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Serotonin transporter deficiency protects mice from mechanical allodynia and heat hyperalgesia in vincristine neuropathy
Neuroscience Letters 495 (2011) 93–97
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Serotonin transporter deficiency protects mice from mechanical allodynia and heat hyperalgesia in vincristine neuropathy Niels Hansen a,∗ , Nurcan Üc¸eyler a , Florian Palm a , Marek Zelenka a , Lydia Biko a , Klaus-Peter Lesch b , Manfred Gerlach c , Claudia Sommer a a b c
Department of Neurology, University of Würzburg, Josef-Schneider-Straße 11, 97080 Würzburg, Germany Molecular Psychiatry, Department of Psychiatry, Psychosomatics and Psychotherapy, University of Würzburg, Füchsleinstraße 15, 97080 Würzburg, Germany Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, University of Würzburg, Füchsleinstraße 15, 97080 Würzburg, Germany
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Article history: Received 24 January 2011 Received in revised form 3 March 2011 Accepted 10 March 2011 Keywords: Heat hyperalgesia Macrophages Mechanical allodynia Peripheral nerve injury Serotonin transporter knockout mice Vincristine neuropathy
a b s t r a c t Painful vincristine (VCR) neuropathy is a frequent and dose-limiting problem in cancer treatment. Here, we investigated how pain behavior is modulated in mice lacking the serotonin transporter (5-HTT−/− mice) after inducing neuropathy by intraperitoneal injections of VCR. We used standard tests for evoked pain, high performance liquid chromatography to measure serotonin (5-HT), and immunohistochemistry of L4/5 dorsal root ganglia (DRG) to assess neuronal injury and inflammation. After injections of VCR, 5-HTT−/− mice did not develop hypersensitivity to heat, in contrast to their wildtype (wt) littermates (p < 0.05). Also, 5-HTT−/− mice recovered faster from mechanical hypersensitivity than wt mice (p < 0.05). 5-HT levels were lower in the peripheral and central nervous tissue of vehicle or VCR-treated 5-HTT−/− mice compared to wt mice. VCR-treated mice had higher numbers of injured neurons as identified by immunostaining for activating transcription factor 3, and more immunoreactive macrophages in the L4/5 DRG than vehicle-treated mice. There was no difference between genotypes. Thus the 5-HTT−/− genotype did not protect mice from VCR-induced neuronal injury and macrophage infiltration in the DRG. Our results suggest that the reduced peripheral 5-HT levels of 5-HTT−/− mice in VCR neuropathy underlie the lack of heat hyperalgesia. Conversely, attenuation of mechanical allodynia in 5-HTT−/− mice may indicate reduced 5-HT-mediated facilitation in the central nervous system. © 2011 Elsevier Ireland Ltd. All rights reserved.
Vincristine (VCR) is an anti-cancer drug used to treat many types of cancer, i.e., leukemias, lymphomas and sarcomas. It belongs to the group of Vinca alkaloids; its antineoplastic action is based on the disaggregation of tubulin monomers in mitosis, which inhibits cell division [17]. VCR treatment in rats results in disorganization of the axonal microtubule cytoskeleton, and swelling of unmyelinated axons and large-diameter sensory neurons in the spinal ganglion, suggestive of impaired axonal transport [32,33]. In humans, painful peripheral neuropathy is a frequent and therapy-limiting complication of VCR treatment [12]. There is evidence that the development of pain behavior in mice is modulated by the efficiency of the serotonin transporter (5-HTT) [23,36]. Serotonin (5-HT) plays several roles in pain processing and
Abbreviations: 5-HT, 5-hydroxytryptamine, serotonin; 5-HTT, serotonin transporter; ATF 3, activating transcription factor 3; CNS, central nervous system; DRG, dorsal root ganglia; HPLC, high performance liquid chromatography; ko, knock out; MAC1, mouse macrophages; PNS, peripheral nervous system; sec, seconds; VCR, vincristine; wt, wildtype. ∗ Corresponding author. Tel.: +49 931 201 23757; fax: +49 931 201 23761. E-mail address: Hansen [email protected] (N. Hansen). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.03.035
modulation [6]. In the periphery, 5-HT exerts direct actions on Cfibers [21] after being released from platelets and mast cells [5]. In the rostro-ventral medulla (RVM), 5-HT is released from descending neurons that facilitate spinal nociception [30]. Mice with a 5-HTT deficiency (5-HTT−/− mice) have an overall reduced tissue content of 5-HT [3,15,36]. These mice have attenuated thermal hyperalgesia in the neuropathic pain model of chronic constriction injury (CCI) of the sciatic nerve and in hind paw inflammation induced by complete Freund’s adjuvant (CFA) [23,36]. Mechanical hypersensitivity in the mouse CCI model was not attenuated in 5HTT−/− mice [36], but was reduced by pharmacological depletion of spinal 5-HT in a spinal nerve ligation model in rats [24]. Because of the growing interest in the 5-HTT (SLC6A4) gene in human disorders [22] and the high incidence of chemotherapy-induced neuropathy, we investigated pain behavior in VCR neuropathy in 5-HTT−/− mice. We hypothesized that concomitant with a reduced peripheral 5-HT content, 5-HTT−/− mice might demonstrate attenuated pain behavior compared to wildtype (wt) littermates. Experiments were performed in 29 adult female mice of C57BL/6J background, 15 wt (5-HTT+/+) and 14 homozygous knockout (5-HTT−/−) littermates. Mean body weight was 35 ± 8 g. The genotype was determined according to Bengel et al. [3]. Mice
