Effluent from ischemic preconditioned hearts confers cardioprotection independent of the number of preconditioning cycles

Coronary effluent collected from ischemic preconditioning (IPC) treated hearts induces myocardial protection in non-ischemic-preconditioned hearts. So far, little is known about the number of IPC cycles required for the release of cardioprotective factors into the coronary effluent to successfully induce cardioprotection. This study investigated the cardioprotective potency of effluent obtained after various IPC cycles in the rat heart. Experiments were performed on isolated hearts of male Wistar rats, mounted onto a Langendorff system and perfused with Krebs-Henseleit buffer. In a first part, effluent was taken before (Con) and after each IPC cycle (Eff 1, Eff 2, Eff 3). IPC was induced by 3 cycles of 5 min of global myocardial ischemia followed by 5 minutes of reperfusion. In a second part, hearts of male Wistar rats were randomized to four groups (each group n = 4–5) and underwent 33 min of global ischemia followed by 60 min of reperfusion. The previously obtained coronary effluent was administered for 10 minutes before ischemia as a preconditioning stimulus. Infarct size was determined at the end of reperfusion by triphenyltetrazoliumchloride (TTC) staining. Infarct size with control effluent was 54±12%. Effluent obtained after IPC confers a strong infarct size reduction independent of the number of IPC cycles (Eff 1: 27±5%; Eff 2: 35±7%; Eff 3: 35±8%, each P<0.05 vs. Con). Effluent extracted after one cycle IPC is comparably protective as after two or three cycles IPC.

Exposition to glucose-based peritoneal dialysis fluids exacerbates adipocyte lipolysis and glycogen storage in rat adipose cells

Glucose absorption during peritoneal dialysis (PD) is suspected to promote visceral fat accretion and weight gain in PD patients. The current study was designed to test the impact of glucose-based PD fluids on adipose cell lipolysis and glycogen content. Rat adipose cells, isolated from epididymal fat pad, were exposed to a 30 vol./70 vol. mixture of glucose-based dialysis solutions (containing 1.36% and 3.86% glucose, Physioneal 35®; Baxter) or Krebs–Henseleit buffer for 4 h. Adipose cells were further incubated with laboratory-made solutions containing 1.36% and 3.86% glucose or mannitol as an osmotic control. Baseline and noradrenaline-stimulated lipolysis was measured, and glycogen content assayed. The glucose-based commercial PD fluids as well as the laboratory-manufactured high glucose solutions exacerbated lipolysis in baseline and noradrenaline conditions and increased glycogen stores in adipose cells. Mannitol solutions (1.36% and 3.86%) used as an osmotic control did not produce such effects. This study provides the first evidence that glucose-based dialysis solutions increase basal as well as stimulated lipolysis in adipocytes, an effect that is directly attributable to high concentrations of glucose per se.

Influence of Hyperglycemia on Dexmedetomidine-Induced Cardioprotection in the Isolated Perfused Rat Heart

Pharmacological preconditioning (PC) and postconditioning (PoC), for example, by treatment with the α2-adrenoreceptor agonist Dexmedetomidine (Dex), protects hearts from ischemia-reperfusion (I/R) injury in experimental studies, however, translation into the clinical setting has been challenging. Acute hyperglycemia adversely affects the outcome of patients with myocardial infarction. Additionally, it also blocks cardioprotection by multiple pharmacological agents. Therefore, we investigated the possible influence of acute hyperglycemia on Dexmedetomidine-induced pre- and postconditioning. Experiments were performed on the hearts of male Wistar rats, which were randomized into 7 groups, placed in an isolated Langendorff system and perfused with Krebs-Henseleit buffer. All hearts underwent 33 min of global ischemia, followed by 60 min of reperfusion. Control (Con) hearts received Krebs-Henseleit buffer (Con KHB), glucose (Con HG) or mannitol (Con NG) as vehicle only. Hearts exposed to hyperglycemia (HG) received KHB, containing 11 mmol/L glucose (an elevated, but commonly used glucose concentration for Langendorff perfused hearts) resulting in a total concentration of 22 mmol/L glucose throughout the whole experiment. To ensure comparable osmolarity with HG conditions, normoglycemic (NG) hearts received mannitol in addition to KHB. Hearts were treated with 3 nM Dexmedetomidine (Dex) before (DexPC) or after ischemia (DexPoC), under hyperglycemic or normoglycemic conditions. Infarct size was determined by triphenyltetrazoliumchloride staining. Acute hyperglycemia had no impact on infarct size compared to the control group with KHB (Con HG: 56 ± 9% ns vs. Con KHB: 56 ± 7%). DexPC reduced infarct size despite elevated glucose levels (DexPC HG: 35 ± 3%, p < 0.05 vs. Con HG). However, treatment with Dex during reperfusion showed no infarct size reduction under hyperglycemic conditions (DexPoC HG: 57 ± 9%, ns vs. Con HG). In contrast, hearts treated with mannitol demonstrated a significant decrease in infarct size compared to the control group (Con NG: 37 ± 3%, p < 0.05 vs. Con KHB). The combination of Dex and mannitol presents exactly opposite results to hearts treated with hyperglycemia. While DexPC completely abrogates infarct reduction through mannitol treatment (DexPC NG: 55 ± 7%, p < 0.05 vs. Con NG), DexPoC had no impact on mannitol-induced infarct size reduction (DexPoC NG: 38 ± 4%, ns vs. Con NG). Acute hyperglycemia inhibits DexPoC, while it has no impact on DexPC. Treatment with mannitol induces cardioprotection. Application of Dex during reperfusion does not influence mannitol-induced infarct size reduction, however, administering Dex before ischemia interferes with mannitol-induced cardioprotection.

