24-h Reperfusion Research Articles

Renal protective effect of nebivolol in rat models of acute renal injury: role of sodium glucose co-transporter 2.

Upregulation of the sodium glucose co-transporter (SGLT2) is implicated in acute renal injury (ARI) progression and is regulated by extracellular signal-regulated kinase (ERK), hypoxia-inducible factor 1 alpha (HIF1α) or prostaglandin E2 (PGE2). This study aimed to assess the possible protective effect of nebivolol on renal ischemia/reperfusion (IR) and glycerol-induced ARI targeting SGLT2 via modulating the ERK-HIF1α pathway. Rats were divided into control, sham, IR or nebivolol-treated group, in which rats were treated with nebivolol (10mg/kg) for 3days prior to the induction of IR. The rats were subjected to renal ischemia by bilateral clamping of the pedicles for 45min, followed by 24h reperfusion. Another group of rats received the vehicle or nebivolol (10mg/kg) for 3days followed by injection of 50% glycerol (8ml/kg, IM) or saline. Kidney function tests, systolic blood pressure (SBP), oxidative stress markers [malondialdehyde (MDA) and NADPH oxidase] and kidney levels of nitric oxide (NO), inducible nitric oxide synthase(iNOS), HIF1α, ERK phosphorylation and PGE2 were determined. Additionally, renal sections were used for histological grading of renal injury and immunological expression of SGLT2. ARI rats showed significantly increased SBP, poor kidney function tests, increased oxidative stress, iNOS, NO, HIF1α levels, decreased PGE2 and ERK phosphorylation and upregulation of SGLT2 expression. Nebivolol treatment protected against the kidney damage both on the biochemical and histological levels. Nebivolol has a direct renoprotective effect, at least in part, by down-regulating SGLT2 possibly via modulating HIF1α, ERK activity and PGE2 production.

Ageing reduces angiotensin II type 1 receptor antagonism mediated pre-conditioning effects in ischemic kidneys by inducing oxidative and inflammatory stress.

Ischemia/reperfusion (I/R) injury is a common cause of acute kidney injury (AKI), which occurs clinically during renal organ transplantation and major cardiac surgeries. Previously, it was demonstrated that angiotensin II type 1 receptor (AT1) receptor antagonism is beneficial in the resolution of AKI episodes in young rats by reducing inflammation and oxidative stress. However, studies have shown that aged kidneys are refractory to surgical ischemic pre-conditioning due to increased oxidative stress, mitochondrial dysfunction, inflammation and apoptosis. Therefore, the present study was designed to evaluate the effects of pharmacologically induced pre-conditioning on I/R induced AKI in aged kidneys. AKI was induced by clamping both renal pedicels for 45min followed by 24h of reperfusion. The AT1 receptor antagonist, losartan was administered for three days prior to I/R injury induction in both aged and young rats. Renal outcomes were assessed by serum creatinine, creatinine clearance and proteinuria, renal antioxidant enzyme assays, membrane Na+K+ATPase activity, inflammatory biomarkers, and histological studies. AKI developed 24h post ischemia, as indicated by elevated serum creatinine levels, proteinuria, oxidative stress, reduced membrane Na+K+ATPase activity, increased inflammatory biomarkers levels and histological damage including cellular infiltration, tubular thickening, tubular dilation and necrosis. Losartan pre-treatment significantly improved renal dysfunction and histological alterations in young rats subjected to I/R injury. However, this treatment did not prevent various AKI manifestations in aged rats due to elevated oxidative and inflammatory stress mediated via tubular dysfunction and damage. We conclude that AT1 receptor antagonism is not beneficial against renal I/R induced AKI in aged rats.

Abstract TP471: Oxygen-Glucose Deprivation and Denser Extracellular Matrix Cause Cerebral Capillary Endothelial Cells to Lose Elasticity

