258 Annals of Vascular Diseases Vol.5, No.3 (2012)

Review Article

Recent Advance in Lymph Dynamic Analysis in Lymphatics and Lymph NodesFumitaka Ikomi, MD, PhD, Yoshiko Kawai, MD, PhD, and Toshio Ohhashi, MD, PhD

Lymphatics are a unidirectional transport system that carries fluid from the interstitial space and back into the blood stream. Initial lymphatics take up not only fluid but also high-molecular-weight substances, such as plasma proteins and hyaluronan; immune cells, such as lymphocytes, macrophages, and dendritic cells; and colloidal particles, such as carbon particles, bacteria, and tattoo dye. Interstitially injected colloidal particles are known to accumulate in the regional lymph nodes. This phenomenon is applied to find sentinel lymph nodes in cancer patients. Lymph flow rate and composition are influenced by interstitial fluid, lymphatic pump activity, and intra-lymphatic pressure. Lymph composition is changed during its flow downstream. In this review, the main focus is on the mechanisms of lymph formation at the initial lymphatics and lymph transport through the collecting lymphatics and lymph nodes. (*English Translation of J Jpn Coll Angiol, 2008, 48: 113-123.)

Keywords: lymphflow,sentinellymphnode,colloidalparticle,drugdeliverysystem(dds),ultrasound contrast medium

IntroductIon

The lymphaticsystemisavascularsystemexistinginparallelwiththebloodvesselsystem,anditspri-

mary function is considered to be transport of materials throughlymphvessels.Thefluid in the lymphvesselsis called lymphor lymphaticfluid and is transportedfrom upstream to downstream in the lymphatic system. Thelymphvesselsfusewiththevenoussystemattheirdownstreamends(thoracicduct-leftvenousangle,rightlymphatic trunk-rightvenousangle).Unlike thebloodvesselsystem,whichisaclosedcirculationsystem,thelymphaticsystemisaone-waychanneloriginatinginthe

initial lymphaticsanddistributed tovarious tissuesofthe body. Because of this structural difference, while the blood circulates by the ejection force of the heart, lymph istransportedbythelymphvesselsthemselvesworkingaspumps.Forthisreason,whilebloodusuallyflowsfromareaswithahigherbloodpressuretoareaswithalowerblood pressure, the internal pressure of the lymphatic system is often higher downstream than upstream.1) Moreover,asacharacteristicofthelymphcirculation,there are lymph nodes in the lymphatic channels, and they functionasfiltersthattrapforeignbodiescarriedbythelymphflow,astransportchannelsandreactionsitesofimmunecellspatrollingthebody,andasprimarysitesof lymph concentration.2)

In the 1990s, VEGF-C and VEGFR-3 (Flt4), a fac-tor that specifically proliferates lymphvessels and itsreceptor, respectively,were identified. In association,knowledgeaboutthedevelopmentofthelymphaticsys-temandtherelationshipoflymphangiogenicfactorswithlymphogenousmetastasisoftumorsincreasedmarkedly.3) At the same time, the concept of sentinel lymph node began to be accepted clinically.4) The sentinel lymph node theory is the concept that there is no metastasis in

Department of Physiology, Shinshu University School of Medi-cine, Matsumoto, Nagano, Japan

Received:April10,2012;Accepted:June15,2012Correspondingauthor:ToshioOhhashi,MD,PhD.DepartmentofPhysiology,ShinshuUniversitySchoolofMedicine,3-1-1,Asahi,Matsumoto,Nagano390-8621,JapanTel:+81-0263-37-2597,Fax:+81-0263-36-5149E-mail:ohhashi@shinshu-u.ac.jp*ThisarticleisEnglishTranslationofJJpnCollAngiol,2008,48:113-123.

AnnVascDisVol.5,No.3;2012;pp258–268©2012 Annals of Vascular Diseases doi:10.3400/avd.ra.12.00046

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lymph nodes farther than sentinel lymph nodes if senti-nel lymph nodes, or lymph nodes that lymph produced aroundtheprimaryfocusoftumorreachesfirst,arefreeofmetastasis.Accordingtothistheory,identificationofsentinel lymph nodes makes possible the determination ofthesurgicalstrategyconcerninglymphnodedissec-tion.5) In these circumstances, much attention has been recently directed to the lymphatic system.

The roles of the lymphatic system includes not only recoveringtissuefluidintothebloodcirculationviathelymphcirculationandservingasarouteoftransportofimmunecellsbut,pathophysiologically,alsoprovidingarouteofmetastasistotumor.Therefore,knowledgeaboutlymphkinetics is indispensable for understanding themechanisms of these functions. In this paper, primarily thelymphflowinanormalstateisdescribedinconnec-tionwiththesignificanceofthelymphaticsystem.

roles of the lymphatIc system and lympho-kInetIcs

Toevaluatethecharacteristicsofthelymphcircula-tion,thesignificanceofthelymphaticsystemandlymphcirculation is discussed serially.

