Warm Ischemia-reperfusion Injury Model Research Articles

#2027 Activation of histone deacetylase-1 in fibroblasts and pericytes induces renal interstitial fibrosis after ischemia-reperfusion injury

Abstract Background and Aims Following an acute kidney ischemic event the chromatin remodelling enzyme, histone deacetylase-1 (HDAC1), is activated in many cell types of the kidney including fibroblasts and pericytes. During kidney injury, the fibroblasts/pericytes differentiate into the myofibroblasts and produce extracellular matrix that can lead to a permanent decline in kidney function. Pharmacological inhibition of HDACs can attenuate ischemia-reperfusion-injury (IRI) mediated interstitial fibrosis. The hypothesis of this study was that fibroblast/pericyte HDAC1 activation promotes myofibroblast differentiation and interstitial fibrosis. Method Tamoxifen inducible, fibroblast/pericyte HDAC1 knockout (KO) mice (HDAC1Fl/Fl; Col1a2-CreEr) and littermate controls (HDAC1Fl/Fl) were used. Male and female mice were given tamoxifen by ip at 8-10 weeks of age and IRI or sham surgery completed after a 2-week tamoxifen washout period. A mild, 18 min, bilateral, warm IRI model was used, and samples collected over 4 weeks. Glomerular filtration rate (GFR) was assessed using transdermal FITC-Sinistrin clearance. In vitro experiments with kidney fibroblast cells (NRK49F) overexpressing empty vector or HDAC1 were used for RNA-sequencing to determine the HDAC1-dependent myofibroblast transcriptome. Results We confirmed that HDAC1 was knockdown in myofibroblast cells from KO mice with co-localization of HDAC1 and PDGFRβ or α-smooth muscle actin in the kidneys of IRI mice. Twenty-four hours post-surgery, all IRI mice exhibited 2-3 times increase in serum creatinine and a ∼50% reduction in GFR compared to sham mice (control IRI = 513 ± 129 μl /min/100 g body mass, control sham = 1010 ± 218, n = 4-5, p = 0.02; KO IRI = 529 ± 116, KO sham = 1187 ± 59 μl/min/100 g body mass, n = 4-5, p = 0.004). Subsequent GFR measurements over the next 4 weeks showed a gradual recovery of renal function of IRI mice starting at week 2 and fully restored to sham values by week 4. However, the male control IRI mice had significant focal interstitial fibrosis 2- and 4-weeks post injury (4-week data: sham = 0.3 ± 0.09 % of cortical area, IRI = 1.2 ± 0.2%, n = 6/group, p = 0.0011) but this was attenuated in the KO male IRI mice (sham = 0.4 ± 0.07% n = 4, IRI = 0.5 ± 0.1%, n = 7, p = 0.93). The female mice, regardless of genotype, had very little kidney damage or interstitial fibrosis at 4 weeks. Transcriptomes of NRK49F cells overexpressing HDAC1 had an upregulation of 55 genes related to pro-inflammatory cytokines and tumour necrosis factor signalling and downregulation of 367 genes related to cell junctions, cytoskeleton proteins, and cell adhesion compared to the empty vector control cells. Fibroblast proliferation rates were not statistically different among the groups, suggesting HDAC1 does not affect myofibroblast proliferation but rather pro-inflammatory gene expression. Conclusion In male mice, fibroblast/pericyte activation of HDAC1 leads to the production of pro-inflammatory cytokines that we predict will lead to immune cell infiltration and crosstalk that leads to the excessive production of interstitial fibrosis following IRI.

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.

