When a Good Flap Turns Bad: A Temporal Predictive Model for Free Flap Complications.

BACKGROUND: Microsurgical cases are complex plastic surgery procedures with a significant risk of acute postoperative complications. In this study, we use a large-scale database to investigate the temporal progression of complications after microsurgical procedures and the risk imparted by acute postoperative complications on subsequent reconstructive outcomes. METHODS: Microsurgery cases were extracted from the National Surgical Quality Improvement Program database by Current Procedural Terminology codes. Postoperative complications were collected for 30 days after surgery and stratified into four temporal periods (postoperative days [PODs] 0-6, 7-13, 14-20, 21-30). Postoperative complication occurrences were incorporated into a weighted multivariate logistic regression model to identify significant predictors of adverse outcomes (p < 0.05). Separately, a regression model was calculated for the time between index operation and reoperation and additional complications. RESULTS: The final cohort comprised 19,517 patients, 6,140 (31.5%) of which experienced at least one complication in the first 30 days after surgery. The occurrence of prior complications in the postoperative period was a significant predictor of future adverse outcomes following the initial week after surgery (p < 0.001). Upon predictive analysis, overall model performance was highest in PODs 7 to 13 (71.1% accuracy and the area under a receiver operating characteristic curve 0.684); 2,578 (13.2%) patients underwent at least one reoperation within the first 2 weeks after surgery. The indication for reoperation (p < 0.001) and number of days since surgery (p = 0.0038) were significant predictors of future complications after reoperation. CONCLUSION: Prior occurrence of complications in an earlier postoperative week, as well as timing and nature of reoperation, were shown to be significant predictors of future complications.

Financial (dis)incentives to surgical management of head and neck cancer care.

BACKGROUND: Patients with head and neck cancer (HNC) often require complex surgical reconstruction. This retrospective, cross-sectional study compares financial factors influencing HNC and breast cancer (BC) care to examine care disparities. METHODS: Pricing data from 2012 to 2021 was abstracted from the CMS Physician Fee Schedule Look-Up Tool. Nonprofit and research support was quantified by searching the NIH, IRS, and GuideStar databases. New York State Department of Health data from 2015 to 2019 was analyzed to compare costs, charges, and payer mix. RESULTS: HNC reconstructive procedures reimburse lower than comparable breast procedures (p < 0.05). Nonprofit and research support for HNC is disproportionately low relative to disease burden. Patients hospitalized for HNC surgical procedures generated higher costs and lower charges than patients with BC (p < 0.05). CONCLUSION: Comparatively low procedure reimbursement, low nonprofit support, and high cost of care for patients with HNC relative to patients with BC may contribute to care disparities for patients with HNC.

Pregnancy-Associated Breast Changes after Nipple-Sparing Mastectomy.

BACKGROUND: Women of reproductive age are chronically underrepresented in breast cancer studies. Recent studies suggest that almost 40% of patients diagnosed with breast cancer who are of reproductive age want to have children after completing treatment. In this study, the authors evaluated patients of reproductive age who had undergone nipple-sparing mastectomy (NSM) and implant-based reconstruction. The authors compared those who became pregnant with those who did not with respect to clinical and radiologic changes that are reported at follow-up. METHODS: Any patient 45 years of age or younger at the time of NSM was determined to be of reproductive age, selected for evaluation, and followed prospectively. The presence or absence of breast examination changes in the setting of pregnancy after NSM was recorded. RESULTS: A total of 36 patients became pregnant after NSM, and 158 patients did not become pregnant after NSM. Of those who became pregnant, nearly half reported some clinical change just before or immediately after delivery. These changes included color change and discharge at the residual nipple-areola complex and palpable nodularity elsewhere. For those with palpable changes, an ultrasound was performed and hypoechoic lesions with variable vascularity were identified. For those who went on to excision, lactational hyperplasia was the most common diagnosis. CONCLUSIONS: Ultrasound is an appropriate first-line investigation of breast changes, which can include hyperplasia of remaining ductal and glandular tissue. Patients who became pregnant after NSM commonly had clinical breast examination changes, but the majority of these changes were found to be benign on further evaluation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, II.

