research article comparison of alcian blue, trypan blue...

Research ArticleComparison of Alcian Blue Trypan Blueand Toluidine Blue for Visualization of the Primo VascularSystem Floating in Lymph Ducts

Da-Un Kim12 Jae Won Han12 Sharon Jiyoon Jung1 Seung Hwan Lee1 Richard Cha12

Byung-Soo Chang3 and Kwang-Sup Soh1

1Nano Primo Research Center Advanced Institute of Convergence Technology Seoul National UniversitySuwon 443-270 Republic of Korea2College of Physical Education The University of Suwon Hwaseong 445-743 Republic of Korea3Department of Cosmetology Hanseo University Seosan 356-706 Republic of Korea

Correspondence should be addressed to Richard Cha wellspring2navercom and Byung-Soo Chang bschanghanseoackr

Received 2 July 2014 Accepted 1 September 2014

Academic Editor Moriya Ohkuma

Copyright copy 2015 Da-Un Kim et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The primo vascular system (PVS) floating in lymph ducts was too transparent to be observed by using a stereomicroscope It wasonly detectable with the aid of staining dyes for instance Alcian blue which was injected into the lymph nodes Some dyes wereabsorbed preferentially by the PVS than the lymph wall It remains a standing problem to know what dyes are absorbed better bythe PVS than the lymph walls Such information would be useful to unravel the biochemical properties of the PVS that are badly inneed for obtaining large amount of PVS specimens In the current work we tried two other familiar dyes which were used in PVSresearch before We found that Trypan blue and toluidine blue did not visualize the PVS Trypan blue was cleared by the naturalwashing Toluidine blue did not stain the PVS but it did leave stained spots in the lymph wall and its surrounding tissues and itleaked out of the lymph wall to stain surrounding connective tissues These completely different behaviors of the three dyes werefound for the first time in the current work and provide valuable information to elucidate the mechanism through which somespecial dyes stained the PVS preferentially compared to the lymphatic wall

1 Introduction

Long threadlike structures floating in large-caliber lymphducts were first observed with the aid of staining with the dyeJanus green B in the lymph ducts stemming from the lumbarnodes near the caudal vena cava in the abdominal cavity of arabbit [1] Since then various other dyes such as fluorescentmagnetic nanoparticles Alcian blue [2 3] and DiI [4] havebeen used for the same purpose with similar protocols [5 6]Subject animals were rabbits [1ndash5] rats [6] and mice [7]The target lymph ducts were thoracic ducts [7] as well asthe above-mentioned lumbar duct and the duct around theepigastric blood vessel in the skin [8 9] The presence ofa lymph-associated primo vascular system (L-PVS) floating

in the flow of lymph was first claimed by Kim in the early1960s [10 11] and was only recently confirmed by variousexperimental groups [1ndash9 12]

One of the important questions concerns the mechanismthrough which the dye preferentially stains the L-PVS ratherthan the lymph wall In answering this question knowingwhich dyes do not stain the L-PVS in a similar process wouldbe helpful If L-PVS affine and nonaffine dyes are comparedvaluable information on themechanismof the staining can beobtained The current work reports on the findings for twodyes Trypan blue and toluidine blue that did not stain theL-PVS when the same protocol as that for the Alcian bluewas used In the first case we injected Alcian blue into theinguinal node of one side and Trypan blue into the inguinal

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 725989 8 pageshttpdxdoiorg1011552015725989

2 Evidence-Based Complementary and Alternative Medicine

node of the other side In the second case toluidine blue andAlcian blue were compared Trypan blue was chosen for thisstudy because it is effective for staining the primo vascularsystem (PVS) on the surfaces of internal organs [13] in thebrain and the spine [14] and around cancer tissues [15ndash18]In the second case toluidine blue was chosen for this studysimply because it is one of the common dyes used in histologyand has been used for PVS histology in connection withtransmission electron microscopy [19]

Effectively visualizing observing and isolating the L-PVS is one of the steps necessary to establish the anatomyand the histology of the L-PVS The current work is oneof various efforts with that purpose The present situationof PVS research is similar to that of the lymph systemabout a decade ago Before the first breakthrough withthe identification of the vascular endothelial growth factorVEGF-C as a specific lymphangiogenic growth factor onlya rudimentary understanding of the molecular biology oflymphatics existed due to technical difficulties in identifyinglymph vessels within tissues and in isolating pure cultures oflymphatic endothelial cells for detailed characterization [20ndash22] A molecular understanding of the PVS which in turnrequires a sufficient amount of a specimen for study is badlyneeded The current work is connected indirectly to ways toobtain sufficient specimens of the L-PVS

