Mapping of the superficial lymphatic network of the breast using indocyanine green injection: an anatomical study

doi:10.21203/rs.3.rs-2251230/v1

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Mapping of the superficial lymphatic network of the breast using indocyanine green injection: an anatomical study

https://doi.org/10.21203/rs.3.rs-2251230/v1

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Purpose

Indocyanine green (ICG) is a fluorophore that allows exploration of the arterial and lymphatic network. The superficial lymphatic network of the breast is particularly vulnerable in breast augmentation surgery. The intraoperative use of ICG could help the plastic surgeon during breast augmentation surgery. The aim of this work is to demonstrate the feasibility of mapping the superficial breast lymphatic network using ICG.

Methods

This is an anatomical study. A peri-areolar injection of indocyanine green was performed on cadaveric subjects. At the end, a mapping of their spatial distribution was carried out according to four quadrants, delimited by two perpendicular lines passing by the areola.

Results

We performed 60 breast injections. The lymphatic network could be identified in 53 of them. A total of 80 vessels were found in the superior-external quadrant, 17 in the superior-internal quadrant, 28 in the inferior-external quadrant and 0 in the inferior-internal quadrant.

Conclusion

This work shows that indocyanine green allows the identification of the superficial lymphatic network of the breast. Its use could represent an intraoperative assistance in breast augmentation surgery. This work also confirms the existing anatomical notions with a predominance of the axillary drainage route.

Indocyanine green

Breast anatomy

Breast lymphatic network

Anatomical study

Breast augmentation

Breast prosthesis

The lymphatic network of the breast is complex; it was described for the first time by Sappey in the 19th century, using ammonium carminate [1]. Since then, new methods of exploration have emerged and proven their effectiveness: lymphography, Patent blue colorimetry, lymphoscintigraphy, and lymphofluoroscopy using indocyanine green (ICG)[2]. ICG is a non radioactive water-soluble fluorophore, approved for clinical use in multiple cases such as retinal vascularization and flap perfusion. When injected, ICG rapidly bounds with plasma proteins with an excitation and emission peak of 780–820 nm when coupled with near infrared imaging [3]. Its use for the exploration of the lymphatic system has widened in the last 10 years, including for sentinel node research in breast and head and neck cancers [4–8].

Several anatomical studies describe the lymphatic distribution of the breast [9–12]. Classically, four areas are described for lymphatic drainage: axillary, internal mammary, supra- and infra-clavicular. The consensus today is that the main drainage of the breast is the axillary zone [1, 11–13]. Although no consensus has been reached yet, the lymphatic distribution of the breast is usually considered to consist of two networks: superficial and deep, interconnecting at the subareolar level. From this subareolar plexus, lymphatic channels provide transport to the main draining lymph nodes [9, 10, 14].

Although the physical properties of ICG may not allow for the identification of vascular structures deeper than 2 cm from the skin surface [15–20], ICG might be of interest to help identifying the superficial lymphatic network of the breast and axillary sentinel nodes. Unlike lymphoscintigraphy, ICG does not require any radiation exposure, and does not cause blue staining of the skin as reported with patent blue.

The ability to highlight the superficial lymphatic network of the breast could prove to be a valuable aid in plastic surgery, as the placement of breast implants using an axillary approach implies a subcutaneous dissection near this superficial network. Thus, the risk of iatrogenic injury of the lymphatic system is higher and this approach can prove to be detrimental to the detection of the sentinel lymph node for the later management of breast cancer. The use of indocyanine green intraoperatively could be valuable to help plastic surgeons identify lymphatic vessels in the axillary area and therefore better preserve these structures.

The purpose of this work is to study the feasibility of mapping the superficial lymphatic network of the breast using ICG.

This is an observational anatomical study of the diffusion of ICG in the breast after injection at the areolar level in fresh and fresh-frozen female and male anatomic specimens.