Dexmedetomidine enhances tolerance to bupivacaine cardiotoxicity in the isolated rat hearts: alpha 2 adrenoceptors were not involved

Background Dexmedetomidine was proved to mitigate bupivacaine-induced cardiotoxicity but mechanism of this ability is still unclear. This study was designed to investigate the direct effects of dexmedetomidine on cardiotoxicity induced by bupivacaine on Langendorff rat heart preparation and the role of alpha 2 adrenoceptors in this process was explored. Methods Hearts of rat were isolated, mounted on a Langendorff system. Five experimental groups were assessed after 10 min Krebs-Henseleit buffer (KHB) infusions as follow: (1) Group Con, only KHB was perfused; (2) Group Dex, KHB was perfused for 5 min, then dexmedetomidine (10 nmol/L) was added; (3) Group Bupi, KHB was perfused for 25 min, then bupivacaine (50 μmol/L) was added; (4) Group Bupi + Dex, KHB was perfused for 5 min, then the dexmedetomidine (10 nmol/L) was added for 20 min, at last a mixture of KHB + dexmedetomidine + bupivacaine were perfused; (5) Group Bupi + Dex + Yoh, a combination of KHB + yohimbine (alpha 2 adrenoceptor antagonists, 1 μmol/L) was perfusion for 5 min, then dexmedetomidine (10 nmol/L) was added for 20 min, at last a mixture of KHB + yohimbine + dexmedetomidine + bupivacaine was perfused. The experimental perfusion was maintained for 35 min in group Con and group Dex, and the experimental perfusion was sustained until asystole in the other three groups. Results Compared with group Bupi, dexmedetomidine significantly increased the time to first arrhythmia (P < 0.001) and time to asystole (P < 0.001) in group Bupi + Dex. In addition, dexmedetomidine also significantly increased the time to 25, 50 and 75% reductions in heart rate (P < 0.001) and the time to 25, 50 and 75% reductions in rate-pressure product (P < 0.001) in group Bupi + Dex. Dexmedetomidine increased the cardiac tissue bupivacaine content when asystole (Bupi + Dex vs. Bupi, 58.5 ± 6.3 vs. 46.8 ± 5.6 nmol/g, P = 0.003). The benefit of dexmedetomidine on bupivacaine-induced cardiotoxicity were not eliminated by yohimbine. Conclusions Dexmedetomidine could delay the occurrence of bupivacaine-induced arrhythmia and asystole in the isolated rat hearts, but the alpha 2 adrenoceptors were not involved in this process.

Comparison of hemodynamic and infarct-limiting effects of preservation solution based on Krebs–Henseleit buffer and HTK solution in the rat model of heterotopic heart transplantation

Objective. To compare the cardioprotective effcacy of a preservation solution based on Krebs–Henseleit buffer and HTK solution in the model of heterotopic heart transplantation in rat.Design and methods. A study was conducted on 12 Wistar rats. The animals were divided into groups depending on the preservation solution used: 1) Krebs–Henseleit buffer-based solution (n = 7), 2) HTK (n = 5). Each experiment consisted of collecting donor heart, preserving it with an appropriate cardioplegic solution, heterotopic transplantation into a recipient rat followed by explantation and evaluation of left ventricular contractility using the Langendorff model and histochemical assessment of the irreversible myocardial damage. Coronary blood flow in the donor heart was assessed in vivo using ultrasound doppler flowmetry. After 3 hours, the donor heart was explanted and connected to the Langendorff apparatus to assess left ventricular contractility, and the myocardium was subjected to histochemical staining with 1 % triphenyltetrazolium chloride for the assessment of the irreversible myocardial damage.Results. In the group of Krebs–Henseleit buffer-based cardioplegic solution, 7 experiments were performed. Myocardial infarct size was 3,5 ± 1,2%, the coronary flow rate was 4,5 ± 1,3 ml/min, and the developed left ventricular pressure of the donor heart was 70 ± 6,3 mmHg at diastolic left ventricular pressure of 10 mmHg. In the HTK solution group (n = 5), in all of the experiments after the start of blood flow, the transplanted heart did not begin to contract, and all 5 hearts remained in an asystole state. Therefore, after the end of the 3-hour reperfusion period, the assessment of the contractility of the left ventricle using the Langendorff apparatus was not carried out. Coronary flow rate was only 0,4 ± 0,1 ml/min, probably resulting from inadequate cardioprotection with HTK solution. Due to the lack of adequate reperfusion, the accurate assessment of the irreversible myocardial damage was impossible in the HTK solution group.Conclusions. The technique of heterotopic heart transplantation in rats is adequate and informative for the study of ischemia-reperfusion myocardial damage as well as for the study of the effectiveness of cardioplegic and cardiac preservation solutions. Cardioplegic solution based on Krebs– Henseleit buffer demonstrated greater cardioprotective effectiveness in our model compared to HTK solution.

