Turkish Neurosurgery 10: 108 - 111, 2000 Çalgiiiier: Possible {neini iierve-tympniiic plexilS comiectioii

OriginStudy

NerveTracer

Fibers of FaciaIPlexus: A

Searching forin the Tympanic

Aranmasi:Kökenli Liflerinçalismasi

SinirIsaretleme

Pleksusta FacialBir

Timpanik

ENGIN ÇALGÜNER, SEMIH KEsKIL, ALPER ATASEVER, RABET GÖZIL

Gazi University Medical Faculty, Department of Anatomy, Besevler (EÇ), Fatih University Medical School, Department ofNeurosurgery, Emek (SK), Hacettepe University Medical Faculty, Department of Anatomy, Sihhiye (RG), Ankara, Turkey

Supported by the Gazi University Research Fund Project No: TF 0195-15.

Received : 10.2.2000 ~ Accepted : 20.10.2000

Abstract: This study investigated a possible connectionbetween the facial nerve andi or its chorda tympanibranch, and the tympanic plexus with the aim of definingsome of the innervation of the middle ear mucosa. The

study involved 20 locally bred male and female Wistaralbino rats that were 2.5 to 3-months old and weighed 200­300 g.. Horseradish peroxidase, a retrograde axonal tracer,was injected into the middle ear mucosa of each rat, andleakage from the injection po int s was carefully removedby wiping with gauze pads or suctioning with amicropipette. The total amount of horseradish peroxidasesolution that remained in the middle ear mucosa was

approximately 3-4 \ll. At 24-72 hours after this application,the animals were anesthetized and perfusion-fixed, andtheir geniculate ganglion, facial nerve, chorda tympani,and superior cervical ganglion were removed bilaterallyunder magnification. The tissues were then frozen,histochemically processed, and examined. Labeled neuroncell bodies were observed in the ipsilateral superiorcervical ganglion, but there were no labeled neurons oraxons in the geniculate ganglion, facial nerve, or chordatympani. The experiment failed to show any connectionbetween the facial nerve, its chorda tympani branch, andthe tympanic plexus.

Özet: Bu çalismanin amaci, fasiyal sinirin ve korda timpanidalinin pleksus timpanikus ile olan baglantilarinidegerlendirip; bunlarin orta kulak mukoza siinervasyonundaki önemini degerlendirmektir. Bu çalismaiçin her iki cinsten, yerelolarak temin edilmis; 2.5-3 aylik,200-300 gram agirliginda Wistar albino siçanlar kullanildi.Retrograd bir aksonal isaretleyici olan horseradishperoxidase, siçanlarin orta kulak mukozasina enjekte edildive enjeksiyon noktalarindan olan sizinti dikkatli bir sekildespançla silinip veya pipetle aspire edilip; orta kulakmukozasinda kalan horseradish peroxidase'in toplam 3-4mikroiitre olmasi saglandi. Bu uygulamadan 24-72 saatsonra hayvanlar anestezi altinda perfüzyonla tespit edildilerve genikülat ganglionlari, korda timpanileri, superiorservikal ganglionlari ve fasiyal sinirleri büyütme altindaçikarilip, histokimyasal isleme tabi tutulduktan soraincelendiler. Her ne kadar uygulama ile bazi hayvanlardaayni taraftaki superior servikal ganglionda isaretli hücrelergörülse de; söz konusu hayvanlarin genikülat ganglion,fasiyal sinir ve korda timpanilerinde hiç bir isaretli sinir lifiveya hücresi gözlenmedi. Çalismamizda fasiyal sinirdenköken alan ve orta kulak mukozasina giden hiç bir sinir lifigösterilernemistir; bunun nedeni pleksus timpanikus ilefasiyal sinirin dallari arasinda herhangi bir baglantininyoklugu olabilir.

Key word s: Chorda tympani, geniculate ganglion, middleear mucosa innervation, rat, retrograde tracing

Anahtar kelimeler: Genikülat ganglion, korda timpani,orta kulak mukozasi inervasyonu, Retrograd isaretlerne,siçan

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Turkish Neurosurgery 10: 108 - 111, 2000

INTRODUCTION

The latest version of a classical human anatomyatlas contains one new figure: a sketching of the"tympanic plexus" (a network containing sensoryfibers that supply the middle and external ear) withsome fine connections from the facial nerve named

the "ramus communicans (eum plexu tympanico)"(13). it is proposed that a few sensorial tympanicbranches of the facial nerve join the tympanic plexusafter passing through the petrous bone from thegeniculate ganglion, where their ceii bodies arefound. "These fibers are embryological remnants ofthe second pharyngeal pouch. Apart from suppIyingthe mucosa, they may extend to the tympanicmembrane and external meatus; thus explaining theoccurrence of vesicles there in focal herpes." (9)

