Physiology & Behavior 147 (2015) 30–37
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The major histocompatibility complex genes impact pain response in DA and DA.1U rats Yuan Guo a, Fan-Rong Yao b, Dong-Yuan Cao c,d, Li Li a, Hui-Sheng Wang a, Wen Xie a, Yan Zhao a,⁎ a
Department of Physiology and Pathophysiology, School of Basal Medical Science, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi 710061, PR China Department of Pharmacology and Toxicology in the Brody School of Medicine at East Carolina University, Greenville, NC, USA Research Center, Stomatological Hospital, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi 710004, PR China d Department of Neural and Pain Sciences, University of Maryland Dental School, 650 West, Baltimore Street, Baltimore, MD 21201, USA b c
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• DA and DA.1U rats, whose genetic background is the same except MHC genes • DA had higher composite pain scores than DA.1U rats in formalin model. • Formalin induced biphasic increase in discharge rates, especially in DA rats. • Formalin induced increase of RT1-B in DA rats, but not in DA.1U rats.
a r t i c l e
i n f o
Article history: Received 15 February 2015 Received in revised form 13 March 2015 Accepted 4 April 2015 Available online 7 April 2015 Keywords: Major histocompatibility complex Pain Formalin DA rats DA.1U rats RT1-B
⁎ Corresponding author. E-mail address: [email protected] (Y. Zhao).
http://dx.doi.org/10.1016/j.physbeh.2015.04.009 0031-9384/© 2015 Elsevier Inc. All rights reserved.
a b s t r a c t Our recent studies have shown that the difference in basal pain sensitivity to mechanical and thermal stimulation between Dark-Agouti (DA) rats and a novel congenic DA.1U rats is major histocompatibility complex (MHC) genes dependent. In the present study, we further used DA and DA.1U rats to investigate the role of MHC genes in formalin-induced pain model by behavioral, electrophysiological and immunohistochemical methods. Behavioral results showed biphasic nociceptive behaviors increased significantly following the intraplantar injection of formalin in the hindpaw of DA and DA.1U rats. The main nociceptive behaviors were lifting and licking, especially in DA rats (P b 0.001 and P b 0.01). The composite pain scores (CPS) in DA rats were significantly higher than those in DA.1U rats in both phases of the formalin test (P b 0.01). Electrophysiological results also showed the biphasic increase in discharge rates of C and Aδ fibers of L5 dorsal root in the two strains, and the net change of the discharge rate of DA rats was significantly higher than that of DA.1U rats (P b 0.05). The mechanical thresholds decreased after formalin injection in both strains (P b 0.01), and the net change in the mechanical threshold in DA was greater than that in DA.1U rats (P b 0.05). The expression of RT1-B, representation of MHC class II molecule, in laminae I-II of L4/5 spinal cord in DA rats was significantly higher than that in DA.1U rats in the respective experimental group (P b 0.05). These results suggested that both DA and DA.1U
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rats exhibited nociceptive responses in formalin-induced pain model and DA rats were more sensitive to noxious chemical stimulus than DA.1U rats, indicating that MHC genes might contribute to the difference in pain sensitivity. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Pain is very complex and widely investigated for many years. Some researches have shown that pain process can be influenced by a variety of factors, including genetic differences [1–3], but the genetic mechanisms of pain are still poorly understood. The major histocompatibility complex (MHC), denoted as RT1 in the rat, is located on chromosomes 20 and spans approximately 4 Mb [4]. The major regions of MHC genes contain class I region (the centromeric class I region is RT1-A and the telomeric class I region is RT1-C/E/M), class II region (RT1-B/D), and class III regions [5,6]. It has been shown that MHC genes are involved in the genetic mechanisms for hyperalgesia induced by peripheral or central nervous system injury [7–9]. Our recent study has suggested that the level of MHC II molecular RT1-B might cause the differences in basal pain sensitivity between Dark-Agouti (DA) and cogenic DA.1U rats, whose genetic background is the same as that of DA rats except for MHC genes [10]. The purpose of the present study was to investigate the role of MHC genes in formalin-induced pain model. The formalin test was used in the present study to observe the pain responses to chemical nociceptive stimulation in the two strains because it is a well-characterized behavioral model of tonic chemogenic pain and widely used in studies on nociceptive processes and analgesic drug effects [11,12]. The behavioral responses including specific and non-specific nociceptive behaviors induced by formalin injection were observed in DA and DA.1U rats. In addition, electrophysiological methods were used to record the afferent discharge rates of C and Aδ fibers from L5 dorsal root. Furthermore, the expression of RT1-B, representation of MHC II molecular, in L4/L5 dorsal horn of spinal cord was also observed to further investigate the role of MHC II molecular in pain sensitivity. 2. Materials and methods 2.1. Animals Experiments were performed on inbred DA (originating from Zentralinstitut Fur Versuchstierzucht, Hannover, Germany) and DA.1U rats (originating from Lond University, Sweden) of either gender. All rats were 12–16 weeks old, and weighed 150–220 g. All experimental procedures were approved by the Institutional Animal Care Committee of Xi'an Jiaotong University, and were in accordance with ethical guidelines of the International Association for the Study of Pain [13]. The animals were kept in three per polystyrene cage in specific pathogen free animal facilities on 12 h light–dark cycles with food and water available ad libitum. 2.2. Behavioral tests Behavioral observations were carried out by the same investigators from 9 to 11 am on experimental days in the same room. The animals were habituated for 30 min before formalin test in a clear 40 × 30 × 30 cm plastic box with a mirror behind the box at a 45° angle to allow an unobstructed view of the opposite paw. The animals were divided into normal saline (NS) group (n = 8 for each rat strain) and formalin (FM) groups (n = 15 for each rat strain). In FM group, formalin (2.5%, 50 μl) was injected into right hindpaw intraplantarly using a 30 g needle. In NS group, NS (50 μl) was injected intraplantarly in the same way.
The nociceptive behavioral responses were quantified by counting the accumulative time (in seconds) spent in specific and non-specific nociceptive behaviors in the injected hindpaw during each 5 min period for 1 h after formalin injection. The specific nociceptive behaviors included favoring, lifting and licking, while non-specific nociceptive behaviors included resting or sleeping, still but alert, walking and grooming. Favoring meant injected paw had little weight and rested on the floor without pressure on the footpad; during locomotion there was a definite limp. Lifting indicated that the injected paw was elevated without touching the floor. Licking indicated that the injected paw was licked or bitten. The composite pain score (CPS) was calculated according to the following numerical scale: 0, no pain (normal weight bearing on the injected paw); 1, favoring; 2, lifting; and 3, licking [14–16]. The nonspecific behaviors for each 5 min block in each of the four behavioral categories were counted simultaneously: 0, rest or sleeping; 1, still but alert; 2, walking; and 3, grooming any part of the body except the injected paw [17]. Then, the CPSs for specific and non-specific nociceptive behaviors, ranging from 0 to 3 were calculated by multiplying the time spent in each category by the category weight, summing these products and dividing by the total time in seconds for each 5 min block of time.
