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Lymphatic transport in patients with chronicvenous insufficiency and venous leg ulcersfollowing sequential pneumatic compressionJohn C. Rasmussen, PhD,a Melissa B. Aldrich, PhD,a I-Chih Tan, PhD,a Chinmay Darne, PhD,a

Banghe Zhu, PhD,a Thomas F. O’Donnell Jr, MD,b Caroline E. Fife, MD,c andEva M. Sevick-Muraca, PhD,a Houston and The Woodlands, Tex; and Boston, Mass

Background:Recent advancements in near-infraredfluorescencelymphatic imaging (NIRFLI) technology provide opportunitiesfor non-invasive, real-time assessment of lymphatic contribu-tion in the etiology and treatment of ulcers. The objective of thisstudy was to assess lymphatics in subjects with venous leg ulcersusing NIRFLI and to assess lymphatic impact of a single sessionof sequential pneumatic compression (SPC).Methods: Following intradermal microdoses of indocyaninegreen (ICG) as a lymphatic contrast agent, NIRFLI was usedin a pilot study to image the lymphatics of 12 subjects withactive venous leg ulcers (Clinical, Etiologic, Anatomic, andPathophysiologic [CEAP] C6). The lymphatics were imagedbefore and after a single session of SPC to assess impact onlymphatic function.Results: Baseline imaging showed impaired lymphatic functionand bilateral dermal backflow in all subjects with chronicvenous insufficiency, even those without ulcer formation in the

the Brown Foundation Institute of Molecular Medicine, The Univer-y of Texas Health Science Center at Houston, Houstona; the DivisionVascular Surgery, Tufts Medical Center, Bostonb; and the Wound

are Clinic, CHI St. Luke’s Health - The Woodlands Hospital, Theoodlands.c

study was funded in parts by Tactile Medical Systems, Inc, and grantsm the National Institutes of Health (R01 HL092923 [E.M.S.M.] and54 CA136404 [E.M.S.M.]).or conflict of interest: J.C.R and E.M.S.M. own stock in a UTHealthrtfolio company, NIRF Imaging, Inc, which seeks to commercializee NIRF lymphatic imaging technology. J.C.R. has also received consul-g fees from NIRF Imaging. T.F.O. is on the scientific advisory board ofactile Medical Systems.ented at the Twenty-seventh Annual Meeting of the American Venousrum, Palm Springs, Calif, February 25-27, 2015.itional material for this article may be found online at www.jvsvenous.org.espondence: Eva M. Sevick-Muraca, PhD, 1825 Pressler St, Houston,X 77030 (e-mail: eva.sevick@uth.tmc.edu).editors and reviewers of this article have no relevant financial relation-ips to disclose per the Journal policy that requires reviewers to declineview of any manuscript for which they may have a conflict of interest.-333Xyright � 2016 The Authors. Published by Elsevier Inc. on behalf ofe Society for Vascular Surgery. This is an open access article undere CC BY-NC-ND license (http://creativecommons.org/licenses/-nc-nd/4.0/).://dx.doi.org/10.1016/j.jvsv.2015.06.001

contralateral limb (C0 and C4 disease). SPC therapy causedproximal movement of ICG away from the active wound in 9 of12 subjects, as indicated by newly recruited functional lymphaticvessels, emptying of distal lymphatic vessels, or proximal move-ment of extravascularfluid. Subjects with the longest duration ofactive ulcers had few visible lymphatic vessels, and proximalmovement of ICG was not detected after SPC therapy.Conclusions: This study provides visible confirmation of lym-phatic dysfunction at an early stage in the etiology of venousulcer formation and demonstrates the potential therapeuticmechanism of SPC therapy in removing excess fluid. Theability of SPC therapy to restore fluid balance through prox-imal movement of lymph and interstitial fluid may explain itsvalue in hastening venous ulcer healing. Anatomical differ-ences between the lymphatics of longstanding and more recentvenous ulcers may have important therapeutic implications.(J Vasc Surg: Venous and Lym Dis 2016;4:9-17.)

Approximately 1% of the total population and 3% to 5%of the population over the age of 65 will suffer from avenous leg ulcer (VLU) in their lifetime.1 VLUs charac-terize the most advanced stages of chronic venous

insufficiency (CVI) as identified by the Clinical, Etiologic,Anatomic, and Pathophysiologic (CEAP) C6 classification.VLUs are often recurrent, can persist for years, and canresult in infection, cellulitis, osteomyelitis, and/or amputa-tion. In the U.S., the financial burden of VLUs is estimatedto be $2 billion annually2,3 and is expected to grow withthe aging population.