Classical neuroanatomic data based on

observations made using magnificatioi:i- in humancadavers and retrograde tracer techniques indicatethat the main trunk of the tympanic plexus isJacobson's nerve (18). This nerve arises from theglossopharyngeal nerve, penetrates the floor of thetympanum, and courses superiorly across thepromontory to project to the tympanic plexus andanastomose with the lesser petrosal nerve (1,5,11,14).

Within the middle ear (ME) mucosa that coversthe promontorium is a fine plexiform network ofnerves, the abovementioned tympanic plexus, whichsupplies sensory fibers to the middle and externalear as well as preganglionic secretomotor fibers tothe petrosal nerves (18).The sympathetic componentsof the ME mucosa innervation arise from the

caroticotympanic nerve branch of the internal carotidsympathetic plexus, which originates mainly in theipsilateral superior cervical ganglion and partly inthe ipsilateral pterygopalatine ganglion, andanastomoses with Jacobson's nerve anteriorly (4,6,7,8,9,11,12,14,16).

The chorda tympani branches oH the facialnerve approximately 6 mm above the stylomastoidforamen. it then ascends anteriorly within a canal topenetrate the posterior wall of the tympanic cavityvia its posterior canalieulus, near the posterior borderof the rnedial aspect of the tympanic membrane andlevel with the upper end of the handIe of the malleus.Passing anteriorly between the fibrous and mucouslayers of the membrane, the nerve erosses rnedial tothe handIe of the malleus and re-enters the bone viaits anterior canaliculus at the rnedial end of thetympanic fissure. The neuronal cellbodies are located

Çalgiiiier: Possib/e facia/ iier<.le-tY17lpmiic p/exus coiwectio/l

in the geniculate ganglion, and receive taste inputsfrom the anterior two-thirds of the tongue. Thechorda tympani also includes some parasympatheticfibers that synapse in the submandibular ganglionand innervate the submandibular and sublingualsalivary glands (18).

Since its introduction, the use of retrogradeaxonal transport of the macromolecular tracerhorseradish peroxidase (HRP) has proven to be areliable and sensitiye method for mapping neuralpathways. HRP labels both axons and cell bodies,thus pinpointing the origin of fibers that project to agiven site within the central or peripheral nervoussystem. This glycoprotein enzyme is injected into theterminal area of the projection, where it is taken upat the synapses and transported in retrogradedirection via the axoplasm, ultimately reaching thecell bodies (2,3,10).

Since the facial nerve passes close to thetympanic plexus, we thought it possible that somenerve fibers from the fadal nerve andi or the chordatympani might innervate the tympanic plexus andthe ME mucosa. Since there is currently somecontroversy surrounding this subject, we sought toinvestigate with a tracer study.

MATERIALS AND METHODS

The investigation involved 20 locally bred maleand female Wistar albino rats that were 2.5 to 3­

months old and weighed 200-300 g. All work wasconducted in accord with the "Guide for the Care

and Use of Laboratory Animals" (National Institutesof Health, Publication No: 86-23). Initially, all the ratswere sedated with ether and were otoscopicallyexamined for the presence of middle- or external eardisease. The 14 rats with disease-free middle ears

were selected for the experiment. These animals weredeeply anesthetized with an intramuscular injectionof 5mg/kg xylazine and 44 mg/kg ketamine. Usingoperating loops (x3.5), 5 iil of 30% HRP (Type VI,Sigma, St. Louis, USA) in water was slowlyintroduced by pressure injection through a thin glasscannula into various points of the mucosa of the leftME cavity. This was accomplished by piercing theeardrum through a small hole made in the tympanicbulla. To reach the site, we ma de a postauricularincision and then retracted the sternocleidomastoidmuscle to expose the tympanic bulla. Care was takennot to injure the tympanic plexus with inadvertentmovements of the cannula. HRP leakage at theinjection sites was carefully removed by wiping with

109

TiiI'kis/i NCIIl'OSIIl'gCl'Y 10: 108 - 111, 2000

gauze pads or suctioning with a micropipette. Thetotal amount of HRP solution that remained in the

ME mucosa was estimated to be 3-4 jll. The openingswere sealed using superglue (Cyanoacrylate, Loctite,UK) to prevent the dye from leaking out of the MEcavity into the surrounding tissue. The wounds werethen sutured using 3/0 Prolene.

