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The effects of N-acetyl-cysteine and acetyl-l -carnitine on neural survival, neuroinflammation and regeneration following spinal cord injury
Neuroscience 269 (2014) 143–151
THE EFFECTS OF N-ACETYL-CYSTEINE AND ACETYL-L-CARNITINE ON NEURAL SURVIVAL, NEUROINFLAMMATION AND REGENERATION FOLLOWING SPINAL CORD INJURY A. KARALIJA, a,b* L. N. NOVIKOVA, a P. J. KINGHAM, a M. WIBERG a,b AND L. N. NOVIKOV a Key words: adult rats, spinal cord, motoneurons, antioxidants, apoptosis.
a Department of Integrative Medical Biology, Section of Anatomy, Umea˚ University, SE-901 87 Umea˚, Sweden b Department of Surgical and Perioperative Science, Section of Hand and Plastic Surgery, Umea˚ University, SE-901 87 Umea˚, Sweden
INTRODUCTION Although traumatic spinal cord injury (SCI) is highly relevant both clinically and epidemiologically, to date, there is no safe and effective enough treatment which is recognized in clinical practice (Rabchevsky et al., 2011; Silva et al., 2014). The previously prevalent use of highdose glucocorticoid treatment, considered to mediate a part of its effect by acting as an antioxidant (Jia et al., 2012), has fallen out of favor due to lack of a convincing clinical effect coupled with suspected increased morbidity and mortality, and is no longer commonly approved (Bydon et al., 2013; Sayer et al., 2006; Short et al., 2000). The inevitable trauma-induced loss of axonal continuity, although debilitating, only accounts for a part of the neurological deficits experienced, and a complete transection of the spinal cord is uncommon since the initial trauma, usually leaves parts of the white matter intact. It is rather the secondary reaction, commencing only minutes after the initial trauma that causes a progressive degradation of the spinal cord (Hall and Springer, 2004). This much studied but poorly understood process is hallmarked by death of neurons and glial cells (Zhang et al., 2012), mitochondrial dysfunction and production of reactive oxygen species (ROS) (Jia et al., 2012; Maragos and Korde, 2004), and a neuroinflammatory response involving, among other cells, the activation of microglia (Donnelly and Popovich, 2008). The events of the secondary reaction lead to the formation of a cavity at the site of the trauma with an eventual astroglial scar formation, preventing the regeneration of axons through the lesion site (Karimi-Abdolrezaee and Billakanti, 2012). Two substances that have lately emerged as potential neuroprotectants and modulators of the neuroinflammatory response are the antioxidants acetylL-carnitine (ALC) and N-acetyl-cysteine (NAC). Both readily cross the blood–brain barrier (Farr et al., 2003; Parnetti et al., 1992) and both have been successfully used in clinical practice (Malaguarnera, 2012; Santos et al., 2011; Waring, 2012). ALC is a small peptide found in the mitochondria, containing a carnitine moiety and an acetyl moiety. The former plays an important role in the oxidation of fatty acids, while the latter contributes to the
Abstract—Traumatic spinal cord injury induces a longstanding inflammatory response in the spinal cord tissue, leading to a progressive apoptotic death of spinal cord neurons and glial cells. We have recently demonstrated that immediate treatment with the antioxidants N-acetyl-cysteine (NAC) and acetyl-L-carnitine (ALC) attenuates neuroinflammation, induces axonal sprouting, and reduces the death of motoneurons in the vicinity of the trauma zone 4 weeks after initial trauma. The objective of the current study was to investigate the effects of long-term antioxidant treatment on the survival of descending rubrospinal neurons after spinal cord injury in rats. It also examines the short- and long-term effects of treatment on apoptosis, inflammation, and regeneration in the spinal cord trauma zone. Spinal cord hemisection performed at the level C3 induced a significant loss of rubrospinal neurons 8 weeks after injury. At 2 weeks, an increase in the expression of the apoptosis-associated markers BCL-2-associated X protein (BAX) and caspase 3, as well as the microglial cell markers OX42 and ectodermal dysplasia 1 (ED1), was seen in the trauma zone. After 8 weeks, an increase in immunostaining for OX42 and the serotonin marker 5HT was detected in the same area. Antioxidant therapy reduced the loss of rubrospinal neurons by approximately 50%. Treatment also decreased the expression of BAX, caspase 3, OX42 and ED1 after 2 weeks. After 8 weeks, treatment decreased immunoreactivity for OX42, whereas it was increased for 5HT. In conclusion, this study provides further insight in the effects of treatment with NAC and ALC on descending pathways, as well as short- and long-term effects on the spinal cord trauma zone. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.
*Correspondence to: A. Karalija, Department of Integrative Medical Biology, Umea˚ University, SE-901 87 Umea˚, Sweden. Tel: +46703234219. E-mail address: [email protected] (A. Karalij. Abbreviations: 5HT, serotonin; ALC, acetyl-L-carnitine; BAX, BCL-2associated X protein; BCL-2, B-cell lymphoma 2; C3bi, complement receptor type 3; DAPI, 4’,6-diamidino-2-phenylindole; DPX, dibutyl phathalate xylene; DRG, dorsal root ganglia; ED1, ectodermal dysplasia 1; EGTA, ethylene glycol tetraacetic acid; NAC, N-acetylcysteine; PIPES, piperazine-1,4-bis-2-ethanesulfonic acid; ROS, reactive oxygen species; SCI, spinal cord injury. http://dx.doi.org/10.1016/j.neuroscience.2014.03.042 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 143
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maintenance of acetyl-CoA levels and production of the anti-oxidant glutathione. ALC has been successfully used in clinical trials for the treatment of neuropathy and neuropathic pain, cognitive disorders and depression (Malaguarnera, 2012). It has also recently been found to contribute to the maintenance of mitochondrial bioenergetics following SCI in rats (Patel et al., 2010). NAC, a derivative of cysteine, similarly exerts a wide range of cellular actions, among others acting as a glutathione precursor (Atkuri et al., 2007) and exhibiting anti-inflammatory actions (Palacio et al., 2011). NAC has been approved for clinical use for many years, being used as a mucolytic agent and for treatment of acetaminophen intoxication (Heard, 2008; Sadowska, 2012; Ziment, 1988). We have previously demonstrated that treatment with NAC and ALC can delay the degeneration of sensory neurons in the dorsal root ganglia (DRG) after peripheral nerve injury, and greatly reduce early retrograde death of spinal motorneurons after ventral root avulsion (Hart et al., 2002; Welin et al., 2009; Wilson et al., 2007; Zhang et al., 2005). In our recent short-term study we have shown that NAC and ALC also attenuate degeneration of spinal motoneurons after lumbar SCI, reduce the activity of microglia and macrophages and promote axonal sprouting in the injured segment of the cord (Karalija et al., 2012). The primary goal of this study is to investigate the long-term effects of NAC and ALC treatment on the survival of descending rubrospinal neurons after cervical SCI and to evaluate the efficacy of antioxidant treatment on apoptotic, inflammatory and regenerative reactions in the trauma zone.
