Posttraumatic Penile Replantation with Minimal Skin Necrosis.

Penile amputation is a surgical emergency where practical and timely perioperative management is crucial for ensuring a successful outcome. Tenuous viability of penile and scrotal skin has been well described in the literature, with a putative mechanism attributed to the transection of distal branches of the external pudendal artery. Although the perforasomes critical to penile replantation have been debated, this case report details a patient who successfully recovered sensation and function with minimal necrosis after penile replantation. Surgically, this was facilitated by intentional drain placement, aggressive debridement beyond the zone of injury, and planned redundancies with dorsal artery/vein anastomoses via interposition grafts of the dorsal penile vessels alone.

Posttraumatic Lip Replantation in a Pregnant Woman.

Traumatic lip amputation is a devastating injury. No other tissue replicates its unique histology, often limiting the reconstructive outcome. Replantation is a technically challenging procedure, requiring extensive postoperative optimization, including systemic anticoagulation, leech therapy, significant blood loss, and antibiosis. Given the rarity of replantation in the context of pregnancy, there are no documented accounts of lip replantation in pregnant patients. We report a case of a 25-year-old pregnant woman who sustained an avulsion injury of the right upper lip from a dog bite. The patient presented with the amputated lip and emergent microvascular replantation was performed. Postoperative course consisted of management of controlled yet significant blood loss through leech therapy and close collaboration with obstetric colleagues. The patient was ultimately discharged with successful cosmetic and functional outcome and, importantly, with maintenance of a healthy pregnancy.

CD4+ T cells are activated in regional lymph nodes and migrate to skin to initiate lymphedema.

T cell-mediated responses have been implicated in the development of fibrosis, impaired lymphangiogenesis, and lymphatic dysfunction in secondary lymphedema. Here we show that CD4+ T cells are necessary for lymphedema pathogenesis by utilizing adoptive transfer techniques in CD4 knockout mice that have undergone tail skin and lymphatic excision or popliteal lymph node dissection. We also demonstrate that T cell activation following lymphatic injury occurs in regional skin-draining lymph nodes after interaction with antigen-presenting cells such as dendritic cells. CD4+ T cell activation is associated with differentiation into a mixed T helper type 1 and 2 phenotype, as well as upregulation of adhesion molecules and chemokines that promote migration to the skin. Most importantly, we find that blocking T cell release from lymph nodes using a sphingosine-1-phosphate receptor modulator prevents lymphedema, suggesting that this approach may have clinical utility.

Topical tacrolimus for the treatment of secondary lymphedema.

Secondary lymphedema, a life-long complication of cancer treatment, currently has no cure. Lymphedema patients have decreased quality of life and recurrent infections with treatments limited to palliative measures. Accumulating evidence indicates that T cells play a key role in the pathology of lymphedema by promoting tissue fibrosis and inhibiting lymphangiogenesis. Here using mouse models, we show that topical therapy with tacrolimus, an anti-T-cell immunosuppressive drug, is highly effective in preventing lymphedema development and treating established lymphedema. This intervention markedly decreases swelling, T-cell infiltration and tissue fibrosis while significantly increasing formation of lymphatic collaterals with minimal systemic absorption. Animals treated with tacrolimus have markedly improved lymphatic function with increased collecting vessel contraction frequency and decreased dermal backflow. These results have profound implications for lymphedema treatment as topical tacrolimus is FDA-approved for other chronic skin conditions and has an established record of safety and tolerability.

Lymph Node Transplantation Decreases Swelling and Restores Immune Responses in a Transgenic Model of Lymphedema.