The excitation and detection of the fluorophore was performed by the Fluobeam® infrared camera from the Fluoptics laboratory. The excitation spectrum of the camera was 750 nm, while the emission spectrum was 800 nm.

This study included 30 anatomical specimens, 20 females and 10 males, and resulted in 60 injections.

Method

Twenty five milligrams of ICG were diluted in 10 ml of 5% glucose solution, and injected subcutaneously. The point of injection was performed 2 cm from the nipple and on 4 points at 12 o'clock, 3 o'clock, 6 o'clock and 9 o'clock (Figs. 1 and 2). Specimens were in supine position.

After ICG injection, a passive diffusion time of 10 minutes was observed, before massaging of the injection area was performed. The massage manipulation was performed continuously for 5 minutes and in a clockwise direction.

Long line diffusion of the fluorophore from the injection site was identified as the presence of a lymphatic vessel.

Four quadrants were identified in each breast: superior-internal (internal mammary), superior-external (axillary), inferior-internal and inferior-external. Each vessel found was counted and mapped accordingly to the sector in which it was identified. This analysis of the spatial distribution of the vessels was carried out by two successive and independent observers, on the videos recorded during the experiment with the Fluobeam camera.

If no such image was visualized, the result was considered to be a manipulation failure, leading to the exclusion of the subject.

This study included 30 anatomical specimens, 20 females and 10 males, and resulted in 60 injections. There were 53 lymphatic networks identified (Fig. 3) and 7 exclusions due to lack of evidence of the lymphatic network (Fig. 4).

In total, the average spatial distribution of the dye after massaging distributes as such (Table 1):

Table 1: Spatial distribution of lymphatic vessels

- Upper-lateral quadrant (axillary): 80 vessels (63.9%) (Fig. 5)

- Upper-medial quadrant (internal mammary): 17 vessels (13.9%)

- Lower-lateral quadrant: 28 vessels (22.2%)

- Lower-medial quadrant: 0 vessels

The aim of our study was to establish the usefulness of ICG for mapping the superficial lymphatic network of the breast. The results of our work, which shows lymphatic diffusion of ICG in 53 of the 60 injections performed, demonstrated the feasibility of identifying mammary lymphatic vessels using ICG injections.

We assumed that the images obtained corresponded to the lymphatic network and not to the arteriovenous system because of previous anatomical studies showing that the latter does not overlap with the lymphatic network [21–24]. Thus, the mapping of the mammary arteriovenous system would be significantly different from the lymphatic system and would not produce superimposable images [25–27].

ICG is now a common tool for the exploration of the arteriovenous system [28–30]. Its use for lymphatic network exploration benefits from the cadaveric study of Shinaoka et al [31] who mapped the lymphatic network of the limbs after subcutaneous injection of the dye in the hands and feet. This anatomical study was then confirmed in vivo by the work of Unno et al. [32, 33]. However, ICG has a low radiation intensity which limits its use for the detection of deep lymphatic vessels because infrared cameras cannot detect radiation deeper than 2 cm [32]. The results obtained in this work are therefore limited to the superficial lymphatic network. Subsequently, the use of ICG seems to be of less interest in obese subjects or in areas with a large amount of fat such as the breast. Therefore, it doesn’t have any use for the detection of deep lymph nodes when searching for the sentinel lymph node.

In order to overcome these limitations, Yamashita et al suggest to apply a transparent material to the study area to enhance the detection of radiation by vitro pressure [34]. Schaafsma et al. suggest the combination of ICG and technetium to form a hybrid tracer providing greater spatial resolution [8].

