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Suppression by intrathecal BmK IT2 on rat spontaneous pain behaviors and spinal c-Fos expression induced by formalin
Brain Research Bulletin 73 (2007) 248–253
Research report
Suppression by intrathecal BmK IT2 on rat spontaneous pain behaviors and spinal c-Fos expression induced by formalin Zhan-Tao Bai c,1 , Tong Liu b,1 , Xue-Yan Pang a , Zhi-Fang Chai b , Yong-Hua Ji a,∗ a
b
School of Life Sciences, Shanghai University, Shang-Da Road 99, Shanghai 200444, PR China Graduate School of the Chinese Academy of Sciences, Institute of Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China c College of Life Sciences, Yanan University, Yanan 716000, PR China Received 17 May 2006; received in revised form 4 February 2007; accepted 14 March 2007 Available online 5 April 2007
Abstract The central anti-nociception of BmK IT2, a sodium channel modulator from scorpion Buthus martensi Karsh (BmK) was investigated in this study. It was found that the formalin-induced rat spontaneous flinches and spinal c-Fos expression could be significantly suppressed by intrathecal BmK IT2 pre- or post-formalin injection in a dose-dependent manner. The time course of inhibitory effect exerted by intrathecal BmK IT2 on spontaneous flinches was longer in the pre-treatment group than in post-treatment group. This was consistent with the stronger suppression on spinal c-Fos expression exerted by intrathecal BmK IT2 pre-treatment. In addition, the suppression by intrathecal BmK IT2 on formalin-induced c-Fos expression in superficial laminae was more significant than that in deeper laminae. These results indicate that BmK IT2 can induce central anti-nociceptive response and might thus be a valuable molecular tool for the understanding of pain mechanisms. © 2007 Elsevier Inc. All rights reserved. Keywords: BmK IT2; Sodium channel modulator; Intrathecal injection; Formalin; Spontaneous pain; c-Fos expression
1. Introduction Physiological and pharmacological studies have demonstrated that sodium channels play a critical role in abnormal hyperexcitability of pain pathway [19,20,32]. The development of novel chemicals targeting nociceptive-specific sodium channels may further promote the understanding of pain mechanisms and therapy. To date, long chain polypeptides modulating the gating properties of sodium channels have been purified from scorpion Buthus martensi Karsch (BmK) venom [14,37]. Polypeptides of one such group, including BmK I and BmK abT [17,18], have been extensively used for the investigation of mechanisms of pain induction [3,6,7,35]. Another group, including BmK IT2 [15] and BmK ASs (BmK AS and BmK AS-1) [16], has been demonstrated to play a key role in anti-nociception. With peripheral administration of BmK AS or BmK AS-1, nociception ∗
Corresponding author. Tel.: +86 21 66135189; fax: +86 21 66135189. E-mail addresses: [email protected], [email protected] (Y.-H. Ji). 1 Both authors made equal contribution in this study. 0361-9230/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2007.03.007
induced in rats by inflammatory chemicals or electrical stimulation could be reduced significantly [4,5,28]. Likewise, peripheral local administration of BmK IT2, a depressant insect-selective toxin [15], was found in rats to suppress formalin-induced spontaneous pain, carrageenan-evoked thermal hyperalgesia and the C-component of the nociceptive reflex flexion evoked by electrical stimulation [29,31,34]. The anti-nociceptive mechanisms of BmK ASs and BmK IT2 may be ascribed to their selective modulation of sodium currents, especially tetrodotoxin-resistant (TTX-R) sodium currents expressed on small dorsal root ganglion neurons (DRG) [4,28,29]. The results pointed out a potent peripheral anti-nociceptive effect of BmK IT2 and BmK ASs. However, the central anti-nociceptive mechanism of these polypeptides has not been investigated so far. The formalin test is a tonic pain model with biphasic responses for the study of peripheral and central mechanisms of nociception as well as for the evaluation of the analgesic effects of endogenous or exogenous chemical agents [13,30]. To examine the central anti-nociceptive activity of BmK IT2, we here investigated the suppressive effect of intrathecal BmK IT2 on rat spontaneous pain behaviors and spinal c-Fos expression induced by formalin, and the possible mechanism of
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formalin-induced biphasic responses mediated by BmK IT2targeted central sodium channels. 2. Materials and methods 2.1. Animals and drug administration All experiments were performed on healthy adult male Sprague–Dawley rats (220–250 g) provided by Shanghai Experimental Animal Center, Chinese Academy of Sciences. A total of 135 of rats were employed for the present experiment. Rats were housed in groups of five per cage with water and food available ad libitum. The animal room was maintained at 21–23 ◦ C, with a 12 h light:12 h dark cycle. Use of the animals was carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised in 1996. Ethical guidelines for investigations of experimental pain in conscious animals were also followed [36]. Fifty microliters of 2.5% formalin were administered subcutaneously (s.c.) into the plantar surface of one rat hind paw. BmK IT2 was purified according to the method described by Ji et al. [15]. BmK IT2 or vehicle (sterile saline) was injected intrathecally by direct lumbar puncture at the L5–L6 spinal levels, as previously described [22]. Briefly, rats were lightly anesthetized with halothane, and the intrathecal injection was made with a 27-gauge, 1-in. sterile disposable needle connected to a 50-l Hamiltom syringe. The injected volume was 10 l. The drug was administered within seconds. Puncture of the dura was indicated by a reflexive flick of tail or formation of an “S” by the tail. After the intrathecal injection, rats recovered from anesthesia within 1–2 min.
