Tamoxifen Attenuates Postsurgical Hindlimb Swelling and Soft-tissue Fibrosis in a Rat Model of Secondary Lymphatic Injury

Tamoxifen Attenuates Postsurgical Hindlimb Swelling and Soft-tissue Fibrosis in a Rat Model of Secondary Lymphatic Injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tamoxifen Attenuates Postsurgical Hindlimb Swelling and Soft-tissue Fibrosis in a Rat Model of Secondary Lymphatic Injury Ertugrul Kargı, Turgay Simsek, Nuh Zafer Canturk, Mustafa Dulger, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8709265/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Introduction: Secondary lymphedema is a common complication after lymph node dissection and is characterized by chronic swelling and progressive soft-tissue fibrosis. Tamoxifen has been reported to exert antifibrotic and antioxidant effects. We investigated whether tamoxifen could attenuate postsurgical limb swelling and fibrosis in a rat model of secondary lymphatic injury and explored potential oxidative mechanisms. Methods Forty Wistar–Albino rats were randomized into four groups (n = 10 each). A standardized right hindlimb lymphadenectomy was performed in all groups. Tamoxifen (2.5 mg/kg/day, oral) was administered with different schedules: 7 days preoperative + 7 days postoperative (Group 2), 14 days postoperative starting immediately (Group 3), and 14 days postoperative starting on day 14 (Group 4). Outcomes at day 30 included limb volume change (water volumetry), lymphoscintigraphy (Tc-99m nanocolloid), histopathologic fibrosis score, tissue hydroxyproline, and oxidative stress markers (MDA, GSH), as well as selected cytokines. Results Lymphoscintigraphic parameters did not differ significantly between groups. However, postoperative volumetric increase of the operated limb was significantly greater in untreated controls than in Groups 2 and 3 (p < 0.05). Histopathologic fibrosis scores were significantly lower in Groups 2 and 3 compared to the controls, supported by lower hydroxyproline levels. Tissue MDA levels were higher and GSH levels lower in controls compared with treated groups, suggesting attenuation of oxidative stress by tamoxifen. Conclusion In this rat model, tamoxifen reduced postsurgical limb swelling and soft-tissue fibrosis, accompanied by lower oxidative stress. Because lymphoscintigraphy did not demonstrate significant between-group differences and a sham-operated control group was not included, the findings support an antifibrotic/antioxidant effect on postoperative tissue changes rather than definitive prevention of lymphedema. antifibrotic effect axillary dissection breast cancer lymphedema rat tamoxifen Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Axillary dissection is commonly performed in women undergoing surgery for breast cancer, Although it does not predict the survival of these patients, axillary lymph node status is still the most valuable prognostic indicator and an effective measure in the control of regional disease. 1 However, one of the most significant complications after axillary dissection is lymphedema of the ipsilateral upper extremity, which can cause functional loss, physical discomfort, and cosmetic disfigurement. 2 Despite the prevalence and morbidity of lymphedema, there are no proven preventative measures, with most currently available treatment options being supportive and noncurative. Although surgical approaches have been described, they have not gained widespread acceptance. Impaired lymphatic regeneration and lymphatic function are a consequence of impaired lymphatic endothelial cell proliferation, an abnormal lymphatic microarchitecture, and lymphatic fibrosis, and they lead to fibrosis of the involved soft tissues. Inhibition of fibrosis using a simple topical dressing can markedly accelerate lymphatic repair and promote regeneration of the normal capillary lymphatics. 3 Tamoxifen is a selective estrogen receptor modulator administered to breast cancer patients following radiotherapy and chemotherapy. It is also an antifibrotic agent and has been used in the treatment of retroperitoneal fibrosis and Riedel’s thyroiditis. 4 , 5 The aim of this study was to evaluate whether tamoxifen attenuates postoperative limb swelling and soft-tissue fibrosis following standardized hindlimb lymphatic injury in rats, and to explore potential mechanisms focusing on oxidative stress and inflammatory mediators.. METHODS Animals and surgical procedure Forty Wistar-Albino rats weighing 200–250 g were handled in compliance with the protocol of the Experimental Medicine and Research Laboratory of Kocaeli University (DETAB), Turkey. This study was approved by the Ethics Committee for Animal Research of Kocaeli University, funded by Kocaeli University Research Fund with the number 2014/0012, and performed in accordance with internationally accepted standard guidelines for the care and use of laboratory animals. Rats were housed in stainless steel cages at a room temperature of 18–26°C and a relative humidity of 40–60%, and were provided with food and water ad libitum. The rats were randomly divided into four groups of 10 as control group (group 1) and experimental groups (groups 2, 3, and 4). Groups 2–4 received the same dose and duration of tamoxifen therapy, but it was administered at different time points to determine the optimal schedule for preventing lymphedema. Tamoxifen was administered through an orogastric tube at a dose of 2.5 mg/kg/day. Group 2 received the drug for 7 days preoperatively and 7 days postoperatively. Group 3 received the drug for 14 days postoperatively, immediately following lymphadenectomy. Group 4 received the drug for 14 days postoperatively, from day 14 to day 30 of the experiment. Animals were anesthetized with 50-mg ketamine HCl (Ketalar, Pfizer)/kg and 5-mg xylazine (Rompun, Bayer)/kg, injected intraperitoneally. The right hind foot of each anesthetized rat was then subcutaneously injected with 0.5 ml of 0.5% methylene blue at neutral pH in saline solution, for visualization of the peripheral lymph nodes. Skin, subcutaneous tissue, and underlying fascia of the right proximal thigh were removed as a 10-mm diameter tissue specimen. Lymph nodes and lymphatic vessels stained by methylene blue were removed segmentally and the proximal and distal borders were ligated. The wound was sutured with 2/0 silk, covered with an antibacterial gel, and bandaged. At the end of the 30 days, lymphedema was evaluated by lymphoscintigraphy and volumetrically (Figure 1 a–d). 6 Lymphoscintigraphy Animals were anesthetized with 50-mg ketamine HCl (Ketalar, Pfizer)/kg and 5-mg xylazine (Rompun, Bayer)/kg, injected intraperitoneally before lymphoscintigraphy Three hundred microliters of 25 μCi Tc-99m nanocolloid (Nanocis, Cisbio, France) were interdigitally injected into both hind limbs. The injected areas were massaged to distribute the Tc-99m nanocolloid within the interdigital soft tissue. The rats were allowed to move freely in their cages for 5 min Dynamic and static images of both lower extremities were obtained with a single-head gamma camera (ADAC Argus Epic, ADAC Laboratories, Milpitas, California, USA) equipped with a low-energy, general-purpose, parallel hole collimator. All images were saved and evaluated quantitatively and qualitatively. Volume estimation The volume of the right thigh of each rat was analyzed preoperatively and at the end of the experiment (day 30). Water volumetry was performed with a water-filled 50-ml graduated cylinder. The right thigh of each rat was immersed in a cylinder filled with 45 ml of water. The amount of water needed to bring the volume to 50 ml was used to calculate the thigh volume. The volume of each right thigh was measured three times. Histopathology Tissue samples were fixed in 10% neutral formalin solution. After routine histopathological procedures, the specimens were embedded in paraffin blocks and cut into 5-µm-thick sections. The tissue sections were stained with hematoxylin and eosin and Mason triple stains and then photographed. Fibrosis was detected using a scoring method that takes into account fibrosis, inflammation, and vascular proliferation, rated on a modified semi-quantitative scale of 0–3. The amount of fibrosis was scored as follows: 0, no fibrosis; 1, minimal, loose fibrosis; 2, moderate fibrosis; and 3, florid dense fibrosis. 