The possible medical significance of the PVS was sug-gested by the observation of abundant immune cells in thePVS with the following population ratios mast cells (20)eosinophils (16) neutrophils (15) lymphocytes (1)immature cells (3) and chromatin cells (03) [12] Thesedata suggest the roles of the PVS in the immune system andin the well-known lymphatic system The L-PVS especiallyis interesting because it resides inside lymph ducts thusindicating a probable interaction between them As is wellknown lymphatics aremajor paths formetastasis in commoncancers such as breast cancer and colorectal carcinomaswhich initially occurs via lymph nodes [23 24] Strongevidence exists that the PVS is another path for metastasisin addition to the lymphatic one [15] Furthermore a primonode might play a role as a haven for cancer stem cells [18]

2 Materials and Methods

21 Animals Rats (Sprague-Dawley male 9 weeks old280sim300 g) were obtained from DooYeol Biotech (SeoulRepublic of Korea) and housed in a temperature-controlledenvironment (23∘C) Seventeen rats were injected with Try-pan blue and twenty-one rats were injected with toluidineblue in the experiments All animals were exposed to a 12-hour light-dark cycle and were provided food and waterad libitum The procedures involving the animals and theircare were in full compliance with current international lawsand policies (Guide for the Care and Use of LaboratoryAnimals National Academy Press 1996) and were approvedby the Institutional Ethics Committee of the AdvancedInstitute of Convergence Technology (Approval NumberWJIACUC20130212-1-07) The rats were anesthetized by

intramuscular injection of a regimen consisting of 15 gkg ofurethane and 20mgkg of xylazine

22 Staining Dye Preparation The staining dye 02 Alcianblue (A5268 Sigma-Aldrich St Louis MO USA) solution inboiled phosphate-buffered saline (PBS pH 74) was preparedand was filtered by using a 022-120583m membrane filter (MerckMilliporeDarmstadt Germany)with a syringe (BD FranklinLakes NJ USA) The staining dye Trypan blue solution(25900048R CORNING Cellgro Manassas VA 20109 USA)04 (wv) in PBS was used The staining dye with a fiducialvolume of 50 toluidine blue working solution was madefrom the stock solution and 1 sodium chloride (NaCl005 gm and distilled water 5mL) The toluidine blue stocksolutionwasmade bymelting 01 gmof toluidine blue powder(Toluidine blue O 198161-5G Sigma-Aldrich St Louis MOUSA) and 10mL of 70 alcohol

23 In-Vivo Surgical Operation and Visualization of the PrimoVascular System A 2 cm skin midline incision up and downfrom the navel along the ventral surface of the abdominalcavity was made and the skin was retracted towards the ratrsquosspinal columnThe inguinal nodes (INs) are bilateral and aresituated close to the bifurcation of the superficial epigastricveins After the left-hand side IN had been exposed theprepared 02 Alcian blue dye was injected into the node at aslow rateThen Trypan blue was injected into the right-handside INThe order of injections was varied and the left and theright sides were alternated from one experiment to anotherThe other experiments with Alcian blue and toluidine bluewere done using similar procedures

The main lymph duct to observe was located along thesuperior epigastric vein and connected the IN to the axillarynode (AN) most of the time it had several branches Naturalcirculation of the lymph fluid had to be promoted in order toensure that the dyeing agent had been thoroughlywashed outTherefore each rat was covered with paper tissue and kept inthe bedding to maintain its body temperature

Two hours after dye injection the IN and the lymphduct were exposed to observe the L-PVS All procedureswere performed under a stereomicroscope (SZX12 OlympusJapan) After the stained L-PVS had been observed the ratswere sacrificed by using an intracardiac injection of urethane(1mL) The skin including the lymph ducts was fixed witha 10 neutral buffered formalin (NBF) solution at 4∘C for 24hours The lymph ducts including the L-PVS were collectedfrom the fixed skin

For the staining of nuclei with 41015840 6-diamidino-2-phenylindole (DAPI) the specimen was stained with 300-nM DAPI (D1306 Invitrogen MO USA) solution for 20minutes After the specimen had been washed it was cov-ered with a mounting solution The stained specimen wasinvestigated under a phase contrast microscope (OlympusU-LH100HG Japan) a microscope with a black and whitecharge-coupled device (CCD Nikon ECLIPSE Ti Japan) acontrast-enhanced microscope (DM2500 Leika Germany)and a confocal laser scanning microscope (CLSM C1 plusNikon Japan)

Evidence-Based Complementary and Alternative Medicine 3

Right subclavian

Right axillary

Brachiocephalic

Superior

Right superficialPopliteal

Left

Left

Left subclavian

Aortic arch

node

mesentericRight renal

inguinal node

Trypan blue injection Alcian blue injection

node

superficialinguinal node

axillary node

Lymph duct

Lymph ductPrimovessel

(a) (b)

(c)

(d)

(e)(f)

(g)

(h)

(i)

