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Experimental evidence for alleviating nociceptive hypersensitivity by single application of capsaicin

Experimental evidence for alleviating nociceptive hypersensitivity by single application of... The single application of high-concentration of capsaicin has been used as an analgesic therapy of persistent pain. However, its effectiveness and underlying mechanisms remain to be further evaluated with experimental approaches. The present study provided evidence showing that the single application of capsaicin dose-dependently alleviated nociceptive hypersensitivity, and reduced the action potential firing in small-diameter neurons of the dorsal root ganglia (DRG) in rats and mice. Pre-treatment with capsaicin reduced formalin-induced acute nocifensive behavior after a brief hyperalgesia in rats and mice. The inhibitory effects of capsaicin were calcium-dependent, and mediated by the capsaicin receptor (transient receptor potential vanilloid type-1). We further found that capsaicin exerted inhibitory effects on the persistent nociceptive hypersensitivity induced by peripheral inflammation and nerve injury. Thus, these results support the long-lasting and inhibitory effects of topical capsaicin on persistent pain, and the clinic use of capsaicin as a pain therapy. Background Neuronal TRPV1 is a homotetrameric, nonselective Topical application of capsaicin has long been clinically ligand-gated cation channel which can be activated by a used to treat persistent pain, such as osteoarthritic pain, wide range of stimuli, including heat, proton, exogenous post-herpetic neuralgia of the trigeminal nerve, migraine compounds such as capsaicin and endovanilloids [17-19]. prophylaxis, diabetic neuropathy, HIV-associated distal Studies with the TRPV1 gene knockout mice show that sensory neuropathy, and intractable pain in cancer pa- TRPV1 is involved in the nociceptive response induced by tients [1-9]. Capsaicin is an agonist of transient receptor noxious heat (>50°C) and inflammation-induced thermal potential cation channel, subfamily V, member 1 (TRPV1), hyperalgesia, although responsiveness to noxious heat which is expressed in small-diameter neurons of the dor- stimuli is not completely lost in TRPV1-deficient mice sal root ganglion (DRG) [10]. Topical treatment with cap- [20-22]. Capsaicin opens TRPV1 channel not only to in- saicin in human initially results in nociceptor firing and a crease the intracellular level of calcium and induce mem- period of enhanced sensitivity to painful heat stimuli, and brane depolarization, but also to initiate the desensitization then a refractory period during which resistant to cap- and downregulation of TRPV1, and the degeneration of saicin and heat but not pinprick stimuli [4,11-14]. epidermal nerve fibers, which is referred as defunctionali- Application of capsaicin can induce long-lasting ther- zation following continuous capsaicin exposure [5,23-26]. mal hypoalgesia in the inflammatory model [15], and The long refractory period of the neurons which were another TRPV1 agonist resiniferatoxin (RTX) allevi- excited previously by capsaicin may result from calcium- ates the thermal nociception in the physiological state dependent conformational changes in TRPV1 protein, ul- and under the inflammatory condition [16]. timately closing the channel pore [27]. However, this effect was presumably temporary and therefore might not ac- count for a persistent pain relief observed clinically. * Correspondence: xu.zhang@ion.ac.cn Studies on the mechanisms for long-lasting analgesia Institute of Neuroscience and State Key Laboratory of Neuroscience, CAS after continuous application of capsaicin suggest that the Center for Excellence in Brain Science, Shanghai Institutes for Biological capsaicin treatment leads to the dysfunctionalization of Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China TRPV1-containing nerve terminals in the skin and periph- School of Life Science and Technology, ShanghaiTech University, Shanghai eral tissues, due to the long-term, functional and pheno- 201210, China typic alternation of neurons [26,28-31]. Capsaicin inhibits Full list of author information is available at the end of the article © 2015 Ma et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ma et al. Molecular Pain (2015) 11:22 Page 2 of 10 action potentials and voltage-gated sodium channels in Formalin injection caused a stereotypic two-phase capsaicin-sensitive DRG neurons, and repeated applica- pattern of nociceptive response shown by the increase tion of capsaicin produced membrane depolarization in flinch number of the hindpaw. The phase I oc- but failed to evoke action potentials [27,32-34]. Re- curred within 0–10 min due to periphery nociceptive peated treatment with capsaicin may reversibly de- response, and the phase II appeared winthin 10– crease the density of epidermal nerve fibers, and 60 min due to central nociceptive reaction (Figure 1B). thus the nociceptive deficiency could be induced To examine the effect of capsaicin on acute pain, cap- several days after treatment and may last for weeks saicin (50 μgor100 μg) was intraplantarly injected [4,5,24,26,29,35-38]. into the hindpaw 120 min before the formalin injec- Recent clinical practice and study show that a single tion (Figure 1B). The formalin-induced nociceptive re- 60-min or 30-min application of 8% capsaicin patch sponse in the phase I and phase II were both inhibited was found to provide significant pain relief for at least by the pretreatment with 100 μg capsaicin but not 12 weeks in patients suffering persistent pain including 50 μg capsaicin (Figure 1B), suggesting that the capsa- peripheral neuropathic pain [30,31,39-43]. However, icin pretreatment could dose-dependently prevent the the experimental evidence for such an application of formalin-induced acute nociceptive hypersensitivity. capsaicin remains to be obtained to evaluate its effect- Similar to the rats, the formalin-induced nociceptive iveness and potential mechanisms for treatment of acute response in mice could be inhibited by the pretreat- and chronic pain. ment with 10 μg capsaicin (Figure 1C). Thepresent studyshowedthatafter abrief excitatory effect on capsaicin-sensitive DRG neurons and a transi- Capsaicin suppresses the excitability of small DRG ent nociceptive response of rats, a single application of neurons capsaicin inhibited the action potential (AP) firing in To further examine whether the excitability of DRG capsaicin-sensitive DRG neurons and alleviated the neuron was regulated by capsaicin, we performed nociceptive hypersensitivity in the acute pain model in- whole-cell patch-clamp recording in small neurons dis- duced by formalin, the inflammatory pain model by sociated from the rat DRGs. The action potentials initi- complete Freund’s adjuvant (CFA) and neuropathic pain ated by single current ramp stimulation were recorded model by spared nerve injury (SNI). This inhibitory ef- before 30 s capsaicin treatment, during the treatment fect of capsaicin was mediated by TRPV1 channel, and and 5 min washout after the treatment, respectively appeared to be dependent on the dosage of capsaicin (Figure 2A). The TRPV1-positive neuron was recog- and calcium influx. These results provide a line of ex- nized once an inward current was induced after puffing perimental evidence of the long-lasting and inhibitory capsaicin. The action potential frequency in small DRG effects of topical capsaicin on persistent pain, and sup- neurons was increased transitorily in response to the port the clinic use of capsaicin as a pain therapy. treatment with 2 μM capsaicin (Figure 2A and B), con- sistent with the acute nociceptive behavior following Results capsaicin treatment. Capsaicin pretreatment induces analgesic effect on the However, after washout with the extracellular solution formalin-induced nociceptive response (ECS) for 5 min, the same neurons no longer fired the We firstly examined the effect of capsaicin on the acute action potentials in response to the same current ramp nociceptive response induced by intraplantar injection stimulation (Figure 2A and B). Furthermore, we found of formalin. As expected, the rats developed nocifen- that this effect was dose-dependent, because 0.25 μM sive behavior after receiving the capsaicin (25, 50 and and 0.5 μM of capsaicin reduced the action potential 100 μg) injection at the hindpaw, as compared to the frequency after 5 