Mária Kürthy

Effect of BRX-220 against peripheral neuropathy and insulin resistance in diabetic rat models.

Abstract: Bimoclomol (BML), a symptomatic antidiabetic agent, has been developed by Biorex R & D Co. to treat diabetic neuropathy and retinopathy. BRX‐220, an orally active member of the BRX family, has been developed to treat diabetic complications and insulin resistance (IR) as a follow‐up compound. The effect of BRX‐220 on peripheral neuropathy was examined in rats with diabetes (type 1) induced by administration of a β‐cell toxin, streptozotocin (STZ, 45 mg/kg iv). Nerve functions were evaluated by electrophysiological measurements of muscle motor and sensory nerve conduction velocities (MNCV and SNCV, respectively). MNCV and SNCV decreased in diabetic rats by 25% (p < 0.001). A 1‐month preventive treatment with BRX‐220 (2.5, 5, 10, and 20 mg/kg po) dose‐dependently improved diabetes‐related deficits in MNCV (51.3%, 71.3%, 86.1%, and 91.3%) and SNCV (48.9%, 68.5%, 86.1%, and 93.2%). Insulin sensitivity was measured using the insulin tolerance test (ITT), both in STZ diabetic and in Zucker diabetic fatty (ZDF) rats (model of type 2 diabetes). Severe IR was detected in STZ diabetic and ZDF rats. This resistance was significantly (p < 0.05) reduced by BRX‐220 treatment.

Expression and Protective Role of Heme Oxygenase-1 in Delayed Myocardial Preconditioning

Abstract:  In the study the authors aimed to demonstrate the expression and protective effect of heme oxygenase‐1 (HO‐1) in the delayed preconditioning (PC) on cultured myocardiac cells. Neonatal rat cardiac myocytes were exposed to ischemic (ischemic medium [IM] for 20 min) and pharmacological (adenosine, epinephrine, opioid) PC. Twenty‐four hours later cells were subjected to a simulated ischemia (SI )—culturing for 3 h in IM, followed by 2‐h reperfusion in normal medium‐–and then lactate dehydrogenase (LDH), live/death ratio, and apoptosis were measured. For demonstrating the protective role of HO‐1, its enzymatic activity was competitively inhibited by administration of zinc protoporphyrin IX (ZnPPIX), and HO‐1 synthesis was blocked with HO‐1 siRNA. Cells in control group were cultured under normoxic conditions. In SI group, cells underwent only an SI without PC. HO‐1 expression in all of the groups was demonstrated with immunostaining. Our results showed a significant decrease of LDH release, apoptosis, and cell death in PC groups versus SI group, which has been risen in ZnPPIX‐ and HO‐1 siRNA‐treated groups. HO‐1 immunostaining showed an appreciable HO‐1 expression in PC groups, which was abolished with HO‐1 siRNA administration, but not in ZnPPIX group. The results therefore suggest that HO‐1 expression increases in both ischemic and pharmacological PC, and HO‐1 has cellular protective effect against cell death and apoptosis in ischemia‐reperfusion‐induced oxidative injury.

Ischaemic postconditioning reduces peroxide formation, cytokine expression and leukocyte activation in reperfusion injury after abdominal aortic surgery in rat model

OBJECTIVE We studied the protective effects of ischaemic postconditioning (PS) on ischemia-reperfusion injury of the lower extremities in a rat model of abdominal aortic intervention. We aimed to examine the evoked oxidative stress, cytokine expression and leukocyte activation after revascularisation surgery. METHODS Anesthetized animals (48 Whistar rats) underwent a 60 min infrarenal aorta cross-clamping. After the ischaemic period, an intermittent 4 times 15 s reperfusion--15 seconds ischaemic episodes--were applied (ischaemic postconditioning: group PS). Then we started a 120 min reperfusion in the aorta. In untreated group animals underwent a long ischaemia (60 min) and the following reperfusion (group IR). Peripherial blood samples were collected before operation, and in early (5, 10, 15, 30, 60 and 120 min) reperfusion periods. Serum peroxide level, TNF-alpha concentration, myeloperoxidase (MPO) activity and PMA-induced leukocyte ROS production were measured. RESULTS In PS group, plasma peroxide level elevation was significantly lower in very early reperfusion (5-30 min) comparing to non-conditioned IR group (10.04+/-1.9 microM/l vs. 16.91+/-3.67 microM/l, p<0.05). PS also reduced serum TNF-alpha concentration (167.41+/-31.26 microg/ml vs. 116.55+/-12.04 microg/ml, p<0.05), MPO activity (1.759+/-0.239 microM/ml vs. 1.22+/-0.126 microM/ml, p<0.05) and leukocyte activation detected by PMA-induced leukocyte ROS production (5.7+/-0.96 AU/10(3) cells vs. 4.63+/-0.69 AU/10(3) cells). CONCLUSIONS Ischaemic postconditioning could reduce ROI production after IR in early reperfusion period, thus limiting ROI mediated tissue lesion, cytokine-leukocyte activation and inflammatory responses. PS seems to be an effective tool in vascular surgery to reduce reperfusion injuries after revascularization interventions.