(1) Recovery of plasma components that have escaped from the blood vascular system into circulating bloodWaterisexchangedbetweenplasmaandtissuefluidvia

thewallofthemicrovasculatureaccordingtoStarling’shypothesis.6) Also, plasma protein leaks out primarily throughthevenularwall.Theamountofalbuminrecov-ered daily from tissue spaces by the lymphatic system is nearlyhalfthetotalamountcontainedinthecirculatingblood.Sinceboththestatichydraulicpressureandplasmaproteinconcentrationarehigherinthebloodvesselsthanin tissue spaces, plasma protein that has leaked into tissue spaces cannot return directly to plasma, because plasma protein causing the colloid osmotic pressure diffusesoutwardevenifwatercanbetransportedintothebloodvesselsduetocolloidosmoticpressuredifferences.Thus,it is necessary to recover plasma protein from tissuespacesbythepumpingactivityofthelymphvessels.7) For this reason, the lymphatic system is considered to becontributingtothehomeostasisof theenvironmentaroundcellsandmaintenanceofcirculatingplasma.

(2) Transport of water, electrolytes, and nutrients absorbed via the digestive tractTheoriginsofthelymphvesselscalledcentrallacte-

alsarepresentinthevilliofthesmallintestineand,indigestionandabsorption,transportlipidsolublematerialsparticularlyaschylomicrons.Moreover,theabsorptionoflipidsisknowntobecloselyrelatedtothegastrointestinalimmune system.8)

(3) Transport of physiologically active materials such as hormones and enzymes released or secreted from cellsFenestratedcapillarieswithastructurefacilitatingthe

transport of macromolecular hormones into plasma, are presentinendocrineglands.Intheseorgans,thelymphvesselsarealsodeveloped,andlymphfromtheadrenalgland,pancreas,thyroid,kidney,andsmallintestinecon-tainshighconcentrationsofcortisol,insulin,thyroxin,renin, and alkaline phosphatase.9)

(4) Elimination of macromolecular materials and particulate components from tissue spacesTattoodyes thathaveentered the tissue spacehave

longbeenknowntobeaccumulatedintheregionallymphnodes.10)Thelymphvesselstakeupandtransportmacro-molecules that constitute tissue space such as hyaluronic acid,micro/nanoparticlessuchascarbonparticles,andforeignbodiessuchasbacteria,andtheseparticlesaredisposed of or accumulated in the lymph nodes. By these activities,homeostasisofthemicroenvironmentofthetissuespacearoundcellsismaintained.Also,utilizingthese characteristics of the lymph vessels and lymphnodes,methodstoidentifysentinellymphnodesusingvariouscolloidparticlesastracershavebeendeveloped.

(5) Channel of movement and site of reaction of anti-gens, antibodies, and immune cells

Lymphocytes, which circulate in the blood stream migrate extravascularly fromhigh endothelial venules(HEV)inlymphnodesandrecognizeantigen-presentingcellsflowinginthroughtheafferentlymphvessels.Themovementsoftheseimmunecellsareregulatedbyvari-ous adhesion molecules and chemokines.11)

(6) Secretion of physiologically active componentsFrom lymph vessel endothelial cells, nitric oxide

(NO) and prostaglandins are released on stimulationof the lymph vessel lumen by the flow and variousdrugs.Thesephysiologicallyactivematerialsreleasedareknowntobeabletoregulatethetensionoftheac-companying blood vessels.12) In the microcirculation system,thearteryandvein,whichflowintheopposite

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directions,areconsideredtoexchangematerials,heat,and information,13)butthelymphvesselalsoflowsintheoppositedirectiontotheaccompanyingarteryandmaybeinvolvedintheregulationofthebloodflowinamannerdependent on the state of the tissue.

(7) Pathophysiologic significanceAbnormalities of the lymphatic system induce edema

andproteinlosingenteropathy.Lymphaticedemaofthelimbs occurs primarily after lymph node dissection or radiationtherapyoftheaxillaorpelvisforbreastcanceror uterine cancer.The site that has developed lymphedemabecomesvulnerabletophlegmonandeventuallypresents a state of elephantiasis. Furthermore, there are life-threateningtypesofedemaincludingbrainedema,whichinducesbrainherniation,laryngealedema/pulmo-naryedema,whichcausesrespiratoryinsufficiency,andexcessive accumulationof pericardial effusion,whichcauses cardiac tamponade. Edema of the myocardium also causes impairment of both contraction and dilation.14)

In contrast with lymphatic edema caused by the arrest ofthelymphflowduetoobstructionofthelymphvessels,amarkedincreaseinthelymphflowisobservedinedemaaccompanyingcongestiveheartfailureorlivercirrhosis.The lymphflow in the human thoracic duct is about1mL/mininaphysiologicstate,butithasbeenreportedtoincrease3–12timesinthesepathologicconditions.15)

The lymphatic system is also known to be a route of tumormetastasis.ToimprovetheQOLoftumorpatients,theaccurateidentificationofthesentinellymphnodeandthedevelopmentofmoreeffectivemethodsforthediag-nosisandtreatmentoflymphnodemetastasisareurgentlyneeded.Forthispurpose,DDStargetedtothelymphaticsystemusingcolloidparticlesarebeingdeveloped.

flows In the lymph Vessels

Lymphisgeneratedastissuefluidispumpedup.Tis-suefluidisfreewaterpresent in the tissuespace.Thecomposition of lymph is known to changeduring theprocesses of its production and transport, and it must be notedthatitisnotthesameastissuefluidwhileitreflectsthepropertiesoftissuefluid.