EXPERIMENTAL PROCEDURES Experimental animals The experiments were performed on adult (10–12 weeks, n = 60) female Sprague–Dawley rats (Taconic Europe A/ S, Denmark). The animal care and experimental procedures were carried out in accordance with Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes and also approved by the Northern Swedish Committee for Ethics in Animal Experiments (No. A3612). All surgical procedures were performed under general anesthesia using a mixture of ketamine (KetalarÒ, Parke-Davis, Pfizer, New York, NY, USA; 100 mg/kg i.v.) and xylazine (RompunÒ, Bayer, Leverkusen, Germany; 10 mg/kg i.v.). After surgery, the rats were given the analgesic Finadyne (ScheringPlough, Denmark; 2.5 mg/kg, s.c.), normal saline (4 ml, s.c.) and benzylpenicillin (Boehringer Ingelheim, Ingelheim am Rhein, Germany; 60 mg, i.m.). Each animal was housed alone in a cage after surgery and exposed to 12-h light/dark cycles, with free access to food and water. SCI Following cervical laminectomy a stab wound was inflicted by inserting a 23-G needle in the dorsal root entry zone of the C3 spinal cervical segment. The
blade of a pair of Vannas spring scissors was then introduced in the perforation and, using the other blade of the scissors, a hemisection of the spinal cord was performed. In the experiments dealing with neuronal survival, rubrospinal neurons were labeled with the non-toxic fluorescent retrograde tracer Fast Blue immediately after SCI. A small pellet prepared from 1 to 2 ll of a 2% aqueous solution of the Fast Blue (FB, EMS-Chemie GmbH, Germany) was placed into the lesion cavity. The opened dura mater was covered with a piece of stretched parafilm and SpongostanÒ. The operation was finished with the closing of the muscles and skin respectively. After the operation, the animals were randomized into one of the following three groups: (i) spinal cord injury without treatment (SCI, n = 18), (ii) SCI and treatment with NAC (n = 13) and (iii) SCI and treatment with ALC (n = 13). Eight uninjured normal rats (used for Western blotting and immunohistochemistry) as well as eight rats at 1 week after Fast Blue labeling served as baseline controls. Treatment with NAC and ALC The treatment with NAC and ALC commenced immediately after performing the SCI by the subcutaneous implantation of an Alzet 2002 osmotic minipump (Alza Corp., Palo Alto, CA, USA) filled with a solution of either the L-stereoisomer of NAC (200 mg/ml; BioPhausia, Stockholm, Sweden) or O-acetyl-L-carnitine hydrochloride (75 mg/ml in normal saline; Sigma–Aldrich, St. Louis, MO, USA) in the neck region. Following L6 laminectomy, a subcutaneous polyethylene catheter (Intermedic, Barcelona, Spain, PE-60) filled with NAC or ALC was inserted into the lower lumbar subarachnoid space with the tip reaching the level of L3–L4 DRG. The tube was fixed to the S1 vertebral bone by HistoacrylÒ glue, the insertion site covered with SpongostanÒ and the catheter secured in place by suturing it to the muscles. The proximal part of the catheter was connected to the osmotic pump, commencing the treatment. The infusion speed corresponded to 2.4 mg/ day for NAC and 0.9 mg/day for ALC, doses derived from previous studies (Karalija et al., 2012). Following 14 days of treatment the empty pump was exchanged for a fresh one containing the same solution. The change of pump was performed every 14 days until the end of the study. The animals were sacrificed at 2 and 8 weeks postoperatively. Tissue processing Following the experiments all animals were euthanized by administering an intraperitoneal overdose of pentobarbital (240 mg/kg, Apoteksbolaget, Sverige). The animals intended for Western blotting underwent harvest of the tissue caudal and rostral to the injury site 2 weeks after initial trauma. The tissue was divided in two halves in the sagittal plane and these two pieces were then divided transversely, separating the tissue rostral to the injury site from the caudal part. The tissue was immediately frozen in liquid nitrogen. The rest of the animals were transcardially perfused using Tyrode’s
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solution followed by 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (pH 7.4). Following the perfusion, the brain stem and the spinal cord segments C1–C3 were harvested and post-fixed in the same paraformaldehyde solution for 2–4 h, after which the tissue underwent cryoprotection in 10% and 20% sucrose for 3 days, and was then frozen in liquid isopentane. The tissue intended for counts of Fast Bluelabeled rubrospinal neurons was cut in 50-lm-thick serial sections on a vibratome (Leica Instruments, Wetzlar, Germany), mounted on gelatin-coated glass slides, air dried, briefly immersed in xylene and coverslipped in dibutyl phathalate xylene (DPX). For immunohistochemical analysis the tissue was cut in 16lm-thick serial sections on a cryomicrotome (Leica Instruments, Wetzlar, Germany), thaw-mounted onto SuperFrostÒPlus slides, dried overnight at room temperature and stored at 85 °C before being processed. Counts of the Fast Blue labeled neurons Counting the nuclear profiles of labeled rubrospinal neurons was performed in all sections through the brainstem using 250 magnification. The total number of profiles was not corrected for split nuclei, since the nuclear diameters were small in comparison with the section thickness used. We have previously demonstrated that the accuracy of this counting technique in estimation of retrograde neuronal cell death is similar to that obtained with the physical dissector method (Ma et al., 2001) and counts of neurons reconstructed from serial sections (Novikov et al., 1997). Immunohistochemistry The serial tissue sections were processed for demonstration of glial cell and neuron markers. After blocking with normal serum, mouse antibodies reacting with C3bi complement receptors (OX42; 1:250, Serotec, Oxford, UK) and rabbit anti-serotonin antibodies (5-HT; 1:500; Sigma–Aldrich, St. Louis, MO, USA) were applied for 2 h at room temperature. After rinsing in PBS, secondary goat anti-mouse and goat anti-rabbit antibodies Alexa FluorÒ 488 and Alexa FluorÒ 568 (1:300; Molecular Probes, Eugene, OR, USA, Invitrogen, Carslsbad, CA, USA) were applied for 1 h at room temperature in the dark. The slides were coverslipped with ProLong mounting media containing 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carslsbad, CA, USA). The staining specificity was tested by omission of primary antibodies.
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quantify the protein level in the samples. Four to five animals per group were combined to create each lysate for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis. An amount of 20 lg of protein was denaturated at 90 °C and, depending on molecular weight, loaded on a 10% or 15% SDS–PAGE. Following transfer to a nitrocellulose membrane the blots were blocked using 5% (w/v) non-fat milk or 1% bovine serum albumin (BSA) (w/v) in Tris buffer saline with Tween (TBST). Following the blocking the blots were incubated with rabbit anti-caspase 3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-BAX antibody (1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), mouse anti-OX42 antibody (1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), mouse anti-ED1 antibody (1:300; Abcam, Cambridge, UK) and the loading control rabbit anti-beta tubulin (1:5000; Abcam, Cambridge, United Kingdom) overnight at 4 °C. After washing the blots in TBS-T 6 5 min they were incubated with mouse or rabbit IgG HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 h. Another series of 6 5-min washes in TBS-T was performed after which the blots were exposed to ECL reagent (GE Healthcare, Little Chalfont, UK) for 1 min and then finally developed onto Kodak XPS films. The developed films were scanned using an Epson Photoscanner.
