The nitrosteroid NCX 1015, a prednisolone derivative, improves recovery of function in rats after spinal cord injury

The nitrosteroid NCX 1015, a prednisolone derivative, improves recovery of function in rats after spinal cord injury

Brain Research 1062 (2005) 16 – 25 www.elsevier.com/locate/brainres Research Report The nitrosteroid NCX 1015, a prednisolone derivative, improves r...

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Brain Research 1062 (2005) 16 – 25 www.elsevier.com/locate/brainres

Research Report

The nitrosteroid NCX 1015, a prednisolone derivative, improves recovery of function in rats after spinal cord injury Alessandra Mallei a, Sadia A. Aden a, Alessia Bachis a, Cinzia Brandoli a, Ennio Ongini b, Italo Mocchetti a,* a

Department of Neuroscience, Georgetown University Medical Center, Research Building, Room EP04, Box 571464 Washington, DC 20057, USA b Nicox Research Institute, Bresso, Milan, Italy Accepted 30 August 2005 Available online 2 November 2005

Abstract Glucocorticoids, given at high-doses, improve recovery of function after spinal cord injury (SCI) in animals. However, side effects combined with a limited efficacy in clinical trials have restricted their usefulness for treatment of SCI patients. Recent studies have shown that incorporation of the nitric oxide releasing moiety into the glucocorticoid structure enhances anti-inflammatory properties and reduces side effects. One compound, a derivative of prednisolone (PRE), (NCX 1015, prednisolone 21 [(4Vnitrooxymethyl)benzoate]), has interesting pharmacological properties. Therefore, we investigated its effects on apoptosis and recovery of function in rats after SCI. Rats received subcutaneously vehicle, NCX 1015 or PRE (37 Amol/kg, each) 3.5 h after a standardized thoracic lesion. The treatment was continued once a day for 3 days and the effect of both steroids on apoptosis was examined by immunohistochemistry 24 h after the last injection. NCX 1015 but not PRE reduced TUNEL and activated caspase 3 in both white and ventral gray matter as well as tumor necrosis factor immunoreactivity in ventral horn motorneurons, suggesting that NCX 1015 reduces SCI-induced apoptosis. The effect of NCX 1015 on motor function was then examined by a standard locomotion rating scale (BBB) starting at 1 day after injury and continuing up to 14 days. NCX 1015 improved significantly locomotor activity by 4 days after injury, whereas PRE had an effect equivalent to that of vehicle, thus providing a correlation between the antiapoptotic effect of NCX1015 and its ability to improve recovery of function. The data suggest that NCX 1015 might be a novel experimental therapeutic compound for recovery of function in SCI patients. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Trauma Keywords: Apoptosis; Caspase-3; Spinal cord injury; Prednisolone; Tumor necrosis factor-a; Ventral motor neuron

1. Introduction

Abbreviations: BBB, Basso, Beattie, Bresnahan locomotor score; DAPI, 4V,6V-diamino-2-phenylindole; iNOS, inducible nitric oxide synthase; LAM, Laminectomy; NCX 1015, prednisolone 21-[(4Vnitrooxymethyl) benzoate]; NO, nitric oxide; PRE, prednisolone; SCI, spinal cord injury; sc, subcutaneously; TNFa, tumor necrosis factor-a; TUNEL, in situ terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling; VEH, vehicle; VMN, ventral motor neurons * Corresponding author. Fax: +1 202 687 0617. E-mail address: [email protected] (I. Mocchetti). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.08.057

Therapies to improve recovery of function after traumatic spinal cord injury (SCI) are still scarce. SCI results in mechanical lesion to tissue at the injury site often accompanied by massive ischemic necrosis. However, it is generally recognized that the initial mechanical injury accounts for only some of the long-term consequences of SCI, as trauma also initiates physiological and molecular events that produce secondary injury (reviewed in [6]). Free radicals, inflammation and

