Anesth Analg 2004;98:1093-1098
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000104580.89717.A2
PAIN MEDICINE
The Effects of Pretreatment with Lidocaine or Bupivacaine on the Spatial and Temporal Expression of c-Fos Protein in the Spinal Cord Caused by Plantar Incision in the Rat
Xiaohui Sun, MD,
Masataka Yokoyama, MD,
Satoshi Mizobuchi, MD,
Ryuji Kaku, MD,
Hideki Nakatsuka, MD,
Toru Takahashi, MD, and
Kiyoshi Morita, MD
Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama City, Okayama, Japan
Address correspondence and reprint requests to Masataka Yokoyama, MD, Department of Anesthesiology and Resuscitology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama City, Okayama 700-8558, Japan. Address e-mail to masayoko{at}cc okayama-u.ac.jp.
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Abstract
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We investigated the spatial and temporal patterns of c-Fos protein (Fos) expression in the dorsal horn of the spinal cord caused by plantar incision in the rat and the effects of pretreatment with local anesthetics. Bupivacaine (0.5%), lidocaine (2%), or saline for control was injected for nerve block and local infiltration before the plantar incision was made under anesthesia. Pain behavior and Fos expression in the L4-L5 segments of the spinal cord were assessed at 1, 3, 6, 24, 48, 72, and 120 h after the incision. The withdrawal threshold to mechanical stimulation decreased significantly at 1 h until 120 h (1–72 h, P < 0.01;120 h, P < 0.05), and pretreatment with local anesthetics increased the threshold significantly at 1 h (both groups: P < 0.01), 3 h (both groups: P < 0.01), and 6 h (bupivacaine, P < 0.01; lidocaine, P < 0.05) in comparison with that in the saline group. In the saline group, Fos expression was detected predominantly in laminae I–II and V–VI, and the total Fos expression was maximal at 1 h and then decreased gradually. Pretreatment with local anesthetics suppressed Fos expression significantly in all layers, and this suppression continued for several days. This study provides evidence of spatial and temporal changes in Fos expression induced by plantar incision. Our results indicate that although pretreatment with local anesthetics suppresses Fos expression for several days in the postoperative period, the analgesic effect is observed only for the expected duration of the local anesthetic used.
IMPLICATIONS: Prevention of early pain by pretreatment with local anesthetics provides little benefit for postoperative pain relief in the plantar incision model, although c-Fos expression is suppressed. The number of c-Fos-expressing neurons is not necessarily correlated with pain behavior.
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Introduction
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Although prevention of pain memory and central sensitization may reduce pain behavior in animal models of persistent pain (1,2), preemptive analgesia provides only minimal relief of postoperative pain in clinical patients (3–5). There are several reasons for the disparity between animal studies and the results of clinical trials of preemptive analgesia. Surgical incision in humans produces tissue injury that likely differs from the chemical irritation, inflammation, or nerve injury produced in animal models. In addition, the time course of persistent pain in animal models compared with postoperative pain and mechanical hyperalgesia in patients is different (6). Plantar incision pain in the rat has been developed by Brennan et al. (7) as a model of postoperative pain. They have shown that an incision through the skin, fascia, and muscle of the plantar surface of the hindpaw produces reproducible and quantifiable mechanical allodynia characterized by hypersensitivity to an innocuous mechanical stimulus.
In addition to behavioral study, molecular biological study is important for clarifying the mechanism by which preemptive analgesia provides only minimal relief of postoperative pain. Immunocytochemical localization of c-Fos protein (Fos) in the spinal cord has been widely used to identify populations of neurons that are activated by peripheral exogenous stimuli (8,9). Tsuda et al. (10) reported that pretreatment with a P2 receptor antagonist attenuated mechanical allodynia and reduced Fos expression caused by plantar incision pain in the rat. More recently, Shimode et al. (11) reported that dexmedetomidine and halothane suppressed Fos induction in the same model. There may be a parallel between the ability of anesthetic and analgesic drugs to preemptively inhibit nociceptive behaviors and to suppress Fos expression in the dorsal horn of the spinal cord (12). However, Tsuda et al. (10) and Shimode et al. (11) investigated Fos expression only at 2 h after surgery, and other investigators (2,13) have reported a dissociation between the analgesic effect on pain behavior and Fos expression in the dorsal horn of the spinal cord in other animal models. Spatial and temporal expression of Fos in the spinal cord caused by plantar incision in the rat has not been studied in detail. Thus, we considered it imperative to investigate the time course of Fos expression in the spinal cord and to clarify changes in neuronal activity in the central pathways of the sensory system after surgical incision.