Quantification of microglial cell reaction and axonal sprouting 5HT-positive raphaespinal axonal arborizations and OX42-positive microglial cells were quantified in the ventral horn of the C2 spinal segments at 8 weeks after SCI. All images were captures at 400 final magnification with a Nikon DS-U2 digital camera. The relative tissue area occupied by immunostained profiles was quantified in five randomly selected sections from each rat in 50 50-lm areas (18.9 pixels per 1-lm tissue length) for 5HT-positive axons and 150 150-lm areas (3.8 pixels per 1-lm tissue length) for microglial cells using Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA).
Image processing For preparation of figures, the captured images were resized, grouped into a single canvas and labeled using Adobe Photoshop CS4 software. Contrast and brightness were adjusted to provide optimal clarity.
Western blotting The spinal cord tissue was homogenized using buffer (pH 6.9) containing 5 mM EGTA, 100 mM piperazine-1,4-bis2-ethanesulfonic acid (PIPES), 5 mM MgCl2, 20% (v/v) glycerol, 0.5% (v/v) Triton X-100 and protease inhibitor cocktail (Sigma). A detergent compatible protein assay (Bio-Rad, Hercules, CA, USA,) was performed in order to
Statistical analysis One-way analysis of variance (ANOVA) followed by a post hoc Newman–Keuls Multiple Comparison Test was used to determine statistical differences between the experimental groups (PrismÒ, GraphPad Software, Inc., La Jolla, CA, USA).
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RESULTS Effects of NAC and ALC on the early reaction at the SCI lesion site In order to evaluate the early neuroprotective effects of NAC and ALC treatment on the local tissue reaction, spinal cord cervical level tissue harvested after 2 weeks was analyzed for the expression of apoptotic markers Bcell lymphoma 2 (BCL-2)-associated X protein (BAX) and caspase 3 using Western blotting. The samples representing each group (CONT, SCI, NAC, and ALC) were made from pooled samples created from tissue harvested from four to five animals. Treatment with NAC and ALC evidently decreased the expression of the proapoptotic protein BAX (Fig. 1). Both treatment with ALC and NAC decreased the levels of caspase 3 in the tissue, although the effects were not as pronounced as with the other markers investigated. (Fig. 1). Treatment however does not seem to exert an equally strong effect on the expression of caspase 3 as it does on the other markers that were investigated in this study. Further, Western blots also revealed an ameliorating effect of antioxidant treatment on the neuroinflammatory reaction. An apparent attenuation of the expression of both OX42 and ED1 was observed in the antioxidant-treated groups, compared with the untreated animals (Fig. 1). Neuroprotective effect of NAC and ALC To investigate the long-term effect of antioxidant treatment, the survival of rubrospinal neurons 8 weeks after SCI was studied. In the control group of uninjured animals 1 week after Fast Blue labeling, the rubrospinal neuronal pool contained 3533 ± 171 labeled cells (mean ± S.E.M., Fig. 2A, F). Eight weeks following SCI, only 61% of labeled rubrospinal neurons remained (Fig. 2B, F). Furthermore, numerous and small Fast
Blue-positive profiles resembling microglial cells were observed in the red nucleus (Fig. 2E). Treatment with ALC significantly ameliorated the cell death, resulting in an 88% neuron survival (P < 0.01; Fig. 2D, F). The neuroprotective effect of NAC was somewhat more modest with the survival of 80% of the neurons (P < 0.05; Fig. 2C, F). Also, an improved preservation of Fast Blue labeling in the proximal dendritic branches was found after ALC and NAC treatments (Fig. 2C, D). Effects of antioxidant treatment on axonal sprouting At 8 weeks after SCI, few 5HT-positive raphaespinal axons grew from the rostral spinal cord into the trauma zone (Fig. 3A, B). At 8 weeks after SCI followed by treatment with NAC and ALC, numerous raphaespinal axonal arborizations were found in the trauma zone, but they did not enter the distal part of the spinal cord (Fig. 3C–F). Quantitative analysis of the raphaespinal terminals in the ventral horn of the C2 segment rostral to the lesion site revealed that SCI induced a significant twofold increase of the area occupied by raphaespinal terminals (P < 0.05; Fig. 4I, A, B). Treatment with NAC and ALC further stimulated axonal sprouting rostral to the lesion site and resulted in a 4.5- and fivefold increase of the area of axonal terminals, respectively (P < 0.001; Fig. 4I, C, D). Effects of antioxidant treatment on the long-term neuroinflammatory response SCI induced a dramatic, almost 40-fold increase in the area occupied by OX42-positive microglia at 8 weeks postoperatively (P < 0.001; Fig. 4J, E, F). Treatment with antioxidants effectively attenuated the microglial response induced by SCI. Administration of both NAC and ALC decreased the OX42-immunoreactivity by 70% (P < 0.001; Fig. 4J, G, H).
DISCUSSION
Fig. 1. Western blots for BAX, Caspase-3, OX-42 and ED1 in the C3 spinal cord segment rostral to the lesion site. Each protein lysate representing an experimental group was produced by combining tissue from four to five animals. Representative Western blots are shown for the normal uninjured rats (CONT), at 2 weeks after C3 spinal cord injury (SCI) and after SCI followed by treatment with Nacetyl-cysteine (NAC) and acetyl-L-carnitine (ALC).
This study shows that treatment with NAC and ALC produces an anti-apoptotic effect in the segments rostral to the injury site, and has a neuroprotective effect on the descending rubrospinal neurons. The results of the study also demonstrate the regenerative properties of ALC and NAC, with an increased sprouting of the descending raphaespinal fibers. Furthermore, an effect on the severity and profile of the neuroinflammatory response is shown, with a particular decreased expression of activated microglia. The attenuating effect on the microglial response was persistent over the two chosen time points. It is well established that SCI causes death of neurons and glial cells in the vicinity of the trauma zone (Zhang et al., 2012) as well as of motor neurons proximal to the injury site, belonging to descending motor pathways (Deumens et al., 2005). However, using immunostaining for neuron-specific protein NeuN, which is present in most CNS and PNS neuronal cell bodies, others have reported that neurons in the red nucleus undergo atrophy rather than cell death following SCI (Kwon et al., 2002). Since
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Fig. 2. Transverse sections through the midbrain (A–E) and histogram (F) showing survival of Fast Blue-labeled neurons in the red nucleus of the normal uninjured rats (A; CONT; n = 8), at 8 weeks after C3 spinal cord injury (B; SCI; n = 10) and after SCI followed by treatment with N-acetylcysteine (C; NAC; n = 5) and acetyl-L-carnitine (D; ALC; n = 5). Arrows in (E) indicate Fast-Blue-loaded microglial cells. Scale bar = 200 lm.
