Experimental Neurology 237 (2012) 260–266 Contents lists available at SciVerse ScienceDirect Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr Regular Article Meteorin reverses hypersensitivity in rat models of neuropathic pain Jesper Roland Jørgensen a,⁎, Xiao-Jun Xu b, H. Moore Arnold c, Gordon Munro d, Jing-Xia Hao b, Blake Pepinsky c, Carol Huang c, Bang Jian Gong c, Zsuzsanna Wiesenfeld-Hallin b, Lars U. Wahlberg a, Teit E. Johansen a a NsGene A/S, Baltorpvej 154, 2750 Ballerup, Denmark Department of Physiology and Pharmacology, Section of Integrative Pain Research, Karolinska Institutet, Stockholm, Sweden Biogen Idec, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA d Department of Pharmacology, NeuroSearch A/S, 93 Pederstrupvej, DK-2750, Ballerup, Denmark b c a r t i c l e i n f o Article history: Received 2 March 2012 Revised 22 May 2012 Accepted 24 June 2012 Available online 2 July 2012 Keywords: Meteorin Neuropathy Pain Hypersensitivity CCI Sciatic nerve a b s t r a c t Neuropathic pain is caused by a lesion or disease to the somatosensory nervous system and current treatment merely reduces symptoms. Here, we investigate the potential therapeutic effect of the neurotrophic factor Meteorin on multiple signs of neuropathic pain in two distinct rat models. In a first study, two weeks of intermittent systemic administration of recombinant Meteorin led to a dose-dependent reversal of established mechanical and cold hypersensitivity in rats after photochemically-induced sciatic nerve injury. Moreover, analgesic efficacy lasted for at least one week after treatment cessation. In rats with a chronic constriction injury (CCI) of the sciatic nerve, five systemic injections of Meteorin over 9 days dose-dependently reversed established mechanical and thermal hypersensitivity as well as weight bearing deficits taken as a surrogate marker of spontaneous pain. The beneficial effects of systemic Meteorin were sustained for at least three weeks after treatment ended and no adverse side effects were observed. Pharmacokinetic analysis indicated that plasma Meteorin exposure correlated well with dosing and was no longer detectable after 24 hours. This pharmacokinetic profile combined with a delayed time of onset and prolonged duration of analgesic efficacy on multiple parameters suggests a disease-modifying mechanism rather than symptomatic pain relief. In sciatic nerve lesioned rats, delivery of recombinant Meteorin by intrathecal injection was also efficacious in reversing mechanical and cold hypersensitivity. Together, these data demonstrate that Meteorin represents a novel treatment strategy for the effective and long lasting relief from the debilitating consequences of neuropathic pain. © 2012 Elsevier Inc. All rights reserved. Introduction Neuropathic pain can arise as a result of lesion to or disease within the somatosensory system and is associated with a diverse range of disease states including diabetes, cancer, autoimmunity, viral infections and stroke (Haanpaa et al., 2011; Treede et al., 2008). Patients typically present with any number of symptoms as typified by spontaneous pain, pain evoked by normally innocuous sensory stimuli (allodynia), or exacerbated pain in response to noxious stimuli (hyperalgesia). Multiple underlying mechanisms (e.g. nociceptor sensitization, local inflammation, ectopic discharges, loss of descending inhibitory controls, spinal disinhibition) contribute to the behavioral manifestation of neuropathic pain (Costigan et al., 2009). In the clinic, antiepileptic and antidepressant drugs form the first line of treatment, albeit they have proven to be at best, symptomatic and only partially effective. Abbreviations: CCI, chronic constriction injury; DRG, dorsal root ganglion; GDNF, glial cell line-derived neurotrophic factor; NGF, Nerve growth factor; SC, subcutaneous; SNL, spinal nerve ligation. ⁎ Corresponding author at: NsGene, Baltorpvej 154, 2750 Ballerup, Denmark. E-mail address: JRJ@NSGENE.DK (J.R. Jørgensen). 0014-4886/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2012.06.027 Neurotrophic factors are critical during development and for maintenance of the adult nervous system. Accordingly, molecules in this group are of clinical interest in relation to neurological disorders including neuropathic pain (Ossipov, 2011; Sah et al., 2005). During development, nerve growth factor (NGF) supports the survival of TrkA-receptor expressing peripheral sensory neurons. Thereafter, a proportion of these neurons lose responsiveness to NGF, instead becoming responsive to glial cell line-derived neurotrophic factor (GDNF). Both neurotrophic factors then continue to play an important role in the normal functioning of the adult nervous system (Pezet and McMahon, 2006). Although NGF has some neuroprotective properties after nerve injury, its clinical use is limited due to well characterized pronociceptive effects of exogenously administered NGF (Apfel et al., 2000), thereby reflecting the complex role of NGF as an endogenous mediator of pain. Accordingly, inhibiting the underlying pronociceptive mechanisms using antibodies against NGF alleviates pain in preclinical and clinical studies, but unfortunately not without side effects in patients (Cattaneo, 2010; Lane et al., 2010). Prolonged intrathecal infusion of GDNF in rats with spinal nerve ligation (SNL) reverses mechanical as well as thermal hypersensitivity (Boucher et al., 