persistent fos protein expression after orofacial deep or cutaneous tissue inflammation in rats:...

Persistent Fos Protein Expression AfterOrofacial Deep or Cutaneous Tissue

Inflammation in Rats: Implications forPersistent Orofacial Pain

QIQI ZHOU, HIROKI IMBE, RONALD DUBNER, AND KE REN*Department of Oral and Craniofacial Biological Sciences,

University of Maryland Dental School, Baltimore, Maryland 21201-1586

ABSTRACTThis study was designed to systematically examine the effects of persistent orofacial

tissue injury on prolonged neuronal activation in the trigeminal nociceptive pathways bydirectly comparing the effects of orofacial deep vs. cutaneous tissue inflammation onbrainstem Fos protein expression, a marker of neuronal activation. Complete Freund’sadjuvant (CFA) was injected unilaterally into the rat temporomandibular joint (TMJ) orperioral (PO) skin to produce inflammation in deep or cutaneous tissues, respectively. Ratswere perfused 2 hours, 24 hours, 3 days, or 10 days following CFA injection. The TMJ and POinflammation-induced Fos expression paralleled the intensity and course of inflammationover the 10-day observation period, suggesting that the increase in intensities and persistenceof Fos protein expression may be associated with a maintained increase in peripheral input.Compared to PO CFA injection, the injection of CFA into the TMJ produced a significantlystronger inflammation associated with a greater Fos expression. In TMJ- but not inPO-inflamed rats, Fos-like immunoreactivity (LI) spread from superficial to deep uppercervical dorsal horn as the inflammation persisted and there was a dominant ipsilateralFos-labeling in the paratrigeminal nucleus. Common to TMJ and PO inflammation, Fos-LIwas induced in the trigeminal subnuclei interpolaris and caudalis, C1–2 dorsal horn, andother medullary nuclei. Substantial bilateral Fos-LI was found in the interpolaris-caudalistrigeminal transition zone. Further analysis revealed that Fos-LI in the ventral transitionzone was equivalent bilaterally, whereas Fos-LI in the dorsal transition zone was predomi-nantly ipsilateral to the inflammation. The differential induction of Fos expression suggeststhat an increase in TMJ C-fiber input after inflammation and robust central neuronalhyperexcitability contribute to persistent pain associated with temporomandibular disorders.J. Comp. Neurol. 412:276–291, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: temporomandibular joint; perioral skin; Freund’s adjuvant; medullary dorsal horn;

paratrigeminal nucleus

Painful temporomandibular disorders involve deep tis-sues and have characteristics different from pain of cutane-ous origin (Dubner, 1991a). Deep pains are diffuse anddifficult to localize and are often described as aching andcramping. In contrast, cutaneous pain is highly localized tothe site of stimulation and is described as sharp, stabbing,or pricking. At spinal levels, C-fiber inputs produce morerobust and longer-lasting neuronal hyperexcitability follow-ing muscle than following cutaneous nerve stimulation(Wall and Woolf, 1984). This has not been demonstrated attrigeminal levels, with the exception of a selective expan-sion of the deep mechanoreceptive fields of trigeminalnociceptive neurons after mustard oil application into the

tongue muscle versus application into the facial skin (Yu etal., 1993). Studies at the spinal level have indicated thatperipheral tissue or nerve injury induces a state of hyper-excitability that leads to, or at least contributes to, thedevelopment of persistent pain and hyperalgesia (Woolf,1983; Dubner, 1991b; Coderre et al., 1993). It is still to be

Grant sponsor: National Institute of Dental and Craniofacial Research,National Institutes of Health; Grant number: DE11964.

*Correspondence to: K. Ren, Ph.D., Department of OCBS, Room 5A26,666 W. Baltimore St., Baltimore, MD 21201-1586.E-mail: [email protected]

Received 24 September 1998; Revised 4 May 1999; Accepted 10 May 1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 412:276–291 (1999)

r 1999 WILEY-LISS, INC.

demonstrated that orofacial deep tissue injury also in-duces prolonged functional changes in the brain, resultingin chronic pain conditions.

One way to study the injury-induced central plasticity isto compare brain neuronal activation in response to identi-fied peripheral stimuli. In this regard, the stimulation-induced expression of Fos, the protein product of animmediate early gene, c-fos, has been widely used as ameasure of nociceptive neuronal activation, particularly atthe lumbar spinal level (Hunt et al., 1987; Menetrey et al.,1989; Bullitt, 1990; Williams et al., 1990; see Munglaniand Hunt, 1995 for a review). Different from the hindpaw-lumbar spinal cord system (Hunt et al., 1987; Menetrey etal., 1989; Presley et al., 1990; Tolle et al., 1990; Williams etal., 1990; Noguchi et al., 1991), stimulation-induced Fosprotein expression is heterogeneous in the trigeminalpathways (Strassman and Vos, 1993; Bereiter et al., 1994;Coimbra and Coimbra, 1994; Sugimoto et al., 1994;Carstens et al., 1995). Some Fos-like immunoreactivity(LI) induced by orofacial noxious stimulation exhibits asomatotopic representation in the trigeminal nuclei, par-ticularly in the subnucleus caudalis, including the parts ofthe upper cervical dorsal horn (Strassman and Vos, 1993;Sugimoto et al., 1994). On the other hand, some Fos-LIresulting from orofacial stimulation is not necessarilytopographically relevant. For example, a portion of Fos-LIinduced by corneal or gingival stimulation is somatotopi-cally inappropriate (Strassman and Vos, 1993; Sugimoto etal., 1994). In addition, there is bilateral Fos expressionafter unilateral stimulation and some Fos-LI is apparentlynot directly related to the stimulus (Strassman and Vos,1993; Bereiter et al., 1994; Coimbra and Coimbra, 1994;Sugimoto et al., 1994; Carstens et al., 1995). Thus, in usingFos protein expression as a marker for studying functionalchanges in the brain associated with orofacial stimulation,special consideration should be given to dissect the func-tional significance of subtle differences in directly stimulus-related vs. indirect Fos expression.

Stimulus-induced Fos protein expression can also beused to study long-term changes in neuronal activity.Bullitt et al. (1992) demonstrated that continuous stimula-tion produces greater Fos-LI in the spinal cord whencompared to brief stimulation. In the spinal cord, theincrease in Fos-LI after chronic nerve injury or peripheralinflammation can persist from days to months (Williams etal., 1991; Abbadie and Besson, 1992; Kajander et al., 1996;Ma and Woolf, 1996; Ren and Ruda, 1996). It is not known,however, whether this long-lasting Fos expression is re-lated to peripheral inflammation or central changes. Mostprevious studies on trigeminal Fos expression have fo-cused on responses to acute or transient stimuli (Lu et al.,1993; Strassman and Vos, 1993; Bereiter et al., 1994;Sugimoto et al., 1994; Carstens et al., 1995; Hathaway etal., 1995). The increase in Fos-LI in the trigeminal com-plex after dental injury (tooth extraction, pulp exposure, orcavity preparation) peaked at 2–4 hours and was resolvedat 24 hours (Wakisaka et al., 1992). In rats receivingchemical stimulation (mustard oil) of the temporomandibu-lar joint (TMJ), the evoked Fos-LI in the subnucleuscaudalis peaks at 2 hours and is diminished significantlyat 5 hours after stimulation (Hathaway et al., 1995). Thelong-lasting effects of persistent orofacial inflammation onFos protein expression at the medullary level requirefurther investigation.

