Abstract
To investigate the possible mechanisms of topical analgesics in relieving pain in an animal model of muscular inflammation.
Adult Sprague-Dawley rats of both sexes were injected with complete Freund's adjuvant to induce inflammation in the anterior tibialis muscle of left hindlimb. One of two types of topical analgesics: Xiaotong Tiegao (XTT), a Tibetan herb compound, or Capzasin (CAP), a cream containing 0.1% capsaicin, was applied to the skin over the inflamed anterior tibialis muscle. The following experiments were performed: pain behavioral tests, evaluation of plasma extravasation in the affected limb, and electrophysiological recordings of afferent nerve fibers.
The behavioral experiments demonstrated that applications of either type of topical analgesic to the skin over the inflamed muscle significantly reduced muscular inflammatory pain, as indicated by the increased weight bearing capacity on the affected hindlimb (with latencies of 10 minutes for XTT and 1–2 hours for CAP). Meanwhile, both analgesics caused plasma extravasation in the affected skin. Electrophysiological recordings from the afferent fibers in the related cutaneous nerve indicated that topical analgesics selectively activated C-fibers, but not A-fibers innervating the same region of receptive field. The latency and duration of C-fiber activation was similar to those of the reduction of muscular inflammatory pain. On the contrary, topical analgesics substantially decreased C-fiber afferent spontaneous firing in the nerve innervating the inflamed muscle. Moreover, denervation of the affected skin blocked the analgesic effects of both topical analgesics in muscular inflammatory pain.
This study suggests that topical analgesics may reduce the nociceptive input from inflamed muscles via a reflex mechanism by activating the cutaneous nociceptive afferents.
Introduction
Topical analgesics have been widely applied to relieve pain since ancient times in China and worldwide. Chinese traditional medicine such as plasters (“Gao-Yao”) are a popular treatment for patients suffering from inflammatory pain in the deep tissue, including injury to the joint ligament, soft tissue contusion, or muscle sprain. In western medicine, pastes or plasters containing capsaicin have also been clinically been proved effective in the treatment of muscular or joint pain . Although a number of reports have been published to confirm the efficacy of pain-relieving topical plasters , there were few study regarding the mechanisms of topical analgesics. In addition, most of the published studies were focused on the ability of drugs in transcutaneous penetration, transdermal absorption, or binding to the cutaneous nociceptive receptors after being applied to the skin . However, transdermal absorption studies have already indicated that the majority of topically applied drugs, regardless the ingredients tested, stay in the epidermis without reaching the dermis layer . Only a minimal concentration of drugs, if any, can penetrate the epidermis and reach the deep tissue. These results, obviously contradictory to the proved efficacy of topical analgesics, disprove the hypothesis that these drugs act directly on deep tissue targets. This study will explore the possible mechanisms of topical analgesics using behavioral and neurophysiological techniques. We propose that topical analgesics relieve pain in the deep tissue by activating cutaneous C-fibers.
Materials and Methods
Establishment and Behavioral Evaluation of an Animal Model of Muscular Inflammatory Pain
A total of 20 male and 20 female adult Sprague-Dawley rats aged 7–8 weeks were used in this study. The experimental procedures were approved by the Animal Use and Care Advisory Committee of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. To induce muscular inflammation, 100 μL of complete Freund's adjuvant (CFA, #F5881, Sigma, St. Louis, MO, USA; composed of inactivated and dried mycobacteria) was injected into the left anterior tibialis muscle of animals under clear consciousness, and 100 μL of sterile saline was injected into the right side as a control.
To evaluate the intensity of inflammatory pain, we measured the differences in weight bearing capacity between the left (experimental) and right (control) hindlimbs on the 3rd day after injection using an incapacitance tester (purchased from the Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical Sciences) . Briefly, rats were placed in an angled clear plastic chamber with each hind paw resting on a separate force plate. The weight bearing capacity (g) of each hind limb was automatically averaged over a 10-second period. Each data point was demonstrated as the difference score (g) over the left (experimental) minus right (control) hindlimb and averaged for three repetitions. The difference scores for weight bearing between the two hindlimbs in naïve rates were close to zero (data not shown). A significant decrease in the difference score (i.e., more negative) after CFA injection indicated that the animal model of chronic muscle inflammatory pain was successfully established.
Evaluation of the Effect of Topical Analgesics
To infer the analgesic effects, topical analgesics were applied to the skin over the inflamed tibialis anterior muscle. We chose two types of widely used topical analgesics with clinically approved code numbers by Chinese State Food and Drug Administration (SFDA) or US Food and Drug Administration: One is a famous topical analgesic in traditional Chinese medicine, Xiaotong Tiegao (XTT, brown traditional medical power and wetting agent adhering to the inside of patches, manufactured by QiZheng Zangyao, Tibet Cheezheng Tibetan Medicine Co., Beijing, China; SFDA Code No. Z54020113) ; Another is Capzasin (CAP), a popular topical analgesic in the United States (cream contain 0.1% capsaicin, manufactured by Chattam, Chattanooga, TN, USA; NDC product code no. 41167-7518) .
