Spinal astrocyte and microglial activation contributes to rat pain-related behaviors induced by the venom of scorpion Buthus martensi Karch

European Journal of Pharmacology 623 (2009) 52–64

Contents lists available at ScienceDirect

European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Neuropharmacology and Analgesia

Spinal astrocyte and microglial activation contributes to rat pain-related behaviors induced by the venom of scorpion Buthus martensi Karch Feng Jiang a,1, Tong Liu b,1, Ming Cheng a, Xue-Yan Pang a, Zhan-Tao Bai c, Jing-Jing Zhou a, Yong-Hua Ji a,⁎ a b c

Lab of Neuropharmacology and Toxicology, Shanghai University, Shanghai, 200444, PR China Department of Anesthesiology, Brigham and Women's hospital, 75, Francis street, Boston, MA, United States College of Life Sciences, Yanan University, Yanan 716000, PR China

a r t i c l e

i n f o

Article history: Received 25 June 2009 Received in revised form 18 August 2009 Accepted 8 September 2009 Available online 24 Septemebr 2009 Keywords: BmK venom Astrocyte Microglia Spontaneous pain Thermal hyperalgesia Mechanical hyperalgesia

a b s t r a c t The present study investigated whether spinal astrocyte and microglia were activated in Buthus martensi Karch (BmK) venom-induced rat pain-related behaviors. The results showed that glial fibrillary acidic protein (GFAP) immunoreactivity indicative astrocyte activation in bilateral spinal cord started to increase by day 3, peaked at day 7 and gradually reversed at day 14 following intraplantar injection of BmK venom. Western blotting analysis confirmed GFAP expression was up-regulated by BmK venom. In contrast, bilateral spinal increase of OX-42 immunoreactivity indicative of microglial activation began at 4 h peaked at day 1 and gradually reversed by days 3 to 7 after the administration of BmK venom. Pretreatment with either intrathecal injection of fluorocitrate or intraperitonial injection of minocycline, and two glial activation inhibitors, suppressed the spontaneous nociceptive responses, and prevented the primary thermal and bilateral mechanical hyperalgesia induced by BmK venom. The post-treatment with fluorocitrate or minocycline could not affect the mechanical hyperalgesia. Moreover, minocycline partially inhibited BmK venom-induced spinal c-Fos expression but lack of effects on BmK venom-induced paw edema. Taken together, the current study demonstrated that spinal astrocyte and microglial activation may contribute to BmK venom-induced rat pain-related behaviors. Thus, spinal glia may represent novel targets for effective treatment of pain syndrome associated with scorpion envenomation. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Scorpion envenomation is a severe public medical problem in many countries of South America, Arabian Peninsula and Africa (Amitai, 1998). Usually, the intense pain is a frequent clinical manifestation caused by scorpion envenomation (Chen et al., 2001). It is noticed that the Asian scorpion Buthus martensi Karsch (BmK) envenomation can cause intense pain at the site of the sting and also at distant sites, skin edema and burning sensation (Balozet, 1971; Chen et al., 2001). To date, there is no established protocol that effectively alleviates the pain produced by scorpion envenomation possibly because of lack of available experimental animal models that mimic scorpion envenomation (Nascimento et al., 2005). A pain animal model was developed by intraplantar injection of BmK venom into rat hind paw aimed to characterize pain-related responses and to investigate mechanisms of scorpion envenomation pain (Bai et al., 2006; Chen et al., 2001, 2002). It was found that this pain model could mimic some clinical manifestations of scorpion BmK envenomation. ⁎ Corresponding author. Lab of Neuropharmacology and Toxicology, Shanghai University, Shang-Da Road 99, Shanghai 200444, PR China. Tel./fax: +86 21 66135189. E-mail address: [email protected] (Y.-H. Ji). 1 Both authors contributed equally to this work. 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.09.028

For example, BmK venom could induce the spontaneous pain lasting for more than 2 h, ipsilateral thermal hyperalgesia more than 72 h, and bilateral mechanical hyperalgesia no less than 14 days (Bai et al., 2006). A series of studies provided valuable information that may help explain the pain induced by scorpion BmK envenomation. Biochemical and electrophysiological studies demonstrated that some neurotoxins purified from BmK venom such as BmK I and BmK abT could delay the inactivation of voltage-sensitive sodium currents in isolated small dorsal root ganglion (DRG) neurons in rats (Chen et al., 2005; Ye et al., 2000). It had been demonstrated that plasma extravasation, mast cells degranulation and histamine release at injury site were involved in BmK venom-induced peripheral sensitization (Chen et al., 2002; Liu et al., 2007a). Dynamic release of excitatory amino acids, phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Fos expression in spinal cord dorsal horn may contribute to BmK venominduced central sensitization (Bai et al., 2002, 2003; Liu et al., 2007b; Pang et al., 2008; Zhang et al., 2002). Efforts made to further elucidate molecular mechanisms underlying BmK venom-induced pain-related behaviors may help us effectively treat the intense pain caused by scorpion envenomation. It is well-documented that spinal glia activation and a subsequent increase of pro-inflammatory cytokines contributed to the induction and maintenance of pain hypersensitivity associated with peripheral

F. Jiang et al. / European Journal of Pharmacology 623 (2009) 52–64

nerve or tissue injury (Colburn et al., 1997; Watkins et al., 2001; Wieseler-Frank et al., 2004). Recently, it was demonstrated that mirror image pain that occurs in association with many clinical pain syndromes, arises from the healthy body region contralateral to the injury site, mediated by spinal glia activation and proinflammatory cytokines (Milligan et al., 2003). Interestingly, unilateral injection of BmK venom not only induces ipsilateral thermal and mechanical hyperalgesia but also mirror-image mechanical hyperalgesia in rats (Bai et al., 2006). Given the pivotal role of glial activation in the creation of mirror-image pain, the present study is designed to investigate whether spinal glial activation is involved in BmK venominduced pain-related behaviors, especially mirror-image hyperalgesia in rats. The up-regulation of the expression of glial fibrillary acidic protein (GFAP) is often considered as a marker of astrocyte activation, whereas the up-regulation of microglial surface antigens including complement receptor 3 (CR3) that is recognized by the antibody OX-42 is widely used as microglial activation maker (Obata et al., 2006). One of the aims of this study was to examine the temporal profile of glial activation in the spinal cord by detecting the increase of GFAP and OX-42 immunoreactivity after BmK venom administration. The second purpose of the present study was to determine the potential roles of glial activation in BmK venom-induced rat pain-related behaviors by using two glia activation inhibitors fluorocitrate and minocycline. 2. Materials and methods 2.1. Experimental animals Adult male Sprague-Dawley rats (220 to 250 g) employed in this experiment were provided by Shanghai Experimental Animal Center, Chinese Academy of Sciences (CAS). The care and treatment of animals used in the present study were approved by the animal committee of CAS. European Community guidelines for the use of experimental animals and guidelines of International Association for the Study of Pain (IASP) for pain research in conscious animals were followed (Zimmermann, 1983). Each cage contains five rats with water and food available ad libitum. The animal room was under a 12-h light–dark cycle at 21–23 °C with 50% humidity. The behavioral observation was performed between 08:00 and 18:00. The rats were acclimatized to the laboratory 5 days before behavioral experiments. 2.2. BmK venom preparation and administration The crude BmK venom collected by electrical stimulation was purchased from an individual scorpion culture farm in Henan Province, China. The venom used in this study was filtered with a Sephadex G-50 column (Yan et al., 1996). BmK venom (50 µg in 50 µl saline) was intraplantar injected into the rat left hind paw. 2.3. Drugs preparation and administration In order to investigate the potential involvement of glia activation in BmK venom-induced pain-related behaviors, fluorocitrate (Sigma, St. Louis, MO), a reversible glial metabolic inhibitor (Qin et al., 2006), or equivolume vehicle was used. Fluorocitrate acts by inhibiting aconitase, an enzyme of the Krebs energy cycle of glia but not neurons (Hassel et al., 1992; Paulsen et al., 1987). Fluorocitrate was dissolved initially in 2 M HCl and then diluted in sterile phosphate buffered saline (PBS) to attain the final concentration, pH 6.0. 1 nmol fluorocitrate or equivolume vehicle (n = 8 for each group) were injected intrathecally (i.t.) by direct lumbar puncture between the L5 and L6 in rats as the previous description (Mestre et al., 1994). Briefly, rats were slightly anesthetized with ether. The intrathecal injection was made with a 27-gauge, 1-inch sterile disposable needle connected to a 50 µl Hamiltom syringe. The injection volume was 20 µl. The drug was administered within seconds.