7 A score of 2 or 3 indicated positivity for fibrosis. Biochemical analysis The rats were euthanized on day 30. Blood collected directly from the heart of each animal was allowed to clot for 1 h at room temperature and then centrifuged to obtain the serum, which was stored at −80°C for later analysis. Tissue samples were frozen and stored at −80°C until biochemical evaluation. Glutathione (GSH) levels were assayed by means of Ellman’s reagent. Malonyldialdehyde ( MDA) levels were measured in a reaction with thiobarbituric acid (TBA) followed by HPLC separation of the MDA-TBA conjugate. 8 Tissue hydroxyproline levels (Biovison), interleukin (IL)-1 and tumor necrosis factor-α (TNF-α) (Invitrogen), and tissue interferon (IFN)-γ levels (Biosource) concentrations were measured with commercial kits at the Alisei Quality System of Kocaeli University Central Laboratory. Statistical analysis The results were expressed as the means ± SD and analyzed using SPSS 17.0. Student's t -test was used in comparisons of the data. When appropriate, analysis of variance and Tukey's test for multiple comparisons were used to analyze the biochemical data. Edema percentages were compared using a χ 2 test. Differences were considered statistically significant at p<0.05. RESULTS At the end of the experiment, on day 30, the volumetric changes in the thighs of control rats were significantly higher than those of rats in experimental groups 2 and 3 (p<0.05). In addition, the thigh volume of rats in group 4 was higher than that of rats in group 2 (Table 1). Although visualization of iliac lymph nodes was used as an indicator of tracer transport through the lymphatic pathway, quantitative and qualitative lymphoscintigraphic assessments did not reveal significant between-group differences at day 30. (Figure 2). Conventional histopathological evaluation showed that fibrosis scores were significantly lower in groups 2 and 3 than in the control group (p=0.005 and p=0.033) (Figure 3). Tissue hydroxyproline levels were lower in group 2 than in the control group (p<0.05) (Table 1), thus providing biochemical support for the histopathological results. Tissue nitric oxide (NO) did not significantly differ among the four groups, nor were there significant differences in blood NO and GSH levels. Tissue GSH levels were significantly higher (p=0.043 and p=0.001) in groups 2 and 3 than in the control group and significantly lower (p<0.005) in group 4 than in group 2 (Table 2). Tissue MDA levels were significantly lower in all experimental groups compared to control animals (p=0.001) and significantly higher in group 4 than in group 2 (p<0.001). Serum MDA levels were significantly lower in groups 2 and 3 than in the control group and significantly higher in group 4 than in groups 2 and 3 (control vs. group 2, p<0.001; control vs. group 3, p<0.001; group 2 vs. group 4, p<0.001) . There was no significant difference in tissue cytokine levels except IL-1, the levels of which were higher in group 2 than in group 3 and significantly lower in group 2 than in the control (Table 3). Serum TNF levels were higher in group 4 than in groups 2 and 3 (Table 4). DISCUSSION Secondary lymphedema is a debilitating complication after lymph node dissection. In experimental settings, postsurgical limb volume changes and progressive soft-tissue fibrosis are considered key features of lymphatic injury–related tissue remodeling. In the present study, lymphoscintigraphic assessments at day 30 did not demonstrate statistically significant differences among groups; therefore, our findings should be interpreted as attenuation of postoperative limb swelling and fibrosis rather than definitive prevention of lymphedema. Chronic lymphedema is a devastating disorder that occurs in 13–27% of patients undergoing lymph node dissection for breast cancer. It has a highly negative impact on the quality of life, by impairing the function and appearance of the involved upper extremity. 9 There is no commonly accepted preventive and therapeutic strategy, 10 although potential, preventative solutions have been described. 11 , 12 Lymphedema develops because of the limited transport capacity of the injured lymph vessels, which leads to an increase in lymphatic volume and pressure and in the osmotic pressure of the interstitial space. The most important pathological changes in the lymphatic system and interstitial tissues of patients with lymphedema are dilatation of lymph vessels, functional and morphological injury of endothelial cells, and fibrosis and sclerosis of regional lymphatic networks. 13 , 14 Clinically, an association between lymphedema and fibrosis has been demonstrated. 15 – 17 In this study, lymphedema was associated with fibrosis and increased levels of hydroxyproline; the latter is indicative of the formation of connective tissue. The risk of lymphedema is higher in the presence of patient- and treatment-related factors that promote fibrosis, such as extensive surgical resection, infection, and radiation therapy. Several potential mechanisms may be responsible for the delayed lymphatic regeneration that occurs in the setting of soft-tissue fibrosis. 18 – 20 Fibrosis and sclerosis of the lymphatics are thought to lead to serious regional oxygen deficiency in lymphedematous tissue, followed by periods of reperfusion. 21 In previous studies, decreases in GSH and increases in non-reduced glutathione (GSSG) and MDA in patients with lymphedema suggested the accelerated production of oxygen free radicals and increased lipid peroxidation in chronic lymphedematous tissue. 22 , 23 In the present study, a comparison of tissue MDA and GSH levels showed significant difference in groups 2 and 3 vs. control animals, consistent with the results of previous studies. Tamoxifen is widely used in the treatment of breast cancer and has been proposed as a prophylactic agent in this disease. 24 It has also been shown to be effective in the regression of desmoid tumors, which have mesenchymal elements similar to those of retroperitoneal fibrosis. 4 , 25 The exact mechanism of action of tamoxifen is unknown but is thought to be related to its anti-inflammatory and anti-angiogenic properties. 4 Tamoxifen is an effective antioxidant that protects membranes and low-density lipoprotein (LDL) particles against oxidative damage. It inhibits the oxidation of lipoproteins such as ceratoid, which has been implicated in the pathogenesis of retroperitoneal fibrosis. 4 The antifibrotic effect of tamoxifen suggested its use in the reduction of lymphedema, which was tested in this study together with experiments aimed at elucidating the underlying mechanism of action. The reduced volumetric changes in groups 2, 3 and 4, treated with tamoxifen, compared to the untreated control group demonstrated the ability of tamoxifen to decrease lymphedema. This finding was confirmed by the fibrosis score and by the tissue hydroxyproline levels. In a previous study, tamoxifen was shown to alter the production of transforming growth factor-β, which modulates fibroblast activity and fibrosis, in soft tissues. 26 , 27 Biochemical analysis of tissue samples showed that the edematous tissues of group 2 rats contained significantly lower concentrations of MDA and significantly higher levels of GSH than those of group 4 rats. This observation is consistent with an antioxidant effect of tamoxifen on lymphedema beginning during the early period of tissue injury. Tamoxifen is currently not prescribed for lymphedema prevention in the immediate preoperative period; however it can be given to patients soon after mastectomy or before they undergo radiation therapy. There was no significant difference in tissue NO levels among the treated and untreated groups. NO decreases the contraction amplitude of the lymphatic pump and inhibits pump activity. 