Tail Lymphedema Model Research Articles

A Novel Dressing Composed of Adipose Stem Cells and Decellularized Wharton’s Jelly Facilitated Wound Healing and Relieved Lymphedema by Enhancing Angiogenesis and Lymphangiogenesis in a Rat Model

Lymphedema causes tissue swelling due to the accumulation of lymphatic fluid in the tissue, which delays the process of wound-healing. Developing effective treatment options of lymphedema is still an urgent issue. In this study, we aim to fabricate tissue-engineered moist wound dressings with adipose stem cells (ASCs) and decellularized Wharton's jelly (dWJ) from the human umbilical cord in order to ameliorate lymphedema. Rat ASCs were proliferated and an apparent layer was observed on dWJ at day 7 and 14. A rat tail lymphedema model was developed to evaluate the efficacy of the treatment. Approximately 1 cm of skin near the base of the rat tail was circularly excised. The wounds were treated by secondary healing (control) (n = 5), decellularized Wharton's jelly (n = 5) and ASC-seeded dWJ (n = 5). The wound-healing rate and the tail volume were recorded once a week from week one to week five. Angiogenesis and lymphangiogenesis were assessed by immunochemistry staining with anti-CD31 and anti-LYVE1. The results showed that the wound-healing rate was faster and the tail volume was lesser in the ASC-seeded dWJ group than in the control group. More CD31+ and LYVE-1+ cells were observed at the wound-healing area in the ASC-seeded dWJ group than in the control group. This proves that tissue-engineered moist wound dressings can accelerate wound-healing and reduce lymphedema by promoting angiogenesis and lymphangiogenesis.

Intravital Microscopy Optimization for Murine Tail Lymphedema Model

Background: Lymphedema is limb swelling caused by lymphatic dysfunction. It occurs in 30% of patients that undergo axillary lymph node dissection in the treatment of breast cancer. It can cause pain, impair function, and decrease quality of life. Lymphedema is treated with compression, excisional procedures and microsurgical physiologic procedures. There is no cure for this disease. The murine tail model of lymphedema is an established animal model for lymphedema. Visualization of lymphatics and functional assessment remains a challenge.
 Project Rationale: Immunohistopathology and qRT-PCR are two commonly used in vitro techniques for molecular assessment of lymphatics in animal tissues. These methods provide incomplete information about the structure/function of lymphatics and introduce the confounder of harvested tissue. Methods of functional evaluation such as lymphoscintigraphy or lymphangiography show transit of dyes through lymphatics without high resolution imaging of the lymphatic vessels. Intravitaltwo-photon microscopy (IVM) addresses these disadvantages through real-time imaging of subcellular level biological processes in live animals. The goal of this project is to optimize IVMmethods for the assessment of functional lymphangiogenesis in the murine tail lymphedemamodel.
 Methodology Development: A full-thickness skin excision is performed near the base of the tail in C57BL/6 mice. The lymphatic trunks are then surgically transected. Gene-based therapy is delivered to the tail at the surgical site. At 10 days post-treatment, a second full-thickness skin excision is made distal to the site of occlusion. FITC-Dextran (2000 kD) is injected at the distal tail for lymphatic uptake. Lymphatic vessels are visualized at the second skin excision site with the Leica SP8 Confocal/Multiphoton Microscope and assessed for number of branching points. Images are captured with Leica Application Suite Advanced Fluorescence Software and analyzed with Imaris Microscopy Image Analysis Software. This results in the ability of functional assessment of lymphatics and visualization of lymphangiogenesis following gene-based therapy.

Lentiviral overexpression of VEGFC in transplanted MSCs leads to resolution of swelling in a mouse tail lymphedema model.

