Lymphatic Reconnection and Restoration of Lymphatic Flow by Nonvascularized Lymph Node Transplantation: Real-Time Fluorescence Imaging Using Indocyanine Green and Fluorescein Isothiocyanate

Abstract

Background:

Lymph node transplantation is being increasingly recognized as a method of reconstruction of the lymphatic system for the treatment of lymphedema. However, the mechanisms regulating the connections between the lymphatic vessels and the lymph nodes remain poorly understood. The objective of this study was to examine whether removal of a popliteal lymph node (PLN) could result in rerouting of lymph flow and whether a single transplanted nonvascularized lymph node could reconnect with a lymphatic vessel and restore lymph flow.

Methods and Results:

Thirty-six mice were allocated to undergo resection of a PLN alone (group 1) or a transplanted lymph node after resection of a PLN (group 2). Changes in lymph flow were observed using indocyanine green dye. The ability of the transplanted lymph node to reconnect with the lymphatic vessels was examined by fluorescein isothiocyanate (FITC)–dextran and immunohistochemical staining. In group 1, the flow of lymph was redirected to an inguinal lymph node (ILN) in 8 of 18 mice and continued to drain to the PLN in 10 mice. In group 2, the lymph continued to drain normally after removal of the PLN, and was also directed to an ILN in two mice. FITC–dextran demonstrated continuity of the transplanted PLN and the lymphatic vessels. Immunohistochemical staining showed that T cell and B cell populations in the transplanted lymph node were preserved.

Conclusion:

Lymphatic flow was rerouted after lymph node resection. A transplanted lymph node can be made viable with normal lymph flow by reconnecting the transplanted lymph node to a lymphatic vessel.

Introduction

The lymphatic system is part of the circulation and includes the lymphatic vessels, lymphoid organs of the immune system, and lymph nodes, which are encapsulated secondary lymphoid tissues. Impairment of lymphatic transport because of development of abnormal vessels or obstruction or obliteration of the lymphatic vessels causes stasis of proteins and movement of fluid into the interstitium, resulting in lymphedema.^1,2 Furthermore, lymphatic vessels also import various antigens and export immune effector cells, so impairment of lymphatic transport can also affect immune surveillance. Recently, Oashi et al. demonstrated that mice with melanoma and lymphedema have a larger burden of lung metastases.³ This finding suggests that local impairment of the lymphatic system is associated with tumor growth and metastasis.

Repair of the damaged lymphatic system is important in the treatment of lymphedema. Lymphovenous anastomosis using supermicrosurgery is a classical technique used in surgical interventions for lymphedema.^4,5 Several studies have demonstrated the usefulness of this method in early-stage lymphedema.^6,7 Recently, lymph node transplantation was introduced as a physiological method for local surgical repair of the lymphatic system.^5,8 Clinical studies have suggested that transplantation of lymph nodes from an unaffected area to the lymphedematous limb may improve lymphatic function.^9,10 Details of the mechanisms by which lymph node transfer can restore lymphatic function have been examined in animal studies. In a study by Tobbia et al., systemic uptake of peripherally injected iodine-125 confirmed reconnection of lymphatic channels after microsurgical transfer of lymph nodes after lymphadenectomy in sheep.¹¹ Furthermore, using technetium-99m uptake in LacZ lymphatic reporter mice, Aschen et al. demonstrated that a transplanted nonvascularized lymph node could reconnect with lymphatic vessels.¹² However, although lymph node transplantation has been shown to improve lymphatic function, the precise mechanisms involved in reconnection of lymph nodes with lymphatic vessels and the actual functional status of the nodes remain unclear.

Tumor cells metastasize through the lymphatic vessels to the sentinel lymph nodes. The sentinel lymph nodes are the first nodes to receive lymph from a primary tumor and are the most common site of initial tumor metastasis.¹³ Sentinel lymph node biopsy is the standard approach used to determine the status of the regional lymph nodes in patients with melanoma and to monitor the outcome of treatment.¹⁴ Using noninvasive near-infrared fluorescence imaging in a mouse model, Kwon et al. demonstrated that resection of a sentinel popliteal lymph node (PLN) was followed by changes in the lymphatic drainage pathway, and thus the pathway for dissemination of tumor cells.¹⁵ Therefore, a change in the flow of lymph may determine the degree of tumor growth and metastasis. If this is the case, restoration of lymph flow after impairment of the local lymphatic system may help to limit tumor growth and metastasis.

