Effects Of Ischemia-reperfusion Injury Research Articles

Effect of Ischemia-Reperfusion Injury on Mitochondrial Malondialdehyde Levels in Kidney Cortex and Medulla of Aged Rats

Effect of Ischemia-Reperfusion Injury on Mitochondrial Malondialdehyde Levels in Kidney Cortex and Medulla of Aged Rats

Novel roles of κ-opioid receptor in myocardial ischemia-reperfusion injury.

Acute heart attack is the primary cause of cardiovascular-related death worldwide. A common treatment is reperfusion of ischemic tissue, which can cause irreversible damage to the myocardium. The number of mitochondria in cardiomyocytes is large, which generate adenosine triphosphate (ATP) to sustain proper cardiac contractile function, and mitochondrial dysfunction plays a crucial role in cell death during myocardial ischemia-reperfusion, leading to an increasing number of studies investigating the impact of mitochondria on ischemia-reperfusion injury. The disarray of mitochondrial dynamics, excessive Ca2+ accumulation, activation of mitochondrial permeable transition pores, swelling of mitochondria, ultimately the death of cardiomyocyte are the consequences of ischemia-reperfusion injury. κ-opioid receptors can alleviate mitochondrial dysfunction, regulate mitochondrial dynamics, mitigate myocardial ischemia-reperfusion injury, exert protective effects on myocardium. The mechanism of κ-OR activation during myocardial ischemia-reperfusion to regulate mitochondrial dynamics and reduce myocardial ischemia-reperfusion injury will be discussed, so as to provide theoretical basis for the protection of ischemic myocardium.

SIRT1 exerts protective effects by inhibiting endoplasmic reticulum stress and NF-κB signaling pathways.

Silent information regulator two homolog 1 (SIRT1), an NAD + -dependent histone deacetylase, plays a pivotal regulatory role in a myriad of physiological processes. A growing body of evidence suggests that SIRT1 can exert protective effects in metabolic disorders and neurodegenerative diseases by inhibiting endoplasmic reticulum (ER) stress and the nuclear factor-κB (NF-κB) inflammatory signaling pathway. This review systematically elucidates the molecular mechanisms and biological significance of SIRT1 in regulating ER stress and the NF-κB pathway. On one hand, SIRT1 can deacetylate key molecules in the ER stress pathway, such as glucose-regulated protein 78 (GRP78), X-box binding protein 1 (XBP1), PKR-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6), thereby alleviating ER stress. On the other hand, SIRT1 can directly or indirectly remove the acetylation modification of the NF-κB p65 subunit, inhibiting its transcriptional activity and thus attenuating inflammatory responses. Through these mechanisms, SIRT1 can ameliorate insulin resistance in metabolic diseases, exert cardioprotective effects in ischemia-reperfusion injury, and reduce neuronal damage in neurodegenerative diseases. However, it is important to note that while these findings are promising, the complex nature of the biological systems involved warrants further investigation to fully unravel the intricacies of SIRT1's regulatory mechanisms. Nevertheless, understanding the regulatory mechanisms of SIRT1 on ER stress and the NF-κB pathway is of great significance for expanding our knowledge of the pathogenesis of related diseases and exploring new preventive and therapeutic strategies targeting SIRT1.

O225: Oxygenated hypothermic machine perfusion (HMPO2) of the pancreas; a porcine circulatory death model to compare a ‘continuous’ and an ‘end-ischaemic’ approach

