Lymphatic transport in patients with chronic venous insufficiency and venous leg ulcers following sequential pneumatic compression

From the American Venous Forum

Lymphatic transport in patients with chronic venous insufficiency and venous leg ulcers following sequential pneumatic compression John 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 and Eva M. Sevick-Muraca, PhD,a Houston and The Woodlands, Tex; and Boston, Mass Background: Recent advancements in near-infrared fluorescence lymphatic imaging (NIRFLI) technology provide opportunities for non-invasive, real-time assessment of lymphatic contribution in the etiology and treatment of ulcers. The objective of this study was to assess lymphatics in subjects with venous leg ulcers using NIRFLI and to assess lymphatic impact of a single session of sequential pneumatic compression (SPC). Methods: Following intradermal microdoses of indocyanine green (ICG) as a lymphatic contrast agent, NIRFLI was used in a pilot study to image the lymphatics of 12 subjects with active venous leg ulcers (Clinical, Etiologic, Anatomic and Pathophysiologic [CEAP] C6). The lymphatics were imaged before and after a single session of SPC to assess impact on lymphatic function. Results: Baseline imaging showed impaired lymphatic function and bilateral dermal backflow in all subjects with chronic venous insufficiency, even those without ulcer formation in the

contralateral limb (C0 and C4 disease). SPC therapy caused proximal movement of ICG away from the active wound in 9 of 12 subjects, as indicated by newly recruited functional lymphatic vessels, emptying of distal lymphatic vessels, or proximal movement of extravascular fluid. Subjects with the longest duration of active ulcers had few visible lymphatic vessels, and proximal movement of ICG was not detected after SPC therapy. Conclusions: This study provides visible confirmation of lymphatic dysfunction at an early stage in the etiology of venous ulcer formation and demonstrates the potential therapeutic mechanism of SPC therapy in removing excess fluid. The ability of SPC therapy to restore fluid balance through proximal movement of lymph and interstitial fluid may explain its value in hastening venous ulcer healing. Anatomical differences between the lymphatics of longstanding and more recent venous ulcers may have important therapeutic implications. (J Vasc Surg: Venous and Lym Dis 2015;-:1-9.)

Approximately 1% of the total population and 3% to 5% of the population over the age of 65 will suffer from a venous leg ulcer (VLU) in their lifetime.1 VLUs characterize 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 can result in infection, cellulitis, osteomyelitis, and/or amputation. In the U.S., the financial burden of VLUs is estimated to be $2 billion annually2,3 and is expected to grow with the aging population. The mechanism of ulcer formation involves a sequence of pathophysiologic steps that are thought to include (1) reduction in venous outflow due to venous obstruction and/or reflux, (2) persistent venous hypertension, and (3) subsequent increased capillary filtration and interstitial fluid load. Typically, excess interstitial fluid is effectively removed by the lymphatics, but if the fluid load overwhelms the lymphatic capacity or if the lymphatics are defective, then the accumulation of interstitial fluid, macromolecules, and cytokines lead to edema (CEAP C3), breakdown of subcutaneous tissue (C4A,B), and formation of ulcers (C6).4,5 Once formed, an ulcer will not heal (C5) unless venous hypertension and the resulting excess capillary fluid filtration are ameliorated. Individuals with severe venous obstruction may undergo venous stenting, and individuals with severe venous insufficiency may benefit from venous ablation or phlebectomy. These procedures are designed to restore venous function by lowering venous hypertension and thereby reduce excess capillary filtration and interstitial edema. Yet correction of venous hypertension may not sufficiently resolve increased interstitial fluid load if lymphatic

From the Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, Houstona; the Division of Vascular Surgery, Tufts Medical Center, Bostonb; and the Wound Care Clinic, CHI St. Luke’s Health - The Woodlands Hospital, The Woodlands.c This study was funded in parts by Tactile Medical Systems, Inc, and grants from the National Institutes of Health (R01 HL092923 [E.M.S.M.] and U54 CA136404 [E.M.S.M.]). Author conflict of interest: J.C.R and E.M.S.M. own stock in a UTHealth portfolio company, NIRF Imaging, Inc, which seeks to commercialize the NIRF lymphatic imaging technology. J.C.R. has also received consulting fees from NIRF Imaging. T.F.O. is on the scientific advisory board of Tactile Medical Systems. Presented at the Twenty-seventh Annual Meeting of the American Venous Forum, Palm Springs, Calif, February 25-27, 2015. Additional material for this article may be found online at www.jvsvenous.org. Reprint requests: Eva M. Sevick-Muraca, PhD, 1825 Pressler St, Houston, TX 77030 (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the Journal policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 2213-333X Copyright Ó 2015 The Authors. Published by Elsevier Inc. on behalf of the Society for Vascular Surgery. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/). http://dx.doi.org/10.1016/j.jvsv.2015.06.001