The major purpose of this study was to systematicallyexamine the effects of persistent orofacial deep versuscutaneous tissue injury on prolonged neuronal activationin the trigeminal nociceptive pathways. We also attemptedto analyze and understand the functional significance ofFos induction in trigeminal as well as other medullarystructures. We used a model of orofacial inflammation thatwas adapted from a hindpaw inflammation model (Renand Dubner, 1996a; Imbe and Ren, 1999). By injecting aninflammatory agent, complete Freund’s adjuvant (CFA),into the rat TMJ1 or perioral (PO) skin, rats developedpersistent behavioral hyperalgesia associated with orofa-cial inflammation, resembling that seen in the hindpawinflammation model (Hargreaves et al., 1988; Iadarola etal., 1988; Millan et al., 1988; Stein et al., 1988). Weextended previous observations by directly comparing thetemporal profiles of Fos expression following deep versuscutaneous orofacial tissue inflammation. We hypothesizedthat there would be a differential distribution of Fosprotein immunoreactivity induced by TMJ versus POtissue inflammation at long time periods. The resultsindicate that there was a persistent trigeminal Fos expres-sion that paralleled the time course of peripheral inflamma-tion; and the induction of Fos-LI after TMJ deep tissueinflammation was more diffuse and quantitatively moreintense than that after PO cutaneous tissue inflammation.Further, we propose to classify brainstem Fos expressionafter orofacial inflammation into several functional catego-ries including direct somatotopically organized nociceptiveactivation and indirect reflexive activity and somatoauto-nomic processing. Portions of these results have beenreported in abstract form (Zhou et al., 1997a,b).

MATERIALS AND METHODS

Animal preparation

A unilateral adjuvant-induced orofacial inflammationand hyperalgesia model was produced in male Sprague-Dawley rats (250–350 g; Harlan, Indianapolis, IN). Com-plete Freund’s adjuvant (Sigma, St. Louis, MO) suspendedin an oil/saline (1:1) emulsion was used as the inflamma-tory agent and was injected into the TMJ or PO skin undermethohexital sodium anesthesia (Brevital sodium, 50mg/kg i.p.). To inject CFA into the TMJ (n 5 18), the site forinjection was identified by palpating the zygomatic archand condyle. A 30-gauge needle was inserted into the pointimmediately inferior to the posteroinferior border of thezygomatic arch. The needle was advanced in an anteriordirection until it contacted the posterolateral aspect of thecondyle. After a gentle aspiration, CFA (0.05 ml, 0.025 mgMycobacterium tuberculosis) was injected. The PO injec-tion (n 5 17) with the same amount of CFA was givensubcutaneously to a spot about 2–5 mm posterolateral tothe lateral labium. This site was just posterioinferior tothe E1 and delta vibrissae (Arvidsson, 1982). An oil/saline(1:1, 0.05 ml) emulsion was injected into the TMJ (n 5 4) orPO skin (n 5 4) as a control for CFA. Because anesthesiaalone may induce trigeminal Fos activation (Strassmanand Vos, 1993), three rats received methohexital anesthe-

1Rodents’ mandibular condyle articulates with the squamosal bone, asthey lack a temporal bone (Gartner et al., 1976). Thus, the squamosoman-dibular joint (SMJ) in rats is equivalent to the temporomandibular joint inhumans. However, the terms SMJ and TMJ have been used interchange-ably in rats in the literature (Noble, 1973).

OROFACIAL INFLAMMATION AND FOS EXPRESSION 277

sia without other treatment to serve as a control for effectof anesthesia. The anesthesia induced by methohexitaltypically dissolves with 1–2 hours. Four naive rats thatdidn’t receive any treatment were also used as a control forbasal level of Fos immunoreactivity.

The animal’s behavior after a unilateral injection of theinflammatory agent into the orofacial regions was care-fully monitored. When compared to naive animals thatwere not injected, there was no apparent difference ingrooming and exploration activity in the injected rats. Theinjection procedure appeared to have temporary nonspe-cific effects on the animal’s feeding behavior. The rats thatreceived either complete Freund’s adjuvant or vehicleinjection stopped gaining weight (but no weight loss) forthe first few days, as compared to noninjected naive rats.One week after inflammation, however, they started togain weight and there was no significant difference inweight increase between the different groups of animals.These animal procedures have been approved by theInstitutional Animal Care and Use Committee of theUniversity of Maryland Dental School.

Verification of inflammation

After injection of CFA into the PO skin, the developmentof inflammation was clearly indicated by the appearance ofredness and edema at the injection site. The TMJ inflam-mation was indicated by swollen skin above the TMJ.Evans’ blue dye extravasation was examined to furtherverify CFA-induced inflammation. Evans’ blue dye (5mg/kg, 0.2% solution) was injected through the tail orfemoral vein at the end of the experiments. The rats wereeuthanized 10 minutes later. The TMJ or PO region wasexposed and the CFA injection site was carefully examinedunder a dissecting microscope for the appearance of ex-travasated dye in the articular capsule, periarticular orPO tissue. For TMJ inflammation, the dye typically ap-peared in the TMJ capsule with minor spread in theperiarticular tissues. In cases of PO inflammation, the dyewas observed in the subcutaneous tissue around theinjection site. To quantify the extravasated Evans’ blue(Harada et al., 1971; Haas et al., 1992), the articular/periarticular and PO tissues were dissected, weighed, and

cut into small blocks. The tissues were then incubatedovernight in a 7:3 mixture of acetone and 35.2 mM sodiumsulphate solution (vol/vol) at room temperature with inter-mittent shaking. After incubation, samples were centri-fuged at 300 rpm (revolutions per minute) for 10 minutesand the supernatant separated. The absorbency of thissuperrnatant at 620 nm was determined in a spectropho-tometer. The recovery of the extravasated dye per gramweight of tissue (µg/g) was calculated by comparing theabsorbency of the supernatant with a standard curve. Thestandard curve was generated from a series of the sameextraction solution mixed with known amounts of Evans’blue dye.