Figure 1 Stereomicroscopic images of lymph ducts in which Alcian blue and Trypan blue had been injected (a) Illustration of the left andthe right lymph ducts along the epigastric blood vessels (thick arrows) in skin (b) Left axillary node (open arrow) which became blue dueto the Alcian blue that flowed in the lymph duct (c) Left inguinal node (open arrow) into which Alcian blue had been injected (d) Mosaicof images of the left lymph duct along the epigastric blood vessel (thick arrows) (e) Magnified image of the rectangular area in (d) The bluethreadlike structure was the primo vessel (arrow) and it was floating in the lymph duct (double arrows) (f) Right axillary node (open arrow)weakly stained by Trypan blue that flowed from the inguinal node through the lymph duct (g) Right inguinal node (open arrow) into whichTrypan blue had been injected (h) Mosaic images of the right lymph duct from the inguinal node to the axillary node in which Trypan blueflowedThe epigastric blood vessel is indicated with thick arrows (i) Magnified image of the rectangular area in (h) The lymph duct (doublearrows) was washed clean and the PVS was not stained More details are presented in Figure 3

3 Results

The position of the relevant lymph duct in the skin fromthe inguinal node to the axillary node along the prominentepigastric blood vessel is illustrated in Figure 1(a) As in aprevious work with Alcian blue [8] the PVS was observed inthis skin lymph duct In Figure 1(b) the left axillary node wasblue due to the Alcian blue which had been injected into theleft inguinal node (Figure 1(c)) and had arrived via the lymphduct A mosaic of the images capturing the whole lymphduct (Figure 1(d)) shows the primo vessel where one part(Figure 1(e)) was magnified to demonstrate that the primovessel was stained well by Alcian blue inside the lymph vesselIn comparison the right axillary node (Figure 1(f)) becameweakly blue due to theTrypan blue that had been injected intothe right inguinal node (Figure 1(g)) and had flowed via thelymph duct (Figure 1(h)) The magnified mosaic image of thelymph duct (Figure 1(i)) showed that the primo vessel was notstained at all This showed that Trypan blue in this injectionmethod was not effective for L-PVS visualization

Compared with Alcian blue and Trypan blue the dyetoluidine blue showed a completely different behavior asshown in Figure 2 Figure 2(a) is the same illustration ofthe rat lymph in skin and the panels from Figures 2(b) to2(e) are for the right axillary node and the right inguinalnode and present a mosaic of images of the lymph ductalong with one magnified image showing the primo vesselin the cleared lymph duct respectively Alcian blue showedno differences in its effectiveness in comparison with Trypanblue and toluidine blue The results with toluidine blue aredepicted in Figures 2(f) to 2(i) The left axillary and inguinalnodes were deeply colored due to toluidine blue (Figures 2(f)

and 2(g)) The magnified images in Figures 2(h) and 2(i)show that the L-PVS was not stained but the lymph duct wasstained at randomly distributed spots in its wall furthermorethe dye leaked out to the surrounding tissue

Figure 3(a) shows the magnified images of the primo ves-sel inside the lymph duct Figure 3(b) shows the transparentlymph duct after washing of the preinjected Trypan blueThe L-PVS was not stained at all so it was not observableFigure 3(c) an enhanced contrast microscope image of thespecimen on a slide glass shows an extracted lymph ductwith the Alcian-blue-stained primo vessel in it A small partof the primo vessel was drawn out on the left upper partof the lymph duct This demonstrates the existence of anindependent threadlike object inside the lymph duct Alsothe primo vessel is seen to branch where the lymph ductbranched

Figure 4(a) shows another Alcian-blue-stained primovessel in comparison with the toluidine blue injection case(Figure 4(b)) in which no primo vessel was observed butin which the lymph duct and the surrounding tissue wereweakly stained In addition strongly stained spots weresporadically distributed The dye leaked from the lymphduct Furthermore some small blood vessels were stronglystained and apparently the dye flowed in the blood vessel(Figure 4(c)) This leaking from the lymph duct and flowingin blood vessels of toluidine blue were observed for the firsttime in this work

The primo node is an oval-shaped tissue packed withvarious cells [12] Figure 5 shows a primo node attached toone primo vessel during preparation Indeed the primonodewas packed with various cell nuclei as seen in Figure 5(b)