A Novel Mangled Upper Extremity Injury Assessment Score.

Managing mangled upper extremity injuries is a challenging problem because multiple tissue components including soft tissue, muscle, tendon, bone, nerves, and vessels are involved. The complexity of these injuries has hindered the development of accurate scoring systems and treatment algorithms. METHODS: Patients with mangled upper extremities presenting to a metropolitan level 1 trauma center in New York City over a 10-year period were identified. A mangled upper extremity was defined as any injury to ≥3 tissue components involving the extremity proximal to the digit. RESULTS: The injuries and outcomes of 76 patients were evaluated and used to create a Mangled Upper Extremity Score (MUES). One point was assigned for each of the following injury characteristics: patient age >40, fasciotomy needed, bony fixation required, bony defect present, revascularization required, crush injury mechanism, degloving or avulsion injury present, and a soft tissue defect >50 cm2. The MUES correlated with the number of complications (P value = 1.96 × 10-7) and length of hospital stay (P value = 3.95 × 10-7). Next, a Mangled Extremity Severity Score (MESS) equivalent was calculated for each patient. There was no correlation between the MESS and the number of complications (P value = 0.92) or length of hospital stay (P value = 0.35). CONCLUSIONS: Existing extremity scoring systems, including the MESS, are not reliable in predicting the success of limb salvage attempts or outcomes of mangled upper extremity injuries. The MUES developed in this study correlates significantly with important outcome measures including the number of hospital complications and length of hospital stay.

Decellularized Lymph Nodes as Scaffolds for Tissue Engineered Lymph Nodes.

BACKGROUND: The lymphatic system is commonly injured during cancer treatment. However, despite the morbidity of these injuries, there are currently no options for replacing damaged lymphatics. The purpose of this study was to optimize methods for decellularization of murine lymph nodes (LN) and to determine if these scaffolds can be used to tissue engineer lymph node-like structures. METHODS AND RESULTS: LNs were harvested from adult mice and subjected to various decellularization protocols. The degree of decellularization and removal of nuclear material was analyzed histologically and quantitatively using DNA isolation. In addition, we analyzed histological architecture by staining for matrix proteins. After the optimal method of decellularization was identified, decellularized constructs were implanted in the renal capsule of syngeneic or allogeneic recipient mice and analyzed for antigenicity. Finally, to determine if decellularized constructs could deliver lymphocytes to recipient animals, the matrices were repopulated with splenocytes, implanted in submuscular pockets, and harvested 14 days later. Decellularization was best accomplished with the detergent sodium dodecyl sulfate (SDS), resulting in negligible residual cellular material but maintenance of LN architecture. Implantation of decellularized LNs into syngeneic or allogeneic mice did not elicit a significant antigenic response. In addition, repopulation of decellularized LNs with splenocytes resulted in successful in vivo cellular delivery. CONCLUSIONS: We show, for the first time, that LNs can be successfully decellularized and that these matrices have preserved extracellular matrix architecture and the potential to deliver leukocytes in vivo. Future studies are needed to determine if tissue engineered lymph nodes maintain immunologic function.

Sterile inflammation after lymph node transfer improves lymphatic function and regeneration.

