Systemic administration of a deoxyribozyme to xylosyltransferase-1 mRNA promotes recovery after a spinal cord contusion injury

Experimental Neurology 237 (2012) 170–179

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Systemic administration of a deoxyribozyme to xylosyltransferase-1 mRNA promotes recovery after a spinal cord contusion injury Martin Oudega a, b, c, Owen Y. Chao d, Donna L. Avison e, Roderick T. Bronson f, William J. Buchser g, Andres Hurtado h, Barbara Grimpe i,⁎ a

Department of Physical Medicine and Rehabilitation, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA Department of Bioengineering, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA d Department of Experimental Psychology, Heinrich Heine University, Düsseldorf, 40225, Germany e Department of Surgery, Leonard M. Miller School of Medicine, University of Miami, Miami, FL 33136, USA f Dana-Faber/Harvard Cancer Center, Boston, MA 02115, USA g Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA h International Center for Spinal Cord Injury, Hugo W. Moser Research Institute at Kennedy Krieger, Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA i Applied Neurobiology, Department of Neurology, Heinrich Heine University, Düsseldorf, 40225, Germany b c

a r t i c l e

i n f o

Article history: Received 27 April 2012 Revised 8 June 2012 Accepted 9 June 2012 Available online 19 June 2012 Keywords: Spinal cord injury Glial scar Chondroitin sulfate proteoglycans DNA enzyme Neurotherapeutics Regeneration Functional recovery

a b s t r a c t After spinal cord injury, proteoglycans with growth-inhibitory glycosaminoglycan (GAG-) side chains in scar tissue limit spontaneous axonal sprouting/regeneration. Interventions that reduce scar-related inhibition facilitate an axonal growth response and possibly plasticity-based spinal cord repair. Xylosyltransferase-1 (XT-1) is the enzyme that initiates GAG-chain formation. We investigated whether intravenous administration of a deoxyribozyme (DNA enzyme) to XT-1 mRNA (DNAXT-1as) would elicit plasticity after a clinically relevant contusion of the spinal cord in adult rats. Our data showed that systemic DNAXT-1as administration resulted in a significant increase in sensorimotor function and serotonergic axon presence caudal to the injury. DNAXT1as treatment did not cause pathological or toxicological side effects. Importantly, intravenous delivery of DNAXT-1as did not exacerbate contusion-induced neuropathic pain. Collectively, our data demonstrate that DNAXT-1as is a safe neurotherapeutic, which holds promise to become an integral component of therapies that aim to improve the quality of life of persons with spinal cord injury. © 2012 Elsevier Inc. All rights reserved.

Introduction Spinal cord injury (SCI) in humans is mostly caused by a contusive impact resulting in immediate neural cell death, axonal severance, and functional impairments. Plasticity-based self-repair is limited in part due to a lesion scar that inhibits axonal sprouting/regeneration (Bradbury and Carter, 2011; Silver and Miller, 2004; Sofroniew, 2009). Facilitating endogenous axonal growth by manipulation of scarrelated inhibitory molecules supports plasticity-based self-repair after a spinal cord contusion (García-Alías et al., 2009; Massey et al., 2006; Onifer et al., 2011).

⁎ Corresponding author at: Applied Neurobiology, Department of Neurology, Heinrich Heine University Düsseldorf, Moorenstrasse 5, D-40225 Düsseldorf, Germany. Fax: + 49 211 81 16282. E-mail addresses: [email protected] (M. Oudega), [email protected] (O.Y. Chao), [email protected] (D.L. Avison), [email protected] (R.T. Bronson), [email protected] (W.J. Buchser), [email protected] (A. Hurtado), [email protected] (B. Grimpe). 0014-4886/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2012.06.006

Scar-related axonal growth-inhibition is in part mediated by glycosaminoglycan (GAG) side chains (Laabs et al., 2007) of proteoglycans (Bush and Silver, 2007; Galtrey and Fawcett, 2007; Jones et al., 2003). Proteoglycan glycosylation is catalyzed by xylosyltransferase (XT-1; Gőtting et al., 2000; Gries et al., 2007). We showed that local intraparenchymal delivery of a deoxyribozyme (DNA enzyme) to XT-1 mRNA (DNAXT-1as; Grimpe and Silver, 2004) leads to XT-1 mRNA digestion causing a decrease in GAG chains and proteoglycans in the extracellular matrix of the glial scar. Also, we observed an increase in axons present in the spinal cord beyond an injury (Hurtado et al., 2008). This proof-of-concept revealed that DNAXT-1as could be employed as a neurotherapeutic to induce plasticity after SCI (Onifer et al., 2011; Priestley, 2007). Deoxyribozymes such as DNAXT-1as are single-strand DNA molecules that cleave targeted mRNA sequences via a catalytic loop structure. After digestion they release the RNA fragments and become available again for continued activity (Grimpe, 2011). Due to their size (30–35 nucleotides) and stability, deoxyribozymes are particularly suitable for minimally invasive intravenous delivery. It is known that the

