Amino acid transport system A is involved in inflammatory nociception in rats

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Research Report

Amino acid transport system A is involved in inflammatory nociception in rats Jing Wanga, c , Bin Genga , Hai-Li Shenb , Xu Xua , Hong Wanga , Cui-Fang Wanga , Jing-Ling Maa , Zhi-Ping Wangc,⁎ a

Institute of Orthopaedics, Second Hospital of Lanzhou University, Lanzhou University, No. 82 Cui Ying Men Street, Lanzhou, Gansu 730030, PR China b Department of Pain, Second Hospital of Lanzhou University, Lanzhou University, No. 82 Cui Ying Men Street, Lanzhou, Gansu 730030, PR China c Institute of Urology, Second Hospital of Lanzhou University, Lanzhou University, No. 82 Cui Ying Men Street, Lanzhou, Gansu 730030, PR China

A R T I C LE I N FO

AB S T R A C T

Article history:

Previous studies have indicated that central sensitization is a state of increased excitability

Accepted 8 February 2012

of nociceptive neurons in the spinal dorsal horn following peripheral tissue injury and/or

Available online 15 February 2012

inflammation and astrocytes play an important role in the central sensitization. The current study investigated the role of amino acid transport system A in central sensitization

Keywords:

and hyperalgesia induced by intraplantar injection of formalin in rats. Formalin (5%, 50 μl)

Amino acid transport system A

injected subcutaneously into the unilateral hindpaw pad induced typical biphase nocicep-

Methylaminoisobutyric acid

tive behaviors, including licking/biting and flinching of the injected paw and an increase of

Glial fibrillary acid protein

glial fibrillary acid protein (GFAP, an activated astrocyte marker) expression in spinal dorsal

Formalin test

horn, and these effects could be attenuated by intrathecal injection of the competitive inhib-

Spinal cord

itor of amino acid system A transporter, methylaminoisobutyric acid (MeAIB, 0.1, 0.3, 0.5, and 0.7 mmol), in a dose-dependent manner. Intrathecal injection of vehicle (PBS) had no effect on the formalin-induced nociceptive behaviors and increase of the GFAP. These findings suggest that amino acid transport system A is involved in inflammation-induced nociception, and inhibition of this transporter system results in inhibition of the central sensitization and hyperalgesia. © 2012 Elsevier B.V. All rights reserved.

1.

Introduction

Previous studies have shown that peripheral injury or inflammation activates both neuronal and non-neuronal (glial) components of the peripheral and central cellular circuitry and that the neuron–glia interactions contribute to pain hypersensitivity in the development of central sensitization

and hyperalgesia (Scholz and Woolf, 2007; Watkins et al., 2007). An important function of astrocytes is mediating the glutamate–glutamine cycle, which is involved in the plasticity underlying the generation of central sensitization, hyperalgesia and chronic pain (Hertz et al., 1999; Muscoli et al., 2010; Tsuboi et al., 2011). The glutamate–glutamine cycle converts glutamate to glutamine by the enzyme glutamine synthetase

⁎ Corresponding author at: Institute of Urology, Second Hospital of Lanzhou University, Lanzhou University, Lanzhou, Gansu 730030, PR China. Fax: +86 931 8942579. E-mail address: [email protected] (Z.-P. Wang). 0006-8993/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2012.02.018

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after glutamate uptake from the synaptic cleft, and the glutamine is extruded from astrocytes via the system N transporter, and is subsequently taken up by neuronal presynaptic terminals through a system A transporter where it is then converted to glutamate and repackaged into synaptic vesicles for release (Chaudhry et al., 1999, 2002). A recent study has demonstrated that the hyperexcitability of trigeminal nociceptive neurons is attenuated by application of methionine sulfoximine (MSO), an inhibitor of astroglial glutamine synthetase that catalyzes the conversion of glutamate to glutamine (Chiang et al., 2007). Importantly, in the glutamate–glutamine cycle, amino acid transport system A is responsible for the accumulation of glutamine by neurons (Armano et al., 2002; Varoqui et al., 2000) and is characterized by its ability to bind and transport N-methylated amino acids (Rae et al., 2003), and therefore may play a critical role in the development of central sensitization. The nonmetabolized amino acid analog methylaminoisobutyric acid (MeAIB) has thus been used in many studies to identify the system transporter A activity and, indeed, appears to be a specific inhibitor of all isoforms of this transporter (Broer and Brookes, 2001). Chiang et al. (2008) have demonstrated that continuous intrathecal superfusion of MeAIB can suppress mustard oil-induced sensitization of trigeminal caudalis nociceptive neurons, but comparable studies in behavioral models of persistent inflammatory pain are not available. Formalin injected into rat hindpaw induced persistent inflammatory nociceptive behaviors believed to be a result of central sensitization (Bittencourt and Takahashi, 1997; Chen and Koyama, 1998; Malmberg and Yaksh, 1992; Qin et al., 2006; Sweitzer et al., 1999). Therefore, the present study was designed to determine whether intrathecal administration of MeAIB reduces the central sensitization induced in the formalin test, and whether such effects are mediated through astroglia activation.

