Postconditioning of the small intestine: which is the most effective algorithm in a rat model?

BACKGROUND: Mesenteric ischemia is a serious clinical condition requiring immediate surgical intervention. The unavoidable ischemic-reperfusion (IR) injury may be ameliorated using the appropriate postconditioning protocol. The aim of the present study was to investigate the optimal postconditioning algorithm in a rat model of intestinal ischemic-reperfusion injury. MATERIALS AND METHODS: Male Wistar rats were randomized into five groups (n = 10), one sham-operated, one IR, and three postconditioned groups, each with different protocols. The animals were subjected to 60 min of mesenteric ischemia, followed by 60 min of reperfusion. Postconditioning was applied at the onset of reperfusion using three different algorithms. Arterial pressure and mucosal microcirculation were monitored throughout the experiment. Mesenteric pH was determined at the early phase of reperfusion. Blood and tissue samples were taken at the end of reperfusion for histologic evaluation, serum lactate dehydrogenase, serum creatine kinase, serum tumor necrosis factor-α, serum interleukin-6, detailed mucosal antioxidant, and scavenger capacity assays. RESULTS: The shorter and intermediate length cycles of postconditioning enhanced mucosal microcirculation and redox state and significantly delayed the normalization of mesenteric pH. Furthermore, milder histopathologic lesions and lower concentrations of serum necroenzymes and proinflammatory cytokines were detected compared with the IR group. The protective effect of postconditioning using longer cycles could only be seen in a tendentious manner. CONCLUSIONS: In a rat model of intestinal ischemia-reperfusion, the shorter and intermediate length cycles of postconditioning proved to be more effective than the use of longer cycles.

Postconditioning of the lower limb--protection against the reperfusion syndrome.

BACKGROUND: Postconditioning-alternating brief cycles of reperfusion/reocclusion applied at the beginning of revascularization-is a potent therapeutic technique, attenuating ischemia-reperfusion injury. Vascular surgery on the lower limb with ischemia-reperfusion injury may give rise to serious systemic complications [organ dysfunction syndrome (MODS), systemic inflammatory response syndrome (SIRS)], a phenomenon called reperfusion-syndrome. MATERIAL AND METHODS: We studied the effects of postconditioning on reperfusion-syndrome in a rodent experimental model. Wistar rats underwent 180 min of bilateral lower limb ischemia using an infrarenal crossclamping of the abdominal aorta. Postconditioning consisted of six cycles of 10-s aortic occlusion/10-s declamping at the beginning of reperfusion. Microcirculation of the lower limb was detected with laser Doppler flowmeter. After 4 h of reperfusion, plasma, urine, and histologic samples were collected. RESULTS: One hundred eighty-minute ischemia resulted in significant hemodynamic changes after reperfusion. Postconditioning affected the character of the microcirculatory flow, the limb circulation stabilized with hyperemia during reperfusion. Postconditioning caused a significant reduction in systemic inflammatory response (TNF-α, oxygen-derived free radicals). The laboratory and histologic samples implied a significant decrease in distant organ (lung and renal) dysfunctions after postconditioning. CONCLUSION: Postconditioning proves to be capable of conferring protection against different organ injuries caused by longer circulatory occlusions during elective major vascular operations.

Reduction of liver ischemia-reperfusion injury via glutamine pretreatment.

BACKGROUND: Surgical methods that reduce bleeding during major hepatic resections lead to warm ischemia-reperfusion (I-R) injury of the liver. This is well known to have a considerable impact on the postoperative outcome. Much research work has been done to develop possible protective techniques. We aimed to investigate the effectivity of L-alanyl-L-glutamine dipeptide pretreatment in an animal model of hepatic I-R injury. MATERIALS AND METHODS: Male Wistar rats underwent normothermic, 60 min segmental liver ischemia followed by 24 h of reperfusion. The animals (n=30) were divided into three experimental groups: sham operated, I-R, and glutamine (Gln) pretreated. Twenty-four h prior to I-R injury, rats in the Gln group received 500 mg/kg Dipeptiven infusion as glutamine pretreatment. Hepatic microcirculation during the first hour of reperfusion was monitored by noninvasive laser Doppler flowmeter. After a 24-h reperfusion period, liver tissue was analyzed by histologic and immunohistochemical assessments. Serum necroenzyme and antioxidant levels were measured. RESULTS: In the Gln group, the integral of the reperfusion curve (RA) and the plateau maximum (PM(10)) of the flow graph showed improving tendency (RA: P=0.096; PM(10): P=0.084). Severity of histologic damage was reduced. Serum necroenzymes (ALT: P=0.042, AST: P=0.044) were significantly lower. Chemiluminescent intensity of liver and plasma was significantly decreased (P=0.0003 and P=0.0496). Further spectrophotometric analysis of liver homogenate samples also showed significant improvement of the redox homeostasis. CONCLUSIONS: Our results suggest that L-alanyl-L-glutamine dipeptide pretreatment given 24 h prior to I-R injury could be an effective method to reduce liver damage caused by hepatic inflow occlusion.