53

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 within 1–2 min from anesthesia. In order to investigate the potential role of microglial activation in BmK venom-induced pain-related behaviors, minocycline was used as a potent and preferential inhibitor on microglia rather than astrocytes or neurons. Minocycline (at doses of 10 mg/kg or 30 mg/kg) (Sigma, St. Louis, MO) or saline vehicle (n = 8 for each group) was intraperitonially (i.p.) injected in rats 1 h before or 7 days after BmK venom administration (Cho et al., 2006).

2.4. Behavioral testing 2.4.1. Measurement of spontaneous nociceptive behaviors A 20 × 20 × 30 cm transparent plexiglas box with a glass floor was placed on a steel frame of 75 cm high above the experimental table covered with a mirror. Before administration of chemical agents, rats were placed in the test box for habituation at least 30 min. After treatment of glia activation inhibitors (fluorocitrate and minocycline), BmK venom was subcutaneously injected in rat hind paw. The Rats were immediately returned to the test box. Spontaneous nociceptive behaviors are determined by counting the number of flinches and the duration spent in lifting and licking the injected hind paw during 5 min interval for 2 h (Chen et al., 1999). After observation of rat spontaneous nociceptive behaviors, each experimental group was examined for the sensitivity to mechanical or radiate heat stimuli. Evaluation of spontaneous nociceptive behaviors was performed by an experimenter unaware of the experimental condition.

2.4.2. Measurement of paw withdrawal mechanical threshold Rats were placed in plexiglas test box (20 × 20× 30 cm) on a mesh floor (1 cm2 openings) for habituation 30 min before examination. Mechanical sensitivity was assayed by using a series of 10 calibrated von Frey filaments with forces from 0.6 to 26 g (58011, Stoelting Co., IL). Filaments were applied from underneath the metal mesh floor to the bilateral hind paws. Each filament was probed for same duration of 2–3 s with an inter-stimulus interval of 10 s. The positive response is indicated by brisk withdrawal or flinching of the tested paw. The rat paw withdrawal mechanical threshold was defined as the lowest force that caused at least five withdrawals out of the ten consecutive applications (Chen et al., 1999). Ahead of the inhibitors used, the rats' paw withdrawal mechanical threshold was measured as the value of baseline. Evaluation of paw withdrawal mechanical threshold was performed by an experimenter unaware of the experimental condition.

2.4.3. Measurement of paw withdrawal thermal latency Rat paw withdrawal thermal latency to radiant heat stimuli was determined as previously described (Hargreaves et al., 1988). Briefly, rat was placed on the surface of a 2 mm thick glass plate covered with the transparent plexiglas test box (20 × 20 × 30 cm) for habituation at least 30 min before testing. Heat stimuli were provided with radiant heat stimulator (RTY-3, Xi'an Fenglan instrumental factory, China). The heat source was a high intensity projector halogen lamp bulb (150 W, 24 V) positioned under the glass floor 2 cm directly beneath the targeting area of hind paw. The diameter of the light spot on the floor surface was about 3 mm. 25 s was set as cutoff time to avoid tissue injury. For one rat, five stimuli were performed and the stimuli interval was 10 min, rat paw withdrawal thermal latency was determined by averaging the last four values. One day before experiment, the rat paw withdrawal thermal latency was measured as the value of baseline. Evaluation of paw withdrawal thermal latency was performed by an experimenter unaware of the experimental condition.

54

F. Jiang et al. / European Journal of Pharmacology 623 (2009) 52–64

Fig. 1. Illustration of quantitative evaluation of GFAP or OX-42 immunoreactivity. (A) As areas of interest, three measuring areas (100 × 100 pixel, about 5625 µm2) were defined. (B–C) Representative microphotographs of GFAP labeled astrocyte and OX-42 labeled microglia. (D) Image of immunostaining in the dorsal horn of spinal segment L4–L5. (E) Softwareassisted evaluation of immunostaining (same figure as shown in D).

2.5. Measurement of paw edema

2.6. Immunohistochemistry

The paw edema was presented as the increase of paw thickness. The dorsal–ventral thickness of rat hind paw at the central plantar surface was determined with a calibrated micrometer (n = 8 for each group). Paw edema was measured three times for each time point, and then averaged (Zhang et al., 2003). Evaluation of paw edema was performed by an experimenter unaware of the experimental condition.

As for GFAP and OX-42 immunostaining, 4 h, 8 h, 1 day, 3 days, 5 days, 7 days and 14 days after BmK venom administration, rats 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% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). For c-Fos expression detection, the same protocol

Fig. 2. BmK venom induced astrocyte activation in the bilateral L4–L5 lumbar spinal cord. Representative photomicrographs of GFAP immunostaining in the spinal cord sections of non-treated control animals (A), the animals administered with BmK venom at 7 day (C); High-magnification image showing astrocytes cellular morphology in non-treated control animals (B) and activated astrocytes in BmK venom-treated animals (D).