2.2. Behavioral tests A 20 cm × 20 cm × 30 cm transparent plexiglas test box with a transparent glass floor was placed on a supporting frame of 75 cm high placed on top of the experimental table. The experimental table was covered with a mirror to allow the experimenters to observe the paws of the animal. The rat was placed in the test box for habituation at least 30 min before administration of the chemical agents. Spontaneous pain behaviors were determined by counting every 5 min the number of flinches occurring in the injected hind paw for 1 h after formalin injection [1,34]. For behavioral observation, rats were randomly divided into: (1) control 1, rats with intrathecal injection of saline or 1 g BmK IT2 (in 10 l sterile saline) alone (n = 6); (2) control 2, rats with intraplantar injection of formalin (n = 8); (3) pre-treatment groups, i.e. treated with intrathecal sterile saline (10 l, n = 8) or BmK IT2 (0.01, 0.1 or 1 g dissolved in 10 l sterile saline, n = 8 per dose) 10 min before intraplantar injection of formalin; (4) post-treatment groups, i.e. treated with intrathecal sterile saline (10 l, n = 8) or BmK IT2 (0.01, 0.1 or 1 g dissolved in 10 l sterile saline, n = 8 per dose) 10 min after the formalin injection. The effects of intrathecal BmK IT2 on the formalin-induced behaviors were monitored for 1 h after formalin injection.
2.3. Detection of spinal c-Fos expression 2.3.1. Animal groups For detection of spinal c-Fos expression, five groups were designed: (1) control 1: na¨ıve rats, without any treatment (n = 4); (2) control 2, rats with intrathecal injection of saline or 1 g BmK IT2 (in 10 l sterile saline) (n = 4); (3) control 3, rats with intraplantar injection of formalin (n = 5); (4) pre-treatment groups, i.e. rats with intrathecal sterile saline (10 l, n = 4) or BmK IT2 (0.01, 0.1 or 1 g dissolved in 10 l sterile saline, n = 5 per dose) 10 min before formalin injection; (5) post-treatment groups, i.e. rats with intrathecal sterile saline (10 l, n = 4) or BmK IT2 (0.01, 0.1 or 1 g dissolved in 10 l sterile saline, n = 5 per dose) 10 min after formalin injection. Then, spinal c-Fos expression at L4–L5 segment was investigated 2 h after intraplantar formalin injection. 2.3.2. Fos immunohistochemistry Two hours after formalin injection, animals were deeply anesthetized with pentobarbital sodium (60 mg/kg body weight, i.p.) and perfused intracardially with 200 ml of sterile saline, followed by 400 ml of fixative containing 4%
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paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The lumbar spinal cord was removed and postfixed in the same fixative for 12 h and then cryoprotected in 0.1 M PB containing 20% sucrose until the tissue sank to the bottom of the container. Frozen serial coronal sections (14 m in thickness) were cut with a cryostat and mounted on gelatin-coated glass slides. The sections were immunostained for c-Fos expression using the avidinbiotin-peroxidase complex (ABC) method, and following the protocol described by Bai et al. [2]. Briefly, the sections were rinsed in 0.01 M phosphate-buffered saline (PBS) and then incubated with a polyclonal antibody raised in a rabbit against a peptide mapped at the amino terminus of human c-Fos P62 (1:2000; Sc-52; Santa Cruz Biotechnology, Santa Cruz, CA) for 48 h at 4 ◦ C. The sections were incubated with biotinylated goat anti-rabbit IgG (1:200; Vector, Burlingame, CA) and ABC solution (1:200; Vector) at room temperature for 2 h. The positive Fos-immunoreactive-like (FIL) product was visualized by the glucose oxidase-diaminobenzidine-nickel method [27]. During the interval between two incubations, the sections were rinsed three times in 0.01 M PBS, each rinse for 10 min. The sections were then dehydrated, cleared, and coverslipped. There was no positive staining when PBS or normal rabbit serum was used instead of the anti-c-Fos antibody. 2.3.3. Quantitative evaluation of Fos immunoreactivity c-Fos expression at L4–L5 segments was evaluated quantitatively by cell counting. Tissue sections with FLI neurons from each animal were selected randomly. To count the number of FLI neurons, we followed the description of the rat spinal cord by Molander et al. [23]. Four regions of the spinal gray matter were defined as follows: the superficial layer of the dorsal horn (laminae I–II); the nucleus proprius (laminae III–IV); the neck of the dorsal horn (laminae V–VI); and the ventral gray, including laminae VII, VIII, and X (laminae VII–X). The number of FLI neurons in different laminar regions was counted regardless of staining intensity. For each animal, the number of FLI neurons in seven to nine sections was counted and averaged as a single data point for statistical analysis. All data were calculated by an observer blind to treatment.
2.4. Statistical analysis Data are presented as mean ± S.E.M. Statistical significance was evaluated using analysis of variance (ANOVA) followed by Turkey test for post hoc analysis of multiple comparisons; level of significance was set at P < 0.05. The number of flinches during the second phase (from 25 to 60 min after formalin injection) was averaged rather than comparing individual time points. To evaluate the decrease of Fos expression in treated groups versus controls, the inhibitory rate of c-Fos expression is presented as follows: inhibitory rate (%) = (1 − B/A) × 100, where A is the number of FLI neurons in the saline-treated control group and B is the number of FLI neurons in BmK IT2-treated groups.
