Vascularized lymph node transplantation successfully reverses lymphedema and maintains immunity in a rat lymphedema model

Abstract Vascularized lymph node transplantation (VLNT) has shown inspiring results for the treatment of lymphedema. Nevertheless, it remains unclear how VLNT restores lymphatic drainage and whether or not immunity recovers after surgery. Hindlimb lymphedema model was created using rats with extensive groin and popliteal lymph node removable following with radiotherapy, and the lymphedema was confirmed using indocyanine green (ICG) lymphangiography and micro‐computer tomography for volume measurement. VLNT was performed 1 month later. Volume measurement, ICG lymphangiography, histology, and immune reaction were done 1 month after surgery. VLNT successfully reduced the volume of the lymphedema hindlimb, restored lymphatic drainage function with proven lymphatic channel, and reduced lymphedema‐related inflammation and fibrosis. It promotes lymphangiogenesis shown from ICG lymphangiography, histology, and enhanced lymphangiogenesis gene expression. Dendritic cell trafficking via the regenerated lymphatic channels was successfully restored, and maintained systemic immune response was proved using dinitrofluorobenzene sensitization and challenge. VLNT effectively reduces lymphedema and promotes lymphatic regeneration in the capillary lymphatic but not the collecting lymphatic vessels. Along with the re‐established lymphatic system was the restoration of immune function locally and systemically. This correlated to clinical experience regarding the reduction of swelling and infection episodes after VLNT in lymphedema patients.

debilitating complications of cancer treatments. It strongly and negatively impacts the quality of life of cancer survivors, and thus establishing a reliable treatment is urgent and crucial. [1][2][3][4][5] The lymphatic system is considered the third circulation system throughout the body and is vital for fluid homeostasis, maintaining immune defense and dietary fat absorption in the intestines. Damage to the lymphatic channels caused by removal of the lymph nodes followed by radiotherapy often results in blockage of lymphatic continuity. This results in disruption of lymphatic fluid and immune cell trafficking, which further damages immune function. Patients often present with localized protein-rich fluid retention, subsequent chronic inflammation and fibrosis, and susceptibility to infection. 6 This suggests that both fluid homeostasis and immune defense are affected by lymphedema.
Vascularized lymph node transfer (VLNT) was initiated in an animal study in 1990 for the purpose of lymphedema treatment and has been popularized in recent years with multiple donor sites explored, various recipient sites used and newly developed equipment allowing for an accurate diagnosis and real-time intraoperative guidance. [7][8][9][10][11][12][13] Although promising results of VLNT have been reported by different groups, it remains unclear whether lymphangiogenesis and the recovery of immune function are established after VLNT. Instead, bypassing lymphatic drainage by diverting it into the venous system (supramicrosurgical lymphatic-venous anastomosis [LVA]) is recommended by some reconstructive surgeons. [14][15][16][17] The conflicts between lymph node transfer and LVA persuaded us to explore the role of lymph nodes in surgical treatments in lymphedema, considering the potential donor site morbidities. 18 Frequent infection is one prominent symptom of lymphedema patients besides the presence of extremity swelling and fibrosis. 19 To understand whether VLNT can restore immune function, we applied an animal model that mirrors the clinical situation of lymphedema, which was then treated with VLNT. 20,21 The recovery of immune defense was investigated by evaluating immune cell trafficking and the systemic immune response in addition to confirming reduced swelling and lymphedema.
F I G U R E 1 Animal model of vascularized lymph node transfer (VLNT). (a) Diagram of the creation of lymphedema followed by regional radiotherapy 3 days postoperatively. Micro-computer tomography (CT) and indocyanine green (ICG) lymphangiography were performed to confirm lymphedema 1 month postoperatively. VLNT was performed 1 month after inducing lymphedema. Micro-CT and ICG lymphangiography were performed 1 month after VLNT. Animals were then sacrificed for immune function and histology evaluation. (b) Representative photo of successfully developed lymphedema with hindlimb swelling. (c) Pattern of dermal backflow type of lymphatic drainage in ICG lymphangiography in rats confirmed the presence of lymphedema. (d) Groin fat pads with skin paddles were harvested as groin lymph node flaps. (e) The recipient site was prepared by gentle dissection of the femoral artery and veins. After vascular preparation, the prepared groin lymph node flap was transferred to the recipient site. (f) After flap inset, a skin paddle was left for perfusion monitoring. (g and h) ICG lymphangiography was performed again before tissue harvest (g), and the transferred lymph node was localized using ICG before tissue harvest and was further confirmed after tissue harvest (h) 2 | RESULTS