Objective: Cerebral ischemia affects the mechano-response of vasculature and the remodeling of extracellular matrix (ECM) components. The present study investigates how oxygen-glucose deprivation (OGD) affects the brain endothelial cell (BEC) stiffness. We further aim to study whether ECM components affect the cellular stiffness in normal and energy deprived conditions. Methods: Primary human BECs received a 16h OGD followed by the reperfusion with normal culture media. Young’s Modulus (YM) and topography in the BEC surface were determined by atomic force microscopy (AFM). To test the effect of ECM composition on the BEC stiffness, various concentrations (5-500 uM) of collagen type IV were used for culture dish coating. BECs on the coating beds underwent 16h OGD and served for YM measurements. Per dish, 10-20 single BECs were measured. Results: BECs under OGD conditions became significantly stiffer (2.1-fold, *P<0.05) than the normoxic control, and the increased stiffness sustained even after the reperfusion for 24h, indicating energy deprivation is a primary factor for the increased stiffness under acute stress conditions. AFM topography confirmed that OGD induced rough and filamentous plasma membrane surface in BECs, suggesting a cytoskeletal change was induced by OGD. BECs grown on a 10x denser collagen type IV (500 uM) layer showed a significant increase (2.3-fold, ***P<0.001) in cellular stiffness, compared with the conventional concentration of 50 uM, suggesting that denser extracellular matrix makes BEC stiffer. Also, in BECs cultured on a denser ECM layer, OGD response was absent in terms of cellular stiffness. Conclusion: The present study found that OGD increased BEC stiffness. In addition, increasing the density of the ECM alone was enough to generate stiffer BECs. These mechanical properties of BECs may be a physical factor mediating endothelial responses after stroke.

Abstract WMP99: Alpha2-Antiplasmin Inactivation Reduces Hemorrhagic Transformation and Ischemic Brain Injury After Tissue Plasminogen Activator Therapy and Mechanical Reperfusion

Tissue plasminogen activator (r-tPA) therapy followed by thrombectomy-mediated, mechanical reperfusion profoundly reduces neurologic disability in appropriately selected patients. Nevertheless, a high proportion of these patients (20-37%) develop brain hemorrhage. We examined the contribution of α2-antiplasmin, a fast-acting plasmin inhibitor, to brain hemorrhage and ischemic injury following r-tPA treatment and mechanical reperfusion. Methods: Mice (C57Bl6) underwent 1 to 4 h of middle cerebral artery occlusion followed by treatment with r-tPA alone (10 mg/kg), r-tPA (10 mg/kg) plus an α2AP inhibitor (α2AP-I, 10 mg/kg) antibody or saline, followed by reperfusion. Mice were assessed by neurobehavioral measures (Bederson score and corner test) and histology 24 h post-ischemic stroke. Analyses were performed in a blinded fashion. Time course studies in r-tPA-treated mice showed that intracerebral hemorrhage (p<0.05), brain infarct volume (p<0.001) and brain swelling (p<0.01) increased progressively with ischemic time, with a plateau at 3 h of ischemia, as compared to saline treated controls. Mice treated at 3 h with r r-tPA + α2AP-I showed significantly (p<0.05) reduced brain hemorrhage, brain infarct volume (p<0.01) and brain swelling (p<0.05). Treatment with r-tPA + α2AP-I also significantly improved neurobehavioral deficits (p<0.01) and sensory motor dysfunction (p<0.01) at 24h of reperfusion. Conclusion: α2AP contributes to the increased brain hemorrhage and ischemic brain injury associated with r-tPA treatment followed by mechanical reperfusion. Targeting a2AP appears beneficial, because specific, monoclonal antibody-mediated inhibition of α2AP markedly reduced brain hemorrhage, infarction, swelling and neurobehavioral disability.

Effects of bone marrow mononuclear cell transplantation on the recovery of nerve function after focal cerebral ischemia and reperfusion

Objective To determine the effect of transplanting bone marrow mononuclear cells (BMMCs) on the expression of glial fibrillary acidic protein (GFAP) and Nogo-A around the ischemic foci after focal cerebral ischemia and reperfusion, and to study any role of BMMCs in nerve function recovery. Methods BMMCs were isolated from the bone marrow of Sprague-Dawley rats. Cerebral ischemia and reperfusion was performed using a nylon thread to occlude the right middle cerebral artery for 2h followed by 24h of reperfusion. The qualified models were selected according to the Longa scale. The 48 models selected were randomly divided into a model group and an observation group, each of 24. Each group was further divided into 7d, 14d and 21d subgroups. 100μl of BMMCs (5×106 /ml) were slowly injected into the ischemic lateral striata of the observation group. The rats in the model group were similarly injected, but with buffered saline solution. The rats were evaluated using the Longa scale after 7d, 14d and 21d. The rats were then sacrificed and the brain was resected. Immunohistochemical assays quantified the expression of GFAP and Nogo-A around the ischemic foci. Results Compared with the model group, the rats in the observation group showed less neurological deficit on the 21st day, significantly greater expression of GFAP and significantly less Nogo-A expression on days 14 and 21. Nogo-A expression on the 21st day was also significantly lower than on the 14th day in the observation group. Conclusion BMMC transplantation can promote recovery from nerve damage after focal cerebral ischemia, which is probably related to enhanced expression of GFAP and restrained expression of Nogo-A in the brain tissues surrounding ischemic lesions. Key words: Bone marrow mononuclear cells; cerebral ischemia; Glial fibrillary acidic protein; Nogo-A