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blood-spinal cord barrier in the contused rat spinal cord remains leaky for several weeks post-injury (Popovich et al., 1996) providing a therapeutic window of opportunity for systemic DNAXT-1as administration. We investigated whether systemic administration of DNAXT-1as facilitates plasticity using an established adult rat spinal cord contusion model system (Kwon et al., 2002). Furthermore, we tested the effects of DNAXT-1as treatment on axon presence beyond the contusion, functional recovery of the hind limbs, and neuropathic pain. Importantly, we comprehensively analyzed potential toxicological and pathological side effects of systemic administered DNAXT-1as. Material and methods Animals: ethics and surgical approval In this single blinded study adult female Fischer rats (n= 187, 200–220 g; Harlan Laboratories, Madison WI, USA) were housed in standard laboratory cages at 21± 0.2 °C and under 12 h light/dark cycle conditions. Water and food was available ad libitum. All procedures were in compliance with federal laws and regulations and approved by the local Institutional Animal Care and Use Committees. The animal facility is fully accredited by the Association of Assessment and Accreditation of Laboratory Animal Care International. Surgical procedures Catheter insertion A day before spinal cord contusion, rats were anaesthetized with a mixture of 1–2% isofluorane in 30% oxygen/70% nitrous oxide; adequate sedation was confirmed by lack of toe-pinch and corneal reflexes. The neck area was shaved and sterilized and an incision was made near the trachea. Sterilized, beveled, saline-filled polyethylene (PE)50 tubing was inserted into the jugular vein and fixed in place with silk sutures. The tubing was guided subcutaneously over the shoulder to leave through an incision in the back of the neck. Spinal cord contusion Rats were anaesthetized as described above and the dorsal aspect of the thoracic (T)9 vertebra was removed without damaging the dura and the exposed T10 spinal cord was contused (10 g, 12.5 mm, NYU (New York University) impactor; Gruner, 1992). To be included in this study the impact velocity needed to be within a 4% margin, the compression between 0.9 and 1.8 mm, and the BBB (Basso–Beattie– Bresnahan) score (Basso et al., 1995) ≤1 at day 1 post-injury. The back muscles were sutured separately using 4.0 Ethilon sutures and

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the skin was closed using metal wound clips. Post-operative care was provided as previously described (Hurtado et al., 2008; Takami et al., 2002). The bladder was manually emptied twice daily at regular times until reflex voiding occurred. Rats were monitored twice daily during survival. DNAXT-1as treatment At 30 min, and at 2, 5, 7, 9, 12, and 14 days post-contusion a total of 400, 1200, or 2000 μg DNAXTas in 1 ml saline or 1 ml saline only (controls) was administered via the jugular catheter over a 1 h period using a hydraulic injection pump (Harvard Apparatus, Holliston, MA, USA). For all but the first treatment, rats were sedated with 0.15 ml Diazepam (5 mg/ml; IM) before DNAXT-1as/saline administration. Five contused rats received biotinylated DNAXT-1as as above and were fixed at 14 days post-contusion to examine the intraspinal distribution of the deoxyribozyme after intravenous administration. Eighteen rats (10%) died during the study due to surgical complications. Assessment of motor function Hind limb performance during over ground walking was assessed using the BBB test (Basso et al., 1995) once a week for 9 weeks postinjury. Each assessment lasted 4 min and was performed by two testers unaware of the treatments. The test was performed on controls (n= 24) and rats treated with 400 μg (n= 18), 1200 μg (n= 13), or 2000 μg (n= 16) DNAXT-1as. All rats were familiarized with the open field and the handling before injury. Higher motor functions of the hind limbs were examined using the BBB sub-score (Lankhorst et al., 1999). For both hind paws we determined position [internal/external at initial contact (IC) and liftoff (0 points), parallel at IC and internal/external at liftoff or vice versa (1), parallel at IC and liftoff (2)], toe clearance [none (0), occasional (1), frequent (2), consistent (3)], tail position [down (0), up (1)], and trunk instability [yes (0), no (1)]. Scores were summed for a possible maximum score of 13. Sensorimotor function of the hind limbs was evaluated at 4 and 8 weeks post-injury using a horizontal ladder walking test (KunkelBagden et al., 1992) performed on controls (n = 24) and rats treated with 400 μg (n = 18), 1200 μg (n = 13), or 2000 μg (n = 16) DNAXT1as. Using video recordings, we determined the number of steps and the number of small (foot or part of foot), medium (foot and part of lower leg), and large (full leg) slips while crossing 60 cm of a 100 cm long horizontal ladder. Baseline values were taken before the

