Empirically, it is known that some interference stimulation applied to the peripheral skin attenuates pain. Kakigi and Watanabe1 reported that various kinds of stimulation, such as vibration, movements, and hot or cold water, affected the amplitude of pain-related somatosensory-evoked brain potentials and visual analog scale scores in normal subjects. Furthermore, the effects and mechanisms of transcutaneous electrical nerve stimulation or vibration have been investigated in both experimental and clinical situations.2,3 However, only a few studies have investigated the effects of nociceptive behaviors on pain relief. Previously, we examined the effects of licking in a formalin test using c-fos immunohistochemistry,4 but the data proved difficult to interpret, because c-fos immunoreactivity after noxious stimulation developed in several types of spinal neurons, including inhibitory interneurons.5 In 1999, Ji et al.6 reported that extracellular signal-regulated kinases (ERK) were rapidly phosphorylated in dorsal horn neurons of the rat spinal cord after noxious stimuli were applied to the hind paw. However, innocuous stimuli did not induce the phosphorylation. The aim of this study was to investigate whether nociceptive behaviors have direct effects of pain relief at the spinal level. Our hypothesis is that phosphorylated ERK (pERK) expression of the dorsal horn is inhibited in the formalin test by licking. If this study confirms this hypothesis, some nonpharmacologic treatment (e.g., massage, etc.) will have further scientific foundations besides improvement of local circulation. We put rats under either free or restricted conditions, injected formalin in their rear paws, and evaluated pERK and c-fos expression using the avidin-biotin peroxidase (ABC) method.
All experimental methods were approved by our Institutional Animal Care Committee. Twenty-four adult male Sprague-Dawley rats weighing 300–350 g were divided into four experimental groups: control (cont group) (n = 6), formalin test under free conditions (free f-test group) (n = 6), restricted conditions (restricted cont group) (n = 6), and formalin test under restricted conditions (restricted f-test group) (n = 6).
Ten percent formalin (3.7% formaldehyde solution, 0.1 mL) was injected subcutaneously with a 26-gauge needle into the plantar surface of the left rear paws of the free f-test and restricted f-test group rats. Observations of the formalin testing were started immediately after the injections. The free f-test group rats were kept in a clear plastic chamber. Their behavior was rated for 25 min; there were three 5-min observation periods separated with 5-min intervals. Rats in the restricted f-test and restricted cont groups were kept in a dark, modified-restraint, pipe-shaped chamber for 25 min (BP-98A, Softron Corp., Tokyo, Japan) (Fig. 1).
All rats were then deeply anesthetized with pentobarbital (60 mg/kg IP) and killed 25 min after formalin injection or after being placed in the restricted chamber. The animals were perfused with 500 mL of phosphate-buffered saline (pH 7.4), followed by 500 mL of 4% paraformaldehyde fixative. After perfusion, the lumbar spinal cord was removed and postfixed in the same fixative for 2 h. Twelve sections (40 μm) were taken at 400-μm intervals from the entire length of the lumbar spinal cord and processed for p-ERK and c-fos immunohistochemistry using the ABC method described by Karim et al.7 and Hsu et al.8 The sections were incubated overnight at 4°C in phosphate-buffered saline containing a polyclonal primary antibody to p-ERK (Phospho-p44/42 MAPK, 1:1000 dilution; Cell Signaling Technology, Beverly, MA) and c-fos (AB-2, 1:1000 dilution; Oncogene Research Products, San Diego, CA). They were then processed according to the usual protocol for the ABC method (Vectastain kit, Vector Laboratories, Burlingame, CA), using diaminobenzidine tetrahydrochloride as a chromogen. We semiquantified the effects of licking on the p-ERK and c-fos staining by counting all the labeled cells plotted on the surface (laminae I–II) and deep (laminae III–VI) layers of the dorsal horn.9 Throughout the data collection phase, the investigator was blinded to each animal's condition.
Data were presented as mean ± sd. Statistical analyses were performed using a two-way analysis of variance (Bonferroni post hoc test) for the immunohistochemical study. A P < 0.05 was considered statistically significant.
Free f-test group rats showed typical nociceptive behaviors, lifting, licking, etc. Total licking time of the three observation periods was 107 ± 21 s, which was approximately the same as in our previous study.4 Cont group rats showed no nociceptive behaviors. Restricted cont group rats remained calm in the restraint chamber throughout the study. The feet and nails of every rat were checked to ensure that, except for formalin injection effects, they were intact. Restricted f-test group rats did not lick the formalin-injected paw before being killed.
Typical p-ERK immunohistochemistry of the four groups is shown in Figure 2. The number of p-ERK positive cells in the restricted f-test group was significantly higher than those in the free f-test, cont, and restricted cont groups on the ipsilateral superficial dorsal horn. There were no significant differences among the four groups in the ipsilateral deep layer or contralateral side (Fig. 3). The c-fos expression of the free f-test and restricted f-test groups was significantly higher than that of the control group in laminae I–II of the ipsilateral dorsal horn (Fig. 4). However, there was no significant difference between the free f-test and restricted f-test groups in the ipsilateral superficial dorsal horn.