F. Jiang et al. / European Journal of Pharmacology 623 (2009) 52–64

55

Fig. 3. Time course of astrocyte activation induced by BmK venom in the L4–L5 lumbar spinal cord. Representative photomicrographs of GFAP immunostaining in the ipsilateral spinal cord sections of non-treated control animals (A), the animals administered with BmK venom at the time points indicated (B–H). It was found that the increase of glial GFAP immunoreactivity indicative astrocyte activation began at 3 day, peaked 7 day and gradually reversed 14 day after BmK venom administration.

was performed 2 h after BmK venom administration. The lumbar spinal cord was removed and postfixed in the same fixative for 12 h and then cryoprotected in 0.1 MPB containing 20% sucrose until the tissue sank to the bottom of the container. Frozen serial coronal sections (14 µm in thickness) were cut with a cryostat and mounted on gelatin-coated glass slides. The avidin–biotin–peroxidase complex (ABC) method was used for GFAP and OX-42 immunostaining. Briefly, the sections were rinsed in 0.01 M phosphate-buffered saline (PBS; 0.01 M PB, pH 7.4) and then incubated with a rabbit polyclonal antibody for GFAP (1:2000; Code-Nr. Z 0334; DakoCytomation, Denmark) or mouse anti-rat CD11b monoclonal antibody (1:500; CHEMICON International, MA) for 48 h at 4 °C. The sections were incubated with biotinylated goat anti-rabbit IgG (1:200; Vector, Burlingame, CA) and ABC complex (1:200; Vector, Burlingame, CA) at room temperature for 2 h. The GFAP and OX-42 immunoreactivity were visualized by the glucose oxidase–diaminobenzidine–nickel method (Shu et al., 1988). 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. Three rats were used for each group. There was no positive staining when PBS or normal rabbit serum was used instead of the GFAP or OX-42 antibody. The stained sections above were pictured. All the images were quantitatively evaluated with image analysis software (Image-Pro Plus 6.0). Statistical clues were used to distinguish background from true immunoreactions. The threshold level for immunostaining against background was defined as the mean background gray value in unstained areas plus three standard deviations as shown in Fig. 1 (Hoheisel et al., 1998; Tenschert et al., 2004). Intensities exceeding the threshold were accepted as true immunostaining. Quantitative

measurements were made ipsi-laterally to the venom-injected side in the spinal dorsal horn. As measuring areas (areas of interest), three fields of 100×100 pixel (about 5625 µm2) in each superficial dorsal horn were defined. Within the squares, the immunoreactive area were measured. The average area in the three defined fields presents the immunoreaction level. Six rats were used for each group. As for c-Fos immunostaining, according to the above protocol, a rabbit polyclonal antibody raised against a peptide mapped at the amino terminus of human c-Fos P62 (1:2,000; Sc-52; Santa Cruz, CA) was used to incubated the sections instead of anti-GFAP or OX-42 antibody. In addition, to count the number of FLI neurons, the segment and organization of the rat spinal cord were determined(Molander et al., 1984). Four regions of the spinal gray matter were defined as follows:

Fig. 4. Spinal segment L4–L5: GFAP immunoreactivity areas in the dorsal horn (ipsilateral to BmK venom injection). Asterisks indicate significant differences between control and venom injected animals; ⁎⁎⁎P b 0.001.

56

F. Jiang et al. / European Journal of Pharmacology 623 (2009) 52–64

2.7. Protein extraction and immunoblotting

Fig. 5. Western blotting analysis of GFAP expression in the L4–L5 lumbar spinal cord following BmK venom administration. (A) Representative western blot of GFAP immunoreactivity (top) at the different time points following intraplantar injection of BmK venom. GAPDH (bottom) was used as a control for sample loading. (B) The intensity of the bands was quantified. The fold change for the density of GFAP bands is calculated after normalization with control. GFAP levels in control group were set at 100% for quantifications. The GFAP immunoreactivity indicative of spinal astrocyte activation began to increase at 3 days and peaked 7 days after BmK venom administration; ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001.

the superficial layer of the dorsal horn (laminae I–II); the proprius nucleus (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. Five rats were used for each group and seven sections per rat were used for counting and averaging. All data were calculated by an observer blind for treatment.

Rats were anesthetized with intraperitoneal injection of sodium pentobarbital (60 mg/kg, i.p.) and decapitated at various time points (4 h, 8 h, 1 days, 3 days, 5 days and 7 days) after the BmK venom injection. Three rats were used in each group. The spinal cord of each rat was removed by pressure expulsion with saline into an ice-cooled glass dish. The lumborsacral enlargement was identified as required. At last, the section was labeled and snap frozen in liquid nitrogen until further treatment. The sectioned tissues were homogenized at 4 °C in radioimmune precipitation buffer [150 mM NaCl, 100 mM Tris (pH 8.0), 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 5 mM EDTA and 10 mM NaF] supplemented with 1 mM sodium vanadate, 2 mM leupeptin, 2 mM aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT, and 2 mM pepstatin A. After centrifugation at 14,000 rpm for 15 min, the supernatant which contained the total cellular protein extract was collected and stored at − 70 °C. The protein concentration was determined using the Brandford assay (Sigma, St. Louis, MO). Western blotting experiments were performed in order to confirm the upregulation of GFAP expression following BmK venom administration. Briefly, sixty micrograms of protein extracts were separated by SDSpolyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a PVDF membrane. The membranes were incubated with following primary antibodies overnight at 4 °C: a rabbit polyclonal antibody against GFAP (1:10000; Code-Nr. Z 0334; DakoCytomation Denmark) or mouse monoclonal antibody for GAPDH (1:10000; Santa Cruz, CA). Protein bands were then detected by incubation with horseradish peroxidaseconjugated antibodies, and immunoblotting signals were visualized by treatment with ECL reagent (PerkinElmer Life Sciences, MA) and exposure to film. Densitometric quantification of immunoreactive bands was performed with GelDoc-2000 Imagine System (Bio-Rad, CA). The bands were captured with the image analysis system and the intensity was quantified. Representative Western Blotting analysis of GFAP expression levels at various time points. Data are representative of at least three independent experiments with similar results. The densitometry data were normalized to GAPDH levels and those of the control.

Fig. 6. BmK venom induced microglial activation in the bilateral L4–L5 lumbar spinal cord. Representative photomicrographs of OX-42 immunostaining in the spinal cord sections of non-treated control animals (A), the animals administered with BmK venom at 1 day (C); High-magnification image showing microglia cellular morphology in non-treated control animals (B) and activated microglia in BmK venom-treated animals (D).

F. Jiang et al. / European Journal of Pharmacology 623 (2009) 52–64

57

Fig. 7. Time course of microglial activation induced by BmK venom in the L4–L5 lumbar spinal cord. Representative photomicrographs of OX-42 immunostaining in the ipsilateral spinal cord sections of non-treated control animals (A), the animals administered with BmK venom at the time points indicated (B–H). It was found that the increase of glial OX-42 immunoreactivity indicative microglial activation began at 4 h, peaked 1 day and gradually reversed 3 day and 7 day after BmK venom administration.

2.8. Statistical analysis All results were expressed as mean ±S.E.M. (standard error of the mean). The quantified data for western blotting and immunostaining was analyzed by one-way ANOVA, followed by the Bonferroni post hoc test. The time course data of spontaneous pain-related behaviors between control and drugs were compared by a non-parametric Mann-Whitney U test. The paw withdrawal thermal latency and mechanical threshold values prior to and at each time point of different treatments after BmK venom administration were compared by using Kruskal-Wallis nonparametric test. Pb 0.05 was considered to be statistically significant. 3. Results

hypertrophied with thick processes (Fig. 2D). The contralateral spinal GFAP expression to BmK venom administration was also enhanced comparable with that of ipsilateral side (Fig. 2C). The temporal expression profile showed that spinal GFAP immunostaining was increased at the 3rd day, peaked at the 7th day and reversed at the 14th day following BmK venom administration (Figs. 3A–H; 4), which suggested that delayed astrocyte activation was induced by BmK venom. To further confirm the immunostaining results, western blotting analysis showed that the increase of spinal GFAP immunoreactivity seemed to start at the 3rd day and peaked at the 7th day following BmK venom administration (Fig. 5A; B). In naïve rats, the level of OX-42 immunostaining was low (Fig. 6A) and OX-42-labeled microglia showed extensive, thinly branches and