| VLNT successfully reduced the volume of the lymphedema hind limb
In accordance with clinical experience, VLNT successfully reduced the volume difference between the lymphedema and contralateral healthy hindlimb. After lymphedema creation, the volume differentiation between the hindlimb of interest and the contralateral healthy hind limb was similar in the lymphedema and VLNT groups (24.54 ± 6.35% vs. 30.02 ± 7.28%, p = 0.1881). Before vascularized lymph node flap transfer, the dermal back flow pattern of lymphatic draining was confirmed (Figure 1b,c). One month after VLNT, volume differentiation between the hindlimb of interest and the contralateral healthy hind limb was significantly different between the VLNT and lymphedema groups (À6.21 ± 2.20% vs. 9.39 ± 6.68%, p = 0.0122). Comparisons within the VLNT group also revealed a significant volume difference before but not 1 month after VLNT (24.54 ± 6.35% vs. À6.21 ± 2.20%, p = 0.001) (Figure 2a-c).

| VLNT effectively reduced adipose deposition and hyperkeratosis in lymphedema
Along with the improvement of the volume difference, the adipose deposition reduced after VLNT, which echoed clinical scenario. From

| VLNT effectively reduced T-cell mediated inflammation
Lymphedema has been proven to be a T cell-mediated inflammatory disease. [25][26][27] Our results revealed more CD3 and CD4 T cell infiltration in the skin from the lymphedema hind limbs than in the skin from the sham control group (CD3: 24 (Figure 3).

| VLNT effectively promotes lymphangiogenesis
According to the ICG lymphangiography performed before sample harvest, ICG can be identified in the transferred lymph node, suggesting that lymphatic drainage was successfully re-established after VLNT (Figure 4a This suggested that more inflammatory reactions were recalled in the VLNT-treated group than in the lymphedema group (Figure 5d-f).

| The transferred lymph nodes maintained a normal structure
The microscopic morphology of the transferred lymph nodes confirmed that they maintained a basic anatomical structure with T cells