This study demonstrated that restricting licking increased pERK in the ipsilateral superficial dorsal horn. Restrictions without the formalin test showed no significant changes in pERK expression. Allowing the rats to lick freely decreased pERK expression of the spinal cord to a level similar to that of the control groups. Licking influences the formalin-induced changes of neural activities in the dorsal horn of the spinal cord.
The ERK are mitogen-activated protein kinases that transduce extracellular stimuli into intracellular posttranslational and posttranscriptional responses.10,11 Within 1 min of an intense noxious peripheral or C-fiber electrical stimulus, many p-ERK positive neurons were observed in the spinal cord; however, low-intensity stimuli, repeated light touches, or Aβ-fiber input did not induce pERK.6 The activation of ERK in response to graded heat, electrical, or mechanical stimuli was intensity dependent.6,12 Furthermore, some nociceptive behaviors (licking, biting, and scratching responses) were attenuated by PD98059 (an ERK inhibitor) in a dose-dependent manner.13,14 Song et al.15 have reported that an intrathecal injection of ERK antisense significantly attenuated chronic constriction injury-induced neuropathic pain and suppressed the increase of chronic constriction injury-induced pERK expression in the spinal cord. Therefore, the phosphorylated form of ERK has been used as an anatomical marker for nociceptor activation.16,17 Thus, we speculated that a decrease of pERK expression in the spinal dorsal horn indicates an attenuation of formalin-induced pain.
The mechanisms of such pain attenuation might be explained by dynamic changes of peripheral tissues, spinal neurons, pain control system of the brainstem, and cerebrum. In peripheral tissues, licking might improve local circulation, decrease the concentration of pain-producing substances (substance P, calcitonin gene-related peptide, etc.), and inhibit the axon reflex. Soft mechanical stimulation, like licking, might inhibit the nociceptive transmission to supraspinal parts at the dorsal horn level by activation of Aβ-fiber (gate control theory). Licking is a complex action including salivary secretion, which is controlled by a parasympathetic nerve. Electrical stimulation of vagus has been reported to show an antinociceptive effect via activation of the nucleus raphe magnus, locus ceruleus, and other nuclei.18 There is the possibility that licking induces excitation of the vagus and some parts of the descending pain modulation system. Furthermore, a pure “psychological” effect, such as distractive mechanisms or feeling of accomplishment, might contribute to the pain attenuation. Conversely, psychological stress due to the licking disturbance might have been related to the high pERK expression in the restricted f-test group. However, the axon reflex does not account for much of the entire nociceptive neurotransmission, licking-induced vagus stimulation should be weaker than direct electrical stimulation, and the clear local effects in the dorsal horn are unexplained by only psychological factors. Therefore, we speculate that modifications at the spinal level very possibly are responsible for the licking-induced pain attenuation.
Because capsaicin-induced ERK activation reached a peak level at 2 min and decreased immediately, the observation period needed to be shortened. However, rats must be allowed to lick their hindpaws enough to evaluate the effect of the nociceptive behavior. Therefore, we selected 25 min, during which licking time reached about 75% of the total amount recorded in our previous study.4 We also observed that c-fos expression showed no significant difference between the free f-test and restricted f-test groups in the ipsilateral superficial dorsal horn. We speculated that the reasons for our conflicting results were due to the short observation period, which interrupted c-fos expression before it had peaked. Shimizu et al.19 have reported a difference in the distribution pattern of pERK and c-fos positive cells in the subnuclei interpolaris-caudalis transition zone after noxious tooth-pulp stimulation. They suggested that the difference in the temporal expression processes of pERK and c-fos accounted for the distribution pattern they observed. Another possibility may have been differences in the original distribution of pERK and c-fos expressions.20 The c-fos promoter basically consists of three main elements: the serum response element, the sis-inducible element, and the cAMP response element. These factors are differently phosphorylated, and the ERKs are related to phosphorylation of the serum response element.21 Because c-fos expression is induced through both ERK-dependent and -independent pathways,22 the results of pERK and c-fos expression might be different in this study. One limitation of this study was that the effects of vehicle solution injection were not investigated. There is the possibility that the physical stimulations of needle and saline induce pERK expression in the dorsal horn. However, the normal saline control group showed very little licking behavior in our previous study.23 Furthermore, c-fos expression of the dorsal horn was scarcely induced by normal saline injection.23 Therefore, we speculate that the results of the cont group and that of normal saline-injected rats are almost the same.
To our knowledge, there are no reports investigating the effects of instinctive pain behaviors on the nociceptive processes of the spinal cord. Licking decreased pERK expression of the dorsal horn in the formalin test. Licking could be interpreted as a pain-attenuating action and a way to protect the injured region. Rubbing or softly touching human behavior might be analogous to rats' licking behavior. If these motions inhibit pERK expression in the human dorsal horn, the repeated soft mechanical stimulation might be revaluated in the field of rehabilitation or terminal care. It was also reported that intrathecal injection of the ERK kinase inhibitor reduced secondary hyperalgesia or mechanical allodynia.24–28 Because licking decreased the number of pERK positive neurons in this study, soft mechanical stimulation after injury or surgery might have the potential of both attenuation of pain and prevention of future pain by inhibiting central sensitization. This study also indicated that results of animal behavioral studies might be affected by both nociceptive stimulations and reactive behaviors.
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