At 24-72 hours after HRP application, we re­anesthetized the animals and intracardially perfusedthem with a rapid bolu s of isotonic saline solutionfollowed by a fixative solution containing 1.25%glutaraldehyde and 1% paraformaldehyde in 0.1 Mphosphate buffer at room temperature (pH 7.4). Thegeniculate ganglion, facial nerve, chorda tympani,and superior cervical ganglion were removedbilaterally under magnification. These tissues wereplaced in 0.1 M phosphate buffer containing 30%sucrose and then stored at 4 oC for 3-5 days. Frozenspecimens were then cut into 30 jlm-thick serialsections on the long axis. These were treated withtetramethylbenzidine in the presence of hydrogenperoxide, and were then counterstained with neutralred (pH 4.8), according to the method of Mesulam(10).

RESULTS

The control outcome was positive in only someof the animals in which HRP injected into the MEmucosa labeled the perikarya and dendrites of thesuperior cervical ganglion neurons ipsilateral to theinjected site (the left side). Diffusion and escape ofthe dye is a well-known problem in tract-tracingstudies. Even though we injected an adequateamount of HRP and there was no tracercontamination of the surrounding tissues, nine of therats showed no HRP labeling in the superior cervicalganglion, possibly due to technical problems.

The superior cervical ganglia of the remainingfive animals exhibited wide variation in the quantitiesof neurons that were labeled. None of these five ratshad any labeled neurons or axons in their geniculateganglia, facial nerves, or chorda tympanii.

DISCUSSION

There are numerous human anatomy reportsof contributions to the tympanic plexus from thetrigeminal and vagal nerves, and from the first fourdorsal root ganglia (4,14,17). In addition, there isreported to be a filament that connects the auricularbranch of the vagus nerve (containing fibers from

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Çnlgi~iit'r: Possible iiicinl iieive-tyiiipnii;c plexiis COllllt'Ctioli

the glossopharyngeal nerve) with the facial nerve,and sometimes with the chorda tympani (11).

When we checked the reference that was cited

in one of the aforementioned human anatomy studiesregarding this connection, it noted an anastomoticbranch from the facial nerve connecting withJacobson's nerve in 86% of cases, but no suchconnection in the remaining 14% (14). Some animalstudies have reported that the tympanic plexus has"functional connections" with the chorda tympani,and a very fine connection with the geniculateganglion, but the authors did not define theseconnections dearly or reliably (17). They may exist,however, and some recent animal studies showedthat a few smaIl nerve cell bodies in the geniculateganglion and in the pterygopalatine ganglion (aparasympathetic ganglion) of the facial nerve werelabeled after a retrograde tracer was introduced intothe ME mucosa 02,16). Although animalexperiments can shed light on these issues, speciesdifferences predude confident extrapolation tohumans.

In this study, although our tracer injections wereadequate, we were not surprised to observe the lackof consistent HRP labeling in some rats. The reasonsfor this may be inappropriate timing of survival ortechnical problems during fixation. In the fiveanimals that exhibited HRP labeling in the ipsilateralsuperior cervical ganglion, we observed no labeledneurons or axons in the geniculate ganglia, facialnerves, or chorda tympanii. The problem with suchnegative findings is that proving the absence of anentity is extremely difficult, if not impossible. Withthis type of result, the only deduction that can bemade, assuming that the technique has been effectiveand that enough data have been gathered, is that theexperiment failed to show any connection betweenthe facial nerve, its chorda tympani branch, and thetympanic plexus.

Our results differ from those of some other

reports in that we were unable to morphologicallydemonstrate that the ME mucosa c0t1tains any fibersof facial nerve origin 02,16). The reason for thisdiscrepancy may be intraspecies or interindividualvariation. it is also possible that these other studiesshowed "false positivity"that actually reflected directuptake by nerve tissue that was damaged duringinjection, or that our results indicate "falsenegativityu in that HRP may not have been taken upby nerve fibers that were, in fact, present.

Turkish Neurosiaga!! 10: LOS - 111, 2000

ACKNOWLEDGEMENTS

This work was slIpported by the Gazi University

Researcli FUlZd Project No: TF 01/ 95-15.

Correspondence: Dr.I.Semih KESKILFethiye Sokak No :4/6Gazi Osman Pasa06700 Ankara TurkeyPhone : 90. 312. 446 45 94Fax: 90. 312. 221 3276EMail: sk06-k@tr.net

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