The part of the lymphatic system that produces lymph in tissues is called the initial lymphatic, and the part that collects lymph from the initial lymphatics and transports itdownstreamiscalledthecollectinglymphatic.Ofthecollectinglymphatics,particularlylargevesselssuchasthelumbartrunk,intestinaltrunk,righttrunk,andtho-

racicduct,whichgatherlymphfromvariousareas,arealsocalledlymphatictrunks.Lymphvesselsthatcarrylymph into lymph nodes are called afferent lymphatics (prenodal lymphatics), and those that drain lymph from lymph nodes are called efferent lymphatics (postnodal lymphatics).16)

Unlike the blood vascular system, the lymphaticsystemhasnosystemthatdrivestheentirecirculationsystem such as the heart. Therefore, the lymphatic system needs special mechanisms for the transport of lymph. The mechanisms primarily of lymph production and transport are described below.

(1) Structure of tissue spaces and properties of tissue fluidThetissuespaceisthegapbetweencellsineachtissue.

Inthispart,highlywater-retentivegelatinousmaterialscalledproteoglycansfillthespacesinthemeshmadeoffibrouscomponentssuchascollagenfibersandelasticfibers(Fig. 1). Cells are also distributed in the mesh of thesefibrouscomponents.Also,proteinsconsideredtobe involved in theadhesionof thesematerialssuchasfibronectin, laminin,tenascin,andvitronectinarealsopresent.Tissuefluid,which is freewater in the tissuespace,existsasverynarrowchannels1mmor less indiameterinthespacesbetweenthesefibers,gel,andcells.Edema occurs primarily due to an increase in free water aswellasswellingofgel.2)

The evaluation of the properties of tissue fluid isdifficult because of the pattern of its distribution. Inmeasuringthehydrostaticpressureoftissuefluid,meth-ods including the porous capsule implantation, cottoncoremethod,andfineglasspipetteinsertionhavebeendevised,resultingintheclarificationthatahydrostaticpressureoftissuefluidintheskinismaintainedatafewmmHglowerthantheatmosphericpressure.2)

(2) Mechanism of lymph productionThe initial lymphatics located most upstream in the

lymphaticsystemaremadeofasinglelayerofendo-thelial cells and a non-continuous basement membrane. A space with a width of a few micrometers is present betweenneighboringendothelialcellscreatedbyfoldsof endothelial cells, and tissue f luid is pumped up through this space.Because of such a structure, notonly macromolecular materials but also particles in a nanometerordercanbetakenintothelymphvessels.Thefoldsofendothelialcellsserveasvalvesthatpre-ventregurgitationoftissuefluidthathasbeentakenup

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(endothelialmicrovalves)(Fig. 2A).Moreover,bundlesofcollagenfiberscalledanchoringfilaments,whichradi-atetosurroundingtissues,areboundtoendothelialcells.Anchoringfilamentsareconsideredtopreventcollapseoftheinitiallymphaticsandevendilatetheminedemadespiteswellingofthetissueandtocontributetodrain-ageoftissuefluid.17)

Intheskin,sincethehydrostaticpressureoftissuefluidisshowntobenegative,andthepressureintheinitiallymphaticstobepositive,somedrivingforceisnecessaryforlymphtobeproducedagainstthispressuregradient.The production of lymph is known to be increased by mechanicalstimulisuchasarterialpulsation,vibration,andmassage.18)Moreover, it is promoted bymassagewhenthetissueisedematous.Inthelightofthesefacts,tissuefluidisconsideredtobetakenupduetothepump-ingeffectcreated in the initial lymphaticsbyperiodicexternalmechanicalstimuli19) (Fig. 2A).

(3) Mechanisms of lymph transportThecollectinglymphaticshavevalvesintheirlumens

(lymphaticintraluminalvalves)andcontainsmoothmus-cleintheirwalls.Asegmentofthecollectinglymphaticbetween2valvesupstreamanddownstreamiscalledalymphangion as a functional unit.16) The permeability ofthewallofthecollectinglymphaticisrelativelyhightowaterandlow-molecular-weightmaterialsbutlowtomacromolecular materials such as protein.20)

In the collecting lymphatics, lymph is driven by 2mechanisms.Oneiscalledpassivetransportdependent

on external forces exerted to the lymph vessels, andtheother isactive transport implementedbyrhythmicspontaneous contraction of the smooth muscle in the lymphvesselwall.21) Lymph is driven downstream ifcompressionorcontractionoccursinalymphagion,anditsregurgitationispreventedbythevalvesinthelumenwhen the lymphangion is dilated or relaxed.Thus, apumpingactionisproducedineachlymphangionduetorepetitionofcompressionandexpansionofthetissueorcontractionandrelaxationofthesmoothmuscleinthelymphvesselwall,resultinginaunidirectionalflowinthecollectinglymphatics(Fig. 2B).Externalforcesofmassage, jointmovements, arterial pulsation, etc. areknowntoinducethispassivetransport.Passivetransportalsoincludesmovementsoflymphduetogravityasintheheadandneckregion.Inrelationtoactivetransport,the basic tension and force and frequency of spontaneous contractionofthesmoothmuscleofthelymphvesselwallareregulatedneurally,humorally,andlocally.22)