2.

Results

2.1. Effect of intrathecal MeAIB on formalin-induced nociceptive behavior As reported previously (Abbott et al., 1995; Xie et al., 2004), intraplantar injection of 5% formalin induced typical biphasic nociceptive behaviors (flinching and licking/biting of the injected paw). The early phase (phase I) began immediately after injection and lasted 5 min. After a short quiescent period (15 min), a prolonged tonic response ensued, persisting for over 45 min (phase II). The peak response appeared approximately 30 min after injection. Intrathecal administration of the vehicle (PBS, 10 μl) did not alter the formalin-induced nociceptive behaviors; the duration of licking/biting and the number of flinches were not significantly different from animals that received the formalin injection alone (P > 0.05, n = 6), either in phase I or phase II (Figs. 1c and d). However, intrathecal administration of MeAIB (0.1, 0.3, 0.5 and 0.7 mmol; 10 μl for each dose; n = 6), an inhibitor of the amino acid system A transporter, 10 min prior to the formalin injection significantly depressed the formalin-induced nociceptive behaviors, in a dose-dependent manner (r = 0.992, P < 0.001 for the licking/biting response; r = 0.942, P = 0.017 for

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the flinching response), during the 60-min observation period with an ED50 of 0.58 mmol (Figs. 1a and b). As shown in Figs. 1c and d, the time course curves for the two different treatments (i.e., for the control (PBS) and the different MeAIB dose groups) were significantly different from each other (F(4, 275) = 10.847, P < 0.001 for duration of licking/biting; F(4, 275) = 11.333, P < 0.001 for number of flinches), across time (F(11, 275) = 56.438, P < 0.001; F(11, 275) = 42.822, P < 0.001) and in their interactions (F(44, 275) = 3.223, P < 0.001; F(44, 275) = 3.500, P < 0.001). Further analyses revealed that a decrease in licking/biting occurred during phase II (P < 0.05), but not during phase I (P > 0.05, Figs. 1c and e), while a decrease in flinching occurred in both phases I and II (P < 0.05, Figs. 1d and f). Detailed comparisons of the individual time points for the different treatments are shown in Figs. 1c and d. In addition, we found that when the dose of MeAIB was 1.0 mmol or higher, the nociceptive behaviors were completely inhibited; and these higher doses were occasionally associated with the appearance of lethargy or convulsions in some rats (data not shown).

2.2. Effect of intrathecal MeAIB on formalin-induced spinal GFAP expression After completion of the behavioral experiments (i.e., 1 h after formalin injection), the expression of GFAP, a marker of activated astrocytes, in the spinal dorsal horn, L4–5, was examined in animals from the various groups. As shown in Fig. 2, GFAP-immunoreactivity was very low in normal control rats, and the astrocytes were evenly distributed in all laminae of the bilateral spinal dorsal horns and possessed small, thin processes (Figs. 2a and a′). However, injection of formalin into the right hindpaw pad significantly increased GFAP expression in the ipsilateral, but not in the contralateral, spinal dorsal horn. GFAP immunoreactivity significantly increased from laminae I to VI, particularly between laminae I and II, and the astrocytes had a significantly hypertrophied morphology, with elongated processes (Figs. 2b and b′). Intrathecal administration of the vehicle (PBS) did not significantly affect the formalin-induced increase in GFAP expression (Fig. 2c). As shown in Fig. 3b, the integrated optical density (IOD) values for the formalin alone and PBS + formalin groups increased to 282.41 and 292.09% of the normal control group value, respectively (H = 12.098, P = 0.002, n = 6 for each group, one-way ANOVA on ranks test). However, intrathecal administration of MeAIB (0.1, 0.3, 0.5 and 0.7 mmol, in 10 μl for each dose, n = 6) 10 min prior to intraplantar formalin injection attenuated the formalininduced increase in GFAP expression, as indicated by the IOD values, in a dose-dependent manner (r = 0.970, P = 0.006, ED50 = 0.54 mmol; Fig. 3a). Astrocytes in the spinal dorsal horn displayed a less hypertrophied morphology and had relatively shorter processes. One-way ANOVA indicated that the GFAP immunoreactivity, assessed using IOD value, was significantly different between the various treatment groups (F(4, 25) = 78.358, P < 0.001). Post hoc comparisons revealed that the 0.3, 0.5 and 0.7 mmol MeAIB groups exhibited significantly lower GFAP immunoreactivity than the PBS + formalin group (P < 0.01 and P < 0.001), but the 0.1 mmol MeAIB group was not significantly different from the PBS + formalin group (P > 0.05), as shown in Fig. 3b.

