Long-Term Follow-Up of Collateral Pathways Established After Lymphadenectomy in Rats

131 Lymphology 50 (2017) 131-135 LONG-TERM FOLLOW-UP OF COLLATERAL PATHWAYS ESTABLISHED AFTER LYMPHADENECTOMY IN RATS Y. Takeno, H. Arita, C. Oshima, A. Mawaki, K. Nakanishi, F. Kurono, E. Fujimoto Department of Nursing (YT,CO,AM,KN,FK,EF), Graduate School of Medicine, Nagoya University, Aichi, and Faculty of Nursing and Social Welfare Sciences (HA), Fukui Prefectural University, Fukui, Japan ABSTRACT Keywords: lymphedema, ICG, lymphography, collateral pathways, rat, long-term follow-up, A collateral pathway established after lymphadenectomy lymphadenectomy could play an important role in long-term lymphedema treatment. Secondary lymphedema is one of the The present study investigated alterations major complications after cancer treatment of lymph dynamics 1 year after lymphadenec- particularly following surgery and radiation tomy using indocyanine green fluorescent therapy for breast and gynecological cancers. lymphography to determine if a collateral Once secondary lymphedema occurs, daily pathway may be used for long-term care for an edematous limb and the skin is lymphedema treatment. Wistar rats were necessary for a lifetime. Secondary lymph- anaesthetized and lymph nodes were excised edema is chronic and non-curable, and at the inguinal and popliteal fossae. The protein-rich fluid accumulated in the inter- treated hind limb was evaluated by fluorescent stitial space could further cause adipose lymphography between 10 weeks and 6 deposition and tissue fibrosis (1). Moreover, months and between 6 months and 1 year the lymph vessels become fibrotic and lose postoperatively. Fluorescent lymphography their normal permeability in the chronic demonstrated a lymphatic pathway to the stage (2). ipsilateral axillary fossa in all rats 1 year after Lymphedema models using animals are lymphadenectomy. Some capillary branches fundamental to understand lymphatic in the paths leading to the ipsilateral axillary mechanisms both in normal and lymphedema fossa were dilated and tortuous. In addition, and could also serve to substitute for clinical areas in which a fluorescent signal was not research. These models can particularly play visible were increased in the thigh. In conclu- an important role in histologic and molecular sion, the collateral pathway did not appear to studies. Although lymphatic flow patterns be only for temporary use to compensate for using laboratory animals have been visualized drainage from the edematous limb but appears in previous studies (3,4), alterations of the more stable as a component of a compensating flow patterns after lymphadenectomy are not lymphatic system. These new dilated vessels, well understood or studied. although functional at this point, may still be We previously produced lymphedema in susceptible to disturbance by further altera- the rat hind limb similar to human lymph- tion to the lymph vessels. edema and elucidated alterations of lymph Permission granted for single print for individual use. Reproduction not permitted with permission of Journal LYMPHOLOGY. 132 flow after inguinal lymph node (LN) dissec- fluorescent images were made between week tion for 10 weeks by indocyanine green (ICG) 10 and 6 months, and between 6 months and fluorescent lymphography. The result showed year 1 after surgery. Indocyanine green that a network-like pattern of the fluorescent lymphography was performed by the same signal interpreted as superficial lymphatics protocol as for surgery. The rat was placed around the incision line appeared within supine for the video recording, and the lens three weeks after loss of original lymphatic of PDE was set to capture an image from the routes in the hind limb and remained until axilla to the hind toes of the rats. All week 10 postoperatively (5). This network conditions of the experimental room and connected to a collecting lymph vessel excitation light emitted from PDE were displayed as a linear signal reaching to the controlled. Fluorescent images obtained by ipsilateral axillary fossa. At the same time, PDE were video-recorded on digital format. swelling gradually decreased. This finding means that escape routes connecting to RESULTS nearby lymph nodes through superficial lymphatics are necessary to prevent or mini- From Week 10 to 6 Months mize edema. The present study sought to determine whether this pathway could be Six months after LNs excision inguinally used for long-term lymphedema control and and popliteally, ICG lymphography shows a investigated alterations of lymph dynamics linear route connecting to the ipsilateral after lymphadenectomy using ICG axillary fossa in all rats, and some rats have fluorescent lymphography for one year. a second route to the contralateral inguinal fossa. There is no new route to other parts of METHODS