Hypothermia and postconditioning after cardiopulmonary resuscitation reduce cardiac dysfunction by modulating inflammation, apoptosis and remodeling

Background: Mild therapeutic hypothermia following cardiac arrest is neuroprotective, but its effect on myocardial dysfunction that is a critical issue following resuscitation is not clear. This study sought to examine whether hypothermia and the combination of hypothermia and pharmacological postconditioning are cardioprotective in a model of cardiopulmonary resuscitation following acute myocardial ischemia. Methodology/Principal Findings: Thirty pigs (28–34 kg) were subjected to cardiac arrest following left anterior descending coronary artery ischemia. After 7 minutes of ventricular fibrillation and 2 minutes of basic life support, advanced cardiac life support was started according to the current AHA guidelines. After successful return of spontaneous circulation (n = 21), coronary perfusion was reestablished after 60 minutes of occlusion, and animals were randomized to either normothermia at 38°C, hypothermia at 33°C or hypothermia at 33°C combined with sevoflurane (each group n = 7) for 24 hours. The effects on cardiac damage especially on inflammation, apoptosis, and remodeling were studied using cellular and molecular approaches. Five animals were sham operated. Animals treated with hypothermia had lower troponin T levels (p<0.01), reduced infarct size (34±7 versus 57±12%; p<0.05) and improved left ventricular function compared to normothermia (p<0.05). Hypothermia was associated with a reduction in: (i) immune cell infiltration, (ii) apoptosis, (iii) IL-1beta and IL-6 mRNA up-regulation, and (iv) IL-1beta protein expression (p<0.05). Moreover, decreased matrix metalloproteinase-9 activity was detected in the ischemic myocardium after treatment with mild hypothermia. Sevoflurane conferred additional protective effects although statistic significance was not reached. Conclusions/Significance: Hypothermia reduced myocardial damage and dysfunction after cardiopulmonary resuscitation possible via a reduced rate of apoptosis and pro-inflammatory cytokine expression

Direct effect of melatonin on Syrian hamster testes: melatonin subtype 1a receptors, inhibition of androgen production, and interaction with the local corticotropin-releasing hormone system

Besides the hypothalamus and pituitary, melatonin action at the testicuiar level has been recently suggested. Therefore, we investigated in the Syrian hamster, a well-characterized seasonal breeder, melatonin action on Leydig cells, testicular expression of melatonergic receptors, and possible interactions between melatonin receptors and the previously identified testicular serotoninergic and CRH systems. In isolated Leydig cells from active testes of adult hamsters kept in a long-day (14 h light, 10 h dark) photoperiod and from regressed testes of adult animals exposed to a short-day photoperiod during 16 wk (6 h light, 18 h dark), melatonin significantly reduced human chorionic gonadotropin-stimulated production of cAMP and the main androgens: testosterone and androstane-3α,17β-diol, respectively, and decreased the expression of steroidogenic acute regulatory protein, P450 side chain cleavage, 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase. In Leydig cells exposed to a short-day photoperiod during 16 wk, melatonin stimulated the conversion of testosterone into 5α-reduced androgens by inducing 5α-reductase isoform 1, and controlled androstane-3α,17β-diol production by inhibiting 3α- hydroxysteroid dehydrogenase expression. Melatonin subtype (mella) receptors were detected in Leydig cells. Although the local serotonin system did not mediate melatonin action on androgen production, melatonergic effect on steroidogenesis involved the interaction between mella receptors and the inhibitory CEH system. Moreover, melatonin significantly increased CRH mRNA levels and production in hamster Leydig cells expressing CRH subtype 1 receptors. Our studies indicate that melatonin may act as a local inhibitor of human chorionic gonadotropin-stimulated cAMP and androgen production through mella receptors, down-regulation of steroidogenic acute regulatory protein, and key steroidogenic enzymes expression and its interaction with the local CRH system.Facultad de Ciencias ExactasInstituto Multidisciplinario de Biología Celula