The mechanism of ulcer formation involves a sequenceof pathophysiologic steps that are thought to include(1) reduction in venous outflow due to venous obstructionand/or reflux, (2) persistent venous hypertension, and(3) subsequent increased capillary filtration and interstitialfluid load. Typically, excess interstitial fluid is effectivelyremoved by the lymphatics, but if the fluid load over-whelms the lymphatic capacity or if the lymphatics aredefective, then the accumulation of interstitial fluid,macromolecules, and cytokines lead to edema (CEAPC3), breakdown of subcutaneous tissue (C4A,B), and for-mation of ulcers (C6).

4,5 Once formed, an ulcer will notheal (C5) unless venous hypertension and the resultingexcess capillary fluid filtration are ameliorated. Individualswith severe venous obstruction may undergo venous stent-ing, and individuals with severe venous insufficiency maybenefit from venous ablation or phlebectomy. These proce-dures are designed to restore venous function by loweringvenous hypertension and thereby reduce excess capillaryfiltration and interstitial edema. Yet correction of venoushypertension may not sufficiently resolve increased

9

JOURNAL OF VASCULAR SURGERY: VENOUS AND LYMPHATIC DISORDERS10 Rasmussen et al January 2016

interstitial fluid load if lymphatic vessels are dysfunctional.Although the lymphatic contribution to VLU formationand healing has long been recognized, recent authoritativereviews on CVI management do not define the lymphaticrole in the outcomes to surgical treatment.6

Complete decongestive therapy is the standard-of-carelymphatic treatment in lymphedema patients and encom-passes a set of therapeutic interventions designed todecrease lymphatic congestion and enhance transport bycombining (1) limb compression, (2) manual lymphaticdrainage (MLD), and, when necessary, (3) multicompart-mental sequential pneumatic compression (SPC). Todate, the adjunctive benefit of lymphatic treatment for pre-venting the formation or promoting the healing of VLUshas not been fully investigated.

The objective of this pilot study was to evaluate, withnear-infrared fluorescence lymphatic imaging (NIRFLI)and off-label, intradermal microdoses of indocyanine green(ICG): (1) the lymphatic anatomy and function in patientswith C6 venous disease and (2) the possible change inlymph and extravascular fluid distribution in response toa single treatment of SPC.

In addition, we sought to provide initial validation ofNIRFLI as a potential diagnostic or prognostic tool forfuture, more definitive clinical studies of venous andlymphatic disease.

METHODS

This study was conducted under the U.S. Food andDrug Administration (IND#: 102,765) and local Institu-tional Review Board approval and in accordance with theHelsinki Declaration.

Clinical criteria. Following informed consent, twelvesubjects with chronic VLUs were enrolled and assigned astudy ID (U01-U12). Subjects were recruited prospec-tively as they presented in the clinic. Inclusion criteriaincluded a minimum age of 18 years, the presence of bothlegs, and the presence of an active VLU. Exclusion criteriaincluded pregnancy or breast feeding; unwillingness to usemedically accepted contraceptives for 1 month followingimaging (for females of child-bearing potential); an allergyto iodine; failing prior compression therapy; inability totolerate compression; or clinically significant arterialdisease, defined by an ankle brachial index <0.7, skinperfusion pressures <30 mm Hg, or transcutaneousoximetry <30 mm Hg with poor response to sea leveloxygen breathing. Subjects with diabetic foot ulcers,exposed bone or tendons, or autoimmune disease withprednisone prescription were excluded.

Near-infrared fluorescence lymphatic imaging(NIRFLI). Prior to NIRFLI, each subject received up to16 intradermal injections of 25 mg of ICG in 0.1 mL salinewith a maximum total dose of 400 mg. Because the ulcers’location and size varied, the exact number and location ofthe injection sites also varied from subject to subject, butgenerally consisted of two injections on the dorsum ofeach foot, injections in the ankles and calves, and an injec-tion in each thigh. To capture the lymphatic components

potentially associated with the ulcer, two to three addi-tional injections were administered distal to the ulcer andin corresponding locations on the contralateral limb.

NIRFLI was conducted immediately following ICGinjection and consisted of illuminating the limbs withdiffuse 785-nm light (<1.9 mW/cm2), which penetratedthe tissues and excited the ICG, and collecting theresultant 830-nm fluorescent signal emanating from thelimbs with a near-infrared-sensitive, Gen III image intensi-fier and a charge-coupled device camera as detailedpreviously.7 Image sequences were acquired with 200-msexposure times, allowing near-real-time visualization oflymphatic contractile function. After imaging for approxi-mately 30 minutes, the affected leg of each subject wasplaced in a SPC garment and underwent therapy for1 hour. The SPC garments are opaque, preventingNIRFLI of the treated limb during therapy. However,the contralateral, untreated limb was imaged duringtherapy to visualize its lymphatic response as past NIRFLIstudies showed that MLD and SPC treatment of symptom-atic limbs stimulated contractile function in contralaterallimbs,8,9 presumably owing to autonomic regulation oflymphatics.10 Following therapy, the treated limb wasagain imaged for approximately 30 minutes.

