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Characterization of a Model of Chronic Orofacial Hyperalgesia in the Rat: Contribution of NAV 1.8
The Journal of Pain, Vol 9, No 6 (June), 2008: pp 522-531 Available online at www.sciencedirect.com
Characterization of a Model of Chronic Orofacial Hyperalgesia in the Rat: Contribution of NAV 1.8 James R. Morgan* and G. F. Gebhart† *Department of Pharmacology, The University of Iowa, Iowa City, Iowa. † The Center for Pain Research, University of Pittsburgh, Pittsburgh, Pennsylvania.
Abstract: The purpose of this study was to develop and characterize a model of orofacial inflammatory hyperalgesia. Injection of complete Freund’s adjuvant (CFA) into the upper lip/whisker pad of the rat produced significant and long-lasting thermal (>14 days) and mechanical (>28 days) hyperalgesia in the area of CFA injection. Both indomethacin and morphine, given systemically, significantly attenuated thermal hyperalgesia; the effect of morphine was shown to be opioid receptormediated. We also examined the contribution of the tetrodotoxin-resistant voltage-gated sodium channel Nav1.8 in CFA-produced orofacial mechanical hypersensitivity. Nav1.8 mRNA was increased >2.5-fold in trigeminal ganglion neurons 1 and 2 weeks after CFA treatment, and Nav1.8 protein was increased in the infraorbital nerve over a similar time course. The changes observed were timedependent and had returned to baseline when examined 2 months after inflammation; there were no changes in Nav1.9 mRNA in trigeminal ganglion neurons after CFA treatment. In support of this, Nav1.8 antisense oligodeoxynucleotide treatment significantly attenuated CFA-produced mechanical hypersensitivity. These results document development of a model of inflammatory orofacial hyperalgesia, which, consistent with other reports, indicate a contribution of tetrodotoxin-resistant, voltage-gated sodium channel Nav1.8. Perspective: Orofacial hypersensitivity develops postoperatively as a routine course of orofacial surgery, and mechanical allodynia is characteristic of temporomandibular joint disorder. The results described in this report are novel with respect to the duration of orofacial hypersensitivity produced and suggest that pharmacological targeting of the voltage-gated sodium channel Nav1.8 may be useful in managing hypersensitivity. © 2008 by the American Pain Society Key words: Orofacial pain, sodium channels, thermal hyperalgesia, mechanical hyperalgesia, orofacial pain model.
Editor’s Note: Guest Editor for this article was Timothy Brennan, MD, PhD, Department of Anesthesia, University of lowa Hospitals and Clinics.
P
ersistent and chronic inflammatory hyperalgesia in the orofacial region is encountered in numerous situations in the day-to-day practice of oral and maxillofacial surgery. Inflammation with accompanying hyperalgesia is seen after surgical procedures both as a
Received September 14, 2007; Revised December 31, 2007; Accepted January 8, 2008. Supported by grant R01 DA 02879. Address reprint requests to Dr. G. F. Gebhart, Director, Center for Pain Research, University of Pittsburgh, W1444 BST, 200 Lothrop Street, Pittsburgh, PA 15213-2536. E-mail: [email protected] 1526-5900/$34.00 © 2008 by the American Pain Society doi:10.1016/j.jpain.2008.01.326
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routine postoperative course, and, in normal complications such as alveolar osteitis, after third molar removal. Inflammation of the synovial lining of the temporomandibular joint is a common feature of symptomatic (hyperalgesic) temporomandibular joints.12,16 The presence in inflamed tissues of inflammatory markers and mediators such as interleukin (IL)-1, IL-6, IL-1, substance P, prostaglandin E2, and tumor necrosis factor (TNF)-␣ has been demonstrated to contribute to the process of sensitization and development of hyperalgesia. For example, the presence of such markers in synovial fluid of temporomandibular joints has been reported to be positively correlated to patients with symptomatic temporomandibular joints.1,23,25,26,27,39 Because pain processing and plasticity after peripheral injury appears to differ in spinal nerves and nerves of a cranial origin, such as the trigeminal nerve,3,34 we undertook
ORIGINAL REPORT/Morgan and Gebhart development of a model of orofacial hyperalgesia in the rat. Several inflammatory or neuropathic models of orofacial pain have been reported and served to guide the present study. The irritant mustard oil has been injected into the temporomandibular joint in rats to produce joint inflammation and orofacial nociception that has been monitored as plasma extravasation,13 increased neck and jaw muscle activity,41 and increased Fos protein expression in trigeminal subnucleus caudails.15 Complete Freund’s adjuvant (CFA) also has been injected into the masseter muscle14,18,19,31 or temporomandibular joint20,31,36,42 to produce a localized inflammation associated, for example, with changes in trigeminal neuron excitability and Fos protein expression. Formalin has been injected into the whisker pad of rats as well as into the temporomandibular joint,32 and wiping and grooming behaviors were monitored as an index of orofacial nociception.6,7 An orofacial neuropathic pain model produced by ligation of the infraorbital nerve in the rat was described by Vos et al,37,38 and Subio et al33 described the consequences of inferior alveolar transection on mechanical sensitivity of the whisker pad area in the rat. These orofacial inflammatory and neuropathic models were based on similar models first established in other tissues, typically the rat hind paw. For the most part, quantification or assessment of the nociceptive quality of the stimulus in the orofacial pain models described above have relied on indirect measures (eg, FOS expression, etc) or changes in grooming behavior. Evocative thermal or mechanical stimuli have not been commonly used. The present study describes and animal model of chronic inflammatory orofacial hyperalgesia that is reliable, easy to measure, and involves no prior surgery. We chose to use CFA injected into the upper lip/whisker pad because of its well-characterized and persistent effects described in a model of hind paw hyperalgesia.18 In addition, because voltage-gated sodium channels, particularly tetrodotoxin-resistant sodium channels, underlie peripheral mechanisms of nociceptor sensitization and have been implicated in various inflammatory and chronic pain states,2,28,40 including painful human dental pulp,30 we subsequently examined the role of NaV 1.8 in the development and maintenance of the inflammatory orofacial hyperalgesia produced by CFA.
Materials and Methods Male-Sprague Dawley rats, weighing 420 to 450g, (Harlan, Indianapolis, IN) were housed 1 per cage with ad libitum access to food and water and maintained on a 12-hour light/dark cycle (lights on 6 AM to 6 PM) in an AAALAC (Association For Assessment and Accreditation of Laboratory Animal Care) accredited animal care facility. All experimental procedures were approved by the Institutional Animal Care and Use Committee, The University of Iowa.
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Figure 1. Complete Freund’s adjuvant (CFA) injected into the upper lip/whisker pad (inset) produces thermal hyperalgesia (decrease in head withdrawal latency) at the site of injection. A, Mean baseline withdrawal latencies for 4 different rates and intensities of heating, which were significantly reduced in the same groups of rats (n ⫽ 6/group) for 3 of the 4 intensities of heating when tested 24 hours after CFA injection (*P ⬍ .05). B, Heat ramps to the different intensities of heating were recorded by a copper-constant in thermocouple placed at the air/skin interface of the right upper lip/whisker pad of an anesthetized rat. Boxes placed on the heat ramps represent the range of withdrawal latencies determined in A for each intensity of heating and correspond to estimated temperatures of 48°, 52°, 55°, and 56°C for heat intensities of 45, 50, 55, and 60, respectively.
Behavioral Testing Rats were gently restrained to measure head withdrawal latency to a radiant heat source or withdrawal threshold to Von Frey–like filaments applied to the orofacial area into which CFA had previously been injected (see Inflammation section below and Fig 1 inset for site of CFA injection and testing). Restraint was necessary to permit access to the snout, upper lip, and whisker pad with vision of the rat blocked. Before any testing, rats were habituated to restraint over several days and allowed to acclimate for at least 15 minutes before testing began. Mechanical and thermal testing, when done in the same animals, was carried out sequentially. Mechanical withdrawal thresholds were determined first with
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Characterization of a Model of Chronic Orofacial Hyperalgesia in the Rat
von Frey–like nylon monofilaments (Stoelting, Chicago, IL). von Frey filaments of logarithmic incremental stiffness (1.5–75 g) were applied perpendicular to the right whisker pad in the intended injection site with enough force to cause the filament to bend. In the absence of a head withdrawal, the filament 1 log value greater was applied. If the head was withdrawn, a positive response was recorded and the filament 1 log value below was applied. After the first “crossover” from a negative to a positive response or vice versa, 5 additional presentations of stimuli were made. The threshold for response was calculated as described by Chaplan et al.5 Ten minutes after completion of mechanical testing, thermal testing was carried out. Radiant heat was provided by a high-intensity projector lamp (8 V, 50 W; Sylvania, Danvers, MA) located 70 mm from the target area projecting through a 10-mm diameter aperture. A rheostat controlled the voltage applied to the lamp and thus intensity of the heat stimulus. A stop watch was started when the lamp was activated and stopped when the rat removed/ flinched its head to escape heat from the lamp. Each rat was tested 3 times with a 5-minute interval between tests. The withdrawal latencies were then averaged to obtain a mean withdrawal latency for each time of testing. As illustrated in the Results section, sensitization did not result from baseline testing (or play a significant role in these experiments) because responses to neither mechanical nor thermal stimuli changed in saline-treated rats that also were subjected to needle puncture of skin and potential mechanical stimulation from the volume injected.