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were housed in groups in a temperature- and humidity-controlled environment with a 12/12 h light/dark cycle and standard rodent nutrition. Experiments were done during the light cycle. All experiments were approved by the Bavarian State authorities and performed in accordance with the European Communities Council Directive of November 1986 (86/609/EEC) for the care and use of laboratory animals. Eight 5-HTT−/− and nine wt mice were treated with VCR i.p. (0.5 mg/kg body weight), and six 5-HTT−/− mice and six wt mice received an equivalent volume of 0.9% saline as placebo treatment. VCR sulfate (Sigma Germany) was dissolved in 0.9% saline to a final concentration of 0.05 mg/ml. VCR was administered for five consecutive days, followed by a two-day pause and subsequent treatment for five more consecutive days. Before the first and sixth injections, the drug dosage was adapted to body weight. The investigator was unaware of the genotype and treatment. Mice were tested on three days to determine baseline thresholds. They were then tested on days 14 and 18 after starting the VCR or saline injections. Both hind paws were tested alternately at an interval of at least 2 min. Mechanical sensitivity was determined by probing the plantar surface of the hind paw with calibrated von Frey hairs with bending forces from 4 mg to 1.48 g. The 50% mechanical withdrawal threshold (the force of the von Frey hair in grams to which an animal reacts in 50% of the presentations) was determined with hairs applied six times on the basis of the up-and-down method as previously described and modified for mice [28]. The time interval between two trials was at least 1 min on the same paw and at least 30 s on the alternate paw. We refer to a significant decrease in the withdrawal threshold compared to baseline as mechanical allodynia. The withdrawal latency to heat stimulation (thermal sensitivity) was tested using a device of Hargreaves et al. [11] purchased from Ugo Basile (Comerio, Italy). The time until the animals reacted by withdrawing from the stimulation by a radiant heat source was determined automatically. Each testing period consisted of at least three presentations for each hind paw. The value of 12 s was assumed if the animal did not react within 12 s, and the stimulation was stopped to prevent tissue damage. A significant decrease in the mean withdrawal latency after injections compared to the control group receiving saline was defined as heat hyperalgesia. Tissue was collected under deep barbiturate anesthesia on day 19 after the first VCR or saline injection. At first we harvested a 1cm piece of each sciatic nerve on both sides from the middle of the thigh. The animals were sacrificed by decapitation; the brain was removed and dissected on an ice-cold glass plate. After removal of the hypothalamus, the brain was bisected sagitally to reveal and dissect the hippocampus and thalamus from each hemisphere. Finally the dorsal root ganglia (DRG) L5 were dissected and a section of the spinal cord was cut just proximally to the L4 and distally to the L5 spinal root. Tissue samples for histology were embedded in Tissue Tek® (Sakura Finetek, Zoeterwoude, The Netherlands), frozen in 2-methylbutan cooled in liquid nitrogen, and stored at −80 ◦ C. Tissue for 5-HT analysis was weighed and immediately frozen at −80 ◦ C. For high performance liquid chromatography (HPLC), samples were thawed, sonicated under argon in ice-cold 150 mM H3 PO4 and 500 M diethylene triamine pentaacetic acid and centrifuged at 35,000 × g for 20 min at +4 ◦ C. The supernatant was filtered through Milipore Ultrafree-MC filter cups at 9,000 × g, 4 ◦ C, for 1–2 h, and 50 l of the filtrate was analyzed for 5-HT by reverse-phase HPLC with electrochemical detection as described previously [36]. Cryosections (10 m) from DRG were mounted on Super Frost Slides from Langenbrinck (Emmerdingen, Germany) and stored at −20 ◦ C. Immunhistochemistry was performed as previously described [26] using primary antibodies to activating transcription factor 3 (ATF 3, 1: 200, rabbit anti-human/rat/mouse, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse macrophages
(MAC-1, 1: 50, rat anti-mouse, Camon, Wiesbaden, Germany). Stains were developed with an ABC method (Vector Laboratories, Burlingame, CA, USA) and 0.02% diaminobenzidine as chromogen. The DRG sections were analyzed using an Axiophot 2 microscope from Zeiss (Oberkochen, Germany), a video camera (DXC 003P) from Sony (Tokyo, Japan), the software SPOT imaging Version 4.5 for Windows XP (SPOT imaging solutions, Burroughs, Sterling Heights, USA) and Image J (Image and Processing Analysing in Java, Wayne R., 1.43r, National Institute of Health, USA, 2010). A counterstain with haemalaun was performed to differentiate anatomical structures. ATF 3 is a sensitive marker of neuronal injury and is activated after diverse noxious chemical stimuli [34]. To determine immunoreactive DRG cells in ATF3 stains, ATF 3 immunoreactive nuclei in DRG neurons were counted and expressed as percent of all neuronal nuclei. To quantify macrophages, the MAC-1 immunoreactive cells were counted in three sections per mouse and expressed as positive cells per DRG section. The total