3. Results 3.1. Suppression by intrathecal BmK IT2 on formalin-induced behavior No significant side effects on rat motor function was found after intrathecal injection of saline or BmK IT2 (1 g). As shown in Fig. 1A, in the first phase, the number of flinches was reduced from 73.75 ± 4.21 (control) to 43.33 ± 4.65 (P < 0.001), 51 ± 2.75 (P < 0.01) or 58.6 ± 3.25 (P < 0.05) with BmK IT2 at the dose of 1, 0.1 or 0.01 g, respectively. The difference of inhibitory potency between different groups treated with BmK IT2 was also statistically significant (0.01 g versus 0.1 g, P < 0.05; 0.01 g versus 1 g, P < 0.01; 0.1 g versus 1 g, P < 0.05). During the second phase, the inhibition by BmK IT2 pretreatment on flinches began at the time point of 25 min for 1 g, 30 min for 0.1 g and 35 min for 0.01 g, and lasted for more than one hour after formalin injection. Compared
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induced flinches could be reduced by pre-treatment of intrathecal BmK IT2 dose-dependently during the first and second phases. The suppressive effect exerted by intrathecal BmK IT2 posttreatment on formalin-induced flinches is shown in Fig. 1B. Similarly to the pre-treatment, the flinches during the second phase could be inhibited dose-dependently by post-treatment with BmK IT2 (Fig. 1B). This effect began at 30 min at the BmK IT2 doses of 0.1 and 1 g, and at 35 min at the 0.01 g dose after formalin injection. Compared with saline post-treatment (51.09 ± 2.6), the average number of flinches in BmK IT2 post-treatment groups during the second phase (25–60 min) was reduced to 42. 21 ± 2.12 for 0.01 g (P < 0.05), 35. 23 ± 3.24 for 0.1 g (P < 0.01) and 28. 25 ± 3.52 for 1 g (P < 0.001), respectively. The difference of inhibitory potency between the different groups post-treated with BmK IT2 was significant likewise (0.01 g versus 0.1 g, P < 0.05; 0.01 g versus 1 g, P < 0.05; 0.1 g versus 1 g, P < 0.05). During the second phase, the inhibitory effect on the number of flinches was almost the same in the BmK IT2 pre- and posttreatment groups. However, the time course of inhibition was longer in the BmK IT2 pre-treated group than in the post-treated animal (Fig. 1A and B). Fig. 1. Suppression exerted by intrathecal BmK IT2 on spontaneous behavior induced by formalin. BmK IT2 or saline (10 l) was administered intrathecally 10 min before or after intraplantar injection of formalin (2.5%). (A) Time course of flinches/5 min in formalin-injected rats with intrathecal saline or BmK IT2 pre-treatment. (B) Time course of flinches/5 min in formalin-injected rats with saline or BmK IT2 post-treatment. Arrow in B indicates the time point of intrathecal injection.
with saline pre-treatment (52.06 ± 3.63), the average number of flinches in BmK IT2 pre-treatment group during the second phase (25–60 min) was reduced to 45.13 ± 2.88 for 0.01 g (P < 0.05), 34.69 ± 3.61 for 0.1 g (P < 0.01) and 23.23 ± 3.4 for 1 g (P < 0.001), respectively. The differences of inhibitory potency among the groups of pre-treated BmK IT2 were also significant (0.01 g versus 0.1 g, P < 0.05; 0.01 g versus 1 g, P < 0.01; 0.1 g versus 1 g, P < 0.05). Thus, the formalin-
3.2. Suppressive effect of intrathecal BmK IT2 on formalin-induced spinal c-Fos expression Intrathecal treatment with saline or BmK IT2 (1 g) alone did not alter the basal level of c-Fos expression in the L4–L5 segments (Fig. 2). c-Fos induced by intraplantar injection of formalin was found to be predominant on the ipsilateral side and only a few cells were Fos-labeled on the contralateral side. Intrathecal injection of saline did not affect the formalininduced spinal c-Fos expression. However, in both the pre- and post-treated groups, formalin-induced ipsilateral spinal c-Fos expression was significantly decreased by intrathecal BmK IT2 administration in a dose-dependent manner (Figs. 3 and 4A and C). The suppressive effect exerted by BmK IT2 on c-Fos expression was more potent in the pre-treatment groups than in
Fig. 2. Microphotographs illustrating spinal c-Fos expression in control untreated rats. (A) After intrathecal control injection of saline (B) or intrathecal injection of 1 g BmK IT2 (C).
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Fig. 3. Microphotographs illustrating the suppression exerted by intrathecal BmK IT2 on formalin-induced spinal c-Fos expression. Saline or BmK IT2 (in 10 l saline) was administered intrathecally 10 min before or after intraplantar injection of formalin (2.5%). (A)–(D) illustrate pre-treatment with saline, 0.01, 0.1 or 1 g BmK IT2, respectively. (E)–(H) illustrate post-treatment of saline, 0.01, 0.1 or 1 g BmK IT2, respectively.
the post-treatment ones (Figs. 3 and 4). Moreover, the magnitude of c-Fos inhibition was higher in the superficial laminae I–II than laminae III–VI (Fig. 4 and Table 1). The contralateral spinal c-Fos expression induced by formalin was not suppressed significantly by BmK IT2, even if the number of FLI neurons in contralateral laminae V–VI and VII–X increased slightly (Fig. 4B and D).
4. Discussion We described in previous studies that BmK IT2, a modulator of sodium channels, can suppress spontaneous pain and hyperalgesia after peripheral local administration [29,31,34]. In the present study we show that intrathecal administration of BmK IT2 can exert a potent suppressive effect on formalin-induced
Fig. 4. Quantitative evaluation of the effect of intrathecal BmK IT2 on formalin-induced spinal c-Fos expression. BmK IT2 was administered intrathecally 10 min before or after intraplantar injection of formalin. (A and B) c-Fos induced by to formalin injection after pre-treatment with BmK IT2 on the ipsilateral (A) and contralateral (B) sides. Similar data are shown in C, D in relation to post-treatment * P < 0.05, ** P < 0.01 and *** P < 0.001 (ANOVA followed by Turkey test) groups treated BmK IT2 vs. group treated with saline (n = 4–5); # P < 0.05, ## P < 0.01 and ### P < 0.001 (ANOVA followed by Turkey test), group treated with 0.01 g BmK IT2 vs. groups with other doses of BmK IT2 (0.1 and 1 g).