Immunohistochemistry

Rats were deeply anesthetized with sodium pentobarbi-tal and perfused transcardially with 4% paraformaldehydein 0.1 M phosphate buffer at pH 7.4. At the beginning of theperfusion, the rat’s head is held in a dorsiflexed position inorder to make the caudal medulla and upper cervical cordnearly coplanar (Strassman and Vos, 1993). Following theperfusion, a single block of tissue from 4 mm rostral and 8mm caudal to the obex was removed, placed in the samefixative for 1–3 hours at 4°C, and transferred to 30%sucrose (w/v) in phosphate buffer for several days forcryoprotection. Thirty-micron-thick sections were cut witha cryostat at 220°C. Every third section was collectedserially in a multiwell tissue culture plate. Free-floatingtissue sections were rinsed in phosphate-buffered saline(0.1 M PBS, pH 7.4) with 0.75% Triton X-100 (Sigma) and0.3% hydrogen peroxide (Sigma) for 1 hour, 3–5% normalgoat serum (NGS) in PBS for 30 minutes, then incubatedwith anti-Fos antibodies (c-fos ab-5, Oncogene, Cam-bridge, MA) in 3% NGS for 48–72 hours with gentleagitation. After two washes in PBS (30 minutes each), anda 60-minute rinse in PBS with 3% NGS, the sections wereincubated with biotinylated secondary IgG (1:400 in PBS,Vector, Burlingame, CA) for 60 minutes. Following rinsesin PBS (30 minutes 3 2) and PBS with 3% NGS (30minutes), the sections were then incubated with avidinand biotinylated horseradish peroxidase complex (1:100 in

Abbreviations

10 dorsal motor nucleus of vagus12 hypoglossal nucleus12n root of hypoglossal nerveI/II laminae I and II of the spinal dorsal hornIII/IV laminae III and IV of the spinal dorsal hornV lamina V of the spinal dorsal hornAmb ambiguus nucleusC1 first cervical ventral rootCFA complete Freund’s adjuvantCG central graycon contralateralCu cuneate nucleuscu cuneate fasciculusDAB diaminobenzidine dihydrochloridedsc dorsal spinocerebellar tractECu external cuneate nucleusFos-LI Fos-like immunoreactivityFos-ir Fos-immunoreactiveGr gracile nucleusgr gracile fasciculusIBN internal basilar nucleusIO inferior olivary nucleusIOC cell group C of the inferior medial olivary nucleus

ipsi ipsilateralLRN lateral reticular nucleusmlf medial longitudinal fasciculusMVe medial vestibular nucleusNMDA N-methyl-D-aspartateNGS normal goat serumNTS nucleus tractus solitariusNRM nucleus raphe magnusoc olivocerebellar tractPa5 paratrigeminal nucleusPBS phosphate-buffered salinePO perioralPy pyramidal tractPyx pyramidal decussationROb nucleus raphe obscurusRPa nucleus raphe pallidusRVL rostroventrolateral reticular nucleusSg substantia gelatinosasol solitary tractSp5 spinal trigeminal tractSP5I subnucleus interpolaris of the spinal trigeminal nucleusSP5C subnucleus caudalis of the spinal trigeminal nucleusTMJ temporomandibular joint

278 Q. ZHOU ET AL.

PBS, Vector, Burlingame, CA) for 30–60 minutes. Afterwashes in PBS, the tissue sections were reacted with0.05% diaminobenzidine dihydrochloride (DAB, Sigma) in0.1 M phosphate buffer containing 0.003% hydrogen perox-ide for 3–6 minutes. The sections were finally rinsed inPBS, mounted onto gelatin-coated slides, dehydrated inalcohols, cleared in xylenes, and coverslipped with Eukitt(Calibrated Instruments Inc., Hawthorne, NY).

As a control for the specificity of the immunostainingprocedure, some sections were incubated in 3% NGSwithout primary antisera. Some sections were incubatedwith primary Fos antibody previously incubated with 0.6mg/ml of the original immunogen (c-Fos 2, Oncogene). Nospecific staining was observed on these sections.

Data analysis

The tissue sections were serially mounted. The appear-ance of the area postrema was followed rostrocaudally andthe first section that showed disappearance of the areapostrema was designated as the obex level. The sectionswere then grouped into 0.45-mm segments rostrocaudallywith reference to the obex. Labeled nuclei were countedonly when structures of the appropriate size and shapedemonstrated clear increases in immunoreactivity whencompared to the background level. The mean number oflabeled cells of individual nuclei per section in eachsegment was calculated for each rat and added up rostro-caudally for the total number of Fos-positive cells. Assuggested by Saper (1996), we did not apply stereologicalmethods (Coggeshall and Lekan, 1996) in our counting ofFos-labeling profiles, because we did not use adjacentsections and the diameter of Fos-labeled nuclei are gener-ally less than 30 µm.

The brainstem structures were identified with referenceto Paxinos and Watson (1986) and Kruger et al. (1995). Intransverse sections, the trigeminal subnucleus interpo-laris was at about 1.0–3.0 mm rostral to the obex. Thetrigeminal interpolaris/caudalis transition zone was about0.2–0.8 mm rostral to the obex. The subnucleus caudalisextended caudally to the C2–3 spinal cord (see Capra andDessem, 1992). For the purpose of analysis, the termsubnucleus caudalis will refer to a portion of the trigemi-nal nucleus between C1 and the interpolaris/caudalistransition zone. The Fos-LI in the rostral cervical cord willbe discussed separately. The C1 spinal cord was identifiedas a 2-mm segment at about 3.0 mm caudal to the obex.The C2 spinal cord was a 3-mm segment centered at about6.0 mm caudal to the obex. Data are presented as mean 6standard error of the mean (S.E.M). Statistical compari-sons were made by analysis of variance (ANOVA; Fisher’sProtected Least Significant Differences or Scheffe F-testfor post-hoc analysis) or Student’s t-test. P , 0.05 wasconsidered significant in all cases.

RESULTS

Quantification of CFA-induced inflammation

The CFA-induced inflammation was verified and quanti-fied by measuring tissue Evans’ blue extravasation (Fig.1A). In TMJ-injected rats (n 5 3), the blue dye wasmeasurable throughout the 10-day observation period.The peak extravasation was found to be at 1–3 daysfollowing CFA injection. In PO-injected rats (n 5 3), theEvans’ blue dye extravasation was measurable within 3days, but was negligible at 10-day post-CFA. There was no

significant difference in the levels of Evans’ blue extravasa-tion at 2 hours between TMJ and PO-inflamed rats (P .0.05). On 1–3 days after CFA, however, the Evans’ blueextravasation in TMJ-inflamed tissue was significantlygreater than that in PO-inflamed tissue (P , 0.01; Fig.1A). These results confirmed that persistent inflammationwas induced after CFA injection and the intensity ofinflammation in deep tissue was higher than that incutaneous tissue.

