Reduce Ischemia-reperfusion Injury Research Articles

A sequential assessment of the preservation injury in porcine intestines

BackgroundClinical and experimental evidence strongly suggest that ischemia–reperfusion injury after intestinal transplantation has deleterious short- and long-term effects and finding means to reduce ischemia–reperfusion injury is a major research area. The anatomical and physiological similarities between the human and porcine digestive tract favor its use as a preclinical model for translational research. Intriguingly, no systematic appraisal of the development of the intestinal preservation injury in pigs is available. Materials and methodsIntestinal procurement was performed in nine pigs using histidine-tryptophan-ketoglutarate solution as preservation fluid. Ileal biopsies were obtained after 8, 14, and 24 h of static cold storage (SCS), and the preservation injury was assessed morphologically (Chiu score) as well as on the molecular level. Tight junction (zonula occludens, claudin-3 and 4, tricellulin, and occludin) and adherens junctions (E-cadherin) proteins were studied using immunofluorescence and Western blot. ResultsEight hours of SCS induced minimal mucosal changes (Chiu grade 1) that advanced to significant subepithelial edema (Chiu grade 3) after 24 h; progressive Goblet cell depletion was also noted. Apoptosis (studied by cleaved caspase-3 staining significantly increased after 24 h of SCS. Significant molecular changes with decreasing expression of zonula occludens, tricellulin, and occludin were evident already after 8 h of SCS and continuously worsened. Claudin-3 and Claudin-4 and E-cadherin expression remained relatively unaltered during SCS. ConclusionsImportant molecular alterations precede histologic changes during SCS of the porcine intestine and may be used as more sensitive injury markers than histologic changes in intestinal ischemia and transplantation.

Heparin and Heparin-Derivatives in Post-Subarachnoid Hemorrhage Brain Injury: A Multimodal Therapy for a Multimodal Disease.

Pharmacologic efforts to improve outcomes following aneurysmal subarachnoid hemorrhage (aSAH) remain disappointing, likely owing to the complex nature of post-hemorrhage brain injury. Previous work suggests that heparin, due to the multimodal nature of its actions, reduces the incidence of clinical vasospasm and delayed cerebral ischemia that accompany the disease. This narrative review examines how heparin may mitigate the non-vasospastic pathological aspects of aSAH, particularly those related to neuroinflammation. Following a brief review of early brain injury in aSAH and heparin’s general pharmacology, we discuss potential mechanistic roles of heparin therapy in treating post-aSAH inflammatory injury. These roles include reducing ischemia-reperfusion injury, preventing leukocyte extravasation, modulating phagocyte activation, countering oxidative stress, and correcting blood-brain barrier dysfunction. Following a discussion of evidence to support these mechanistic roles, we provide a brief discussion of potential complications of heparin usage in aSAH. Our review suggests that heparin’s use in aSAH is not only safe, but effectively addresses a number of pathologies initiated by aSAH.

Salvage of an Octogenarian Liver Graft Using Normothermic Perfusion: A Case Report

Liver transplantation with very old donors is safe, but is associated with an increased incidence of ischemic-type biliary lesions and delayed graft function. Normothermic machine perfusion (NMP) is a novel technique for preservation of liver grafts and has the potential to reduce ischemia-reperfusion injury. A case is reported here of a liver transplantation (LT) with a graft from an 83-year-old brain-dead donor. Procurement was with dual perfusion and en bloc, modified fast technique. Donor kidneys were not transplanted due to severe atherosclerosis and poor perfusion. The liver was shipped to the transplantation center and underwent NMP with a blood-based perfusate. During machine perfusion lactates decreased, vascular flow was stable, and bile production restored, and the graft was considered suitable for transplantation. The postoperative course was uneventful and 4 months after surgery the patient is in good clinical condition with normal liver function. To date, few LTs have been performed with NMP in humans, but its preliminary results are promising. NMP allows functional evaluation of the graft and possibly reduction of post-transplantation complications when extended-criteria donor grafts are used.

Opportunities for Therapeutic Intervention During Machine Perfusion.

There is a vast discrepancy between the number of patients waiting for organ transplantation and the available donor organs. Ex vivo machine perfusion (MP) has emerged in an effort to expand the donor pool, by improving organ preservation, providing diagnostic information, and more recently, acting as a platform for organ improvement. This article reviews the current status of MP with a focus on its role in organ preconditioning and therapeutic interventions prior to transplantation. MP has allowed longer organ preservation compared to conventional static cold storage and allowed the use of organs that might otherwise have been discarded. Moreover, experimental studies have investigated the role of MP in reducing ischemia reperfusion injury of lungs, kidneys and livers by applying mesenchymal stem cells (MSCs), anti-inflammatory agents, cytotopic anticoagulants, and defatting cocktails. MP has opened a new era in the field of organ transplantation and tissue medication.