SPC. SPC therapy was accomplished using theFlexitouch system (Tactile Systems Technology, Inc,Minneapolis, Minn), a programmable pneumatic compres-sion device specifically designed to treat lymphedema.11

The garments were fitted to each subject per manufac-turer’s instructions, and placed around the trunk, abdomen,and the affected leg. SPC treatment began with initial,truncal decongestion lasting approximately 12 minutes,during which a gradient pressure was applied to theabdominal areas to stimulate and clear the regional lymphnodes. Next, SPC treatment continued with initial unilateralleg preparation, lasting approximately 18 minutes, duringwhich a gradient pressure was applied to the more proximalareas of the leg.The treatment culminatedwith leg and trunkdrainage, lasting approximately 30 minutes, during whichconcerted cycles of mild variable pressures were appliedsequentially to the limb and trunk in a distal-to-proximalmanner. To prevent contamination, each wound wasbandaged prior to SPC and, following treatment, the SPCgarments were disinfected with a >60% isopropyl alcoholsolution per manufacturer’s recommendations.

Follow-up. Clinical follow-up was performed for eachsubject at 18 to 24 hours after ICG injection to ascertainadverse events (eg, local skin reaction, fever, or allergy). Inthis pilot study, neither ulcer healing nor any other clinicaloutcome was followed after the single SPC session, norwere NIRFLI results used in patient care.

Image analysis. Lymphatic function and anatomy, asobserved in NIRFLI images, were assessed in both treatedand contralateral limbs. The study was designed to measurewhether SPC therapy resulted in improved lymphatic functionas determined by (1) the presence of newly recruited lym-phatics (lymphatic vessels or draining tissues which were notfluorescent prior to SPC, but were fluorescent after), (2) the

Table I. Demographic information for subject with venous leg ulcers (VLUs)

SubjectID Gender

Age,years BMI

Race,ethnicity

WoundLymphedemaand stagea

Duration ofcurrent wound(s),

years

Skin perfusionpressure,mm Hg

TotalICG dose,

mgHistory Current Healed

U01 M 54 46.2 AA B R-C5L-C6

R B e II 7-8 RD-69 400

U02 M 69 40.5 C U R-C6L-C4

N N 2 RD-55 400

U03 F 26 61.6 AA B L-C6R-C5

R B e III 13 LD-67LP-54

400

U04 M 50 47.3 AA B R-C6L-C5

L B e II 3 RD-67RP-69

350

U05 M 53 38.0 AA B R-C6L-C5

L B e II 27 RD-65RP-65LD-62LP-64

300

U06 M 57 32.3 AI B R-C6L-C5

L B e I 5 RD-43RP-61

350

U07 F 32 34.5 C B R-C6L-C5

L B e I 0.42 RD-49LD-44

400

U08 F 60 31.1 C B R-C6L-C5

L B e II 6 RD-63 350

U09 F 52 29.8 C, H B R-C6L-C6

L N 0.5 LD-99 300

U10 F 65 49.2 C U L-C6R-C0

N B e II 0.42 LD-98 350

U11 M 59 49.5 AA U R-C6L-C4

N B e (I-II) 0.17 R-79 350

U12 M 56 31.7 C U L-C6R-C0

N N 1 L-70 350

AA, African American; AI, American Indian; B, bilateral; BMI, body mass index; C, Caucasian; H, Hispanic; ICG, indocyanine green; L, left; LD, left dorsal;LP, left plantar; N, none; R, right; RD, right dorsal; RP, right plantar; U, unilateral.aLymphedema staging (I, II, or III) is consistent with the International Society of Lymphology.

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emptying of ICG from lymphatic vessels as a result of SPC,and/or (3) the change in contractile function in each subject.

RESULTS

Table I displays the demographic and anthropomor-phic characteristics of the seven males and five femalesenrolled. The median age was 55 years (range, 26-69 years;

Table II. Imaging results in treated and untreated legs of pat

SubjectID

Lymphatic architecture,immediate vicinity

of wound

Lymphatic architecturalabnormalities,treated leg

Lymphaabnun

U01 DB DB, T DBU02 DB DB, T, Possible D DB, T,U03 No DB DBU04 No DB, T DB, TU05 No DB, T, D DB, TU06 DB DB DBU07 DB DB, D T, DU08 DB DB, D DB, DU09 DB DB, D DBU10 DB DB, Possible D DBU11 DB DB, D DBU12 DB DB DB

D, Dilated lymphatics; DB, dermal backflow; E, emptying; LR, lymphatic reflux;compression; T, tortuous lymphatics.aDue to technical difficulties, the image acquisition rate was severely inhibited an

mean, 53 years), and the median body mass index (BMI)was 39.3 (range, 29.8-61.6; mean, 41.0). In five subjects,the ulcer(s) had been present for 1 year or less, thoughmost subjects had a multiple-year history of bilateral ulcer-ation. During this study, the subjects’ contralateral limbshad (1) no clinical signs of disease (C0, 2 subjects),(2) healed ulcers (C5 disease, 7 subjects), (3) skin changes

ients with venous leg ulcers (VLUs)

tic architectureormalities,treated leg

Lymphatic responseto SPC

Lymphatic contractilefunction

None LimitedD R, E Limited

None LimitedR LimitedNone LimitedR, Possible E LimitedR YesR Noa

R, RW, E YesE Yes before, No after SPCR, RW, E YesE Yes, LR

R, recruitment; RW, recruitment within wound; SPC, sequential pneumatic

d may have prevented the observation of lymphatic contractile function.