Impact of Anesthetics on Cardioprotection Induced by Pharmacological Preconditioning

Anesthetics, especially propofol, are discussed to influence ischemic preconditioning. We investigated whether cardioprotection by milrinone or levosimendan is influenced by the clinically used anesthetics propofol, sevoflurane or dexmedetomidine. Hearts of male Wistar rats were randomised, placed on a Langendorff system and perfused with Krebs–Henseleit buffer (KHB) at a constant pressure of 80 mmHg. All hearts underwent 33 min of global ischemia and 60 min of reperfusion. Three different anesthetic regimens were conducted throughout the experiments: propofol (11 μM), sevoflurane (2.5 Vol%) and dexmedetomidine (1.5 nM). Under each anesthetic regimen, pharmacological preconditioning was induced by administration of milrinone (1 μM) or levosimendan (0.3 μM) 10 min before ischemia. Infarct size was determined by TTC staining. Infarct sizes in control groups were comparable (KHB-Con: 53 ± 9%, Prop-Con: 56 ± 9%, Sevo-Con: 56 ± 8%, Dex-Con: 53 ± 9%; ns). Propofol completely abolished preconditioning by milrinone and levosimendan (Prop-Mil: 52 ± 8%, Prop-Lev: 52 ± 8%; ns versus Prop-Con), while sevoflurane did not (Sevo-Mil: 31 ± 9%, Sevo-Lev: 33 ± 7%; p < 0.05 versus Sevo-Con). Under dexmedetomidine, results were inconsistent; levosimendan induced infarct size reduction (Dex-Lev: 36 ± 6%; p < 0.05 versus Dex-Con) but not milrinone (Dex-Mil: 51 ± 8%; ns versus Dex-Con). The choice of the anesthetic regimen has an impact on infarct size reduction by pharmacological preconditioning.

Amino Acid-Containing Krebs-Henseleit Buffer Protects Rat Liver in a Long-Term Organ Perfusion Model

Background: The liver is vulnerable to the toxicity induced by xenobiotics. On the other hand, it has been found that several endogenously-found amino acids have hepatoprotective properties. The current study was designed to evaluate the effect of taurine, glycine, and histidine on the liver function in an ex vivo model of prolonged organ perfusion. Methods: Rat liver was isolated and perfused with a hemoglobin- and albumin-free Krebs‑Henseleit buffer (KBH). Liver injury biomarkers were monitored at scheduled time intervals. Results: The perfusate level of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and the potassium ion (K+) were gradually increased in control (Only KBH) group. The histopathological evaluation also revealed significant necrosis, sinusoidal dilation, and pyknosis in control liver. Moreover, significant increase in lipid peroxidation and depletion of hepatic glutathione stores were detected in the control group. It was found that taurine (5, 10 and 20 mM) and glycine (5, 10 and 20 mM)-containing KBH buffer significantly decreased the perfusate level of liver injury biomarkers. Furthermore, lower liver tissue pathological changes, decreased lipid peroxidation, and higher glutathione content was detected in amino acid-treated groups. Histidine administration showed no significant protective effect on liver injury in the current study. On the other hand, combination amino acid administration (glycine and taurine) showed a better hepatoprotective profile. Conclusion: The data obtained from the current study might help to provide safe hepatoprotective agents against xenobiotics-induced hepatotoxicity or preserve liver functionality outside the body.

Molecular hydrogen potentiates beneficial anti-infarct effect of hypoxic postconditioning in isolated rat hearts: a novel cardioprotective intervention

Generation of free radicals through incomplete reduction of oxygen during ischemia–reperfusion (I/R) is well described. On the other hand, molecular hydrogen (H2) reduces oxidative stress due to its ability to react with strong oxidants and easily penetrate cells by diffusion, without disturbing metabolic redox reactions. This study was designed to explore cardioprotective potential of hypoxic postconditioning (HpostC) against I/R (30 min global I – 120 min R) in isolated rat hearts using oxygen-free Krebs-Henseleit buffer (KHB). Furthermore, the possibility to potentiate the effect of HpostC by H2using oxygen-free KHB saturated with H2(H2+ HpostC) was tested. HPostC was induced by 4 cycles of 1-minute perfusion with oxygen-free KHB intercepted by 1-minute perfusion with normal KHB, at the onset of reperfusion. H2+ HPostC was applied in a similar manner using H2-enriched oxygen-free KHB. Cardioprotective effects were evaluated on the basis of infarct size (IS, in % of area at risk, AR) reduction, post-I/R recovery of heart function, and occurrence of reperfusion arrhythmias. HPostC significantly reduced IS/AR compared with non-conditioned controls. H2present in KHB during HPostC further decreased IS/AR compared with the effect of HPostC, attenuated severe arrhythmias, and significantly restored heart function (vs. controls). Cardioprotection by HpostC can be augmented by molecular hydrogen infusion.