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vessels are dysfunctional. Although the lymphatic contribution to VLU formation and healing has long been recognized, recent authoritative reviews on CVI management do not define the lymphatic role in the outcomes to surgical treatment.6 Complete decongestive therapy is the standard-of-care lymphatic treatment in lymphedema patients and encompasses a set of therapeutic interventions designed to decrease lymphatic congestion and enhance transport by combining (1) limb compression, (2) manual lymphatic drainage (MLD), and, when necessary, (3) multicompartmental sequential pneumatic compression (SPC). To date, the adjunctive benefit of lymphatic treatment for preventing the formation or promoting the healing of VLUs has not been fully investigated. The objective of this pilot study was to evaluate, with near-infrared fluorescence lymphatic imaging (NIRFLI) and off-label, intradermal microdoses of indocyanine green (ICG): (1) the lymphatic anatomy and function in patients with C6 venous disease and (2) the possible change in lymph and extravascular fluid distribution in response to a single treatment of SPC. In addition, we sought to provide initial validation of NIRFLI as a potential diagnostic or prognostic tool for future, more definitive clinical studies of venous and lymphatic disease. METHODS This study was conducted under the U.S. Food and Drug Administration (IND#: 102,765) and local Institutional Review Board approval and in accordance with the Helsinki Declaration. Clinical criteria. Following informed consent, twelve subjects with chronic VLUs were enrolled and assigned a study ID (U01-U12). Subjects were recruited prospectively as they presented in the clinic. Inclusion criteria included a minimum age of 18 years, the presence of both legs, and the presence of an active VLU. Exclusion criteria included pregnancy or breast feeding; unwillingness to use medically accepted contraceptives for 1 month following imaging (for females of child-bearing potential); an allergy to iodine; failing prior compression therapy; inability to tolerate compression; or clinically significant arterial disease, defined by an ankle brachial index <0.7, skin perfusion pressures <30 mm Hg, or transcutaneous oximetry <30 mm Hg with poor response to sea level oxygen breathing. Subjects with diabetic foot ulcers, exposed bone or tendons, or autoimmune disease with prednisone prescription were excluded. Near-infrared fluorescence lymphatic imaging (NIRFLI). Prior to NIRFLI, each subject received up to 16 intradermal injections of 25 mg of ICG in 0.1 mL saline with a maximum total dose of 400 mg. Because the ulcers’ location and size varied, the exact number and location of the injection sites also varied from subject to subject, but generally consisted of two injections on the dorsum of each foot, injections in the ankles and calves, and an injection in each thigh. To capture the lymphatic components