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ultimately apoptosis are key events that occur during secondary injury and play a crucial role in the loss of cells within the spinal cord [4,18,32,36]. Without pharmacological intervention, secondary injury continues to exacerbate cell and tissue damage and functional impairment initially triggered by mechanical insult. Thus, limiting apoptosis is a strategy to ameliorate recovery of function following SCI. Apoptosis seen in SCI can be reduced by antiinflammatory drugs such as the glucocorticoids [12,43]. The synthetic glucocorticoid methylprednisolone, given systemically at high doses, is widely used to improve recovery of function following SCI in humans [11,57]. In rats, however, the benefits of methylprednisolone have been recently questioned [42,52] despite promising earlier findings showing a positive outcome on motor and sensory function following SCI [9,21,38]. Moreover, when given at high doses, glucocorticoids produce several severe side effects including reduction of hormone release and inhibition of natural immunoresponses, thus limiting their therapeutic application. In addition, experimental findings as well as recent clinical evidence have questioned the use of high doses of methylprednisolone for acute SCI based on the negative relationship between improved neurological recovery and deleterious effects [48]. Thus, from a clinical perspective, additional studies are urgently needed to develop pharmacological compounds that, in addition to reducing inflammatory responses and apoptosis, do not exhibit side effects. Recently, nitric oxide (NO)-releasing derivatives of conventional steroidal anti-inflammatory drugs have been shown to reduce inflammatory responses, as well as apoptosis, without possessing some of the typical side effects of glucocorticoids [5,37,41]. One such example is prednisolone 21-[(4Vnitrooxymethyl)benzoate] (NCX 1015), a derivative of prednisolone (PRE) that reduces experimental colitis or arthritis [23,40]. Most importantly, compounds that increase NO levels have been shown to block apoptosis induced by neurotoxins such as glutamate, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, tumor necrosis factor-a (TNFa) and Zn2+ [26,50]. These properties make NCX 1015 a compound of interest to improve recovery of function after SCI. The purpose of the present study was to examine the efficacy of NCX 1015 using a well-characterized rat model of incomplete SCI [24,55] on apoptosis and shortterm recovery of motor behavior. Moreover, to gain crucial information about whether the NO-donating moiety or the steroid portion of the molecule has a positive effect for SCI, we have compared the pharmacological profiles of NCX 1015 with that of the parent drug PRE. We report evidence of a relationship between the ability of NCX 1015 to reduce apoptotic cell death in both white and gray matter and its effectiveness in improving recovery of motor function.

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2. Materials and methods 2.1. Spinal cord injury Adult female Sprague –Dawley rats (230 – 250 g, Taconic Farms, Germantown, NY) were housed in temperaturecontrolled (18 – 20 -C) and light-controlled (12 h light/dark cycle) rooms with access to food and water ad libitum. All surgical procedures were carried out in strict accordance with the Laboratory Animal Welfare Act, Guide for the Care and Use of Laboratory Animals (NIH, DHEW Pub. No 7823, Revised, 1978) only after review and approval by the Animal Care and Use Committee of Georgetown University Medical Center. Contusive injury was produced using a standardized weight-drop model as previously described [24,54,55]. Briefly, animals were deeply anesthetized with ketamine and xylazine (80 and 10 mg/kg, intraperitoneal, respectively), and a dorsal laminectomy was performed at the T8 vertebral level to expose the spinal cord. The vertebral column was stabilized by clamping the spinous processes of the T7 and T9 vertebral bodies, the impounder tip (ArmaLab, Bethesda, MD) lowered onto the dura, and a 10-g weight dropped 2.5 cm onto the impounder to produce a spinal cord contusion. Laminectomized (LAM) rats had the injury device lowered onto the dura but no weight was dropped. This model of contusive injury has previously been characterized in terms of functional deficits produced [24,55], growth factor induction [12,34] and quantitative histopathology [45,54]. After surgery, the overlying muscles were sutured, the skin was closed with wound clips, and the animals were hydrated with 5 ml of Ringer’s solution and were allowed to recover in warmed cages. To limit bacterial infection animals received penicillin G (40,000 U/kg, i.p.) (Sigma, St. Louis, MO, USA) the night before and after the operation, thereafter animals received, orally, a suspension of sulfamethoxazole (20 mg/kg) and trimethoprim (4 mg/kg) (Hi-Tech Pharmacal Co., Inc., Amityville, NY) twice a day for 2 days. Manual expression of bladders was performed twice daily. Post-operative care also included housing the rats in pairs (to reduce isolation-induced stress), and using highly-absorbent bedding. 2.2. Animal treatments LAM or injured rats received subcutaneously (sc), either NCX 1015, PRE (both from the Nicox Research Institute, Bresso, Italy) or vehicle used to dissolve the drugs [(40% DMSO, 35% PEG 400, and 25% ethanol (vol/vol)] (VEH). For the histological studies, NCX 1015 and PRE were used at equimolar concentrations (37 Amol/ kg) starting 3.5 h after injury. One group of animals was sacrificed 24 h later, the other group received either compounds daily for 3 more days. These animals were then sacrificed 24 after the last injection. A group of animals received PRE at 74 Amol/kg, sc for 4 days.

