Mechanisms of Lymphatic Regeneration after Tissue Transfer

Lymphedema is the chronic swelling of an extremity that occurs commonly after lymph node resection for cancer treatment. Recent studies have demonstrated that transfer of healthy tissues can be used as a means of bypassing damaged lymphatics and ameliorating lymphedema. The purpose of these studies was to investigate the mechanisms that regulate lymphatic regeneration after tissue transfer.Nude mice (recipients) underwent 2-mm tail skin excisions that were either left open or repaired with full-thickness skin grafts harvested from donor transgenic mice that expressed green fluorescent protein in all tissues or from LYVE-1 knockout mice. Lymphatic regeneration, expression of VEGF-C, macrophage infiltration, and potential for skin grafting to bypass damaged lymphatics were assessed.Skin grafts healed rapidly and restored lymphatic flow. Lymphatic regeneration occurred beginning at the peripheral edges of the graft, primarily from ingrowth of new lymphatic vessels originating from the recipient mouse. In addition, donor lymphatic vessels appeared to spontaneously re-anastomose with recipient vessels. Patterns of VEGF-C expression and macrophage infiltration were temporally and spatially associated with lymphatic regeneration. When compared to mice treated with excision only, there was a 4-fold decrease in tail volumes, 2.5-fold increase in lymphatic transport by lymphoscintigraphy, 40% decrease in dermal thickness, and 54% decrease in scar index in skin-grafted animals, indicating that tissue transfer could bypass damaged lymphatics and promote rapid lymphatic regeneration.Our studies suggest that lymphatic regeneration after tissue transfer occurs by ingrowth of lymphatic vessels and spontaneous re-connection of existing lymphatics. This process is temporally and spatially associated with VEGF-C expression and macrophage infiltration. Finally, tissue transfer can be used to bypass damaged lymphatics and promote rapid lymphatic regeneration

Surgical management of lymphedema : past, present, and future

Recent advances in surgical management of lymphedema have provided options for patients who have failed conservative management with manual lymphatic massage and/or compression garments. The purpose of this review is to provide a historical background to the surgical treatment of lymphedema and how these options have evolved over time. In addition, we aim to delineate the various types of surgical approaches available, indications for surgery, and reported outcomes. Our goal is to increase awareness of these options and foster research to improve their outcomes.9 page(s

Correction: CD4+ Cells Regulate Fibrosis and Lymphangiogenesis in Response to Lymphatic Fluid Stasis

INTRODUCTION: Lymphedema is a chronic disorder that occurs commonly after lymph node removal for cancer treatment and is characterized by swelling, fibrosis, inflammation, and adipose deposition. Although previous histological studies have investigated inflammatory changes that occur in lymphedema, the precise cellular make up of the inflammatory infiltrate remains unknown. It is also unclear if this inflammatory response plays a causal role in the pathology of lymphedema. The purpose of this study was therefore to characterize the inflammatory response to lymphatic stasis and determine if these responses are necessary for the pathological changes that occur in lymphedema. METHODS: We used mouse-tail lymphedema and axillary lymph node dissection (ANLD) models in order to study tissue inflammatory changes. Single cell suspensions were created and analyzed using multi-color flow cytometry to identify individual cell types. We utilized antibody depletion techniques to analyze the causal role of CD4+, CD8+, and CD25+ cells in the regulation of inflammation, fibrosis, adipose deposition, and lymphangiogenesis. RESULTS: Lymphedema in the mouse-tail resulted in a mixed inflammatory cell response with significant increases in T-helper, T-regulatory, neutrophils, macrophages, and dendritic cell populations. Interestingly, we found that ALND resulted in significant increases in T-helper cells suggesting that these adaptive immune responses precede changes in macrophage and dendritic cell infiltration. In support of this we found that depletion of CD4+, but not CD8 or CD25+ cells, significantly decreased tail lymphedema, inflammation, fibrosis, and adipose deposition. In addition, depletion of CD4+ cells significantly increased lymphangiogenesis both in our tail model and also in an inflammatory lymphangiogenesis model. CONCLUSIONS: Lymphedema and lymphatic stasis result in CD4+ cell inflammation and infiltration of mature T-helper cells. Loss of CD4+ but not CD8+ or CD25+ cell inflammation markedly decreases the pathological changes associated with lymphedema. In addition, CD4+ cells regulate lymphangiogenesis during wound repair and inflammatory lymphangiogenesis

Lymphatic regeneration after tissue transfer is associated with expression of VEGF-C.

<p><b>A.</b> VEGF-C expression in skin-grafted tails 2 weeks after surgery. Representative low power (2x; upper panel) photomicrographs encompassing the skin-grafted area and distal/proximal portions of the recipient mouse-tail are shown. High power (20x) views of the distal and proximal junctions between recipient tissues and skin grafts are shown below. Black arrow shows large number of VEGF-C⁺ cells in the distal junction. Dashed box delineates skin-grafted area. <b>B.</b> VEGF-C expression in skin-grafted tails 6 weeks after surgery. Representative low power (2x; upper panel) and high power (20x) photomicrographs encompassing the skin-grafted area and distal/middle/proximal portions of the recipient mouse tails are shown. Dashed box delineates skin-grafted area. Note small amount of wound/skin graft contracture after repair. <b>C.</b> Cell counts per high power field of VEGF-C⁺ cells in the various tail regions (D = distal, M = middle, P = proximal) 2 and 6 weeks after surgery. Cell counts are means ± SD of at least 4 high power fields/mouse/time point. At least 6 mice were analyzed in each group (*<i>p</i><0.05; <i>#</i><0.01).</p

Lymphatic regeneration after tissue transfer is associated with infiltration of macrophages.