Right subclavian

Right axillary

Brachiocephalic

Superior

Left

Left subclavian

Aortic arch

node


Right superficialinguinal node

Poplitealnode

Leftsuperficial

inguinal node

axillary node

(a)(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

Lymph duct

Lymph duct

Primovessel

Toluidine blue injectionAlcian blue injection

Figure 2 Stereomicroscopic images of lymph ducts in which Alcian blue and toluidine blue had been injected (a) Illustration of the left andthe right lymph ducts along the epigastric blood vessels in skin (thick arrows) (b) Right axillary node which became blue due to the Alcianblue that flowed through the lymph duct (c) Right inguinal node into which Alcian blue had been injected (d) Mosaic of images of the rightlymph duct along the epigastric blood vessel (thick arrows) (e) Magnified image of the rectangular area in (d) The blue threadlike structurewas the primo vessel and it was floating in the lymph duct (double arrows) (f) Left axillary node stained with toluidine blue that flowed fromthe inguinal node through the lymph duct (g) Left inguinal node into which toluidine blue had been injected (h) Combined images of theright lymph duct from the inguinal node to the axillary node in which toluidine blue flowed (i) Magnified image of the rectangular area in(h) The lymph duct (double arrows) and its surrounding tissue were stained blue but the PVS was not visible More details are presented inFigure 4

Primo vessel

Lymph duct

(a)

Lymph duct

(b)

Primo vesselLymph duct

(harr)

(c)

Figure 3 Comparison of stereomicroscopic images of the lymph ducts into whichAlcian blue and Trypan blue had been injected (a) A primovessel emerged in a lymph duct (double arrows) after a two-hour washing of the injected Alcian blue This image corresponds to Figure 1(e)(b) The lymph duct after a two-hour washing of the injected Trypan blue became clear again without any hint of the presence of the primovessel This showed that the L-PVS was selectively stained by the Alcian blue not by the Trypan blue (c) Contrast-enhanced microscopeimage of a branched lymph duct (double arrows) that was extracted and put on a slide Connective tissues wrapped the lymph duct Thestained L-PVS also branched


Primo vessel

Lymph duct (harr)

(a)

Lymph duct (harr)

(b)

Blood vessel stained by

toluidine blue

Lymph duct (harr)

(c)

Figure 4 Comparison of lymph ducts into which Alcian blue and toluidine blue had been injected (a) Two branches of lymph ducts (one ofthem indicated with double arrows) and their L-PVS stained with Alcian blue (b) Toluidine blue left stained spots in the lymph wall (arrowheads) It also leaked from the wall and stained the surrounding tissues However the L-PVS was not stained (c) A thin blood vessel wasdeeply stained by using toluidine blue How it flowed into the blood vessel is not known

Primo node

Primo vessel

(a)

Primo node

Rod-shaped nuclei

Primo vessel

(b)

Figure 5 Images of a primo node extracted from the lymph duct (a) A primo node stained by Alcian blue was extracted from a lymph vesselOne of the primo vessels connected to the node was cut off during the extraction process and only one side was kept The primo vessel was abundle of two or more subvessels (b) DAPI images of the same primo node as in (a) which contained many nuclei Two rod-shaped nuclei(arrows) can be seen in the primo vessel

The minimal criterion to confirm the presence of the L-PVS is the observation of a distribution of the rod-shapednuclei by DAPI staining Figure 5(b) shows the presence oflongitudinally distributed rod-shaped nucleimore are shownin Figure 6

Because the distribution of nuclei and their shapes arecritical we present one more specimen with DAPI staining

A phase contrast microscopic image of the extracted primovessel is shown in Figure 6(a) The DAPI image was takenwith a sensitive black and white CCD (Figure 6(b)) and dis-tinguished the part with the rod-shaped nuclei from the partwith the round-shaped nuclei of aggregated lymphocytesTherod-shapednuclei were located inside the primo vessel whichwas confirmed by a series of confocal microscope images of


(a) (b)

Rod-shapednucleus

(c)

Rod-shapednucleus

(d)

Rod-shapednucleus

(e) (f)

Figure 6 Rod-shaped nuclei in the primo vessel (a) Phase contrast microscope image of a primo vessel that was extracted from a lymphduct (b) Fluorescence microscopic image of the boxed area in (a) The boxed area was magnified and the part with rod-shaped nuclei wasthe primo vessel The round-shaped nuclei were aggregated lymphocytes (cndashf) Confocal laser scanning microscope images of the circledarea in (b) These panels were consecutive optical sections from top to bottom separated by 2120583m in each step The same rod-shaped nucleusappeared (panel (c)) looked long (panels (d) and (e)) and then nearly disappeared (panel (f)) which implies that this particular endothelialnucleus was inside the primo vessel and therefore had not aggregated from outside

optical sections (Figures 6(c) to 6(f)) For verification wechose a particular nucleus (arrow in Figures 6(d) and 6(e))

4 Discussion

To the present time several dyes have been used for thevisualization of the L-PVS in lymph ducts Janus green B [1]fluorescent magnetic nanoparticles Alcian blue [2 3 5 68] and DiI [4] Among them Alcian blue has been mostextensively used Even though all these dyes are effective andhave different merits the mechanism through which theystain preferentially the L-PVS has not yet been uncoveredBefore we attempted to determine the exact mechanism weexamined the efficacies of two commonmaterials for the visu-alization of the L-PVS the results of which should providemore information useful in investigating the mechanism

