σ1Receptors Are Involved in the Visceral Pain Induced by Intracolonic Administration of Capsaicin in Mice

VISCERAL pain is the most frequent type of pathological pain and one of the main reasons for patients to seek medical assistance.1,2However, most of our knowledge about pain mechanisms derives from experimental studies of somatic (principally cutaneous) pain rather than visceral pain.3,4The characteristics, pathophysiological mechanisms, and response to drug treatment of visceral and somatic pain are different; consequently, it is not valid to indiscriminately extrapolate findings from the somatic–cutaneous to visceral domain.5–10Most existing animal models of visceral pain are either technically complex (e.g. , require surgery) or combine visceral and somatic mechanisms of peritoneal pain (e.g. , writhing test). However, a technically simple and purely visceral mouse model of chemical stimulation of the colon by capsaicin has been developed.11This model permits examination of both acute visceral pain and referred mechanical hyperalgesia to the abdominal wall, which is an important clinical feature of visceral pain.11,12The intracolonic capsaicin model in mice may represent an appropriate translational model of visceral pain, because application of capsaicin to the human gut evokes intense (acute) pain and referred mechanical hyperalgesia and is a well-validated human model of visceral pain.13–15Sigma receptors have been classified into two distinct subtypes, sigma-1 (σ1) and sigma-2 (σ2), although only the σ1receptor has been cloned.16,17σ1Receptors are highly expressed in the central nervous system, including important areas for pain control such as the superficial layers of the spinal cord dorsal horn, periaqueductal gray matter, locus coeruleus, and rostroventral medulla.18,19The role of σ1receptors in somatic pain control is well documented. Thus, σ1receptor knockout (σ1-KO) mice20–22and wild-type (WT) mice treated with σ1receptor antagonists22–29showed a marked reduction of pain-related behaviors in different models of somatic pain. However, it is not known whether σ1receptors also play a role in models of pure visceral pain.To test this possibility, we performed studies using the intracolonic capsaicin model in mice with the following objectives: to compare the behavioral responses to chemical stimulation of the colon and the referred hyperalgesia to abdominal mechanical stimulation after the intracolonic instillation of capsaicin between naïve σ1-KO and WT mice; and to evaluate the effects of the systemic administration of σ1receptor antagonists (BD-1063, NE-100, and S1RA) and control drugs (morphine and ketoprofen) on the spontaneous pain-related behaviors and referred mechanical hyperalgesia induced by intracolonic capsaicin in both mouse types.Experiments were performed in female WT (Charles River, Barcelona, Spain) and σ1-KO (Laboratorios Esteve, Barcelona, Spain) CD-1 mice weighing 25–30 g. The σ1-KO mice were generated on a CD-1 background as described previously.22Animals were acclimated in our animal facilities for at least 1 week before testing, housed in colony cages in temperature- and light-controlled rooms (22 ± 1°C, lights on at 8:00 AM and off at 8:00 PM, air replacement every 20 min). A standard laboratory diet (Harlan Teklad Research Diets, Madison, WI) and tap water were available ad libitum until the beginning of the experiments. Testing took place during the light phase (from 9:00 AM to 3:00 PM). Mice were handled in accordance with the European Communities Council Directive of November 24, 1986 (86/609/ECC), and the experimental protocol was approved by the University of Granada Research Ethics Committee (Granada, Spain).We used the selective σ1receptor antagonists BD-1063 (1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydrochloride), supplied by Tocris Cookson Ltd. (Bristol, UK); NE-100 (N,N -dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine hydrochloride), synthesized as reported previously30; and S1RA (4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl] morpholine),27supplied by Laboratorios Esteve. Morphine hydrochloride (General Directorate of Pharmacy and Drugs, Spanish Ministry of Health, Madrid, Spain) was used as a control drug against visceral pain, and ketoprofen (Sigma-Aldrich Química S.A., Madrid, Spain) served as a negative control. All of these drugs were dissolved in sterile physiologic saline, with the exception of ketoprofen, which was dissolved in 10% absolute ethanol and 90% saline. Drug solutions were prepared immediately before the start of the experiments, and 5 ml/kg of the drug or its solvent was injected subcutaneously into the interscapular area.Capsaicin (Sigma-Aldrich), which was used to induce visceral pain, was prepared to make up a 1% (weight/volume) stock solution in