(4) Integration of lymph production and transportLymphstagnatesinperipheralregionsifitsproduction

isincreased,butthepumpingactivityofthecollectinglymphatics does not increase proportionally. On the other hand,thelymphflowcannotbeincreasedbystrength-eningthepumpingactivityofthecollectinglymphaticsalone if the lymph production is not increased. Thus, the lymph production and transport must be harmonized to efficientlymaintainthelymphflow.Here,thestimulithatpromotepassivetransportmentionedaboveallcontrib-

Fig. 1 Schematicrepresentationofinterstitium.

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utetothelymphproductionaswell.Inactivetransport,however,stretchingofthewallisknowntoenhancetheforce of spontaneous contraction, and the lymph transport is increased in proportion to the lymph production by a mechanismsimilartoStarling’slawoftheheart.21)

To dynamically evaluate the relationship betweenthe lymph production and lymph transport, a method similartotheanalyticalmethodusingthevenousreturncurveandcardiacoutputcurveinbloodcirculationbyGuyton can be applied.23) Specifically, the pressure-flowrelationshipisevaluatedbymakingantegradeandretrogradecannulationatapointinalymphvessel,andtherelationshipbetweentheoutflowpressureandflow(lymphformationcurve)andtherelationshipbetweentheinfusionpressureandflow(lymphtransportcurve)areplotted on the same coordinates (Fig. 3A). If the pressure inthelymphvesselishigh,thelymphproductionfromup-stream is suppressed, and the lymph transport downstream is promoted. On the other hand, if the pressure is low, the situation is reversed.Therefore, theamountof theflowagreesatthepointofthepressureshownastheintersec-tionofthethese2curves(equilibriumpoint)23) (Fig. 3A). Forexample,whenlimbmovementsareincreased,thepumpingactivityofboththeinitialandcollectinglym-phaticsisenhanced,thelymphformationcurveshifts

upward,thelymphtransportcurveshiftstotheleft,andequilibrium is reached at a new point of intersection (unpublisheddata).Byusingthisanalyticalmethod,theequilibriumpointmovesprimarilyupward,andthemechanismoftheincreaseintheflowwithoutmuchchangeinthepressurecanbeunderstood24) (Fig. 3B).Tocomprehensivelyunderstandthelymphkinetics,it

isnecessarytoexhaustivelycollectinformationinclud-ingthepressureandflowdistributions,permeabilityofvariousparts,andcompositionof thelymphproducedin the lymphatic system of the body by an approach that maybecalledsystemlymphology.Thisapproachmakessimulation and more detailed interpretation of the effects oflocalchangesinthelymphvesselfunctionontheentirelymphdynamics,effectsoflymphvesselobstructiondueto lymph node dissection, etc. on lymph dynamics up-streamanddownstream,andchangesinlymphdynamicscausedbygeneralizeddisordersincludingheartfailureandlivercirrhosispossible.Suchintegratedinterpretationof lymph dynamics is a future issue.

flow at lymph nodes

(1) Mechanism of lymph concentrationLymph is gradually concentrated, and its protein

Fig. 2 Effects of physical stimulation on pump function that causes lymph formation (A) and lymph transport (B).A:Initiallymphatic(crosssection)B:Collectinglymphatic(longitudinalsection)©2000 Shinshu Igakukai.All RightsReserved. Ikomi, F.ColloidalTransport through theLymphaticSystem:LymphCirculationandDrugTargeting.TheShinshuMedicalJournal2000;48:305-318.(inJapanese)24)

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concentrationincreases,asitflowsdownstreamalongthelymphvessels.7) Concentration of lymph occurs not only inthelymphvesselsbuthasbeenreportedtobemorenotable in the lymph nodes. In the 1980s, Guyton et al. evaluatedtherelationshipofthecolloidosmoticpressureand hydrostatic pressure between plasma and lymph in detail usingperfused samples of dogpopliteal lymphnode.Theresultsstronglysuggested:(1)Watermoves,but protein does not, between blood and lymph in the lymphnode,and(2)themovementofwaterbetweenbloodand lymph is conducted by a mechanism similar to the mechanismofwatermovementbetweenthecapillaryandtissuespaceaccordingtotheStarling’shypothesis.7) The

observationthatproteinisconcentratedaslymphtravelsthroughthelymphaticsysteminaphysiologicstateisinagreementwith the abovediscussion. In the transportofmaterials,2factors, i.e., theconcentrationandflowrate,mustbeconsidered.However,fromtheviewpointofrecoveryofplasmaproteinintothecirculatingblood,theefficiencyoftransportissufficientlyimprovedevenwhenthelossduetotheincreaseintheviscosityassoci-ated with concentration is taken into consideration.25)