CPS ¼
X
score for each behavior
duration of each behavior in every 5 min=300 2.3. Electrophysiological experiment Rats were anesthetized initially with urethane (1.0 g/kg i.p.) and supplemental doses (0.05 g/kg/h) were given as needed to maintain areflexia. Hair on the back was shaved off and a skin incision was made longitudinally along the median line. A T13–L3 laminectomy was performed in order to expose L5 dorsal root and cut it proximally. A pool was formed by raising the skin flaps and filling the space with warm paraffin oil (37 °C). Rectal temperature was maintained at approximately 37 ± 0.5 °C using a servo-controlled heating blanket. The distal end of the L5 dorsal root was placed on platinum bipolar electrodes for recording. On a small platform, the nerve was mechanically desheathed and teased apart under a dissecting microscope. Small filaments were repeatedly split with sharpened watchmaker forceps until single unit activity was obtained. Neural activity was recorded, then amplified (Biophysical Amplifier AVB-11A, Nihon Kohden, Japan), filtered (30–3000 Hz), and displayed on an oscilloscope (VC-11, Nihon Kohden, Japan) for monitoring the action potential's waveform and amplitude. The signals were also fed into a computer based data acquisition system (Spike2, Cambridge Electronic Design Limited, Cambridge, UK) that allowed continuous monitoring of discharges as well as off-line data analysis. The mechanical threshold (MT) of each unit was measured with a set of calibrated von Frey's filaments (Stoelting Company, Wood Dale, IL, USA), with bending forces from 3.92 mN to 254.8 mN, applied to the unit's receptive field (RF) and expressed as the minimum force (mN) needed to evoke a response in ≥ 50% of the trials [18–22]. The location of most sensitive point of RF to mechanical stimulation was marked and targeted for drug injection. The 28 g needle with PE10 tubing was inserted into the most sensitive point and connected to microinfusion pump (WZ-50, Zhejiang Medical university, China). After the spontaneous discharge became stable, the 5 min discharge
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was recorded as baseline control. Then formalin (2.5%, 10 μl) was microinjected for 5 min and discharge rates were continuously recorded for 1 h. After recording, the MT was measured again to compare with that before injection. After MT examined, a single electrical stimulus with progressively increasing intensity (0.1–1 mA, duration 0.5 ms) was applied to the sensitive point of RF. Conduction velocity (CV) was calculated by dividing the distance from the stimulating to the recording electrode by the latency of the action potential. Units with CVs less than 2.0 m/s were classified as C fibers, 2.0–30.0 m/s as Aδ fibers, and 30.1–70.0 m/s as Aβ fibers [19–23].
2.4. Immunohistochemistry The rats were divided into three groups. In control group, no treatment was applied on rats. In NS and FM groups, the rats were intraplantarly injected NS (50 μl) or formalin (2.5%, 50 μl), respectively. Rats were deeply anesthetized with sodium pentobarbital (80 mg/kg, i.p.) 90 min after injection. Then the rats were perfused through the left ventricle with 100 ml saline (37 °C) followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4, 4 °C). The L4/L5 spinal cord segments were cut, post-fixed, and cryoprotected in 30% sucrose in 0.1 M PB (pH 7.4) for 24–36 h.
Fig. 1. Nociceptive behaviors induced by intraplantar injection of formalin in DA and DA.1U rats. A–D: Cumulative histogram of all kinds of specific nociceptive behaviors (A, C) and nonspecific nociceptive behaviors (B, D) in DA rats (A, B) and in DA.1U rats (C, D); E and F: The comparison in each specific (E) and non-specific (F) nociceptive behavior between DA and DA.1U rats in FM group; G and H: The comparison in time course of composite pain score (CPS) for specific (G) and non-specific nociceptive behaviors (H). NS: normal saline group; FM: formalin group. **P b 0.01 vs NS group in each rat strain; ## P b 0.01 vs DA.1U in each group; two way RM ANOVA.
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Tissues were embedded in OCT compound (Sakura Finetechnical Co., Ltd., Tokyo, 103, Japan) and frozen at −20 °C and then sectioned coronally into 40-μm slices (Leica CM1900, Germany). Every third section was selected for immunohistochemical staining. After washing with 0.01 M phosphate-buffered saline (PBS, pH 7.4), the sections were pre-treated with 0.3% H2O2 (10 min, at room temperature) and 10% normal goat serum (NGS, 1 h, at room temperature) in PBS with 0.3% Triton X-100 (Sigma, St Louis, MI, USA). Then sections were incubated with monoclonal antibody OX-6 (staining for RT1-B, 1:100; gifts from Prof. SM Lu, Department of Biochemistry and Molecular Biology, Xi'an Jiaotong University Health Science Center) in 5% NGS-PBS for 48 h at 4 °C. Subsequently, the sections were incubated overnight with biotinylated goat anti-mouse staining for RT1-B at 4 °C, and further processed using avidin biotin peroxidase complex according to manufacturer instructions (Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China). Finally, the sections were reacted with 0.02% 3,3′diaminobenzidine (Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China) for 10–15 min. The sections were then mounted onto gelatin-coated glass slides, air dried, dehydrated through a graded alcohol series, cleaned with dimethylbenzene, and coverslipped with neutral balsam. All sections were observed under a light microscope (BX-51; Olympus, Tokyo, Japan) and images were captured using a SensiCam digital camera (SPOT-Insight QE, Diagnostic Instruments Inc., Sterling Heights, MI, USA). Images were imported and analyzed, as TIFF files, using SigmaScan Pro Image Analysis Software (SPOT-Insight QE, Diagnostic Instruments Inc., Sterling Heights, MI, USA). To discriminate positive immunostaining from the background, the cells showing a two-fold more intense staining than the average background were considered as RT1-B-positive cells and counted by the software Image-Pro Plus 4.5 (Media Cybernetics, Silver Spring, MD, USA). In the negative control experiments, the primary antibodies were replaced with NGS; no positive staining for the replaced antibodies was detected.
