Hepatic Warm Ischemia-reperfusion Injury Research Articles

Study of R2* value to evaluate regulation of liver regeneration by hepatic warm ischemia-reperfusion injury after partial hepatectomy in rabbits

Objective: To investigate the effect of R2* value on the evaluation of different degrees of hepatic warm ischemia-reperfusion injury (WIRI) and liver regeneration after partial hepatectomy in rabbits. Methods: Thirty healthy adult male New Zealand White rabbits were randomly divided into five groups. Hepatic caudal lobectomy was performed in both the control and the warm ischemia time-dependent variation group. After reperfusion, routine MRI and BOLD MRI scans were performed for each group at 6 h, 3 d, 7 d, 14 d and 30 d, respectively, and then R2* value and liver regeneration rate (LRR) were measured and calculated. After 30 days of scanning, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), myeloperoxidase (MPO), tumor necrosis factor - α (TNF - α), interleukin-6 (IL-6) and proliferating cell nuclear antigen (PCNA) were detected in frozen rabbit liver tissues, and the pathological sections were collected. Repeated measures analysis of variance was used to evaluate the changes of R2* value, LRR and its influencing factors at different follow-up time and warm ischemia time in each group. Pearson's or Spearman's correlation analysis was used to evaluate the correlation of R2* value with LRR and various biochemical indexes. Results: The interaction between different follow-up time and warm ischemia time (F = 24.600, P < 0.001) and the single effect of the both on the R2* value had statistically significant difference (P < 0.05). The interaction of different follow-up time and different warm ischemia time had no effect on LRR, and the difference was not statistically significant (F = 0.925, P = 0.528), but the difference in the main effect of the both on LRR was statistically significant (P < 0.05). At the same follow-up time, except for the 40-min ischemia group, the R2* values ​​were significantly positively correlated with LRR (3, 7, 14, 30 days after operation, r = 0.510, 0.681, 0.612, 0.541 respectively, P < 0.05). At the same warm ischemia time, the R2* value were significantly negatively correlated with LRR (3, 7, 14, 30 and 40 days after operation, r = - 0.800, -0.852, -0.893, -0.648, -0.853, respectively, P < 0.05). There was no correlation between R2 * value and biochemical indexes at 30 days after operation (P > 0.05). Conclusion: The R2* value may be used for noninvasive and quantitative evaluation of microstructural changes of WIRI and affect liver regeneration after partial hepatectomy in rabbits. A certain degree of WIRI (≤30 min) after partial hepatectomy can promote liver regeneration in rabbits. Furthermore, as the warm ischemia time prolongs, the promoting effect becomes more pronounced, and if the warm ischemic time exceeds 30 minutes, the promoting effect is significantly reduced.

Alteration of connexins gene expression by silibinin during hepatic warm ischemia-reperfusion injury in the rat model

Background: Ischemia-reperfusion injury (IRI) is an injurious phenomenon that is the primary determinant of liver dysfunction after surgery and transplantation. The present evidence demonstrated that connexin 43 (Cx43), Cx32, and Cx26 are the essential gap junction proteins involved in the liver IRI. This study aimed to characterize the beneficial effects of silibinin on Cx43, Cx32, and Cx26 gene expression during warm hepatic ischemia-reperfusion (IR). Materials and Methods: A total of 32 male Wistar rats weighing 250-300 g were randomly divided into four equal groups of eight animals in each group as follows: 1) control group (laparotomy+normal saline), 2) laparotomy+silibinin (30 mg/kg) (SILI), 3) liver IR procedure+normal saline (IR), and 4) liver IR procedure+silibinin (30 mg/kg) (IR+SILI). After 1 h of ischemia followed by 3 h of reperfusion, blood samples and tissue sections were gathered to assess the serum liver markers and evaluate the liver histological changes as well as gene expression, respectively. Results: The obtained data proved no considerable differences between control and SILI groups in all experiments. Furthermore, the gene expression of Cx26, Cx32, and Cx43 was significantly induced in the IR group, compared to the control group. Silibinin markedly reduced Cx26 and Cx32 mRNA expression, whereas increased Cx43 mRNA expression. Moreover, serum alanine aminotransferase and aspartate aminotransferase levels were markedly elevated in the IR group (P&lt;0.001), compared to the control group. However, in the IR+SILI group, silibinin could significantly decline these elevations, compared to the IR group. In addition, silibinin diminished hepatic tissue damages during IR. Conclusion: Silibinin could attenuate liver injury through better cell-to-cell communication via lowering Cx32 and Cx26, as well as increasing Cx43 gene expression, respectively.

