosphatases (PTPs) act as receptors for CSPGs to restrict axonal extension

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(Coles et al., 2011; Shen et al., 2009). The PTP 2a subtypes leukocyte antigen-related PTP (LAR) and PTP bind to CSPGs through N-terminal immunoglobulin-like domains to mediate the effects of proteoglycans. Knockout of PTP enables axonal extension against a concentration gradient of CSPGs in vitro and promotes axonal regeneration and functional recovery after SCI in vivo (Lang et al., 2014; Shen et al., 2009). Neuronal axons of LAR knockout mice can grow in CSPG-containing medium, and the LAR inhibitor peptide encourages axonal extension. Signaling downstream of C-ABC might therefore involve a PTP-/LAR-dependent mechanism. In our study, WGA tracing of the CST revealed regrowing CST fibers near the lesion epicenter during the chronic phase of SCI, as previously reported in moderate contusion injury (Fabian and Coulter, 1985). Interestingly, extending WGA-labeled CST fibers were found in closer proximity to the epicenter in the C-ABC group than in the vehicle control and no-treatment groups. These observations indicate that CST axons can regenerate even in the chronically injured spinal cord, as long as favorable environmental conditions (e.g., CSPG deficiency) are provided.

C-ABC ameliorates the microenvironment of the caudal spinal cord Despite extensive cavitation at the lesion epicenter after severe and chronic SCI, in our animal model a small amount of spared tissue remained, particularly at sites rostral and caudal to the lesion epicenter. Significantly larger amounts of spared neural tissue were observed in the C-ABC group than in the vehicle control and no-treatment groups, which likely contributed to functional motor recovery (Fig. 3D-F). Nonetheless,

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scarcely any WGA-labeled CST fibers crossed over the lesion epicenter from the rostral to the caudal side (Fig. 7), supporting the idea that functional recovery via the extension of rostral fibers or the connection of rostral and caudal fibers is limited, especially in the CST (Abematsu et al., 2010). An increase in caudal fibers after SCI is partially due to the reconstruction of the neural structure by changes in the plasticity of the perineuronal net (PNN) in spinal cord tissue (Berardi et al., 2004; Celio et al., 1998; Kadomatsu and Sakamoto, 2014; Takahashi-Iwanaga et al., 1998). Among the CSPGs, aggrecan, brevican, neurocan, and versican exhibit an N-terminal G1 domain and a C-terminal G3 domain, and are collectively termed lecticans. Lecticans bind to hyaluronic acid via the G1 domain, and to tenascin-R via the G3 domain. The resultant hyaluronic acid/CSPG/tenascin-R complex makes up the PNN to form a specific matrix around inhibitory interneurons. Administration of C-ABC into the primary visual cortex revives ocular dominance plasticity, presumably by destruction of the PNN (Berardi et al., 2004). Similarly, degradation of the PNN by C-ABC selectively rendered acquired fear memories susceptible to erasure (Gogolla et al., 2009). Furthermore, removal of the PNN modulated synaptic short-term plasticity by synaptic exchange of postsynaptic glutamate receptors (Frischknecht et al., 2009). These reports suggest that the PNN, which is mainly constructed of CSPGs, regulates neural plasticity. Although the bridging of rostral fibers was insufficient in the present study, caudal neuronal fibers were enriched and motor function was expedited in the C-ABC group, suggesting that C-ABC administration permitted PNN reconstruction and recovery of the propriospinal circuit. In the present study, motor function was promoted immediately after the