2000). Unfortunately, significant side effects are also associated with GDNF treatment likely reflecting the relatively broad J.R. Jørgensen et al. / Experimental Neurology 237 (2012) 260–266 distribution of its receptor GFRα1. Artemin (Baloh et al., 1998) belongs to the GDNF family and its receptor, GFRα3, is found almost exclusively on nociceptive afferents (Orozco et al., 2001). As the ligand specificity of GDNF and Artemin is mediated by GFRα1 and GFRα3 respectively (Carmillo et al., 2005) but the RET receptor tyrosine kinase is a common signaling component (Airaksinen and Saarma, 2002), neurotrophic support by Artemin may be expected to have fewer off-target effects than GDNF. Consistent with this interpretation, repeated intermittent systemic injection of Artemin in SNL rats, dose-dependently reverses mechanical and thermal hypersensitivity concomitant with normalization of neurochemical as well as morphological features of primary sensory neurones (Gardell et al., 2003). Importantly no adverse effects were observed in the studies by Gardell et al. even at high doses and with continued treatment of SNL rats. Even though the beneficial effects of GDNF and Artemin persist only for the duration of the administration regimen, these studies demonstrate the potential of neurotrophic factors in relation to peripheral neuropathy. Meteorin (Nishino et al., 2004) and the recently described molecule Cometin (Jorgensen et al., 2012) constitute a unique family of neurotrophic factors. This family is unrelated to the GDNF family and other known neurotrophic factors. During mouse development, Meteorin is widely expressed within the nervous system, including the dorsal root ganglia (DRG). In primary DRG explant cultures Meteorin promotes extensive neurite outgrowth of small and intermediate nociceptive sensory neurons (Jorgensen et al., 2009; Nishino et al., 2004). Interestingly, this seems to be a glia mediated effect rather than a direct neuronal effect. However, the receptor and exact mechanism of action is currently unknown but Meteorin has been shown to use the gp130 co-receptor as an upstream transducer of Jak-STAT3 signalling (Lee et al., 2010). In the central nervous system, Meteorin protects against quinolinic acid-mediated excitoxicity (Jorgensen et al., 2010) and has furthermore been reported to participate in cerebral angiogenesis (Park et al., 2008) as well as neurogenesis (Wang et al., 2012). On this basis, we investigated the therapeutic potential of Meteorin in relation to peripheral neuropathy and associated pain. In the current set of experiments, we demonstrate that intermittent systemic administration of recombinant Meteorin dose-dependently reverses hypersensitivity in two distinct rat models of neuropathic pain. Following a slow onset, the beneficial effects last for weeks after cessation of treatment, which together with the pharmacological profile indicate a modification of the underlying neuropathy rather than symptomatic pain relief. Hence, Meteorin is a new candidate for treatment of neuropathic pain through a novel mechanism. Materials and methods Recombinant Meteorin Recombinant Meteorin was manufactured in collaboration with R&D Systems Inc. (Minneapolis, MN). Briefly, the sequence encoding mouse Meteorin (Q8C1Q4) was cloned and expressed in an NS0 mouse myeloma cell line. The recombinant mouse Meteorin was purified from the conditioned medium by ion exchange, hydrophobic interaction and size exclusion chromatography. The buffer was exchanged into PBS and the protein solution stored at − 80 °C. The purity of the preparation, as judged by densitometry scan, was 95%. Photochemically induced sciatic nerve injury All procedures involving animals were reviewed and approved by Institutional Animal Care and Use Committees of the respective institutions (Karolinska Institutet and Biogen Idec), and were in accordance with the US National Institutes of Health guidelines. Photochemically induced sciatic nerve injured rats are known to develop allodynia to both mechanical and cold stimulation (Kupers et al., 1998). For this model, 280–330 g male Sprague–Dawley rats 261 (Taconic, Denmark) were used. Briefly, under general anesthesia (chloral hydrate 300 mg/kg), the left sciatic nerve was exposed at mid-thigh level and irradiated for 1.5 min with an argon laser operating at 514 nm at an average power of 0.17 W. Erythrosin B (32.5 mg/kg dissolved in 0,9% saline) was injected intravenously through the tail vein just prior to irradiation. The resulting local ischemic damage to the sciatic nerve leads to a highly reproducible allodynia within 7 days. Hypersensitive animals were next randomly divided into four groups (n = 8) and subcutaneously (s.c.) injected with either saline as the negative control or Meteorin at 0.05, 0.2 or 0.8 mg/kg. Each rat received six injections over a two week period on days 7, 9, 11, 14, 17 and 21 counting from the time of nerve injury. Behavioral assessments were conducted before each injection during the treatment period and on days 28 and 35. In a second study, male Sprague–Dawley rats (Harlan, The Netherlands) weighing 400–450 g were fitted with a chronic intrathecal catheter with the tip at the lumbar enlargement (Storkson et al., 1996). Proper location of the catheter was verified by intrathecal injection of 10 μl lidocaine (Xylocain 50 mg/ml, Astra, Sweden) and a corresponding transient block of sensory and motor function. Three to