Mild hypothermia alone or in combination with anesthetic post-conditioning reduces expression of inflammatory cytokines in the cerebral cortex of pigs after cardiopulmonary resuscitation

Introduction: Hypothermia improves survival and neurological recovery after cardiac arrest. Pro-inflammatory cytokines have been implicated in focal cerebral ischemia/reperfusion in-jury. It is unknown whether cardiac arrest also triggers the release of cerebral inflammatory molecules, and whether therapeutic hypothermia alters this inflammatory response. This study sought to examine whether hypothermia or the combination of hypothermia with anes-thetic postconditioning with sevoflurane affect cerebral inflammatory response after cardio-pulmonary resuscitation. Methods: Thirty pigs (28 - 34kg) were subjected to cardiac arrest following temporary coro-nary artery occlusion. After 7 minutes of ventricular fibrillation and 2 minutes of basic life support, advanced cardiac life support was started according to the current AHA guidelines. Return of spontaneous circulation was achieved in 21 animals who were randomized to ei-ther normothermia at 38degreesC, hypothermia at 33degreesC or hypothermia at 33degreesC combined with se-voflurane (each group: n = 7) for 24 hours. The effects of hypothermia and the combination of hypothermia with sevoflurane on cerebral inflammatory response after cardiopulmonary resuscitation were studied using tissue samples from the cerebral cortex of pigs euthanized after 24 hours and employing quantitative RT-PCR and ELISA techniques. Results: Global cerebral ischemia following resuscitation resulted in significant upregulation of cerebral tissue inflammatory cytokine mRNA expression (mean +/- SD; interleukin (IL)-1beta 8.7 +/- 4.0, IL-6 4.3 +/- 2.6, IL-10 2.5 +/- 1.6, tumor necrosis factor (TNF)alpha 2.8 +/- 1.8, intercellular adhesion molecule-1 (ICAM-1) 4.0 +/- 1.9-fold compared with sham control) and IL-1beta protein concentration (1.9 +/- 0.6-fold compared with sham control). Hypothermia was associated with a significant (P <0.05 versus normothermia) reduction in cerebral inflammatory cytokine mRNA expression (IL-1beta 1.7 +/- 1.0, IL-6 2.2 +/- 1.1, IL-10 0.8 +/- 0.4, TNFalpha 1.1 +/- 0.6, ICAM-1 1.9 +/- 0.7-fold compared with sham control). These results were also confirmed for IL-1beta on protein level. Experimental settings employing hypothermia in combination with sevoflurane showed that the volatile anesthetic did not confer additional anti-inflammatory effects com-pared with hypothermia alone. Conclusions: Mild therapeutic hypothermia resulted in decreased expression of typical ce-rebral inflammatory mediators after cardiopulmonary resuscitation. This may confer, at least in part, neuroprotection following global cerebral ischemia and resuscitation

Human monocytes subjected to ischaemia/reperfusion inhibit angiogenesis and wound healing in vitro

Objectives The sequence of initial tissue ischaemia and consecutive blood flow restoration leads to ischaemia/reperfusion (I/R) injury, which is typically characterized by a specific inflammatory response. Migrating monocytes seem to mediate the immune response in ischaemic tissues and influence detrimental as well as regenerative effects during I/R injury. Materials and methods To clarify the role of classical monocytes in I/R injury, isolated human monocytes were subjected to I/R in vitro (3 hours ischaemia followed by 24 hours of reperfusion). Cellular resilience, monocyte differentiation, cytokine secretion, as well as influence on endothelial tube formation, migration and cell recovery were investigated. Results We show that I/R supported an enhanced resilience of monocytes and induced intracellular phosphorylation of the prosurvival molecules Erk1/2 and Akt. FACS analysis showed no major alteration in monocyte subtype differentiation and surface marker expression under I/R. Further, our experiments revealed that I/R changes the cytokine secretion pattern, release of angiogenesis associated proteins and MMP-9 activity in supernatants of monocytes exposed to I/R. Supernatants from monocytes subjected to I/R attenuated endothelial tube formation as indicator for angiogenesis as well as endothelial cell migration and recovery. Conclusion In summary, monocytes showed no significant change in cellular integrity and monocyte subtype after I/R. Functionally, monocytes might have a rather detrimental influence during the initial phase of I/R, suppressing endothelial cell migration and neoangiogenesis