In vivo multiparametric magnetic resonance imaging study for differentiating the severity of hepatic warm ischemia-reperfusion injury in a rabbit model

ObjectivesTo assess the value of multiparametric magnetic resonance imaging including intravoxel incoherent motion (IVIM), diffusion tensor imaging (DTI) and blood oxygen level dependent (BOLD) MRI in differentiating the severity of hepatic warm ischemia-reperfusion injury (WIRI) in a rabbit model. MethodsFifty rabbits were randomly divided into a sham-operation group and four test groups (n = 10 for each group) according to different hepatic warm ischemia times. IVIM, DTI and BOLD MRI were performed on a 3 T MR scanner with 11 b values (0 to 800 s/mm2), 2 b values (0 and 500 s/mm2) on 12 diffusion directions, multiple-echo gradient echo (GRE) sequences (TR/TE, 75/2.57–24.25 ms), respectively. IVIM, DTI and BOLD MRI parameters, hepatic biochemical and histopathological parameters were compared. Pearson and Spearman correlation methods were performed to assess the correlation between these MRI parameters and laboratory parameters. Furthermore, receiver operating characteristic (ROC) curves were compiled to determine diagnostic efficacies. ResultsTrue diffusion (Dslow), pseudodiffusion (Dfast), perfusion fraction (PF), mean diffusivity (MD) significantly decreased, while R2* significantly increased with prolonged warm ischemia times, and significant differences were found in all of biochemical and histopathological parameters (all P < 0.05). Dslow, PF, and R2* correlated significantly with all of biochemical and histopathological parameters (all |r| = 0.381–0.746, all P < 0.05). ROC analysis showed that the area under the ROC curve (AUC) of IVIM across hepatic WIRI groups was the largest among IVIM, DTI and BOLD. ConclusionsMultiparametric MRI may be helpful with characterization of early changes and determination of severity of hepatic WIRI in a rabbit model.

Multiparametric MRI Evaluation of Liposomal Prostaglandins E1 Intervention on Hepatic Warm Ischemia-Reperfusion Injury in Rabbits.

Liposomal prostaglandin E1 (Lipo-PGE1) treatment should protect against hepatic warm ischemia-reperfusion injury (WIRI). Improved methods are needed for the noninvasive evaluation of hepatic responses to prophylactic Lipo-PGE1 pretreatment approaches. To demonstrate that multiparametric MRI measurements permit noninvasive differentiation of Lipo-PGE1 treatment outcomes in a hepatic WIRI animal model. Animal study. Seventy rabbits were randomly divided into a sham-operated group (A0), warm ischemia groups experiencing increasing periods of ischemia (A1-A3), and corresponding intervention groups (I1-I3) (n = 10 for each group). Imaging was performed at 3T using a multiecho gradient echo (GRE) sequence (repetition time / echo time [TR/TE], 75/2.57-24.25 msec) for R2* blood oxygenation level-dependent (BOLD) measurements, free-breathing single-shot echo-planar imaging (ss-EPI) sequence with two b-values (0 and 500 s/mm2 ) in 12 diffusion directions for diffusion tensor imaging (DTI), and a free-breathing ss-EPI sequence with eight b-values (0 to 800 s/mm2 ) for intravoxel incoherent motion (IVIM) measurements. The BOLD-derived parameter (R2*), DTI-derived parameters (ADC, FA), and IVIM-derived parameters (Dslow, Dfast, and PF) were calculated for comparisons between treatment groups and correlation to ALT, AST, and LDH levels. One-way analysis of variance (ANOVA), independent sample t-test, Spearman correlation, and receiver operating characteristic (ROC) analysis were performed. Histopathology confirmed the validity of the WIRI model and the efficacy of intervention with clear structure and morphology differences between the different ischemia times and between the Lipo-PGE1 treatment and control groups. Prolonged warm ischemia times resulted in higher R2* and FA values and gradually lower ADC, Dslow, Dfast, and PF values (all P < 0.05). The R2* and FA values were lower, and the ADC, Dslow, Dfast, and PF values were higher in the Lipo-PGE1 intervention groups compared with those in the warm ischemia group for each paired time. However, none of the parameters reached the levels of the A0 group (all P < 0.05). As the warm ischemia time increased, additional parameters demonstrated significant differences between warm ischemia time groups and corresponding intervention groups. At the shortest (30 min), middle (40 min), and longest (60 min) ischemia times, three, four, and five parameters were significantly different between the WIRI and intervention groups, respectively (all P < 0.05). ADC, Dslow, Dfast, and PF values were negatively correlated, while R2* and FA values were positively correlated with serum ALT (|r| = 0.312-0.606) and AST (|r| = 0.432-0.602) (all P < 0.05). ADC and Dfast values showed negative correlations, and R2* showed positive correlations with serum LDH (|r| = 0.323-0.542, all P < 0.05). ROC analysis showed that DTI yielded the strongest diagnostic performance for evaluating the improvement of WIRI. Multiparametric MRI can serve as a noninvasive radiologic evaluation for monitoring the protective impact of Lipo-PGE1 therapy on hepatic WIRI. 1 Technical Efficacy Stage: 2 J. Magn. Reson. Imaging 2020;52:217-228.