Alcian blue Trypan blue and toluidine blue showeddifferent behaviors in this experiment Alcian blue is used for

the staining of acidic polysaccharides in histology [25] It wasinitially chosen with the expectation that it would stain thehyaluronic acid that is known to be abundant in the primofluid [11] Until now whether the successful application ofAlcian blue in many previous experiments [2 3 5 6 8] wasdue to its affinity to hyaluronic acid is not yet clear In thecurrent work Alcian blue was used as a reference materialfor the Trypan blue and toluidine blue

The dye Trypan blue is used in histology for stainingconnective tissues such as collagen muscle and cornifiedepithelium [25] Another common use is to discriminatedead cells from live ones Trypan blue is also useful for vivi-staining of vitereoretinal membranes in ophthalmic surgery[26] In PVS-visualization work Trypan blue was first usedas a visualizing agent in vivo and in situ not as a staining dyeof tissue specimens for histological purposes By using it wewere able to make the following significant contributions (1)a web-like network of PVS was observed on the omentum


and the visceral peritoneum [13] (2) the PVS exists aroundcancer tissues [15ndash18] and (3) the PVS exists in the brainand the spine [14] In the current work we compared Alcianblue and Trypan blue for their abilities to visualize the L-PVS Surprisingly the Trypan blue was not absorbed eitherby the lymphwall or the L-PVSWe therefore conjecture thatTrypan bluemight have been absorbed by the dead cells of thePVS which had been exposed to air during the experimentalprocedure However further study is needed before a definiteconclusion can be drawn about the mechanism of Trypan-blue absorption by the PVS in previous experiments

The behavior of the toluidine blue was beyond our expec-tation in the sense that it leaked out through the lymphaticwall and left randomly-distributed dots in the lymph walland its surrounding tissues In addition the dye flowed insome venules around the lymph duct and strongly stained thevascular wall but it was not absorbed by the L-PVS Theseresults were newly found in this experiment Toluidine bluehas often been used in the staining of PVS tissues for lightmicroscopic imaging in transmission electron microscopicstudies of PVS tissues [19]

The possible medical significance of the PVS is suggestedby the observation of abundant immune cells in the PVS [12]The data suggest the roles of the PVS in immune systems andas an extrahematopoietic source of immune cells and bonemarrow Other suggested functions of the PVS are a path formetastasis [15] and a haven for cancer stem cells [18] whichis worth deeper study

The present work was limited in that it only compared theefficacies of the three dyes in visualizing the L-PVS and didnot uncover the biochemical mechanism for their differentbehaviors The understanding of this mechanism is left for afuture study For this purpose it is valuable to know the dyesthat were used to stain lymph vessels For example Evansblue and guanylate cyclase that was used to stain dermallymphatic capillary [27]

In conclusion Trypan blue and toluidine blue were quitedifferent from Alcian blue in their abilities to visualize theL-PVS Trypan blue was cleared completely by a two-hournatural washing and did not stain either the lymphwall or theL-PVS To the contrary toluidine blue left scattered stronglystained dots in the lymph wall and surrounding tissues afterleaking out from the lymph duct It did not however stainthe L-PVS at all Sometimes it entered some small veins andstained the vessel strongly These were first observed in thisexperiment but this phenomenon in terms of biochemicalprocesses is still not understood

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Da-Un Kim and Jae Won Han contributed equally to thispaper

Acknowledgment

This research was supported in part by the Basic ScienceResearch Program through the National Research Founda-tion of Korea (NRF) funded by the Ministry of Science ICTamp Future Planning (Grant no 2013R1A1A2008343)

References

[1] B-C Lee J S Yoo K Y Baik K W Kim and K-S Soh ldquoNovelthreadlike structures (Bonghan ducts) inside lymphatic vesselsof rabbits visualized with a Janus Green B staining methodrdquoAnatomical RecordmdashPart B New Anatomist vol 286 no 1 pp1ndash7 2005

[2] C Lee S-K Seol B-C Lee Y-K Hong J-H Je and K-SSoh ldquoAlcian blue stainingmethod to visualize Bonghan threadsinside large caliber lymphatic vessels and X-ray microtomog-raphy to reveal their microchannelsrdquo Lymphatic Research andBiology vol 4 no 4 pp 181ndash189 2006

[3] Y-I Noh M Rho Y-M Yoo S J Jung and S-S Lee ldquoIsolationand morphological features of primo vessels in rabbit lymphvesselsrdquo JAMS Journal of Acupuncture andMeridian Studies vol5 no 5 pp 201ndash205 2012

[4] B-C Lee and K-S Soh ldquoContrast-enhancing optical methodto observe a Bonghan duct floating inside a lymph vessel of arabbitrdquo Lymphology vol 41 no 4 pp 178ndash185 2008