3.1. Activation of spinal glial cells induced by BmK venom To explore whether spinal glial cells were activated and the temporal expression profile of glia activation induced by BmK venom, the immunostaining was performed to evaluate spinal astrocyte and microglia activation based on cellular morphology and intensity of immunoreactivity of GFAP (an astrocyte activation marker) and OX-42 (a microglial activation marker) at different time points following peripheral administration of BmK venom. In naïve rats, the intensity of GFAP immunostaining in the spinal cord was low, indicating no apparent signs of astrocyte activation (Fig. 2A; B); whereas in BmK venom-treated rats, spinal GFAP expression was upregulated by BmK venom (Fig. 2C; D). As shown in Fig. 2B, GFAPimmunoreactive astrocyte showed fine branches and were sparsely distributed in non-treated rats. At day 7 after BmK venom administration, a large number of GFAP-immunoreactive astrocyte occurred and

Fig. 8. Spinal segment L4–L5: OX–42 immunoreactivity areas in the dorsal horn (ipsilateral to BmK venom injection). Asterisks indicate significant differences between control and BmK venom injected animals; ⁎P b 0.05; ⁎⁎⁎P b 0.001.

58

F. Jiang et al. / European Journal of Pharmacology 623 (2009) 52–64

Fig. 9. Intrathecal injection of fluorocitrate suppress BmK venom-induced various pain-related behaviors in rats. Intrathecal injection of vehicle or fluorocitrate (1 nmol in 20 µl vehicle) was performed 30 min before intraplantar injection of BmK venom. The number of paw flinches (A) and duration of paw lifting and licking (C) per 5 min induced by BmK venom were suppressed by intrathecal administration of 1 nmol fluorocitrate; The total number of the rat paw flinches (B) and total duration of paw lifting and biting (D) within 2 h after BmK venom injection were suppressed. At 5 h and 8 h after BmK venom injection, ipsilateral mechanical hyperalgesia (E) contralateral mechanical hyperalgesia (F) and ipsilateral thermal hyperalgesia (G) were suppressed by fluorocitrate. Rat contralateral paw withdrawal thermal latency was not affected by fluorocitrate (H). One day before experiment, the rat paw withdrawal mechanical threshold and thermal threshold were measured as the value of baseline. Rat hind paw injected with BmK venom was considered as ipsilateral side and the other side was named as contralateral side. All data were presented as mean±S.E.M. ⁎Pb 0.05, ⁎⁎Pb 0.01, ⁎⁎⁎Pb 0.001 group treated with fluorocitrate compared with group treated with vehicle (n=8 for each group).

F. Jiang et al. / European Journal of Pharmacology 623 (2009) 52–64

were well spaced in the L4–L5 lumbar spinal cord, indicating some features of resting state (Fig. 6B). In contrast, the number of OX-42immunoreactive microglia cells remarkably increased in bilateral spinal cord of BmK venom-treated rats compared with non-treated rats (Fig. 6C). The OX-42-labeled microglia cell revealed morphology characteristics of activation state, namely microglia exhibited short, thick processes and enlarged cell bodies, which differ from that of resting microglia (Fig. 6D). As for the time course of microglia activation, it was found that the OX-42 immunoreactivity began to increase at 4 h, and peaked at 1 day and gradually reversed from 3 day after BmK venom administration (Figs. 7A–H; 8). 3.2. Suppression by intrathecal injection of fluorocitrate on BmK venominduced spontaneous nociceptive behaviors and cutaneous hyperalgesia in rats Fluorocitrate was employed as a reversible glial metabolic inhibitor to investigate the potential involvement of glia activation in BmK venom-induced pain-related behaviors (Qin et al., 2006). Fluorocitrate or vehicle was intrathecally injected 30 min before the intraplantar administration of BmK venom. Intrathecal injection of vehicle did not affect BmK venom induced pain-related behaviors in rats (data not shown). Compared with the vehicle group, 1 nmol fluorocitrate suppressed the spontaneous nociceptive behaviors significantly (Fig. 9A; C). The suppression by fluorocitrate on flinches lasted for 2 h after the BmK venom injection. The total number of flinches in 2 h was decreased by pre-treatment with fluorocitrate from 909.5 ± 36.6 to 514.5 ± 24.8 (P b 0.001) (Fig. 9B). The total duration of lifting and licking of the injected hind paw in 2 h was also decreased from 1512.5 ± 71.8 s to 545.4 ± 99.9 s (P b 0.001) (Fig. 9D). BmK venom-induced bilateral mechanical hypersensitivity was suppressed significantly by pre-treatment with fluorocitrate at the time point of 5 h and 8 h. 1 nmol fluorocitrate partially inhibited bilateral mechanical hypersensitivity induced by BmK venom (Fig. 9E; F). Ipsilateral paw withdrawal mechanical thresholds were increased by intrathecal fluorocitrate from 11.6 ± 0.9 g to 18.8 ±1.9 g (P b 0.01) at 5 h; from 7.0±0.4 g to 16.8 ± 2.1 g (P b 0.001) at 8 h. Contralateral paw withdrawal mechanical thresholds were increased by intrathecal fluorocitrate from 17.9 ±2.3 g to 24.9 ± 1.1 g (P b 0.001) at 5 h; from 15.3 ± 2.0 g to 22.8 ±2.1 g (P b 0.001) at 8 h. BmK venom-induced ipsilateral thermal hypersensitivity was also suppressed by pre-treatment with fluorocitrate at the time point of 5 h and 8 h significantly. Moreover, 1 nmol fluorocitrate prevented the development of the ipsilateral thermal hypersensitivity (Fig. 9G). Ipsilateral paw withdrawal thermal latencies were increased by intrathecal fluorocitrate from 5.6 ±0.2 s to 7.6 ±0.4 s (P b 0.001) at 5 h; from 5.6 ±0.2 s to 8.2 ±0.4 s (P b 0.001) at 8 h, While the contralateral paw thermal withdrawal latency displayed no significant alternation by intrathecal administration of fluorocitrate (Fig. 9H). 3.3. BmK venom-induced diverse pain-related behaviors could be suppressed by systemic administration of minocycline In order to investigate the potential role of microglial activation in BmK venom-induced pain-related behaviors, minocycline was used as a potent and preferential inhibitor on microglia rather than astrocytes or neurons. Minocycline or saline was injected intraperitonially 1 h before the intraplantar administration of BmK venom. Intraperitonial injection of saline did not affect BmK venom induced pain-related behaviors in rats (data not shown). Compared with the vehicle group, the two doses of minocycline (10 mg/kg and 30 mg/kg) suppressed the spontaneous nociceptive behaviors significantly (Fig. 10A; C). The suppression by minocycline on flinches lasted for 2 h after BmK venom injection. Treated with minocycline at the dose of 10 mg/kg and 30 mg/kg, the total number of flinches in 2 h was decreased from 1031.9 ± 25.1 to 707.9 ± 56.2 (P b 0.001) and 622.1 ± 39.6 (P b 0.001),