After depilation on both hindlimbs, a sticky patch containing the drug power or cream was applied to the anterior tibial skin of the experimental side, and recorded the changes in weight; distribution and difference score were recorded at different time points: −20, −10, 0, 10, 20, 30, 40, 50, and 60 minutes. The value of each time point was averaged of three repeated measurements. Preliminary experiments using the same type of patch containing no drugs on the skin showed no effects on the difference score in naïve or CFA-injected rats (data not shown).
An additional denervation experiment was performed to find out whether the drugs take effect by physical penetration or by activating nerve terminals in the skin. In a subgroup of animals (N = 14), the anterior tibial skin was denervated by excising the lateral cutaneous branches of the superficial peroneal nerve. After the surgery, the anterior tibial skin failed to respond to nociceptive mechanical stimuli (such as a pinch by tweezers), indicating a successful denervation. CFA injection was then performed 3 days after the denervation surgery, followed by the behavioral tests described above.
Orthodromic Recordings of Afferent Nerve Microfilaments
In order to study whether drugs applied topically can influence activities in the cutaneous or muscular afferents, we recorded the action potential firings from afferent nerve fibers innervating the skin of the target area and the anterior tibial muscle as described previously . Briefly, 5–7 days after CFA injection, the rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). The common sciatic nerve was exposed at the middle of the thigh, covered by a pool of mineral oil, and kept at 35 ± 1°C. Single afferent fibers were recorded extracellularly from the desheathed sciatic nerve 20 mm proximal to the CFA-injected site using silver wire electrodes, a custom-made low-noise differential amplifier, 100-Hz high-pass and 1-kHz low-pass filters, and an analog-digital interface with software running on a personal computer (Biological Functional System, BL-420B, made by Chengdu Taimeng, Sichuan, China) for online analysis. Single electrical pulses were delivered via a pair of bipolar silver stimulating electrodes placed around the nerve distal to the recording site to measure the conduction velocity (CV) of the recorded fiber. According to the CV and the response to the light touch of skin, the recorded neurons were classified into C-fiber (CV < 1.5 m/s) or A-fiber (CV ≥ 1.5 m/s).
Qualitative Measurements of Evan's Blue Extravasation
Plasma extravasation of Evan's Blue dye has been validated as a method to measure local inflammatory responses to nociceptive transmitters released from C-fiber nociceptors . Briefly, rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), injected with Evan's Blue (Sigma, i.v. 0.5%, 5 ml/kg b.w. in saline) and perfused transcardially with 0.1 M phosphate-buffered saline (pH 7.4) 20 minutes later. The limbs were photographed, and the difference in the extent of plasma extravasations of Evans blue was compared between limbs with applied vs nonapplied topical analgesics.
Data Analysis and Statistics
Behavioral data were compared using nonparametric repeated measures analysis of variance with Bonferroni posttest. Student's t-test was used to compare data in all other experiments. A value of P < 0.05 was considered significant. N refers to the number of animals used or fibers sampled in a given group. Data were expressed as means ± standard error of the mean.
Results
Injection of CFA into the Muscle Elicited a Reliable Inflammatory Pain
Starting from the 3rd day after CFA injection, the weight bearing capacity of the inflammatory hindlimb declined, and the difference score decreased significantly. An anti-inflammatory medicine, indometacin (12 mg/kg, p.o.), produced a significant increase in the different score as demonstrated in Figure 0001 (N = 6, P < 0.05), indicating that the inflammatory pain model was successfully established via a CFA injection of the muscle.
Complete Freund's adjuvant (CFA)-induced inflammatory pain in muscle was inhibited by indometacin. *P < 0.05, compared with vehicle. The vertical axis in the histogram indicates the difference score (g) between the weight bearing of two hindlimbs measured from the experimental (left) side vs the control (right) side. The black square over the “zero” line on the vertical axis indicates the range of 2 × standard error of the mean(SEM) of the difference score between the two hindlimbs in the naïve animals. *P< 0.05, compared with averages before treatment.
Effects of Topical Analgesia on Inflammatory Muscular Pain
Both types of topical analgesics applied on the skin over the left (experiment side) anterior tibialis muscle of the modeled animals were able to induce a significant increase in the difference score after a latency ranging from 8 to 20 minutes for XTT and 1–2 hours for CAP (Figure 0002), indicating the effectiveness in relieving inflammatory pain. After XTT application, the pain relief effects peaked at around 30–50 minutes followed by a wave-like degrading pattern that lasted for 4–6 hours (Figure 0002A). CAP application induced similar results as XTT, but with a longer latency of up to 2 hours (Figure 0002B).