potentially associated with the ulcer, two to three additional injections were administered distal to the ulcer and in corresponding locations on the contralateral limb. NIRFLI was conducted immediately following ICG injection and consisted of illuminating the limbs with diffuse 785-nm light (<1.9 mW/cm2), which penetrated the tissues and excited the ICG, and collecting the resultant 830-nm fluorescent signal emanating from the limbs with a near-infrared-sensitive, Gen III image intensifier and a charge-coupled device camera as detailed previously.7 Image sequences were acquired with 200-ms exposure times, allowing near-real-time visualization of lymphatic contractile function. After imaging for approximately 30 minutes, the affected leg of each subject was placed in a SPC garment and underwent therapy for 1 hour. The SPC garments are opaque, preventing NIRFLI of the treated limb during therapy. However, the contralateral, untreated limb was imaged during therapy to visualize its lymphatic response as past NIRFLI studies showed that MLD and SPC treatment of symptomatic limbs stimulated contractile function in contralateral limbs,8,9 presumably owing to autonomic regulation of lymphatics.10 Following therapy, the treated limb was again imaged for approximately 30 minutes. SPC. SPC therapy was accomplished using the Flexitouch system (Tactile Systems Technology, Inc, Minneapolis, Minn), a programmable pneumatic compression device specifically designed to treat lymphedema.11 The garments were fitted to each subject per manufacturer’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 the abdominal areas to stimulate and clear the regional lymph nodes. Next, SPC treatment continued with initial unilateral leg preparation, lasting approximately 18 minutes, during which a gradient pressure was applied to the more proximal areas of the leg. The treatment culminated with leg and trunk drainage, lasting approximately 30 minutes, during which concerted cycles of mild variable pressures were applied sequentially to the limb and trunk in a distal-to-proximal manner. To prevent contamination, each wound was bandaged prior to SPC and, following treatment, the SPC garments were disinfected with a >60% isopropyl alcohol solution per manufacturer’s recommendations. Follow-up. Clinical follow-up was performed for each subject at 18 to 24 hours after ICG injection to ascertain adverse events (eg, local skin reaction, fever, or allergy). In this pilot study, neither ulcer healing nor any other clinical outcome was followed after the single SPC session, nor were NIRFLI results used in patient care. Image analysis. Lymphatic function and anatomy, as observed in NIRFLI images, were assessed in both treated and contralateral limbs. The study was designed to measure whether SPC therapy resulted in improved lymphatic function as determined by (1) the presence of newly recruited lymphatics (lymphatic vessels or draining tissues which were not fluorescent prior to SPC, but were fluorescent after), (2) the

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

Age, years

BMI

Race, ethnicity

History

Current

Healed

Lymphedema and stagea

Gender

U01

M

54

46.2

AA

B

R

B e II

M

69

40.5

C

U

N

N

U03

F

26

61.6

AA

B

R

U04

M

50

47.3

AA

B

U05

M

53

38.0

AA

B

R-C5 L-C6 R-C6 L-C4 L-C6 R-C5 R-C6 L-C5 R-C6 L-C5

U02

U06

M

57

32.3

AI

B

U07

F

32

34.5

C

B

U08

F

60

31.1

C

B

U09

F

52

29.8

C, H

B

U10

F

65

49.2

C

U

U11

M

59

49.5

AA

U

U12

M

56

31.7

C

U

R-C6 L-C5 R-C6 L-C5 R-C6 L-C5 R-C6 L-C6 L-C6 R-C0 R-C6 L-C4 L-C6 R-C0

Duration of current wound(s), years

Skin perfusion pressure, mm Hg

Total ICG dose, mg

7-8

RD-69

400

2

RD-55

400

B e III

13

400

L

B e II

3

L

B e II

27

L

BeI

5

L

BeI

0.42

L

B e II

6

LD-67 LP-54 RD-67 RP-69 RD-65 RP-65 LD-62 LP-64 RD-43 RP-61 RD-49 LD-44 RD-63

350

L

N

0.5

LD-99

300

N

B e II

0.42

LD-98

350

N

B e (I-II)

0.17

R-79

350

N

N

1

L-70

350

350 300

350 400

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. a Lymphedema staging (I, II, or III) is consistent with the International Society of Lymphology.

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 anthropomorphic characteristics of the seven males and five females enrolled. The median age was 55 years (range, 26-69 years;

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, though most subjects had a multiple-year history of bilateral ulceration. During this study, the subjects’ contralateral limbs had (1) no clinical signs of disease (C0, 2 subjects), (2) healed ulcers (C5 disease, 7 subjects), (3) skin changes

Table II. Imaging results in treated and untreated legs of patients with venous leg ulcers (VLUs) Subject ID

Lymphatic architecture, immediate vicinity of wound

Lymphatic architectural abnormalities, treated leg

Lymphatic architecture abnormalities, untreated leg

Lymphatic response to SPC

U01 U02 U03 U04 U05 U06 U07 U08 U09 U10 U11 U12

DB DB No No No DB DB DB DB DB DB DB

DB, DB, DB DB, DB, DB DB, DB, DB, DB, DB, DB

DB DB, T, D DB DB, T DB, T DB T, D DB, D DB DB DB DB

None R, E None R None R, Possible E R R R, RW, E E R, RW, E E

T T, Possible D T T, D D D D Possible D D

Lymphatic contractile function Limited Limited Limited Limited Limited Limited Yes Noa Yes Yes before, No after SPC Yes Yes, LR