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way ANOVA, P b 0.001 and P b 0.01). For the time course following formalin injection, the CPSs for specific and non-specific nociceptive behaviors increased at all time points in both DA and DA.1U rats in FM group compared with those in NS group (two way RM ANOVA, P b 0.01, Fig. 1G, H). The CPSs for specific nociceptive behaviors of DA rats in FM group were significantly higher than those of DA.1U rats in both phases (two way RM ANOVA, P b 0.01, Fig. 1G). The CPSs for non-specific nociceptive behaviors of DA rats in FM group were significantly higher than those of DA.1U rats at 35–45 min (two way RM ANOVA, P b 0.01, Fig. 1H). 3.2. The sensitivity of C and Aδ units to formalin stimulation in DA rats was higher than DA.1U rats
3. Results
The basal physiological properties of C and Aδ units in DA and DA.1U rats were shown in Table 1. There were no obvious differences in CV and background discharge rates between DA and DA.1U rats, while the basal MTs of C and Aδ units in DA rats were significantly lower than those in DA.1U rats (P b 0.01, t-test, Table 1). After formalin injection, the MTs of C and Aδ units decreased in both DA and DA.1U rats (P b 0.01, paired t-test, Fig. 2A and B); however, the net changes in MTs of C and Aδ units after formalin injection in DA rats were much higher than those in DA.1U rats (P b 0.05, t-test, Fig. 2A and B). These results suggested that DA rats were more sensitive to formalin than DA.1U rats. The discharge rates of C units increased biphasically in both DA and DA.1U rats. The first phase was at first 5 min after formalin injection, and the second phase was from 20 to 60 min. In DA rats, the discharge rates of C units increased at all time points after formalin injection (P b 0.01, two way RM ANOVA, Fig. 2C). In DA.1U rats, the discharge rates of C units increased at the first 5 min and from 30 to 45 min after formalin injection (P b 0.01, two way RM ANOVA, Fig. 2C). The discharge rates in DA rats were significantly higher than those in DA.1U rats in both phases (P b 0.01, two way RM ANOVA, Fig. 2C). The right panel of Fig. 2C showed the original discharge of C units in DA (upper) and DA.1U rat (lower). As for Aδ units, the discharge rates in both DA and DA.1U rats significantly increased after formalin injection, and the discharge rates in DA rats were also significantly higher than those in DA.1U rats (P b 0.01, two way RM ANOVA, Fig. 2D). These electrophysiological results suggested that DA rats were more sensitive to formalin than DA.1U rats, which were in accordance with the duration of lifting and licking in behavioral test.
3.1. More spontaneous nociceptive behaviors were observed in DA rats than that in DA.1U rats after formalin injection
3.3. The expression of RT1-B in spinal cord in DA was higher than that in DA.1U rats
No obvious spontaneous nociceptive behaviors were observed in NS group in both DA and DA.1U rats. There was no difference in single behavior and CPSs for specific and non-specific nociceptive behaviors between DA and DA.1U rats in NS group (two way ANOVA, P N 0.05). After intraplantar injection of formalin, obvious biphasic nociceptive behaviors were observed in both DA and DA.1U rats. The first phase is the first 5 min, and the second phase is from 20 min to 60 min after formalin injection. The main specific nociceptive behaviors were lifting and licking, the main non-specific behaviors were still but alert (Fig. 1A–D). The duration of favoring in DA rats was significantly shorter than that in DA.1U rats (t-test, P b 0.001, Fig. 1E), while the durations of lifting and licking in DA rats were significantly longer than those in DA.1U rats (t-test, P b 0.001 and P b 0.01, respectively, Fig. 1E). There was no difference in the duration of each non-specific behavior between DA and DA.1U rats (t-test, P N 0.05, Fig. 1F). The average CPSs for specific and non-specific nociceptive behaviors of DA rats in FM group were 2.028 ± 0.026 and 1.461 ± 0.019, respectively, which were significantly higher than those in NS group (0.014 ± 0.009 and 0.270 ± 0.061, two way ANOVA, P b 0.001), and also significantly higher than those in DA.1U rats in FM group (1.681 ± 0.039 and 1.275 ± 0.064, two
The RT1-B expression was observed mainly in both sides of lamina I-II in L4/5 spinal cord. The location of RT1-B expression in DA.1U rats was similar to that of DA rats. In agreement with our recent study [10], we confirmed that basal RT1-B expressions in DA rats (61.53 ± 6.83
2.5. Statistical analysis All data were presented as mean ± SEM and analyzed by SigmaStat 2.0 software. One way ANOVA, two way (RM) ANOVA, t-test or paired t-test are used as appropriate. P b 0.05 was considered to be statistically significant.
Table 1 The basal physiological properties of C and Aδ units from L5 dorsal root in DA and DA.1U rats. Units
Physiological properties
DA rats
DA.1U rats
C
n Mechanical thresholds (mN) Background discharge rates (imp/5 min) Conduction velocity (m/s) n Mechanical thresholds (mN) Background discharge rates (imp/5 min) Conduction velocity (m/s)
15 84.93 ± 7.75 12.93 ± 0.78
17 139.51 ± 11.99⁎⁎ 6.29 ± 1.17⁎⁎⁎
1.32 ± 0.07 24 80.44 ± 6.7 9.58 ± 1.23
1.36 ± 0.16 28 121.45 ± 10.98⁎⁎ 8.86 ± 0.96
7.16 ± 1.25
9.95 ± 1.72
Aδ
⁎⁎ P b 0.01. ⁎⁎⁎ P b 0.001, vs DA rats.
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and 53.87 ± 5.47 for right and left side) were more than those in DA.1U rats (26.00 ± 2.38 and 27.20 ± 3.60). Formalin injection in right hindpaw increased RT1-B expression in lamina I–II in L4/5 spinal cord of both sides in DA rats (161.67 ± 7.17 and 136.00 ± 8.48 for right and left sides, respectively, P b 0.001, two way ANOVA, Fig. 3A, B and E), but not in DA.1U rats (32.27 ± 3.16 and 23.20 ± 2.501, Fig. 3C–E). And there was no difference between two sides (P N 0.05, two way ANOVA, Fig. 3E). Injection of saline did not change the RT1-B expression in both DA and DA.1U rats (P N 0.05, two way ANOVA, Fig. 3E).
4. Discussion In the present study, behavioral results showed that formalin induced typical biphasic pain responses in both DA and DA.1U rats, but the CPS for specific nociceptive behavior in DA rats were significantly higher than that of DA.1U rats, especially the duration of lifting and licking. Electrophysiological results also showed that formalin sensitized biphasically the C and Aδ units of primary sensory nerve in both DA and DA.1U rats, but the sensitivity of DA rats was higher than DA.1U
Fig. 2. The changes in mechanical thresholds (MT) and discharge rates in DA and DA.1U rats in formalin model. A and B: The changes in MT of C units (A) and Aδ units (B. **, P b 0.01 vs pre-injection, paired t-test; #, P b 0.05, ##, P b 0.01 vs DA rats, t-test); C and D: The comparison in discharge rates of C units (C) and Aδ units (D). Left panels are the time course curves of discharge rates and right panels are the samples of original discharge. con: pre-injection of formalin. ** P b 0.01 vs con; ## P b 0.01 vs DA.1U rats.