Dysfunction of the lymphatic system following injury, disease, or cancer treatment can lead to lymphedema, a debilitating condition with no cure. Despite the various physical therapy and surgical options available, most treatments are palliative and fail to address the underlying lymphatic vascular insufficiency driving lymphedema progression. Stem cell therapy provides a promising alternative in the treatment of various chronic diseases with a wide range of therapeutic effects that reduce inflammation, fibrosis, and oxidative stress, while promoting lymphatic vessel (LV) regeneration. Specifically, stem cell transplantation is suggested to promote LV restoration, rebuild lymphatic circulation, and thus potentially be utilized towards an effective lymphedema treatment. In addition to stem cells, studies have proposed the administration of vascular endothelial growth factor C (VEGFC) to promote lymphangiogenesis and decrease swelling in lymphedema. Here, we seek to combine the benefits of stem cell therapy, which provides a cellular therapeutic approach that can respond to the tissue environment, and VEGFC administration to restore lymphatic drainage. Specifically, we engineered mesenchymal stem cells (MSCs) to overexpress VEGFC using a lentiviral vector (hVEGFC MSC) and investigated their therapeutic efficacy in improving LV function and tissue swelling using near infrared (NIR) imaging, and lymphatic regeneration in a single LV ligation mouse tail lymphedema model. First, we showed that overexpression of VEGFC using lentiviral transduction led to an increase in VEGFC protein synthesis in vitro. Then, we demonstrated hVEGFC MSC administration post-injury significantly increased the lymphatic contraction frequency 14-, 21-, and 28-days post-surgery compared to the control animals (MSC administration) in vivo, while also reducing tail swelling 28-days post-surgery compared to controls. Our results suggest a therapeutic potential of hVEGFC MSC in alleviating the lymphatic dysfunction observed during lymphedema progression after secondary injury and could provide a promising approach to enhancing autologous cell therapy for treating lymphedema.

A High-Fat Diet in the Absence of Obesity Increases Lymphangiogenesis by Inducing VEGF-C in a Murine Lymphedema Model.

Many researchers have attempted to induce lymphangiogenesis for the treatment of lymphedema. However, most previous studies had limited clinical usefulness. A high-fat diet (HFD) increases serum β-hydroxybutyrate (β-OHB) levels, which can stimulate lymphangiogenesis. The authors hypothesized that an HFD will ameliorate lymphedema through enhanced lymphangiogenesis. The effects of β-OHB on the lymphangiogenic process in human dermal lymphatic endothelial cells were analyzed. A mouse tail lymphedema model was used to evaluate the effects of an HFD on lymphedema. Experimental mice were fed an HFD (45% kcal as fat, 20% as protein, and 35% as carbohydrates) for 4 weeks. Tail volume was measured using the truncated cone formula. Biopsy specimens were taken 6 weeks after surgical induction of lymphedema. In human dermal lymphatic endothelial cells, treatment with 20 mM of β-OHB increased cell viability ( P = 0.008), cell migration ( P = 0.011), tube formation ( P = 0.005), and VEGF-C mRNA and protein expression ( P < 0.001) compared with controls. HFD feeding decreased tail volume by 14.3% and fibrosis by 15.8% ( P = 0.027), and increased the lymphatic vessel density ( P = 0.022) and VEGF-C protein expression ( P = 0.005) compared with those of operated, standard chow diet-fed mice. The authors' findings demonstrated that β-OHB promoted lymphatic endothelial cell function and increased VEGF-C mRNA and protein expression. When mice with tail lymphedema were fed an HFD, volume and fibrosis of the tail decreased. Therefore, the authors' findings suggest that an HFD can be a successful novel dietary approach to treating lymphedema. Lymphatic regeneration after vascularized lymph node transfer can be augmented when a high-fat diet is used in conjunction with vascularized lymph node transfer.

Hydrogen Sulfide Attenuates Lymphedema Via the Induction of Lymphangiogenesis Through a PI3K/Akt-Dependent Mechanism.

Background Accumulating evidence suggests that hydrogen sulfide ( H2S ), an endogenously produced gaseous molecule, plays a critical role in the regulation of cardiovascular homeostasis. However, little is known about its role in lymphangiogenesis. Thus, the current study aimed to investigate the involvement of H2S in lymphatic vessel growth and lymphedema resolution using a murine model and assess the underlying mechanisms. Methods and Results A murine model of tail lymphedema was created both in wild-type mice and cystathionine γ-lyase-knockout mice, to evaluate lymphedema up to 28 days after lymphatic ablation. Cystathionine γ-lyase-knockout mice had greater tail diameters than wild-type mice, and this phenomenon was associated with the inhibition of reparative lymphangiogenesis at the site of lymphatic ablation. In contrast, the administration of an H2S donor, diallyl trisulfide, ameliorated lymphedema by inducing the formation of a considerable number of lymphatic vessels at the injured sites in the tails. In vitro experiments using human lymphatic endothelial cells revealed that diallyl trisulfide promoted their proliferation and differentiation into tube-like structures by enhancing Akt (protein kinase B) phosphorylation in a concentration-dependent manner. The blockade of Akt activation negated the diallyl trisulfide-induced prolymphangiogenic responses in lymphatic endothelial cells. Furthermore, the effects of diallyl trisulfide treatment on lymphangiogenesis in the tail lymphedema model were also negated by the inhibition of phosphoinositide 3'-kinase (P13K)/Aktsignaling. Conclusions H2S promotes reparative lymphatic vessel growth and ameliorates secondary lymphedema, at least in part, through the activation of the Akt pathway in lymphatic endothelial cells. As such, H2S donors could be used as therapeutics against refractory secondary lymphedema.

