Model Of Ischemia-reperfusion Injury In Mice Research Articles

Bilateral Renal Ischemia-Reperfusion Model for Acute Kidney Injury in Mice.

Acute kidney injury (AKI) is defined as a rapid decline in renal function, in which persistent kidney dysfunction gradually progresses to chronic kidney disease (CKD) due to the irreversible loss of nephrons and their maladaptive repair. In recent years, the incidence of AKI has been increasing concerning diverse etiologies, including volume depletion, sepsis, nephrotoxicity, muscle injury, and major trauma, in which ischemia-reperfusion injury (IRI)accounts for most episodes. Development of the IRI model in mice is induced by surgical clamping of the renal pedicles, which provides powerful and controllable tools for preclinical models of AKI. Importantly, the IRI model is deployed at different stages of the AKI development, especially in the processes of AKI to CKD. Despite the IRI model being widely practiced in many laboratories, a series of variables still influence the results of this model. Here, we describe the procedure of IRI model development to provide a repeatable and reliable method for researchers to explore the underlying pathogenesis in the development of AKI and the progression of AKI to CKD.

Purinergic P2Y2 receptor-induced activation of endothelial TRPV4 channels mediates lung ischemia-reperfusion injury.

Lung ischemia-reperfusion injury (IRI), characterized by inflammation, vascular permeability, and lung edema, is the major cause of primary graft dysfunction after lung transplantation. Here, we investigated the cellular mechanisms underlying lung IR-induced activation of endothelial TRPV4 channels, which play a central role in lung edema and dysfunction after IR. In a left lung hilar-ligation model of IRI in mice, we found that lung IRI increased the efflux of ATP through pannexin 1 (Panx1) channels at the endothelial cell (EC) membrane. Elevated extracellular ATP activated Ca2+ influx through endothelial TRPV4 channels downstream of purinergic P2Y2 receptor (P2Y2R) signaling. P2Y2R-dependent activation of TRPV4 channels was also observed in human and mouse pulmonary microvascular endothelium in ex vivo and in vitro models of IR. Endothelium-specific deletion of P2Y2R, TRPV4, or Panx1 in mice substantially prevented lung IRI-induced activation of endothelial TRPV4 channels and lung edema, inflammation, and dysfunction. These results identify endothelial P2Y2R as a mediator of the pathological sequelae of IRI in the lung and show that disruption of the endothelial Panx1-P2Y2R-TRPV4 signaling pathway could be a promising therapeutic strategy for preventing lung IRI after transplantation.

Gα13-Mediated β2 Integrin Outside-in Signaling in Neutrophil Migration

Transendothelial migration of neutrophils requires chemoattractant signals and also integrin family of adhesion receptors, particularly the β 2 family of integrins, including Mac-1 and LFA-1. Signals transmitted by G protein-coupled receptors (GPCR) for chemoattractants and cytokines induces inside-out signaling activating the ligand binding function of integrins. Conversely ligand binding to integrins stimulates outside-in signaling, leading to cell spreading, retraction and migration. The heterotrimeric G protein subunit, Gα13, is important for GPCR signaling leading to RhoA activation but also binds to integrins, including β2 integrins to stimulate outside-in signaling. To study the roles of Gα13 in neutrophil migration, we tested the effect of Gα13 knockout on transendothelial migration of neutrophils stimulated by chemoattractant fMIVIL. We demonstrate that transendothelial migration of Gα13 knockout neutrophils was significantly but partially reduced as compared with wild type mice. Transendothelial migration of Gα13 knockout neutrophils is similar to wild type neutrophil migration neutralized with an anti-Mac1 (anti-αm) antibody, and was not further inhibited by the anti-Mac1 antibody, suggesting that transendothelial migration mediated by integrin αmβ2 was predominantly Gα13-dependent. Interestingly, either anti-β2 antibody or anti-LFA1 (anti-αL) antibody appeared to inhibit transendothelial migration of not only wild type neutrophils, but also to a degree, Gα13-knockout neutrophils, suggesting a minor LFA1-dependent but Gα13-independent component of transendothelial migration in addition to the Gα13-dependent transendothlial migration. Furthermore, even though the fibrinogen and ICAM-1 are both β2 ligands, we show that more neutrophils migrated through ICAM-1-coated transwells than fibrinogen-coated transwells, and only ICAM-1-mediated neutrophil migration is Gα13 dependent, suggesting that Gα13-dependent neutrophil migration is selective for certain β2 integrin ligand (ICAM-1). Importantly, Gα13 knockout selectively inhibited the velocity of neutrophil migration on integrin ligand ICAM-1, but had no effect on the directionality of neutrophil migration which requires GPCR-dependent chemoattactant signaling. To understand whether and how Gα13 regulate integrin signaling, we show that Gα13 knockout did not affect the static adhesion of neutrophils to ICAM1, but significantly inhibited neutrophil spreading on ICAM-1. Furthermore, Gα13 bound to β2 integrins in neutrophils adherent on ICAM-1, and this binding was inhibited by the ExE motif peptide MB2mP6 derived from the Gα13 binding site of β2 integrin cytoplasmic domain. MB2mP6 also inhibited transendothelial cell migration similarly as Gα13 knockout. These data suggest that Gα13 plays an important role in promoting β2-integrin dependent neutrophil transendothelial migration mainly by mediating integrin outside-in signaling. Consistent with previous findings of the role of Gα13-dependent outside-in signaling in negative regulation of RhoA in other integrin subtypes, both Gα13 knockout and MB2mP6 abolished the transient inhibition in RhoA during adhesion of neutrophils on ICAM-1. These data suggest that Gα13 mediates outside-in signaling and transient inhibition of RhoA, and thus promotes neutrophil spreading and migration on integrin ligands. To test the role of Gα13 in neutrophil migration in vivo, we showed that neutrophil infiltration in vivo was reduced in leukocyte-selective Gα13 knockout mice using both thioglycolate-induced peritoneal neutrophil infiltration model and LPS-induced neutrophil lung infiltration model in vivo. Furthermore, MB2mP6 inhibited neutrophil infiltration in cardiac tissues in the cardiac ischemia-reperfusion injury model in mice. These data suggest that Gα13-integrin interaction plays an essential role in the integrin-dependent transendothelial migration and is likely to be important in neutrophils' immune and inflammatory functions. DisclosuresNo relevant conflicts of interest to declare.