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Table 1 Comparison of the inhibitory efficacy of pre- and post-treated intrathecal BmK IT2 on the ipsilateral c-Fos expression induced by formalin Treatment with BmK IT2
Laminae I–II
III–IV
V–VI
1 g
Pre Post
88.3 ± 1.4 64.7 ± 2.0*
63.7 ± 2.1 31.0 ± 3.4*
61.0 ± 3.3 37.5 ± 3.8*
0.1 g
Pre Post
57.2 ± 1.5 41.5 ± 3.0*
25.7 ± 3.1 20.8 ± 6.0*
37.4 ± 4.8 20.5 ± 3.0*
0.01 g
Pre Post
39.8 ± 3.7 23.5 ± 2.9*
4.5 ± 1.4 4.4 ± 1.2
8.3 ± 0.8 3.7 ± 1.6*
All data are presented as mean ± S.E.M. (n = 4–5). The rate of inhibition of c-Fos expression was presented as follows: the rate of inhibition of c-Fos expression is presented as follows: inhibitory rate (%) = (1 − B/A) × 100, where A is the number of FLI neurons in the saline-treated control group and B is the number of FLI neurons in BmK IT2-treated groups. * P < 0.05, pre-treatment versus post-treatment of BmK IT2 at the same dose and laminae (ANOVA followed by Turkey test), respectively.
biphasic behavior and spinal c-Fos expression. Thus, the present findings indicate that BmK IT2 is effective for anti-nociception at spinal level. A wealth of evidence indicates that the intraplantar injection of formalin can induce both peripheral and central biphasic responses [11,12,21,25]. The first phase of the response is driven directly by formalin on peripheral nociceptors, thereby producing an acute barrage of activity in the dorsal horn. The second phase is considered to be a result of the first phase, representing the consequence of ongoing afferent input maintained by inflammatory mediators acting on peripheral nociceptors [10,21,24–26]. The inhibition exerted by intrathecal BmK IT2 on the biphasic spontaneous flinches and spinal c-Fos expression found in the present study may be ascribed to the modulation of BmK IT2 on the TTX-R and TTX-S sodium channels located in the cell bodies and/or central terminals of primary afferent neurons as well as in the spinal cord [29]. Thereby, BmK IT2 could have caused a suppression of the ongoing afferent inputs and spinal hypersensitization induced by formalin. In contrast to BmK IT2, lidocaine suppresses formalininduced biphasic behavior only when intrathecal pretreatment is performed [8,9,33], suggesting that BmK IT2 may be more effective than lidocaine. The underlying mechanisms may be attributed to the selectivity of BmK IT2 on TTX-R sodium currents [29]. The partial, but not complete suppressive effect exerted by BmK IT2 on formalin-induced biphasic behavior was well consistent with the previously reported partial suppression exerted by BmK IT2 on TTX-R sodium currents [29]. The longer time course of inhibition on spontaneous flinches (Fig. 1) and more potent suppressive effect on spinal c-Fos expression (Figs. 3 and 4 and Table 1) of intrathecal BmK IT2 in the pre-treatment group than in the post-treatment one indicated that the suppression exerted by BmK IT2 pre-treatment on the first phasic responses may inhibit the development of the second phasic responses. The pattern of the inhibition exerted by intrathecal BmK IT2 pre-treatment or post-treatment on formalin-induced pain
behavior was found to be similar to that of peripheral BmK IT2 [34]. The present study shows that the suppression by pretreatment with intrathecal BmK IT2 on formalin-induced spinal c-Fos expression was more potent than that by post-treatment. However, in the case of intraplantar BmK IT2 [34], the suppression exerted by BmK IT2 pre-treatment on formalin-induced spinal c-Fos expression was found to be weaker than that by post-treatment. These results suggest that BmK IT2 may thus be a promising molecular tool used for understanding the correlation of pain behaviors and neuronal activities. As for the controversial inhibitory effect of peripheral and intrathecal BmK IT2 on formalin-induced c-Fos expression, it might be attributed to either there are more active sites and/or the higher expressing abundance of BmK IT2-targeted sodium channels located at spinal level than those at peripheral terminals, and/or formalininduced inflammation developed during the later phase after formalin injection makes BmK IT2 to easily access target channels resulting in the inhibition of BmK IT2 post administration stronger than BmK IT2 pre-treatment on c-Fos expression. We found in the present study that suppression of intrathecal BmK IT2 on formalin-induced spinal c-Fos expression in the superficial laminae was higher than in the deeper laminae, indicating that BmK IT2-sensitive targets in the superficial laminae might be more abundant than in the deeper laminae. It is consistent well with the previous electrophysiological recordings that the inhibition of BmK IT2 on sodium currents of small DRG neurons was stronger than that of larger DRG neurons [29]. In summary, the present study reports that formalin-induced spontaneous pain behavior and spinal c-Fos expression could be effectively suppressed by either pre- or post-treatment with intrathecal BmK IT2. The results indicated that intrathecal administration of BmK IT2 resulted in potent central antinociception, and that sodium channels located in central pain pathway might be potential targets for anti-nociception exerted by BmK IT2. Therefore, BmK IT2 could provide a valuable tool for understanding pain mechanisms not only peripherally but also centrally. Acknowledgements This study was supported by National Basic Research Program of China (2006CB500801), a grant from National Nature Sciences Foundation of China (30370446), and partially by Shaanxi Provincial Nature Sciences Foundation (03JK220 and 2004C2 07), China. References [1] F.V. Abbott, K.B.J. Franklin, R.F. Westbrook, The formalin test: scoring properties of the first and second phases of pain responses in rats, Pain 60 (1995) 91–102. [2] Z.T. Bai, B. Chen, X.Y. Zhang, G.L. Fan, Y.H. Ji, c-Fos expression in rat spinal cord induced by scorpion BmK venom via subcutaneous plantar injection, Neurosci. Res. 44 (2002) 447–454. [3] Z.T. Bai, X.Y. Zhang, Y.H. Ji, Fos expression in rat spinal cord induced by peripheral injection of BmK I, an alpha-like scorpion neurotoxin, Toxicol. Appl. Pharmacol. 192 (2003) 78–85.