[5] S J Jung S Y Cho K-H Bae et al ldquoProtocol for theobservation of the primo vascular system in the lymph vesselsof rabbitsrdquo JAMS Journal of Acupuncture and Meridian Studiesvol 5 no 5 pp 234ndash240 2012

[6] S J Jung K-H Bae M-H Nam H M Kwon Y-K Song andK-S Soh ldquoPrimo vascular system floating in lymph ducts ofratsrdquo JAMS Journal of Acupuncture andMeridian Studies vol 6no 6 pp 306ndash318 2013

[7] I H Choi H K Chung and Y K Hong ldquoDetection of theprimo vessels in the rodent thoracic lymphatic ductsrdquo in ThePrimo Vascular System K S Soh K A Kang and D HarrisonEds pp 121ndash126 Springer New York NY USA 2011

[8] S H Lee K-H Bae G O Kim et al ldquoPrimo vascular systemin the lymph vessel from the inguinal to the axillary nodesrdquoEvidence-based Complementary and Alternative Medicine vol2013 Article ID 472704 6 pages 2013

[9] S J Jumg S H Lee K H Bae H M Kwon Y K Songand K S Soh ldquoVisualization of the primo vascular systema putative cancer metastasis thread afloat in a lymph ductrdquoProtocol Exchange 2013

[10] B H Kim ldquoOn the Kyungrak systemrdquo Journal of the Academyof Medical Sciences of the Democratic Peoplersquos Republic of Koreavol 90 pp 1ndash41 1963

[11] B H Kim ldquoThe Kyungrak systemrdquo Journal of Jo Sun Medicinevol 108 pp 1ndash38 1965 (Korean)

[12] B S Kwon C M Ha S Yu B-C Lee J Y Ro and S HwangldquoMicroscopic nodes and ducts inside lymphatics and on thesurface of internal organs are rich in granulocytes and secretorygranulesrdquo Cytokine vol 60 no 2 pp 587ndash592 2012

[13] B-C Lee K W Kim and K-S Soh ldquoVisualizing the networkof bonghan ducts in the omentum and peritoneum by usingtrypan bluerdquo JAMS Journal of Acupuncture and MeridianStudies vol 2 no 1 pp 66ndash70 2009

[14] J Dai B-C Lee P An et al ldquoIn situ staining of the primovascular system in the ventricles and subarachnoid space of the


brain by trypan blue injection into the lateral ventriclerdquo NeuralRegeneration Research vol 6 no 28 pp 2171ndash2175 2011

[15] J S Yoo M Hossein Ayati H B Kim W-B Zhang andK-S Soh ldquoCharacterization of the primo-vascular system inthe abdominal cavity of lung cancer mouse model and itsdifferences from the lymphatic systemrdquo PLoS ONE vol 5 no4 Article ID e9940 2010

[16] W Akers Y Liu G Sudlow et al ldquoIdentification of primovascular system in murine tumors and viscerardquo in The PrimoVascular System pp 179ndash183 Springer New York NY USA2012

[17] M Y Hong S S Park H K Do G-J Jhon M Suh and Y LeeldquoPrimo vascular system ofmurinemelanoma and heterogeneityof tissue oxygenation of themelanoma rdquo Journal of Acupunctureand Meridian Studies vol 4 no 3 pp 159ndash163 2011

[18] M A Islam S D Thomas S Slone H Alatassi and D MMiller ldquoTumor-associated primo vascular system is derivedfrom xenograft not hostrdquo Experimental and Molecular Pathol-ogy vol 94 no 1 pp 84ndash90 2013

[19] B-C Lee J S Yoo V Ogay et al ldquoElectron microscopic studyof novel threadlike structures on the surfaces of mammalianorgansrdquo Microscopy Research and Technique vol 70 no 1 pp34ndash43 2007

[20] M J Karkkanien T Makinen and K Alitalo ldquoLymphaticendothelium a new frontier of metastasis researchrdquoNature CellBiology vol 4 pp E2ndashE5 2002

[21] M J Karkkainen and K Alitalo ldquoLymphatic endothelial regu-lation lymphoedema and lymph node metastasisrdquo Seminars inCell and Developmental Biology vol 13 no 1 pp 9ndash18 2002

[22] L Jussila and K Alitalo ldquoVascular growth factors and lymphan-giogenesisrdquo Physiological Reviews vol 82 no 3 pp 673ndash7002002

[23] K Alitalo and P Carmeliet ldquoMolecular mechanisms of lym-phangiogenesis in health and diseaserdquo Cancer Cell vol 1 no3 pp 219ndash227 2002

[24] GOliver andMDetmar ldquoThe rediscovery of the lymphatic sys-tem old and new insights into the development and biologicalfunction of the lymphatic vasculaturerdquoGenes and Developmentvol 16 no 7 pp 773ndash783 2002

