Histologic evaluation of inferior alveolar lymphatics:an anatomic studyMasoud Yaghmaei, DMD, MS,a Fatemeh Mashhadiabbas, DDS, MS,b Samaneh Shahabi, DDS,c

Akram Zafarbakhsh, DDS,d Saeideh Yaghmaei, DDS,e and Arash Khojasteh, DMD, MS,f

Tehran, IranDENTAL RESEARCH CENTER, SCHOOL OF DENTISTRY, SHAHID BEHESHTI UNIVERSITY OF MEDICALSCIENCES

The purpose of this study was to investigate the presence of a lymphatic system within the inferior alveolarcanal, using specific immunohistochemical staining. Lymphatic capillaries are characterized by a thin wall with anirregular profile. They are superficial to organs and collect the lymph. Currently, the lymphatic system of the mandibleand the floor of the mouth are attributed to the periosteum. The inferior alveolar canal within the mandible housesthe inferior alveolar nerve, artery, and vein. Owing to the surgical vulnerability of the canal, several recent studieshave focused on defining the exact contents and arrangements within the canal as well as precise measurements of thediameter of its components. No study has been performed on the existence of a lymphatic system within the inferioralveolar canal, the presence of which has significant importance for surgical management of malignant lesions.Presence of lymph channels would make the inferior alveolar nerve a potential route for spreading malignant cells. Inthis study, podoplanin-positive thin-walled vessels, considered to be lymphatic vessels, were traced within the inferior

alveolar canal. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;xx:xxx)

The lymphatic structure of the facial skeleton is notfully understood. The oldest definition of the lymphaticdrainage was found in the literature as depicted bySappey in 1874.1 It is suggested that the lymphaticdrainage of the intraoral structures and the floor of themouth communicates with the periosteal lymphaticdrainage of the mandible2. Lymphatics of the anteriorneck lay above the platysma muscle and course inhorizontal, oblique, and upward patterns toward themandible. It is suggested that these pathways also join

This study was supported by a grant-in-aid of the Dental ResearchCenter, Shahid Beheshti University of Medical Sciences.aProfessor and Head, Department of Oral and Maxillofacial Surgery,Dental Research Center, School of Dentistry, Shahid Beheshti Uni-versity of Medical Sciences, Tehran, Iran.bAssistant Professor, Department of Oral and Maxillofacial Pa-thology, Dental Research Center, School of Dentistry, ShahidBeheshti University of Medical Sciences, Tehran, Iran.cSenior Dental Student, School of Dentistry, Dental Research Center,Shahid Beheshti University of Medical Sciences, Tehran, Iran.dSenior Dental Student, School of Dentistry, Dental Research Center,Shahid Beheshti University of Medical Sciences, Tehran, Iran.ePrivate Practice, School of Dentistry, Dental Research Center, Sha-hid Beheshti University of Medical Sciences, Tehran, Iran.fAssistant Professor, Department of Oral and Maxillofacial Surgery,Dental Research Center, School of Dentistry, Shahid Beheshti Uni-versity of Medical Sciences, Tehran, Iran.Received for publication Aug. 3, 2010; returned for revision Nov. 2,2010; accepted for publication Nov. 12, 2010.1079-2104/$ - see front matter© 2011 Mosby, Inc. All rights reserved.

doi:10.1016/j.tripleo.2010.11.013

the periosteal lymphatic drain of the mandible.3 Peri-osteal lymphatic drainage has great importance whendefining the surgical margins of a lesion. Perineuralinvasion has been reported to be responsible for 76% oflocal recurrences.4 The inferior alveolar canal (IAC) isconsidered to be the most important anatomic featurewithin the body of the mandible, containing the inferioralveolar nerve (IAN), artery, and vein. Several studieshave investigated the pattern of the IAN, its correlationto dental roots, and its distance from the outer cortex ofthe mandible.5-12 The anatomic configuration of theIAC has been clearly described in recent years: Theartery is usually a solitary compartment, positionedlingual to the nerve, and the vein appears to be mostlya multiple structure and lies superior to the nerve.13

However, the presence of the inferior alveolar lym-phatic (IAL) has never been investigated before. Re-cently, podoplanin, a specific lymphatic microvesselmarker, has enabled selective immunostaining of thelymphatic microvessels in paraffin-embedded sec-tions.14-16 The purpose of the present study was first, toassess the presence of a lymphatic draining systemwithin the IAC and second, to define its correlationwith the IAN.