Abstract Pancreas and islet cell transplantation are used in the treatment of patients with diabetes complications. During static cold storage (SCS) ATP is depleted, and by-products of anaerobic respiration accumulate. Ischaemia-reperfusion injury (IRI) affects quantity and viability of islets and it is characterised by acinar necrosis, oedema, and endothelial disruption (graft pancreatitis). The introduction of HMPO2 in liver & kidney preservation has demonstrated a significant reduction in the consequences of IRI. Pancreas HMPO2 was shown to be feasible in pre-clinical studies. We aimed to compare a ‘continuous’ to an ‘end-ischaemic’ approach of HMPO2 in pancreases using a porcine circulatory death model. Porcine pancreases were either started on HMPO2 for the totality of the cold storage time (‘Continuous’ group, n=6) or for the last two hours of the cold storage time (‘End’ group, n=6). All pancreases then underwent normothermic reperfusion (NR) to mimic transplantation. Glucose stimulated insulin secretion (GSIS) was measured using Mercodia human insulin ELISA (n=3 in each group). Both groups had no significant change of wet-to-dry ratio during the experiment, despite an increase in gross weight. Flows were higher during NR in the Continuous group. Amylase, Lipase and LDH increased throughout the study for all pancreases and showed no statistically significant difference between both groups. The Continuous group had a significantly greater insulin secretion in response to glucose stimulation and followed a biphasic pattern. Continuous HMPO2 preserved islet function and was a superior mode of preservation, showing no statistically significant difference in oedema or markers of damage, with improved perfusion parameters.

Effect of ischemia-reperfusion injury on elafin levels in rat liver.

The aim of this study was to quantify serum levels of elafin, a serine protease inhibitor, and to assess its effects on histopathological and biochemical parameters in hepatic ischemia-reperfusion injury. Forty female Wistar albino rats were divided into five groups: Group 1 served as the control group. Liver ischemia was induced for 30 minutes in the other four groups. An additional 1-hour, 2-hour, and 3-hour reperfusion was induced in Groups 3, 4, and 5, respectively. At the end of the experiment, intracardiac blood samples were obtained for biochemical examination, and tissue samples from the liver were taken for histopathological examination. Levels of elafin, ischemia-modified albumin (IMA), total antioxi-dant status (TAS), and total oxidant status (TOS) were also examined. Serum elafin levels decreased beginning from Group 2, with the lowest level reached in Group 5 (p<0.01). The IMA level was the lowest in the control group and the highest in Group 5 (p<0.01). TOS, aspartate aminotransferase (AST), and alanine amino-transferase (ALT) levels were lowest in the control group and highest in Group 5 (p<0.01). Group 5 had the highest IMA/albumin ratio, although no significant differences were found between these four groups. The lowest TAS level was found in the control group, but a stable and significant increase was not detected in the other groups. No significant differences were found between the groups in terms of alkaline phosphatase (ALP) and albumin levels. A negative correlation was observed between serum elafin levels and AST, ALT, and TOS levels (p<0.01). The number of Grade 1 histopathological results was found to be higher in the groups with reperfusion (Groups 3, 4, 5). In histopathological subgroup analysis, while the elafin level was lower in Grade 1 group, AST, ALT, and TOS levels were higher (p<0.01). Additionally, the IMA/albumin ratio was found to be higher in the Grade 1 group (p=0.02). In hepatic ischemia-reperfusion injury, elafin levels decreased as the reperfusion time increased. As the reperfusion time increased, both hepatocyte damage and oxidant capacity increased, with a negative correlation observed between these findings and elafin levels. Therefore, elafin may play a protective role in hepatic ischemia-reperfusion injury and could assist clinicians in assessing liver injury.

The Effect of Ischemia-Reperfusion Injury on Mitochondrial Reduced Glutathione Levels in Kidney Cortex and Medulla from Aging Female Lewis Rats

The Effect of Ischemia-Reperfusion Injury on Mitochondrial Reduced Glutathione Levels in Kidney Cortex and Medulla from Aging Female Lewis Rats

KUS121, an ATP-Retaining VCP Modulator, Exerts Renoprotective Effects in Ischemia-Reperfusion Injury With Enhancing Endoplasmic Reticulum-Associated Degradation

KUS121, an ATP-Retaining VCP Modulator, Exerts Renoprotective Effects in Ischemia-Reperfusion Injury With Enhancing Endoplasmic Reticulum-Associated Degradation

Systemic and Renal Dynamics of Free Sulfhydryl Groups during Living Donor Kidney Transplantation.