min washout to 0.25 (0.25 ± 0.07)-fold rats received the vehicle injection (Figure 1A). The and 0.18 (0.18 ± 0.13)-fold of that before capsaicin treat- flinch number of the hindpaw was increased to a peak ment, respectively, while 2 μM of capsaicin completely level 10 min after the injection of 25, 50 and 100 μg suppressed the action potential firing (Figure 2C). At capsaicin, and then gradually decreased 20–60 min the same time interval, the membrane potential of the after the capsaicin injection (Figure 1A). Most time capsaicin-stimulated neurons returned to the basal points between vehicle group and capsaicin group levels, and the second treatment with 2 μMcapsaicin showed significant difference (10, 20, 30, 40, 60 and could induce inward current with ~48% of the ampli- 70 min between vehicle group and 25 μggroup;10, 20, tude of first one (n = 5). Taken together, the treatment 30, 40, 50 and 70 min between vehicle group and 50 μg with capsaicin could result in a dose-dependent inhibi- group; 10, 30, 40, 50 and 60 min between vehicle group tory effect on the excitability of small DRG neurons, and 100 μg group). The increased flinch behavior dis- consistent with the reduction in the nocicptive behavior appeared 90 min after capsaicin injection. after capsaicin pretreatment observed in rats. Ma et al. Molecular Pain (2015) 11:22 Page 3 of 10 Figure 1 Capsaicin treatment causes acute algetic responses and a subsequent analgesic effect. (A) Time course of 90 min following intraplantar injection of 25 (n = 9 rats), 50 (n = 16) and 100 μg (n = 12) capsaicin or vehicle (n = 14) (p < 0.001 for 25 μg, p < 0.001 for 50 μg and p < 0.001 for 100 μgcapsaicin versus vehicle control group, ANOVA). The quantitative analysis showed that the number of flinches was increased at capsaicin-injected hindpaw. (B) Intraplantar pre-injection of 100 μg capsaicin (n = 13) but not 50 μg capsaicin (n = 8) or vehicle (n = 12) induced an analgesic effect in the pain model induced by 2% formalin (p < 0.001 for 100 μg capsaicin versus vehicle group, ANOVA). The time course showed that the inhibitory effect of capsaicin on the formalin-induced flinch in the phase II was lasted for 30 min. The number of flinches in the phase I and phase II was reduced in the rats with capsaicin pre-treatment. (C) Intraplantar pre-injection of 10 μg capsaicin (n = 15) reduced the licking time of mice induced by 2% formalin, compared to vehicle group (n = 15) (p < 0.001, ANOVA). The licking time in both phase I and phase II was decreased in mice. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, versus vehicle group. TRPV1 is required for the capsaicin-induced inhibition the action potential frequency after 5 min washout in To examine whether this capsaicin-induced inhibition small DRG neurons (Figure 3A). Furthermore, we per- was specifically mediated via TRPV1, neurons dissoci- formed whole-cell patch-clamp recording in small ated from the rat DRGs were incubated with capsaze- DRG neurons dissociated from the DRGs of TRPV1 −/− pine, the antagonist of TRPV1, for 30 min before the gene knock-out (TRPV1 ) and wild-type littermate +/+ treatment with 2 μM capsaicin. This pre-blockade of (TRPV1 ) mice. The 30 s pretreatment with 2 μM TRPV1 prevented the capsaicin-induced reduction in capsaicin and following 5 min washout in the Ma et al. Molecular Pain (2015) 11:22 Page 4 of 10 Figure 2 Pre-treatment with capsaicin reduces the excitability of small DRG neurons of rats. (A) Whole-cell patch-clamp recording in small neurons acutely dissociated from the rat DRGs showed that the firing frequency of action potentials induced by a current ramp (1 s duration; peak ranging from 100–500 pA) was firstly increased, and was then decreased after 5 min washout of capsaicin. (B) The action potential frequency during capsaicin treatment was increased to 3.12 (3.12 ± 0.65)-folds of that recorded before treatment, and such a change was attenuated in the same neuron after 5 min washout. **p < 0.01, ***p < 0.001, versus the same group of neurons before capsaicin treatment (n = 15 neurons). (C) The capsaicin-induced suppression of action potential was dose-dependent. The action potential frequency after 5 min washout reduced to 0.25-fold and 0.18-fold of that before 0.25 μM (n = 26) and 0.5 μM (n = 9) capsaicin treatment, respectively, and was almost totally suppressed with 2 μMcapsaicin (n = 11). capsaicin-sensitive small neurons (n = 10/34) from data suggest that TRPV1 is required for the capsaicin- +/+ TRPV1 mice reduced the action potential frequency induced inhibition. to 0.08 (0.08 ± 0.08)-fold of that before capsaicin treatment, while the capsaicin-insensitive neurons had Capsaicin-induced inhibition is calcium-dependent no obvious changes (Figure 3B and C). In contrast, the Considering TRPV1 as a non-selective cation channel, we −/− small DRG neurons (n = 31) from TRPV1 mice did next asked whether calcium influx induced by activation of not show any changes in the action potential fre- TRPV1 was involved in the capsaicin-induced analgesia. quency (Figure 3B and C). We also examined the Small DRG neurons dissociated from rats were incubated effect of capsaicin at a higher concentration. The 30 s in the calcium-free ECS, and were then stimulated by a pretreatment with 1 mM capsaicin and following current ramp. We found that in the absence of extra- 5 min washout reduced the action potential frequency cellular calcium, 2 μM capsaicin treatment reduced to 0.02 (0.02 ± 0.02)-fold of that before capsaicin the action potential frequency after 5 min washout to treatment in the capsaicin-sensitive small neurons 0.66 (0.66 ± 0.07)-fold of that before capsaicin treat- +/+ (n = 8/22) from TRPV1 mice, while the capsaicin- ment (Figure 4A). This result suggests that the influx insensitive neurons from wild-type mice and the of extracellular calcium is required for the capsaicin- −/− small DRG neurons (n = 10) from TRPV1 mice induced inhibition on nociceptive afferent neurons. did not show any changes in the action potential fre- To test whether the extracellular calcium affect quency (Figure 3D). Thus, TRPV1 in small DRG the capsaicin-induced analgesia, 50 μg capsaicin was neurons is required for the capsaicin-induced inhib- mixed with 10 mM calcium was injected 120 min ition of excitability. before formalin injection. This mixture significantly We further examined the effect of capsaicin on the reduced the nociceptive response in the phase II of −/− formalin-induced nociceptive response in TRPV1 formalin test and also caused a tendency of de- mice. The capsaicin-induced inhibition on the phase I creased response in the phase I, whereas 50 μgcap- and phase II of the formalin-induced nociceptive re- saicin and 10 mM calcium alone did not affect the −/− sponse was lost in TRPV1 mice (Figure 3E). These nociceptive response (Figure 4B and C). Taken Ma et al. Molecular Pain (2015) 11:22 Page 5 of 10 Figure 3 TRPV1 is required for the inhibitory effect of capsaicin on the neuronal excitability and formalin-induced nociceptive response. (A) Treatment with TRPV1 antagonist, capsazepine, attenuated the capsaicin-induced reduction in action potential frequency in small DRG neurons (n = 13). (B, C, D) Whole-cell patch-clamp recording showed that the capsaicin-induced reduction of action potential frequency in capsaicin-sensitive small DRG neurons (for 2 μM capsaicin, 10 positive neurons out of 34; for 1 mM capsaicin, 8 positive neurons out of 22) +/+ −/− dissociated from TRPV1 littermates was absent in small DRG neurons from TRPV1 mice (for 2 μM capsaicin, n = 31; for 1 mM capsaicin, n = 10). (E) The licking time in both phase I and phase II of mice induced by 2% formalin was not affected by intraplantar pre-injection of 10 μg capsaicin (n = 8), compared to vehicle group (n = 8). Data are shown as mean ± SEM. together, the capsaicin-induced inhibition on the noci- the vehicle-injected control group, while the analgesic ceptive response could be regulated by the influx of effect of 100 μg capsaicin lasted for at least 24 h extracellular calcium. (Figure 5A). Furthermore, small DRG neurons dissociated from the CFA-treated rats were recorded to examine the Capsaicin reduces the nociceptive hypersensitivity neuronal excitability. A ramp current was injected into the induced by peripheral inflammation and nerve injury neuron to induce action potential before and after the cap- Using chronic inflammatory pain model induced by saicin application. After