The aims of this study were to demonstrate rerouting of the lymphatic vessels after lymph node resection, to observe reconnection between a nonvascularized transplanted lymph node and the lymphatic vessels directly, and to investigate dynamic modulation of the restored lymph flow.

Materials and Methods

Experimental model

Thirty-six male C57BL/6N mice (age 6–8 weeks, weight 18–20 g) were purchased from Sankyo Labo Service (Tokyo, Japan). All experiments were performed under general anesthesia, which was induced and maintained by isoflurane inhalation at a flow rate of 2.5%. Five microliters of 2% patent blue dye was injected into the subcutaneous tissue of the left paw in each animal to identify the PLN and lymphatic vessels. Using sharp dissecting scissors, a 1 cm incision was then made through the skin of the thigh with the animal in the supine position. The blue dye was confirmed to be distributed in the PLNs in the biceps femoris (Fig. 1A, B). The 36 mice were allocated to undergo resection of a PLN alone (group 1) or an autotransplanted, nonvascularized, resected PLN (group 2) as shown in Figure 1C. In animals that underwent PLN transplantation (group 2), the removed PLN was then placed back into its original position (autotransplantation). In groups 1 and 2, the lymphatic vessels were left unsutured and the incised biceps femoris was sutured with 10-0 nylon (Fig. 1D). These procedures were performed using a microsurgical technique under a microscope. The incised skin on the thigh was sutured with 5-0 nylon. Four weeks after surgery, the mice were euthanized and the tissues were collected for immunohistochemical staining. The protocols used in this study were approved by the Institutional Animal Care and Use Committee, Hokkaido University School of Medicine, and conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals, Hokkaido University.

FIG. 1.

Surgical procedure used in this study. (A) The PLN was identified in the biceps femoris in the supine position. The size of the PLN was about 1–2 mm. (B) Enlarged image of the PLN. (C) The tissue surrounding the PLN was excised along the surface of the lymph node. The PLN was then removed. (D) In animals that only underwent resection of a PLN, the biceps femoris is sutured with 10-0 nylon. In animals that underwent PLN transplantation, the removed PLN was then placed back into its original position. PLN, popliteal lymph node.

Indocyanine green fluorescence near-infrared video camera system

The lymphatic vessels were observed using a photodynamic eye (PDE) system (Hamamatsu Photonics, Hamamatsu, Japan) equipped with a charge-coupled device camera as a fluorescence detector, a low-cut filter less than 820 nm, and 760 nm light-emitting diodes as a light source to monitor emission of indocyanine green (ICG). This PDE system has been reported to be able to detect sentinel lymph nodes located ∼2 cm from the body surface.¹⁶ The fluorescence images were digitalized using a standard personal computer.

Infrared imaging

ICG solution (2 μL, 1 mg/mL) was injected subcutaneously into the foot pads of both hindlimbs in each of the anesthetized mice. Body hair was removed using depilatory cream to avoid light scattering and autofluorescence from the hair. The upper surface of each hindlimb was then observed using the PDE camera system as previously reported.⁸ Twenty minutes after the injection, fluorescence images were acquired with the mice in the supine and right lateral positions. Follow-up observations were carried out at weekly intervals for 4 weeks.

Fluorescein isothiocyanate–dextran foot pad injection and preparation of specimens

In group 2, 10 μL of 0.5 mg/mL fluorescein isothiocyanate (FITC)–dextran (molecular weight 2000 kDa; Molecular Probes, Eugene, OR) was injected subcutaneously into the footpad of each hindlimb. Dextran of this size is too large to enter the bloodstream but does enter the lymphatic vessels and is used widely in studies of the lymphatic system.^17,18 Following a previously reported method,¹⁹ the mice were fixed by perfusion with Zamboni's fixative (2% formaldehyde, 15% picric acid, 0.1 mol/L phosphate buffer, pH 6.9) through the left ventricle. After fixation, the animals were perfused with cryoprotectant (10% sucrose in phosphate-buffered saline, pH 7.2). The PLNs were then removed from both sides, embedded in tissue freezing medium, and flash frozen in partially frozen isopentane. Cryosections (20 μm thick) were obtained from each specimen for scanning and observation by confocal microscopy.