Effects of silibinin on hepatic warm ischemia-reperfusion injury in the rat model.

Objective(s):Liver ischemia-reperfusion injuries (I/RI) are typically the main causes of liver dysfunction after various types of liver surgery especially liver transplantation. Radical components are the major causes of such direct injuries. We aimed to determine the beneficial effects of silibinin, a potent radical scavenger on liver I/RI.Materials and Methods:Thirty-two rats were divided into 4 groups. Group I: VEHICLE, the rats underwent laparotomy and received DMSO, group II: SILI, laparotomy was done and silibinin was administered. Group III: I/R, the rats received DMSO and were subjected to a liver I/R procedure and group IV: I/R+SILI, the animals underwent the I/R procedure and received silibinin. After 1 hr of ischemia followed by 3 hr reperfusion, blood was collected to evaluate the serum marker of liver injuries. Hepatic tissue was harvested to investigate glycogen content, histological changes, and vasoregulatory gene expression.Results:Results showed that serum AST, ALT, LDH, GGT, ALP, and hyaluronic acid (HA) increased significantly in I/R group compared with the VEHICLE group. Silibinin reduced this elevation except for GGT. Silibinin inhibited hepatocyte vacuolization and degeneration, endothelium damages, sinusoidal congestion and inflammation, and glycogen depletion during I/R. ET-1 mRNA was overproduced in the I/R group compared with the VEHICLE group which was decreased by silibinin. KLF2 and eNOS expression was reduced during I/R compared with the VEHICLE group. Silibinin elevated KLF2 expression but had no meaningful effect on eNOS expression.Conclusion:Silibinin protected the liver from I/RI. Silibinin could improve liver circulation by preventing sinusoidal congestion, inflammation, and perhaps modification of the vasoregulatory gene expression.

The Protective Effects of Carbon Monoxide Against Hepatic Warm Ischemia–Reperfusion Injury in MHC-Inbred Miniature Swine

BackgroundThe development of treatment strategies to protect against ischemia–reperfusion injury (IRI) to livers is important not only for liver surgeries but also in regard to increasing the utilization of livers from marginal donors. In this study, we examined whether inhalational carbon monoxide (CO) therapy reduced IRI after a 45-min (min) warm ischemia (WI) in a miniature swine model. Materials and MethodsSix CLAWN miniature swine underwent a 45-min hepatic WI induced by clamping the portal vein and proper hepatic artery. Three animals were subjected to control conditions while the remaining three were treated with CO inhalation for a total of 345-min, including 120-min after reperfusion to maintain a concentration of CO-Hb under 15% (CO-treated group). IRI of the livers was evaluated by liver function tests, serum pro-inflammatory cytokines, and liver biopsies. ResultsAll controls had statistically significant increased levels of liver enzymes compared to the CO-treated group (p < 0.05). In controls, liver biopsies at 2 h after reperfusion showed marked histological changes including diffuse hemorrhage, congestion, necrosis, vacuolization, and neutrophil infiltration with apoptosis. In contrast, the CO-treated group showed less obvious or only minimal histological changes. Furthermore, increases in high-mobility group box 1, TNF-α, and IL-6 in sera that were induced by IRI in controls were markedly inhibited by the CO treatment. ConclusionWe demonstrated that low-dose CO inhalation reduces hepatic warm IRI, potentially through downregulation of pro-inflammatory mediators and activation of anti-apoptotic pathways. To our knowledge, this is the first report demonstrating CO inhalation attenuated hepatic IRI following WI in a large animal model.

Sevoflurane has postconditioning as well as preconditioning properties against hepatic warm ischemia-reperfusion injury in rats.

Ischemia-reperfusion (IR) injury is inevitable after liver transplantation and liver resection with inflow occlusion. Sevoflurane has been widely used during hepatobiliary surgery and was reported to exhibit preconditioning (PreC) properties against hepatic IR injury; however, its postconditioning (PostC) properties remain unknown. This study examined whether a clinically applicable dose of sevoflurane has PostC and PreC properties against hepatic IR injury and roles of heme oxygenase-1 (HO-1). Warm ischemia was induced in male Wistar rats, excluding the sham group, for 1h, followed by 3h of reperfusion. Group C received propofol from 60min before ischemia until the end of the experimental procedure. In the SPreC and SPostC groups, propofol was replaced by 2.5% sevoflurane for 30min from 35min before ischemia in the SPreC group and for 30min from 5min before reperfusion in the SPostC group. The SPreC+Z and SPostC+Z groups received a HO-1 inhibitor, zinc protoporphyrin (Znpp), 60min before ischemia, and sevoflurane PreC and PostC were induced. Serum aspartate aminotransferase, alanine aminotransferase, and lactic dehydrogenase levels, and histological damage scores in the SPreC and SPostC groups were significantly lower than those in group C. Inhibiting HO-1 with Znpp partially blocked these protective effects of sevoflurane. Sevoflurane PreC and PostC significantly increased the number of HO-1-positive Kupffer cells in comparison with group C, and Znpp prevented sevoflurane-induced HO-1 expression. PostC and PreC by sevoflurane at a clinically applicable dose have equally protective effects against hepatic IR injury by increasing HO-1 expression.

Protective Effects of Ischemic Preconditioning Protocols on Ischemia-Reperfusion Injury in Rat Liver

Background: Hepatic ischemia reperfusion injury often leads to increased complications and mortality after surgery. Although ischemic preconditioning is used as a convenient and effective method to protect the liver from warm ischemia reperfusion injury, the optimal protocol is currently unclear. Therefore, in this study, we sought to identify ideal conditions and methods for ischemic preconditioning. Materials and methods: We compared several preconditioning protocols of the ischemia/reperfusion (I/R) cycle in 30 male Sprague-Dawley rats (5 groups, n = 6), including relevant sham and I/R injury (no preconditioning) controls. Experimental group conditions included: (1) ischemia for 5 min/reperfusion for 10 min (ischemic preconditioning 1, IPC-1); (2) ischemia for 5 min/reperfusion for 5 min, repeated three times (IPC-2); and (3) ischemia for 10 min/reperfusion for 10 min (IPC-3). Readouts included transaminase activity levels measured from collected sera, and histopathological changes, liver cell apoptosis, superoxide dismutase (SOD) activity, glutathione (GSH) levels, and malondialdehyde (MDA) levels measured from collected liver tissue segments subjected to warm ischemia (that is from the 70% of the liver mass that had been deprived from blood flow during the ischemia phase). Results: Compared to the I/R control group, the IPC-1, IPC-2, and IPC-3 groups all showed significant decreases in liver transaminase activity levels, alleviation of pathological injury-associated changes, and a decrease in liver cell apoptosis. Moreover, SOD activity and GSH content were increased while MDA content was decreased in the three experimental groups. Compared to the IPC-1 and IPC-3 groups, the changes in the IPC-2 group were the most significant (P < 0.05). Conclusions: Ischemic preconditioning can reduce hepatic warm ischemia reperfusion injury in rats. The IPC-2 protocol, involving ischemia for 5 min and reperfusion for 5 min, repeated three times, provided the optimal protection against hepatic ischemia reperfusion injury among the protocols studied.

3.0T intravoxel incoherent motion magnetic resonance imaging evaluation for the hepatic warm-ischemia injury in a rabbit model

Objective To investigate the feasibility and diagnostic efficacy of 3.0T intravoxel incoherent motion (IVIM) magnetic resonance imaging (MRI) in the evaluation of hepatic warm ischemia-reperfusion injury (WIRI) in a rabbit model. Methods Fifty healthy, adult, male New Zealand white rabbits were randomly divided into sham-operation group (n=10) and four test groups (each n=10), which depended on different warm ischemia time (ie, 10, 20, 30, and 40 min group). Hepatic WIRI was induced by occluding hepatic inflow for different minute and reperfusion for 6 hours. Traditional MRI and IVIM were performed on a 3.0T clinical magnetic resonance (MR) scanner, four diffusion parameters, that is apparent diffusion coefficient (ADC), true diffusion (Dslow), pseudodiffusion (Dfast) and perfusion fraction (PF), were calculated from IVIM image. Rabbits were sacrificed for biochemical and histomorphological analysis after MR scanning, which including alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH) of serum, and malondialdehyde (MDA), superoxide dismutase (SOD) and myeloperoxidase (MPO) of liver tissue. Statistical analysis was performed using one-way analysis of variance (ANOVA) or non-parametric test for multiple group comparisons of the IVIM parameters and biochemical parameters. The Pearson’s or Spearman’s correlation coefficient was calculated to assess the correlations between IVIM parameters and biochemical parameters. Receiver operating characteristic (ROC) curves were used to determine the diagnostic accuracy of IVIM parameters for predicting the hepatic WIRI. Results Compared with the sham group, the hepatic ADC, Dslow, Dfast, and PF were reduced in the test groups. Meanwhile, the values of IVIM parameters gradually decreased as prolonged warm ischemia time (ADC was 1.36±0.15, 1.28±0.07, 1.10±0.13, 1.10±0.15 and 0.98±0.18, Dslow was 1.29±0.14, 1.24±0.07, 1.08±0.11, 1.03±0.18 and 0.94±0.15, Dfast was 31.33±6.44, 25.53±2.10, 24.90±1.84, 23.00±1.59 and 23.76±1.41, PF was 30.44±2.80, 26.26±4.41, 24.50±2.24, 19.97±3.39 and 17.42±3.25, respectively) (P=0.005). IVIM parameters corresponded well with biochemical parameters (P=0.005). The area under curve (AUC) of ADC, Dslow, Dfast, and PF was 0.876, 0.824, 0.897 and 0.937, respectively. Histopathological analysis showed hepatocyte edema, inflammatory cell infiltration, sinusoids and central vein congestion, and different levels of fibrous tissue proliferation in the test groups. Conclusion 3.0T IVIM imaging is a noninvasive and quantitative technique in assessing the hepatic WIRI in a rabbit model, which implies its further application for dynamic monitoring of hepatic WIRI, and preclinical assessment of new pharmaceutics. Key words: Hepatic warm ischemia-reperfusion injury; Diffusion weighted imaging; Intravoxel incoherent motion; Magnetic resonance imaging

Morphological alterations and redox changes associated with hepatic warm ischemia-reperfusion injury.

AIMTo study the effects of warm ischemia-reperfusion (I/R) injury on hepatic morphology at the ultrastructural level and to analyze the expression of the thioredoxin (TRX) and glutaredoxin (GRX) systems.METHODSEleven patients undergoing liver resection were subjected to portal triad clamping (PTC). Liver biopsies were collected at three time points; first prior to PTC (baseline), 20 min after PTC (post-ischemia) and 20 min after reperfusion (post-reperfusion). Electron microscopy and morphometry were used to study and quantify ultrastructural changes, respectively. Additionally, gene expression analysis of TRX and GRX isoforms was performed by quantitative PCR. For further validation of redox protein status, immunogold staining was performed for the isoforms GRX1 and TRX1.RESULTSPost-ischemia, a significant loss of the liver sinusoidal endothelial cell (LSEC) lining was observed (P = 0.0003) accompanied by a decrease of hepatocyte microvilli in the space of Disse. Hepatocellular morphology was well preserved apart from the appearance of crystalline mitochondrial inclusions in 7 out of 11 patients. Post-reperfusion biopsies had similar features as post-ischemia with the exception of signs of a reactivation of the LSECs. No changes in the expression of redox-regulatory genes could be observed at mRNA level of the isoforms of the TRX family but immunoelectron microscopy indicated a redistribution of TRX1 within the cell.CONCLUSIONAt the ultrastructural level, the major impact of hepatic warm I/R injury after PTC was borne by the LSECs with detachment and reactivation at ischemia and reperfusion, respectively. Hepatocytes morphology were well preserved. Crystalline inclusions in mitochondria were observed in the hepatocyte after ischemia.

Multiparametric functional magnetic resonance imaging for evaluation of hepatic warm ischemia-reperfusion injury in a rabbit model

BackgroundTo assess the feasibility of noninvasive and quantitative evaluation of hepatic pathophysiological changes in rabbit hepatic warm ischemia-reperfusion injury (WIRI) models by using intravoxel incoherent motion (IVIM), diffusion tensor imaging (DTI) and blood oxygen level dependent (BOLD) MRI.MethodsTwenty rabbits were randomly divided into hepatic WIRI model group and sham-operation group (n = 10 for each group). Hepatic WIRI was induced in rabbit by occluding hepatic inflow for 30 min and reperfusion for 6 h. The control group only underwent laparotomy and liver ligament dissection. IVIM with 11 b values (0 to 800 s/mm2), DTI with 2 b values (0 and 500 s/mm2) on 12 diffusion directions, and BOLD MRI with 9 TE (2.57 to 24.25 ms) were performed at 3 T clinical MR scanner. Rabbits were sacrificed for biochemical and histopathological analysis after MR scanning. All of functional MR, biochemical and histopathological parameters were analyzed by independent sample t test, Mann-Whitney U test, Pearson and Spearman correlation methods.ResultsAll of MR parameters showed moderate to excellent interobserver reproducibility. True diffusion (Dslow), pseudodiffusion (Dfast), perfusion fraction (PF), and mean diffusitivity (MD) were lower in WIRI models than in control rabbits (P < 0.01), R2* was higher in WIRI models than in control rabbits (P < 0.001), while fractional anisotropy (FA) showed no statistical difference. There were significant differences in I score and all of biochemical parameters between the two groups (P < 0.01). Functional MR parameters corresponded well with all of biochemical parameters and some of histopathological parameters (P < 0.05). Histopathological analysis showed the structure and morphology of hepatic lobule was normal and clear in control rabbits, while diffuse hepatocyte swelling, central vein and sinusoids congestion, and inflammatory cell infiltration in WIRI models.ConclusionsIVIM, DTI, and BOLD MRI are noninvasive and useful techniques for assessing the microenvironment changes of hepatic WIRI in rabbit models.

Effect of astilbin on HO-1 expression in hepatic warm ischemia-reperfusion injury

Effect of astilbin on HO-1 expression in hepatic warm ischemia-reperfusion injury

Effects of a Preconditioning Oral Nutritional Supplement on Pig Livers after Warm Ischemia

Background. Several approaches have been proposed to pharmacologically ameliorate hepatic ischemia/reperfusion injury (IRI). This study was designed to evaluate the effects of a preconditioning oral nutritional supplement (pONS) containing glutamine, antioxidants, and green tea extract on hepatic warm IRI in pigs. Methods. pONS (70 g per serving, Fresenius Kabi, Germany) was dissolved in 250 mL tap water and given to pigs 24, 12, and 2 hrs before warm ischemia of the liver. A fourth dose was given 3 hrs after reperfusion. Controls were given the same amount of cellulose with the same volume of water. Two hours after the third dose of pONS, both the portal vein and the hepatic artery were clamped for 40 min. 0.5, 3, 6, and 8 hrs after reperfusion, heart rate (HR), mean arterial pressure (MAP), central venous pressure (CVP), portal venous flow (PVF), hepatic arterial flow (HAF), bile flow, and transaminases were measured. Liver tissue was taken 8 hrs after reperfusion for histology and immunohistochemistry. Results. HR, MAP, CVP, HAF, and PVF were comparable between the two groups. pONS significantly increased bile flow 8 hrs after reperfusion. ALT and AST were significantly lower after pONS. Histology showed significantly more severe necrosis and neutrophil infiltration in controls. pONS significantly decreased the index of immunohistochemical expression for TNF-α, MPO, and cleaved caspase-3 (P < 0.001). Conclusion. Administration of pONS before and after tissue damage protects the liver from warm IRI via mechanisms including decreasing oxidative stress, lipid peroxidation, apoptosis, and necrosis.

Meloxicam, a COX-2 Inhibitor, Ameliorates Ischemia/Reperfusion Injury in Non-Heart-Beating Donor Livers

Background/Aims: Chronic organ donor shortage has led to the consideration to expand the donor pool with livers from non-heart-beating donors (NHBD), although a higher risk of graft dys- or nonfunction is associated with these livers. We examined the effects of selective cyclooxygenase-2 (COX-2) inhibition on hepatic warm ischemia (WI) reperfusion (I/R) injury of NHBD. Methods: Male Wistar rats were used as donors and meloxicam (5 mg/kg body weight) was administered into the preservation solution. Livers were excised after 60 min of WI in situ, flushed and preserved for 24 h at 4°C. Reperfusion was carried out in vitro at a constant flow for 45 min. During reperfusion (5, 15, 30 and 45 min), enzyme release of alanine aminotransferase and glutamate lactate dehydrogenase were measured as well as portal venous pressure, bile production and oxygen consumption. The production of malondialdehyde was quantified and TUNEL staining was performed. Quantitative PCR analyzed COX-2 mRNA. COX-2 immunohistochemistry and TxB₂ detection completed the measurements. Results: Meloxicam treatment led to better functional recovery concerning liver enzyme release, vascular resistance and metabolic activity over time in all animals. Oxidative stress and apoptosis were considerably reduced. Conclusion: Cold storage using meloxicam resulted in significantly better integrity and function of livers retrieved from NHBD. Selective COX-2 inhibition is a new therapeutic approach achieving improved preservation of grafts from NHBD.

Implication of Protein Kinase A for a Hepato-Protective Mechanism of Milrinone Pretreatment

We have previously reported that an increase of adenosine 3',5'-cyclic monophosphate (cAMP) in liver tissue after an administration of milrinone, a phosphodiesterase-3 inhibitor attenuates hepatic warm ischemia-reperfusion injury. The aim of this study was to determine whether cAMP-dependent protein kinase (protein kinase A) activation was involved in the milrinone-induced hepatoprotective effect on an ischemia-reperfusion injury in an in vivo model. Male Lewis rats were allocated into 3 groups. In Group M, milrinone was administrated before ischemia; in Group I, a protein kinase A inhibitor, adenosine 3',5'-cyclic monophosphorothioate, 8-bromo-, Rp-isomer, sodium salt (Rp-8-Br-cAMPS), was injected prior to the administration of milrinone; and in Group C, the control group, there was no pretreatment. After pretreatment, all rats were exposed to a 45-min total hepatic inflow occlusion. After milrinone administration, liver cAMP concentrations and protein kinase A activity ratios were elevated. They protected the liver from ischemia-reperfusion injury. Rp-8-Br-cAMPS suppressed protein kinase A activation without affecting cAMP elevating responses to milrinone. Compared with Group C, hepatocellular necrosis, neutrophil infiltration, and congestion were ameliorated, and serum tumor necrosis factor-alpha, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase levels were significantly suppressed in Group M. Rp-8-Br-cAMPS canceled this effect, showing histological damages in Group I as severe as in Group C. The levels of tumor necrosis factor-alpha, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were the same in Groups C and I. Activation of protein kinase A might play an important role in the mechanism of milrinone-induced ischemic tolerance in the liver.

Possible Protection of Sinusoidal Endothelial Cells by Endothelin B Receptor During Hepatic Warm Ischemia–Reperfusion

Endothelins (ETs) are important regulators of the hepatic microcirculation. We investigated the pure biological roles of endothelin B receptors (ETB-Rs) on hepatic warm ischemia-reperfusion (I/R) injury using ETB-R deficient spotting lethal (sl) rats. Homozygous (sl/sl) and wild-type (+/+) rats were exposed to 60 min of 92% partial hepatic ischemia and then were killed at 2, 6, and 24 h, and 3 and 7 days after reperfusion. We measured the serum alanine aminotransferase (ALT) levels to assess hepatocyte injury, and the serum hyaluronic acid (HA) levels and factor VIII-related antigen (FVIIIRAg) staining to assess sinusoidal endothelial cell (SEC) injury. We also measured the concentrations of ET-1 and nitrite (NO2-) and nitrate (NO3-) of liver tissue samples. Although no significant difference was observed in the ALT levels, the HA levels were significantly elevated at an early stage after reperfusion in the sl/sl rats. Regarding FVIIIRAg staining, positive SECs were enhanced in the sl/sl rats. The ET-1 levels were also significantly elevated at an early stage after reperfusion in the sl/sl rats. Regarding the NO2- and NO3- levels, no significant difference was observed. Endothelin B receptor was shown to have a protective effect on SECs through the inhibition of ET-1 during hepatic warm I/R injury.

Dynamic Changes of Post-Ischemic Hepatic Microcirculation Improved by a Pre-Treatment of Phosphodiesterase-3 Inhibitor, Milrinone

Phosphodiesterase-3 inhibition has been shown to attenuate hepatic warm ischemia-reperfusion injury. The aim of this study was to investigate the effect of milrinone, phosphodiesterase-3 inhibitor, on post-ischemic microcirculation of rat livers by intravital microscopy. Male Wistar rats were randomly assigned to three groups; group A, milrinone pre-treatment; group B, ischemic pre-conditioning; and group C, no pre-treatment. All animals underwent a 60-min warm ischemia of the left lateral liver lobe. Microvascular perfusion and leukocyte-endothelial interaction were observed by intravital videomicroscopy. Hepatocellular viability and cellular damage were quantified by adenosine triphosphate tissue concentration as well as alanine aminotransferase and lactate dehydrogenase blood levels, respectively. In groups A and B, cyclic AMP hepatic tissue concentration was elevated significantly. After reperfusion, microvascular perfusion in hepatic sinusoids was significantly better maintained, and the number of adherent leukocytes was reduced in sinusoids and in post-sinusoidal venules in these rats. Serum transaminase blood levels were suppressed significantly in these groups compared with controls. The demonstrated improvement of hepatic microcirculation is certainly derived from milrinone induced cell protection in ischemia reperfusion of the liver. This effect is outlined by improved energy status and reduced liver enzyme liberation and mimics the effect of ischemic pre-conditioning.