the body established between week 10 and 6 months post-surgery, and the routes for each The protocol to create hind limb rat has are identical to those observed 10 lymphedema using a rat has been described weeks after surgery. previously (5) and the experiments approved In all rats, a network-like pattern of by the Animal Experiment Committee of fluorescent signals at the treated thigh, the Nagoya University. Briefly, nine male Wistar incision line, and the lower abdomen appears rats were anesthetized with isoflurane to be almost the same as observed at week 10. inhalation, and 0.2 ml of a 1 mg/ml solution Furthermore, all rats have no sign of diffuse of ICG was injected subcutaneously into the or spotted fluorescent signals known as dorsum of the hindpaw and the medial and dermal backflow. Compared to images taken the lateral ankle to detect lymph nodes and at week 10, some segments without any lymph vessels. After circumferential incision fluorescent signals are observed in common. at the right groin, fluorescent lymph nodes (LNs) were excised at the inguinal fossa and From 6 Months to 1 Year the popliteal fossa, and fluorescent lymph vessels were ligated under optical imaging. One year after LNs excision, ICG The skin edges were then sutured end to end. lymphography shows a route to the ipsilateral axillary fossa in all rats, and some have an Evaluation of Limb Edema additional route to the contralateral inguinal fossa. There is no new route to other areas The treated hind limb was evaluated by created between 6 months and year 1 post- ICG lymphography from Day 3 post-surgery surgery, and the routes for each rat remain and one year thereafter at the specific the same as those observed 6 months after periods of time. These time comparisons of surgery. Permission granted for single print for individual use. Reproduction not permitted with permission of Journal LYMPHOLOGY. 133 Fig. 1. Collateral pathways from the right hindlimb both to the ipsilateral axillary fossa and to the contralateral inguinal fossa established after lymphadenectomy at the inguinal fossa are depicted by ICG lymphography after (a) 10 weeks, (b) 6 months and (c) 1 year following operation. No differences were observed between (a) and (b) but network signal decrease (white arrow) and some branches became dilated and torturous (black arrow) in (c). Compared to images taken at 6 months, completely within 10 weeks after surgery. a decrease of network-like signals which were Thus, it is suggested that these routes are not expanded at the thigh, the incision line, and only for temporary use to compensate for the lower abdomen is observed. Some drainage from the edematous limb but may capillary branches disappear or blur and in function as a reconstituted lymphatic system. their place dilated and tortuous fluorescent Not all patients who have risk factors for branches are clearly observed in the paths to lymphedema develop it in humans. It is the ipsilateral axillary and the contralateral considered that those who do not develop it inguinal fossa. There are enlarged segments could have such collateral pathways from the followed by narrow segments on the branches limb that possibly develop lymphedema, and all over the path. In addition, areas in which this study provides some evidence for this a fluorescent signal is invisible increase at theory. Generally, superficial lymphatic the thigh as compared to the incision line and anastomosis is blocked between watersheds the lower abdomen (Fig. 1). on umbilical and midline areas. However, under conditions of increased intralymphatic DISCUSSION pressure, the flow of the lymph across the watersheds from one territory to another is The present study revealed that collateral facilitated (6). Akita reported an inter- routes to the ipsilateral and contralateral territorial pathway from the edematous axillary fossa developed from the treated hind limb to the axilla in a patient with chronic limb following LN excision in the inguinal lymphedema using scintigraphy (7). In fossa after the first postoperative year. In varicose veins, venous valves fail to function addition, dermal backflow signs were not and lead to significant reflux under venous observed during this period. According to hypertension (8). As with lymphatics, Takeno and Fujimoto, collateral pathways to excessive intralymphatic pressure could cause other LNs have been established within three valve dysfunction and lead to lymphatic weeks after LNs excision at the groin and anastomosis between watersheds. Our the popliteal fossa (5). After the completion findings show that network signals, i.e., of the pathways, dermal backflow signs lymph capillaries, bridge over the incision gradually decreased and then disappeared line from the treated hindlimb to lymph Permission

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