Histology

On day 28 after surgery, the PLNs in group 2 were removed on both sides microsurgically, fixed in 4% paraformaldehyde, and embedded in paraffin. Sections (4 μm thick) were then incubated with purified rat antimouse CD3 (CD3-12; GeneTex, Irvine, CA), purified rat antimouse CD45R/B220 (RA3-6B2; WuXi Biosciences, San Diego, CA), and rabbit antimouse lymphatic vessel endothelial receptor 1 (LYVE-1) antibody (Abcam, Cambridge, MA). The primary antibodies were detected using a Histofine MAX-PO kit (Nichirei Bioscience, Tokyo, Japan) followed by observation of CD3 and LYVE-1 staining using 3,3-diaminobenzidine substrate. An avidin-biotin complex kit (Elite, Vector Laboratories Burlingame, CA) was used to detect the primary antibody followed by observation of the substrate for CD45R/B220 staining by 3,3-diaminobenzidine. Verhoeff's elastic staining was used to observe the elastic fibers.

Results

Lymphatic drainage after PLN removal and after PLN implantation

Because ICG administered to the hind paw of the mouse drains to the PLN, then to the ischial, lumbar, and renal lymph nodes, and finally to the thoracic duct, we imaged mice after injection of 2 μL of ICG into both hind paws. Figures 2 –5 show fluorescent images of the ventral (Fig. 2A–D) and right lateral (Fig. 2E–H) aspects in group 1 (PLN resection only) mice (Figs. 2 and 3) and group 2 (PLN transplantation) mice (Figs. 4 and 5) at 7 (Figs. 4A, E and 5A, E), 14 (Figs. 4B, F and 5B, F), 21 (Figs. 4C, G and 5C, G), and 28 (Figs. 4D, H and 5D, H) days after removal of the PLN. The right limb that did not undergo surgery served as the control limb for each mouse.

FIG. 2.

Fluorescent images from the ventral side (A–D) and right lateral position (E, F) in an animal that underwent resection of a PLN at 7 (A, E), 14 (B, F), 21 (C, G), and 28 (D, H) days after surgery. Lymph drained into the site of the resected PLN (10/18). The area of the resected PLN did not fluoresce in the 3 weeks after surgery. The red square indicates the treated (left) side and the white arrow indicates the area from which the PLN was resected. A schema of the image from the ventral side is shown (I).

FIG. 3.

Fluorescent images from the ventral side (A–D) and right lateral position (E, F) in an animal that underwent resection of a PLN at 7 (A, E), 14 (B, F), 21 (C, G), and 28 (D, H) days after surgery. The lymph drainage was rerouted to an ILN (8/18). The red square indicates the treated (left) side and the white arrowhead and the black arrowhead indicate the ILN. A schema of the image from the ventral side is shown (I).

FIG. 4.

Fluorescent images of the ventral side (A–D) and right lateral position (E, F) in an animal that underwent transplantation of a PLN at 7 (A, E), 14 (B, F), 21 (C, G), and 28 (D, H) days after surgery. Flow of lymph to the PLN was observed for up to 28 days after removal of the PLN, indicating normalization of lymph flow (16/18). The red square indicates the treated (left) side and the white arrow indicates the PLN resection area. A schema of the image from the ventral side is shown (I).

FIG. 5.

Fluorescent images of the ventral side (A–D) and right lateral position (E, F) in an animal that underwent transplantation of a PLN at 7 (A, E), 14 (B, F), 21 (C, G), and 28 (D, H) days after surgery. Flow of lymph to the ILN as well as the PLN was observed for up to 28 days after removal of the PLN (2/18). The red square indicates the treated (left) side, the white arrow and the black arrow indicate the PLN resection area, and the white arrowhead indicates the inguinal lymph node. A schema of the image from the ventral side is shown (I).

In group 1, lymph drained to the PLN in the right (control) limb in a constant manner in all mice but drained to the PLN in the left limb in only 10 of the 18 mice. Pooling of lymph fluid was observed in group 1 at the site of the removed PLN for up to 14 days postoperatively; by day 21, the pooling had resolved and lymph was observed to be draining to an ischial lymph node (Fig. 2). In group 1, drainage of lymph was observed to be diverted to an inguinal lymph node (ILN) in the left limb in 8 of the 18 mice at 28 days postoperatively (Fig. 3). Flow of lymph to the PLN was observed for up to 28 days after removal of the PLN in the left limb in all group 2 mice, indicating normalization of lymph flow (Fig. 4). In addition, flow of lymph was also directed to an ILN in the left limb in only 2 of the 18 mice in group 2 (Fig. 5).

Reconnection of transplanted PLN and lymphatic vessels

Both afferent and efferent lymphatic vessels were observed on the caudal and cephalic sides under a microscope after injection of patent blue dye in group 2 mice (Fig. 6A–C).

FIG. 6.