Inflammation After baseline withdrawal latency and threshold measurements at the intended site of inflammation, rats were briefly sedated with halothane and 50 L of CFA (Sigma Chemical Co., St. Louis, MO) or saline was injected into the right upper lip/whisker pad, lateral to the nostrils, with a 30-gauge needle. Follow-up measurements were made from 24 hours to 4 weeks, depending on the experimental protocol. An arbitrary cutoff time of 20 seconds was used for heat stimuli to limit tissue damage.
Drugs The effects of indomethacin (0.5, 1.0, 5.0 or 10 mg/kg intraperitoneally in volumes of 1 mL/kg; Sigma), a nonsteroidal anti-inflammatory drug with the same mechanism of action as ibuprofen, morphine sulfate (0.5, 1.0, or 5 mg/kg subcutaneously in volumes of 1 mL/kg; Spectrum, Gardena, CA), a -opioid receptor agonist, and vehicle (saline) were tested 24 hours after CFA-injection into the upper lip/whisker pad. Drugs or vehicle were given 30 minutes before testing for withdrawal from the radiant heat source. Naloxone (1.0 mg/kg; Research Biochemicals International; Natick, MA) was used to establish the opioid receptor-mediated action of morphine.
RT-PCR Two trigeminal ganglia ipsilateral to the site of CFA or saline injection in the right whisker pad were removed
from sodium pentobarbital (Nembutal; Abbott Laboratories, North Chicago, IL)-anesthetized rats at various times after treatment (24 hours to 4 weeks; 2 rats/time after treatment) and pooled for RT-PCR. The ganglia were immersed into RNAlater (Ambion, Austin, TX), an RNA stabilization solution until processing. The tissue was then homogenized in TRIzol reagent and total RNA was extracted. cDNA from the respective tissue samples were reverse-transcribed from 500 ng of total RNA. PCR primers targeted specific and unique regions of the sodium channels of interest as determined by a blast gene search and defined amplicons of the following sizes: NaV1.8 to 513 bp and NaV1.9 to 391 bp. Thermal cycling parameters were as follows: 45 minutes/48°C, 2 min/94°C (1 cycle); 30 seconds/94°C, 1 minute/66°C, 2 minutes/68°C (25 cycles); 10 minutes/68°C (1 cycle). cDNA from each tissue was also PCR amplified using primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to demonstrate template viability and act as an internal control for total RNA. PCR amplicons from trigeminal ganglia were confirmed by nucleotide sequence analysis.
Immunohistochemistry Two rats injected with either CFA or saline into the right whisker pad were deeply anesthetized with 10% chloral hydrate and perfused transcardially with 200 mL of 0.1 M phosphate buffered saline (PBS) followed by 500 mL of 10% formalin. The right infraorbital nerve (ION) was dissected out and postfixed in 10% formalin for 4 to 5 hours at 4°C followed by immersion in 20% sucrose in 0.1 M PBS at 4°C overnight. The tissue was embedded in optimal cutting temperature embedding medium (OCT), cut on a cryostat at 40-M thickness, and placed on a glass slide. The sections were preincubated in 3% normal donkey serum and 0.3% triton X-100 for 1 hour at room temperature and then incubated in primary NaV1.8 antibody at 1:500 in the blocking solution. The sections were washed in PBS and then incubated in biotinylated antirabbit IgG (1:500), followed by incubation in Cy3-streptavidin complex (1:1000) for 45 minutes at room temperature. The slides were washed in PBS, coverslipped with Vectashield mounting medium, and stored at 4°C in the dark until viewed. There was no staining in the absence of primary antibody.
Oligodeoxynucleotides Both antisense and mismatch oligodeoxynucleotides (ODNs) were studied. Antisense ODN (5’-TCCTCTGTGCTTGGTTCTGGCCT-3’) complementary to nucleotides 107-129 of the coding region of rat Nav1.8 and a corresponding mismatch ODN sequence (5’-TCCTTCGTGCTGTGTTCGTG CCT3’) were synthesized as phosphodiester ODNs by HPLC purification (Integrated DNA Technologies, Inc., Coralville, IA). The mismatch sequences were derived from the respective antisense sequences by scrambling 6 bases, giving rise to a 25% mismatch with the target sequences. This level of mismatch was sufficient to abolish the selectivity of the ODN for the target sequence. These sequences were screened with the BLAST (basic local align-
ORIGINAL REPORT/Morgan and Gebhart ment search tool) algorithm against the Genbank Database. Search results indicated that there was no significant complementarity (equal or greater than 70% as defined by the mismatch control) with known sequences for other ion channels. The fluorescence labeling of the antisense and mismatch ODNs was carried out by conjugation of the fluorophor TAMRA to the 5’ end of the ODN, and the fluorescence-labeled ODNs were then purified with HPLC (Integrated DNA Technologies, Inc.). All ODNs were reconstituted in nuclease-free, ultrapure water to a final concentration of 9 g/L. Intra– cisterna magna injections of ODNs by indwelling cannula targeting the brainstem overlying the trigeminal ganglion were made daily for either 2 or 5 days in a volume of 5 L followed by a 10-L saline flush. Evaluation of the uptake of ODN by the trigeminal ganglion was evaluated at 12 hours after ODN administration. Rats were deeply anesthetized with 10% chloral hydrate and perfused transcardially with 200 mL of 0.1 M PBS followed by 500 mL of 10% formalin. The right trigeminal ganglion was dissected out and postfixed in 10% formalin for 4 to 5 hours at 4°C followed by immersion in 20% sucrose in 0.1 M PBS at 4°C overnight. The tissue was embedded in OCT embedding medium, cut on a cryostat at 30-M thickness, and placed on a glass slide, coverslipped with Vectashield (Vector, Burlingame, CA), and viewed with a fluorescent microscope.
525 rates of heating were used, the estimated temperatures producing withdrawal do not represent temperature at the receptive ending in skin. Twenty-four hours after injection of CFA into the upper lip/whisker pad, withdrawal latencies to thermal stimulation were significantly reduced (ie, CFA produced a thermal hyperalgesia; Fig 1A). The magnitude and duration of CFA-produced thermal and mechanical hyperalgesia were examined in subsequent experiments.
CFA Injection Into the Upper Lip/Whisker Pad Produces Persistent Hyperalgesia We chose to use heating intensities of 50 and 55 because they produced consistent head withdrawal latencies and afforded the opportunity to evaluate and quantify hyperalgesia as well as antinociception. CFA but not saline injection into the upper lip/whisker pad produced significant, persistent thermal hyperalgesia. As illus-
Data Analysis Data were analyzed by parametric or nonparametric ANOVAs as appropriate with follow-up parametric or nonparametric post hoc analyses. Group sizes in these behavioral experiments were typically 6. Baseline measurements were taken only at time or day 0 (zero). Subsequent testing was done after either saline or CFA treatment and compared with baseline values taken on time/ day 0. A value of P ⬍ .05 was considered statistically significant.
Results Response Latency Is Intensity Dependent Applying different lamp intensities that produced different rates of heating and maximum temperatures, each rat (n ⫽ 5) was subjected to a random ordering of the 4 different lamp intensities both before and 24 hours after CFA injection (Fig 1A). Head withdrawal latency was clearly intensity-dependent: 14.4 ⫾ 0.8, 11.5 ⫾ 0.6, 9.7 ⫾ 0.5, and 6.5 ⫾ 0.7 seconds at lamp intensities of 45, 50, 55, and 60, respectively (Fig 1B). The estimated temperatures at which head withdrawal was produced were determined in a naive, anesthetized rat with a thermocouple attached to the right upper lip to record temperature at the air/hairy skin interface. Lamp intensities of 45, 50, 55, and 60 resulted in estimated withdrawal temperatures of ⬃ 48°, 52°, 55°, and 56°C, respectively, based on mean withdrawal latencies and extrapolating from the heating curves (Fig 1B). Because temperatures at the air/hairy skin interface (and not temperature at the receptive endings in skin) and different
Figure 2. Complete Freund’s adjuvant (CFA) injected into the upper lip/whisker pad produces long-lasting thermal hyperalgesia (decrease in head withdrawal latency) at the site of injection. A, Mean withdrawal latencies for 2 different rates and intensities of heating after saline injection into the upper lip/whisker pad (n ⫽ 6). B, Mean withdrawal latencies for 2 different rates and intensities of heating after CFA injection into the upper lip/whisker pad (n ⫽ 6). Significant thermal hyperalgesia was evident when first tested 24 hours after CFA injection and persisted for 1 or 2 weeks at different intensities of heating (*P ⬍ .05 vs baseline).
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Characterization of a Model of Chronic Orofacial Hyperalgesia in the Rat
trated in Fig 2A, saline injection into the upper lip produced no changes (relative to baseline, b) in thermal withdrawal latencies at either of the 2 lamp intensities tested, 50 and 55. CFA, however, produced a significant and robust thermal hyperalgesia at both lamp intensities that persisted for 1 week and was evident for 2 weeks at a lamp intensity of 55; later times were not tested. In other groups of rats (n ⫽ 6), CFA but not saline injection into the upper lip/whisker pad produced a significant mechanical hypersensitivity that was maintained for at least 4 weeks (Fig 3). The median withdrawal force was significantly reduced from 28.84 g at baseline to forces ranging between 2.75 and 7.21 g over the 4 weeks of testing; later times were not tested.