Reduction of inflammatory pain following Xiaotong Tiegao (XTT) (A) or Capzasin (CAP) (B) application. *P< 0.05, compared with averages before treatment. Other legends are the same as Figure 0001.
Cutaneous Innervation is the Decisive Factor in the Topical Analgesic Effect
In the last experiment, we confirmed that both XTT and CAP have similar pain relieving effects. However, there has been no experimental evidence supporting the notion that topical analgesics take effect by penetrating into deep tissue. Due to the strong irritation effects of most topical analgesics such as XTT and CAP, we hypothesize that these drugs act by stimulating the cutaneous nociceptive nerve terminals. Based on this hypothesis, the effects of topical analgesics would be diminished after denervation of the target skin. Therefore, we designed a subset of experiments in which the lateral cutaneous branches of the superficial peroneal nerve innervating the anterior tibial skin were transected 3 days before the CFA injection. As demonstrated in Figure 0003, application of CAP (N = 6, Figure 0003A) or XTT (N = 8, Figure 0003B) on the denervated skin could no longer relieve the inflammatory pain, as indicated by the lack of changes in the difference score. These experiments strongly suggest that topical analgesics take effect by acting on cutaneous nerve terminals rather than penetrating directly to the muscle.
Capzasin (CAP) (A) or Xiaotong Tiegao (XTT) (B) failed to relieve the muscular inflammatory pain after the denervation of the skin. Other legends are the same as Figure 0001.
Topical Analgesics Selectively Activate Cutaneous C-Afferents
Using the Evan's blue labeling technique, we were able to discover the plasma extravasation in the skin over the inflamed muscle after the application of topical analgesics, but not in those without drug application (Figure 0004A). These results indicate that the drugs can activate the cutaneous C-fiber to release inflammatory substances. Electrophysiological microfilament recordings from the nerve fibers innervating the skin revealed that neither the ongoing nor evoked activities of A-β fibers were affected by topical analgesics (Figure 0004B), indicating a lack of effects of these drugs on the A-fiber afferents. However, application of XTT induced spontaneous discharge from C-fibers which were silent before the drug application (e.g., see Figure 0004B, N = 16). The shortest latency for XTT to induce C-fiber discharges is 5 minutes, and the discharge frequency is around 5–8 Hz. Figure 0004B represents a typical recording of C-fiber spontaneous discharges induced by XTT, while the touch-evoked discharges of A-fiber remained unchanged.
Topical application of Xiaotong Tiegao (XTT) induced plasma extravasation and C-fiber discharges. (A) Extravasation of Evan's blue observed on the skin region over the inflamed muscle, indicated with dotted lines, and was corresponding to the shadowed area applied with XTT on the right side (schematic diagram of the hind limb). (B) XTT selectively elicited discharges of C-fibers but not A-fibers in the superficial peroneal nerve. No spontaneous firing was recorded before XTT application. The horizontal bars underlying the recording traces indicate light touch to the skin.
The possible cooling effects of the solvents which may evoke activities from cooling- or cold-sensitive C-fibers could be excluded by the fact that such stimuli usually have a much shorter latency than the 5 minutes observed in the drug application in our experiments. The extravasation of Evan's blue observed on the drug-applied skin region also indicates the activities of nociceptive C-fibers.
Inhibitory Effects of Topical Analgesics on the Inflammatory Muscular C-Fiber Afferent Activities
In the nerve innervating normal muscles, we did not observe any spontaneous afferent activities of C-fibers despite a large amount of A-fiber muscle spindle activities. However, in the animal models of CFA-induced inflammatory muscular pain which showed pain-related behaviors, tonic C-fiber spontaneous discharges of about 4–9 Hz were recorded from the deep peroneal nerve innervating the anterior tibial muscle. In these preparations (N = 10), XTT was applied to the skin in the background of C-fiber's tonic afferent discharges. After a latency of about 20–40 minutes, the spontaneous discharges of C-fibers gradually decreased, then almost stopped or remained sporadic (Figure 0005). This experiment demonstrated the dynamic process through which topical application of XTT to the skin inhibited the C-fibers' spontaneous afferent activities coming from the inflamed muscles. Because the skin and muscle have been separated via the formation of skin pool containing mineral oil, it would be unlikely that topical drugs applied to the skin penetrate directly into the inflamed muscles.
Inhibition of Xiaotong Tiegao (XTT) to afferent C-fiber discharges recorded from the deep peroneal nerve innervating the tibialis anterior muscle after complete Freund's adjuvant (CFA)-induced inflammation. The inset (at right upper corner) indicates C-fiber conduction velocity (0.54 m/s) of the unit recorded.