BACKGROUND: The aim of this study was to determine whether sterile inflammatory reactions can serve as a physiologic means of augmenting lymphangiogenesis in transplanted lymph nodes using a murine model. METHODS: The authors used their previously reported model of lymph node transfer to study the effect of sterile inflammation on lymphatic regeneration. Mice were divided into three groups: group 1 (controls) underwent lymphadenectomy followed by immediate lymph node transplantation without inflammation; group 2 (inflammation before transfer) underwent transplantation with lymph nodes harvested from donor animals in which a sterile inflammatory reaction was induced in the ipsilateral donor limb; and group 3 (inflammation after transfer) underwent transplantation with lymph nodes and then inflammation was induced in the ipsilateral limb. Lymphatic function, lymphangiogenesis, and lymph node histology were examined 28 days after transplantation and compared with those of normal lymph nodes. RESULTS: Animals that had sterile inflammation after transplantation (group 3) had significantly improved lymphatic function (>2-fold increase) on lympho scintigraphy, increased perinodal lymphangiogenesis, and functional lymphatics compared with the groups with no inflammation and inflammation before transplantation (p<0.01). Inflammation after transplantation was associated with a more normal lymph node architecture, expansion of B-cell zones, and decreased percentage of T cells compared with the other experimental groups. CONCLUSIONS: Sterile inflammation is a potent method of augmenting lymphatic function and lymphangiogenesis after lymph node transplantation and is associated with maintenance of lymph node architecture. Induction of inflammation after transplantation is the most effective method and promotes maintenance of normal lymph node B- and T-cell architecture.

Lymph node transplantation results in spontaneous lymphatic reconnection and restoration of lymphatic flow.

BACKGROUND: Although lymph node transplantation has been shown to improve lymphatic function, the mechanisms regulating lymphatic vessel reconnection and functional status of lymph nodes remains poorly understood. METHODS: The authors developed and used LacZ lymphatic reporter mice to examine the lineage of lymphatic vessels infiltrating transferred lymph nodes. In addition, the authors analyzed lymphatic function, expression of vascular endothelial growth factor (VEGF)-C, maintenance of T- and B-cell zone, and anatomical localization of lymphatics and high endothelial venules. RESULTS: Reporter mice were specific and highly sensitive in identifying lymphatic vessels. Lymph node transfer was associated with rapid return of lymphatic function and clearance of technetium-99 secondary to a massive infiltration of recipient mouse lymphatics and putative connections to donor lymphatics. T- and B-cell populations in the lymph node were maintained. These changes correlated with marked increases in the expression of VEGF-C in the perinodal fat and infiltrating lymphatics. Newly formed lymphatic channels in transferred lymph nodes were in close anatomical proximity to high endothelial venules. CONCLUSIONS: Transferred lymph nodes have rapid infiltration of functional host lymphatic vessels and maintain T- and B-cell populations. This process correlates with increased endogenous expression of VEGF-C in the perinodal fat and infiltrating lymphatics. Anatomical proximity of newly formed lymphatics and high endothelial venules supports the hypothesis that lymph node transfer can improve lymphedema by exchanges with the systemic circulation.

Obesity impairs lymphatic fluid transport and dendritic cell migration to lymph nodes.

INTRODUCTION: Obesity is a major cause of morbidity and mortality resulting in pathologic changes in virtually every organ system. Although the cardiovascular system has been a focus of intense study, the effects of obesity on the lymphatic system remain essentially unknown. The purpose of this study was to identify the pathologic consequences of diet induced obesity (DIO) on the lymphatic system. METHODS: Adult male wild-type or RAG C57B6-6J mice were fed a high fat (60%) or normal chow diet for 8-10 weeks followed by analysis of lymphatic transport capacity. In addition, we assessed migration of dendritic cells (DCs) to local lymph nodes, lymph node architecture, and lymph node cellular make up. RESULTS: High fat diet resulted in obesity in both wild-type and RAG mice and significantly impaired lymphatic fluid transport and lymph node uptake; interestingly, obese wild-type but not obese RAG mice had significantly impaired migration of DCs to the peripheral lymph nodes. Obesity also resulted in significant changes in the macro and microscopic anatomy of lymph nodes as reflected by a marked decrease in size of inguinal lymph nodes (3.4-fold), decreased number of lymph node lymphatics (1.6-fold), loss of follicular pattern of B cells, and dysregulation of CCL21 expression gradients. Finally, obesity resulted in a significant decrease in the number of lymph node T cells and increased number of B cells and macrophages. CONCLUSIONS: Obesity has significant negative effects on lymphatic transport, DC cell migration, and lymph node architecture. Loss of T and B cell inflammatory reactions does not protect from impaired lymphatic fluid transport but preserves DC migration capacity. Future studies are needed to determine how the interplay between diet, obesity, and the lymphatic system modulate systemic complications of obesity.