(2) Cell dynamics in lymph nodesLymphocytes in the circulating bloodmigrate ex-

travascularlythroughtheHEVofthelymphnodesandreturntobloodviaefferentlymphvessels.Thisreleaseoflymphocytesfromtheefferentlymphvesselsisknownto be affected by adrenaline, histamine, serotonin, corti-costeroids,andprostaglandins.26) Recently, stimulation by sphingosine-1-phosphate (S1P) receptors has beenshown to be essential to the release of lymphocytes from thelymphnodestotheefferentlymphvessels.27) In clas-sicexperimentsinwhichalymphvesselwasperfusedwith diluted plasma components, and lymphocytes in the collected lymph were counted, the possibility that lipidmediatorsincludingS1Pandotherphysiologicallyactivematerialscontainedintraceamountsinthelymphaffectedtheresultscannotbeexcluded.Recently, theadventof themultiphotonmicroscope

hasmadepossible invivovisualizationof thestateofrelatively deep tissues. Studies using thismicroscopehavealsograduallyclarifiedthecelldynamicsinlymphnodes.28,29) Furthermore, analysis of the relationship betweenthedistributionofchemokinesandmovementsoflymphocytesisalsoadvancing.11)However,howthelymphflow is involved in thedynamicsof these cellsremains mostly unknown.

(3) Contraction and relaxation of lymph nodesThe smooth muscle present in the capsule and trabeculae

ofthelymphnodeisknowntocontractandrelaxunderdrugorneuralstimulation.30)Asexamples,Fig. 4 shows typicalresponsesofresectedspecimensofdogandmon-keyaxillary,tracheobronchial,mesenteric,andinguinallymph nodes to adrenaline, histamine, serotonin, corti-costeroids,andprostaglandins.Althoughthesignificanceofthiscontractionandrelaxationisunclear,itmaybe(1)regulationoftheresistancetothelymphflow,(2)promo-tionofthepumpingfunctionforlymphandlymphocytes,and(3)regulationofthevolumeofthelymphnode.Seean-otherreviewfordetailsofcontraction/relaxationresponses

Fig. 3 Lymph dynamic analysis. The equilibrium point, whichisthecrossingpointofalymphformationcurve (broken line) anda lymph transport curve(solid line), determines lymphatic pressure and lymphflowratesimultaneously(A). The equilib-riumpointischangedbyshiftingofthetwocurves(B).©2000ShinshuIgakukai.AllRightsReserved.Iko-mi,F.ColloidalTransportthroughtheLymphaticSystem:LymphCirculation andDrugTargeting.TheShinshuMedical Journal2000;48:305-318.(inJapanese)24)

A

B

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of the smooth muscle in the lymph node capsule and trabeculae.31)

transport of partIcles and Its applIcatIon to the dds

(1) Uptake of particles into lymph vesselsFolds of endothelial cells, which constitute endothelial

microvalvesin theinitial lymphatics,creategapswithawidth reaching severalmicrometers, andparticulatematerials injected into the tissue space are taken up into lymphvessels via these gaps (Fig. 5A). Particles in tissue spaces are known to be taken up into lymph vesselsdirectly(extracellulartransport)orindirectlybymacrophages, etc.,whichphagocytose themandenterthe lymph vessels (intracellular transport).32) When thesetwomodesofparticleuptakearecomparedusingfluorocarbonemulsion,extracellulartransport,ordirectuptake,ispredominant.Also,extracellulartransportismost notable immediately after particle injection and decreasesgraduallythereafter,butintracellulartransportisminimallyobserved immediately after the injectionbutcontinuesevenafter1week,showingadifferenceintime-dependence.32) In addition, the particle concentra-tioninlymphandlymphflowincreased10–100timesbymassagingtheinjectionsite.19,32)Theseobservationssug-

gestthattheuptakeofparticlesbythelymphaticsystemincreasesmarkedlybyanenhancementofthepumpingfunction of the initial lymphatics.Theuptakeofparticlesbythelymphvesselsisalso

known to be affected by the diameter and surface prop-ertiesoftheparticles.Evenparticleswithadiameterofseveralmicrometersareshowntobetransferredintothelymphvesselsthroughtissuespaces.Also,theuptakeofparticlesbythelymphvesselsincreasesastheparticlesize is smaller.However, the uptake of fluorocarbonemulsionconsistingofparticleswithasimilarsizehasbeen reported to changewith the surfactant coveringthe particles.33) Therefore, at least, the size and surface properties of the particles are considered to affect their uptake of particles by the initial lymphatics.