a solvent comprising 10% absolute ethanol (Panreac Química SA, Barcelona, Spain), 10% Tween 80 (Sigma-Aldrich), and 80% sterile saline. The capsaicin solution was prepared once per week and stored at −20°C in aliquots, which were thawed and diluted at the appropriate concentrations on the day of the experiment. The capsaicin solution (50 µl) was instilled into the colon by introducing through the anus a fine round-tip cannula (external diameter, 0.61 mm; length, 4 cm) connected to a 1710 TLL Hamilton microsyringe (Teknokroma, Barcelona, Spain). Control animals were intracolonically instilled with the same volume of capsaicin solvent.Spontaneous pain-related behaviors and referred mechanical hyperalgesia induced by intracolonic capsaicin were tested following a previously described protocol,11with small modifications. Mice were housed in individual transparent plastic boxes (7 × 7 × 13 cm) on an elevated platform with a wire mesh floor (small mirrors behind and below the chambers enhanced observation of the animals). After a 40-min habituation period, animals were removed from the compartments, and a capsaicin solution (or its solvent) was instilled intracolonically after application of petroleum jelly on the perianal area to avoid stimulation of somatic areas through contact with the algogen. The animal was immediately returned to the compartment, where the number of pain-related behaviors (licking of the abdomen, stretching of the abdomen, and abdominal retractions) were counted by direct observation for 20 min.The presence of capsaicin-induced referred hyperalgesia was determined by measuring the withdrawal response to a punctate mechanical stimulation of the abdomen at 20 min after the instillation of capsaicin (or its solvent). Forces ranging from 0.02 to 2 g (0.19–19.6 mN) were applied to the abdomen with a series of calibrated von Frey filaments (Touch-Test Sensory Evaluators; North Coast Medical Inc., Gilroy, CA) using the up–down paradigm.31Perianal and external genitalia areas were avoided, concentrating the stimulation on the lower and mid abdomen, as reported previously.11Filaments were applied three times for 2–3 s, with interapplication intervals of 5 s. Testing was initiated with the 0.4-g (3.92-mN) von Frey filament (i.e. , the middle of the range). In each consecutive test, if there was no response to the filament, a stronger stimulus was then selected; if there was a positive response, a weaker one was then used. The response to the filament was considered positive if immediate licking/scratching of the application site, sharp retraction of the abdomen, or jumping was observed.The experimenter who evaluated the behavioral responses was blinded to the treatment and genotype of experimental subjects. In all cases, experiments in WT or σ1-KO groups, solvent- or capsaicin-treated groups, and saline- or drug-treatment groups were run in parallel. Each animal (n = 12 WT or n = 10 σ1-KO mice per group were tested in all experiments) was used only once and received a single concentration of capsaicin (or its solvent) and a single dose of one drug (or its solvent).WT and σ1-KO mice were administered different capsaicin concentrations (0.01–1%) intracolonically, and the pain-related behaviors and referred hyperalgesia to abdominal mechanical stimulation induced by each concentration were recorded consecutively in the same animal following the procedure described earlier in the first and second paragraphs of the General Procedures for Evaluating Intracolonic Capsaicin-evoked Visceral Pain and Referred Hyperalgesia section. This allowed the construction of concentration–response curves (concentration vs. number of behaviors or mechanical threshold) and identification of the optimal concentrations of capsaicin for the pharmacologic studies (see the next section for details).Two experimental approaches were used to evaluate the effect of the drugs on capsaicin-induced visceral pain and referred hyperalgesia. First, we tested the effect of a fixed dose of a σ1receptor antagonist on the responses induced by different concentrations of capsaicin. Second, we tested the effects of several doses of various σ1receptor antagonists and control drugs on the pain-related behavior and referred hyperalgesia induced by a fixed concentration of capsaicin.In the first experimental approach, BD-1063 (32 mg/kg) or its solvent was administered subcutaneously 30 min before the intracolonic instillation of different concentrations (0.01–1%) of capsaicin, and the number of pain-related behaviors was counted for 20 min after the capsaicin instillation. Immediately afterward (i.e. , 50 min after injection of the drug), its effect on the capsaicin-induced referred hyperalgesia was assessed in the same animal by stimulation of the abdomen with von Frey filaments as described