During ischemia–reperfusion injury (IRI), reactive oxygen species are produced that can be scavenged by free sulfhydryl groups (R-SH, free thiols). In this study, we hypothesized that R-SH levels decrease as a consequence of renal IRI and that R-SH levels reflect post-transplant graft function. Systemic venous, arterial, renal venous, and urinary samples were collected in donors and recipients before, during, and after transplantation. R-SH was measured colorimetrically. Systemic arterial R-SH levels in recipients increased significantly up to 30 sec after reperfusion (p < 0.001). In contrast, renal venous R-SH levels significantly decreased at 5 and 10 min compared to 30 sec after reperfusion (both p < 0.001). This resulted in a significant decrease in delta R-SH (defined as the difference between renal venous and systemic arterial R-SH levels) till 30 sec after reperfusion (p < 0.001), indicating a net decrease in R-SH levels across the transplanted kidney. Overall, these results suggest trans-renal oxidative stress as a consequence of IRI during kidney transplantation, reflected by systemic and renal changes in R-SH levels in transplant recipients.

Preserving and rejuvenating old organs for transplantation: novel treatments including the potential of senolytics.

Older donors have the potential to close the gap between demand and supply in solid organs transplantation. Utilizing older organs, at the same time, has been associated with worse short- and long-term outcomes. Here, we introduce potential mechanisms on how treatments during machine perfusion (MP) may safely improve the utilization of older organs. Consequences of ischemia reperfusion injury (IRI), a process of acute, sterile inflammation leading to organ injury are more prominent in older organs. Of relevance, organ age and IRI seem to act synergistically, leading to an increase of damage associated molecular patterns that trigger innate and adaptive immune responses. While cold storage has traditionally been considered the standard of care in organ preservation, accumulating data support that both hypothermic and normothermic MP improve organ quality, particularly in older organs. Furthermore, MP provides the opportunity to assess the quality of organs while adding therapeutic agents. Experimental data have already demonstrated the potential of applying treatments during MP. New experimental show that the depletion of senescent cells that accumulate in old organs improves organ quality and transplant outcomes. As the importance of expanding the donor pool is increasing, MP and novel treatments bear the potential to assess and regenerate older organs, narrowing the gap between demand and supply.

Study on the pharmacodynamic effect of Rhizoma Dioscoreae polysaccharides on cerebral ischemia-reperfusion injury in rats and the possible mechanism

Ethnopharmacological relevanceRhizoma Dioscoreae (RD) is the rhizome of Dioscorea opposita Thunb., a traditional Chinese medicine, which can treat hypertension, diabetes, cerebral vasospasm headache and Alzheimer's disease. Meanwhile, RD is the main component of Liuwei Dihuang pill, a Chinese patent medicine. Rhizoma Dioscoreae polysaccharides (RDPS) are the primary active ingredient of RD. Modern medical research confirmed RDPS has multiple pharmacological effects, including neuroprotection, immunoregulation, antioxidant effect in many organs. The primary ischemia/hypoxia injury and secondary reperfusion injury are mainly caused by oxidative stress, which caused by hypoxia, such as free radical generation, energy metabolism disorder, intracellular calcium overload, excitatory amino acid release and inflammatory reaction. Aim of the studyWe have investigated the pharmacodynamic effect of RDPS on cerebral ischemia-reperfusion (IR) injury in rats and the possible mechanism in vitro. Materials and methodsThe pharmacodynamic effect of RDPS on IR injury in rats was studied by the construction of the occlusion of middle cerebral artery (MCAO) model, measuring the volume of cerebral infarct area, the content of oxidation index, inflammatory cytokines, and the expression of CaMMKβ in brain tissue. The in vitro study was explored by oxygen-glucose deprivation/glycogen reoxygenation (OGD/R) model, construction of the CaMMKβ interference sequence, measuring the expression of CaMMKβ in BV2 cells before and after inhibition of CaMMKβ, and the influence of RDPS on Nrf2/HO-1 signal pathway, in order to investigate the possible mechanism. ResultsCompared with the model group, the present study showed that RDPS with high-dose and low-dose groups could significantly reduce the volume of cerebral infarction. The content of MDA decreased and the activities of GSH and SOD increased in the two dose groups of RDPS. We confirmed that after RDPS treatment, the levels of IL-6, IL-1 β and TNF-α in brain tissue were lower than those in model group, and the expression of CaMMKβ in brain tissue of rats decreased in the model group, but increased in the groups of RDPS. In the in vitro study, compared with the control group, RDPS could regulate the OGD/R-induced apoptosis of BV2 cells and increase the level of CaMMKβ, Nrf2 and HO-1 induced by OGD/R. To our surprise, these therapeutic effects are no longer present after the inhibition of CaMMKβ protein. The activity of BV2 induced by OGD/R could not be enhanced by RDPS after the inhibition of CaMMKβ protein. ConclusionsRDPS has the pharmacodynamic effect in IR injury, which reduce the area of cerebral infarction, up-regulate the activity of anti-oxidant kinase, and down-regulate the inflammatory cytokine. Additionally, RDPS could affect the activation of Nrf2/HO-1 signaling pathway by regulating the expression of CaMMKβ. Our observations justify the RDPS could be a new strategy for IR injury therapy, and the mechanism may be related to the improvement of antioxidant enzyme activity and inhibition of inflammatory reaction.