the red nucleus contains about 23,000 neurons (Paxinos, 1995), and only about 3500–4000 of them send their axons to the spinal cord (Novikov et al., 2002; Novikova et al., 2000), even a 50% loss among Fast Blue-labeled rubrospinal neurons will correspond to the less than 10% total cell loss in the red nucleus. Although some of the cells close to the injury site will inevitably perish due to necrosis soon after the initial trauma, the extensive and prolonged cell death following is caused by apoptosis (Donnelly and Popovich, 2008). The complex mechanisms of apoptosis are known to be regulated by, among others, the pro-apoptotic protein BAX and the anti-apoptotic protein BCL-2. These antagonistic proteins are known to control the downstream effector caspases, in particular caspase 3 (Adams and Cory, 1998; Rudel, 1999). Caspase 3, in turn, plays a central role in the induction of apoptosis in general (Porter and Janicke, 1999; Snigdha et al., 2012), and not least in the nervous system (Springer et al., 2001) where caspase
inhibitors have been shown to prevent neural loss (Charriaut-Marlangue, 2004). Both BAX and caspase 3 are well known to be up-regulated early after SCI while an increased ratio of BCL-2/BAX is generally associated with increased survival of cells (Fan et al., 2006; Hou et al., 2003; Song et al., 2012; Vukojevic et al., 2008; Yin et al., 2012). More specifically, mitochondrial impairment has been linked with the activation of caspase 3 as well as other pro-apoptotic proteins (Hagberg, 2004). In accordance, our study demonstrated that the antioxidants ALC and NAC, acting as promoters of mitochondrial function, decreased the production of the pro-apoptotic BAX and caspase 3. The attenuating effect on the activation of death-promoting pathways may at least in part account for the increased survival of cells after SCI, seen 8 weeks after initial trauma. This indicates continuous beneficial anti-apoptotic effects of antioxidant treatment on motorneurons belonging to the important descending motor pathways. As such, antioxidants may have a legit-
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Fig. 3. Horizontal sections through the C3 spinal cord segment showing raphaespinal 5HT-positive axons in the trauma zone at 8 weeks after C3 spinal cord injury (SCI) and after SCI followed by treatment with N-acetyl-cysteine (NAC) and acetyl-L-carnitine (ALC). Dotted line indicates the rostral border of the trauma zone. Boxed areas in A, C and E are enlarged in B, D and F, respectively. Scale bars = 100 lm (A, C, E) and 25 lm (B, D, F).
imate place as anti-apoptotic agents in the treatment of SCI. At 8 weeks after initial injury, an increase in the sprouting of the raphaespinal fibers belonging to the descending pathways was seen. Treatment with NAC and ALC induced an ingrowth of these axons in the trauma zone. Indeed, treatment with ALC 4 weeks after SCI on the L1/L2 segments has recently been shown to help preserve the locomotor circuitry rostral to the injury site, and induce functional improvement of long-term hind-limb function in rats (Patel et al., 2012). These findings raise hope that treatment with antioxidants may contribute to the development of a novel neural pathway that could play a role in the functional recovery after SCI. The reactive neuroinflammatory response commences at a very early time point after initial SCI, and is always associated with the activation of microglial cells. Additionally, an influx of monocytes, with the potential of differentiating into ED1-positive macrophages, occurs over the following days (Benowitz and Popovich, 2011; Donnelly and Popovich, 2008). This study shows that
treatment with ALC and NAC immediately after the induction of SCI reduces the general presence of microglia at an early time point (2 weeks) around the trauma site. Members of the microglial cell population, however, exhibit different phenotypical and functional features, with some secreting pro-inflammatory factors while others produce neurotrophic factors. Our findings indicate an early antiinflammatory effect targeting activated ED1-positive microglia, which have been associated with cytotoxic properties (Kullberg et al., 2001). It is appealing to speculate that a reduction of microglial cells could be associated with a reduction of microglia bearing neurotoxic properties. Further immunohistochemical investigation of the trauma zone at the later time point (8 weeks) revealed that antioxidant treatment exerts a strong and persisting long-term effect on the microglial activity. These findings indicate the potential of NAC and ALC as therapeutic compounds, exhibiting a long-term effect on the neuroinflammatory response after SCI. While we have previously shown this effect in short-term 4-week experiments (Karalija et al., 2012), this study is to our
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Fig. 4. Transverse sections through the C2 cervical spinal segments (A–H) and histograms (I, J) showing sprouting of 5HT-positive raphaespinal terminals (A–D) and the density of OX42-immunolabeled microglial cells in C2 cervical ventral horn (E–H) of the normal uninjured rats (CONT; n = 4), at 8 weeks after C3 spinal cord injury (SCI; n = 5) and after SCI followed by treatment with N-acetyl-cysteine (NAC; n = 4) and acetyl-Lcarnitine (ALC; n = 4). Error bars show S.E.M. P < 0.05 is indicated by *, P < 0.01 is indicated by ** and P < 0.001 is indicated by ***. Scale bars = 20 lm (A–D) and 100 lm (E–H).
knowledge the first study describing the long-term effect of NAC and ALC treatment on the neuroinflammatory response. The two antioxidants investigated in this study share some biological features, but also have some unique properties. Both NAC and ALC promote the creation of glutathione (Atkuri et al., 2007; McEwen et al., 2011), and as such contribute to the creation of ROS scavenging enzymes (Circu and Aw, 2008). ALC is also important for the transport of fatty acids into the mitochondria, thus helping to sustain the b-oxidation and subsequent ATP production (Ribas et al., 2014). Additionally, ALC also possess the ability to enter the citric acid cycle and, acting as an energy substrate, provide the mitochondria with an alternative source of energy after SCI (Patel et al., 2010). Due to the similarities in effect on neuroinflammation and neuronal survival, additional beneficial effects of a combinational treatment may be possible. The two antioxidants, however, do not share the same optimal pH. Further, according to the manufacturers of ALC and NAC, both molecules are highly sensitive and unstable. The result of mixing ALC and NAC in the same solution is not known, and may affect the efficacy of the drug(s). For these reasons the possible benefits of a combinational treatment of ALC and NAC, present in the same pump, were not investigated in this study.
Novel and intriguing pharmacological approaches in the treatment of SCI are emerging (Priestley et al., 2012; Silva et al., 2014). Several antioxidants have lately been used to target the mitochondrial dysfunction and oxidative stress component of SCI, with promising results (Bains and Hall, 2012; Jia et al., 2012; McEwen et al., 2011). Although addressing a part of the pathophysiological events following SCI most probably will not provide the final remedy, the antioxidants may prove valuable as part of a combinational treatment. In the light of our findings concerning the anti-inflammatory and neuroprotective properties of NAC and ALC, both antioxidants are interesting candidates for further investigation.
CONCLUSION This study demonstrates the anti-apoptotic, antiinflammatory and pro-regenerative effects of NAC and ALC treatment after injury to the spinal cord. These effects are observed at an early point after initial trauma and also persist over a period of several weeks postoperatively. In our experience, NAC and ALC seem to exert similar positive effects on neuroinflammation and neuronal survival after SCI. Both antioxidants may have a place in the future pharmacological management of SCI.
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CONFLICTS OF INTEREST None. Acknowledgments—We would like to thank Mrs G Folkesson and Mrs G Hellstro¨m for their excellent technical assistance. This study was supported by the Swedish Medical Research Council, European Union, Umea˚ University, County of Va¨sterbotten, A˚ke Wibergs Stiftelse, Magn. Bergvalls Stiftelse, Clas Groschinskys Minnesfond and the Gunvor and Josef Aner Foundation.
