The protective effect of 1400W against ischaemia and reperfusion injury is countered by transient medullary kidney endothelial dysregulation.

Renal ischaemia and reperfusion (I/R) is caused by a sudden temporary impairment of the blood flow. I/R is a prevalent cause of acute kidney injury. As nitric oxide generated by inducible nitric oxide synthase (iNOS) has detrimental effects during I/R, the pharmacological blockade of iNOS has been proposed as a potential strategy to prevent I/R injury. The aim of this study was to improve the understanding of 1400W (an iNOS inhibitor) on renal I/R as a pharmacological strategy against kidney disease. BALB/c mice received 30 min of bilateral ischaemia, followed by 48 h or 28 days of reperfusion. Vehicle or 1400W (10 mg/kg) was administered 30 min before inducing ischaemia. We found that after 48 h of reperfusion 1400W decreased the serum creatinine, blood urea nitrogen, neutrophil gelatinase-associated lipocalin and proliferating cell nuclear antigen 3 in the I/R animals. Unexpectedly, we observed mRNA upregulation of genes involved in kidney injury, cell-cycle arrest, inflammation, mesenchymal transition and endothelial activation in the renal medulla of sham animals treated with 1400W. We also explored if 1400W promoted chronic kidney dysfunction 28 days after I/R and did not find significant alterations in renal function, fibrosis, blood pressure or mortality. The results provide evidence that 1400W may have adverse effects in the renal medulla. Importantly, our data point to 1400W-induced endothelial dysfunction, establishing therapeutic limitations for its use. KEY POINTS: Acute kidney injury is a global health problem associated with high morbidity and mortality. The pharmacological blockade of inducible nitric oxide synthase (iNOS) has been proposed as a potential strategy to prevent AKI induced by ischaemia and reperfusion (I/R). Our main finding is that 1400W, a selective and irreversible iNOS inhibitor with low toxicity that is proposed as a therapeutic strategy to prevent kidney I/R injury, produces aberrant gene expression in the medulla associated to tissue injury, cell cycle arrest, inflammation, mesenchymal transition and endothelial activation. The negative effect of 1400W observed in the renal medulla at 48 h from drug administration, is transient as it did not translate into a chronic kidney disease condition.

Evaluation of the therapeutic effects of oestradiol on the systemic inflammatory response and on lung injury caused by the occlusion of the proximal descending aorta in male rats.

OBJECTIVES: Ischaemia and reperfusion-induced microvascular dysfunction is a serious problem encountered during a variety surgical procedures, leading to systemic inflammation and affecting remote organs, specially the lungs. 17ß-Oestradiol reduces pulmonary repercussions from various acute lung injury forms. Here, we focused on the 17ß-oestradiol therapeutic effects after aortic ischaemia and reperfusion (I/R) by evaluating lung inflammation. METHODS: Twenty-four Wistar rats were submitted to I/R by insufflation of a 2-F catheter in thoracic aorta for 20 min. Reperfusion took 4 h and 17ß-oestradiol (280 µg/kg, i.v.) was administered after 1 h of reperfusion. Sham-operated rats were controls. Bronchoalveolar lavage was performed and lung samples were prepared for histopathological analysis and tissue culture (explant). Interleukin (IL)-1ß, IL-10 and tumour necrosis factor-α were quantified. RESULTS: After I/R, higher number of leukocytes in bronchoalveolar lavage were reduced by 17ß-oestradiol. The treatment also decreased leukocytes in lung tissue. I/R increased lung myeloperoxidase expression, with reduction by 17ß-oestradiol. Serum cytokine-induced neutrophil chemoattractant 1 and IL-1ß increased after I/R and 17ß-oestradiol decreased cytokine-induced neutrophil chemoattractant 1. I/R increased IL-1ß and IL-10 in lung explants, reduced by 17ß-oestradiol. CONCLUSIONS: Our results showed that 17ß-oestradiol treatment performed in the period of reperfusion, modulated the systemic response and the lung repercussions of I/R by thoracic aortic occlusion. Thus, we can suggest that 17ß-oestradiol might be a supplementary approach leading the lung deterioration after aortic clamping in surgical procedures.

Role of the antioxidant pathway in the healing of peptic ulcers induced by ischemia-reperfusion in male and female rats treated with Eugenia punicifolia.

Ischaemia and reperfusion (I/R)-induced gastrointestinal disorders are caused by free radicals, resulting in organ damage and functional disarrangement. This study aimed to investigate the healing effects of hydroalcoholic extracts from the leaves of Eugenia punicifolia (Kunth) DC. (HEEP) in male and female Wistar rats with I/R-induced peptic injuries, and the role of antioxidants in improving this response. After I/R-induced gastric and duodenal injuries, male and female [intact (INT) and ovariectomized (OVZ)] rats were orally treated with HEEP for 6 days. Biochemical analysis was used to determine the catalase (CAT), superoxide dismutase (SOD), and myeloperoxidase (MPO) activities, as well as malondialdehyde and reduced glutathione levels, to measure the gastric and duodenal healing process. Six days of HEEP treatment significantly decreased the I/R-induced gastric [male (73.68%), INT (52.83%), and OVZ (43.13%)] and duodenal damage [male (57.03%), INT (56.04%), and OVZ (54.83%)] in all groups. In OVZ rats, the healing effect of HEEP occurred because of the increased activity of SOD (2x) and CAT (1.16x) in the gastric mucosa. In the duodenal mucosa of INT rats, the extract reduced MPO (20.83%) activity. The 6-day HEEP treatment improved the healing of I/R-induced peptic ulcer injury, with the system acting differently in males and females. The antioxidant system is an important component of the HEEP activity during post-I/R mucosal recovery. This result revealed the importance of antioxidant compounds in minimizing the severity of I/R-related events.