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Fig. 1 – Inhibitory effects of intrathecal injection of MeAIB on formalin-induced nociceptive behaviors. (a, b) Dose–response curves showing that intrathecal injection of different doses of MeAIB depresses formalin-induced licking/biting (a) and flinching (b) behaviors during the 60-min observation period, in a dose-dependent manner, with an ED50 of 0.58 mmol. (c, d) Time-course plots showing the effects of intrathecal injection of different doses of MeAIB on formalin-evoked licking/ biting (c) and flinching (d) behaviors. (e, f) Bar graphs showing mean duration of licking/biting (e) and number of flinches (f) during phase I and phase II, respectively. *P < 0.05, compared with the PBS + formalin group; #P < 0.05, compared with the 0.1 mmol MeAIB + formalin group; △P < 0.05, △△P < 0.01 and △△△P < 0.001, compared with the 0.3 mmol MeAIB + formalin group (n = 6 for each group) (two-way RM ANOVA followed by a Bonferroni t-test). Abbreviation: F, formalin.

3.

Discussion

Glutamate is an important neurotransmitter, responsible for spinal excitatory synaptic transmission and the generation and maintenance of central hypersensitivity via activation of glutamate receptors (Basbaum and Woolf, 1999; Tao et al., 2005). The glutamate–glutamine shuttle is a key function of astrocytes (Bacci et al., 2002; Waagepetersen et al., 2005; Yang and Shen, 2005), and it plays an important role in central sensitization (Sessle, 2000). This shuttle comprises glutamate release from neurons into the synaptic cleft, uptake by

astrocytes where glutamate is converted into glutamine via a series of enzymatic reactions, glutamine release from astrocytes, and its uptake by neurons for the synthesis of glutamate (Broer et al., 2004; Fonseca et al., 2005; Keyser and Pellmar, 1997; Rae et al., 2003). Amino acid transport system A, an important component of the glutamate–glutamine cycle, is responsible for the accumulation of glutamine by neurons (Armano et al., 2002; Varoqui et al., 2000), and is characterized by its ability to bind and transport N-methylated amino acids (Rae et al., 2003). Our present results indicate that intrathecal injection of MeAIB, for inhibiting the activity of amino acid transport system A, significantly depresses

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Fig. 2 – Formalin-induced GFAP expression in the dorsal horn. The panels are representative photomicrograph of the L4–5 dorsal horns in the different treatment groups: (a) ipsilateral dorsal horn in the normal group, (a′) contralateral dorsal horn in the normal group, (b) ipsilateral dorsal horn in the formalin alone group, (b′) contralateral dorsal horn in the formalin alone group, (c) PBS + formalin group, (d) 0.1 mmol MeAIB + formalin group, (e) 0.3 mmol MeAIB + formalin group, (f) 0.5 mmol MeAIB + formalin group, and (g) 0.7 mmol MeAIB + formalin group. Only the ipsilateral dorsal horn is shown for the PBS and different MeAIB dose groups. Magnification: 200× (the graph in the lower right corner is 400×), scale bar = 50 μm for all dorsal horn micrographs, and bar = 20 μm for those in the lower right corner.

Fig. 3 – GFAP-positive astrocytes in the ipsilateral dorsal horn (I–VI). Intrathecal administration of 0.1, 0.3, 0.5 and 0.7 mmol MeAIB decreased GFAP expression in all laminae of the L4–5 dorsal horns, especially laminae I–II. (a) Dose–response curve showing the percent (%) reductions in GFAP immunoreactivity, indicated by IOD values, in the different MeAIB dose groups; “0” dose represents the PBS group. (b) Bar graph showing the percent (%) changes in GFAP immunoreactivity IOD values in the different treatment groups (normal control group set at 100%). ##P < 0.01, compared with the normal control group (Kruskal–Wallis ANOVA on ranks test); **P < 0.01 and ***P < 0.001, compared with the PBS + formalin groups (n = 6 for each group, one-way ANOVA).