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(Accepted 19 March 2014) (Available online 27 March 2014)
THE EFFECTS OF N-ACETYL-CYSTEINE AND ACETYL-L-CARNITINE ON NEURAL SURVIVAL, NEUROINFLAMMATION AND REGENERATION FOLLOWING SPINAL CORD INJURY A. KARALIJA, a,b* L. N. NOVIKOVA, a P. J. KINGHAM, a M. WIBERG a,b AND L. N. NOVIKOV a Key words: adult rats, spinal cord, motoneurons, antioxidants, apoptosis.
a Department of Integrative Medical Biology, Section of Anatomy, Umea˚ University, SE-901 87 Umea˚, Sweden b Department of Surgical and Perioperative Science, Section of Hand and Plastic Surgery, Umea˚ University, SE-901 87 Umea˚, Sweden
INTRODUCTION Although traumatic spinal cord injury (SCI) is highly relevant both clinically and epidemiologically, to date, there is no safe and effective enough treatment which is recognized in clinical practice (Rabchevsky et al., 2011; Silva et al., 2014). The previously prevalent use of highdose glucocorticoid treatment, considered to mediate a part of its effect by acting as an antioxidant (Jia et al., 2012), has fallen out of favor due to lack of a convincing clinical effect coupled with suspected increased morbidity and mortality, and is no longer commonly approved (Bydon et al., 2013; Sayer et al., 2006; Short et al., 2000). The inevitable trauma-induced loss of axonal continuity, although debilitating, only accounts for a part of the neurological deficits experienced, and a complete transection of the spinal cord is uncommon since the initial trauma, usually leaves parts of the white matter intact. It is rather the secondary reaction, commencing only minutes after the initial trauma that causes a progressive degradation of the spinal cord (Hall and Springer, 2004). This much studied but poorly understood process is hallmarked by death of neurons and glial cells (Zhang et al., 2012), mitochondrial dysfunction and production of reactive oxygen species (ROS) (Jia et al., 2012; Maragos and Korde, 2004), and a neuroinflammatory response involving, among other cells, the activation of microglia (Donnelly and Popovich, 2008). The events of the secondary reaction lead to the formation of a cavity at the site of the trauma with an eventual astroglial scar formation, preventing the regeneration of axons through the lesion site (Karimi-Abdolrezaee and Billakanti, 2012). Two substances that have lately emerged as potential neuroprotectants and modulators of the neuroinflammatory response are the antioxidants acetylL-carnitine (ALC) and N-acetyl-cysteine (NAC). Both readily cross the blood–brain barrier (Farr et al., 2003; Parnetti et al., 1992) and both have been successfully used in clinical practice (Malaguarnera, 2012; Santos et al., 2011; Waring, 2012). ALC is a small peptide found in the mitochondria, containing a carnitine moiety and an acetyl moiety. The former plays an important role in the oxidation of fatty acids, while the latter contributes to the
Abstract—Traumatic spinal cord injury induces a longstanding inflammatory response in the spinal cord tissue, leading to a progressive apoptotic death of spinal cord neurons and glial cells. We have recently demonstrated that immediate treatment with the antioxidants N-acetyl-cysteine (NAC) and acetyl-L-carnitine (ALC) attenuates neuroinflammation, induces axonal sprouting, and reduces the death of motoneurons in the vicinity of the trauma zone 4 weeks after initial trauma. The objective of the current study was to investigate the effects of long-term antioxidant treatment on the survival of descending rubrospinal neurons after spinal cord injury in rats. It also examines the short- and long-term effects of treatment on apoptosis, inflammation, and regeneration in the spinal cord trauma zone. Spinal cord hemisection performed at the level C3 induced a significant loss of rubrospinal neurons 8 weeks after injury. At 2 weeks, an increase in the expression of the apoptosis-associated markers BCL-2-associated X protein (BAX) and caspase 3, as well as the microglial cell markers OX42 and ectodermal dysplasia 1 (ED1), was seen in the trauma zone. After 8 weeks, an increase in immunostaining for OX42 and the serotonin marker 5HT was detected in the same area. Antioxidant therapy reduced the loss of rubrospinal neurons by approximately 50%. Treatment also decreased the expression of BAX, caspase 3, OX42 and ED1 after 2 weeks. After 8 weeks, treatment decreased immunoreactivity for OX42, whereas it was increased for 5HT. In conclusion, this study provides further insight in the effects of treatment with NAC and ALC on descending pathways, as well as short- and long-term effects on the spinal cord trauma zone. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.
*Correspondence to: A. Karalija, Department of Integrative Medical Biology, Umea˚ University, SE-901 87 Umea˚, Sweden. Tel: +46703234219. E-mail address: [email protected] (A. Karalij. Abbreviations: 5HT, serotonin; ALC, acetyl-L-carnitine; BAX, BCL-2associated X protein; BCL-2, B-cell lymphoma 2; C3bi, complement receptor type 3; DAPI, 4’,6-diamidino-2-phenylindole; DPX, dibutyl phathalate xylene; DRG, dorsal root ganglia; ED1, ectodermal dysplasia 1; EGTA, ethylene glycol tetraacetic acid; NAC, N-acetylcysteine; PIPES, piperazine-1,4-bis-2-ethanesulfonic acid; ROS, reactive oxygen species; SCI, spinal cord injury. http://dx.doi.org/10.1016/j.neuroscience.2014.03.042 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 143
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maintenance of acetyl-CoA levels and production of the anti-oxidant glutathione. ALC has been successfully used in clinical trials for the treatment of neuropathy and neuropathic pain, cognitive disorders and depression (Malaguarnera, 2012). It has also recently been found to contribute to the maintenance of mitochondrial bioenergetics following SCI in rats (Patel et al., 2010). NAC, a derivative of cysteine, similarly exerts a wide range of cellular actions, among others acting as a glutathione precursor (Atkuri et al., 2007) and exhibiting anti-inflammatory actions (Palacio et al., 2011). NAC has been approved for clinical use for many years, being used as a mucolytic agent and for treatment of acetaminophen intoxication (Heard, 2008; Sadowska, 2012; Ziment, 1988). We have previously demonstrated that treatment with NAC and ALC can delay the degeneration of sensory neurons in the dorsal root ganglia (DRG) after peripheral nerve injury, and greatly reduce early retrograde death of spinal motorneurons after ventral root avulsion (Hart et al., 2002; Welin et al., 2009; Wilson et al., 2007; Zhang et al., 2005). In our recent short-term study we have shown that NAC and ALC also attenuate degeneration of spinal motoneurons after lumbar SCI, reduce the activity of microglia and macrophages and promote axonal sprouting in the injured segment of the cord (Karalija et al., 2012). The primary goal of this study is to investigate the long-term effects of NAC and ALC treatment on the survival of descending rubrospinal neurons after cervical SCI and to evaluate the efficacy of antioxidant treatment on apoptotic, inflammatory and regenerative reactions in the trauma zone.