Mitochondrial permeability transition in cardiac ischemia-reperfusion: whether cyclophilin D is a viable target for cardioprotection?

Growing number of studies provide strong evidence that the mitochondrial permeability transition pore (PTP), a non-selective channel in the inner mitochondrial membrane, is involved in the pathogenesis of cardiac ischemia-reperfusion and can be targeted to attenuate reperfusion-induced damage to the myocardium. The molecular identity of the PTP remains unknown and cyclophilin D is the only protein commonly accepted as a major regulator of the PTP opening. Therefore, cyclophilin D is an attractive target for pharmacological or genetic therapies to reduce ischemia-reperfusion injury in various animal models and humans. Most animal studies demonstrated cardioprotective effects of PTP inhibition; however, a recent large clinical trial conducted by international groups demonstrated that cyclosporine A, a cyclophilin D inhibitor, failed to protect the heart in patients with myocardial infarction. These studies, among others, raise the question of whether cyclophilin D, which plays an important physiological role in the regulation of cell metabolism and mitochondrial bioenergetics, is a viable target for cardioprotection. This review discusses previous studies to provide comprehensive information on the physiological role of cyclophilin D as well as PTP opening in the cell that can be taken into consideration for the development of new PTP inhibitors.

The Authors' Reply.

Thank you very much for your comment on our manuscript “Results of the TOP Study: Prospectively Randomized Multicenter Trial of an Ex Vivo Tacrolimus Rinse Before Transplantation in EDC Livers.”1 We would like to take the opportunity to respond to the valuable comments of Kobayashi.2 In the tacrolimus organ perfusion (TOP) study marginal liver grafts exhibiting ≥ 2 extended donor criteria (EDC) were randomly flushed ex situ with Tacrolimus (20 ng/mL) dissolved in histidine tryptophane ketoglutarate (HTK) solution or with HTK only (control) to reduce ischemia reperfusion injury. Kobayashi suspects a potential warming of the grafts and harmful effects through this procedure. Facing this criticism, the authors state that the temperature of the rinse solution was 4°C at the beginning of the treatment (though it was not measured at the end of the perfusion as suggested by Kobayashi) and the procedure took on an average of 18 minutes only. Although the plastic bags containing the rinse solution were not cooled during the procedure (analogously with the systemic perfusion at organ harvesting), grafts undergoing the study treatment were continuously stored on ice until the beginning of implantation. Therefore, the authors assume a warming of the graft and a subsequent initiation of the hepatic metabolism to be extremely unlikely. Moreover, Kobayashi questions the anastomosis time and assumes that warm ischemia could counteract the protective effects of a rinse treatment. In this respect, he suggests a new in situ technique of organ perfusion. The authors would like to state that changes in the surgical procedure were beyond the scope of the TOP Study. Moreover, the rinse treatment itself was not the point of interest in our study, but to investigate the effects of Tacrolimus in a rinse of 1000 ml HTK compared to 1000 ml HTK only. Cava sparing transplantation including anastomoses was performed after the rinse treatment according to center specific standards in both groups. Therefore, the study treatment did not affect the time for vascular reconstruction. To the authors' best knowledge, a simple study treatment in combination with routine surgery is required to generate valid data in a multicentric trial in liver transplantation. Kobayashi presents an interesting concept of an in-situ perfusion. The authors greatly appreciate any attempts (ie, organ preperfusion) to improve the utilization of EDC grafts. In this respect, an additional in situ flush of the graft may have protective effects. Nonetheless, this procedure does not represent an established procedure and would need to be evaluated in a prospective trial. Since the TOP study failed to show protective effects of Tacrolimus compared with control, this concept must be reevaluated and the authors suggest further analyses of this substance due to its proven anti-inflammatory effects. Therefore, an adjunct of Tacrolimus to an in-situ perfusion as described by Kobayashi or to machine perfusion could be an option to reduce ischemia reperfusion injury especially in EDC grafts thereby increasing the limited donor pool, which represents the most pressing problem in today’s transplantation medicine.

Effects of age factors on hypothermia-induced reduction of ischemia-reperfusion injury in isolated rat hearts