Images of transplanted (A–C) PLNs. (A) An afferent lymphatic vessel is confirmed with patent blue dye on the caudal side of the implanted PLN. (B) An efferent lymphatic vessel is confirmed with patent blue dye on the cephalic side of the implanted PLN. (C) Both afferent and efferent lymphatic vessels are connected with the implanted PLN.

FITC–dextran was used to observe the continuity between the transplanted PLN and the afferent lymphatic vessel because it flows selectively into lymphatic vessels when injected subcutaneously. In a normal PLN, a thin outer layer of FITC fluorescence was observed primarily in the marginal sinuses, indicating that lymph was being received from the afferent lymphatic vessels. Most of the FITC fluorescence was concentrated in the medullary region, from which lymph exits through efferent lymphatic vessels.²⁰ Fluorescence in this area, therefore, confirmed that the lymph node was normal (Fig. 7A). A thin outer layer of FITC fluorescence was also observed in a transplanted PLN, confirming that this lymph node was also normal (Fig. 7B). Fluorescence in the marginal sinuses was increased in the transplanted PLN when compared with the normal PLN and was thought to indicate inflammation. Fluorescence of the transplanted PLN confirmed reconnection between this lymph node and afferent lymphatic vessels.

FIG. 7.

Fluorescein isothiocyanate–dextran fluorescence images. (A) Architecture of a normal PLN. (B) Architecture of a transplanted PLN. Scale bar, 100 μm.

Transplanted PLN retained T cells, B cells, and lymphatic endothelial cells

LYVE-1 is expressed on the luminal surface of lymphatic endothelium, immunostaining of which represents lymphatic flow. LYVE-1 was found to be positive in the marginal area of a normal PLN (Fig. 8A) and in the marginal area of a transplanted PLN (Fig. 9A). Therefore, immunohistochemical staining results showing reconnection between the transplanted PLN and afferent lymphatic vessels were consistent with the results of FITC–dextran fluorescence.

FIG. 8.

Immunohistochemistry staining of a normal PLN. (A) LYVE-1, (B) CD3, (C) B220, and (D) Verhoeff's elastic stain. Scale bar, 100 μm. LYVE-1, lymphatic vessel endothelial receptor 1.

FIG. 9.

Immunohistochemical staining of a transplanted PLN. (A) LYVE-1, (B) CD3, (C) B220, and (D) Verhoeff's elastic stain. Scale bar, 100 μm.

The transplanted PLNs were stained with antibodies directed against T cells (CD3) and B cells (B220) to determine whether the immune components of the lymph node survived after transplantation. T cell populations are usually distributed in the T cell zones of the paracortex and B cell populations in the B cell follicles of the cortex (Fig. 8B, C). Our analysis demonstrated that the histological architecture of a transplanted PLN was almost the same as that of a normal PLN. The T cell and B cell populations survived at the paracortex and cortex, respectively, after transplantation (Fig. 9B, C).

The transplanted PLNs were also stained with Verhoeff's elastic stain to detect inflammation. The elastic fibers in the vicinity of each normal PLN did not stain, indicating no inflammation (Fig. 8D). However, there was staining of elastic fibers in the vicinity of each transplanted PLN, reflecting an inflammatory change (Fig. 9D). This change was in accordance with the thickening of the marginal sinuses seen on FITC–dextran fluorescence.

Discussion

The main findings of our study were that removal of a PLN resulted in rerouting of lymph flow and transplantation of a nonvascularized PLN normalized lymph flow; FITC–dextran fluorescence could demonstrate reconnection between a transplanted lymph node and afferent lymphatic vessels; the histological architecture of a transplanted nonvascularized PLN remained unchanged, with the usual distribution of lymphatic endothelial cells, T cells, and B cells.

Several other research groups have used ICG fluorescence to identify changes in the pattern of lymph flow after PLN removal in mice.^15,21,22 Kwon et al. demonstrated rerouting of lymphatic drainage to the ILN after removal of a PLN, and mentioned the possibility of increased risk of metastasis in the context of melanoma.¹⁵ Furthermore, Blum et al. emphasized that the extent of invasive surgery could determine the degree of change in the lymphatic drainage pattern.²¹ In this study, we observed a change in the pattern of lymphatic drainage after surgical resection of a single PLN. Blum et al. mentioned that a new network of collateral vessels to reroute lymph could develop in just 3 weeks. In our mouse model, it took 3 weeks for a stable lymphatic pattern to be restored. After removal of a PLN, fluorescence indicated lymphatic pooling at the location of the removed PLN in group 1 mice, but this had disappeared after 3 weeks. We speculate that, in the process of regeneration, a space is created after removal of a PLN and ICG flows into that space, resulting in a pooling phenomenon; then, as time passes, the space diminishes and is gradually replaced by regenerating tissue. In contrast with group 1, in which lymph drainage was rerouted to the ILNs, group 2 showed normalization of lymphatic drainage.