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formalin-induced nociceptive behaviors in a dose-dependent manner, suggesting that this transport system may play a key role in astrocyte glutamate–glutamine shuttle-mediated central sensitization in the spinal cord caused by persistent inflammatory stimulation. This result provides the first behavioral support for electrophysiological studies showing that continuous intrathecal superfusion of MeAIB suppresses mustard oil-induced sensitization of trigeminal caudalis nociceptive neurons (Chiang et al., 2008). In the present study, formalin injection into the rat hindpaw pad significantly increased GFAP expression in the lumbar spinal dorsal horn 1 h after formalin injection. Because GFAP is a marker of astrocyte activation, its increased expression suggests that astroglia are hyperactivated during persistent inflammatory pain state. Our result is consistent with the work of Qin et al. (2006). These investigators found that GFAP-immunoreactive astrocytes were expressed in the period between 15 and 120 min after subcutaneous formalin injection, which is earlier than microglial activation in the spinal dorsal horn. However, many studies have shown that upregulation of astroglial markers (GFAP), examined using immunostaining and reverse transcription-PCR assays, was at the subacute (4 h) and chronic (4–14 d) phases of inflammation induced by intra-plantar or intra-articular injection of complete Freund's adjuvant (Raghavendra et al., 2004; Sun et al., 2007, 2008), and after trigeminal sensory nerve injury (Piao et al., 2006), which are later than microglial activation. The discrepancy in astrocytic and microglial expression may be related to the use of different animal models. Taken together with the results of previous studies, our present observations suggest that astroglial hyperactivation plays an important role in the development and maintenance of central sensitization and hyperalgesia under persistent inflammatory and neuropathic states. The early phase (phase I) of formalin-induced biphasic nociceptive behavior is believed to be a result of direct activation of peripheral nociceptors, whereas the later phase (phase II) is mediated by a combination of low-level ongoing activity in the primary afferents and increased sensitivity of spinal cord neurons (Bittencourt and Takahashi, 1997; Malmberg and Yaksh, 1992). There is evidence that during formalininduced persistent inflammation, the threshold of response to mechanical and thermal stimuli is reduced in the injected paw, and this is associated with spinal astrocyte and microglial activation (Qin et al., 2006; Sweitzer et al., 1999). In addition, the neuronal response to peripheral noxious stimuli is significantly enhanced in the spinal dorsal horn ipsilateral to the injection paw (Chen and Koyama, 1998), indicating that hyperalgesia and central sensitization have occurred. Therefore, although in the present study, GFAP expression and the influence of MeAIB on GFAP expression were observed only 1 h after formalin injection, it is easy to understand the inhibitory effect of intrathecal administration of MeAIB on formalin-induced phase II nociceptive behavior, based on the fact that GFAP expression occurred 15–120 min after formalin injection (Qin et al., 2006). However, in our present study, phase I nociceptive flinching (but not licking/ biting) behavior was also attenuated by intrathecal injection of MeAIB. Because there is no evidence that astrocytes are activated in the phase I nociceptive behavior period, it is not

possible to exclude the possibility that the inhibitory effect of MeAIB on phase I flinching behavior is unrelated to the activity of the amino acid transport system A. This issue requires further research for clarification. In this study, intrathecal administration of MeAIB dosedependently attenuated the formalin-induced increase in astroglial activation in the lumbar spinal dorsal horn. This was correlated with a depression of formalin-induced chronic nociceptive behavior, as well as a diminishment of mustard oil-induced hyperexcitability of nociceptive neurons in the medullar dorsal horn (Chiang et al., 2008), and suggests that the amino acid transport system A plays an important role in the development and maintenance of central sensitization and hyperalgesia. Blockade of this transport system results in lack of neuronal glutamate, which in turn inhibits central sensitization and produces analgesia (Chiang et al., 2008). The reduction in GFAP expression in astrocytes in the spinal dorsal horn may be due to MeAIB interrupting the transport of glutamine from astrocytes to neurons, resulting in the accumulation of glutamine in astrocytes and lack of glutamate in neurons. The increased level of glutamine may be toxic to astrocytes, or decreased neuronal activity may inhibit astrocyte activation. The observed correlations and the underlying mechanisms require further investigation. In conclusion, our findings suggest that amino acid transport system A is involved in mediating persistent inflammationinduced nociception (i.e., central sensitization). Inhibition of this transport system results in attenuation of astrocyte activation in the spinal dorsal horn and has an antinociceptive effect.

4.

Experimental procedures

4.1.