28 – 31 In a previous study using lymphangiography and intravital microscopy to quantify lymph flow, we found that lymph flow is decreased in endothelial nitric oxide synthase (eNOS)-knockout mice. 31 , 32 Since, in this study, measurements of NO levels in tissue and in blood did not show significant difference between any of the groups with surgically induced lymphedema, the effect of tamoxifen is probably not related to NO content, although NO is known to effect the function of the microlymphatic system. We also investigated the effects of tamoxifen on TNF-α levels in the four groups of rats. Tissue TNF-α levels in the tamoxifen-treated groups did not differ significantly from those of the control group. However, serum TNF-α levels were significantly lower in groups 2 and 3 than in group 4. In the previous study, molecular characterization of whole-tissue homogenates from mice with lymphedema revealed high levels of inflammatory mediators such as TNF-α. 33,34 Based on our results, tamoxifen treatment was associated with smaller postoperative limb volume increase and reduced histopathologic fibrosis, supported by hydroxyproline measurements. The lower MDA and higher GSH levels in tamoxifen-treated groups are consistent with reduced lipid peroxidation and improved antioxidant capacity, which may contribute to decreased fibrotic remodeling. Tamoxifen is traditionally administered to patients after chemotherapy and radiotherapy. While this protocol may be slow to change, our results strongly suggest that tamoxifen therapy should be started in the immediate postoperative period. This study did not include a sham-operated group (skin incision and tissue handling without lymphadenectomy), which would have helped distinguish nonspecific postoperative edema from lymphatic insufficiency–related swelling. In addition, measurements were obtained at baseline and day 30; serial assessments would better characterize the temporal course of swelling and tissue remodeling. Finally, lymphoscintigraphy did not reveal significant differences, which limits conclusions regarding lymphatic transport; thus, the observed benefits likely reflect antifibrotic/antioxidant effects on postoperative tissue changes. Tamoxifen attenuated postsurgical limb swelling and soft-tissue fibrosis in this rat lymphatic injury model, accompanied by reduced oxidative stress markers; however, definitive prevention of lymphedema cannot be concluded from the current assessments. Our findings remain to be confirmed in further experimental and clinical studies. Declarations Author Contribution EK, Researcher, Author, StatisticianTS, Researcher, AuthorNZC, Mentor, Corresponding Author, Researcher,MD, Mentor, ResearcherHD, Researcher for Nuclear Medicine MC, Laboratory researchCE, Pathological evaluation Acknowledgement We thank Dr.Atilla Soran, MD (Director of the Lymphedema Program from Magee-Womens Hospital of UPMC, Pittsburgh, PA) for his support and encouragement during the planning project. We also thank Dr. Jennifer Erdrich for preparing, criticizing, and editing the manuscript as a native English speaker. This study was supported by Kocaeli University Research Fund with project ID 4883 and project code 2014/0012. References Guo W, Zhang L, Ji J, Gao W, Liu J, Tong M. Evaluation of the benefit of using blue dye in addition to indocyanine green fluorescence for sentinel lymph node biopsy in patients with breast cancer. World J Surg Oncol 2014; 12:290. Jung SY, Shin KH, Kim M, Chung SH, Lee S, Kang HS, et al. 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Inflammatory manifestations of experimental lymphatic insufficiency. PLoS Med 2006; 3(7):e254. Nakamura K, Radhakrishnan K, Wong YM, Rockson SG. Anti-inflammatory pharmacotherapy with ketoprofen ameliorates experimental lymphatic vascular insufficiency in mice. PLoS One 2009; 4(12):e8380. Tables Table 1: Lymphedema in tamoxifen-treated (groups 2–4) and untreated control (group 1) rats; n=10 rats/group. Scintigraphy a Volumetric changes Fibrosis b Hydroxyproline +/- +/- Group 1 6/4 2.70±0.75 10/0 0.84±0.15 Group 2 7/3 1.10±0.61 4/6 0.68±0.23 Group 3 4/5 1.72±0.44 5/4 1.10±0.40 Group 4 4/6 2.25±0.68 7/3 0.92±0.22 a Number of rats positive or negative for scintigraphy determined by transport capacity of the injured lymph vessels b Number of rats positive or negative for fibrosis determined by the amount of fibrosis was scored as follows: 0, no fibrosis; 1, minimal, loose fibrosis; 2, moderate fibrosis; and 3, florid dense fibrosis. A score of 2 or 3 indicated positivity for fibrosis. Fibrosis: Control vs. group 2, p=0.005; control vs. group 3, p=0.033 Hydroxyproline: Control vs. group 2, p<0.05; group 2 vs. group 3, p<0.01 Volumetric change:. Control vs. group 2, p<0.001; control vs. group 3, p=0.01; group 2 vs. group 4, p=0.002 Table 2: Tissue levels of nitric oxide (NO), malondialdehyde (MDA), and reduced glutathione (GSH) in tamoxifen-treated (groups 2–4) and untreated control (group 1) rats; n=10 rats/group NO (nanomol/ g) MDA (nanomol/ g) GSH (nanomol/ g) Group 1 98.43±14.00 68.29±18.2 3.52±1.47 Group 2 84.55±27.48 33.15±11.55 6.46±1.65 Group 3 107.23±22.41 31.98±13.77 5.46±.53 Group 4 86.67±19.71 37.13±9.66 4.12±1.10 MDA: Control vs. group 2, p<0.001; control vs. group 3, p<0.001; group 2 vs. group 4, p<0.001 GSH; Control vs. group 3, p<0.043; control vs. group 2, p<0.001; group 2 vs. group 4, p<0.005 Table 3: Tissue cytokine levels in tamoxifen-treated (groups 2–4) and untreated control (group 1) rats; n=10 rats/group IFN (IU/ml) IL-1 (picogram/ml) TNF (picogram/ml) Group 1 1.70±0.83 152.20±48.38 99.74±38.29 Group 2 1.015±0.53 109.12±27.03 85.24±15.24 Group 3 1.49±0.58 146.54±47.29 116.76±42.72 Group 4 1.52±0.65 167.46±38.46 98.09±39.68 IL-1; Group 2 vs. group 4, p<0.001 Table 4: Blood nitric oxide (NO), malonyldialdehyde (MDA), reduced glutathione (GSH), and tumor necrosis factor-α (TNF- α) levels in tamoxifen-treated (groups 2–4) and untreated control (group 1) rats; n=10 rats/group NO (nanomol/ml plasma) MDA (nanomol/ ml plasma) GSH (nanomol/ ml plasma) TNF (picogram/ml plasma) Group 1 36.52±14.66 6.50±1.44 1.16±0.44 47.46±18.68 Group 2 31.38±9.88 3.90±1.13 0.97±0.28 41.18±15.76 Group 3 36.62±14.10 3.86±1.44 0.90±0.42 38.77±7.72 Group 4 30.24±10.07 6.96±2.14 1.30±0.46 59.33±13.03 MDA: Control vs. group 2, p<0.004; control vs. group 3, p<0.005; group 3 vs. group 4, p<0.001; group 2 vs. group 4, p<0.001 TNF: Group 2 vs. group 4, p<0.047; group 3 vs. group 4, p<0.02 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 16 Feb, 2026 Reviewers agreed at journal 15 Feb, 2026 Reviews received at journal 14 Feb, 2026 Reviewers agreed at journal 14 Feb, 2026 Reviews received at journal 14 Feb, 2026 Reviewers agreed at journal 14 Feb, 2026 Reviewers agreed at journal 13 Feb, 2026 Reviews received at journal 12 Feb, 2026 Reviewers agreed at journal 12 Feb, 2026 Reviewers invited by journal 12 Feb, 2026 Editor assigned by journal 02 Feb, 2026 Submission checks completed at journal 02 Feb, 2026 First submitted to journal 27 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8709265","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":591932985,"identity":"386e37aa-0678-4ef6-a1ad-e005fb232aaa","order_by":0,"name":"Ertugrul Kargı","email":"","orcid":"","institution":"Kocaeli University","correspondingAuthor":false,"prefix":"","firstName":"Ertugrul","middleName":"","lastName":"Kargı","suffix":""},{"id":591932997,"identity":"9b555370-d0ec-4563-992c-0392a4b3cc0d","order_by":1,"name":"Turgay Simsek","email":"","orcid":"","institution":"Kocaeli University","correspondingAuthor":false,"prefix":"","firstName":"Turgay","middleName":"","lastName":"Simsek","suffix":""},{"id":591932999,"identity":"db3cc97d-08a6-4b55-9259-93c0d9fe66c3","order_by":2,"name":"Nuh Zafer Canturk","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYPCCBAZ+HjDDAkQZEFLO2ADSItkD5khAtSQQocXgDEQLA0Et/O1njz/m3ZNmb3zm8MEPPyokZBjYm7dJMP64h1OLxJm8xGaeZznMZmfbkiV7zgAdxnOsTIIhoRinFgOGHMNmngMVbGbnecwYeNuAWiRyzIBacLvMgP8NWAuPcT+PGePff0At8m8IaJEA25IjYcDbY8bM2wCyhQe/FokbbwxnzjmQZiBx5liytMwxCR42nrRii4Q03Fr4+3MMPrw5kGzP35N88OObGht7fvbDG298sMGtBROwgQhSNIyCUTAKRsEowAQArYBHXAkt+FwAAAAASUVORK5CYII=","orcid":"","institution":"Kocaeli University","correspondingAuthor":true,"prefix":"","firstName":"Nuh","middleName":"Zafer","lastName":"Canturk","suffix":""},{"id":591933001,"identity":"3e29e732-5259-499e-bc46-9b0377a84f36","order_by":3,"name":"Mustafa Dulger","email":"","orcid":"","institution":"Kocaeli University","correspondingAuthor":false,"prefix":"","firstName":"Mustafa","middleName":"","lastName":"Dulger","suffix":""},{"id":591933004,"identity":"51c7726b-11e3-4a67-9015-e0d66ce45c9a","order_by":4,"name":"Hakan Demir","email":"","orcid":"","institution":"Kocaeli University","correspondingAuthor":false,"prefix":"","firstName":"Hakan","middleName":"","lastName":"Demir","suffix":""},{"id":591933010,"identity":"b7380e07-d295-4193-b850-370981f5dc93","order_by":5,"name":"Mustafa Cekmen","email":"","orcid":"","institution":"Kocaeli University","correspondingAuthor":false,"prefix":"","firstName":"Mustafa","middleName":"","lastName":"Cekmen","suffix":""},{"id":591933017,"identity":"ea1691bd-3b98-484a-99c4-2d7ea64d73fe","order_by":6,"name":"Cengiz Ercin","email":"","orcid":"","institution":"Kocaeli University","correspondingAuthor":false,"prefix":"","firstName":"Cengiz","middleName":"","lastName":"Ercin","suffix":""}],"badges":[],"createdAt":"2026-01-27 10:39:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8709265/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8709265/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102933685,"identity":"dc3dea8d-a143-4820-bf70-6335db2ea49f","added_by":"auto","created_at":"2026-02-18 15:37:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6268070,"visible":true,"origin":"","legend":"\u003cp\u003ea-b)Skin, subcutaneous tissue, and fascia from the proximal thigh were removed. c) Lymph nodes and vessels, which had been stained with methylene blue, were removed segmentally and ligated, d) a sample of developed lymphedema at the end of the 30th day.