Objective To evaluate the effects of age factors on hypothermia-induced reduction of ischemia-reperfusion(I/R)injury in isolated rat hearts. Methods Pathogen-free healthy aged male Sprague-Dawley rats, aged 18-20 months, weighing 400-600 g, and young rats, aged 4-6 months, weighing 280-350 g, were used in the study.After the animals were anesthetized with intraperitoneal chloral hydrate and heparinized, their hearts were excised and perfused with K-H solution in a Langendorff apparatus.Twenty-four isolated hearts of aged rats were assigned into 2 groups(n=12 each)using a random number table: I/R group(group AI/R)and hypothermia group(group AH). Twenty-four isolated hearts of young rats were assigned into 2 groups(n=12 each)using a random number table: I/R group(group YI/R)and hypothermia group(group YH). Perfusion was suspended for 30 min followed by 120 min reperfusion to establish the model of I/R.The temperature was maintained at 37 ℃ during the whole process in AI/R and YI/R groups.The hearts were perfused with 34 ℃ K-H solution until 120 min of reperfusion starting from onset of reperfusion in AH and YH groups.At 30 min of equilibration(T0)and 15, 30, 60 and 120 min of reperfusion(T1-4), heart rate(HR), left ventricular developed pressure(LVDP), the maximum rate of increase in left ventricular pressure(+ dp/dtmax), and the minimum rate of increase in left ventricular pressure(+ dp/dtmin)were recorded.Six hearts from each group were randomly selected at T4, and myocardial specimens were obtained for determination of ATP, superoxide dismutase(SOD)and malondialdehyde(MDA)levels and myocardial infarct size(IS). Results Compared with group YI/R, HR was significantly decreased at T1-4, ATP and SOD levels were increased, and the MDA content and myocardial IS were decreased in group YH, and the HR, LVDP, + dp/dtmax and + dp/dtmin at T0 and ATP and SOD levels at T4 were significantly decreased, and the MDA content and myocardial IS were increased in group AI/R(P<0.05). Compared with group YH, HR, LVDP, + dp/dtmax and + dp/dtmin at T0 and ATP and SOD levels at T4 were significantly decreased, and the MDA content and myocardial IS were increased in group AH(P<0.05). Compared with group AH, the levels of ATP and SOD were significantly decreased, and the MDA content and myocardial IS were increased in group AI/R(P<0.05). Conclusion Age factors affect the efficacy of hypothermia in reducing I/R injury in isolated rat hearts, and hypothermia provides better cardioprotection for young rats than for aged rats. Key words: Age factors; Hypothermia, induced; Myocardial reperfusion injury

The endothelial glycocalyx and perioperative lung injury.

Ventilator-induced lung injury is a major contributor to perioperative lung injury. The end-expiratory lung volume, regional lung overdistension, and tidal recruitment are known to be the main factors causing subsequent alveolar damage and inflammation. The alveolar-capillary membrane including the endothelial glycocalyx as an integral part of the vascular endothelium seems to play a major role in different kinds of lung injury. Recent studies underline the pivotal importance of the endothelial glycocalyx in lung injury. The glycocalyx regulates and modulates plasma endothelial cell interactions. Several triggers are known to deteriorate the gylcocalyx such as fluid overload, ischemia, and TRALI. The clinical manifestation is inflammation, capillary leak, and edema formation. Breakdown of the endothelial gylcocalyx is of gaining importance in the context of one-lung ventilation, known to be a major risk factor for postoperative lung injury. Studies suggest that volatile anesthetics may have a protective influence on the endothelial glycocalyx of pulmonary capillaries and reduce ischemia-reperfusion injury. This might be of clinical relevance for postoperative outcome. This review focuses on the involvement of the pulmonary endothelial glycocalyx in the context of perioperative lung injury. The pathophysiological mechanisms and trigger factors of glycocalyx deterioration are discussed, and prevention strategies are taken into consideration.

Mild hypothermia pretreatment protects hepatocytes against ischemia reperfusion injury via down-regulating miR-122 and IGF-1R/AKT pathway

BackgroundMild hypothermia has been well known as an effective way to reduce ischemia reperfusion injury (IRI), while the mechanisms are still unclear. More and more evidences have indicated that miRNAs should been involved in the regulation of IRI and expecially some miRNAs have shown temp-responsiveness for temperature variation. Therefore, the role of miR-122 in mild hypothermia pretreatment after IRI was investigated. MethodsWe established a LO2 cell anoxia-reoxygenation injury model to simulate liver IRI. Five groups of differently pretreated L02 cells were studied. ALT, AST and LDH as well as cell viability were measured. Flow cytometric analysis was used to evaluate the apoptosis. The expression of miR-122 was quantified by qRT-PCR. Insulin-like growth factor 1 receptor (IGF-1R), protein kinase B (p-AKT), AKT, forkhead box O3a (p-FOXO3a) and Caspase3 were examined using western blot analysis. ResultsWe found that mild hypothermia pretreatment could reduce the hepatocellular injury and induce a significant down-regulation in miR-122 expression after IRI. However, those effects of protection were attenuated by overexpressed miR-122 blockade. We further demonstrated that down-regulation of miR-122 promoted IGF-1R translation and AKT activity, suppressed FOXO3a activity and Caspase3 expression after mild hypothermia pretreatment, which was abrogated by miR-122 mimic. ConclusionOur data clearly demonstrate that mild hypothermia pretreatment can down-regulate miR-122 to protect hepatocytes against IRI through activation IGF-1R/AKT signaling pathway and inhibit cells apoptosis.