<p><b>A.</b> F4/80 localization in skin-grafted tails 2 weeks after surgery. Representative low power (2x; upper panel) photomicrographs encompassing the skin-grafted area and distal/proximal portions of the recipient mouse-tail are shown. High power (20x) views of the distal and proximal junctions between recipient tissues and skin grafts are shown below. Dashed box delineates skin-grafted area. <b>B.</b> F4/80 localization in skin-grafted tails 6 weeks after surgery. Representative low power (2x; upper panel) and high power (20x) photomicrographs encompassing the skin-grafted area and distal/proximal portions of the recipient mouse tails are shown. Dashed box delineates skin-grafted area. Note small amount of wound/skin graft contracture after repair. <b>C.</b> Cell counts per high power field of F4/80⁺ cells in various regions of the tail 2 and 6 weeks after surgery. Cell counts are means ± SD of at least 4 high power fields/mouse/time point. At least 6 mice were analyzed in each group (*<i>p</i><0.05).</p

Spontaneous regeneration of lymphatics after tissue transfer can be used to bypass damaged lymphatics.

<p><b>A.</b> Gross photographs comparing nude mice that had undergone tail excision with (right) and without (left) skin grafting are shown 6 weeks after surgery. Note obvious difference in tail swelling. <b>B.</b> Tail volume measurements in nude mice that had undergone tail excision with or without skin grafting. Data are presented as percent change from baseline (i.e. preoperatively) with mean ± SD (*<i>p</i><0.05). <b>C, D.</b> Representative lymphoscintigraphy of nude mice that had undergone tail excision with or without skin grafting. <b>E.</b> Representative photomicrograph (5x) of H&E stained tails sections from nude mice treated with (left) or without (right) 6 weeks after surgery. Dashed box delineates area of skin graft. Note decreased inflammation (cellularity) and dermal thickness in skin-grafted mice distal (to the left) of the wound. <b>F.</b> High power (40x) photomicrographs of tail skin harvested 5 mm distal to the excision site. Note decreased cellularity in skin-grafted section (left) as compared with excision section (right; arrow). Also note decreased dermal thickness. <b>G.</b> Dermal thickness measurements and representative figures (40x) in nude mice that had undergone tail excision with or without skin grafting 6 weeks following surgery (*<i>p</i><0.05). <b>H.</b> Scar index measurements in tail tissues localized just distal to the site of lymphatic injury 6 weeks after treatment with excision with or without skin grafting. Representative Sirius red birefringence images are shown to the right. Orange-red is indicative of scar; yellow-green is consistent with normal (i.e. non-fibrosed) tissue (*<i>p</i><0.01).</p

Autogenous tissue lymphangiogenesis is associated with spontaneous reconnection of local lymphatics and infiltration of new lymphatic capillaries.

<p><b>A.</b> Skin grafts harvested from GFP transgenic mice express GFP at high levels and for a long period of time. A representative fluorescent picture of a mouse (top) with higher magnification of the tail (bottom) is shown 6 weeks after surgery. The skin-grafted GFP portion of the tail can easily be seen. <b>B.</b> Gross photographs of mouse tails treated with skin grafts harvested from GFP transgenic mice. Note the rapid incorporation and ingrowth of hair follicles at the 6-week time point. <b>C.</b> Microlymphangiography performed 2 (left) or 6 (right) weeks following skin graft. Distal portion of the tail is shown at the bottom. Note the flow of fluorescent dye by interstitial flow after 2 weeks. In contrast, lymphatic flow can be seen in the skin-grafted area at the 6-week time point. Representative figures of triplicate experiments are shown. <b>D.</b> Higher power photograph of microlymphangiography 2 and 6 weeks after surgery demonstrating a few honeycomb-like dermal lymphatics (white arrows) in the skin graft at the 6-week time point. <b>E.</b> Representative LYVE-1 (pink) and GFP (green) co-localization in skin-grafted mouse tails 6 weeks after surgery. Low power (5x; left) and high power (20x; right) views are shown. Note connection between GFP^- (recipient) and GFP⁺ (donor) lymphatic vessel. <b>F.</b> Representative photomicrograph (2x) demonstrating GFP (left), LYVE-1 (middle), and co-localization (right) of skin-grafted mouse tails 6 weeks after surgery. Note ingrowth of GFP^-/LYVE-1⁺ vessels (yellow circle) from the distal (yellow dotted line) portion of the wound. <b>G.</b> High power (20x) view of section shown in <b>F</b>. Note the presence of both recipient (GFP^-/LYVE-1⁺) and donor (GFP⁺/LYVE-1⁺) lymphatics at the distal margin of the wound.</p