puffing 2 μM capsaicin for 30 s, CFA and the neuropathic pain model induced by SNI, the action potential frequency was completely suppressed we evaluated whether capsaicin could alleviate persist- after 5 min washout (Figure 5B). ent mechanical allodynia. The von Frey test was used We also evaluated the effect of capsaicin treatment to measure the mechanical threshold of rats. The on the mechanical allodynia in the SNI model. Seven mechanical allodynia developed 2 days after intraplan- days after SNI, the intraplantar injection of 100 μg cap- tar injection of CFA at left hindpaw. We found that the saicin could mildly reduce the mechanical allodynia intraplantar injection of 50 μg capsaicin reduced (Figure 5C). Whole-cell patch-clamp recording showed mechanical allodynia in subsequent 2 h compared to that in small DRG neurons dissociated from rats on Ma et al. Molecular Pain (2015) 11:22 Page 6 of 10 Figure 4 Calcium is involved in the capsaicin-induced inhibition on neuronal excitability and analgesia. (A) whole-cell patch-clamp recording showed that in the absence of extracellular calcium, the ratio of action potential frequency was only partially inhibited in capsaicin-pretreated small DRG neurons. ***p < 0.001, versus the same group of neurons before capsaicin treatment (n = 30). (B, C) Behavior test showed that 50 μg capsaicin or 10 mM calcium or vehicle (n = 9) alone could not reduce the nociceptive response in the phase II of formalin test. However, the injection of a mixture of 50 μg capsaicin with 10 mM calcium (n = 7) could inhibit the reaction in the phase II (p < 0.001 for rats treated with capsaicin mixed with 10 mM calcium versus vehicle group, ANOVA). Data are shown as mean ± SEM. **p < 0.01, versus vehicle group. post-SNI day 7, 2 μM capsaicin for 30 s almost com- of 8% capsaicin patch significantly relieves pain for pletely suppressed the action potential frequency after many weeks in patients [30,31,39-43]. However, the ex- 5 min washout (Figure 5D). Taken together, capsaicin perimental evidence for the capsaicin-induced analgesia treatment alleviates the persistent nociceptive hyper- and the related mechanism has been insufficient to sup- sensitivity induced by peripheral inflammation and port this clinical application of capsaicin. In the present nerve injury. study, we analyzed the analgesic effect of capsaicin in the animal models of acute and chronic pain, including Discussion the formalin test, CFA model of chronic inflammatory The present study showed that a brief and topical cap- pain and SNI model of chronic neuropathic pain. Our saicin treatment could lead to long-lasting inhibition of results showed that a single application of capsaicin neuronal excitability in a dose- and calcium-dependent reduced the nociceptive hypersensitivity induced by manner, and alleviation of both acute and persistent acute noxious stimulation and peripheral inflammation nociceptive responses. These results suggest that a sin- or nerve injury. Notably, the analgesic effect of capsaicin gle application of capsaicin could produce a therapeutic wasmorepronounced in theinflammation modelthan approach to effectively treat persistent pain. that in the neuropathic pain model, which may be due Clinical practice suggests that capsaicin can be used to the elevated expression of TRPV1 in inflammatory as an analgesic to relief pain [1-9]. Particularly, recent condition and the reduced expression of TRPV1 in neuro- reports show that a single 60-min or 30-min application pathic pain model [44,45]. Thus, the single injection of Ma et al. Molecular Pain (2015) 11:22 Page 7 of 10 Figure 5 Capsaicin alleviates the nociceptive hypersensitivity induced by peripheral inflammation or nerve injury. (A) Intraplantar injection of 100 μg capsaicin could reduce the mechanical allodynia 2 days after CFA injection (n = 12 for vehicle, n = 9 for 50 μg capsaicin treatment and n = 11 for 100 μg capsaicin treatment). The significant analgesic effect of 100 μg capsaicin began 2 h after capsaicin injection and maintained for 24 h, while 50 μg showed a shorter analgesic effect (p < 0.05 for 50 μg and p < 0.001 for 100 μg capsaicin versus vehicle group, ANOVA). (B) Whole-cell patch-clamp recording showed that puffing 2 μM capsaicin for 30 s, the action potential frequency was totally suppressed after 5 min washout in small DRG neurons dissociated from rats injected CFA for 2 days. ***p < 0.001, versus the same group of neurons before capsaicin treatment (n = 11). (C) Intraplantar injection of 100 μg capsaicin could mildly reduce mechanical allodynia 7 days after SNI (n = 8 for vehicle and n = 9 for capsaicin treatment) (p < 0.01 for 100 μg capsaicin versus vehicle group, ANOVA). (D) Whole-cell patch-clamp recording showed that treatment with 2 μM capsaicin for 30 s almost totally suppressed the action potential firing after 5 min washout in small DRG neurons dissociated from rats on post-SNI day 7. ***p < 0.001, versus the same group of neurons before capsaicin treatment (n = 17). Data are shown as mean ± SEM. capsaicin may be more effective in managing inflamma- long refractory period of the neurons after capsaicin tory pain than neuropathic pain. treatment may result from calcium-dependent conform- Previous studies showed that capsaicin desensitizes ational changes in TRPV1 protein [27], suggesting that capsaicin-sensitive nociceptors by inhibiting the gener- the refractory period of capsaicin-sensitive neurons also ation of action potentials through the indirect block accounts for the relief of persistent pain. of voltage-gated sodium channels [27,32-34]. In the The refractory period of capsaicin-sensitive neurons present study, we found that a brief treatment with cap- could be attributed to a desensitization state of TRPV1 saicin could induce long-lasting suppressive effects on or the depolarized state of DRG neuron after TRPV1 the generation of action potentials in capsaicin-sensitive opening [2,42]. The present electrophysiological exper- DRG neurons in a dose- and calcium-dependent man- iments showed that there were a short period of ner, consistent with the inhibitory effects of a single TRPV1 desensitization and membrane depolarization capsaicin treatment on the acute and persistent noci- following capsaicin treatment. However, after 5 min ceptive hypersensitivity. Our finding that high-dose washout, DRG neurons were re-sensitized to capsaicin capsaicin produced more pronounced inhibition is and the membrane potential returned to the basal consistent with the clinical observation that high- levels, whereas the neuronal excitability at the same concentration topical capsaicin is required to treat time interval was suppressed. Therefore, the analgesia neuropathic pain [30,31,39,40,43,46]. Our present re- induced by a single application of capsaicin might be sults show that the capsaicin-induced calcium influx in- not attributed to the TRPV1 desensitization or the volves the acute regulation of both neuronal excitability membrane depolarization of sensory neurons. An al- and nociceptive behavior induced by a single injection ternative explanation could be a reduced excitability of of capsaicin. This is consistent with the notion that the primary sensory neurons. Capsaicin was found to Ma et al. Molecular Pain (2015) 11:22 Page 8 of 10 inhibit the generation of action potentials and voltage- pore-loop region was disrupted [20]. The heterozy- −/− gated sodium channels in the DRG neurons that were gotes were bred to obtain TRPV1 mice and their +/+ −/− previously excited by capsaicin [27,32-34]. Moreover, wild-type littermates (TRPV1 ). TRPV1 mice were capsaicin induces sodium influx, which would indir- viable and fertile. Capsaicin and capsazepine were pur- ectly induce the membrane depolarization and inacti- chased from Tocris Bioscience. vation of voltage-gated sodium channels [32-34]. Therefore, a single application of capsaicin may Animal models and behavior tests desensitize nociceptors by inhibiting the generation of For acute nociceptive response of capsaicin, rats were action potentials. divided into several groups after habituated in Perspex Previous studies showed that heat responses, but chamber with a wire mesh floor for at least 2 h, ob- not mechanical hyperalgesia or allodynia, were atten- served in the chamber, and then injected with vehicle uated by ablation of TRPV1-lineage neurons using or different dose of capsaicin intraplantarly. The num- genetic approach in