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However, this treatment was discontinued because of side effects. For the behavioral studies, NCX 1015 was used at concentrations of 9, 37 and 74 Amol/kg, sc. 2.3. Immunohistochemistry At appropriate survival times, rats were deeply anesthetized with ketamine/xylazine and intracardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The spinal cord was removed and post-fixed in the same fixative for 2 h, then transferred into buffered graded sucrose (10% and 20%) and serial coronal sections (20 Am) were made from a 1.5 cm length of spinal cord centered on the injury epicenter. Representative sections were stained with hematoxylin, ECRC (eriochrome cyanine), and eosin and phyloxine to assess tissue morphology and determine the injury epicenter, as previously described [54]. Serial sections (1 section every 200 Am) obtained from 2 and 3 mm of tissue rostral and caudal to the epicenter were used for immunohistochemical analysis of apoptosis by in situ terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) or activated caspase-3 as previously described [3]. In brief, sections were post-fixed in ethanol/acetic acid 2:1, washed, and equilibrated according to the instructions of the manufacturer (ApopTag, Serological Corporation, Norcross, GA). Sections were then incubated with TdT enzyme in the presence of digoxigenin-labeled dNTP, followed by antidigoxigenin (fluorescein conjugate) antibody, to visualize TUNEL-positive cells. For caspase-3, sections were incubated with antibodies against activated caspase-3 (1:100; Cell Signaling, Beverly, MA) for 48 h at 4 -C followed by fluorescein isothiocyanate conjugated secondary antibody. Slides were then mounted using Vectashield Mounting Medium with 4V,6V-diamino-2-phenylindole (DAPI) as counterstaining (Vector Laboratories, Burlingame, CA). For NeuN staining, sections were incubated with NeuN antibody (1:500, Chemicon, Temecula, CA) overnight at 4 -C followed by Texas red conjugated antibody (Vector Laboratories). For tumor necrosis factor a (TNFa) immunostaining, sections were exposed to microwave irradiation for 15 min in 10 mM sodium citrate (pH 6) before incubation overnight at 4 -C with a TNFa antibody (1:10, R&D System Inc., Minneapolis, MN). Sections were then washed, incubated with fluorescein-conjugated secondary antibody (1:500, Chemicon) followed by NeuroTracei red fluorescent Nissl stain (1:150, Molecular Probes, Eugene, OR) for 20 min at room temperature, to visualize TNFa and neurons, respectively, and mounted with Vectashield Mounting Medium with DAPI. Images of immunoreactive cells were analyzed using the Zeiss fluorescent microscope Axioplan. 2.4. Cell counting and statistical analysis All morphological analyses were done with tissue identified only by animal number. The evaluators were

blind to the treatment group until after the primary data were collected. Caspase-3, NeuN and TNFa-positive cells were analyzed using the Zeiss fluorescent microscope Axioplan. Sections representing 2 and 3 mm of tissue from the injury epicenter of animals at 1 and 4 days post injury and LAM controls were used to quantify immunoreactive cells. Cell counting was carried out in 10 sections (5 rostral and 5 caudal to the injury epicenter) per segment per animal in a 1 mm2 area of the lateral and ventral white matter, and ventral grey matter using MethaMorph\ software (Universal Imaging Corporationi, Downingtown, PA). Apoptotic cells in the white matter regions were counted in two randomly chosen sections (Fig. 1A) per 1 mm length of spinal cord, and the numbers were averaged. These regions were chosen because the tissue was well preserved even after SCI. Sections in which the primary antibody or polymerase (for TUNEL) was omitted were used as negative control to assess the background of the fluorescence conjugated secondary antibodies. Positive cells were defined as those with a bright fluorescence staining above background. Cells were counted only based on staining, not shape, size, or other measurable quantities. Data were analyzed by one-way ANOVA followed by all

Fig. 1. NCX 1015 decreases the number of TUNEL-positive cells. SCI rats received a single injection of VEH, NCX 1015 or PRE (37 Amol each, sc) 3.5 h after the injury and were sacrificed the day after (one). Another group of rats received VEH, NCX 1015 or PRE 3.5 h after the injury and the treatment was continued daily for 3 days. Rats were then sacrificed 24 h after the last injection (four). TUNEL-positive cells were counted in the white matter areas indicated by the squares, in sections from both 2 and 3 mm segments, caudal to the injury epicenter (5 sections each segment, each treatment). Data from the lateral and ventral white matter were combined. Scatterplots with mean values refer to three animals per group, each treatment.