Fos-LI in the trigeminal nucleus

Compared to naive animals (n 5 4) in which very fewFos-positive cells were seen, Fos-LI was induced in thetrigeminal subnuclei interpolaris and caudalis and rostralspinal dorsal horn following CFA-induced TMJ and POinflammation. Fos-immunoreactive reaction product ap-peared as distinctive light to dark brown staining incellular nuclei. The labeled nuclei were round to oval andoften exhibited unstained nucleoli. In laminated sub-nucleus caudalis and cervical dorsal horn, Fos-positivecells were mainly distributed in laminae I/II and V/VI; fewFos-labeled neurons were found in laminae III/IV. Photomi-crographs in Figure 2 illustrate examples of Fos-labeledcells in upper cervical dorsal horn, the spinal trigeminalnucleus, and paratrigeminal nucleus after CFA injection.

Acute Fos-LI after orofacial inflammation (Figs.

3,4). Two hours after the injection of CFA into one side ofthe TMJ and PO skin, Fos-immunoreactive nuclei ap-peared throughout the trigeminal subnucleus interpolaristo the C2–3 cervical spinal dorsal horn. There were noFos-positive cells in the trigeminal subnucleus oralis atthis time point. In TMJ-inflamed rats, the Fos-LI exhibiteda bimodal distribution with a large peak centered at theperiobex level and a small peak at about the C2 spinallevel. The caudal peak of Fos-labeling in PO-inflamed ratswas not as clear as in TMJ-inflamed rats. Compared toFos-LI after TMJ inflammation, PO-inflammation-in-duced Fos-LI was more restricted rostrocaudally at 2hours after CFA injection. In addition to the side of thebrainstem ipsilateral to the inflammation, a number ofcells located on the contralateral side also exhibited Fos-LI. However, the mean number of Fos-labeled neuronsipsilateral to inflammation was significantly higher thanthat of the contralateral side (ANOVA, TMJ-inflamed:F

1,1205 28.24, P , 0.0001; PO-inflamed: F1,108 5 592.80,

P , 0.0001). Post-hoc analysis revealed significant differ-ences in the number of Fos-immunoreactive neurons at theperiobex and upper cervical levels (Fig. 4a, b) betweenipsilateral and contralateral sides.

In TMJ vehicle- (oil/saline) injected rats, a few Fos-labeled cells were observed in the middle portion of theipsilateral superficial dorsal horn in the segments between2.70 mm to 5.80 mm caudal to the obex 2 hours afterinjection (n 5 2). Two hours after PO vehicle- (oil/saline)injection (n 5 2), a few scattered Fos-labeled cells wereobserved in the laminae III/IV of the upper cervical dorsalhorn. At the periobex level, some Fos-labeled cells weredetected primarily in the ventral pole of the trigeminalnucleus in vehicle-injected rats, which was very similar tothat in rats receiving anesthesia alone. The total numbersof Fos-labeled neurons on the side ipsilateral to vehicleinjection were 46.8 and 51.4 in PO- and TMJ-injected rats,respectively (Fig. 1B).


Persistent Fos-LI after orofacial inflammation (Figs.

3,4). Compared to the 2-hour time point, there was asignificant increase in Fos-LI in the spinal trigeminalnucleus at 24 hours and 72 hours following CFA injection(Fig. 1B) and Fos-LI spread further rostrally and caudally(Fig. 4a,b). The overall Fos-LI peaked between 24 and 72hours post-CFA (Fig. 1B). In TMJ-inflamed rats, a distinc-tive feature of Fos-LI during 24–72 hours after CFA wasthat in the upper cervical dorsal horn, the Fos-LI gradu-ally spread from the superficial to the deep dorsal horn(Figs. 3a, 5A). As shown in Figure 5A, within 24 hoursafter inflammation, the number of Fos-positive cells in thesuperficial dorsal horn (laminae I/II) was significantlyhigher than that in the deep dorsal horn (P , 0.01).Seventy-two hours after inflammation, however, Fos-positive cells were predominantly located in the deepdorsal horn (laminae V/VI) due to a dramatic increase inFos-labeled cells in the deep dorsal horn. In PO-inflamedrats, the number of Fos-positive cells also progressivelyincreased in the deep dorsal horn between 2 hours and 72hours following inflammation (Fig. 5B). However, theFos-LI in the superficial dorsal horn (laminae I/II) was alsoincreased and remained significantly higher than that inthe deep dorsal horn (P , 0.01) from 2 hours to 72 hoursafter CFA (Fig. 5B).

Ten days (240 hours) after TMJ inflammation, there wasstill residual Fos-LI in the subnuclei interpolaris andcaudalis, and rostral cervical spinal dorsal horn (Figs. 3a,4a). The bimodal distribution of Fos-LI was still identifi-able at 10-day post-CFA (Fig. 4a). Compared to TMJinflammation, the PO inflammation-induced Fos-LI didnot persist as long. Very few Fos-positive cells were foundon 10 days after PO inflammation. The other interestingdifference was that TMJ, but not PO, inflammation in-duced significant Fos-LI in the ipsilateral paratrigeminalnucleus (Figs. 2C, D, and 3a). Fos-labeled neurons ap-peared in the paratrigeminal nucleus 2 hours after CFA,peaked at 72 hours, and persisted over the 10-day observa-tion period. There were no Fos-labeled cells in the paratri-geminal nucleus after PO inflammation (Fig. 3b).

The overall Fos-LI in the trigeminal nucleus after TMJand PO inflammation was similar at 2-hour post-CFA (P .0.05). At 24–72 hours following inflammation, however,TMJ-inflamed rats exhibited significantly higher levels ofFos-LI than that in PO-inflamed rats (Fig. 1B). Interest-ingly, the time course of Fos-LI in TMJ- and PO-inflamedrats closely paralleled the development of inflammation(Comparing Fig. 1A and 1B). Thus, the same amount ofCFA induced significantly higher levels of Fos-proteinexpression when injected into the TMJ, suggesting that

Fig. 1. A: The intensity of complete Freund’s adjuvant (CFA)-induced temporomandubular joint (TMJ) and perioral (PO) inflamma-tion as indicated by plasma extravasation. Evans’ blue dye wasextracted from the inflamed tissue and measured with a spectropho-tometer at 620 nm. B: Comparison of the total number of Fos-immunoreactive (ir) neurons in the spinal trigeminal complex ipsilat-

eral to TMJ and PO inflammation, vehicle injection, and methohexitalanesthesia alone. A log10 scale is used on the abscissa. The time pointsshown are 2-hour, 1-day, 3-day, and 10-day, respectively. Asterisksindicate significant differences between TMJ- and PO-inflamed rats:**P , 0.01; ***P , 0.001.