Contribution of angiotensin II in hepatic ischemia /reperfusion induced lung injury: Acute versus chronic usage of captopril

BackgroundAcute lung injury is one of the most popular consequences of hepatic ischemia/reperfusion (I/R) injury. Recently it was documented that renin-angiotensin system plays a key role in tissue inflammation, generation of reactive oxygen species (ROS) and tumor necrosis factor-alpha (TNF-α) (the principal liver injury mediators) during I/R. Material and methodsWe investigated the effect of acute versus chronic usage of angiotensin converting enzyme inhibitor (captopril) on liver inflammation and lung injury caused by hepatic ischemia for 1h followed by 24h reperfusion. Forty adult Wistar male rats were divided into sham, I/R, I/R-acute captopril (100 mg/kg, 24 and 1.5 h before surgery) and I/R-chronic captopril (10 mg/kg/day for 28 days before surgery) groups. ResultsWe found captopril pretreatment significantly decreased liver damage indices, adhesion molecules, and TNF-α level in hepatic and tracheal tissues. Histologically, acute captopril pretreatment significantly decreased hepatic Kupffer cells number and lung α-smooth muscle actin expression more than chronic pretreatment. Increased tracheal tone, in response to acetylcholine, was suppressed by acute and chronic captopril pretreatment. ConclusionAngiotensin II plays a key role in tissue inflammation and airway hyperresponsiveness (AHR) via enhancing production of TNF-α. With more protection observed in lung, acute captopril could attenuate liver-induced lung injury via lowering TNF-α; a suggested possible mediator of airway hyperreactivity.

Deletion of Mitochondrial Uncoupling Protein 2 Exacerbates Mitochondrial Damage in Mice Subjected to Cerebral Ischemia and Reperfusion Injury under both Normo- and Hyperglycemic Conditions.

Deletion of mitochondrial uncoupling protein 2 (UCP2) has been shown to aggravate ischemic damage in the brain. However, the underlying mechanisms are not fully understood. The objective of this study is to explore the impact of homozygous UCP2 deletion (UCP2-/-) on mitochondrial fission and fusion dynamic balance in ischemic mice under normo- and hyperglycemic conditions. UCP2-/- and wildtype mice were subjected to a 60 min middle cerebral artery occlusion (MCAO) and allowed reperfusion for 6h, 24h and 72h. Our results demonstrated that deletion of UCP2 enlarged infarct volumes and increased numbers of cell death in both normo- and hyperglycemic ischemic mice compared with their wildtype counterparts subjected to the same duration of ischemia and reperfusion. The detrimental effects of UCP deletion were associated with increased ROS production, elevated mitochondrial fission markers Drp1 and Fis1 and suppressed fusion markers Opa1 and Mfn2 in UCP2-/- mice. Electron microscopic study demonstrated a marked mitochondrial swolling after 6h of reperfusion in UCP2-/- mice, contrasting to a mild mitochondrial swolling in wildtype ischemic animals. It is concluded that the exacerbating effects of UCP2-/- on ischemic outcome in both normo- and hyperglycemic animals are associated with increased ROS production, disturbed mitochondrial dynamic balance towards fission and early damage to mitochondrial ultrastructure.

Antioxidant effect of p-coumaric acid on interleukin 1-β and tumor necrosis factor-α in rats with renal ischemic reperfusion