We chose to include both male and female subjects in this study. Although anatomical studies of the male breast are rare in the literature, they show that the vascularity of the breast is similar between men and women [26, 35]. In addition, axillary prosthetic breast augmentation surgery is equally applicable to women for cosmetic purposes as it is to men during sex reassignment surgery. The topographical analysis of the superficial lymphatic vessels revealed by the ICG injection shows that they are predominantly distributed in the superior-external quadrant, corresponding to the axillary zone. This analysis is consistent with previous anatomical studies of the mammary lymphatic network [1, 11, 14, 36]. Previous anatomical studies describe two lymphatic networks in the breast: superficial and deep [10, 11, 14]. Our findings strongly suggest the predominance of the superficial lymphatic network of the breast, which seems to be the main contributor to lymphatic drainage to the axillary lymphnode, confirming Sappey’s findings. Sappey described a primary centripetal lymphatic drainage to the subareolar plexus via the deep network, then, secondarily, the lymph travels to the superficial network to reach the axillary lymph nodes [36, 37].

We noticed during the experiments that the vessels belonging to the upper external quadrant run in the direction of the axillary fossa, without actually ever reaching it.

This phenomenon confirms various studies [38–41] reporting an anatomical zone located behind the lateral edge of the pectoralis major, near the lower pole of the axillary fossa, where the lymph nodes responsible for breast drainage are concentrated: the "Soft Tissue Triangle" (Fig. 6 and Fig. 7). This area is located at a distance from the axillary approach, which is used for prosthetic augmentation.

As this is an original study of mammary lymphatic circulation in the cadaveric subject, there is no previous work regarding the optimal amount to be injected. We based our study on the results of Murawa et al [6] who showed that the level of fluorescence obtained was directly correlated to the dose injected. Therefore, as this was a cadaveric study, we chose to inject a whole dose of infracyanin, i.e. 25 mg of product, in order to potentiate the fluorescence capabilities of the ICG. This amount may need to be adjusted for in vivo lymph nodes detection.

The study of lymphatic circulation in anatomical subjects suffers from an important limitation related to the absence of tissue perfusion pressure. In vivo, lymph flow through vessels is governed by both intrinsic factor, the spontaneous contractility of endothelial cells and extrinsic factors: muscle contraction, perfusion pressure, respiratory amplitude for thoracic lymph vessels [42]. We based our protocol on various previous works using massage to enhance ICG diffusion in cadavers [17, 18, 31, 33] and described passive diffusion time after dye injection during which the diffusion of ICG within the lymphatic vessels was related solely to the mechanical pressure induced by the syringe. Massaging of the injection site was performed afterwards to theoretically reproduce the effect exerted by the cardiac pump under physiological conditions.

However, several limitations to this study should be noted. Amongst the 60 breast injections performed, only 53 were usable. There was some heterogeneity among the subjects. The sex ratio was unbalanced (10 men and 20 women), and the state of preservation was not identical amongst all subjects. The study included only 8 "fresh" subjects, i.e. deceased for less than 72 hours and never frozen.

The 7 excluded breasts in which it was not possible to detect lymphatic vessels were characterized by the formation of a diffusion "cloud" at the dye injection site (Fig. 4). Two hypothesis can be proposed: a degradation of the mammary lymphatic network in the cadaver or a handling mistake during the injection leading to a deeper penetration of the dye not allowing its absorption by the subareolar plexus of the anatomical subjects.

Interestingly, one of the excluded breasts belonged to a subject in which a satisfactory result was obtained on the contralateral breast, inciting us not to neglect the possibility of a handling mistake.

The value of ICG for the study of skin perfusion is now well recognized and its use for the study of lymphatic vessels is in full development. Our work shows that ICG is an effective tracer for mapping the superficial lymphatic network of the breast and our results regarding lymphatic networks of the breast are consistent with those of the existing literature. Its intraoperative use in breast augmentation surgery would allow for better control of subcutaneous dissection and help preserve the integrity of the lymphatic network. However, the inherent limitations of any cadaveric study do not allow us to extrapolate these results to the living. An in vivo study will be required to confirm these results.

The authors have no personal, Financial or institutional interest in any of the drugs, materials or devices describes in this article

Acknowledgments

The authors would like to thank the people who donated their bodies for making this study possible and for improving medical knowledge.

Ethical Approval :

Not applicable

Competing interests :

The authors declare that they have no ties of interest.