five days after catheter implantation, ischemic sciatic nerve injury was produced as described above. Baseline responses were evaluated after catheter implantation and again before sciatic nerve irradiation. Rats that developed hypersensitivity to mechanical and cold stimulation 7 days after nerve injury were randomly divided into four groups (n = 8) which were given saline as negative control or recombinant Meteorin (0.5, 2 or 6 μg) in a volume of 10 μl intrathecally. Each rat received six injections over a two week period (on days 7, 9, 11, 14, 16 and 18 counting from the time of nerve injury). Behavioral testing was conducted prior to intrathecal injection on respective treatment days and furthermore on days 21, 25, 28 and 35 following treatment cessation. For evaluation of mechanical hypersensitivity, a set of calibrated nylon monofilaments (von Frey hairs, Stoelting, IL) was applied to the glabrous skin of the paws in ascending order from the lowest to the highest monofilament used. Each monofilament was applied 5 times at successively increasing force and the withdrawal threshold was determined as the force at which the animal withdrew the paw from at least 3 out of 5 consecutive stimuli of the same force. The response to cold was tested with ethyl chloride, which was briefly (b1 s) sprayed on the plantar surface of the hindpaw. The response was scored as the following: 0 = no response, 1 = startle-like response, no hindpaw withdrawal (normal), 2 = brief withdrawal of the stimulated hindpaw (mild response), 3 = sustained or repeated withdrawal of the stimulated hindpaw, brief licking or shaking (strong response) (Wu et al., 2006). Animals were observed and body weight followed throughout the study. All tests were performed by an experimenter who was blind with respect to the experimental conditions. No animals were removed from the study. Chronic constriction injury (CCI) Thirty male Sprague–Dawley rats weighing 250–280 g underwent surgery to produce a chronic constriction of the left sciatic nerve (Bennett and Xie, 1988). Rats were anesthetized via inhalation of isofluorane gas and received a skin incision just caudal to the biceps femoris at mid-thigh level on the left hindlimb. A small incision was then made into the underlying muscle layer and separated gently using hemostats with care taken not to disturb the sciatic nerve. The sciatic nerve was freed of adhering tissue and slightly elevated using 45° angle forceps. Four pieces of 4‐0 chromic gut suture material (previously washed in sterile saline) were brought under the nerve and then each loosely tied around the nerve into a square knot allowing for a chronic constriction of the nerve without cutting off the blood supply. The knots were spaced 1 mm apart. Muscle 262 J.R. Jørgensen et al. / Experimental Neurology 237 (2012) 260–266 layers were sutured closed with 4‐0 vicryl suture and skin was closed with wound clips. On day 0 of the experiment, prior to surgery, all rats were tested for mechanical hypersensitivity using Von Frey filaments as described above. In addition, thermal hypersensitivity was evaluated using the Hargreaves' method (Hargreaves et al., 1988) and differential weight bearing between the injured and non injured limb was determined using an incapacitance meter (Columbus Instruments, Columbus, OH). Based on scores at day 10, 24 hypersensitive rats were selected to continue in the study and randomly divided into four treatment groups (n = 6). Animals were injected five times subcutaneously (s.c.) with either vehicle or 0.1, 0.5 or 1.8 mg/kg Meteorin protein on post surgical days 10, 12, 14, 17 and 19. Animals were tested for mechanical and thermal hypersensitivity as well as weight bearing differences at days 10, 12, 14, 17, 19, 21, 26, 32 and 39 post surgery. Behavioral analysis was done prior to injection of Meteorin in order to exclude immediate analgesic effects and to focus on long lasting effects. Animals were observed and body weight followed throughout the study. The experimenter was blinded to treatment condition and no animals were removed from the study once dosing was initiated. Pharmacokinetic analysis Six male Sprague–Dawley rats weighing 250–300 g were implanted with a jugular catheter (Charles River Laboratories, Wilmington, MA) and placed in an automated blood sampling system. Three animals were dosed with 0.5 mg/kg and three were dosed with 2.0 mg/kg (s.c.) and blood samples were collected 0, 0.5, 2, 4, and 7 h after dosing. After seven hours, rats were returned to their homecage. At 24, 31, 48, and 72 h post dose blood was withdrawn via the jugular catheter. Rat serum samples were assayed by Meteorin ELISA (R&D Systems, DY3475). Statistics For mechanical and thermal hypersensitivity, weight bearing and body weight, one way analysis of variance was used with multiple comparisons versus a control group. Data on cold hypersensitivity were evaluated using the Wilcoxon single-rank test. Data are shown as mean ± SEM. p b 0.05 was considered as statistically significant. Results Systemic injection of Meteorin reverses established mechanical and cold hypersensitivity in rats with a photochemically induced sciatic nerve lesion Given the neurotrophic effect of Meteorin on peripheral nerves (Jorgensen et al., 2009; Nishino et al., 2004) we hypothesized that the molecule may have therapeutic effects in relation to neuropathic pain. Therefore, a photochemically induced sciatic