Blockade of microglial activation reduces mechanical allodynia in rats with compression of the trigeminal ganglion

Progress in Neuro-Psychopharmacology & Biological Psychiatry 36 (2012) 52–59

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Blockade of microglial activation reduces mechanical allodynia in rats with compression of the trigeminal ganglion Seung R. Han a, 1, Gwi Y. Yang a, 1, Myung H. Ahn a, Min J. Kim a, Jin S. Ju a, Yong C. Bae b, Dong K. Ahn a,⁎ a b

Department of Oral Physiology, School of Dentistry, Kyungpook National University, Daegu (700-412), Republic of Korea Department of Oral Anatomy, School of Dentistry, Kyungpook National University, Daegu (700-412), Republic of Korea

a r t i c l e

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Article history: Received 12 June 2011 Received in revised form 7 October 2011 Accepted 7 October 2011 Available online 13 October 2011 Keywords: Antinociception Microglia p38 MAPK Trigeminal ganglion Trigeminal neuralgia

a b s t r a c t The present study investigated the role of microglia and p38 MAPK in the development of mechanical allodynia in rats with compression of the trigeminal ganglion. Male Sprague–Dawley rats weighing 250–260 g were used. Under pentobarbital sodium anesthesia, the animals were mounted onto a stereotaxic frame and given injections of 4% agar solution (10 μL) to compress the trigeminal ganglion. The air-puff thresholds significantly decreased after compression of the trigeminal ganglion. On postoperative day 14, immunoreactivity to both OX-42 and p-p38 MAPK was up-regulated in the medullary dorsal horn as compared to the sham group. P-p38 MAPK was found to be co-localized with OX-42, but not with NeuN, a neuronal cell marker, or with GFAP, an astroglial cell marker. Intracisternal administration of 100 μg of minocycline significantly inhibited both mechanical allodynia and activation of microglia produced by compression of the trigeminal ganglion. Intracisternal administration of 0.1, 1, or 10 μg of SB203580, a p38 MAPK inhibitor, also significantly decreased mechanical allodynia and p38 MAPK activation in the trigeminal ganglion-compressed group. These results suggest that activation of p38 MAPK in the microglia is an important step in the development of mechanical allodynia in rats with compression of the trigeminal ganglion and that the targeted blockade of microglial p38 MAPK pathway is a potentially important new treatment strategy for trigeminal neuralgia-like nociception. © 2011 Elsevier Inc. All rights reserved.

1. Introduction The mitogen-activated protein kinases (MAPKs) are a family of signaling molecules able to transduce extracellular stimuli into intracellular responses in a wide variety of contexts. Recently, p38 MAPK activation in the central nervous system, induced by a plantar incision (Wen et al., 2009), spinal nerve transection (Ito et al., 2009), spinal nerve ligation (Jin et al., 2003; Tsuda et al., 2004), trigeminal sensory nerve injury (Lim et al., 2007; Piao et al., 2006), or carrageenan paw injection (Hua et al., 2005), has been found to contribute to pain hypersensitivity. These results suggested the fundamental involvement of p38 MAPK pathways in central nociceptive processing. P-p38 MAPK expression in the central nervous system, both spinal neurons and neuroglia, could be induced by nociceptive stimuli and inflammatory mediators, following tissue and nerve damage (Ji,

Abbreviations: MAPK, Mitogen-Activated Protein Kinase; DRG, dorsal root ganglion. ⁎ Corresponding author at: Department of Oral Physiology, School of Dentistry, Kyungpook National University, 188-1 Sam Deok 2ga, Chung-gu, Daegu (700-412), Republic of Korea. Tel.: + 82 53 660 6840; fax: + 82 53 421 4077. E-mail address: [email protected] (D.K. Ahn). 1 These authors contributed equally to this study as a first author. 0278-5846/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2011.10.007