Effect of Ischemia-Reperfusion injury and Preconditioning on Lung Parenchyma after Acute Limb Ischemia

Introduction. Acute limb ischemia, a sudden decrease of perfusion to the extremities, can compromise the survival of the limbs. Medical intervention and surgery are often needed to return perfusion. However, reperfusion injury can trigger oxidative stress and inflammatory response, leading to local and remote tissue damage, such as the lungs, which increases morbidity and mortality. This research aims to study the effects of hypothermia and remote ischemic preconditioning (RIPC) on the lung parenchyma after being exposed to reperfusion after acute limb ischemia in rabbits.

A novel VCP modulator KUS121 exerts renoprotective effects in ischemia-reperfusion injury with retaining ATP and restoring ERAD-processing capacity.

Acute kidney injury (AKI) is a life-threatening condition and often progresses to chronic kidney disease or the development of other organ dysfunction even after recovery. Despite the increased recognition and high prevalence of AKI worldwide, there has been no established treatment so far. The aim of this study was to investigate the renoprotective effect of Kyoto University substance 121 (KUS121), a novel valosin-containing protein modulator, on AKI. In in vitro experiments, we evaluated cell viability and ATP levels of proximal tubular cells with or without KUS121 under endoplasmic reticulum (ER) stress conditions. In in vivo experiments, the effects of KUS121 were examined in mice with AKI caused by ischemia-reperfusion injury. ER-associated degradation (ERAD)-processing capacity was evaluated by quantification of the ERAD substrate CD3delta-YFP. KUS121 protected proximal tubular cells from cell death under ER stress. The apoptotic response was mitigated as indicated by the suppression of C/EBP homologous protein expression and caspase-3 cleavage, with maintained intracellular ATP levels by KUS121 administration. KUS121 treatment suppressed the elevation of serum creatinine and neutrophil gelatinase-associated lipocalin levels and attenuated renal tubular damage after ischemia-reperfusion. The expression of inflammatory cytokines in the kidney was also suppressed in the KUS121-treated group. Valosin-containing protein expression levels were not altered by KUS121 both in vitro and in vivo. KUS121 treatment restored ERAD-processing capacity associated with potentiation of its upstream pathway, phosphorylated inositol-requiring enzyme-1α, and spliced X box-binding protein-1. In conclusion, these findings indicate that KUS121 can protect renal tubular cells from ER stress-induced injury, suggesting that KUS121 could be a novel and promising therapeutic compound for ischemia-associated AKI.NEW & NOTEWORTHY Novel findings of this study are as follows: 1) Kyoto University substance 121 (KUS121), a novel valosin-containing protein (VCP) modulator, can reduce ATP consumption of VCP; 2) KUS121 reduced endoplasmic reticulum (ER) stress and improved cell viability in proximal tubular cells; 3) KUS121 exerted renoprotective effects against ischemia-reperfusion injury; and 4) KUS121 may prevent ischemic acute kidney injury with ATP retention and restoring ER-associated degradation capacity.