MATERIALS AND METHODSTen right hemimandibles were selected from the

forensic anatomy hall, after being ethically approved bythe Iranian forensic medicine ethical committee. Har-

vested hemimandibles were either dentate or partially

1

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edentulous. Samples were frozen at �18°C. Using afissure bur, the body of each hemimandible was cut into3 parts: central to canine, canine to second premolar,and second premolar to the most posterior tooth,named, respectively, anterior, middle, and posteriorsections. Sections were then fixed in a 10% formalinsolution for 10 days, rinsed, decalcified in a 10% nitricacid for 30 days, dehydrated using a graded concentra-tion of ethanol, and embedded in paraffin wax. Anultrathin microtome was used to prepare 5-�m-thickcross-sections along the mandibular canal and mentalforamen.

One-half of the cross-sectioned specimens werestained with hematoxylin-eosin (H&E) and examinedunder the light microscope (400E-Eclipse; Nikon, To-kyo, Japan). The remaining sections were prepared forpodoplanin (11-003; Angio Bio, Del Mar, CA, USA)antibody staining (NovoCastra System, Newcastle,UK) according to the process described by Liang etal.16 A lymphatic was defined as a podoplanin-positiveendothelial-lined structure. For this purpose, sampleswere heated for 16 hours in 37°C and then for 1 hour in60°C in an oven. To reactivate the antigen, sampleswere heated with nitrate buffer solution in microwavesfor 5 minutes (700 W) and 15 minutes (450 W), whichwas followed by a double wash in Tris-buffered salinesolution (TBS), each for 5 minutes. Then samples wereincubated at room temperature for 20 minutes withperoxidase and rinsed for another 5 minutes in TBS.Next, samples were incubated with the antibody againstpodoplanin as a primary antibody with 1:50 dilution for60 minutes at room temperature. The samples werethen incubated with the secondary antibody for 30minutes and washed twice for 5 minutes in TBS, afterwhich the samples were incubated with Novolink poly-mer (NovoCastra) for 30 minutes. It was followed byanother double wash with TBS, each for 5 minutes.Finally, samples were incubated with 3,3=-diaminoben-zidine tetrahydrochloride solution for 10 minutes,washed in tap water for 5 minutes, and then counter-stained with Mayer hematoxylin. Negative control sec-tions were stained by omitting the primary antibody.External positive control samples were lymphangiomasand lingual varicosities. The internal positive controlwas the presence of lymphatic vessel. Slides were eval-uated by light microscope. Data collection was carriedout through observations of a single operator, and allthe findings were recorded manually.

RESULTSH&E staining confirmed the presence of the IAN,

inferior alveolar artery, and inferior alveolar veinwithin the mandibular canal (Fig. 1). Podoplanin-

positive thin-walled channels, considered to be lym-

phatic vessels, were also seen within the IAC alongwith the veins. The lymphatic system was seen ininterfascicular area as well as within the proximity ofthe arterioles and venules (Figs. 2 and 3). There wasno significant difference in the distribution of thelymphatic vessels on either side of the canal. Thelymphatic system around the main trunk of the IAN

Fig. 1. H&E staining of the body of mandible (�10). Sagittalsection showing the inferior alveolar artery (A), the inferioralveolar vein (B), and the inferior alveolar nerve bundle (C).

Fig. 2. Positive podoplanin staining around the inferior alve-olar artery. Arrows: Lymphatic-containing vessels (�40).

was small, and there was no evidence of large lym-

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phatic trunks. The distribution of the lymphatic wallsin the anterior, middle, and posterior sections wasfollowing the same pattern. Five of the middle sec-tions were edentulous. No qualitative histologic dif-ferences were found regarding the presence or dis-tribution of the lymphatic channels between thedentate and edentulous parts of the mandible.