Our Strategy to Overcome Hepatic Ischemia Reperfusion Injury -From Bench to Bedside-

Background: In the field of liver surgery, especially transplantation, ischemia reperfusion injury (IRI) causes approximately 10% of early graft failure and leads to a higher incidence of acute and chronic rejection. It is an urgent issue to overcome IRI from the viewpoint of organ preservation. This study aims to reveal the molecular mechanism and develop the effective protective way to prevent the liver IRI for a safer liver surgery. Methods :We applied the established partial warm hepatic IRI model in mice and investigated changes of immune responses due to IR, and compared the efficacy of various biological molecules (neutrophil elastase inhibitor, recombinant Galectin-9, recombinant thrombomodulin and so on), antioxidant agents (vitamins C and E) and maneuvers (preoperative short-term fasting). The liver damage was assessed by serum alanine aminotransferase (sALT) level, histological observations, change in expression profile of various cytokines and analysis of intracellular molecules. Results: The most effective way to improve IRI was the preoperative short-term (12 hours) fasting. It exhibited a remarkable therapeutic effect compared with any other drug or diet we have so far examined. The up-regulation of Forkhead Box O1 (FOXO-1) induced by the raised acetylated histone and β-hydroxybutyric acid was a crucial factor for the short-term fasting to ameliorate the liver injury due to IR. Conclusion: Preoperative short-term dietary restriction might play an important role to overcome liver IRI and have a therapeutic potential for clinical setting.

SNHG16 accelerates the proliferation of primary cardiomyocytes by targeting miRNA-770-5p.

The present study aimed to clarify the influence of long non-coding RNA small nuclear host gene 16 (lncRNA SNHG16) on cardiomyocyte proliferation following ischemia/reperfusion injury (IRI) and the potential mechanism. An IRI model in mice was established by performing ligation of the anterior descending coronary artery (LAD). Primary cardiomyocytes were isolated from newborn mice and subjected to H2O2 treatment to mimic in vitro IRI. Relative levels of SNHG16 and miRNA-770-5p in both in vivo and in vitro IRI models were examined. The regulatory effects of SNHG16 and miRNA-770-5p on the proliferative ability of H2O2-treated cardiomyocytes were assessed by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assay. The binding relationship between SNHG16 and miRNA-770-5p was verified through dual-luciferase reporter gene assay. It is found that SNHG16 was time-dependently downregulated in the IRI models. Overexpression of SNHG16 enhanced the proliferative ability of the cardiomyocytes. miRNA-770-5p was found to be a direct target of SNHG16. Moreover, SNHG16 was able to negatively regulate the miRNA-770-5p level. Overexpression of miRNA-770-5p partially reversed the role of SNHG16 on accelerating cardiomyocyte proliferation. Collectively, SNHG16 accelerates the proliferative ability of cardiomyocytes following IRI by negatively regulating miRNA-770-5p.