Advantage of Ischemic Preconditioning for Hepatic Resection in Pigs

Ischemic preconditioning (IP) and intermittent inflow occlusion (IO) have provided beneficial outcomes in hepatic resection. However, comparison of these two procedures against warm hepatic ischemia-reperfusion injury has not been studied enough. Pigs that had undergone 65% hepatectomy were subjected to Control (120 min continuous ischemia, n = 6), IP (10 min ischemia and 10 min reperfusion, followed by 120 min continuous ischemia, n = 6), and IO (120 min ischemia in the form of eight successive periods of 15 min ischemia and 5 min reperfusion, n = 6). We evaluated hepatocyte injury by aspartate aminotransferase, lactate dehydrogenase and hepaplastin test, hepatic microcirculation by hepatic tissue blood flow (HTBF) and endothelin (ET)-1, inflammatory response by tumor necrosis factor-alpha (TNF-alpha), and histopathology after reperfusion. IP prevented hepatocyte injury, HTBF disturbance, and hepatocyte necrosis in histopathology as well as IO. These two groups showed significantly better outcomes than Control. IP produced significantly less ET-1 and TNF-alpha than IO. IP ameliorated hepatic warm ischemia-reperfusion injury. Furthermore, IP gained more advantages in preventing chemokine production such as ET-1 and inflammatory response over IO. IP could take the place of IO for hepatectomy.

MCI-186 (edaravone), a free radical scavenger, attenuates hepatic warm ischemia-reperfusion injury in rats

Hepatic warm ischemia-reperfusion injury (IRI) during hepatectomy and liver transplantation is a major cause of liver dysfunction in which the pathologic role of free radicals is a major concern. To assess the effect of MCI-186 (edaravone) on hepatic IRI, male Wistar rats were subjected to partial hepatic ischemia for 60 min after pretreatment with vehicle (group C) or MCI-186 (group M), or after both MCI-186 pretreatment and additional administration of MCI-186 12 h after reperfusion (group MX). Groups M and MX showed significantly lower levels of serum alanine aminotransferase and hepatic lipid peroxidation than group C, and also significantly lower expression levels of mRNA for cytokines, chemokines and intercellular adhesion molecule-1. There were fewer tissue monocytes and neutrophils in groups M and MX than in group C. These effects were more marked in group MX than in group M. Our findings suggest that treatment with MCI-186 attenuates hepatic IRI in this rat in vivo model.

Dual Role of Vascular Endothelial Growth Factor in Hepatic Ischemia-Reperfusion Injury

Vascular endothelial growth factor (VEGF), a major angiogenic factor, mediates a variety of disease conditions through promotion of angiogenesis. It also plays a critical role as a potent proinflammatory cytokine in a variety of physiologic and pathologic immune responses. In the present study, we evaluated the expression of VEGF in hepatic warm ischemia-reperfusion (I/R) injury and examined the effect of recombinant human (rh)VEGF administration in an established murine model. The expression of VEGF in the liver was assessed by quantitative real-time polymerase chain reaction and immunohistochemistry during I/R injury using 70% partial hepatic ischemia model. The effect of rhVEGF administration on I/R injury was evaluated by measuring liver function and histology. In addition, local inducible nitric oxide synthase (iNOS) and endothelial NO synthase expressions were examined to address the underlying mechanisms. The local expression of VEGF was significantly up-regulated at 2 hours after reperfusion after 60 minutes of ischemia compared with that in the naive liver. VEGF was expressed predominantly in CD11b+ cells infiltrating into the ischemic liver. The administration of rhVEGF had a significant protective effect on ischemic injury in the liver. This effect was associated with the up-regulation of iNOS expression in the rhVEGF-treated liver. We demonstrate a dual role of VEGF in hepatic warm I/R injury. Although endogenous VEGF is expressed and functional to initiate hepatic I/R injury, exogenous rhVEGF has a beneficial effect on the ischemic liver. These data may provide new insights into the role of VEGF as well as pathophysiology of hepatic I/R injury.