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(C4 disease, 2 subjects), or (4) active bilateral C6 disease(1 subject). It is noteworthy that in this study, all subjectswere obese, a factor that predisposes for CVI and isalso well known to be associated with lymphedema.At enrollment, lymphedema was confirmed in nine sub-jects, based upon a positive Stemmer’s sign. The venousdisease etiology was post-thrombic, but anatomical andpathophysiologic classifications were not assessed in thispilot study. No adverse events were reported.

Table II summarizes the results for each subject,including brief summaries of the lymphatic anatomy inthe immediate vicinity of the wound, the SPC-treatedleg, and the untreated, contralateral leg. Observationsof lymphatic contractile function and lymphatic vesselrecruitment and/or emptying as a result of SPC arenoted.

Abnormal lymphatic anatomy. In past NIRFLIstudies (NCT000833599: “Imaging lymphatic function innormal subjects and in persons with lymphatic disorders”),healthy subjects (Fig 1, A) showed well-defined lymphatic

Fig 1. A, Normal lymphatics as seen in the lower anterior legwith extensive dermal backflow and tortuous vessels as seen iprimary lymphedema; C,Diseased lymphatics with lymphatic pin U09; and D, extensive dermal backflow and tortuous vess

vasculature that actively “pumped” ICG-laden lymph(Video 1, online only)12 proximally to the inguinal lymphnodes,13 while subjects with lower extremity, primarylymphedema (Fig 1, B), showed dilated lymphatic vesselswith dermal backflow, extravascular fluorescence, and/orlimited contractile activity.14 Because NIRFLI dependsupon detecting ICG taken up by lymphatics, non-functional and/or obstructed lymphatics may not bedetected with the technique. In all 12 subjects with CVI,NIRFLI showed dermal backflow and extravascular fluo-rescence that was visualized as “pooling” of ICG-ladenlymph (1) near the ulcers, as seen in U09 with bilateralC6 disease (Fig 1, C), (2) at distant sites away from theulcers (U02; Fig 1,D), and/or (3) in the asymptomatic legsof subjects with unilateral ulcers (U10-U12). Dermalbackflow (Fig 2, A) was observed in the immediate vicinityof the wound in nine subjects, two of which (U09 andU11)had fluorescence within the ulcer itself (Fig 2, B). Threesubjects (U03-U05) with long duration of active ulcer(s)(3, 13, and 27 years) did not show dermal backflow or other

s of a 57-year-old healthy subject; B, Diseased lymphaticsn the back, lower right leg of a 49-year-old subject withooling as evidenced by the dermal backflow in the anklesels as seen the lower right leg of U02.

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fluorescence uptake in the immediate vicinity of the woundas shown in Fig 2, C. In these cases, the lack of uptake nearthe wound may be related to the distance from the injectionsites to the wound, as we were reluctant to inject into newlyhealed regions (color inset in Fig 2,C). Consistent with pastwork in subjects with the longest duration of cancer-relatedlymphedema,15 we found fewer functional lymphatics andgreater dermal backflow in those study subjects with thelongest duration of active ulceration.

In the limbs of two subjects (U02 and U11) with C4A

disease, dermal backflow was observed to follow thehemosiderin stains on the subjects’ ankles and lower legsas demonstrated in Fig 2, D and E. In addition to dermalbackflow, tortuous and/or dilated conducting lymphaticvessels were visualized in all but two subjects (U03 andU06).

Interestingly, in U12, with an active ulcer of 1-yearduration located on the back of the calf, we observed

Fig 2. Images of fluorescent uptake in the lymphatics near thlack of fluorescence typically observed within the wound siteof wound inset) while (B) shows an example of a wound thinset). C, Illustrates the lack of fluorescence uptake from thwound inset). D, Color image of the hemosiderin staining inimage overlaid on a color image illustrating the presence of flU11. F, Color image of the wound on the back of the lowoverlaid on a color image illustrating the brightly fluorescent lleg of U12 while the lymphatics in the right leg are faintly v

substantial lymphatic drainage proximal to the ulcer(Fig 2, F and G), whereas no symmetric, functionallymphatic vessels were visualized on the contralateral C0

leg. In our past experience with NIRFLI in healthysubjects, we typically visualized functional lymphaticssymmetrically located on the anterior areas of each limb(Fig 1, A) and paralleling the great saphenous vein.Hence, the lack of functional vessels in the unaffectedleg indicates that the lymphatics visualized in the affectedleg may be specifically associated with the ulcer. In addi-tion, we observed “lymphatic reflux” in the ulcer-associated lymphatics in this subject (Video 2,online only).