INTRODUCTION: Secondary lymphedema is a common complication of cancer treatment and recent studies have demonstrated that lymph node transplantation (LNT) can decrease swelling, as well as the incidence of infections. However, although these results are exciting, the mechanisms by which LNT improves these pathologic findings of lymphedema remain unknown. Using a transgenic mouse model of lymphedema, this study sought to analyze the effect of LNT on lymphatic regeneration and T cell-mediated immune responses. METHODS: We used a mouse model in which the expression of the human diphtheria toxin receptor is driven by the FLT4 promoter to enable the local ablation of the lymphatic system through subdermal hindlimb diphtheria toxin injections. Popliteal lymph node dissection was subsequently performed after a two-week recovery period, followed by either orthotopic LNT or sham surgery after an additional two weeks. Hindlimb swelling, lymphatic vessel regeneration, immune cell trafficking, and T cell-mediated immune responses were analyzed 10 weeks later. RESULTS: LNT resulted in a marked decrease in hindlimb swelling, fibroadipose tissue deposition, and decreased accumulation of perilymphatic inflammatory cells, as compared to controls. In addition, LNT induced a marked lymphangiogenic response in both capillary and collecting lymphatic vessels. Interestingly, the resultant regenerated lymphatics were abnormal in appearance on lymphangiography, but LNT also led to a notable increase in dendritic cell trafficking from the periphery to the inguinal lymph nodes and improved adaptive immune responses. CONCLUSIONS: LNT decreases pathological changes of lymphedema and was shown to potently induce lymphangiogenesis. Lymphatic vessels induced by LNT were abnormal in appearance, but were functional and able to transport antigen-presenting cells. Animals treated with LNT have an increased ability to mount T cell-mediated immune responses when sensitized to antigens in the affected hindlimb.

Diphtheria toxin-mediated ablation of lymphatic endothelial cells results in progressive lymphedema.

Development of novel treatments for lymphedema has been limited by the fact that the pathophysiology of this disease is poorly understood. It remains unknown, for example, why limb swelling resulting from surgical injury resolves initially, but recurs in some cases months or years later. Finding answers for these basic questions has been hampered by the lack of adequate animal models. In the current study, we used Cre-lox mice that expressed the human diphtheria toxin receptor (DTR) driven by a lymphatic-specific promoter in order to noninvasively ablate the lymphatic system of the hind limb. Animals treated in this manner developed lymphedema that was indistinguishable from clinical lymphedema temporally, radiographically, and histologically. Using this model and clinical biopsy specimens, we show that the initial resolution of edema after injury is dependent on the formation of collateral capillary lymphatics and that this process is regulated by M2-polarized macrophages. In addition, we show that despite these initial improvements in lymphatic function, persistent accumulation of CD4+ cells inhibits lymphangiogenesis and promotes sclerosis of collecting lymphatics, resulting in late onset of edema and fibrosis. Our findings therefore provide strong evidence that inflammatory changes after lymphatic injury play a key role in the pathophysiology of lymphedema.

Obesity-induced lymphatic dysfunction is reversible with weight loss.

KEY POINTS: Obesity induces lymphatic leakiness, decreases initial lymphatic vessel density, impairs collecting vessel pumping and decreases transport of macromolecules. Obesity results in perilymphatic inducible nitric oxide synthase (iNOS) expression and accumulation of T cells and macrophages. Deleterious effects of obesity on the lymphatic system correlate with weight gain. Weight loss restores lymphatic function in obese animals and decreases perilymphatic iNOS and inflammatory cell accumulation. ABSTRACT: Although clinical and experimental studies have shown that obesity results in lymphatic dysfunction, it remains unknown whether these changes are permanent or reversible with weight loss. In the current study, we used a mouse model of diet-induced obesity to identify putative cellular mechanisms of obesity-induced lymphatic dysfunction, determine whether there is a correlation between these deleterious effects and increasing weight gain, and finally examine whether lymphatic dysfunction is reversible with diet-induced weight loss. We report that obesity is negatively correlated with cutaneous lymphatic collecting vessel pumping rate (r = -0.9812, P < 0.0005) and initial lymphatic vessel density (r = -0.9449, P < 0.005). In addition, we show a significant positive correlation between weight gain and accumulation of perilymphatic inflammatory cells (r = 0.9872, P < 0.0005) and expression of inducible nitric oxide synthase (iNOS; r = 0.9986, P < 0.0001). Weight loss resulting from conversion to a normal chow diet for 8 weeks resulted in more than a 25% decrease in body weight and normalized cutaneous lymphatic collecting vessel pumping rate, lymphatic vessel density, lymphatic leakiness, and lymphatic macromolecule clearance (all P < 0.05). In addition, weight loss markedly decreased perilymphatic inflammation and iNOS expression. Taken together, our findings show that obesity is linearly correlated with lymphatic dysfunction, perilymphatic inflammation and iNOS expression, and that weight loss via dietary modification effectively reverses these deleterious effects.