nerve lesion was introduced (Kupers et al., 1998) and hypersensitive rats subsequently systemically injected six times over two weeks with recombinant Meteorin at different doses or with saline as a negative control. Fig. 1A shows that the baseline paw withdrawal threshold to mechanical stimulation before surgery (t = 0) was between 24 and 31 g for the four experimental groups. Seven days after nerve injury, all rats developed significant mechanical hypersensitivity evident as a reduced paw withdrawal threshold to between 5 and 8 g. Injection of saline or a 0.05 mg/kg dose of Meteorin did not affect the response to mechanical stimulation, while 0.2 mg/kg Meteorin produced a partial but non-significant reversal of mechanical hypersensitivity. In contrast, repeated injection of 0.8 mg/kg Meteorin produced a robust reversal of mechanical hypersensitivity. Notably, this reversal became Fig. 1. Systemically administered Meteorin reverses neuropathic hypersensitivity in photochemically induced sciatic nerve injured rats. Arrows indicate treatment days where animals were injected with 0.05 mg/kg, 0.2 mg/kg or 0.8 mg/kg recombinant Meteorin or with vehicle as negative control. A) Ipsilateral hindpaw withdrawal threshold to mechanical stimulation with von Frey hairs. B) Ipsilateral hind paw response score to cold stimulation with ethyl chloride. 0 is no response, 1 corresponds to a startle-like response seen in normal rats whereas 2 and 3 indicate mild and severe pain-like reactions. Note that Meteorin treatment dose-dependently alleviates both mechanical and cold hypersensitivity. The data are shown as mean±SEM. *, #: pb 0.05 for 0.8 mg/kg and 0.2 mg/kg Meteorin respectively compared to vehicle. significant after the third injection of Meteorin and was maintained for at least one week after cessation of treatment. The response to cold was evaluated in the same animals by briefly spraying ethyl chloride onto the plantar surface of the hind paw. The baseline cold score was 1.0 in all groups corresponding to a normal startle-like response. Seven days after nerve injury, rats developed a marked cold hypersensitivity evident as an increase in the cold score to approximately 2.5. By day 9 cold hypersensitivity had increased slightly further in the saline group and then remained stable for the rest of the study. Whereas cold hypersensitivity was unaffected by 0.05 mg/kg Meteorin, it was dose-dependently alleviated by treatment with 0.2 mg/kg and 0.8 mg/kg Meteorin, with the onset of significant reversal apparent after the second injection. Cold hypersensitivity was gradually re-established after treatment cessation but the 0.8 mg/kg Meteorin group remained significantly different from vehicle for at least one week. All animals gained weight normally throughout the study and no immediate behavioral side effects were observed. Systemic injection of Meteroin reverses evoked and non-evoked pain-like behaviors in CCI rats We also assessed effects of Meteroin in CCI rats which exhibit a behavioral repertoire consistent with clinical facets of trauma-induced neuropathic pain (Bennett and Xie, 1988). In this model, allodynia is typically established within 10 days after surgery. Over the following nine days hypersensitive animals were treated with five subcutaneous injections of Meteorin at different doses up to 1.8 mg/kg or saline as negative control. Mechanical and thermal hypersensitivity as well as weight-bearing deficits were examined throughout the study including three weeks after treatment cessation (Fig. 2). Prior to injury rats had a baseline withdrawal latency of approximately 14 g in response to hind paw von Frey stimulation, and this was reduced to 1.5 g ten days after surgery indicative of a marked mechanical hypersensitivity (Fig. 2A). Comparison with vehicle treatment throughout the duration of the experiment revealed that no increase in the withdrawal threshold occurred following repeated dosing with 0.1 mg/kg Meteorin, whereas a small but non-significant reversal was obtained J.R. Jørgensen et al. / Experimental Neurology 237 (2012) 260–266 263 Fig. 2. Systemically administered Meteorin reverses neuropathic hypersensitivity in CCI rats. Arrows indicate treatment days where animals were injected with 0.1 mg/kg, 0.5 mg/ kg or 1.8 mg/kg recombinant Meteorin or with vehicle as negative control. A) Ipsilateral hind paw withdrawal threshold to mechanical stimulation with von Frey hairs. B) Ipsilateral hind paw withdrawal latency to noxious heat stimulation. C) Weight bearing difference between the injured (ipsilateral) and non injured (contralateral) limb expressed as percent. D) Body weights measured throughout the duration of the study. Data are shown as means ± SEM. *, #, $: p b 0.05 for 1.8 mg/kg, 0.5 mg/kg and 0.1 mg/kg Meteorin respectively compared to vehicle. Note that Meteorin significantly and dose-dependently reduced all pain phenotypes without causing any weight loss. with 0.5 mg/kg Meteorin. However, a robust reversal of mechanical hypersensitivity was observed after treatment with 1.8 mg/kg Meteorin. This positive effect become statistically significant after three doses of Meteorin and was maintained for 13 days following treatment cessation with a trend towards reduced hypersensitivity still evident at day 39 post-injury. A marked thermal hypersensitivity also developed following CCI surgery (Fig. 2B), as revealed by a reduction in the hind paw withdrawal