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initiation of rehabilitation in both the vehicle control and C-ABC groups. Functional recovery was secondarily boosted in the C-ABC group at a later time point. Several reports have documented various mechanisms supporting recovery after rehabilitation, including increased production and release of neurotrophic factors, changes in metabolic status and electrical properties, and reconstruction of neural circuits (Girgis et al., 2007; Ke et al., 2011; Madinier et al., 2014; Petruska et al., 2007; Plunet et al., 2008; Tashiro et al., 2014; Ying et al., 2005). The first phase of recovery we observed suggests that even in chronic SCI a rapid mechanistic reaction may occur. However, rehabilitation can elicit unfavorable effects during the chronic phase, as the reconstruction of neural circuits driven by rehabilitation relies primarily on neural plasticity (Ishikawa et al., 2015; Krajacic et al., 2010; Wang et al., 2011). The effective period for rehabilitation-facilitated reconstruction of neural circuits was thus presumably achieved through changes in neural plasticity, while the second C-ABC-stimulated recovery phase relied on plasticity-associated modifications. In conclusion, the present study showed that C-ABC together with treadmill rehabilitation promotes tissue regeneration through the extension of neuronal fibers and/or changes in neural plasticity. With an eye to assessing the potential clinical application of this approach, we combined physical rehabilitation with C-ABC in an animal model of chronic severe contusional SCI. Nevertheless, further studies of combinatorial therapies encompassing C-ABC, rehabilitation, scaffolding materials, and neural stem cell transplantation are needed to maximize the likelihood of enhanced axonal regeneration and improved functional recovery after chronic severe SCI.

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Acknowledgments We thank Drs. T. Ikegami, N. Nagoshi, O. Tsuji, M. Mukaino, F. Renault-Mihara, T. Harada, and K. Yasutake for their technical assistance and scientific discussions and the members of the Okano laboratory for encouragement and generous support.

Conflict of interest H.O. is a paid scientific advisory board member of SanBio Co. Ltd. Other authors have no conflicts of interests to declare.

Role of authors All authors had full access to all the data in the study and take responsibility for the integrity of the data and accuracy of the data analysis. Study concept and design: MS, KF Acquisition of data: MS, ST, KK, HF Analysis and interpretation of data: MS, KF, ST, KK, HF Drafting the manuscript: MS Critical revision of the manuscript for important intellectual content: MN, HO Statistical analysis: MS, ST Obtained funding: MN, HO Administrative, technical, and material support: AI, SS, HF Study supervision: AI, SS, MN, HO

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Figure Legends Fig. 1. Experimental design of behavioral and histological analyses. Contusion spinal cord injury (SCI) was induced at T10, and motor function was evaluated using the Basso, Beattie, and Bresnahan (BBB) scoring scale. At 6 weeks post-SCI, chondroitinase ABC (C-ABC) was intrathecally administered for 1 week in the C-ABC group, whereas inactivated C-ABC was administered in the vehicle control and no-treatment groups. Rehabilitation by treadmill training was provided to the C-ABC and vehicle control groups from weeks 6–14 post-SCI. The locomotor activity of all animals was evaluated until euthanasia at 14 weeks post-SCI for histological examination. For trans-synaptic wheat germ agglutinin (WGA) tracing, WGA was injected into the motor cortex at 14 weeks post-SCI in the C-ABC, vehicle control, and no-treatment groups, and the animals were euthanized 4 days later. For high-performance liquid chromatography (HPLC) analysis of chondroitin sulfate proteoglycan in spinal cord tissue, rats in the C-ABC and vehicle control groups were euthanized just after the administration of C-ABC or inactivated C-ABC at 7 weeks post-SCI.

Fig. 2. Behavioral scores. (A) BBB scores of the C-ABC (n=7), vehicle control (n=8), and no-treatment (n=9) groups. All rats demonstrated a slight natural recovery, reaching a plateau for the BBB score at 3–3.5 at approximately 5 weeks (5w) after SCI. In both the C-ABC and vehicle control with rehabilitation groups, the animals showed another increase in BBB scores due to treadmill rehabilitation, reaching a second plateau at approximately 8–9 weeks after SCI. Rats in the C-ABC group demonstrated a third

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increase in BBB scores from 12 weeks onwards. * P < 0.05 vs. the no-treatment group. (B) The threshold of sensory response against von Frey stimuli. Six weeks after severe contusional injury, approximately half of the rats showed hypersensitivity, resulting in a moderate average of the thresholds in all groups. The control group temporarily showed less hypersensitivity, but there was no significant difference.

