Figure 6

Morphological stages of lymphatic heart regeneration following complete excision. (a) Normal lymphatic heart (LH) (magenta arrowheads) with its supplying nerve (blue arrowheads), the trunci lymphatici longitudinales lateralis (TLyLL) (black arrowheads) and venous lateralis (VL) (red arrowheads). LH function was confirmed by collection of rhodamine-dextran (Scale bars = 100 μm). (b) The tissue defect immediately after LH excision. A segment of the TLyLL, VL and the supplying nerve, all of which are intimately associated with the LH were also resected. (c) Stage 1 Post Operative (PO) Day 5–10: Clot formation was followed by accumulation of a soft, fragile tissue mass beneath the wound epidermis. (d) Stage 2 PO Day 28–40: Angiogenesis of several small blood vessels interconnecting the cut ends of the VL and the other major blood vessels. No lymphangiogenesis seen at the injury site. Histology showed regenerating muscle and fibroblasts but no functional lymphatics with no rhodamine-dextran collection. (e) Stage 3 PO Day 50–60: Maturation of blood vessels restoring their original size and structure and restoration of VL continuity. Small lymphatic vessels were observed sprouting from the edges of the wound towards the injury zone. (f) Stage 4 PO Day 80–100: Lymphatic vessel maturation with active flow and collection of dye observed as a cystic mass in the region of the original LH, but no pulsations. (g) Stage 5 PO Day 100–130: Complete LH regeneration with pulsation. The regenerated LHs had fully functional input and output valves and uptake of rhodamine-dextran confirming they were fully functional.