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Research report
Suppression by intrathecal BmK IT2 on rat spontaneous pain behaviors and spinal c-Fos expression induced by formalin Zhan-Tao Bai c,1 , Tong Liu b,1 , Xue-Yan Pang a , Zhi-Fang Chai b , Yong-Hua Ji a,∗ a
b
School of Life Sciences, Shanghai University, Shang-Da Road 99, Shanghai 200444, PR China Graduate School of the Chinese Academy of Sciences, Institute of Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China c College of Life Sciences, Yanan University, Yanan 716000, PR China Received 17 May 2006; received in revised form 4 February 2007; accepted 14 March 2007 Available online 5 April 2007
Abstract The central anti-nociception of BmK IT2, a sodium channel modulator from scorpion Buthus martensi Karsh (BmK) was investigated in this study. It was found that the formalin-induced rat spontaneous flinches and spinal c-Fos expression could be significantly suppressed by intrathecal BmK IT2 pre- or post-formalin injection in a dose-dependent manner. The time course of inhibitory effect exerted by intrathecal BmK IT2 on spontaneous flinches was longer in the pre-treatment group than in post-treatment group. This was consistent with the stronger suppression on spinal c-Fos expression exerted by intrathecal BmK IT2 pre-treatment. In addition, the suppression by intrathecal BmK IT2 on formalin-induced c-Fos expression in superficial laminae was more significant than that in deeper laminae. These results indicate that BmK IT2 can induce central anti-nociceptive response and might thus be a valuable molecular tool for the understanding of pain mechanisms. © 2007 Elsevier Inc. All rights reserved. Keywords: BmK IT2; Sodium channel modulator; Intrathecal injection; Formalin; Spontaneous pain; c-Fos expression
1. Introduction Physiological and pharmacological studies have demonstrated that sodium channels play a critical role in abnormal hyperexcitability of pain pathway [19,20,32]. The development of novel chemicals targeting nociceptive-specific sodium channels may further promote the understanding of pain mechanisms and therapy. To date, long chain polypeptides modulating the gating properties of sodium channels have been purified from scorpion Buthus martensi Karsch (BmK) venom [14,37]. Polypeptides of one such group, including BmK I and BmK abT [17,18], have been extensively used for the investigation of mechanisms of pain induction [3,6,7,35]. Another group, including BmK IT2 [15] and BmK ASs (BmK AS and BmK AS-1) [16], has been demonstrated to play a key role in anti-nociception. With peripheral administration of BmK AS or BmK AS-1, nociception ∗
Corresponding author. Tel.: +86 21 66135189; fax: +86 21 66135189. E-mail addresses: [email protected], [email protected] (Y.-H. Ji). 1 Both authors made equal contribution in this study. 0361-9230/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2007.03.007
induced in rats by inflammatory chemicals or electrical stimulation could be reduced significantly [4,5,28]. Likewise, peripheral local administration of BmK IT2, a depressant insect-selective toxin [15], was found in rats to suppress formalin-induced spontaneous pain, carrageenan-evoked thermal hyperalgesia and the C-component of the nociceptive reflex flexion evoked by electrical stimulation [29,31,34]. The anti-nociceptive mechanisms of BmK ASs and BmK IT2 may be ascribed to their selective modulation of sodium currents, especially tetrodotoxin-resistant (TTX-R) sodium currents expressed on small dorsal root ganglion neurons (DRG) [4,28,29]. The results pointed out a potent peripheral anti-nociceptive effect of BmK IT2 and BmK ASs. However, the central anti-nociceptive mechanism of these polypeptides has not been investigated so far. The formalin test is a tonic pain model with biphasic responses for the study of peripheral and central mechanisms of nociception as well as for the evaluation of the analgesic effects of endogenous or exogenous chemical agents [13,30]. To examine the central anti-nociceptive activity of BmK IT2, we here investigated the suppressive effect of intrathecal BmK IT2 on rat spontaneous pain behaviors and spinal c-Fos expression induced by formalin, and the possible mechanism of
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formalin-induced biphasic responses mediated by BmK IT2targeted central sodium channels. 2. Materials and methods 2.1. Animals and drug administration All experiments were performed on healthy adult male Sprague–Dawley rats (220–250 g) provided by Shanghai Experimental Animal Center, Chinese Academy of Sciences. A total of 135 of rats were employed for the present experiment. Rats were housed in groups of five per cage with water and food available ad libitum. The animal room was maintained at 21–23 ◦ C, with a 12 h light:12 h dark cycle. Use of the animals was carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised in 1996. Ethical guidelines for investigations of experimental pain in conscious animals were also followed [36]. Fifty microliters of 2.5% formalin were administered subcutaneously (s.c.) into the plantar surface of one rat hind paw. BmK IT2 was purified according to the method described by Ji et al. [15]. BmK IT2 or vehicle (sterile saline) was injected intrathecally by direct lumbar puncture at the L5–L6 spinal levels, as previously described [22]. Briefly, rats were lightly anesthetized with halothane, and the intrathecal injection was made with a 27-gauge, 1-in. sterile disposable needle connected to a 50-l Hamiltom syringe. The injected volume was 10 l. The drug was administered within seconds. Puncture of the dura was indicated by a reflexive flick of tail or formation of an “S” by the tail. After the intrathecal injection, rats recovered from anesthesia within 1–2 min.