Fig. 3. Glial scarring and tissue atrophy. (A) HPLC-quantified CSPG content in the spinal cord tissue of the C-ABC (n=5) and vehicle control (n=6) groups just after administration of C-ABC/vehicle. The C-ABC group showed decreased CSPG levels, indicative of successful proteoglycan digestion. (B and C) CS56-immunoreactive and CSPG-positive areas within midsagittal sections at 8 weeks after the administration of active/inactive C-ABC and 14 weeks post-SCI in the C-ABC (n=4) and vehicle control (n=3) groups. The reduction in CSPG content suggests the long-term efficacy of C-ABC-facilitated proteoglycan digestion. Scale bar = 1000 m. (D) Axial sections showing H&E/LFB staining at 14 weeks post-SCI in the C-ABC (n=3), vehicle control (n=5), and no-treatment (n=4) groups. With the present severe contusion model, the injured spinal cord was markedly atrophic. Especially within the lesion epicenter, the dorsal tissue was almost completely replaced with fibrous granulation tissue or a large cavity, and only a small amount of residual white matter remained. Sites rostral to the lesion epicenter revealed extensive atrophic neural tissue and cavitation with diminished white matter content. Sites caudal to the epicenter showed diminished gray and white matter. Scale bar = 500 m. (E and F) Quantification of residual H&E/LFB-positive area. A significantly larger residual area was observed in the C-ABC vs. the no-treatment group. * P < 0.05, ** P < 0.01 vs. the indicated treatment.

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Fig. 4. Scatterplots between BBB score and HE/LFB staining. (A, B, and C) Scatterplots between BBB scores and H&E staining (n=12). At the rostral and epicenter areas, there is no significant correlation between BBB score and H&E staining, whereas a positive correlation was revealed in the caudal area. (D, E and F) Scatterplots between BBB score and LFB staining (n=12). A significant correlation was revealed in the epicenter area. ** P < 0.01 vs. the indicated treatment.

Fig. 5. Residual and regenerating RT-97-positive neuronal fibers. (A) Neuronal fibers were immunostained with the monoclonal anti-neurofilament 200 antibody clone RT-97 in C-ABC (n=4), vehicle control (n=3), and no-treatment (n=4) groups. RT-97 staining was considerably diminished within the lesion epicenter for all three groups, and fractional residual longitudinal fibers were found in the dorsal area of the injured spinal cord. Scale bar = 500 m. (B) Quantification of the RT-97-positive area. The C-ABC vs. vehicle-control/no-treatment group showed a larger RT-97-positive area, which is indicative of a greater number of residual and/or regenerating fibers, caudal to the lesion epicenter. * P < 0.05, ** P < 0.01 vs. the indicated treatment.

Fig. 6. Renewed fibers. (A) Regenerated neuronal fibers immunostained for GAP-43 at the rostral edge of the cavity in C-ABC (n=4), vehicle control (n=3), and no-treatment (n=4) groups. Scale bar = 100 m. (B) The C-ABC group showed a significantly larger GAP-43-positive area compared with the vehicle control and no-treatment groups. (C) 5-HT-positive serotonergic fibers 0.4 mm rostral to the epicenter in C-ABC (n=4), vehicle control (n=3), and no-treatment (n=4) groups. Scale bar = 20 m. (D)

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Quantification of 5-HT-positive fibers. The C-ABC group showed a significant 5-HT-positive area just rostral to the lesion and at the epicenter. * P < 0.05, ** P < 0.01 vs. the indicated treatment.

Fig. 7. WGA tracing of CST fibers. (A) WGA-labeled CST fibers in the midsagittal sections in C-ABC (n=3), vehicle control (n=5), and no-treatment (n=4) groups. The white dashed line shows the lesion epicenter, which was measured when the spinal cord tissue was harvested. The WGA-labeled CST was disrupted by cavitation. Scale bar = 1 mm. (B) Quantification of the WGA-positive area. The C-ABC group demonstrated an extension of CST-positive fibers in close proximity to the cavity, but there were scarcely any labeled fibers or cells at the epicenter or caudal to the lesion. * P < 0.05, ** P < 0.01 vs. the indicated treatment.

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