The Correlation of Lipid Peroxidation and Antioxidant Capacity on Perioperative Outcomes in On-Pump CABG in Adults.

Background: Cardiac surgeries in adults usually use cardiopulmonary bypass (CPB) for cardiac protection and provide a blood-free field for operation. However, due to changes in tissue perfusion and ischemia-reperfusion injury (IRI), there are some side effects for CPB operations. Lipid peroxidation and compromised antioxidant defense are consequences of IRI. This can, in turn, cause organ dysfunction and lead to unwanted biochemical and clinical changes. Methods: In a cross-sectional study 107 patients with the ages of 35 to 79 years old matching the inclusion criteria with indication for elective on-pump CABG were studied. Renal function, serum malondialdehyde (MDA) and total antioxidant capacity (TAC), and clinical outcomes were studied until 24 hours after intensive care unit (ICU) admission. Correlations between MDA and TAC and other outcomes were tested. Between-group comparisons was one-way ANOVA with repeated measures was used for inferring changes in the plasma TAC and MDA levels, creatinine, and BUN over time. Correlations were investigated using regression models. Results: Preoperative EF was inversely correlated with TAC at post- CPB time (r= -0.262, p= 0.031). Hyperlipidemia (HLP) was directly associated with higher MDA at post- CPB time (r= 0.267, p= 0.017. Cross-clamp and CPB duration were inversely correlated with the systemic MDA concentration at 24 hours post-ICU admission (r= -0.314, p= 0.005 and r= -0.312, p= 0.005, respectively). Preoperative TAC was inversely correlated with lactate at ICU admission (r= -0.294, p= 0.011). Creatine phosphokinase (CPK) and TAC were directly correlated with post-CPB time (r= -0.327, p= 0.006). Conclusion: According to the findings, a direct correlation between TAC and myocardial protection during CPB exists. Reduced TAC during CPB is associated with elevation of muscle damage marker CPK. Preoperative HLP is associated with higher circulatory MDA content at the post-CPB time.

Intraarterial anti-leptin therapy via ICA protects ipsilateral CA1 neurons subjected to ischemia and reperfusion.

BackgroundBrain reperfusion following an ischemic event is essential for tissue viability, however, it also involves processes that promote neuronal cell death. We have recently shown that local expression of the hormone leptin in cardiovascular organs drives deleterious remodeling. As cerebral ischemia-reperfusion (IR) lesions derive expression of both the leptin hormone and its receptor, we hypothesized that blocking leptin activity in the injured brain area will reduce the deleterious effects of IR injury.MethodsC57BL6 male mice underwent bilateral common carotid artery and external carotid artery ligation. The right hemisphere was reperfused after 12 minutes, followed by intraarterial injection of either a low-dose leptin antagonist or saline solution via the ipsilateral ICA. The left common carotid artery remained ligated. Fifteen IR/leptin antagonist-injected and fourteen IR/saline-injected mice completed the experiment. Five days after surgery brains were collected and samples of the hippocampal CA1 region were analyzed for cell viability (H&E) and apoptosis (TUNEL and caspase3), for neuroinflammation (Iba1), and for signaling pathways of pSTAT3 and pSmad2.ResultsThe right hemisphere hippocampal CA1 region subjected to IR and saline injection exhibited increased apoptosis and necrosis of pyramidal cells. Also, increased density of activated microglia/macrophages was evident around the CA1 region. Comparatively, leptin antagonist treatment at reperfusion reduced apoptosis and necrosis of pyramidal cells, as indicated by increased number of viable cells (p < 0.01), and reduced TUNEL (p < 0.001) and caspase3-positive cells (p<0.05). Furthermore, this treatment reduced the density of activated microglia/macrophages (p < 0.001) in the CA1 region. Signaling pathway analysis revealed that while pSTAT3 and pSmad2-positive cells were found surrounding the stratum pyramidal in saline-treated animals, pSTAT3 signal was undetected and pSmad2 was greatly reduced in this territory following leptin antagonist treatment (p < 0.01).ConclusionsInhibition of leptin activity in hemispheric IR injury preserved the viability of ipsilateral hippocampal CA1 neurons, likely by preventing apoptosis and local inflammation. These results indicate that intraarterial anti-leptin therapy may have clinical potential in reducing hemispheric brain IR injury.

