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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Pain. Author manuscript; available in PMC Nov 1, 2009.
Published in final edited form as:
PMCID: PMC2632609
NIHMSID: NIHMS78699

Partial infraorbital nerve ligation as a model of trigeminal nerve injury in the mouse: Behavioral, Neural and Glial reactions

Abstract

Trigeminal nerve damage often leads to chronic pain syndromes including trigeminal neuralgia, a severely debilitating chronic orofacial pain syndrome. Options for treatment of neuropathic pain are limited in effectiveness and new approaches based on a better understanding of the underlying pathologies are required. Partial ligation has been shown to effectively mimic many of the qualities of human neuropathic pain syndromes. We have devised a mouse model of trigeminal neuralgia using a partial ligation of the infraorbital nerve (pIONL) that induces persistent pain behaviors and morphological changes in the brainstem. We found that the pIONL effectively induced mechanical allodynia lasting for over 3 weeks. Cell proliferation (BrdU), activation of astrocytes and microglia in the ipsilateral caudal medulla, and persistent satellite cell reaction in the ipsilateral ganglion were observed. Neurochemical markers CGRP, substance P were decreased in medullary dorsal horn ipsilateral to the injury side, whereas substance P receptor NK1 expression was increased after 8 days. Nerve injury marker ATF3 was markedly increased in ipsilateral trigeminal ganglion neurons at 8 days after pIONL. The data indicate that partial trigeminal injury in mice produces many persistent anatomical changes in neuropathic pain, as well as mechanical allodynia.

Perspective

The study describes the development of a new mouse model of trigeminal neuropathic pain. Our goal is to devise better treatments of trigeminal pain, and this will be facilitated by characterization of the underlying cellular and molecular neuropathological mechanisms in genetically designed mice.

Keywords: infraorbital nerve, caudal medulla, allodynia, neuron, glia

Introduction

Orofacial pain disorders encompass a wide range of conditions including trigeminal neuralgia, temporomandibular joint disorders, periodontal pain, and atypical face pain. Great advances in the understanding of the mechanisms that underlie neuropathic pain states have come from the development of animal models such as the partial sciatic nerve ligation model of Seltzer’s et al.42 For the trigeminal nerve there is the infraorbital chronic constriction injury (CCI) with loose ligatures in rat, developed by Vos et al.52, 53 and adapted from the sciatic CCI model of Bennett and Xie.5 These models reproduce important aspects of trigeminal neuralgia, including signs of abnormal spontaneous pain-related behavior, mechanical allodynia 51, 52 and heat hyperalgesia.24 There are also studies of complete transection or crush injury of infraorbital, or alveolar or lingual nerves to analyze deafferentation or regeneration mechanisms in rats.6, 22, 23, 25, 37, 39, 41, 47 Abnormal spontaneous discharges from trigeminal cell bodies and upregulated glial fibrillary acidic protein (GFAP) expression in satellite cells have been detected in both mandibular and maxillary divisions in rats as long as 2 months after inferior alveolar (maxillary) nerve crush10 with some persistent behavioral consequences.12

The generation of a trigeminal pain model for studies using knock-out and transgenic mice would offer a promising approach to the identification of novel biochemical factors that contribute to persistent trigeminal pain conditions. Behavioral approaches to the study of nociceptive pain have been used in mouse,50 but to our knowledge, reliable and readily performed mouse models of neuropathic trigeminal pain have not been published. We think that this is an important goal because of some unique features of trigeminal nerve organization, the special tissues that it innervates, and special clinical problems of orofacial pain.43

We have adapted the partial sciatic nerve ligation model of Seltzer et al. (1990) 42 to the infraorbital branch of the trigeminal nerve of mice. Partial sciatic nerve injury results in a decrease in substance P (Sub P), calcitonin gene-related peptide (CGRP) and an increase in neurokinin-1 (NK-1/Sub P) receptor immunoreactivity in the dorsal horn of rats 17, 19, 27 and mice.33 Glial cells in the central nervous system also respond to the peripheral insults, both microglia and astrocytes are activated following inferior alveolar nerve and mental nerve transaction in medullary dorsal horn in rats40 and sciatic nerve ligation in mice.55, 56 Here we investigate the behavioral and anatomical changes in wild type C57B1/6 mice with partial infraorbital nerve ligation. This model provides an approach for future studies with mice in which genetic dissection of mechanisms for trigeminal neuropathic pain would be possible.

Material and methods

Animals

C57Bl/6 male adult mice (Charles River Laboratories, Wilmington, MA) weighing 22–32 g (12–16 weeks old) were used in these experiments. Mice were group-housed, in self-standing plastic cages (28 cm L × 16cm W × 13 cm H) within the animal core facility at the University of Washington, and maintained in a specific pathogen-free housing unit. Mice were transferred 1 week prior to behavior testing into a colony room adjacent to the testing room to acclimatize to the testing environment. Housing rooms were illuminated on a 12-h light-dark cycle with lights on at 0700. Food pellets and water were available ad libitum. Procedures with mice were approved by the Institutional Animal Care and Use Committee in accordance with the 1996 NIH Guide for the Care and Use of Laboratory Animals.