Authors contributions :

1 : Y CLAUDIC : Data collection, writing and protocol development

2 : A PERRUISSEAU-CARRIER : Manuscrit editing

3 : Charlie DEMARTELEIRE : Manuscrit editing

4 : Weiguo HU : Manuscrit editing and protocol development

5 : Romuald SEIZEUR : Manuscrit editing and protocol development

Funding :

Not applicable

Availability of data and materials

The data are available upon request to the corresponding author

Salmon RJ, Montemagno S, Laki F, Alran S, Charitansky H, Fourchotte V, et al. Réseaux lymphatiques de la glande mammaire : l’identification du ganglion sentinelle revue à la lumière des anciens anatomistes. Journal de Chirurgie 2007;144:72–4. https://doi.org/10.1016/S0021-7697(07)89473-7.
Riquet M, Becker C, Mordant P, Arrivé L. Explorations du système lymphatique : épreuve au bleu, lymphographies directes, lymphoscintigraphies et autres méthodes 2019:13.
Benson RC, Kues HA. Fluorescence properties of indocyanine green as related to angiography. Phys Med Biol 1978;23:159–63. https://doi.org/10.1088/0031-9155/23/1/017.
Struk S, Honart J-F, Qassemyar Q, Leymarie N, Sarfati B, Alkhashnam H, et al. Utilisation du vert d’indocyanine en chirurgie sénologique et reconstruction mammaire. Annales de Chirurgie Plastique Esthétique 2018;63:54–61. https://doi.org/10.1016/j.anplas.2017.09.008.
Verbeek FPR, Troyan SL, Mieog JSD, Liefers G-J, Moffitt LA, Rosenberg M, et al. Near-infrared fluorescence sentinel lymph node mapping in breast cancer: a multicenter experience. Breast Cancer Res Treat 2014;143:333–42. https://doi.org/10.1007/s10549-013-2802-9.
Murawa D, Hirche C, Dresel S, Hünerbein M. Sentinel lymph node biopsy in breast cancer guided by indocyanine green fluorescence. British Journal of Surgery 2009;96:1289–94. https://doi.org/10.1002/bjs.6721.
Guo J, Yang H, Wang S, Cao Y, Liu M, Xie F, et al. Comparison of sentinel lymph node biopsy guided by indocyanine green, blue dye, and their combination in breast cancer patients: a prospective cohort study. World J Surg Onc 2017;15:196. https://doi.org/10.1186/s12957-017-1264-7.
Schaafsma BE, Verbeek FPR, Rietbergen DDD, Hiel B, Vorst JR, Liefers GJ, et al. Clinical trial of combined radio- and fluorescence-guided sentinel lymph node biopsy in breast cancer. British Journal of Surgery 2013;100:1037–44. https://doi.org/10.1002/bjs.9159.
Pan W-R, Rozen WM, Stella DL, Ashton MW. A Three-Dimensional Analysis of the Lymphatics of a Bilateral Breast Specimen: A Human Cadaveric Study. Clinical Breast Cancer 2009;9:86–91. https://doi.org/10.3816/CBC.2009.n.016.
Olivier J-B, Verhaeghe J-L, Butarelli M, Marchal F, Houvenaeghel G. Anatomie fonctionnelle du drainage lymphatique du sein : apport de la technique du lymphonœud sentinelle. Annales de Chirurgie 2006;131:608–15. https://doi.org/10.1016/j.anchir.2006.06.011.
Suami H, Pan W-R, Taylor GI. Historical review of breast lymphatic studies. Clin Anat 2009;22:531–6. https://doi.org/10.1002/ca.20812.
Fowler JC, Solanki CK, Ballinger JR, Ravichandran D, Douglas-Jones A, Lawrence D, et al. Axillary Lymph Node Drainage Pathways from Intradermal and Intraparenchymal Breast Planes. Journal of Surgical Research 2010;161:69–75. https://doi.org/10.1016/j.jss.2009.01.003.