2.2. Behavioral tests A 20 cm × 20 cm × 30 cm transparent plexiglas test box with a transparent glass floor was placed on a supporting frame of 75 cm high placed on top of the experimental table. The experimental table was covered with a mirror to allow the experimenters to observe the paws of the animal. The rat was placed in the test box for habituation at least 30 min before administration of the chemical agents. Spontaneous pain behaviors were determined by counting every 5 min the number of flinches occurring in the injected hind paw for 1 h after formalin injection [1,34]. For behavioral observation, rats were randomly divided into: (1) control 1, rats with intrathecal injection of saline or 1 g BmK IT2 (in 10 l sterile saline) alone (n = 6); (2) control 2, rats with intraplantar injection of formalin (n = 8); (3) pre-treatment groups, i.e. treated with intrathecal sterile saline (10 l, n = 8) or BmK IT2 (0.01, 0.1 or 1 g dissolved in 10 l sterile saline, n = 8 per dose) 10 min before intraplantar injection of formalin; (4) post-treatment groups, i.e. treated with intrathecal sterile saline (10 l, n = 8) or BmK IT2 (0.01, 0.1 or 1 g dissolved in 10 l sterile saline, n = 8 per dose) 10 min after the formalin injection. The effects of intrathecal BmK IT2 on the formalin-induced behaviors were monitored for 1 h after formalin injection.
2.3. Detection of spinal c-Fos expression 2.3.1. Animal groups For detection of spinal c-Fos expression, five groups were designed: (1) control 1: na¨ıve rats, without any treatment (n = 4); (2) control 2, rats with intrathecal injection of saline or 1 g BmK IT2 (in 10 l sterile saline) (n = 4); (3) control 3, rats with intraplantar injection of formalin (n = 5); (4) pre-treatment groups, i.e. rats with intrathecal sterile saline (10 l, n = 4) or BmK IT2 (0.01, 0.1 or 1 g dissolved in 10 l sterile saline, n = 5 per dose) 10 min before formalin injection; (5) post-treatment groups, i.e. rats with intrathecal sterile saline (10 l, n = 4) or BmK IT2 (0.01, 0.1 or 1 g dissolved in 10 l sterile saline, n = 5 per dose) 10 min after formalin injection. Then, spinal c-Fos expression at L4–L5 segment was investigated 2 h after intraplantar formalin injection. 2.3.2. Fos immunohistochemistry Two hours after formalin injection, animals were deeply anesthetized with pentobarbital sodium (60 mg/kg body weight, i.p.) and perfused intracardially with 200 ml of sterile saline, followed by 400 ml of fixative containing 4%
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paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The lumbar spinal cord was removed and postfixed in the same fixative for 12 h and then cryoprotected in 0.1 M PB containing 20% sucrose until the tissue sank to the bottom of the container. Frozen serial coronal sections (14 m in thickness) were cut with a cryostat and mounted on gelatin-coated glass slides. The sections were immunostained for c-Fos expression using the avidinbiotin-peroxidase complex (ABC) method, and following the protocol described by Bai et al. [2]. Briefly, the sections were rinsed in 0.01 M phosphate-buffered saline (PBS) and then incubated with a polyclonal antibody raised in a rabbit against a peptide mapped at the amino terminus of human c-Fos P62 (1:2000; Sc-52; Santa Cruz Biotechnology, Santa Cruz, CA) for 48 h at 4 ◦ C. The sections were incubated with biotinylated goat anti-rabbit IgG (1:200; Vector, Burlingame, CA) and ABC solution (1:200; Vector) at room temperature for 2 h. The positive Fos-immunoreactive-like (FIL) product was visualized by the glucose oxidase-diaminobenzidine-nickel method [27]. During the interval between two incubations, the sections were rinsed three times in 0.01 M PBS, each rinse for 10 min. The sections were then dehydrated, cleared, and coverslipped. There was no positive staining when PBS or normal rabbit serum was used instead of the anti-c-Fos antibody. 2.3.3. Quantitative evaluation of Fos immunoreactivity c-Fos expression at L4–L5 segments was evaluated quantitatively by cell counting. Tissue sections with FLI neurons from each animal were selected randomly. To count the number of FLI neurons, we followed the description of the rat spinal cord by Molander et al. [23]. Four regions of the spinal gray matter were defined as follows: the superficial layer of the dorsal horn (laminae I–II); the nucleus proprius (laminae III–IV); the neck of the dorsal horn (laminae V–VI); and the ventral gray, including laminae VII, VIII, and X (laminae VII–X). The number of FLI neurons in different laminar regions was counted regardless of staining intensity. For each animal, the number of FLI neurons in seven to nine sections was counted and averaged as a single data point for statistical analysis. All data were calculated by an observer blind to treatment.
2.4. Statistical analysis Data are presented as mean ± S.E.M. Statistical significance was evaluated using analysis of variance (ANOVA) followed by Turkey test for post hoc analysis of multiple comparisons; level of significance was set at P < 0.05. The number of flinches during the second phase (from 25 to 60 min after formalin injection) was averaged rather than comparing individual time points. To evaluate the decrease of Fos expression in treated groups versus controls, the inhibitory rate of c-Fos expression is presented as follows: inhibitory rate (%) = (1 − B/A) × 100, where A is the number of FLI neurons in the saline-treated control group and B is the number of FLI neurons in BmK IT2-treated groups.