280 Q. ZHOU ET AL.

the difference in Fos-LI after TMJ and PO CFA injectionmay be due to a difference in the intensity of inflammationinduced in cutaneous and deep orofacial tissues.

Very few Fos-positive cells were seen in the trigeminalnucleus 72 hours following vehicle injection into the TMJ(n 5 2; Fig. 1B). Three days after vehicle injection into thePO skin, some Fos-positive cells were seen in the interme-diate portion of the ipsilateral trigeminal nucleus at thelevel of the interpolaris/caudalis transition zone (n 5 2).The total numbers of Fos-labeled neurons on the sideipsilateral to vehicle injection were 49.0 and 11.9 in PO-and TMJ-injected rats, respectively (Fig. 1B).

Bilateral Fos-LI in the trigeminal nucleus

A unilateral injection of CFA into the TMJ or PO skininduced Fos-LI on both sides of the medulla (Figs. 3 and 4).We further analyzed this bilateral Fos-LI by comparingthe number of Fos-positive neurons in the subdivisions ofthe trigeminal nucleus. Because contralateral Fos-LI wasmost prominent surrounding the interpolaris/caudalis tran-sition zone,we focused our analysis in a segment from 2.7mm rostral to 0.9 mm caudal to the obex. The trigeminalnucleus was arbitrarily divided into dorsal two-thirds andventral one-third subdivisions. Within the trigeminal tran-

sition zone, the dorsal two-thirds of the trigeminal nucleusincludes mainly a portion of the subnucleus caudalis, andthe ventral one-third of the trigeminal nucleus contains amajor portion of the subnucleus interpolaris and only asmall part of the ventromedial subnucleus caudalis. Impor-tantly, this analysis revealed that in the dorsal portion ofthe trigeminal nucleus, the inflammation-induced Fos-LIwas predominantly ipsilateral, whereas the Fos-LI in theventral portion of the trigeminal nucleus was equivalentbilaterally. This was seen in both TMJ- and PO-inflamedrats (Fig. 6). In rats receiving methohexital anesthesiaonly (n 5 3), an induction of Fos-LI was also observed, butit was predominantly located in the ventral portion of thetrigeminal nucleus at the periobex, but not upper cervical,level at 2 hours after anesthesia (Fig. 7). This was alsoseen in vehicle-treated rats at 2 hours, but not at 72 hours,after injection.

Fos-LI in other medullary nuclei

Inflammation of the TMJ and PO skin also inducedFos-LI in other medullary nuclei in the medulla. Thedistribution of Fos-positive cells in the following medullarynuclei was qualitatively the same after TMJ and POinflammation. The Fos-LI in the nucleus tractus solitarius

Fig. 2. Photomicrographs showing examples of inflammation-induced Fos-like immunoreactivity (LI). A: Fos-labeling in the ipsilat-eral superficial dorsal horn at the C1 level 2 hours after completeFreund’s adjuvant (CFA) injection into the temporomandubular joint(TMJ). B: Montage of Fos-LI in the spinal trigeminal nucleus at the

obex level 3 days following CFA injection into the TMJ. C: Fos-LI in theparatrigeminal nucleus 2 hours after CFA injection into the TMJ.D: Fos-LI in the paratrigeminal nucleus 3 days after CFA injectioninto the TMJ. Scale bars 5 50 µm. Scale bar in D applies to A, C.


(NTS) was the most prominent among nontrigeminalnuclei. Fos-labeled nuclei clustered within bilateral NTS,sparing the solitary tract. It peaked at the 2-hour timepoint and was reduced, but still persisted, 24–72 hoursafter CFA. Fos-labeled cells could be identified in thesubdivisions of the NTS. At about 1.0 mm rostral to theobex, some Fos-positive cells accumulated in the lateralNTS; the other Fos-positive cells were scattered in themedial and gelatinous portions of the NTS. A group ofFos-labeled cells was often found located in the parasoli-tary nucleus ipsilateral to inflammation. From the caudalbrainstem at the level of pyramidal decussation, Fos-LIappeared in bilateral cell group B of the inferior medialolivary nucleus (IOM) and then rostrally in cell group C(IOC) of the IOM (Fig. 3). The Fos-LI in the IOM peaked atthe obex and gradually disappeared about 0.5 mm rostralto the obex. Lateral reticular nucleus (LRN) exhibitedFos-labeled cells bilaterally within 1.0 mm rostral to theobex. Most Fos-positive cells in the LRN were scatteredthroughout the parvicellular portion of the LRN. As manyas 30 labeled cells could be seen on one side of the LRN on

one section. Some Fos-positive cells were also seen in thetrigeminal extension of the parvicellular reticular forma-tion, a part of the interstitial structures of the spinaltrigeminal tract. The nucleus raphe obscurus (ROb) con-tained a few labeled cells at the level of the subnucleusinterpolaris. The nucleus raphe pallidus (RPa) exhibitedFos-labeled nuclei throughout its rostrocaudal extentionwith a peak at about 2.0 mm rostral to the obex. ScatteredFos-labeled cells were observed in the nucleus raphemagnus (NRM) at the level of subnucleus oralis. As manyas 20 labeled cells could be seen in the NRM territory onone section. Scattered Fos-positive cells were also found inthe caudal medullary reticular field, C1 adrenaline cellgroup, external cuneate nucleus (ECu), and medial vestibu-lar nucleus (MVe).

Methohexital anesthesia alone also resulted in Fosexpression in other medullary nuclei including the NTS,caudal ventrolateral medulla, and IOM. Compared to TMJor PO inflammation, the anesthesia-induced Fos labelingwas weaker in other medullary nuclei and only appearedat 2 hours after inflammation. The Fos-labeling in vehicle-

Fig. 3. A,B: Schematic illustration of inflammation-induced Fos-like immunoreactivity (LI) in the rat caudal brainstem at 2 hours, 24hours, 3 days, and 10 days after injection of complete Freund’sadjuvant (CFA) into the temporomandubular joint (TMJ; A) or perioral(PO) skin (B). The brightfield and darkfield microscopic images weredigitized, input into a graphic application (Macintosh Clarisdraw),and traced. The brain structures and outlines of the nuclei were

identified by comparing the images under darkfield illumination withreference to Paxinos and Watson (1986), Kruger et al. (1995), andMolander et al. (1989). Each dot represents one Fos-labeled nucleus.The numbers in the middle column indicate the rostrocaudal distance(mm) from the obex. Data for each time point are from one individualrat. For abbreviations, see list.

282 Q. ZHOU ET AL.

treated rats with methohexital anesthesia was very simi-lar to that induced by anesthesia alone at 2-hour timepoint. Seventy-two hours after vehicle injection, very fewFos-labeled cells were seen in other medullary nuclei.Thus, any Fos-labels seen in vehicle-treated animals werealmost entirely due to anesthesia.