Surgical preparation: Partial Ligation of the Infraorbital Nerve (pIONL)

The unilateral partial ligation to the right ION was performed under direct visual control using a Zeiss surgical microscope (x10–25) (Fig 1A). The animals were anesthetized with sodium pentobarbital (Nembutal, 80 mg/kg i.p.). They were kept warm with a heat lamp and foil blanket, their eyes were treated with lubricating ophthalmic ointment (Akorn, Buffalo Grove, IL). The mouse was taped to a sterilized cork board, the skin along the top of the snout was shaved, iodine treated, and a mid-line incision was made to expose nasal and maxillary bone. All tools were gas sterilized prior to surgery and then washed and heat-treated (glass beads at 250°C) for each animal. The infraorbital part of the right ION was initially exposed 1–2mm rostral to infraorbital fissure on the maxillary bone using blunt dissection with small scissors. The ION was gently isolated using fine forceps without damaging nearby facial nerve branches. Approximately 1/3 to 1/2 the diameter of the nerve was tightly ligated with 7-0 silk suture (Surgical Specialties Corporation, Reading, PA) by passing the suture needle completely under the lateral aspect of the nerve and then up through the middle. The incision was closed using silk sutures (5–0) after confirming hemostasis. For the sham-operated mice, the ION was exposed on the right side using the same procedure, but the ION was not touched or ligated. The mice received one analgesic (buprenorphine, 0.05 mg/kg) treatment at the end of the surgery. The operated mice were able to eat and drink unaided soon after waking up, the body weight returned back to or exceeded preoperative weights after the first week.

Fig 1
Images showing the surgical approach used during partial infraorbital nerve ligation (pIONL) and mechanical allodynia produced following pIONL

Behavioral testing

Stimulus-evoked responses: Mechanical allodynia

The mice were tested one day before surgery, daily during the first postoperative week, and on alternate days after that for a period of 25 days. All experiments were carried out in a quiet room between 0800 and 1200 hr to avoid diurnal variations. Body weight was measured every time before testing. On the day of testing, mice were habituated to handling and testing equipment 20–30 min before experiments. A graded series of von Frey filaments (Semmes-Weinstein monofilaments, Stoelting, Wood Dale, IL) was used for mechanical stimulation of ipsilateral infraorbital nerve territory. The filaments produced a bending force of 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1.0 and 1.4 gm. The mice stood on a metal mesh with a porous plastic cup (diameter: 8cm) covering them. Von Frey hairs were then inserted from below through the mesh when the animal’s head was steady resting or alert status.8 The stimuli were applied within the infraorbital nerve territory, near the center of the vibrissa pad, on the hairy skin of the right (ipsilateral) side and involved brief bending of the filament. The threshold was taken with a response of a brisk withdrawal of the head followed by an uninterrupted series of at least three face grooming strokes directed to the stimulated facial area. The time period between each filament stimulus was about 2–3 minutes. The stimulation always began with the filament producing the lowest force and stopped when threshold was found. The threshold response had to occur within 2 seconds to be considered a head withdrawal response. Unresponsive mice received a maximal score of 1.4 gm.

Non-evoked Behaviors: Face grooming behavior

Mice were placed individually in small transparent plastic cages (14cm × 16cm × 13cm) without bedding. A video camera was placed 0.8m at the side of the cage and positioned so that the image of the mouse head was observed. Mice were habituated in the cage for 15 min and then recorded 15 min per day for 7 days. Duration of face grooming actions was recorded when the forelimbs contact facial region and ear grasps.

Immunohistochemistry in brainstem and trigeminal ganglion sections

Mice were anesthetized with sodium pentobarbital (Nembutal, 100 mg/kg i.p.) and intracardially perfused with 4% para-formaldehyde in PB (phosphate buffer, 0.1 M sodium phosphate, pH 7.4). The brainstem and trigeminal ganglia (TG) were dissected, postfixed 2 hours, cryoprotected with solution of 30% (w/v) sucrose in PB at 4°C overnight and cut into series of 40 µm sections (brainstem) or 20 µm sections (TG) with microtome. Briefly, sections were washed 3 times in PBS (phosphate buffer saline), blocked in PBS containing 0.1% Triton X-100 and 4% normal goat serum for 1 hr, and incubated overnight with primary antibodies. Primary antibody concentrations were as follows: rabbit anti-CGRP (1:5000, Chemicon, Temecula CA), rabbit anti-NK1 receptor (1:1000, Chemicon, Temecula CA), rabbit anti-Substance P (1:5000, Chemicon, Temecula CA), rabbit anti-ATF3 (1:50, Santa Cruz Biotechnology, Santa Cruz, CA), rat anti-CD11b (1:200, Serotac, Oxford, UK), rabbit anti-GFAP(1:3000, Dakoplatts, Denmark), and mouse anti-BrdU (6µg/ml, Chemicon, Temecula CA) or rat anti-BrdU (1: 50 Abcam Cambridge, MA). For BrdU staining, the animals were treated with i.p. injection of BrdU 100mg/kg once per day for 7 days before perfusion. The brain stem sections were treated with 2N HCl for 60 min at 37°C, followed by two 10 min washes in 0.1 M borate buffer before incubation overnight with primary antibody. Sections were then washed with PBS, and detection was carried out using the rhodamine or fluorescein conjugated fluorescent secondary antibodies (1:250; Jackson ImmunoResearch, West Grove, PA). Antibodies were diluted in a solution containing 0.1% Triton X-100 and 4% normal goat serum in PBS. The sections were rinsed in PBS for 30 min, and then mounted on gelatin-coated slides with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and sealed with nail polish for microscopy. The sections were viewed with a Nikon Eclipse E600 fluorescence microscope (Tokyo, Japan) or a Leica SL confocal microscope located in the W.M. Keck Imaging Facility at the University of Washington. Relative staining intensities were analyzed using NIH Image J version 1.62 software (National Institutes of Health, Bethesda, MD). Values were expressed as ratios of labeling intensity over background.