Regulation of adipogenesis by lymphatic fluid stasis: part I. Adipogenesis, fibrosis, and inflammation.

BACKGROUND: Although fat deposition is a defining clinical characteristic of lymphedema, the cellular mechanisms that regulate this response remain unknown. The goals of this two-part study were to determine the effect of lymphatic fluid stasis on adipogenesis and inflammation (part I) and how these changes regulate the temporal and spatial expression of fat differentiation genes (part II). METHODS: Adult female mice underwent tail lymphatic ablation and were euthanized 6 weeks after surgery (n = 20). Fat deposition, fibrosis, and inflammation were then analyzed in the regions of the tail exposed to lymphatic fluid stasis as compared with normal lymphatic flow. RESULTS: Lymphatic fluid stasis in the tail resulted in significant subcutaneous fat deposition, with a 2-fold increase in fat thickness (p < 0.01). In addition, lymphatic stasis was associated with subcutaneous fat fibrosis and collagen deposition. Adipogenesis in response to lymphatic fluid stasis was associated with a marked mononuclear cell inflammatory response (5-fold increase in CD45 cells; p < 0.001). In addition, the authors noted a significant increase in the number of monocytes/macrophages as identified by F4/80 immunohistochemistry (p < 0.001). CONCLUSIONS: The mouse-tail model has pathologic findings that are similar to clinical lymphedema, including fat deposition, fibrosis, and inflammation. Adipogenesis in response to lymphatic fluid stasis closely resembles this process in obesity. This model therefore provides an excellent means with which to study the molecular mechanisms that regulate the pathophysiology of lymphedema.

Regulation of adipogenesis by lymphatic fluid stasis: part II. Expression of adipose differentiation genes.

BACKGROUND: Although fat deposition is a defining clinical characteristic of lymphedema, the cellular mechanisms that regulate this response remain unknown. The goal of this study was to determine how lymphatic fluid stasis regulates adipogenic gene activation and fat deposition. METHODS: Adult female mice underwent tail lymphatic ablation and were euthanied at 1, 3, or 6 weeks postoperatively (n = 8 per group). Samples were analyzed by immunohistochemistry and Western blot analysis. An alternative group of mice underwent axillary dissections or sham incisions, and limb tissues were harvested 3 weeks postoperatively (n = 8 per group). RESULTS: Lymphatic fluid stasis resulted in significant subcutaneous fat deposition and fibrosis in lymphedematous tail regions (p < 0.001). Western blot analysis demonstrated that proteins regulating adipose differentiation including CCAAT/enhancer-binding protein-α and adiponectin were markedly up-regulated in response to lymphatic fluid stasis in the tail and axillary models. Expression of these markers increased in edematous tissues according to the gradient of lymphatic stasis distal to the wound. Immunohistochemical analysis further demonstrated that adiponectin and peroxisome proliferator-activated receptor-γ, another critical adipogenic transcription factor, followed similar expression gradients. Finally, adiponectin and peroxisome proliferator-activated receptor-γ expression localized to a variety of cell types in newly formed subcutaneous fat. CONCLUSIONS: The mouse-tail model of lymphedema demonstrates pathologic findings similar to clinical lymphedema, including fat deposition and fibrosis. The authors show that lymphatic fluid stasis potently up-regulates the expression of fat differentiation markers both spatially and temporally. These studies elucidate mechanisms regulating abnormal fat deposition in lymphedema pathogenesis and therefore provide a basis for developing targeted treatments.

Toll-like receptor deficiency worsens inflammation and lymphedema after lymphatic injury.