Turner-Warwick RT. The lymphatics of the breast. British Journal of Surgery 2005;46:574–82. https://doi.org/10.1002/bjs.18004620004.
Suami H, Pan W-R, Mann GB, Taylor GI. The Lymphatic Anatomy of the Breast and its Implications for Sentinel Lymph Node Biopsy: A Human Cadaver Study. Ann Surg Oncol 2008;15:863–71. https://doi.org/10.1245/s10434-007-9709-9.
Desmettre T. Particularités métaboliques de la fluorescéine. J Fr Ophtalmol n.d.:15.
Weiler M, Kassis T, Dixon JB. Sensitivity analysis of near-infrared functional lymphatic imaging. J Biomed Opt 2012;17:066019. https://doi.org/10.1117/1.JBO.17.6.066019.
Groenlund JH, Telinius N, Skov SN, Hjortdal V. A Validation Study of Near-Infrared Fluorescence Imaging of Lymphatic Vessels in Humans. Lymphatic Research and Biology 2017;15:227–34. https://doi.org/10.1089/lrb.2016.0061.
Yamamoto T, Narushima M, Yoshimatsu H, Yamamoto N, Oka A, Seki Y, et al. Indocyanine Green Velocity: Lymph Transportation Capacity Deterioration With Progression of Lymphedema. Annals of Plastic Surgery 2013;71:591–4. https://doi.org/10.1097/SAP.0b013e318255168a.
Gashev AA, Nagai T, Bridenbaugh EA. Indocyanine Green and Lymphatic Imaging: Current Problems. Lymphatic Research and Biology 2010;8:127–30. https://doi.org/10.1089/lrb.2010.0005.
Ogata F, Azuma R, Kikuchi M, Koshima I, Morimoto Y. Novel Lymphography Using Indocyanine Green Dye for Near-Infrared Fluorescence Labeling. Annals of Plastic Surgery 2007;58:652–5. https://doi.org/10.1097/01.sap.0000250896.42800.a2.
van Deventer PV, Page BJ, Graewe FR. Vascular Anatomy of the Breast and Nipple-Areola Complex: Plastic and Reconstructive Surgery 2008;121:1860–1. https://doi.org/10.1097/PRS.0b013e31816b14d5.
Ellis H, Colborn GL, Skandalakis JE. Surgical Embryology and Anatomy of the Breast and its Related Anatomic Structures. Surgical Clinics of North America 1993;73:611–32. https://doi.org/10.1016/S0039-6109(16)46077-9.
Cunningham L. The anatomy of the arteries and veins of the breast. J Surg Oncol 1977;9:71–85. https://doi.org/10.1002/jso.2930090112.
Corduff N, Rozen WM, Taylor GI. The superficial venous drainage of the breast: A clinical study and implications for breast reduction surgery. Journal of Plastic, Reconstructive & Aesthetic Surgery 2010;63:809–13. https://doi.org/10.1016/j.bjps.2009.02.055.
Andres A-C, Djonov V. The Mammary Gland Vasculature Revisited. J Mammary Gland Biol Neoplasia 2010;15:319–28. https://doi.org/10.1007/s10911-010-9186-9.
Ding N, Yu N, Dong R, Kong L, Xue H, Long X, et al. Blood supply of the male breast nipple-areola complex evaluated by CTA. Journal of Plastic, Reconstructive & Aesthetic Surgery 2021;74:2588–95. https://doi.org/10.1016/j.bjps.2021.02.006.
Orguc S, Basara I, Coskun T, Pekindil G. 3D vascular mapping of breast using contrast enhanced mri: association of unilateral increased vascularity with ipsilateral breast cancer. Diagn Interv Radiol 2012. https://doi.org/10.4261/1305-3825.DIR.5280-11.2.
Blondeel PN. One hundred free DIEP flap breast reconstructions: a personal experience. British Journal of Plastic Surgery 1999;52:104–11. https://doi.org/10.1054/bjps.1998.3033.