Reduction of lymphatic contractile function. Wehave previously measured 0.4 6 0.3 lymphatic contrac-tions/min in normal subjects.16 In this study, however,both affected and contralateral limbs exhibited diminishedcontractile events too infrequent to be quantified within

e venous leg ulcers (VLUs). A, Shows an example of thewhile surrounded by dermal backflow (U07, color imageat contained fluorescence (U11, color image of wounde injection sites below the wound (U05, color image ofthe lower left leg of U11. E, Near-infrared fluorescenceuorescence within hemosiderin stain on lower left leg ofer left leg of U12. G, Near-infrared fluorescence imageymphatics draining the wound area on the back of the leftisible.

Fig 3. Images illustrating lymphatic recruitment following sequential pneumatic compression (SPC) therapy. A, Colorimage of the right medial leg of U02 with wound on medial ankle and near-infrared fluorescence images acquired(B) before and (C) after treatment. D, Color image of the right medial leg of U04 with open wound on the lowerlateral right calf (inset) and near-infrared fluorescence images acquired (E) before and (F) after treatment. The pink skinaround lower leg of (D) shows the previous extent of wound, and the darker skin tones in (A) illustrate hemosiderinstaining commonly associated with venous insufficiency. The yellow ellipses denote the approximate field of view in thenear-infrared fluorescence images.

JOURNAL OF VASCULAR SURGERY: VENOUS AND LYMPHATIC DISORDERS14 Rasmussen et al January 2016

the confines of our standard period of observation, whichwas constrained by the need to scan the entirety of bothlegs before and after SPC. With the exception of U08,where technical difficulties may have prevented observationof contractile function, this diminished, contractilelymphatic function was observed in all subjects includingthe three study subjects (U02, U09, and U12) not diag-nosed with lymphedema. After a single session of SPC,there was no apparent increase in the contractile functionin any of the subjects.

Fluorescence distribution pre- and post-SPCtherapy. Following SPC, we observed newly fluorescentlymphatics in seven subjects (U02, U04, U06-U09, andU11). Fig 3 presents images illustrating the movementof fluorescence in the few, but dilated lymphatics inU02 and U04 before and after SPC. In both subjects,SPC resulted in intravascular and extravascular move-ment of fluorescence towards the inguinal region. We

also observed the emptying of fluorescence from intra-vascular and/or extravascular spaces in at least fivesubjects (U02, U06 (possible), and U09-U12) afterSCP, with example images shown in Fig 4. Fig 4, A-Cillustrate the reduction of fluorescence signal in theinitial lymphatics and extravascular spaces proximal tothe ulcer in U09. Fig 4, D-F illustrate the reduction offluorescence signal in the initial lymphatics and extra-vascular spaces below the knee and proximal to thewound in U10. Thus, it would appear that a major routeof lymph fluid clearance with SPC occurs through theinterstitial space as well as through functional lymphaticvessels. SPC did not appear to cause movement ofICG from the intravascular to extravascular tissuecompartment, but rather moved ICG in their respectivecompartments proximally. Finally, we observed noproximal ICG movement after SPC in the three subjects(U01, U03, and U05) who had the longest duration

Fig 4. Images illustrating lymphatic emptying following sequential pneumatic compression (SPC) therapy. A, Colorimage of the wound on the left medial ankle of U09 and near-infrared fluorescence images acquired (B) before and(C) after treatment. D, Color image of the legs of U10 with an open wound on the lower left shin and near-infraredfluorescence images acquired (E) before and (F) after treatment. The yellow ellipse in (D) denotes the approximatefield of view in the near-infrared fluorescence images (E) and (F).

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(7-8, 13, and 27 years) of active ulceration, infection,and inflammation.

DISCUSSION

This pilot study demonstrates that lymphatic functionand anatomy were abnormal in patients with CVI andVLUs as compared to healthy subjects previously studiedwith NIRFLI.7,8,16,17 NIRFLI consistently demonstrateddermal backflow in all the lower extremities of VLU pa-tients, even those with C0 and C4 disease. These resultsare consistent with indirect lymphangiography studies,which have shown dermal backflow proximal to areas ofthe skin marked by hemosiderin staining and lipoderma-tosclerosis in early C4 disease, later stage C6, and C5 dis-ease.18 Furthermore, in prior studies of normal subjectswith BMIs approaching 40, we did not observe localizeddermal backflow as seen in these VLU patients, poten-tially suggesting that localized dermal backflow at the an-kles could be part of the etiology of and a diagnostic forulcer formation. In addition, these NIRFLI data provide

the first evidence that lymphatic dysfunction may occur inearly C0 disease and could precede clinically overt ulcerformation.