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rats. The electrophysiological results were consistent with the duration of lifting and licking, suggesting that DA rats had higher sensitivity in formalin induced pain. The formalin model first described by Dubuisson and Dennis [14] is usually used as a pain model of acute tissue injury-induced pain to characterize nociceptive processes and analgesic drug effects in rodents [14–16,24–32]. Various nociceptive behaviors observed after injection of formalin include the flinches [33,34], lifting, and licking [30,35,36]. Meanwhile, the CPS is also derived by applying the amount of time that the animal spends in a given behavioral category to a prior assigned category weights and summing the products, which is termed the weighted scores technique [14–16,37]. Sometime the non-specific nociceptive responses including sleeping or resting, being still but alerting, walking, and grooming were also observed [17,38,39]. In the present study, the behavioral responses in both DA and DA.1U rats were consistent with the previous studies showing that formalin induced typical biphasic nociceptive behaviors, mainly lifting and licking [12,16,24,25,28, 34,40,41]. As for the single behavior, the duration of favoring in DA rats was shorter than that in DA.1U rats, while the durations of lifting and licking in DA rats were longer than those in DA.1U rats. Another index,
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CPS for specific nociceptive behaviors was higher in DA rats than DA.1U rats. About these inconsistent results between single behaviors, it should be related to the intensity of pain. It was reported that the single behavior was related to the intensity of pain. Favoring was thought to reflect an absence of pain experience in the animal, lifting was thought to reflect intermediate pain experience, and licking was thought to reflect an intense pain experience [16]. Intense pain response was observed in DA rats, so the durations of lifting and licking were longer and the duration of favoring was shorter in DA rats. Combined with the result of CPS, we concluded that formalin injection induced nociceptive behaviors in both DA and DA.1U rats. DA rats had higher sensitivity to formalin. In addition, CPS might be a more robust measure of pain than any single behavior [15,16,24]. Our electrophysiological results also showed that formalin sensitized the C and Aδ units from the primary sensory nerve, which was consistent with previous studies [42]. It has been shown that in the periphery, primary afferent nociceptive fibers become sensitized through the release of pro-inflammatory substances from C and Aδ fiber terminals and surrounding cells. The consequent increase of spontaneous firing then initiates a second phase [43–45]. In addition,
Fig. 3. The expression of RT1-B in dorsal horn of L4/5 spinal cord induced by formalin in DA (A and B) and DA.1U rats (C and D). A and B: The expression of RT1-B after formalin injection in DA rats in left (A) and right (B) sides. C and D: The expression of RT1-B after formalin injection in DA.1U rats in left (C) and right (D) sides; E: the comparison in number of RT1-B positive cells. Con: normal control group without any treatment; NS: normal saline group; FM: formalin group. ***P b 0.001 vs Con; ###P b 0.001 vs DA rats, two way ANOVA.
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this intense activation of nociceptive afferents by formalin leads to increase excitability of neurons in the central nervous system [46]. In the present study, the sensitization of C and Aδ units from primary sensory nerve was consistent with the increase of lifting and licking, indicating that the activation of primary sensory nerve might contribute to these two behaviors induce by formalin. Pain can be affected by many factors including environment and genetics. It is widely accepted that genetic factors are important in individual differences in pain sensitivity [47,48]. The present study was performed on DA and cogenic DA.1U rats. DA.1U rats are derived by initially crossing female DA (a-haplotype) rats with male E3 (u-haplotype) rats using the speed congenic technique [49]. The difference between DA and DA.1U rats is that DA.1U rats express u alleles whereas DA rats express a alleles in MHC genes [50]. So we conclude that rats carrying a alleles in MHC genes are more susceptible to formalin in comparison to rats carrying u alleles. The genomic sequence of rat MHC genes, denoted as RT1 on chromosome 20 was identified [4]. It has been reported that MHC genes are upregulated after sensory nerve injury [51], and mechanical allodynia occurs following peripheral nerve transection [52]. MHC class II contributes to hyperalgesia induced by peripheral and central nervous system injury [7–9,52,53]. Experiments about different MHC haplotypes in a model for post-herpetic pain further provide support for the role of MHC in the development of pain [54]. Moreover, MHC class II knockout mice exhibit an attenuation of nerve injury-induced allodynia [52]. Our previous studies also showed that basal expression of RT1-B, representation of MHC class II DQ subregion was higher in DA rats than that in DA.1U rats [10], which suggested that MHC class II was involved in the basal pain sensitivity. In the present study, the expression of RT1-B increased after formalin injection in DA rats, suggesting that MHC genes might be involved in formalininduced pain. However, the expression of RT1-B did not increase in DA.1U rats following injection of formalin, which was inconsistent with the increase of nociceptive behaviors and the sensitization of C and Aδ units from primary sensory nerves observed in DA.1U rats. Accordingly, although MHC genes might be involved in formalininduced pain, the importance and mechanism underlying the role of MHC genes in the process need to be investigated in the future. In addition, other genomic structures such as RT1-A for MHC class I, RT1-D for MHC class II, and RT1-C/E/M for the non-classical MHC class I region should be investigated in the future study. Acknowledgments Thanks for the help from Prof. She-Min Lu (Department of Genetics Molecular Biology, School of Basal Medical Science, Xi'an Jiaotong University Health Science Center) in providing animals and primary antibody for RT1-B. This study was supported by the National Natural Science Foundation of China (No. 81200604). References [1] W.R. Lariviere, S.G. Wilson, T.M. Laughlin, A. Kokayeff, E.E. West, S.M. Adhikari, et al., Heritability of nociception. III. Genetic relationships among commonly used assays of nociception and hypersensitivity, Pain 97 (2002) 75–86. [2] J.S. Mogil, S.G. Wilson, K. Bon, S.E. Lee, K. Chung, P. Raber, et al., Heritability of nociception I: responses of 11 inbred mouse strains on 12 measures of nociception, Pain 80 (1999) 67–82. [3] J.S. Mogil, S.G. Wilson, K. Bon, S.E. Lee, K. Chung, P. Raber, et al., Heritability of nociception II. ‘Types’ of nociception revealed by genetic correlation analysis, Pain 80 (1999) 83–93. [4] P. Hurt, L. Walter, R. Sudbrak, S. Klages, I. Muller, T. Shiina, et al., The genomic sequence and comparative analysis of the rat major histocompatibility complex, Genome Res. 14 (2004) 631–639. [5] R. Dressel, L. Walter, E. Gunther, Genomic and functional aspects of the rat MHC, the RT1 complex, Immunol. Rev. 184 (2001) 82–95. [6] E. Gunther, L. Walter, The major histocompatibility complex of the rat (Rattus norvegicus), Immunogenetics 53 (2001) 520–542. [7] C.A. Dominguez, L. Li, O. Lidman, T. Olsson, Z. Wiesenfeld-Hallin, F. Piehl, et al., Both MHC and non-MHC genes regulate development of experimental neuropathic pain in rats, Neurosci. Lett. 442 (2008) 284–286.