Statistical Analysis

We have used a total of 72 male mice in the study. Sample size was 4–6 brainstem sections from each of 4 mice to assess the anatomical changes, and 4–8 mice per group were used to assess behavioral changes. Mechanical allodynia data were analyzed by ANOVA (non-parametric). For all groups, pre- and post-operative behavior for intra-animal comparisons as well as group comparisons were made. Statistical significance determined by ANOVA were then further analyzed with Student-Newman–Keuls test or Student's t test for significant pair-wise comparisons. Response data are presented as means ± SEM of the animal treatment group, with significance set at p < 0.05.

Results

Behavioral responses

Stimulus-Evoked mechanical allodynia following pIONL

We applied von Frey fibers to the anterior right snout to test for mechanical allodynia in the operated mice. Ligated and sham operated mice showed similar responses to the von Frey filament that was applied with a stimulus of 1.4g before pIONL surgery. Mice showed significant allodynia from day 1 that lasted for over three weeks after the pIONL surgery. After day 9, ligated mice began recovery toward baseline, but still showed significant allodynia compared with sham mice until day 23 (Fig. 1B) (p < 0.05). These results support the hypothesis that pIONL induces persistent trigeminal neuropathy.

Non-evoked behavior (isolated face grooming) and body weight changes following pIONL

We video recorded mouse spontaneous activity for 15 min each day, including once before the surgery (pre-study baseline). The face grooming duration was analyzed from the videos by an independent observer, blind to treatment. There was a significant increase in face grooming in mice following pIONL compared to sham operated mice one day after surgery (Fig. 2A) (p < 0.05), but face grooming returned to normal by 3 days post surgery. We also found that during the first week following pIONL, the body weights of injured mice were slightly below pre-injury baseline (Fig. 2B), but the difference did not reach statistical significance (p>0.05) between pIONL and sham operated mice. The body weight of operated mice increased above the pre-injury baseline during the second week after pIONL.

Fig 2
Change in the duration of face grooming behaviors and body weight after pIONL

Neuronal Responses after pIONL injury in brainstem

In our study, we found that pIONL produced a marked decrease in CGRP-IR and Sub P-IR and an increase in NK-1 receptor expression in the ipsilateral side of caudal medullary dorsal horn 8 day after pIONL (Fig. 3A–G). NK-1 receptors localized densely in superficial lamina I–II and in deeper area lamina III–V (Fig. 3C, D). Sham-operated mice showed no change in CGRP, NK-1 receptor or Sub P IR (Fig. 3H–J). The decrease in CGRP-IR extended into the cervical spinal cord (C1) (Fig. 3A) as expected.34, 35

Fig 3
Neuronal expression of CGRP-IR, sub P-IR and NK1R-IR in the cervical C1 spinal cord and caudal medulla at 8 days after pIONL

Glial Reactions to pIONL in brainstem

We evaluated alterations in microglia (CD11b) and astrocytes (GFAP) in caudal medulla. In the contralateral side of pIONL mice and the sham-operated mice, CD11b-IR was uniformly distributed and had modest intensity throughout caudal medulla (Fig. 4A). The stained resident microglia had long, finely branched processes that extended in all directions from the cell soma (Fig. 4B). In the ipsilateral side of pIONL mice, microglia had profound CD11b-IR at 1 day after pIONL and appeared to be in an activated state with enlarged cell bodies and thicker processes than on the contralateral side (Fig. 4 C,D). After 8 days following pIONL injury, ipsilateral CD11b-IR was reduced and was no longer different from that in the contralateral side (Fig. 4E–G). The most extensive expression of activated microglia after pIONL was observed in the medial portion of superficial lamina, and microglial activation was not found in normal or sham-operated mice.

Fig 4
Microglial responses after pIONL

Activated astrocytes were visualized with anti-GFAP immunostaining to assess the effects of pIONL. GFAP-IR in caudal medulla was homogeneous and modest throughout the superficial lamina on the contralateral side of pIONL mice and on both sides in sham-operated mice (Fig. 5A–D). However, the ipsilateral side had strong GFAP-IR 8 days following pIONL, and these cells had hypertrophic cell bodies and long processes (Fig. 5E). The greatest astrocytic activation was observed in the medial portion of the superficial lamina of the ipsilateral side (Fig. 5A,E). GFAP-IR was significantly increased in the ipsilateral caudal medulla compared with contralateral side and sham operated mice on 8 day after pIONL (Fig. 5F) (p < 0.05).