2004). Indeed, continuous intrathecal pre- and post-treatment with SB203580, a p38 MAPK inhibitor, decreased spinal nerve ligationinduced mechanical allodynia (Jin et al., 2003; Tsuda et al., 2004). In the orofacial area, intracisternal treatments with SB203580 have been shown to significantly inhibit mechanical allodynia in rats with chronic constriction of the infraorbital nerve (Lim et al., 2007). These results suggest that activation of central p38 MAPK also plays important roles in the development of neuropathic pain, including the orofacial area. Chronic compression of the trigeminal ganglion produced prolonged nociceptive behavior in rats (Ahn et al., 2009b), similar to symptoms of patients with trigeminal neuralgia. Although compression of the trigeminal ganglion produced nociceptive behavior, participation of microglia and p38 MAPK in the development of trigeminal neuropathic pain produced by compression of the trigeminal ganglion has not been well known. Our hypothesis was that the compression of the trigeminal ganglion activates microglia and p38 MAPK in the medullary dorsal horn and that blockade of activation of microglia and p38 MAPK attenuates mechanical allodynia. Thus, the present study investigated up-regulation of OX-42, a microglial cell marker, and p38 MAPK in the medullary dorsal horn following compression of the trigeminal ganglion. We also evaluated the effects exerted on mechanical allodynia by minocycline, a selective inhibitor of microglial cell activation, and SB203580, a p38 MAPK inhibitor, when these agents had been intracisternally injected.

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2. Materials and methods

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for the air-puffs was 40 psi. Naïve rats did not respond to a pressure of less than 40 psi.

2.1. Animals 2.4. Intracisternal administration of minocycline and SB203580 Experiments were carried out on male Sprague–Dawley rats weighing between 250 and 260 g. The rats were housed at a density of one animal per cage (26 × 50 × 18 cm) and maintained under constant temperature and lighting conditions with a 12 h light/dark cycle. Food and water were freely available. All procedures involving the use of animals were approved by the Institutional Animal Care and Use Committee of the School of Dentistry, Kyungpook National University, and were carried out in accordance with the ethical guidelines for investigation of experimental pain in conscious animals, issued by the International Association for the Study of Pain. All behavioral responses were measured in a blind fashion. 2.2. Compression of the trigeminal ganglion Compression of the trigeminal ganglion was induced as previously described (Ahn et al., 2009b). Surgical procedures were performed under pentobarbital sodium anesthesia (40 mg/kg, i.p.). Anesthetized rats were mounted onto a stereotaxic frame (Model 1404, David Kopf Instruments, Tujunga, CA). After a small hole was made in the skull, a guide cannula (21 gauge) was implanted into the left trigeminal ganglion (3.4 mm posterior to the bregma, 3.5 mm lateral from the midline, and 8.0 mm ventral from the surface of the skull). These coordinates are located in the maxillary branch of the trigeminal ganglion. To compress the trigeminal ganglion, a 4% agar solution (10 μl) was injected into the left trigeminal ganglion through a stainless steel injector (24 gauge). The injector was connected to a 100 μl Hamilton syringe with a polyethylene tube (PE 50; Clay Adams, Parsippany, NJ), which was preheated in a warm water bath (38 °C). After both the injector and guide cannula were removed, a local anesthetic ointment was applied to the sutured wounds. The sham-treated animals were subjected to all surgical procedures, including implantation of a guide cannula and injector, without the agar injection. However, the sham-treated animals did not show any significant behavioral differences as compared with the naïve rats. These results suggested that the cannula implantation (sham operation) did not affect the rats' pain threshold nor produced nociceptive behavior (Ahn et al., 2009b). At the end of experiments, the animals were deeply anesthetized and perfused transcardially with heparinized normal saline, followed by 10% buffered formalin. After perfusion, the compression site of the trigeminal ganglion was identified by stereoscope. Only data from rats with injection sites clearly within the trigeminal ganglion were used for the final analysis. 2.3. Evaluation of facial mechanical allodynia To evaluate facial mechanical allodynia, each rat was placed in a customized observation cage, which was then placed in a darkened and noise-free room. The animals were then acclimated for at least 30 min. Withdrawal behavior, produced by 10 successive trials of constant air-puff pressure (4 s duration, 10 s intervals), was examined in freely moving rats, as described previously (Ahn et al., 2009a,b; Yang et al., 2009). The intensity and intervals of the airpuff pressure were controlled with a pneumatic pump module (BH2 system; Harvard Apparatus, Holliston, MA). The air-puffs were applied through a 26-gauge metal tube (length: 10 cm) located 1 cm from the skin at a 90° angle. We searched for the most sensitive area after compression of the trigeminal ganglion, and then applied air-puff stimulation to that area. Allodynia was defined as the decrease in the intensity of the air-puff threshold, when rats attempted to escape or showed an aggressive behavioral response to air-puff stimulation. Thresholds were determined by the air-puff pressure at which each rat responded in 50% of the trials. The cut-off pressure