latency from 16.5 s to approximately 7 s 10 days post injury. While vehicle treated animals remained hypersensitive throughout the duration of the experiment, treatment with Meteorin at 1.8 mg/kg significantly reversed the paw withdrawal latency by day 14. This positive effect remained significant throughout the dosing period and endured for as long as three weeks after treatment cessation. The 0.5 mg/kg dose of Meteorin resulted in a moderate decrease in thermal hypersensitivity becoming significant at days 19 and 21 post-injury while repeated dosing with 0.1 mg/kg Meteorin had no significant effect compared to vehicle treatment. Prior to injury all groups of rats distributed their weight equally between their hindlimbs (Fig. 2C). However, by day 10 a marked weight bearing difference of approximately 60% was evident, indicative of the presence of spontaneous pain. The weight bearing difference was evident in the vehicle group throughout the duration of the study. In contrast, systemic treatment with 0.5 and 1.8 mg/kg Meteorin significantly reduced the difference within a few days, and in both cases the improvement was maintained for at least three weeks following treatment cessation. A statistically significant reduction of the weight bearing difference was also seen with the low dose of Meteorin at day 19. Generally, from day 26 onwards until the end of the experiment, the weight bearing difference in all Meteorin treated groups settled at steady levels lower than the vehicle group. Where the vehicle control group remained above 60%, the average weight bearing difference for the treated groups was approximately 55%, 48% and 40% respectively for 0.1, 0.5 and 1.8 mg/kg Meteorin. In summary, Meteorin dose-dependently reversed multiple signs of neuropathic hypersensitivity in CCI rats and the positive effects lasted for several weeks after cessation of treatment. Importantly, no overt behavioral side effects were observed and all animals gained weight normally throughout the study (Fig. 2D). Meteorin pharmacokinetics We next investigated the pharmacokinetic profile of systemically administered Meteorin. Naïve rats were dosed with 0.5 and 2.0 mg/kg Fig. 3. Pharmacokinetic profile of Meteorin in rat serum. Naïve rats were subcutaneously administered 0.5 mg/kg or 2.0 mg/kg Meteorin. Subsequently, serum was collected over time and the concentration of Meteorin determined by ELISA analysis. Note that Meteorin is no longer detectable after 24 hr. 264 J.R. Jørgensen et al. / Experimental Neurology 237 (2012) 260–266 Meteorin and serum collected at time points ranging from 0 to 72 hours. As can be seen in Fig. 3, a good relationship between dose and serum concentration was observed. The high dose of Meteorin (2.0 mg/kg) reached a Cmax of 692±85 ng/ml at 4 hours and the lower dose (0.5 mg/kg) reached a Cmax of 240±14 ng/ml at 2 hours post injection. In both cases, Meteorin was no longer detectable in serum 24 hours after administration, Intrathecal injection of Meteorin also reverses experimental neuropathic pain As demonstrated, systemic injection of Meteorin can mediate effective reversal of multiple pain phenotypes. We next examined whether intrathecal injection of Meteorin could also reverse neuropathic hypersensitivity after nerve injury. As seen in Fig. 4A, the baseline paw withdrawal threshold to mechanical stimulation was typically 50 g and this was reduced to approximately 8 g seven days after photochemically induced sciatic nerve injury. From this time point, rats received intrathecal injections of either vehicle or Meteorin (0.5 μg, 2 μg or 6 μg) over a two week period. From Fig. 4A, it is evident that Meteorin dose-dependently reduced mechanical hypersensitivity, which then gradually re-established after treatment cessation. Animals treated with 6 μg Meteorin were significantly different from control a week after treatment ended while the effect of 2 μg only lasted as long as administration was maintained. As seen in Fig. 4B, the baseline response to ethyl chloride stimulation of the hind paw was 1.0 corresponding to a normal startle-like response. Seven days after nerve injury, rats developed a cold hypersensitivity evident by an increase in cold score response of the injured paw. Vehicle administration had no effect on cold hypersensitivity and there was no significant effect of the low Meteorin dose. In contrast, intrathecal treatment with 2 μg or 6 μg Meteorin quickly reversed the hypersensitivity and both treatment groups had a near normal response to cold during the treatment period. This reversal was maintained for three additional days in both groups before cold hypersensitivity was fully re-established. Discussion Our data clearly demonstrate that Meteorin effectively mediates a dose-dependent long-lasting reversal of pain-like behaviors in two Fig. 4. Intrathecal Meteorin reverses neuropathic hypersensitivity in photochemicallyinduced sciatic nerve injured rats. Arrows indicate time points for intrathecal injection of 0.5, 2 or 6 μg recombinant Meteorin or saline as negative control. A) Ipsilateral hind paw withdrawal threshold to mechanical stimulation with von Frey hairs. B) Ipsilateral hind paw withdrawal score to cold stimulation with ethyl chloride. Data are shown as means ± SEM. *pb 0.05 between vehicle and 6 μg Meteorin; #p b 0.05 between vehicle and 2 μg Meteorin. separate rat models of neuropathic pain. In the general experimental design, once