In this study, we investigated the spatial and temporal patterns of Fos expression in the dorsal horn of the spinal cord in response to plantar incision in the rat. We also investigated the effects of pretreatment with both short- and long-acting local anesthetics on Fos expression.
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Methods
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This study was reviewed and approved by the Animal Care and Use Committee of Okayama University Medical School. The animals were treated in accordance with the Ethical Guidelines for Investigation of Experimental Pain in Conscious Animals issued by the International Association for the Study of Pain (14). Experiments were performed on adult (weight 250–350 g) male Sprague-Dawley rats housed in pairs before incision and individually after incision. Food and water were available ad libitum.
On the day of the experiment, rats were placed individually on an elevated plastic mesh floor covered with a clear plastic cage top (21 x 27 x 15 cm2), and the baseline withdrawal threshold to punctate mechanical stimulation was determined. The mechanical stimulus was applied from underneath the mesh (openings 12 x 12 mm2) to the plantar aspect of the proximal part of the heel by means of an Electro von Frey (Transducer Indicator, model 1601; IITC Inc., Woodland Hills, CA). It has the advantage over conventional von Frey filaments in that it measures the paw-withdrawal threshold at once, and the result is shown digitally on a display screen. Moreover, the limitations of variability in the filament’s strength and the need for multiple stimulations of the paw for a single data point are overcome in this model (15). The lowest force from three tests that produced a response was considered the withdrawal threshold.
After assessment of baseline pain behavior, animals were anesthetized with a mixture of 2% isoflurane and oxygen that was maintained during pretreatment and surgery. Rats were randomly assigned to one of three groups (n = 6 each) depending on the pretreatment. To block afferent input from the plantar region, three injections were used for combined nerve blockade and local anesthetic infiltration (16). In the bupivacaine group, 0.5% bupivacaine was injected at the medial and lateral aspect of the ankle (0.1 mL each) posterior to the malleoli to anesthetize the sural and tibial nerves. This was followed by subcutaneous infiltration of 0.3 mL of bupivacaine at the intended incision site. The lidocaine group received 2% lidocaine, and the saline group received saline at the same locations and same volumes as in the bupivacaine group. Fifteen minutes after pretreatment, a plantar incision was made according to the procedure described by Brennan et al. (7). The plantar surface of the left hindpaw was prepared with 70% ethanol, and a 1-cm longitudinal incision was made through the skin and fascia, starting 0.5 cm from the edge of the heel and extending toward the toes. The plantaris muscle was elevated with forceps and incised longitudinally. The wound was closed with two simple sutures of braided silk. Gentamicin solution was applied to the wound, and rats were allowed to awaken from anesthesia. Pain behavior was assessed at 1, 3, 6, 24, 48, 72, and 120 h after the plantar incision.
Baseline Fos expression was determined in naive control rats at 1 h after isoflurane anesthesia (no pretreatment and no incision). According to the pain behavioral study, rats were randomly assigned to one of three groups depending on the pretreatment. Fos expression was studied at 1, 3, 6, 24, 48, 72, and 120 h after plantar incision in 126 rats (each time and each group: n = 6). Animals were deeply anesthetized with sodium pentobarbital (60 mg/kg intraperitoneally) and perfused intracardially with 250 mL of phosphate-buffered saline 0.1 M (PBS) followed by 300 mL of 4% paraformaldehyde in 0.1 M phosphate buffer. After perfusion, the lumbar spinal cord was removed, immersed for 4 h in the same fixative, and cryoprotected overnight in 30% sucrose in phosphate buffer. Cords were embedded in OCT compound and kept at -70°C until cryostat sectioning. Coronal 30- to 35-µm sections corresponding to the L4 and L5 levels were obtained from each cord and collected in 0.02 M PBS for immunocytochemical analysis. The tissue was immunostained for Fos by the avidin-biotin-peroxidase complex (ABC) method of Hsu et al. (17). The tissue sections were washed with a solution of 0.02 M PBS containing 1% normal goat serum and then incubated for 1 h at room temperature, followed by incubation in a blocking solution of 0.1% NaN3 with 0.3% H2O2 for 10 min at room temperature. The sections were incubated for 48–72 h at 4°C in primary antibody solution at 1:10,000 (rabbit polyclonal antibody for c-Fos; Oncogene Research Products, San Diego, CA). After incubation, tissue sections were washed and incubated for 12 h at 4°C in a biotinylated secondary antibody solution at 1:400 (goat anti-rabbit immunoglobulin G; Kirkegaard & Perry Labs, Gaithersburg, MD) and ABC complex (ABC kit; Vector Laboratories, Burlingame, CA). The reaction product was visualized with 0.01% hydrogen peroxide and 0.05% diaminobenzidine as the chromogen (9).