The decreased air-puff thresholds indicated a maximal response on post operative day 14. We performed additional analyses at this time point after compression of the trigeminal ganglion. Hence, the effects of minocycline and SB203580 on mechanical allodynia were evaluated on postoperative day 14. For intracisternal injections, individual rats were anesthetized and mounted onto a stereotaxic frame. A polyethylene tube (PE 10; Clay Adams, Parsippany, NJ) was implanted on postoperative day 12, as described previously (Ahn et al., 1998, 2005; Wang et al., 2002; Yaksh and Rudy, 1976). The polyethylene tube was inserted through a tiny hole made in the atlanto-occipital membrane and dura with a 27-gauge syringe needle. The tip of the cannula was placed at the obex level. The polyethylene tube was guided subcutaneously to the top of the skull and secured in place by a stainless steel screw and dental acrylic resin. After recovery for 2 days, we intracisternally administered minocycline (25, 50 or 100 μg in 10 μL), an inhibitor of microglial cell activation, or SB203580 (0.1, 1 or 10 μg in 10 μL), a p38 MAPK inhibitor. The effects of intracisternal administration of minocycline or SB203580 on changes in the air-puff thresholds were subsequently measured at 10, 30, 60, 120, 180, 360 min, and 24 h. Changes in the expression of OX42/Iba1 and p-p38 MAPK were investigated using immunohistochemical staining and western blotting analysis, 6 h after intracisternal administration of minocycline (100 μg) or SB253580 (10 μg). Minocycline was purchased from Sigma (St. Louis, MO) and dissolved in sterile saline. SB203580 was purchased from Calbiochem (La Jolla, CA) and dissolved in 70% DMSO and 30% sterile saline. A vehicle solution (saline or 70% DMSO/30% saline) was administered as a control. 2.5. Immunohistochemical staining On postoperative day 14, the rats (n = 5 per group) were transcardially perfused with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M PB (pH 7.4). The caudal medulla was then removed, postfixed in the same fixative at 4 °C overnight, and replaced with 30% sucrose in 0.1 M PB overnight. It was then frozen, cut into transverse sections of 30 μm thickness, and blocked with 5% goat serum in PBS containing 0.2% Triton X-100 for 1 h at room temperature. Subsequently, the sections were incubated at 4 °C overnight with rabbit polyclonal anti-phospho p38 antibody (1:300; Cell Signaling Technology, Danvers, MA). The sections were then incubated with Cy3-conjugated anti-rabbit IgG antibody (1:300, Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature. For double immunofluorescence, the sections were incubated with a mixture of p-p38 and NeuN (neuronal marker, 1:1000, Millipore, Temecula, CA), OX-42 (microglial marker, 1:100, Millipore) or GFAP (glial fibrillary acidic protein, 1:1000, Millipore) overnight at 4 °C, followed by a mixture of Cy3-conjugated rabbit IgG and FITCconjugated anti-mouse IgG for 2 h at room temperature. The stained sections were then examined under a fluorescence microscope (BX 41 and U-RFL-T; Olympus, Tokyo, Japan). 2.6. Western blotting The rats (n = 5 per group) were sacrificed by decapitation on postoperative day 14. The dorsal part of caudal medulla was then rapidly removed and frozen in liquid nitrogen. These specimens were sonicated with Biorupture (Cosmo Bio., Tokyo, Japan) in a lysis buffer containing protease and a phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL). Protein concentrations in the samples were measured using a fluorometer (Invitrogen, Carlsbad, CA).

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Total proteins (30 μg) were separated in a 4–12% gradient NuPAGE Novex Bis–Tris gel (Invitrogen, Carlsbad, CA) and transferred onto PVDF membranes using the iBlot Dry blotting system (Invitrogen, Carlsbad, CA). The membranes were subsequently blocked with 5% non-fat milk in TBS containing 0.1% Tween 20 for 1 h at room temperature, and then incubated with either p-p38 (1:1000, Cell Signaling Technology) or a total p38 loading control (1:1000, Cell Signaling Technology) at 4 °C overnight. We also used Iba1 (1:500, WAKO Pure Chemical Industries, Ltd., Osaka, Japan) antibody to detect microglia. The GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) antibody was used as a loading control for Ibal (Ionized Calcium-Binding Adapter Molecule 1). The blots were then incubated with goat anti-rabbit horseradish peroxidase for 1 h at room temperature. Membranes were developed using the SuperSignal West Femto substrate (Pierce, Rockford, IL), and exposed to X-ray films. We used the ImageJ analysis system (NIH, Bethesda, MD) for quantification of specific bands.

