Imaging Lymphatics

Abstract

The lymphatic system drains fluid and metabolic waste from tissues and provides a route for antigen and antigen presenting cells to move from tissue to lymph nodes where systemic immune responses can be initiated. When the lymphatic system becomes dysfunctional, immune function is impaired and the ability to maintain fluid balance and tissue homeostasis is compromised. Both lymphatic vessels and lymph nodes are of paramount importance in keeping the system fully operational. The role of the lymphatic system has been implicated in a wide array of clinically relevant diseases, including inflammatory settings like bacterial infections, lymphedema, cancer and cancer metastasis. To do this end, the research presented in this thesis addresses several critical unmet needs by developing and implementing novel imaging methods for studying both lymphatic vessels and lymph nodes in health and disease. In chapter 2, we describe the microsurgical preparation and implantation of a chronic lymph node window (CLNW) of the murine inguinal lymph node to facilitate imaging of the lymph node in physiological and pathological settings for up to 14 days. Measurements of transvascular transport have been proven invaluable in studying numerous in vivo processes, including vascular abnormalities and treatments in disease states like cancer and inflammation. In chapter 3, we summarize methods to measure transvascular transport as we describe a method for estimating effective vascular permeability (EVP) using 2D imaging, as well a recently developed 3D method using multiphoton microscopy which we optimized. This 3D method yields a more realistic measurement of EVP. Accurate in vivo measurements of lymph flow velocity and lymph volumetric flow will be critical in understanding disease processes. Several methods have been developed to date to study lymphatic function, however they intrinsically change the parameters to be measured by their obligatory use of exogenous labels and cannot directly quantify lymph flow. Therefore, in chapter 4, we developed a method that allows direct label-free measurement of lymph flow in vivo by Doppler optical coherence tomography (DOCT). In chapter 5 we describe additional modifications to the DOCT experimental setup, thereby extending the technique to support the simultaneous measurement of flow and contraction in three neighboring lymphangions and the status (i.e., open vs closed) of the valves present in-between each lymphangion. In chapter 6, we utilized the CLNW and the 3D method to measure EVP to investigate physiologic lymph node EVP and whether altering EVP can improve chemotherapy penetration in the murine lymph node. We performed the first measurements of physiologic lymph node EVP and found that lymph node EVP can be increased ~fourfold by VEGF-A treatment, although not resulting in a significant increase in the maximum drug concentrations of chemotherapeutic drugs as measured by high-performance liquid chromatography. Chapter 7 describes how Methicillin-Resistant Staphylococcus aureus (MRSA), a frequent cause of skin and soft tissue infections in patientsresulting in high morbidity and costs, causes chronic decreased lymphatic function. Several MRSA exotoxins controlled by the accessory gene regulator (agr) operon are identified to be responsible for lymphatic muscle cell loss and resulting aberrant lymphatic vessel function.