Fig 5
Astrocytic responses in caudal medulla 8d after pIONL (A). GFAP staining was compared in ipsi and contraleteral regions of the caudal medulla of sham operated mice (B, C) and following pIONL (D, E). There was an striking increase of GFAP-IR on the ipsilateral ...

Cellular Proliferation after pIONL (BrdU staining) in brainstem

We found that at 8 day after pIONL, the number of bromodeoxyuridine (BrdU)-positive cells was increased 5.6-fold in the ipsilateral side of the caudal medulla compared with contralateral side (Fig. 6A, B, p<0.05). The BrdU immunoreactive cells were preferentially located in the dorsal lamina I–III, where microglia and astrocytes were activated by pIONL. To characterize the cell types that were BrdU-positive after pIONL, we performed dual-labeling of BrdU and GFAP, BrdU and CD11b, BrdU and NeuN (neuronal marker), or BrdU and NG2 (oligodendrocytes precursor marker). A portion of BrdU-positive cells were double labeled with the microglial marker CD11b-IR (Fig. 6C–E) or astrocytic marker GFAP-IR (Fig. 6F–H). The majority (~70%) of the BrdU-positive cells were nestin-positive stem cells (Fig. 6I–K). We did not detect BrdU-positive cells that double labeled with NG2 (Fig 6L–N) or NeuN (data not shown), suggesting that proliferation of oligodendrocytes or neurons were not evident 8 d following pIONL.

Fig 6
Cellular proliferation in caudal medulla 8d after pIONL was detected following daily BrdU administration (100 mg/kg intraperitoneally, once daily for 8 days)

Anatomical changes in trigeminal ganglia (TG)

We counted the number of neurons with reactive satellite cells in the TG as shown by GFAP-IR (Fig. 7). Neuronal cell body distribution in trigeminal ganglion were shown in Fig 7A: (I): Maxillary infraorbital neurons. (II): Maxillary dental neurons. (III): Mandibular neurons. We found that the number of GFAP-IR labeled satellite cells was significantly increased in the ipsilateral TG ganglia compared with contralateral side and sham operated mice 8 day after pIONL (Fig. 7B–E) (p <0.05). The increased GFAP-IR was returning to normal by the second week (Fig. 7B, F–H). Neuronal expression of ATF3, which is normally minimal, is upregulated after peripheral nerve injury and acts as a marker of nerve injury.48 We found that ATF3 staining was significantly increased in the ipsilateral trigeminal ganglia 8 days after pIONL (p<0.05) (Fig. 8A–E). ATF3 expression occurred within neurons in the infraorbital region of TG (Fig 8D).

Fig 7
GFAP positive satellite cell changes in trigeminal ganglion 1wk and 2wks after pIONL
Fig 8
Immunohistochemical expression of ATF3 in trigeminal ganglia 8 days after pIONL

Discussion

Trigeminal neuralgia is a form of neuropathic pain characterized by severe lancinating pain in orofacial regions innervated by the trigeminal nerve. Most cases of trigeminal neuralgia are caused by sensory nerve root compression. 26 Vos et al 52 developed a rat model of trigeminal neuropathic pain produced by chronic constriction of the infraorbital nerve (CCI). These orofacial CCI injury-induced sensory changes resemble those seen in neuropathic pain of the hind limb following injury to sciatic or L5/L6 spinal nerves in rats, but the CCI surgery is prohibitively difficult in mice. In the present study, we developed and characterized a feasible mouse model of partial infraorbital nerve ligation (pIONL) that is a combination of the partial nerve ligation (dental maxillary nerve fibers and medial ION fibers are not injured) and spared nerve injury (trigeminal mandibular and ophthalmic branches are intact) models of chronic pain. We demonstrated that pIONL in mice produced prolonged mechanical allodynia and a transient change of grooming time. pIONL in the mouse produced persistent changes in peptide neurotransmitter and receptor expression in the caudal medulla region of the brainstem. Microglia and astrocytes were also activated in this region following pIONL injury. ATF3 staining showed that specific neurons in TG were also injured by pIONL. These results established this model as a reliable and objective model of trigeminal neuropathic pain in the mouse.

The trigeminal system shares many neuropathic features with spinal nerves but also differs in important ways.43 The special features make it likely that persistent pain mechanisms in the trigeminal system may have unique features, and better animal models are needed to study the effects of trigeminal nerve injury. So far, partial trigeminal nerve injury studies have mainly used the infraorbital chronic constriction injury with loose ligatures in rats.52,53 Some other animal models for persistent trigeminal pain and plasticity involve peripheral inflammation, 13, 14, 45, 50 nerve transection,22, 23, 25, 37, 39, 40 or chronic tooth infection.7 There are no reports of partial ligation of trigeminal nerves using mice. One study used complete transection of the ION at the foramen that caused large changes in neuronal and glial properties in the ganglion at one week later; however behavioral data were not reported.9 Our present study is adapted from these preceding studies in rats and mice to give a new, reliable and easily applied partial nerve ligation of the mouse trigeminal nerve. We found that pIONL induced persistent anatomical and behavioral changes including mechanical allodynia lasting for over 3 weeks.