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paired multiple comparison procedure (Holm-Sidak method) using SigmaStat\ software (Systat Software, Inc, Point Richard, CA). 2.5. Behavioral assessment Behavioral outcome after SCI was assessed by open field locomotion score. This test measures recovery of hindlimb function in rats during free open field locomotion as described by Basso et al. [8] and is therefore referred to as the BBB score. Rats were tested preoperatively in an open field (110  110 cm) to acclimate to the testing environment. A total of 60 rats (20 VEH, 20 NCX 1015 and 20 PRE) were used for the behavioral analysis. These animals were tested at various time points starting at 1 day and up to 14 days after SCI. Behavioral analysis was carried out at appropriate time points by two investigators, blind to the treatment. Each animal was observed for a period of 4 min. For the dose –response study, 5 animals per dose were used. A score of 21 indicates normal locomotion, a score of 0 indicates no locomotion. 2.6. Statistical analysis of behavior Behavioral data were analysed using a repeated measures ANOVA after transforming BBB scores into ranks. According to the longitudinal study design, the objective of the analysis was to test the pairwise differences between treatments using the rate of progression from day 1 to the study end. With this purpose, the response time profiles were portioned into polynominal components (linear, quadratic and so on) and the mean squares due to interaction ‘‘linear component of the profile-by-treatment-group’’ [17] were used to test the difference in the progression rate between the treatments. Results are reported in graph as adjusted means (least-square estimates) with SE.

3. Results 3.1. NCX 1015 reduces apoptosis in the white matter The role of apoptosis in the long-term neurological deficits associated with SCI has been established [18,32,36]. NCX 1015 has been shown to reduce inflammatory events and apoptosis in non-neuronal cells [23,37]. Thus, we investigated whether NCX 1015 and its parental compound PRE reduces apoptosis after SCI. Moreover, previous studies have shown that SCI triggers apoptotic cell death of oligodendrocytes associated with white matter tracts within 3 –4 days after injury [51]. Thus, we first established whether apoptosis follows a similar temporal profile in our experimental model of SCI. Rats were sacrificed at 1 or 4 days after LAM or SCI. Sections were analyzed for TUNEL-positive cells. LAM rats control showed an average of 3 T 1 TUNEL-positive cells

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per mm2 section independently on whether the section was at 1, 2 or 3 mm rostral or caudal to T8 or taken at 1 or 4 days after LAM (data not shown). In SCI rats instead, there was a dramatic increase in TUNEL-positive cells within the dorsal white matter especially around the lesion epicenter, accompanied by tissue loss, which is characteristic of this model of SCI [12,45]. Therefore, we focused the analysis of TUNELpositive cells in a 1-mm2 area of the lateral and ventral white matter (Fig. 1A) of sections taken at 2 and 3 mm caudal to the epicenter. At day one, the white matter exhibited fewer TUNEL-positive cells than at day 4 (Fig. 1B) both at 2 and 3 mm segments. At 4 days, we found no dramatic difference between the number of TUNEL-positive cells in the lateral white matter versus the ventral white matter (22 T 2, 19 T 2, respectively). Therefore data from lateral and ventral white matter were combined. The effect of NCX 1015 and PRE was then evaluated using a concentration of 37 Amol/kg (equivalent to 20 mg/ kg of NCX 1015 and 13.3 mg/kg of PRE), based on our previous experience with the positive outcome of synthetic glucocorticoids for SCI [12]. To establish the acute versus the sub-chronic effect, SCI rats received a single injection of VEH, NCX 1015 or PRE at 3.5 h after the surgery and were sacrificed the day after. Another group of animals received a single injection 3.5 h after the surgery but the treatment was continued daily for 3 days. Animals were sacrificed the day after the last injection. At 1 day, the average number of TUNEL-positive cells was similar in VEH, NCX 1015 and PRE-treated rats (Fig. 1B). Instead, at 4 days, the average of TUNEL-positive cells in NCX 1015-treated rats was lower than that in VEH or PRE-treated rats at both 2 and 3 mm (Fig. 1B) suggesting that NCX 1015 but not PRE might reduce apoptotic cell death. A group of animals received PRE at 74 Amol/kg for 4 days to establish whether a higher concentration of this steroid may have a positive effect. Indeed, steroids are usually given at high concentrations to improve recovery of function after SCI. At a higher concentration, PRE-treated rats showed body weight loss, a generalized deterioration of health status, and a survival rate ¨30%. Thus, this concentration was discontinued following the veterinarian’s request. 3.2. NCX 1015 reduces activated caspase-3 immunoreactivity TUNEL alone is not considered a marker of apoptosis. Caspase-3 plays a crucial role in SCI-mediated apoptotic cell death in the white matter [15,51]. Therefore, we analyzed the number of caspase-3-positive cells in the white matter in 1 mm2 areas as described in Fig. 1A. Moreover, to better examine the effect of SCI at the rostrocaudal extent, sections were taken at 2 and 3 mm rostral and caudal to the epicenter and analyzed separately. At day 1 after injury, few positive cells were present confirming the data obtained with TUNEL (data not shown). By 4 days,