Effects of different ischemic preconditioning strategies on physiological and cellular mechanisms of intestinal ischemia/reperfusion injury: Implication from an isolated perfused rat small intestine model

Background Intestinal ischemia/reperfusion (I/R)-injury often results in sepsis and organ failure and is of major importance in the clinic. A potential strategy to reduce I/R-injury is the application of ischemic preconditioning (IPC) during which repeated, brief episodes of I/R are applied. The aim of this study was to evaluate physiological and cellular effects of intestinal I/R-injury and to compare the influence of in-vivo IPC (iIPC) with ex-vivo IPC (eIPC), in which blood derived factors and nerval regulations are excluded. Results I/R-injury decreased intestinal galactose uptake (iIPC group: p<0.001), increased vascular perfusion pressure (iIPC group: p<0.001; eIPC group: p<0.01) and attenuated venous flow (iIPC group: p<0.05) while lactate-to-pyruvate ratio (iIPC group, eIPC group: p<0.001), luminal flow (iIPC group: p<0.001; eIPC group: p<0.05), goblet cell ratio (iIPC group, eIPC group: p<0.001) and apoptosis (iIPC group, eIPC group: p<0.05) were all increased. Application of iIPC prior to I/R increased vascular galactose uptake (P<0.05) while eIPC had no significant impact on parameters of I/R-injury. On cellular level, I/R-injury resulted in a reduction of the phosphorylation of several MAPK signaling molecules. Application of iIPC prior to I/R increased phosphorylation of JNK2 and p38δ while eIPC enhanced CREB and GSK-3α/β phosphorylation. Conclusion Intestinal I/R-injury is associated with major physiological and cellular changes. However, the overall influence of the two different IPC strategies on the acute phase of intestinal I/R-injury is rather limited

Direct effect of melatonin on Syrian hamster testes: melatonin subtype 1a receptors, inhibition of androgen production, and interaction with the local corticotropin-releasing hormone system

Besides the hypothalamus and pituitary, melatonin action at the testicuiar level has been recently suggested. Therefore, we investigated in the Syrian hamster, a well-characterized seasonal breeder, melatonin action on Leydig cells, testicular expression of melatonergic receptors, and possible interactions between melatonin receptors and the previously identified testicular serotoninergic and CRH systems. In isolated Leydig cells from active testes of adult hamsters kept in a long-day (14 h light, 10 h dark) photoperiod and from regressed testes of adult animals exposed to a short-day photoperiod during 16 wk (6 h light, 18 h dark), melatonin significantly reduced human chorionic gonadotropin-stimulated production of cAMP and the main androgens: testosterone and androstane-3α,17β-diol, respectively, and decreased the expression of steroidogenic acute regulatory protein, P450 side chain cleavage, 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase. In Leydig cells exposed to a short-day photoperiod during 16 wk, melatonin stimulated the conversion of testosterone into 5α-reduced androgens by inducing 5α-reductase isoform 1, and controlled androstane-3α,17β-diol production by inhibiting 3α- hydroxysteroid dehydrogenase expression. Melatonin subtype (mella) receptors were detected in Leydig cells. Although the local serotonin system did not mediate melatonin action on androgen production, melatonergic effect on steroidogenesis involved the interaction between mella receptors and the inhibitory CEH system. Moreover, melatonin significantly increased CRH mRNA levels and production in hamster Leydig cells expressing CRH subtype 1 receptors. Our studies indicate that melatonin may act as a local inhibitor of human chorionic gonadotropin-stimulated cAMP and androgen production through mella receptors, down-regulation of steroidogenic acute regulatory protein, and key steroidogenic enzymes expression and its interaction with the local CRH system.Facultad de Ciencias ExactasInstituto Multidisciplinario de Biología Celula