3. Results 3.1. Suppression by intrathecal BmK IT2 on formalin-induced behavior No significant side effects on rat motor function was found after intrathecal injection of saline or BmK IT2 (1 g). As shown in Fig. 1A, in the first phase, the number of flinches was reduced from 73.75 ± 4.21 (control) to 43.33 ± 4.65 (P < 0.001), 51 ± 2.75 (P < 0.01) or 58.6 ± 3.25 (P < 0.05) with BmK IT2 at the dose of 1, 0.1 or 0.01 g, respectively. The difference of inhibitory potency between different groups treated with BmK IT2 was also statistically significant (0.01 g versus 0.1 g, P < 0.05; 0.01 g versus 1 g, P < 0.01; 0.1 g versus 1 g, P < 0.05). During the second phase, the inhibition by BmK IT2 pretreatment on flinches began at the time point of 25 min for 1 g, 30 min for 0.1 g and 35 min for 0.01 g, and lasted for more than one hour after formalin injection. Compared
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induced flinches could be reduced by pre-treatment of intrathecal BmK IT2 dose-dependently during the first and second phases. The suppressive effect exerted by intrathecal BmK IT2 posttreatment on formalin-induced flinches is shown in Fig. 1B. Similarly to the pre-treatment, the flinches during the second phase could be inhibited dose-dependently by post-treatment with BmK IT2 (Fig. 1B). This effect began at 30 min at the BmK IT2 doses of 0.1 and 1 g, and at 35 min at the 0.01 g dose after formalin injection. Compared with saline post-treatment (51.09 ± 2.6), the average number of flinches in BmK IT2 post-treatment groups during the second phase (25–60 min) was reduced to 42. 21 ± 2.12 for 0.01 g (P < 0.05), 35. 23 ± 3.24 for 0.1 g (P < 0.01) and 28. 25 ± 3.52 for 1 g (P < 0.001), respectively. The difference of inhibitory potency between the different groups post-treated with BmK IT2 was significant likewise (0.01 g versus 0.1 g, P < 0.05; 0.01 g versus 1 g, P < 0.05; 0.1 g versus 1 g, P < 0.05). During the second phase, the inhibitory effect on the number of flinches was almost the same in the BmK IT2 pre- and posttreatment groups. However, the time course of inhibition was longer in the BmK IT2 pre-treated group than in the post-treated animal (Fig. 1A and B). Fig. 1. Suppression exerted by intrathecal BmK IT2 on spontaneous behavior induced by formalin. BmK IT2 or saline (10 l) was administered intrathecally 10 min before or after intraplantar injection of formalin (2.5%). (A) Time course of flinches/5 min in formalin-injected rats with intrathecal saline or BmK IT2 pre-treatment. (B) Time course of flinches/5 min in formalin-injected rats with saline or BmK IT2 post-treatment. Arrow in B indicates the time point of intrathecal injection.
with saline pre-treatment (52.06 ± 3.63), the average number of flinches in BmK IT2 pre-treatment group during the second phase (25–60 min) was reduced to 45.13 ± 2.88 for 0.01 g (P < 0.05), 34.69 ± 3.61 for 0.1 g (P < 0.01) and 23.23 ± 3.4 for 1 g (P < 0.001), respectively. The differences of inhibitory potency among the groups of pre-treated BmK IT2 were also significant (0.01 g versus 0.1 g, P < 0.05; 0.01 g versus 1 g, P < 0.01; 0.1 g versus 1 g, P < 0.05). Thus, the formalin-
3.2. Suppressive effect of intrathecal BmK IT2 on formalin-induced spinal c-Fos expression Intrathecal treatment with saline or BmK IT2 (1 g) alone did not alter the basal level of c-Fos expression in the L4–L5 segments (Fig. 2). c-Fos induced by intraplantar injection of formalin was found to be predominant on the ipsilateral side and only a few cells were Fos-labeled on the contralateral side. Intrathecal injection of saline did not affect the formalininduced spinal c-Fos expression. However, in both the pre- and post-treated groups, formalin-induced ipsilateral spinal c-Fos expression was significantly decreased by intrathecal BmK IT2 administration in a dose-dependent manner (Figs. 3 and 4A and C). The suppressive effect exerted by BmK IT2 on c-Fos expression was more potent in the pre-treatment groups than in
Fig. 2. Microphotographs illustrating spinal c-Fos expression in control untreated rats. (A) After intrathecal control injection of saline (B) or intrathecal injection of 1 g BmK IT2 (C).
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Fig. 3. Microphotographs illustrating the suppression exerted by intrathecal BmK IT2 on formalin-induced spinal c-Fos expression. Saline or BmK IT2 (in 10 l saline) was administered intrathecally 10 min before or after intraplantar injection of formalin (2.5%). (A)–(D) illustrate pre-treatment with saline, 0.01, 0.1 or 1 g BmK IT2, respectively. (E)–(H) illustrate post-treatment of saline, 0.01, 0.1 or 1 g BmK IT2, respectively.
the post-treatment ones (Figs. 3 and 4). Moreover, the magnitude of c-Fos inhibition was higher in the superficial laminae I–II than laminae III–VI (Fig. 4 and Table 1). The contralateral spinal c-Fos expression induced by formalin was not suppressed significantly by BmK IT2, even if the number of FLI neurons in contralateral laminae V–VI and VII–X increased slightly (Fig. 4B and D).
4. Discussion We described in previous studies that BmK IT2, a modulator of sodium channels, can suppress spontaneous pain and hyperalgesia after peripheral local administration [29,31,34]. In the present study we show that intrathecal administration of BmK IT2 can exert a potent suppressive effect on formalin-induced
Fig. 4. Quantitative evaluation of the effect of intrathecal BmK IT2 on formalin-induced spinal c-Fos expression. BmK IT2 was administered intrathecally 10 min before or after intraplantar injection of formalin. (A and B) c-Fos induced by to formalin injection after pre-treatment with BmK IT2 on the ipsilateral (A) and contralateral (B) sides. Similar data are shown in C, D in relation to post-treatment * P < 0.05, ** P < 0.01 and *** P < 0.001 (ANOVA followed by Turkey test) groups treated BmK IT2 vs. group treated with saline (n = 4–5); # P < 0.05, ## P < 0.01 and ### P < 0.001 (ANOVA followed by Turkey test), group treated with 0.01 g BmK IT2 vs. groups with other doses of BmK IT2 (0.1 and 1 g).