This study has also demonstrated reduced lymphaticcontractile function in all the lower limbs. This is consis-tent with past NIRFLI results showing that lymphedemapatients with unilateral disease, unrelated to venous hyper-tension, were characterized by reduced lymphatic contrac-tile function in both symptomatic and asymptomatic limbsas compared with healthy subjects. While neither lympho-scintigraphy nor lymphangiography can directly measurecontractile lymphatic function, our results are consistentwith previously described measurements of radiotracer“transit time,” where prolonged radiotracer transit wasrelated to increasing severity of CVI in both ulceratedand non-ulcerated legs.19-22

It is notable that, in U12, with unilateral C6 disease of 1-year duration, substantial numbers of lymphatic vessels wereobserved in the back of the affected leg, suggesting that, atleast in this stage of the disease, the ulcer-associated lym-phatics became more functional, presumably to compensate

JOURNAL OF VASCULAR SURGERY: VENOUS AND LYMPHATIC DISORDERS16 Rasmussen et al January 2016

for increased interstitial fluid load. Using lymphoscintigra-phy, Cariati21 likewise reported the compensatory effort ofincreased lymphatic transport in patients with acute venousthrombi as measured by transit times. Using NIRFLI,compensatory lymphatic pumping in rats has been shownto increase after stressing with salt-induced hypertension.23

However, the proximal drainage capacity of the ulcer-associated lymphatics associated in U12 may be limiteddue to the observed reflux. Like the veins, lymphatic vesselshave one-way valves that keep lymph moving proximally to-wards the thoracic duct, where it is returned to the blood.Deterioration of lymphatic valves may result in lymphaticreflux and in some patients, as found in past correlation ofDuplex ultrasound and lymphoscintigraphy studies,24 mayoccur in concert with the deterioration of venous valvescontributing to CVI.

Previously we used NIRFLI to show that MLD stimu-lated lymphatic contractile activity in primary and cancer-related lymphedema and in healthy subjects.8 While SPCis designed to mimic MLD, we were unable to quantifyor qualitatively demonstrate increased lymphatic contrac-tility in a single SPC session in VLU patients due to theinfrequent contractile events observed for each region ofinterest.

While we did not observe evidence that SPC movedICG from the intravascular to extravascular spaces, thereis a potential mechanism for extravascular deposition ofICG from the initial lymphatics in the dermis. One couldspeculate that the stagnation of lymph in conducting ves-sels, owing to an insufficient “lymphatic pump” or obstruc-tion could cause lymph to backflow into the initiallymphatics and ultimately out of the lymphatics into theextravascular space. Because our results show that SPCmoves intravascular lymph proximally, one may expectminimal movement of fluid from the intravascular to extra-vascular space with SPC therapy, though further investiga-tion is needed to confirm this hypothesis.

One could also speculate that the inflammatoryresponse to an active VLU25 may reduce lymphatic pumpactivity as shown to occur following cytokine storm26 orin rheumatoid arthritis.27 Interestingly, lymphatic refluxsimilar to that observed in U12 has been shown in micein response to tumor necrosis factor-alpha, interleukin 6,and interleukin 1-beta,26 the former of which has beenimplicated in ulceration in CVI.25 If lymphatic insufficiencyand inflammation are together found to be a significantconfounder of venous disease, then new pharmacologicalstrategies to mediate inflammation and increase lymphatictransport could improve wound healing and reducereoccurrence.

These data demonstrate that even a single SPC sessionwas effective by promoting the emptying and filling ofproximal lymphatic vessels and the proximal movementof ICG-laden extravascular fluid in most CVI patients.However, those subjects with the longest duration of activeulceration did not respond to a single SPC session as simi-larly observed in subjects with advanced breast cancer-related lymphedema.9 Whether mediation of interstitial

fluid load and inflammation by repeated sessions of SPCor MLD can improve local fluid efflux in these patientsremains to be investigated. Histological analysis of dermallymphatics in VLU specimens shows that, while there aremore lymphatic vessels per unit area than in control speci-mens, a greater percentage of these vessels have collapsedlumina28 that could potentially be re-opened by movinginterstitial fluids away from the wound reducing interstitialpressure. The restoration of fluid balance by intravascularand extravascular movement of fluid, as well as stimulationof the lymphatic pump, could be mechanisms by whichMLD and SPC may prevent formation or hasten healingof ulcers.