(2) Accumulation of particles in the lymph nodesThelymphnodehasafilterfunctionforforeignbod-

ies, and it accumulates and disposes of the captured foreignbodiesinit.Findingsconcerningtheparticle-size-dependence of the passage of particles throughthe lymphnode,whichcorresponds to thefilter size,havebeenaccumulatedrecently.Whenthepassageofmicrospheresthroughtherabbitpopliteallymphnodewasmeasured,fewparticles2mmorgreaterindiam-eterpassed through the lymphnode,but thepassage

Fig. 4 Representativerecordingsoftheeffectsofvasoactiveaminesinisolatedcanineandmonkeylymphnodes.NA:noradrenaline;His:histamine;5-HT:5-hydroxytryptamine;Adr:adrenaline;Ax:axillarylymphnode;Tb:tracheobron-chiallymphnode;Me:mesentericlymphnode;In:inguinallymphnode

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of smaller particles increased with decreases in their diameterinahyperbolicmanner.Alongwiththesizebar-rier,thepassageoflymphocyteswiththesamesizewasfoundtoincreasesignificantlybydepletingmacrophagesinthelymphnodewithliposomescontainingCL2MBP (unpublished data).Toconcentrateamaximumnumberofparticlesinthe

lymphnodesbyaDDS,itisnecessarytoincreaseatleasteither the uptake of particles from the tissue spaces or the capture of particles in the lymph nodes (Fig. 5B and 5C). Here, factors that affect the capture of particles at the lymphnodeareconsideredtoincludebiologicalfactorssuchasthedistributionandactivityofphagocytesandtheexpressionofadhesivemoleculesaswellasphysicalfactors such as the diameter and surface properties of the particles,buttheclarificationoftheseproblemsislefttofuture studies.

(3) Search for sentinel lymph nodesAsaDDSfromthetissuespacetothelymphnode,the

searchforsentinellymphnodesusingradioactivecolloidis conducted clinically at present.4,34)Weareengaginginthedevelopmentofamethodforthesearchofsentinellymphnodesusingultrasounddiagnosticdevices.35) Here, Figure 6 shows the results of a preliminary study in which a lymphnodewas examined in theDopplermodebyinjectingacontrastagentintoaprenodallymphvessel.If it becomes possible to detect an ultrasound contrast agentinjectedintothetissuespaceoveralongtimeinlymphnodes,thetechniqueisexpectedtobeextremelyusefulforthediagnosis.

Fig. 5 Schematicrepresentationofcolloidalmovementfrominterstitialspaceto lymph node (A),uptakerate(redlines)andtrappingrate(bluelines)(B), and accumulation rate (C). There may be an appropriate particle diameter formaximal accumulation in the lymphnode.To increasethe accumulation rate, it may be important to increase uptake rate and trappingrate(brokenlines).

A

B

C

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BIology of the lymph flow and lymphatIc system

(1) Regulation of the lymph vessel smooth muscleAneffectofthelymphflowisshearstressonlymph

vessel endothelial cells.Theflow-dependentNOpro-ductionandsecretionhavebeennotedinthelymphaticsystemaswellasinthebloodvesselsystem.36)Inanex-perimentusingthedogthoracicduct,flow-dependentNOproductionandsecretionweresuppressedbyL-arginine,which is a substrate of NO production, and NG-nitro-L-arginine,which is an inhibitor ofNO synthesis. Inthisexperiment,also,noflow-dependentprostaglandinproductionorsecretionwasobserved.36)

(2) Regulation of lymph vessel endothelial cellsShear stress applied to lymph vessel endothelial

cells not only induces flow-dependentNO secretionasobservedabove,butwehaverecentlydemonstratedthatitalsoenhancesthegeneandproteinexpressionof

endothelial constitutiveNOsynthase (ecNOS), anNOsynthesizingenzymeandarete-controllingenzymeinthis reaction.37)We also confirmed that the release ofCa2+ from the Ca2+storesiteandactivationoftheCa2+-dependent K+channelareinvolvedinthisaction.37)

(3) Role in the lymphangiogenesisWhen the initial lymphatics are damaged, theflow

oflymphthathasleakedfromtheirseveredendsinthetissue space has been shown to play an important role intherepairoflymphvessels.38)Wehavealsoobtainedsimilarresultsusingtherabbitcollectinglymphatics.39) Whentherabbitpopliteallymphnodeisexcised,thecol-lectinglymphaticsrecanalize,andcollateralsdevelop,atthesiteofexcisedlymphnodeinacourseofafewweeks.However,therecanalizationofthecollectinglymphaticsispreventedwhenthelymphpooledatthesiteofexcisedlymphnodeisdrainedearlyaftersurgery.Inaddition,theimportanceoflymphangiogenicfactorshasbeensug-gested.40) Details of the mechanism of the formation and

Fig. 6 ColorDopplerimageoftherabbitpopliteallymphnode.Ultrasonographic(US)contrastmediumwasinjectedinto one of the popliteal afferent lymphatics.©2000ShinshuIgakukai.AllRightsReserved.IkomiF,NagaiT,SuzukiS,etal.Functionalinsightsforde-cisionandregulationoflymphaticmetastasisofcarcinomacells-Developmentoflymphcirculation-relatedDDS-.Lymphology2007;30:13-16.35)

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recanalizationofthecollectinglymphatics,e.g.,whetherthephysicaleffectofthelymphflowinthetissuespaceorgrowthfactorsorprecursorcellsofendothelialcells,etc.inlymphareimportant,awaitfutureclarification.