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caspase-3-positive cells were observed at both 2 and 3 mm (Fig. 2) supporting previous results that SCI activates a caspase-3-dependent apoptotic pathway in glial cells [15,51]. In SCI animals, the number of activated caspase3-positive cells in segments taken at 2 (Fig. 2A) and 3 mm (Fig. 2B) caudal to the injury epicenter was comparable. Instead, the rostral portion contained less caspase-3-positive cells than the caudal portion (Fig. 2). This was not surprising because other studies have reported that cells caudal to the injury epicenter exhibit more apoptosis compared to the cells rostral to the epicenter [15]. In SCI rats receiving the experimental treatments, analysis of caspase-3 immunoreactivity revealed that NCX 1015 decreased the number of caspase-3-positive cells (P < 0.001 vs. VEH; P < 0.001 vs. PRE) in the white matter caudal to the injury epicenter in both 2 and 3 mm segments while PRE and

VEH did not (Fig. 2). A similar effect by NCX 1015 was observed in segments rostral to the injury epicenter (P < 0.001 vs. VEH; P < 0.001 vs. PRE) (Fig. 2). The anti-caspase-3 activity of NCX 1015 is not attributable to a non-specific effect of the VEH used to dissolve the drug because neither VEH nor PRE changed the total number of caspase-3-positive cells (Fig. 2). Moreover, no difference in the number of activated caspase-3-positive cells between LAM rats treated with VEH, NCX 1015 or PRE were found (data not shown). 3.3. NCX decreases apoptosis in the gray matter and helps preserving ventral motor neuron morphology The ability of NCX 1015 to reduce activated caspase-3 in the white matter prompted us to examine whether this drug reduces apoptosis in the ventral horn grey matter. Sections from 4-day SCI rats representing the 2 and 3 mm segments rostral and caudal to the injury epicenter were analyzed for activated caspase-3. As expected, numerous caspase-3positive cells were detected in SCI rats treated with vehicle (Fig. 3). As for the white matter, there was no difference in caspase-3-positive cells between 2 and 3 mm segments (Fig. 3). However, the caudal portions contained more caspase-3positive cells than the rostral portion (Fig. 3). The number of caspase-3-positive cells was significantly reduced by NCX 1015 in both 2 and 3 mm segments taken from the caudal and rostral portions (P < 0.001 vs. VEH; P < 0.001 vs. PRE) (Fig. 3). PRE showed no effect (Fig. 3). Therefore, it appears that NCX 1015 might interfere with the caspasedependent apoptotic pathway. Nevertheless, it is too premature to speculate whether NCX-1015 has a direct inhibitory effect on caspase-3 activation. 3.4. NCX 1015 does not affect the number of neurons

Fig. 2. NCX 1015 decreases the number of caspase-3-positive cells in the white matter. SCI rats received VEH, NCX 1015 or PRE (37 Amol each) beginning at 3.5 h after injury and daily for 3 days thereafter. Animals were perfused 24 h after the last injection. Serial sections were prepared throughout the thoracic spinal cord and processed for activated caspase-3 immunoreactivity. Determination of the number of caspase-3-positive cells was carried out in areas of 1 mm2 of white matter (see Fig. 1A) in sections taken at 2 (A) and 3 mm (B) caudal and rostral from the injury epicenter. Data, expressed as average number of activated caspase-3-positive cells per section (5 sections both rostral and caudal to the injury epicenter per animal) are the mean T SEM of six animals per group. *P < 0.001 vs. VEH caudal, or VEH rostral.

We next examined whether NCX 1015 affects the number of ventral motor neurons (VMN). SCI animals were treated with VEH or NCX 1015 for 4 days and were sacrificed 24 h after the last injection. We selected sections of cord tissue from 3 mm rostral and caudal to the epicenter to stain for NeuN immunoreactivity. NeuN-positive cells were then defined as VMN based on their localization [mostly below the deep apex (Fig. 4A)], and exhibiting a diameter between 30 and 70 Am, as previously established [54]. No significant changes in the number of VMN were observed between VEH [15 T 4 per section (square in Fig. 4A)] and NCX 1015treated rats (16 T 4). However, in contrast to NCX 1015treated rats (Fig. 4C), VMN in the VEH group exhibited an atypical morphology with an irregular body and shape, and undefined nucleus and nucleolus (Fig. 4B). This effect might be followed by shrinking of the body at a later time. Thus, it appears that NCX 1015 may help preserving VMN morphology and perhaps survival that otherwise is altered by the injury. However, future studies are needed to examine the long term (e.g., 15 and 30 days) effect of NCX 1015 on neuronal and tissue sparing.