NIRFLI may have several advantages over lymphoscin-tigraphy and lymphangiography for evaluating thelymphatic contribution to CVI, including the use oftrace doses of non-radioactive ICG to provide almostimmediate visualization of dynamic lymphatic draining ina “point-of-care” setting. When used as a lymphaticcontrast agent, ICG does not require the cannulation oflymphatic vessels or ultrasound-guided injection intoregional lymph nodes as is customary in direct methodsof lymphangiography.17 As a result, ICG can be safelyadministered repeatedly over the course of extended treat-ments to visualize lymphatic efflux. NIRFLI has beenshown to provide diagnostic data in patients with subclin-ical lymphedema7,16 and to reveal lymphatic abnormalitiesotherwise undetected when directly compared with con-ventional lymphoscintigraphy.29 In addition, lymphaticpumping can be directly visualized with NIRFLI, due tothe high temporal resolution of the technique.30 This tech-nique can also be used to detect lymphatic reflux that cancomplement duplex ultrasound measurements of venousreflux. Both intravascular and extravascular dye can be visu-alized at depths as great as 3 to 4 centimeters,31 and,because the intradermal injection sites provide a continualsource of ICG, lymphatic drainage can be observed forover 4 hours without re-administering the dye. NIRFLIshows that SPC effectively moves lymph and extravascularICG-laden fluid proximally away from ulcers, possiblyexplaining a mechanism for SPC-mediated hastening of ul-cer healing32 and suggesting that lymphatic treatmentcould be complementary to surgical management in orderto prevent progression of C3 and C4A,B disease to C6 dis-ease and to hasten the regression from C6 to C5 disease.

CONCLUSIONS

This pilot study demonstrates that lymphatic abnor-malities compromise fluid transport in VLU and that, ina single session, SPC can move accumulated fluid proxi-mally, potentially providing a means for reducing intersti-tial fluid loads to achieve both prevention and healing ofvenous ulcers. The study also suggests that NIRFLI couldprovide a new tool to assess lymphatic dysfunction in indi-vidual patients in order to discern the role of lymphaticdysfunction in CVI and VLU. Further studies areneeded to longitudinally image the lymphatics in the pro-gression of CVI to indicate whether lymphatic dysfunction

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contributes to the etiology of VLUs or is the result ofVLUs. If future studies indicate lymphatic dysfunction con-tributes to the etiology of VSUs, then new managementstrategies that focus both upon the lymphatic and venouscontributions could potentially prevent ulcers and ulcerreoccurrence, and improve outcomes. Whether treatmentsfocused upon lymphatic dysfunction precede or occur intandem with conventional endovascular and surgical treat-ments to improve outcomes in patients with CVI willrequire additional investigation.

The authors acknowledge the helpful review andcritique by Alan Hirsch, as well as the clinical assistanceof Erik A. Maus and Renie Guilliod.

AUTHOR CONTRIBUTIONS

Conception and design: CF, ESAnalysis and interpretation: JR, MA, IT, CD, BZ, TO, CF,

ESData collection: JR, MA, IT, CD, BZWriting the article: JR, MA, IT, CD, BZ, TO, CF, ESCritical revision of the article: JR, TO, CF, ESFinal approval of the article: JR, MA, IT, CD, BZ, TO, CF,

ESStatistical analysis: Not applicableObtained funding: ESOverall responsibility: ES

REFERENCES

1. O’Meara S, Cullum NA, Nelson EA. Compression for venous legulcers. Cochrane Database Syst Rev 2009:CD000265.

2. Valencia IC, Falabella A, Kirsner RS, Eaglstein WH. Chronic venousinsufficiency and venous leg ulceration. J Am Acad Dermatol 2001;44:401-21; quiz 422-4.

3. Etufugh CN, Phillips TJ. Venous ulcers. Clin Dermatol 2007;25:121-30.

4. Poeartsch H, Lee B. Phlebology and lymphology - a family affair.Phlebology 2014;29:645-7.

5. Mortimer PS. Implications of the lymphatic system in CVI-associatededema. Angiology 2000;51:3-7.

6. Eberhardt RT, Raffetto JD. Chronic venous insufficiency. Circulation2014;130:333-46.

7. Rasmussen JC, Tan IC, Marshall MV, Fife CE, Sevick-Muraca EM.Lymphatic imaging in humans with near-infrared fluorescence. CurrOpin Biotechnol 2009;20:74-82.

8. Tan IC, Maus EA, Rasmussen JC, Marshall MV, Adams KE, Fife CE,et al. Assessment of lymphatic contractile function after manuallymphatic drainage using near-infrared fluorescence imaging. Am JPhys Med Rehabil 2011;92:756-764.e1.

9. Adams KE, Rasmussen JC, Darne C, Tan IC, Aldrich MB,Marshall MV, et al. Direct evidence of lymphatic function improvementafter advanced pneumatic compression device treatment of lymphe-dema. Biomed Opt Express 2010;1:114-25.

10. Howarth D, Burstal R, Hayes C, Lan L, Lantry G. Autonomic regu-lation of lymphatic flow in the lower extremity demonstrated on lym-phoscintigraphy in patients with reflex sympathetic dystrophy. ClinNucl Med 1999;24:383-7.

11. Mayrovitz HN. Interface pressures produced by two different types oflymphedema therapy devices. Phys Ther 2007;87:1379-88.