Down-regulation of nuclear HMGB1 reduces ischemia-induced HMGB1 translocation and release and protects against liver ischemia-reperfusion injury

Hepatocyte-specific HMGB1 deletion has been found to worsen the injury and inflammation in liver ischemia-reperfusion injury (IRI), highlighting a role for intracellular HMGB1 in cellular protection. Down-regulation of nuclear HMGB1 by small interfering RNA (siRNA) might not only decrease its injurious extracellular role by reducing its release but also serve to maintain its beneficial intracellular role, thus protecting against IRI. We established a non-lethal liver IRI model in mice via segmental hepatic warm ischemia for 1 h and reperfusion for 6 h. HMGB1-siRNA achieved a reduction of ~60–70% in the nuclear HMGB1 expression in the liver at 48 h post-treatment. Knockdown of nuclear HMGB1 expression dramatically reduced both the degree of nuclear-cytoplasmic translocation of HMGB1 during hepatic ischemia and of HMGB1 release after hepatic reperfusion, resulting in significant preservation of liver function and a marked reduction in pathological damage. Also, HMGB1-siRNA pretreatment markedly inhibited the increases in hepatic expression of TLR4, TLR2, RAGE, TNF-α, IL-1β, IL-6, MCP-1, iNOS, and COX-2 seen in control mice after hepatic reperfusion. We demonstrated for the first time that down-regulation of nuclear HMGB1 reduces ischemia-induced HMGB1 release and protects against liver IRI, which is helpful for better understanding the role of HMGB1 in organ IRI.

Outer Balloon Ligation Increases Success Rate of Ischemia-Reperfusion Injury Model in Mice.

BackgroundCoronary artery disease is a growing public health problem and a major cause of morbidity and mortality. Experimental animal models provide valuable tools for studying myocardial ischemia reperfusion (I/R) injury in vivo.ObjectiveThe purpose of this study was to describe a new method (outer balloon ligation) to induce myocardial I/R injury in mice.MethodsNinety-nine male C57BL/6J mice were randomly divided into three groups: sham group, classic method group (I/R-C) and the new method group (I/R-N). The surgical procedure and recovery time were recorded. The levels of TNF-α, IL-6, cTnT and LDH were detected by ELISA kits. Hematoxylin-eosin staining was applied to assess neutrophil infiltration. Moreover, surgical survival, myocardial infarction areas, and cardiac function measurements were also recorded.ResultsThe reperfusion operation time in the I/R-N group were markedly less than the I/R-C group (14.73±2.86 vs. 168.60±33.01 sec, p <0.0001). Similarly, the recovery time in I/R-N group was shorter than the I/R-C group (45.39±15.39 vs. 101.70±19.33 min, p <0.0001). The levels of TNF-α and IL-6 in I/R-N group were also markedly lower than in I/R-C group (136.5±22.21 vs. 170.5±24.79 pg/ml, p <0.05 and 100.3±23.74 vs. 144.40±22.24 pg/ml, p <0.001). Compared I/R-N group with I/R-C group, the levels of neutrophil infiltration, cTnT and LDH had no significant differences. Surgical survival rate was 96.7% in the I/R-N group, which was significantly improved compared to the rate of 80% in the I/R-C group. However, there were no significant differences in the areas of myocardial infarction and cardiac function between the two groups.ConclusionsCompared with the classic method, our new method of inducing myocardial I/R injury has higher efficiency and less tissue damage in mice, but achieves the same modeling effects.

Evaluation of Vitamin C Supplementation on Kidney Function and Vascular Reactivity Following Renal Ischemic Injury in Mice

Background/Aims: Renal ischemia/reperfusion injury (IRI) is a very common clinical event and usually leads to ischemic acute renal failure (ARF). In the present study, we investigated the protective role of vitamin C in renal function and renal arterial relaxation following ischemic injury. Methods: IRI model in mice was used. Various biochemical parameters including nitric oxide (NO), reduced glutathione (GSH), total reactive oxygen species (ROS) level and superoxide dismutase (SOD) were examined. Doppler was used to investigate renal arterial resistance. The isolated renal arterial rings served for hypoxia/reoxygenation analysis. Acetylcholine (ACh) and sodium nitroprusside (SNP)-induced relaxations of isolated renal arterial rings were exerted. Results: Vitamin C supplementation preserved kidney morphology and renal function following IRI. It was shown that pretreatment with vitamin C for mice subjected to IRI significantly elevated renal NO and GSH levels after reperfusion. Meanwhile, vitamin C administration decreased resistance index of renal artery and ameliorated oxidative stress secondary to IRI. The total ROS level in renal artery was decreased whereas the renal arterial SOD expression was increased by vitamin C supplementation following IRI. Pretreatment with vitamin C significantly potentiated the ACh and SNP-induced relaxations in both control and hypoxic renal arterial rings. Conclusion: Vitamin C protects kidney function and renal arterial reactivity against IRI. The protective role of vitamin C is linked to ROS, SOD, GSH and NO levels in renal ischemic injury.