Fig. 1. Intravenously delivered DNAXT-1as is active and effective in the contused spinal cord segment. (A) Biotinylated DNAXT-1as was found in spinal cord tissue surrounding the contusion. The dotted line marks the distance of diffusion of the biotinylated DNAXT-1as. (B) Immunostaining with CS-56 antibodies showed a decrease in GAG‐chain presence at the contusion site in DNAXT-1as-treated rats but not in (C) saline-treated controls at 14 days after start of the treatment. Scale bar: 100 μm.

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start of the experiment. Slips were expressed as a percentage of the number of total steps.

Assessment of neuropathic pain Allodynia (i.e., pain resulting from an innocuous stimulus) was determined once a week for 9 weeks post-contusion using a mechanical stimulus to the hind paws by a Semmes–Weinstein von Frey monofilament (Stoelting, Wood Dale, IL, USA) set at 15 mN (Bruce et al., 2002). Baseline testing was performed before the start of the experiment. Rats were acclimated within a Plexiglas box (8 × 3.5 × 3.5 in) with a mesh floor for 5 min before measurements were taken. Ten stimuli (3 s each, 5 s intervals) were applied perpendicular to the plantar surface of each hind paw. Avoidance responses (flinching, escape, paw withdrawal, paw licking, vocalization, and abnormal aggressive behavior) were recorded and averaged for both hind paws. Hyperalgesia (i.e., increased sensitivity to a painful stimulus) was determined at 4 and 8 weeks after contusion using a tail-flick test. Rats were restrained in a conical polypropylene tube and their tail immersed 3 times at 20 min intervals into a 52 ± 0.2 °C water bath. Baseline values were determined before the start of the experiment. The times until the rat flicked its tail out of the water bath was recorded and averaged. Both allodynia tests were performed on controls (n= 16) and rats treated with 400 μg (n= 5), 1200 μg (n= 8), or 2000 μg (n= 11) DNAXT-1as.

Toxicological assessment To evaluate possible toxicological effects of DNAXT-1as, 0.75 ml blood was drawn through the jugular catheter at 1 day before and from the tail vein at 1, 2, 3, 5, 7, and 9 weeks after the start of DNAXT-1as treatment from controls (n = 15) and rats that received 400 μg (n = 15), 1200 μg (n = 13), or 2000 μg (n = 10) DNAXT-1as. The blood was used for complete blood count (CBC) on platelets, white blood cell (WBC), red blood cell (RBC), mean cell volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), nucleated red blood cell (NRBC), and hemoglobin (0.6 ml) and chemical profiling for glucose, blood urea nitrogen (BUN), creatinine (CREA), sodium, potassium, chloride, amylase, calcium, phosphorus, cholesterol, total protein, albumin, albumin/globulin (A/G) ratio, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, and total bilirubin (0.15 ml). Tests were performed by people unaware of the treatments at the Comparative Pathology Laboratory at the University of Miami in Miami, FL, which also provided the normal values. The data was arranged using Spotfire (TIBCO, Palo Alto, CA, USA).

Pathology assessment Pathology of DNAXT-1as treatment was examined in spinal cord, brain, eyes, tongue, cervical lymph nodes, mesenteric lymph nodes, salivary glands, esophagus, trachea, muscle, heart, lung, liver, pancreas, spleen, kidney, stomach, duodenum, small intestine, large intestine, colon, rectum, ovarian, uterus, and bladder. Tissue was taken from 56 rats from all experimental groups, embedded in paraffin, cut into 10 μm-thick sections, and stained with hematoxylin/eosin for microscopical evaluation.