Enhancement of Lymphangiogenesis by Human Mesenchymal Stem Cell Sheet.

Preparation of human mesenchymal stem cell (hMSC) suspension for lymphedema treatment relies on conventional enzymatic digestion methods, which severely disrupts cell-cell and cell-extracellular matrix (ECM) connections, and drastically impairs cell retention and engraftment after transplantation. The objective of the present study is to evaluate the ability of hMSC-secreted ECM to augment lymphangiogenesis by using an in vitro coculturing model of hMSC sheets with lymphatic endothelial cells (LECs) and an in vivo mouse tail lymphedema model. Results demonstrate that the hMSC-secreted ECM augments the formation of lymphatic capillary-like structure by a factor of 1.2-3.6 relative to the hMSC control group, by serving as a prolymphangiogenic growth factor reservoir and facilitating cell regenerative activities. hMSC-derived ECM enhances MMP-2 mediated matrix remodeling, increases the synthesis of collagen IV and laminin, and promotes lymphatic microvessel-like structure formation. The injection of rat MSC sheet fragments into a mouse tail lymphedema model confirms the benefits of the hMSC-derived ECM by stimulating lymphangiogenesis and wound closure.

Piezo1-Regulated Mechanotransduction Controls Flow-Activated Lymphatic Expansion.

Mutations in PIEZO1 (Piezo type mechanosensitive ion channel component 1) cause human lymphatic malformations. We have previously uncovered an ORAI1 (ORAI calcium release-activated calcium modulator 1)-mediated mechanotransduction pathway that triggers lymphatic sprouting through Notch downregulation in response to fluid flow. However, the identity of its upstream mechanosensor remains unknown. This study aimed to identify and characterize the molecular sensor that translates the flow-mediated external signal to the Orai1-regulated lymphatic expansion. Various mutant mouse models, cellular, biochemical, and molecular biology tools, and a mouse tail lymphedema model were employed to elucidate the role of Piezo1 in flow-induced lymphatic growth and regeneration. Piezo1 was found to be abundantly expressed in lymphatic endothelial cells. Piezo1 knockdown in cultured lymphatic endothelial cells inhibited the laminar flow-induced calcium influx and abrogated the flow-mediated regulation of the Orai1 downstream genes, such as KLF2 (Krüppel-like factor 2), DTX1 (Deltex E3 ubiquitin ligase 1), DTX3L (Deltex E3 ubiquitin ligase 3L,) and NOTCH1 (Notch receptor 1), which are involved in lymphatic sprouting. Conversely, stimulation of Piezo1 activated the Orai1-regulated mechanotransduction in the absence of fluid flow. Piezo1-mediated mechanotransduction was significantly blocked by Orai1 inhibition, establishing the epistatic relationship between Piezo1 and Orai1. Lymphatic-specific conditional Piezo1 knockout largely phenocopied sprouting defects shown in Orai1- or Klf2- knockout lymphatics during embryo development. Postnatal deletion of Piezo1 induced lymphatic regression in adults. Ectopic Dtx3L expression rescued the lymphatic defects caused by Piezo1 knockout, affirming that the Piezo1 promotes lymphatic sprouting through Notch downregulation. Consistently, transgenic Piezo1 expression or pharmacological Piezo1 activation enhanced lymphatic sprouting. Finally, we assessed a potential therapeutic value of Piezo1 activation in lymphatic regeneration and found that a Piezo1 agonist, Yoda1, effectively suppressed postsurgical lymphedema development. Piezo1 is an upstream mechanosensor for the lymphatic mechanotransduction pathway and regulates lymphatic growth in response to external physical stimuli. Piezo1 activation presents a novel therapeutic opportunity for preventing postsurgical lymphedema. The Piezo1-regulated lymphangiogenesis mechanism offers a molecular basis for Piezo1-associated lymphatic malformation in humans.