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the grey matter was then determined in sections 3 mm rostral and caudal to the injury epicenter. In rats receiving VEH (Fig. 4D), TNFa immunoreactivity in the grey matter was higher than that in NCX 1015-treated rats (Fig. 4E) in both caudal and rostral segments (Fig. 5). The effect of NCX 1015 is specific because PRE failed to alter TNFa immunoreactivity (Fig. 5). This finding supports previous data showing that glucocorticoids do not change TNFa expression in SCI [53]. Thus, the ability of NCX 1015 to reduce TNFa immunoreactivity may help explaining why this compound has a pharmacological profile different from that of PRE. 3.6. NCX 1015 but not PRE improves recovery of locomotor function Reducing apoptosis may not necessarily improve recovery of function. The effect of NCX 1015 on recovery of function after SCI was evaluated according to an expanded 21-point locomotor scale (BBB) that is currently

Fig. 3. Caspase-3 immunoreactivity is reduced in the gray matter of NCX 1015-treated rats. Caspase-3 immunoreactivity was examined in an area of 1 mm2 of the ventral gray matter (see also Fig. 4) in sections obtained as described in Fig. 2. Data, expressed as average number of activated caspase-3-positive cells per section (5 sections both rostral and caudal to the injury epicenter per rat), are the mean T SEM of six animals per group. *P < 0.001 vs. VEH caudal, or VEH rostral.

3.5. NCX 1015 but not PRE decreases TNFa immunoreactivity The role of proinflammatory cytokines in SCI-evoked secondary injury is well established [10]. Recently, attention has focused on TNFa, a pro-inflammatory cytokine whose levels increase in SCI [7,56] and is implicated in both neuronal and glial apoptosis [1,29,31]. Therefore, we examined whether NCX 1015 decreases TNFa levels. LAM and SCI animals received VEH or NCX 1015 for 4 days and were sacrificed 24 h after the last injection. In both LAM rats receiving VEH or NCX 1015, TNFa immunoreactivity was barely detectable in both white and grey matter (data not shown). Nevertheless, there was no difference in TNFa immunoreactivity between VEH and NCX 1015-treated rats (data not shown), suggesting that NCX 1015 per se does not affect the basal levels of TNFa. Instead, TNFa immunoreactivity was easily detected in SCI rats, mainly in the grey matter in Nissl-positive cells, suggesting neurons. The number of TNFa-positive cells in

Fig. 4. NCX 1015 preserves VMN morphology and attenuates TNFa immunoreactivity. SCI rats received VEH or NCX 1015 for 4 days and were perfused 24 h after the last injection. Sections (3 mm rostral or caudal to the injury epicenter) were processed for NeuN or TNFa. Examples of photomicrographs of the ventral horn (square in A) showing the morphology of VMN in VEH (B) and NCX 1015-treated rats (C). Please note in B the presence of neurons displaying an atypical morphology with an irregular cell body shape (e.g., green arrow) and undefined nucleus and nucleolus (e.g., white arrow). D and E, TNFa immunoreactivity (green) in Nissl-positive cells (red) of the ventral horns from SCI rats treated with VEH or NCX 1015, respectively. Note the decrease in TNFa immunoreactivity (yellow, overlay green and red) in NCX 1015-treated rats. DAPI (blue). Scale bar: B and C = 50 Am, D and E = 150 Am.

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Fig. 5. Quantification of TNFa-positive cells. Relative quantification of TNFa-positive neurons was carried out by counting TNFa- and Nisslpositive cells in the ventral horns (see Fig. 4) in sections taken 3 mm rostral and caudal to the injury site. At least 10 sections (5 rostral and 5 caudal to the injury epicenter) per animal per group were analyzed. Data are presented as mean T SEM of four separate animals per group. *P < 0.01 vs. control.

being widely used to allow easy inter-laboratory comparison of results [8]. We first established that LAM rats receiving NCX 1015 did not show any overt behavioral outcome when compared to LAM controls receiving VEH. In fact, both groups scored 21 on the BBB at each time point (data not shown). Therefore, we report only data obtained in SCI rats. All injured rats receiving the experimental treatments exhibited a severe locomotion deficit one day after surgery (Fig. 6). For example, the mean BBB score in VEH group was 0.8 T 0.2 compared to 1.3 T 0.5 in the NCX 1015 group. Recovery of function improved slowly but steadily over the course of the two-week observation in both animal groups.

Fig. 6. Effect of synthetic glucocorticoids on locomotor activity. SCI rats received VEH, NCX 1015 or PRE beginning at 3.5 h after injury and daily for 3 days thereafter. The ability to use limbs in open field locomotion was measured by the BBB score at the indicated days after injury. Animals tested before surgery showed a score of 21. BBB score was significantly better in the NCX 1015 group (P < 0.001 vs. VEH or PRE). Data are the mean T SEM of 20 animals per group.

