The Basic Research Of The Severed Limbs Preservation Using A Novel Method: In Vitro Simulation Of The Physiological Environment

Background：The success of limb replantation is directly related to the method of limbpreservation, the traditional methods of limb preservation include low-temperature coldstorage, low-temperature perfusion, hyperbaric oxygen treatment and temporary ectopicimplantation. All of methods mentioned above have limitation and side-injury. Theherein-described technique of in vitro simulation of the physiological environment is a newmethod established by author. The in vitro system includes an extracorporeal circulationpump, a membrane lung and a hemodialysis device. which can provide blood to the severedlimb.This method can extend the limb viability time. This method has not been reported athome and abroad by sci-tech novelty retrieval.Objective: To investigate if the in vitro physiological environment fostering limbs betterthan the traditional preservation methods through RT-PCR and Western Blotting. Find themost appropriate temperature of this method by investigate the effects of differenttemperatures on the system of in vitro physiological environment fostering limbsMethods: Experiment1:Six Bama minipigs (24limbs) in Guangxi province wererandomly divided into two groups: the ischemia group (I group) and perfusion group (Pgroup). The limbs in the P group underwent in vitro limb perfusion experiments using bloodat0,3,6,9, and12hours. The limbs in the I group underwent ischemia at the same timepoints. The mRNA and protein levels of apoptosis and inflammatory factors were detected byreverse transcription quantitative polymerase chain reaction and western blotting,respectively.Experiment2: Twenty-four limbs were harvested from6adult Bama mini pigs and wererandomly divided into4groups (n=6) according to different temperatures: limbs were placedin vitro physiological environment fostering limbs at26℃(group A),4℃(group B),10℃(group C), and18℃(group D). After12hours of perfusion, the morphology detection wasdone to observe structure and ultra-structure changes of the muscle cells by light microscopyand transmission electron microscopy. The mRNA levels of tumor necrosis factor α (TNF-α)and interleukin1β (IL-1β) were detected by real time fluorescent quantitative PCR(RT-qPCR). Results: Experiment1:In the P group, the levels of interleukin-1(IL-1), TNF-, andcaspase-3significantly decreased at all time points compared with those in the I group, andboth mRNA and protein levels of these factors were low after12hours of perfusion.Experiment2:Light microscope and Transmission electron microscope results showedthe cells of group10℃had normal morphology and a large number of muscle fibers arrangedin order and regularity with clear sarcomere. RT-qPCR results showed that the expressions ofinflammatory factor TNF-α and IL-1β mRNA in group10℃were significantly lower thanthose in groups B, C, and D.Conclusion: These results suggest that in vitro simulation of the physiologicenvironment may extend the in vitro limb viability time. The mechanism might be that it cancontrol both the release of inflammatory factor and apoptosis factor and so on.In the system of in vitro physiological environment fostering limbs, temperature plays animportant role in the preservation of amputated limbs. It is suggested that10℃cansignificantly attenuated the reperfusion-induced skeletal muscle cell injuries in this system.More importantly this applied research has been applied to clinical for2cases, and has beenshown to be useful.