cross-sectional areas of MAC1 and ATF3 sections did not differ between groups (t-test: p > 0.25). We used Predictive Analytics Software (PASW Version 18.0; IBM SPSS, New York, 2009) for statistical analysis. Results are presented as mean ± standard error of the mean (SEM). The normal distribution of data was shown by a Kolmogorov–Smirnov test. To compare the behavioral data between groups, test sides and test days, a three-way repeated measures ANOVA was used for parametrical analysis, followed by an LSD post hoc analysis. For nonparametric analysis of the von Frey thresholds, a Mann–Whitney U test with a Bonferroni correction for multiple testing was used to compare data between test days. A one-way ANOVA followed by an LSD post hoc analysis was used to compare 5-HT levels between groups. A Student’s t-test was used to compare ATF3 and MAC-1 immunostaining data between groups. Differences were considered statistically significant if p < 0.05. The withdrawal latencies to thermal and the withdrawal threshold to mechanical stimuli did not differ between the right and left hind paws in all treatment conditions; we therefore pooled the data. Baseline values for withdrawal latencies to thermal stimuli did not differ between wt and 5-HTT−/− mice treated (n = 8–10 per genotype). On day 14 after the start of VCR injections wt but not 5-HTT−/− mice revealed heat hypersensitivity with a further reduction in withdrawal latencies on day 18 (LSD post hoc test: baseline vs. day 14, p = 0.05, baseline vs. day 18, p < 0.001; Fig. 1A). Baseline withdrawal thresholds to von Frey hairs did not differ between genotypes (n = 8–10 per genotype). Withdrawal thresholds decreased significantly in both genotypes on day 14 after the start of VCR injections (Mann–Whitney U test: 5-HTT−/− mice and wt mice vs. baseline: p < 0.05; Fig. 1B). On day 18 withdrawal thresholds were back to normal in 5-HTT−/− mice (Fig. 1B), whereas wt mice still showed pronounced mechanical hypersensitivity (Mann–Whitney U test: VCR-treated wt vs. baseline, p < 0.05 vs. baseline, Fig. 1B). 5-HT levels were lower in hippocampus, hypothalamus, thalamus, and L4/5 spinal cord in VCR and saline-treated 5-HTT−/− mice as well as in the sciatic nerve in VCR-treated 5-HTT−/− mice compared to wt mice (LSD post hoc test: p < 0.05; Fig. 2A–E). 5-HT levels did not differ between mice with saline and VCR injections within genotypes (Fig. 2A–E). Quantification of immunohistochemistry revealed a significant increase in ATF 3 positive nuclei in DRG of both wt and 5-HTT−/− mice treated with VCR compared to saline (t-test: p < 0.05; Fig. 3A). The number of macrophages per DRG section was significantly higher in wt mice treated with VCR than in saline-treated mice (t-test: p < 0.05). Macrophage numbers did not increase in the 5HTT−/− mice after VCR (t-test: p = 0.7; Fig. 3B). We provide evidence supporting the hypothesis that the 5HTT−/− genotype protects mice from pain behavior induced by
N. Hansen et al. / Neuroscience Letters 495 (2011) 93–97
Fig. 1. Lack of heat hypersensitivity and attenuation of mechanical hypersensitivity in 5-HTT−/− mice. (A) VCR injection induced reduction in thermal withdrawal latencies in wt mice (LSD post hoc test: **p < 0.001 VCR wt mice on day 18 vs. baseline and *p < 0.05 VCR vs. NaCl-treated wt mice). Withdrawal latencies in VCR- treated 5-HTT−/− mice did not decrease over time and were different from VCR treated wt mice (LSD post hoc test: VCR-treated 5-HTT−/− vs. VCR-treated wt mice, +p < 0.05). (B) Withdrawal thresholds of wt and 5-HTT−/− mice to von Frey hairs decreased significantly on day 14 after VCR injection (Mann–Whitney U test: *p < 0.05 VCR-treated wt vs. baseline, +p < 0.05 VCR-treated 5-HTT−/− mice vs. baseline). On day 18 mechanical withdrawal thresholds were back to normal in 5-HTT−/− mice, whereas wt mice showed even more pronounced mechanical hypersensitivity (Mann–Whitney U test, *p < 0.05 vs. baseline).
VCR. The modulation of both mechanical and heat sensitivity in 5HTT−/− mice first shown in VCR neuropathy was associated with a reduced 5-HT tissue content in the peripheral nervous system (PNS) and central nervous system (CNS). Functionally ablated 5-HTT in 5-HTT−/− mice entails reduced tissue 5-HT concentrations, as 5-HTT is needed for 5-HT to be taken up into the cells. Accordingly, 5-HTT−/−mice had lower levels of 5HT than wt mice in the PNS and CNS. This confirms previous studies showing reduced 5-HT in peripheral [23,36] and central nervous tissue. No difference in thermal nociception was found between naive 5-HTT−/− and wt mice, suggesting that the loss of 5-HTT did not lead to altered thermal pain sensation by itself [13]. Nociceptive nerve fibers [2,29] and injured axons in particular [18,19] can be sensitized by 5-HT at the receptor site on the sensory nerve terminal. Thus we hypothesize that 5-HT in the injured nerve after VCR injection may sensitize nociceptive fibers to heat stimuli, which leads to heat hyperalgesia. The low 5-HT tissue concentration in 5-HTT−/− mice treated with VCR may thus underlie their diminished heat hyperalgesia. Reduced thermal hyperalgesia in 5-HTT−/− mice with low 5-HT content has been shown in two other models of neuropathic pain [24,36]. Reduced expression of 5-HT1A,B and especially 5-HT2A receptors may further contribute to decreased thermal hyperalgesia in 5-HTT−/− mice [7,25].