Prolymphangiogenic Effects of 9-cis Retinoic Acid Are Enhanced at Sites of Lymphatic Injury and Dependent on Treatment Duration in Experimental Postsurgical Lymphedema.

Background: Patients undergoing surgical treatment for solid tumors are at risk for development of secondary lymphedema due to intraoperative lymphatic vessel injury. The damaged lymphatic vessels fail to adequately regenerate and lymphatic obstruction leads to fluid and protein accumulation in the interstitial space and chronic lymphedema develops as a result. There are currently no effective pharmacological agents that reduce the risk of developing lymphedema or treat pre-existing lymphedema, and management is largely palliative. The present study investigated the efficacy of various 9-cis retinoic acid (9-cis RA) dosing strategies in reducing postsurgical lymphedema by utilizing a well-established mouse tail lymphedema model. Methods and Results: Short-duration treatment with 9-cis RA did not demonstrate a significant reduction in postoperative tail volume, nor an improvement in lymphatic clearance. However, long-term treatment with 9-cis RA resulted in decreased overall tail volume, dermal thickness, and epidermal thickness, with an associated increase in functional lymphatic clearance and lymphatic vessel density, assessed by LYVE-1 immunostaining, compared with control. These effects were seen at the site of lymphatic injury, with no significant changes observed in uninjured sites such as ear skin and the diaphragm. Conclusions: Given the reported results indicating that 9-cis RA is a potent promoter of lymphangiogenesis and improved lymphatic clearance at sites of lymphatic injury, investigation of postoperative 9-cis RA administration to patients at high risk of developing lymphedema may demonstrate positive efficacy and reduced rates of postsurgical lymphedema.

A Murine Tail Lymphedema Model.

Lymphedema is extremity swelling caused by lymphatic dysfunction. The affected limb enlarges because of accumulation of fluid, adipose, and fibrosis. There is no cure for this disease. A mouse tail model that uses a focal full thickness skin excision near the base of the tail, resulting in tail swelling, has been used to study lymphedema. However, this model may result in vascular comprise and consequent tail necrosis and early tail swelling resolution, limiting its clinical translatability. The chronic murine tail lymphedema model induces sustained lymphedema over 15 weeks and a reliable perfusion to the tail. Enhancements of the traditional murine tail lymphedema model include 1) precise full thickness excision and lymphatic clipping using a surgical microscope, 2) confirmation of post-operative arterial and venous perfusion using high resolution laser speckle, and 3) functional assessment using indocyanine green near infrared laser lymphangiography. We also use tissue nanotransfection technology (TNT) for novel non-viral, transcutaneous, focal delivery of genetic cargo to the mouse tail vasculature.

Abstract 1644: Detection and quantification of lymphedema using optical coherence tomography lymphangiography

Abstract Purpose: Imaging of the lymphatic system is important for monitoring and quantification of the vasculature of tumours and in lymphatic disorders (e.g. lymphedema, an obstruction of the lymphatic system that occurs as a side-effect of cancer treatments such as radiation and/or surgery). Traditional methods of lymphangiography require injection of a contrast agent to visualize the lymphatic system, which may be suboptimal when there is lymphatic obstruction. Thus, there is a need to develop intrinsic-contrast, high-resolution volumetric (3D) non-invasive quantitative in vivo imaging systems for both pre-clinical and potentially clinical use. Methods: An emerging non-invasive 3D imaging technology called optical coherence tomography (OCT) lymphangiography was optimized to visualize lymphatic vasculature and edema within tissues of C57BL/6 mice under normal and surgically-induced lymphedematous conditions. A murine tail lymphedema model was created by micro-surgical obstruction of the superficial and deep lymphatic networks under a dissecting microscope. OCT imaging was validated against fluorescent microscopy, an established but contrast-agent-dependent lymphangiography technique, as well as histology. Fluorescent microscopy was performed with intradermal injection of FITC-dextran followed by imaging with the AxioZoom.V16 Stereo Zoom Microscope. Immunofluorescence staining was performed with an anti-LYVE1 (Lymphatic Vessel Endothelial Receptor 1) antibody. Results: Comparing 3D OCT images and their 2D projections under normal and lymphedematous conditions showed significantly different networks. Quantification analysis revealed increased lymphatic vasculature dilation (~4X in vessel diameters, p&amp;lt;0.01) and increased fluid accumulation (~3X in edematous regions, p&amp;lt;0.05) under lymphedema conditions. Lymphatic vascular dilation and edema were confirmed by fluorescent microscopy and histology. Conclusions: OCT is an emerging non-invasive volumetric imaging modality that can be adapted to visualize lymphatic vasculature within normal and lymphedematous tissues without the use of vascular contrast agents. Furthermore, OCT has the ability to quantify the degree of vessel dilation and edema accumulation. Citation Format: Jennifer Y. Kwan, Valentin Demidov, Blake Jones, Wei Shi, Justin Williams, Costel Flueraru, Kenneth W. Yip, Fei-Fei Liu, Alex Vitkin. Detection and quantification of lymphedema using optical coherence tomography lymphangiography [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1644.