a robust neuropathic hypersensitivity was established this was followed by 5–6 systemic injections of recombinant Meteorin over 9–12 days. During the dosing period, pain-like behaviors were examined prior to Meteorin administration in order to focus on long lasting treatment effects rather than acute effects of the neurotrophic factor. Using this dosing regimen significant effects of Meteorin were typically observed after the second or third administration during the first week of dosing which continued to improve during the second week. The peak anti-hyperalgesic efficacy obtained with Meteorin in response to hindpaw mechanical stimulation was at least as good as that typically obtained with other standard of care drugs such as gabapentin in rat models of peripheral nerve injury. Moreover, noxious heat and cold hypersensitivity and non-evoked weight bearing deficits were also reversed by Meteorin. Remarkably, the reduction in pain behaviors produced by Meteorin was sustained for weeks after treatment cessation even though the protein was undetectable in serum within 24 hours after administration. Also importantly, we did not observe any weight loss or behavioral side effects. The above findings collectively suggest that Meteorin has disease modifying properties and that the long lasting effects on behavior may reflect a normalization or restoration of neuronal function similar to the actions reported for GDNF and Artemin (Boucher et al., 2000; Gardell et al., 2003). While the mechanism of action employed by the GDNF family is relatively well understood (Airaksinen and Saarma, 2002), the specific receptor(s) for Meteorin remains unknown. Nonetheless, recent studies suggest that gp130 is recruited and mediates intracellular signalling primarily through the Jak-STAT3 pathway (Lee et al., 2010). Interestingly, gp130/Jak-STAT3 is also the common signalling mechanism for the IL-6 family of cytokines. However, IL-6 is a major inflammatory mediator producing both thermal and mechanical hypersensitivity after intrathecal injection (DeLeo et al., 1996). Correspondingly, intrathecally applied IL-6 neutralizing antibodies alleviate hypersensitivity after L5 spinal nerve ligation (Arruda et al., 2000). Thus, although they share a similar transduction cascade, Meteorin and IL-6 have paradoxical effects on nociceptive transmission after neuropathic injury. Although intrathecal overexpression of anti-inflammatory cytokines such as IL-4 and IL-10 has shown beneficial effects in animal models of neuropathic pain (Hao et al., 2006; Milligan et al., 2005) these molecules do not signal through gp130. Unlike NGF which signals through TrkA/p75, systemic injection of Meteorin does not seem to induce any acute pain. Therefore, the prominent effects of Meteorin in our study appear to engage a novel mechanism for alleviation of neuropathic pain which is clearly different from that mediated by the GDNF family of ligands, the interleukins and NGF. The precise location and the pathological mechanisms targeted by Meteorin after nerve injury require further exploration. It is known that Meteorin mediates neurotrophic effects on nociceptive DRG neurons through satellite glial cells (Nishino et al., 2004). Again, this is different from GDNF and Artemin which acts directly on DRG neurons expressing the relevant receptors to restore neurochemical phenotypes and diminish electrical excitability of injured primary afferent fibres (Boucher et al., 2000; Gardell et al., 2003). Schwann cells in the sciatic nerve express Meteorin at P2 (Nishino et al., 2004) and the transcript is also found in DRGs during development (Jorgensen et al., 2012) but in the adult state Meteorin is not detected in the peripheral nervous system (Jorgensen et al., 2009). This is in line with our general working hypothesis that neurotrophic factors distinctively expressed during development of a specific part of the nervous system may be of therapeutic benefit when re-applied to the same specific part of the adult nervous system if this becomes diseased or damaged. Interestingly, we show that intrathecal injection of Meteorin is also efficacious suggesting that it might directly interfere with the pathological interactions between neurons, glia and possibly immune cells involved in the development and maintenance of central sensitization within spinal dorsal horn pain circuits underpinning neuropathic hyperexcitability (Scholz and Woolf, 2007; von Hehn et al., 2012). However, the spinal J.R. Jørgensen et al. / Experimental Neurology 237 (2012) 260–266 cord is unlikely to have been the primary site of action of systemically administered recombinant Meteorin given the difficulty such a protein would have crossing the blood brain barrier. Although we cannot rule out that Meteorin might have leaked from the intrathecal space to mediate modulatory effects within the DRGs via satellite glia the same argument should also hold true regards crossing the blood brain barrier. Again, the absence of the molecular target for Meteorin precludes a more definitive examination of these possibilities. An important aspect of our study is that we have shown near identical effects of systemically administered Meteorin in two separate rat models of trauma-induced nerve injury. These models recapitulate many of the behavioral signs and symptoms observed in human neuropathic pain patients (Maier et al., 2010) and were performed in independent laboratories