Oxaloacetate Protects Rat Liver From Experimental Warm Ischemia/Reperfusion Injury by Improving Cellular Energy Metabolism

Liver ischemia/reperfusion injury (IRI) is an important cause of liver damage especially early after liver transplantation, following liver resection, and in other clinical situations. Using rat experimental models, we identified oxaloacetate (OAA) as a key metabolite able to protect hepatocytes from hypoxia and IRI. In vitro screening of metabolic intermediates beneficial for hepatocyte survival under hypoxia was performed by measures of cell death and injury. In vivo, the effect of OAA was evaluated using the left portal vein ligation (LPVL) model of liver ischemia and a model of warm IRI. Liver injury was evaluated in vivo by serum transaminase levels, liver histology, and liver weight (edema). Levels and activity of caspase 3 were also measured. In vitro, the addition of OAA to hepatocytes kept in a hypoxic environment significantly improved cell viability (P < 0.01), decreased cell injury (P < 0.01), and improved energy metabolism (P < 0.01). Administration of OAA significantly reduced the extent of liver injury in the LPVL model with lower levels of alanine aminotransferase (ALT; P < 0.01), aspartate aminotransferase (AST; P < 0.01), and reduced liver necrosis (P < 0.05). When tested in a warm IRI model, OAA significantly decreased ALT (P < 0.001) and AST levels (P < 0.001), prevented liver edema (P < 0.001), significantly decreased caspase 3 expression (P < 0.01), as well as histological signs of cellular vesiculation and vacuolation (P < 0.05). This was associated with higher adenosine triphosphate (P < 0.05) and energy charge levels (P < 0.01). In conclusion, OAA can significantly improve survival of ischemic hepatocytes. The hepatoprotective effect of OAA was associated with increased levels of liver bioenergetics both in vitro and in vivo. These results suggest that it is possible to support mitochondrial activity despite the presence of ischemia and that OAA can effectively reduce ischemia-induced injury in the liver.

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.

Down-Regulation of MicroRNA-146a in the Early Stage of Liver Ischemia-Reperfusion Injury

BackgroundMicroRNAs (miRNAs), 21-23-nucleotide noncoding RNAs, act as regulators of gene expression transcriptionally. MicroRNA-146a(miR-146a) has been demonstrated to be one of the key molecules in oncogenesis and inflammatory responses. Few data describe the expression of miR-146a in liver ischemia-reperfusion (IR) injury. The present study sought to explore the relationship of miR-146a to Toll-like receptor 4 (TLR4) signaling pathways in a rat model of warm IR injury. MethodsThe expression of miR-146a was detected by real-time reverse-transcriptase polymerase chain reaction using a partial warm hepatic IR injury model. The expression of TLR4, tumor necrosis factor receptor-associated factor 6 (TRAF6), and interleukin-1 receptor-associated kinase (IRAK 1) protein was assessed by Western blotting as well as the signaling pathways induced by TLR4. ResultsThe expression of hepatic miR-146a was down-regulated in IR injury during the 24 hours after reperfusion, reaching the lowest level at 6 hours after reperfusion. Increases in TLR4, TRAF6, and IRAK1 were accompanied by decreased miR-146a during the 24 hours after reperfusion, peaking at 6 hours. Immunohistochemistry showed cytoplasmic expression of cells positive for TLR4, and nuclear expression of cells positive for nuclear factor κB p65 and c-jun to be increased among IR groups after reperfusion. ConclusionmiR-146a was down-regulated in the early stage of liver IR injury.

The Protective Role of CD59 and Pathogenic Role of Complement in Hepatic Ischemia and Reperfusion Injury

Hepatic ischemia-reperfusion injury (IRI) is a major factor influencing graft outcome in liver transplantation, but its mechanism is not well defined. Although complement, including the membrane attack complex (MAC), a terminal product of complement activation, is thought to be involved in the multiple reactions subsequent to the ischemia-reperfusion (IR) process, the role of MAC in the pathogenesis of hepatic IRI requires further investigation. We used a warm ischemia-reperfusion injury model in mice and a syngeneic orthotopic liver transplantation model in rats to define the role of complement, including MAC, in hepatic IR. CD59-deficient mice had more severe liver dysfunction, evidenced by increased aspartate aminotransferase levels and increased injury of liver parenchymal and nonparenchymal cells than did CD59-sufficient mice during warm hepatic IR. Furthermore, complement depletion in CD59-deficient mice by pretreatment with cobra venom factor (CVF) or the genetic introduction of C3 deficiency partially protected against development of the severe liver dysfunction that occurred in CD59-deficient mice. Severity of liver dysfunction correlated with MAC deposition, apoptotic cells, and increased inflammatory mediators such as tumor necrosis factor α. Moreover, depletion of complement with CVF in orthotopic liver transplantation recipient rats attenuated IRI of the donor livers. Taken together, these results highlight the protective role of CD59 and pathogenic role of complement, including MAC, in the pathogenesis of hepatic IRI.

Kupffer-cell activity is essential for thyroid hormone rat liver preconditioning

We studied the role of Kupffer cell functioning in T3 liver preconditioning against ischemia-reperfusion (IR) injury using the macrophage inactivator gadolinium chloride (GdCl3) previous to T3 treatment. Male Sprague-Dawley rats given a single i.p. dose of 0.1 mg T3/kg were subjected to 1 h ischemia followed by 20 h reperfusion, in groups of animals pretreated with 10 mg GdCl3/kg i.v. 72 h before T(3) or with the respective vehicles. IR resulted in significant enhancement of serum aspartate aminotransferase (3.3-fold increase) and tumor necrosis factor-alpha (93% increase) levels, development of liver damage, and diminished nuclear factor-kappaB DNA binding over control values. These changes, which were suppressed by the T3 administration prior to IR, persisted in animals given GdCl3 before T3 treatment, under conditions of complete elimination of ED2+ Kupffer cells achieved in a time window of 72 h. It is concluded that Kupffer cell functioning is essential for T3 liver preconditioning, assessed in a warm IR injury model by hepatic macrophage inactivation.

Carbon monoxide reduces pulmonary ischemia–reperfusion injury in miniature swine

Carbon monoxide is produced endogenously as a by-product of heme catalysis and has been shown to reduce ischemia-reperfusion injury in a variety of organs in murine models. The aims of this translational research were to establish an in situ porcine lung model of warm ischemia-reperfusion injury and to evaluate the cytoprotective effects of low-dose inhaled carbon monoxide in this model. Warm ischemia was induced for 90 minutes by clamping the left pulmonary artery and veins in 8 Clawn miniature swine (Japan Farm CLAWN Institute, Kagoshima, Japan). The left main bronchus was also dissected and reanastomosed just before reperfusion. Four animals were treated with inhaled carbon monoxide at a concentration of approximately 250 ppm throughout the procedure. Lung function and structure were serially accessed via lung biopsy, chest x-ray films, and blood gas analysis. Carbon monoxide inhalation dramatically decreased the lung injury associated with ischemia and reperfusion. Two hours after reperfusion, the arterial oxygen tension of the carbon monoxide-treated group was 454 +/- 34 mm Hg, almost double the arterial oxygen tension of the control group (227 +/- 57 mm Hg). There were fewer pathologic changes seen on chest x-ray films and in biopsy samples from animals in the carbon monoxide-treated group. Animals in the carbon monoxide-treated group also had fewer inflammatory cell infiltrates and a markedly smaller increase in serum concentrations of the proinflammatory cytokines interleukin 1beta, interleukin 6, and high-mobility group box 1 after ischemia-reperfusion injury. The perioperative administration of low-dose inhaled carbon monoxide decreases warm ischemia-reperfusion injury in lungs in miniature swine. This protective effect is mediated in part by the downregulation of proinflammatory mediators.

Causal role of oxidative stress in liver preconditioning by thyroid hormone in rats

Hepatic ischemia–reperfusion (IR) injury, a major clinical drawback during surgery, is abolished by l-3,3′,5-triiodothyronine (T 3) administration. Considering that the triggering mechanisms are unknown, the aim of this study is to assess the role of oxidative stress in T 3 preconditioning using N-acetylcysteine (NAC) before T 3 administration. Male Sprague–Dawley rats given a single dose of 0.1 mg of T 3/kg were subjected to 1 h ischemia followed by 20 h reperfusion, in groups of animals pretreated with 0.5 g of NAC/kg 0.5 h before T 3 or with the respective control vehicles. At the end of the reperfusion period, blood and liver samples were taken for analysis of serum aspartate aminotransferase (AST) and hepatic histology, glutathione (GSH) and protein carbonyl contents, and nuclear factor-κB (NF-κB) and activating protein 1 (AP-1) DNA binding. The IR protocol used led to a 4.5-fold increase in serum AST levels and drastic changes in liver histology, with significant GSH depletion and enhancement of protein carbonyl levels and of the protein carbonyl/GSH content ratio, whereas NF-κB and AP-1 DNA binding was decreased and enhanced, respectively. In a time window of 48 h, T 3 exerted protection against hepatic IR injury, with 88% reduction in the protein carbonyl/GSH ratio and normalization of NF-κB and AP-1 DNA binding, changes that were suppressed by NAC administration before T 3. Data presented suggest that a transient increase in the oxidative stress status of the liver is an important trigger for T 3 preconditioning, evidenced in a warm IR injury model through antioxidant intervention.

Do contrast media (iomeprol, gadopentetate dimeglumine) deteriorate ischemia/reperfusion injury of the liver?

Hepatic microcirculation is a main determinant of reperfusion injury and graft quality in liver transplantation. One of the important diagnostic procedures to recognize reperfusion failure is contrast-enhanced computed tomography or magnetic resonance imaging. To examine the additional effect of contrast media (iomeprol and gadopentetate dimeglumine) on hepatic microcirculation and hepatic cellular damage in the phase of early ischemia/reperfusion injury of the rat liver. The partial warm ischemia-reperfusion injury model of rat liver was used. Microcirculation and leukocyte-endothelium interaction were measured by intravital microscopy. Hepatic cellular damage was indicated by liver enzyme activity in the sera. The evaluation parameters were measured at baseline and at 30, 60, and 90 min after reperfusion. The contrast media (iomeprol group, n = 6; gadopentetate dimeglumine group, n = 6) or Ringer's solution (control group, n = 8) were applied after 30 min of reperfusion. No additional injury to the ischemia/reperfusion injury of the liver after intravenous application of radiographic contrast media was found. Some protective effect was even recorded after application of iodinated contrast media. The use of contrast media during diagnostic procedure of the liver seems to be relatively safe, even in the stage of early reperfusion after liver transplantation.

Cellular infiltrates and injury evaluation in a rat model of warm pulmonary ischemia–reperfusion

IntroductionBeside lung transplantation, cardiopulmonary bypass, isolated lung perfusion and sleeve resection result in serious pulmonary ischemia–reperfusion injury, clinically known as acute respiratory distress syndrome. Very little is known about cells infiltrating the lung during ischemia–reperfusion. Therefore, a model of warm ischemia–reperfusion injury was applied to differentiate cellular infiltrates and to quantify tissue damage.MethodsFifty rats were randomized into eight groups. Five groups underwent warm ischemia for 60 min followed by 30 min and 1–4 hours of warm reperfusion. An additional group was flushed with the use of isolated lung perfusion after 4 hours of reperfusion. One of two sham groups was also flushed. Neutrophils and oedema were investigated by using samples processed with hematoxylin/eosin stain at a magnification of ×500. Immunohistochemistry with antibody ED-1 (magnification ×250) and antibody 1F4 (magnification ×400) was applied to visualize macrophages and T cells. TdT-mediated dUTP nick end labelling was used for detecting apoptosis. Statistical significance was accepted at P < 0.05.ResultsNeutrophils were increased after 30 min until 4 hours of reperfusion as well as after flushing. A doubling in number of macrophages and a fourfold increase in T cells were observed after 30 min until 1 and 2 hours of reperfusion, respectively. Apoptosis with significant oedema in the absence of necrosis was seen after 30 min to 4 hours of reperfusion.ConclusionsAfter warm ischemia–reperfusion a significant increase in infiltration of neutrophils, T cells and macrophages was observed. This study showed apoptosis with serious oedema in the absence of necrosis after all periods of reperfusion.