Animals

All experiments were performed on healthy adult male Sprague–Dawley rats (200–250 g) provided by the Experimental Animal Center of Gansu College of Traditional Chinese Medicine, China. Rats were housed in individual cages at room temperature (22 °C) on a 12-h light/dark cycle (6 am/6 pm), and were given food and water ad libitum. The experimental protocol was in accordance with the guidelines of the International Association for the Study of Pain (Zimmermann, 1983) and was approved by the Animal Care and Ethics Committee of the University of Lanzhou. All efforts were made to minimize the number of animals used and their suffering. 4.2.

Chemicals and administration

The main chemicals used in this study included MeAIB, GFAP and phosphate-buffered saline (PBS, pH 7.4, 0.01 mol/l), all from Sigma-Aldrich; and formalin (5%) and halothane from Lanzhou Chemical Agent Company, China. MeAIB or PBS was injected intrathecally by direct lumbar puncture at the L5–6 spinal levels, as previously described (Gao et al., 2009; Hylden and Wilcox, 1980; Mestre et al., 1994). Briefly, rats were lightly anesthetized with halothane, intrathecal injection was made with a 30-gauge needle, and 10 μl of drug or vehicle was administered within 10 s. Puncture of the dura was indicated by a reflexive flick or formation of an “S” by

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the tail. After the intrathecal injection, rats recovered from anesthesia within 1–2 min. 4.3.

Behavioral tests

On the day of testing, each rat was moved to the laboratory and placed in a hexagonal polycarbonate chamber (16 cm × 10 cm × 8 cm), with a mirror at a 45° angle for unobstructed observation of the rats' paws, for about 15–20 min to acclimate them to the experimental environment. The criterion for adequate acclimation was that rats did not freeze or defecate in the test chamber and rarely exhibited spontaneous activity (Abbott et al., 1995). Then, the rat was removed from this chamber and 5% formalin (50 μl) was administered subcutaneously (s.c.) into the right hindpaw pad. The rat was immediately replaced in the chamber. Spontaneous nociceptive behaviors were assessed by measuring the duration of licking/biting and the number of flinches every 5 min in the injected hindpaw over a period of 1 h after formalin injection by two experimenters, as reported previously (Noble et al., 1995; Przewlocka et al., 1999; Xie et al., 2004). For behavioral observations, rats were randomly divided into three groups: (1) Formalin alone—rats with intraplantar injection of formalin only (n = 6); (2) PBS—rats with intrathecal injection of PBS (10 μl) and intraplantar injection of formalin (n = 6); and (3) Treatment—rats with intrathecal injection of MeAIB (0.1, 0.3, 0.5 or 0.7 mmol; freshly dissolved in PBS at pH 7.4; n = 6 for each dose) 10 min before intraplantar injection of formalin. 4.4.

Astrocyte activation in the spinal dorsal horn

4.4.1. Immunohistochemical staining After the completion of behavioral testing (i.e., 1 h after formalin injection), all rats were sacrificed for GFAP staining using immunohistochemistry. Rats were deeply anesthetized with intraperitoneal pentobarbital sodium (i.p. 100 mg/kg) and perfused intracardially with 200 ml 0.01 M PBS (pH 7.4) followed by 500 ml of ice-cold fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The spinal cord, L4–5, was removed, post-fixed for 2 h, and cryoprotected in 30% sucrose until the tissue sank to the bottom of the container. Fifteen micron sections were cut on a freezing microtome (CM1950; Leitz, Germany). Serial sections were collected into three dishes containing 0.01 M PBS. Every third section was collected for each of the three complete sets. All sections were washed carefully with 0.01 M PBS. The sections in the first dish were used for immunohistochemical staining of GFAP using the avidin–biotin-peroxidase (ABC) method (Hsu et al., 1981). Briefly, the sections were washed in 0.01 M PBS (pH 7.4), and incubated sequentially with (1) mouse antiserum against GFAP (G3893, 1:800 dilution; Sigma-Aldrich, USA) in 0.01 M PBS containing 5% (v/v) normal goat serum (NGS), 0.3% (v/v) Triton X-100, 0.05% (w/v) NaN3, and 0.25% (w/v) carrageenan (PBS-NGS, pH 7.4) for 48–72 h at 4 °C; (2) biotinylated goat anti-mouse IgG (1:200 dilution; Vector, Burlingame, CA) in PBS-NGS overnight at 4 °C; and (3) ABC Elite complex (1:100; Vector) in 0.01 M PBS (pH 7.4) containing 0.3% (v/v) Triton X-100 (PBS-X) for 2 h at room temperature. Bound peroxidase was visualized by incubation