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Fig. 2. 5-HT tissue content measured by HPLC. After ten days of VCR or saline injection, 5-HT tissue content was lower in the hippocampus (A), hypothalamus (B), thalamus (C) and spinal cord (D) of 5-HTT−/− mice compared with wt mice (LSD post hoc test: p < 0.05). In the sciatic nerve (E) 5-HT tissue content was lower in 5-HTT−/− than wt mice only after VCR but not saline injection (LSD post hoc test: p < 0.05). Data are expressed as mean ± standard deviation. *p < 0.05.
Punctate mechanical allodynia depends on central sensitization mediated by A␦-primary sensory neurons, as exemplified in the CCI model [9]. Descending facilitation and loss of inhibition are mechanisms of central sensitization [35]. Descending pathways are known to be sources of 5-HT in the spinal cord [10,19]. The actions of these descending serotoninergic pathways have long been associated with suppression of nociceptive inputs [1,6], but evidence has accumulated supporting a facilitatory effect with regard to nociceptive input [6,20]. When 5-HT is released from descending neurons from the RVM, it may activate 5-HT3 receptors localized on the nerve terminals of a subpopulation of small-diameter afferents, thereby enhancing neurotransmitter release in the dorsal horn [30]. Remarkably, a direct activation of silent glutamatergic synapses between the afferent fibers and spinal dorsal horn neurons by 5HT has been demonstrated in rats, indicating a further mechanism of central sensitization via 5-HT [16]. Assuming that 5-HT in VCRtreated mice exerts a predominantly facilitatory influence on pain transmission, especially on the mechanically evoked responses of dorsal horns neurons [31], the reversed mechanical allodynia in 5HTT−/− mice may be attributed to a loss in spinal facilitation due to a lack of spinal 5-HT. There are several possible reasons why the
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increased ATF 3 expression one day after the last VCR injection and 13 days after the first vincristine injection being concordant with the time course of ATF 3 expression over two weeks in a model of axonal sciatic nerve injury [34] similar to the axonal damage in VCR neuropathy [27]. Nevertheless, recent evidence indicates that the distal axon is more vulnerable than the perikaryon to VCR’s toxic effect in the DRG-cell culture of rats [27]. Histological sections of the sciatic nerves revealed axonal degeneration and demyelination in rats with VCR-induced neuropathy [8]. Our data and findings reported in the literature support the view that VCR’s toxic effects affect both neuronal somata in the DRG and axons. Unlike our earlier results in the CFA model [23], the 5-HTT−/− genotype did not protect murine DRG neurons from VCR-induced injury in DRG, indicating that the 5-HTT−/− genotype has different functional implications in toxic as opposed to inflammatory neuropathies. The higher number of invading macrophages found in DRG of VCR-treated mice may contribute to VCR-induced hyperalgesia, as macrophages release pro-inflammatory cytokines possibly involved in the development and maintenance of neuropathic pain. Macrophage-derived interleukin-6, for example, has been shown to play a critical role in VCR-induced mechanical hypersensitivity [14]. However, the 5-HTT−/− genotype is not protected from macrophage infiltration in the DRG, indicating that the prevention of heat hyperalgesia and reversal of mechanical allodynia in 5-HTT−/− mice is independent of inflammation due to macrophage influx. Taken together, the 5-HTT−/− genotype may protect from VCRinduced heat and mechanical hypersensitivity due to lower 5-HT levels in the PNS and CNS. Furthermore, our data suggest that peripheral nerve injury and macrophage infiltration in the DRG are not modified by the 5-HTT−/− genotype in VCR-induced neuropathy. Contributions NH, CS and NU wrote the manuscript and performed statistical analysis; FP, LB, NU and MZ conducted the experiments; KPL and MG critically revised this manuscript for important intellectual content. Acknowledgements Fig. 3. Morphometric determination of ATF 3 (A) and macrophage (B) immunoreactivity in DRG neurons. (A) DRG from VCR-treated mice have a higher percentage of positive nuclei than saline treated rats (t-test: p < 0.05). (B) Macrophage numbers are increased per DRG of VCR-treated wt mice compared to saline-treated wt mice (t-test: p < 0.05). *p < 0.05.