Sections with Fos-positive components from each animal were coded, and sections were selected randomly from the L4 and L5 segments. Locations of the c-Fos-like immunoreactive (Fos-ir) neurons were plotted with a camera attachment at 10 x 20 magnification under a bright-field microscope. The total number of Fos-ir neurons per section and the numbers in laminae I–II, III–IV, and V–VI were counted. At least three sections per animal (n = 6) were averaged so that mean numbers of regional and total Fos-ir neurons were obtained for each animal.
Statistical analysis was performed by the Kruskal-Wallis test followed by Dunn’s method. Differences were considered statistically significant at P < 0.05.
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Results
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The withdrawal threshold in the saline group was decreased significantly in comparison to the baseline value at 1 h after incision until 120 h (1, 3, 6, 24, 48, and 72 h, P < 0.01; 120 h, P < 0.05) (Fig. 1). The withdrawal thresholds were decreased significantly in comparison with the baseline value at 3 h until 72 h in the bupivacaine group (3 h, P < 0.05; 6, 24, 48, and 72 h, P < 0.01) and the lidocaine group (3, 6, 24, 48, and 72 h; P < 0.01). Withdrawal threshold values in the bupivacaine and lidocaine groups were significantly greater than values in the saline group at 1 h (both groups: P < 0.01), 3 h (both groups: P < 0.01), and 6 h (bupivacaine, P < 0.01; lidocaine, P < 0.05).
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Figure 1. Changes in the withdrawal threshold to punctate mechanical stimulation from 1 to 120 h after plantar incision. Mean values ± SD are shown; n = 6. ¶P < 0.05 versus baseline; ¶¶P < 0.01 versus baseline; *P < 0.05 versus the saline group; **P < 0.01 versus the saline group.
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Example photomicrographs of immunoreactive Fos neurons are shown in Figure 2. Fos expression was rarely seen in the dorsal horn of the spinal cord in the naive control rat at 1 h after isoflurane anesthesia. In the saline group, peak total Fos expression was seen at 1 and 3 h, after which Fos expression decreased gradually (Fig. 3). At 1 and 3 h, Fos expression was detected predominantly in the superficial (laminae I–II) and the deeper (laminae V–VI) laminae (Fig. 4). In laminae I–II, peak Fos expression was seen at 1 h; expression then decreased sharply, and Fos immunoreactivity was seen in only a couple of neurons after 24 h (Fig. 4A). In laminae III–IV, the peak was seen at 1 and 3 h; expression then decreased very slowly, and Fos immunoreactivity was still observed at 120 h (Fig. 4B). In laminae V–VI, the peak was seen at 1 and 3 h; Fos expression then decreased gradually (Fig. 4C).
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Figure 2. Photomicrographs of the distribution of immunoreactive Fos neurons in the ipsilateral spinal dorsal horn. (A) Photograph obtained at 1 h after anesthesia from a naive control rat. (B–D) Photographs obtained at 1 h after incision from a rat that underwent pretreatment. The rats were given saline (B), lidocaine (C), or bupivacaine (D).
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Figure 3. Changes in the total c-Fos protein expression in the dorsal horn of the spinal cord from 1 to 120 h after plantar incision. Mean values ± SD are shown; n = 6. *P < 0.05 versus the saline group; **P < 0.01 versus the saline group; ##P < 0.01 versus the lidocaine group.
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Figure 4. A, Changes in c-Fos protein expression in laminae I–II from 1 to 120 h after plantar incision. B, Changes in c-Fos protein expression in laminae III–IV from 1 to 120 h after plantar incision. C, Changes in c-Fos protein expression in laminae V–VI from 1 to 120 h after plantar incision. Mean values ± SD are shown; n = 6. *P < 0.05 versus the saline group; **P < 0.01 versus the saline group; #P < 0.05 versus the lidocaine group; ##P < 0.01 versus the lidocaine group.
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Total Fos expression was significantly suppressed at 1 h until 48 h with lidocaine (1, 3, 6, and 48 h, P < 0.01; 24 h, P < 0.05) and bupivacaine (1, 3, 6, 24, and 48 h; P < 0.01) pretreatment in comparison to that in the saline group (Fig. 3). Total Fos expression in the bupivacaine group was significantly less than that in the lidocaine group at 1 and 48 h (P < 0.01) (Fig. 3). In laminae I–II, Fos expression was significantly suppressed in the lidocaine and bupivacaine groups at 1, 3, and 6 h (P < 0.01) compared with that in the saline group (Fig. 4A). In laminae III–IV, Fos expression was significantly suppressed at 1 h until 72 h in the lidocaine group (1, 3, 24, and 72 h, P < 0.01; 6 and 48 h, P < 0.05) and the bupivacaine group (1, 3, 6, 24, and 48 h, P < 0.01; 72 h, P < 0.05) compared with that in the saline group (Fig. 4B). Fos expression at 1 h in the bupivacaine group was decreased significantly in comparison to that in the lidocaine group (P < 0.01). In laminae V–VI, Fos expression was decreased significantly in the lidocaine group at 1 h (P < 0.01), 3 h (P < 0.01), and 24 h (P < 0.05) (Fig. 4C), and Fos expression was significantly decreased in the bupivacaine group at 1 h until 48 h (1, 3, 24, and 48 h, P < 0.01; 6 h, P < 0.05) in comparison with that in the saline group. Fos expression at 24 and 48 h in the bupivacaine group was decreased significantly in comparison with that in the lidocaine group (P < 0.05).