The recurrent episodes of asymmetric face grooming directed to the territory of the injured nerve after chronic constriction of the infraorbital nerve have been interpreted as behavioral signs of “spontaneous” neuropathic facial pain.52 The results of the present study showed that mice with pIONL displayed significantly more face grooming time compared to sham operated mice during the first day after nerve injury. These results suggest that spontaneous facial pain was only increased during the acute phase of pIONL. This is different from previous rat studies showing that chronic constriction of the infraorbital nerve (CCI) caused an increased face grooming lasting as long as 130 days.52 Therefore, when using grooming activity as a criterion for facial pain, there may be important species differences in the responses to neuropathic pain.

Rats with chronic constriction injury of the ION were found to have lower average daily weight gain than sham operated animals.52 In contrast, Lim et al (2007)32 showed that body weight gain was not significantly decreased after ION chronic constriction injury. In our study, we found that in the first week following pIONL, the body weights of injured mice were slightly below pre-injury baseline, but the difference did not reach statistical significance between pIONL and sham operated mice. All groups gained weight steadily in the second week after pIONL.

Injury to a peripheral nerve not only produces profound behavioral signs of persistent pain, but it also alters peptide expression in the CNS. For example, sciatic nerve injury results in a decrease in CGRP, Sub P and causes an increase in neurokinin-1 (NK1/Sub P) receptor-IR in the dorsal horn of rat spinal cord.17, 19, 28 The decrease of Sub P and CGRP that we observed in caudal medulla after pIONL was likely to reflect changes in expression of these peptides in primary afferents. The NK-1 receptor, on which Sub P acts, is expressed by most of the lamina I neurons in the spinal dorsal horn46 and trigeminal subnucleus caudalis.31 The increase in NK-1 receptor expression after the pIONL suggested that this response was triggered by the injury barrage generated by the nerve ligation. Consistent with this, the Sub P and CGRP system reorganization only occurred in the dorsal part of the caudal medulla, which receives primary afferent input from the trigeminal nerves. This observation is similar to the results from studies in the rat after complete transection of the sciatic nerve.1, 17, 19, 28 Using the chronic sciatic nerve constriction model, Cameron and colleagues9 found a significant relationship between allodynia and peptide expression levels. We also found that the anatomical and behavioral changes were correlated. These results suggest that nerve injury-induced allodynia is associated with the neurochemical reorganization of caudal medulla neurons and primary afferents.

Partial injury to peripheral nerves has been shown to induce persistent changes in nerve, ganglion, central neurons, and glial cells that are correlated with the development of pain behaviors. Microglia and astrocytes are activated by peripheral insults such as peripheral nerve injury 18, 29 and peripheral inflammation15, spinal glial activation might be a causal factor in the pain hypersensitivity at the spinal level.54 Consistent with these studies, glial activation was also induced in our pIONL model. The regions showing the greatest glial activation were in the caudal medulla. It is interesting to note that temporal changes in the activation of microglia are different from those of astrocytes; microglia were activated earlier than astrocytes by pIONL. This is consistent with results in the CNS where microglia are the initial responders to trauma, ischemia, tumors, and inflammation.30 It is likely that activated microglia may subsequently lead to the activation of astrocytes following pIONL, because Minocycline, an inhibitor of microglia was previously shown to attenuate the development of neuropathic pain following trigeminal sensory nerve injury.40

Incorporation of BrdU following nerve ligation has revealed robust cell proliferation in the spinal cord following partial sciatic nerve ligation in mice.36, 55 Consistent with these results, we found that astrocyte and microglia proliferation was induced by pIONL. At 8 days after pIONL, BrdU-positive cells on the ipsilateral side of the caudal medulla were increased 7 fold compared with the contralateral side in nerve-ligated mice. In addition, BrdU-positive cells were partially co-localized with GFAP and CD11b within the caudal medulla.

Previous studies have shown that injury to neurons in the maxillary division by tooth injury induced GFAP expression and satellite cell reactions around specifically injured (Di-I labeled) dental neurons and in neighboring neurons in that division as well as in mandibular regions.44 Similarly, injury to the mandibular neurons by inferior alveolar nerve crush caused GFAP reactions in satellite cells around those cell bodies.2, 11 Some interesting persisting neuronal or glial responses have been found in neurons and in their satellite cells in the trigeminal ganglion.3 In our study, we showed that GFAP-IR satellite cells increased one week after pIONL. This is consistent with previous reports that up-regulated GFAP expression in satellite cells could be detected in both mandibular and maxillary divisions after inferior alveolar nerve crush.11 Changes in the trigeminal ganglion cells are probably caused by the change of extracellular ionic concentrations or injury-related factors, such as growth factors released following pIONL injury. Future studies combining intracellular recording from ganglion neurons and immunohistochemical methods may resolve these questions.