[25] G Clark Staining Procedures Williams andWilkins BaltimoreMd USA 4th edition 1980

[26] M FarahMMaia B Furlani et al ldquoCurrent concepts of trypanblue in chromovitrectomyrdquo Developments in Ophthalmologyvol 42 pp 91ndash100 2008

[27] S Nishida and M Ohkuma ldquoStaining of dermal lymphaticcapillary by guanylatecylaserdquo Lymphology vol 26 no 4 pp195ndash199 1993

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


node of the other side In the second case toluidine blue andAlcian blue were compared Trypan blue was chosen for thisstudy because it is effective for staining the primo vascularsystem (PVS) on the surfaces of internal organs [13] in thebrain and the spine [14] and around cancer tissues [15ndash18]In the second case toluidine blue was chosen for this studysimply because it is one of the common dyes used in histologyand has been used for PVS histology in connection withtransmission electron microscopy [19]

Effectively visualizing observing and isolating the L-PVS is one of the steps necessary to establish the anatomyand the histology of the L-PVS The current work is oneof various efforts with that purpose The present situationof PVS research is similar to that of the lymph systemabout a decade ago Before the first breakthrough withthe identification of the vascular endothelial growth factorVEGF-C as a specific lymphangiogenic growth factor onlya rudimentary understanding of the molecular biology oflymphatics existed due to technical difficulties in identifyinglymph vessels within tissues and in isolating pure cultures oflymphatic endothelial cells for detailed characterization [20ndash22] A molecular understanding of the PVS which in turnrequires a sufficient amount of a specimen for study is badlyneeded The current work is connected indirectly to ways toobtain sufficient specimens of the L-PVS

The possible medical significance of the PVS was sug-gested by the observation of abundant immune cells in thePVS with the following population ratios mast cells (20)eosinophils (16) neutrophils (15) lymphocytes (1)immature cells (3) and chromatin cells (03) [12] Thesedata suggest the roles of the PVS in the immune system andin the well-known lymphatic system The L-PVS especiallyis interesting because it resides inside lymph ducts thusindicating a probable interaction between them As is wellknown lymphatics aremajor paths formetastasis in commoncancers such as breast cancer and colorectal carcinomaswhich initially occurs via lymph nodes [23 24] Strongevidence exists that the PVS is another path for metastasisin addition to the lymphatic one [15] Furthermore a primonode might play a role as a haven for cancer stem cells [18]

2 Materials and Methods

21 Animals Rats (Sprague-Dawley male 9 weeks old280sim300 g) were obtained from DooYeol Biotech (SeoulRepublic of Korea) and housed in a temperature-controlledenvironment (23∘C) Seventeen rats were injected with Try-pan blue and twenty-one rats were injected with toluidineblue in the experiments All animals were exposed to a 12-hour light-dark cycle and were provided food and waterad libitum The procedures involving the animals and theircare were in full compliance with current international lawsand policies (Guide for the Care and Use of LaboratoryAnimals National Academy Press 1996) and were approvedby the Institutional Ethics Committee of the AdvancedInstitute of Convergence Technology (Approval NumberWJIACUC20130212-1-07) The rats were anesthetized by

intramuscular injection of a regimen consisting of 15 gkg ofurethane and 20mgkg of xylazine

22 Staining Dye Preparation The staining dye 02 Alcianblue (A5268 Sigma-Aldrich St Louis MO USA) solution inboiled phosphate-buffered saline (PBS pH 74) was preparedand was filtered by using a 022-120583m membrane filter (MerckMilliporeDarmstadt Germany)with a syringe (BD FranklinLakes NJ USA) The staining dye Trypan blue solution(25900048R CORNING Cellgro Manassas VA 20109 USA)04 (wv) in PBS was used The staining dye with a fiducialvolume of 50 toluidine blue working solution was madefrom the stock solution and 1 sodium chloride (NaCl005 gm and distilled water 5mL) The toluidine blue stocksolutionwasmade bymelting 01 gmof toluidine blue powder(Toluidine blue O 198161-5G Sigma-Aldrich St Louis MOUSA) and 10mL of 70 alcohol

23 In-Vivo Surgical Operation and Visualization of the PrimoVascular System A 2 cm skin midline incision up and downfrom the navel along the ventral surface of the abdominalcavity was made and the skin was retracted towards the ratrsquosspinal columnThe inguinal nodes (INs) are bilateral and aresituated close to the bifurcation of the superficial epigastricveins After the left-hand side IN had been exposed theprepared 02 Alcian blue dye was injected into the node at aslow rateThen Trypan blue was injected into the right-handside INThe order of injections was varied and the left and theright sides were alternated from one experiment to anotherThe other experiments with Alcian blue and toluidine bluewere done using similar procedures