VCR-treated 5HTT−/− mice revealed a degree of mechanical allodynia on day 14. One reason may be due to the different mechanisms responsible for the initiation and maintenance state of mechanical allodynia in 5-HTT−/− mice. Recent data showed that the RVM is important for time-dependent facilitation, but not the initiation of neuropathic pain, indicating that descending serotonergic modulation may alter pain processing after a certain time delay [4]. Unfortunately, we could not directly analyze the 5-HT content in RVM samples, as the murine RVM is too small to be dissected with enough precision to analyze 5-HT content reliably. The finding of increased numbers of ATF 3 immunoreactive nuclei in DRG neurons in VCR-treated mice indicates that peripheral injury in VCR-induced neuropathy did indeed have an impact on the neuronal somata of DRG neurons. The amount and time course of ATF 3 expression differs between injury models [34]. The time of tissue harvesting was determined by behavioral testing. A limit of our study is that we did not use additional mice to test ATF 3 expression at different time points. However, we observed an
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Serotonin transporter deficiency protects mice from mechanical allodynia and heat hyperalgesia in vincristine neuropathy Niels Hansen a,∗ , Nurcan Üc¸eyler a , Florian Palm a , Marek Zelenka a , Lydia Biko a , Klaus-Peter Lesch b , Manfred Gerlach c , Claudia Sommer a a b c
Department of Neurology, University of Würzburg, Josef-Schneider-Straße 11, 97080 Würzburg, Germany Molecular Psychiatry, Department of Psychiatry, Psychosomatics and Psychotherapy, University of Würzburg, Füchsleinstraße 15, 97080 Würzburg, Germany Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, University of Würzburg, Füchsleinstraße 15, 97080 Würzburg, Germany
a r t i c l e
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Article history: Received 24 January 2011 Received in revised form 3 March 2011 Accepted 10 March 2011 Keywords: Heat hyperalgesia Macrophages Mechanical allodynia Peripheral nerve injury Serotonin transporter knockout mice Vincristine neuropathy
a b s t r a c t Painful vincristine (VCR) neuropathy is a frequent and dose-limiting problem in cancer treatment. Here, we investigated how pain behavior is modulated in mice lacking the serotonin transporter (5-HTT−/− mice) after inducing neuropathy by intraperitoneal injections of VCR. We used standard tests for evoked pain, high performance liquid chromatography to measure serotonin (5-HT), and immunohistochemistry of L4/5 dorsal root ganglia (DRG) to assess neuronal injury and inflammation. After injections of VCR, 5-HTT−/− mice did not develop hypersensitivity to heat, in contrast to their wildtype (wt) littermates (p < 0.05). Also, 5-HTT−/− mice recovered faster from mechanical hypersensitivity than wt mice (p < 0.05). 5-HT levels were lower in the peripheral and central nervous tissue of vehicle or VCR-treated 5-HTT−/− mice compared to wt mice. VCR-treated mice had higher numbers of injured neurons as identified by immunostaining for activating transcription factor 3, and more immunoreactive macrophages in the L4/5 DRG than vehicle-treated mice. There was no difference between genotypes. Thus the 5-HTT−/− genotype did not protect mice from VCR-induced neuronal injury and macrophage infiltration in the DRG. Our results suggest that the reduced peripheral 5-HT levels of 5-HTT−/− mice in VCR neuropathy underlie the lack of heat hyperalgesia. Conversely, attenuation of mechanical allodynia in 5-HTT−/− mice may indicate reduced 5-HT-mediated facilitation in the central nervous system. © 2011 Elsevier Ireland Ltd. All rights reserved.
Vincristine (VCR) is an anti-cancer drug used to treat many types of cancer, i.e., leukemias, lymphomas and sarcomas. It belongs to the group of Vinca alkaloids; its antineoplastic action is based on the disaggregation of tubulin monomers in mitosis, which inhibits cell division [17]. VCR treatment in rats results in disorganization of the axonal microtubule cytoskeleton, and swelling of unmyelinated axons and large-diameter sensory neurons in the spinal ganglion, suggestive of impaired axonal transport [32,33]. In humans, painful peripheral neuropathy is a frequent and therapy-limiting complication of VCR treatment [12]. There is evidence that the development of pain behavior in mice is modulated by the efficiency of the serotonin transporter (5-HTT) [23,36]. Serotonin (5-HT) plays several roles in pain processing and
Abbreviations: 5-HT, 5-hydroxytryptamine, serotonin; 5-HTT, serotonin transporter; ATF 3, activating transcription factor 3; CNS, central nervous system; DRG, dorsal root ganglia; HPLC, high performance liquid chromatography; ko, knock out; MAC1, mouse macrophages; PNS, peripheral nervous system; sec, seconds; VCR, vincristine; wt, wildtype. ∗ Corresponding author. Tel.: +49 931 201 23757; fax: +49 931 201 23761. E-mail address: Hansen [email protected] (N. Hansen). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.03.035
