Neuroscience 167 (2010) 1168 –1174 ACETYL-L-CARNITINE INCREASES ARTEMIN LEVEL AND PREVENTS NEUROTROPHIC FACTOR ALTERATIONS DURING NEUROPATHY E. VIVOLI,a L. DI CESARE MANNELLI,a* A. SALVICCHI,a A. BARTOLINI,a A. KOVERECH,b R. NICOLAI,b P. BENATTIb AND C. GHELARDINIa allodynia or ongoing pain can persist in the absence of visible injury or clinically measurable inflammation (Woolf and Mannion, 1999; Gilron et al., 2006; Baron, 2006). Actual therapies for pain improvement are not able to revert the nervous alteration or to induce tissue regeneration. The most clinically used compound gabapentin is active on pain but it is ineffective on neuroprotection or neuroregeneration. On the other hand, many growth factors for the nervous system do not relieve pain. Nerve growth factor (NGF), the prototypical neurotrophic factor, maintains the survival of sympathetic and sensory neurons as well as neurite outgrowth but it also exerts profound biological effect on nociceptors that express high-affinity NGF receptors. NGF is upregulated by inflammatory states and by peripheral nerve injury with the ensuing Wallerian degeneration (Ramer et al., 1997); it is able to sensitize peripheral nociceptive terminals inducing nociceptor gene expression. Moreover, NGF is retrogradely transported to sensory neuron soma and regulates genes involved in pain processing (Sah et al., 2003; Pezet and McMahon, 2006). Therefore NGF acts as pain mediator and its administration in rats results in pronounced mechanical and thermal hyperalgesia (Levin et al., 1994). Similar hyperalgesic effects are referable also to BDNF and, at a lesser extent, to NT3, other members of the neurotrophin family (Sah et al., 2003). A different profile has been described for the glial cell line-derived neurotrophic factor (GDNF)-related family, in particular for GDNF and Artemin (ARTN). These factors are notable for their ability to promote growth and survival of neurons, are neuroprotective and support regeneration after nervous tissue damage (Chen et al., 2001; Wang et al., 2008); nevertheless, they are also able to normalize pain threshold. GDNF has been reported to reduce mechanical hyperalgesia and ectopic discharges within sensory neurons after nerve injury (Boucher and McMahon, 2001). Porreca and co-workers (Gardell et al., 2003; Wang et al., 2008) described that a repeated ARTN administration prevented pain behaviour after spinal nerve ligation. The acetyl ester of L-Carnitine isomer (ALCAR) is able to raise the pain threshold, showing an analgesic effect in acute pain conditions (Ghelardini et al., 2002; Galeotti et al., 2004) and an anti-hyperalgesic effect both in animal and human neuropathic conditions (Di Cesare Mannelli et al., 2009; Osio et al., 2006). Moreover, ALCAR shows a protective and regenerative action profile on the nervous tissue after toxic (Pisano et al., 2003) or traumatic injuries (Fernandez et al., 1989; Hart et al., 2002; McKay-Hart et al., 2002; Di Cesare Mannelli et al., 2007). a Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139, Florence, Italy b Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Via Pontina km 30,400, I-00040 Pomezia, Rome, Italy Abstract—Damages to the nervous system are the primarily cause of neuropathy and chronic pain. Current pharmacological treatments for neuropathic pain are not able to prevent or revert morphological and molecular consequences of tissue injury. On the other hand, many neurotrophins, like nerve growth factor (NGF), paired off restorative effects with hyperalgesia. Interestingly, the glial cell line– derived neurotrophic factors GDNF and Artemin (ARTN) seem to support neuron survival and to normalize abnormal pain behaviour. In the present research protein levels of NGF, GDNF and ARTN were evaluated in a rat model of peripheral neuropathy, the chronic constriction injury (CCI). NGF was increased by CCI in the ipsilateral dorsal root ganglia (DRG), in the spinal cord and in the periaqueductal grey matter (PAG). On the contrary, ARTN was decreased bilaterally in DRG, spinal cord and PAG. GDNF levels decreased in ipsilateral DRG, whereas the constriction did not modify its expression in the central nervous system districts. Repeated treatments with the antihyperalgesic and neuroregenerative compound acetyl-L-carnitine (ALCAR; 100 mgkgⴚ1 i.p. twice daily for 15 days) was able to prevent the increase of NGF levels. In conditions of pain relief ALCAR normalized peripheral and central alterations of GDNF and ARTN levels. Characteristically, sham animals that underwent the same ALCAR treatment, showed increased levels of ARTN both in the DRG and in the spinal cord. These data offer a new point of view on the mechanism of the antihyperalgesic as well as the neuroprotective effect of ALCAR. © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: artemin, GDNF, NGF, pain, neurorestoration. Neuropathic pain is a chronic algic sensation, a characteristic symptom of neuropathies along altered sensibility and loss of functionality. Lesions to the central or peripheral nervous system are the causes of this syndrome, which may result from traumatic events or metabolic or toxic insults. In these conditions the protective role of pain is lost, it does not offer biological advantage and cause suffering and distress; the signalling is altered and hyperalgesia, *Corresponding author. Tel: ⫹39-0554271316; fax: ⫹39-0554271280. E-mail address: lorenzo.mannelli@unifi.it (L. Di Cesare Mannelli). Abbreviations: ALCAR, acetyl-L-carnitine; ARTN, artemin; CCI, chronic constriction injury; DRG, dorsal root ganglia; GDNF, glial cell line-derived neurotrophic factor; NGF, nerve growth factor; PAG, periaqueductal grey matter. 0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2010.03.017 1168 E. Vivoli et al. / Neuroscience 167 (2010) 1168 –1174 Aimed to study the complex relationship between pain relieve and neuroprotection and to explore the pharmacodynamic profile of ALCAR, we characterized a rat model of chronic constriction injury in terms of ARTN levels in comparison with GDNF and NGF levels, both in the peripheral and central nervous system. The effect of repeated administration of ALCAR on pain and on neurotrophic factor expressions was analyzed. EXPERIMENTAL PROCEDURES Animals Male Sprague–Dawley rats from Harlan-Italia (Varese, Italy) were used. Four rats were housed per cage (size 26⫻41 cm2) and placed in the experimental room for acclimatization 24 h before the test. The animals were fed with standard laboratory diet and with tap water ad libitum, and kept at 23⫾1 °C with a 12 h light/dark cycle, light at 7 AM. All animal manipulations were carried out according to the European Community guidelines for animal care (DL 116/92, application of the European Communities Council Directive of November 24, 1986 (86/609/EEC)). Ethical policy of the University of Florence complies with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH Publication No. 85-23, revised 1996; University of Florence assurance number: A5278-01). All efforts were made to minimize animal suffering and to reduce the number of animals used. Peripheral rat mononeuropathy Neuropathy was induced according to the procedure described by Bennett and Xie (1988). Briefly, rats were anaesthetized with 400 mg/kg i.p. chloral hydrate (Merck, Darmstadt, Germany). Under aseptic conditions, the right common sciatic nerve was exposed at the level of the middle thigh by blunt dissection. Proximal to the trifurcation, the nerve was carefully freed from the surrounding connective tissue and four chromic cat gut ligatures (4 – 0, Ethicon, Norderstedt, Germany) were tied loosely around it with about 1 mm spacing. After hemostasis was confirmed, the incision was closed in layers. The animals were allowed to recover from surgery and then housed one per cage with free access to water and standard laboratory chow. Another group of rats were subjected to sham surgery in which the sciatic nerve was only exposed but not ligated. Drug administration ALCAR was provided from Sigma-Tau (Pomezia, Italy) and the administration was performed i.p. twice daily for 15 consecutive days. Paw pressure test The nociceptive threshold in the rat was determined with an analgesimeter (Ugo Basile, Varese, Italy), according to the method described by Leighton et al. (1988). Briefly, a constantly increasing pressure was applied to a small area of the dorsal surface of the paw using a blunt conical probe by a mechanical device to a small area of the paw. Mechanical pressure was increased until vocalization or a withdrawal reflex occurred while rats were lightly restrained. Vocalization or withdrawal reflex thresholds were expressed in grams. Rats scoring below 40 g or over 75 g during the test before drug administration (25%) were rejected. An arbitrary cut-off value of 250 g was adopted. The data were collected by an observer who was blinded to the protocol. 1169 Tissue explants After treatment, animals were sacrificed by decapitation and the ipsilateral (right) and contralateral (left) dorsal root ganglia (DRG), the entire spinal cord and periaqueductal grey matter (PAG) were explanted. Tissue extracted was approximately 10, 400 and 100 mg respectively. Western blot Tissue was mechanically homogenized on ice with lysis buffer containing 1 M Tris–HCl pH 7.5, 10% sodium dodecyl sulfate (SDS), 40 mM p-Nitrophenyl Phosphate, 57 mM Phenylmethylsulfonyl fluoride, 100 mM Sodium Orthovanadate, 100 mM Sodium Pyrophosphate, 1.4 mg/ml Aprotinin, 2 mg/ml Leupeptine, 1 M NaCl, 100 mM EGTA and 50 mM EDTA. The samples were centrifuged at 10000⫻g for 15 min at 4 °C. The supernatant was collected and