The effect of lithium tetraborate as a novel cardioprotective agent after renal ischemia-reperfusion injury

Abstract Epidemiological studies suggest that acute kidney injury has certain effect on myocardial function. In this study, for the first time, we tested a boron compound namely lithium tetraborate an act as an anti-oxidant and anti-inflammatory agent in ischemia-reperfusion injury. For this, we employed an in vivo rat model with kidney ischemia reperfusion injury to evaluate cardiac injury to clarify the mechanisms of lithium tetraborate. The evaluation of cardiac injury through kidney artery occlusion and reperfusion rat model indicated that lithium tetraborate could (1) reduce oxidative stress-induced endothelial dysfunction; (2) attenuate the inflammatory response of cardiac cells; and (3) alleviate the apoptosis and necrosis of myocytes. In summary, lithium tetraborate demonstrates significant therapeutic properties that contribute to the amelioration of cardiac damage, and it could be a promising candidate for future applications in myocardial dysfunction.

3,5-Diiodothyronine protects against cardiac ischaemia-reperfusion injury in male rats.

NEW FINDINGS: What is the central question of this study? 3,5-Diiodothyronine (3,5-T2) administration increases resting metabolic rate, prevents or treats liver steatosis in rodent models, and ameliorates insulin resistance: what are its effects on cardiac electrical and contractile properties and autonomic regulation? What is the main finding and its importance? Chronic 3,5-T2 administration has no adverse effects on cardiac function. Remarkably, 3,5-T2 improves the autonomous control of the rat heart and protects against ischaemia-reperfusion injury. ABSTRACT: The use of 3,5,3'-triiodothyronine (T3) and thyroxine (T4) to treat metabolic diseases has been hindered by potential adverse effects on liver, lipid metabolism and cardiac electrical properties. It is recognized that 3,5-diiodothyronine (3,5-T2) administration increases resting metabolic rate, prevents or treats liver steatosis in rodent models and ameliorates insulin resistance, suggesting 3,5-T2 as a potential therapeutic tool. However, a comprehensive assessment of cardiac electrical and contractile properties has not been made so far. Three-month-old Wistar rats were daily administered vehicle, 3,5-T2 or 3,5-T2+T4 and no signs of atrial or ventricular arrhythmia were detected in non-anaesthetized rats during 90 days. Cardiac function was preserved as heart rate, left ventricle diameter and shortening fraction in 3,5-T2-treated rats compared to vehicle and 3,5-T2+T4 groups. Power spectral analysis indicated an amelioration of the heart rate variability only in 3,5-T2-treated rats. An increased baroreflex sensitivity at rest was observed in both 3,5-T2-treated groups. Finally, 3,5-T2 Langendorff-perfused hearts presented a significant recovery of left ventricular function and remarkably smaller infarction area after ischaemia-reperfusion injury. In conclusion, chronic 3,5-T2 administration ameliorates tonic cardiac autonomic control and confers cardioprotection against ischaemia-reperfusion injury in healthy male rats.

Protective effect of naringin in rat model of renal ischemia reperfusion injury / Efecto protector de la naringina en modelo de rata con lesión por reperfusión de isquemia renal

ABSTRACT Objective: We aimed to research that naringin whether protects from renal ischemia/reperfusion induced renal damage in rats. Methods: Twenty-four Wistar albino female rats randomly were divided into three groups: 1) control group, in which the rats were only performed right nephrectomy; 2) a second group received right nephrectomy and left kidney ischemia (1 h) and reperfusion (24 h) group ischemia/reperfusion (I/R); 3) a third group received 50 mg/kg naringin orally once a day for two weeks before ischemia/reperfusion (I/R/N). Expression of cyclooxygenase-2 (COX-2), cytosolic phospholipase A2 (cPLA2), inducible nitric oxide synthase (iNOS), caspase-3, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated x protein (Bax), serum creatinine (Cr), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) were measured by using enzyme-linked immunosorbent assay (ELISA). Results: Naringin-treated rats that performed renal ischemia/reperfusion demonstrated significant decrease in Cr, IL-6 and TNF-α levels when compared to the only renal ischemia/reperfusion performed rats. While renal ischemia/reperfusion caused a decrease of bcl-2 (1.72 ± 0.20 pg/ml) levels, while an increase of COX-2 (11882 ± 642 pg/ml), cPLA2 (2448 ± 139 pg/ml), iNOS (4331 ± 438 IU/ml), cleaved caspase-3 (7.33 ± 0.76 ng/ml) and Bax (2.33 ± 0.44 ng/ml) levels. The treatment of naringin reversed these kidney effects (7.47 ± 60.35 pg/ml; 9299 ± 327 pg/ml; 2001 ± 78 pg/ml; 3112 ± 220 IU/ml; 3.38 ± 0.54 ng/ml; 2.33 ± 0.44 ng/ml, respectively) (p <0.05). Conclusion: This study showed that naringin treatment attenuated renal damage induced by ischemia/reperfusion in rats.