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8709265/v1/d72b1a7ede253babeca2755b.png"},{"id":102933683,"identity":"311217f6-506a-4538-92ee-23d059d56d25","added_by":"auto","created_at":"2026-02-18 15:37:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":326367,"visible":true,"origin":"","legend":"\u003cp\u003ea-b. A sample of a) positive lymphoscintigraphy and b) negative lymphoscintigraphy\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8709265/v1/2f40068b30510ae121cea670.png"},{"id":102933684,"identity":"fac794a9-2fb5-4bcf-902f-bcff14aa52df","added_by":"auto","created_at":"2026-02-18 15:37:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2269944,"visible":true,"origin":"","legend":"\u003cp\u003ea-b: Histopathological samples for lymphedema\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8709265/v1/322321ddc8a329e21f912e0a.png"},{"id":103050001,"identity":"c669cc7d-d8a1-4c4c-bfda-01b7e1d6c576","added_by":"auto","created_at":"2026-02-20 07:47:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10145813,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8709265/v1/be3fdc87-e5d5-4dce-9ce0-c858da3321f3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eTamoxifen Attenuates Postsurgical Hindlimb Swelling and Soft-tissue Fibrosis in a Rat Model of Secondary Lymphatic Injury\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAxillary dissection is commonly performed in women undergoing surgery for breast cancer, Although it does not predict the survival of these patients, axillary lymph node status is still the most valuable prognostic indicator and an effective measure in the control of regional disease.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e However, one of the most significant complications after axillary dissection is lymphedema of the ipsilateral upper extremity, which can cause functional loss, physical discomfort, and cosmetic disfigurement.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Despite the prevalence and morbidity of lymphedema, there are no proven preventative measures, with most currently available treatment options being supportive and noncurative. Although surgical approaches have been described, they have not gained widespread acceptance. Impaired lymphatic regeneration and lymphatic function are a consequence of impaired lymphatic endothelial cell proliferation, an abnormal lymphatic microarchitecture, and lymphatic fibrosis, and they lead to fibrosis of the involved soft tissues. Inhibition of fibrosis using a simple topical dressing can markedly accelerate lymphatic repair and promote regeneration of the normal capillary lymphatics.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Tamoxifen is a selective estrogen receptor modulator administered to breast cancer patients following radiotherapy and chemotherapy. It is also an antifibrotic agent and has been used in the treatment of retroperitoneal fibrosis and Riedel\u0026rsquo;s thyroiditis.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e The aim of this study was to evaluate whether tamoxifen attenuates postoperative limb swelling and soft-tissue fibrosis following standardized hindlimb lymphatic injury in rats, and to explore potential mechanisms focusing on oxidative stress and inflammatory mediators..\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"METHODS","content":"\u003ch2\u003eAnimals and surgical procedure\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eForty Wistar-Albino rats weighing 200\u0026ndash;250 g were handled in compliance with the protocol of the Experimental Medicine and Research Laboratory of Kocaeli University (DETAB), Turkey. This study was approved by the Ethics Committee for Animal Research of Kocaeli University, funded by Kocaeli University Research Fund with the number 2014/0012, and performed in accordance with internationally accepted standard guidelines for the care and use of laboratory animals. Rats were housed in stainless steel cages at a room temperature of 18\u0026ndash;26\u0026deg;C and a relative humidity of 40\u0026ndash;60%, and were provided with food and water ad libitum. The rats were randomly divided into four groups of 10 as control group (group 1) and experimental groups (groups 2, 3, and 4). Groups 2\u0026ndash;4 received the same dose and duration of tamoxifen therapy, but it was administered at different time points to determine the optimal schedule for preventing lymphedema. Tamoxifen was administered through an orogastric tube at a dose of 2.5 mg/kg/day. Group 2 received the drug for 7 days preoperatively and 7 days postoperatively. Group 3 received the drug for 14 days postoperatively, immediately following lymphadenectomy. Group 4 received the drug for 14 days postoperatively, from day 14 to day 30 of the experiment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnimals were anesthetized with 50-mg ketamine HCl (Ketalar, Pfizer)/kg and 5-mg xylazine (Rompun, Bayer)/kg, injected intraperitoneally. The right hind foot of each anesthetized rat was then subcutaneously injected with 0.5 ml of 0.5% methylene blue at neutral pH in saline solution, for visualization of the peripheral lymph nodes. Skin, subcutaneous tissue, and underlying fascia of the right proximal thigh were removed as a 10-mm diameter tissue specimen. Lymph nodes and lymphatic vessels stained by methylene blue were removed segmentally and the proximal and distal borders were ligated. The wound was sutured with 2/0 silk, covered with an antibacterial gel, and bandaged. At the end of the 30 days, lymphedema was evaluated by lymphoscintigraphy and volumetrically (Figure 1 a\u0026ndash;d).\u003csup\u003e6\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eLymphoscintigraphy\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eAnimals were anesthetized with 50-mg ketamine HCl (Ketalar, Pfizer)/kg and 5-mg xylazine (Rompun, Bayer)/kg, injected intraperitoneally before lymphoscintigraphy Three hundred microliters of 25 \u0026mu;Ci Tc-99m nanocolloid (Nanocis, Cisbio, France) were interdigitally injected into both hind limbs. The injected areas were massaged to distribute the Tc-99m nanocolloid within the interdigital soft tissue. The rats were allowed to move freely in their cages for 5 min Dynamic and static images of both lower extremities were obtained with a single-head gamma camera (ADAC Argus Epic, ADAC Laboratories, Milpitas, California, USA) equipped with a low-energy, general-purpose, parallel hole collimator. All images were saved and evaluated quantitatively and qualitatively.\u003c/p\u003e\n\u003ch2\u003eVolume estimation\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe volume of the right thigh of each rat was analyzed preoperatively and at the end of the experiment (day 30). Water volumetry was performed with a water-filled 50-ml graduated cylinder. The right thigh of each rat was immersed in a cylinder filled with 45 ml of water. The amount of water needed to bring the volume to 50 ml was used to calculate the thigh volume. The volume of each right thigh was measured three times.\u003c/p\u003e\n\u003ch2\u003eHistopathology\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eTissue samples were fixed in 10% neutral formalin solution. After routine histopathological procedures, the specimens were embedded in paraffin blocks and cut into 5-\u0026micro;m-thick sections. The tissue sections were stained with hematoxylin and eosin and Mason triple stains and then photographed. Fibrosis was detected using a scoring method that takes into account fibrosis, inflammation, and vascular proliferation, rated on a modified semi-quantitative scale of 0\u0026ndash;3. The amount of fibrosis was scored as follows: 0, no fibrosis; 1, minimal, loose fibrosis; 2, moderate fibrosis; and 3, florid dense fibrosis.