| DISCUSSION
Considering the limitations of understanding recovery mechanism of lymphedema in clinical cases after VLNT, we applied a surgical animal F I G U R E 7 Gene expression indicating lymphatic regeneration after vascularized lymph node transfer. Relative mRNA levels of the following genes in the harvested skin and subcutaneous tissue: (a) VEGF-C, (b) podoplanin, (c) LYVE-1, (d) Prox-1, (e) FLT4/VEGFR3, (f) Sox-18, and (g) FOXC-2 (n = 6) model of lymphedema followed by VLNT to investigate its effect. 28 Our results revealed that VLNT treated lymphedema with recovery of volume reduction in micro-CT, lymphatic transportation in ICG lymphangiography, and reductions in the histological hallmarks of lymphedema. Furthermore, the maintenance of immunity can be identified after VLNT, including dendritic cell trafficking and systemic immune reactions. Re-establishment of the lymphatic channel is the main purpose of physiological treatment of lymphedema. Our study provides evidence that lymphangiogenesis can be promoted and lymphatic channel can be re-established after VLNT.
ICG lymphangiography revealed a dermal back flow in the distal hindlimb and more lymphatics with a linear pattern can be seen around the transferred lymph node, suggesting that there was more lymphangiogenesis around the transferred lymph node than the distal hindlimb ( Figure 4a). Besides, dendritic cell trafficking can be seen from the distal hindlimb to the transferred groin lymph node, and a few of the cells are subsequently transported to the axillary lymph nodes ( Figure 5).
For the mechanism of lymphedema recovery from VLNT, drainage via natural lymphaticovenous connections inside the lymph node has been suggested. 29 However, the route for dendritic cell trafficking provided evidence that the re-establishment of lymphatic drainage seemed to be more than just from the natural lymphaticovenous connections inside the lymph nodes. Our results suggested that VLNT promoted lymphangiogenesis, with more podoplanin(+) lymphatic vessels present in the VLNT group (Figure 4d 32 Our results also highlighted the increased presence of Sox-18 in the VLNT group, which echoed lymphangiogenesis for capillary lymphatics. Interestingly, FOXC-2, which is not a key factor in the development of capillary lymphatics but is crucial for patterning lymphatics into collecting lymphatic vessels, was downregulated in our results. This may explain why lymphangiogenesis following VLNT was mainly associated with capillary lymphatics.
In addition to drainage, frequent infection is one prominent feature symptom of lymphedema patients beyond the presence of extremity swelling and fibrosis. The recovery of immune defense should be considered equally important to successful fluid transport, including antigen presentation and effective immune response by naïve T cell differentiation. 33,34 After confirming the improvements in characteristics of swelling and lymphedema, we next investigated immune function after VLNT.
Our results showed that dendritic cell trafficking and the systemic immune response can both be restored after VLNT. A few of the dendritic cells can further travel to the axillary lymph nodes, which are the next stream of lymph nodes to the groin lymph nodes, suggesting the connection from the transferred lymph node to the next ones.
The challenge of DNFB, followed by DNFB stimulation from remote areas (ears) other than the hindlimb, provides evidence of the recovery of systemic immune reactions. Similar results have been reported earlier in non VLNT in a mouse model. 35 The present study further confirmed the restoration of immune function in a VLNT model, providing evidence of immune recovery in a clinical scenario and that patients receiving vascularized lymph nodes present with less cellulitis than patients who have lymphedema without treatment. 36 Interestingly, while the transferred vascularized lymph node maintained its basic structure with a typical distribution of immune cells, the flow cytometry results confirmed that the numbers of CD3 (+) and CD4(+) T cells were significantly higher in the transferred lymph nodes than in the normal/nonoperated lymph nodes. Conversely, CD45R(+) B cells were more abundant in the normal lymph nodes ( Figure 6). The exact reason for this remains unknown. It can likely be from the surgery itself or because of the recipient site.
Lymphedema is a T cell-mediated inflammatory disease. [25][26][27] Additional study will be required to investigate this in depth.

| METHODS
This study was completed using a rat animal model. Hind limb lymphedema was created surgically by removing the groin and popliteal lymph nodes followed by postoperative radiation to mimic the clinical scenario. The presence of lymphedema was confirmed with microcomputer tomography (micro-CT) and indocyanine green (ICG) lymphangiography 1 month after radiotherapy, identifying the presence of volume differences and confirmation of the dermal back flow. The animals were grouped into VLNT and control lymphedema groups. The final evaluation was performed another month later with a volume measurement using micro-CT and ICG lymphangiography. Immune recovery was surveyed. The animals were then euthanized, and histological evaluation was completed (Figure 1a). In addition, animals receiving sham surgery with an oblique incision over the groin area with primary closure were prepared as the control group (n = 6 in each group).

| Animals
All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC number: 20151804) of Chang Gung Memorial Hospital and were performed in accordance with the institution's animal research guidelines. Male Lewis rats weighing 400 to 450 g were obtained from BioLASCO Taiwan Co., Ltd. The animal surgeries were conducted under anesthesia using 2.5% isoflurane inhalation via a nose cone. After confirmation of the depth of anesthesia, the skin was prepared by removing the fur from an area 150% larger than the incision area. The skin was then prepared in an aseptic manner.

| Micro-CT imaging
The volume of the hind limbs was measured by using Nano-SPECT/ CT (NanoSPECT/CT, Bioscan) with the animals in a supine position 1 month after radiotherapy. The rats were under general anesthesia as previously described. 20,21 The scan was taken for 15 min, and the images were reconstructed with filtered back-projection into a 3Dimage volume with pixels sized 0.2 mm in both the transverse and axial directions. All images were saved in DICOM format and later analyzed with PMOD software (PMOD Technologies Ltd.).
The volume differentiation was determined by the following equation: (Volume of the hind limb of interest À Volume of the contralateral healthy hind limb)/Volume of the contralateral healthy hind limb Â 100%.
A volume differentiation greater than 5% was considered swelling from lymphedema.

