Anesth Analg 2006;102:811-814
© 2006 International Anesthesia Research Society
doi: 10.1213/01.ane.0000197690.19075.bd
ANESTHETIC PHARMACOLOGY
Licking and C-Fos Expression in the Dorsal Horn of the Spinal Cord After the Formalin Test
Taeko Fukuda, MD,
Kazuhiro Watanabe, MD,
Setsuji Hisano, PhD, and
Hidenori Toyooka, MD
Department of Anesthesiology, Institute of Clinical Medicine; Tsukuba University Hospital; Laboratory of Neuroendocrinology, Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, Tsukuba University, Tsukuba-city, Ibaraki, Japan
Address correspondence and reprint requests to Taeko Fukuda, MD, Department of Anesthesiology, Institute of Clinical Medicine; Tsukuba University, Tsukuba-city, Ibaraki 305-8575, Japan. Address e-mail to taekof{at}md.tsukuba.ac.jp.
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Abstract
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We investigated whether c-fos expression in the dorsal horn is affected by licking in the formalin test. Thirty adult Sprague-Dawley rats were divided into 5 groups of 6 rats each: a free condition control (Free Cont) group, formalin test under free condition (Free F-test) group, scrub stimulation under free condition (Free Scrub) group, restrained condition control (Restricted Cont) group, and formalin test under restrained condition (Restricted F-test) group. Animals in the three free condition groups and two restricted groups were put in a clear plastic chamber and a restraining chamber, respectively. Ten percent formalin was injected into the left rear paw in the Free and Restricted F-test groups. Animals in the Free Scrub group were scrubbed on the left rear paw with a wet cotton swab. The Free Cont, Restricted Cont, and Free Scrub groups showed little c-fos expression. The number of c-fos positive cells in the ipsilateral surface dorsal horn of the Restricted F-test group was significantly less than that of the Free F-test group (P < 0.05). The results indicated that the licking action increased c-fos expression of the lumbar dorsal horn in the formalin test.
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Introduction
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The formalin test has been widely used as an animal model of nociception (1,2). Although this test consists of assessments of the pain-related behaviors of experimental animals, the c-fos immunocytochemistry technique is also applied frequently to evaluate pain and/or pain control in the formalin test. It has been reported that the majority of c-fos expression in the superficial layers of the dorsal horn is induced by noxious stimuli (3,4). However, Todd et al. (5) reported that some inhibitory neurons in the spinal cord expressed c-fos immunoreactivity after formalin injection. We speculated that the c-fos expression of gamma-aminobutyric acid or glycine neurons in their study might have been induced by licking. It is conceivable that licking mitigates the pain by activating inhibitory neurons. Therefore, the aim of the present study was to investigate whether c-fos expression is affected by licking.
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Methods
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All experimental methods were approved by our institutional animal care committee. Thirty adult male Sprague-Dawley rats weighing 300–350 g were divided into 5 experimental groups: a free condition control (Free Cont; n = 6) group, formalin test under free condition (Free F-test; n = 6) group, scrub stimulation under free condition (Free Scrub; n = 6) group, restrained condition control (Restricted Cont; n = 6) group, and formalin test under restrained condition (Restricted F-test; n = 6) group. The Free Cont, Free F-test, and Free Scrub group rats were put in a clear plastic chamber. The Restricted Cont and Restricted F-test group rats were put in a modified-restraining, dark-colored pipe-shaped chamber (BP-98A; Softron Corp., Tokyo, Japan) and observed through the front mesh window for 2 h. 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 paw of Free F-test and Restricted F-test group rats. Observations of formalin testing were started immediately after the formalin injections. Behavior was rated for 1 h, 6 times, and each observation period was 5 min. Both licking and biting were counted as licking. Animals in the Free Scrub group were scrubbed on the left rear paw with a warm wet cotton swab 59 times, which was the average number of times that the animals of the Free F-test group licked their paws. The Restricted F-test group rats were monitored for 2 h to confirm that they did not lick their formalin-injected paw.
Two hours after formalin injection or placement in the chambers, all rats were deeply anesthetized with pentobarbital (60 mg/kg intraperitoneally) and killed. Swelling of the formalin-injected paw was classified into 3 categories: none, moderate, and severe. 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 c-fos immunohistochemistry using the avidin-biotin peroxidase method described by Hsu et al. (6). The sections were incubated overnight at 4°C in phosphate-buffered saline containing a polyclonal primary antibody to c-fos (Ab-2, 1:1000 dilution; Oncogene Research Products, San Diego, CA). They were then processed according to the usual protocol for the avidin-biotin peroxidase method (Vectastain kit; Vector Laboratories, Burlingame, CA), using diaminobenzidine tetrahydrochloride as a chromogen. Every tissue was reacted for the same period with the same reagents. We quantified the effects of licking on c-fos staining by counting all labeled cells on the surface of the dorsal horn (laminae I-II) and in the deep dorsal horn (laminae III-VI). Throughout the data collection phase, the investigator was blinded as 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 licking behavior and the immunohistochemical study. Values of P < 0.05 were considered to indicate statistical significance.