59

respectively (Fig. 10B). The total duration of lifting and licking of the injected hind paw in 2 h was also decreased from 1485.1 ± 188.5 s to 662.4 ± 113.4 s (P b 0.01), and 463.0 ± 74.7 (P b 0.001), respectively (Fig. 10D). However, the inhibitory potency between different doses was not statistically significant. BmK venom-induced bilateral mechanical hypersensitivity was suppressed significantly by pre-treatment with minocycline at the time point of 5 h and 8 h. Both doses of minocycline (10 mg/kg and 30 mg/kg) partially inhibited the development of bilateral mechanical hypersensitivity (Fig. 10E; F). Ipsilateral paw withdrawal mechanical thresholds were increased by minocycline from 7.3 ± 0.3 g to 11.0 ± 1.0 g (10 mg/kg; P b 0.05) and 14.3 ± 2.0 g (30 mg/kg; P b 0.05) at 5 h; from 4.3± 0.2 g to 7.3 ± 0.5 g (10 mg/kg; P b 0.01) and 14.0 ± 2.1 g (30 mg/kg; P b 0.01) at 8 h. Contralateral paw withdrawal mechanical thresholds were increased by minocycline from 9.8 ± 0.9 g to 19.7 ± 2.3 g (10 mg/kg; P b 0.05) and 21.5 ± 2.3 g (30 mg/kg; P b 0.01) at 5 h; from 6.7 ± 0.3 g to 17.0 ± 2.3 g (10 mg/kg; P b 0.01) and 21.5 ± 2.3 g (30 mg/kg; P b 0.001) at 8 h. BmK venom-induced ipsilateral thermal hypersensitivity was also suppressed by pre-treatment with minocycline at the time point of 5 h and 8 h significantly. Both doses of minocycline (10 mg/kg and 30 mg/kg) prevented the development of the ipsilateral thermal hypersensitivity (Fig. 10G). Ipsilateral paw withdrawal thermal latencies were increased by intrathecal minocycline from 6.1 ± 0.2 s to 7.1 ± 0.5 s (10 mg/kg; P N 0.05) and 8.5 ± 0.3 s (30 mg/kg; P b 0.001) at 5 h; from 5.4 ± 0.2 s to 6.9 ± 0.3 s (10 mg/kg; P b 0.001) and 9.9 ± 0.6 s (30 mg/kg; P b 0.001) at 8 h. However, minocycline could not alter the contralateral paw thermal withdrawal latency (Fig. 10H). In addition, it was also found that intraperitonial injection of minocycline lacked effects on BmK venom-induced paw edema at the dose of 30 mg/kg (Fig. 11). 3.4. Partial inhibition by systemic administration of minocycline on BmK venom-induced spinal c-Fos expression in rats C-Fos, an immediate early gene protein, is extensively used as a functional marker of spinal neuron activation induced by noxious stimuli (Coggeshall, 2005). Previous studies have demonstrated that BmK venom could induce spinal c-Fos expression in rats (Bai et al., 2002). Thereby, the effects of systemic administration of minocycline on BmK venom-induced spinal c-Fos were investigated in the current study. The minocycline was administrated intraperitonially 1 h before BmK venom injection. In accordance with previous study, c-Fos expression was found mainly in the ipsilateral L4–L5 spinal cord with only a few on the contralateral side 2 h after BmK venom administration. Intraperitonially injection of saline or minocycline alone could not alter the basal level of c-Fos expression in the L4–L5 segments (data not shown). And intraperitonially injection of saline was ineffective on BmK venom-induced spinal c-Fos expression (Fig. 12A; B). However, BmK venom-induced ipsilateral spinal c-Fos expression in all laminas was significantly decreased by both doses of minocycline (10 and 30 mg/kg) (Fig. 12C–E). 3.5. BmK venom-induced pain related behavior could not be affected by post-administration of minocycline or fluorocitrate The paw withdrawal mechanical thresholds appeared to have no significant changes by post-administration of either minocycline (10 mg/kg and 30 mg/kg) or fluorocitrate (1 nmol) on day 7 as shown in Figs. 13A; B and 14. 4. Discussion The results found in the present study indicated that spinal microglia activation precedes astrocyte activation following BmK venom

60

F. Jiang et al. / European Journal of Pharmacology 623 (2009) 52–64

F. Jiang et al. / European Journal of Pharmacology 623 (2009) 52–64

61

Fig. 11. BmK venom-induced paw edema was not affected by minocycline. The increase of paw thickness was indicative of paw edema. All data were presented as mean ± S.E.M. The results showed that intraperitonial injection of minocycline lacked effects on BmK venominduced paw edema at dose of 30 mg/kg.

administration. The extent of spinal astrocyte and microglia activation on the contralateral side was comparable on the ipsilateral side. The suppression of the spontaneous nociceptive responses, prevented the primary thermal and bilateral mechanical hyperalgesia by either fluorocitrate (i.t.) or minocycline (i.p.) suggested that spinal astrocyte and microglia activation may contribute to BmK venom-induced rat painrelated behaviors. Astrocytes are often shown with delayed activation after peripheral nerve or tissue injury, thus astrocytes are thought to contribute to the maintenance of chronic pain in these models (Raghavendra et al., 2004; Tanga et al., 2004). After various types of nerve injury, spinal astrocytes proliferate and up-regulate GFAP expression (Colburn et al., 1999; Winkelstein and DeLeo, 2002). The present study firstly examined the time course of spinal glial activation by detecting the increase of GFAP and OX-42 immunoreactivity after BmK venom administration (Garrison et al., 1991; Hayward, 2006). Our results show that BmK venom-induced astrocyte activation occurs in bilateral spinal cord and the extent of activation in the contralateral side is comparable with that in ipsilateral side to BmK venom administration. It is well correlated with similar intensity of bilateral mechanical hyperalgesia induced by BmK venom (Bai et al., 2006). The time course of spinal astrocyte activation suggested that the delayed activation of astrocyte could be induced by BmK venom. Previous studies showed that BmK venom-induced bilateral mechanical hyperalgesia could last for more than 2 weeks (Bai et al., 2006), thus indicating that spinal astrocyte activation may mainly contribute to the maintenance of BmK venom-induced bilateral mechanical hyperalgesia. Although there are some similarities in the spinal microglial activation among neuropathic and inflammatory pain models, the extent and time course of spinal microglial activation seems to be different (Colburn et al., 1997; Coyle, 1998; Fu et al., 1999; Raghavendra et al., 2003). In the present study, the bilateral spinal microglial activation was found to start at 4 h, peaked 1 day and gradually reversed at 3 day and 7 day after BmK venom administration (Figs. 7; 8). Unlike the delayed astrocyte activation induced by BmK venom, microglial activation seemed to be quicker but lasted shorter than that of astrocyte. Therefore, the time course of microglial activation was not well correlated with the development of BmK venom-induced bilateral mechanical hyperalgesia (Bai et al., 2006). A disconnection between microglial activation and mechanical hyperalgesia was also observed in

Fig. 12. Systemic administration of minocycline inhibited c-Fos expression in the spinal cord induced by BmK venom. (A) Representative immunohistochemical staining of BmK venom induced c-Fos expression in the spinal dorsal horn of rats; (B–D) Representative immunohistochemical staining of c-Fos expression in the spinal dorsal horn of rats induced by BmK venom following systemic administration of saline (B), 10 mg/kg (C) or 30 mg/kg (D) minocycline; (E) Quantitative data indicating the number of c-Fos positive neurons in all laminaes of spinal cord in each group. Five rats were used for each group. The data presented as the mean number FLI neurons random from 7 sections of each animal. All data were presented as mean± S.E.M. ⁎⁎⁎P b 0.001 (10 mg/kg minocycline) and ###P b 0.001 (30 mg/kg minocycline) group treated with minocycline compared with group treated with saline.