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with 0.05% 3,3-diaminobenzidine tetrahydrochloride (DAB; Dojin, Kumamoto, Japan) and 0.003% H2O2 in 0.05 M Tris– HCl buffer (pH 7.6) for 20–30 min. The sections were rinsed at least 3 times in 0.01 M PBS (pH 7.4) for at least 10 min after each incubation. The sections were then mounted onto gelatin-coated glass slides, air dried, dehydrated and cleaned, coverslipped with DPX, and observed under the light microscope (BX-60; Olympus, Tokyo, Japan). The microphotographs were taken with a digital camera (DP-70; Olympus) attached to a microscope. The sections in the second dish were mounted onto gelatin-coated glass slides and processed for Nissl staining. The sections in the third dish were used for control tests. In the control experiments, the primary antibodies were omitted or replaced with normal guinea pig serum; no positive staining for the omitted or replaced antibodies was detected. Six normal rats were used for baseline staining for quantitative analysis. The sections from formalin alone and PBS controls were always processed together for comparison under the same conditions. 4.4.2. Evaluation of astrocyte morphological changes Every third section was labeled with GFAP and observed under low and high magnification to evaluate astrocyte morphological changes. We used the semi-quantitative assessment of activation, as described previously (Colburn et al., 1997; Lin et al., 2007; Wu et al., 2004). Briefly, these criteria are as follows: (1) Baseline staining—astrocytes are extensively ramified with fine processes that are evenly distributed throughout the dorsal horn (Figs. 2a, a′, b′, g); (2) Mild increase— astrocytes are ramified and evenly spaced with a slight increase in the number or intensity of cells, both medially and superficially in the dorsal horn (Fig. 2f); (3) Moderate and localized—astrocytes have lengthened and clumpy processes and more are found densely packed in the medial dorsal horn. Astrocyte activation spreads laterally and dorsoventrally, and leaves little or no space between cells (Fig. 2e); (4) Maximal staining—dark brown-stained dots in the dorsal horn can be seen without a microscope. Under the microscope, astrocytes exhibit extremely swollen bodies and thick processes with extensive overlap. Astrocyte activation is spread into the deep dorsal horn, extending to the central canal (Figs. 2b, c, d). 4.4.3. Quantitative evaluation of astrocyte GFAP immunoreactivity Because the morphology of astrocytes is complex and immunoreactive staining includes both cell bodies and their processes, cell counts may not appropriately represent activation. To quantitatively evaluate GFAP immunoreactivity, as described previously by Fu et al. (1999), the IOD values of GFAP immunoreactivities (GFAP-IR) within the spinal dorsal horn, including laminae I–VI of L4–5, were automatically measured with a computer-based image analysis system (image pro-plus (IPP) version 6.0 software) at six successive sections for each animal. The density of GFAP staining in images from normal rats was designated the baseline value and was used to determine the relative densities in the different treatment groups. All data were measured by an observer blind to the treatment.

44 4.5.

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Statistical analysis

Data are presented as mean ± S.E.M. Two-way repeated measures of analysis of variance (two-way RM ANOVA) followed by a post hoc multiple comparison (Bonferroni t-test) was used to analyze differences in formalin-induced nociceptive behavior among the different treatment groups. One-way analysis of variance (one-way ANOVA) followed by a post hoc multiple comparison (Bonferroni t-test) was used to analyze the difference in IOD values of GFAP-IR between different treatment groups; if the normality test failed, a Kruskal– Wallis ANOVA by rank test was used. Percent reduction of the nociceptive behavior and the IOD value of GFAP immunoreactivity in the different MeAIB treatment groups was calculated using the formula [(control value − test value) / control value × 100%], and a linear regression was performed to analyze the correlation between the MeAIB dose and effect, as well as to determine the ED50. Level of significance was set at P < 0.05. Sigmastat software v.3.5 was used for all analyses.

Acknowledgments The authors wish to thank Professor J.S. Tang in Xi'an Jiaotong University School of Medicine for his expert help in preparing the manuscript. The study was supported by the National Natural Science Foundation of China (No. 30800333), the Fundamental Research Funds for the Central Universities (No. lzujbky-2010-148) and the Research Funds for the Second Hospital of Lanzhou University (No. YJ-2010-38). There is no conflict of interest in the studies reported in the paper.