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Physiology & Behavior journal homepage: www.elsevier.com/locate/phb
The major histocompatibility complex genes impact pain response in DA and DA.1U rats Yuan Guo a, Fan-Rong Yao b, Dong-Yuan Cao c,d, Li Li a, Hui-Sheng Wang a, Wen Xie a, Yan Zhao a,⁎ a
Department of Physiology and Pathophysiology, School of Basal Medical Science, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi 710061, PR China Department of Pharmacology and Toxicology in the Brody School of Medicine at East Carolina University, Greenville, NC, USA Research Center, Stomatological Hospital, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi 710004, PR China d Department of Neural and Pain Sciences, University of Maryland Dental School, 650 West, Baltimore Street, Baltimore, MD 21201, USA b c
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• DA and DA.1U rats, whose genetic background is the same except MHC genes • DA had higher composite pain scores than DA.1U rats in formalin model. • Formalin induced biphasic increase in discharge rates, especially in DA rats. • Formalin induced increase of RT1-B in DA rats, but not in DA.1U rats.
a r t i c l e
i n f o
Article history: Received 15 February 2015 Received in revised form 13 March 2015 Accepted 4 April 2015 Available online 7 April 2015 Keywords: Major histocompatibility complex Pain Formalin DA rats DA.1U rats RT1-B
⁎ Corresponding author. E-mail address: [email protected] (Y. Zhao).
http://dx.doi.org/10.1016/j.physbeh.2015.04.009 0031-9384/© 2015 Elsevier Inc. All rights reserved.
a b s t r a c t Our recent studies have shown that the difference in basal pain sensitivity to mechanical and thermal stimulation between Dark-Agouti (DA) rats and a novel congenic DA.1U rats is major histocompatibility complex (MHC) genes dependent. In the present study, we further used DA and DA.1U rats to investigate the role of MHC genes in formalin-induced pain model by behavioral, electrophysiological and immunohistochemical methods. Behavioral results showed biphasic nociceptive behaviors increased significantly following the intraplantar injection of formalin in the hindpaw of DA and DA.1U rats. The main nociceptive behaviors were lifting and licking, especially in DA rats (P b 0.001 and P b 0.01). The composite pain scores (CPS) in DA rats were significantly higher than those in DA.1U rats in both phases of the formalin test (P b 0.01). Electrophysiological results also showed the biphasic increase in discharge rates of C and Aδ fibers of L5 dorsal root in the two strains, and the net change of the discharge rate of DA rats was significantly higher than that of DA.1U rats (P b 0.05). The mechanical thresholds decreased after formalin injection in both strains (P b 0.01), and the net change in the mechanical threshold in DA was greater than that in DA.1U rats (P b 0.05). The expression of RT1-B, representation of MHC class II molecule, in laminae I-II of L4/5 spinal cord in DA rats was significantly higher than that in DA.1U rats in the respective experimental group (P b 0.05). These results suggested that both DA and DA.1U
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rats exhibited nociceptive responses in formalin-induced pain model and DA rats were more sensitive to noxious chemical stimulus than DA.1U rats, indicating that MHC genes might contribute to the difference in pain sensitivity. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Pain is very complex and widely investigated for many years. Some researches have shown that pain process can be influenced by a variety of factors, including genetic differences [1–3], but the genetic mechanisms of pain are still poorly understood. The major histocompatibility complex (MHC), denoted as RT1 in the rat, is located on chromosomes 20 and spans approximately 4 Mb [4]. The major regions of MHC genes contain class I region (the centromeric class I region is RT1-A and the telomeric class I region is RT1-C/E/M), class II region (RT1-B/D), and class III regions [5,6]. It has been shown that MHC genes are involved in the genetic mechanisms for hyperalgesia induced by peripheral or central nervous system injury [7–9]. Our recent study has suggested that the level of MHC II molecular RT1-B might cause the differences in basal pain sensitivity between Dark-Agouti (DA) and cogenic DA.1U rats, whose genetic background is the same as that of DA rats except for MHC genes [10]. The purpose of the present study was to investigate the role of MHC genes in formalin-induced pain model. The formalin test was used in the present study to observe the pain responses to chemical nociceptive stimulation in the two strains because it is a well-characterized behavioral model of tonic chemogenic pain and widely used in studies on nociceptive processes and analgesic drug effects [11,12]. The behavioral responses including specific and non-specific nociceptive behaviors induced by formalin injection were observed in DA and DA.1U rats. In addition, electrophysiological methods were used to record the afferent discharge rates of C and Aδ fibers from L5 dorsal root. Furthermore, the expression of RT1-B, representation of MHC II molecular, in L4/L5 dorsal horn of spinal cord was also observed to further investigate the role of MHC II molecular in pain sensitivity. 2. Materials and methods 2.1. Animals Experiments were performed on inbred DA (originating from Zentralinstitut Fur Versuchstierzucht, Hannover, Germany) and DA.1U rats (originating from Lond University, Sweden) of either gender. All rats were 12–16 weeks old, and weighed 150–220 g. All experimental procedures were approved by the Institutional Animal Care Committee of Xi'an Jiaotong University, and were in accordance with ethical guidelines of the International Association for the Study of Pain [13]. The animals were kept in three per polystyrene cage in specific pathogen free animal facilities on 12 h light–dark cycles with food and water available ad libitum. 2.2. Behavioral tests Behavioral observations were carried out by the same investigators from 9 to 11 am on experimental days in the same room. The animals were habituated for 30 min before formalin test in a clear 40 × 30 × 30 cm plastic box with a mirror behind the box at a 45° angle to allow an unobstructed view of the opposite paw. The animals were divided into normal saline (NS) group (n = 8 for each rat strain) and formalin (FM) groups (n = 15 for each rat strain). In FM group, formalin (2.5%, 50 μl) was injected into right hindpaw intraplantarly using a 30 g needle. In NS group, NS (50 μl) was injected intraplantarly in the same way.
The nociceptive behavioral responses were quantified by counting the accumulative time (in seconds) spent in specific and non-specific nociceptive behaviors in the injected hindpaw during each 5 min period for 1 h after formalin injection. The specific nociceptive behaviors included favoring, lifting and licking, while non-specific nociceptive behaviors included resting or sleeping, still but alert, walking and grooming. Favoring meant injected paw had little weight and rested on the floor without pressure on the footpad; during locomotion there was a definite limp. Lifting indicated that the injected paw was elevated without touching the floor. Licking indicated that the injected paw was licked or bitten. The composite pain score (CPS) was calculated according to the following numerical scale: 0, no pain (normal weight bearing on the injected paw); 1, favoring; 2, lifting; and 3, licking [14–16]. The nonspecific behaviors for each 5 min block in each of the four behavioral categories were counted simultaneously: 0, rest or sleeping; 1, still but alert; 2, walking; and 3, grooming any part of the body except the injected paw [17]. Then, the CPSs for specific and non-specific nociceptive behaviors, ranging from 0 to 3 were calculated by multiplying the time spent in each category by the category weight, summing these products and dividing by the total time in seconds for each 5 min block of time.