EXPERIMENTAL PROCEDURES Experimental animals The experiments were performed on adult (10–12 weeks, n = 60) female Sprague–Dawley rats (Taconic Europe A/ S, Denmark). The animal care and experimental procedures were carried out in accordance with Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes and also approved by the Northern Swedish Committee for Ethics in Animal Experiments (No. A3612). All surgical procedures were performed under general anesthesia using a mixture of ketamine (KetalarÒ, Parke-Davis, Pfizer, New York, NY, USA; 100 mg/kg i.v.) and xylazine (RompunÒ, Bayer, Leverkusen, Germany; 10 mg/kg i.v.). After surgery, the rats were given the analgesic Finadyne (ScheringPlough, Denmark; 2.5 mg/kg, s.c.), normal saline (4 ml, s.c.) and benzylpenicillin (Boehringer Ingelheim, Ingelheim am Rhein, Germany; 60 mg, i.m.). Each animal was housed alone in a cage after surgery and exposed to 12-h light/dark cycles, with free access to food and water. SCI Following cervical laminectomy a stab wound was inflicted by inserting a 23-G needle in the dorsal root entry zone of the C3 spinal cervical segment. The
blade of a pair of Vannas spring scissors was then introduced in the perforation and, using the other blade of the scissors, a hemisection of the spinal cord was performed. In the experiments dealing with neuronal survival, rubrospinal neurons were labeled with the non-toxic fluorescent retrograde tracer Fast Blue immediately after SCI. A small pellet prepared from 1 to 2 ll of a 2% aqueous solution of the Fast Blue (FB, EMS-Chemie GmbH, Germany) was placed into the lesion cavity. The opened dura mater was covered with a piece of stretched parafilm and SpongostanÒ. The operation was finished with the closing of the muscles and skin respectively. After the operation, the animals were randomized into one of the following three groups: (i) spinal cord injury without treatment (SCI, n = 18), (ii) SCI and treatment with NAC (n = 13) and (iii) SCI and treatment with ALC (n = 13). Eight uninjured normal rats (used for Western blotting and immunohistochemistry) as well as eight rats at 1 week after Fast Blue labeling served as baseline controls. Treatment with NAC and ALC The treatment with NAC and ALC commenced immediately after performing the SCI by the subcutaneous implantation of an Alzet 2002 osmotic minipump (Alza Corp., Palo Alto, CA, USA) filled with a solution of either the L-stereoisomer of NAC (200 mg/ml; BioPhausia, Stockholm, Sweden) or O-acetyl-L-carnitine hydrochloride (75 mg/ml in normal saline; Sigma–Aldrich, St. Louis, MO, USA) in the neck region. Following L6 laminectomy, a subcutaneous polyethylene catheter (Intermedic, Barcelona, Spain, PE-60) filled with NAC or ALC was inserted into the lower lumbar subarachnoid space with the tip reaching the level of L3–L4 DRG. The tube was fixed to the S1 vertebral bone by HistoacrylÒ glue, the insertion site covered with SpongostanÒ and the catheter secured in place by suturing it to the muscles. The proximal part of the catheter was connected to the osmotic pump, commencing the treatment. The infusion speed corresponded to 2.4 mg/ day for NAC and 0.9 mg/day for ALC, doses derived from previous studies (Karalija et al., 2012). Following 14 days of treatment the empty pump was exchanged for a fresh one containing the same solution. The change of pump was performed every 14 days until the end of the study. The animals were sacrificed at 2 and 8 weeks postoperatively. Tissue processing Following the experiments all animals were euthanized by administering an intraperitoneal overdose of pentobarbital (240 mg/kg, Apoteksbolaget, Sverige). The animals intended for Western blotting underwent harvest of the tissue caudal and rostral to the injury site 2 weeks after initial trauma. The tissue was divided in two halves in the sagittal plane and these two pieces were then divided transversely, separating the tissue rostral to the injury site from the caudal part. The tissue was immediately frozen in liquid nitrogen. The rest of the animals were transcardially perfused using Tyrode’s
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solution followed by 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (pH 7.4). Following the perfusion, the brain stem and the spinal cord segments C1–C3 were harvested and post-fixed in the same paraformaldehyde solution for 2–4 h, after which the tissue underwent cryoprotection in 10% and 20% sucrose for 3 days, and was then frozen in liquid isopentane. The tissue intended for counts of Fast Bluelabeled rubrospinal neurons was cut in 50-lm-thick serial sections on a vibratome (Leica Instruments, Wetzlar, Germany), mounted on gelatin-coated glass slides, air dried, briefly immersed in xylene and coverslipped in dibutyl phathalate xylene (DPX). For immunohistochemical analysis the tissue was cut in 16lm-thick serial sections on a cryomicrotome (Leica Instruments, Wetzlar, Germany), thaw-mounted onto SuperFrostÒPlus slides, dried overnight at room temperature and stored at 85 °C before being processed. Counts of the Fast Blue labeled neurons Counting the nuclear profiles of labeled rubrospinal neurons was performed in all sections through the brainstem using 250 magnification. The total number of profiles was not corrected for split nuclei, since the nuclear diameters were small in comparison with the section thickness used. We have previously demonstrated that the accuracy of this counting technique in estimation of retrograde neuronal cell death is similar to that obtained with the physical dissector method (Ma et al., 2001) and counts of neurons reconstructed from serial sections (Novikov et al., 1997). Immunohistochemistry The serial tissue sections were processed for demonstration of glial cell and neuron markers. After blocking with normal serum, mouse antibodies reacting with C3bi complement receptors (OX42; 1:250, Serotec, Oxford, UK) and rabbit anti-serotonin antibodies (5-HT; 1:500; Sigma–Aldrich, St. Louis, MO, USA) were applied for 2 h at room temperature. After rinsing in PBS, secondary goat anti-mouse and goat anti-rabbit antibodies Alexa FluorÒ 488 and Alexa FluorÒ 568 (1:300; Molecular Probes, Eugene, OR, USA, Invitrogen, Carslsbad, CA, USA) were applied for 1 h at room temperature in the dark. The slides were coverslipped with ProLong mounting media containing 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carslsbad, CA, USA). The staining specificity was tested by omission of primary antibodies.
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quantify the protein level in the samples. Four to five animals per group were combined to create each lysate for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis. An amount of 20 lg of protein was denaturated at 90 °C and, depending on molecular weight, loaded on a 10% or 15% SDS–PAGE. Following transfer to a nitrocellulose membrane the blots were blocked using 5% (w/v) non-fat milk or 1% bovine serum albumin (BSA) (w/v) in Tris buffer saline with Tween (TBST). Following the blocking the blots were incubated with rabbit anti-caspase 3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-BAX antibody (1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), mouse anti-OX42 antibody (1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), mouse anti-ED1 antibody (1:300; Abcam, Cambridge, UK) and the loading control rabbit anti-beta tubulin (1:5000; Abcam, Cambridge, United Kingdom) overnight at 4 °C. After washing the blots in TBS-T 6 5 min they were incubated with mouse or rabbit IgG HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 h. Another series of 6 5-min washes in TBS-T was performed after which the blots were exposed to ECL reagent (GE Healthcare, Little Chalfont, UK) for 1 min and then finally developed onto Kodak XPS films. The developed films were scanned using an Epson Photoscanner.