REFERENCES

Abbott, F.V., Franklin, K.B., Westbrook, R.F., 1995. The formalin test: scoring properties of the first and second phases of the pain response in rats. Pain 60, 91–102. Armano, S., Coco, S., Bacci, A., Pravettoni, E., Schenk, U., Verderio, C., Varoqui, H., Erickson, J.D., Matteoli, M., 2002. Localization and functional relevance of system a neutral amino acid transporters in cultured hippocampal neurons. J. Biol. Chem. 277, 10467–10473. Bacci, A., Sancini, G., Verderio, C., Armano, S., Pravettoni, E., Fesce, R., Franceschetti, S., Matteoli, M., 2002. Block of glutamate–glutamine cycle between astrocytes and neurons inhibits epileptiform activity in hippocampus. J. Neurophysiol. 88, 2302–2310. Basbaum, A.I., Woolf, C.J., 1999. Pain. Curr. Biol. 9, R429–R431. Bittencourt, A.L., Takahashi, R.N., 1997. Mazindol and lidocaine are antinociceptives in the mouse formalin model: involvement of dopamine receptor. Eur. J. Pharmacol. 330, 109–113. Broer, S., Brookes, N., 2001. Transfer of glutamine between astrocytes and neurons. J. Neurochem. 77, 705–719. Broer, A., Deitmer, J.W., Broer, S., 2004. Astroglial glutamine transport by system N is upregulated by glutamate. Glia 48, 298–310. Chaudhry, F.A., Reimer, R.J., Krizaj, D., Barber, D., Storm-Mathisen, J., Copenhagen, D.R., Edwards, R.H., 1999. Molecular analysis of system N suggests novel physiological roles in nitrogen metabolism and synaptic transmission. Cell 99, 769–780. Chaudhry, F.A., Schmitz, D., Reimer, R.J., Larsson, P., Gray, A.T., Nicoll, R., Kavanaugh, M., Edwards, R.H., 2002. Glutamine

uptake by neurons: interaction of protons with system a transporters. J. Neurosci. 22, 62–72. Chen, J., Koyama, N., 1998. Differential activation of spinal dorsal horn units by subcutaneous formalin injection in the cat: an electrophysiological study. Exp. Brain Res. 118, 14–18. Chiang, C.Y., Wang, J., Xie, Y.F., Zhang, S., Hu, J.W., Dostrovsky, J.O., Sessle, B.J., 2007. Astroglial glutamate–glutamine shuttle is involved in central sensitization of nociceptive neurons in rat medullary dorsal horn. J. Neurosci. 27, 9068–9076. Chiang, C.Y., Li, Z., Dostrovsky, J.O., Hu, J.W., Sessle, B.J., 2008. Glutamine uptake contributes to central sensitization in the medullary dorsal horn. Neuroreport 19, 1151–1154. 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. Fonseca, L.L., Monteiro, M.A., Alves, P.M., Carrondo, M.J., Santos, H., 2005. Cultures of rat astrocytes challenged with a steady supply of glutamate: new model to study flux distribution in the glutamate–glutamine cycle. Glia 51, 286–296. 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. Gao, Y.J., Zhang, L., Samad, O.A., Suter, M.R., Yasuhiko, K., Xu, Z.Z., Park, J.Y., Lind, A.L., Ma, Q., Ji, R.R., 2009. JNK-induced MCP-1 production in spinal cord astrocytes contributes to central sensitization and neuropathic pain. J. Neurosci. 29, 4096–4108. Hertz, L., Dringen, R., Schousboe, A., Robinson, S.R., 1999. Astrocytes: glutamate producers for neurons. J. Neurosci. Res. 57, 417–428. Hsu, S.M., Raine, L., Fanger, H., 1981. Use of avidin–biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577–580. Hylden, J.L., Wilcox, G.L., 1980. Intrathecal morphine in mice: a new technique. Eur. J. Pharmacol. 67, 313–316. Keyser, D.O., Pellmar, T.C., 1997. Regional differences in glial cell modulation of synaptic transmission. Hippocampus 7, 73–77. Lin, T., Li, K., Zhang, F.Y., Zhang, Z.K., Light, A.R., Fu, K.Y., 2007. Dissociation of spinal microglia morphological activation and peripheral inflammation in inflammatory pain models. J. Neuroimmunol. 192, 40–48. Malmberg, A.B., Yaksh, T.L., 1992. Antinociceptive actions of spinal nonsteroidal anti-inflammatory agents on the formalin test in the rat. J. Pharmacol. Exp. Ther. 263, 136–146. 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. Muscoli, C., Doyle, T., Dagostino, C., Bryant, L., Chen, Z., Watkins, L.R., Ryerse, J., Bieberich, E., Neumman, W., Salvemini, D., 2010. Counter-regulation of opioid analgesia by glial-derived bioactive sphingolipids. J. Neurosci. 30, 15400–15408. Noble, F., Blommaert, A., Fournie-Zaluski, M.C., Roques, B.P., 1995. A selective CCKB receptor antagonist potentiates, mu-, but not delta-opioid receptor-mediated antinociception in the formalin test. Eur. J. Pharmacol. 273, 145–151. Piao, Z.G., Cho, I.H., Park, C.K., Hong, J.P., Choi, S.Y., Lee, S.J., Lee, S., Park, K., Kim, J.S., Oh, S.B., 2006. Activation of glia and microglial p38 MAPK in medullary dorsal horn contributes to tactile hypersensitivity following trigeminal sensory nerve injury. Pain 121, 219–231. Przewlocka, B., Mika, J., Labuz, D., Toth, G., Przewlocki, R., 1999. Spinal analgesic action of endomorphins in acute, inflammatory and neuropathic pain in rats. Eur. J. Pharmacol. 367, 189–196. 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.