stored at ⫺80 °C. Protein concentrations in the supernatant were measured by Bradford’s method (Protein assay kit, Bio-Rad Laboratories, Milan, Italy). Protein homogenates were separated on a 10% SDS-polyacrylamide gel by electrophoresis and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Milan, Italy). Membranes were blocked with 5% non-fat dry milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBST) and then probed overnight with primary antibodies specific versus NGF, GDNF, ARTN (Santa Cruz Biotechnology Inc, CA, USA) and used in PBST/5% non fat dry milk at concentration 1:1000 for NGF and GDNF and 1:500 for ARTN. After washing with PBST, the membranes were incubated for 2 h in PBST/milk containing the appropriate horse radish peroxidaseconjugated secondary antibody (1:5000). Blots were then extensively washed according to the manufacture’s instruction and developed using a colorimetric method (OPTI 4 CN substrate kit, Bio-Rad Laboratories, Milan, Italy). Densitometric analysis was performed on a Macintosh Imac computer. Measurements in control samples (sham, saline) were assigned a relative value of 100%. ␤-actin was used as loading control. Statistical analysis All experimental results are given as mean⫾SEM analysis of variance ANOVA, followed by Fisher’s post hoc comparison, was used to verify significance between two means. Data were analyzed with StatView software for the Macintosh (1992). P-values of less than 0.05 were considered significant. RESULTS The chronic constriction injury (CCI) model induced a pain syndrome characterized by hyperalgesia and nerve tissue alterations well demonstrated 15 days after nerve injury. Following this period the hyperalgesic behaviour of rats that underwent the right sciatic nerve ligation were confirmed by Paw pressure test. As shown in Table 1 repeated treatments with ALCAR (100 mgkg⫺1 i.p. twice per day) for Table 1. Paw-Pressure test—15 d after nerve ligation ALCAR (100 mg kg⫺1 s.c. twice daily) Saline Left paw Right paw Left paw Right paw 60.9⫾4.7 34.1⫾4.3* 118.4⫾6.7** 58.9⫾6.2** Each value represents the mean of two experiments with 12 rats per group. * P⬍0.01 versus Saline left nerve. ** P⬍0.01 versus Saline. 1170 E. Vivoli et al. / Neuroscience 167 (2010) 1168 –1174 15 days starting on the day of the operation, was able to induce an antihyperalgesic effect, evaluated on the right paw. The same treatment was able to induce an analgesic effect evaluated on the left unoperated paw. At day 15, after the measurement of the pain threshold, rats were sacrified and the expression levels of NGF, GDNF and ARTN were analyzed by Western blot in the left (contralateral) and in the right (ipsilateral) DRG, in the spinal cord and in the PAG. In CCI saline-treated animals NGF was increased in the ipsilateral DRG (150%), in the spinal cord (350%) and in the PAG (180%) in respect to the sham saline-treated group arbitrarily assigned a relative value of 100% (Fig. 1–CCI, saline). In all districts ALCAR administration was able to prevent NGF increase (Fig. 1–CCI, ALCAR) in CCI-treated rats whereas it did not modify NGF expression profile of sham rats (Fig. 1—sham, ALCAR). In Fig. 2—panels A–D the expression of GDNF was shown. The loose ligation of the sciatic nerve decreased GDNF expression in the DRG of the ipsilateral side in respect to the sham saline-treated group (70%; Fig. 2 panel A–CCI, saline). Repeated treatment with ALCAR restored the control level (110%; Fig. 2 panel A–CCI, ALCAR). No alterations were observed in the contralateral DRG as well as in the spinal cord and in the PAG (Fig. 2, panels B–D). ALCAR did not has a per se effect on GDNF expression in sham animals (Fig. 2—sham, ALCAR). The nerve injury was able to induce a decrease of ARTN expression bilaterally in the DRG of CCI, salinetreated rats, up to 50% in the ipsilateral and at a lesser extent in the contralateral (75%) (Fig. 3, panel A–CCI, saline). Analogous reduction was measured in the spinal cord (60%) (Fig. 3, panel B–CCI, saline). Both in DRG and spinal cord of CCI the effects of ALCAR administration consisted of a complete prevention in the ARTN decrease (the expression level in DRG was bilaterally about 120% and 150% in the spinal cord; Fig. 3, panels A, B–CCI, ALCAR). ALCAR treatment was also able to increase ARTN levels in DRG and spinal cord of sham animals (160% and 240% respectively; Fig. 3, panel A, B—sham, ALCAR). Fig. 3 panel C shows the pattern of expression of Fig. 1. NGF protein expression levels. Rats underwent to surgical process with (CCI) or without (sham) the loose ligation of the sciatic nerve; the administration of saline or ALCAR (100 mgkg⫺1 i.p., twice per day) was started on the day of the operation and continued for 15 days. (A) The right (ipsilateral) and the left (contralateral) DRG were analyzed by Western blot: a