RESUMEN Objetivo: Nuestro objetivo fue investigar si la naringina protege del daño en los riñones provocado por isquemia-reperfusión renal en ratas. Material y métodos: De forma aleatoria, dividimos 24 ratas albinas Wistar hembras en tres grupos: 1) grupo control, en el que solo se les realizó a las ratas una nefrectomía derecha; 2) un segundo grupo isquemia-reperfusión, con nefrectomía derecha e isquemia de riñón izquierdo (1 h) y reperfusión (24 h); 3) un tercer grupo al que se le administró 50 mg/kg de naringina por vía oral una vez al día durante dos semanas antes de la isquemia-reperfusión. Por medio de un ensayo inmunoabsorbente ligado a enzimas (ELISA), se midieron las siguientes expresiones: ciclooxigenasa-2 (COX-2), fosfolipasa citosólica A2 (cPLA2), óxido nítrico sintetasa inducible (ONSi), caspasa-3, linfoma de células B2 (Bcl-2), proteína X asociada a Bcl-2 (Bax), creatinina sérica (Cr), factor de necrosis tumoral alfa (FNT-α) e interleucina 6 (IL-6). Resultados: Las ratas tratadas con naringina por isquemia-reperfusión renal mostraron un descenso significativo en los niveles de Cr, IL-6 y FNT-α en comparación con las ratas a las que se les indujo isquemia-reperfusión renal pero que no se les suministró naringina. La isquemia-reperfusión renal provocó un descenso de los niveles de Bcl-2 (1,72 ± 0,20 pg/ml) y un ascenso en los niveles de COX-2 (11882 ± 642 pg/ml), cPLA2 (2448 ± 139 pg/ml), ONSi (4331 ± 438 UI/ml), caspasa-3 escindida (7,33 ± 0,76 ng/ml) y Bax (2,33 ± 0.,44 ng/ml). El tratamiento con naringina diminuyó estos efectos en el riñón (7,47 ± 60,35 pg/ml; 9299 ± 327 pg/ml; 2001 ± 78 pg/ml; 3112 ± 220 UI/ml; 3.38 ± 0.54 ng/ml; 2.33 ± 0,44 ng/ml, respectivamente) (p <0,05). Conclusión: En este estudio se demostró que el tratamiento con naringina atenuó el daño renal producido por isquemia-reperfusión en ratas.

The Effects of Lung Ischaemia/Reperfusion on TRPM Gene Expression

ABSTRACT Objective: Transient receptor potential melastatin (TRPM) are integral membrane proteins that have broad range of cellular functions. Roles of TRPM2, TRPM3, TRPM4 and TRPM7 among these channels are very important, and their roles in lung ischaemia/reperfusion injury have not been evaluated yet. The aim of this study is to investigate the contribution of these genes in lung ischaemia/reperfusion injury and evaluate histopathology of tissues. Methods: A total of 40 Wistar albino rats were enrolled for the study. Ischaemia was performed by the application of an atramvatic clamp to pulmonary artery. Gene expressions were determined by the semi-quantitative reverse transcription-polymerase chain reaction method. Histopatholical evaluations were held by a standard haematoxyline-eosin staining. Results: The major histopathological tissue damage was observed in ischaemia performed groups, and expression of TRPM channels was found to be obviously downregulated. Substantial changes were determined between TRPM2, TRPM3, TRPM4 and TRPM7 and lung ischaemia/reperfusion injury. In particular, expression of TRPM2 and TRPM7 was reversibly downregulated in ischaemia. Yet, the expression of TRPM3 and TRPM4 was irreversibly down-regulated after ischaemia. Conclusion: Consequently, these results indicate that TRPM family of cation channels may have significant roles in the lung ischaemia/reperfusion injury.

Chemical chaperones improve the functional recovery of stunned myocardium by attenuating the endoplasmic reticulum stress.

AIM: Myocardial ischaemia/reperfusion (I/R) produces structural and functional alterations depending on the duration of ischaemia. Brief ischaemia followed by reperfusion causes reversible contractile dysfunction (stunned heart) but long-lasting ischaemia followed by reperfusion can result in irreversible injury with cell death. Events during I/R can alter endoplasmic reticulum (ER) function leading to the accumulation of unfolded/misfolded proteins. The resulting ER stress induces activation of several signal transduction pathways, known as unfolded protein response (UPR). Experimental evidence shows that UPR contributes to cell death in irreversible I/R injury; however, there is still uncertainty for its occurrence in the stunned myocardium. This study investigated the ER stress response and its functional impact on the post-ischaemic cardiac performance of the stunned heart. METHODS: Perfused rat hearts were subjected to 20 minutes of ischaemia followed by 30 minutes of reperfusion. UPR markers were evaluated by qRT-PCR and western blot. Post-ischaemic mechanical recovery was measured in absence and presence of two chemical chaperones: tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyric acid (4-PBA). RESULTS: Analysis of mRNA and protein levels of various ER stress effectors demonstrated that different UPR signalling cascades, involving both pro-survival and pro-apoptotic pathways, are activated. Inhibition of the UPR with chemical chaperones improved the post-ischaemic recovery of cardiac mechanical function without affecting the I/R-induced increase in oxidative stress. CONCLUSION: Our results suggest that prevention of ER stress by chemical chaperones could be a therapeutic tool to limit deterioration of the contractile function in clinical settings in which the phenomenon of myocardial stunning is present.