2.7. Rota-rod test Changes in motor performance after intracisternal administration of either minocycline or SB203580 were monitored using the Rotarod (Ugo Basile, Comerio-Varese, Italy), as described previously (Ahn et al., 2007; Erichsen et al., 2005). The Rota-rod speed was set at 12 rpm with a maximum time spent on the rod set at 180 s. The rats received two or three training trials on two separate days prior to testing for acclimatization. On the testing day, the resting response was examined. After intracisternal administration of either minocycline or SB203580, the time course of the motor performance was assessed.

2.8. Statistical analysis Statistical analyses of the behavioral data were carried out using repeated measured analysis of variance (RM-ANOVA), followed by LSD post-hoc analysis. Comparisons between the two means were performed using the Student's t-test. In all statistical comparisons, P b 0.05 was used as the criterion for statistical significance. All data values are the mean ± standard error (SEM).

3. Results Our present results demonstrated that compression of the trigeminal ganglion produced prolonged mechanical allodynia in the trigeminal territory of the facial area in the rats. This in turn produced a dramatic increase in the responses to mechanical stimulation of the face. Fig. 1 shows the expression profile of OX-42, a microglial cell marker, in the medullary dorsal horn on postoperative day 14. After compression, OX-42 expression was significantly increased in the ipsilateral medullary dorsal horn, as compared to the sham treatment (Fig. 1A). However, a statistically significant increase in OX-42 expression was not detected on the contralateral side (data not shown). Western blot analysis further showed up-regulation of Iba1, a microglial cell marker, in comparison with sham group (P b 0.05, Fig. 1B,C), thus indicating that compression of the trigeminal ganglion significantly increased the expression of this factor. On postoperative day 14, the expression of p-p38 MAPK immunoreactivity in the medullary dorsal horn was found to be increased on the ipsilateral side after compression (Fig. 2A), as shown in Fig. 2. We identified the enhanced expression of p-p38 MAPK using western blot analysis and found that this was caused by compression of the

Fig. 1. Changes in microglial immunoreactivity in the medullary dorsal horn of rats with compression of the trigeminal ganglion. On postoperative day 14, OX-42 immunoreactivity was increased in the ipsilateral side of the medullary dorsal horn, as compared to the sham group (A). Western blot analysis revealed increases in the Iba1 protein levels following compression of the trigeminal ganglion (B). Quantification of the Iba1 protein levels by western blotting is shown in (C). The GAPDH levels were used as a loading control. Scale bars: 200 μm. *P b 0.05, compared with the sham group.

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Fig. 2. Changes in p38 MAPK immunoreactivity in the medullary dorsal horn of rats with compression of the trigeminal ganglion. On postoperative day 14, p-p38 MAPK immunoreactivity increased in the medullary dorsal horn, as compared to the sham groups (A). Western blot analysis revealed increases in the p-p38 MAPK levels following compression of the trigeminal ganglion (B). Quantification of the p-p38 MAPK levels on immunoblots is shown in (C). The total p38 MAPK levels were used as the loading control. Scale bars: 200 μm. *P b 0.05, compared with the sham group.