Quantification of microglial cell reaction and axonal sprouting 5HT-positive raphaespinal axonal arborizations and OX42-positive microglial cells were quantified in the ventral horn of the C2 spinal segments at 8 weeks after SCI. All images were captures at 400 final magnification with a Nikon DS-U2 digital camera. The relative tissue area occupied by immunostained profiles was quantified in five randomly selected sections from each rat in 50 50-lm areas (18.9 pixels per 1-lm tissue length) for 5HT-positive axons and 150 150-lm areas (3.8 pixels per 1-lm tissue length) for microglial cells using Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA).
Image processing For preparation of figures, the captured images were resized, grouped into a single canvas and labeled using Adobe Photoshop CS4 software. Contrast and brightness were adjusted to provide optimal clarity.
Western blotting The spinal cord tissue was homogenized using buffer (pH 6.9) containing 5 mM EGTA, 100 mM piperazine-1,4-bis2-ethanesulfonic acid (PIPES), 5 mM MgCl2, 20% (v/v) glycerol, 0.5% (v/v) Triton X-100 and protease inhibitor cocktail (Sigma). A detergent compatible protein assay (Bio-Rad, Hercules, CA, USA,) was performed in order to
Statistical analysis One-way analysis of variance (ANOVA) followed by a post hoc Newman–Keuls Multiple Comparison Test was used to determine statistical differences between the experimental groups (PrismÒ, GraphPad Software, Inc., La Jolla, CA, USA).
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RESULTS Effects of NAC and ALC on the early reaction at the SCI lesion site In order to evaluate the early neuroprotective effects of NAC and ALC treatment on the local tissue reaction, spinal cord cervical level tissue harvested after 2 weeks was analyzed for the expression of apoptotic markers Bcell lymphoma 2 (BCL-2)-associated X protein (BAX) and caspase 3 using Western blotting. The samples representing each group (CONT, SCI, NAC, and ALC) were made from pooled samples created from tissue harvested from four to five animals. Treatment with NAC and ALC evidently decreased the expression of the proapoptotic protein BAX (Fig. 1). Both treatment with ALC and NAC decreased the levels of caspase 3 in the tissue, although the effects were not as pronounced as with the other markers investigated. (Fig. 1). Treatment however does not seem to exert an equally strong effect on the expression of caspase 3 as it does on the other markers that were investigated in this study. Further, Western blots also revealed an ameliorating effect of antioxidant treatment on the neuroinflammatory reaction. An apparent attenuation of the expression of both OX42 and ED1 was observed in the antioxidant-treated groups, compared with the untreated animals (Fig. 1). Neuroprotective effect of NAC and ALC To investigate the long-term effect of antioxidant treatment, the survival of rubrospinal neurons 8 weeks after SCI was studied. In the control group of uninjured animals 1 week after Fast Blue labeling, the rubrospinal neuronal pool contained 3533 ± 171 labeled cells (mean ± S.E.M., Fig. 2A, F). Eight weeks following SCI, only 61% of labeled rubrospinal neurons remained (Fig. 2B, F). Furthermore, numerous and small Fast
Blue-positive profiles resembling microglial cells were observed in the red nucleus (Fig. 2E). Treatment with ALC significantly ameliorated the cell death, resulting in an 88% neuron survival (P < 0.01; Fig. 2D, F). The neuroprotective effect of NAC was somewhat more modest with the survival of 80% of the neurons (P < 0.05; Fig. 2C, F). Also, an improved preservation of Fast Blue labeling in the proximal dendritic branches was found after ALC and NAC treatments (Fig. 2C, D). Effects of antioxidant treatment on axonal sprouting At 8 weeks after SCI, few 5HT-positive raphaespinal axons grew from the rostral spinal cord into the trauma zone (Fig. 3A, B). At 8 weeks after SCI followed by treatment with NAC and ALC, numerous raphaespinal axonal arborizations were found in the trauma zone, but they did not enter the distal part of the spinal cord (Fig. 3C–F). Quantitative analysis of the raphaespinal terminals in the ventral horn of the C2 segment rostral to the lesion site revealed that SCI induced a significant twofold increase of the area occupied by raphaespinal terminals (P < 0.05; Fig. 4I, A, B). Treatment with NAC and ALC further stimulated axonal sprouting rostral to the lesion site and resulted in a 4.5- and fivefold increase of the area of axonal terminals, respectively (P < 0.001; Fig. 4I, C, D). Effects of antioxidant treatment on the long-term neuroinflammatory response SCI induced a dramatic, almost 40-fold increase in the area occupied by OX42-positive microglia at 8 weeks postoperatively (P < 0.001; Fig. 4J, E, F). Treatment with antioxidants effectively attenuated the microglial response induced by SCI. Administration of both NAC and ALC decreased the OX42-immunoreactivity by 70% (P < 0.001; Fig. 4J, G, H).
DISCUSSION
Fig. 1. Western blots for BAX, Caspase-3, OX-42 and ED1 in the C3 spinal cord segment rostral to the lesion site. Each protein lysate representing an experimental group was produced by combining tissue from four to five animals. Representative Western blots are shown for the normal uninjured rats (CONT), at 2 weeks after C3 spinal cord injury (SCI) and after SCI followed by treatment with Nacetyl-cysteine (NAC) and acetyl-L-carnitine (ALC).
This study shows that treatment with NAC and ALC produces an anti-apoptotic effect in the segments rostral to the injury site, and has a neuroprotective effect on the descending rubrospinal neurons. The results of the study also demonstrate the regenerative properties of ALC and NAC, with an increased sprouting of the descending raphaespinal fibers. Furthermore, an effect on the severity and profile of the neuroinflammatory response is shown, with a particular decreased expression of activated microglia. The attenuating effect on the microglial response was persistent over the two chosen time points. It is well established that SCI causes death of neurons and glial cells in the vicinity of the trauma zone (Zhang et al., 2012) as well as of motor neurons proximal to the injury site, belonging to descending motor pathways (Deumens et al., 2005). However, using immunostaining for neuron-specific protein NeuN, which is present in most CNS and PNS neuronal cell bodies, others have reported that neurons in the red nucleus undergo atrophy rather than cell death following SCI (Kwon et al., 2002). Since
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Fig. 2. Transverse sections through the midbrain (A–E) and histogram (F) showing survival of Fast Blue-labeled neurons in the red nucleus of the normal uninjured rats (A; CONT; n = 8), at 8 weeks after C3 spinal cord injury (B; SCI; n = 10) and after SCI followed by treatment with N-acetylcysteine (C; NAC; n = 5) and acetyl-L-carnitine (D; ALC; n = 5). Arrows in (E) indicate Fast-Blue-loaded microglial cells. Scale bar = 200 lm.