Cardioprotective action of urocortin in early pre- and postconditioning

Abstract:  Pre‐ and postconditioning are powerful endogenous adaptive phenomenon of the organism whereby different stimuli enhance the tolerance against various types of stress. Urocortin (Ucn), member of the corticotropin‐releasing factor (CRF) family has potent effects on the cardiovascular system. The aim of this article was to investigate the action of Ucn on cultured cardiomyocytes in the process of pre‐ and postconditioning. Isolated neonatal rat ventricular myocytes were preconditioned with adenosine, simulated ischemia, and Ucn (10‐min treatment followed by 10‐min reperfusion/recovery). For detecting the effect of alternative types of preconditioning, necrosis enzyme (lactate dehydrogenase [LDH]) release, vital staining (trypan blue), and ratio of apoptosis/necrosis were examined after cardiac cells were exposed to 3‐h sustained ischemia and 2‐h reperfusion. Same parameters were measured in the postconditioned groups (30‐ or 60‐min ischemia followed by postconditioning with 10‐min ischemic stimulus or Ucn and 2‐h reperfusion). Cells exposed to 3‐h ischemia followed by 2‐h reperfusion were shown as control. Our results show that LDH release a number of trypan blue‐stained dead cells and the ratio of apoptotized and necrotized cells was decreased in all preconditioned groups compared with control group. In postconditioned groups LDH content of culture medium, trypan blue‐positive cardiomyocytes, and the rate of apoptotic/necrotic cells was reduced contrasted with non‐postconditioned group. We can conclude that preconditioning with Ucn induced such a powerful cell protective effect as adenosine and ischemia. Furthermore, postconditioning with Ucn after 60‐min ischemia was more cardioprotective than ischemic postconditioning.

Effect of vitamin E on reperfusion injuries during reconstructive vascular operations on lower limbs.

INTRODUCTION The challenge against reperfusion injury and tissue oxidative stress, especially in vascular surgical interventions has an essential importance to reach the optimal clinical result. Numerous experimental attempts have proved the positive antioxidant effect of vitamin E in both chronic and acute phase models. In our study we monitored the effect of continuous preoperative treatment with vitamin E, on oxidative stress and tissue inflammation reactions developed after reconstructive operations. PATIENTS AND METHODS 32 patients have been involved in a randomized, prospective study, all suffering from AFS occlusion proved by angiography, and all undergone supragenual reconstruction. Duration of ischemia and amount of tissues under vascular clamping were almost the same in all patients. In the group treated with E-vitamin, we administered 1 x 200 mg of vitamin E p/o from the preoperative day till the 7th post operative day. Patients of the second group did not receive vitamin E. MATERIALS AND METHODS Peripheral blood samples were collected immediately before operation and at the end of the second reperfusion hour (early reperfusion period). Late reperfusion period has been monitored by analyzing blood samples taken at 24th hour and 7th day next to the operative ischemia. Among oxidative stress parameters, direct measurement of reactive oxygen intermediator (ROI) and determination of antioxidant state (GSH, Total-SH group, SOD) have been performed. Malondialdehyde was chosen as marker for lipidperoxidation. Inflammation reactions were monitored up on expression of adhesion molecules (CD11a and CD18). We also controlled the oscillation of myeloperoxidase (MPO) activity. RESULTS Our study has proved that preoperative (from the preoperative day till the 7th post operative day) administration of 200 mg vitamin E could reduce the level of oxidative stress developed after ischemic-reperfusion insult (lipidproxidation, antioxidant enzymes). According to our results, the prooxidant-antioxidant imbalance also diminished in the group with E-vitamin treatment. We proved that elective administration of vitamin E could decrease the WBC activity (MPO activity, free radicals production, expression of adhesion molecules) and its consequential local inflammation process, during early reperfusion.