BR A I N R ES E A RCH 1 4 49 ( 20 1 2 ) 3 8 –45

Rae, C., Hare, N., Bubb, W.A., McEwan, S.R., Broer, A., McQuillan, J.A., Balcar, V.J., Conigrave, A.D., Broer, S., 2003. Inhibition of glutamine transport depletes glutamate and GABA neurotransmitter pools: further evidence for metabolic compartmentation. J. Neurochem. 85, 503–514. Raghavendra, V., Tanga, F.Y., DeLeo, J.A., 2004. Complete Freunds adjuvant-induced peripheral inflammation evokes glial activation and proinflammatory cytokine expression in the CNS. Eur. J. Neurosci. 20, 467–473. Scholz, J., Woolf, C.J., 2007. The neuropathic pain triad: neurons, immune cells and glia. Nat. Neurosci. 10, 1361–1368. Sessle, B.J., 2000. Acute and chronic craniofacial pain: brainstem mechanisms of nociceptive transmission and neuroplasticity, and their clinical correlates. Crit. Rev. Oral Biol. Med. 11, 57–91. Sun, S., Cao, H., Han, M., Li, T.T., Pan, H.L., Zhao, Z.Q., Zhang, Y.Q., 2007. New evidence for the involvement of spinal fractalkine receptor in pain facilitation and spinal glial activation in rat model of monoarthritis. Pain 129, 64–75. Sun, S., Cao, H., Han, M., Li, T.T., Zhao, Z.Q., Zhang, Y.Q., 2008. Evidence for suppression of electroacupuncture on spinal glial activation and behavioral hypersensitivity in a rat model of monoarthritis. Brain Res. Bull. 75, 83–93. Sweitzer, S.M., Colburn, R.W., Rutkowski, M., DeLeo, J.A., 1999. Acute peripheral inflammation induces moderate glial activation and spinal IL-1 beta expression that correlates with pain behavior in the rat. Brain Res. 829, 209–221. Tao, Y.X., Gu, J., Stephens Jr., R.L., 2005. Role of spinal cord glutamate transporter during normal sensory transmission and pathological pain states. Mol. Pain 1, 30.

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Tsuboi, Y., Iwata, K., Dostrovsky, J.O., Chiang, C.Y., Sessle, B.J., Hu, J.W., 2011. Modulation of astroglial glutamine synthetase activity affects nociceptive behaviour and central sensitization of medullary dorsal horn nociceptive neurons in a rat model of chronic pulpitis. Eur. J. Neurosci. 34, 292–302. Varoqui, H., Zhu, H., Yao, D., Ming, H., Erickson, J.D., 2000. Cloning and functional identification of a neuronal glutamine transporter. J. Biol. Chem. 275, 4049–4054. Waagepetersen, H.S., Qu, H., Sonnewald, U., Shimamoto, K., Schousboe, A., 2005. Role of glutamine and neuronal glutamate uptake in glutamate homeostasis and synthesis during vesicular release in cultured glutamatergic neurons. Neurochem. Int. 47, 92–102. Watkins, L.R., Hutchinson, M.R., Milligan, E.D., Maier, S.F., 2007. “Listening” and “talking” to neurons: implications of immune activation for pain control and increasing the efficacy of opioids. Brain Res. Rev. 56, 148–169. Wu, Y., Willcockson, H.H., Maixner, W., Light, A.R., 2004. Suramin inhibits spinal cord microglia activation and long-term hyperalgesia induced by formalin injection. J. Pain 5, 48–55. Xie, Y.F., Wang, J., Huo, F.Q., Jia, H., Tang, J.S., 2004. Mu but not delta and kappa opioid receptor involvement in ventrolateral orbital cortex opioid-evoked antinociception in formalin test rats. Neuroscience 126, 717–726. Yang, J., Shen, J., 2005. In vivo evidence for reduced cortical glutamate–glutamine cycling in rats treated with the antidepressant/antipanic drug phenelzine. Neuroscience 135, 927–937. Zimmermann, M., 1983. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16, 109–110.