the red nucleus contains about 23,000 neurons (Paxinos, 1995), and only about 3500–4000 of them send their axons to the spinal cord (Novikov et al., 2002; Novikova et al., 2000), even a 50% loss among Fast Blue-labeled rubrospinal neurons will correspond to the less than 10% total cell loss in the red nucleus. Although some of the cells close to the injury site will inevitably perish due to necrosis soon after the initial trauma, the extensive and prolonged cell death following is caused by apoptosis (Donnelly and Popovich, 2008). The complex mechanisms of apoptosis are known to be regulated by, among others, the pro-apoptotic protein BAX and the anti-apoptotic protein BCL-2. These antagonistic proteins are known to control the downstream effector caspases, in particular caspase 3 (Adams and Cory, 1998; Rudel, 1999). Caspase 3, in turn, plays a central role in the induction of apoptosis in general (Porter and Janicke, 1999; Snigdha et al., 2012), and not least in the nervous system (Springer et al., 2001) where caspase
inhibitors have been shown to prevent neural loss (Charriaut-Marlangue, 2004). Both BAX and caspase 3 are well known to be up-regulated early after SCI while an increased ratio of BCL-2/BAX is generally associated with increased survival of cells (Fan et al., 2006; Hou et al., 2003; Song et al., 2012; Vukojevic et al., 2008; Yin et al., 2012). More specifically, mitochondrial impairment has been linked with the activation of caspase 3 as well as other pro-apoptotic proteins (Hagberg, 2004). In accordance, our study demonstrated that the antioxidants ALC and NAC, acting as promoters of mitochondrial function, decreased the production of the pro-apoptotic BAX and caspase 3. The attenuating effect on the activation of death-promoting pathways may at least in part account for the increased survival of cells after SCI, seen 8 weeks after initial trauma. This indicates continuous beneficial anti-apoptotic effects of antioxidant treatment on motorneurons belonging to the important descending motor pathways. As such, antioxidants may have a legit-
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Fig. 3. Horizontal sections through the C3 spinal cord segment showing raphaespinal 5HT-positive axons in the trauma zone at 8 weeks after C3 spinal cord injury (SCI) and after SCI followed by treatment with N-acetyl-cysteine (NAC) and acetyl-L-carnitine (ALC). Dotted line indicates the rostral border of the trauma zone. Boxed areas in A, C and E are enlarged in B, D and F, respectively. Scale bars = 100 lm (A, C, E) and 25 lm (B, D, F).
imate place as anti-apoptotic agents in the treatment of SCI. At 8 weeks after initial injury, an increase in the sprouting of the raphaespinal fibers belonging to the descending pathways was seen. Treatment with NAC and ALC induced an ingrowth of these axons in the trauma zone. Indeed, treatment with ALC 4 weeks after SCI on the L1/L2 segments has recently been shown to help preserve the locomotor circuitry rostral to the injury site, and induce functional improvement of long-term hind-limb function in rats (Patel et al., 2012). These findings raise hope that treatment with antioxidants may contribute to the development of a novel neural pathway that could play a role in the functional recovery after SCI. The reactive neuroinflammatory response commences at a very early time point after initial SCI, and is always associated with the activation of microglial cells. Additionally, an influx of monocytes, with the potential of differentiating into ED1-positive macrophages, occurs over the following days (Benowitz and Popovich, 2011; Donnelly and Popovich, 2008). This study shows that
treatment with ALC and NAC immediately after the induction of SCI reduces the general presence of microglia at an early time point (2 weeks) around the trauma site. Members of the microglial cell population, however, exhibit different phenotypical and functional features, with some secreting pro-inflammatory factors while others produce neurotrophic factors. Our findings indicate an early antiinflammatory effect targeting activated ED1-positive microglia, which have been associated with cytotoxic properties (Kullberg et al., 2001). It is appealing to speculate that a reduction of microglial cells could be associated with a reduction of microglia bearing neurotoxic properties. Further immunohistochemical investigation of the trauma zone at the later time point (8 weeks) revealed that antioxidant treatment exerts a strong and persisting long-term effect on the microglial activity. These findings indicate the potential of NAC and ALC as therapeutic compounds, exhibiting a long-term effect on the neuroinflammatory response after SCI. While we have previously shown this effect in short-term 4-week experiments (Karalija et al., 2012), this study is to our
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Fig. 4. Transverse sections through the C2 cervical spinal segments (A–H) and histograms (I, J) showing sprouting of 5HT-positive raphaespinal terminals (A–D) and the density of OX42-immunolabeled microglial cells in C2 cervical ventral horn (E–H) of the normal uninjured rats (CONT; n = 4), at 8 weeks after C3 spinal cord injury (SCI; n = 5) and after SCI followed by treatment with N-acetyl-cysteine (NAC; n = 4) and acetyl-Lcarnitine (ALC; n = 4). Error bars show S.E.M. P < 0.05 is indicated by *, P < 0.01 is indicated by ** and P < 0.001 is indicated by ***. Scale bars = 20 lm (A–D) and 100 lm (E–H).
knowledge the first study describing the long-term effect of NAC and ALC treatment on the neuroinflammatory response. The two antioxidants investigated in this study share some biological features, but also have some unique properties. Both NAC and ALC promote the creation of glutathione (Atkuri et al., 2007; McEwen et al., 2011), and as such contribute to the creation of ROS scavenging enzymes (Circu and Aw, 2008). ALC is also important for the transport of fatty acids into the mitochondria, thus helping to sustain the b-oxidation and subsequent ATP production (Ribas et al., 2014). Additionally, ALC also possess the ability to enter the citric acid cycle and, acting as an energy substrate, provide the mitochondria with an alternative source of energy after SCI (Patel et al., 2010). Due to the similarities in effect on neuroinflammation and neuronal survival, additional beneficial effects of a combinational treatment may be possible. The two antioxidants, however, do not share the same optimal pH. Further, according to the manufacturers of ALC and NAC, both molecules are highly sensitive and unstable. The result of mixing ALC and NAC in the same solution is not known, and may affect the efficacy of the drug(s). For these reasons the possible benefits of a combinational treatment of ALC and NAC, present in the same pump, were not investigated in this study.
Novel and intriguing pharmacological approaches in the treatment of SCI are emerging (Priestley et al., 2012; Silva et al., 2014). Several antioxidants have lately been used to target the mitochondrial dysfunction and oxidative stress component of SCI, with promising results (Bains and Hall, 2012; Jia et al., 2012; McEwen et al., 2011). Although addressing a part of the pathophysiological events following SCI most probably will not provide the final remedy, the antioxidants may prove valuable as part of a combinational treatment. In the light of our findings concerning the anti-inflammatory and neuroprotective properties of NAC and ALC, both antioxidants are interesting candidates for further investigation.
CONCLUSION This study demonstrates the anti-apoptotic, antiinflammatory and pro-regenerative effects of NAC and ALC treatment after injury to the spinal cord. These effects are observed at an early point after initial trauma and also persist over a period of several weeks postoperatively. In our experience, NAC and ALC seem to exert similar positive effects on neuroinflammation and neuronal survival after SCI. Both antioxidants may have a place in the future pharmacological management of SCI.
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CONFLICTS OF INTEREST None. Acknowledgments—We would like to thank Mrs G Folkesson and Mrs G Hellstro¨m for their excellent technical assistance. This study was supported by the Swedish Medical Research Council, European Union, Umea˚ University, County of Va¨sterbotten, A˚ke Wibergs Stiftelse, Magn. Bergvalls Stiftelse, Clas Groschinskys Minnesfond and the Gunvor and Josef Aner Foundation.
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(Accepted 19 March 2014) (Available online 27 March 2014)