Validation of laser scanning confocal microscopy as a diagnostic method for lymphedema using a rat model

Most previous diagnostic methods for lymphedema are invasive. Laser scanning confocal microscopy (LSCM) combines laser and computer image processing technology, is capable of increasing the resolution of optical microscopy by 30-40%, and boasts a comparable resolution to that of histological examination. We constructed the rat tail lymphedema model to simulate secondary lymphedema and to validate the noninvasive technique of in vivo reflectance LSCM for the diagnosis of lymphedema. The rat tail lymphedema model was constructed by cutting and ligating the lymphatic vessels in the rat tail. Lymphedema in the postoperative rat tail was assessed by a comprehensive range of methods including the change of rat tail diameter, lymphocytic radionuclide imaging, LSCM, and immunohistochemistry using a specific lymphatic vessel marker, prospero homeobox protein 1 (PROX1). The noninvasive LSCM method along with other techniques validated the rat tail lymphedema model. LSCM was used to perform qualitative and quantitative evaluation of the state and extent of lymphedema in the rat tail model. Receiver operating characteristic (ROC) curve analysis, which provided an area under the curve (AUC) value of 0.861, supported the feasibility of using LSCM as a reliable diagnostic technique for lymphedema. The rat tail lymphedema model can be successfully constructed by cutting and ligating the lymphatic vessels in the rat tail. Although LSCM cannot replace the method of skin biopsy examination, it offers a painless and noninvasive alternative for diagnosing lymphedema. Thus, LSCM can potentially be adopted in clinical practice as a supporting method to be used in combination with other techniques.

Endogenous Calcitonin Gene–Related Peptide Deficiency Exacerbates Postoperative Lymphedema by Suppressing Lymphatic Capillary Formation and M2 Macrophage Accumulation

Lymphedema is a chronic condition caused by disruption of lymphatic vessels, which often occurs after invasive surgery. Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide produced by alternative splicing of the primary transcript of the calcitonin/CGRP gene (Calca). CGRP was initially identified as a neuropeptide released primarily from sensory nerves and involved in regulating pathophysiological nociceptive pain. However, recent studies have shown CGRP is also released from a variety of other cells and possesses multiple functions. In this study, CGRP knockout (-/-) mice were used to show the actions of endogenous CGRP in postoperative lymphedema. After generating a mouse postoperative tail lymphedema model, the edema was observed to be more severe in CGRP-/- mice than in wild-type mice. Numbers of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1)-positive lymphatic capillaries were decreased and lymphatic capillary formation-related factors were down-regulated in CGRP-/- mice. In addition, accumulation of M2 but not M1 macrophages was selectively reduced in the edematous tissue of CGRP-/- mice. Selective depletion of M2 macrophages decreased lymphatic capillary formation and worsened lymphedema in wild-type mice but not CGRP-/- mice, where numbers of M2 macrophages were already diminished. These findings suggest that endogenous CGRP acts to ameliorate postoperative lymphedema by enhancing lymphatic capillary formation and that M2 macrophages play critical roles. CGRP may be a useful therapeutic target for the treatment of postoperative lymphedema.

A novel mouse tail lymphedema model for observing lymphatic pump failure during lymphedema development

It has been suggested that many forms of secondary lymphedema in humans are driven by a progressive loss of lymphatic pump function after an initial risk-inducing event. However, the link between pump failure and disease progression has remained elusive due to experimental challenges in the clinical setting and a lack of adequate animal models. Using a novel surgical model of lymphatic injury, we track the adaptation and functional decline of the lymphatic network in response to surgery. This model mimics the histological hallmarks of the typical mouse tail lymphedema model while leaving an intact collecting vessel for analysis of functional changes during disease progression. Lymphatic function in the intact collecting vessel negatively correlated with swelling, while a loss of pumping pressure generation remained even after resolution of swelling. By using this model to study the role of obesity in lymphedema development, we show that obesity exacerbates acquired lymphatic pump failure following lymphatic injury, suggesting one mechanism through which obesity may worsen lymphedema. This lymphatic injury model will allow for future studies investigating the molecular mechanisms leading to lymphedema development.