Effect of ischemic preconditioning on the intestinal microvascular perfusion and oxygenation in reperfusion injury of the intestine

Background. Ischemia reperfusion (IR) injury of the intestine is inevitable in small bowel transplantation. Ischemic preconditioning (IPC) provides a way of protecting the intestine from damage inflicted by IR injury. This study was designed to evaluate the effect of IPC on the intestinal microvascular perfusion (IMP) and the intestinal tissue oxygenation. Methods. Rats were allocated into (a) sham group, (b) IR group consisting of 30 min of intestinal ischemia followed by 2 h of reperfusion, and (c) IPC group, as in IR group, but preceded by 10 min of ischemia and 10 min of reperfusion. Heart rate (HR), arterial oxygen saturation (SaO 2), and mean arterial pressure (MAP) were monitored continuously. IMP was continuously monitored by laser Doppler flowmetry (LDF). Near Infrared reflectance spectroscopy (NIRS) was used to monitor intestinal oxygenation, namely oxyhemoglobin (HbO 2) and cytochrome oxidase Cu A redox state (Cyt Ox) continuously. At the end of the reperfusion, blood samples for lactate dehydrogenase (LDH) levels and biopsies of ileum for histopathological examination were obtained. Data were analyzed using one-way analysis of variance (ANOVA) and Bonferroni adjustment for multiple comparisons were used with Prism® software. Results. HR and SaO 2 did not change statistically throughout the experiment ( P > 0.05). During ischemia there was an increase of MAP from the baseline in both IR (87 ± 3.6 to 111.5 ± 4.8 mmHg, P < 0.01) and IPC groups (87.7 ± 6.5 to 111.7 ± 4.6 mmHg, P < 0.01).There was a significant improvement in IMP with IPC ( P < 0.01 versus IR). IPC also improved the HbO 2and Cyt Ox significantly ( P < 0.001 versus IR) at the end of the reperfusion. Serum LDH levels were significantly reduced ( P < 0.001) in IPC (667.1 ± 86.8) as compared to Group IR (1973.8 ± 306.5). Histological evaluation demonstrated that IPC lowered the intestinal mucosal injury grade ( P < 0.01 versus IR). Conclusion. This study demonstrates that IPC improves the intestinal microvascular perfusion and tissue oxygenation in a model of warm IR injury of the intestine. These findings suggest that IPC may be applied during small bowel transplantation to reduce IR injury.

Warm Hepatic Ischemia in Pigs: Effects of L-Arginine and Oligotide Treatment

Reperfusion injury represents a key event leading to graft nonfunction. Maintaining adequate nitric oxide levels and stimulating vasodilator synthesis can probably minimize endothelial damage. The aim of this study was to investigate the effect of L-arginine, a substrate of nitric oxide synthesis, and oligotide, a promoter of prostacyclin synthesis, on liver function and morphology after warm ischemia-reperfusion injury. After constructing a side-to-side portacaval shunt, ischemia was induced by clamping the hepatic hilum for 2 h above the shunt, in 19 female pigs divided into a control group ( n = 7), an L-arginine treatment group ( n = 6), and an oligotide treatment group ( n = 6). Liver function tests and measurements of serum and red blood cell malondialdehyde and plasma nitric oxide levels were performed before reperfusion and at 1, 10, 60, and 120 min after reperfusion. Liver biopsies, taken before reperfusion and at 30 min and 7 days after reperfusion, were analyzed for tissue malondialdehyde, histological-ultrastructural features, and apoptosis evaluation. Thirty minutes after reperfusion, liver malondialdehyde, sinusoidal congestion, necrosis, and apoptosis were significantly lower in the L-arginine group than in the controls ( p < .05). On postoperative day 7, tissue malondialdehyde decreased, while plasma nitric oxide and hepatocyte glycogen content were increased in the L-arginine group compared to controls ( p < .05). This study demonstrates the protective effect of L-arginine on hepatic lipoperoxidation and liver morphology in a pig model of warm ischemia-reperfusion injury. The increased plasma levels of nitric oxide a week after ischemia-reperfusion injury support the hypothesis that it has a role in preventing liver damage. The same beneficial effect was not confirmed for oligotide.