mice [47]. TRPV1 may distribute ber of flinches was counted every 10 min during in both peptidergic and non-peptidergic neurons of 90 min after injection. rats [48]. Selective blocking sodium channels in the For tonic inflammatory formalin test, rats were ha- TRPV1-expressing neurons of rats showed reduced bituated in Perspex chamber for at least 2 h. The rats response to both noxious mechanical and thermal were intraplantarly pretreated with vehicle or capsaicin stimuli [49]. We found that intraplantar injection of for 2 h. After that, 50 μlof2% formalin(Sigma- capsaicin attenuated mechanical hyperalgesia in the Aldrich) in saline was injected into the rat left hindpaw CFA model and mechanical allodynia in SNI model intraplantarly. The number of flinches was counted of the rat. One explanation is that high-dose capsa- based on the first phase (1–10 min) and the second icin activated TRPV1 and then indirectly suppressed phase (10–60 min) [50,51]. +/+ −/− the sodium channels in nociceptive afferent neurons For formalin test in TRPV1 and TRPV1 mice, to reduce mechanical hypersensitivity. the mice were intraplantarly pretreated with vehicle or Taken together, the present study showed that the 10 μg capsaicin for 2 h. Then, mice were injected with generation of action potentials was attenuated in the 20 μl of 2% formalin into the dorsal surface of the left capsaicin-sensitive DRG neurons that were previously hindpaw. The time that mice licked the injected paw excited by a single and brief treatment of capsaicin. A was recorded during the phase I (1–10 min) and II single injection of capsaicin significantly reduced the (10–40 min). nociceptive hypersensitivity in both the rat model of As chronic inflammatory pain model, rats were peripheral inflammation and the model of neuropathic injected with 100 μl CFA (Sigma-Aldrich) at the hind- pain. However, its effect on the inflammatory nocicep- paw intraplantarly. To determine the threshold for tive response is more pronounced than that in the mechanical pain response, the von Frey test was per- neuropathic pain model. Thus, our study provides ex- formed 2 days after injection. For electrophysiological perimental evidence supporting the single application experiment, CFA-treated rats were sacrificed 2 days of high-dose capsaicin as a clinical approach to treat after injection. persistent pain. As chronic neuropathic pain model, the SNI model was performed on Sprague–Dawley rats as previously Methods described. Two of the three branches of the sciatic Animals and drugs nerve (common peroneal nerve and tibial nerve) were All experiments were approved by the Committee of injured, leaving the remaining sural nerve intact Use of Laboratory Animals and Common Facility, In- [52,53]. The von Frey test and electrophysiological ex- stitute of Neuroscience, Chinese Academy of Sciences periment were performed on 7 d after SNI surgery. and carried out according to the guidelines of the To measure mechanical threshold, the rats were ha- International Association for the Study of Pain. All bituated in Perspex chamber for more than 2 h before animals were housed under a 12-h light/dark cycle at tests, and then graded von Frey filaments were applied 22-26°C, with free access to food and water. Sprague– to the lateral area of the hindpaw, using the up-and- Dawley male rats were bought from Shanghai SLAC down testing paradigm [54]. The minimum bending Laboratory Animal CO. LTD (SLAC). TRPV1 knock- force with three positive responses out of five stimuli −/− out (TRPV1 ) micewereboughtfromthe Jackson with von Frey filaments presented perpendicular to the Laboratory (JAX). The strain name is B6.129X1- plantar surface of the rat hindpaw was determined as tm1jul TRPV1 /J and the stock number is 003770. An the mechanical threshold. exon encoding part of the fifth and all of the sixth All behavioral tests were all performed double- putative trans-membrane domains together with the blindly. Ma et al. Molecular Pain (2015) 11:22 Page 9 of 10 Cell culture and electrophysiology was considered to reach statistical significance when p < L4 and L5 DRGs were acutely dissected from approxi- 0.05. Levels of significance are indicated by the number of mately 100 g Sprague–Dawley rats or approximately 20 g p value, e.g., *, 0.01 < p < 0.05; **, 0.001 < p < 0.01; ***, p < +/+ −/− TRPV1 and TRPV1 mice after they were anesthetized 0.001. Data are present as mean ± SEM. by injection of pentobarbital sodium (80 mg/kg). The con- Abbreviations nective tissue was digested by 0.4 mg/ml trypsin type I AP: Action potential; CFA: Complete Freund’s adjuvant; SNI: Spared nerve (Sigma-Aldrich), 1 mg/ml collagenase type 1A (Sigma-Al- injury; DRG: Dorsal root ganglion; ECS: Extracellular solution; RTX: Resiniferatoxin; TRPV1: Transient receptor potential cation channel, drich), and 0.1 mg/ml DNase I (Sigma-Aldrich) in DMEM −/− +/+ subfamily V, member 1; TRPV1 : TRPV1 gene knock-out; TRPV1 : TRPV1 Medium (Gibco-Invitrogen, Carlsbad, CA, USA) at 37°C wild-type. followed by three washes in ECS. DRGs were then mechan- Competing interests ically dissociated with a set of flame-polished Pasteur pi- The authors declare that they have no competing interests. pettes. Dissociated cells were placed on glass coverslips at room temperature and patch clamp recording was per- Authors’ contributions formed within 2–12 h. XLM performed cell culture, electrophysiological recording and behavior tests. FXZ instructed and designed experiments. FD performed some Neuronal excitability was examined with long suprathres- electrophysilogical recording. XLM, LB and XZ designed experiments and hold current pulse (1 s). The amount of injecting current wrote the manuscript. All authors read and approved the final manuscript. (100–500 pA) was individually determined for each neuron Acknowledgements by repeated injections that evoke multiple action potentials. This work was supported by National Natural Science Foundation of China The TRPV1-positive neurons were selected by observing (31130066) and the Strategic Priority Research Program (B) of Chinese immediate inward current when capsaicin was applied. Academy of Sciences (XDB01020300). We thank Dr. Xiaoyang Cheng for instruction on action potential recording and analysis. Once an inward current was induced after puffing capsa- icin, we recognized this neuron as a TRPV1-positive one. Author details To compare the effect of single application of capsaicin on Institute of Neuroscience and State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science, Shanghai Institutes for Biological neuronal excitability, the number of action potential was Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai normalized to that before capsaicin treatment for each 200031, China. State Key Laboratory of Cell Biology, Institute of Biochemistry neuron. The ratio of action potential frequency was calcu- and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. School of Life Science and lated from the number of action potential after 5 min wash- Technology, ShanghaiTech University, Shanghai 201210, China. out versus that before capsaicin treatment for each neuron. Normal ECS contained following: 150 mM NaCl, 5 mM Received: 30 December 2014 Accepted: 10 April 2015 KCl, 2.5 mM CaCl ,1mM MgCl , 10 mM HEPES and 2 2 10 mM glucose, then adjusted to pH 7.4 with NaOH. To References 2+ prepare ECS containing 0 mM Ca , 2.5 mM CaCl was 1. Jara-Oseguera A, Simon SA, Rosenbaum T. TRPV1: on the road to pain relief. replaced by 2.5 mM MgCl . Electrodes had a resistance of Curr Mol Pharmacol. 2008;1(3):255–69. 2. Szallasi A, Sheta M. Targeting TRPV1 for pain relief: limits, losers and laurels. 2–4MΩ when filled with the pipette solution, which con- Expert Opin Investig Drugs. 2012;21(9):1351–69. tained the following: 140 mM KCl, 1 mM MgCl ,2.5 mM 3. Backonja M, Wallace MS, Blonsky ER, Cutler BJ, Malan Jr P, Rauck R, et al. CaCl , 5 mM EGTA, 10 mM HEPES, 2 mM Na-ATP and NGX-4010, a high-concentration capsaicin patch, for the treatment of postherpetic neuralgia: a randomised, double-blind study. Lancet Neurol. 0.3 mM Na-GTP, then adjusted to pH 7.3 [55]. 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Experimental evidence for alleviating nociceptive hypersensitivity by single application of capsaicin

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Springer Journals
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Copyright © 2015 by Ma et al.; licensee BioMed Central.