The number of clinical reports on the benefits of lymph node transfer as a treatment for lymphedema is increasing.^5,10,23,24 However, corresponding experimental studies remain limited^8,11,12,25 and none has shown the time course of lymphatic normalization after lymph node transplantation in detail. Interstitial flow is essential for restoration of normal lymph flow.^19,26 However, as we described in our animals that underwent removal of a PLN, a new lymphatic drainage pattern was formed in advance of bridging the gap between afferent and efferent lymphatic vessels. Transplantation of a lymph node was effective for bridging this gap even though the lymph node was nonvascularized. In our study, the results in animals that underwent PLN transplantation confirmed that the transplanted lymph node functioned as a duct to reconnect the afferent and efferent lymphatic vessels relatively soon after transplantation. In two mice in group 2, lymph flow to both PLN and ILN was confirmed. This could indicate that a new lymphatic drainage pattern was formed subsequent to the surgical intervention even though the transplanted lymph node functioned as a duct.

FITC–dextran was useful for observing the reconnection between the implanted lymph node and the lymphatic vessels. Because dextran used in our study enters the lymphatic capillaries, but not the rest of the circulation, after subcutaneous injection, it is possible to observe the lymph flow pattern in real time by following the distribution of dextran.¹⁹ In a normal lymph node, the fluorescence at the subcapsular sinus of the lymph node was clear, and even clearer at the hilum of the lymph node on the concave side. This was thought to be reflective of the structure whereby the lymph flows into the medullary sinuses after the subcapsular sinus, and finally flows into the efferent lymph vessels at the hilum of the node. In our study, the fluorescence in a transplanted lymph node was similar to that in a normal lymph node, confirming the pathway of lymph flow to the medullary sinuses. The margin of the transplanted lymph node was a little thickened although part of the subcapsular sinus fluoresced clearly. The other part was thickened and obscured. This is probably attributable to the inflammatory changes that occurred after surgery. The immunohistochemical staining of LYVE-1 and Verhoeff's elastic staining were also consistent with the results of FITC–dextran fluorescence.

Although lymph nodes have complex functions, the most relevant with respect to neoplasms is the management of antigen-primed dendritic cells that migrate from the tumor environment and present tumor-derived antigens to naive T cells in the regional lymph nodes.^13,27 Functional connections between the lymphatic vessels and the subcapsular sinus of a lymph node are important given that these connections are known to maintain immune cell homeostasis.²⁸ We have now demonstrated continuity between the lymphatic vessels and a transplanted lymph node and preservation of the T cell and B cell populations therein. Durable regeneration of these connections opens up the possibility of preserving the role of lymph nodes in tumor immunity. However, further research is needed to clarify the mechanisms involved in normal functioning of immune cells in a transplanted lymph node.

In conclusion, we have demonstrated rerouting of lymphatic vessels after lymph node resection. Furthermore, we successfully transplanted a nonvascularized PLN, reconnected it with the lymphatic vessels, and normalized the pattern of lymph flow after removal of a lymph node. The T cell and B cell populations associated with tumor immunity were maintained in the transplanted lymph node.

Footnotes

Acknowledgment

This study was funded by a Grant-in-Aid for Scientific Research B from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (No. 15H05001).

Author Disclosure Statement

No competing financial interests exist.

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Lymphatic Reconnection and Restoration of Lymphatic Flow by Nonvascularized Lymph Node Transplantation: Real-Time Fluorescence Imaging Using Indocyanine Green and Fluorescein Isothiocyanate–Dextran

Abstract

Abstract

Background:

Methods and Results:

Conclusion:

Introduction

Materials and Methods

Experimental model

Indocyanine green fluorescence near-infrared video camera system

Infrared imaging

Fluorescein isothiocyanate–dextran foot pad injection and preparation of specimens

Histology

Results

Lymphatic drainage after PLN removal and after PLN implantation

Reconnection of transplanted PLN and lymphatic vessels

Transplanted PLN retained T cells, B cells, and lymphatic endothelial cells

Discussion

Footnotes

Acknowledgment

Author Disclosure Statement

References