Measuring Water Distribution in the Heart: Preventing Edema Reduces Ischemia-Reperfusion Injury.

BackgroundEdema is present in many heart diseases, and differentiation between intracellular (ICW) and extracellular (ECW) myocardial water compartments would be clinically relevant. In this work we developed a magnetic resonance imaging–based method to differentiate ICW and ECW and applied it to analyze ischemia–reperfusion–induced edema.Methods and ResultsIsolated rat hearts were perfused with gadolinium chelates as a marker of extracellular space. Total water content was measured by desiccation. Gadolinium quantification provided ECW, and ICW was calculated by subtraction of ECW from total water content. In separate experiments, T1, T2, diffusion‐weighted imaging and proton‐density parameters were measured in isolated saline‐perfused hearts. In in‐situ rat hearts, ECW and ICW were 79±10 mL and 257±8 mL of water per 100 g of dry tissue, respectively. After perfusion for 40 minutes, ECW increased by 92.4±3% without modifying ICW (−1±3%). Hyposmotic buffer (248 mOsm/L) increased ICW by 16.7±2%, while hyperosmotic perfusion (409 mOsm/L) reduced ICW by 26.5±3%. Preclinical imaging showed good correlation between T2 and diffusion‐weighted imaging with ECW, and proton‐density correlated with total water content. Ischemia–reperfusion resulted in marked myocardial edema at the expense of ECW, because of cellular membrane rupture. When cell death was prevented by blebbistatin, water content and distribution were similar to normoxic perfused hearts. Furthermore, attenuation of intracellular edema with hyperosmotic buffer reduced cell death.ConclusionsWe devised a method to determine edema and tissue water distribution. This method allowed us to demonstrate a role of edema in reperfusion‐induced cell death and could serve as a basis for the study of myocardial water distribution using magnetic resonance imaging.

Allopurinol as a therapeutic option in cardiovascular disease.

Epidemiological studies indicate that hyperuricaemia is an independent risk factor for cardiovascular disease. Alongside uric acid formation, increased xanthine oxidase activity also results in the formation of oxidative free radicals and superoxide particles. Oxidative stress significantly contributes to the development of cardiovascular disease, including endothelial cell dysfunction, atherosclerosis, vascular calcification and impaired myocardial energetics. Allopurinol, a competitive xanthine oxidase inhibitor, in addition to reducing serum uric acid levels, can act as a free radical scavenger. Although traditionally used for the management of gout, there has been renewed interest in the role of allopurinol in the management of cardiovascular disease. In this review, we summarise the role of the xanthine oxidase pathway in the generation of oxidative stress and evaluate the current body of evidence assessing the clinical effects of allopurinol in patients with cardiovascular disease. A number of small clinical studies have shown a beneficial effect of allopurinol in reducing ischemia-reperfusion injury in the setting of bypass surgery and coronary angioplasty. Additionally, studies in heart failure indicate a potential favourable effect of allopurinol on endothelial dysfunction, LV function and haemodynamic indices, particularly in those with raised serum uric acid levels. Whilst this cheap and readily available pharmacological option may offer a very cost effective therapeutic option, large-scale prospective studies are required to better delineate its role in reducing hard clinical end-points.

Limb Remote Ischemic Postconditioning Reduces Ischemia-Reperfusion Injury by Inhibiting NADPH Oxidase Activation and MyD88-TRAF6-P38MAP-Kinase Pathway of Neutrophils.

Limb remote ischemic postconditioning (LRIP) has been confirmed to reduce the ischemia-reperfusion injury but its mechanisms are still not clear. This study clarified the mechanism of LRIP based on the nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase and Myeloid differentiation factor 88 (MyD88)-Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6)-P38 pathway of neutrophils. Rat middle cerebral artery occlusion (MCAO) model was used in this study. Ischemia-reperfusion injury was carried out by MCAO 1.5 h followed by 24 h reperfusion. LRIP operation was performed to the left femoral artery at 0, 1 or 3 h after reperfusion. Behavioral testing, including postural reflex test, vibrissae-elicited forelimb placing test and tail hang test, showed that LRIP operated at 0 h of reperfusion could significantly ameliorate these behavioral scores. Pathological examinations, infarct size, Myeloperoxidase (MPO) activity showed that LRIP operated at 0 h of reperfusion could significantly ameliorate the pathological scores, reduce the infarct size and MPO activity in the brain and increase the MPO activity in the left leg. By using Neutrophil counting, immunofluorescence and real-time PCR techniques, we found that LRIP operated at 0 h of reperfusion could reduce neutrophil counts in the peripheral blood and downregulate the activation of neutrophil in the peripheral blood and rat brain. Western blots revealed that MyD88, TRAF6, p38 mitogen-activated protein kinase (p38-MAPK) in neutrophils and the phosphorylation of p47phox (Ser 304 and Ser 345) in neutrophil could be downregulated by LRIP. Our study suggests that LRIP inhibits the number and activation of neutrophils in the rat brain and peripheral blood linked to down-regulating the activation of NADPH oxidase in neutrophils by MyD88/TRAF6/p38-MAPK pathway.