ATF3 is a member of the activating transcription factor/cAMP-responsive element binding protein family (ATF/CREB family) and is induced in response to stress signals in many different tissues.20 Tsujino et al. 48 first reported that ATF3 was a reliable and sensitive marker for axotomized neurons, and it has now become widely used as an indicator of nerve injury.4, 38, 49 In our study, neuronal expression of ATF3, which is normally minimal, was robustly increased only in ipsilateral side of ION region of TG, but not in contralateral side or in sham operated mice 8 days after pIONL. This finding suggests that upregulated ATF3 in TG is caused by direct injury to ION, rather than by the surgical procedure, ATF3 expression is significantly lower in TG neurons of other regions, on the contralateral side following pIONL, or in TG of sham-ligated mice. Recent studies showed that many neurons in the L4 DRG also exhibited phenotypic plasticity of mRNA or protein expression for a variety of neurotransmitters, neurotrophic factors, ion channels and receptors after L5 SNL.16, 21 In our experimental model, increased ATF3 expression was only seen in ION region. This is likely because of the anatomical differences between trigeminal nerve and sciatic nerve. Trigeminal nerve is surrounded by bone structure, and all branches are isolated by bone canals. Thus, there is likely less damage to adjacent neurons in our pIONL model than the sciatic nerve injury model. In summary, the pIONL model of trigeminal nerve injury is a robust and feasible tool for future studies describing the cellular and molecular mechanisms of neuropathic pain.

Acknowledgements

We thank Dr. Margaret R. Byers for helpful advice during initial experiment planning and for comments on the manuscript, Dr. GW Terman for his helpful advice and discussions and Mr. Dan Messinger for help with the mouse colony. This work was supported by USPHS grant DA11672 from the National Institute of Health (NIH) to CC, by the Treuer Foundation (CC, MX) and by the UW-Anesthesia Research and Training Fund (MRB). MA was supported by a fellowship from the Japan Society for the Promotion of Science.