FK506 reduces calpain-regulated calcineurin activity in both the cytoplasm and the nucleus.

Excessive immune responses induced by ischemia-reperfusion injury (IRI) are known to lead to necrotic and apoptotic cell death, and calcineurin plays a major role in this process. Calcineurin dephosphorylates the nuclear factor of activated T-cells (NFAT), permitting its translocation into the nucleus. As a result, calcineurin promotes the release of pro-inflammatory cytokines, such as tumor necrosis factor-α. The overproduction of pro-inflammatory cytokines causes renal cell death. Calcineurin activity is regulated by calpain, a cysteine protease present in the nucleus. Calpain-mediated proteolysis increases the phosphatase activity of calcineurin, resulting in NFAT dephosphorylation. This process has been studied in cardiomyocytes but its role in renal IRI is unknown. Thus, we examined whether calpain regulates calcineurin in renal tubule nuclei. We established an in vivo renal IRI model in mice and identified the protective role of a calcineurin inhibitor, FK506, in this process. Calcineurin is expressed in the nucleus, where it is present in its calpain-cleaved form. FK506 reduced nuclear expression of calcineurin and prevented calcineurin-mediated NFAT activation. Our study shows clearly that FK506 reduces calpain-mediated calcineurin activity. Consequently, calcineurin could not maintain NFAT activation. FK506 reduced renal cell death by suppressing the transcription of pro-inflammatory cytokine genes. This study provides evidence that FK506 protects against inflammation in a renal IRI mouse model. We also provided a mechanism of calcineurin action in the nucleus. Therefore, FK506 could improve renal function by decreasing calcineurin activity in both the cytoplasm and the nucleus of renal tubule cells.

Defining the Nonreturn Time for Intestinal Ischemia Reperfusion Injury in Mice

Among the abdominal organs, the intestine is probably the most sensitive to ischemia reperfusion injury (IRI), a phenomenon that occurs in many intestinal disorders. Few studies have reported in detail the impact of intestinal ischemia time in mice. We evaluated the effect of various warm intestinal ischemia times in an intestinal IRI model in mice. Adult male Balb/c mice were divided into 4 groups that differed in intestinal ischemia time: G1, 30; minutes; G2, 35 minutes; G3, 40 minutes; and G4, 45 minutes. Histological evaluation showed average Park scores as follows: G1 0.6 ± 0.55; G2 1.8 ± 0.45; G3 4.8 ± 2.25; and G4 5 ± 1.79. All animals from G1 survived 30 hours. G2 animals showed intermediate behavior with all succumbing between 18 and 30 hours postprocedure. G3 and G4 displayed similar survival results with animals succumbing before 6 hours after intestinal reperfusion. These data showed that Park index scores of 3 or higher were related to early death. We concluded that the 5 minutes between 35 and 40 minutes is the critical limit, after which all mice die after reperfusion. This result may represent a valuable tool for future research in mice.

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.

Gender Differences in the Susceptibility to Renal Ischemia-Reperfusion Injury in BALB/c Mice

Recent studies have shown the remarkable gender differences in the susceptibility or expression of many diseases. The mechanism underlying the gender differences is unclear. In the present study, we evaluated the effects of gender differences and different ischemia time on the renal ischemia-reperfusion injury (IRI). The IRI was induced in the bilateral kidneys of 156 male and 30 female BALB/c mice. Renal function, serum creatinine and blood urea nitrogen, and pathology of the kidneys were examined at 24 hr after IRI. Renal IRI was generated successfully in 182 of 186 mice with a 97.85% success rate. The levels of serum creatinine and blood urea nitrogen were significantly increased in male mice subjected to 30 min, 35 min, or 45 min of renal ischemia and in female mice subjected to 75 min of renal ischemia, compared to the control group at 24 hr after operation. In males following 35 min or 45 min of ischemia and in females following 75 min of ischemia, typical acute tubular necrosis was found in the areas of corticomedullary junction and the histopathologic scores, which represent the degree of renal tissue injuries, were significantly increased. In view of our data, the kidneys of male are much more susceptible to IRI than those of female. The optimal ischemia time of kidney is 35-45 min in males and 75 min in females for generating a stable model of IRI in mice. Investigation of the gender differences might provide a new area for mechanistic study of renal IRI.