modulation [6]. In the periphery, 5-HT exerts direct actions on Cfibers [21] after being released from platelets and mast cells [5]. In the rostro-ventral medulla (RVM), 5-HT is released from descending neurons that facilitate spinal nociception [30]. Mice with a 5-HTT deficiency (5-HTT−/− mice) have an overall reduced tissue content of 5-HT [3,15,36]. These mice have attenuated thermal hyperalgesia in the neuropathic pain model of chronic constriction injury (CCI) of the sciatic nerve and in hind paw inflammation induced by complete Freund’s adjuvant (CFA) [23,36]. Mechanical hypersensitivity in the mouse CCI model was not attenuated in 5HTT−/− mice [36], but was reduced by pharmacological depletion of spinal 5-HT in a spinal nerve ligation model in rats [24]. Because of the growing interest in the 5-HTT (SLC6A4) gene in human disorders [22] and the high incidence of chemotherapy-induced neuropathy, we investigated pain behavior in VCR neuropathy in 5-HTT−/− mice. We hypothesized that concomitant with a reduced peripheral 5-HT content, 5-HTT−/− mice might demonstrate attenuated pain behavior compared to wildtype (wt) littermates. Experiments were performed in 29 adult female mice of C57BL/6J background, 15 wt (5-HTT+/+) and 14 homozygous knockout (5-HTT−/−) littermates. Mean body weight was 35 ± 8 g. The genotype was determined according to Bengel et al. [3]. Mice
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were housed in groups in a temperature- and humidity-controlled environment with a 12/12 h light/dark cycle and standard rodent nutrition. Experiments were done during the light cycle. All experiments were approved by the Bavarian State authorities and performed in accordance with the European Communities Council Directive of November 1986 (86/609/EEC) for the care and use of laboratory animals. Eight 5-HTT−/− and nine wt mice were treated with VCR i.p. (0.5 mg/kg body weight), and six 5-HTT−/− mice and six wt mice received an equivalent volume of 0.9% saline as placebo treatment. VCR sulfate (Sigma Germany) was dissolved in 0.9% saline to a final concentration of 0.05 mg/ml. VCR was administered for five consecutive days, followed by a two-day pause and subsequent treatment for five more consecutive days. Before the first and sixth injections, the drug dosage was adapted to body weight. The investigator was unaware of the genotype and treatment. Mice were tested on three days to determine baseline thresholds. They were then tested on days 14 and 18 after starting the VCR or saline injections. Both hind paws were tested alternately at an interval of at least 2 min. Mechanical sensitivity was determined by probing the plantar surface of the hind paw with calibrated von Frey hairs with bending forces from 4 mg to 1.48 g. The 50% mechanical withdrawal threshold (the force of the von Frey hair in grams to which an animal reacts in 50% of the presentations) was determined with hairs applied six times on the basis of the up-and-down method as previously described and modified for mice [28]. The time interval between two trials was at least 1 min on the same paw and at least 30 s on the alternate paw. We refer to a significant decrease in the withdrawal threshold compared to baseline as mechanical allodynia. The withdrawal latency to heat stimulation (thermal sensitivity) was tested using a device of Hargreaves et al. [11] purchased from Ugo Basile (Comerio, Italy). The time until the animals reacted by withdrawing from the stimulation by a radiant heat source was determined automatically. Each testing period consisted of at least three presentations for each hind paw. The value of 12 s was assumed if the animal did not react within 12 s, and the stimulation was stopped to prevent tissue damage. A significant decrease in the mean withdrawal latency after injections compared to the control group receiving saline was defined as heat hyperalgesia. Tissue was collected under deep barbiturate anesthesia on day 19 after the first VCR or saline injection. At first we harvested a 1cm piece of each sciatic nerve on both sides from the middle of the thigh. The animals were sacrificed by decapitation; the brain was removed and dissected on an ice-cold glass plate. After removal of the hypothalamus, the brain was bisected sagitally to reveal and dissect the hippocampus and thalamus from each hemisphere. Finally the dorsal root ganglia (DRG) L5 were dissected and a section of the spinal cord was cut just proximally to the L4 and distally to the L5 spinal root. Tissue samples for histology were embedded in Tissue Tek® (Sakura Finetek, Zoeterwoude, The Netherlands), frozen in 2-methylbutan cooled in liquid nitrogen, and stored at −80 ◦ C. Tissue for 5-HT analysis was weighed and immediately frozen at −80 ◦ C. For high performance liquid chromatography (HPLC), samples were thawed, sonicated under argon in ice-cold 150 mM H3 PO4 and 500 M diethylene triamine pentaacetic acid and centrifuged at 35,000 × g for 20 min at +4 ◦ C. The supernatant was filtered through Milipore Ultrafree-MC filter cups at 9,000 × g, 4 ◦ C, for 1–2 h, and 50 l of the filtrate was analyzed for 5-HT by reverse-phase HPLC with electrochemical detection as described previously [36]. Cryosections (10 m) from DRG were mounted on Super Frost Slides from Langenbrinck (Emmerdingen, Germany) and stored at −20 ◦ C. Immunhistochemistry was performed as previously described [26] using primary antibodies to activating transcription factor 3 (ATF 3, 1: 200, rabbit anti-human/rat/mouse, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse macrophages