D, Dilated lymphatics; DB, dermal backflow; E, emptying; LR, lymphatic reflux; R, recruitment; RW, recruitment within wound; SPC, sequential pneumatic compression; T, tortuous lymphatics. a Due to technical difficulties, the image acquisition rate was severely inhibited and 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 subjects were obese, a factor that predisposes for CVI and is also well known to be associated with lymphedema. At enrollment, lymphedema was confirmed in nine subjects, based upon a positive Stemmer’s sign. The venous disease etiology was post-thrombic, but anatomical and pathophysiologic classifications were not assessed in this pilot study. No adverse events were reported. Table II summarizes the results for each subject, including brief summaries of the lymphatic anatomy in the immediate vicinity of the wound, the SPC-treated leg, and the untreated, contralateral leg. Observations of lymphatic contractile function and lymphatic vessel recruitment and/or emptying as a result of SPC are noted. Abnormal lymphatic anatomy. In past NIRFLI studies (NCT000833599: “Imaging lymphatic function in normal subjects and in persons with lymphatic disorders”), healthy subjects (Fig 1, A) showed well-defined lymphatic

vasculature that actively “pumped” ICG-laden lymph (Video 1, online only)12 proximally to the inguinal lymph nodes,13 while subjects with lower extremity, primary lymphedema (Fig 1, B), showed dilated lymphatic vessels with dermal backflow, extravascular fluorescence, and/or limited contractile activity.14 Because NIRFLI depends upon detecting ICG taken up by lymphatics, nonfunctional and/or obstructed lymphatics may not be detected with the technique. In all 12 subjects with CVI, NIRFLI showed dermal backflow and extravascular fluorescence that was visualized as “pooling” of ICG-laden lymph (1) near the ulcers, as seen in U09 with bilateral C6 disease (Fig 1, C), (2) at distant sites away from the ulcers (U02; Fig 1, D), and/or (3) in the asymptomatic legs of subjects with unilateral ulcers (U10-U12). Dermal backflow (Fig 2, A) was observed in the immediate vicinity of the wound in nine subjects, two of which (U09 and U11) had fluorescence within the ulcer itself (Fig 2, B). Three subjects (U03-U05) with long duration of active ulcer(s) (3, 13, and 27 years) did not show dermal backflow or other

Fig 1. A, Normal lymphatics as seen in the lower anterior legs of a 57-year-old healthy subject; B, Diseased lymphatics with extensive dermal backflow and tortuous vessels as seen in the back, lower right leg of a 49-year-old subject with primary lymphedema; C, Diseased lymphatics with lymphatic pooling as evidenced by the dermal backflow in the ankles in U09; and D, extensive dermal backflow and tortuous vessels as seen the lower right leg of U02.

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fluorescence uptake in the immediate vicinity of the wound as shown in Fig 2, C. In these cases, the lack of uptake near the wound may be related to the distance from the injection sites to the wound, as we were reluctant to inject into newly healed regions (color inset in Fig 2, C). Consistent with past work in subjects with the longest duration of cancer-related lymphedema,15 we found fewer functional lymphatics and greater dermal backflow in those study subjects with the longest duration of active ulceration. In the limbs of two subjects (U02 and U11) with C4A disease, dermal backflow was observed to follow the hemosiderin stains on the subjects’ ankles and lower legs as demonstrated in Fig 2, D and E. In addition to dermal backflow, tortuous and/or dilated conducting lymphatic vessels were visualized in all but two subjects (U03 and U06). Interestingly, in U12, with an active ulcer of 1-year duration located on the back of the calf, we observed

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substantial lymphatic drainage proximal to the ulcer (Fig 2, F and G), whereas no symmetric, functional lymphatic vessels were visualized on the contralateral C0 leg. In our past experience with NIRFLI in healthy subjects, we typically visualized functional lymphatics symmetrically 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 unaffected leg indicates that the lymphatics visualized in the affected leg may be specifically associated with the ulcer. In addition, we observed “lymphatic reflux” in the ulcerassociated lymphatics in this subject (Video 2, online only). Reduction of lymphatic contractile function. We have previously measured 0.4 6 0.3 lymphatic contractions/min in normal subjects.16 In this study, however, both affected and contralateral limbs exhibited diminished contractile events too infrequent to be quantified within