CPS ¼
X
score for each behavior
duration of each behavior in every 5 min=300 2.3. Electrophysiological experiment Rats were anesthetized initially with urethane (1.0 g/kg i.p.) and supplemental doses (0.05 g/kg/h) were given as needed to maintain areflexia. Hair on the back was shaved off and a skin incision was made longitudinally along the median line. A T13–L3 laminectomy was performed in order to expose L5 dorsal root and cut it proximally. A pool was formed by raising the skin flaps and filling the space with warm paraffin oil (37 °C). Rectal temperature was maintained at approximately 37 ± 0.5 °C using a servo-controlled heating blanket. The distal end of the L5 dorsal root was placed on platinum bipolar electrodes for recording. On a small platform, the nerve was mechanically desheathed and teased apart under a dissecting microscope. Small filaments were repeatedly split with sharpened watchmaker forceps until single unit activity was obtained. Neural activity was recorded, then amplified (Biophysical Amplifier AVB-11A, Nihon Kohden, Japan), filtered (30–3000 Hz), and displayed on an oscilloscope (VC-11, Nihon Kohden, Japan) for monitoring the action potential's waveform and amplitude. The signals were also fed into a computer based data acquisition system (Spike2, Cambridge Electronic Design Limited, Cambridge, UK) that allowed continuous monitoring of discharges as well as off-line data analysis. The mechanical threshold (MT) of each unit was measured with a set of calibrated von Frey's filaments (Stoelting Company, Wood Dale, IL, USA), with bending forces from 3.92 mN to 254.8 mN, applied to the unit's receptive field (RF) and expressed as the minimum force (mN) needed to evoke a response in ≥ 50% of the trials [18–22]. The location of most sensitive point of RF to mechanical stimulation was marked and targeted for drug injection. The 28 g needle with PE10 tubing was inserted into the most sensitive point and connected to microinfusion pump (WZ-50, Zhejiang Medical university, China). After the spontaneous discharge became stable, the 5 min discharge
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was recorded as baseline control. Then formalin (2.5%, 10 μl) was microinjected for 5 min and discharge rates were continuously recorded for 1 h. After recording, the MT was measured again to compare with that before injection. After MT examined, a single electrical stimulus with progressively increasing intensity (0.1–1 mA, duration 0.5 ms) was applied to the sensitive point of RF. Conduction velocity (CV) was calculated by dividing the distance from the stimulating to the recording electrode by the latency of the action potential. Units with CVs less than 2.0 m/s were classified as C fibers, 2.0–30.0 m/s as Aδ fibers, and 30.1–70.0 m/s as Aβ fibers [19–23].
2.4. Immunohistochemistry The rats were divided into three groups. In control group, no treatment was applied on rats. In NS and FM groups, the rats were intraplantarly injected NS (50 μl) or formalin (2.5%, 50 μl), respectively. Rats were deeply anesthetized with sodium pentobarbital (80 mg/kg, i.p.) 90 min after injection. Then the rats were perfused through the left ventricle with 100 ml saline (37 °C) followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4, 4 °C). The L4/L5 spinal cord segments were cut, post-fixed, and cryoprotected in 30% sucrose in 0.1 M PB (pH 7.4) for 24–36 h.
Fig. 1. Nociceptive behaviors induced by intraplantar injection of formalin in DA and DA.1U rats. A–D: Cumulative histogram of all kinds of specific nociceptive behaviors (A, C) and nonspecific nociceptive behaviors (B, D) in DA rats (A, B) and in DA.1U rats (C, D); E and F: The comparison in each specific (E) and non-specific (F) nociceptive behavior between DA and DA.1U rats in FM group; G and H: The comparison in time course of composite pain score (CPS) for specific (G) and non-specific nociceptive behaviors (H). NS: normal saline group; FM: formalin group. **P b 0.01 vs NS group in each rat strain; ## P b 0.01 vs DA.1U in each group; two way RM ANOVA.
Y. Guo et al. / Physiology & Behavior 147 (2015) 30–37
Tissues were embedded in OCT compound (Sakura Finetechnical Co., Ltd., Tokyo, 103, Japan) and frozen at −20 °C and then sectioned coronally into 40-μm slices (Leica CM1900, Germany). Every third section was selected for immunohistochemical staining. After washing with 0.01 M phosphate-buffered saline (PBS, pH 7.4), the sections were pre-treated with 0.3% H2O2 (10 min, at room temperature) and 10% normal goat serum (NGS, 1 h, at room temperature) in PBS with 0.3% Triton X-100 (Sigma, St Louis, MI, USA). Then sections were incubated with monoclonal antibody OX-6 (staining for RT1-B, 1:100; gifts from Prof. SM Lu, Department of Biochemistry and Molecular Biology, Xi'an Jiaotong University Health Science Center) in 5% NGS-PBS for 48 h at 4 °C. Subsequently, the sections were incubated overnight with biotinylated goat anti-mouse staining for RT1-B at 4 °C, and further processed using avidin biotin peroxidase complex according to manufacturer instructions (Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China). Finally, the sections were reacted with 0.02% 3,3′diaminobenzidine (Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China) for 10–15 min. The sections were then mounted onto gelatin-coated glass slides, air dried, dehydrated through a graded alcohol series, cleaned with dimethylbenzene, and coverslipped with neutral balsam. All sections were observed under a light microscope (BX-51; Olympus, Tokyo, Japan) and images were captured using a SensiCam digital camera (SPOT-Insight QE, Diagnostic Instruments Inc., Sterling Heights, MI, USA). Images were imported and analyzed, as TIFF files, using SigmaScan Pro Image Analysis Software (SPOT-Insight QE, Diagnostic Instruments Inc., Sterling Heights, MI, USA). To discriminate positive immunostaining from the background, the cells showing a two-fold more intense staining than the average background were considered as RT1-B-positive cells and counted by the software Image-Pro Plus 4.5 (Media Cybernetics, Silver Spring, MD, USA). In the negative control experiments, the primary antibodies were replaced with NGS; no positive staining for the replaced antibodies was detected.
