Fibrosis Worsens Chronic Lymphedema in

30 Secondary lymphedema in humans is a common consequence of lymph node dissection (LND) 31 to treat breast cancer. A peculiar characteristic of the disease is that life-long swelling often 32 precipitously appears several years following the surgical treatment, often due to an 33 inflammatory stimulus. Although the incidence of secondary lymphedema dramatically 34 increases following radiation therapy, the relationship between fibrotic scarring and the eventual 35 appearance of lymphedema remains unclear. In order to clarify the role of fibrosis in secondary 36 lymphedema initiation, we chemically increased fibrosis in rodent tissues with bleomycin and 37 assessed the ability of the local lymphatic system to prevent lymphedema, either acutely or in a 38 chronic state induced by inflammation. We found that bleomycin injections exacerbated fibrotic 39 matrix deposition in an acute mouse tail lymphedema model (p<0.005), reduced wound closure 40 (p<0.005), and impaired the ability of the tail lymphatics to regenerate (p<0.005) and reduce the 41 swelling (p<0.05). When fibrosis was worsened with bleomycin following axillary LND in the 42 rat foreleg, the ability of the foreleg lymphatic system to reduce the chronic state swelling 43 induced by stimulated inflammation was severely impaired (p<0.005). Indocyanine Green (ICG) 44 lymphography in the ALND-recovered rat forelegs revealed a worsened lymphatic drainage due 45 to the inflammation and bleomycin pre-treatment. Although inflammation reduced the drainage 46 of dextran fluid tracer from the control forelegs (p<0.05), the reduction in fluid drainage was 47 more severe post-ALND when fibrosis was first increased (p<0.005). The findings demonstrate 48 that fibrosis reduces the lymphatic capacity to functionally regenerate and prevent the chronic 49 appearance of lymphedema. 50 51 52