Exercise training improves obesity-related lymphatic dysfunction.

KEY POINTS: Obesity results in perilymphatic inflammation and lymphatic dysfunction. Lymphatic dysfunction in obesity is characterized by decreased lymphatic vessel density, decreased collecting lymphatic vessel pumping frequency, decreased lymphatic trafficking of immune cells, increased lymphatic vessel leakiness and changes in the gene expression patterns of lymphatic endothelial cells. Aerobic exercise, independent of weight loss, decreases perilymphatic inflammatory cell accumulation, improves lymphatic function and reverses pathological changes in gene expression in lymphatic endothelial cells. ABSTRACT: Although previous studies have shown that obesity markedly decreases lymphatic function, the cellular mechanisms that regulate this response remain unknown. In addition, it is unclear whether the pathological effects of obesity on the lymphatic system are reversible with behavioural modifications. The purpose of this study, therefore, was to analyse lymphatic vascular changes in obese mice and to determine whether these pathological effects are reversible with aerobic exercise. We randomized obese mice to either aerobic exercise (treadmill running for 30 min per day, 5 days a week, for 6 weeks) or a sedentary group that was not exercised and analysed lymphatic function using a variety of outcomes. We found that sedentary obese mice had markedly decreased collecting lymphatic vessel pumping capacity, decreased lymphatic vessel density, decreased lymphatic migration of immune cells, increased lymphatic vessel leakiness and decreased expression of lymphatic specific markers compared with lean mice (all P < 0.01). Aerobic exercise did not cause weight loss but markedly improved lymphatic function compared with sedentary obese mice. Exercise had a significant anti-inflammatory effect, resulting in decreased perilymphatic accumulation of inflammatory cells and inducible nitric oxide synthase expression. In addition, exercise normalized isolated lymphatic endothelial cell gene expression of lymphatic specific genes, including VEGFR-3 and Prox1. Taken together, our findings suggest that obesity impairs lymphatic function via multiple mechanisms and that these pathological changes can be reversed, in part, with aerobic exercise, independent of weight loss. In addition, our study shows that obesity-induced lymphatic endothelial cell gene expression changes are reversible with behavioural modifications.

Inhibition of Inflammation and iNOS Improves Lymphatic Function in Obesity.

Although recent studies have shown that obesity decreases lymphatic function, the cellular mechanisms regulating this response remain unknown. In the current study, we show that obesity results in perilymphatic accumulation of inflammatory cells and that local inhibition of this response with topical tacrolimus, an inhibitor of T cell differentiation, increases lymphatic vessel density, decreases perilymphatic iNOS expression, increases lymphatic vessel pumping frequency, and restores lymphatic clearance of interstitial fluid to normal levels. Although treatment of obese mice with 1400W, a selective inhibitor of iNOS, also improved lymphatic collecting vessel contractile function, it did not completely reverse lymphatic defects. Mice deficient in CD4(+) cells fed a high fat diet also gained weight relative to controls but were protected from lymphatic dysfunction. Taken together, our findings suggest that obesity-mediated lymphatic dysfunction is regulated by perilymphatic accumulation of inflammatory cells and that T cell inflammatory responses are necessary to initiate this effect.

Lymphatic Function Regulates Contact Hypersensitivity Dermatitis in Obesity.

Obesity is a major risk factor for inflammatory dermatologic diseases, including atopic dermatitis and psoriasis. In addition, recent studies have shown that obesity impairs lymphatic function. As the lymphatic system is a critical regulator of inflammatory reactions, we tested the hypothesis that obesity-induced lymphatic dysfunction is a key regulator of cutaneous hypersensitivity reactions in obese mice. We found that obese mice have impaired lymphatic function, characterized by leaky capillary lymphatics and decreased collecting vessel pumping capacity. In addition, obese mice displayed heightened dermatitis responses to inflammatory skin stimuli, resulting in both higher peak inflammation and a delayed clearance of inflammatory responses. Injection of recombinant vascular endothelial growth factor-C remarkably increased lymphangiogenesis, lymphatic function, and lymphatic endothelial cell expression of chemokine (C-C motif) ligand 21, while decreasing inflammation and expression of inducible nitrous oxide synthase. These changes resulted in considerably decreased dermatitis responses in both lean and obese mice. Taken together, our findings suggest that obesity-induced changes in the lymphatic system result in an amplified and a prolonged inflammatory response.