Figure 4. Indomethacin significantly attenuated thermal hyperalgesia at 2 dosages (5 and 10 mg/kg; *P ⬍ .05 vs vehicle; n ⫽ 6/treatment group) at 2 intensities of heating when tested 24 hours after complete Freund’s adjuvant (CFA) injection into the upper lip/whisker pad.
Pharmacological Modulation of Orofacial Hyperalgesia
Figure 3. Complete Freund’s adjuvant (CFA) injected into the upper lip/whisker pad produces long-lasting mechanical hypersensitivity (decrease in von Frey filament withdrawal threshold) at the site of injection. A, Median withdrawal thresholds after saline injection into the upper lip/whisker pad (n ⫽ 6). B, Median withdrawal thresholds after CFA injection into the upper lip/whisker pad (n ⫽ 6). Significant mechanical hypersensitivity was evident when first tested 24 hours after CFA injection and persisted for 4 weeks (*P ⬍ .05 vs baseline [b] and vs saline treatment). Boxes represent the 25th and 75th percentiles and whiskers the 10th and 90th percentiles.
To further characterize this model of orofacial hyperalgesia in the rat, the antihyperalgesic/antinociceptive effects of indomethacin and morphine were tested 24 hours after CFA injection into the upper lip/ whisker pad. Indomethacin significantly attenuated thermal hyperalgesia at the greatest doses tested (5 and 10 mg/kg; Fig 4). Morphine also significantly reversed CFA-produced thermal hyperalgesia at all doses tested, and at the greatest dose tested (5 mg/kg) produced a significant antinociception (Fig 5). There were no differences in the efficacy of morphine at the 2 lamp intensities tested, 50 and 55. To verify that the effects of morphine were receptor-mediated, naloxone was shown to reverse the significant antinociception produced by 5 mg/kg morphine (Fig 6). Interestingly, naloxone alone produced no change in head withdrawal latency compared with vehicle administration, suggesting that there is no contribution in this model by endogenous opioids (Fig 5).
ORIGINAL REPORT/Morgan and Gebhart
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Nav1.8 Immunoreactivity Increases in the ION After CFA Injection Rats that received a CFA injection into the right upper lip/whisker pad exhibited an increase in immunoreactivity for Navl.8 protein in the infraorbital nerve (ION) 48 hours and 1 week after CFA treatment (Fig 7B), revealing transport of Nav1.8 protein to the periphery where it has been established as critical to nociceptive processing.4 There was very little evidence of Nav1.8 in the ION from untreated (naive) rats and no staining in the absence of primary antibody (not shown).
Confirmation of Antisense Delivery Into the Trigeminal Ganglion Twelve hours after a bolus injection of TAMRA-labeled Nav1.8 antisense ODN (n ⫽ 2), many cell bodies in the trigeminal ganglion were highly fluorescent, indicating uptake of the antisense ODN (Fig 7C). Although we did not measure the size of cell profiles, the fluorescence intensity appeared not to distribute among trigeminal ganglion cells based on cell size, suggesting that neither a specific subset of cell nor that all cells took up the labeled ODN, and also that the labeling was not due to some nonspecific diffusion or optical artifact. Control animals injected with saline (n ⫽ 2) exhibited a low level of autofluorescence.
Attenuation of Behavioral Mechanical Hypersensitivity With Antisense But Not Mismatch ODNs Figure 5. Morphine significantly attenuated thermal hyperalgesia at all dosages (*P ⬍ .05 vs vehicle) at 2 intensities of heating when tested 24 hours after complete Freund’s adjuvant (CFA) injection into the upper lip/whisker pad (n ⫽ 6/treatment group). The greatest dose of morphine (5 mg/kg) was antinociceptive at both intensities of heating. Naloxone (1 mg/kg) had no effect on CFA-produced thermal hyperalgesia.
Antisense administration into the cisterna magna directly over the trigeminal ganglion before injection of CFA into the right upper lip/whisker pad did not significantly attenuate mechanical hyperalgesia in the 4 rats
NaV1.8 mRNA Content in the Trigeminal Ganglion Increases After CFA Injection Compared with rats that received a saline injection into the right trigeminal ganglion distribution, rats that received CFA exhibited an upregulation of the Nav1.8 sodium channel transcript that peaked at 1 to 2 weeks and returned to near baseline levels at 4 weeks as determined by RT-PCR (Fig 7A). We used imaging software (Kodak Imager, Seattle, WA) to pixelate the blots into quantitative values on replicate samples; the increase in pixelation density 24 hours after CFA injection was found to be 1.2 times baseline. It was 1.9 times baseline at 48 and 72 hours, 2.8 times at 1 week, 2.5 times at 2 weeks, and 1.1 times at 4 weeks after CFA treatment. No increase in message content was observed for another tetrodotoxin-resistant sodium channel (Nav1.9) at any time after CFA injection compared with saline (Fig 7A).
Figure 6. Naloxone (1 mg/kg) reversed the antihyperalgesic and antinociceptive effect of morphine (5 mg/kg) in rats 24 hours after complete Freund’s adjuvant injection into the upper lip/whisker pad (represented by the vertical shaded bar) (*P ⬍ .05 vs 5 mg/kg morphine; n ⫽ 6/treatment group).
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Characterization of a Model of Chronic Orofacial Hyperalgesia in the Rat
Discussion The present study describes development and characterization of a reliable and useful model of orofacial thermal hyperalgesia and mechanical hypersensitivity in the rat. Thermal withdrawal responses were appropriately intensity-dependent; faster withdrawal latencies were associated with more rapid and greater increases in applied heat. The thermal hyperalgesia and mechanical hypersensitivity produced by CFA injected into the upper lip/whisker pad is robust, persistent and subject to appropriate pharmacologic modulation. NaV 1.8 is implicated in the development and maintenance of the orofacial hyperalgesia by its upregulation in the infraorbital nerve and trigeminal ganglion after CFA injection and modulation of mechanical hypersensitivity by antisense ODN treatment.
Figure 7. A, NaV 1.8 (top) and 1.9 (bottom) message in trigeminal ganglia from naive and complete Freund’s adjuvant (CFA)treated rats (n ⫽ 2 ganglia pooled for each treatment time). The message for NaV 1.8 increases after CFA injection into the upper lip/whisker pad before returning to baseline at 1 month (see text for details). There was no change in NaV 1.9 message after CFA treatment. B, Immunohistochemistry for NaV 1.8 protein in the infraorbital nerve (ION), which increases after CFA injection into the upper lip/whisker pad. (C) NaV 1.8 antisense ODN accesses trigeminal ganglion neurons after intracisternal injection.
tested. There was a nonsignificant increase to 8.19 g in the mechanical withdrawal threshold on day 3 in antisense ODN-treated rats from a median 3.97 g withdrawal threshold on days 1 and 3 in mismatch ODN-treated rats (Fig 8A). To examine whether Navl.8 was involved in the maintenance of orofacial mechanical hypersensitivity, antisense or mismatch ODN administration was initiated after development of mechanical hypersensitivity (day 5 after CFA injection into the upper lip/whisker pad). A significant attenuation of the mechanical hypersensitivity (withdrawal threshold increased from a median 4.34 g to 11.95 g) was noted in Nav1.8 antisense (but not mismatch) ODN-treated rats on day 7; Fig 8B). After discontinuation of antisense ODN treatment on day 7, mechanical hypersensitivity was fully restored when tested on day 11.
Figure 8. Antisense (AS) but not mismatch (MM) ODN reverses complete Freund’s adjuvant (CFA)-produced mechanical hypersensitivity. Median withdrawal thresholds are illustrated in A and B for 2 ODN treatment protocols. The onset and termination of ODN administration is illustrated below the box plots; boxes represent the 25th and 75th percentiles and whiskers the 10th and 90th percentiles. A, Median withdrawal thresholds before (⫺2 days), at baseline (b; day 0) and on days 1, 3, and 7 after CFA injection into the upper lip/whisker pad (n ⫽ 4). B, Median withdrawal thresholds at baseline (b; day 0) and on days 4, 7, and 11 after CFA injection into the upper lip/whisker pad (n ⫽ 6). *P ⬍ .05 vs MM on the same day of testing.
