Research On The Flap Ischemia-reperfusion Injury With Hyperbaric Oxygen Preconditioning Hydrogen-rich Saline Treatment

Background:Skin flaps are widely used in the field of difficult wound repair and organ reconstruction, to which one of the major threats is Ischemia/Reperfusion (I/R) injury. Therefore people have made great efforts to find ways to avoid this injury for many years. Literature shows that Hyperbaric Oxygen (HBO) preconditioning helps improve the success rate of flap operation, while another popular gas molecule, Hydrogen gas (H2), has recently been proven to have potential abilities to save I/R flaps. No research on the subject of the combined application of HBO and H2has been published to this day.Objective:The objective of this study is to build a new animal model of I/R skin flaps, by which we can estimate and compare the protective effects of HBO preconditioning, H2treatment, and their combined applications. Furthermore, this can be used as a tool to study the mechanisms of H2therapy.Methods:Dorsal large flaps (6×9cm) based on the cutaneous branches of the left iliolumbar artery of male SD rats were constructed with a5h ischemia intervention achieved by bulldog clamps. HBO preconditioning began from the third day before the operation with the method of hyperbaric oxygenation, and postoperative H2treatment was actualized by intraperitoneal injection of Hydrogen-rich Saline (HS). Laser Doppler Flowmeter Imager (LDFI) was used to monitor and compare the perfusion of the flaps, while H-E and TUNEL staining procedures were carried on to evaluate the level of inflammation and apoptosis of these dorsal flaps, respectively. We also made use of immunohistochemistry, Western blot (protein) and RT-qPCR (gene expression) techniques to confirm and expound our results on the tissue level.Results:From our preliminary results, all or most areas of the rat dorsal large flaps based on the cutaneous branches of the left iliolumbar artery could survive and present excellent perfusion in normal conditions, but when subjected to severe I/R injury, the necrosis ratio of the flaps increased and the perfusion decreased. HBO preconditioning improved the perfusion of flaps at either24h or72h postoperative, and HS intraperitoneal injection could only improve the state of flaps at72h postoperative. When combined, it showed a further improvement of the perfusion of the flaps. The outcome of H-E and TUNEL staining revealed that both HBO and HS treatment helped to reduce the level of inflammation and apoptosis of the flaps. HS presented a better effect in the aspect of resistance to apoptosis than HBO, and combined application showed the best results. The outcome of immunohistochemistry, Western blot and RT-qPCR showed that both HBO and HS treatment inhibited the expression of the inflammation associated protein NF-κB, pro-apoptotic protein Bax and pJNK, and also promoted the expression of apoptosis suppressor gene Bcl-2on the level of gene expression. And as stated previously, HS presented a better effect than HBO, and combined application presented the best results.Conclusions:Either HBO preconditioning or HS intraperitoneal injection can remarkably reduce the I/R injury in rat skin flaps.The two methods of treatment have synergistic effects, which were reflected in the further improvement of perfusion and remission of inflammation and apoptosis of the flaps. These results might be related to the koinonia of high oxygen and perfusion environment constructed by HBO preconditioning and the inhibition of the ROSâ†'pASK1â†'pJNKâ†'Bcl-2â†'Baxâ†'Cyt-Câ†'Caspaseâ†'Apoptosis pathway caused by HS. In conclusion, the clinical combined application of HBO preconditioning and H2in the field of skin flap transplantation should have a bright future.