DISCUSSION

Complete Freund’s adjuvant has been used as an inflam-matory agent to induce inflammation and hyperalgesia inthe hindpaw of the rat (Iadarola et al., 1988; Stein et al.,1988; Hylden et al., 1989). We have adapted this method toproduce inflammation and hyperalgesia in the orofacialregions (Ren and Dubner, 1996a; Imbe and Ren, 1999). Thepresent study showed that CFAproduced persistent inflam-mation and protracted trigeminal neuronal activation thatlasted over a week. Mustard oil has also been injected intothe TMJ to produce inflammation (Haas et al., 1992; Yu etal., 1995). After mustard oil injection, the TMJ inflamma-tion peaks within 1 hour and electromyographic (EMG)activity of the jaw muscles is facilitated and resolvedwithin less than 1 hour (Yu et al., 1995). Compared tomustard oil injection, the CFA-produced inflammation

provides a model that mimics more closely orofacial chronicpathologic conditions and persistent pain.

Persistent orofacial inflammation andprolonged Fos protein expression

Most previous studies on trigeminal Fos expression havefocused on responses to acute or transient stimuli (Lu etal., 1993; Strassman and Vos, 1993; Bereiter et al., 1994;Sugimoto et al., 1994; Carstens et al., 1995; Hathaway etal. 1995). Trigeminal Fos-LI typically reaches the maxi-mum at 2 hours after acute orofacial stimulation and wasresolved within 24 hours (Wakisaka et al., 1992; Hatha-way et al., 1995). Different from previous studies, persis-tent Fos protein expression was found following injectionof CFA into the TMJ and PO skin in the present study. Itwas further found that the TMJ and PO inflammation-induced Fos expression paralleled the intensity and courseof inflammation over the 10-day observation period. Thisobservation suggests that the increase in intensities andpersistence of Fos protein expression may be associatedwith a maintained increase in peripheral neural inputfrom the site of injury. The greater Fos expression afterTMJ CFA may reflect a difference in peripheral inflamma-tory response after deep and cutaneous CFA injection.

Figure 3 (Continued)


The maintained peripheral neural barrage is also asource of central sensitization after tissue injury. Indeed,deep tissue inflammation produces stronger central sensi-tization (Woolf and Wall, 1986). We cannot rule out thepossibility that increased central hyperexcitability alsocontributes to greater Fos induction after TMJ inflamma-tion. N-methyl-D-aspartate (NMDA) receptor activationplays an important role in development and maintenanceof central hyperexcitability after injury (Woolf and Thomp-son, 1991; Ren et al., 1992). Trigeminal Fos activationafter noxious stimulation is selectively attenuated byadministration of NMDA receptor antagonists (Bereiter etal., 1996; Mitsikostas et al., 1998), suggesting an NMDAreceptor-related central sensitization at the trigeminal

level. Our recent results indicate that trigeminal nocicep-tive neurons with hyperexcitability after persistent TMJinflammation were more sensitive to NMDA receptorantagonist-produced suppression when compared to neu-rons after PO cutaneous inflammation (Miki et al., 1999).It is likely that greater central excitation also occurred inthe TMJ-inflamed animal as a consequence of intenseperipheral inflammation.

Temporomandibular joint versus perioralskin inflammation

Inflammation of the TMJ and PO skin mimics injury tothe orofacial deep and cutaneous tissues, respectively. TheTMJ is innervated mainly by the articular branch of the

Fig. 4. a,b: Summary of the induction of Fos-like immunoreactiv-ity (LI) in the trigeminal nucleus and rostral cervical spinal cordfollowing inflammation of temporomandubular joint (TMJ; a) andperioral (PO; b) skin. Mean number of Fos-positive neurons per sectionis obtained from every 0.45-mm segment of the brainstem-spinaltissue block from 4.05 mm rostral to 6.75 caudal to the obex. The

relative location of the section is indicated on the abscissa. 0 5 obex;positive 5 rostral; negative 5 caudal. Note that the scale for theordinate is reduced in b. The asterisks indicate significant differencesin Fos-labeled neurons between ipsilateral and contralateral sides:*P , 0.05; **P , 0.01. CFA, complete Freund’s adjuvant.

284 Q. ZHOU ET AL.

auriculotemporal nerve from the mandibular division ofthe trigeminal nerve and the PO skin is innervated bybranches of both maxillary and mandibular divisions ofthe trigeminal nerve (Greene, 1955). The central represen-tation of primary afferents from these two regions hasdistinct patterns, although there are some similarities(Jacquin et al., 1983; Takemura et al., 1987; Arvidsson etal., 1992). Although the Fos-LI to joint inflammation haslimited utility for topographic analysis of primary afferentinput, comparison from a functional perspective providessome insight into the mechanisms underlying TMJ inflam-mation and associated disorders.

Common to inflammation of both TMJ and PO skin,major Fos expression was exhibited at the periobex andupper cervical levels. Very few Fos-LI was found beyondthe rostral subnucleus interpolaris. In the laminatedportion of the subnucleus caudalis, Fos-labeled cells weremainly distributed in laminae I/II and V/VI; few Fos-labeled neurons were found in laminae III/IV. This result

suggests that the Fos-labeling was primarily a response toafferent input carried by high-threshold nociceptive fibers.Most high-threshold trigeminal nociceptive afferents donot project rostral to the subnucleus interpolaris and theymainly terminate in the superficial (laminae I/II) and deepdorsal horn (laminae V/VI) but not the nucleus proprius(laminae III/IV) where low- threshold large fibers termi-nate (see Capra and Dessem, 1992). Together with verifica-tion of the Evans’ blue dye extravasation, the laminarspecific distribution of Fos-LI also ruled out the possibilitythat the major branches of the mandibular nerve weredirectly affected by injection of CFA into the TMJ.

An interesting finding of this study was that a dominantipsilateral Fos induction was found in the paratrigeminalnucleus after TMJ but not PO inflammation. By using insitu hybridization histochemistry, we have found a similardifferential effect of TMJ and PO inflammation on prepro-dynorphin mRNAexpression in the paratrigeminal nucleus

Figure 4 (Continued)


(Imbe and Ren, 1999). The injection of mustard oil into theTMJ also results in Fos-labeling in the ipsilateral paratri-geminal nucleus (Hathaway et al., 1995). The paratrigemi-nal nucleus consists of a group of neurons and neuropilwithin the dorsal part of the spinal trigeminal tractthroughout the caudal half of the subnucleus interpolaris(Chan-Palay, 1978; Takemura et al., 1987; Phelan andFalls, 1989) and receives convergent sensory input fromthe head and neck as well as from the cranial portion of thealimentary tract (Armstrong and Hopkins, 1998). Ourfindings indicate that this input is from deep tissuesexclusively. The involvement of the paratrigeminal nucleus

in trigeminal nociceptive transmission and pain requiresfurther investigation.