Mattison GL, Lewis PG, Gupta SC, Kim HY. SPY Imaging Use in Postmastectomy Breast Reconstruction Patients: Preventative or Overly Conservative? Plastic and Reconstructive Surgery 2016;138:15e–21e. https://doi.org/10.1097/PRS.0000000000002266.
Holm C, Dornseifer U, Sturtz G, Ninkovic M. Sensitivity and Specificity of ICG Angiography in Free Flap Reexploration. J Reconstr Microsurg 2010;26:311–6. https://doi.org/10.1055/s-0030-1249314.
Shinaoka A, Koshimune S, Yamada K, Kumagishi K, Suami H, Kimata Y, et al. A Fresh Cadaver Study on Indocyanine Green Fluorescence Lymphography: A New Whole-Body Imaging Technique for Investigating the Superficial Lymphatics. Plastic and Reconstructive Surgery 2018;141:1161–4. https://doi.org/10.1097/PRS.0000000000004315.
Unno N, Nishiyama M, Suzuki M, Yamamoto N, Inuzuka K, Sagara D, et al. Quantitative Lymph Imaging for Assessment of Lymph Function using Indocyanine Green Fluorescence Lymphography. European Journal of Vascular and Endovascular Surgery 2008;36:230–6. https://doi.org/10.1016/j.ejvs.2008.04.013.
Unno N, Nishiyama M, Suzuki M, Tanaka H, Yamamoto N, Sagara D, et al. A novel method of measuring human lymphatic pumping using indocyanine green fluorescence lymphography. Journal of Vascular Surgery 2010;52:946–52. https://doi.org/10.1016/j.jvs.2010.04.067.
Yamashita M, Yamamoto T, Yamamoto N, Furuya M, Ishiura R, Hayashi A. Diascopic indocyanine green lymphography for deep lymphatic visualization. Journal of Plastic, Reconstructive & Aesthetic Surgery 2014;67:e293–4. https://doi.org/10.1016/j.bjps.2014.07.029.
Andersen JA, Gram JB. MALE BREAST AT AUTOPSY. Acta Pathologica Microbiologica Scandinavica Series A :Pathology 2009;90A:191–7. https://doi.org/10.1111/j.1699-0463.1982.tb00081_90A.x.
Sappey C. Description et iconographie des vaisseaux lymphatiques, considérés chez l’homme et les vertébrés. Paris, Delahaye. 1885.
Sappey MPC. Anatomie, physiologie, pathologie des vaisseaux lymphatiques considérés chez l’homme et les vertébrés. Adrien Delahaye. 1874.
Mottura AA, Del Castillo R. Transaxillary Breast Augmentation: Two Breast Cancer Patients with Successful Sentinel Lymph Node Diagnosis. Aesth Plast Surg 2007;31:544–9. https://doi.org/10.1007/s00266-006-0260-z.
Huang G. Sentinel lymph node biopsy in the augmented breast: Role of the transaxillary subpectoral approach. Aesthetic Surgery Journal 2003;23:184–7. https://doi.org/10.1067/maj.2003.38.
Graf RM, Canan LW, Romano GG, Tolazzi ARD, Cruz GA de O e. Implications of Transaxillary Breast Augmentation: Lifetime Probability for the Development of Breast Cancer and Sentinel Node Mapping Interference. Aesth Plast Surg 2007;31:322–4. https://doi.org/10.1007/s00266-006-0204-7.
Sado HN, Graf RM, Canan LW, Romano GG, Timi JR, Matias JE, et al. Sentinel Lymph Node Detection and Evidence of Axillary Lymphatic Integrity After Transaxillary Breast Augmentation: A Prospective Study Using Lymphoscintography. Aesth Plast Surg 2008;32:879–88. https://doi.org/10.1007/s00266-008-9212-0.
Riquet M. Physiologie de la circulation lymphatique 2019:12.

No competing interests reported.

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Mapping of the superficial lymphatic network of the breast using indocyanine green injection: an anatomical study

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