Comparison of approaches for microscopic imaging of skin lymphatic vessels

Assessment of skin lymphatic vessels is of great significance in understanding their roles in many pathological conditions. Our aim was to identify the optimal approach for investigation of cutaneous lymphatic system. We performed comparative studies on skin lymphatic vessels using immunohistochemistry of tissue sections, computer graphic reconstruction method together with immunohistochemically stained serial sections and whole mount fluorescence in human lower limb. Lymphatic vessels were identified with podoplanin antibody. The relative merits and drawbacks of each method in evaluation of structure, spatial organization, and distribution of cutaneous lymphatic vessels were described. Immunohistology of tissue sections enabled the investigation of the structure and distribution of the whole cutaneous lymphatic system in two-dimensional slices, whereas three-dimensional morphology of only the most superficial lymph capillary network immediately under the epidermis could be evaluated with the whole mount technique. Meanwhile, only little segmentation of skin lymphatic vessel from five immunohistochemically stained serial sections was reconstructed and evaluated due to expense and special skills required using computer graphic three-dimensional reconstruction. Furthermore, a great number of artifacts and special skills required in its processes leaded to less accurate structure of skin lymphatic vessels. Our findings demonstrated that the use of either of the proposed techniques alone could not allow a comprehensive analysis of the skin lymphatic system due to their relative drawbacks. Combination of immunohistology of tissue sections and three-dimensional whole-mount preparations appears to be the best candidate for comprehensive evaluation of skin lymphatic system.