Footnotes

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REFERENCES

1. Abbadie C, Brown JL, Mantyh PW, Basbaum AI. Spinal cord substance P receptor immunoreactivity increases in both inflammatory and nerve injumry models of persistent pain. Neuroscience. 1996;70:201–209. [PubMed]
2. Anderson LC, von Bartheld CS, Byers MR. NGF depletion reduces ipsilateral and contrlateral trigeminal satellite cell reactions after inferior alveolar nerve injury in adult rats. Exp Neurol. 1998;150:312–320. [PubMed]
3. Anderson LC, Vakoula A, Veinote R. Inflammatory hypersensitivity in a rat model of trigeminal neuropathic pain. Arch Oral Biol. 2003;48:161–169. [PubMed]
4. Averill S, Michael GJ, Shortland PJ, Leavesley RC, King VR, Bradbury EJ, McMahon SB, Priestley JV. NGF and GDNF ameliorate the increase in ATF3 expression which occurs in dorsal rootganglion cells in response to peripheral nerve injury. Eur. J. Neurosci. 2004;19:1437–1445. [PubMed]
5. Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain. 1988;33:87–107. [PubMed]
6. Bongenhielm U, Boissonade FM, Westermark A, Robinson PP, Fried K. Sympathetic nerve sprouting fails to occur in the trigeminal ganglion after peripheral nerve injury in the rat. Pain. 1999;99:567–578. [PubMed]
7. Byers MR, Chudler EH, Iadarola MJ. Chronic tooth pulp inflammation causes transient and persistent expression of Fos in dynorphin-rich regions of rat brainstem. Brain Res. 2000;861:191–207. [PubMed]
8. Callahan BL, Gil AS, Levesque A, Mogil JS. Modulation of mechanical and thermal nociceptive sensitivity in the laboratory mouse by behavioral state. J Pain. 2008;9:174–184. [PubMed]
9. Cameron AA, Cliffer KD, Dougherty PM, Garrison CJ, Willis WD, Carlton SM. Time course of degenerative and regenerative changes in the dorsal horn in a rat model of peripheral neuropathy. J Comp Neurol. 1997;379:428–442. [PubMed]
10. Cherkas PS, Tian-Ying Huang, Pannicke T, Tal M, Richenbach A, Hanani M. The effects of axotomy on neurons and satellite glial cells in mouse trigeminal ganglion. Pain. 2004;110:290–298. [PubMed]
11. Chudler EH, Anderson LC, Byers MR. Trigeminal neuronal activity and glial fibrillary acidic protein immunoreactivity after inferior alveolar nerve crush in the adult rat. Pain. 1997;73:141–149. [PubMed]
12. Chudler EH, Anderson LC. Behavioral and electrophysiological consequences of deafferentation following chronic constriction of the infraorbital nerve in adult rats. Arch Oral Biol. 2002;47:165–172. [PubMed]
13. Clemente JT, Parada CA, Veiga MC, Gear RW, Tambeli CH. Sexual dimorphism in the antinociception mediated by kappa opioid receptors in the rat temporomandibular joint. Neurosci Lett. 2004;372:250–255. [PubMed]
14. Dubner R, Ren K. Brainstem mechanisms of persistent pain following injury. J Orofac Pain. 2004;18:299–305. [PubMed]
15. Fu KY, Light AR, Matsushima GK, Maixner W. Microglial reactions after subcutaneous formalin injection into the rat hind paw. Brain Res. 1999;825:59–67. [PubMed]
16. Fukuoka T, Noguchi K. Contribution of the spared primary afferent neurons to the pathomechanisms of neuropathic pain. Mol. Neurobiol. 2002;26:57–67. [PubMed]
17. Gardell LR, Vanderah TW, Gardell SE, Wang R, Ossipov MH, Lai J, Porreca F. Enhanced evoked excitatory transmitter release in experimental neuropathy requires descending facilitation. J Neurosci. 2003;23:8370–8379. [PubMed]
18. Garrison CJ, Dougherty PM, Kajander KC, Carlton SM. Staining of glial fibrillary acidic protein (GFAP) in lumbar spinal cord increases following a sciatic nerve constriction injury. Brain Res. 1991;565:1–7. [PubMed]
19. Garrison CJ, Dougherty PM, Carlton SM. Quantitative analysis of substance P and calcitonin gene-related peptide immunohistochemical staining in the dorsal horn of neuropathic MK-801 treated rats. Brain Res. 1993;607:205–214. [PubMed]
20. Hai T, Hartman MG. The molecular biology and nomenclature of the activating transcription factor/cAMP responsive element binding family of transcription factors: activating transcription factor proteins and homeostasis. Gene. 2001;273:1–11. [PubMed]
21. Hammond DL, Ackerman L, Holdsworth R, Elzey B. Effects of spinal nerve ligation on immunohistochemically identified neurons in the L4 and L5 dorsal root ganglia of the rat. J. Comp. Neurol. 2004;475:575–589. [PubMed]
22. Henry MA, Freking AR, Johnson LR, Levinson SR. Increased sodium channel immunofluorescence at myelinated and demyelinated sites following an inflammatory and partial axotomy lesion of the rat infraorbital nerve. Pain. 2006;124:222–233. [PubMed]
23. Holland GR. Experimental trigeminal nerve injury. Crit Rev Oral Biol Med. 1996;7:237–258. [PubMed]
24. Imamura Y, Kawamoto H, Nakanishi O. Characterization of heat-hyperalgesia in an experimental trigeminal neuropathy in rats. Exp Brain Res. 1997;116:97–103. [PubMed]
25. Iwata K, Tsuboi Y, Shima A, Harada T, Ren K, Kanda K, Kitagawa J. Central neuronal changes after nerve injury: neuroplastic influences of injury and aging. J Orofac Pain. 2004;18:293–298. [PubMed]
26. Jannetta PJ. Arterial compression of the trigeminal nerve at the pons in patients with trigeminal neuralgia. J Neurosurg. 1967;26:159–162. [PubMed]
27. Jessell T, Tsunoo A, Kanazawa I, Otsuka M. Substance P: depletion in the dorsal horn of rat spinal cord after section of the peripheral processes of primary sensory neurons. Brain Res. 1979;168:247–259. [PubMed]
28. Kajander KC, Xu J. Quantitative evaluation of calcitonin gene-related peptide and substance P levels in rat spinal cord following peripheral nerve injury. Neurosci Lett. 1995;186:184–188. [PubMed]
29. Kalla R, Liu Z, Xu S, Koppius A, Imai Y, Kloss CU, Kohsaka S, Gschwendtner A, Moller JC, Werner A, Raivich G. Microglia and the early phase of immune surveillance in the axotomized facial motor nucleus: impaired microglial activation and lymphocyte recruitment but no effect on neuronal survival or axonal regeneration in macrophage-colony stimulating factor-deficient mice. J Comp Neurol. 2001;436:182–201. [PubMed]
30. Kreutzberg GW. Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 1996;19:312–318. [PubMed]
