An Intravital Study Of The Protective Effects Of Ischemic Pre-conditioning Or Post-conditioning On Hepatic Microcirculation Ischemia Reperfusion Injury

Objective: The aim of this study is to establish an in vivo real time method to clarify the three-dimension visualization and fluid dynamics characteristics of hepatic microcirculation and peribiliary blood plexus (PBP).We discuss the characteristic representation and the significance of ischemic pre-conditioning (IPC) and ischemic post-conditioning (I-postC) on hepatic microcirculation ischemia-reperfusion injury, and provide a theoretical basis for clinical prevention and treatment of ischemic reperfusion injury(IRI).Methods: First, we established an in vivo real time method that microcirculation laser scanning Confocal microscopy (MLSCM) was used to observe the three-dimension visualization morphological structure and the flow dynamics of hepatic microcirculation and PBP in rat's hepatohilar bile duct after hepatic artery was perfused with Indocyanine Green( ICG) which provided a strong background contrast to microvessels. The parameters were microvascular flow velocity (MFV), microvascular diameter (MVD) and the density of functional capillaries (DFC) which were filled with red blood cells.Second, we discussed the characteristic representation and its significance of IPC and I-postC on hepatic microcirculation ischemia-reperfusion injury with the intravital study method. IR model was established with 60-minute inflow occlusion according to Nauta's method. Wistar rats were divided into four groups, i.e. 1,Sham group; 2,IR group, 60-minute warm ischemia only; 3,IPC group, with 10-minute ischemia followed by 10-minute reperfusion prior to IR; 4,I-postC group, with 30-second reperfusion followed by 30-second occlusion for three times at the onset of reperfusion after ischemia. Blood samples were collected and intravital hepatic microcirculation study was proceeded at 2h,6h, 5d,7d after hepatic reperfusion. The indicators included: 1,MVD and DFC measured by microcirculation laser scanning Confocal; 2,MFV by laser Doppler analyze of blood flow; 3,the morphological structure observed by histopathological examination and ESEM examination; 4,the hepatic tissue ATPase activity and serum transaminase; 5,The levels of PO₂,O₂SATand pH value of hepatic vein blood; 6,the apoptotic cells detected by TUNEL method. All data were expressed in mean±SD.Results: We found that the three-dimension visualization microcirculation study method could give a real time, clear representation of hepatic microcirculation by which the MFV, MVD and DFC could be measured exactly. We found the normal hepatic microcirculation blood flow was smooth and fluent, and the hilar PVP composed of three layers. The outer layer vessels with sparse and straight branches were dendritic and located within the ductal wall at scanning depth 0-60μm from the adventitia. The intermediate layer vessels with numerous and tortuous branches were intricate and located at scanning depth 40-80μm. The inner layer vessels with uniform diameter were adjacent to the epithelium and formed hexagonal ring with 120°intersecting angle.In the second part of this paper, the characteristic representation of hepatic microcirculation under the condition of ischemia-reperfusion with or without ischemic conditioning showed that: 1,At 2h after reperfusion, the FCD was decreased, MVD was diminished, MFV stepped down, the volume of blood flow reduced, the levels of PO₂. O₂SAT and pH and tissue ATPase became fall-off, while serum ALT,AST levels was increased, sinus endotheliocytes were swelling and began to apoptosis. 2,At 6h after reperfusion, neutrophil cells adhered to the wall of the sinusoids, Leukocytes infiltrated around microvessels, part sinusoids were blockage and their flows were stagnated, FCD was obviously decreased, MFV kept on stepping down, the volume of blood flow was obviously reduced, the levels of PO2. O2SAT and pH was continuously fall-off, the levels of ALT,AST was obviously increased, and more endotheliocytes apoptosis could be seen. But the levels of ATPase were increased. 3,At 5d after reperfusion, the FCD was increased, MFV step up, the volume of blood flow was backswing, the levels of PO₂,O₂SAT,pH and tissue ATPase increased, the levels of ALT and AST fall-off, the number of apoptosis sinus endotheliocytes was lessened. 4,At 2h,6h and 5d after reperfusion, the IR group compared with the IPC group and the I-postC group showed significantly amelioration .Conclusions:1,Microcirculation laser scanning Confocal microscopy could lively and clearly show the three-dimension visualization of liver and PBP microcirculation after ICG fluorescence staining was used.2,The intravital characteristic performance of hepatic microcirculation of ischemia-reperfusion injury showed that: at 2h after reperfusion, effective circulation volume was reduced; it became so worse that Leukocytes infiltrated around microvessel and part sinusoids were blockage at 6h;it was inclined to recover at 5d after reperfusion.3,Both IPC and I-postC treatment show a protective effects against ischemia-reperfusion injury by the representation of hepatic microcirculation observation. Both of them wound become useful and valuable methods for clinical prevention and treatment of ischemic reperfusion injury(IRI) .The ameliorated changes of hepatic microcirculation might be one of the mechanisms of their protective effects against IRI.