Subject
Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology
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1744-8069
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1744-8069
DOI
10.1186/s12990-015-0019-0
pmid
25896608
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Abstract

The single application of high-concentration of capsaicin has been used as an analgesic therapy of persistent pain. However, its effectiveness and underlying mechanisms remain to be further evaluated with experimental approaches. The present study provided evidence showing that the single application of capsaicin dose-dependently alleviated nociceptive hypersensitivity, and reduced the action potential firing in small-diameter neurons of the dorsal root ganglia (DRG) in rats and mice. Pre-treatment with capsaicin reduced formalin-induced acute nocifensive behavior after a brief hyperalgesia in rats and mice. The inhibitory effects of capsaicin were calcium-dependent, and mediated by the capsaicin receptor (transient receptor potential vanilloid type-1). We further found that capsaicin exerted inhibitory effects on the persistent nociceptive hypersensitivity induced by peripheral inflammation and nerve injury. Thus, these results support the long-lasting and inhibitory effects of topical capsaicin on persistent pain, and the clinic use of capsaicin as a pain therapy. Background Neuronal TRPV1 is a homotetrameric, nonselective Topical application of capsaicin has long been clinically ligand-gated cation channel which can be activated by a used to treat persistent pain, such as osteoarthritic pain, wide range of stimuli, including heat, proton, exogenous post-herpetic neuralgia of the trigeminal nerve, migraine compounds such as capsaicin and endovanilloids [17-19]. prophylaxis, diabetic neuropathy, HIV-associated distal Studies with the TRPV1 gene knockout mice show that sensory neuropathy, and intractable pain in cancer pa- TRPV1 is involved in the nociceptive response induced by tients [1-9]. Capsaicin is an agonist of transient receptor noxious heat (>50°C) and inflammation-induced thermal potential cation channel, subfamily V, member 1 (TRPV1), hyperalgesia, although responsiveness to noxious heat which is expressed in small-diameter neurons of the dor- stimuli is not completely lost in TRPV1-deficient mice sal root ganglion (DRG) [10]. Topical treatment with cap- [20-22]. Capsaicin opens TRPV1 channel not only to in- saicin in human initially results in nociceptor firing and a crease the intracellular level of calcium and induce mem- period of enhanced sensitivity to painful heat stimuli, and brane depolarization, but also to initiate the desensitization then a refractory period during which resistant to cap- and downregulation of TRPV1, and the degeneration of saicin and heat but not pinprick stimuli [4,11-14]. epidermal nerve fibers, which is referred as defunctionali- Application of capsaicin can induce long-lasting ther- zation following continuous capsaicin exposure [5,23-26]. mal hypoalgesia in the inflammatory model [15], and The long refractory period of the neurons which were another TRPV1 agonist resiniferatoxin (RTX) allevi- excited previously by capsaicin may result from calcium- ates the thermal nociception in the physiological state dependent conformational changes in TRPV1 protein, ul- and under the inflammatory condition [16]. timately closing the channel pore [27]. However, this effect was presumably temporary and therefore might not ac- count for a persistent pain relief observed clinically. * Correspondence: xu.zhang@ion.ac.cn Studies on the mechanisms for long-lasting analgesia Institute of Neuroscience and State Key Laboratory of Neuroscience, CAS after continuous application of capsaicin suggest that the Center for Excellence in Brain Science, Shanghai Institutes for Biological capsaicin treatment leads to the dysfunctionalization of Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China TRPV1-containing nerve terminals in the skin and periph- School of Life Science and Technology, ShanghaiTech University, Shanghai eral tissues, due to the long-term, functional and pheno- 201210, China typic alternation of neurons [26,28-31]. Capsaicin inhibits Full list of author information is available at the end of the article © 2015 Ma et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ma et al. Molecular Pain (2015) 11:22 Page 2 of 10 action potentials and voltage-gated sodium channels in Formalin injection caused a stereotypic two-phase capsaicin-sensitive DRG neurons, and repeated applica- pattern of nociceptive response shown by the increase tion of capsaicin produced membrane depolarization in flinch number of the hindpaw. The phase I oc- but failed to evoke action potentials [27,32-34]. Re- curred within 0–10 min due to periphery nociceptive peated treatment with capsaicin may reversibly de- response, and the phase II appeared winthin 10– crease the density of epidermal nerve fibers, and 60 min due to central nociceptive reaction (Figure 1B). thus the nociceptive deficiency could be induced To examine the effect of capsaicin on acute pain, cap- several days after treatment and may last for weeks saicin (50 μgor100 μg) was intraplantarly injected [4,5,24,26,29,35-38]. into the hindpaw 120 min before the formalin injec- Recent clinical practice and study show that a single tion (Figure 1B). The formalin-induced nociceptive re- 60-min or 30-min application of 8% capsaicin patch sponse in the phase I and phase II were both inhibited was found to provide significant pain relief for at least by the pretreatment with 100 μg capsaicin but not 12 weeks in patients suffering persistent pain including 50 μg capsaicin (Figure 1B), suggesting that the capsa- peripheral neuropathic pain [30,31,39-43]. However, icin pretreatment could dose-dependently prevent the the experimental evidence for such an application of formalin-induced acute nociceptive hypersensitivity. capsaicin remains to be obtained to evaluate its effect- Similar to the rats, the formalin-induced nociceptive iveness and potential mechanisms for treatment of acute response in mice could be inhibited by the pretreat- and chronic pain. ment with 10 μg capsaicin (Figure 1C). Thepresent studyshowedthatafter abrief excitatory effect on capsaicin-sensitive DRG neurons and a transi- Capsaicin suppresses the excitability of small DRG ent nociceptive response of rats, a single application of neurons capsaicin inhibited the action potential (AP) firing in To further examine whether the excitability of DRG capsaicin-sensitive DRG neurons and alleviated the neuron was regulated by capsaicin, we performed nociceptive hypersensitivity in the acute pain model in- whole-cell patch-clamp recording in small neurons dis- duced by formalin, the inflammatory pain model by sociated from the rat DRGs. The action potentials initi- complete Freund’s adjuvant (CFA) and neuropathic pain ated by single current ramp stimulation were recorded model by spared nerve injury (SNI). This inhibitory ef- before 30 s capsaicin treatment, during the treatment fect of capsaicin was mediated by TRPV1 channel, and and 5 min washout after the treatment, respectively appeared to be dependent on the dosage of capsaicin (Figure 2A). The TRPV1-positive neuron was recog- and calcium influx. These results provide a line of ex- nized once an inward current was induced after puffing perimental evidence of the long-lasting and inhibitory capsaicin. The action potential frequency in small DRG effects of topical capsaicin on persistent pain, and sup- neurons was increased transitorily in response to the port the clinic use of capsaicin as a pain therapy. treatment with 2 μM capsaicin (Figure 2A and B), con- sistent with the acute nociceptive behavior following Results capsaicin treatment. Capsaicin pretreatment induces analgesic effect on the However, after washout with the extracellular solution formalin-induced nociceptive response (ECS) for 5 min, the same neurons no longer fired the We firstly examined the effect of capsaicin on the acute action potentials in response to the same current ramp nociceptive response induced by intraplantar injection