Fig 2. Images of fluorescent uptake in the lymphatics near the venous leg ulcers (VLUs). A, Shows an example of the lack of fluorescence typically observed within the wound site while surrounded by dermal backflow (U07, color image of wound inset) while (B) shows an example of a wound that contained fluorescence (U11, color image of wound inset). C, Illustrates the lack of fluorescence uptake from the injection sites below the wound (U05, color image of wound inset). D, Color image of the hemosiderin staining in the lower left leg of U11. E, Near-infrared fluorescence image overlaid on a color image illustrating the presence of fluorescence within hemosiderin stain on lower left leg of U11. F, Color image of the wound on the back of the lower left leg of U12. G, Near-infrared fluorescence image overlaid on a color image illustrating the brightly fluorescent lymphatics draining the wound area on the back of the left leg of U12 while the lymphatics in the right leg are faintly visible.

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Fig 3. Images illustrating lymphatic recruitment following sequential pneumatic compression (SPC) therapy. A, Color image 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 lower lateral right calf (inset) and near-infrared fluorescence images acquired (E) before and (F) after treatment. The pink skin around lower leg of (D) shows the previous extent of wound, and the darker skin tones in (A) illustrate hemosiderin staining commonly associated with venous insufficiency. The yellow ellipses denote the approximate field of view in the near-infrared fluorescence images.

the confines of our standard period of observation, which was constrained by the need to scan the entirety of both legs before and after SPC. With the exception of U08, where technical difficulties may have prevented observation of contractile function, this diminished, contractile lymphatic function was observed in all subjects including the three study subjects (U02, U09, and U12) not diagnosed with lymphedema. After a single session of SPC, there was no apparent increase in the contractile function in any of the subjects. Fluorescence distribution pre- and post-SPC therapy. Following SPC, we observed newly fluorescent lymphatics in seven subjects (U02, U04, U06-U09, and U11). Fig 3 presents images illustrating the movement of fluorescence in the few, but dilated lymphatics in U02 and U04 before and after SPC. In both subjects, SPC resulted in intravascular and extravascular movement of fluorescence towards the inguinal region. We

also observed the emptying of fluorescence from intravascular and/or extravascular spaces in at least five subjects (U02, U06 (possible), and U09-U12) after SCP, with example images shown in Fig 4. Fig 4, A-C illustrate the reduction of fluorescence signal in the initial lymphatics and extravascular spaces proximal to the ulcer in U09. Fig 4, D-F illustrate the reduction of fluorescence signal in the initial lymphatics and extravascular spaces below the knee and proximal to the wound in U10. Thus, it would appear that a major route of lymph fluid clearance with SPC occurs through the interstitial space as well as through functional lymphatic vessels. SPC did not appear to cause movement of ICG from the intravascular to extravascular tissue compartment, but rather moved ICG in their respective compartments proximally. Finally, we observed no proximal ICG movement after SPC in the three subjects (U01, U03, and U05) who had the longest duration

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Fig 4. Images illustrating lymphatic emptying following sequential pneumatic compression (SPC) therapy. A, Color image 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-infrared fluorescence images acquired (E) before and (F) after treatment. The yellow ellipse in (D) denotes the approximate field of view in the near-infrared fluorescence images (E) and (F).

(7-8, 13, and 27 years) of active ulceration, infection, and inflammation. DISCUSSION This pilot study demonstrates that lymphatic function and anatomy were abnormal in patients with CVI and VLUs as compared to healthy subjects previously studied with NIRFLI.7,8,16,17 NIRFLI consistently demonstrated dermal backflow in all the lower extremities of VLU patients, even those with C0 and C4 disease. These results are consistent with indirect lymphangiography studies, which have shown dermal backflow proximal to areas of the skin marked by hemosiderin staining and lipodermatosclerosis in early C4 disease, later stage C6, and C5 disease.18 Furthermore, in prior studies of normal subjects with BMIs approaching 40, we did not observe localized dermal backflow as seen in these VLU patients, potentially suggesting that localized dermal backflow at the ankles could be part of the etiology of and a diagnostic for ulcer formation. In addition, these NIRFLI data provide