Development and Evaluation of a New In Vivo Volume Measuring System in Mouse Tail Lymphedema Model.

Backgrounds: Secondary lymphedema is a common complication of parasitization and breast or gynecologic cancer therapy; however, options for the treatment of lymphedema are ineffective and limited. A mouse tail model is one of the most successful animal models for a lymphatic study. Lymphedema of the mouse tail is characterized by increases in the volume of the extremity caused by accumulation of tissue fluid, proliferation of fibroblasts and adipocytes, and excessive production of collagen. However, the study of lymphedema using mouse has been plagued with difficulty in directly assessing physiologic changes owing to limitations in the measurement of the mouse tail volume. Furthermore, the mouse tail volume cannot be obtained using the general in vivo measurement method such as volumetric water displacement. Methods and Results: Lymphatic researchers have used the truncated cone formula to approximate the volume as used in the numerical approximation of a cylindrical figure. Although this formula is simple and easy to use, it has difficulties of repeatability and accuracy because the measurement procedure is highly subjective and the accuracy depends on the number of divided segments on the tail. In this article, two novel volumetric measurement methods for the mouse tail model were introduced. The methods were evaluated and compared using three mice with surgically created lymphedema on the tails. Conclusions: The two continuous measuring methods showed a possibility to improve the conventional method by continuous measurement using visual and physical detecting methods. The proposed methods facilitate the extraction of longitudinal section-specific information, which can be an important clue in a lymphatic study.

An Important Role of VEGF-C in Promoting Lymphedema Development

Secondary lymphedema is a common complication after cancer treatment, but the pathomechanisms underlying the disease remain unclear. Using a mouse tail lymphedema model, we found an increase in local and systemic levels of the lymphangiogenic factor vascular endothelial growth factor (VEGF)-C and identified CD68+ macrophages as a cellular source. Surprisingly, overexpression of VEGF-C in a transgenic mouse model led to aggravation of lymphedema with increased immune cell infiltration and vascular leakage compared with wild-type littermates. Conversely, blockage of VEGF-C by overexpression of soluble VEGF receptor-3 reduced edema development, diminishing inflammation and blood vascular leakage. Similar findings were obtained in a hind limb lymph node excision lymphedema model. Flow cytometry analyses and immunofluorescence stainings in lymphedematic tissue showed that VEGF receptor-3 expression was restricted to lymphatic endothelial cells. Our data suggest that endogenous VEGF-C causes blood vascular leakage and fluid influx into the tissue, thus actively contributing to edema formation. These data may provide the basis for future clinical therapeutic approaches.

T regulating lymphedema

Adoptive T regulatory cell therapy lessens disease severity in a surgical mouse tail lymphedema model.

Prominent Lymphatic Vessel Hyperplasia with Progressive Dysfunction and Distinct Immune Cell Infiltration in Lymphedema

Lymphedema is a common complication that occurs after breast cancer treatment in up to 30% of the patients undergoing surgical lymph node excision. It is associated with tissue swelling, fibrosis, increased risk of infection, and impaired wound healing. Despite the pronounced clinical manifestations of the disease, little is known about the morphological and functional characteristics of the lymphatic vasculature during the course of lymphedema progression. We used an experimental murine tail lymphedema model where sustained fluid stasis was generated on disruption of lymphatic flow, resulting in chronic edema formation with fibrosis and adipose tissue deposition. Morphological analysis of the lymphatic vessels revealed a dramatic expansion during the course of the disease, with active proliferation of lymphatic endothelial cells at the early stages of lymphedema. The lymphatic capillaries exhibited progressively impaired tracer filling and retrograde flow near the surgery site, whereas the collecting lymphatic vessels showed a gradually decreasing contraction amplitude with unchanged contraction frequency, leading to lymphatic contraction arrest at the later stages of the disease. Lymphedema onset was associated with pronounced infiltration by immune cells, predominantly Ly6G(+) and CD4(+) cells, which have been linked to impaired lymphatic vessel function.