Mechanisms regulating lymphedema pathogenesis remain unknown. Recently, we have shown that lymphatic fluid stasis increases endogenous danger signal expression, and these molecules influence lymphatic repair (Zampbell JC, et al. Am J Physiol Cell Physiol 300: C1107-C1121, 2011). Endogenous danger signals activate Toll-like receptors (TLR) 2, 4, and 9 and induce homeostatic or harmful responses, depending on physiological context. The purpose of this study was to determine the role of TLRs in regulating tissue responses to lymphatic fluid stasis. A surgical model of lymphedema was used in which wild-type or TLR2, 4, or 9 knockout (KO) mice underwent tail lymphatic excision. Six weeks postoperatively, TLR KOs demonstrated markedly increased tail edema compared with wild-type animals (50-200% increase; P < 0.01), and this effect was most pronounced in TLR4 KOs (P < 0.01). TLR deficiency resulted in decreased interstitial and lymphatic transport, abnormal lymphatic architecture, and fewer capillary lymphatics (40-50% decrease; P < 0.001). Lymphedematous tissues of TLR KOs demonstrated increased leukocyte infiltration (P < 0.001 for TLR4 KOs), including higher numbers of infiltrating CD3+ cells (P < 0.05, TLR4 and TLR9 KO), yet decreased infiltrating F4/80+ macrophages (P < 0.05, all groups). Furthermore, analysis of isolated macrophages revealed twofold reductions in VEGF-C (P < 0.01) and LYVE-1 (P < 0.05) mRNA from TLR2-deficient animals. Finally, TLR deficiency was associated with increased collagen type I deposition and increased transforming growth factor-ß1 expression (P < 0.01, TLR4 and TLR9 KO), contributing to dermal fibrosis. In conclusion, TLR deficiency worsens tissue responses to lymphatic fluid stasis and is associated with decreased lymphangiogenesis, increased fibrosis, and reduced macrophage infiltration. These findings suggest a role for innate immune responses, including TLR signaling, in lymphatic repair and lymphedema pathogenesis.

Mechanisms of lymphatic regeneration after tissue transfer.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Temporal and spatial patterns of endogenous danger signal expression after wound healing and in response to lymphedema.

While acute tissue injury potently induces endogenous danger signal expression, the role of these molecules in chronic wound healing and lymphedema is undefined. The purpose of this study was to determine the spatial and temporal expression patterns of the endogenous danger signals high-mobility group box 1 (HMGB1) and heat shock protein (HSP)70 during wound healing and chronic lymphatic fluid stasis. In a surgical mouse tail model of tissue injury and lymphedema, HMGB1 and HSP70 expression occurred along a spatial gradient relative to the site of injury, with peak expression at the wound and greater than twofold reduced expression within 5 mm (P < 0.05). Expression primarily occurred in cells native to injured tissue. In particular, HMGB1 was highly expressed by lymphatic endothelial cells (>40% positivity; twofold increase in chronic inflammation, P < 0.001). We found similar findings using a peritoneal inflammation model. Interestingly, upregulation of HMGB1 (2.2-fold), HSP70 (1.4-fold), and nuclear factor (NF)-κß activation persisted at least 6 wk postoperatively only in lymphedematous tissues. Similarly, we found upregulation of endogenous danger signals in soft tissue of the arm after axillary lymphadenectomy in a mouse model and in matched biopsy samples obtained from patients with secondary lymphedema comparing normal to lymphedematous arms (2.4-fold increased HMGB1, 1.9-fold increased HSP70; P < 0.01). Finally, HMGB1 blockade significantly reduced inflammatory lymphangiogenesis within inflamed draining lymph nodes (35% reduction, P < 0.01). In conclusion, HMGB1 and HSP70 are expressed along spatial gradients and upregulated in chronic lymphatic fluid stasis. Furthermore, acute expression of endogenous danger signals may play a role in inflammatory lymphangiogenesis.