Non-thermal Infrared Light Treatment of Ischemia/Reperfusion Injury and Subsequent Analysis of Macrophage Differentiation.

Tissue damage and necrosis from inflammatory processes are a consequence of ischemia reperfusion injury (IRI). In skeletal muscle, ischemia reduces the aerobic energy capacity of muscle cells, leading to adverse biochemical alterations and inflammation. The goal of this study is to show that exposure to near-infrared light (NIR) during a period of ischemia reduces IRI by decreasing necrosis and inflammation in addition to decreasing proinflammatory M1 and increasing protective M2 macrophages. C57/Bl6 mice underwent unilateral tourniquet-induced hindlimb ischemia for 3 h followed by reperfusion for either 15 or 30 min. Mice were randomly assigned to 3 groups. Group 1 underwent IRI with 30 min reperfusion. Group 2 underwent IRI with a 15 min reperfusion. Each group consisted of 50% no-NIR and 50% NIR-treated mice with exposure of 50 mW/cm2 for 5 min/1 h after tourniquet closure. Group 3 were sham animals anesthetized for 3 h omitting IRI. Laser doppler flow imaging was performed on all mice to confirm ischemia and reperfusion. Flow data were expressed as the ratio of ischemic limb and the contralateral control. The mice were euthanized after reperfusion, and the quadriceps and gastrocnemius were harvested. Immunoprecipitation and western blot of macrophage-markers CD68 (M1) and CD206 (M2) were performed and normalized to CD14 expression. The expression of the inflammatory markers CXCL1 and CXCL5 was significantly reduced by NIR in the IRI group. A significant decrease in CD68 and an increase in CD206 expression was observed in animals receiving IR and NIR. Tissue necrosis was decreased by NIR in the IRI group, as visualized by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The findings demonstrate that exposure to NIR reduced IRI and improved tissue survival. NIR reduced inflammation, decreased proinflammatory M1, and increased protective M2 macrophages. Exposure to NIR reduced inflammation and enhanced regeneration, leading to tissue protection following ischemia.

Comparative evaluation of empagliflozin, canagliflozin and sitagliptin cardioprotective properties in rats with experimental type 2 diabetes mellitus

Background: Myocardial infarction (MI) is one of the leading causes of mortality in patients with type 2 diabetes mellitus (DM), therefore it is essential to give preference to a glucose-lowering drug having optimal cardioprotective properties. A comparative study of the various sodium-glucose co-transporter inhibitors representatives’ protective effects in experimental MI was not carried out within the framework of one study.Aim: To evaluate the influence of empagliflozin (EMPA) and canagliflozin (CANA), in comparison with sitagliptin (SITA), on hemodynamic parameters and myocardial damage area in rats with diabetes type 2 model in experimental MI.Materials and methods: Type 2 DM was modelled in Wistar rats by means of 4-week high-fat diet followed by nicotinamide 230 mg/kg and streptozotocin 60 mg/kg administration. 4 weeks after DM induction the following groups were made: «DM+SITA» — treatment with SITA 50 mg/kg, «DM+EMPA» — treatment with EMPA 2 mg/kg, «DM+CANA» — treatment with CANA 25 mg/kg per os once daily for 8 weeks. Animals in «DM» group remained untreated for the following 8 weeks. Rats in control group were fed with standard chow. 16 weeks after the experiment beginning transient global myocardial ischemia was modelled in all rats. Hemodynamic parameters and myocardium necrosis area were evaluated.Results: The necrosis area was larger in «DM» group, than in control one (p=0.018). Infarction size in «DM+SITA» did not differ from that in «DM» group (62.92(41.29;75.84) and 57.26(45.51;70.08)%, р=0.554). Necrosis area in «DM+EMPA» and «DM+CANA» groups was smaller than in «DM» group (37.90(20.76;54.66)%, 46.15(29.77;50.55) vs 57.26(45.51;70.08)%, р=0.008 and р=0.009, respectively). Necrosis size did not differ between «DM+EMPA» and «DM+CANA» groups (p=0.630). Ischemic contracture in «DM+CANA» group was less prominent than under the use of all other glucose-lowering drugs. We observed increase of coronary blood flow in «DM+EMPA» group, in comparison with «DM», «DM+CANA» and «DM+SITA» groups.Conclusions: SITA does not have cardioprotective effect in ischemia-reperfusion injury in diabetic rats. EMPA and CANA have similarly prominent infarct-limiting properties. EMPA is able to increase coronary blood flow, whereas cardioprotective action of CANA is associated with ischemic contracture diminishing.