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Results
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The Free F-test group rats showed a typical biphasic nociceptive pattern of licking (Fig. 1). Neither the Restricted Cont nor the Restricted F-test group rats hesitated to enter the restraining chamber. The Restricted Cont group rats remained calm in the chamber throughout the study. The Restricted F-test group rats struggled after the formalin injection. However, they did not lick the formalin-injected paws before being killed.
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Figure 1. Time course of the licking after formalin injection in the Free F-test group (formalin test under free condition). The upper graph shows the number of licks and the lower graph shows the sum of licking times (s). Each data point represents the amount of time that the animals licked the injected paw during a 5-min observation period.
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Every rat in the Free F-test and the Restricted F-test groups showed severe swelling in the formalin-injected paw. Both formalin-injected groups (Free F-test and Restricted F-test) showed higher c-fos expression than did the other nonformalin-injected groups in all dorsal horn areas except the contralateral side of the surface dorsal horn (P < 0.05) (Fig. 2). The number of c-fos positive cells in the Restricted F-test group (179 ± 78) was significantly less than that of the Free F-test group (304 ± 68) in the ipsilateral surface dorsal horn (laminae I and II). The c-fos expression ratios of the Restricted F-test group to the Free F-test group in laminae I-II and laminae III-VI of the ipsilateral dorsal horn were 59% (P < 0.05) and 67% (P = 0.09), respectively. The Free Cont, Free Scrub, and Restricted Cont groups showed very few c-fos positive cells in laminae I-II (3 ± 3, 3 ± 2, and 6 ± 5, respectively). Figure 3 shows typical results of the c-fos staining in the ipsilateral dorsal horn for each group.
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Figure 2. Number of c-fos immunoreactive neurons in the lumbar dorsal horn (12 sections). I-II and III-VI indicate the superficial dorsal horn (laminae I-II) and the deep dorsal horn (laminae III-VI), respectively. Free Cont, Free F-test, Free Scrub, Restricted Cont, and Restricted F-test indicate the free condition control, formalin test under free condition, scrub stimulation under free condition, restrained condition control, and formalin test under restrained condition groups, respectively. * P < 0.05 versus the Free Cont, Free Scrub, and Restricted Cont groups.# P < 0.05 versus the Restricted F-test group.
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Figure 3. Typical c-fos staining in the ipsilateral dorsal horn. A: Free Cont (free condition control); B: Free F-test (formalin test under free condition); C: Free Scrub (scrub stimulation under free condition); D: Restricted Cont (restrained condition control); and E: Restricted F-test (formalin test under restrained condition). Scale Bar = 100 µm.
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Discussion
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The present study showed that physically restricting rats from licking themselves decreased the c-fos expression in the surface laminae to 59% of that in the unrestricted rats in the formalin test. Because the Restricted F-test group rats struggled after formalin injection, the rats must have experienced some mental stress. It has been reported that immobilization stress increases c-fos expression in the hypothalamus, pituitary, locus coeruleus, and amygdala and sometimes reduces the expression in some parts of the hippocampus and cortex (7,8). However, there has been no report investigating the effects of restraint stress on c-fos expression of the spinal cord. The Restricted Cont group rats showed neither behaviors indicative of discomfort nor prominent c-fos expression in the spinal dorsal horn. There was no significant difference of c-fos expression between the Free Cont and the Restricted Cont groups. Therefore, we think that restraint stress per se did not decrease c-fos expression of the dorsal horn in the present study.
Licking could be interpreted as a pain-attenuating action as well as an action to protect the injured region. We speculated that neurons of the dorsal horn react differently to licking stimulation in painful or painless situations. In other words, non-noxious manipulation such as licking causes few c-fos positive cells in a normal (painless) condition, as described by Hunt et al. (3). However, licking, a non-noxious stimulation, might increase c-fos expression in a painful situation (formalin test) in which the excitement of the primary sensory neurons affects the surrounding neuron thresholds. Although we did not perform double-staining for c-fos and inhibitory neurons, c-fos positive cells of the Free F-test group might include some interneurons containing inhibitory neurotransmitters; for example, gamma-aminobutyric acid, glycine, enkephalin, or dynorphin (5,9,10). Reduction in the number of c-fos positive cells in the present study is consistent with the results of Todd et al. (5).
In conclusion, licking after the formalin test plays an important role in modulating c-fos expression of the dorsal horn, especially in the ipsilateral superficial laminae.
The authors thank Yumi Isaka and Yasuyuki Baba for their technical assistance.
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Footnotes
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Accepted for publication October 20, 2005.
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References
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Abstract
Introduction