Th2 cytokines inhibit lymphangiogenesis.

Lymphangiogenesis is the process by which new lymphatic vessels grow in response to pathologic stimuli such as wound healing, inflammation, and tumor metastasis. It is well-recognized that growth factors and cytokines regulate lymphangiogenesis by promoting or inhibiting lymphatic endothelial cell (LEC) proliferation, migration and differentiation. Our group has shown that the expression of T-helper 2 (Th2) cytokines is markedly increased in lymphedema, and that these cytokines inhibit lymphatic function by increasing fibrosis and promoting changes in the extracellular matrix. However, while the evidence supporting a role for T cells and Th2 cytokines as negative regulators of lymphatic function is clear, the direct effects of Th2 cytokines on isolated LECs remains poorly understood. Using in vitro and in vivo studies, we show that physiologic doses of interleukin-4 (IL-4) and interleukin-13 (IL-13) have profound anti-lymphangiogenic effects and potently impair LEC survival, proliferation, migration, and tubule formation. Inhibition of these cytokines with targeted monoclonal antibodies in the cornea suture model specifically increases inflammatory lymphangiogenesis without concomitant changes in angiogenesis. These findings suggest that manipulation of anti-lymphangiogenic pathways may represent a novel and potent means of improving lymphangiogenesis.

Regulation of inflammation and fibrosis by macrophages in lymphedema.

Lymphedema, a common complication of cancer treatment, is characterized by inflammation, fibrosis, and adipose deposition. We have previously shown that macrophage infiltration is increased in mouse models of lymphedema. Because macrophages are regulators of lymphangiogenesis and fibrosis, this study aimed to determine the role of these cells in lymphedema using depletion experiments. Matched biopsy specimens of normal and lymphedema tissues were obtained from patients with unilateral upper extremity breast cancer-related lymphedema, and macrophage accumulation was assessed using immunohistochemistry. In addition, we used a mouse tail model of lymphedema to quantify macrophage accumulation and analyze outcomes of conditional macrophage depletion. Histological analysis of clinical lymphedema biopsies revealed significantly increased macrophage infiltration. Similarly, in the mouse tail model, lymphatic injury increased the number of macrophages and favored M2 differentiation. Chronic macrophage depletion using lethally irradiated wild-type mice reconstituted with CD11b-diphtheria toxin receptor mouse bone marrow did not decrease swelling, adipose deposition, or overall inflammation. Macrophage depletion after lymphedema had become established significantly increased fibrosis and accumulation of CD4(+) cells and promoted Th2 differentiation while decreasing lymphatic transport capacity and VEGF-C expression. Our findings suggest that macrophages home to lymphedematous tissues and differentiate into the M2 phenotype. In addition, our findings suggest that macrophages have an antifibrotic role in lymphedema and either directly or indirectly regulate CD4(+) cell accumulation and Th2 differentiation. Finally, our findings suggest that lymphedema-associated macrophages are a major source of VEGF-C and that impaired macrophage responses after lymphatic injury result in decreased lymphatic function.

Lymphaticovenous bypass decreases pathologic skin changes in upper extremity breast cancer-related lymphedema.