trigeminal ganglion (P b 0.05, Fig. 2B,C). Compression of the trigeminal ganglion increased OX-42 and p-p38 MAPK immunoreactivity in both the deep and superficial lamina. The effects of minocycline, a selective inhibitor of microglial cell activation, on mechanical allodynia are illustrated in Fig. 3. Neither the intracisternal administration of vehicle nor 25 μg of minocycline affected the air-puff thresholds on the ipsilateral and contralateral sides. In addition, although 50 μg of minocycline inhibited mechanical allodynia, the differences were not statistically significant. In contrast, intracisternal administration of 100 μg of minocycline significantly inhibited mechanical allodynia produced by compression of the trigeminal ganglion on the ipsilateral (F(3, 25) = 5.669, P b 0.05, Fig. 3A), as well as the contralateral side (F(3, 25) = 5.667, P b 0.05, Fig. 3B). Similar to the behavioral responses, intracisternal administration of 100 μg of minocycline inhibited the expression of OX-42 in the medullary dorsal horn produced by compression of the trigeminal ganglion (Fig. 3C). Moreover, western blotting revealed that treatment with 100 μg of minocycline significantly attenuated Iba1 expression, compared with the vehicle-treated group (P b 0.05; Fig. 3D,E). Intracisternal administration of 0.1, 1, or 10 μg of SB203580, a p38 MAPK inhibitor, was also found to inhibit the suppression of ipsilateral air-puff thresholds in the trigeminal ganglion-compression group, compared to the vehicle-treated group (F(3, 25) = 3.169, P b 0.05, Fig. 4A). However, intracisternal administration of vehicle did not affect the air-puff thresholds. Intracisternal administration of all doses of SB203580 also inhibited the suppression of the contralateral airpuff thresholds (F(3, 25) = 3.288, P b 0.05; Fig. 4B). Similar to behavioral responses, intracisternal administration of 10 μg of SB203580 inhibited the expression of p-p38 MAPK in the medullary dorsal horn produced by compression of the trigeminal ganglion (Fig. 4C). Moreover, immunoblotting analysis showed that treatment with SB203580 significantly attenuated p-p38MAPK expression (P b 0.05, Fig. 4D,E).

On postoperative day 14, double immunofluorescence analysis was performed and indicated that p-p38 MAPK was co-localized with OX-42, a microglial marker, but not GFAP, an astrocyte marker or NeuN, a neuronal marker (Fig. 5). To evaluate whether minocycline or SB203580 was associated with motor dysfunction, the Rota-rod test was performed after drugs were administered. No motor dysfunction was observed, however, after these treatments. 4. Discussion The results of our present study first demonstrate that compression of the trigeminal ganglion increases microglial activation and p38 MAPK expression in the medullary dorsal horn. P-p38 MAPK co-localizes with OX-42, a microglial marker. The intracisternal administration of either minocycline, a selective inhibitor of microglial cell activation, or SB203580, a p38 MAPK inhibitor, attenuated mechanical allodynia. These results suggest that activation of p38 MAPK in the microglia is associated with trigeminal neuralgia-like nociception produced by compression of the trigeminal ganglion. Trigeminal neuralgia (also known as tic douloureux) is experienced in one or more divisions of the trigeminal distribution and is a severe chronic pain syndrome characterized by brief, but excruciatingly intense stabbing or electrical shock-like paroxysmal pain (Devor et al., 2002; Kitt et al., 2000; Sindrup and Jensen, 2002). Since 1967, when microvascular compression was first recognized as a cause of trigeminal neuralgia by Jannetta (2007), the etiology of most cases of trigeminal neuralgia has been strongly supported by clinical observation, whereby vascular contact occurred in a high proportion of patients with trigeminal neuralgia in clinical studies (Haines et al., 1980; Jannetta, 1980; McLaughlin et al., 1999; Meaney et al., 1995). Moreover, prolonged pain relief could be obtained by surgical microvascular decompression (Lovely and Jannetta, 1997; McLaughlin et al., 1999; Zakrzewska et al., 2005). The results of our present

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Fig. 3. Effects of intracisternal injection of minocycline, a selective inhibitor of microglial cell activation, on mechanical allodynia (A) and mirror-image mechanical allodynia (B) in rats with compression of the trigeminal ganglion, as measured on postoperative day 14. Vehicle (saline) did not affect mechanical allodynia, whereas intracisternal treatment with 100 μg of minocycline significantly reduced mechanical allodynia both ipsilateral and contralateral to the compression of trigeminal ganglion. There were eight animals in each group. Minocycline (100 μg) inhibited OX-42 expression in the medullary dorsal horn produced by compression of the trigeminal ganglion (C). Western blot analysis revealed that minocycline attenuated Iba1 expression, compared with the vehicle-treated group (D,E). Scale bars: 200 μm. *P b 0.05 vs. vehicle-treated group.