Thioredoxin-1 is required for the cardioprotecive effect of sildenafil against ischaemia/reperfusion injury and mitochondrial dysfunction in mice.

Sildenafil is a phosphodiesterase type 5 inhibitor which confers cardioprotection against myocardial ischaemia/reperfusion (I/R) injury. The aim of this study was to determine if Trx1 participates in cardioprotection exerted by sildenafil in an acute model of I/R, and to evaluate mitochondrial bioenergetics and cellular redox status. Langendorff-perfused hearts from wild type (WT) mice and a dominant negative (DN-Trx1) mutant of Trx1 were assigned to placebo or sildenafil (0.7 mg/kg i.p.) and subjected to 30 min of ischaemia followed by 120 min of reperfusion. WT + S showed a significant reduction of infarct size (51.2 ± 3.0% vs. 30 ± 3.0%, p < .001), an effect not observed in DN-Trx. After I/R, sildenafil preserved state 3 oxygen consumption from WT, but had a milder effect in DN-Trx1 only partially protecting state 3 values. Treatment restored respiratory control (RC) after I/R, which resulted 8% (WT) and 24% (DN-Trx1) lower than in basal conditions. After I/R, a significant increase in H2O2 production was observed both for WT and DN-Trx (WT: 1.17 ± 0.13 nmol/mg protein and DN-Trx: 1.38 ± 0.12 nmol/min mg protein). With sildenafil, values were 21% lower only in WT I/R. Treatment decreased GSSG levels both in WT and DN-Trx1. In addition, GSSG/GSH2 ratio was partially restored by sildenafil. Also, an increase in p-eNOS/eNOS even before the myocardial ischaemia was observed with sildenafil, both in WT (14%, p > .05) and in DN-Trx (35%, p < .05). Active Trx1 is required for the onset of the cardioprotective effects of sildenafil on I/R injury, together with the preservation of cellular redox balance and mitochondrial function.

Acute administration of oestradiol or progesterone in a spinal cord ischaemia-reperfusion model in rats.

OBJECTIVES: Despite research into protective pharmacological adjuncts, paraplegia persists as a dreaded complication after thoracic and thoracoabdominal aortic interventions. Reports on gender-related neurological outcomes after ischaemic and traumatic brain injuries have led to increased interest in hormonal neuroprotective effects and have generated other studies seeking to prove the neuroprotective effects of the therapeutic administration of 17ß-oestradiol and of progesterone. We hypothesised that acute administration of oestradiol or progesterone would prevent or attenuate spinal cord ischaemic injury induced by occlusion of the descending thoracic aorta. METHODS: Male rats were divided into groups receiving 280 µg/kg of 17ß-oestradiol or 4 mg/kg of progesterone or vehicle 30 min before transitory endovascular occlusion of the proximal descending thoracic aorta for 12 min. Hindlimb motor function was assessed by a functional grading scale (that of Basso, Beattie and Bresnahan) for 14 days after reperfusion. On the 14th day, a segment of the thoracolumbar spinal cord was harvested and prepared for histological and immunohistochemical analyses. RESULTS: There was significant impairment of the motor function of the hindlimb in the 3 study groups, with partial improvement noticed over time, but no difference was detected between the groups. On Day 1 of assessment, the 17ß-oestradiol group had a functional score of 9.8 (0.0-16.5); the progesterone group, a score of 0.0 (0-17.1) and the control group, a score of 6.5 (0-16.9); on the 14th day, the 17ß-oestradiol group had a functional score of 18.0 (4.4-19.4); the progesterone group had a score of 7.5 (0-18.5) and the control group had a score of 17.0 (0-19.9). Analysis of the grey matter showed that the number of viable neurons per section was not different between the study groups on the 14th day. Immunostaining of the spinal cord grey matter was also similar among the 3 groups. CONCLUSIONS: Acute administration of oestradiol or of progesterone 30 min before transitory occlusion of the proximal descending thoracic aorta of male rats could not prevent or attenuate spinal cord ischaemic injury based on an analysis of functional and histological outcomes.

Histological, morphometric, protein and gene expression analyses of rat retinas with ischaemia-reperfusion injury model treated with sildenafil citrate.