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Table 1 Comparison of the inhibitory efficacy of pre- and post-treated intrathecal BmK IT2 on the ipsilateral c-Fos expression induced by formalin Treatment with BmK IT2
Laminae I–II
III–IV
V–VI
1 g
Pre Post
88.3 ± 1.4 64.7 ± 2.0*
63.7 ± 2.1 31.0 ± 3.4*
61.0 ± 3.3 37.5 ± 3.8*
0.1 g
Pre Post
57.2 ± 1.5 41.5 ± 3.0*
25.7 ± 3.1 20.8 ± 6.0*
37.4 ± 4.8 20.5 ± 3.0*
0.01 g
Pre Post
39.8 ± 3.7 23.5 ± 2.9*
4.5 ± 1.4 4.4 ± 1.2
8.3 ± 0.8 3.7 ± 1.6*
All data are presented as mean ± S.E.M. (n = 4–5). The rate of inhibition of c-Fos expression was presented as follows: the rate of inhibition of c-Fos expression is presented as follows: inhibitory rate (%) = (1 − B/A) × 100, where A is the number of FLI neurons in the saline-treated control group and B is the number of FLI neurons in BmK IT2-treated groups. * P < 0.05, pre-treatment versus post-treatment of BmK IT2 at the same dose and laminae (ANOVA followed by Turkey test), respectively.
biphasic behavior and spinal c-Fos expression. Thus, the present findings indicate that BmK IT2 is effective for anti-nociception at spinal level. A wealth of evidence indicates that the intraplantar injection of formalin can induce both peripheral and central biphasic responses [11,12,21,25]. The first phase of the response is driven directly by formalin on peripheral nociceptors, thereby producing an acute barrage of activity in the dorsal horn. The second phase is considered to be a result of the first phase, representing the consequence of ongoing afferent input maintained by inflammatory mediators acting on peripheral nociceptors [10,21,24–26]. The inhibition exerted by intrathecal BmK IT2 on the biphasic spontaneous flinches and spinal c-Fos expression found in the present study may be ascribed to the modulation of BmK IT2 on the TTX-R and TTX-S sodium channels located in the cell bodies and/or central terminals of primary afferent neurons as well as in the spinal cord [29]. Thereby, BmK IT2 could have caused a suppression of the ongoing afferent inputs and spinal hypersensitization induced by formalin. In contrast to BmK IT2, lidocaine suppresses formalininduced biphasic behavior only when intrathecal pretreatment is performed [8,9,33], suggesting that BmK IT2 may be more effective than lidocaine. The underlying mechanisms may be attributed to the selectivity of BmK IT2 on TTX-R sodium currents [29]. The partial, but not complete suppressive effect exerted by BmK IT2 on formalin-induced biphasic behavior was well consistent with the previously reported partial suppression exerted by BmK IT2 on TTX-R sodium currents [29]. The longer time course of inhibition on spontaneous flinches (Fig. 1) and more potent suppressive effect on spinal c-Fos expression (Figs. 3 and 4 and Table 1) of intrathecal BmK IT2 in the pre-treatment group than in the post-treatment one indicated that the suppression exerted by BmK IT2 pre-treatment on the first phasic responses may inhibit the development of the second phasic responses. The pattern of the inhibition exerted by intrathecal BmK IT2 pre-treatment or post-treatment on formalin-induced pain
behavior was found to be similar to that of peripheral BmK IT2 [34]. The present study shows that the suppression by pretreatment with intrathecal BmK IT2 on formalin-induced spinal c-Fos expression was more potent than that by post-treatment. However, in the case of intraplantar BmK IT2 [34], the suppression exerted by BmK IT2 pre-treatment on formalin-induced spinal c-Fos expression was found to be weaker than that by post-treatment. These results suggest that BmK IT2 may thus be a promising molecular tool used for understanding the correlation of pain behaviors and neuronal activities. As for the controversial inhibitory effect of peripheral and intrathecal BmK IT2 on formalin-induced c-Fos expression, it might be attributed to either there are more active sites and/or the higher expressing abundance of BmK IT2-targeted sodium channels located at spinal level than those at peripheral terminals, and/or formalininduced inflammation developed during the later phase after formalin injection makes BmK IT2 to easily access target channels resulting in the inhibition of BmK IT2 post administration stronger than BmK IT2 pre-treatment on c-Fos expression. We found in the present study that suppression of intrathecal BmK IT2 on formalin-induced spinal c-Fos expression in the superficial laminae was higher than in the deeper laminae, indicating that BmK IT2-sensitive targets in the superficial laminae might be more abundant than in the deeper laminae. It is consistent well with the previous electrophysiological recordings that the inhibition of BmK IT2 on sodium currents of small DRG neurons was stronger than that of larger DRG neurons [29]. In summary, the present study reports that formalin-induced spontaneous pain behavior and spinal c-Fos expression could be effectively suppressed by either pre- or post-treatment with intrathecal BmK IT2. The results indicated that intrathecal administration of BmK IT2 resulted in potent central antinociception, and that sodium channels located in central pain pathway might be potential targets for anti-nociception exerted by BmK IT2. Therefore, BmK IT2 could provide a valuable tool for understanding pain mechanisms not only peripherally but also centrally. Acknowledgements This study was supported by National Basic Research Program of China (2006CB500801), a grant from National Nature Sciences Foundation of China (30370446), and partially by Shaanxi Provincial Nature Sciences Foundation (03JK220 and 2004C2 07), China. References [1] F.V. Abbott, K.B.J. Franklin, R.F. Westbrook, The formalin test: scoring properties of the first and second phases of pain responses in rats, Pain 60 (1995) 91–102. [2] Z.T. Bai, B. Chen, X.Y. Zhang, G.L. Fan, Y.H. Ji, c-Fos expression in rat spinal cord induced by scorpion BmK venom via subcutaneous plantar injection, Neurosci. Res. 44 (2002) 447–454. [3] Z.T. Bai, X.Y. Zhang, Y.H. Ji, Fos expression in rat spinal cord induced by peripheral injection of BmK I, an alpha-like scorpion neurotoxin, Toxicol. Appl. Pharmacol. 192 (2003) 78–85.
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