earlier in the General Procedures for Evaluating Intracolonic Capsaicin-evoked Visceral Pain and Referred Hyperalgesia section. Experiments were performed in both WT and σ1-KO mice.In the second experimental approach, different doses of BD-1063 (16–64 mg/kg), S1RA (32–128 mg/kg), NE-100 (8–64 mg/kg), morphine (1–16 mg/kg), ketoprofen (32–128 mg/kg), or their solvents were administered subcutaneously at 30 min before the intracolonic instillation of 1% capsaicin, and the number of pain-related behaviors was counted for 20 min after administration of capsaicin. This concentration of capsaicin was selected because it produces the maximum number of pain-related behaviors in WT and σ1-KO mice (fig. 1A) and therefore offers the maximum window for observing any reductions in this response. In separate experiments, we tested the effect of the same doses of the σ1receptor antagonists and the control drugs on the referred hyperalgesia induced by a fixed concentration of capsaicin. In these experiments, the drug under study or its solvent was injected subcutaneously at 30 min before the intracolonic instillation of 0.1% capsaicin and, 20 min after the instillation, the response of the animal to abdominal stimulation with von Frey filaments was tested using the up–down method, as described earlier under General Procedures for Evaluating Intracolonic Capsaicin-evoked Visceral Pain and Referred Hyperalgesia. A capsaicin concentration of 0.1% was selected for these experiments because it reaches the maximum reduction in the mechanical threshold for referred hyperalgesia in WT and σ1-KO mice (fig. 1B).The degree of referred pain, expressed as the mechanical threshold that produces 50% of responses, was calculated using the formula of Dixon32: 50% mechanical threshold (g) = [(10(Xf + κδ))/10,000], where X f = value (in logarithmic units) of the final von Frey filament used, κ = tabular value for the pattern of positive/negative responses, and δ = mean difference (in log units) between stimuli.The number of behaviors and mechanical thresholds were compared across experimental groups with one-way ANOVA or two-way non–repeated measures ANOVA (factors were genotype and algogen, or drug and algogen, or genotype and drug, depending on the experiment performed) followed by the Bonferroni test, using the SigmaPlot 12.0 program (Systat Software Inc., San Jose, CA). ED50values (dose producing half of the maximal response) and their standard errors were calculated using nonlinear regression analysis of the equation for a sigmoid plot and were compared by means of Snedecor’s F test using the GraphPad Prism 5.00 program (GraphPad Software Inc., San Diego, CA). No data were missing for any of the variables. A value of P < 0.05 was considered statistically significant.In both WT and σ1-KO mice, intracolonic administration of the capsaicin solvent elicited a small number of abdominal licking behaviors that could be clearly differentiated from normal grooming activity (fig. 1A), whereas intracolonic instillation of capsaicin-induced multiple types of pain-related behavior (e.g. , licking, stretching, and contraction of the abdomen). The number of pain-related behaviors induced by capsaicin (0.01–1%) increased in a concentration-dependent manner in both WT and σ1-KO mice (fig. 1A), but the pattern of this response differed markedly between them (P < 0.001, two-way ANOVA). In WT mice, the maximum number of pain responses (46 ± 4.2) was evoked at the highest concentration tested (1%); in σ1-KO mice, the maximum number of pain responses, which was half that in WT mice, reached a plateau from a concentration of 0.1% (fig. 1A). The difference in number of pain responses between WT and σ1-KO animals was statistically significant at concentrations of 0.3% and 1% (P < 0.01) (fig. 1A).In contrast, there were no significant differences between WT and σ1-KO mice in the intracolonic capsaicin-induced referred mechanical hyperalgesia as evaluated with von Frey filaments (fig. 1B). Instillation of capsaicin (0.01–1%) reduced the mechanical threshold in a concentration-dependent manner in both groups, which showed almost identical stimulus–response curves (fig. 1B). The difference between these two groups and solvent-treated mice reached statistical significance at a capsaicin concentration of 0.03% (fig. 1B). No significant difference was found between the 50% mechanical thresholds in animals treated with capsaicin solvent (0.54 ± 0.08 and 0.52 ± 0.05 g in WT and σ1-KO mice, respectively) and those observed in naïve animals without any instillation (0.53 ± 0.09 and 0.51 ± 0.10 g in WT and σ1-KO mice, respectively).In summary, the intracolonic administration of capsaicin evoked concentration-dependent visceral pain-related behaviors and referred mechanical hyperalgesia in both WT and σ1-KO mice. The