closIng remarks

Asmentionedabove,thelymphvesselsareconsideredtobeanetworkofpumpscoveringtheentirebody.Lymphis produced and transported by this pumping effect.Thecompositionoflymphchangesduringitstransport.Lymphcontainsvariousphysiologicallyactivemateri-als and immune cells as well as water, electrolytes, and plasmaproteins.Knowledgeoflymphdynamicsisneces-sarytounderstandtheirtransport.Moreover,knowledgeabout lymph dynamics is considered to contribute to the controlofimmunefunctionsandthedesigningofDDSforthediagnosisandtreatmentoflymphnodemetastasisoftumors.Ihopethatthisarticleprovidesaviewpointfromthelymphdynamicstomanycolleaguesengagedinangiology.

acknowledgments

Theultrasoundcontrastagentandliposomescontain-ingCL2MBPwereprovidedbyProfessorKojiAbe,Sec-tionofMacromoleculeProcessing andDisintegration,Department of FunctionalMacromolecules, ShinshuUniversityFacultyofTextileScienceandTechnology.Cooperation was obtained from Messrs. Akira Teramoto andTakayukiYanagawaofthesamesectioninpreparingthesedrugs.

Part of this research was subsidized by a Grand-in-AidforScientificResearchbytheJapanSocietyforthePromotionofScience(15500315,19500384,19209044,22249052).

Part of this study was carried out in the Project for theResearch andDevelopment of IntelligentSurgicalInstruments.

references

1)CalnanJS,PflugJJ,ReisND,etal.Lymphaticpres-suresandtheflowoflymph.BrJPlastSurg1970;23:305-17.[Medline] [CrossRef]

2)AdairTH,GuytonAC:Introductiontothelymphaticsystem.In:JohnstonMG,ed.Experimentalbiologyofthe lymphatic circulation. 1st edition ed. The Nether-lands:ElsevierSciencePublishers,B.V.Amsterdam,1985, 1-12.

3) Jussila L,AlitaloK.Vascular growth factors andlymphangiogenesis.PhysiolRev2002;82:673-700.[Medline]

4)KitagawaY,FujiiH,MukaiM,etal.Radio-guidedsentinelnodedetectionforgastriccancer.BrJSurg2002;89:604-8.[Medline] [CrossRef]

5)MortonDL,WenDR,WongJH,etal.Technicaldetailsof intraoperative lymphaticmappingforearlystagemelanoma.ArchSurg1992;127: 392-9. [Medline] [CrossRef]

6)StarlingEH.On theAbsorptionofFluids from theConnectiveTissueSpaces.JPhysiol(Lond)1896;19:312-26.[Medline]

7)AdairTH,GuytonAC.Lymphformationanditsmodi-ficationinthelymphaticsystem.In:JohnstonMG,ed.Experimentalbiologyofthelymphaticcirculation.1steditioned.TheNetherlands:ElsevierSciencePublish-ers,B.V.Amsterdam,1985,13–44.

8)TsuzukiY,MiyazakiJ,MatsuzakiK,etal.Differentialmodulation in the functions of intestinal dendritic cells bylong-andmedium-chainfattyacids.JGastroenterol2006;41:209-16.[Medline] [CrossRef]

9) Daniel PM, Gale MM, Pratt OE. Hormones and re-latedsubstancesinthelymphleavingfourendocrineglands–thetestis,ovary,adrenal,andthyroid.Lancet1963;1:1232-4.[Medline]

10)FloreyHW,Gowans JL. Chapter 5 The reticulo-endothelial system. The omentum. The lymphatic system.Thelymphocyte.In:FloreyHW,ed.Generalpathology.W.B.SaundersCompany,Philadelphia,1962,128-166.

11)MiyasakaM,TanakaT.Lymphocytetraffickingacrosshighendothelialvenules:dogmasandenigmas.NatRevImmunol2004;4:360-70.[Medline] [CrossRef]

12)SteenbergenJM,BohlenHG.Sodiumhyperosmolar-ityofintestinallymphcausesarteriolarvasodilationinpartmediatedbyEDRF.AmJPhysiol1993;265:H323-8. [Medline]

13)KobayashiH,PelsterB,PiiperJ,etal.SignificanceoftheBohreffectfortissueoxygenationinamodelwithcounter-currentbloodflow.RespirPhysiol1989;76:277-88.[Medline] [CrossRef]

14)Pratt JW, Schertel ER, Schaefer SL, et al.Acutetransient coronary sinus hypertension impairs left ventricularfunctionandinducesmyocardialedema.AmJPhysiol1996;271:H834-41.[Medline]

15) Dumont AE, Clauss RH, Reed GE, et al. Lymph drainage in patientswith congestive heart failure.comparisonwithfindingsinhepaticcirrhosis.NEnglJMed1963;269:949-52.[Medline] [CrossRef]

16)Schmid-SchönbeinGW.Microlymphaticsandlymphflow.PhysiolRev1990;70:987-1028.[Medline]

17)LeakLV,BurkeJF.Finestructureof the lymphaticcapillaryandtheadjoiningconnectivetissuearea.AmJAnat1966;118:785-809.[Medline] [CrossRef]

18) IkomiF,Schmid-SchönbeinGW.Lymphpumpme-chanicsintherabbithindleg.AmJPhysiol1996;271 (HeartCircPhysiol,40):H173-H183.