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way ANOVA, P b 0.001 and P b 0.01). For the time course following formalin injection, the CPSs for specific and non-specific nociceptive behaviors increased at all time points in both DA and DA.1U rats in FM group compared with those in NS group (two way RM ANOVA, P b 0.01, Fig. 1G, H). The CPSs for specific nociceptive behaviors of DA rats in FM group were significantly higher than those of DA.1U rats in both phases (two way RM ANOVA, P b 0.01, Fig. 1G). The CPSs for non-specific nociceptive behaviors of DA rats in FM group were significantly higher than those of DA.1U rats at 35–45 min (two way RM ANOVA, P b 0.01, Fig. 1H). 3.2. The sensitivity of C and Aδ units to formalin stimulation in DA rats was higher than DA.1U rats
3. Results
The basal physiological properties of C and Aδ units in DA and DA.1U rats were shown in Table 1. There were no obvious differences in CV and background discharge rates between DA and DA.1U rats, while the basal MTs of C and Aδ units in DA rats were significantly lower than those in DA.1U rats (P b 0.01, t-test, Table 1). After formalin injection, the MTs of C and Aδ units decreased in both DA and DA.1U rats (P b 0.01, paired t-test, Fig. 2A and B); however, the net changes in MTs of C and Aδ units after formalin injection in DA rats were much higher than those in DA.1U rats (P b 0.05, t-test, Fig. 2A and B). These results suggested that DA rats were more sensitive to formalin than DA.1U rats. The discharge rates of C units increased biphasically in both DA and DA.1U rats. The first phase was at first 5 min after formalin injection, and the second phase was from 20 to 60 min. In DA rats, the discharge rates of C units increased at all time points after formalin injection (P b 0.01, two way RM ANOVA, Fig. 2C). In DA.1U rats, the discharge rates of C units increased at the first 5 min and from 30 to 45 min after formalin injection (P b 0.01, two way RM ANOVA, Fig. 2C). The discharge rates in DA rats were significantly higher than those in DA.1U rats in both phases (P b 0.01, two way RM ANOVA, Fig. 2C). The right panel of Fig. 2C showed the original discharge of C units in DA (upper) and DA.1U rat (lower). As for Aδ units, the discharge rates in both DA and DA.1U rats significantly increased after formalin injection, and the discharge rates in DA rats were also significantly higher than those in DA.1U rats (P b 0.01, two way RM ANOVA, Fig. 2D). These electrophysiological results suggested that DA rats were more sensitive to formalin than DA.1U rats, which were in accordance with the duration of lifting and licking in behavioral test.
3.1. More spontaneous nociceptive behaviors were observed in DA rats than that in DA.1U rats after formalin injection
3.3. The expression of RT1-B in spinal cord in DA was higher than that in DA.1U rats
No obvious spontaneous nociceptive behaviors were observed in NS group in both DA and DA.1U rats. There was no difference in single behavior and CPSs for specific and non-specific nociceptive behaviors between DA and DA.1U rats in NS group (two way ANOVA, P N 0.05). After intraplantar injection of formalin, obvious biphasic nociceptive behaviors were observed in both DA and DA.1U rats. The first phase is the first 5 min, and the second phase is from 20 min to 60 min after formalin injection. The main specific nociceptive behaviors were lifting and licking, the main non-specific behaviors were still but alert (Fig. 1A–D). The duration of favoring in DA rats was significantly shorter than that in DA.1U rats (t-test, P b 0.001, Fig. 1E), while the durations of lifting and licking in DA rats were significantly longer than those in DA.1U rats (t-test, P b 0.001 and P b 0.01, respectively, Fig. 1E). There was no difference in the duration of each non-specific behavior between DA and DA.1U rats (t-test, P N 0.05, Fig. 1F). The average CPSs for specific and non-specific nociceptive behaviors of DA rats in FM group were 2.028 ± 0.026 and 1.461 ± 0.019, respectively, which were significantly higher than those in NS group (0.014 ± 0.009 and 0.270 ± 0.061, two way ANOVA, P b 0.001), and also significantly higher than those in DA.1U rats in FM group (1.681 ± 0.039 and 1.275 ± 0.064, two
The RT1-B expression was observed mainly in both sides of lamina I-II in L4/5 spinal cord. The location of RT1-B expression in DA.1U rats was similar to that of DA rats. In agreement with our recent study [10], we confirmed that basal RT1-B expressions in DA rats (61.53 ± 6.83
2.5. Statistical analysis All data were presented as mean ± SEM and analyzed by SigmaStat 2.0 software. One way ANOVA, two way (RM) ANOVA, t-test or paired t-test are used as appropriate. P b 0.05 was considered to be statistically significant.
Table 1 The basal physiological properties of C and Aδ units from L5 dorsal root in DA and DA.1U rats. Units
Physiological properties
DA rats
DA.1U rats
C
n Mechanical thresholds (mN) Background discharge rates (imp/5 min) Conduction velocity (m/s) n Mechanical thresholds (mN) Background discharge rates (imp/5 min) Conduction velocity (m/s)
15 84.93 ± 7.75 12.93 ± 0.78
17 139.51 ± 11.99⁎⁎ 6.29 ± 1.17⁎⁎⁎
1.32 ± 0.07 24 80.44 ± 6.7 9.58 ± 1.23
1.36 ± 0.16 28 121.45 ± 10.98⁎⁎ 8.86 ± 0.96
7.16 ± 1.25
9.95 ± 1.72
Aδ
⁎⁎ P b 0.01. ⁎⁎⁎ P b 0.001, vs DA rats.
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and 53.87 ± 5.47 for right and left side) were more than those in DA.1U rats (26.00 ± 2.38 and 27.20 ± 3.60). Formalin injection in right hindpaw increased RT1-B expression in lamina I–II in L4/5 spinal cord of both sides in DA rats (161.67 ± 7.17 and 136.00 ± 8.48 for right and left sides, respectively, P b 0.001, two way ANOVA, Fig. 3A, B and E), but not in DA.1U rats (32.27 ± 3.16 and 23.20 ± 2.501, Fig. 3C–E). And there was no difference between two sides (P N 0.05, two way ANOVA, Fig. 3E). Injection of saline did not change the RT1-B expression in both DA and DA.1U rats (P N 0.05, two way ANOVA, Fig. 3E).
4. Discussion In the present study, behavioral results showed that formalin induced typical biphasic pain responses in both DA and DA.1U rats, but the CPS for specific nociceptive behavior in DA rats were significantly higher than that of DA.1U rats, especially the duration of lifting and licking. Electrophysiological results also showed that formalin sensitized biphasically the C and Aδ units of primary sensory nerve in both DA and DA.1U rats, but the sensitivity of DA rats was higher than DA.1U
Fig. 2. The changes in mechanical thresholds (MT) and discharge rates in DA and DA.1U rats in formalin model. A and B: The changes in MT of C units (A) and Aδ units (B. **, P b 0.01 vs pre-injection, paired t-test; #, P b 0.05, ##, P b 0.01 vs DA rats, t-test); C and D: The comparison in discharge rates of C units (C) and Aδ units (D). Left panels are the time course curves of discharge rates and right panels are the samples of original discharge. con: pre-injection of formalin. ** P b 0.01 vs con; ## P b 0.01 vs DA.1U rats.