number of pain-related behaviors was lower in the σ1-KO mice, but the mechanical hyperalgesia did not differ between the mouse types.The subcutaneous administration of the selective σ1antagonist BD-1063 (32 mg/kg) at 30 min before the intracolonic instillation of 0.1–1% capsaicin reduced the number of pain-related behaviors in WT mice (P < 0.001, two-way ANOVA) (fig. 2A) but not in σ1-KO mice (fig. 2B). The maximum number of 1% capsaicin–induced pain-related behaviors was highly similar between WT mice treated with BD-1063 (22 ± 3.9) and σ1-KO mice treated with saline (22 ± 2.9) or with BD-1063 (20 ± 2.8). BD-1063 (32 mg/kg) also inhibited the referred mechanical hyperalgesia in WT mice, promoting a shift to the right of the concentration–response curve of capsaicin (P < 0.05, two-way ANOVA) (fig. 3A), but it had no effect on the referred hyperalgesia in σ1-KO mice (fig. 3B).To determine whether the effects of BD-1063 were dose-dependent and shared by other σ1antagonists, we administered various doses of the selective σ1antagonists BD-1063, S1RA, and NE-100 at 30 min before instillation of the capsaicin concentrations found to induce the maximum number of pain-related behaviors and maximum referred hyperalgesia (1% and 0.1% capsaicin, respectively). The subcutaneous administration of BD-1063 (16–64 mg/kg), S1RA (32–128 mg/kg), and NE-100 (8–64 mg/kg) induced a dose-dependent inhibition of capsaicin-induced pain responses in WT mice but produced no change in σ1-KO mice (fig. 4). None of the σ1antagonists abolished pain-related behaviors in WT mice; however, at the highest doses tested, all of them reduced the number of behaviors in WT mice to the same number observed in capsaicin-treated σ1-KO mice (fig. 4).In WT mice, the referred mechanical hyperalgesia induced by 0.1% capsaicin was almost completely by with the highest doses of BD-1063 or S1RA (fig. a 50% mechanical threshold ± g and ± g in BD-1063 capsaicin and S1RA capsaicin groups, respectively) that was to the threshold in capsaicin solvent-treated animals (0.53 ± 0.05 The subcutaneous administration of NE-100 also the capsaicin-induced mechanical hyperalgesia in WT mice (fig. However, NE-100 was than BD-1063 and S1RA, and the 50% maximal mechanical threshold was lower in the capsaicin-treated mice with the highest dose mg/kg) of NE-100 ± than in the mice treated with capsaicin solvent (0.53 ± 0.05 (fig. NE-100 doses than produced and that with the mechanical threshold not the subcutaneous administration of morphine (1–16 mg/kg) produced a dose-dependent inhibition of capsaicin-induced pain-related behavioral responses and mechanical referred hyperalgesia in WT and σ1-KO mice In both mouse the pain-related behaviors induced by intracolonic 1% capsaicin were reduced by morphine at doses of than or to and completely abolished by a dose of (fig. Morphine administration the referred mechanical hyperalgesia and a and in both mouse types (fig. a mechanical threshold than that observed in mice treated with capsaicin solvent (0.53 ± 0.05 g and 0.52 ± g in WT and σ1-KO mice, respectively). In at the highest morphine doses tested and mg/kg), most of the mice no response to the stimulus The effect of morphine on the referred mechanical hyperalgesia evoked by 0.1% capsaicin was statistically different in σ1-KO compared with WT mice (P < 0.001, two-way ANOVA) (fig. In the effect of morphine mg/kg) was in σ1-KO mice than in WT mice (P < (fig. The subcutaneous administration of ketoprofen (32–128 mg/kg) had no effect on the pain-related responses or referred mechanical hyperalgesia at any dose tested in either σ1-KO or WT mice main findings of this study were that the pain-related behaviors induced by intracolonic capsaicin were reduced markedly in σ1-KO mice and that the of σ1receptors inhibited the capsaicin-induced pain-related behaviors and mechanical referred hyperalgesia in WT mice but not in σ1-KO This is the first to of a role for the σ1receptor in a pure visceral pain with found that intracolonic capsaicin evoked concentration-dependent visceral pain-related behaviors and referred mechanical hyperalgesia. The number of capsaicin-induced pain-related behaviors was lower in σ1-KO mice than in WT mice. has been that the acute visceral pain evoked by intracolonic capsaicin is to the direct of in the this our group previously reported that the response in the first phase of pain, also to the direct of was by in σ1-KO to the reduction in the number of pain-related behaviors observed in the σ1-KO mechanical threshold was similar between naïve WT and σ1-KO animals (0.53 ± 0.09 and 0.51 ± that σ1-KO animals and to a punctate mechanical stimulus applied to the This is in with various that the of σ1receptors in naïve σ1-KO animals not with the of mechanical and applied to the or with the response to these referred