stimulation (Figure 2A and B). Furthermore, we found of formalin. As expected, the rats developed nocifen- that this effect was dose-dependent, because 0.25 μM sive behavior after receiving the capsaicin (25, 50 and and 0.5 μM of capsaicin reduced the action potential 100 μg) injection at the hindpaw, as compared to the frequency after 5 min washout to 0.25 (0.25 ± 0.07)-fold rats received the vehicle injection (Figure 1A). The and 0.18 (0.18 ± 0.13)-fold of that before capsaicin treat- flinch number of the hindpaw was increased to a peak ment, respectively, while 2 μM of capsaicin completely level 10 min after the injection of 25, 50 and 100 μg suppressed the action potential firing (Figure 2C). At capsaicin, and then gradually decreased 20–60 min the same time interval, the membrane potential of the after the capsaicin injection (Figure 1A). Most time capsaicin-stimulated neurons returned to the basal points between vehicle group and capsaicin group levels, and the second treatment with 2 μMcapsaicin showed significant difference (10, 20, 30, 40, 60 and could induce inward current with ~48% of the ampli- 70 min between vehicle group and 25 μggroup;10, 20, tude of first one (n = 5). Taken together, the treatment 30, 40, 50 and 70 min between vehicle group and 50 μg with capsaicin could result in a dose-dependent inhibi- group; 10, 30, 40, 50 and 60 min between vehicle group tory effect on the excitability of small DRG neurons, and 100 μg group). The increased flinch behavior dis- consistent with the reduction in the nocicptive behavior appeared 90 min after capsaicin injection. after capsaicin pretreatment observed in rats. Ma et al. Molecular Pain (2015) 11:22 Page 3 of 10 Figure 1 Capsaicin treatment causes acute algetic responses and a subsequent analgesic effect. (A) Time course of 90 min following intraplantar injection of 25 (n = 9 rats), 50 (n = 16) and 100 μg (n = 12) capsaicin or vehicle (n = 14) (p < 0.001 for 25 μg, p < 0.001 for 50 μg and p < 0.001 for 100 μgcapsaicin versus vehicle control group, ANOVA). The quantitative analysis showed that the number of flinches was increased at capsaicin-injected hindpaw. (B) Intraplantar pre-injection of 100 μg capsaicin (n = 13) but not 50 μg capsaicin (n = 8) or vehicle (n = 12) induced an analgesic effect in the pain model induced by 2% formalin (p < 0.001 for 100 μg capsaicin versus vehicle group, ANOVA). The time course showed that the inhibitory effect of capsaicin on the formalin-induced flinch in the phase II was lasted for 30 min. The number of flinches in the phase I and phase II was reduced in the rats with capsaicin pre-treatment. (C) Intraplantar pre-injection of 10 μg capsaicin (n = 15) reduced the licking time of mice induced by 2% formalin, compared to vehicle group (n = 15) (p < 0.001, ANOVA). The licking time in both phase I and phase II was decreased in mice. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, versus vehicle group. TRPV1 is required for the capsaicin-induced inhibition the action potential frequency after 5 min washout in To examine whether this capsaicin-induced inhibition small DRG neurons (Figure 3A). Furthermore, we per- was specifically mediated via TRPV1, neurons dissoci- formed whole-cell patch-clamp recording in small ated from the rat DRGs were incubated with capsaze- DRG neurons dissociated from the DRGs of TRPV1 −/− pine, the antagonist of TRPV1, for 30 min before the gene knock-out (TRPV1 ) and wild-type littermate +/+ treatment with 2 μM capsaicin. This pre-blockade of (TRPV1 ) mice. The 30 s pretreatment with 2 μM TRPV1 prevented the capsaicin-induced reduction in capsaicin and following 5 min washout in the Ma et al. Molecular Pain (2015) 11:22 Page 4 of 10 Figure 2 Pre-treatment with capsaicin reduces the excitability of small DRG neurons of rats. (A) Whole-cell patch-clamp recording in small neurons acutely dissociated from the rat DRGs showed that the firing frequency of action potentials induced by a current ramp (1 s duration; peak ranging from 100–500 pA) was firstly increased, and was then decreased after 5 min washout of capsaicin. (B) The action potential frequency during capsaicin treatment was increased to 3.12 (3.12 ± 0.65)-folds of that recorded before treatment, and such a change was attenuated in the same neuron after 5 min washout. **p < 0.01, ***p < 0.001, versus the same group of neurons before capsaicin treatment (n = 15 neurons). (C) The capsaicin-induced suppression of action potential was dose-dependent. The action potential frequency after 5 min washout reduced to 0.25-fold and 0.18-fold of that before 0.25 μM (n = 26) and 0.5 μM (n = 9) capsaicin treatment, respectively, and was almost totally suppressed with 2 μMcapsaicin (n = 11). capsaicin-sensitive small neurons (n = 10/34) from data suggest that TRPV1 is required for the capsaicin- +/+ TRPV1 mice reduced the action potential frequency induced inhibition. to 0.08 (0.08 ± 0.08)-fold of that before capsaicin treatment, while the capsaicin-insensitive neurons had Capsaicin-induced inhibition is calcium-dependent no obvious changes (Figure 3B and C). In contrast, the Considering TRPV1 as a non-selective cation channel, we −/− small DRG neurons (n = 31) from TRPV1 mice did next asked whether calcium influx induced by activation of not show any changes in the action potential fre- TRPV1 was involved in the capsaicin-induced analgesia. quency (Figure 3B and C). We also examined the Small DRG neurons dissociated from rats were incubated effect of capsaicin at a higher concentration. The 30 s in the calcium-free ECS, and were then stimulated by a pretreatment with 1 mM capsaicin and following current ramp. We found that in the absence of extra- 5 min washout reduced the action potential frequency cellular calcium, 2 μM capsaicin treatment reduced to 0.02 (0.02 ± 0.02)-fold of that before capsaicin the action potential frequency after 5 min washout to treatment in the capsaicin-sensitive small neurons 0.66 (0.66 ± 0.07)-fold of that before capsaicin treat- +/+ (n = 8/22) from TRPV1 mice, while the capsaicin- ment (Figure 4A). This result suggests that the influx insensitive neurons from wild-type mice and the of extracellular calcium is required for the capsaicin- −/− small DRG neurons (n = 10) from TRPV1 mice induced inhibition on nociceptive afferent neurons. did not show any changes in the action potential fre- To test whether the extracellular calcium affect quency (Figure 3D). Thus, TRPV1 in small DRG the capsaicin-induced analgesia, 50 μg capsaicin was neurons is required for the capsaicin-induced inhib- mixed with 10 mM calcium was injected 120 min ition of excitability. before formalin injection. This mixture significantly We further examined the effect of capsaicin on the reduced the nociceptive response in the phase II of −/− formalin-induced nociceptive response in TRPV1 formalin test and also caused a tendency of de- mice. The capsaicin-induced inhibition on the phase I creased response in the phase I, whereas 50 μgcap- and phase II of the formalin-induced nociceptive re- saicin and 10 mM calcium alone did not affect the −/− sponse was lost in TRPV1 mice (Figure 3E). These nociceptive response (Figure 4B and C). Taken Ma et al. Molecular Pain (2015) 11:22 Page 5 of 10 Figure 3 TRPV1 is required for the inhibitory effect of capsaicin on the neuronal excitability and formalin-induced nociceptive response. (A) Treatment with TRPV1 antagonist, capsazepine, attenuated the capsaicin-induced reduction in action potential frequency in small DRG neurons (n = 13). (B, C, D) Whole-cell patch-clamp recording showed that the capsaicin-induced reduction of action potential frequency in capsaicin-sensitive small DRG neurons (for 2 μM capsaicin, 10 positive neurons out of 34; for 1 mM capsaicin, 8 positive neurons out of 22) +/+ −/− dissociated from TRPV1 littermates was absent in small DRG neurons from TRPV1 mice (for 2 μM capsaicin, n = 31; for 1 mM capsaicin, n = 10). (E) The licking time in both phase I and phase II of mice induced by 2% formalin was not affected by intraplantar pre-injection of 10 μg capsaicin (n = 8), compared to vehicle group (n = 8). Data are shown as mean ± SEM. together, the