The main lymph duct to observe was located along thesuperior epigastric vein and connected the IN to the axillarynode (AN) most of the time it had several branches Naturalcirculation of the lymph fluid had to be promoted in order toensure that the dyeing agent had been thoroughlywashed outTherefore each rat was covered with paper tissue and kept inthe bedding to maintain its body temperature

Two hours after dye injection the IN and the lymphduct were exposed to observe the L-PVS All procedureswere performed under a stereomicroscope (SZX12 OlympusJapan) After the stained L-PVS had been observed the ratswere sacrificed by using an intracardiac injection of urethane(1mL) The skin including the lymph ducts was fixed witha 10 neutral buffered formalin (NBF) solution at 4∘C for 24hours The lymph ducts including the L-PVS were collectedfrom the fixed skin

For the staining of nuclei with 41015840 6-diamidino-2-phenylindole (DAPI) the specimen was stained with 300-nM DAPI (D1306 Invitrogen MO USA) solution for 20minutes After the specimen had been washed it was cov-ered with a mounting solution The stained specimen wasinvestigated under a phase contrast microscope (OlympusU-LH100HG Japan) a microscope with a black and whitecharge-coupled device (CCD Nikon ECLIPSE Ti Japan) acontrast-enhanced microscope (DM2500 Leika Germany)and a confocal laser scanning microscope (CLSM C1 plusNikon Japan)


Right subclavian

Right axillary

Brachiocephalic

Superior


Left

Left

Left subclavian

Aortic arch

node


inguinal node


node


axillary node

Lymph duct


(a) (b)

(c)

(d)

(e)(f)

(g)

(h)

(i)


3 Results








Right subclavian

Right axillary

Brachiocephalic

Superior

Left

Left subclavian

Aortic arch

node



Poplitealnode

Leftsuperficial

inguinal node

axillary node

(a)(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

Lymph duct

Lymph duct

Primovessel



Primo vessel

Lymph duct

(a)

Lymph duct

(b)


(harr)

(c)



Primo vessel

Lymph duct (harr)

(a)

Lymph duct (harr)

(b)


toluidine blue

Lymph duct (harr)

(c)


Primo node

Primo vessel

(a)

Primo node

Rod-shaped nuclei

Primo vessel

(b)






(a) (b)

Rod-shapednucleus

(c)

Rod-shapednucleus

(d)

Rod-shapednucleus

(e) (f)



4 Discussion















Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Right subclavian

Right axillary

Brachiocephalic

Superior


Left

Left

Left subclavian

Aortic arch

node


inguinal node


node


axillary node

Lymph duct


(a) (b)

(c)

(d)

(e)(f)

(g)

(h)

(i)


3 Results








Right subclavian

Right axillary

Brachiocephalic

Superior

Left

Left subclavian

Aortic arch

node



Poplitealnode

Leftsuperficial

inguinal node

axillary node

(a)(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

Lymph duct

Lymph duct

Primovessel



Primo vessel

Lymph duct

(a)

Lymph duct

(b)


(harr)

(c)



Primo vessel

Lymph duct (harr)

(a)

Lymph duct (harr)

(b)


toluidine blue

Lymph duct (harr)

(c)


Primo node

Primo vessel

(a)

Primo node

Rod-shaped nuclei

Primo vessel

(b)






(a) (b)

Rod-shapednucleus

(c)

Rod-shapednucleus

(d)

Rod-shapednucleus

(e) (f)



4 Discussion















Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Right subclavian

Right axillary

Brachiocephalic

Superior

Left

Left subclavian

Aortic arch

node



Poplitealnode

Leftsuperficial

inguinal node

axillary node

(a)(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

Lymph duct

Lymph duct

Primovessel



Primo vessel

Lymph duct

(a)

Lymph duct

(b)


(harr)

(c)



Primo vessel

Lymph duct (harr)

(a)

Lymph duct (harr)

(b)


toluidine blue

Lymph duct (harr)

(c)


Primo node

Primo vessel

(a)

Primo node

Rod-shaped nuclei

Primo vessel

(b)






(a) (b)

Rod-shapednucleus

(c)

Rod-shapednucleus

(d)

Rod-shapednucleus

(e) (f)



4 Discussion















Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Primo vessel

Lymph duct (harr)

(a)

Lymph duct (harr)

(b)


toluidine blue

Lymph duct (harr)

(c)


Primo node

Primo vessel

(a)

Primo node

Rod-shaped nuclei

Primo vessel

(b)






(a) (b)

Rod-shapednucleus

(c)

Rod-shapednucleus

(d)

Rod-shapednucleus

(e) (f)



4 Discussion















Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

Rod-shapednucleus

(c)

Rod-shapednucleus

(d)

Rod-shapednucleus

(e) (f)



4 Discussion















Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article comparison of alcian blue, trypan blue...

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