Olivér Rosero

Attenuation of Skeletal Muscle and Renal Injury to the Lower Limb following Ischemia-Reperfusion Using mPTP Inhibitor NIM-811

Introduction Operation on the infrarenal aorta and large arteries of the lower extremities may cause rhabdomyolysis of the skeletal muscle, which in turn may induce remote kidney injury. NIM-811 (N-metyl-4-isoleucine-cyclosporine) is a mitochondria specific drug, which can prevent ischemic-reperfusion (IR) injury, by inhibiting mitochondrial permeability transition pores (mPTP). Objectives Our aim was to reduce damages in the skeletal muscle and the kidney after IR of the lower limb with NIM-811. Materials and methods Wistar rats underwent 180 minutes of bilateral lower limb ischemia and 240 minutes of reperfusion. Four animal groups were formed called Sham (receiving vehicle and sham surgery), NIM-Sham (receiving NIM-811 and sham surgery), IR (receiving vehicle and surgery), and NIM-IR (receiving NIM-811 and surgery). Serum, urine and histological samples were taken at the end of reperfusion. NADH-tetrazolium staining, muscle Wet/Dry (W/D) ratio calculations, laser Doppler-flowmetry (LDF) and mean arterial pressure (MAP) monitoring were performed. Renal peroxynitrite concentration, serum TNF-α and IL-6 levels were measured. Results Less significant histopathological changes were observable in the NIM-IR group as compared with the IR group. Serum K+ and necroenzyme levels were significantly lower in the NIM-IR group than in the IR group (LDH: p<0.001; CK: p<0.001; K+: p = 0.017). Muscle mitochondrial viability proved to be significantly higher (p = 0.001) and renal function parameters were significantly better (creatinine: p = 0.016; FENa: p<0.001) in the NIM-IR group in comparison to the IR group. Serum TNF-α and IL-6 levels were significantly lower (TNF-α: p = 0.003, IL-6: p = 0.040) as well as W/D ratio and peroxynitrite concentration were significantly lower (p = 0.014; p<0.001) in the NIM-IR group than in the IR group. Conclusion NIM-811 could have the potential of reducing rhabdomyolysis and impairment of the kidney after lower limb IR injury.

Adenosine and inosine exert cytoprotective effects in an in vitro model of liver ischemia-reperfusion injury

Liver ischemia represents a common clinical problem. In the present study, using an in vitro model of hepatic ischemia-reperfusion injury, we evaluated the potential cytoprotective effect of the purine metabolites, such as adenosine and inosine, and studied the mode of their pharmacological actions. The human hepatocellular carcinoma-derived cell line HepG2 was subjected to combined oxygen-glucose deprivation (COGD; 0-14-24 h), followed by re-oxygenation (0-4-24 h). Adenosine or inosine (300–1,000 μM) were applied in pretreatment. Cell viability and cytotoxicity were measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and lactate dehydrogenase methods, respectively. The results showed that both adenosine and inosine exerted cytoprotective effects, and these effects were not related to receptor-mediated actions, since they were not prevented by selective adenosine receptor antagonists. On the other hand, the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA, 10 μM) markedly and almost fully reversed the protective effect of adenosine during COGD, while it did not influence the cytoprotective effect of inosine in the same assay conditions. These results suggest that the cytoprotective effects are related to intracellular actions, and, in the case of adenosine also involve intracellular conversion to inosine. The likely interpretation of these findings is that inosine serves as an alternative source of energy to produce ATP during hypoxic conditions. The protective effects are also partially dependent on adenosine kinase, as the inhibitor 4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido[2,3-d]pyrimidine, 2HCl (ABT 702, 30 μM) significantly reversed the protective effect of both adenosine and inosine during hypoxia and re-oxygenation. Collectively, the current results support the view that during hypoxia, adenosine and inosine exert cytoprotective effects via receptor-independent, intracellular modes of action, which, in part, depend on the restoration of cellular bioenergetics. The present study supports the view that testing of inosine for protection against various forms of warm and cold liver ischemia is relevant.

Levosimendan: a cardiovascular drug to prevent liver ischemia-reperfusion injury?

Introduction Temporary occlusion of the hepatoduodenal ligament leads to an ischemic-reperfusion (IR) injury in the liver. Levosimendan is a new positive inotropic drug, which induces preconditioning-like adaptive mechanisms due to opening of mitochondrial KATP channels. The aim of this study was to examine possible protective effects of levosimendan in a rat model of hepatic IR injury. Material and Methods Levosimendan was administered to male Wistar rats 1 hour (early pretreatment) or 24 hours (late pretreatment) before induction of 60-minute segmental liver ischemia. Microcirculation of the liver was monitored by laser Doppler flowmeter. After 24 hours of reperfusion, liver and blood samples were taken for histology, immuno- and enzyme-histochemistry (TUNEL; PARP; NADH-TR) as well as for laboratory tests. Furthermore, liver antioxidant status was assessed and HSP72 expression was measured. Results In both groups pretreated with levosimendan, significantly better hepatic microcirculation was observed compared to respective IR control groups. Similarly, histological damage was also reduced after levosimendan administration. This observation was supported by significantly lower activities of serum ALT (pearly = 0.02; plate = 0.005), AST (pearly = 0.02; plate = 0.004) and less DNA damage by TUNEL test (pearly = 0.05; plate = 0.034) and PAR positivity (pearly = 0.02; plate = 0.04). Levosimendan pretreatment resulted in significant improvement of liver redox homeostasis. Further, significantly better mitochondrial function was detected in animals receiving late pretreatment. Finally, HSP72 expression was increased by IR injury, but it was not affected by levosimendan pretreatment. Conclusion Levosimendan pretreatment can be hepatoprotective and it could be useful before extensive liver resection.