ORIGINAL REPORT/Morgan and Gebhart Injection of small volumes of CFA to induce inflammatory hyperalgesia limited to the area of its administration has been commonly employed to produce both thermal and mechanical hypersensitivity of the hind paw.17,18 Animals injected with CFA do not immediately develop edema or erythema; however, by 12 to 24 hours, a pronounced swelling usually accompanied by redness can be detected at the injection site. The hyperalgesia that develops after CFA injection into animals is consistent with the initial diffuse throbbing and persistent thermal (7 days) and mechanical (84 days) hypersensitivity produced in humans.11 Although we limited thermal testing to 2 weeks and mechanical testing to 4 weeks after CFA injection into the upper lip/whisker pad, the mechanical hypersensitivity appeared to persist longer than the thermal hyperalgesia. This is consistent with the initial characterization of this model in the rat hindpaw.18 We further characterized the model by examining its sensitivity to pharmacological modulation by drugs representative of the major classes of analgesics used in dental practice, an opioid and a nonsteroidal antiinflammatory drug. In this study, slowing of the faster, hyperalgesic head withdrawal latency was seen with the systemic administration of both indomethacin and morphine. The dosages at which the antihyperalgesic effects were produced are consistent with effective dosages of morphine in related orofacial pain studies. For example, dosages of 2.5 and 5.0 mg/kg morphine attenuated to about 25% and 5% the chewing and grooming behaviors produced by mustard oil injected into the temporomandibular joint of the rat but were less efficacious in reducing Fos expression in subnucleus caudalis (to ⬃75% and ⬃50%, respectively) in the same study.15 After injection of formalin into the vibrissal pad of the rat, Eisenberg et al9 reported that 1.0 mg/kg morphine administered systemically 30 minutes before formalin reduced significantly both the early (to 4% of control) and late (to 29% of control) phases of formalin-induced face grooming. Notably, in the present study, the greatest dose of morphine tested (5 mg/kg) was not only antihyperalgesic but antinociceptive as well, producing an almost doubling of the thermal withdrawal latency relative to the baseline withdrawal latency in saline-treated rats, an effect that was opioid receptor mediated. Indomethacin has also been tested in orofacial pain models in rats. Thut et al36 studied feeding behavior in rats after CFA-produced inflammation of the temporomandibular joint (TMJ) or masseter muscle and reported that 4 mg/kg indomethacin reversed the inflammation-induced increase in time between food rewards in an operant setting. In a similar experiment, Ro31 inflamed the masseter muscles bilaterally with CFA, which reduced bite force; indomethacin (4 mg/kg) prevented CFA-induced changes in bite force. CFA injection as used here also produces an increase in both Nav1.8 message in trigeminal ganglion neurons and protein in the infraorbital nerve. The increase in expression of Nav1.8 mRNA after CFA injection ap-
529 pears to peak about 1 week before returning to preCFA injection expression at 4 weeks. This effect of CFA was limited to Nav1.8; no change in Nav1.9 mRNA expression was found after inflammation at any time measured. Qualitatively, there was also an increased transport of Nav1.8 protein toward peripheral nerve terminals when examined in the infraorbital nerve 48 hour and 7 days after CFA injection into the upper lip/whisker pad. These observations are consistent with reports of others. For example, Tanaka et al35 and Porreca et al29 demonstrated increased Nav 1.8 message and protein expression, respectively, in dorsal root ganglion neurons 4 days after CFA inflammation of the rat hind paw. Coggeshall et al8 reported a significant increase in the proportion of Nav1.8 labeled myelinated axons (6-fold) and a smaller increase in Nav1.8 labeled unmyelinated axons (1.5-fold) 48 hours after hind paw inflammation. In contrast, the percentages of Nav1.9 labeled axons either decreased or did not change. Evidence that Nav1.8 contributes to the hypersensitivity documented here is provided by the effects of Nav1.8 antisense ODN on mechanical withdrawal thresholds after CFA injection. Although the effects of Nav1.8 antisense ODN treatment were modest and statistically significant in only 1 of the 2 experiments, we believe that Nav1.8 significantly contributes to the hypersensitivity produced in this model for several reasons. First, CFA increased both Nav1.8 message and protein in a time-dependent and reversible manner. Second, the TAMRA-labeled Nav1.8 antisense ODN was shown to be present in trigeminal ganglion neurons after intracisternal administration. Third, antisense ODN and not mismatch ODN reversed established mechanical hypersensitivity, which was fully restored after cessation of antisense ODN administration. Finally, others have established the efficacy of Nav1.8 antisense treatment in other models.10,21,24 Khasar et al22 documented an attenuation of CFA-induced behavioral hyperalgesia and the mechanical hypersensitivity induced by direct injection of prostaglandin E2 into the rat hind paw. Others have also documented attenuation of inflammatory hyperalgesia by Nav1.8 antisense treatment.21,29 Similarly, Nav1.8 antisense treatment effectively attenuates the allodynia and mechanical hypersensitivity in models of neuropathic pain.10,21,24,29 The magnitude of effect of Nav1.8 antisense treatment varies in the different studies, most of which delivered the antisense ODN into the intrathecal space of rats, where it has direct access to dorsal root ganglion neurons. In the present experiments, we delivered the antisense ODN in the cisterna magna superior to the right trigeminal ganglion, and the modest magnitude of effect we observed probably reflects that the concentration of antisense ODN at its intended site of action was less than in other studies where it was delivered intrathecally. The model of chronic inflammatory orofacial pain described in this report is associated with long-lasting thermal and mechanical hypersensitivity. Appropriately, the hypersensitivity is modulated by analgesic pharmacolog-
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Characterization of a Model of Chronic Orofacial Hyperalgesia in the Rat
ical strategies. Like other models of inflammatory hypersensitivity, the tetrodotoxin-resistant, voltage-gated sodium channel Nav1.8 plays an important role in the increased sensitivity to applied stimuli that develops after orofacial inflammation.
Acknowledgments The authors gratefully acknowledge Michael Burcham for preparation of the graphics and Joanne Hirt for secretarial assistance.
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2. Amir R, Argoff CE, Bennett GJ, Cummins TR, Durieux ME, Gerner P, Gold MS, Porreca F, Strichartz G: The Role of sodium channels in chronic inflammatory and neuropathic pain. J Pain 7:S1-S29, 2006 3. Bonegenhielm 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 82:283-288, 1999 4. Brock JA, McLachlan EM, Belmonte C: Tetrodotoxin-resistant impulses in single nociceptor nerve terminals in guineapig cornea. J Physiol 512:211-217, 1998 5. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL: Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Meth 53:55-63,1994 6. Clavelou P, Dallel R, Oriiaguet T, Woda A, Roboisson P: The orofacial formalin test in rats: Effects of different formalin concentrations. Pain 62:295-301, 1995 7. Clavelou P, Pajot J, Dallel R, Raboisson P: Application of the formalin test to the study of orofacial pain in the rat. Neurosci Lett 103:349-353, 1989 8. Coggeshall RE, Tate S, Carlton SM: Differential expression of tetrodotoxin-resistant sodium channels Na v1.8 and Nav1.9 in normal and inflamed rats. Neurosci Lett 355:45-48, 2004 9. Eisenberg E, Vos BP, Stassman AM: The peripheral antinociceptive effect of morphine in a rat model of facial pain. Neurosci 72:519-525, 1996 10. Gold MS, Weinreich D, Kim C, Wang R, Treanor J, Porreca F, Lai J: Redistribution of NaV1.8 in uninjured axons enables neuropathic pain. J Neurosci 23:158-166, 2003 11. Gould HJ: Complete Freund’s adjuvant-induced hyperalgesia: A human perception. Pain 85:301-303, 2000 12. Gynther GW, Dijkgraaf LC, Reinholt FP, Holmlund AB, Liem RS, de Bont LG: Synovial inflammation in arthroscopically obtained biopsy specimens from the temporomandibular joint: A review of the literature and a proposed histological grading system. J Oral Maxillofac Surg 56:1281-1287, 1998 13. Haas DA, Nakanishi O, MacMillan RE, Jordan RC, Hu JW: Development of an orofacial model of acute inflammation in the rat. Arch Oral Biol 37:417-422, 1992 14. Harriott AM, Dessem D, Gold MS: Inflammation increases the excitability of masseter muscle afferents. Neuroscience 141:433-442, 2006 15. Hartwig AC, Mathias SL, Law AS, Gebhart GF: Characterization and opioid modulation of inflammatory temporo-
17. Hylden JLK, Nahin RL, Traub RJ, Dubner R: Expansion of receptive fields of spinal lamina I projection neurons in rats with unilateral adjuvant-induced inflammation: The contribution of dorsal horn mechanisms. Pain 37:229-243, 1989 18. Iadarola MJ, Brady LS, Draisci G, Dubner R: Enhancement of dynorphin gene expression in spinal cord following experimental inflammation: Stimulus specificity, behavioral parameters and opioid receptor binding. Pain 35:313-326, 1988 19. Ikeda T, Terayama R, Jue SS, Sugiyo S, Dubner R, Ren K: Differential rostral projections of caudal brainstem neurons receiving trigeminal input after masseter inflammation. J Comp Neurol 465:220-233, 2003 20. Imbea H, Iwata K, Zhou QQ, Zou S, Dubner R, Ren K: Orofacial deep and cutaneous tissue inflammation and trigeminal neuronal activation: Implications for persistent temporomandibular pain. Cell Tiss Org 169:238-247, 2001 21. Joshi SK, Mikusa JP, Hernandez G, Baker S, Shieh C-C, Neelands T, Zhang Z-F, Niforatos W, Kage K, Han P, Krafte D, Faltynek C, Sullivan JP, Harvis MF, Honoré P. Involvement of the TTX-resistant sodium channel Nav 1.8 in inflammatory and neuropathic, but not post-operative, pain states. Pain 123:75-82, 2006 22. Khasar SG, Gold MS, Levine JD: A tetrodotoxin-resistant sodium current mediates inflammatory pain in the rat. Neurosci Lett 256:17-20, 1998 23. Kubota E, Kubota T, Matsumoto J, Shibata T, Muradami KI: Synovial fluid cytokines and proteinases as markers of temporomandibular joint disease. J Oral Maxillofac Surg 56: 192-198, 1998 24. Lai J, Gold MS, Kim C-S, Biana D, Ossipov MH, Hunter JC, Porreca F. Inhibition of neuropathic pain by decreased expression of the tetrodotoxin-resistant sodium channel, NaV1.8. Pain 95:143-152, 2002 25. Murakami KI, Shibata T, Kubota E, Maeda H: Intra-articular levels of prostaglandin E2, hyaluronic acid, and chondroitin-4 and ⫺6 sulfates in the temporomandibular joint synovial fluid of patients with internal derangement. J Oral Maxillofac Surg 56:199-203, 1998 26. Nordahl S, Alstergren P, Eliasson S, Kopp S: Interleukin-1 beta in plasma and synovial fluid in relation to radiographic changes in arthritic temporomandibular joints. J Oral Sci 106:559-563, 1998 27. Nordahl S, Alstergren P, Kopp S: Tumor necrosis factoralpha in synovial fluid and plasma from patients with chronic connective tissue disease and its relation to temporomandibular joint pain. J Oral Maxillofac Surg 58:525530, 2000