study in the rat demonstrate that compression of the trigeminal ganglion produces mechanical allodynia. Neither normal naïve rats nor sham-treated rats showed mechanical allodynia. However, in the trigeminal ganglion-compressed animal group, the air-puff threshold was significantly lower than that in the sham-operated group. These findings suggest that prolonged nociceptive behavior, produced by compression of the trigeminal ganglion, may mimic symptoms of patients with trigeminal neuralgia, as previously described in a report from our laboratory (Ahn et al., 2009b). Our current data first demonstrate that compression of the trigeminal ganglion increases OX-42 immunoreactivity in the medullary dorsal horn and enhances the expression of Iba1, a microglial cell marker. Moreover, intracisternal administration of minocycline, a selective inhibitor of microglial cell activation, was found to reduce mechanical allodynia following compression of the trigeminal ganglion. Although there was no evidence for participation of glia in the suppression of trigeminal ganglion-induced mechanical allodynia, these results are consistent with results from previous studies showing

that inactivation of microglia by minocycline inhibits the development of neuropathic pain. In those earlier reports, repeated intraperitoneal administration of minocycline significantly attenuated both allodynia and cold hyperalgesia in rats with chronic constriction injury (Mika et al., 2007), and intrathecal administration of minocycline inhibited mechanical allodynia in the neuropathic pain induced by sciatic nerve inflammation (Ledeboer et al., 2005). Taken together, our present results and previous data suggest that activation of microglia cells in the medullary dorsal horn may reflect its important role in the development of mechanical allodynia produced by compression of the trigeminal ganglion. The possible involvement of p38 MAPK in spinal cord and dorsal root ganglion (DRG) cells in the development of peripheral neuropathic pain is well known. Under conditions of either tissue or nerve damage, ERK and p38 MAPK are activated by nociceptive activity or inflammatory mediators in primary sensory neurons of both peripheral and central nervous systems. Mechanical allodynia and thermal hyperalgesia, which are associated with the phosphorylation

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Fig. 4. Effects of intracisternal injection of SB203580, a p38 MAPK inhibitor, on mechanical allodynia (A) and mirror-image mechanical allodynia (B) in rats with compression of the trigeminal ganglion, as measured on postoperative day 14. Injection with vehicle (70% DMSO/30% sterile saline) did not affect mechanical allodynia. Intracisternal treatments with 0.1, 1 or 10 μg of SB203580 significantly reduced mechanical allodynia both ipsilateral and contralateral to the compression of trigeminal ganglion. There were eight animals in each group. SB203580 (10 μg) inhibited the expression of p-p38 MAPK in the medullary dorsal horn produced by compression of the trigeminal ganglion (C). Western blot analysis showed that SB203580 attenuated p-p38MAPK expression, compared with the vehicle-treated group (D,E). Scale bars: 200 μm. *P b 0.05 vs. vehicle-treated group.

of p38 MAPK in the dorsal root ganglion (Obata et al., 2004; Tsuda et al., 2004) or in the superficial dorsal horn (Zhuang et al., 2005), were found previously to be reversed by intrathecal treatment with p38 MAPK inhibitors in a rat model of spinal nerve ligation (Obata et al., 2004; Tsuda et al., 2004; Zhuang et al., 2005). The results of our present study also demonstrate that expression of p38 MAPK increases in the ipsilateral medullary dorsal horn of rats, when the trigeminal ganglion is compressed. The participation of MAPK pathways in the trigeminal nociceptive processing has already been postulated. Subcutaneous injection of formalin into the peri-oral skin of the upper lip of mice significantly increased the number of activated p44/42 MAPK-like immunoreactive neurons in lamina I and II of the trigeminal subnucleus caudalis (Huang et al., 2000). Intracisternal pretreatment with SB203580, a p38 MAPK inhibitor, significantly inhibited mechanical allodynia following infraorbital nerve ligation in rats (Lim et al., 2007). Our current study further demonstrates that mechanical allodynia, produced by compression of the trigeminal ganglion, is sensitive to treatment with p38

MAPK inhibitors. Thus, taken together with the results of previous studies, our present data suggest that the central p38 MAPK pathways contribute to the processing of orofacial nociceptive information, including trigeminal neuralgia-like nociception produced by compression of the trigeminal ganglion. The present study demonstrated that compression of the trigeminal ganglion increased OX-42 and p-p38 MAPK immunoreactivity in both the deep and superficial lamina. However, a previous study in the peripheral neuropathic animal model showed activation of glial cell in the superficial lamina (Piao et al., 2006). These contrasting results may be caused by different animal models. The present study used rats with compression of the trigeminal ganglion that produced prolonged mechanical allodynia and hyperalgesia, but not thermal hypersensitivity. It is well known that low threshold mechanical and pressure primary afferents project to the deep lamina (Gwak and Hulsebosch, 2009). Our EM study supported the present data showing activation of OX-42 and p38 MAPK in the deep lamina. EM study revealed that compression of the trigeminal