the first evidence that lymphatic dysfunction may occur in early C0 disease and could precede clinically overt ulcer formation. This study has also demonstrated reduced lymphatic contractile function in all the lower limbs. This is consistent with past NIRFLI results showing that lymphedema patients with unilateral disease, unrelated to venous hypertension, were characterized by reduced lymphatic contractile function in both symptomatic and asymptomatic limbs as compared with healthy subjects. While neither lymphoscintigraphy nor lymphangiography can directly measure contractile lymphatic function, our results are consistent with previously described measurements of radiotracer “transit time,” where prolonged radiotracer transit was related to increasing severity of CVI in both ulcerated and non-ulcerated legs.19-22 It is notable that, in U12, with unilateral C6 disease of 1year duration, substantial numbers of lymphatic vessels were observed in the back of the affected leg, suggesting that, at least in this stage of the disease, the ulcer-associated lymphatics became more functional, presumably to compensate

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for increased interstitial fluid load. Using lymphoscintigraphy, Cariati21 likewise reported the compensatory effort of increased lymphatic transport in patients with acute venous thrombi as measured by transit times. Using NIRFLI, compensatory lymphatic pumping in rats has been shown to increase after stressing with salt-induced hypertension.23 However, the proximal drainage capacity of the ulcerassociated lymphatics associated in U12 may be limited due to the observed reflux. Like the veins, lymphatic vessels have one-way valves that keep lymph moving proximally towards the thoracic duct, where it is returned to the blood. Deterioration of lymphatic valves may result in lymphatic reflux and in some patients, as found in past correlation of Duplex ultrasound and lymphoscintigraphy studies,24 may occur in concert with the deterioration of venous valves contributing to CVI. Previously we used NIRFLI to show that MLD stimulated lymphatic contractile activity in primary and cancerrelated lymphedema and in healthy subjects.8 While SPC is designed to mimic MLD, we were unable to quantify or qualitatively demonstrate increased lymphatic contractility in a single SPC session in VLU patients due to the infrequent contractile events observed for each region of interest. While we did not observe evidence that SPC moved ICG from the intravascular to extravascular spaces, there is a potential mechanism for extravascular deposition of ICG from the initial lymphatics in the dermis. One could speculate that the stagnation of lymph in conducting vessels, owing to an insufficient “lymphatic pump” or obstruction could cause lymph to backflow into the initial lymphatics and ultimately out of the lymphatics into the extravascular space. Because our results show that SPC moves intravascular lymph proximally, one may expect minimal movement of fluid from the intravascular to extravascular space with SPC therapy, though further investigation is needed to confirm this hypothesis. One could also speculate that the inflammatory response to an active VLU25 may reduce lymphatic pump activity as shown to occur following cytokine storm26 or in rheumatoid arthritis.27 Interestingly, lymphatic reflux similar to that observed in U12 has been shown in mice in response to tumor necrosis factor-alpha, interleukin 6, and interleukin 1-beta,26 the former of which has been implicated in ulceration in CVI.25 If lymphatic insufficiency and inflammation are together found to be a significant confounder of venous disease, then new pharmacological strategies to mediate inflammation and increase lymphatic transport could improve wound healing and reduce reoccurrence. These data demonstrate that even a single SPC session was effective by promoting the emptying and filling of proximal lymphatic vessels and the proximal movement of ICG-laden extravascular fluid in most CVI patients. However, those subjects with the longest duration of active ulceration did not respond to a single SPC session as similarly observed in subjects with advanced breast cancerrelated lymphedema.9 Whether mediation of interstitial