N-acetylglucosaminidase participation during the hamster gametes interaction

La interacción entre gametas está mediada por los oligosacáridos de la cubierta del ovocito (la zona pelúcida, ZP) y proteínas complementarias del espermatozoide. En este trabajo se estudió la participación de los carbohidratos en las distintas etapas del proceso de interacción entre el espermatozoide de hámster y ZP. La presencia de GlcNAc fue capaz de inhibir la unión del espermatozoide a la ZP, revelando que es el azúcar más relevante en este proceso de interacción. El monosacárido también inhibió la reacción acrosomal (RA) espontánea e inducida por ZP. Usando un modelo que nos permitió tener capacitación y unión a ZP sin RA, pudimos verificar que la GlcNAc está involucrada en la unión primaria a través de sitios de unión para el azúcar en el espermatozoide. Utilizando ensayos de fertilización in vitro (FIV), analizamos la participación de GlcNAc en los diferentes pasos de la interacción espermatozoide-ovocito, pero no hallamos evidencias de su participación más allá de la unión primaria. La ZP fue reconocida como un ligando por la β-N-acetilglucosaminidasa (NAG) de espermatozoides de hámster. Esta enzima estaría distribuida en dos poblaciones: una acrosomal y otra débilmente asociada a la membrana plasmática. La actividad total de NAG disminuyó como consecuencia de la activación y liberación de un modulador luego de la capacitación y RA. Ligandos de NAG fueron capaces de inhibir la unión primaria del espermatozoide a la ZP. En ensayos de FIV, no se detectó efecto alguno cuando un inhibidor de NAG fue agregado luego de que la unión primaria tuviera lugar. Por otro lado, el agregado de NAG exógena fue capaz de inhibir la interacción entre gametas. En conclusión, los resultados presentados en este trabajo indican que una NAG de membrana del espermatozoide actuaría como proteína de unión para los residuos GlcNAc terminales de la ZP, mediando de esta manera la unión primaria entre ambas gametas.Gamete interaction is mediated by the oligosaccharides of the egg extracellular coat (the zona pellucida, ZP) and complementary proteins in the spermatozoa. In this study, the participation of carbohydrates in the different steps of hamster sperm interaction with the ZP was analyzed. The presence of GlcNAc inhibited sperm binding to the ZP, revealing that it is the most relevant sugar in this interaction process. The monosaccharide also inhibited the spontaneous and ZP-induced acrosome reaction (AR). Taken advantage of a method that allowed capacitation and ZP binding without AR, the involvement of sperm GlcNAc binding sites in primary binding to the ZP was verified. Carrying out in vitro fertilization (IVF) assays, the participation of GlcNAc in the different steps involved in sperm-oocyte interaction was analyzed, but no evidences for its involvement beyond primary binding was found. Soluble ZP was recognized as a ligand by the hamster sperm β-N-acetylglucosaminidase (NAG). This enzyme seems to be distributed in two populations in sperm: one within the acrosome and another one weakly associated to the plasma membrane. Total NAG activity diminished as a consequence of the activation and release of a regulatory molecule after capacitation and AR. NAG ligands were able to inhibit sperm primary binding to ZP. During IVF assays, no effect was detected when a NAG inhibitor was added after primary binding took place. Additionally, exogenous NAG was able to inhibit gamete interaction. In conclusion, results suggest that a membrane sperm NAG would be acting as a binding protein for the ZP terminal GlcNAc residues during primary binding between gametes.Fil: Zitta, Karina S.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina