Volume 39, Issue 3, Pages 341-348 (September 2003) Atrial natriuretic peptide preconditioning protects against hepatic preservation injury by attenuating necrotic and apoptotic cell death  Tobias Gerwig, Herbert Meiβner, Manfred Bilzer, Alexandra K Kiemer, Hans Arnholdt, Angelika M Vollmar, Alexander L Gerbes  Journal of Hepatology  Volume 39, Issue 3, Pages 341-348 (September 2003) DOI: 10.1016/S0168-8278(03)00240-X

Fig. 1 ANP and 8-Br-cGMP preconditioning reduce caspase-3-like activity. At indicated time points (0′: shortly perfused, no ischemia; 24 h isch.: 24 h of cold ischemia; 45′ rep.: 45 min of reperfusion; 120′ rep.: 120 min of reperfusion) control livers and pre-treated livers (ANP 200nM: panel A, 8-Br-cGMP 50 μM: panel B) were snap frozen and caspase-3-like activity was measured as described under Section 2. Data are expressed as percentage of the highest activity. Columns show mean±SEM of two to three independent experiments with four to five rat livers. *P<0.05 versus control, #P<0.05 versus 0′. Journal of Hepatology 2003 39, 341-348DOI: (10.1016/S0168-8278(03)00240-X)

Fig. 2 Reduced processing of caspase-3 in ANP pre-treated livers. At indicated time points (0′: shortly perfused, no ischemia; 24 h isch.: 24 h of cold ischemia; 120′ rep.: 120 min of reperfusion) control livers and pre-treated livers (ANP 200nM) were snap frozen. Western blot with caspase-3 antibody was performed as described under Section 2. CPP32: caspase-3 precursor; p17: proteolytic active subunit. Data show one representative blot out of three independent experiments (mean±SEM) with two to three rat livers. Panel A: Western blot; panel B: densitometric analysis of CPP32; panel C: densitometric analysis of p17. *P<0.01 versus control, #P<0.05 versus 0′. Journal of Hepatology 2003 39, 341-348DOI: (10.1016/S0168-8278(03)00240-X)

Fig. 3 (A) Morphological characteristics of apoptotic cell death. As described in detail under Section 2, apoptotic cell death was determined by biochemical and morphological techniques in order to clearly discriminate from necrotic cell death. The picture shows one representive TUNEL staining after 24 h of cold ischemia. Morphological criteria of apoptosis in TUNEL stained hepatocytes were cell shrinkage, chromatin condensation and margination, and the occurrence of apoptotic bodies. Original magnification: 400-fold. (B) ANP and 8-Br-cGMP preconditioning decrease proportion of apoptotic cells after ischemia. At indicated time points (0′: shortly perfused, no ischemia; 24 h isch.: 24 h of cold ischemia; 45′ rep.: 45 min of reperfusion; 120′ rep.: 120 min of reperfusion) control livers and pre-treated livers (ANP 200nM: panel A, 8-Br-cGMP 50 μM: panel B) were snap frozen and prepared for TUNEL analysis as described under Section 2. Columns show numbers of apoptotic cells (mean±SEM, with mean in numerical values) in four to five rat livers. Ten high power fields (1.96 mm2, approx. 4000 hepatocytes) were counted at a magnification of 400-fold. *P<0.05 versus control, #P<0.05 versus 0′. Journal of Hepatology 2003 39, 341-348DOI: (10.1016/S0168-8278(03)00240-X)

Fig. 4 Degenerative changes of hepatocytes are decreased by ANP pretreatment. After 24 h of preservation in UW solution, liver slices were fixed in formalin solution (1.5%) and embedded in paraffin. HE staining was performed using a standard protocol. (A) Arrows show cytoplasmatic vacuolization as an indicator of degenerative cell damage in a representative control liver. (B) Representative picture of an ANP-pretreated liver (200nM). Original magnification: 400-fold. Journal of Hepatology 2003 39, 341-348DOI: (10.1016/S0168-8278(03)00240-X)

Fig. 5 Necrotic damage during reperfusion is reduced by ANP preconditioning in hepatocytes. After 24 h of preservation in UW solution and 2 h of reperfusion with KH buffer, livers were perfused with trypan blue (0.2 mmol/l) for 10 min and fixed with formalin (1.5%) during a 5-min perfusion as described under Section 2. Panel A: percentage of necrotic hepatocytes in periportal and pericentral areas, panel B: percentage of necrotic endothelial cells. Columns show the percentage of trypan blue stained cells around randomly selected central and portal veins. Magnification 400-fold (mean±SEM of four to five different rat livers). *P<0.001 versus control. Journal of Hepatology 2003 39, 341-348DOI: (10.1016/S0168-8278(03)00240-X)

Fig. 6 No influence of ANP on liver proliferation. At indicated time points (0′: shortly perfused, no ischemia; 24 h isch.: 24 h of cold ischemia; 45′ rep.: 45 min of reperfusion; 120′ rep.: 120 min of reperfusion) control livers and pre-treated liver slices (ANP 200nM) were fixed in formalin solution (1.5%) and embedded in paraffin. Histochemical detection of the proliferation marker Ki67 was performed as described under Section 2. Data (mean±SEM) show percentage of Ki67-positive cells per 10 midpower fields (approximately 10,000 hepatocytes). Four to five different rat livers were counted. There were no significant differences versus untreated control livers. #P<0.01 versus 0′. Journal of Hepatology 2003 39, 341-348DOI: (10.1016/S0168-8278(03)00240-X)