Axonal tracing Axonal tracing was performed in rats that received 1200 μg DNAXT1-as (n= 5) or saline (n = 6). At 6 weeks post-injury, rats were sedated with ketamine (40–75 mg/kg) and xylazine (5–10 mg/kg) and placed within a stereotaxic apparatus (Stoelting). The skull was exposed and 0.15 μl dextran tetramethylrhodamine (10kD, Invitrogen, Carlsbad, CA) injected bilateral in the red nucleus via two burr holes (±0.6 μm lateral from Bregma, 7.0 μm deep) over a 3 min period using a Hamilton syringe with a pulled glass needle attached (tip diameter: 150 μm) fixed in a micromanipulator. The needle was kept in place for an additional 2 min before it was slowly retracted. The skin was closed and the rats survived for additional 3 weeks. Histological procedures Rats were fixed with 4% paraformaldehyde and 0.2% glutaraldehyde in phosphate buffered saline (PBS: 0.1 M, pH7.4, Grimpe and Silver, 2004). Spinal cords were dissected out and the segments centered on the contusion removed and kept in the same fixative overnight. Tissue was processed for gelatin embedding (Grimpe and Silver, 2004), deeply frozen, and cut in 40 μm-thick horizontal sections. Sections were collected in PBS. Some were mounted onto glass slides and stained with luxol fast blue and hematoxylin/eosin to assess myelin presence and cytoarchitecture. Immunohistochemistry Every sixth section was pre-incubated in PBS with 0.1% bovine serum albumin and 5% normal goat serum at room temperature for 30 min and then incubated overnight at 4 °C with a monoclonal antibody against chondroitin sulfate chains (clone CS-56, mouse; Sigma, St. Louis, MO) or with a polyclonal antibody against 5hydroxytryptamine (serotonin, 5-HT, rabbit; Biologo, Kronshagen, Germany), tyrosine hydroxylase (TH, rabbit; Abcam, Cambridge, MA), or calcitonin-gene related protein (CGRP, goat; AbD Serotec, Raleigh, NC). The primary antibody was diluted in PBS with 0.1% bovine serum albumin and 5% normal goat serum. Next, sections were rinsed in PBS 3 times for a total of 20 min, incubated for 2 h at room temperature with a secondary antibody conjugated to Alexa 568 or Alexa 488 (Invitrogen), rinsed in PBS three times for a total of 20 min, and covered with a glass slip with Citifluor (Ted Pella, Redding, CA). Staining specificity was confirmed by omitting the primary antibody. A Zeiss LSM 510 microscope was used for analysis. Axon quantification Traced rubrospinal axons and stained 5HT-, TH-, and CGRP axons were quantified at 9 weeks post-contusion/treatment in rats with 1200 μg/ml DNAXTas (n = 5) or saline (n = 6) treatment. In every sixth section, virtual lines were placed perpendicularly to the rostral– caudal axis of the spinal cord at the mid-point of the contusion and at 7 mm rostral and caudal to the epicenter. All traced/stained axons that crossed the lines were quantified in all sections, summed per animal, and then averaged per group. To account for variability in tracing, the numbers of traced axons at the mid-point and caudal to the contusion were expressed relative to the number of axons rostral to the contusion.

Fig. 2. Systemic DNAXT-1as presence does not cause short-term or long-term toxicological effects. In these heat maps of the DNAXT-1as effects blue represents normal values, shades of green represents increased values, and shades of red represent decreased values. Gray areas represent missing data points. (A) CBC and chemistry data per treatment throughout the experiment. (B) CBC and chemistry data per week for all treatments. The heat maps reveal the lack of short- and long-term toxicological effects following systemic DNAXT-1as treatment.

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Fig. 3. Systemic DNAXT-1as presence does not cause pathology. Low power microscopic images of the liver (A, A′), kidney (B, B′), lung (C, C′), and brain (D, D′) from rats treated with 1200 μg/ml DNAXT-1as (A–D) or saline (A′–D′). Tissues were embedded in paraffin, sectioned, and stained with hematoxylin/eosin. The insets are higher power (40×) microscopic images. The expert analyses revealed a lack of DNAXT-1as-induced pathology in any of the examined tissues.

Hematoxylin/eosin-stained sections were used to determine spared tissue volume in the contused spinal cord segment using Neurolucida® software (MicroBrightField Inc., Colchester, VA, USA). Following the rules of stereology, sections (40 μm) were analyzed from control rats (n= 7) and from rats treated with 400 μg (n= 10), 1200 μg (n= 8), or 2000 μg (n= 9) DNAXT-1as. In each rat, the volumes of the analyzed segment and that of lost and damaged nervous tissue were measured; the difference represents the volume of spared tissue (Nandoe Tewarie et al., 2009; Takami et al., 2002).

weeks of post-treatment. The amounts were used to index the state of the bladder as full (index 4), medium (3), small (2), drop (1), empty (0). This was performed for control rats (n= 26) and rats that received 400 μg (n= 23), 1200 μg (n= 17), or 2000 μg (n= 13) DNAXT-1as. Values were averaged per four-day intervals. The effect of DNAXT-1as treatment on bodyweight was assessed by weighing rats before and 3, 10, 13, 17, 31, and 59 days after contusion. Bodyweight was determined for control rats (n = 24) and rats that received 400 μg (n = 18), 1200 μg (n = 13), or 2000 μg (n = 16) DNAXT-1as. Bodyweight was expressed relative to the weight of the rat just before surgery.