Fig. 7. Dose-dependent effect of NCX 1015. SCI rats received NCX 1015 at the indicated concentrations, beginning at 3.5 h after injury and daily for 3 days thereafter. BBB score was then analyzed at day 7 after injury as indicated in Fig. 6. *P < 0.05 vs. VEH (non-parametric Dunnett’s t test), n = 5 each group.

PRE-treated rats exhibited BBB scores similar to VEHtreated rats at each time point tested (Fig. 6). Instead, performance of the NCX 1015-treated group in the BBB test was significantly higher compared to the PRE- or VEHtreated groups, starting at 4 days after surgery and continuing up to 14 days (F [1,57] = 16.4, P < 0.0002, NCX vs. VEH; F [1,57] = 10,29, P < 0.0022, NCX vs. PRE). For instance, by 7 days after surgery, some NCX 1015treated rats were able to support their weight on the dorsal part of the paws while stationary or stepping, other exhibited frequent to consistent steps with or without coordination (average BBB score of 10, Fig. 6). In contrast, PRE or VEH-treated rats only displayed slightly or extensive movement of all three joints with occasional plantar placement, corresponding to an average BBB score of 7. By 14 days, all NCX 1015-treated rats exhibited frequent to consistent steps and some showed also step coordination (BBB average 12) while the control group showed an average BBB score of 9 (Fig. 6). Dose– response studies were carried out to assess the relative pharmacological potency of NCX 1015. Rats received NCX 1015 (9, 37 and 74 Amol/kg, sc) and were tested up to 7 days. At a lower dose (9 Amol/kg) NCX 1015 did not significantly changed BBB when compared to VEHtreated rats (Fig. 7), whereas at a higher concentration (74 Amol/kg), NCX 1015 improved recovery of function but its effect was not significantly different from the dose of 37 Amol/kg (Fig. 7). Thus, although SCI rats treated with NCX 1015 failed to exhibit a BBB comparable to uninjured animals, they show evidence of improved recovery of function. Future studies will investigate whether NCX 1015 has a long-lasting positive effect.

4. Discussion Valid therapies for SCI in humans are at present limited. Methylprednisolone, given at high doses to SCI patients,

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has been shown to produce beneficial effects; however its therapeutic efficacy is restricted to a small percentage of patients [11,57]. Neurotrophic factors may constitute another potential therapy for SCI as these polypeptides exert neurotrophic and neuroprotective activity in experimental SCI alone [2,46,54] or in conjunction with cell transplant [14]. However, their efficacy appears to be limited to selected neuronal populations or pathways. Moreover, given the fact that a variety of mechanical factors contribute to different types of injury, it is reasonable to believe that a beneficial outcome after SCI may be improved by different therapies. In the present study, we report that the NO-releasing PRE derivative NCX 1015, but not PRE itself, improves recovery of locomotor function in a rodent model of SCI that reproduces the human pathology [24,55]. The positive outcome correlates with the ability of NCX 1015 to reduce apoptotic cell death. Although the behavioral and biochemical assessment of the long-term positive effect of NCX 1015 has not yet been carried out, it appears that NCX 1015 may have a promising therapeutic property for SCI. Most importantly, NCX 1015 is more potent as an antiinflammatory agent than PRE but does not exhibit some of the typical side effects of the classical glucocorticoids [40]. SCI is characterized by at least three phases (acute, secondary and chronic). The acute tissue damage, caused by the mechanical trauma itself, is irreversible. The secondary phase can lead to massive additional cell loss but its progression is preventable. Therefore, in an attempt to characterize the molecular and cellular mechanisms of NCX 1015, we focused our investigation on the secondary pathological processes. These are not confined to the injury epicenter, occur and evolve over a few hours to few days after injury. We reasoned that in order to be relevant therapeutically and preserve neurological function, a compound must reduce secondary processes within a few days rather then weeks. Our data show that NCX 1015 reduces apoptosis and improve locomotor function by 4 days after injury. There are several mechanisms that may account for the positive effect of NCX 1015. These may include antioxidant and anti-inflammatory effects as it has been shown for methylprednisolone [27]. Though, in this study, we did not characterize the primary mechanism of action of NCX 1015, the results presented here show that NCX 1015 may have a broad pharmacological profile. In fact, NCX 1015 may possess a number of properties therapeutically important for SCI including its ability to reduce caspase-3, a final effector caspase involved in the activation and execution of apoptotic pathways [58]. Indeed, various experimental models of SCI have revealed that apoptosis, which occurs in both neurons and oligodendrocytes within a few hours after the initial trauma, is responsible for a delayed cell loss [18,32]. This process may continue for several months, leading to massive tissue loss. Apoptosis is also seen in postmortem spinal cord of human SCI patients [20]. Blocking apoptosis with inhibitors of protein synthesis [32] or calpain [44,47] in experimental SCI has been shown