ORIGINAL REPORT/Morgan and Gebhart 28. Novakovic SD, Tzoumaka E, McGivern JG, Haraguchi M, Sangameswaran L, Gogas KR, Eglen RM, Hunter JC: Distribution of the tetrodotoxin-resistant sodium channel PN3 in rat sensory neurons in normal and neuropathic conditions. J Neurosci 18:2174-2187, 1998 29. Porreca F, Lai J, Biau D, Wegert S, Ossipov MH, Eglen LRM, Kassotakis L, Novakovic S, Robert DK, Sangameswanran L, Hunter JC: A comparison of the potential role of the tetrodotoxin-insensitive sodium channels, PN3/SNS and NaN/SNS2, in rat models of chronic pain. Proc Natl Acad Sci U S A 96:7640-7644, 1999
531 JA, Waxman SG: SNS Na channel expression increases in dorsal root ganglion neurons in the carrageenan inflammatory pain model. NeuroReport 9:967-972, 1998 36. Thut PD, Hermanstyne TO, Flake NM, Gold MS: An operant conditioning model to assess changes in feeding behavior associated with temporomandibular joint inflammation in the rat. J Orofacial Pain 21:7-18, 2007 37. 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 76:173-178, 1998
30. Renton T, Yiangou Y, Plumpton C, Tate S, Bountra C, Anand P: Sodium channel Nav1.8 immunoreactivity in painful human dental pulp. BMC Oral Health 5: DOI: 10.1186/ 1472-6831-5-5, 2005
38. Vos BP, Strassman AM, Maciewicz RJ: Behavioral evidence of trigeminal neuropathic pain following chronic constriction injury to the rat’s infraorbital nerve. J Neurosci 14: 2708-2723, 1994
31. Ro JY: Bite force measurement in awake rats: A behavioral model for persistent orofacial muscle pain and hyperalgesia. J Orofacial Pain 19:159-167, 2005
39. Yoshida H, Fujita S, Nishida M, Iizuka T: The expression of substance P in human temporomandibular joint samples: An immunohistochemical study. J Oral Rehabil 26:338-344, 1999
32. Roveroni RC, Parada CA, Ceciìlia M, Veiga FA, Tambeli CH: Development of a behavioral model of TMJ pain in rats: The TMJ formalin test. Pain 94:185-191, 2001 33. Subio Y, Rakeda 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 111:323-334, 2004 34. Tal M, Devor M: Ectopic discharge in injured nerves: Comparison of trigeminal and somatic afferents. Brain Res 579:148-151, 1992 35. Tanaka M, Cummins TR, Ishikawa K, Dib-Hajj SD, Black
40. Yoshimura N, Seki S, Novakovic SD, Tzoumaka E, Erickson VL, Erickson KA, Chancellor MB, de Groat WC: The involvement of the tetrodotoxin-resistant sodium channel NaV1.8 (PN3/SNS) in a rat model of visceral pain. J Neurosci 21:8690-8696, 2001 41. Yu XM, Sessle BJ, Vernon H, Hu JW: Effects of inflammatory irritant application to the rat temporomandibular joint on jaw and neck muscle activity. Pain 60:143-149, 1995 42. Zhou Q, Imbe H, Dubner R, Ren K: Persistent Fos protein expression after orofacial deep or cutaneous tissue inflammation in rats: Implications for persistent orofacial pain. J Comp Neurol 412:276-291, 1999
Characterization of a Model of Chronic Orofacial Hyperalgesia in the Rat: Contribution of NAV 1.8 James R. Morgan* and G. F. Gebhart† *Department of Pharmacology, The University of Iowa, Iowa City, Iowa. † The Center for Pain Research, University of Pittsburgh, Pittsburgh, Pennsylvania.
Abstract: The purpose of this study was to develop and characterize a model of orofacial inflammatory hyperalgesia. Injection of complete Freund’s adjuvant (CFA) into the upper lip/whisker pad of the rat produced significant and long-lasting thermal (>14 days) and mechanical (>28 days) hyperalgesia in the area of CFA injection. Both indomethacin and morphine, given systemically, significantly attenuated thermal hyperalgesia; the effect of morphine was shown to be opioid receptormediated. We also examined the contribution of the tetrodotoxin-resistant voltage-gated sodium channel Nav1.8 in CFA-produced orofacial mechanical hypersensitivity. Nav1.8 mRNA was increased >2.5-fold in trigeminal ganglion neurons 1 and 2 weeks after CFA treatment, and Nav1.8 protein was increased in the infraorbital nerve over a similar time course. The changes observed were timedependent and had returned to baseline when examined 2 months after inflammation; there were no changes in Nav1.9 mRNA in trigeminal ganglion neurons after CFA treatment. In support of this, Nav1.8 antisense oligodeoxynucleotide treatment significantly attenuated CFA-produced mechanical hypersensitivity. These results document development of a model of inflammatory orofacial hyperalgesia, which, consistent with other reports, indicate a contribution of tetrodotoxin-resistant, voltage-gated sodium channel Nav1.8. Perspective: Orofacial hypersensitivity develops postoperatively as a routine course of orofacial surgery, and mechanical allodynia is characteristic of temporomandibular joint disorder. The results described in this report are novel with respect to the duration of orofacial hypersensitivity produced and suggest that pharmacological targeting of the voltage-gated sodium channel Nav1.8 may be useful in managing hypersensitivity. © 2008 by the American Pain Society Key words: Orofacial pain, sodium channels, thermal hyperalgesia, mechanical hyperalgesia, orofacial pain model.
Editor’s Note: Guest Editor for this article was Timothy Brennan, MD, PhD, Department of Anesthesia, University of lowa Hospitals and Clinics.
P
ersistent and chronic inflammatory hyperalgesia in the orofacial region is encountered in numerous situations in the day-to-day practice of oral and maxillofacial surgery. Inflammation with accompanying hyperalgesia is seen after surgical procedures both as a
Received September 14, 2007; Revised December 31, 2007; Accepted January 8, 2008. Supported by grant R01 DA 02879. Address reprint requests to Dr. G. F. Gebhart, Director, Center for Pain Research, University of Pittsburgh, W1444 BST, 200 Lothrop Street, Pittsburgh, PA 15213-2536. E-mail: [email protected] 1526-5900/$34.00 © 2008 by the American Pain Society doi:10.1016/j.jpain.2008.01.326
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routine postoperative course, and, in normal complications such as alveolar osteitis, after third molar removal. Inflammation of the synovial lining of the temporomandibular joint is a common feature of symptomatic (hyperalgesic) temporomandibular joints.12,16 The presence in inflamed tissues of inflammatory markers and mediators such as interleukin (IL)-1, IL-6, IL-1, substance P, prostaglandin E2, and tumor necrosis factor (TNF)-␣ has been demonstrated to contribute to the process of sensitization and development of hyperalgesia. For example, the presence of such markers in synovial fluid of temporomandibular joints has been reported to be positively correlated to patients with symptomatic temporomandibular joints.1,23,25,26,27,39 Because pain processing and plasticity after peripheral injury appears to differ in spinal nerves and nerves of a cranial origin, such as the trigeminal nerve,3,34 we undertook
ORIGINAL REPORT/Morgan and Gebhart development of a model of orofacial hyperalgesia in the rat. Several inflammatory or neuropathic models of orofacial pain have been reported and served to guide the present study. The irritant mustard oil has been injected into the temporomandibular joint in rats to produce joint inflammation and orofacial nociception that has been monitored as plasma extravasation,13 increased neck and jaw muscle activity,41 and increased Fos protein expression in trigeminal subnucleus caudails.15 Complete Freund’s adjuvant (CFA) also has been injected into the masseter muscle14,18,19,31 or temporomandibular joint20,31,36,42 to produce a localized inflammation associated, for example, with changes in trigeminal neuron excitability and Fos protein expression. Formalin has been injected into the whisker pad of rats as well as into the temporomandibular joint,32 and wiping and grooming behaviors were monitored as an index of orofacial nociception.6,7 An orofacial neuropathic pain model produced by ligation of the infraorbital nerve in the rat was described by Vos et al,37,38 and Subio et al33 described the consequences of inferior alveolar transection on mechanical sensitivity of the whisker pad area in the rat. These orofacial inflammatory and neuropathic models were based on similar models first established in other tissues, typically the rat hind paw. For the most part, quantification or assessment of the nociceptive quality of the stimulus in the orofacial pain models described above have relied on indirect measures (eg, FOS expression, etc) or changes in grooming behavior. Evocative thermal or mechanical stimuli have not been commonly used. The present study describes and animal model of chronic inflammatory orofacial hyperalgesia that is reliable, easy to measure, and involves no prior surgery. We chose to use CFA injected into the upper lip/whisker pad because of its well-characterized and persistent effects described in a model of hind paw hyperalgesia.18 In addition, because voltage-gated sodium channels, particularly tetrodotoxin-resistant sodium channels, underlie peripheral mechanisms of nociceptor sensitization and have been implicated in various inflammatory and chronic pain states,2,28,40 including painful human dental pulp,30 we subsequently examined the role of NaV 1.8 in the development and maintenance of the inflammatory orofacial hyperalgesia produced by CFA.