\u003csup\u003e7\u003c/sup\u003e A score of 2 or 3 indicated positivity for fibrosis.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eBiochemical analysis\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe rats were euthanized on day 30. Blood collected directly from the heart of each animal was allowed to clot for 1 h at room temperature and then centrifuged to obtain the serum, which was stored at \u0026minus;80\u0026deg;C for later analysis. Tissue samples were frozen and stored at \u0026minus;80\u0026deg;C until biochemical evaluation. Glutathione (GSH) levels were assayed by means of Ellman\u0026rsquo;s reagent.\u003csup\u003e\u0026nbsp;\u003c/sup\u003eMalonyldialdehyde\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003eMDA) levels were measured in a reaction with thiobarbituric acid (TBA) followed by HPLC separation of the MDA-TBA conjugate.\u003csup\u003e8\u003c/sup\u003e Tissue hydroxyproline levels (Biovison), interleukin (IL)-1 and tumor necrosis factor-\u0026alpha; (TNF-\u0026alpha;) (Invitrogen), and tissue interferon (IFN)-\u0026gamma; levels (Biosource) concentrations were measured with commercial kits at the Alisei Quality System of Kocaeli University Central Laboratory.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe results were expressed as the means \u0026plusmn; SD and analyzed using SPSS 17.0. Student\u0026apos;s \u003cem\u003et\u003c/em\u003e-test was used in comparisons of the data. When appropriate, analysis of variance and Tukey\u0026apos;s test for multiple comparisons were used to analyze the biochemical data. Edema percentages were compared using a \u0026chi;\u003csup\u003e2\u003c/sup\u003e test. Differences were considered statistically significant at p\u0026lt;0.05.\u0026nbsp;\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eAt the end of the experiment, on day 30, the volumetric changes in the thighs of control rats were significantly higher than those of rats in experimental groups 2 and 3 (p\u0026lt;0.05). In addition, the thigh volume of rats in group 4 was higher than that of rats in group 2 (Table 1).\u0026nbsp;Although visualization of iliac lymph nodes was used as an indicator of tracer transport through the lymphatic pathway, quantitative and qualitative lymphoscintigraphic assessments did not reveal significant between-group differences at day 30.\u0026nbsp;(Figure 2). Conventional histopathological evaluation showed that fibrosis scores were significantly lower in groups 2 and 3 than in the control group (p=0.005 and p=0.033) (Figure 3).\u0026nbsp;Tissue hydroxyproline levels were lower in group 2 than in the control group (p\u0026lt;0.05) (Table 1), thus providing biochemical support for the histopathological results.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTissue nitric oxide (NO) did not significantly differ among the four groups, nor were there significant differences in blood NO and GSH levels. Tissue GSH levels were significantly higher (p=0.043 and p=0.001) in groups 2 and 3 than in the control group and significantly lower (p\u0026lt;0.005) in group 4 than in group 2 (Table 2). Tissue MDA levels were significantly lower in all experimental groups compared to control animals (p=0.001) and significantly higher in group 4 than in group 2 (p\u0026lt;0.001). Serum MDA levels were significantly lower in groups 2 and 3 than in the control group and significantly higher in group 4 than in groups 2 and 3 (control vs. group 2, p\u0026lt;0.001; control vs. group 3, p\u0026lt;0.001; group 2 vs. group 4, p\u0026lt;0.001)\u003csub\u003e.\u0026nbsp;\u003c/sub\u003eThere was no significant difference in tissue cytokine levels except IL-1, the levels of which were higher in group 2 than in group 3 and significantly lower in group 2 than in the control (Table 3). Serum TNF levels were higher in group 4 than in groups 2 and 3 (Table 4).\u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSecondary lymphedema is a debilitating complication after lymph node dissection. In experimental settings, postsurgical limb volume changes and progressive soft-tissue fibrosis are considered key features of lymphatic injury\u0026ndash;related tissue remodeling. In the present study, lymphoscintigraphic assessments at day 30 did not demonstrate statistically significant differences among groups; therefore, our findings should be interpreted as attenuation of postoperative limb swelling and fibrosis rather than definitive prevention of lymphedema. Chronic lymphedema is a devastating disorder that occurs in 13\u0026ndash;27% of patients undergoing lymph node dissection for breast cancer. It has a highly negative impact on the quality of life, by impairing the function and appearance of the involved upper extremity.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e There is no commonly accepted preventive and therapeutic strategy,\u003csup\u003e10\u003c/sup\u003e although potential, preventative solutions have been described.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Lymphedema develops because of the limited transport capacity of the injured lymph vessels, which leads to an increase in lymphatic volume and pressure and in the osmotic pressure of the interstitial space. The most important pathological changes in the lymphatic system and interstitial tissues of patients with lymphedema are dilatation of lymph vessels, functional and morphological injury of endothelial cells, and fibrosis and sclerosis of regional lymphatic networks.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Clinically, an association between lymphedema and fibrosis has been demonstrated.\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e In this study, lymphedema was associated with fibrosis and increased levels of hydroxyproline; the latter is indicative of the formation of connective tissue.\u003c/p\u003e \u003cp\u003eThe risk of lymphedema is higher in the presence of patient- and treatment-related factors that promote fibrosis, such as extensive surgical resection, infection, and radiation therapy. Several potential mechanisms may be responsible for the delayed lymphatic regeneration that occurs in the setting of soft-tissue fibrosis.\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Fibrosis and sclerosis of the lymphatics are thought to lead to serious regional oxygen deficiency in lymphedematous tissue, followed by periods of reperfusion.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e In previous studies, decreases in GSH and increases in non-reduced glutathione (GSSG) and MDA in patients with lymphedema suggested the accelerated production of oxygen free radicals and increased lipid peroxidation in chronic lymphedematous tissue.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e In the present study, a comparison of tissue MDA and GSH levels showed significant difference in groups 2 and 3 vs. control animals, consistent with the results of previous studies.\u003c/p\u003e \u003cp\u003eTamoxifen is widely used in the treatment of breast cancer and has been proposed as a prophylactic agent in this disease.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e It has also been shown to be effective in the regression of desmoid tumors, which have mesenchymal elements similar to those of retroperitoneal fibrosis.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e The exact mechanism of action of tamoxifen is unknown but is thought to be related to its anti-inflammatory and anti-angiogenic properties.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Tamoxifen is an effective antioxidant that protects membranes and low-density lipoprotein (LDL) particles against oxidative damage. It inhibits the oxidation of lipoproteins such as ceratoid, which has been implicated in the pathogenesis of retroperitoneal fibrosis.