Figure 5

Physiological changes in lymphatic circulation following lymphatic heart excision. (a) Lymphatic hearts (LH) excision in newts did not cause edema, histological changes, nor death (Scale bars = 1 mm). There was no significant difference in the 1-week post operative (PO) limb diameters (proximal t(9) = 0.729, p = 0.485, middle t(9) = − 0.504, p = 0.627, distal t(9) = 0.297, p = 0.773) between LH excision newts and controls with good inter-rater reliability ICC = 0.893, 95% CI (0.825, 0.934). (b) Micro-CT lymphangiography showed reduced lymphatic contrast in the blood circulation in LH excision newts at 90 days PO, with Trunci lymphatici longitudinales parabdominales (TLyLPab) collateral flow seen (yellow arrowheads). Vena cava posterior (VCP) analysis showed LH excision newts had a significantly lower mean grey value (t(34) = − 3.156, p = 0.003) than controls. (c) Rhodamine-dextran fluorescence lymphangiography analysis of lymphatics to vein flow (Scale bars = 100 μm) also showed a significant decrease in the density of blood vessels receiving lymphatic fluid (t(188) = 13.057, p < 0.001) at 28 days post excision of all the LHs but this returned to normal at 120 days PO (t(161) = − 0.854, p = 0.394). (d) Ultra-high frequency ultrasonography showed a significant increase (t(12) = − 2.707, p = 0.019) in the LH rate in newts that had bilateral posterior LH excision. (e) Collateral flow maintained lymphatic circulation after excision of LHs. TLyLPab collateral is shown (yellow arrowheads) with connections to the TvIL (magenta arrowhead). These results confirmed that LH excision successfully terminated peripheral lymphatic to vein flow for 90 days following which normal lymphatic to vein flow was restored by 120 days PO. Data represent mean ± standard deviation (error bars).