DISCUSSIONThe IAN is the largest branch of the mandibular

division of the trigeminal nerve. It enters the lower jawfrom the mandibular foramen, travels through the bodyof the mandible, and divides into 2 terminal branches(incisive and mental nerves) at the mental foramen.17

Recent studies have focused on the arrangements andexact diameters of the contents of the canal. The meandiameters of the IAN, inferior alveolar artery, and in-ferior alveolar vein have been measured to be 2.52 mm,1.84 mm, and 0.42-0.58 mm, respectively.18 Thesestudies have mostly dealt with the canal position and itsproximities, space relationship between the IAN andthe lower dental roots, or the surgical vulnerability ofthe canal. Nothing, however, could be found in theliterature regarding the presence of a lymphatic systemwithin the IAC.

Until now, the periosteum of the mandible has beenknown to be the main source of the lymphatic drainageand has been overemphasized when treating carcino-mas of the floor of the mouth. Internal structures withinthe mandible have never been recognized as potentialroutes to carry exotoxins or malignant cells. Nerve

Fig. 3. Positive podoplanin staining around the inferior alve-olar nerve (arrows, �40).

invasion by malignant tumors, especially salivary gland

tumors, was thought to be the etiologic factor for localmetastasis.4

The pathogenesis of perineural invasion is not fullyelucidated. In 1905, it was theorized that tumor cellsspread along nerve trunks via perineural lymphatics.However, the literature did not provide histologic evi-dence, and suggestions were based on this theory thatall vascular bundles contain lymphatic vessels.19 Lar-son et al.20 demonstrated that the lymphatic channelshave no role in the local recurrences of the adenoidcystic carcinoma. They believed that tumor cells spreadthrough nerve sheaths. Feasel et al.21 reviewed theclinical pattern of perineural invasion. They studied thepresence of tumor cells adjacent to epineural bundlesand around venules and noted the perineural lymphaticsand perineural sheath spaces as important routes for thespread of cutaneous malignancies. Presence of tumorcells within and around the IAC at the so-called safemargins of resection would be crucial in changing thecurrent concept of locoregional spread, which wouldthen warrant a more radical resection to reduce theincidence of recurrences. Considering the IAL as apotential route to spread malignant cells may changethe surgical treatment protocols in preserving or resect-ing the IAN. Several specific lymphatic endothelial cellmarkers have become available, such as podoplanin,lymphatic vessel endothelial hyaluronan receptor 1,vascular endothelium growth factor receptor 3, D2-40,and Prox-1.22-26

The significance of the present findings regarding thepresence of a lymphatic system in the body of themandible is not yet clear, and further studies are re-quired to verify the exact role of the lymphatic vesselsin the inflammatory responses of the pulp, local metas-tasis of the malignant tumors, distribution of odonto-genic tumors, and resistance to surgical trauma.

The lymphatic pathway is attributed to collectingveins. The mammalian lymphatic vascular system orig-inates solely from venous endothelial cells. After arte-rial and venous endothelial cells differentiate, a sub-population of venous endothelial cells is thought tobecome competent to acquire lymphatic endothelialcell characteristics.27 The maxilla and mandible arecomponents of the facial skeleton which contain theteeth. Having a vital pulp, teeth are considered to bevital organs. Each dental root has its own feeding andcollecting vessels that drain into the IAC. A lymphaticsystem is also found within the pulp.28 Recent studieshave shown that lymphatic drainage in dental pulp hasa major role in the equilibrium of interstitial fluid.29

Lymphatic networks have also been identified in humanperiodontal tissues with a higher density in the radicular

section.30

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CONCLUSIONSEvidence for the existence of a lymphatic system

within the IAC may contribute in postulating new an-atomic, physiologic, and functional properties for theIAC of the mandible.

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Reprint requests:

Arash Khojasteh, DMD, MSAssistant Professor, Director of Basic Science ResearchDepartment of Oral and Maxillofacial SurgerySchool of DentistryShahid Beheshti University of Medical SciencesTehranIran

arashkhojasteh@gmail.com