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e The antifibrotic effect of tamoxifen suggested its use in the reduction of lymphedema, which was tested in this study together with experiments aimed at elucidating the underlying mechanism of action. The reduced volumetric changes in groups 2, 3 and 4, treated with tamoxifen, compared to the untreated control group demonstrated the ability of tamoxifen to decrease lymphedema. This finding was confirmed by the fibrosis score and by the tissue hydroxyproline levels. In a previous study, tamoxifen was shown to alter the production of transforming growth factor-β, which modulates fibroblast activity and fibrosis, in soft tissues.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Biochemical analysis of tissue samples showed that the edematous tissues of group 2 rats contained significantly lower concentrations of MDA and significantly higher levels of GSH than those of group 4 rats. This observation is consistent with an antioxidant effect of tamoxifen on lymphedema beginning during the early period of tissue injury. Tamoxifen is currently not prescribed for lymphedema prevention in the immediate preoperative period; however it can be given to patients soon after mastectomy or before they undergo radiation therapy.\u003c/p\u003e \u003cp\u003eThere was no significant difference in tissue NO levels among the treated and untreated groups. NO decreases the contraction amplitude of the lymphatic pump and inhibits pump activity.\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e In a previous study using lymphangiography and intravital microscopy to quantify lymph flow, we found that lymph flow is decreased in endothelial nitric oxide synthase (eNOS)-knockout mice.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Since, in this study, measurements of NO levels in tissue and in blood did not show significant difference between any of the groups with surgically induced lymphedema, the effect of tamoxifen is probably not related to NO content, although NO is known to effect the function of the microlymphatic system. We also investigated the effects of tamoxifen on TNF-α levels in the four groups of rats. Tissue TNF-α levels in the tamoxifen-treated groups did not differ significantly from those of the control group. However, serum TNF-α levels were significantly lower in groups 2 and 3 than in group 4. In the previous study, molecular characterization of whole-tissue homogenates from mice with lymphedema revealed high levels of inflammatory mediators such as TNF-α.\u003csup\u003e33,34\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBased on our results, tamoxifen treatment was associated with smaller postoperative limb volume increase and reduced histopathologic fibrosis, supported by hydroxyproline measurements. The lower MDA and higher GSH levels in tamoxifen-treated groups are consistent with reduced lipid peroxidation and improved antioxidant capacity, which may contribute to decreased fibrotic remodeling. Tamoxifen is traditionally administered to patients after chemotherapy and radiotherapy. While this protocol may be slow to change, our results strongly suggest that tamoxifen therapy should be started in the immediate postoperative period. This study did not include a sham-operated group (skin incision and tissue handling without lymphadenectomy), which would have helped distinguish nonspecific postoperative edema from lymphatic insufficiency\u0026ndash;related swelling. In addition, measurements were obtained at baseline and day 30; serial assessments would better characterize the temporal course of swelling and tissue remodeling. Finally, lymphoscintigraphy did not reveal significant differences, which limits conclusions regarding lymphatic transport; thus, the observed benefits likely reflect antifibrotic/antioxidant effects on postoperative tissue changes. Tamoxifen attenuated postsurgical limb swelling and soft-tissue fibrosis in this rat lymphatic injury model, accompanied by reduced oxidative stress markers; however, definitive prevention of lymphedema cannot be concluded from the current assessments. Our findings remain to be confirmed in further experimental and clinical studies.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eEK, Researcher, Author, StatisticianTS, Researcher, AuthorNZC, Mentor, Corresponding Author, Researcher,MD, Mentor, ResearcherHD, Researcher for Nuclear Medicine MC, Laboratory researchCE, Pathological evaluation\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Dr.Atilla Soran, MD (Director of the Lymphedema Program from Magee-Womens Hospital of UPMC, Pittsburgh, PA) for his support and encouragement during the planning project. We also thank Dr. Jennifer Erdrich for preparing, criticizing, and editing the manuscript as a native English speaker. This study was supported by Kocaeli University Research Fund with project ID 4883 and project code 2014/0012.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGuo W, Zhang L, Ji J, Gao W, Liu J, Tong M. Evaluation of the benefit of using blue dye in addition to indocyanine green fluorescence for sentinel lymph node biopsy in patients with breast cancer. World J Surg Oncol 2014; 12:290.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eJung SY, Shin KH, Kim M, Chung SH, Lee S, Kang HS, et al. Treatment factors affecting breast cancer-related lymphedema after systemic chemotherapy and radiotherapy in stage II/III breast cancer patients. Breast Cancer Res Treat 2014;148(1):91-8.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKootstra JJ, Dijkstra PU, Rietman H, de Vries J, Baas P, Geertzen JH, et al. A longitudinal study of shoulder and arm morbidity in breast cancer survivors 7 years after sentinel lymph node biopsy or axillary lymph node dissection. Breast Cancer Res Treat 2013; 139(1):125-34.\u003c/li\u003e\n \u003cli\u003eBrandt AS, Kamper L, Kukuk S, Haage P, Roth S. Tamoxifen monotherapy in the treatment of retroperitoneal fibrosis. Urol Int 2014; 93(3):320-5.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePritchyk K, Newkirk K, Garlich P, Deeb Z. Tamoxifen therapy for Riedel\u0026apos;s thyroiditis. Laryngoscope 2004; 114(10):1758-60.\u003c/li\u003e\n \u003cli\u003ePark HS, Jung IM, Choi GH, Hahn S, Yoo YS, Lee T. Modification of a rodent hindlimb model of secondary lymphedema: surgical radicality versus radiotherapeutic ablation. Biomed Res Int 2013; 2013:208912.\u003c/li\u003e\n \u003cli\u003eErsoy E, Ozturk V, Yazgan A, Ozdogan M, Gundogdu H. Comparison of the two types of bioresorbable bariers to prevent intra-abdominal adhesions in rats. J Gastrointest Surg 2009; 13(2):282-6.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHe LF, Chen JG. DNA damage, apoptosis and cell cycle changes induced by fluoride in rat oral mucosal cells and hepatocytes. World J Gastroenterol 2006; 12(7):1144-8.\u003c/li\u003e\n \u003cli\u003eSmile TD, Tendulkar R, Schwarz G, Arthur D, Grobmyer S, Valente S, Vicini F, Shah C. A Review of Treatment for Breast Cancer-Related Lymphedema: Paradigms for Clinical Practice. Am J Clin Oncol. 2018;41(2):178-190.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSchaverien MV, Coroneos CJ. Surgical Treatment of Lymphedema. Plast Reconstr Surg 2019;144(3):738-758.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWarren AG, Brorson H, Borud LJ, Slavin SA. Lymphedema: a comprehensive review. Ann Plast Surg 2007; 59(4):464-72.\u003c/li\u003e\n \u003cli\u003eKareh AM, Xu KY. Surgical Management of Lymphedema. Mo Med. 2020;117(2):143-148. (PMID: 32308240).\u003c/li\u003e\n \u003cli\u003eRockson SG. Lymphedema. Am J Med 2001; 110(4):288-95.\u003c/li\u003e\n \u003cli\u003eRockson SG. Diagnosis and management of lymphatic vascular disease. J Am Coll Cardiol 2008; 52(10):799-806.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDaroczy J. Pathology of lymphedema. Clin Dermatol 1995; 13(5):433-44.\u003c/li\u003e\n \u003cli\u003eTalarico F, Brunetto D, Scialabba \u0026nbsp;M, Pernice I, Valenti G, Benvegna S, et al. \u0026nbsp; Fibrosclerotic lymphedema: pathophysiology and therapy. \u0026nbsp;Lymphology 1991; 24(1):11-5.\u003c/li\u003e\n \u003cli\u003eKataru RP, Wiser I, Baik JE, Park HJ, Rehal S, Shin JY, Mehrara BJ. Fibrosis and secondary lymphedema: chicken or egg? Transl Res 2019; 209:68-76.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHinrich CS, Watroba NL, Rezaishiraz H, Giese W, Hurd T, Fassl KA, et al. Lymphedema secondary to postmastectomy radiation: Incidence and risk factors. Ann Surg Oncol 2004; 11(6):573-80.\u003c/li\u003e\n \u003cli\u003eAbu-Rustum NR, Alektiar K, Iasonos A, Lev G, Sonoda Y, Aghajanian C, et al. The incidence of symptomatic lower-extremity lymphedema following treatment of uterine corpus malignancies: a 12-year experience at Memorial Sloan-Kettering Cancer Center. Gynecol Oncol 2006 ; 103(2):714-8.