Ikomi F, et al.

268 Annals of Vascular Diseases Vol.5, No.3 (2012)

19) Ikomi F,Hunt J,HannaG, et al. Interstitial fluid,plasma protein, colloid, and leukocyte uptake into initial lymphatics. JApplPhysiol1996;81:2060-7.[Medline]

20)OnoN,MizunoR,OhhashiT.Effectivepermeabilityof hydrophilic substances throughwalls of lymphvessels: roles of endothelial barrier.Am J PhysiolHeartCircPhysiol2005;289:H1676-82.[Medline] [CrossRef]

21)OhhashiT,AzumaT,SakaguchiM.Activeandpassivemechanicalcharacteristicsofbovinemesentericlym-phatics.AmJPhysiol1980;239:H88-95.[Medline]

22) Ohhashi T, Mizuno R, Ikomi F, et al. Current topics of physiology and pharmacology in the lymphaticsystem.PharmacolTher2005;105:165-88.[Medline] [CrossRef]

23) IkomiF,OhhashiT.Effectsoflegrotationonlymphflowandpressureinrabbitlumbarlymphcirculation:in vivo experiments and graphical analysis. ClinHemorheolMicrocirc2000;23:329-33.[Medline]

24) IkomiF.ColloidalTransportthroughtheLymphaticSystem:LymphCirculationandDrugTargeting.TheShinshuMedicalJournal2000;48:305-18.(inJapa-nese)

25)TakahashiT,ShibataM,KamiyaA.Mechanismofmacromoleculeconcentrationincollectinglymphaticsin ratmesentery.MicrovascRes1997;54:193-205.[Medline] [CrossRef]

26)MooreTC.Neurovascularimmunology:a25yearod-yssey.AnnNYAcadSci1992;650:30-9.[Medline] [CrossRef]

27)MandalaS,HajduR,BergstromJ,etal.Alterationoflymphocyte trafficking by sphingosine-1-phosphatereceptoragonists.Science2002;296:346-9.[Medline] [CrossRef]

28)BoussoP,RobeyE.DynamicsofCD8+Tcellprimingby dendritic cells in intact lymph nodes. Nat Immunol 2003;4:579-85.[Medline] [CrossRef]

29)MempelTR,HenricksonSE,VonAndrianUH.T-cellprimingbydendriticcellsinlymphnodesoccursinthreedistinctphases.Nature2004;427:154-9.[Med-

line] [CrossRef]30)FloreyH.Observationsonthecontractilityoflacteals:

PartII.JPhysiol(Lond)1927;63:1-18.[Medline] 31) Ikomi F, Ohhashi T. Control mechanisms of lymph

circulationwithin lymphnodes.Kekkan 2000;23:55-64.(inJapanese)

32) IkomiF,HannaGK,Schmid-SchönbeinGW.Mecha-nism of colloidal particle uptake into the lymphatic system:basicstudywithpercutaneouslymphography.Radiology1995;196:107-13.[Medline]

33) IkomiF,HannaGK,Schmid-SchönbeinGW.Size-and surface-dependent uptake of colloid particles into thelymphaticsystem.Lymphology1999;32:90-102.[Medline]

34) IkomiF,MizunoR,KawaiY,etal.Sentinellymphnodeconcept;fromlymphdynamicspointsofview.ClinicalSurgery2004;59:535-43.(inJapanese)

35) IkomiF,NagaiT,SuzukiS,etal.Functionalinsightsfordecisionandregulationoflymphaticmetastasisofcarcinomacells-Developmentoflymphcirculation-relatedDDS-.Lymphology2007;30:13-6.(inJapa-nese)

36)TsunemotoH, IkomiF,OhhashiT. Flow-mediatedreleaseofnitricoxidefromlymphaticendothelialcellsofpressurizedcaninethoracicduct.JpnJPhysiol2003;53:157-63.[Medline] [CrossRef]

37)KawaiY,YokoyamaY,KaidohM,etal.Shearstress-inducedATP-mediatedendothelialconstitutivenitricoxide synthase expression in human lymphatic en-dothelialcells.AmJPhysiol,CellPhysiol2010;298:C647-55.[Medline] [CrossRef]

38)BoardmanKC,SwartzMA.Interstitialflowasaguidefor lymphangiogenesis.CircRes 2003;92: 801-8.[Medline] [CrossRef]

39) IkomiF,YokoyamaY,OgiwaraN,etal.Recanalizationofthecollectinglymphaticsinrabbithindleg.Micro-circulation2006;13:365-76.[Medline] [CrossRef]

40) IkomiF,KawaiY,NakayamaJ,etal.CriticalrolesofVEGF-C-VEGF receptor 3 in reconnection of the col-lectinglymphvesselsinmice.Microcirculation2008;15:591-603.[Medline] [CrossRef]