INTRODUCTION: Recent advances in microsurgery such as lymphaticovenous bypass (LVB) have been shown to decrease limb volumes and improve subjective symptoms in patients with lymphedema. However, to date, it remains unknown if these procedures can reverse the pathological tissue changes associated with lymphedema. Therefore, the purpose of this study was to analyze skin tissue changes in patients before and after LVB. METHODS AND RESULTS: Matched skin biopsy samples were collected from normal and lymphedematous limbs of 6 patients with unilateral breast cancer-related upper extremity lymphedema before and 6 months after LVB. Biopsy specimens were fixed and analyzed for inflammation, fibrosis, hyperkeratosis, and lymphangiogenesis. Six months following LVB, 83% of patients had symptomatic improvement in their lymphedema. Histological analysis at this time demonstrated a significant decrease in tissue CD4(+) cell inflammation in lymphedematous limb (but not normal limb) biopsies (p<0.01). These changes were associated with significantly decreased tissue fibrosis as demonstrated by decreased collagen type I deposition and TGF-ß1 expression (all p<0.01). In addition, we found a significant decrease in epidermal thickness, decreased numbers of proliferating basal keratinocytes, and decreased number of LYVE-1(+) lymphatic vessels in lymphedematous limbs after LVB. CONCLUSIONS: We have shown, for the first time, that microsurgical LVB not only improves symptomatology of lymphedema but also helps to improve pathologic changes in the skin. These findings suggest that the some of the pathologic changes of lymphedema are reversible and may be related to lymphatic fluid stasis.

Obesity increases inflammation and impairs lymphatic function in a mouse model of lymphedema.

Although obesity is a major clinical risk factor for lymphedema, the mechanisms that regulate this effect remain unknown. Recent reports have demonstrated that obesity is associated with acquired lymphatic dysfunction. The purpose of this study was to determine how obesity-induced lymphatic dysfunction modulates the pathological effects of lymphatic injury in a mouse model. We used a diet-induced model of obesity in adult male C57BL/6J mice in which experimental animals were fed a high-fat diet and control animals were fed a normal chow diet for 8-10 wk. We then surgically ablated the superficial and deep lymphatics of the midportion of the tail. Six weeks postoperatively, we analyzed changes in lymphatic function, adipose deposition, inflammation, and fibrosis. We also compared responses to acute inflammatory stimuli in obese and lean mice. Compared with lean control mice, obese mice had baseline decreased lymphatic function. Lymphedema in obese mice further impaired lymphatic function and resulted in increased subcutaneous adipose deposition, increased CD45(+) and CD4(+) cell inflammation (P < 0.01), and increased fibrosis, but caused no change in the number of lymphatic vessels. Interestingly, obese mice had a significantly increased acute inflammatory reaction to croton oil application. In conclusion, obese mice have impaired lymphatic function at baseline that is amplified by lymphatic injury. This effect is associated with increased chronic inflammation, fibrosis, and adipose deposition. These findings suggest that obese patients are at higher risk for lymphedema due to impaired baseline lymphatic clearance and an increased propensity for inflammation in response to injury.

IL-6 regulates adipose deposition and homeostasis in lymphedema.

Lymphedema (LE) is a morbid disease characterized by chronic limb swelling and adipose deposition. Although it is clear that lymphatic injury is necessary for this pathology, the mechanisms that underlie lymphedema remain unknown. IL-6 is a known regulator of adipose homeostasis in obesity and has been shown to be increased in primary and secondary models of lymphedema. Therefore, the purpose of this study was to determine the role of IL-6 in adipose deposition in lymphedema. The expression of IL-6 was analyzed in clinical tissue specimens and serum from patients with or without LE, as well as in two mouse models of lymphatic injury. In addition, we analyzed IL-6 expression/adipose deposition in mice deficient in CD4(+) cells (CD4KO) or IL-6 expression (IL-6KO) or mice treated with a small molecule inhibitor of IL-6 or CD4 depleting antibodies to determine how IL-6 expression is regulated and the effect of changes in IL-6 expression on adipose deposition after lymphatic injury. Patients with LE and mice treated with lymphatic excision of the tail had significantly elevated tissue and serum expression of IL-6 and its downstream mediator. The expression of IL-6 was associated with adipose deposition and CD4(+) inflammation and was markedly decreased in CD4KO mice. Loss of IL-6 function resulted in significantly increased adipose deposition after tail lymphatic injury. Our findings suggest that IL-6 is increased as a result of adipose deposition and CD4(+) cell inflammation in lymphedema. In addition, our study suggests that IL-6 expression in lymphedema acts to limit adipose accumulation.