The Search For A Better View

  Normal wound repair and tissue homeostasis depends upon an orchestrated balance between (i) the venous circulatory system that drains water and small proteins and (ii) the lymphatic circulatory system that additionally transports macromolecules, cells, bacteria, and debri from sites of tissue inflammation. If venous insufficiency occurs, then the lymphatic circulation must compensate to remove excess fluid filtered from hypertensive venous circulation. If the lymphatic circulation becomes overwhelmed or, it is dysfunctional to begin with, then in addition to excess fluid, large proteins and macromolecules accumulate within tissues. This accumulation results in the sequelae of inflammatory responses that compromises wound healing and can constitute the disease of lymphedema.

Lymphedema

  Lymphedema is an incurable disease that is often acquired following lymph node removal and radiation for cancer treatment in western countries (usually referred to as “secondary” lymphedema). Lymphedema impacts an estimated 3-5 million of the 12 million cancer survivors alive in the US. A small percentage of patients in the US have primary lymphedema due to congenital defects in lymphatic formation. There is recent evidence to suggest that similar to heritable primary lymphedema^1-5, there may also be a genetic predisposition for acquiring lymphedema following trauma or cancer treatment⁶. Lymphedema that arises from underlying venous disorders (such as chronic venous insufficiency (CVI)), impairs normal wound healing and contributes to chronic wounds, including venous stasis ulcers. Each year, venous stasis ulcers impact 0.5 million of the 13 million Americans who suffer from CVI.

Management and Treatment Methods

  Regardless of whether venous or lymphatic origin, management of lymphedema and chronic wounds depend upon improving lymphatic function through manual lymphatic drainage (MLD) and compression therapy. Unfortunately, there are no diagnostic methods available to directly and quantitatively assess the efficacy of these and other emerging treatments to improve lymphatic function. In other words, there is currently no study that allows the visualization of the anatomy of the lymphatic system as well as “real time” measurement of how much lymph flows through it. Recently, the U.S. Centers for Medicare and Medicaid Services (CMS) challenged the adequacy of available evidence to support diagnostic and treatment methods currently used in lymphedema management⁷. Current diagnostic measures (including lymphoscintigraphy and limb volume measurements) and standards of clinical treatment (including MLD and several marketed pneumatic compression devices (PCDs)) were deemed to have insufficient evidence of benefit.

Pilot Results

  Recently, we have demonstrated pilot results showing that investigational near-infrared NIR fluorescence lymphatic imaging in humans may enable diagnosis of aberrant lymphatic function as well as personalized evaluation of the efficacy of MLD and PCDs at the point-of-care^8-11. The technique depends upon simple 0.1 cc intradermal injection of indocyanine green (ICG), an agent that has been used for over 50 years for assessing hepatic clearance, cardiovascular function testing, and retinal angiography on the basis of its dark green color. ICG happens also to be fluorescent when excited by NIR light and associates with proteins, making it a feasible fluorescence-imaging agent for assessing lymphatic function and protein transport. Following intradermal injection of ICG at 1,000 times less dose than currently approved for intravenous administration, we found the dye transits immediately into normal initial lymphatics and is actively transported to draining lymph node basins. Using a laser diode (similar to that found in the grocery store scanner, but less visible to the human eye), tissue surfaces are dimly illuminated with NIR excitation light. The excitation light harmlessly penetrates several centimeters into tissue, activating ICG. The ICG fluoresces, and fluorescent light propagates out through the tissues and is collected by a custom camera outfitted with military grade night vision technology¹² (Figure 1). In other words the camera uses the same technology as military “night vision” goggles, allowing the “glow in the dark” dye to be seen right through the skin.

  Owing to the sensitivity of the custom camera system, we acquire images on the order of 50–200 milliseconds enabling a compilation of “movies” depicting lymphatic propulsive function8 and in the case of dysfunction, lymphatic reflux9. We have seen for the first time, the lymphatics actually “pumping” fluid.