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Discussion
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This study in a rat model of postoperative pain provides experimental evidence of spatial and temporal changes in Fos expression induced by plantar incision and also reveals the effect of pretreatment with local anesthetics on Fos expression. Although pretreatment with local anesthetics suppressed Fos expression significantly for several days, the analgesic effect lasted only as long as the expected duration of the local anesthetic used.
Most of our behavioral results were similar to those reported by Pogatzki et al. (16). Injection of local anesthetics before incision decreased mechanical hyperalgesia until several hours afterward, but one day after incision, pain behavior did not differ between rats injected with saline and those injected with local anesthetic. Therefore, prevention of early pain by pretreatment did not have a long-lasting effect on subsequent hyperalgesia in this model. Prevention of pain signals in the early stage can lead to a prolonged antiallodynic effect (18). However, pretreatment in the plantar incision model does not provide a prolonged antiallodynic effect (6,16). In a neurophysiological study, Pogatzki et al. (16) showed that continuing afferent input is critical to sensitization of dorsal horn neurons in the plantar incision model, because blocking afferent input has no effect on processes that persist longer than the anticipated drug action. This may explain why pretreatment analgesic strategies offer little benefit for postoperative pain relief.
Pretreatment with 0.5% bupivacaine or 2% lidocaine had a similar effect on mechanical hyperalgesia. The potencies of 0.5% bupivacaine and 2% lidocaine are similar, but bupivacaine is effective for a longer period than lidocaine. If we had checked pain behavior between 6 and 24 hours, the withdrawal threshold might have appeared to differ between the two groups.
The withdrawal threshold was measured in this study by Electro von Frey, not conventional von Frey, filaments. However, changes in the withdrawal threshold were similar to those in a previously reported study (16) in which conventional von Frey filaments were used. The Electro von Frey system precludes the need for the up-down method of finding the threshold and thus is advantageous over the conventional von Frey filament system.
To clarify changes in neuronal activity in the central pathways of the sensory system after surgical incision, we investigated the spatial and temporal patterns of Fos expression in the spinal cord. Sensory inputs, particularly nociceptive inputs, are reported to produce increased expression of the immediate early gene c-fos and its protein product Fos (8,9). There may be a parallel between the ability of anesthetics and analgesics to preemptively inhibit nociceptive behaviors and suppress Fos expression in the spinal cord dorsal horn (19). Several reports, however, describe dissociation between analgesics on pain behaviors and Fos expression in the spinal cord dorsal horn (2,13). In this study, Fos expression in the untreated group was observed mainly at one and three hours after incision in laminae I–II and V–VI, which contain neurons activated by A
- and C-fibers (20). This finding is in accordance with recent electrophysiological findings that increases in the activities of A- and C-fibers occur after incision of the rat hindpaw (21). Fos expression was drastically decreased in laminae I–II at one day after incision even in the untreated group, and there was little Fos expression in any layer at three days after incision when mechanical hyperalgesia remained. Furthermore, pretreatment with local anesthetics suppressed Fos expression significantly in all layers during the early period after incision, and this suppression continued for several days. These results indicate that the number of Fos-expressing neurons is not necessarily correlated with pain behavior in the plantar incision model.
Although there was no particular difference between the lidocaine and bupivacaine groups with regard to Fos expression, expression tended to be more suppressed in the bupivacaine group. Pretreatment with long-acting local anesthetics may be of more benefit, not only for the duration of the analgesic effect, but also for concurrent suppression of Fos induction.
In clinical situations, nerve block and local infiltration with local anesthetics before surgery offer little benefit for postoperative pain relief. However, Pogatzki et al. (16) suggest that certain chronic pain states, such as postamputation phantom limb pain caused by surgery, may be reduced by preemptive analgesia. Our results do not exclude the possibility that persistent postoperative pain at several weeks or months after surgery might be reduced by preemptive treatment that significantly suppresses Fos expression in the early postoperative phase.
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Accepted for publication October 16, 2003.
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Abstract
Introduction
