Protective effect of normothermic mechanical perfusion with red blood cells from humanized genetically modified pigs on severed human limb

Objective To evaluate the preservation effect of normothermic mechanical perfusion with red blood cells from humanized genetically modified pig on severed human limbs.

Methods Severed human limbs were perfused with red blood cells from humanized genetically modified pigs for 6 h. Perfusion solution was taken every hour to measure the oxygen partial pressure, Na⁺, K⁺, Ca²⁺, pH value, glucose, lactic acid and creatine kinase levels. Superficial flexor muscle was sampled to detect the changes of tumor necrosis factor (TNF)-α, interleukin (IL)-2 and IL-1 levels. At 0 and 6 h after perfusion, the superficial flexor muscles of the forearm were taken for pathological examination. Intercellular space and glycogen consumption of skeletal muscles were observed. An appropriate amount of forearm vessels was collected every 2 h to detect the apoptosis of vascular endothelial cells. X-ray angiography was performed before perfusion and 6 h after perfusion to observe the filling degree of finger-tip peripheral vessels.

Results The oxygen partial pressure was observed in the normal range throughout the perfusion. Na⁺ concentration peaked at 1 h, reaching 138.7 mmol/L, and then fluctuated within the normal range. K⁺ level peaked at 2 h up to 6.08 mmol/L, then decreased and fluctuated within the normal range. Ca²⁺ concentration reached the peak at 4 h, up to 1.03 mmol/L. Glucose level was gradually decreased at the beginning of perfusion, reaching the lowest value of 17.7 mmol/L at 2 h after perfusion, and then maintained a dynamic balance. The pH value was decreased to 7.28 at 6 h after perfusion. The lactic acid level was increased to 9.6 mmol/L at 1 h after perfusion, and then gradually decreased. The creatine kinase level was increased at the start of perfusion, reached the peak at 2 h up to 20 030 U/L, then decreased and remained stable at the end of perfusion. At the end of perfusion, the morphology of muscle fibers was normal, the gap among muscle fibers was expanded slightly, and the glycogen of skeletal muscles was not significantly accumulated. At 0 h perfusion, the number of apoptotic cells in vascular endothelial cells was large, which was declined at 6 h perfusion. Evident vascular filling was observed at 0 h, and the filling degree of some finger-tip vessels was decreased at 6 h.

Conclusions Normothermic mechanical perfusion of severed human limbs with red blood cells from humanized genetically modified pigs may continuously and stably supply energy and oxygen, adjust the ion pH balance of perfusion solution, maintain normal cellular metabolism and exerts certain protective effect upon severed human limbs.