Impaired Intestinal Mucosal Barrier upon Ischemia-Reperfusion: “Patching Holes in the Shield with a Simple Surgical Method”

Mesenteric ischemia-reperfusion (IR) is associated with impairment of the gut barrier function and the initiation of a proinflammatory cascade with life-threatening results. Therefore methods directed to ameliorate IR injury are of great importance. We aimed at describing the effects of postconditioning (PC) on the alterations of the intestinal mucosal function and the inflammatory response upon mesenteric IR. Methods. Male Wistar rats were gavaged with green fluorescent protein-expressing E. coli suspensions. Animals were randomized into three groups (n = 15), sham-operated, IR-, and PC-groups, and underwent 60 minutes of superior mesenteric artery occlusion, followed by 6 hours of reperfusion. Postconditioning was performed at the onset of reperfusion. Blood and tissue samples were taken at the end of reperfusion, for histological, bacteriological, and plasma examinations. Results. The PC-group presented a more favorable claudin-2, claudin-3, claudin-4, and zonula occludens-1 membrane expression profile, and significantly lower rates of bacterial translocation to distant organs and plasma D-lactate levels compared to the IR-group. Histopathological lesions, plasma I-FABP, IL-6, and TNF-α levels were significantly lower in the PC-group compared to the IR-group. Conclusion. The use of postconditioning improved the integrity of the intestinal mucosal barrier upon mesenteric IR, and thus reduced the incidence of bacterial translocation and development of a systemic inflammatory response.

Therapeutic option for managing lung injury induced by infrarenal aortic cross-clamping

BACKGROUND Operations on the infrarenal aorta can cause ischemic-reperfusion (IR) injury in local tissues, which could result in remote organ (e.g., lung) damage. Treatment of such injuries remains an unresolved problem. OBJECTIVES Our aim was to reduce remote lung damage after lower limb IR by means of postconditioning. MATERIALS AND METHODS Male Wistar rats were divided into three groups: Sham-operated, IR, and Postconditioned (PostC). In the latter two groups rats underwent 180 min of exclusion of the infrarenal aorta. The reperfusion time was 4 h. Serum-free radical levels, tumor necrosis factor-α and interleukin-6 concentrations, histologic changes in the lung, wet/dry-ratio, myeloperoxidase activity, heat shock protein 72 level and blood gas changes were investigated. RESULTS Postconditioning reduced histological damage in the lung (P < 0.05). Free radical levels and tumor necrosis factor-α concentrations were significantly lower in the PostC group than in the IR group (P < 0.05 and P < 0.01, respectively). Interleukin-6 concentrations did not significantly differ in the PostC group. Compared with the IR group, lung myeloperoxidase activity was lower in the PostC group. Decreased pulmonary heat shock protein 72 level was observed in the PostC group compared with the IR group and the wet/dry-ratio was also significantly lower in the PostC group (P < 0.05). A noticeably higher arterial pO2 level was manifest in the PostC group after 2 and 4 h of reperfusion (P < 0.05). CONCLUSIONS Postconditioning reduced lung damage under experimental conditions, in the early period of reperfusion after lower limb IR injury.

Improvement of small intestinal microcirculation by postconditioning after lower limb ischemia

BACKGROUND Major lower limb vascular surgeries may result in severe, remote injury of the gastrointestinal system, which has high mortality rates. Postconditioning is a technique with potential capability of reducing remote gastrointestinal complications. Our aim was to assess the remote macro- and micro-hemodynamic changes of the small intestine following an infrarenal aortic occlusion and to evaluate the effects of postconditioning on these alterations. METHODS Rats underwent 3h of infrarenal aortic occlusion followed by 4h of reperfusion. In one group, postconditioning was applied. Blood pressure, superior mesenteric artery flow and mucosal microcirculation of the duodenum, jejunum and ileum were assessed. Samples were taken from each intestinal segment for histological examinations. RESULTS Superior mesenteric artery flow, as well as microcirculation of the duodenum, jejunum and ileum showed significant impairment in the IR group, while histological damage was significantly worsened. Postconditioning was able to limit flow reduction in all three small bowel segments and in the superior mesenteric artery, and was able to significantly reduce histological damage. Strong negative correlation was found between microcirculatory values and histological damage. CONCLUSIONS Microcirculatory impairment might be responsible for remote intestinal injury following infrarenal aortic occlusion. Postconditioning was able to reduce this remote intestinal damage.