The effects of melatonin and colchicine on ischemia–reperfusion injury in experimental rat testicular torsion model

PurposeThe aim of the present study is to investigate the efficiency of colchicine and melatonın in an experimental rat testicular torsion model in the light of histological and biochemical data. MethodsA total of 34 Wistar albino male rats were randomly divided into 5 groups as: Group C (control, n=6), Group S (sham; underwent only left scrotal exploration, n=7), Group TD (torsion and detorsion; 6h of ischemia and 7days of reperfusion, n=7), Group TD/M (TD+Melatonin; 6h of ischemia and 7days of reperfusion and 7days of 17mg/kg intraperitoneal melatonin per day, n=7), group TD/Col (TD+Colchicine; 6h of ischemia and 7days of reperfusion and 7days of 1mg/kg oral colchicine per day, n=7). Histopathologic evaluation of seminiferous tubule deterioration was performed by Johnsen's scoring system. Total antioxidant status (TAS), total oxidant status (TOS), IL-6, TNF alpha levels were analyzed in each group. ResultsThe histopathologic scores, total antioxidant status (TAS), total oxidant status (TOS), IL-6, TNF alpha levels in groups C and TD/Col were significantly lower than groups TD and TD/M (P<.001). ConclusionOur study results revealed that colchicine reduced testicular ischemia–reperfusion injury in experimental rat testis torsion model. Although detorsion of testis is crucial for the preserving the testicular viability, antioxidant and anti-inflammatory treatment modalities like colchicine might help to reduce ischemia–reperfusion injury in detorsed testis.

Effect of panax notoginseng saponins on efficacy and hemorrhagic transformation of rt-PA intravenous thrombolysis in patients with acute ischemic stroke

Objective To study the effect of panax notoginseng saponins (PNS) on the efficacy and hemorrhagic transformation (HT) of recombinant tissue-type plasminogen activator (rt-PA) intravenous thrombolysis in patients with acute ischemic stroke. Methods A total of 200 patients with early acute ischemic stroke (the length of time between attack and hospital admission < 4.50 h) were divided into 2 groups according to random number table method: treatment group (N = 100) and control group (N = 100). The control group was treated with routine rt-PA intravenous thrombolysis treatment, and the treatment group was treated with rt-PA intravenous thrombolysis plus PNS injection. The ischemia-reperfusion injury index [malondialdehyde (MDA) and superoxide dismutase (SOD)], hemorrhagic transformation prediction index [matrix metalloproteinase-9 (MMP-9) and fibronectin (FN)] and nerve function index [National Institutes of Health Stroke Scale (NIHSS) and Barthel Index (BI)] were measured and compared before treatment, 24 h after thrombolysis and 14 d after thrombolysis. Adverse drug reactions and hemorrhagic transformation rate were observed 14 d after thrombolysis, and the prognosis (mortality and BI) was evaluated 12 months after thrombolysis. Results Compared with control group, serum SOD ( P = 0.000) and BI ( P = 0.000) in treatment group were significantly higher, while serum MDA ( P = 0.001), MMP-9 ( P = 0.001), plasma FN ( P = 0.000) and NIHSS score ( P = 0.006) were significantly lower. In treatment group, 24 h after rt-PA intravenous thrombolysis plus PNS injection, serum MDA ( P = 0.000), MMP-9 ( P = 0.000) and BI ( P = 0.000) were significantly increased, while NIHSS score ( P = 0.000) was significantly decreased; 14 d after treatment, serum MDA ( P = 0.000) and MMP-9 ( P = 0.000) were decreased, serum SOD ( P = 0.000) and BI ( P = 0.000) were continuously increased, plasma FN ( P = 0.000) and NIHSS score ( P = 0.000) were continuously decreased. On the 14th day after thrombolysis, hemorrhagic transformation rate of treatment group was lower than that of control group [9 cases (9%) vs 19 cases (19%); χ 2 = 4.153, P = 0.042]. There was no significant difference in the incidence of adverse drug reactions between 2 groups [14 cases (14%) vs 11 cases (11%); χ 2 = 0.411, P = 0.521]. Twelve months after thrombolysis, there were 5 cases of death (5% ) in control group and one case (1% ) of death in treatment group. There was no significant difference in the incidence of mortality between 2 groups ( χ 2 = 1.546, P = 0.241). The BI of treatment group was significantly higher than that of control group (88.51 ± 11.49 vs 84.47 ± 9.83; t = 2.451, P = 0.015). Conclusions PNS reduces ischemia-reperfusion injury after rt-PA intravenous thrombolysis in patients with acute ischemic stroke. It can reduce the rate of hemorrhagic transformation after rt-PA intravenous thrombolysis and improve the prognosis with good safety. DOI: 10.3969/j.issn.1672-6731.2016.11.012

Adenosine Receptor Activation in the “Trigger” Limb of Remote Pre-Conditioning Mediates Human Endothelial Conditioning and Release of Circulating Cardioprotective Factor(s)

Remote ischemic pre-conditioning (rIPC) has emerged as a potential mechanism to reduce ischemia-reperfusion injury. Clinical data, however, have been mixed, and its physiological basis remains unclear, although it appears to involve release of circulating factor(s) and/or neural pathways. Here, the authors demonstrate that adenosine receptor activation is an important step in initiating human pre-conditioning; that pre-conditioning liberates circulating cardioprotective factor(s); and that exogenous adenosine infusion is able to recapitulate release of this factor. However, blockade of adenosine receptors in ischemic tissue does not block the protection afforded by pre-conditioning. These data have important implications for defining the physiology of human pre-conditioning and its translation to future clinical trials.

Relationship of clusterin with renal inflammation and fibrosis after the recovery phase of ischemia-reperfusion injury.

BackgroundLong-term outcomes after acute kidney injury (AKI) include incremental loss of function and progression towards chronic kidney disease (CKD); however, the pathogenesis of AKI to CKD remains largely unknown. Clusterin (CLU) is a chaperone-like protein that reduces ischemia-reperfusion injury (IRI) and enhances tissue repair after IRI in the kidney. This study investigated the role of CLU in the transition of IRI to renal fibrosis.MethodsIRI was induced in the left kidneys of wild type (WT) C57BL/6J (B6) versus CLU knockout (KO) B6 mice by clamping the renal pedicles for 28 min at the body temperature of 32 °C. Tissue damage was examined by histology, infiltrate phenotypes by flow cytometry analysis, and fibrosis-related gene expression by PCR array.ResultsReduction of kidney weight was induced by IRI, but was not affected by CLU KO. Both WT and KO kidneys had similar function with minimal cellular infiltration and fibrosis at day 14 of reperfusion. After 30 days, KO kidneys had greater loss in function than WT, indicated by the higher levels of both serum creatinine and BUN in KO mice, and exhibited more cellular infiltration (CD8 cells and macrophages), more tubular damage and more severe tissue fibrosis (glomerulopathy, interstitial fibrosis and vascular fibrosis). PCR array showed the association of CLU deficiency with up-regulation of CCL12, Col3a1, MMP9 and TIMP1 and down-regulation of EGF in these kidneys.ConclusionOur data suggest that CLU deficiency worsens renal inflammation and tissue fibrosis after IRI in the kidney, which may be mediated through multiple pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12882-016-0348-x) contains supplementary material, which is available to authorized users.

A Translational Study of a New Therapeutic Approach for Acute Myocardial Infarction: Nanoparticle-Mediated Delivery of Pitavastatin into Reperfused Myocardium Reduces Ischemia-Reperfusion Injury in a Preclinical Porcine Model

BackgroundThere is an unmet need to develop an innovative cardioprotective modality for acute myocardial infarction, for which interventional reperfusion therapy is hampered by ischemia-reperfusion (IR) injury. We recently reported that bioabsorbable poly(lactic acid/glycolic acid) (PLGA) nanoparticle-mediated treatment with pitavastatin (pitavastatin-NP) exerts a cardioprotective effect in a rat IR injury model by activating the PI3K-Akt pathway and inhibiting inflammation. To obtain preclinical proof-of-concept evidence, in this study, we examined the effect of pitavastatin-NP on myocardial IR injury in conscious and anesthetized pig models.Methods and ResultsEighty-four Bama mini-pigs were surgically implanted with a pneumatic cuff occluder at the left circumflex coronary artery (LCx) and telemetry transmitters to continuously monitor electrocardiogram as well as to monitor arterial blood pressure and heart rate. The LCx was occluded for 60 minutes, followed by 24 hours of reperfusion under conscious conditions. Intravenous administration of pitavastatin-NP containing ≥ 8 mg/body of pitavastatin 5 minutes before reperfusion significantly reduced infarct size; by contrast, pitavastatin alone (8 mg/body) showed no therapeutic effects. Pitavastatin-NP produced anti-apoptotic effects on cultured cardiomyocytes in vitro. Cardiac magnetic resonance imaging performed 4 weeks after IR injury revealed that pitavastatin-NP reduced the extent of left ventricle remodeling. Importantly, pitavastatin-NP exerted no significant effects on blood pressure, heart rate, or serum biochemistry. Exploratory examinations in anesthetized pigs showed pharmacokinetic analysis and the effects of pitavastatin-NP on no-reflow phenomenon.ConclusionsNP-mediated delivery of pitavastatin to IR-injured myocardium exerts cardioprotective effects on IR injury without apparent adverse side effects in a preclinical conscious pig model. Thus, pitavastatin-NP represents a novel therapeutic modality for IR injury in acute myocardial infarction.