Bladder functions and body weight

Statistical analysis

To assess whether DNAXT-1as treatment affected bladder function, we determined the daily amount of urine produced during the first

Statistical analyses were performed using SPSS (vs. 18.0, SPSS Inc, Chicago, IL, USA) and Prism (vs. 4.0, GraphPad Software, La Jolla, CA,

Spared tissue volume

Fig. 4. Intravenous DNAXT-1as injection improves sensorimotor recovery. (A) Bar graph shows that rats treated with 1200 μg/ml DNAXT-1as had significantly less slips per steps on the horizontal ladder compared to rats treated with saline at 4 and 8 weeks. Also, rats with 1200 μg/ml DNAXT-1as performed better than rats with 2000 μg/ml DNAXT-1as at 4 weeks and with 400 μg/ml DNAXT-1as at 8 weeks post-injury. (B) Intravenous administration of DNAXT-1as did not affect over ground walking. The asterisk at 3 weeks indicated a significant difference (P = 0.008) between rats that received 1200 μg/ml DNAXT-1as and rats that received saline. (C) Intravenous DNAXT-1as injection did not affect higher motor functions. Data represent mean ± s.e.m.

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examination of 25 different tissues from the body did not reveal any anatomical signs of disease (Fig. 3). The liver (Fig. 3A vs. A′) and kidney (Fig. 3B vs. B′) which typically fail first after systemic delivery of knock-down agents were unaffected by intravenous DNAXT-1as treatment. The data indicated that intravenous delivery of 400– 2000 μg DNAXT-1as after spinal cord contusion does not elicit acute or long-term toxicological or pathological side effects. DNAXT-1as treatment improves sensorimotor function

Fig. 5. Intravenously delivered DNAXT-1as does not exacerbate neuropathic pain. Mechanical allodynia was tested using the von Frey monofilament and found to be similar in all experimental groups. Data represent mean ± s.e.m.

USA). The BBB, horizontal ladder, hyperalgesia test, and body weight were analyzed by repeated two-way analysis of variance (ANOVA) for treatment and time followed by one-way ANOVA for treatment with Bonferroni post-hoc test. The BBB sub-score, allodynia, and bladder function were analyzed using the Kruskal–Wallis test followed by the Friedman test with Dunn's multiple comparisons for post-hoc testing. One-way ANOVA for treatment was conducted to analyze the number of 5HT-positive and traced rubrospinal axons. Mann– Whitney U tests were conducted to analyze the number of TH- and CGRP-positive axons. Spared tissue volumes were analyzed using two-way ANOVA with Bonferroni post-hoc test. For all statistical comparisons the difference was considered significant with P b 0.05 (two-tailed). All values are expressed as mean ± s.e.m. Results Intravenously delivered DNAXT-1as is effective in the contused spinal cord DNAXT-1as was delivered intravenously during the first 2 weeks post-contusion while the blood-spinal cord barrier (Popovich et al., 1996) is still open. Biotinylated DNAXT-1as penetrated about 1.2 mm into the nervous tissue surrounding the contusion (Fig. 1A; diffusion distance is marked by dotted line). This particular area exhibited a decreased staining for GAG chains (Fig. 1B) compared to controls (Fig. 1C) at 14 days after injury and start of DNAXT-1as treatment. The data revealed that intravenous administration of DNAXT1as, as per our protocol, reduces GAG‐chain presence in the nervous tissue surrounding the contusion epicenter. Intravenous delivery of DNAXT-1 is safe To examine potential side effects of intravenous DNAXT-1as delivery we performed CBC and chemical profiling of blood samples taken before and after start of the experiment. We did not find differences over time in any of the treatment groups (Fig. 2A) nor between treatment groups at particular time points (Fig. 2B). A pathological

Table 1 Average response time (sec ± s.e.m.) in the tail-flick test. Group

Pretest

4 weeks

8 weeks

Saline DNAXTas (400 μg/ml) DNAXTas (1200 μg/ml) DNAXTas (2000 μg/ml)

18.50 ± 4.21 13.50 ± 2.20 15.50 ± 1.50 14.60 ± 0.68

17.20 ± 1.16 13.83 ± 0.69 15.33 ± 1.47 15.50 ± 1.34

15.57 ± 0.90 15.33 ± 1.33 16.25 ± 1.93 13.33 ± 2.73

We investigated whether DNAXT-1as treatment improves functional recovery. At 4 and 8 weeks after contusion/treatment, there was a significant effect of time (F2,138 = 103.73, P = 0.000), treatment (F4,69 = 11.30, P = 0.000), and time x treatment (F8,138 = 7.72, P = 0.000) in the horizontal ladder test. Treatment with 1200 μg DNAXT-1as resulted in a significant decrease in slips per steps compared to controls at 4 (33%, P = 0.015) and 8 (46%, P = 0.000) weeks post-contusion (Fig. 4A). Compared to the 1200 μg DNAXT-1as, rats treated with 400 μg DNAXT-1as performed similar at 4 but not 8 (P = 0.002) weeks and rats treated with 2000 μg DNAXT-1as performed similar at 8 but not 4 (P= 0.011) weeks (Fig. 4A), suggesting a plateau effect of DNAXT-1as. There were no statistically significant differences in open field locomotor scores (Fig. 4B) and in higher motor functions (Fig. 4C). DNAXT-1as treatment does not exacerbate neuropathic pain It was shown that reduction in GAG-chain presence might aggravate neuropathic pain (Christensen et al., 1996). We assessed whether DNAXT-1as would affect allodynia for a mechanical stimulus and/or hyperalgesia for a thermal stimulus. Using a von Frey monofilament we did not find significant differences in mechanical allodynia between experimental groups (Fig. 5). The tail-flick test revealed that thermal hyperalgesia was similar between groups (Table 1). Thus, acute intravenous administration of 400–2000 μg DNAXT-1as does not exacerbate neuropathic pain after spinal cord contusion. DNAXT-1 treatment improves axon presence in the contused spinal cord We showed that intravenous delivery of 1200 μg DNAXT-1as reduces the presence of GAG chains in the contused spinal cord (Fig. 1) and improved sensorimotor function (Fig. 4A). The effect on the presence of descending and ascending axons was quantitatively examined. We found significantly more 5HT-positive axons (Fig. 6A) rostral (P = 0.007), at the mid-point (P = 0.005), and caudal (P = 0.023) to the contusion in DNAXT-1as-treated rats compared to controls (Fig. 6A′). We also found that the number of TH-positive axons (Fig. 6B) was increased, but not significantly (P= 0.051, Fig. 6B′) at the mid-point and caudal to the contusion in treated rats compared to controls. Significant differences in dextran tetramethylrhodaminelabeled rubrospinal axons (Fig. 6C) and CGRP-positive axons (Fig. 6D) were not found at any of the levels examined. DNAXT-1as treatment does not affect spared nervous tissue volume Because differences in axon numbers between treated and control rats could result from differences in amounts of nervous tissue at and near the lesion. Therefore, we examined whether DNAXT-1as affected nervous tissue sparing in the contused spinal cord segment using Neurolucida® software (MicroBrightField Inc.). We found that spared tissue volume was similar across experimental groups (Fig. 7A), indicating that acute administration of DNAXT-1as does not affect nervous tissue loss in the contused adult rat spinal cord.

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Fig. 6. DNAXT-1as increases serotonergic axon presence in the contused spinal cord. Schematic drawings of spinal cord are not to scale. Pictures represent the outlined boxes in the schematic drawings. Traced and immunostained axons were quantified at, rostral and caudal to the contusion epicenter. (A) 5-HT-positive axons at the contusion epicenter (a), and 1 (b) and 2.5 (c) mm caudal to the lesion cavity. Scale bar: 100 μm in panels (a) and (b), and 40 μm in panel (c). (B) TH-positive axons at the contusion epicenter (a), and 1.5 (b) and 3.5 (c) mm caudal to the lesion cavity. Scale bar: 100 μm in all panels. Bar graphs showing that significantly more 5-HT-positive axons were found at all levels examined in DNAXT-1astreated rats compared to controls (A′) and that the numbers of TH-positive axons (B′), rubrospinal axons (C), and CGRP-positive axons (D) did not significantly differ between DNAXT1as-treated and control rats. Data represent mean ± s.e.m.

Fig. 7. DNAXT-1as treatment does not affect nervous tissue loss, bodyweight, and bladder function. (A) Systemic administration of DNAXT-1as does not affect the volume of spared tissue in the contused spinal cord segment. (B) Intravenous injection of DNAXT-1as does not affect contusion-induced changes in bodyweight. (C) DNAXT-1as treatment does not affect bladder function after contusion; similar amounts of urine were produced over the first 19 days after contusion/treatment in all experimental groups. State of the bladder is indexed as full (index 4), medium (3), small (2), drop (1), empty (0). Data in (A) and (B) represent mean ± s.e.m.

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DNAXT-1as does not affect bodyweight or bladder function after a contusion The bodyweight of both DNAXT-1as-treated and control rats was 10–14% less at 10 days post-contusion, remained similar during the next 3 weeks, and increased by 25% over the next 4 weeks. The average bodyweight at the end of the experiment was similar between groups and 10–16% larger compared to that just before the start of the experiment (Fig. 7B). Bladder function was similar across groups throughout the entire experiment (Fig. 7C). The data showed that systemic DNAXT-1as administration does not affect contusion-related changes in bodyweight and bladder function. Discussion We show that systemic DNAXT-1as administration after a spinal cord contusion results in reduced GAG‐chain presence, enhanced serotonergic axon presence beyond the injury, and improved functional recovery. Systemic DNAXT-1as administration does not cause short- or long-term toxicological or pathological side effects, nor does it exacerbate neuropathic pain, spinal cord nervous tissue loss, bladder function, or bodyweight beyond what is normally observed after a contusion. Collectively, our data indicate that DNAXT-1as is a safe and promising neurotherapeutic for facilitating anatomical and functional plasticity after spinal cord contusion. Intravenous administration of DNAXT-1as promoted serotonergic axon presence beyond the contusion, which may have been facilitated by the DNAXT-1as-mediated reduction in GAG‐chain presence at the contusion site. These findings concur with earlier studies showing that a DNAXT-1as-mediated decrease in GAG chains is associated with increased axon presence (Grimpe and Silver, 2004; Hurtado et al., 2008). We show here that DNAXT-1as treatment does not affect nervous tissue loss in the contused segment suggesting that serotonergic axon numbers were not influenced by treatment-mediated tissue sparing. After SCI, brainstem neurons express regeneration-associated genes (Kwon et al., 2007; Schmitt et al., 2003) and their (damaged) axons sprout near the injury (Barron et al., 1989; Hill et al., 2001). However, lengthy axonal regeneration is typically absent in large part because of scar-related growth-obstructive GAG chains (Bush and Silver, 2007; Galtrey and Fawcett, 2007). Enzymatic digestion of GAG chains with chondroitinase ABC (ChABC) results in higher serotonergic axon numbers in the injured spinal cord which can be further enhanced with concomitant administration of neurotrophin-3 (Lee et al., 2010) or transplantation of a peripheral nerve graft (Alilain et al., 2011). Interestingly, ChABC treatment alone elicited a small increase in serotonergic axons in Lee et al. (2010) and Alilain et al. (2011) despite the use of a hemisection which generally leads to less accumulation of CSPGs within a smaller volume of nervous tissue (Iseda et al., 2008) and less secondary damage (Hill et al., 2001; Iseda et al., 2004) compared to a contusive injury as used in the present study. We now show that treatment with DNAXT1-as alone promotes a significant increase in serotonergic axon presence in the contused adult rat spinal cord. Further research will be necessary to elucidate whether regeneration/sprouting and/or sparing were responsible for the increase in serotonergic axons. It is possible that ChABC treatment-mediated increases in serotonergic axon presence in the injured spinal cord are limited due to stub-antigens on the core proteoglycans, which are known to have growth-inhibitory activity (Lemons et al., 2003). Although typical for ChABC treatment, this would not occur with DNAXT-1as treatment as it prevents GAG‐chain glycosylation rather than digests GAG chains. The mode of administration may also play a role in the efficacy of DNAXT-1as and ChABC (Iseda et al., 2008; Lee et al., 2010). In the present study, our systemic administration paradigm led to DNAXT-1as presence in the contusion site for at least the first 2 weeks after injury. However, a single ChABC injection (Iseda et al., 2008) or release from