capsaicin-induced inhibition on the noci- the vehicle-injected control group, while the analgesic ceptive response could be regulated by the influx of effect of 100 μg capsaicin lasted for at least 24 h extracellular calcium. (Figure 5A). Furthermore, small DRG neurons dissociated from the CFA-treated rats were recorded to examine the Capsaicin reduces the nociceptive hypersensitivity neuronal excitability. A ramp current was injected into the induced by peripheral inflammation and nerve injury neuron to induce action potential before and after the cap- Using chronic inflammatory pain model induced by saicin application. After puffing 2 μM capsaicin for 30 s, CFA and the neuropathic pain model induced by SNI, the action potential frequency was completely suppressed we evaluated whether capsaicin could alleviate persist- after 5 min washout (Figure 5B). ent mechanical allodynia. The von Frey test was used We also evaluated the effect of capsaicin treatment to measure the mechanical threshold of rats. The on the mechanical allodynia in the SNI model. Seven mechanical allodynia developed 2 days after intraplan- days after SNI, the intraplantar injection of 100 μg cap- tar injection of CFA at left hindpaw. We found that the saicin could mildly reduce the mechanical allodynia intraplantar injection of 50 μg capsaicin reduced (Figure 5C). Whole-cell patch-clamp recording showed mechanical allodynia in subsequent 2 h compared to that in small DRG neurons dissociated from rats on Ma et al. Molecular Pain (2015) 11:22 Page 6 of 10 Figure 4 Calcium is involved in the capsaicin-induced inhibition on neuronal excitability and analgesia. (A) whole-cell patch-clamp recording showed that in the absence of extracellular calcium, the ratio of action potential frequency was only partially inhibited in capsaicin-pretreated small DRG neurons. ***p < 0.001, versus the same group of neurons before capsaicin treatment (n = 30). (B, C) Behavior test showed that 50 μg capsaicin or 10 mM calcium or vehicle (n = 9) alone could not reduce the nociceptive response in the phase II of formalin test. However, the injection of a mixture of 50 μg capsaicin with 10 mM calcium (n = 7) could inhibit the reaction in the phase II (p < 0.001 for rats treated with capsaicin mixed with 10 mM calcium versus vehicle group, ANOVA). Data are shown as mean ± SEM. **p < 0.01, versus vehicle group. post-SNI day 7, 2 μM capsaicin for 30 s almost com- of 8% capsaicin patch significantly relieves pain for pletely suppressed the action potential frequency after many weeks in patients [30,31,39-43]. However, the ex- 5 min washout (Figure 5D). Taken together, capsaicin perimental evidence for the capsaicin-induced analgesia treatment alleviates the persistent nociceptive hyper- and the related mechanism has been insufficient to sup- sensitivity induced by peripheral inflammation and port this clinical application of capsaicin. In the present nerve injury. study, we analyzed the analgesic effect of capsaicin in the animal models of acute and chronic pain, including Discussion the formalin test, CFA model of chronic inflammatory The present study showed that a brief and topical cap- pain and SNI model of chronic neuropathic pain. Our saicin treatment could lead to long-lasting inhibition of results showed that a single application of capsaicin neuronal excitability in a dose- and calcium-dependent reduced the nociceptive hypersensitivity induced by manner, and alleviation of both acute and persistent acute noxious stimulation and peripheral inflammation nociceptive responses. These results suggest that a sin- or nerve injury. Notably, the analgesic effect of capsaicin gle application of capsaicin could produce a therapeutic wasmorepronounced in theinflammation modelthan approach to effectively treat persistent pain. that in the neuropathic pain model, which may be due Clinical practice suggests that capsaicin can be used to the elevated expression of TRPV1 in inflammatory as an analgesic to relief pain [1-9]. Particularly, recent condition and the reduced expression of TRPV1 in neuro- reports show that a single 60-min or 30-min application pathic pain model [44,45]. Thus, the single injection of Ma et al. Molecular Pain (2015) 11:22 Page 7 of 10 Figure 5 Capsaicin alleviates the nociceptive hypersensitivity induced by peripheral inflammation or nerve injury. (A) Intraplantar injection of 100 μg capsaicin could reduce the mechanical allodynia 2 days after CFA injection (n = 12 for vehicle, n = 9 for 50 μg capsaicin treatment and n = 11 for 100 μg capsaicin treatment). The significant analgesic effect of 100 μg capsaicin began 2 h after capsaicin injection and maintained for 24 h, while 50 μg showed a shorter analgesic effect (p < 0.05 for 50 μg and p < 0.001 for 100 μg capsaicin versus vehicle group, ANOVA). (B) Whole-cell patch-clamp recording showed that puffing 2 μM capsaicin for 30 s, the action potential frequency was totally suppressed after 5 min washout in small DRG neurons dissociated from rats injected CFA for 2 days. ***p < 0.001, versus the same group of neurons before capsaicin treatment (n = 11). (C) Intraplantar injection of 100 μg capsaicin could mildly reduce mechanical allodynia 7 days after SNI (n = 8 for vehicle and n = 9 for capsaicin treatment) (p < 0.01 for 100 μg capsaicin versus vehicle group, ANOVA). (D) Whole-cell patch-clamp recording showed that treatment with 2 μM capsaicin for 30 s almost totally suppressed the action potential firing after 5 min washout in small DRG neurons dissociated from rats on post-SNI day 7. ***p < 0.001, versus the same group of neurons before capsaicin treatment (n = 17). Data are shown as mean ± SEM. capsaicin may be more effective in managing inflamma- long refractory period of the neurons after capsaicin tory pain than neuropathic pain. treatment may result from calcium-dependent conform- Previous studies showed that capsaicin desensitizes ational changes in TRPV1 protein [27], suggesting that capsaicin-sensitive nociceptors by inhibiting the gener- the refractory period of capsaicin-sensitive neurons also ation of action potentials through the indirect block accounts for the relief of persistent pain. of voltage-gated sodium channels [27,32-34]. In the The refractory period of capsaicin-sensitive neurons present study, we found that a brief treatment with cap- could be attributed to a desensitization state of TRPV1 saicin could induce long-lasting suppressive effects on or the depolarized state of DRG neuron after TRPV1 the generation of action potentials in capsaicin-sensitive opening [2,42]. The present electrophysiological exper- DRG neurons in a dose- and calcium-dependent man- iments showed that there were a short period of ner, consistent with the inhibitory effects of a single TRPV1 desensitization and membrane depolarization capsaicin treatment on the acute and persistent noci- following capsaicin treatment. However, after 5 min ceptive hypersensitivity. Our finding that high-dose washout, DRG neurons were re-sensitized to capsaicin capsaicin produced more pronounced inhibition is and the membrane potential returned to the basal consistent with the clinical observation that high- levels, whereas the neuronal excitability at the same concentration topical capsaicin is required to treat time interval was suppressed. Therefore, the analgesia neuropathic pain [30,31,39,40,43,46]. Our present re- induced by a single application of capsaicin might be sults show that the capsaicin-induced calcium influx in- not attributed to the TRPV1 desensitization or the volves the acute regulation of both neuronal excitability membrane depolarization of sensory neurons. An al- and nociceptive behavior induced by a single injection ternative explanation could be a reduced excitability of of capsaicin. This is consistent with the notion that the primary sensory neurons. Capsaicin was found to Ma et al. Molecular Pain (2015) 11:22 Page 8 of 10 inhibit the generation of action potentials and voltage- pore-loop region was disrupted [20]. The heterozy- −/− gated sodium channels in the DRG neurons that were gotes were bred to obtain TRPV1 mice and their +/+ −/− previously excited by capsaicin [27,32-34]. Moreover, wild-type littermates (TRPV1 ). TRPV1 mice were capsaicin induces sodium influx, which would indir- viable and fertile. Capsaicin and capsazepine were pur- ectly induce the membrane depolarization and inacti- chased from Tocris Bioscience. vation of voltage-gated sodium channels [32-34]. Therefore, a single application of capsaicin may Animal models and behavior tests desensitize nociceptors by inhibiting the generation of For acute nociceptive response of capsaicin, rats were action potentials. divided into several groups after habituated in Perspex Previous studies showed that heat responses, but chamber with a wire mesh floor for at least 2 h, ob- not mechanical hyperalgesia or allodynia, were atten- served in the chamber, and then injected with vehicle uated by ablation of TRPV1-lineage neurons using or different dose of capsaicin intraplantarly. The num- genetic approach in mice [47]. TRPV1 may distribute ber of flinches was counted every 10 min during in both peptidergic and non-peptidergic neurons of 90 min after injection. rats [48]. Selective blocking sodium channels in the For tonic inflammatory formalin test, rats were ha- TRPV1-expressing neurons of rats showed reduced bituated in Perspex chamber for at least 2 h. The rats response to both noxious mechanical and thermal were intraplantarly pretreated with vehicle or capsaicin stimuli [49]. We found that intraplantar injection of for 2 h. After that, 50 μlof2% formalin(Sigma- capsaicin attenuated mechanical hyperalgesia in the Aldrich) in saline was injected into the rat left hindpaw CFA model and mechanical allodynia in SNI model intraplantarly. The number of flinches was counted of the rat. One explanation is that high-dose capsa- based on the first phase (1–10 min) and the second icin activated TRPV1 and then indirectly suppressed phase (10–60 min) [50,51]. +/+ −/− the sodium channels in nociceptive afferent neurons For formalin test in TRPV1 and TRPV1 mice, to reduce mechanical hypersensitivity. the mice were intraplantarly pretreated with vehicle or Taken together, the present study showed that the 10 μg capsaicin for 2 h. Then, mice were injected with generation of action potentials was attenuated in the 20 μl of 2% formalin into the dorsal surface of the left capsaicin-sensitive DRG neurons that were previously hindpaw. The time that mice licked the injected paw excited by a single and brief treatment of capsaicin. A was recorded during the phase I (1–10 min) and II single injection of capsaicin significantly reduced the (10–40 min). nociceptive hypersensitivity in both the rat model of As chronic inflammatory pain model, rats were peripheral inflammation and the model of neuropathic injected with 100 μl CFA (Sigma-Aldrich) at the hind- pain. However, its effect on the inflammatory nocicep- paw intraplantarly. To determine the threshold for tive response is more pronounced than that in the mechanical pain response, the von Frey test was per- neuropathic pain model. Thus, our study provides ex- formed 2 days after injection. For electrophysiological perimental evidence supporting the single application experiment, CFA-treated rats were sacrificed 2 days of high-dose capsaicin as a clinical approach to treat after injection. persistent pain. As chronic neuropathic pain model, the SNI model was performed on Sprague–Dawley rats as previously Methods described. Two of the three branches of the sciatic Animals and drugs nerve (common peroneal nerve and tibial nerve) were All experiments were approved by the Committee of injured, leaving the remaining sural nerve intact Use of Laboratory Animals and Common Facility, In- [52,53]. The von Frey test and electrophysiological ex- stitute of Neuroscience, Chinese Academy of Sciences periment were performed on 7 d after SNI surgery. and carried out according to the guidelines of the To measure mechanical threshold, the rats were ha- International Association for the Study of Pain. All bituated in Perspex chamber for more than 2 h before animals were housed under a 12-h light/dark cycle at tests, and then graded von Frey filaments were applied 22-26°C, with free access to food and water. Sprague– to the lateral area of the hindpaw, using the up-and- Dawley male rats were bought from Shanghai SLAC down testing paradigm [54]. The minimum bending Laboratory Animal CO. LTD (SLAC). TRPV1 knock- force with three positive responses out of five stimuli −/− out (TRPV1 ) micewereboughtfromthe Jackson with von Frey filaments presented perpendicular to the Laboratory (JAX). The strain name is B6.129X1- plantar surface of the rat hindpaw was determined as tm1jul TRPV1 /J and the stock number is 003770. An the mechanical threshold. exon encoding part of the fifth and all of the sixth All behavioral tests were all performed double- putative trans-membrane domains together with the blindly. Ma et al. Molecular Pain (2015) 11:22 Page 9 of 10 Cell culture and electrophysiology was considered to reach statistical significance when p < L4 and L5 DRGs were acutely dissected from approxi- 0.05. Levels of significance are indicated by the number of mately 100 g Sprague–Dawley rats or approximately 20 g p value, e.g., *, 0.01 < p < 0.05; **, 0.001 < p < 0.01; ***, p < +/+ −/− TRPV1 and TRPV1 mice after they were anesthetized 0.001. Data are present as mean ± SEM. by injection of pentobarbital sodium (80 mg/kg). The con- Abbreviations nective tissue was digested by 0.4 mg/ml trypsin type I AP: Action potential; CFA: Complete Freund’s adjuvant; SNI: Spared nerve (Sigma-Aldrich), 1 mg/ml collagenase type 1A (Sigma-Al- injury; DRG: Dorsal root ganglion; ECS: Extracellular solution; RTX: Resiniferatoxin; TRPV1: Transient receptor potential cation channel, drich), and 0.1 mg/ml DNase I (Sigma-Aldrich) in DMEM −/− +/+ subfamily V, member 1; TRPV1 : TRPV1 gene knock-out; TRPV1 : TRPV1 Medium (Gibco-Invitrogen, Carlsbad, CA, USA) at 37°C wild-type. followed by three washes in ECS. DRGs were then mechan- Competing interests ically dissociated with a set of flame-polished Pasteur pi- The authors declare that they have no competing interests. pettes. Dissociated cells were placed on glass coverslips at room temperature and patch clamp recording was per- Authors’ contributions formed within 2–12 h. XLM performed cell culture, electrophysiological recording and behavior tests. FXZ instructed and designed experiments. FD performed some Neuronal excitability was examined with long suprathres- electrophysilogical recording. XLM, LB and XZ designed experiments and hold current pulse (1 s). The amount of injecting current wrote the manuscript. All authors read and approved the final manuscript. (100–500 pA) was individually determined for each neuron Acknowledgements by repeated injections that evoke multiple action potentials. This work was supported by National Natural Science Foundation of China The TRPV1-positive neurons were selected by observing (31130066) and the Strategic Priority Research Program (B) of Chinese immediate inward current when capsaicin was applied. Academy of Sciences (XDB01020300). We thank Dr. Xiaoyang Cheng for instruction on action potential recording and analysis. Once an inward current was induced after puffing capsa- icin, we recognized this neuron as a TRPV1-positive one. Author details To compare the effect of single application of capsaicin on Institute of Neuroscience and State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science, Shanghai Institutes for Biological neuronal excitability, the number of action potential was Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai normalized to that before capsaicin treatment for each 200031, China. State Key Laboratory of Cell Biology, Institute of Biochemistry neuron. The ratio of action potential frequency was calcu- and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. School of Life Science and lated from the number of action potential after 5 min wash- Technology, ShanghaiTech University, Shanghai 201210, China. out versus that before capsaicin treatment for each neuron. 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Published: Apr 22, 2015

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