23

to improve the neurological outcome. Therefore, preventing apoptosis in SCI patients is a target for clinical intervention. However, there are few CNS penetrating antiapoptotic compounds that are ready for clinical trials. In this context, the lipophilic structure of NCX 1015 and its limited side effect profile are appealing properties that make this compound suitable for human therapy. Apoptosis is an active process involving activation of caspase-3. Traumatic injury to the spinal cord has been shown to activate this caspase both in neurons and oligodendrocytes and to be a crucial component of secondary injury. Indeed, reduction of caspase activity by synthetic peptide-based inhibitors has been demonstrated to attenuate neuronal apoptosis and the spread of secondary damage in animal models of CNS trauma [22,35,39]. In this study, we confirm a widespread induction of caspase-3 in SCI, supporting the notion that the rat spinal cord contains the molecular apparatus necessary for activation and execution of the caspase-3 apoptotic pathway. NCX 1015 reduced activated caspase-3 immunoreactivity in both the white and gray matter, indicating that NCX 1015 should be added to the list of potential anti-caspase-3 compounds that can be used to limit the spread of secondary injury. However, in addition to the anti-caspase properties shown in this report, NCX 1015 may also exhibit other pharmacological effects that are important for SCI. NCX 1015 is a NO-releasing derivative of PRE. NO is a ubiquitous messenger produced from converting l-arginine to l-citrulline by either inducible (iNOS) or constitutive NO synthases [13]. NO plays multiple roles in different physiological systems, including the brain, where it acts as a retrograde transmitter [25]. Moreover, NO is known to exert neuroprotective activities against inflammatory cytokine-induced apoptosis [19,30,33] and to downregulate the expression of TNFa [49], which can induce cell death after SCI [1,29,31]. Thus, TNFa, whose levels are increased after SCI could be a critical component of the apoptotic cascade pathway. NCX 1015, but not PRE, decreased TNFa immunoreactivity in VMN of injured rats, suggesting that it is the NO-releasing properties of NCX 1015 which may have a therapeutic importance for SCI. Consistent with this suggestion, VMN of NCX 1015-treated rats displayed a well preserved and defined nucleus and nucleolus and an overall better neuronal morphology than those of VEH group. On the other hand, NCX 1015 did not block completely the increase in TNFa caused by the contusive injury. Consequently, we did not observed a significant effect of NCX 1015 in increasing the number of surviving neurons at 4 days after injury. However, based on the role of TNFa in promoting apoptosis, our data suggest that by reducing the expression of TNFa, NCX 1015 may promote the long-term survival of VMN. Future studies will test this hypothesis. Although NO normally functions as a physiological neuronal mediator, activation of iNOS especially after stimulation of NMDA receptors and excessive production of NO can cause neuronal cell death [13]. Thus, selective

24

A. Mallei et al. / Brain Research 1062 (2005) 16 – 25

inhibitors of iNOS may find a role in the treatment of neuronal degeneration. Pharmacological levels of NO as those obtained by NO donors have been shown to inhibit NOS and in particular iNOS [16]. Expression of iNOS has been detected in both glia cells and neurons after trauma, in chronic neurodegenerative diseases or aging (reviewed in [28]). Inhibition of iNOS prevents neuronal apoptosis. Thus, iNOS may contribute to neuronal damage in acute and chronic neurodegenerative diseases. NCX 1015, as well as other NO-donors [37], has been shown to inhibit iNOS [23], which might explain in part the ability of NCX 1015 to limit neuronal damage after SCI. Future experiments will test this hypothesis by assessing whether a relationship exists between the NO releasing property of NCX 1015 and its ability to down-regulate iNOS expression/function after SCI. In conclusion, NCX 1015 appears to combine the antiinflammatory with the neuroprotective effects that are needed to attenuate apoptosis processes set in motion by SCI. More studies are needed to investigate in more detail whether NCX 1015 affects other cells that are primarily producing TNFa after injury, such as microglia or oligodendrocytes. Moreover, the long term effectiveness of this compound must be established. In addition, future studies will identity whether NCX 1015 affects other secondary events crucial for SCI including hemorrhage, excitotoxicity and mitochondrial disturbances.

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We thank Dr. E. Bonizzoni for statistical analysis. The study was supported by grant from Nicox Research Institute.

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