Nanoparticles-Mediated Targeting of Pioglitazone Reduces Myocardial Ischemia-Reperfusion Injury and Cardiac Remodeling by Antagonizing Monocyte/Macrophage-mediated Inflammation in Preclinical Animal Models

Background: Monocyte-mediated inflammation is a major mechanism of myocardial ischemia-reperfusion (IR) injury and cardiac remodeling. However, no decisive anti-inflammatory therapy has been developed for clinical setting. Pioglitazone, a peroxisome proliferator-activated receptor (PPAR)γ agonist, has unique anti-inflammatory effects on monocyte/macrophage. Here we tested the hypothesis that nanoparticle-mediated targeting of pioglitazone into cardiomyocytes and inflammatory monocytes ameliorates IR injury and cardiac remodeling in preclinical animal models. Methods and Results: We formulated poly(lactic acid/glycolic acid) nanoparticle containing pioglitazone (Pio-NPs). In mouse IR model, nanoparticles were delivered predominantly to circulating monocytes and to cardiomyocytes and macrophages in the IR heart. Intravenous treatment with Pio-NPs at the time of reperfusion reduced IR injury, which was canceled by the pretreatment with PPARγ antagonist GW9662. In contrast, pioglitazone solution showed no therapeutic effects. Pio-NPs reduced inflammatory gene expression and inhibited the recruitment of Ly6Chigh inflammatory monocytes into IR heart. In mouse MI model, intravenous treatment with Pio-NPs for 3 days after LAD ligation reduced recruitment of macrophage and polarized macrophages toward an anti-inflammatory M2 phenotype, which, leads to improvement of cardiac remodeling and mortality. Finally, in a conscious mini-pig model of myocardial IR injury, Pio-NPs induced cardioprotection from IR injury, indicating the pre-clinical proof of concept. Conclusion: Nanoparticle-mediated targeting of pioglitazone can be developed as a novel modality by antagonizing monocyte-mediated inflammation in myocardial infarction. Background: Monocyte-mediated inflammation is a major mechanism of myocardial ischemia-reperfusion (IR) injury and cardiac remodeling. However, no decisive anti-inflammatory therapy has been developed for clinical setting. Pioglitazone, a peroxisome proliferator-activated receptor (PPAR)γ agonist, has unique anti-inflammatory effects on monocyte/macrophage. Here we tested the hypothesis that nanoparticle-mediated targeting of pioglitazone into cardiomyocytes and inflammatory monocytes ameliorates IR injury and cardiac remodeling in preclinical animal models. Methods and Results: We formulated poly(lactic acid/glycolic acid) nanoparticle containing pioglitazone (Pio-NPs). In mouse IR model, nanoparticles were delivered predominantly to circulating monocytes and to cardiomyocytes and macrophages in the IR heart. Intravenous treatment with Pio-NPs at the time of reperfusion reduced IR injury, which was canceled by the pretreatment with PPARγ antagonist GW9662. In contrast, pioglitazone solution showed no therapeutic effects. Pio-NPs reduced inflammatory gene expression and inhibited the recruitment of Ly6Chigh inflammatory monocytes into IR heart. In mouse MI model, intravenous treatment with Pio-NPs for 3 days after LAD ligation reduced recruitment of macrophage and polarized macrophages toward an anti-inflammatory M2 phenotype, which, leads to improvement of cardiac remodeling and mortality. Finally, in a conscious mini-pig model of myocardial IR injury, Pio-NPs induced cardioprotection from IR injury, indicating the pre-clinical proof of concept. Conclusion: Nanoparticle-mediated targeting of pioglitazone can be developed as a novel modality by antagonizing monocyte-mediated inflammation in myocardial infarction.