12. Burrows PE, Gonzalez-Garay ML, Rasmussen JC, Aldrich MB,Guilliod R, Maus EA, et al. Lymphatic abnormalities are associatedwith RASA1 gene mutations in mouse and man. Proc Natl Acad Sci US A 2013;110:8621-6.

13. Rasmussen JC, Herbst KL, Aldrich MB, Darne C, Tan I-C, Zhu B,et al. An abnormal lymphatic phenotype is associated with subcutane-ous adipose tissue deposits in Dercum’s disease. Obesity 2014;22:2186-92.

14. Agollah GD, Gonzalez-Garay ML, Rasmussen JC, Tan IC,Aldrich MB, Darne C, et al. Evidence for SH2 domain-containing5’-inositol phosphatase-2 (SHIP2) contributing to a lymphaticdysfunction. PLoS One 2014;9:e112548.

15. Aldrich M, Guilliod R, Fife CE, Maus EA, Smith LA, Rasmussen JC,et al. Lymphatic abnormalities in the normal contralateral arms ofsubjects with breast cancer-related lymphedema as assessed by near-infrared fluorescent imaging. Biomed Opt Express 2012;3:1256-65.

16. Rasmussen JC, Tan IC, Marshall MV, Adams KE, Kwon S, Fife CE,et al. Human lymphatic architecture and dynamic transport imagedusing near-infrared fluorescence. Transl Oncol 2010;3:362-72.

17. Sevick-Muraca EM, Kwon S, Rasmussen JC. Emerging lymphatic im-aging technologies for mouse and man. J Clin Invest 2014;124:905-14.

18. Partsch H, Stoberl C, Urbanek A, Wenzel-Hora BI. Clinical use ofindirect lymphography in different forms of leg edema. Lymphology1988;21:152-60.

19. Bull RH, Gane JN, Evans JE, Joseph AE, Mortimer PS. Abnormallymph drainage in patients with chronic venous leg ulcers. J Am AcadDermatol 1993;28:585-90.

20. Mortimer PS. Evaluation of lymphatic function: abnormal lymphdrainage in venous disease. Int Angiol 1995;14(3 Suppl 1):32-5.

21. Cariati A. Investigation of the leg lymphatic function in patients withleg acute venous thrombi and in patients with leg post-thromboticsyndrome. Open Circ Vasc J 2010;3:67-71.

22. Raju S, Owen S Jr, Neglen P. Reversal of abnormal lymphoscintigraphyafter placement of venous stents for correction of associated venousobstruction. J Vasc Surg 2001;34:779-84.

23. Kwon S, Agollah GD, Chan W, Sevick-Muraca EM. Alteredlymphatic function and architecture in salt-induced hypertensionassessed by near-infrared fluorescence imaging. J Biomed Opt2012;17;080504-1.

24. Mellor RH, Brice G, Stanton AW, French J, Smith A, Jeffery S, et al.Mutations in FOXC2 are strongly associated with primary valve failurein veins of the lower limb. Circulation 2007;115:1912-20.

25. Liu YC, Margolis DJ, Isseroff RR. Does inflammation have a role in thepathogenesis of venous ulcers? A critical review of the evidence. J InvestDermatol 2011;131:818-27.

26. Aldrich MB, Sevick-Muraca EM. Cytokines are systemic effectors oflymphatic function in acute inflammation. Cytokine 2013;64:362-9.

27. Zhou Q, Wood R, Schwarz EM, Wang YJ, Xing L. Near-infraredlymphatic imaging demonstrates the dynamics of lymph flow andlymphangiogenesis during the acute versus chronic phases of arthritis inmice. Arthritis Rheum 2010;62:1881-9.

28. Fernandez AP, Miteva M, Roberts B, Ricotti C, Rouhani P,Romanelli P. Histopathologic analysis of dermal lymphatic alterationsin chronic venous insufficiency ulcers using D2-40. J Am Acad Der-matol 2011;64:1123.e1-1123.e12.

29. Mihara M, Hara H, Araki J, Kikuchi K, Narushima M, Yamamoto T,et al. Indocyanine green (ICG) lymphography is superior to lympho-scintigraphy for diagnostic imaging of early lymphedema of the upperlimbs. PLoS One 2012;7:e38182.

30. Zhu B, Sevick-Muraca EM. A review of performance of near-infraredfluorescence imaging devices used in clinical studies. Br J Radiol2015;88:20140547.

31. Sevick-Muraca EM, Sharma R, Rasmussen JC, Marshall MV,Wendt JA, Pham HQ, et al. Imaging of lymph flow in breast cancerpatients after microdose administration of a near-infrared fluorophore:feasibility study. Radiology 2008;246:734-41.

32. Nelson EA, Mani R, Thomas K, Vowden K. Intermittent pneumaticcompression for treating venous leg ulcers. Cochrane Database SystRev 2011:CD001899.

Submitted Apr 20, 2015; accepted Jun 2, 2015.

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