densitometric evaluation is shown. (B) NGF levels in the total spinal cord and (C) in the PAG. (D) Representative Western blots in respect to ␤-actin expression. Results are expressed as the mean⫾SEM of five different animals. Measurements in control samples (sham, saline) were assigned a relative value of 100%. * P⬍0.05, significantly different from sham, saline; ^ P⬍0.05 significantly different from CCI, saline. E. Vivoli et al. / Neuroscience 167 (2010) 1168 –1174 1171 Fig. 2. GDNF, protein expression levels. (A) contralateral and ipsilateral DRG were analyzed using a specific antibody. Effects of saline or ALCAR treatment (100 mgkg⫺1 i.p., twice per day) were also evaluated in (B) spinal cord and (C) PAG. Western blot was performed 15 days after the operation, (D) representative results are shown. Results are expressed as the mean⫾SEM of five different animals; ␤-actin normalization was performed for each sample. Measurements in sham, saline samples were assigned a relative value of 100%. * P⬍0.05, significantly different from sham, saline; ^ P⬍0.05 significantly different from CCI, saline. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article. ARTN in PAG: animals suffered a decrease up to 60% in consequence of CCI also in this area (CCI, saline). Conversely, in the ALCAR-treated group a 190% expression level of ARTN was measurable (Fig. 3, panel C–CCI, ALCAR). ALCAR did not increase ARTN in PAG of sham rats (Fig. 3C—sham, ALCAR). DISCUSSION Chronic pain developed in consequence of lesions to the nervous system is the most observable phenomena of a complex syndrome founded on dysfunctional signalling. Pathological responses include neurotrophic factor expression. The role of NGF, GDNF and ARTN on pain has been extensively studied but the relationship between their expression levels and pain is not well established. The gray matter located in the midbrain around the cerebral aqueduct plays a key role in nociceptive processing (Behbehani, 1995). It receives afferent projections from a number of brainstem and spinal areas which are known to be involved in the modulation and conduction of nociception. The PAG is also a major component of the endogenous pain control system that is able to inhibit and facilitate pain processing. The present research describes an increase in NGF expression levels in DRG during a painful neuropathy induced by traumatic injury to the sciatic nerve. This alteration dramatically raises in the central nervous system, in particular in the spinal cord as well as in the PAG. Blocking the action of NGF provides highly effective pain relief in many animal models of acute and chronic pain (Li et al., 2003; Gwak et al., 2003; Zahn et al., 2004; Hefti et al., 2006). Current options about NGF-related therapies being explored involve the use of protein such as antibodies, peptibodies and small protein domains, which either sequester NGF or prevent the binding to its receptor TrkA (Watson et al., 2008). On the contrary, GDNF and ARTN, members of another family of growth factors primarily related to glia cells, have an antihyperalgesic effect. Their characteristic actions on pain and neuron survival 1172 E. Vivoli et al. / Neuroscience 167 (2010) 1168 –1174 Fig. 3. ARTN. Protein expression levels were analyzed in CCI rats in respect to sham and the effect of ALCAR (100 mgkg⫺1 i.p. twice per day) was evaluated after administrations repeated for 15 days. Densitometric analysis of (A) contralateral and ipsilateral DRG; (B) total spinal cord; (C) PAG. Panel (D) shows representative Western blot performed in the different areas with an ARTN specific antibody; a colorimetric method was used to visualize the peroxidase-coated bands. Results are expressed as the mean⫾SEM of five different animals; ␤-actin normalization was performed for each sample. Measurements in control samples (sham, saline) were assigned a relative value of 100%. * P⬍0.05, significantly different from sham, saline; ^ P⬍0.05 significantly different from CCI, saline. strongly suggest these factors as therapeutic agents in neuropathy. Our data showed a GDNF decrease in the peripheral (DRG) but not in the CNS of CCI rats. On the other hand, in the same rat model Nagano et al. (2003) showed that the GDNF content in DRG was markedly decreased at day 7 after the operation and stayed at low levels at day 14; comparable reductions of GDNF levels were observed in DRG on the injured side at 14 postoperative days in a model of spinal nerve ligation. GDNF decrease seems to be not specifically related to neuropathic pain since GDNF was down-regulated in both dorsal root ganglia and spinal cord of rats with chronic inflammation induced by injection of complete Freund’s adjuvant (Fang et al., 2003). Spinal infusion of GDNF prevents abnormal pain behaviour in the L5 spinal nerve ligation