  The imaging may be more sensitive than lymphoscintigraphy, which depends upon the administration of radioactive colloid that upon decay releases a single gamma photon from each molecule of radionuclide for collection using a gamma camera typically housed in nuclear medicine departments. Several minutes or hours after radiocolloid administration, collection of single gamma photons occur over several minutes to produce a low-resolution image depicting main lymphatic vessels and draining lymph nodes. It takes a significant amount of time to acquire nuclear scintigraphy images so it is not possible to image the rapid contractions responsible for lymphatic motion. NIR imaging is sensitive enough to capture lymphatic pumping. The enhanced sensitivity of NIR fluorescence imaging depends upon the ability to repeatedly excite ICG with tissue-penetrating NIR excitation light, but can be compromised by tissue scatter. ICG (and other potential NIR excited fluorophores) can repeatedly be activated and emit fluorescent photons as often as every nanosecond. Consequently each dye molecule can emit as many as 100 million NIR photons per second. Compared with the single gamma photon emitted from the radiocolloid used in nuclear scintigraphy, NIR fluorescence imaging offers greater sensitivity, improved resolution, lack of radioactivity, and compact instrumentation that may facilitate point-of-care use.

  Figure 2 is an example of the investigational NIR fluorescence imaging of the normal and aberrant lymphatics in the legs of in the right and left legs of a 44 y.o. women who developed unilateral secondary lymphedema after experiencing a twisted ankle 5 years after radical hysterectomy (supplemental movie shows the dynamics of lymphatic movement in each leg). In the normal lymphatics of the right leg, the NIR fluorescence imaging movies shows the propulsion of indocyanine green dye-laden lymph “packets” to sequential lymphangions while the affected leg shows typical extravascular deposition of ICG as well as dense dermal lymphatic capillaries that is typical of progressing or advanced disease⁸. By using the rate of lymph propulsion as a quantifiable measure of normal lymphatic function, we have shown that in some diagnosed cases of unilateral “secondary” lymphedema developing after cancer surgery or trauma, lymphatic function was often compromised not only in the afflicted limb, but in the unafflicted, contralateral limb as well. These results suggest that lymphatic dysfunction may be pre-existing and that regional symptoms of lymphedema occur after local challenge of trauma or infection.

  Using NIR fluorescence lymphatic imaging, we also quantitatively assessed improvement of lymphatic function and recruitment of new lymphatic vessels in response to manual lymphatic drainage¹³ and a specific pneumatic compression device¹⁰. By evaluating lymphatic function, before and after therapy in a single session, we hypothesize NIR lymphatic imaging may enable us to tailor treatments for an individual patient. Continuing development of 3-D imaging NIR fluorescence tomography and the future use of brighter NIR imaging agents (not yet tested in humans) promises to improve lymph-imaging quality. Nonetheless, the current state of the NIR fluorescence imaging offers for the first time, the ability for to assess lymphatic function for effective management of lymphedema and chronic wounds.

  To view a short video of the imaging of lymphatic function please visit www.youtube.com/watch?v=2RCzS6Wnvzw.

  Eva M. Sevick-Muraca, Ph.D. is Professor and Cullen Chair of Molecular Medicine and John C. Rasmussen, Ph.D. is Assistant Professor at the Brown Foundation Institute of Molecular Medicine at the University of Texas Health Science Center in Houston. Both are members of the Center for Molecular Imaging and faculty in the National Cancer Institute’s Network for Translational Research. Drs. Sevick and Rasmussen are engineers who have translated near-infrared fluorescence imaging for assessment of the lymphedema subjects in the clinic of Dr. Caroline Fife, M.D. Currently, the team is using near-infrared fluorescence to phenotype families with heritable lymphatic disorders in order to discover genetic causes of the disease. Dr. Sevick can be reached at eva.sevick@uth.tmc.edu and Dr. Rasmussen at John.Rasmussen@uth.tmc.edu.

  Dr. Caroline Fife, is currently co-editor of Today’s Wound Clinic, is the director of Clinical Research at the Memorial Hermann Center for Wound Healing, Houston, Tex., and Chief Medical Officer of Intellicure, Inc. She can be reached at cfife@intellicure.com.