31. Li JL, Ding YQ, Shigemoto R, Mizuno N. Distribution of trigeminothalamic and spinothalamic-tract neurons showing substance P receptor-like immunoreactivity in the rat. Brain Res. 1996;719:207–212. [PubMed]
32. Lim EJ, Jeon HJ, Yang GY, Lee MK, Ju JS, Han SR, Ahn DK. Intracisternal administration of mitogen-activated protein kinase inhibitors reduced mechanical allodynia following chronic constriction injury of infraorbital nerve in rats. Prog Neuropsychopharmacol Biol Psychiatry. 2007;15(31):1322–1329. [PubMed]
33. Malmberg AB, Basbaum AI. Partial sciatic nerve injury in the mouse as a model of neuropathic pain: behavioral and neuroanatomical correlates. Pain. 1998;76:215–222. [PubMed]
34. Marfurt CF, Rajchert DM. Trigeminal primary afferent projections to "non-trigeminal" areas of the rat central nervous system. J Comp Neurol. 1991;303:489–511. [PubMed]
35. Mørch CD, Hu JW, Arendt-Nielsen L, Sessle BJ. Convergence of cutaneous, musculoskeletal, dural and visceral afferents onto nociceptive neurons in the first cervical dorsal horn. Eur J Neurosci. 2007;26:142–154. [PubMed]
36. Narita M, Yoshida T, Nakajima M, Narita M, Miyatake M, Takagi T, Yajima Y, Suzuki T. Direct evidence for spinal cord microglia in the development of a neuropathic pain-like state in mice. J Neurochem. 2006;97:1337–1348. [PubMed]
37. Nomura H, Ogawa A, Tashiro A, Morimoto T, Hu JW, Iwata K. Induction of Fos protein-like immunoreactivity in the trigeminal spinal nucleus caudalis and upper cervical cord following noxious and non-noxious mechanical stimulation of the whisker pad of the rat with an inferior alveolar nerve transection. Pain. 2002;95:225–238. [PubMed]
38. Obata K, Yamanaka H, Fukuoka T, Yi D, Tokunaga A, Hashimoto N, Yoshikawa H, Noguchi K. Contribution of injured and uninjured dorsal root ganglion neurons to pain behavior and the changes in gene expression following chronic constriction injury of the sciatic nerve in rats. Pain. 2003;101:65–77. [PubMed]
39. Ogawa A, Dai Y, Yamanaka H, Iwata K, Niwa H, Noguchi K. Ca(2+)/calmodulin-protein kinase IIalpha in the trigeminal subnucleus caudalis contributes to neuropathic pain following inferior alveolar nerve transection. Exp Neurol. 2005;192:310–319. [PubMed]
40. Piao ZG, Cho IH, Park CK, Hong JP, Choi SY, Lee SJ, Lee S, Park K, Kim JS, Oh SB. Activation of glia and microglial p38 MAPK in medullary dorsal horn contributes to tactile hypersensitivity following trigeminal sensory nerve injury. Pain. 2006;121:219–231. [PubMed]
41. Robinson PP, Boissonade FM, Loescher AR, Smith KG, Yates JM. Peripheral mechanisms for the initiation of pain following trigeminal nerve injury. J Orofacial Pain. 2004;18:287–292. [PubMed]
42. Seltzer Z, Dubner R, Shir Y. A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain. 1990;43:205–218. [PubMed]
43. Sessle BJ. Orofacial pain. In: Merskey H, Loeser JD, Dubner R, editors. The Paths of Pain 1975–2005. Seattle: IASP Press; 2005. pp. 131–151.
44. Stephenson JS, Byers MR. GFAP immunoreactivity in trigeminal ganglion satellite cells after tooth injury in rats. Exp. Neurol. 1995;131:11–22. [PubMed]
45. Sugiyo S, Takemura M, Dubner R, Ren K. Trigeminal transition zone/rostral ventromedial medulla connections and facilitation of orofacial hyperalgesia after masseter inflammation in rats. J Comp Neurol. 2005;493:510–523. [PubMed]
46. Todd AJ, Puskar Z, Spike RC, Hughes C, Watt C, Forrest L. Projection neurons in lamina I of rat spinal cord with the neurokinin 1 receptor are selectively innervated by substance p-containing afferents and respond to noxious stimulation. J Neurosci. 2002;22:4103–4113. [PubMed]
47. Tsuboi Y, Takeda M, Tanimoto T, Ikeda M, Matsumoto S, Kitagawa J, Teramoto K, Simizu K, Yamazaki Y, Shima A, Ren K, Iwata K. Alteration of the second branch of the trigeminal nerve activity following inferior alveolar nerve transection in rats. Pain. 2004;111:323–334. [PubMed]
48. Tsujino H, Kondo E, Fukuoka T. Activating transcription factor 3 (ATF3) induction by axotomy in sensory and motoneurons: a novel neuronal marker of nerve injury. Mol Cell Neurosci. 2000;15:170–182. [PubMed]
49. Tsuzuki K, Fukuoka T, Sakagami M, Noguchi K. Increase of preprotachykinin mRNA in the uninjured mandibular neurons after rat infraorbital nerve transection. Neurosci Lett. 2003;345:57–60. [PubMed]
50. Vahidy WH, Ong WY, Farooqui AA, Yeo JF. Effects of intracerebroventricular injections of free fatty acids, lysophospholipids, or platelet activating factor in a mouse model of orofacial pain. Exp Brain Res. 2006;174:781–785. [PubMed]
51. Vos BP, Maciewicz RJ. Behavioral changes following ligation of the infraorbital nerve in rat: an animal model of trigeminal neuropathic pain. In: Besson JM, Guilbaud G, editors. Lesions of Primary Afferent Fibers as a Tool for the Study of Clinical Pain. Elsevier: Amsterdam; 1991. pp. 147–158.
52. Vos BP, Strassman AM, Maciewicz RJ. Behavioral evidence of trigeminal neuropathic pain following chronic constriction injury to the rat’s infraorbital nerve. J Neurosci. 1994;14:2708–2723. [PubMed]
53. Vos BP, Hans G, Adriaensen H. Behavioral assessment of facial pain in rats: face grooming patterns after painful and non-painful sensory disturbances in the territory of the rat's infraorbital nerve. Pain. 1998;76:173–178. [PubMed]
54. Watkins LR, Milligan ED, Maier SF. Spinal cord glia: new players in pain. Pain. 2001;93:201–205. [PubMed]
55. Xu M, Bruchas MR, Ippolito DL, Gendron L, Chavkin C. Sciatic nerve ligation-induced proliferation of spinal cord astrocytes is mediated by kappa opioid activation of p38 mitogen-activated protein kinase. J Neurosci. 2007;27:2570–2581. [PMC free article] [PubMed]
56. Xu M, Petraschka M, McLaughlin JP, Westenbroek RE, Caron MG, Lefkowitz RJ, Czyzyk TA, Pintar JE, Terman GW, Chavkin C. Neuropathic pain activates the endogenous kappa opioid system in mouse spinal cord and induces opioid receptor tolerance. J Neurosci. 2004;24:4576–4584. [PMC free article] [PubMed]
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