[Investigation of postconditioning in intestinal ischemia-reperfusion experimental models]. / Posztkondicionálás kísérletes vizsgálata vékonybél ischaemiás-reperfusiós modelljében.

INTRODUCTION: The ischemic-reperfusion injury of the intestine, which occurs as a consequence of circulatory redistribution or occlusion of the superior mesenteric artery, is associated with high mortality rates. Postconditioning may reduce ischemic-reperfusion damage in such cases. Effects of this new surgical method were investigated in rats. METHODS: Male Wistar rats underwent 60 minutes of superior mesenteric artery occlusion in four groups: sham-operated, control and two postconditioned groups with different algorithms. Postconditioning was performed immediately at the beginning of reperfusion, by repetitive cycles of reperfusion and reocclusion. 3 cycles of 1 minute and 6 cycles of 10 seconds were applied according to groups. Intestinal microcirculation was followed by laser Doppler flowmetry. Blood and tissue samples were taken after 60 minutes of reperfusion. Histological analayses of the small intestine, measurement of serum necroenzyme levels and IL-6, mesenterial venous blood gas analyses were preformed and antioxidant state of the mucosa was investigated. RESULTS: The microcirculation during the reperfusion showed significant improvement in both postconditioned groups. Histological damage, necroenzyme and IL-6 levels were significantly reduced, while antioxidant state was improved in the postconditioned groups. CONCLUSION: Postconditioning was capable of increasing the guts chance to survive ischemic-reperfusion injury caused by superior mesenteric artery occlusion.

[Long ischemic period of the lower limb--study of skeletal muscle viability in experimental animal models]. / Hosszú ideju végtagi veroér-elzáródás és izomszövet-életképesség vizsgálata kísérletes állatmodellben.

INTRODUCTION: Surgical treatment for acute limb ischemia is revascularization or - when the limb is in a critical stage - amputation. Correct staging of the disease is relatively difficult, therefore complication and mortality rates are extremely high. Our aim was to invesitigate acute critical ischemia in rats and to test postconditioning on this model. METHODS: Experiment I: male Wistar rats underwent 4, 6, 8 hours of bilateral lower limb ischemia without reperfusion. Experiment II: suspected critical ischemia was followed by 2 hours of reperfusion with or without postconditioning. Histological samples were collected for routine staining and nitroblue-tetrazolium (NBT) enzyme-histochemistry. In Experiment II the microcirculatory changes were measured by laser Doppler flowmetry and blood samples were collected for laboratory testing (kreatin-kinase, CK). RESULTS: Experiment I: After an eight-hour-obstruction, severe ischemic lesions were detectable, with rutine and NBT stainings, therefore 8 hours of ischemia was chosen for further testing. Experiment II: The CK levels showed significant (p < 0.05) drop, quantitative evaluation of enzyme-histochemisty resulted in significantly (p < 0.001) less viability depletion and microcirculation showed significant (p < 0.05) amelioration of the reperfusion parameters in the postconditioned group compared to the control. CONCLUSIONS: Eight hours of lower limb ischemia is a suitable model to investigate acute critical ischemia in rats. Postconditioning could be a feasible technique to reduce IR injury associated with acute lower limb ischemia.

[Effect of postconditioning in major vascular operations on rats]. / Posztkondicionálás kísérletes alkalmazása aortakirekesztés kapcsán.