References

1. Karkkainen, M.J., R.E. Ferrell, E.C. Lawrence, M.A. Kimak, K.L. Levinson, M.A. McTigue, K. Alitalo, and D.N. Finegold, Missense mutations interfere with VEGFR-3 signaling in primary lymphoedema. Nat Genet, 2000. 25(2): p. 153-9. PMID: 10835628

2. Irrthum, A., K. Devriendt, D. Chitayat, G. Matthijs, C. Glade, P.M. Steijlen, J.P. Fryns, M.A. Van Steensel, and M. Vikkula, Mutations in the transcription factor gene SOX18 underlie recessive and dominant forms of hypotrichosis-lymphedema-telangiectasia. Am J Hum Genet, 2003. 72(6): p. 1470-8. PMID: 12740761; PMCID: PMC1180307

3. Fang, J., S.L. Dagenais, R.P. Erickson, M.F. Arlt, M.W. Glynn, J.L. Gorski, L.H. Seaver, and T.W. Glover, Mutations in FOXC2 (MFH-1), a forkhead family transcription factor, are responsible for the hereditary lymphedema-distichiasis syndrome. Am J Hum Genet, 2000. 67(6): p. 1382-8. PMID: 11078474; PMCID: PMC1287915

4. Brice, G., S. Mansour, R. Bell, J.R. Collin, A.H. Child, A.F. Brady, M. Sarfarazi, K.G. Burnand, S. Jeffery, P. Mortimer, and V.A. Murday, Analysis of the phenotypic abnormalities in lymphoedema-distichiasis syndrome in 74 patients with FOXC2 mutations or linkage to 16q24. J Med Genet, 2002. 39(7): p. 478-83. PMID: 12114478; PMCID: PMC1735188

5. Kriederman, B.M., T.L. Myloyde, M.H. Witte, S.L. Dagenais, C.L. Witte, M. Rennels, M.J. Bernas, M.T. Lynch, R.P. Erickson, M.S. Caulder, N. Miura, D. Jackson, B.P. Brooks, and T.W. Glover, FOXC2 haploinsufficient mice are a model for human autosomal dominant lymphedema-distichiasis syndrome. Hum Mol Genet, 2003. 12(10): p. 1179-85. PMID: 12719382

6. Ferrell RE, Kimak MA, Lawrence EC, Finegold DN, “Candidate gene analysis in primary lymphedema,” Lymphat Res. Biol., 6(2): 69-76, 2008.

7. Oremus, M., Diagnosis and treatment of secondary lymphedema. 2009, Medicare Evidence Development & Coverage Advisory Committee meeting, Centers for Medicare & Medicaid, 7500 Security Boulevard, Baltimore, MD 21244 USA, https://www.cms.hhs.gov/mcd/viewmcac.asp?where=index&mid=51.

8. Rasmussen, J.C., Tan, I-C., Marshall, M.V., Fife, C.E., and E.M. Sevick-Muraca, “Lymphatic imaging in humans with near-infrared fluorescence,” Current Opinion in Biotechnology, 20: 1-9, 2009. PMID: 19233639; PMCID: PMC2692490

9. Rasmussen J.C., Tan, I-C, Marshall, M.V., Adams, K.E., Kwon, S., Fife, C.E., Maus, E.A., Smith, L., Covington, K.R., and E.M. Sevick-Muraca, “Human lymphatic architecture and (dys)function imaged using NIR fluorescence,” Translational Oncology, in press.

10. Adams, K.E., Rasmussen, J.C., Darne, C., Tan, I-C., Aldrich, M.B., Marshall, M.V., Fife, C.E., Maus, E.A., Smith, L.A., Guilloid, R., Hoy, S., and E.M. Sevick-Muraca, “A method to assess lymphatic response to advanced pneumatic compression device treatment of lymphedema,” Biomedical Optics Express, 1, 114-125, 2010.

11. Maus, E.A., Tan, I-C., Rasmussen, J.C., Marshall, M.V., Fife, C.E., Smith, L.A., and E.M. Sevick-Muraca, “Near-infrared fluorescence lymphatic imaging of head and neck lymphedema,” Head and Neck, in press.

12. Marshall, M.V., Rasmussen, J.C., Tan, I-C., Aldrich, M.B., Adams, K.E., Wang, X., Fife, C.E., Maus, E.A., Smith, L.A., and E.M. Sevick-Muraca,” Near-infrared fluorescence imaging in humans with indocyanine green: a review and update,” The Open Surgical Oncology Journal, 2: 12-25, 2010.

13. Tan, I-C., Maus, E.A., Rasmussen, J.C., Marshall, M.V., Fife, C.E., Smith, L.A., and E.M. Sevick-Muraca, “NIR fluorescence imaging of improved lymphatic propulsion and transport following manual lymphatic drainage,” in review.