The aim of this study was to better understand the role of apoptosis in a retinal ischaemia-reperfusion injury model and to determine whether sildenafil citrate treatment can prevent retinal cell apoptosis. Thirty-six rats were divided into a control group (n = 6) and two experimentally induced ischaemia-reperfusion groups (7 and 21 days; n = 15 per group). The induced ischaemia-reperfusion groups were treated with sildenafil for 7 and 21 days (n = 10 per group), and 10 animals were treated with a placebo for the same period (n = 5 per group). Paracentesis of the anterior chamber was performed with a 30-G needle attached to a saline solution (0.9%) bag positioned at a height of 150 cm above the eye for 60 min. Intraocular pressure was measured by rebound tonometer (TonoVet® ). The eyes were analysed by histology and morphometry, and by immunohistochemistry and qRT-PCR for expression of Caspase-7, Caspase-6, Caspase-9, Tnf-r2, Fas-l, Bcl-2 and Bax. Sildenafil-treated animals showed lower levels of histopathological changes (inflammatory, cellular and tissue) than their placebo-treated counterparts at both 7 and 21 days. The retinal ganglion cell layer (RGC) was preserved in the sildenafil groups (SG), with a cell count closer to control than in the placebo groups (PG). Caspase-7 expression was significantly higher in both treated groups at 7 days compared to controls. Gene expression levels in both treatment groups differed from the controls, but in SG Bax and Caspase-6 expression levels were similar to control animals. These results suggest that the main mechanism of retinal cell death in this model is apoptosis, as there is an increase in pro-apoptotic factors and decrease in the anti-apoptotic ones. Also, sildenafil seems to protect the retinal ganglion cell layer from apoptosis. Cell survival was evident in the histological and morphometric analyses, and sildenafil treatment had a protective effect in the apoptosis pathways, with gene expression levels in SG similar to the controls.

Angiotensin II-preconditioning is associated with increased PKCε/PKCδ ratio and prosurvival kinases in mitochondria.

Angiotensin II-preconditioning (APC) has been shown to reproduce the cardioprotective effects of ischaemic preconditioning (IPC), however, the molecular mechanisms mediating the effects of APC remain unknown. In this study, Langendorff-perfused rat hearts were subjected to IPC, APC or both (IPC/APC) followed by ischaemia-reperfusion (IR), to determine translocation of PKCε, PKCδ, Akt, Erk1/2, JNK, p38 MAPK and GSK-3ß to mitochondria as an indicator of activation of the protein kinases. In agreement with previous observations, IPC, APC and IPC/APC increased the recovery of left ventricular developed pressure (LVDP), reduced infarct size (IS) and lactate dehydrogenase (LDH) release, compared to controls. These effects were associated with increased mitochondrial PKCε/PKCδ ratio, Akt, Erk1/2, JNK, and inhibition of permeability transition pore (mPTP) opening. Chelerythrine, a pan-PKC inhibitor, abolished the enhancements of PKCε but increased PKCδ expression, and inhibited Akt, Erk1/2, and JNK protein levels. The drug had no effect on the APC- and IPC/APC-induced cardioprotection as previously reported, but enhanced the post-ischaemic LVDP in controls. Losartan, an angiotensin II type 1 receptor (AT1-R) blocker, abolished the APC-stimulated increase of LVDP and reduced PKCε, Akt, Erk1/2, JNK, and p38. Both drugs reduced ischaemic contracture and LDH release, and abolished the inhibition of mPTP by the preconditioning. Chelerythrine also prevented the reduction of IS by APC and IPC/APC. These results suggest that the cardioprotection induced by APC and IPC/APC involves an AT1-R-dependent translocation of PKCε and survival kinases to the mitochondria leading to mPTP inhibition. In chelerythrine-treated hearts, however, alternate mechanisms appear to maintain cardiac function.

Cardioprotection by the transfer of coronary effluent from ischaemic preconditioned rat hearts: identification of cardioprotective humoral factors.

Ischaemic preconditioning (IPC) provides myocardial resistance to ischaemia/reperfusion (I/R) injuries. The protection afforded by IPC is not limited to the target tissue but extends to remote tissues, suggesting a mechanism mediated by humoral factors. The aim of the present study was to identify the humoral factors that are responsible for the cardioprotection induced by the coronary effluent transferred from IPC to naïve hearts. Isolated rat hearts were submitted to IPC (three cycles of 5 min I/R) before 30-min global ischaemia and 60-min reperfusion. The coronary effluent (Efl_IPC) collected during IPC was fractionated by ultrafiltration in different molecular weight ranges (<3, 3-5, 5-10, 10-30, 30-50, and >50 kDa) and evaluated for cardioprotective effects by perfusion before I/R in naïve hearts. Only the <3, 5-10 and <10 kDa fractions of hydrophobic eluate reduced I/R injuries. The cardioprotective effect of the 5-10 fraction was blocked by KATP channel blockers and a PKC inhibitor. An Efl_IPC proteomic analysis revealed 14 cytoprotection-related proteins in 4-12 kDa peptides. HSP10 perfusion protected the heart against I/R injuries. These data provide insights into the mechanisms of cardioprotection in humoral factors released by IPC. Cardioprotection is afforded by hydrophobic peptides in the 4-12 kDa size range, which activate pathways that are dependent on PKC and KATP. Fourteen 4-12 kDa peptides were identified, suggesting a potential therapeutic role for these molecules in ischaemic diseases. One of these, HSP10, identified by mass spectrometry, reduced I/R injuries and may be a potential candidate as a therapeutic target.