chronic constriction nerve injury (CCI) model in rats (Colburn et al., 1997). It strongly indicated that spinal microglial activation might play a critical role in the induction but not in the maintenance of BmK venom-

Fig. 10. Systemic administration of minocycline suppressed BmK venom-induced pain-related behaviors in rats. Systemic administration of minocycline (10 or 30 mg/kg) or saline was performed 1 h before intraplantar injection of BmK venom. The number of paw flinches (A) and duration of paw lifting and licking (C) per 5 min induced by BmK venom were suppressed by systemic administration of minocycline; The total number of the rat paw flinches (B) and total duration of paw lifting and biting (D) within 2 h after BmK venom injection were suppressed. At 5 h and 8 h after BmK venom injection, ipsilateral mechanical hyperalgesia (E) contralateral mechanical hyperalgesia (F) and ipsilateral thermal hyperalgesia (G) were suppressed by minocycline. Rat contralateral paw withdrawal thermal latency was not affected by minocycline (H). One day before experiment, the rat paw withdrawal mechanical threshold and thermal threshold were measured as the value of baseline. Rat hind paw injected with BmK venom was considered as ipsilateral side and the other side was named as contralateral side. All data were presented as mean ± S.E.M. ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 (10 mg/kg minocycline) and #P b 0.05, ##P b 0.01, ###P b 0.001 (30 mg/kg minocycline) group treated with minocycline compared with group treated with saline vehicle (n = 8 for each group).

62

F. Jiang et al. / European Journal of Pharmacology 623 (2009) 52–64

Fig. 13. Systemic administration of minocycline on day 7 after venom injection could not suppress BmK venom-induced pain-related behaviors in rats. Systemic administration of minocycline (10 or 30 mg/kg) or saline were performed 7 days after intraplantar injection of BmK venom. The ipsilateral mechanical hyperalgesia (A) and contralateral mechanical hyperalgesia (B) could not be affected by minocycline. Before minocycline injection, the rat paw withdrawal mechanical threshold was measured as day 7 base line. All data were presented as mean±S.E.M. group treated with minocycline compared with their base line (n=8 for each group).

Fig. 14. Intrathecal injection of fluorocitrate on day 7 after venom injection could not suppress BmK venom-induced pain-related behaviors in rats. Intrathecal administration of 1 nmol fluorocitrate or saline were performed 7 days after intraplantar injection of BmK venom. The ipsilateral mechanical hyperalgesia (left) and contralateral mechanical hyperalgesia (right) could not be affected by fluorocitrate. Before fluorocitrate injection, the rat paw withdrawal mechanical threshold was measured as day 7 base line. All data were presented as mean± S.E.M. group treated with minocycline compared with their base line (n=8 for each group).

induced bilateral mechanical hyperalgesia. Finally, it may allow us to reach a conclusion that spinal microglial and astrocyte activation might play different roles in the induction and maintenance of BmK venominduced bilateral mechanical hyperalgesia. Previous studies have shown that 1 nmol of fluorocitrate functionally and morphologically suppresses glial cell activation in vivo (Fonnum et al., 1997). In the present study, it was found that pre-treatment with fluorocitrate suppressed the BmK venom-induced rat spontaneous nociceptive responses and prevented the primary thermal and bilateral mechanical hyperalgesia. Considering the increase of GFAP expression was not significant within 1 day after BmK venom administration, the effects of fluorocitrate may attribute to its inhibition on microglia. Indeed, minocycline as a specific microglial activation inhibitor could suppress BmK venom-induced rat spontaneous nociceptive responses, and prevented the primary thermal and bilateral mechanical hyperalgesia. However, the other possibility cannot be excluded. For example, the upregulation of GFAP expression is not a good marker for astrocyte activation, namely activities of astrocyte had been enhanced before upregulation of GFAP expression. It has been demonstrated that formalin-induced c-Fos expression could be inhibited by systemic administration of minocycline. In line with these results, the current study demonstrated that systemic administration of minocycline could reduce BmK venom-induced c-Fos expression in all laminaes of spinal cord (Fig. 12). However, systemic administration of minocycline reduced formalin-induced paw edema significantly, but not BmK venom-induced paw edema. The detailed mechanisms underlying the anti-edematous effects of minocycline in the periphery is unclear so far. Nevertheless, the results suggested that BmK venom-induced edema had distinct mechanisms from formalininduced paw edema. Previous studies demonstrated that once repetitive synaptic communication occurs, there is an increase in the responsiveness of spinal nociceptive neurons to the stimuli, which is known as central sensitization (Gebhart, 2004; Hucho and Levine, 2007; Kawasaki et al., 2008; Millan, 2002; Petrenko et al., 2003). Under this condition, NMDA receptor could be activated by the co-release of glutmamate and other neurotransmitters such as substance P and calcitonin gene-related peptide (CGRP). As a result, the voltage-gated Ca2+ current (VGCC) is activated subsequently (McMahon et al., 2005). Additionally, mitogenactivated protein kinases (MAPK) such as extracellular signal-regulated kinase (ERK) (Ji et al., 1999; Pang et al., 2008) and p38 (Ji et al., 2002; Kim et al., 2002; Svensson et al., 2003b), and other cell signaling pathways are activated. Astrocyte and microglia express several neurotransmitter receptors. They could be activated by glutamate (Queiroz et al., 1997; Svensson et al., 2003a), substance P (Svensson et al., 2003b), CGRP (Priller et al., 1995) and other factors. Continually excitation can induce ERK, p38 and c-Jun N-terminal kinase (JUK) activation in glial cells (Panenka et al., 2001; Suzuki et al., 2004). These signaling pathways lead to an increasing synthesis of inflammatory factors, such as interleukin 1β (IL-1β), tumour-necrosis factor (TNFα) and NO (Yang et al., 2004). Under this glia-neuron cross talk mechanism, the responsiveness of spinal nociceptive neurons to the stimuli is further enhanced. In the present study, The pre-treatment with glial activation inhibitors could significantly suppress BmK venom-induced mechanical hyperalgesia, but not by the postadministration. This result may suggest that astrocyte and microglia are involved in the facilitation of central sensitization induced by BmK venom, instead of in conducting the synaptic nociceptive signals directly. As critical mediators, astrocyte and microglia might mediate the bilateral mechanical hyperalgesia indirectly. In summary, spinal astrocyte and microglia in bilateral spinal cord are activated with different time courses following BmK venom administration. Pre-treatment with glia activation inhibitors fluorocitrate and minocycline suppressed the BmK venom-induced spontaneous nociceptive responses, prevented the primary thermal and bilateral mechanical hyperalgesia in rats, while post-treatment with these two inhibitors could not alter the development of mechanical hyperalgesia.

F. Jiang et al. / European Journal of Pharmacology 623 (2009) 52–64

The results indicate that spinal glia activation may contribute to BmK venom-induced various pain-related behaviors in an indirect manner and the inhibition of glial cell activity in the spinal cord may represent a promising approach for treatment of scorpion sting pain.