Background and aimsRenal ischemia-reperfusion occurs in some clinical conditions such as kidney surgery that can leads to acute renal failure. The aim of this study was to investigate the effect of p-coumaric acid (CA) on ischemia reperfusion (I/R) injury. MethodsThirty rats were randomly divided into five groups; control, CA (100mg/kg), I/R, propylene glycol (10%)+I/R and CA+I/R, (n=6 each). CA and propylene glycol were administered orally for 2 weeks. Then, the rats were subjected to bilateral renal ischemia for 45min and followed by reperfusion for 24h. All rats were killed and kidney function tests, tissue malondialdehyde and activity of antioxidant enzymes were determined. Histopathological evaluations were also performed. In addition, renal expression of the tumor necrosis factor-α and interleukin-1β were determined using enzyme-linked immunosorbent assay and immunohistochemistry. ResultsCA significantly improved the Cr and BUN levels in CA+I/R group compared to I/R group (p<0.005 and p<0.001, respectively). Reduction of tissue superoxide dismutase, glutathione peroxidase and catalase, were significantly improved by CA (p<0.01, p<0.01 and p<0.05). Treatment with CA also resulted in significant reduction in tissue MDA (p<0.05), TNF-α (p<0.001) and interleukin-1β expression (p<0.001) that were increased by renal I/R. Also, the rats treated with CA had nearly normal structure of the kidney. ConclusionsThe present findings suggest that, CA protects the kidneys against I/R injury via its antioxidant and anti-inflammatory effects.

Salvianolic acid B protects against myocardial ischaemia-reperfusion injury in rats via inhibiting high mobility group box 1 protein expression through the PI3K/Akt signalling pathway

Salvianolic acid B (Sal B) has a significant protective effect on myocardial ischaemia-reperfusion (I/R) injury. Therefore, the aims of this study were to determine the effects of Sal B on myocardial ischaemic-reperfusion (I/R) injury in rats and to explore whether its underlying mechanism of cardioprotection occurs through activating the expression of the phosphoinositide 3-kinase/protein, kinase B (PI3K/Akt) and inhibiting the expression of high mobility group protein 1 (HMGB1). Ninety Sprague-Dawley rats were randomized into five groups: group 1 (sham-operated), group 2 (myocardial I/R), group 3 (low dose of Sal B+I/R), group 4 (high dose of Sal B+I/R), and group 5 (high dose of Sal B+I/R+LY294002, which is a specific PI3k inhibitor). All I/R rats received 30 min myocardial ischaemia followed by 24-h reperfusion. Cardiac function, infarct size, myocardial injury marker levels, inflammatory response and cardiomyocyte apoptosis as well as Bcl-2, Bax, P-Akt, HMGB1 and TLR4 expression were measured. In the current study, Sal B significantly ameliorated myocardial I/R injury in a dose-dependent manner, ameliorated cardiac function, reduced myocardial infarction size, decreased myocardial injury marker expression, decreased inflammatory responses, reduced apoptosis, activated PI3K/Akt expression and inhibited HMGB1 expression. However, all effects of Sal B were significantly reversed by LY294002. Overall, the present study indicated that Sal B attenuated myocardial I/R injury by activating PI3K/Akt and inhibiting the release of HMGB1 in rats.

Neuroprotective effect of Crocus sativus against cerebral ischemia in rats.

The present study aimed to investigate the role of vascular endothelial growth factor (VEGF) in the neuroprotective effect of Crocus sativus (saffron) against cerebral ischemia/reperfusion injury (I/R) in rats. Four groups of a total forty I/R rats with 60-min occlusion followed by 48h reperfusion or sham surgery were used. The sham and left-brain I/R control groups where treated with normal saline. The rats of the other two groups received saffron extract (100 or 200mg/kg, ip, respectively) for 3 successive weeks prior to left-brain I/R. Other four doses of saffron extract were received by the rats of the last 2 groups 60min prior to operation, during the surgery, and on days 1 and 2 following reperfusion. I/R group showed marked neurobehavioral, neurochemical and histopathological alterations. The results revealed a significant reduction in neurological deficit scores in the saffron-treated rats at both doses. Saffron significantly attenuated lipid peroxidation, decreased NO and brain natriuretic peptide (BNP) contents in I/R-brain tissue. On the other hand, saffron reversed the depletion of GSH in the injured brain. Moreover, saffron treatment evidently reduced apoptosis as revealed by a decrease in caspase-3 and Bax protein expression with a marked decrease in the apoptotic neuronal cells compared to I/R group. In addition, saffron administration effectively upregulated the expression of VEGF in I/R-brain tissue. In conclusion, saffron treatment offers significant neuroprotection against I/R damage possibly through diminishing oxidative stress and apoptosis and enhancement of VEGF.

Protective effect of hydrogen sulfide on experimental testicular ischemia reperfusion in rats

Testicular torsion is an urgent urological condition. Ischemia-reperfusion (I/R) processes that occur after detorsion as a treatment for torsion are caused by testicular injury. The purpose of our study is investigating the protecting effect of hydrogen sulfide (H2S) on the testicular ischemia reperfusion injury. Thirty-eightWistar-Albino rats were divided randomly into 6 different groups: Control (6); sham (6); IR-E (6)-2h of torsion and 4h of reperfusion; IR-E+H2S (6)-in addition to the IR-E group, 75μmol/kg of sodium hydrogen sulfide (NaHS) was administered intraperitoneally 30min before reperfusion; IR-L (7)-2h of torsion and 24h of reperfusion; IR-L+H2S (7)-in addition to the IR-L group, 75μmol/kg NaHS was administered intraperitoneally 30min before reperfusion. Biochemically, nitric oxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD), reductive glutathione (GSH), and tumor TNF-α levels were measured in the testis. Serum TNF-α levels were also measured. Hematoxylin and eosin (H & E) was used for histopathologicalstaining and microscopic findings were examined. The Johnsen score was performed to assess spermatogenesis activity in the testis. Apoptosis protease activating factor-1 (Apaf-1) and inducible nitric oxide synthase (iNOS) activity were evaluated immunohistochemically as well. Statistical analyses were made by the Chi-squared test and one-way analysis of variance. MDA and NO levels were significantly increased in the IR-L group compared with sham and which decreased by the addition of H2S treatment to the IR-L group (p<0.05) in biochemical evaluation. GSH vs SOD levels were decreased in the IR-L group compared with sham and which increased by the addition of H2S treatment to the IR-L group, but this correlations were not statistically significant (p>0.05). Tissue and serum TNF-α levels were significantly increased in the IR-E group compared with sham and which decreased by the addition of H2S treatment to the IR-E group. Johnsen score was the lowest in IR-L group (p<0.05). Apaf-1 and iNOS activity were significantly increased in the IR-L group compared with sham and which decreased by the addition of H2S treatment to the IR-L group (p<0.05) in immunohistochemical evaluation. First, the authors would like to say that H2S treatment is protective and it is againstischemia reperfusion injury in testicular torsion. The anti-inflammatory, antioxidant, and antiapoptotic properties of H2S caused protective effect as shown in this study.

EPA plus DHA improves survival related to a decrease of injury after extended liver ischemia in Sprague-Dawley rats

Introduction and objectivesThe omega-3 fatty acids (ω3), EPA and DHA, have been described for their beneficial effects on metabolism and inflammation. In addition, they are interesting tools in the treatment of acute liver disease. This investigation was conducted to assess the effect of EPA+DHA administration before partial ischemia (IR) on survival and liver injury. Materials and methodsMale Sprague-Dawley rats were supplemented for 7 days with ω3 [EPA (270mg/kg) and DHA (180mg/kg)]; controls received saline solution. After EPA+DHA supplementation, liver IR was induced by temporarily occluding the blood supply for 1h, followed up by 48h of reperfusion. Control animals were subjected to sham laparotomy. ResultsPrevious to IR, the EPA+DHA administration improved the rate and prolonged the survival time by decreasing the AST and ALT levels and improving liver degenerative changes generated by the IR, which decreased TNF-α and IL-1β. In addition, IL-10 increased at 20h with a tendency to normalize at 48h. The IR group had no differences in the IL-10 levels compared to controls. ConclusionsThe ω3 supplementation could prevent and promote the restoration of the liver tissue and significantly improve the survival rate in rats at 48h.

Intraluminal fluid infusion in a rat jejunum ischemia/reperfusion model is associated with improved tissue perfusion and less mucosal damage

This study used an experimental model mimicking early postoperative enteral feeding after the transfer of free jejunal flap and tested the hypothesis that jejunal infusion with dextrose or saline is associated with improved tissue perfusion and/or less mucosal damage after ischemia/reperfusion (IR) injury. Thirty-five male Sprague Dawley rats were randomly divided into five groups: sham group (no IR and no intraluminal infusion); IR control group (IR but not intraluminal infusion); IR plus intraluminal 0.9% NaCl infusion or 5% dextrose or 10% dextrose infusion groups. A jejunal segment of each rat was isolated. The animals had jejunal ischemia for 40min, reperfusion, and intestinal infusion on the basis of their allocation. Jejunal tissue perfusion was measured with laser Doppler flowmetry at one hour and two hours after reperfusion, after which the animals were sacrificed and tissue samples were obtained for the scoring of histological damage at superficial and cryptic epithelium, villus structure, and inflammatory cell infiltration and tissue nitric oxide (NO), interleukin (IL)-1, IL-6, and matrix metalloproteinase-1 (MMP) level measurements. At 1h of reperfusion, IR plus 5% dextrose and 10% dextrose groups both had significantly higher perfusion rates than the IR control group (384.8±26.7 and 462.4±44.7 versus 270.3±34.2 PU, respectively, p<0.05 for both). These differences were maintained at 2h of reperfusion (p<0.05 for both). Saline infusion, however, resulted in improved tissue perfusion only at the early phase of reperfusion. Intraluminal infusion with dextrose solution, either 5% or 10%, was associated with higher tissue NO, IL-1, and IL-6 levels than that in the sham group (p<0.05 for all). In addition, intraluminal infusion of any fluid resulted in less severe histological damage (8.1±0.9 versus 5.8±1.0, 5.4±0.9, and 5.2±1.9, for IR plus saline, 5% dextrose and 10% dextrose groups, respectively, p<0.05 for all). Intraluminal infusion of fluids, particularly dextrose solutions, may be protective against IR injury as demonstrated by improved tissue perfusion and less histological damage. In addition, increases in tissue NO, IL-1, and IL-6 levels in association with dextrose infusion may be explained by the activation of pro-inflammatory and anti-inflammatory protective pathways. These support early enteral feeding after free jejunum flap transfers; however, further studies are warranted.

Effect of renal ischaemia/reperfusion-induced acute kidney injury on pharmacokinetics of midazolam in rats.

This study aimed to investigate the effects of renal ischaemia/reperfusion (I/R)-induced acute kidney injury (AKI) on the distribution of midazolam (MDZ), a probe drug for cytochrome P450 3A (CYP3A) activity. We established an AKI model inducing ischaemia of both renal pedicles for 60min followed by 24-h reperfusion. MDZ was administered intravenously (i.v.) to the rats via the jugular vein, and then, blood samples were collected to determine the plasma concentration of MDZ. While the plasma concentration of MDZ after i.v. administration was decreased in the I/R rats, the tissue concentration was not altered. In addition, the tissue-to-plasma (T/P) ratio of MDZ was increased in the I/R rats. The unbound fraction of MDZ and the level of indoxyl sulphate (IS) in plasma were elevated in the I/R rats. Furthermore, the unbound fraction of MDZ was significantly increased by the addition of IS. These results indicated that the displacement of albumin-bound MDZ by IS changed the unbound fraction of MDZ and elevated the T/P ratio of MDZ in I/R rats.

FUNCTIONAL ASSESSMENT OF DCD HEARTS FOLLOWING NORMOTHERMIC REGIONAL PERFUSION CAN PREDICT POST-TRANSPLANTATION CONTRACTILITY

Procurement of DCD hearts using normothermic regional perfusion (NRP) leads to rapid organ reperfusion and allows for a complete cardiac assessment in situ, ensuring only suitable hearts are procured. We aimed to identify which parameters measured following NRP could predict myocardial performance post-transplantation. Donor pigs underwent hypoxic circulatory arrest followed by 15min of warm ischemia and resuscitation with NRP. They underwent 3h of cold ischemia, transplantation, and reperfusion for 3h. We assessed cardiac function using left ventricular (LV) pressure-volume loops, right ventricular (RV) catheterization, and echocardiography following NRP and at 3h of reperfusion. Lactate levels at the time of arrest and time from withdrawal of care to arrest were measured. We performed linear regressions to identify correlations post-transplant cardiac performance. Predictors and outcomes were analyzed as absolute and relative (% from baseline) values. Relative LV fractional area change was the best predictor of relative preload recruitable stroke work (R2=0.754; p=0.002). Lactate concentration immediately before NRP also was a good predictor of relative stroke work (R2=0.788; p < 0.001). Relative and absolute RV stroke work index (SWI) and RV S’, and time to arrest were predictors of recovery in RV function. Only RV functional parameters were capable of predicting global myocardial performance following transplantation. RV S’ showed the strongest correlation with absolute (R2=0.540; p=0.01) and relative (R2=0.500; p=0.022) cardiac index (Table 1). Contractility and biochemical markers measured before heart procurement seemed to predict myocardial performance accurately. Functional parameters demonstrated a more significant number of correlations and provided a better evaluation following NRP. It is of critical importance to also assess DCD heart function to determine organ suitability before transplantation.