\u003c/li\u003e\n \u003cli\u003eSakorafas GH, Peros G, Cataliotti L, Vlastos G. Lymphedema \u0026nbsp;following axillary lymph node dissection for breast cancer. Surg Oncol 2006; 15(3):153-65.\u003c/li\u003e\n \u003cli\u003eWitte WH, Bernas MJ, Martin CP, Witte CL. Lymphangiogenesis and lymphangiodysplasia: from molecular to clinical lymphology. Microsc Res Tech 2001; 55(2):122-45.\u003c/li\u003e\n \u003cli\u003eSiems WG,Brenke R, Sommerburg O, Grune T. Improved antioxidative protection in winter swimmers. QJM 1999; 92:193-8.\u003c/li\u003e\n \u003cli\u003eSiems WG, Brenke R, Beier A, Grune T. Oxidative stress in chronic lymphedema. QJM 2002; 95(12):803-9.\u003c/li\u003e\n \u003cli\u003eHuang B, Warner M, Gustafsson JA. Estrogen receptors in breast carcinogenesis and endocrine therapy. Mol Cell Endocrinol 2014; pii: S0303-7207(14)00371-2.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBocale D, Rotelli MT, Cavallini A, Altomare DF. Anti-oestrogen therapy in the treatment of desmoid tumours: a systematic review. Colorectal Dis 2011; 13(12):e388-95.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGilkeson GS, Allen NB. Retroperitoneal fibrosis. A true connective tissue disease. Rheum Dis Clin North Am 1996; 22(1):23-38.\u003c/li\u003e\n \u003cli\u003eWakabayashi K, Hamada C, Kanda R, Nakano T, Io H, Horikoshi S, et al. Oral Astaxanthin Supplementation Prevents Peritoneal Fibrosis in Rats. Perit Dial Int 2015; 35(5):506-16.\u003c/li\u003e\n \u003cli\u003eMizuno R, Koller A, Kaley G. Regulation of the vasomotor activity of lymph microvessels by nitric oxide and prostaglandins. Am J Physiol 1998; 274(3 Pt 2):R790-6.\u003c/li\u003e\n \u003cli\u003eShirasawa Y, Ikomi F, Ohhashi T. Physiological roles of endogenous nitric oxide in lymphatic pump activity of rat mesentery in vivo. Am J Physiol Gastrointest Liver Physiol 2000; 278(4):G551-6.\u003c/li\u003e\n \u003cli\u003eGashew AA, Davis MJ, Zawieja DC. Inhibition of active lymph pumps by flow in rat mesenteric lymphatics and thoracic duct. J Physiol 2002; 540(Pt 3):1023-37.\u003c/li\u003e\n \u003cli\u003eHagendoorn J, Padera TP, Fukumura D, Jain RK. Molecular regulation of micro lymphatic formation and function: Role of nitric oxide. Trends Cardiovasc Med 2005; 15:169-73.\u003c/li\u003e\n \u003cli\u003eHagendoorn J, Padera TP, Kashiwagi S, Isaka N, Noda F, Lin MI, et al. Endothelial nitric oxide synthase regulates microlymphatic flow via collecting lymphatics. Circ Res 2004; 95(2):204-9.\u003c/li\u003e\n \u003cli\u003eTabibiazar R, Cheung L, Han J, Swanson J, Bailhack A, Dadras SS, et al. Inflammatory manifestations of experimental lymphatic insufficiency. PLoS Med 2006; 3(7):e254.\u003c/li\u003e\n \u003cli\u003eNakamura K, Radhakrishnan K, Wong YM, Rockson SG. Anti-inflammatory pharmacotherapy with ketoprofen ameliorates experimental lymphatic vascular insufficiency in mice. PLoS One 2009; 4(12):e8380.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1: Lymphedema in tamoxifen-treated (groups 2\u0026ndash;4) and untreated control (group 1) rats; n=10 rats/group.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"662\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eScintigraphy\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eVolumetric changes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eFibrosis\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eHydroxyproline\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e+/-\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e+/-\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eGroup \u0026nbsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e6/4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e2.70\u0026plusmn;0.75\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e10/0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e0.84\u0026plusmn;0.15\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eGroup 2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e7/3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e1.10\u0026plusmn;0.61\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e4/6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e0.68\u0026plusmn;0.23\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eGroup 3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e4/5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e1.72\u0026plusmn;0.44\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e5/4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e1.10\u0026plusmn;0.40\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eGroup 4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e4/6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e2.25\u0026plusmn;0.68\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e7/3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e0.92\u0026plusmn;0.22\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eNumber of rats positive or negative for scintigraphy determined by transport capacity of the injured lymph vessels\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003csup\u003eb\u003c/sup\u003eNumber of rats positive or negative for fibrosis determined by the amount of fibrosis was scored as follows: 0, no fibrosis; 1, minimal, loose fibrosis; 2, moderate fibrosis; and 3, florid dense fibrosis. A score of 2 or 3 indicated positivity for fibrosis. \u0026nbsp;Fibrosis: Control vs. group 2, p=0.005; control vs. group 3, p=0.033 \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHydroxyproline: Control vs. group 2, p\u0026lt;0.05; group 2 vs. group 3, p\u0026lt;0.01\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVolumetric change:. Control vs. group 2, p\u0026lt;0.001; control vs. group 3, p=0.01; group 2 vs. group 4, p=0.002\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eTable 2: Tissue levels of nitric oxide (NO), malondialdehyde (MDA), and reduced glutathione (GSH) in tamoxifen-treated (groups 2\u0026ndash;4) and untreated control (group 1) rats; n=10 rats/group\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"591\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eNO\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(nanomol/ g)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003eMDA\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(nanomol/ g)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003eGSH\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(nanomol/ g)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003eGroup 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e98.43\u0026plusmn;14.00\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e68.29\u0026plusmn;18.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e3.52\u0026plusmn;1.47\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003eGroup 2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e84.55\u0026plusmn;27.48\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e33.15\u0026plusmn;11.55\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e6.46\u0026plusmn;1.65\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003eGroup 3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e107.23\u0026plusmn;22.41\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e31.98\u0026plusmn;13.77\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e5.46\u0026plusmn;.53\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003eGroup 4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e86.67\u0026plusmn;19.71\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e37.13\u0026plusmn;9.66\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e4.12\u0026plusmn;1.10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eMDA:\u003c/strong\u003e Control vs. group 2, p\u0026lt;0.001; control vs. group 3, p\u0026lt;0.001; group 2 vs. group 4, p\u0026lt;0.001 \u003cstrong\u003eGSH;\u003c/strong\u003e Control vs. group 3, p\u0026lt;0.043; control vs. group 2, p\u0026lt;0.001; group 2 vs. group 4, p\u0026lt;0.005\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3: Tissue cytokine levels in tamoxifen-treated (groups 2\u0026ndash;4) and untreated control (group 1) rats; n=10 rats/group\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eIFN\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(IU/ml)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eIL-1 (picogram/ml)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eTNF\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(picogram/ml)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eGroup 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e1.70\u0026plusmn;0.83\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e152.20\u0026plusmn;48.38\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e99.74\u0026plusmn;38.29\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eGroup 2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e1.015\u0026plusmn;0.53\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e109.12\u0026plusmn;27.03\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e85.24\u0026plusmn;15.24\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eGroup 3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e1.49\u0026plusmn;0.58\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e146.54\u0026plusmn;47.29\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e116.76\u0026plusmn;42.72\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eGroup 4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e1.52\u0026plusmn;0.65\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e167.46\u0026plusmn;38.46\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e98.09\u0026plusmn;39.68\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eIL-1;\u003c/strong\u003e Group 2 vs. group 4, p\u0026lt;0.001\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4: Blood nitric oxide (NO), malonyldialdehyde (MDA), reduced glutathione (GSH), and tumor necrosis factor-\u0026alpha; (TNF- \u0026alpha;) levels in tamoxifen-treated (groups 2\u0026ndash;4) and untreated control (group 1) rats; n=10 rats/group\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"671\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNO\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(nanomol/ml plasma)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMDA\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(nanomol/ ml plasma)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGSH\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(nanomol/ ml plasma)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTNF\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(picogram/ml plasma)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eGroup 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e36.52\u0026plusmn;14.66\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e6.50\u0026plusmn;1.44\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e1.16\u0026plusmn;0.44\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e47.46\u0026plusmn;18.68\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eGroup 2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e31.38\u0026plusmn;9.88\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e3.90\u0026plusmn;1.13\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.97\u0026plusmn;0.28\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e41.18\u0026plusmn;15.76\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eGroup 3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e36.62\u0026plusmn;14.10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e3.86\u0026plusmn;1.44\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.90\u0026plusmn;0.42\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e38.77\u0026plusmn;7.72\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eGroup 4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e30.24\u0026plusmn;10.07\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e6.96\u0026plusmn;2.14\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e1.30\u0026plusmn;0.46\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e59.33\u0026plusmn;13.03\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eMDA: Control vs. group 2, p\u0026lt;0.004; control vs. group 3, p\u0026lt;0.005; group 3 vs. group 4, p\u0026lt;0.001; group 2 vs. group 4, p\u0026lt;0.001 \u0026nbsp;TNF:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eGroup 2 vs. group 4, p\u0026lt;0.047; group 3 vs. group 4, p\u0026lt;0.02\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"antifibrotic effect, axillary dissection, breast cancer, lymphedema, rat, tamoxifen","lastPublishedDoi":"10.21203/rs.3.rs-8709265/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8709265/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction:\u003c/h2\u003e \u003cp\u003eSecondary lymphedema is a common complication after lymph node dissection and is characterized by chronic swelling and progressive soft-tissue fibrosis. Tamoxifen has been reported to exert antifibrotic and antioxidant effects. We investigated whether tamoxifen could attenuate postsurgical limb swelling and fibrosis in a rat model of secondary lymphatic injury and explored potential oxidative mechanisms.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eForty Wistar\u0026ndash;Albino rats were randomized into four groups (n\u0026thinsp;=\u0026thinsp;10 each). A standardized right hindlimb lymphadenectomy was performed in all groups. Tamoxifen (2.5 mg/kg/day, oral) was administered with different schedules: 7 days preoperative\u0026thinsp;+\u0026thinsp;7 days postoperative (Group 2), 14 days postoperative starting immediately (Group 3), and 14 days postoperative starting on day 14 (Group 4). Outcomes at day 30 included limb volume change (water volumetry), lymphoscintigraphy (Tc-99m nanocolloid), histopathologic fibrosis score, tissue hydroxyproline, and oxidative stress markers (MDA, GSH), as well as selected cytokines.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eLymphoscintigraphic parameters did not differ significantly between groups. However, postoperative volumetric increase of the operated limb was significantly greater in untreated controls than in Groups 2 and 3 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Histopathologic fibrosis scores were significantly lower in Groups 2 and 3 compared to the controls, supported by lower hydroxyproline levels. Tissue MDA levels were higher and GSH levels lower in controls compared with treated groups, suggesting attenuation of oxidative stress by tamoxifen.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn this rat model, tamoxifen reduced postsurgical limb swelling and soft-tissue fibrosis, accompanied by lower oxidative stress. Because lymphoscintigraphy did not demonstrate significant between-group differences and a sham-operated control group was not included, the findings support an antifibrotic/antioxidant effect on postoperative tissue changes rather than definitive prevention of lymphedema.\u003c/p\u003e","manuscriptTitle":"Tamoxifen Attenuates Postsurgical Hindlimb Swelling and Soft-tissue Fibrosis in a Rat Model of Secondary Lymphatic Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-18 15:37:29","doi":"10.21203/rs.3.rs-8709265/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-16T07:28:40+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"32214503466194825260419700191436315955","date":"2026-02-15T06:36:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-14T21:49:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16693065866414228249304216257742399407","date":"2026-02-14T21:48:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-14T12:00:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259854965313669662751153726380271275132","date":"2026-02-14T08:29:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"206119293588519540534004409494827958162","date":"2026-02-13T08:11:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-12T18:51:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113262106383205945673250807456312164289","date":"2026-02-12T16:57:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-12T15:31:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-02T09:25:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-02T09:19:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Naunyn-Schmiedeberg's Archives of Pharmacology","date":"2026-01-27T10:04:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"69e5bb09-9db7-4e8a-8e4a-af1e332eb33a","owner":[],"postedDate":"February 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T14:11:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-18 15:37:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8709265","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8709265","identity":"rs-8709265","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}