Myostatin Deficiency Protects C2C12 Cells from Oxidative Stress by Inhibiting Intrinsic Activation of Apoptosis.

Ischemia reperfusion (IR) injury remains an important topic in clinical medicine. While a multitude of prophylactic and therapeutic strategies have been proposed, recent studies have illuminated protective effects of myostatin inhibition. This study aims to elaborate on the intracellular pathways involved in myostatin signaling and to explore key proteins that convey protective effects in IR injury. We used CRISPR/Cas9 gene editing to introduce a myostatin (Mstn) deletion into a C2C12 cell line. In subsequent experiments, we evaluated overall cell death, activation of apoptotic pathways, ROS generation, lipid peroxidation, intracellular signaling via mitogen-activated protein kinases (MAPKs), cell migration, and cell proliferation under hypoxic conditions followed by reoxygenation to simulate an IR situation in vitro (hypoxia reoxygenation). It was found that mitogen-activated protein kinase kinase 3/6, also known as MAPK/ERK Kinase 3/6 (MEK3/6), and subsequent p38 MAPK activation were blunted in C2C12-Mstn−/− cells in response to hypoxia reoxygenation (HR). Similarly, c-Jun N-terminal kinase (JNK) activation was negated. We also found the intrinsic activation of apoptosis to be more important in comparison with the extrinsic activation. Additionally, intercepting myostatin signaling mitigated apoptosis activation. Ultimately, this research validated protective effects of myostatin inhibition in HR and identified potential mediators worth further investigation. Intercepting myostatin signaling did not inhibit ROS generation overall but mitigated cellular injury. In particular, intrinsic activation of apoptosis origination from mitochondria was alleviated. This was presumably mediated by decreased activation of p38 caused by the diminished kinase activity increase of MEK3/6. Overall, this work provides important insights into HR signaling in C2C12-Mstn−/− cells and could serve as basis for further research.

Effects of hyperoxia and cardiovascular risk factors on myocardial ischaemia-reperfusion injury: a randomized, sham-controlled parallel study.

What is the central question of this study? The beneficial effects of supplemental oxygen in patients with acute myocardial infarction are still uncertain: what are the effects of ischaemia-reperfusion injury during hyperoxia and normoxia in mature rats with and without cardiovascular risk factors? What is the main finding and its importance? Despite elevated baseline oxidative stress in rodents with cardiovascular risk factors, hyperoxic reperfusion limited myocardial necrosis and anti/pro-oxidant imbalance in spontaneously hypertensive and Zucker rats. In contrast, this effect was exacerbated in healthy Wistar rats. These results suggest that oxygen supplementation may not be harmful in patients with acute myocardial injury. Recent studies on O2 supplementation in acute coronary syndrome patients are equivocal. We tested the hypothesis that oxidative stress is increased in rodents with cardiovascular risk factors and enhances ischaemia-reperfusion injury in the presence of hyperoxia. A total of 43 Wistar rats (WR), 30 spontaneously hypertensive rats (SHR) and 33 obese Zucker rats (ZR) were randomized in a sham procedure (one-third) or underwent a left anterior descending ligation of the coronary artery for 60min (two-thirds). This was followed by 3h of reperfusion while animals were randomized either in a hyperoxic (HR) or a normoxic reperfusion (NR) group. Myocardial infarction size and oxidative stress biomarkers (myeloperoxidase (MPO), malondialdehyde and total free thiols) were assessed in blood samples. Baseline troponin T was higher in SHR and ZR than in WR (both P<0.001). Baseline total MPO was elevated in ZR in comparison to SHR and WR (both P<0.001). SHR had lower thiol concentration compared to WR and ZR (P<0.000001). HR was associated with a lower troponin T rise in SHR and ZR than in NR (both P<0.001), while the reverse occurred in WR (P<0.001). In SHR, HR limited total MPO increase as compared to NR (P=0.0056) and the opposite effect was observed with total MPO in WR (P=0.013). NR was associated with a drastic reduction of total thiols as compared to HR both in SHR and in ZR (both P<0.001). Despite a heightened baseline oxidative stress level, HR limited myocardial necrosis and anti/pro-oxidant imbalance in SHR and ZR whereas this effect was exacerbated in healthy WR.