Ischaemic postconditioning reduces serum and tubular TNF-α expression in ischaemic-reperfused kidney in healthy rats.

OBJECTIVE We studied the protective effects of postconditioning (PS) in healthy and hypercholesterolemic rats after renal ischaemia-reperfusion (IR) injury. We aimed to examine cytokine expression and apoptosis in tissue damage after revascularisation (TNF-α levels in serum and tissue). METHODS Male Wistar rats (n = 32) were divided into four groups. The animals of normal feed groups (NF) were fed with normal rat chow and the cholesterol feed groups (CF) were fed with 1.5% cholesterol containing diet for 8 weeks. Anaesthetized rats underwent a 45-min cross-clamping in both kidney pedicles. Ischaemia was followed by 120-min reperfusion with or without PS protocol (group PS vs. IR). Postconditioning was induced by four intermittent periods of ischaemia-reperfusion of 15-s duration each. Serum cholesterol, triglyceride, urea and creatinine levels were determined. Proinflammation was characterized by the measurement of serum TNF-α. Tissue injury in kidney was determined by formaline-fixed, paraffin-embedded tissue sections. Tissue TNF-α levels were determined by immunohistochemistry. RESULTS Significant elevation was observed in serum TNF-α level after IR injury in normal feed groups, which was reduced by PS. In CF group neither the elevation nor the postconditioning induced reduction were as significant as in the NF groups. In normal feed group PS caused a significant reduction in tissue TNF-α level which was significantly higher in CF. CONCLUSIONS Ischaemic postconditioning proved to be an effective defense against IR in NF groups, but it was ineffective in CF groups in kidney tissue.

Comparison of the Extrapancreatic Action of BRX‐220 and Pioglitazone in the High‐Fat Diet‐Induced Insulin Resistance

Abstract: A new Biorex molecule, BRX‐220, has been shown to be effective in animal models of diabetic neuro‐ and retinopathy. Recent in vitro studies showed that it might also have an insulin‐sensitizing action. Therefore, the effect of BRX‐220 on insulin sensitivity was compared with the action of pioglitazone (PGZ) in high fat (HF) diet‐induced insulin resistance (IR) of rats. Methods—Male Wistar rats were fed for 3 weeks a standard chow (PD) or the HF (70‐cal%) diet. The HF‐fed rats were also given daily BRX‐220 (20 mg/kg BW) or PGZ (6 mg/kg BW) by gavage. In vivo insulin action was assessed by the euglycemic hyperinsulinemic clamp. Glucose, insulin, FFA, triglyceride (TG), and glycerol levels in blood were also measured, as well as tissue TG content. Results—Increased levels of fed TG in circulation after HF diet (PD: 2.0±0.2 vs. HF: 5.0±0.8 mmol/L) were partially corrected by BRX‐220 (HF + BRX: 3.8±0.3) and normalized by PGZ (HF + PGZ: 2.6±0.3). Both molecules prevented the increase in fed serum FFA levels after HF diet (PD: 0.5±0.06; HF: 1.8±0.2 mmol/L), with a more pronounced effect of PGZ (HF + BRX: 1.2±0.1; HF + PGZ: 0.7±0.06). Tissue TG levels increased significantly in response to HF feeding in both liver (HF: 16±3.0; PD: 6.4±1.1 μmol/g) and skeletal muscle (HF: 7.7±1.2; PD: 2.4±0.4). This increase was completely normalized by both agents in the liver (HF + BRX: 8.8±0.8; HF + PGZ: 8.8±1.0), and only partially in the skeletal muscles. HF diet‐induced in vivo IR (PD: 25.4±0.5; HF: 15.7±0.5 mg/kg/min) was significantly reduced by BRX‐220 (HF + BRX: 18.7±0.3) and PGZ (HF + PGZ: 22.8±0.4) treatment. Conclusions—(1) Subchronic administration of BRX‐220 leads to an improvement of in vivo insulin action. (2) This insulin‐sensitizing effect is, however, not as pronounced as that of PGZ. (3) It is accompanied by a decrease of circulating TG and FFA levels in the postprandial state and (4) by lower TG content in liver and skeletal muscle.