Materials and Methods Male-Sprague Dawley rats, weighing 420 to 450g, (Harlan, Indianapolis, IN) were housed 1 per cage with ad libitum access to food and water and maintained on a 12-hour light/dark cycle (lights on 6 AM to 6 PM) in an AAALAC (Association For Assessment and Accreditation of Laboratory Animal Care) accredited animal care facility. All experimental procedures were approved by the Institutional Animal Care and Use Committee, The University of Iowa.
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Figure 1. Complete Freund’s adjuvant (CFA) injected into the upper lip/whisker pad (inset) produces thermal hyperalgesia (decrease in head withdrawal latency) at the site of injection. A, Mean baseline withdrawal latencies for 4 different rates and intensities of heating, which were significantly reduced in the same groups of rats (n ⫽ 6/group) for 3 of the 4 intensities of heating when tested 24 hours after CFA injection (*P ⬍ .05). B, Heat ramps to the different intensities of heating were recorded by a copper-constant in thermocouple placed at the air/skin interface of the right upper lip/whisker pad of an anesthetized rat. Boxes placed on the heat ramps represent the range of withdrawal latencies determined in A for each intensity of heating and correspond to estimated temperatures of 48°, 52°, 55°, and 56°C for heat intensities of 45, 50, 55, and 60, respectively.
Behavioral Testing Rats were gently restrained to measure head withdrawal latency to a radiant heat source or withdrawal threshold to Von Frey–like filaments applied to the orofacial area into which CFA had previously been injected (see Inflammation section below and Fig 1 inset for site of CFA injection and testing). Restraint was necessary to permit access to the snout, upper lip, and whisker pad with vision of the rat blocked. Before any testing, rats were habituated to restraint over several days and allowed to acclimate for at least 15 minutes before testing began. Mechanical and thermal testing, when done in the same animals, was carried out sequentially. Mechanical withdrawal thresholds were determined first with
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von Frey–like nylon monofilaments (Stoelting, Chicago, IL). von Frey filaments of logarithmic incremental stiffness (1.5–75 g) were applied perpendicular to the right whisker pad in the intended injection site with enough force to cause the filament to bend. In the absence of a head withdrawal, the filament 1 log value greater was applied. If the head was withdrawn, a positive response was recorded and the filament 1 log value below was applied. After the first “crossover” from a negative to a positive response or vice versa, 5 additional presentations of stimuli were made. The threshold for response was calculated as described by Chaplan et al.5 Ten minutes after completion of mechanical testing, thermal testing was carried out. Radiant heat was provided by a high-intensity projector lamp (8 V, 50 W; Sylvania, Danvers, MA) located 70 mm from the target area projecting through a 10-mm diameter aperture. A rheostat controlled the voltage applied to the lamp and thus intensity of the heat stimulus. A stop watch was started when the lamp was activated and stopped when the rat removed/ flinched its head to escape heat from the lamp. Each rat was tested 3 times with a 5-minute interval between tests. The withdrawal latencies were then averaged to obtain a mean withdrawal latency for each time of testing. As illustrated in the Results section, sensitization did not result from baseline testing (or play a significant role in these experiments) because responses to neither mechanical nor thermal stimuli changed in saline-treated rats that also were subjected to needle puncture of skin and potential mechanical stimulation from the volume injected.
Inflammation After baseline withdrawal latency and threshold measurements at the intended site of inflammation, rats were briefly sedated with halothane and 50 L of CFA (Sigma Chemical Co., St. Louis, MO) or saline was injected into the right upper lip/whisker pad, lateral to the nostrils, with a 30-gauge needle. Follow-up measurements were made from 24 hours to 4 weeks, depending on the experimental protocol. An arbitrary cutoff time of 20 seconds was used for heat stimuli to limit tissue damage.
Drugs The effects of indomethacin (0.5, 1.0, 5.0 or 10 mg/kg intraperitoneally in volumes of 1 mL/kg; Sigma), a nonsteroidal anti-inflammatory drug with the same mechanism of action as ibuprofen, morphine sulfate (0.5, 1.0, or 5 mg/kg subcutaneously in volumes of 1 mL/kg; Spectrum, Gardena, CA), a -opioid receptor agonist, and vehicle (saline) were tested 24 hours after CFA-injection into the upper lip/whisker pad. Drugs or vehicle were given 30 minutes before testing for withdrawal from the radiant heat source. Naloxone (1.0 mg/kg; Research Biochemicals International; Natick, MA) was used to establish the opioid receptor-mediated action of morphine.
RT-PCR Two trigeminal ganglia ipsilateral to the site of CFA or saline injection in the right whisker pad were removed
from sodium pentobarbital (Nembutal; Abbott Laboratories, North Chicago, IL)-anesthetized rats at various times after treatment (24 hours to 4 weeks; 2 rats/time after treatment) and pooled for RT-PCR. The ganglia were immersed into RNAlater (Ambion, Austin, TX), an RNA stabilization solution until processing. The tissue was then homogenized in TRIzol reagent and total RNA was extracted. cDNA from the respective tissue samples were reverse-transcribed from 500 ng of total RNA. PCR primers targeted specific and unique regions of the sodium channels of interest as determined by a blast gene search and defined amplicons of the following sizes: NaV1.8 to 513 bp and NaV1.9 to 391 bp. Thermal cycling parameters were as follows: 45 minutes/48°C, 2 min/94°C (1 cycle); 30 seconds/94°C, 1 minute/66°C, 2 minutes/68°C (25 cycles); 10 minutes/68°C (1 cycle). cDNA from each tissue was also PCR amplified using primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to demonstrate template viability and act as an internal control for total RNA. PCR amplicons from trigeminal ganglia were confirmed by nucleotide sequence analysis.
Immunohistochemistry Two rats injected with either CFA or saline into the right whisker pad were deeply anesthetized with 10% chloral hydrate and perfused transcardially with 200 mL of 0.1 M phosphate buffered saline (PBS) followed by 500 mL of 10% formalin. The right infraorbital nerve (ION) was dissected out and postfixed in 10% formalin for 4 to 5 hours at 4°C followed by immersion in 20% sucrose in 0.1 M PBS at 4°C overnight. The tissue was embedded in optimal cutting temperature embedding medium (OCT), cut on a cryostat at 40-M thickness, and placed on a glass slide. The sections were preincubated in 3% normal donkey serum and 0.3% triton X-100 for 1 hour at room temperature and then incubated in primary NaV1.8 antibody at 1:500 in the blocking solution. The sections were washed in PBS and then incubated in biotinylated antirabbit IgG (1:500), followed by incubation in Cy3-streptavidin complex (1:1000) for 45 minutes at room temperature. The slides were washed in PBS, coverslipped with Vectashield mounting medium, and stored at 4°C in the dark until viewed. There was no staining in the absence of primary antibody.
Oligodeoxynucleotides Both antisense and mismatch oligodeoxynucleotides (ODNs) were studied. Antisense ODN (5’-TCCTCTGTGCTTGGTTCTGGCCT-3’) complementary to nucleotides 107-129 of the coding region of rat Nav1.8 and a corresponding mismatch ODN sequence (5’-TCCTTCGTGCTGTGTTCGTG CCT3’) were synthesized as phosphodiester ODNs by HPLC purification (Integrated DNA Technologies, Inc., Coralville, IA). The mismatch sequences were derived from the respective antisense sequences by scrambling 6 bases, giving rise to a 25% mismatch with the target sequences. This level of mismatch was sufficient to abolish the selectivity of the ODN for the target sequence. These sequences were screened with the BLAST (basic local align-
ORIGINAL REPORT/Morgan and Gebhart ment search tool) algorithm against the Genbank Database. Search results indicated that there was no significant complementarity (equal or greater than 70% as defined by the mismatch control) with known sequences for other ion channels. The fluorescence labeling of the antisense and mismatch ODNs was carried out by conjugation of the fluorophor TAMRA to the 5’ end of the ODN, and the fluorescence-labeled ODNs were then purified with HPLC (Integrated DNA Technologies, Inc.). All ODNs were reconstituted in nuclease-free, ultrapure water to a final concentration of 9 g/L. Intra– cisterna magna injections of ODNs by indwelling cannula targeting the brainstem overlying the trigeminal ganglion were made daily for either 2 or 5 days in a volume of 5 L followed by a 10-L saline flush. Evaluation of the uptake of ODN by the trigeminal ganglion was evaluated at 12 hours after ODN administration. Rats were deeply anesthetized with 10% chloral hydrate and perfused transcardially with 200 mL of 0.1 M PBS followed by 500 mL of 10% formalin. The right trigeminal ganglion was dissected out and postfixed in 10% formalin for 4 to 5 hours at 4°C followed by immersion in 20% sucrose in 0.1 M PBS at 4°C overnight. The tissue was embedded in OCT embedding medium, cut on a cryostat at 30-M thickness, and placed on a glass slide, coverslipped with Vectashield (Vector, Burlingame, CA), and viewed with a fluorescent microscope.