fluid load and inflammation by repeated sessions of SPC or MLD can improve local fluid efflux in these patients remains to be investigated. Histological analysis of dermal lymphatics in VLU specimens shows that, while there are more lymphatic vessels per unit area than in control specimens, a greater percentage of these vessels have collapsed lumina28 that could potentially be re-opened by moving interstitial fluids away from the wound reducing interstitial pressure. The restoration of fluid balance by intravascular and extravascular movement of fluid, as well as stimulation of the lymphatic pump, could be mechanisms by which MLD and SPC may prevent formation or hasten healing of ulcers. NIRFLI may have several advantages over lymphoscintigraphy and lymphangiography for evaluating the lymphatic contribution to CVI, including the use of trace doses of non-radioactive ICG to provide almost immediate visualization of dynamic lymphatic draining in a “point-of-care” setting. When used as a lymphatic contrast agent, ICG does not require the cannulation of lymphatic vessels or ultrasound-guided injection into regional lymph nodes as is customary in direct methods of lymphangiography.17 As a result, ICG can be safely administered repeatedly over the course of extended treatments to visualize lymphatic efflux. NIRFLI has been shown to provide diagnostic data in patients with subclinical lymphedema7,16 and to reveal lymphatic abnormalities otherwise undetected when directly compared with conventional lymphoscintigraphy.29 In addition, lymphatic pumping can be directly visualized with NIRFLI, due to the high temporal resolution of the technique.30 This technique can also be used to detect lymphatic reflux that can complement duplex ultrasound measurements of venous reflux. Both intravascular and extravascular dye can be visualized at depths as great as 3 to 4 centimeters,31 and, because the intradermal injection sites provide a continual source of ICG, lymphatic drainage can be observed for over 4 hours without re-administering the dye. NIRFLI shows that SPC effectively moves lymph and extravascular ICG-laden fluid proximally away from ulcers, possibly explaining a mechanism for SPC-mediated hastening of ulcer healing32 and suggesting that lymphatic treatment could be complementary to surgical management in order to prevent progression of C3 and C4A,B disease to C6 disease and to hasten the regression from C6 to C5 disease. CONCLUSIONS This pilot study demonstrates that lymphatic abnormalities compromise fluid transport in VLU and that, in a single session, SPC can move accumulated fluid proximally, potentially providing a means for reducing interstitial fluid loads to achieve both prevention and healing of venous ulcers. The study also suggests that NIRFLI could provide a new tool to assess lymphatic dysfunction in individual patients in order to discern the role of lymphatic dysfunction in CVI and VLU. Further studies are needed to longitudinally image the lymphatics in the progression of CVI to indicate whether lymphatic dysfunction

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contributes to the etiology of VLUs or is the result of VLUs. If future studies indicate lymphatic dysfunction contributes to the etiology of VSUs, then new management strategies that focus both upon the lymphatic and venous contributions could potentially prevent ulcers and ulcer reoccurrence, and improve outcomes. Whether treatments focused upon lymphatic dysfunction precede or occur in tandem with conventional endovascular and surgical treatments to improve outcomes in patients with CVI will require additional investigation. The authors acknowledge the helpful review and critique by Alan Hirsch, as well as the clinical assistance of Erik A. Maus and Renie Guilliod. AUTHOR CONTRIBUTIONS Conception and design: CF, ES Analysis and interpretation: JR, MA, IT, CD, BZ, TO, CF, ES Data collection: JR, MA, IT, CD, BZ Writing the article: JR, MA, IT, CD, BZ, TO, CF, ES Critical revision of the article: JR, TO, CF, ES Final approval of the article: JR, MA, IT, CD, BZ, TO, CF, ES Statistical analysis: Not applicable Obtained funding: ES Overall responsibility: ES REFERENCES 1. O’Meara S, Cullum NA, Nelson EA. Compression for venous leg ulcers. Cochrane Database Syst Rev 2009:CD000265. 2. Valencia IC, Falabella A, Kirsner RS, Eaglstein WH. Chronic venous insufficiency 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-associated edema. Angiology 2000;51:3-7. 6. Eberhardt RT, Raffetto JD. Chronic venous insufficiency. Circulation 2014;130:333-46. 7. Rasmussen JC, Tan IC, Marshall MV, Fife CE, Sevick-Muraca EM. Lymphatic imaging in humans with near-infrared fluorescence. Curr Opin 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 manual lymphatic drainage using near-infrared fluorescence imaging. Am J Phys 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 improvement after advanced pneumatic compression device treatment of lymphedema. Biomed Opt Express 2010;1:114-25. 10. Howarth D, Burstal R, Hayes C, Lan L, Lantry G. Autonomic regulation of lymphatic flow in the lower extremity demonstrated on lymphoscintigraphy in patients with reflex sympathetic dystrophy. Clin Nucl Med 1999;24:383-7. 11. Mayrovitz HN. Interface pressures produced by two different types of lymphedema 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 associated with RASA1 gene mutations in mouse and man. Proc Natl Acad Sci U S A 2013;110:8621-6.

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