Acknowledgements The authors thank Mr. Jian-Wei Zhang and Ms. Xue-Qin Shu for their excellent technical assistance. This study was supported by National Basic Research Program of China (2006CB500801), partially by a grant from Science and Technology Commission of Shanghai Municipality (08JC1409500) and the Innovation Fund Project for Graduate Student of Shanghai University (SHUCX080224).

References Amitai, Y., 1998. Clinical manifestations and management of scorpion envenomation. Public Health Rev. 26, 257–263. Bai, Z.T., Chen, B., Zhang, X.Y., Fan, G.L., Ji, Y.H., 2002. c-Fos expression in rat spinal cord induced by scorpion BmK venom via plantar subcutaneous injection. Neurosci. Res. 44, 447–454. Bai, Z.T., Zhang, X.Y., Ji, Y.H., 2003. Fos expression in rat spinal cord induced by peripheral injection of BmK I, an alpha-like scorpion neurotoxin. Toxicol. Appl. Pharmacol. 192, 78–85. Bai, Z.T., Liu, T., Chai, Z.F., Pang, X.Y., Ji, Y.H., 2006. Rat pain-related responses induced by experimental scorpion BmK sting. Eur. J. Pharmacol. 552, 67–77. Balozet, L., 1971. Scorpionism in the old world. In: Bucherl, W., Buckley, E.E. (Eds.), Venomous Animal and Their Venoms, vol. 3. Academic Press, New York, pp. 349–371. Chen, J., Luo, C., Li, H.L., Chen, H.S., 1999. Primary hyperalgesia to mechanical and heat stimuli following subcutaneous bee venom injection into the plantar surface of hindpaw in the conscious rat: a comparative study with the formalin test. Pain 83, 67–76. Chen, B., Wang, C.Y., Ji, Y.H., 2001. Scorpion BmK venom induces nociceptive response of rats by plantar injection. Neurotoxicol. Teratol. 23, 675–679. Chen, B., Zhuo, X.P., Wang, C.Y., Ji, Y.H., 2002. Asian scorpion BmK venom induces plasma extravasation and thermal hyperalgesia in the rat. Toxicon 40, 527–533. Chen, J., Tan, Z.Y., Zhao, R., Feng, X.H., Shi, J., Ji, Y.H., 2005. The modulation effects of BmK I, an alpha-like scorpion neurotoxin, on voltage-gated Na+ currents in rat dorsal root ganglion neurons. Neurosci. Lett. 390, 66–71. Cho, I.H., Chung, Y.M., Park, C.K., Park, S.H., Li, H.Y., Kim, D., Piao, Z.G., Choi, S.Y., Lee, S.J., Park, K., Kim, J.S., Jung, S.J., Oh, S.B., 2006. Systemic administration of minocycline inhibits formalin-induced inflammatory pain in rat. Brain Res. 1072, 208–214. Coggeshall, R.E., 2005. Fos, nociception and the dorsal horn. Prog. Neurobiol. 77, 299–352. Colburn, R.W., DeLeo, J.A., Rickman, A.J., Yeager, M.P., Kwon, P., Hickey, W.F., 1997. Dissociation of microglial activation and neuropathic pain behaviors following peripheral nerve injury in the rat. J. Neuroimmunol. 79, 163–175. Colburn, R.W., Rickman, A.J., DeLeo, J.A., 1999. The effect of site and type of nerve injury on spinal glial activation and neuropathic pain behavior. Exp. Neurol. 157, 289–304. Coyle, D.E., 1998. Partial peripheral nerve injury leads to activation of astroglia and microglia which parallels the development of allodynic behavior. Glia 23, 75–83. Fonnum, F., Johnsen, A., Hassel, B., 1997. Use of fluorocitrate and fluoroacetate in the study of brain metabolism. Glia 21, 106–113. Fu, K.Y., Light, A.R., Matsushima, G.K., Maixner, W., 1999. Microglial reactions after subcutaneous formalin injection into the rat hind paw. Brain Res. 825, 59–67. Garrison, C.J., Dougherty, P.M., Kajander, K.C., Carlton, S.M., 1991. Staining of Glial Fibrillary Acidic Protein (GFAP) in lumbar spinal-cord increases following a sciatic-nerve constriction injury. Brain Res. 565, 1–7. Gebhart, G.F., 2004. Descending modulation of pain. Neurosci. Biobehav. Rev. 27, 729–737. Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J., 1988. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32, 77–88. Hassel, B., Paulsen, R.E., Johnsen, A., Fonnum, F., 1992. Selective-inhibition of glial-cell metabolism invivo by fluorocitrate. Brain Res. 576, 120–124. Hayward, P., 2006. Microglial role in neuropathic pain. Lancet Neurol. 5, 118–119. Hoheisel, U., Kaske, A., Mense, S., 1998. Relationship between neuronal activity and substance P-immunoreactivity in the rat spinal cord during acute and persistent myositis. Neurosci. Lett. 257, 21–24. Hucho, T., Levine, J.D., 2007. Signaling pathways in sensitization: toward a nociceptor cell biology. Neuron 55, 365–376. Ji, R.R., Baba, H., Brenner, G.J., Woolf, C.J., 1999. Nociceptive-specific activation of ERK in spinal neurons contributes to pain hypersensitivity. Nat. Neurosci. 2, 1114–1119. Ji, R.R., Samad, T.A., Jin, S.X., Schmoll, R., Woolf, C.J., 2002. p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron 36, 57–68. Kawasaki, Y., Zhang, L., Cheng, J.K., Ji, R.R., 2008. Cytokine mechanisms of central sensitization: distinct and overlapping role of interleukin-1 beta, interleukin-6, and tumor necrosis factor-beta in regulating synaptic and neuronal activity in the superficial spinal cord. J. Neurosci. 28, 5189–5194. Kim, S.Y., Bae, J.C., Kim, J.Y., Lee, H.L., Lee, K.M., Kim, D.S., Cho, H.J., 2002. Activation of p38 MAP kinase in the rat dorsal root ganglia and spinal cord following peripheral inflammation and nerve injury. NeuroReport 13, 2483–2486.