mechanical hyperalgesia did not differ between WT and σ1-KO mice, in to a by our group that capsaicin was completely to induce mechanical to a punctate stimulus in σ1-KO and are both by an enhanced of in the central nervous differ in the mechanisms that the mouse spinal are in and dorsal and are and 1 the are found at dorsal and a of them are than are or this may be to the distinct of the after the and intracolonic administration of capsaicin. In this a abdominal referred hyperalgesia was reported in knockout mice than in WT mice after intracolonic capsaicin, whereas mechanical hyperalgesia after capsaicin was similar in the two mouse the differences between somatic and visceral mechanical induced by capsaicin are by the in our the maximum mechanical pain in WT mice was after the administration of 1 of capsaicin (20 of a capsaicin 50 of intracolonic capsaicin (50 of a 0.1% capsaicin was to the maximum referred mechanical hyperalgesia in the the of acute pain induced by capsaicin is than that of intracolonic capsaicin (20 min). it could be that after intracolonic administration of capsaicin, the stimulus and may be to a of referred mechanical hyperalgesia in σ1-KO mice in with σ1-KO mice treated with capsaicin the subcutaneous administration of the σ1receptor antagonists BD-1063, S1RA, and NE-100 inhibited the number of pain-related behaviors and the referred mechanical hyperalgesia induced by intracolonic capsaicin in WT mice but not in σ1-KO mice. to these the effects of the σ1antagonists are by their with The highest doses of all of the σ1receptor antagonists tested reduced the number of capsaicin-induced pain-related behaviors in WT mice to the same number observed in σ1-KO that these doses of BD-1063, S1RA, and NE-100 are to the of σ1receptors to the pain-related The response to capsaicin in σ1-KO and WT animals treated with the highest doses of σ1receptor antagonists that mechanisms other than σ1receptor are also in the acute visceral pain induced by capsaicin in both mouse was previously reported that σ1receptor antagonists had no effect on acute pain induced by or mechanical to the almost completely abolished the first phase of in the reduced capsaicin-induced visceral pain-related data that the of σ1receptor antagonists may on the type of pain or and on the stimulus applied or referred mechanical hyperalgesia induced by intracolonic capsaicin in WT mice was almost by with BD-1063 and S1RA but only by The effect of NE-100 was not to a difference of because the same was NE-100 mg/kg) was administered at 10 or 30 min before intracolonic capsaicin mechanical ± 0.02 and ± respectively). However, we a difference between NE-100 and the other two any the effect of BD-1063, S1RA, and NE-100 in WT mice was to their with their of effect in σ1-KO mice. The role of σ1receptors in pain in somatic pain models in WT mice has been has been reported that σ1receptor antagonists the pain responses in the second phase of the mechanical induced by the pain responses in various is an between the normal of capsaicin-induced referred hyperalgesia in σ1-KO mice and the inhibition of this hyperalgesia in WT mice treated with σ1receptor This may be to the of mechanisms in σ1-KO mice WT a in knockout there have been of effects and between knockout animals and experiments in different areas such as of role of in the role of A receptors in against the visceral pain induced by intracolonic capsaicin, administration of doses that were up to and than those to be in the writhing test mg/kg) and in the second phase of the test mg/kg) in the of effect of ketoprofen in our study be to the of doses of the drug and to that are not in capsaicin-induced visceral pain. contrast, morphine abolished the pain-related behaviors and the mechanical referred hyperalgesia induced by intracolonic capsaicin in both WT and σ1-KO mice. with that morphine inhibited intracolonic in the spinal the visceral pain induced by intracolonic we found that morphine produced a similar reduction in the number of pain-related behaviors in both types of mouse ± and ± in σ1-KO and WT mice, P but was (P < Snedecor’s F against pain induced by mechanical stimulation of the abdominal in σ1-KO mice ± mg/kg) than in WT mice ± in models of pain induced by previously an of morphine by σ1receptor antagonists or by against the study the first that σ1receptors in a visceral pain increased number of capsaicin were reported in from patients with one of the most human pain and the of with the of abdominal data may therefore have clinical because the visceral pain induced by intracolonic instillation of capsaicin to mice this through the of a role for σ1receptor antagonists in these findings that σ1receptors play a role in visceral pain, an important and pain that σ1receptor may represent a for this pain (Granada, Spain), for of the