| Harvesting the vascularized lymph node flap
Vascularized groin lymph node flaps were harvested from the donor rats' inguinal areas. 37,38 Briefly, an incision was made along the groin area. Subcutaneous dissection was carried out carefully, and all of the subcutaneous fat pads were preserved. The fat pad, including the lymph nodes, was then dissected carefully along with its supply vessels, mainly the superficial epigastric artery and vein. The vessels were further dissected to include part of the femoral artery and vein to facilitate microvascular anastomosis (Figure 1d. The donor wounds were then closed. Postoperative pain medication was given immediately after the surgery.

| Transferring a vascularized lymph node flap
An incision was made in the groin area of the hind limb of the lymphedema rats. The femoral artery and vein were explored after layer-bylayer dissection. The vessels were prepared carefully by dissection of a sufficient length for vascular anastomosis. The adventitia was removed as much as possible. After vascular preparation, the prepared groin lymph node flap was transferred to the recipient site, and anastomosis was performed in an end-to-end manner with 11-0 nylon.
The vascularized lymph node flap was inset into the recipient site and the wound closed (Figure 1e,f).

| ICG lymphography
The use of near-infrared images with ICG injection have been developed for lymphatic identification, lymphedema evaluation and intraoperative guidance for lymphatic surgeries. 39   Nuclear staining was performed using DAPI.

| DNFB sensitization and IHC
The rats were sensitized to 2,4-dinitrofluorobenzene by painting the foot paw and distal of the lymphedema hindlimb with 500 μl 0.25% DNFB (Sigma Aldrich, Merck) in acetone/olive oil (4:1, Sigma Aldrich) 5 and 6 days before sacrifice. Their ears were challenged with 100 μl DNFB 1 day before sacrifice. The ears were harvested to evaluate inflammatory reactions.
The inflammation of the DNFB-challenged ear was analyzed by

| Flow cytometry
The lymph nodes were removed into Petri dishes containing 5 ml PBS, rubbed, and broken to make cell suspensions. The suspension was centrifuged at 1500 rpm for 5 min. The supernatant was discarded, and 2 ml ammonium-chloride-potassium (ACK) lysis buffer was added and incubated at room temperature for 1 min. Then, 2 ml PBS was added to neutralize the ACK lysing buffer. The suspension was centrifuged at 1500 rpm for 5 min. The supernatant was discarded, and then 2 ml PBS was added to resuspend the cells. The suspension was then filtered through a 100 μm strainer (Falcon, Corning) to obtain a single cell suspen-

| Real-time qRT-PCR
The harvested skin and subcutaneous tissue were overlaid with TRIzol (Invitrogen) and stored. RNA was isolated using the RNeasy kit (QIAGEN) according to the manufacturer's protocol. One microgram of RNA was reverse transcribed with a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). FastStart SYBR Green master mix (Roche, Basel, Switzerland) was used for the quantitative PCR assays and analyzed with a StepOnePlus Real-Time PCR System (Applied Biosystems). All gene expression values were normalized to the GAPDH (mouse) levels for each sample. Table 1 shows the primers used in the qRT-PCR.

| Statistics
Statistical analysis was performed using GraphPad Prism software (GraphPad Software). Comparisons were performed using Student's t-test or one-way ANOVA, depending on the grouping, for comparison. A p-value less than 0.05 was considered to indicate a significant difference.

| CONCLUSIONS
In summary, this study provides evidence at the histological and molecular levels to confirm the effectiveness of VLNT in treating lymphedema, which is not possible to collect from clinical cases. More importantly, our study showed that the transferred vascularized lymph node can successfully restore immune function by providing adequate drainage function and restore the adaptive immune system. These findings provide valuable information on the clinical application of VLNT. It also provides fundamental knowledge to continue further lymphatic research.