Near Normothermic Ex Situ Perfusion of Human Limbs for 24 Hours: N/A - Not a clinical study

There is no financial information to disclose. Currently vascularized composite allografts (VCA) are cold preserved (4°C) until transplantation.1Hartzell T.L. Benhaim P. Imbriglia J.E. Shores J.T. Goitz R.J. Balk M. et al.Surgical and technical aspects of hand transplantation: is it just another replant?.Hand Clin. 2011; 27: 521-530Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar This process is time limited, as the tissue has to be revascularized within 4-6 hours to minimize ischemia reperfusion (IR) injury. Normothermic perfusion was proposed as an alternative method of preservation in solid organ transplantation.2Rojas-Pena A. Reoma J.L. Krause E. Boothman E.L. Padiyar N.P. Cook K.E. et al.Extracorporeal support: improves donor renal graft function after cardiac death.Am J Transpl. 2010; 10: 1365e-1374eCrossref PubMed Scopus (33) Google Scholar This method helps to avoid complications associated with cold preservation and maintains tissue viability without inducing IR injury.3Murata S. Miniati D.N. Kown M.H. Koransky M.L. Lijkwan M.A. Balsam L.B. et al.Superoxide dismutase mimetic m40401 reduces ischemia-reperfusion injury and graft coronary artery disease in rodent cardiac allografts.Transplantation. 2004; 78: 1166-1171Crossref PubMed Scopus (27) Google Scholar Using this method, previous investigators demonstrated its potential to prolong swine forelimb allograft survival up to 24 hours.4Ozer K. Rojas-Pena A. Mendias C.L. Bryner B. Toomasian C. Bartlett R.H. Ex-situ limb perfusion system to extend vascularized composite tissue allograft survival.Transplantation. 2015; 99: 2095-2101Crossref PubMed Scopus (37) Google Scholar, 5Ozer K. Rojas-Pena A. Mendias C.L. Bryner B. Toomasian C. Bartlett R.H. The effect of ex situ perfusion in a swine limb vascularized composite tissue allograft on survival up to 24 hours.J Hand Surg Am. 2016; 41: 3-12Abstract Full Text Full Text PDF Scopus (45) Google Scholar In this study, we aimed to test this system on human limb allografts. Four human forearms were procured from brain-dead adult donors under tourniquet control. Following elbow disarticulation, the brachial artery was cannulated. The limb was flushed with heparinized saline and connected to a temperature controlled (30-33°C) ex situ perfusion system (Figure) for 24 hours. The perfusate consisted of plasma and red blood cells with a target hemoglobin (Hb) concentration of 4-6 g/dL. Muscle biopsies (flexor carpi radialis) were obtained at 0, 12, and 24 hours. Average warm ischemia time was 76 minutes. Average arterial systolic pressure was 98±2 mmHg, perfusion flow 319±18 mL/min (∼6-8% of the donor’s estimated cardiac output), and vascular resistance 158±18 mmHg/mL/min. Perfusate had an average pH of 7.43±0.04, pCO2 32±1 mmHg, pO2 317±18 mmHg, and Hb 4.8±0.4 g/dL. Electrolytes (sodium, potassium, chloride) remained within a physiologic range. Lactate started to increase steadily throughout the experiment; however, neuromuscular electrical stimulation revealed ongoing contraction throughout the experiment and H& E staining showed mild to moderate fatty infiltration on some myocytes at 24 hours. •All limbs appeared viable after 24 hours of near-normothermic ex situ perfusion as evidenced by ongoing neuromuscular stimulation.•While no conclusions can be drawn about the long-term function of the extremity, this approach could help extend VCA transplantation to a wider geographic area.•It also has the potential to circumvent complications associated with cold preservation.

Inhibition of Plexin C1 Protects Against Hepatic Ischemia-Reperfusion Injury.

Hepatic ischemia-reperfusion injury is a disease pattern that is associated with an acute inflammatory reaction. It is well known that neutrophils play an essential role in the early phase of hepatic ischemia-reperfusion injury and determine the extent of tissue damage. Hepatic ischemia-reperfusion injury can result in organ failure, which is linked to high mortality. Recent data indicate that the neuronal guidance receptor Plexin C1 is involved in the control of the acute inflammatory response and, as such, modulates the transmigration of neutrophils. Hence, we investigated the functional role of Plexin C1 in a mouse model of early hepatic ischemia-reperfusion injury. Animal study. University experimental laboratory. Wild-type, PLXNC1 and chimeric mice. Hepatic ischemia-reperfusion injury or sham operation. We found that the functional inhibition of Plexin C1 in wild-type mice treated with an anti-Plexin C1 antibody and a Semaphorin 7A peptide reduced hepatic ischemia-reperfusion injury, as measured by the levels of lactate dehydrogenase, aspartate, and alanine aminotransferase. This reduction in ischemia-reperfusion injury was accompanied by reduced numbers of neutrophils in ischemic hepatic tissue and reduced serum levels of inflammatory cytokines. Experiments using Plexin C1 receptor-deficient (PLXNC1) mice also demonstrated decreased hepatic ischemia-reperfusion injury. Studies of chimeric mice revealed that the hematopoietic Plexin C1 knockout is crucial for reducing the extent of hepatic ischemia-reperfusion injury. These results describe a role for Plexin C1 during ischemia-reperfusion injury, highlight the role of hematopoietic Plexin C1 in the development of hepatic ischemia-reperfusion injury, and suggest that Plexin C1 is a potential drug target.