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Fig. 5. Double-labeled immunofluorescent analysis of p-p38 (red), with OX-42, a microglial marker (A, D, green), GFAP, an astrocyte marker (B, E, green), and NeuN, a neuronal marker (C, F, green) was performed on postoperative day 14. The signals demonstrated that p-p38 MAPK exclusively co-localizes with OX-42 (A, D, yellow). D–F is magnification of the rectangle area in A–C, respectively. Scale bars: A–C = 200 μm; D–F = 50 μm, respectively.

ganglion produced focal demyelination of the myelinated fiber in the trigeminal ganglion (not published). These results suggest that demyelination produced by compression of the trigeminal ganglion plays an important role in the development of nociception. Moreover, compression of the trigeminal ganglion might affect more myelinated A-fibers projecting to the deep lamina than small unmyelinated C-fibers projecting to the superficial lamina, suggesting that our subjected animals produced activation of microglial and p38 MAPK in the deep lamina, as well. Our present findings provide evidence that p-p38 MAPK expression is predominantly co-localized in the microglia, and not in the neurons or astrocytes. These observations suggest that p38 MAPK activation in the microglia plays a crucial role in the development of trigeminal neuralgia-like pain. Increasing evidence from animal models of neuropathic pain shows that p38 MAPK is likely to contribute to the development of pain hypersensitivity through spinal microglia (Crown et al., 2008; Jin et al., 2003; Piao et al., 2006). Moreover, systemic administration of minocycline inhibited p38 MAPK activation in the microglia, and significantly attenuated the development of pain hypersensitivity following inferior alveolar nerve and mental nerve transection (Piao et al., 2006). These previous results, together with our present data, suggest that p38 MAPK located in hyperactive microglia in the medullary dorsal horn contributes to pain hypersensitivity in trigeminal neuralgia-like nociception. In addition, in our current analyses, compression of the trigeminal ganglion produced a mirror-image mechanical allodynia on the contralateral side. This mirror-image mechanical allodynia was apparently not caused by the sham surgery, because control rats did not develop contralateral mechanical sensitivity until 40 days postoperatively (Ahn et al., 2009b). Several studies have already reported the development of mirror-image pain in neuropathic pain animal models (Kim and Chung, 1992; Lim et al., 2007; Milligan et al.,

2003; Vos et al., 1994). Recently, Milligan et al. (2003) provided evidence that mirror-image inflammatory neuropathic pain is created through glial and proinflammatory cytokine activities (Milligan et al., 2003). In our present study, although the air-puff threshold was found to be increased on both the ipsilateral and contralateral sides in rats with compression of the trigeminal ganglion, the activation of microglia or p38 MAPK was only altered on the ipsilateral medullary dorsal horn, but not on contralateral side. These findings suggested that activation of p38 MAPK in the microglia did not cause mirror image mechanical allodynia in our rat model. Recently, intrathecal administration of carbenoxolone, a gap junction de-coupler in astrocytes, abolished mirror-image mechanical allodynia in rats with sciatic inflammatory neuropathy, without affecting the ipsilateral allodynia (Spataro et al., 2004). These results suggest that astrocytes are involved in the development of mirror-image mechanical allodynia following compression of the trigeminal ganglion.

5. Conclusion In summary, rats with compression of the trigeminal ganglion demonstrated activated microglia and up-regulation of p-p38 MAPK in the ipsilateral medullary dorsal horn. P-p38 MAPK expression was found to be predominantly co-localized in the microglia, but not in the neurons or astrocytes. Intracisternal administration of minocycline, a selective inhibitor of microglial cell activation, or of SB203580, a p38 MAPK inhibitor, attenuated mechanical allodynia in the trigeminal neuralgia. These results suggest that activation of microglial p38 MAPK in the medullary dorsal horn may reflect the important role of this signaling pathway in the development of mechanical allodynia in trigeminal neuralgia-like nociception.

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