525 rates of heating were used, the estimated temperatures producing withdrawal do not represent temperature at the receptive ending in skin. Twenty-four hours after injection of CFA into the upper lip/whisker pad, withdrawal latencies to thermal stimulation were significantly reduced (ie, CFA produced a thermal hyperalgesia; Fig 1A). The magnitude and duration of CFA-produced thermal and mechanical hyperalgesia were examined in subsequent experiments.
CFA Injection Into the Upper Lip/Whisker Pad Produces Persistent Hyperalgesia We chose to use heating intensities of 50 and 55 because they produced consistent head withdrawal latencies and afforded the opportunity to evaluate and quantify hyperalgesia as well as antinociception. CFA but not saline injection into the upper lip/whisker pad produced significant, persistent thermal hyperalgesia. As illus-
Data Analysis Data were analyzed by parametric or nonparametric ANOVAs as appropriate with follow-up parametric or nonparametric post hoc analyses. Group sizes in these behavioral experiments were typically 6. Baseline measurements were taken only at time or day 0 (zero). Subsequent testing was done after either saline or CFA treatment and compared with baseline values taken on time/ day 0. A value of P ⬍ .05 was considered statistically significant.
Results Response Latency Is Intensity Dependent Applying different lamp intensities that produced different rates of heating and maximum temperatures, each rat (n ⫽ 5) was subjected to a random ordering of the 4 different lamp intensities both before and 24 hours after CFA injection (Fig 1A). Head withdrawal latency was clearly intensity-dependent: 14.4 ⫾ 0.8, 11.5 ⫾ 0.6, 9.7 ⫾ 0.5, and 6.5 ⫾ 0.7 seconds at lamp intensities of 45, 50, 55, and 60, respectively (Fig 1B). The estimated temperatures at which head withdrawal was produced were determined in a naive, anesthetized rat with a thermocouple attached to the right upper lip to record temperature at the air/hairy skin interface. Lamp intensities of 45, 50, 55, and 60 resulted in estimated withdrawal temperatures of ⬃ 48°, 52°, 55°, and 56°C, respectively, based on mean withdrawal latencies and extrapolating from the heating curves (Fig 1B). Because temperatures at the air/hairy skin interface (and not temperature at the receptive endings in skin) and different
Figure 2. Complete Freund’s adjuvant (CFA) injected into the upper lip/whisker pad produces long-lasting thermal hyperalgesia (decrease in head withdrawal latency) at the site of injection. A, Mean withdrawal latencies for 2 different rates and intensities of heating after saline injection into the upper lip/whisker pad (n ⫽ 6). B, Mean withdrawal latencies for 2 different rates and intensities of heating after CFA injection into the upper lip/whisker pad (n ⫽ 6). Significant thermal hyperalgesia was evident when first tested 24 hours after CFA injection and persisted for 1 or 2 weeks at different intensities of heating (*P ⬍ .05 vs baseline).
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Characterization of a Model of Chronic Orofacial Hyperalgesia in the Rat
trated in Fig 2A, saline injection into the upper lip produced no changes (relative to baseline, b) in thermal withdrawal latencies at either of the 2 lamp intensities tested, 50 and 55. CFA, however, produced a significant and robust thermal hyperalgesia at both lamp intensities that persisted for 1 week and was evident for 2 weeks at a lamp intensity of 55; later times were not tested. In other groups of rats (n ⫽ 6), CFA but not saline injection into the upper lip/whisker pad produced a significant mechanical hypersensitivity that was maintained for at least 4 weeks (Fig 3). The median withdrawal force was significantly reduced from 28.84 g at baseline to forces ranging between 2.75 and 7.21 g over the 4 weeks of testing; later times were not tested.
Figure 4. Indomethacin significantly attenuated thermal hyperalgesia at 2 dosages (5 and 10 mg/kg; *P ⬍ .05 vs vehicle; n ⫽ 6/treatment group) at 2 intensities of heating when tested 24 hours after complete Freund’s adjuvant (CFA) injection into the upper lip/whisker pad.
Pharmacological Modulation of Orofacial Hyperalgesia
Figure 3. Complete Freund’s adjuvant (CFA) injected into the upper lip/whisker pad produces long-lasting mechanical hypersensitivity (decrease in von Frey filament withdrawal threshold) at the site of injection. A, Median withdrawal thresholds after saline injection into the upper lip/whisker pad (n ⫽ 6). B, Median withdrawal thresholds after CFA injection into the upper lip/whisker pad (n ⫽ 6). Significant mechanical hypersensitivity was evident when first tested 24 hours after CFA injection and persisted for 4 weeks (*P ⬍ .05 vs baseline [b] and vs saline treatment). Boxes represent the 25th and 75th percentiles and whiskers the 10th and 90th percentiles.
To further characterize this model of orofacial hyperalgesia in the rat, the antihyperalgesic/antinociceptive effects of indomethacin and morphine were tested 24 hours after CFA injection into the upper lip/ whisker pad. Indomethacin significantly attenuated thermal hyperalgesia at the greatest doses tested (5 and 10 mg/kg; Fig 4). Morphine also significantly reversed CFA-produced thermal hyperalgesia at all doses tested, and at the greatest dose tested (5 mg/kg) produced a significant antinociception (Fig 5). There were no differences in the efficacy of morphine at the 2 lamp intensities tested, 50 and 55. To verify that the effects of morphine were receptor-mediated, naloxone was shown to reverse the significant antinociception produced by 5 mg/kg morphine (Fig 6). Interestingly, naloxone alone produced no change in head withdrawal latency compared with vehicle administration, suggesting that there is no contribution in this model by endogenous opioids (Fig 5).
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Nav1.8 Immunoreactivity Increases in the ION After CFA Injection Rats that received a CFA injection into the right upper lip/whisker pad exhibited an increase in immunoreactivity for Navl.8 protein in the infraorbital nerve (ION) 48 hours and 1 week after CFA treatment (Fig 7B), revealing transport of Nav1.8 protein to the periphery where it has been established as critical to nociceptive processing.4 There was very little evidence of Nav1.8 in the ION from untreated (naive) rats and no staining in the absence of primary antibody (not shown).
Confirmation of Antisense Delivery Into the Trigeminal Ganglion Twelve hours after a bolus injection of TAMRA-labeled Nav1.8 antisense ODN (n ⫽ 2), many cell bodies in the trigeminal ganglion were highly fluorescent, indicating uptake of the antisense ODN (Fig 7C). Although we did not measure the size of cell profiles, the fluorescence intensity appeared not to distribute among trigeminal ganglion cells based on cell size, suggesting that neither a specific subset of cell nor that all cells took up the labeled ODN, and also that the labeling was not due to some nonspecific diffusion or optical artifact. Control animals injected with saline (n ⫽ 2) exhibited a low level of autofluorescence.
Attenuation of Behavioral Mechanical Hypersensitivity With Antisense But Not Mismatch ODNs Figure 5. Morphine significantly attenuated thermal hyperalgesia at all dosages (*P ⬍ .05 vs vehicle) at 2 intensities of heating when tested 24 hours after complete Freund’s adjuvant (CFA) injection into the upper lip/whisker pad (n ⫽ 6/treatment group). The greatest dose of morphine (5 mg/kg) was antinociceptive at both intensities of heating. Naloxone (1 mg/kg) had no effect on CFA-produced thermal hyperalgesia.
Antisense administration into the cisterna magna directly over the trigeminal ganglion before injection of CFA into the right upper lip/whisker pad did not significantly attenuate mechanical hyperalgesia in the 4 rats
NaV1.8 mRNA Content in the Trigeminal Ganglion Increases After CFA Injection Compared with rats that received a saline injection into the right trigeminal ganglion distribution, rats that received CFA exhibited an upregulation of the Nav1.8 sodium channel transcript that peaked at 1 to 2 weeks and returned to near baseline levels at 4 weeks as determined by RT-PCR (Fig 7A). We used imaging software (Kodak Imager, Seattle, WA) to pixelate the blots into quantitative values on replicate samples; the increase in pixelation density 24 hours after CFA injection was found to be 1.2 times baseline. It was 1.9 times baseline at 48 and 72 hours, 2.8 times at 1 week, 2.5 times at 2 weeks, and 1.1 times at 4 weeks after CFA treatment. No increase in message content was observed for another tetrodotoxin-resistant sodium channel (Nav1.9) at any time after CFA injection compared with saline (Fig 7A).