Mesenchymal stromal cell treatment of donor kidneys during ex vivo normothermic machine perfusion: A porcine renal autotransplantation study.

Normothermic machine perfusion (NMP) of injured kidneys offers the opportunity for interventions to metabolically active organs prior to transplantation. Mesenchymal stromal cells (MSCs) can exert regenerative and anti-inflammatory effects in ischemia-reperfusion injury. The aims of this study were to evaluate the safety and feasibility of MSC treatment of kidneys during NMP using a porcine autotransplantation model, and examine potential MSC treatment-associated kidney improvements up to 14days posttransplant. After 75min of kidney warm ischemia, four experimental groups of n=7 underwent 14h of oxygenated hypothermic machine perfusion. In three groups this was followed by 240min of NMP with infusion of vehicle, 10million porcine, or 10million human adipose-derived MSCs. All kidneys were autotransplanted after contralateral nephrectomy. MSC treatment did not affect perfusion hemodynamics during NMP or cause adverse effects at reperfusion, with 100% animal survival. MSCs did not affect plasma creatinine, glomerular filtration rate, neutrophil gelatinase-associated lipocalin concentrations or kidney damage assessed by histology during the 14days, and MSCs retention was demonstrated in renal cortex. Infusing MSCs during ex vivo NMP of porcine kidneys was safe and feasible. Within the short posttransplant follow-up period, no beneficial effects of ex vivo MSC therapy could be demonstrated.

Organ Restoration With Normothermic Machine Perfusion and Immune Reaction.

Liver transplantation is the only recognized effective treatment for end-stage liver disease. However, organ shortages have become the main challenge for patients and physicians within the transplant community. Waiting list mortality remains an issue with around 10% of patients dying whilst waiting for an available organ. The post-transplantation period is also associated with an adverse complication rate for these specific cohorts of high-risk patients, particularly regarding patient and graft survival. Ischaemia reperfusion injury (IRI) has been highlighted as the mechanism of injury that increases parenchymal damage, which eventually lead to significant graft dysfunction and other poor outcome indicators. The consequences of IRI in clinical practice such as reperfusion syndrome, primary non-function of graft, allograft dysfunction, ischaemic biliary damage and early biliary complications can be life-threatening. IRI dictates the development of a significant inflammatory response that drives the pathway to eventual cell death. The main mechanisms of IRI are mitochondrial damage due to low oxygen tension within the hepatic micro-environment and severe adenosine triphosphate (ATP) depletion during the ischaemic period. After the restoration of normal blood flow, this damage is further enhanced by reoxygenation as the mitochondria respond to reperfusion by releasing reactive oxygen species (ROS), which in turn activate Kupffer cells within the hepatic micro-environment, leading to a pro-inflammatory response and eventual parenchymal cell apoptosis and associated tissue degradation. Machine perfusion (MP) is one emergent strategy considered to be one of the most important advances in organ preservation, restoration and transplantation. Indeed, MP has the potential to rescue frequently discarded organs and has been shown to limit the extent of IRI, leading to suppression of the deleterious pro-inflammatory response. This immunomodulation reduces the prevalence of allograft rejection, the use of immunosuppression therapy and minimizes post-transplant complications. This review aims to update the current knowledge of MP with a focus on normothermic machine liver perfusion (NMLP) and its potential role in immune response pathways.