by investigators blinded to treatment. Both nerve injury models have been rigorously validated with standard of care medications used in the clinical treatment of neuropathic pain and the CCI model has been estimated to have predictive validity up to 88% (Kontinen and Meert, 2003). However, while drugs like gabapentin typically reverse neuropathic hypersensitivity with a level of efficacy similar to that shown here for Meteorin invariably they do so only for the duration of plasma and CNS exposure. In this respect it is particularly alluring that Meteorin was eliminated from the peripheral circulation within 24 hours following administration yet the prolongation of action lasted for weeks beyond the final dose. Although we cannot rule out accumulation of Meteorin within specific tissue compartments, we think this unlikely to account for maintained analgesic efficacy weeks after the final dosing. As alluded to earlier, anti-inflammatory or anti-microglial mechanisms could also contribute to the observed effects. Future studies should focus on the mechanism of action and especially the possible disease modifying aspects of Meteorin. This should include electrophysiological as well as detailed morphological investigations. In relation to this, it would be interesting to address how long following nerve injury treatment can be postponed, yet still reverse neuronal damage, as a neurotrophic mechanism would likely require at least some residual plasticity in the system to be effective. Furthermore, the role of Meteorin in the communication between glia and neurons should be clarified and identification of the specific receptor(s) for Meteorin is naturally also an important task. Knowing the specific receptor and its expression pattern at the cellular level could furthermore guide a toxicological evaluation to clarify if Meteorin could act on other body systems. Conclusion Systemic administration of recombinant Meteorin dose-dependently reversed neuropathic hypersensitivity in two distinct rat models of peripheral nerve injury without any observed side effects. The delay of onset and the maintained efficacy following cessation of Meteorin treatment, coupled with the pharmacokinetic profile in plasma suggest an effect on the underlying neuropathy rather than symptomatic pain relief. Conflict of interest statement JRJ, LUW and TEJ are employed by NsGene holding patents on Meteorin. Acknowledgments We appreciate the collaboration with R&D Systems Inc. (Minneapolis, MN) on the production of in vivo grade recombinant Meteorin for these studies. We thank Ellen Rohde and Robin Caputo for assistance with collecting blood for the PK analysis. Studies were partially funded by NsGene. 265 References Airaksinen, M.S., Saarma, M., 2002. The GDNF family: signalling, biological functions and therapeutic value. Nat. Rev. Neurosci. 3, 383–394. Apfel, S.C., Schwartz, S., Adornato, B.T., Freeman, R., Biton, V., Rendell, M., Vinik, A., Giuliani, M., Stevens, J.C., Barbano, R., Dyck, P.J., 2000. Efficacy and safety of recombinant human nerve growth factor in patients with diabetic polyneuropathy: a randomized controlled trial. rhNGF Clinical Investigator Group. JAMA 284, 2215–2221. Arruda, J.L., Sweitzer, S., Rutkowski, M.D., DeLeo, J.A., 2000. Intrathecal anti-IL-6 antibody and IgG attenuates peripheral nerve injury-induced mechanical allodynia in the rat: possible immune modulation in neuropathic pain. Brain Res. 879, 216–225. Baloh, R.H., Tansey, M.G., Lampe, P.A., Fahrner, T.J., Enomoto, H., Simburger, K.S., Leitner, M.L., Araki, T., Johnson Jr., E.M., Milbrandt, J., 1998. Artemin, a novel member of the GDNF ligand family, supports peripheral and central neurons and signals through the GFRalpha3-RET receptor complex. Neuron 21, 1291–1302. Bennett, G.J., Xie, Y.K., 1988. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33, 87–107. Boucher, T.J., Okuse, K., Bennett, D.L., Munson, J.B., Wood, J.N., McMahon, S.B., 2000. Potent analgesic effects of GDNF in neuropathic pain states. Science 290, 124–127. Carmillo, P., Dago, L., Day, E.S., Worley, D.S., Rossomando, A., Walus, L., Orozco, O., Buckley, C., Miller, S., Tse, A., Cate, R.L., Rosenblad, C., Sah, D.W., Gronborg, M., Whitty, A., 2005. Glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFR alpha 1) is highly selective for GDNF versus artemin. Biochemistry 44, 2545–2554. Cattaneo, A., 2010. Tanezumab, a recombinant humanized mAb against nerve growth factor for the treatment of acute and chronic pain. Curr. Opin. Mol. Ther. 12, 94–106. Costigan, M., Scholz, J., Woolf, C.J., 2009. Neuropathic pain: a maladaptive response of the nervous system to damage. Annu. Rev. Neurosci. 32, 1–32. DeLeo, J.A., Colburn, R.W., Nichols, M., Malhotra, A., 1996. Interleukin-6-mediated hyperalgesia/allodynia and increased spinal IL-6 expression in a rat mononeuropathy model. J. Interferon Cytokine Res. 16, 695–700. Gardell, L.R., Wang, R., Ehrenfels, C., Ossipov, M.H., Rossomando, A.J., Miller, S., Buckley, C., Cai, A.K., Tse, A., Foley, S.F., Gong, B., Walus, L., Carmillo, P., Worley, D., Huang, C., Engber, T., Pepinsky, B., Cate, R.L., Vanderah, T.W., Lai, J., Sah, D.W., Porreca, F., 2003. Multiple actions of systemic artemin in experimental neuropathy. Nat. Med. 9, 1383–1389. Haanpaa, M., Attal, N., Backonja, M., Baron, R., Bennett, M., Bouhassira, D., Cruccu, G., Hansson, P., Haythornthwaite, J.A., Iannetti, G.D., Jensen, T.S., Kauppila, T., Nurmikko, T.J., Rice, A.S., Rowbotham, M., Serra, J., Sommer, C., Smith, B.H., Treede, R.D., 2011. NeuPSIG guidelines on neuropathic pain assessment. Pain 152, 14–27. Hao, S., Mata, M., Glorioso, J.C., Fink, D.J., 2006. HSV-mediated expression of interleukin-4 in dorsal root ganglion neurons reduces neuropathic pain. Mol. Pain 2, 6. Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J., 1988. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32, 77–88. Jorgensen, J.R., Thompson, L., Fjord-Larsen, L., Krabbe, C., Torp, M., Kalkkinen, N., Hansen, C., Wahlberg, L., 2009. Characterization of Meteorin—an evolutionary conserved neurotrophic factor. J. Mol. Neurosci. 39, 104–116. Jorgensen, J.R., Emerich, D.F., Thanos, C., Thompson, L.H., Torp, M., Bintz, B., FjordLarsen, L., Johansen, T.E., Wahlberg, L.U., 2010. Lentiviral delivery of Meteorin protects striatal neurons against excitotoxicity and reverses motor deficits in the quinolinic acid rat model. Neurobiol. Dis. 41, 160–168. Jorgensen, J.R., Fransson, A., Fjord-Larsen, L., Thompson, L.H., Houchins, J.P., Andrade, N., Torp, M., Kalkkinen, N., Andersson, E., Lindvall, O., Ulfendahl, M., Brunak, S., Johansen, T.E., Wahlberg, L.U., 2012. Cometin is a novel neurotrophic factor that promotes neurite outgrowth and neuroblast migration in vitro and supports survival of spiral ganglion neurons in vivo. Exp. Neurol. 233, 172–181. Kontinen, V.K., Meert, T.F., 2003. Predictive validity of neuropathic pain models in pharmacological studies with a behavioural outcome in the rat: a systematic review. In: Dostrovsky, J.O., Carr, D.B., Koltzenburg, M. (Eds.), Proceedings of 10th World Congress on Pain. IASP Press, Seattle, pp. 489–498. Kupers, R., Yu, W., Persson, J.K., Xu, X.J., Wiesenfeld-Hallin, Z., 1998. Photochemically-induced ischemia of the rat sciatic nerve produces a dose-dependent and highly reproducible mechanical, heat and cold allodynia, and signs of spontaneous pain. Pain 76, 45–59. Lane, N.E., Schnitzer, T.J., Birbara, C.A., Mokhtarani, M., Shelton, D.L., Smith, M.D., Brown, M.T., 2010. Tanezumab for the treatment of pain from osteoarthritis of the knee. N. Engl. J. Med. 363, 1521–1531. Lee, H.S., Han, J., Lee, S.H., Park, J.A., Kim, K.W., 2010. Meteorin promotes the formation of GFAP-positive glia via activation of the Jak-STAT3 pathway. J. Cell Sci. 123, 1959–1968. Maier, C., Baron, R., Tolle, T.R., Binder, A., Birbaumer, N., Birklein, F., Gierthmuhlen, J., Flor, H., Geber, C., Huge, V., Krumova, E.K., Landwehrmeyer, G.B., Magerl, W., Maihofner, C., Richter, H., Rolke, R., Scherens, A., Schwarz, A., Sommer, C., Tronnier, V., Uceyler, N., Valet, M., Wasner, G., Treede, R.D., 2010. Quantitative sensory testing in the German Research Network on Neuropathic Pain (DFNS): somatosensory abnormalities in 1236 patients with different neuropathic pain syndromes. Pain 150, 439–450. Milligan, E.D., Sloane, E.M., Langer, S.J., Cruz, P.E., Chacur, M., Spataro, L., WieselerFrank, J., Hammack, S.E., Maier, S.F., Flotte, T.R., Forsayeth, J.R., Leinwand, L.A., Chavez, R., Watkins, L.R., 2005. Controlling neuropathic pain by adenoassociated virus driven production of the anti-inflammatory cytokine, interleukin-10. Mol. Pain 1, 9. Nishino, J., Yamashita, K., Hashiguchi, H., Fujii, H., Shimazaki, T., Hamada, H., 2004. Meteorin: a secreted protein that regulates glial cell differentiation and promotes axonal extension. EMBO J. 23, 1998–2008. 266 J.R. Jørgensen et al. / Experimental Neurology 237 (2012) 260–266 Orozco, O.E., Walus, L., Sah, D.W., Pepinsky, R.B., Sanicola, M., 2001. GFRalpha3 is expressed predominantly in nociceptive sensory neurons. Eur. J. Neurosci. 13, 2177–2182. Ossipov, M.H., 2011. Growth factors and neuropathic pain. Curr. Pain Headache Rep. 15, 185–192. Park, J.A., Lee, H.S., Ko, K.J., Park, S.Y., Kim, J.H., Choe, G., Kweon, H.S., Song, H.S., Ahn, J.C., Yu, Y.S., Kim, K.W., 2008. Meteorin regulates angiogenesis at the gliovascular interface. Glia 56, 247–258. Pezet, S., McMahon, S.B., 2006. Neurotrophins: mediators and modulators of pain. Annu. Rev. Neurosci. 29, 507–538. Sah, D.W., Ossipov, M.H., Rossomando, A., Silvian, L., Porreca, F., 2005. New approaches for the treatment of pain: the GDNF family of neurotrophic growth factors. Curr. Top. Med. Chem. 5, 577–583. Scholz, J., Woolf, C.J., 2007. The neuropathic pain triad: neurons, immune cells and glia. Nat. Neurosci. 10, 1361–1368. Storkson, R.V., Kjorsvik, A., Tjolsen, A., Hole, K., 1996. Lumbar catheterization of the spinal subarachnoid space in the rat. J. Neurosci. Meth. 65, 167–172. Treede, R.D., Jensen, T.S., Campbell, J.N., Cruccu, G., Dostrovsky, J.O., Griffin, J.W., Hansson, P., Hughes, R., Nurmikko, T., Serra, J., 2008. Neuropathic pain: redefinition and a grading system for clinical and research purposes. Neurology 70, 1630–1635. von Hehn, C.A., Baron, R., Woolf, C.J., 2012. Deconstructing the neuropathic pain phenotype to reveal neural mechanisms. Neuron 73, 638–652. Wang, Z., Andrade, N., Torp, M., Wattananit, S., Arvidsson, A., Kokaia, Z., Jorgensen, J.R., Lindvall, O., 2012. Meteorin is a chemokinetic factor in neuroblast migration and promotes stroke-induced striatal neurogenesis. J. Cereb. Blood Flow Metab. 32, 387–398. Wu, W.P., Hao, J.X., Fredholm, B.B., Wiesenfeld-Hallin, Z., Xu, X.J., 2006. Effect of acute and chronic administration of caffeine on pain-like behaviors in rats with partial sciatic nerve injury. Neurosci. Lett. 402, 164–166.