The present results show that in the laminated sub-nucleus caudalis and cervical dorsal horn, Fos-labeledneurons spread from the superficial into deep dorsal hornas the inflammation persisted. Further, the spread ofFos-LI into the deep dorsal horn was much greater inTMJ-inflamed rats. These results are very similar to thosein a spinal model of arthritis (Abbadie and Besson, 1992)and chronic constriction injury to the infraorbital nerve(Vos and Strassman, 1995). Comparing the time course ofinflammation and Fos-LI, the first phase of Fos expressionin the superficial laminae may be a response to a phasicnociceptive input, and the late induction of Fos-LI a resultof persistent or tonic nociceptive input (Munglani andHunt, 1995). The persistent increase in Fos-LI in the deeplaminae may reflect injury-induced neuroplasticity that isinvolved in the development of persistent hyperalgesia. Ithas been shown that application of inflammatory media-tors to deep cranial structures sensitizes trigeminal sen-sory neurons that are mainly located in lamina V of thesubnucleus caudalis (Hu et al., 1992; Burstein et al., 1998).

Compared to cutaneous CFA injection, the injection ofCFA into the TMJ produced a significantly stronger inflam-mation associated with a greater Fos expression. TheFos-LI after TMJ inflammation persisted longer, was moreintense, particularly in the superficial laminae, and morewidespread rostrocaudally. Thus, the same amount ofirritant produces a much stronger effect in deep than incutaneous orofacial tissue. As there is heavy innervation ofthe TMJ by unmyelinated nerve endings (Broton et al.,1988; Sessle and Hu, 1991, review), strong nociceptiveprimary afferent barrage is expected following inflamma-tion. An increase in TMJ C-fiber input after inflammationand strong central neuronal activation may initiate cen-tral hyperexcitability and contribute to persistent painassociated with temporomandibular disorders.

Functional and topographical considerations

The present results indicate that orofacial inflammation-induced Fos expression spread over a long segment of theipsilateral trigeminal nucleus and upper cervical dorsalhorn, appeared on the side contralateral to the injury, andwas induced in many nontrigeminal nuclei. This is consis-tent with the observation that trigeminal primary affer-ents terminate in widespread and functionally diverseareas of the rat central nervous system (Marfurt andRajchert, 1991). This is somewhat different from thespinal-hindlimb system where a clear somatotopic relation-ship can be defined for nociceptive primary afferent inputand spinal Fos protein expression (see Munglani andHunt, 1995, for a review). Because the Fos-LI induced byorofacial stimulation cannot be fully explained by puresomatotopic analysis and based on the fact that Fosprotein expression can be induced both mono- and poly-synaptically (Hunt et al., 1987; Sagar et al., 1988), wepropose to divide the overall orofacial inflammation-induced Fos-LI into the following functional categories.

Somatotopically organized nociceptive activation.

The caudal peak of Fos-labeling after TMJ and PO inflam-mation matched terminal fields of TMJ and PO primaryafferents (Arvidsson, 1982; Jacquin et al., 1983) and thusmay be a direct response to nociceptive input that origi-nated from the site of injury (also see Strassman and Vos,1993; Hathaway et al., 1995). The paratrigeminal Fos-

Fig. 5. A,B: Comparison of laminar distribution of inflammation-induced Fos-like immunoreactivity (LI) in laminated subnucleuscaudalis and rostral cervical dorsal horn. The mean of the sum ofFos-labeled neurons in the segments from 2.70 mm to 6.75 mm caudalto the obex is shown. The Fos-LI was distributed primarily in thesuperficial (laminae I,II) dorsal horn after orofacial inflammation.Three days after temporomandubular joint (TMJ) inflammation,however, Fos-positive cells were predominantly located in the deepdorsal horn (laminae V/VI) due to a dramatic increase in Fos-labeledcells in that region. A log10 scale is used on the abscissa. The timepoints after complete Freund’s adjuvant (CFA) injection are 2-hour,1-day, 3-day, and 10-day, respectively. Each group includes 3–6 rats.Asterisks denote significant differences between TMJ- and perioral-(PO) inflamed rats; P , 0.01.

286 Q. ZHOU ET AL.

labeling after TMJ inflammation is another example of adirect response to nociceptive input, which is consistentwith a dense trigeminal primary afferent projection, includ-ing auriculotemporal nerve that innervates the TMJ, tothe paratrigeminal nucleus (Jacquin et al., 1983; Take-mura et al., 1987; Marfurt and Rajchert, 1991; Sugimoto etal., 1997). The rostral peak of Fos-labeling at the interpo-laris/caudalis transition zone is more complex. Primaryafferent input from the TMJ and PO regions terminate inthe dorsal portion, but not the ventral portion of thetransition zone (Klineberg, 1971; Capra, 1987; Takemuraet al., 1987; Pfaller and Arvidsson, 1988). Our analysisindicated that the Fos-LI was predominantly ipsilateralonly in the dorsal portion of the trigeminal nucleus that

includes mainly the subnucleus caudalis. Thus, at thelevel of the trigeminal transition zone, only a portion ofFos-positive cells, primarily in the dorsal part of thecaudalis, are directly activated by the primary afferentbarrage.

Reflexive activity. Fos-labeling was found on the sidecontralateral to TMJ and PO inflammation in the presentstudy (also see Strassman and Vos, 1993; Bereiter et al.,1994; Coimbra and Coimbra, 1994; Sugimoto et al., 1994;Carstens et al., 1995). Although a portion of the mandibu-lar primary afferents, some of which may innervate the POregion, projects directly to the contralateral subnucleuscaudalis and cervical dorsal horn via the posterior commis-sure, most crossing fibers innervate midline divisions

Fig. 6. Summary of inflammation-induced Fos-like immunoreactiv-ity (LI) in the trigeminal interpolaris/caudalis transition zone. Thetrigeminal nucleus at the transition zone was divided into dorsaltwo-thirds and ventral third subdivisions (see text). The mean of thesum of Fos-labeled cells in the segments between 2.7 mm rostral and0.9 mm caudal to the obex is plotted against the time post-inflammation. Asterisks denote significant differences between dorsal

and ventral divisions; P , 0.01. Significant difference was onlydetected in the dorsal transition zone. Note that the Fos-LI waspredominantly ipsilateral in the dorsal division but equivalent bilater-ally in the ventral division. A log10 scale is used on the abscissa. Thetime points after complete Freund’s adjuvant (CFA) are 2-hour, 1-day,3-day, and 10-day, respectively. Each group includes 3–6 rats.