Reperfusion injury and inflammatory responses following acute lower limb revascularization surgery

After revascularization of an acute arterial occlusion the development of a serious ischaemic-reperfusion injury is a menacing challenge and a hard task in peripheral vascular surgery. A whale of evidences point to oxidative stress, as an important trigger, in the complex chain of events leading to reperfusion injury. In the present study authors aimed to examine oxidative stress parameters, antioxidant-prooxidant state and leukocyte adhesion molecules (CD11a and CD18) expression following acute revascularization surgery of lower limb.10 patients were examined in the prospective randomized study. Peripheral blood sample was collected in ischaemic period, and after reperfusion in the 2nd and 24th hours, and on 7th day. Superoxide-dismutase activity, reduced glutathion concentration and leukocytes free radical production were measured. The degree of lipidperoxidation was marked with the quantity of malondialdehyde. The expressions of adhesion molecules were measured with flowcytometry.The speed and rate of free radical production significantly increased in the early reperfusion (p<0.05). The level of antioxidant enzymes decreased after revascularization. The CD11a and CD18 expression of the granulocytes significantly (p<0.05) decreased right after the revascularization, but with a gradual elevation until the 7th day they exceed the ischaemic value. Our results showed a time specific turnover of the sensitive antioxidant-prooxidant balance after revascularization operation.

Preconditioning is a method that may reduce the negative side-effect of pneumoperitoneum

Abstract Increased intra-abdominal pressure during laparoscopy leads to hypoxia due to reduced blood flow. Aim of our study was to investigate whether preconditioning can reduce this negative effect of the pneumoperitoneum. Fifty female Wistar rats were used, divided into 5 groups. I: Sham operation (Sham), II: conventional pneumoperitoneum (PP), III: transvaginal pneumoperitoneum (TV), IV: preconditioning for 2.5 minutes in two cycles (Pre 2.5), V: preconditioning for 5 minutes (Pre 5). Malondialdehyde (MDA), reduced glutathione (GSH), sulfhydrylgroup (SH-) concentrations, superoxide-dismutase (SOD) and mieloperoxidase (MPO) activity, and anti-apoptotic pathway marker p-AKT level and inflammatory cytokine TNF-α were measured. SOD activity and GSH concentration were decreased in PP and TV groups comparing to Sham and preconditioning groups. MPO activity was decreased also in PP and TV groups comparing to the Sham group but in the preconditioning groups it has remained high. MDA concentration in plasma was...

Investigation of the Oxidative Stress, the Altered Function of Platelets and Neutrophils, in thePatients with Peripheral Arterial Disease

Ischemia reperfusion injury (I/R) is a relevant problem in case of myocardial infarction (Moens AL, Claeys MJ et al. 2005.), stroke, (Kato H and Kogure K 1999), coronary bypass surgery, (Bakkaloglu C, and Soyagir B, 2006), under thrombolysis, (Krumholz HM and Goldberger 2006), revascularization surgery of lower limb (Arato et al 2006., Laird IR 2003), balloon angioplasty (Weissand A.G. and Zahger AT 1999) and in every cases, when the physiological blood flow in the occluded vessels are restored (Falkensammer J and Oldenburg WA 2006), (Ferencz A et al 2004). Vessel closure and hypoxia can be caused by embolism (thrombus, tumour, fat, foreign body) stenotic arteriopathy, arterial spasm, compression, arterial thrombus, trauma, etc. During the exclusion of a segment of the vessels from the circulation, ischemia and acidosis appeared in the surrounding tissues. In case of the heart, when oxygen supply is inadequate, the respiration shift from aerobic fatty acid consumption and metabolism to anaerobic glycolysis, resulting in a reduced ATP production. The results of hypoxia in the metabolically active tissues (cardiac, skeletal muscle and neuronal tissues) are more profound than in other cell types. The cells are exposed to hypoxia try to adapt to the absence of oxygen, by switching their metabolism from aerobic to anaerobic. Finally this strategy leads to tissue damages and loss of cells too, as it can be seen in acute or chronic occlusive diseases, as well. The measures of the tissue injuries depend on the duration of hypoxia, the mass of tissues are involved, the ATP requirement of the cell types and the blood pressure of the patients. Under hypoxic condition the generations of reactive oxygen species (ROS), such as O2.-, H2O2, are increased. During normoxyc, physiological condition mitochondria generate low level of ROS by the respiratory chain. These are managed by natural antioxidants, such as manganese superoxid dismutase (SOD) in the mitochondria, or copper-zinc SOD in the inter-membrane space in the mitochondria, and in the cytosol, making the dismutation of superoxide anion