(MAC-1, 1: 50, rat anti-mouse, Camon, Wiesbaden, Germany). Stains were developed with an ABC method (Vector Laboratories, Burlingame, CA, USA) and 0.02% diaminobenzidine as chromogen. The DRG sections were analyzed using an Axiophot 2 microscope from Zeiss (Oberkochen, Germany), a video camera (DXC 003P) from Sony (Tokyo, Japan), the software SPOT imaging Version 4.5 for Windows XP (SPOT imaging solutions, Burroughs, Sterling Heights, USA) and Image J (Image and Processing Analysing in Java, Wayne R., 1.43r, National Institute of Health, USA, 2010). A counterstain with haemalaun was performed to differentiate anatomical structures. ATF 3 is a sensitive marker of neuronal injury and is activated after diverse noxious chemical stimuli [34]. To determine immunoreactive DRG cells in ATF3 stains, ATF 3 immunoreactive nuclei in DRG neurons were counted and expressed as percent of all neuronal nuclei. To quantify macrophages, the MAC-1 immunoreactive cells were counted in three sections per mouse and expressed as positive cells per DRG section. The total cross-sectional areas of MAC1 and ATF3 sections did not differ between groups (t-test: p > 0.25). We used Predictive Analytics Software (PASW Version 18.0; IBM SPSS, New York, 2009) for statistical analysis. Results are presented as mean ± standard error of the mean (SEM). The normal distribution of data was shown by a Kolmogorov–Smirnov test. To compare the behavioral data between groups, test sides and test days, a three-way repeated measures ANOVA was used for parametrical analysis, followed by an LSD post hoc analysis. For nonparametric analysis of the von Frey thresholds, a Mann–Whitney U test with a Bonferroni correction for multiple testing was used to compare data between test days. A one-way ANOVA followed by an LSD post hoc analysis was used to compare 5-HT levels between groups. A Student’s t-test was used to compare ATF3 and MAC-1 immunostaining data between groups. Differences were considered statistically significant if p < 0.05. The withdrawal latencies to thermal and the withdrawal threshold to mechanical stimuli did not differ between the right and left hind paws in all treatment conditions; we therefore pooled the data. Baseline values for withdrawal latencies to thermal stimuli did not differ between wt and 5-HTT−/− mice treated (n = 8–10 per genotype). On day 14 after the start of VCR injections wt but not 5-HTT−/− mice revealed heat hypersensitivity with a further reduction in withdrawal latencies on day 18 (LSD post hoc test: baseline vs. day 14, p = 0.05, baseline vs. day 18, p < 0.001; Fig. 1A). Baseline withdrawal thresholds to von Frey hairs did not differ between genotypes (n = 8–10 per genotype). Withdrawal thresholds decreased significantly in both genotypes on day 14 after the start of VCR injections (Mann–Whitney U test: 5-HTT−/− mice and wt mice vs. baseline: p < 0.05; Fig. 1B). On day 18 withdrawal thresholds were back to normal in 5-HTT−/− mice (Fig. 1B), whereas wt mice still showed pronounced mechanical hypersensitivity (Mann–Whitney U test: VCR-treated wt vs. baseline, p < 0.05 vs. baseline, Fig. 1B). 5-HT levels were lower in hippocampus, hypothalamus, thalamus, and L4/5 spinal cord in VCR and saline-treated 5-HTT−/− mice as well as in the sciatic nerve in VCR-treated 5-HTT−/− mice compared to wt mice (LSD post hoc test: p < 0.05; Fig. 2A–E). 5-HT levels did not differ between mice with saline and VCR injections within genotypes (Fig. 2A–E). Quantification of immunohistochemistry revealed a significant increase in ATF 3 positive nuclei in DRG of both wt and 5-HTT−/− mice treated with VCR compared to saline (t-test: p < 0.05; Fig. 3A). The number of macrophages per DRG section was significantly higher in wt mice treated with VCR than in saline-treated mice (t-test: p < 0.05). Macrophage numbers did not increase in the 5HTT−/− mice after VCR (t-test: p = 0.7; Fig. 3B). We provide evidence supporting the hypothesis that the 5HTT−/− genotype protects mice from pain behavior induced by
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Fig. 1. Lack of heat hypersensitivity and attenuation of mechanical hypersensitivity in 5-HTT−/− mice. (A) VCR injection induced reduction in thermal withdrawal latencies in wt mice (LSD post hoc test: **p < 0.001 VCR wt mice on day 18 vs. baseline and *p < 0.05 VCR vs. NaCl-treated wt mice). Withdrawal latencies in VCR- treated 5-HTT−/− mice did not decrease over time and were different from VCR treated wt mice (LSD post hoc test: VCR-treated 5-HTT−/− vs. VCR-treated wt mice, +p < 0.05). (B) Withdrawal thresholds of wt and 5-HTT−/− mice to von Frey hairs decreased significantly on day 14 after VCR injection (Mann–Whitney U test: *p < 0.05 VCR-treated wt vs. baseline, +p < 0.05 VCR-treated 5-HTT−/− mice vs. baseline). On day 18 mechanical withdrawal thresholds were back to normal in 5-HTT−/− mice, whereas wt mice showed even more pronounced mechanical hypersensitivity (Mann–Whitney U test, *p < 0.05 vs. baseline).
VCR. The modulation of both mechanical and heat sensitivity in 5HTT−/− mice first shown in VCR neuropathy was associated with a reduced 5-HT tissue content in the peripheral nervous system (PNS) and central nervous system (CNS). Functionally ablated 5-HTT in 5-HTT−/− mice entails reduced tissue 5-HT concentrations, as 5-HTT is needed for 5-HT to be taken up into the cells. Accordingly, 5-HTT−/−mice had lower levels of 5HT than wt mice in the PNS and CNS. This confirms previous studies showing reduced 5-HT in peripheral [23,36] and central nervous tissue. No difference in thermal nociception was found between naive 5-HTT−/− and wt mice, suggesting that the loss of 5-HTT did not lead to altered thermal pain sensation by itself [13]. Nociceptive nerve fibers [2,29] and injured axons in particular [18,19] can be sensitized by 5-HT at the receptor site on the sensory nerve terminal. Thus we hypothesize that 5-HT in the injured nerve after VCR injection may sensitize nociceptive fibers to heat stimuli, which leads to heat hyperalgesia. The low 5-HT tissue concentration in 5-HTT−/− mice treated with VCR may thus underlie their diminished heat hyperalgesia. Reduced thermal hyperalgesia in 5-HTT−/− mice with low 5-HT content has been shown in two other models of neuropathic pain [24,36]. Reduced expression of 5-HT1A,B and especially 5-HT2A receptors may further contribute to decreased thermal hyperalgesia in 5-HTT−/− mice [7,25].