These studies showed that application of the topical analgesics such as XTT or CAP to the skin can reduce the afferent activities of the C-fibers innervating the muscles. This inhibitory effect is possibly not due to the results of direct penetration of drugs to the inflamed muscle, but rather to the activation of cutaneous nociceptors. At the same time, the topical analgesics enhanced the C-fiber afferent activities from the skin and reduced the C-fiber afferent activities from the muscle. Therefore, we speculate that inputs from the cutaneous nociceptive afferents may be able to inhibit the dorsal root reflexes of C-fiber afferents from the inflamed muscles, thereby reducing the release of inflammatory factors in the deep tissue as well as the nociceptive inputs from the damaged tissue.
Discussion
Topical analgesics are traditional drugs of growing interest. They have been widely applied for relieving various types of pain in the deep tissue, and are considered to be convenient, effective, with low risk, and low cost . They are easily accessible in drug stores and supermarkets as popular over-the-counter painkillers. There are certain common features of all kinds of topical analgesics: they have some irritation effects which may trigger skin inflammation or even blistering; they are usually unsuitable for oral use; in most cases, they are only applied for relieving pain in deep tissue such as chronic or inflammatory pain in the muscle and joints, and are rarely used in relieving pain in the skin. Although topical analgesics have been widely used for thousands of years, the mechanisms of relieving pain in deeper tissues are not fully understood yet. Most previous research works proposed that topical analgesics can pass through the skin and reach the pain-originating site in deep tissues, and some manage to promote the efficacy via facilitating the penetration of topical analgesics through the skin . In fact, these studies indicate that the concentration of the drugs reaching the pain-originating site is far below the threshold required to silence the nociceptors. In the present study, we found that once the skin was denervated, the inflammatory pain in the muscle was not relieved by the application of XTT or CAP, indicating that the pain relief in muscle tissue provided by topical analgesics was closely related to the integrity of the innervation of local skin and was possibly a result of topical analgesics-evoked inputs of C-fibers. In the experiment of recording the afferent fibers from the inflamed muscle, though the skin receiving the topical analgesics was almost completely separated from the muscle by liquid paraffin in order to prevent the drugs from penetrating into the muscle, the topic analgesic could still effectively reduce the spontaneous C-fiber afferent firings from the inflammatory muscle. Both of the above results mentioned above suggested that the analgesic effect was neither due to the direct penetration of the topical analgesic, nor to any potential effects on the excitability of A-fibers, but was rather a result of cutaneous C-fiber afferent firings induced selectively by topical analgesics, at least in the cases of XXT and CAP. The duration of C-fiber-evoked firing is quite consistent with that of the pain relief which demonstrates the behavior indicating a possible causality relationship. We speculate that the afferent inputs from nociceptors of the skin could inhibit evoked C-fiber afferent from the deep tissue.
The noxious afferent from different parts of the body may interact in the center nervous system. Le Bars et al. have successfully demonstrated a diffuse noxious inhibitory control in dorsal horn convergent neurons, i.e., noxious afferents from one side or a certain area of the body can diffusely inhibit the responses of pain-transmitting neurons in the spinal dorsal horn from the other side or other areas. Such “diffuse inhibition” only occurs in central nervous system (CNS), not in peripheral nervous system. However, in the present study, we found that topical analgesics applied on the surface of skin significantly inhibited the spontaneous afferent firings of the C-fibers innervating the inflammatory pain muscle. Such interactions between the noxious afferent firings of the peripheral nerves are probably involved in the dorsal root reflex . The afferent signals of C-fiber from the skin may inhibit the dorsal root reflex in the nerve fibers supplying the inflamed muscle, therefore decreasing the antidromic release of inflammatory factors in the muscle. As a result, the C-fiber afferent activities from the inflamed muscle are reduced together with the pain-related behaviors.
Our proposed mechanisms of topical analgesics could be used to explain some phenomena in clinical practice. For instance, some topical analgesics with strong irritative effects such as skin inflammation or blistering can produce unexpected relief of pain in the deep tissue, while mild topical analgesics induce only weak pain relief. In traditional Chinese medicine, the efficacy of moxibustion (a treatment that involves putting burning moxa on target acupuncture points to relieve pain) is dependent on the temperature of the burning moxa, i.e., within the acceptable temperature range, a higher temperature produces stronger curative effects. Moreover, the stimulating temperature has to reach above the threshold of C-fiber's activation in order to achieve a satisfactory effect . In modern western medicine, topical capsaicin has been proved effective in the treatment of neuropathic pain, postherpetic pain, and arthritis pain . However, the side effects of this drug, such as a burning pain on the skin application site, restricted its application . We speculate that the so-called “side effect” of skin irritation, caused by these topical analgesics, may actually play a key role in the relieving of deep tissue pain.
Acknowledgments
Supported by: National Natural Science Foundation of China, Grant no. 39830150. We thank Professor Chao Ma for English editing.