63

Liu, T., Bai, Z.T., Pang, X.Y., Chai, Z.F., Jiang, F., Ji, Y.H., 2007a. Degranulation of mast cells and histamine release involved in rat pain-related behaviors and edema induced by scorpion Buthus martensi Karch venom. Eur. J. Pharmacol. 575, 46–56. Liu, T., Pang, X.Y., Bai, Z.T., Chai, Z.F., Jiang, F., Ji, Y.H., 2007b. Intrathecal injection of glutamate receptor antagonists/agonist selectively attenuated rat pain-related behaviors induced by the venom of scorpion Buthus martensi Karsch. Toxicon 50, 1073–1084. McMahon, S.B., Cafferty, W.B.J., Marchand, F., 2005. Immune and glial cell factors as pain mediators and modulators. Exp. Neurol. 192, 444–462. Mestre, C., Pelissier, T., Fialip, J., Wilcox, G., Eschalier, A., 1994. A method to perform direct transcutaneous intrathecal injection in rats. J. Pharmacol. Toxicol. Methods 32, 197–200. Millan, M.J., 2002. Descending control of pain. Prog. Neurobiol. 66, 355–474. Milligan, E.D., Twining, C., Chacur, M., Biedenkapp, J., O'Connor, K., Poole, S., Tracey, K., Martin, D., Maier, S.F., Watkins, L.R., 2003. Spinal glia and proinflammatory cytokines mediate mirror-image neuropathic pain in rats. J. Neurosci. 23, 1026–1040. Molander, C., Xu, Q., Grant, G., 1984. The cytoarchitectonic organization of the spinalcord in the rat. 1. The lower thoracic and lumbosacral cord. J. Comp. Neurol. 230, 133–141. Nascimento, E.B., Costa, K.A., Bertollo, C.A., Oliveira, A.C.P., Rocha, L.T.S., Souza, A.L.S., Gloria, M.B.A., Moraes-Santos, T., Coelho, M.M., 2005. Pharmacological investigation of the nociceptive response and edema induced by venom of the scorpion Tityus serrulatus. Toxicon 45, 585–593. Obata, H., Eisenach, J.C., Hussain, H., Bynum, T., Vincler, M., 2006. Spinal glial activation contributes to postoperative mechanical hypersensitivity in the rat. J. Pain 7, 816–822. Panenka, W., Jijon, H., Herx, L.M., Armstrong, J.N., Feighan, D., Wei, T., Yong, V.W., Ransohoff, R.M., MacVicar, B.A., 2001. P2X7-like receptor activation in astrocytes increases chemokine monocyte chemoattractant protein-1 expression via mitogenactivated protein kinase. J. Neurosci. 21, 7135–7142. Pang, X.Y., Liu, T., Jiang, F., Ji, Y.H., 2008. Activation of spinal ERK signaling pathway contributes to pain-related responses induced by scorpion Buthus martensi Karch venom. Toxicon 51, 994–1007. Paulsen, R.E., Contestabile, A., Villani, L., Fonnum, F., 1987. An in vivo model for studying function of brain-tissue temporarily devoid of glial-cell metabolism — the use of fluorocitrate. J. Neurochem. 48, 1377–1385. Petrenko, A.B., Yamakura, T., Baba, A., Shimoji, K., 2003. The role of N-methyl-Daspartate (NMDA) receptors in pain: a review. Anesth. Analg. 97, 1108–1116. Priller, J., Haas, C.A., Reddington, M., Kreutzberg, G.W., 1995. Calcitonin gene-related peptide and ATP induce immediate early gene expression in cultured rat microglial cells. Glia 15, 447–457. Qin, M., Wang, J.J., Cao, R., Zhang, H., Duan, L., Gao, B., Xiong, Y.F., Chen, L.W., Rao, Z.R., 2006. The lumbar spinal cord glial cells actively modulate subcutaneous formalin induced hyperalgesia in the rat. Neurosci. Res. 55, 442–450. Queiroz, G., GebickeHaerter, P.J., Schobert, A., Starke, K., VonKugelgen, I., 1997. Release of ATP from cultured rat astrocytes elicited by glutamate receptor activation. Neuroscience 78, 1203–1208. Raghavendra, V., Tanga, F., DeLeo, J.A., 2003. Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy. J. Pharmacol. Exp. Ther. 306, 624–630. Raghavendra, V., Tanga, R.Y., DeLeo, J.A., 2004. Complete Freunds adjuvant-induced peripheral inflammation evokes glial activation and proinflammatory cytokine expression in the CNS. Eur. J. NeuroSci. e20, 467–473. Shu, S.Y., Ju, G., Fan, L.Z., 1988. The glucose–oxidase dab nickel method in peroxidase histochemistry of the nervous-system. Neurosci. Lett. 85, 169–171. Suzuki, T., Hide, I., Ido, K., Kohsaka, S., Inoue, K., Nakata, Y., 2004. Production and release of neuroprotective tumor necrosis factor by P2X(7) receptor-activated microglia. J. Neurosci. 24, 1–7. Svensson, C.I., Hua, X.Y., Protter, A.A., Powell, H.C., Yaksh, T.L., 2003a. Spinal p38 MAP kinase is necessary for NMDA-induced spinal PGE(2) release and thermal hyperalgesia. NeuroReport 14, 1153–1157. Svensson, C.I., Marsala, M., Westerlund, A., Calcutt, N.A., Campana, W.M., Freshwater, J.D., Catalano, R., Feng, Y., Protter, A.A., Scott, B., Yaksh, T.L., 2003b. Activation of p38 mitogen-activated protein kinase in spinal microglia is a critical link in inflammationinduced spinal pain processing. J. Neurochem. 86, 1534–1544. Tanga, F.Y., Raghavendra, V., DeLeo, J.A., 2004. Quantitative real-time RT-PCR assessment of spinal microglial and astrocytic activation markers in a rat model of neuropathic pain. Neurochem. Int. 45, 397–407. Tenschert, S., Reinert, A., Hoheisel, U., Mense, S., 2004. Effects of a chronic myositis on structural and functional features of spinal astrocytes in the rat. Neurosci. Lett. 361, 196–199. Watkins, L.R., Milligan, E.D., Maier, S.F., 2001. Glial activation: a driving force for pathological pain. Trends Neurosci. 24, 450–455. Wieseler-Frank, J., Maier, S.F., Watkins, L.R., 2004. Glial activation and pathological pain. Neurochem. Int. 45, 389–395. Winkelstein, B.A., DeLeo, J.A., 2002. Nerve root injury severity differentially modulates spinal glial activation in a rat lumbar radiculopathy model: considerations for persistent pain. Brain Res. 956, 294–301. Yan, L., Ren, H.M., Ji, Y.H., Ohishi, T., Mochizuki, T., Hoshino, M., Yanaihara, N., 1996. Purification and the partial amino acid sequence of a novel activator of ryanodine receptor (BmK AS-1) from mammalian skeletal muscle. Biomed. Res. (Tokyo) 17, 451–455. Yang, L.Q., Blumbergs, P.C., Jones, N.R., Manavis, J., Sarvestani, G.T., Ghabriel, M.N., 2004. Early expression and cellular localization of proinflammatory cytokines interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha in human traumatic spinal cord injury. Spine 29, 966–971.

64

F. Jiang et al. / European Journal of Pharmacology 623 (2009) 52–64

Ye, J.G., Wang, C.Y., Li, Y.J., Tan, Z.Y., Yan, Y.P., Li, C., Chen, J., Ji, Y.H., 2000. Purification, cDNA cloning and function assessment of BmK abT, a unique component from the Old World scorpion species. FEBS Lett. 479, 136–140. Zhang, X.Y., Zhang, J.W., Chen, B., Bai, Z.T., Shen, J., Ji, Y.H., 2002. Dynamic determination and possible mechanism of amino acid transmitter release from rat spinal dorsal horn induced by the venom and a neurotoxin (BmK 1) of scorpion Buthus martensi Karsch. Brain Res. Bull. 58, 27–31.

Zhang, Y.H., Chen, Y., Zhao, Z.Q., 2003. Resiniferatoxin reversibly blocks adjuvantinduced thermal hyperalgesia in the rat. Eur. J. Pharmacol. 481, 301–304. Zimmermann, M., 1983. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16, 109–110.