Tourniquet-induced ischaemia-reperfusion injury: the comparison of antioxidative effects of small-dose propofol and ketamine / Lesão de isquemia-reperfusão induzida por torniquete: comparação dos efeitos antioxidantes de propofol e cetamina em doses baixas

Abstract Objectives: The aim of the present study was to investigate the preventive effects of propofol and ketamine as small dose sedation during spinal anaesthesia on tourniquet-induced ischaemia-reperfusion injury. Methods: 30 patients were randomly assigned into two groups of 15 patients. In the propofol group, sedation was performed with propofol 0.2 mg·kg-1 followed by infusion at a rate of 2 mg·kg-1·h-1. In the ketamine group, a continuous infusion of ketamine 0.5 mg·kg-1·h-1 was used until the end of surgery. Intravenous administration of midazolam was not used in any patients. Ramsay sedation scale was used for assessing the sedation level. Venous blood samples were obtained before propofol and ketamine infusion (T1), at 30 minutes (min) of tourniquet ischaemia (T2), and 5 min after tourniquet deflation (T3) for malondialdehyde (MDA) measurements. Results: No differences were noted between the groups in haemodynamic (p > 0.05) and demographic data (p > 0.05). There was no statistically significant difference between the two groups in terms of T1, T2 and T3 periods (p > 0.05). There was a statistically increase observed in MDA values respectively both in Group P and Group K between the reperfusion period (1.95 ± 0.59, 2.31 ± 0.48) and pre-ischaemia (1.41 ± 0.38, 1.54 ± 0.45), and ischaemia (1.76 ± 0.70, 1.71 ± 0.38) (µmoL-1) periods (p < 0.05). Conclusions: Small-dose propofol and ketamine has similar potential to reduce the oxidative stress caused by tourniquet-induced ischaemia-reperfusion injury in patients undergoing arthroscopic knee surgery under spinal anaesthesia.

Resumo Objetivos: O objetivo do presente estudo foi investigar os efeitos preventivos de propofol e cetamina em sedação com doses baixas durante a raquianestesia sobre lesão de isquemia-reperfusão induzida por torniquete. Métodos: 30 pacientes foram randomicamente alocados em dois grupos de 15 pacientes cada. No grupo propofol, a sedação foi feita com 0,2 mg.kg-1 de propofol seguida por infusão a uma taxa de 2 mg.kg-1.h-1. No grupo cetamina, uma infusão contínua de 0,5 mg.kg-1.h-1 de cetamina foi usada até o final da cirurgia. Midazolam intravenoso não foi administrado em nenhum dos pacientes. A Escala de Sedação de Ramsay (ESR) foi usada para avaliar o nível de sedação. Amostras de sangue venoso foram colhidas antes da administração de propofol e infusão de cetamina (T1), aos 30 minutos (min) de isquemia do torniquete (T2) e 5 min após a desinsuflação do torniquete (T3), para medir os valores de malondialdeído (MDA). Resultados: Não observamos diferenças entre os grupos em relação à hemodinâmica (p > 0,05) e dados demográficos (p > 0,05). Não houve diferença estatisticamente significativa entre os dois grupos nos períodos T1, T2 e T3 (p > 0,05). Um aumento estatisticamente significativo foi observado nos valores de MDA, respectivamente, no Grupo P e Grupo C entre os períodos de reperfusão (1,95 ± 0,59, 2,31 ± 0,48) e pré-isquemia (1,41 ± 0,38, 1,54 ± 0,45) e isquemia (1,76 ± 0,70, 1,71 ± 0,38) (µmoL-1) (p < 0,05). Conclusões: Propofol e cetamina em doses baixas apresentam potencial semelhante para reduzir o estresse oxidativo causado pela lesão de isquemia-reperfusão induzida por torniquete em pacientes submetidos à artroscopia de joelho sob raquianestesia.

[Tourniquet-induced ischaemia-reperfusion injury: the comparison of antioxidative effects of small-dose propofol and ketamine]. / Lesão de isquemia­reperfusão induzida por torniquete: comparação dos efeitos antioxidantes de propofol e cetamina em doses baixas.

OBJECTIVES: The aim of the present study was to investigate the preventive effects of propofol and ketamine as small dose sedation during spinal anesthesia on tourniquet-induced ischemia-reperfusion injury. METHODS: 30 patients were randomly assigned into two groups of 15 patients. In the propofol group, sedation was performed with propofol 0.2mg.kg-1 followed by infusion at a rate of 2mg.kg-1.h-1. In the ketamine group, a continuous infusion of ketamine 0.5mg.kg-1.h-1 was used until the end of surgery. Intravenous administration of midazolam was not used in any patients. Ramsay sedation scale was used for assessing the sedation level. Venous blood samples were obtained before propofol and ketamine infusion (T1), at 30minutes (min) of tourniquet ischemia (T2), and 5min after tourniquet deflation (T3) for malondialdehyde (MDA) measurements. RESULTS: No differences were noted between the groups in hemodynamic (p>0.05) and demographic data (p>0.05). There was no statistically significant difference between the two groups in terms of T1, T2 and T3 periods (p>0.05). There was a statistically increase observed in MDA values respectively both in Group P and Group K between the reperfusion period (1.95±0.59, 2.31±0.48) and pre-ischemia (1.41±0.38, 1.54±0.45), and ischemia (1.76±0.70, 1.71±0.38) (µmoL-1) periods (p<0.05). CONCLUSIONS: Small-dose propofol and ketamine has similar potential to reduce the oxidative stress caused by tourniquet-induced ischemia-reperfusion injury in patients undergoing arthroscopic knee surgery under spinal anesthesia.

Presence of arteriolar hyalinosis in post-reperfusion biopsies represents an additional risk to ischaemic injury in renal transplant.

AIM: The role of post-reperfusion biopsy findings as a predictor of early and long-term graft function and survival is still a target of research. METHODS: We analyzed data from 136 post-reperfusion biopsies performed in deceased donor renal transplanted patients from November 2008 to May 2012. We analyzed the presence of acute tubular necrosis (ATN), arteriolar hyalinosis (AH), intimal thickness (IT), interstitial fibrosis (IF) and glomerulosclerosis (GS). We also analyzed the impact of donor features on the following outcomes: delayed graft function (DGF) and chronic allograft dysfunction defined as eGFR < 60 mL/min at 1 year. RESULTS: The mean donor age was 41 years, 26% of whom were extended criteria donors (ECD), 33% had hypertension and 50% had cerebral vascular accident (CVA) as the cause of death. ATN was present in 87% of these biopsies, AH in 31%, IF in 21%, IT in 27% and GS in 32%. DGF occurred in 80% and chronic allograft dysfunction was present in 53%. AH was the only histological finding associated with DGF and chronic allograft dysfunction at 1 year. Patients with AH had a lower eGFR at 1 year than patients without it (49.8 mL/min × 64.5 mL/min, P = 0.02). In the multivariate analysis, risk variables for development of chronic graft dysfunction were male sex (odds ratio [OR] = 3.159 [CI: 1.22-8.16]; P = 0.018), acute rejection (OR = 8.91 [CI: 2.21-35.92]; P = 0.002), donor hypertension (OR = 2.94 [CI: 1.10-7.84]; P = 0.031), AH (OR = 3.96 [CI: 1.46-10.70]; P = 0.007) and eGFR at discharge (OR = 0.96 [CI: 0.93-0.98]; P = 0.005). In multivariate analysis, risk factors for AH were donor age ≥ 50 years (OR = 2.46 [CI: 1.10-5.44]; P = 0.027) and CVA as the cause of donor death (OR = 2.33 [CI: 1.05-5.15]; P = 0.007). CONCLUSION: The presence of AH in post-reperfusion biopsies is a marker of ageing and vascular disease and was associated with DGF and a one year poorer renal function. AH in donor biopsies superimposed to long ischaemic time is a predictor of renal function. The management of immunosuppression based on the presence of AH in post-reperfusion biopsy could be useful to improve long term graft function.

Overnourishment during lactation induces metabolic and haemodynamic heart impairment during adulthood.

AIM: In this study, the effects of postnatal overfeeding on heart energy homoeostasis and cardiac haemodynamics in adult male Swiss mice were examined. METHODS AND RESULTS: During the suckling period, the mice were divided into four groups of control or overfed pups in combination with baseline or ischaemia/reperfusion treatments (control group baseline, CGBL; overfed group baseline, OGBL; control group ischaemia/reperfusion, CGIR; and overfed group ischaemia/reperfusion, OGIR). End diastolic pressure (EDP), heart contraction speed (Max dP/dt), relaxation speed (Min dP/dt), isovolumetric relaxation time (Tau) and frequency by beats per minute (BPM) were measured. During baseline and ischaemia/reperfusion, key proteins such as AKT1, AKT2, AKT3, pAKT, adenosine monophosphate-activated protein kinase (AMPK), pAMPK, insulin receptor beta (IRß), protein tyrosine phosphatase 1B (PTP1B), insulin receptor substrate 1 (IRS1), fatty acid binding protein (FABP), CD36, phosphoinositide 3-kinase (PI3K) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were studied. The expression of atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), carnitine palmitoyltransferase 1 (CPT1) and uncoupling protein 3 (UCP3) was studied as a marker of cardiac hypertrophy and energetic metabolism. Cardiac fibrosis was analyzed by quantifying collagen deposition, which is increased in the OGBL and OGIR groups compared with the control groups. CONCLUSIONS: The OGBL group showed reduced EDP compared with the CGBL group and high Max dP/dt compared with the OGBL group. Ischaemia/reperfusion increased EDP and Min dP/dt in the intragroup comparison. By contrast, Tau and frequency were not significantly different among groups. The OGIR mice showed significant alterations in heart metabolism proteins, including AKT2, pAKT/AKT1, pAKT/AKT2, AMPK, pAMPK/AMPK, PTP1B, IRS1, FABP and CD36. Furthermore, alterations in ANP, BNP, CPT1 and UCP3 messenger RNA (mRNA) expression indicated hypertrophy and reduction in their efficiency, such that exclusive overnutrition in childhood induces a long-term effect on haemodynamics, metabolism and heart remodelling.

Effects of 3-methyladenine on isolated left atria subjected to simulated ischaemia-reperfusion.