CONTRACTILITY VERSUS METABOLIC CARDIAC ASSESSMENT DURING EX SITU HEAR PERFUSION: A PRE-CLINICAL TRANSPLANT STUDY

Assessment of DCD hearts during ex situ perfusion (ESP) is required to determine suitability of these organs for transplant. To date, metabolic and contractility parameters have not been compared using a full transplant model. We aimed to identify which parameters measured during ESP could predict myocardial performance post-transplantation. Porcine hearts were procured from beating-heart donations (n=6) or following hypoxic circulatory arrest and 15min of warm ischemia (n=6). They underwent 4h of ESP, transplantation, and reperfusion for 3h. Cardiac function was assessed during ESP using left (LV) and right (RV) ventricular pressure-volume loops, and metabolic parameters. We performed linear regressions to identify significant correlations. Several parameters correlated with recovery of cardiac function (Table 1). LV developed pressure was the best predictor of LV Max dP/dt (R2=0.523; p=0.018) while myocardial oxygen consumption (MVO2) at 30min of ESP better predicted LV stroke work (R2=0.625; p=0.006). RV Tau during ESP was the best predictor of post-transplant RV function (R2=0.615; p=0.021). Several metabolic parameters predicted cardiac index. LV Preload Recruitable Stroke Work was the only contractile measurement to predict cardiac index (R2=0.467; p=0.021), while MVO2 at 30min was the best predictor (R2=0.578; p=0.007). Both contractility and metabolic markers measured during ESP were able to predict myocardial performance following transplantation. Assessment of metabolic parameters demonstrated a more significant number of correlations and provided a better evaluation of cardiac index. This is contradictory with previous reports showing contractile evaluation would provide a more accurate estimation of early transplant function. However, contractility parameters were better to evaluate isolated LV and RV function post-transplant. Further studies are needed to clarify this question.

TGF-β2/Smad3 Signaling Pathway Activation Through Enhancing VEGF and CD34 Ameliorates Cerebral Ischemia/Reperfusion Injury After Isoflurane Post-conditioning in Rats.

Evidence has shown the therapeutic potential of isoflurane (ISO) in cerebral stroke. The present study investigated the mechanism of ISO on vascular endothelial growth factor (VEGF) and CD34 expression in a rat model of stroke. Transient focal cerebral ischemia was established by middle cerebral artery occlusion (MCAO) for 1h followed by reperfusion for 24h in rats. ISO was administered for 1.5h when the reperfusion was initiated. Neurologic deficit scores, infarct volumes, HE staining, Nissl staining, and TUNEL staining were evaluated at 24h after reperfusion. The levels of transforming growth factor (TGF)-β2, Smad3, p-Smad3, VEGF, and CD34 proteins were detected by immunofluorescence (IF) staining and Western blot assay. Administration of ISO significantly reduced the neurologic deficit scores, infarct volumes, and damaged and apoptotic cells after cerebral ischemia/reperfusion (I/R) injury (P < 0.05). Meanwhile, ISO post-conditioning significantly increased the expression levels of TGF-β2, p-Smad3, VEGF, and CD34 (P < 0.05), whereas the expression of Smad3 showed no difference (P > 0.05). However, Pirfenidone, a TGF-β2 inhibitor, decreased the expression levels of TGF-β2, p-Smad3, VEGF, and CD34 (P < 0.05). Moreover, the protective effects of ISO post-conditioning were negated by the inhibitor. The present study indicated that ISO attenuates brain damage by activating the TGF-β2/Smad3 signaling pathway and increasing the protein expression of VEGF and CD34 in the rat MCAO model.

The propolis and boric acid can be highly suitable, alone/or as a combinatory approach on ovary ischemia-reperfusion injury