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rats. The electrophysiological results were consistent with the duration of lifting and licking, suggesting that DA rats had higher sensitivity in formalin induced pain. The formalin model first described by Dubuisson and Dennis [14] is usually used as a pain model of acute tissue injury-induced pain to characterize nociceptive processes and analgesic drug effects in rodents [14–16,24–32]. Various nociceptive behaviors observed after injection of formalin include the flinches [33,34], lifting, and licking [30,35,36]. Meanwhile, the CPS is also derived by applying the amount of time that the animal spends in a given behavioral category to a prior assigned category weights and summing the products, which is termed the weighted scores technique [14–16,37]. Sometime the non-specific nociceptive responses including sleeping or resting, being still but alerting, walking, and grooming were also observed [17,38,39]. In the present study, the behavioral responses in both DA and DA.1U rats were consistent with the previous studies showing that formalin induced typical biphasic nociceptive behaviors, mainly lifting and licking [12,16,24,25,28, 34,40,41]. As for the single behavior, the duration of favoring in DA rats was shorter than that in DA.1U rats, while the durations of lifting and licking in DA rats were longer than those in DA.1U rats. Another index,
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CPS for specific nociceptive behaviors was higher in DA rats than DA.1U rats. About these inconsistent results between single behaviors, it should be related to the intensity of pain. It was reported that the single behavior was related to the intensity of pain. Favoring was thought to reflect an absence of pain experience in the animal, lifting was thought to reflect intermediate pain experience, and licking was thought to reflect an intense pain experience [16]. Intense pain response was observed in DA rats, so the durations of lifting and licking were longer and the duration of favoring was shorter in DA rats. Combined with the result of CPS, we concluded that formalin injection induced nociceptive behaviors in both DA and DA.1U rats. DA rats had higher sensitivity to formalin. In addition, CPS might be a more robust measure of pain than any single behavior [15,16,24]. Our electrophysiological results also showed that formalin sensitized the C and Aδ units from the primary sensory nerve, which was consistent with previous studies [42]. It has been shown that in the periphery, primary afferent nociceptive fibers become sensitized through the release of pro-inflammatory substances from C and Aδ fiber terminals and surrounding cells. The consequent increase of spontaneous firing then initiates a second phase [43–45]. In addition,
Fig. 3. The expression of RT1-B in dorsal horn of L4/5 spinal cord induced by formalin in DA (A and B) and DA.1U rats (C and D). A and B: The expression of RT1-B after formalin injection in DA rats in left (A) and right (B) sides. C and D: The expression of RT1-B after formalin injection in DA.1U rats in left (C) and right (D) sides; E: the comparison in number of RT1-B positive cells. Con: normal control group without any treatment; NS: normal saline group; FM: formalin group. ***P b 0.001 vs Con; ###P b 0.001 vs DA rats, two way ANOVA.
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this intense activation of nociceptive afferents by formalin leads to increase excitability of neurons in the central nervous system [46]. In the present study, the sensitization of C and Aδ units from primary sensory nerve was consistent with the increase of lifting and licking, indicating that the activation of primary sensory nerve might contribute to these two behaviors induce by formalin. Pain can be affected by many factors including environment and genetics. It is widely accepted that genetic factors are important in individual differences in pain sensitivity [47,48]. The present study was performed on DA and cogenic DA.1U rats. DA.1U rats are derived by initially crossing female DA (a-haplotype) rats with male E3 (u-haplotype) rats using the speed congenic technique [49]. The difference between DA and DA.1U rats is that DA.1U rats express u alleles whereas DA rats express a alleles in MHC genes [50]. So we conclude that rats carrying a alleles in MHC genes are more susceptible to formalin in comparison to rats carrying u alleles. The genomic sequence of rat MHC genes, denoted as RT1 on chromosome 20 was identified [4]. It has been reported that MHC genes are upregulated after sensory nerve injury [51], and mechanical allodynia occurs following peripheral nerve transection [52]. MHC class II contributes to hyperalgesia induced by peripheral and central nervous system injury [7–9,52,53]. Experiments about different MHC haplotypes in a model for post-herpetic pain further provide support for the role of MHC in the development of pain [54]. Moreover, MHC class II knockout mice exhibit an attenuation of nerve injury-induced allodynia [52]. Our previous studies also showed that basal expression of RT1-B, representation of MHC class II DQ subregion was higher in DA rats than that in DA.1U rats [10], which suggested that MHC class II was involved in the basal pain sensitivity. In the present study, the expression of RT1-B increased after formalin injection in DA rats, suggesting that MHC genes might be involved in formalininduced pain. However, the expression of RT1-B did not increase in DA.1U rats following injection of formalin, which was inconsistent with the increase of nociceptive behaviors and the sensitization of C and Aδ units from primary sensory nerves observed in DA.1U rats. Accordingly, although MHC genes might be involved in formalininduced pain, the importance and mechanism underlying the role of MHC genes in the process need to be investigated in the future. In addition, other genomic structures such as RT1-A for MHC class I, RT1-D for MHC class II, and RT1-C/E/M for the non-classical MHC class I region should be investigated in the future study. Acknowledgments Thanks for the help from Prof. She-Min Lu (Department of Genetics Molecular Biology, School of Basal Medical Science, Xi'an Jiaotong University Health Science Center) in providing animals and primary antibody for RT1-B. This study was supported by the National Natural Science Foundation of China (No. 81200604). References [1] W.R. Lariviere, S.G. Wilson, T.M. Laughlin, A. Kokayeff, E.E. West, S.M. Adhikari, et al., Heritability of nociception. III. Genetic relationships among commonly used assays of nociception and hypersensitivity, Pain 97 (2002) 75–86. [2] J.S. Mogil, S.G. Wilson, K. Bon, S.E. Lee, K. Chung, P. Raber, et al., Heritability of nociception I: responses of 11 inbred mouse strains on 12 measures of nociception, Pain 80 (1999) 67–82. [3] J.S. Mogil, S.G. Wilson, K. Bon, S.E. Lee, K. Chung, P. Raber, et al., Heritability of nociception II. ‘Types’ of nociception revealed by genetic correlation analysis, Pain 80 (1999) 83–93. [4] P. Hurt, L. Walter, R. Sudbrak, S. Klages, I. Muller, T. Shiina, et al., The genomic sequence and comparative analysis of the rat major histocompatibility complex, Genome Res. 14 (2004) 631–639. [5] R. Dressel, L. Walter, E. Gunther, Genomic and functional aspects of the rat MHC, the RT1 complex, Immunol. Rev. 184 (2001) 82–95. [6] E. Gunther, L. Walter, The major histocompatibility complex of the rat (Rattus norvegicus), Immunogenetics 53 (2001) 520–542. [7] C.A. Dominguez, L. Li, O. Lidman, T. Olsson, Z. Wiesenfeld-Hallin, F. Piehl, et al., Both MHC and non-MHC genes regulate development of experimental neuropathic pain in rats, Neurosci. Lett. 442 (2008) 284–286.
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