Although autophagy is a prominent feature of myocardial ischaemia and reperfusion, its functional significance is unclear and controversial. In order to gain a deeper insight into the role of autophagy in myocardial ischaemia-reperfusion, we explored the effects of the pharmacological inhibitor of autophagy 3-methyladenine (3-MA). Isolated rat atria subjected to simulated 75-min ischaemia/75-min reperfusion (Is-Rs) in the presence or absence of 3-MA were used. The LC3-II/LC3-I ratio, an indicator of autophagosome formation, did not increase after ischaemia either in the presence or absence of 3-MA, but there was significant enhancement during reperfusion, which was prevented by the presence of 3-MA. The autophagy inhibitor also increased p62 protein, one of the specific substrates degraded through the autophagy-lysosomal pathway. Electron micrographs showed double membrane autophagosome-like structures during reperfusion, which were absent in atria subjected to Is-Rs in the presence of 3-MA. These findings suggest that this agent inhibited the autophagic flux under the present experimental conditions. Inhibition of autophagy during Is-Rs was accompanied by a high incidence of tachyarrhythmias during reperfusion, and a decrease in the maximal inotropic response to ß-adrenergic and to calcium stimulation at the end of Is-Rs. Deterioration of mitochondrial morphology and function, without affecting cell viability, was observed in atria subjected to Is-Rs in the presence of 3-MA. The present results suggest an association between the inhibition of autophagy and functional alterations of the cells that have undergone sublethal stress, and have been able to recover in this experimental model of ischaemia-reperfusion.

Hypotonic swelling promotes nitric oxide release in cardiac ventricular myocytes: impact on swelling-induced negative inotropic effect.

AIMS: Cardiomyocyte swelling occurs in multiple pathological situations and has been associated with contractile dysfunction, cell death, and enhanced propensity to arrhythmias. We investigate whether hypotonic swelling promotes nitric oxide (NO) release in cardiomyocytes, and whether it impacts on swelling-induced contractile dysfunction. METHODS AND RESULTS: Superfusing rat cardiomyocytes with a hypotonic solution (HS; 217 mOsm), increased cell volume, reduced myocyte contraction and Ca(2+) transient, and increased NO-sensitive 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) fluorescence. When cells were exposed to HS + 2.5 mM of the NO synthase inhibitor l-NAME, cell swelling occurred in the absence of NO release. Swelling-induced NO release was also prevented by the nitric oxide synthase 1 (NOS1) inhibitor, nitroguanidine, and significantly reduced in NOS1 knockout mice. Additionally, colchicine (inhibitor of microtubule polymerization) prevented the increase in DAF-FM fluorescence induced by HS, indicating that microtubule integrity is necessary for swelling-induced NO release. The swelling-induced negative inotropic effect was exacerbated in the presence of either l-NAME, nitroguandine, the guanylate cyclase inhibitor, ODQ, or the PKG inhibitor, KT5823, suggesting that NOS1-derived NO provides contractile support via a cGMP/PKG-dependent mechanism. Indeed, ODQ reduced Ca(2+) wave velocity and both ODQ and KT5823 reduced the HS-induced increment in ryanodine receptor (RyR2, Ser2808) phosphorylation, suggesting that in this context, cGMP/PKG may contribute to preserve contractile function by enhancing sarcoplasmic reticulum Ca(2+) release. CONCLUSIONS: Our findings suggest a novel mechanism for NO release in cardiomyocytes with putative pathophysiological relevance determined, at least in part, by its capability to reduce the extent of contractile dysfunction associated with hypotonic swelling.

N-acetylcysteine administration confers lung protection in different phases of lung ischaemia-reperfusion injury.

OBJECTIVES: To verify the effects of N-acetylcysteine (NAC) administered before and after ischaemia in an animal model of lung ischaemia-reperfusion (IR) injury. METHODS: Twenty-four Wistar rats were subjected to an experimental model of selective left pulmonary hilar clamping for 45 min followed by 2 h of reperfusion. The animals were divided into four groups: control group (SHAM), ischaemia-reperfusion, N-acetylcysteine-preischaemia (NAC-Pre) and NAC-postischaemia (NAC-Post). We recorded the haemodynamic parameters, blood gas analysis and histology. We measured the thiobarbituric acid reactive substances concentration; the expression of superoxide dismutase (SOD), inducible nitric oxide synthase (iNOS), nitrotyrosine, cleaved caspase 3, nuclear factor κB (NF-κB), NF-kappa-B inhibitor alpha (IκB-α), tumour necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß); myeloperoxidase activity (MPO). RESULTS: No significant differences were observed in the haemodynamic parameters, blood gas analysis and SOD activity among the groups. Lipid peroxidation was significantly higher in the IR and NAC-Pre groups (P < 0.01). The expression of nitrotyrosine, cleaved caspase 3, NF-κB, IκB-α, TNF-α and IL-1ß were significantly higher in the IR group when compared with the SHAM and NAC groups (P < 0.01). The NAC-Pre group showed a significantly higher expression of these proteins when compared with the SHAM and NAC-Post groups (P < 0.05). After reperfusion, the expression of iNOS increased almost uniformly in all groups when compared with the SHAM group (P < 0.01). The histological analysis showed fewer inflammatory cells in the NAC groups. CONCLUSIONS: The intravenous administration of NAC demonstrated protective properties against lung IR injury. The use of NAC immediately after reperfusion potentiates its protective effects.