Ovarian ischemia-reperfusion (IR) damage continues to be a serious infertility problem. The oxidative stress plays central role in the development of IR injuries. Activation of antioxidants decreases IR injuries; however, the efficacy of antioxidant agents remains controversial. Unfortunately, there has been no evidence for medicinal use of boric acid (BA) and propolis (Prop) on ovarian IR injury on rats so far. This study will provide to reveal the potential applications of the Prop and BA in ovarian IR therapy. The Sprague-Dawley rats were randomized into five groups: I-control, II-IR, 3h of ischemia and 3h of reperfusion, III and IV-a signal dose of oral BA (7mg/kg) and Prop (100mg/kg) alone 1h before induction of IR, V-Prop and BA together 1h before induction of IR. SOD (superoxide dismutase), CAT (catalase), GSH (glutathione), MPO (myeloperoxidase), MDA (malondialdehyde), and IL-6 (interleukin-6) levels were quantified by ELISA and the TNF-α (tumor necrosis factor-α), 8-OHdG (8-hydroxylo-2'-deoxyguanosin) and Caspase-3 expressions were performed by immunohistochemical analyses. BA and Prop pretreatment significantly reduced MPO, MDA, and IL-6 levels and pathologic score in IR rats, with no effects in control group. These agents used in therapy also decreased TNF-α, 8-OHdG and Caspase-3 protein expressions increased by IR. Furthermore, BA and Prop combination showed significant ameliorative effects on ovary injury caused by IR through acting as an antioxidant, anti-inflammatory and antiapoptotic agent. BA and Prop alone and especially in combination could be developed as therapeutic agents against ovary IR injury.

Ginkgetin attenuates cerebral ischemia-reperfusion induced autophagy and cell death via modulation of the NF-κB/p53 signaling pathway.

Background: Cerebral ischemia–reperfusion (I/R) injury is the key to fatality in cerebrovascular accident, hence further endeavor is warranted to delineate the mechanism underlying its lethal aggravation procedure. In the present study, we aimed to elucidate the anti-autophagy and anti-apoptosis effects of ginkgetin via nuclear factor κB (NF-κB)/p53 pathway in cerebral I/R rats.Methods: Rats were administrated 2-h occlusion of right middle cerebral artery before the 24-h reperfusion followed. There were three doses of ginkgetin (25, 50, 100 mg/kg) given intraperitoneally (i.p.) after the 2-h ischemia, and Pifithrin-α (PFT-α, p53 inhibitor), SN50 (NF-κB inhibitor) and 3-methyladenine (3-MA, autophagy inhibitor) was administered 20 min before the ischemia, respectively.Results: The neurological deficits decreased significantly with the administration of ginkgetin. The concentrations of microtubule-associated protein 1 light chain 3-II and p53 were significantly decreased by PFT-α, 3-MA and ginkgetin. The concentrations of Beclin 1, damage-regulated autophagy modulator, cathepsin B and cathepsin D were significantly decreased due to the administration of PFT-α, ginkgetin and SN50. Furthermore, the concentrations of Bax and p53-upregulated modulator of apoptosis were significantly decreased with that of Bcl-2 being significantly increased by administration of SN50, PFT-α and ginkgetin.Conclusion: Ginkgetin can alleviate cerebral ischemia/reperfusion induced autophagy and apoptosis by inhibiting the NF-κB/p53 signaling pathway.

DPP-4 inhibition by linagliptin prevents cardiac dysfunction and inflammation by targeting the Nlrp3/ASC inflammasome.

We compared the effects of linagliptin (Lina, a DPP4 inhibitor) and GLP-1 receptor activation by exenatide followed by exendin-4 in an infusion pump (EX) on infarct size (IS), post-infarction activation of the inflammasome and remodeling in wild-type (WT) and db/db diabetic mice. Mice underwent 30min ischemia followed by 24h reperfusion. IS was assessed by TTC. Additional mice underwent permanent coronary artery occlusion. Echocardiography was performed 2w after infarction. Activation of the inflammasome in the border zone of the infarction was assessed by rt-PCR and ELISA 2w after reperfusion. Further in vitro experiments were done using primary human cardiofibroblasts and cardiomyocytes exposed to simulated ischemia-reoxygenation. Lina and EX limited IS in both the WT and the db/db mice. Lina and EX equally improved ejection fraction in both the WT and the db/db mice. mRNA levels of ASC, NALP3, IL-1β, IL-6, Collagen-1, and Collagen-3 were higher in the db/db mice than in the WT mice. Infarction increased these levels in the WT and db/db mice. Lina more than EX attenuated the increase in ASC, NALP3, IL-1β, IL-6, Collagen-1 and Collagen-3, TNFα and IL-1β, and decreased apoptosis, especially in the db/db mice. In vitro experiments showed that Lina, but not EX, attenuated the increase in TLR4 expression, an effect that was dependent on p38 activation with downstream upregulation of Let-7i and miR-146b levels. Lina and EX had similar effects on IS and post-infarction function, but Lina attenuated the activation of the inflammasome and the upregulation of collagen-1 and collagen-3 more than direct GLP-1 receptor activation. This effect depends on p38 activation with downstream upregulation of miR-146b levels that suppresses TLR4 expression.