Anti-inflammatory and lymphangiogenetic effects of low-level laser therapy on lymphedema in an experimental mouse tail model.

The aim of the present study was to investigate the therapeutic mechanism of low-level laser therapy (LLLT) in the mouse tail lymphedema model. Six-week-old female mice were classified into the laser treatment group, sham treatment group, and surgical control group (10 mice per group). LLLT was administered daily for 10 min from the surgical day to 11 days (12 times). Macrophage activation and lymphatic vessel regeneration were evaluated through immunohistochemical staining with anti-F4/80 and anti-LYVE-1 antibodies, respectively, at 12 days post-procedure. Quantitative real-time polymerase chain reaction (qPCR) was performed to measure messenger RNA (mRNA) expression of vascular endothelial growth factor A, B, C, R1, R2, and R3 (VEGF-A, VEGF-B, VEGF-C, VEGFR1, VEGFR2, and VEGFR3) at 12 days post-procedure. Student's t and one-way ANOVA tests were performed for statistical analyses. Significance was defined as p < 0.05. The thickness of the tail rapidly increased until 6 days in the laser and sham groups. The mice in the laser group showed a significantly decreased thickness compared with the sham group at 10 and 12 days. Immunohistochemistry assay revealed that LLLT reduced inflammation and induced new lymphatic vessel growth. qPCR showed that expressions of VEGFR3 were (p = 0.002) increased in the laser group. These results suggest that LLLT has anti-inflammatory and lymphangiogenetic effects for the management of lymphedema.

Impact of platelet rich plasma and adipose stem cells on lymphangiogenesis in a murine tail lymphedema model

BackgroundLymphedema is an underdiagnosed pathology which in industrialized countries mainly affects cancer patients that underwent lymph node dissection and/or radiation. Currently no effective therapy is available so that patients' life quality is compromised by swellings of the concerned body region. This unfortunate condition is associated with body imbalance and subsequent osteochondral deformations and impaired function as well as with an increased risk of potentially life threatening soft tissue infections. MethodsThe effects of PRP and ASC on angiogenesis (anti-CD31 staining), microcirculation (Laser Doppler Imaging), lymphangiogenesis (anti-LYVE1 staining), microvascular architecture (corrosion casting) and wound healing (digital planimetry) are studied in a murine tail lymphedema model. ResultsWounds treated by PRP and ASC healed faster and showed a significantly increased epithelialization mainly from the proximal wound margin. The application of PRP induced a significantly increased lymphangiogenesis while the application of ASC did not induce any significant change in this regard. ConclusionsPRP and ASC affect lymphangiogenesis and lymphedema development and might represent a promising approach to improve regeneration of lymphatic vessels, restore disrupted lymphatic circulation and treat or prevent lymphedema alone or in combination with currently available lymphedema therapies.

Fibrosis worsens chronic lymphedema in rodent tissues.

Secondary lymphedema in humans is a common consequence of lymph node dissection (LND) to treat breast cancer. A peculiar characteristic of the disease is that lifelong swelling often precipitously appears several years after the surgical treatment, often due to an inflammatory stimulus. Although the incidence of secondary lymphedema dramatically increases after radiation therapy, the relationship between fibrotic scarring and the eventual appearance of lymphedema remains unclear. To clarify the role of fibrosis in secondary lymphedema initiation, we chemically increased fibrosis in rodent tissues with bleomycin and assessed the ability of the local lymphatic system to prevent lymphedema, either acutely or in a chronic state induced by inflammation. We found that bleomycin injections exacerbated fibrotic matrix deposition in an acute mouse tail lymphedema model (P < 0.005), reduced wound closure (P < 0.005), and impaired the ability of tail lymphatics to regenerate (P < 0.005) and reduce the swelling (P < 0.05). When fibrosis was worsened with bleomycin after axillary LND in the rat foreleg, the ability of the foreleg lymphatic system to reduce the chronic state swelling induced by stimulated inflammation was severely impaired (P < 0.005). Indocyanine green lymphography in axillary LND-recovered rat forelegs revealed a worsened lymphatic drainage due to inflammation and bleomycin pretreatment. Although inflammation reduced the drainage of dextran fluid tracer from control forelegs (P < 0.05), the reduction in fluid drainage was more severe after axillary LND when fibrosis was first increased (P < 0.005). These findings demonstrate that fibrosis reduces the lymphatic capacity to functionally regenerate and prevent the chronic appearance of lymphedema.