and partial sciatic nerve ligation models in the rat (Boucher et al., 2000; Boucher and McMahon, 2001). McMahon and coworkers (Pezet et al., 2006) also evaluated the possibility to administer GDNF by the use of lentiviral vectors. The GDNF-expressing vectors were injected unilaterally into the spinal dorsal horn 5 weeks before a spinal nerve ligation producing a partial but significant reversal of thermal and mechanical hyperalgesia. Despite these effects of GDNF on experimentally induced neuropathic pain, an important caveat regarding GDNF therapy is that it elicits clinically unacceptable side effects in vivo, such as weight loss and allodynia (Nutt et al., 2003; Kordower et al., 1999). On the other hand this strategy might increase the risk of cancer because constant activation of its functional receptor Ret by continuously GDNF can lead to malignancy (Bespalov and Saarma, 2007). Along GDFN, ARTN is the neurotrophic factor mainly involved in the strategy of pain treatment. In 2003, Porreca and coworkers demonstrated that systematically administration of ARTN normalized the behavioural hypersensitivity to mechanical and thermal stimuli in rat that underwent to spinal nerve ligation (Gardell et al., 2003). ARTN treatment was able to restore sensorimotor functions and improve morphological and neurochemical features of the injury state induced by spinal nerve axotomy (Bennett et E. Vivoli et al. / Neuroscience 167 (2010) 1168 –1174 al., 2006) and dorsal root crush (Wang et al., 2008). Trophic effect of ARTN seems orientated to sensory neurons where its receptor GFR␣3 is mainly expressed. Thus ARTN could be a valuable tool to affect neuropathic pain without having a broader effects on other organs and tissues (Sah et al., 2003). For the first time, at our knowledge, in the present article a measure of ARTN levels was performed in DRG, spinal cord and PAG. In respect to GDNF, ARTN levels was more involved in the CCI-induced alterations. ARTN expression was reduced in DRG, not only ipsilaterally but also in the contralateral part. On the other hand the peripheral injury was able to induce ARTN decrease at spinal and supraspinal level. A mirror effect in respect to NGF increase. In this pathological condition ALCAR repeated treatments displayed an antihyperalgesic effect. Contrary to the great part of the compounds clinically-used to treat neuropathic pain ALCAR joints this effect with neurorestorative properties and a good safety profile. On the other hand, ALCAR shows any analgesic or antihyperalgesic effect after a single administration (data not shown) allowing to hypothesize a relationship among the efficacy on pain relief and the neuroprotective profile observed after a repeated treatment. Clinical studies on diabetic peripheral neuropathy show that ALCAR reduces pain sensation and improves nerve conduction velocities and regeneration (Evans et al., 2008). Open studies involving HIV-positive patients have shown that a chronic treatment with ALCAR ameliorates pain symptoms related to peripheral polyneuropathy (Scarpini et al., 1997). Hart and colleagues (2002) report that 6 months of oral ALCAR treatment results in peripheral nerve regeneration of small sensory fibers as observed from skin biopsies in patients with distal symmetrical polyneuropathy. Finally, in our laboratory it has been highlighted that the repeated treatment with ALCAR prevents the apoptotic cascade in the peripheral nerve after CCI (Di Cesare Mannelli et al., 2007, 2009). Only the antiapoptotic effect has been related to a specific pharmacodynamic property of ALCAR (Di Cesare Mannelli et al., 2009) while the neuroregenerative profile has been associated to the important ALCAR role for energetics in the brain. ALCAR transports fatty acids from the cell cytoplasm into the mitochondria where they provide a substrate for ATP generation via oxidative phosphorylation (Kidd, 2008). The evidences shown in the present research suggest the capability of ALCAR to restore altered levels of all the evaluated neurotrophic factors: it prevents NGF increase as well as GDNF and ARTN decrease. At least for NGF and GDNF this effect seems strictly related to the injury since ALCAR did not increase these factors in sham rats. On the other hand an ALCAR normalizing effect on altered neurotrophic factors has been described. In condition of NGF depletion, as aged rats or rats subjected to total fimbria–fornix transection, ALCAR upregulated the expression of the NGF and of its receptor, p75NGFR, in the CNS (Piovesan et al., 1994; Foreman et al., 1995). 1173 Characteristically, ALCAR is able to increase ARTN levels in a pathology-independent manner since it increases ARTN levels both in the peripheral and CNS of sham animals in respect to saline-treated group. 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