BACKGROUND: Postconditioning - using alternating brief cycles of reperfusion/reocclusion applied just at the very beginning of reperfusion - has recently been described as a potent therapeutic technique, attenuating ischaemia-reperfusion injury. In vascular surgery, certain elective interventions involve cross-clamping of major arteries, resulting in temporary ischaemia in large peripheral organs, which thus suffer ischaemia-reperfusion injury. Patients undergoing these operations may develop also serious systemic complications such as multiple distant organ dysfunctions, SIRS, detrimental redistribution of the circulation or even shock, a phenomenon called reperfusion-syndrome. We studied the effects of postconditioning on reperfusion-syndrome in a rodent experimental model. MATERIAL AND METHODS: Anaesthetized male Wistar rats underwent 180 minutes of bilateral lower limb ischaemia and 4 hours of reperfusion using an infrarenal cross-clamping of the abdominal aorta. Control animals underwent no additional intervention. Postconditioning consisted of 6 cycles of 10-second aortic occlusion/10-second declamping starting at the beginning of reperfusion. Haemodynamic parameters were observed with invasive arterial manometer, microcirculation of the lower limb was detected with laser-Doppler-flowmeter. After 4 hours of reperfusion serum, urine, and histological samples were collected. RESULTS: 180-minute ischaemia resulted in significant haemodynamic changes after reperfusion. Postconditioning affected the character of the microcirculatory flow curves, the limb circulation stabilized with hyperaemia after reperfusion. Postconditioning caused a significant reduction in systemic inflammatory response (TNF-alpha, oxygen-derived free radicals). The laboratory and histological samples implied a significant decrease in remote organ (lung and renal) dysfunctions after postconditioning. CONCLUSION: Postconditioning proves to be capable in conferring protection against different organ injuries caused by longer circulatory occlusions during elective major vascular surgeries.

Short-term alanyl-glutamine dipeptide pretreatment in liver ischemia-reperfusion model: effects on microcirculation and antioxidant status in rats.

BACKGROUND & AIMS: Ischemia-reperfusion (I-R) injury is responsible for the morbidity associated with liver surgery. Production of toxic free radicals influences the microcirculation. The aim of our study was to examine the effect of glutamine (Gln) supplementation--adminstered in alanyl-glutamine dipeptide form--on liver function, immuno/histopathology and the oxidative state of the liver after injury. METHODS: Two-hundred and fifty grams male Wistar rats underwent normothermic, 60 min, segmental liver ischemia followed by 6 h of reperfusion. The animals (n = 45) were divided into three groups: sham operated, I-R and parenteral Gln pretreatment. Hepatic microcirculation was monitored by laser Doppler flowmetry. At the 6 h of reperfusion, histological alterations, TUNEL reaction, active caspase-3 reaction, serum and liver tissue antioxidant levels, serum ALAT, ASAT and TNF-alpha levels were measured. RESULTS: Upon reperfusion, the Gln group had significantly (p<0.05) higher flow rates than the I-R group and, at the end of the 6h of reperfusion, significantly (p<0.05) lower serum ALAT and ASAT levels. The liver chemiluminescent intensity was lower, free SH-groups were elevated, while the reducing power was decreased in the Gln-pretreated group. Positive staining for caspase-3 after Gln pretreatment was significantly increased in contrast to the control tissues. CONCLUSION: Glutamine pretreatment is beneficial in supporting hepatic microcirculation and can prevent hepatocellular necrosis in liver reperfusion injury.

Levosimendan Administration in Limb Ischemia: Multicomponent Signaling Serving Kidney Protection.

AIMS AND OBJECTIVES: Acute renal failure is a severe complication of lower extremity major arterial reconstructions, which could even be fatal. Levosimendan is a dual-acting positive inotropic and vasodilatory agent, which is suspected to have protective effects against cardiac ischemia. However, there is no data available on lower limb or remote organ ischemic injuries therefore the aim of the study was to investigate the effect of levosimendan on lower limb ischemia-reperfusion injury and the corollary renal dysfunction. METHODS: Male Wistar rats underwent 180 min bilateral lower limb ischemia followed by 4 or 24 hours of reperfusion. Intravenous Levosimendan was administered continuously (0.2µg/bwkg/min) throughout the whole course of ischemia and the first 3h of reperfusion. Results were compared with sham-operated and ischemia-reperfusion groups. Hemodynamic monitoring was performed by invasive arterial blood pressure measurement. Kidney and lower limb muscle microcirculation was registered by a laser Doppler flowmeter. After 4h and 24h of reperfusion, serum, urine and histological samples were collected. RESULTS: Systemic hemodynamic parameters and microcirculation of kidney and the lower limb significantly improved in the Levosimendan treated group. Muscle viability was significantly preserved 4 and 24 hours after reperfusion. At the same time, renal functional laboratory tests and kidney histology demonstrated significantly less expressive kidney injury in Levosimendan groups. TNF-α levels were significantly less elevated in the Levosimendan group 4 hours after reperfusion. CONCLUSION: The results claim a protective role for Levosimendan administration during major vascular surgeries to prevent renal complications.

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-(6­morpholino-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 (p early = 0.02; p late = 0.005), AST (p early = 0.02; p late = 0.004) and less DNA damage by TUNEL test (p early = 0.05; p late = 0.034) and PAR positivity (p early = 0.02; p late = 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.