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microtubes within an agarose gel (Lee et al., 2010) may fall short providing sufficient ChABC for long enough to lead to increased axon presence. This may also explain why additional stimuli such as neurotrophin-3 (Lee et al., 2010) or a peripheral nerve graft (Alilain et al., 2011) were needed to obtain a serotonergic response. We found that systemic administration of DNAXT-1as after a spinal cord contusion significantly improved horizontal ladder walking. Here, as well as in an earlier study (Ritfeld et al., 2012), we show that this particular motor behavior is strongly correlated with serotonergic axon presence. Serotonergic axons are involved in the initiation and modulation of hind limb motor function (Barbeau and Rossignol, 1991; Jacobs et al., 2002; Schmidt and Jordan, 2000). Treatmentmediated increase in serotonergic axon numbers caudal to a spinal cord lesion typically is associated with improved recovery in motor functions (Lu et al., 2001; Tysseling et al., 2010). The specific role of serotonergic axons in the execution of walking across a horizontal ladder is currently unknown and requires further investigations. Our data indicate that DNAXT-1as induced plasticity in the contused adult rat spinal cord as evidenced by increased axon numbers and motor improvements. Facilitating plasticity after SCI is deemed an important means of spinal cord repair (Onifer et al., 2011; Priestley, 2007) but holds the possibility of aggravating chronic neuropathic pain such as allodynia and hyperalgesia. Enhancement of these normally occurring pain sensations after SCI would diminish the potential of any treatment for clinical application. We examined mechanical allodynia and thermal hyperalgesia in rats treated with DNAXT-1as and did not find any changes over untreated rats suggesting that DNAXT-1as-induced plasticity did not led to aggravation of contusioninduced neuropathic pain. Previously, plasticity-enhancing treatment with ChABC or anti-Nogo were also shown not to affect neuropathic pain after SCI (Barritt et al., 2006; Galtrey and Fawcett, 2007; KarimiAbdolrezaee et al., 2010). On the other hand, plasticity-enhancement by neurotrophins may exacerbate neuropathic pain (Romero et al., 2000; Suter et al., 2007) and cause other side effects such as weight loss (Lebrun et al., 2006; Suter et al., 2007) or autonomic dysreflexia (Cameron et al., 2006). Unwanted side effects, especially aggravation of neuropathic pain, need to be taken into consideration before clinical translation (Baron, 2006) because of its significant impact on the quality of life of SCI patients. We demonstrate here that systemic administration of DNAXT-1as facilitates plasticity without worsening contusion-induced neuropathic pain. In addition, we also show that the deoxyribozyme does not affect bladder function and weight loss beyond what is normally observed after spinal cord contusion injury. A clinically relevant aspect of our study is that DNAXT-1as was effective following intravenous administration. In general, intravenous injection would be preferred over surgically more invasive treatment modes such as intrathecal or intraspinal infusion. However, intravenous DNAXT-1as delivery might affect proteoglycan levels in other tissues in the body. A minority (5%) of proteoglycans has a half-life of about 8 days (Mankin and Lippiello, 1969; Maroudas, 1975) while the majority has a half-life of over 100 days (Baron, 2006). Thus, our 14-day treatment with DNAXT-1as could have only affected a small portion of proteoglycans, which is unlikely to elicit significant side effects. To exclude the presence of any specific or non-specific unwanted responses, we conducted a detailed toxicology and pathology examination on blood and tissues from treated and untreated rats and found no conspicuous undesired side effects. We also assessed bladder function and weight loss which are noticeable and measurable consequences of SCI and found that they were not adversely affected by intravenous DNAXT-1as administration. Our data show that it is safe to administer DNAXT-1as systemically after a spinal cord contusion. The promising results showing that intravenous administration of DNAXT-1as after a spinal cord contusion improves plasticity-based motor function recovery do not conceal the need to further enhance motor outcomes to achieve more meaningful functional restoration

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after contusive SCI. It is generally accepted that a combination therapy will be required for such increased spinal cord repair (Boulenguez and Vinay, 2009). The effect of DNAXT-1as on spinal cord plasticity and functional recovery could be enhanced with additional interventions that further facilitate plasticity such as anti-Nogo treatment (Harel et al., 2010; Li et al., 2005; Raineteau et al., 1999; Thallmair et al., 1998). Other possibilities are combining systemic DNAXT-1as delivery with rehabilitative strategies (García-Alías et al., 2009; Girgis et al., 2007; Wang et al., 2011) or neuroprotective treatments. Acknowledgments We thank the Animal/Behavioral and Histology Core Facilities, Dr. Alex Marcillo, and Dr. Beata Frydel of the Miami Project to Cure Paralysis, and Heidrun Podini and Julia Domke. 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