Collateral circulation of the rat lower limb and its significance in ischemia–reperfusion studies

AbstractPurpose Rats are the most commonly used animal model for studies of acute lower limb ischemia–reperfusion. The ischemia induced by arterial clamping may cause milder damage than the application of a tourniquet if the presence of a possible collateral system is considered.MethodsMale Wistar rats were randomized into three groups: in group A, the muscle weight affected by ischemia was measured; in group B, the severity of muscle damage caused by the application of a tourniquet and by infrarenal aortic occlusion was examined. Blood and muscle samples were taken from group B to assess the serum necroenzyme, potassium and TNF-α levels, as well as the muscle fiber viability and for histological examinations. In group C, the identification of the lower limb collateral system was performed using corrosion casting.ResultsTourniquet application affected the lower muscle mass and resulted in significantly more severe injury compared to infrarenal aortic occlusion. This difference was reflected in the serum necroenzyme, potassium and TNF-α levels. The histological examination and viability assay confirmed these findings. The corrosion casts showed several anastomoses capable of supplying the lower limb.ConclusionTourniquet application proved to be capable of inducing absolute lower limb ischemia, in contrast to infrarenal aortic ligation, where a rich collateral system is considered to help mitigate the injury.

Postconditioning: “Toll-erating” mesenteric ischemia-reperfusion injury?

Background. Postconditioning may prove to be a suitable method to decrease ischemia‐reperfusion injury of intestine after mesenteric arterial occlusion. Toll‐like‐receptor‐4 is involved in the pathophysiology of organ damage after ischemia‐reperfusion; therefore, the aim of our study was to investigate the effect of postconditioning on the mucosal expression of toll‐like‐receptor‐4. Methods. Male Wistar rats (n = 10/group) underwent 60 minutes of superior mesenteric artery occlusion followed by 6 hours of reperfusion in 3 groups: sham‐operated, ischemia‐reperfusion, and a postconditioned group. Postconditioning was performed by 6 alternating cycles of 10 seconds of reperfusion/reocclusion. Blood and tissue samples were collected at the end of reperfusion. Intestinal histopathologic changes and immunohistochemical expression of mucosal caspase‐3, antioxidant status, and protein levels of high‐mobility group box‐1 and toll‐like‐receptor‐4 were assessed. Immunofluorescent labeling and confocal microscopic analysis of toll‐like‐receptor‐4 were performed. Mucosal and serum levels of interleukin‐6 and tumor necrosis factor‐&agr; protein were measured. Results. Histologic alterations in the postconditioned group were associated with decreased caspase‐3 positivity, less toll‐like‐receptor‐4 mRNA, and less protein expression of high‐mobility group box‐1 and toll‐like‐receptor‐4 in the intestinal villi compared with the ischemia‐reperfusion group. Furthermore, a significantly improved antioxidant state of the intestinal mucosa and less mucosal and serum protein levels of interleukin‐6 and tumor necrosis factor‐&agr; were detected in the postconditioned group. Conclusion. Small intestinal ischemia‐reperfusion injury in male Wistar rats caused by the occlusion of the superior mesenteric artery was ameliorated by the use of postconditioning, showing a more favorable inflammatory response, which may be attributed to the decreased mucosal expression of toll‐like‐receptor‐4.

[Bacterial translocation: gap in the shield].

The gastrointestinal tract is not only regarded as a system where nutrient absorption takes place, but also as a vital barrier against intraluminal pathogens entering the circulation and the maintenance of immune homeostasis. Bacterial translocation is defined as the penetration of viable bacteria or bacterial compounds from the gastrointestinal tract to extraintestinal sites. This disorder has been described in several clinical conditions. The main promoting factors for bacterial translocation have been proposed to be changes in the intestinal microflora, mucosal barrier failure and defects in host immunity. The presence of bacterial translocation has been associated with higher complications and mortality rates; therefore it should be taken into account in the therapeutic strategies of patients with predisposing factors.

Acute mesenteric ischemia: Do biomarkers contribute to diagnosis?

Az akut mesenterialis ischaemia azonnali beavatkozást igénylő sürgősségi állapot. A sebészet és intenzív terápia területén az utóbbi években bekövetkezett komoly fejlődés ellenére a kórkép halálozása továbbra is kiemelkedően magas. A klinikai kép sokszínűsége és a nem specifi kus laboratóriumi eltérések gyakran késleltetik a diagnózist, ami az ischaemiás károsodás progresszióját és a beteg túlélési esélyeinek csökkenését vonja maga után. Mindezek miatt egyre nagyobb az igény olyan, a mesenterialis ischaemiát korán és megbízhatóan jelző szerológiai markerre, amely képes lerövidíteni a diagnosztikus folyamatot. A szerzők áttekintik a mesenterialis ischaemia korai diagnózisában használt tradicionális, valamint a jelenleg még kísérleti stádiumban lévő új típusú biomarkereket. Orv. Hetil., 2014, 155(41), 1615–1623.