(Jacquin et al., 1982,1990; Takemura et al., 1987). Primaryafferent nerves with unilateral peripheral receptive fieldssuch as the auriculotemporal branch of the mandibularnerve do not project to the contralateral brainstem andcervical cord (Jacquin et al., 1983; Takemura et al., 1987).In addition, the decussation fibers of trigeminal primaryafferents may mainly consist of non-nociceptive fibers(Jacquin et al., 1990). Thus, it is likely that most contralat-eral Fos-labeling found in the present study was not aresponse to direct primary afferent input from the in-flamed side (also see Sugimoto et al., 1994). Instead,contralateral neurons may be excited through polysynap-tic mechanisms. In the cat, medullary and cervical dorsalhorn neurons do project intrasegmentally to contralateraldorsal horn (Hockfield and Gobel, 1982). The contralateralneuronal activation may be important for reflexive adapta-tion of head-neck movement after inflammatory injury ofthe orofacial tissues.

Significant Fos-LI was found in the LRN and IOM afterTMJ and PO inflammation. Previous studies have shownFos protein induction in the LRN and IOM followingnoxious stimulation of the rat hindpaw (Jones and Blair,1995) and injection of mustard oil into the TMJ (Hathawayet al., 1995). Because the LRN and IOM do not receivedirect primary afferents from the orofacial region, theFos-LI in these nuclei must be a result of polysynapticactivation. The LRN in the caudal ventrolateral medullareceives bilateral input from cervical neurons and sendsmajor afferent fibers to the cerebellar cortex (see Ruigrokand Cella, 1995). The IOM receives afferent connectionsfrom somatosensory pathways originating from spinalcord, spinal trigeminal nucleus, and dorsal column nuclei(see Ruigrok and Cella, 1995). The inferior olive is the solesource of cerebellar climbing fibers (Desclin, 1974). The

increased neuronal activity in these cerebellar pathwaysmay facilitate coordination of nocifensive behaviors suchas frequent licking and facial grooming that require fine-controlled forelimb and head movement.

Somatoautonomic processing. Orofacial sensorystimulation also leads to changes in autonomic function(Bereiter, 1993; Lu et al., 1993; Allen and Pronych, 1997).Somatovisceral pathways consist of trigeminal primaryafferents, the NTS, the trigeminal nuclei, the parabrachialnuclei, and other brainstem nuclei (Bereiter, 1993; Feiland Herbert, 1995; Allen and Pronych, 1997). Some Fos-labeling after orofacial inflammation may reflect neuronalactivity that is associated with autonomic control. Trigemi-nal primary afferents terminate in the NTS (Takemura etal., 1987; Pfaller and Arvidsson, 1988; Marfurt and Ra-jchert, 1991). The NTS has extensive efferent connectionswith other parts of the nervous system and is a keystructure in somatovisceral processing (see Randich andMaixner, 1984; Ren et al., 1990; Lima et al., 1994).Although a variety of noxious stimuli evoke Fos-LI in theNTS (Bullitt, 1990; Hammond et al., 1992; Lu et al., 1993;Strassman and Vos, 1993; Lanteri-Mintet et al., 1994;Hathaway et al., 1995; Jones and Blair, 1995; presentstudy), many nonnoxious stimuli also induce Fos-labelingin NTS (Erickson and Millhorn, 1991; Strassman and Vos,1993; Monnikes et al., 1997; Teppema et al., 1997; Uemuraet al., 1997; Wu and Ling, 1998). It appears that neuronalactivation in the NTS is a general response to somatic orvisceral input that is not necessarily selective to noxiousstimulation. However, Fos-labeling in the NTS may beindirectly associated with noxious manipulation in theorofacial region in a given experimental setting.

The Fos-labeling in the ventral transition zone wasequivalent bilaterally. Interestingly, anesthesia alone caninduce Fos-labeling at this level, primarily located in theventral portion of the trigeminal nucleus (Strassman andVos, 1993; present study). Bereiter et al. (1994) havedemonstrated in cats that neurons in the ventrolateralpole of the spinal trigeminal nucleus within the transitionare required for corneal-evoked changes in autonomicfunction. It appears that the Fos-labeling observed in theventral trigeminal transition zone may be involved inautonomic responses after orofacial inflammation.

Descending modulation. Peripheral inflammation ac-tivates endogenous pain control (Ren and Dubner, 1996b).The Fos-labeling in the NRM, a major nucleus thatproduces descending modulation of nociception (Fields andBasbaum, 1978), suggests an activation of descendingcontrol after orofacial inflammation. Fos-labeling in thenucleus raphe obscurus may also belong to this category(Tseng et al., 1995). Because the LRN and NTS are alsoinvolved in nociceptive modulation (Gebhart and Ossipov,1986; Ren et al., 1990), some Fos-LI in these nuclei mayalso be a result of neuronal activation within pain-modulating circuitry (also see Jones and Blair, 1995;Bellavance and Beitz, 1996; Yamashiro et al., 1997).However, it is difficult to separate the Fos-LI related todescending modulation from those related to other activi-ties (see above). Persistent orofacial stimulation may alsoactivate neurons in more rostral structures of the nervoussystem. After formalin injection into the face of the cat, Fosprotein is induced in the anterior cingulate, anteriorinsula, thalamic nuclei, and hypothalamus (Kuroda et al.,1995). Twenty-four hours after experimental tooth move-ment, Fos-labeling was found in the amygdala, hypothala-

Fig. 7. Effect of methohexital anesthesia alone on Fos proteinexpression in the trigeminal transition zone. Fos-labeling was primar-ily located bilaterally in the ventral portion of the transition zone. Therelative location of the section is indicated on the abscissa. 0 5 obex;positive 5 rostral; negative 5 caudal. Right and left refer to right andleft side of the brainstem, respectively. **Significantly different fromLeft/Dorsal and Right/Dorsal; P , 0.01. ##Significantly different fromRight/Dorsal; P , 0.01.

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mus, and thalamus in rats (Yamashiro et al., 1998). Thelong-term effect of persistent orofacial inflammation oncentral nociceptive pathways and endogenous pain controlneeds to be further investigated.

Thus, a full spectrum of nociceptive activity includessomatotopically organized nociceptive activation, reflexactivity, autonomic response, and endogenous pain modu-lation. The complexity of Fos induction after orofacialnoxious stimulation suggests that Fos is not an exclusivemarker for somatotopically organized neuronal activationafter noxious orofacial stimulation, and involves complexbrainstem neural networks participating in the animal’sreaction to persistent tissue injury.

ACKNOWLEDGMENTS

We thank Dr. N. Capra for critical reviewing of an earlierdraft of this manuscript, Drs. L. Gartner and J. Hiatt fordiscussion on the anatomy of the temporomandibularjoint, and Ms. E. Wade and Ms. S. Zou for technicalassistance.

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