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Fig. 2. 5-HT tissue content measured by HPLC. After ten days of VCR or saline injection, 5-HT tissue content was lower in the hippocampus (A), hypothalamus (B), thalamus (C) and spinal cord (D) of 5-HTT−/− mice compared with wt mice (LSD post hoc test: p < 0.05). In the sciatic nerve (E) 5-HT tissue content was lower in 5-HTT−/− than wt mice only after VCR but not saline injection (LSD post hoc test: p < 0.05). Data are expressed as mean ± standard deviation. *p < 0.05.
Punctate mechanical allodynia depends on central sensitization mediated by A␦-primary sensory neurons, as exemplified in the CCI model [9]. Descending facilitation and loss of inhibition are mechanisms of central sensitization [35]. Descending pathways are known to be sources of 5-HT in the spinal cord [10,19]. The actions of these descending serotoninergic pathways have long been associated with suppression of nociceptive inputs [1,6], but evidence has accumulated supporting a facilitatory effect with regard to nociceptive input [6,20]. When 5-HT is released from descending neurons from the RVM, it may activate 5-HT3 receptors localized on the nerve terminals of a subpopulation of small-diameter afferents, thereby enhancing neurotransmitter release in the dorsal horn [30]. Remarkably, a direct activation of silent glutamatergic synapses between the afferent fibers and spinal dorsal horn neurons by 5HT has been demonstrated in rats, indicating a further mechanism of central sensitization via 5-HT [16]. Assuming that 5-HT in VCRtreated mice exerts a predominantly facilitatory influence on pain transmission, especially on the mechanically evoked responses of dorsal horns neurons [31], the reversed mechanical allodynia in 5HTT−/− mice may be attributed to a loss in spinal facilitation due to a lack of spinal 5-HT. There are several possible reasons why the
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increased ATF 3 expression one day after the last VCR injection and 13 days after the first vincristine injection being concordant with the time course of ATF 3 expression over two weeks in a model of axonal sciatic nerve injury [34] similar to the axonal damage in VCR neuropathy [27]. Nevertheless, recent evidence indicates that the distal axon is more vulnerable than the perikaryon to VCR’s toxic effect in the DRG-cell culture of rats [27]. Histological sections of the sciatic nerves revealed axonal degeneration and demyelination in rats with VCR-induced neuropathy [8]. Our data and findings reported in the literature support the view that VCR’s toxic effects affect both neuronal somata in the DRG and axons. Unlike our earlier results in the CFA model [23], the 5-HTT−/− genotype did not protect murine DRG neurons from VCR-induced injury in DRG, indicating that the 5-HTT−/− genotype has different functional implications in toxic as opposed to inflammatory neuropathies. The higher number of invading macrophages found in DRG of VCR-treated mice may contribute to VCR-induced hyperalgesia, as macrophages release pro-inflammatory cytokines possibly involved in the development and maintenance of neuropathic pain. Macrophage-derived interleukin-6, for example, has been shown to play a critical role in VCR-induced mechanical hypersensitivity [14]. However, the 5-HTT−/− genotype is not protected from macrophage infiltration in the DRG, indicating that the prevention of heat hyperalgesia and reversal of mechanical allodynia in 5-HTT−/− mice is independent of inflammation due to macrophage influx. Taken together, the 5-HTT−/− genotype may protect from VCRinduced heat and mechanical hypersensitivity due to lower 5-HT levels in the PNS and CNS. Furthermore, our data suggest that peripheral nerve injury and macrophage infiltration in the DRG are not modified by the 5-HTT−/− genotype in VCR-induced neuropathy. Contributions NH, CS and NU wrote the manuscript and performed statistical analysis; FP, LB, NU and MZ conducted the experiments; KPL and MG critically revised this manuscript for important intellectual content. Acknowledgements Fig. 3. Morphometric determination of ATF 3 (A) and macrophage (B) immunoreactivity in DRG neurons. (A) DRG from VCR-treated mice have a higher percentage of positive nuclei than saline treated rats (t-test: p < 0.05). (B) Macrophage numbers are increased per DRG of VCR-treated wt mice compared to saline-treated wt mice (t-test: p < 0.05). *p < 0.05.
VCR-treated 5HTT−/− mice revealed a degree of mechanical allodynia on day 14. One reason may be due to the different mechanisms responsible for the initiation and maintenance state of mechanical allodynia in 5-HTT−/− mice. Recent data showed that the RVM is important for time-dependent facilitation, but not the initiation of neuropathic pain, indicating that descending serotonergic modulation may alter pain processing after a certain time delay [4]. Unfortunately, we could not directly analyze the 5-HT content in RVM samples, as the murine RVM is too small to be dissected with enough precision to analyze 5-HT content reliably. The finding of increased numbers of ATF 3 immunoreactive nuclei in DRG neurons in VCR-treated mice indicates that peripheral injury in VCR-induced neuropathy did indeed have an impact on the neuronal somata of DRG neurons. The amount and time course of ATF 3 expression differs between injury models [34]. The time of tissue harvesting was determined by behavioral testing. A limit of our study is that we did not use additional mice to test ATF 3 expression at different time points. However, we observed an
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