Figure 6. Naloxone (1 mg/kg) reversed the antihyperalgesic and antinociceptive effect of morphine (5 mg/kg) in rats 24 hours after complete Freund’s adjuvant injection into the upper lip/whisker pad (represented by the vertical shaded bar) (*P ⬍ .05 vs 5 mg/kg morphine; n ⫽ 6/treatment group).
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Characterization of a Model of Chronic Orofacial Hyperalgesia in the Rat
Discussion The present study describes development and characterization of a reliable and useful model of orofacial thermal hyperalgesia and mechanical hypersensitivity in the rat. Thermal withdrawal responses were appropriately intensity-dependent; faster withdrawal latencies were associated with more rapid and greater increases in applied heat. The thermal hyperalgesia and mechanical hypersensitivity produced by CFA injected into the upper lip/whisker pad is robust, persistent and subject to appropriate pharmacologic modulation. NaV 1.8 is implicated in the development and maintenance of the orofacial hyperalgesia by its upregulation in the infraorbital nerve and trigeminal ganglion after CFA injection and modulation of mechanical hypersensitivity by antisense ODN treatment.
Figure 7. A, NaV 1.8 (top) and 1.9 (bottom) message in trigeminal ganglia from naive and complete Freund’s adjuvant (CFA)treated rats (n ⫽ 2 ganglia pooled for each treatment time). The message for NaV 1.8 increases after CFA injection into the upper lip/whisker pad before returning to baseline at 1 month (see text for details). There was no change in NaV 1.9 message after CFA treatment. B, Immunohistochemistry for NaV 1.8 protein in the infraorbital nerve (ION), which increases after CFA injection into the upper lip/whisker pad. (C) NaV 1.8 antisense ODN accesses trigeminal ganglion neurons after intracisternal injection.
tested. There was a nonsignificant increase to 8.19 g in the mechanical withdrawal threshold on day 3 in antisense ODN-treated rats from a median 3.97 g withdrawal threshold on days 1 and 3 in mismatch ODN-treated rats (Fig 8A). To examine whether Navl.8 was involved in the maintenance of orofacial mechanical hypersensitivity, antisense or mismatch ODN administration was initiated after development of mechanical hypersensitivity (day 5 after CFA injection into the upper lip/whisker pad). A significant attenuation of the mechanical hypersensitivity (withdrawal threshold increased from a median 4.34 g to 11.95 g) was noted in Nav1.8 antisense (but not mismatch) ODN-treated rats on day 7; Fig 8B). After discontinuation of antisense ODN treatment on day 7, mechanical hypersensitivity was fully restored when tested on day 11.
Figure 8. Antisense (AS) but not mismatch (MM) ODN reverses complete Freund’s adjuvant (CFA)-produced mechanical hypersensitivity. Median withdrawal thresholds are illustrated in A and B for 2 ODN treatment protocols. The onset and termination of ODN administration is illustrated below the box plots; boxes represent the 25th and 75th percentiles and whiskers the 10th and 90th percentiles. A, Median withdrawal thresholds before (⫺2 days), at baseline (b; day 0) and on days 1, 3, and 7 after CFA injection into the upper lip/whisker pad (n ⫽ 4). B, Median withdrawal thresholds at baseline (b; day 0) and on days 4, 7, and 11 after CFA injection into the upper lip/whisker pad (n ⫽ 6). *P ⬍ .05 vs MM on the same day of testing.
ORIGINAL REPORT/Morgan and Gebhart Injection of small volumes of CFA to induce inflammatory hyperalgesia limited to the area of its administration has been commonly employed to produce both thermal and mechanical hypersensitivity of the hind paw.17,18 Animals injected with CFA do not immediately develop edema or erythema; however, by 12 to 24 hours, a pronounced swelling usually accompanied by redness can be detected at the injection site. The hyperalgesia that develops after CFA injection into animals is consistent with the initial diffuse throbbing and persistent thermal (7 days) and mechanical (84 days) hypersensitivity produced in humans.11 Although we limited thermal testing to 2 weeks and mechanical testing to 4 weeks after CFA injection into the upper lip/whisker pad, the mechanical hypersensitivity appeared to persist longer than the thermal hyperalgesia. This is consistent with the initial characterization of this model in the rat hindpaw.18 We further characterized the model by examining its sensitivity to pharmacological modulation by drugs representative of the major classes of analgesics used in dental practice, an opioid and a nonsteroidal antiinflammatory drug. In this study, slowing of the faster, hyperalgesic head withdrawal latency was seen with the systemic administration of both indomethacin and morphine. The dosages at which the antihyperalgesic effects were produced are consistent with effective dosages of morphine in related orofacial pain studies. For example, dosages of 2.5 and 5.0 mg/kg morphine attenuated to about 25% and 5% the chewing and grooming behaviors produced by mustard oil injected into the temporomandibular joint of the rat but were less efficacious in reducing Fos expression in subnucleus caudalis (to ⬃75% and ⬃50%, respectively) in the same study.15 After injection of formalin into the vibrissal pad of the rat, Eisenberg et al9 reported that 1.0 mg/kg morphine administered systemically 30 minutes before formalin reduced significantly both the early (to 4% of control) and late (to 29% of control) phases of formalin-induced face grooming. Notably, in the present study, the greatest dose of morphine tested (5 mg/kg) was not only antihyperalgesic but antinociceptive as well, producing an almost doubling of the thermal withdrawal latency relative to the baseline withdrawal latency in saline-treated rats, an effect that was opioid receptor mediated. Indomethacin has also been tested in orofacial pain models in rats. Thut et al36 studied feeding behavior in rats after CFA-produced inflammation of the temporomandibular joint (TMJ) or masseter muscle and reported that 4 mg/kg indomethacin reversed the inflammation-induced increase in time between food rewards in an operant setting. In a similar experiment, Ro31 inflamed the masseter muscles bilaterally with CFA, which reduced bite force; indomethacin (4 mg/kg) prevented CFA-induced changes in bite force. CFA injection as used here also produces an increase in both Nav1.8 message in trigeminal ganglion neurons and protein in the infraorbital nerve. The increase in expression of Nav1.8 mRNA after CFA injection ap-
529 pears to peak about 1 week before returning to preCFA injection expression at 4 weeks. This effect of CFA was limited to Nav1.8; no change in Nav1.9 mRNA expression was found after inflammation at any time measured. Qualitatively, there was also an increased transport of Nav1.8 protein toward peripheral nerve terminals when examined in the infraorbital nerve 48 hour and 7 days after CFA injection into the upper lip/whisker pad. These observations are consistent with reports of others. For example, Tanaka et al35 and Porreca et al29 demonstrated increased Nav 1.8 message and protein expression, respectively, in dorsal root ganglion neurons 4 days after CFA inflammation of the rat hind paw. Coggeshall et al8 reported a significant increase in the proportion of Nav1.8 labeled myelinated axons (6-fold) and a smaller increase in Nav1.8 labeled unmyelinated axons (1.5-fold) 48 hours after hind paw inflammation. In contrast, the percentages of Nav1.9 labeled axons either decreased or did not change. Evidence that Nav1.8 contributes to the hypersensitivity documented here is provided by the effects of Nav1.8 antisense ODN on mechanical withdrawal thresholds after CFA injection. Although the effects of Nav1.8 antisense ODN treatment were modest and statistically significant in only 1 of the 2 experiments, we believe that Nav1.8 significantly contributes to the hypersensitivity produced in this model for several reasons. First, CFA increased both Nav1.8 message and protein in a time-dependent and reversible manner. Second, the TAMRA-labeled Nav1.8 antisense ODN was shown to be present in trigeminal ganglion neurons after intracisternal administration. Third, antisense ODN and not mismatch ODN reversed established mechanical hypersensitivity, which was fully restored after cessation of antisense ODN administration. Finally, others have established the efficacy of Nav1.8 antisense treatment in other models.10,21,24 Khasar et al22 documented an attenuation of CFA-induced behavioral hyperalgesia and the mechanical hypersensitivity induced by direct injection of prostaglandin E2 into the rat hind paw. Others have also documented attenuation of inflammatory hyperalgesia by Nav1.8 antisense treatment.21,29 Similarly, Nav1.8 antisense treatment effectively attenuates the allodynia and mechanical hypersensitivity in models of neuropathic pain.10,21,24,29 The magnitude of effect of Nav1.8 antisense treatment varies in the different studies, most of which delivered the antisense ODN into the intrathecal space of rats, where it has direct access to dorsal root ganglion neurons. In the present experiments, we delivered the antisense ODN in the cisterna magna superior to the right trigeminal ganglion, and the modest magnitude of effect we observed probably reflects that the concentration of antisense ODN at its intended site of action was less than in other studies where it was delivered intrathecally. The model of chronic inflammatory orofacial pain described in this report is associated with long-lasting thermal and mechanical hypersensitivity. Appropriately, the hypersensitivity is modulated by analgesic pharmacolog-
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Characterization of a Model of Chronic Orofacial Hyperalgesia in the Rat
ical strategies. Like other models of inflammatory hypersensitivity, the tetrodotoxin-resistant, voltage-gated sodium channel Nav1.8 plays an important role in the increased sensitivity to applied stimuli that develops after orofacial inflammation.
Acknowledgments The authors gratefully acknowledge Michael Burcham for preparation of the graphics and Joanne Hirt for secretarial assistance.
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