Postischemic Reperfusion Period Research Articles

SOCS3 Ablation in Leptin Receptor-Expressing Cells Causes Autonomic and Cardiac Dysfunctions in Middle-Aged Mice despite Improving Energy and Glucose Metabolism

Leptin resistance is a hallmark of obesity. Treatments aiming to improve leptin sensitivity are considered a promising therapeutical approach against obesity. However, leptin receptor (LepR) signaling also modulates several neurovegetative aspects, such as the cardiovascular system and hepatic gluconeogenesis. Thus, we investigated the long-term consequences of increased leptin sensitivity, considering the potential beneficial and deleterious effects. To generate a mouse model with increased leptin sensitivity, the suppressor of cytokine signaling 3 (SOCS3) was ablated in LepR-expressing cells (LepR∆SOCS3 mice). LepR∆SOCS3 mice displayed reduced food intake, body adiposity and weight gain, as well as improved glucose tolerance and insulin sensitivity, and were protected against aging-induced leptin resistance. Surprisingly, a very high mortality rate was observed in aging LepR∆SOCS3 mice. LepR∆SOCS3 mice showed cardiomyocyte hypertrophy, increased myocardial fibrosis and reduced cardiovascular capacity. LepR∆SOCS3 mice exhibited impaired post-ischemic cardiac functional recovery and middle-aged LepR∆SOCS3 mice showed substantial arhythmic events during the post-ischemic reperfusion period. Finally, LepR∆SOCS3 mice exhibited fasting-induced hypoglycemia and impaired counterregulatory response to glucopenia associated with reduced gluconeogenesis. In conclusion, although increased sensitivity to leptin improved the energy and glucose homeostasis of aging LepR∆SOCS3 mice, major autonomic/neurovegetative dysfunctions compromised the health and longevity of these animals. Consequently, these potentially negative aspects need to be considered in the therapies that increase leptin sensitivity chronically.

Reduction of blood cholesterol and ischemic injury in the hypercholesteromic rabbits with modified resveratrol, logevinex

The present study examined the efficacy of using longevinex, a commercially available resveratrol formulation, to lower blood cholesterol in hypercholesteromic rabbits. New Zealand white rabbits were randomly divided into two groups (n = 6 per group), one group was given high cholesterol diet for 3 months while the other group fed regular diet served as control. The high cholesterol diet fed group was further subdivided into two groups (n = 6 per group), one group was given longevinex resveratrol while the other group given vehicle only served as control. Longevinex was given by gavaging up to a period of 6 months. Longevinex-treated rabbits exhibited lowering of plasma cholesterol level. Inhibition of arterial plaque formation was noticed even after 1 month. Longevinex-treated hearts demonstrated improved ventricular recovery when isolated working hearts were subjected to 30 min of ischemia followed by 2 h of reperfusion. Aortic flow and developed pressure during post-ischemic reperfusion period were significantly higher for the longevinex-treated hearts compared to those in control group of hearts. Myocardial infarct size was also lower in the treated group compared to that for the untreated group. These results indicate cholesterol-lowering ability of longevinex, which appears to reflect in its ability to protect the hypercholesteromic hearts from ischemic reperfusion injury.

Past and present course of cardioprotection against ischemia- reperfusion injury

Despite tremendous advances in cardiovascular research and clinical therapy, ischemic heart disease remains the leading cause of serious morbidity and mortality in western society and is growing in developing countries. For the past 5 decades, many scientists have studied the pathophysiology of myocardial ischemia-reperfusion (I/R) injury leading to infarction. With the exception of reperfusion therapy, attempts to salvage the myocardium during an acute myocardial infarction showed disappointing results in directly decreasing infarct size. Nevertheless, the phenomena of ischemic preconditioning and ischemic postconditioning show a consistent and robust cardioprotective effect in every used experimental animal model. As a result, many studies have focused on the intracellular protective signaling pathways that are involved in preconditioning and postconditioning. More recently, it has been suggested that components of the reperfusion injury salvage kinases pathway, protein kinase B, and the extracellular signal-regulated kinases can induce cardioprotection against I/R injury when they are activated during the postischemic reperfusion period. In addition, inhibition of mitochondrial permeability transition during postischemic reperfusion also shows a strong cardioprotective effect against I/R injury. The present mini-review highlights a short summary of the historical and present course of research into cardioprotection against myocardial I/R injury.

Increased state 4 mitochondrial respiration and swelling in early post-ischemic reperfusion of rat heart

Isolated rat hearts were exposed to 30 min ischemia or to 30 min ischemia followed by 2, 5 or 40 min reperfusion and mitochondria were isolated at these different time points. ADP-stimulated, succinate-dependent respiration rate (state 3) was not significantly changed at the different time points examined. In contrast, state 4 (non-ADP-stimulated) respiration rate was significantly increased after 30 min ischemia, and it increased further during the first post-ischemic reperfusion period. Mitochondrial swelling, as evaluated under conditions of the major controlled ion channels (i.e. permeability transition pore and ATP-dependent mitochondrial K + channel) closed, significantly increased in parallel. It is suggested that the inner mitochondrial membrane permeability is increased under exposure of the heart to ischemia and early reperfusion, and that the phenomenon is reversible upon subsequent long periods of reperfusion.

Electrical responses in hippocampal slices after prolonged global ischemia: effects of neuroprotectors

A simple and reproducible animal model of global ischemia, induced by decapitation in 30-day-old Wistar rats, has been developed. It allows to perform electrophysiological analysis of the postischemic reperfusion period in the brain slices. Periods of ischemia up to 40 min increase population spikes measured in the CA1 area of the hippocampus during 2–5 h of reperfusion. Thus after 30-min decapitation-induced ischemia (at t ischem=25°C), the mean amplitude of the recorded maximum orthodromic population spikes was 159% of the control obtained in the non-ischemic animals. Longer ischemic episodes result in the depression of the population spikes. After 2 h of ischemia, the amplitude of population spikes was about 89% of control. After 3 h of decapitation ischemia, the neurons could not be reactivated. The duration of ischemic episode needed for the irreversible depression of the electrical activity of the brain neurons drastically depends on the temperature at which the ischemic brain is maintained. Thus, only 2 h were needed at 30°C as compared to nearly 3 h at 25°C. We have found that intraperitoneal injection of neuroprotectors which precedes decapitation enables reactivation of the post-ischemic neurons even after very long periods of global ischemia. Thus, MK-801, a non-competitive NMDA receptors antagonist, or NBQX, a blocker of AMPA receptors, administrated 15 min before the long-term (90 min) decapitation ischemia (30°C), induced dose-dependent recovery of population spike with ED 50 values 0.2 mg/kg and 3 mg/kg respectively. Our results demonstrate that, in spite of the high vulnerability of hippocampal neurons to hypoxia and ischemia, their electrical activity can be restored after prolonged (more then 1 h) decapitation ischemia. Administration of NMDA or AMPA antagonists enhances recovery.

The cardioprotective effects of a new 1,4-benzothiazepine derivative, JTV519, on ischemia/reperfusion-induced Ca2+ overload in isolated rat hearts.

A new 1,4-benzothiazepine derivative, JTV519 (JTV), has strong protective effects against isoproterenol-induced myocardial injury. We investigated the effects of JTV on Ca2+ overload and on functional recovery during ischemia/reperfusion in isolated coronary-perfused rat hearts. After 30 minutes of reperfusion following 30 min of global ischemia, the % recovery of LV developed pressure was improved in a concentration-dependent manner when JTV (0.3-3.0 microM) was administered either 5 min before induction of ischemia or for 5 min at the time of reperfusion only JTV showed a negative inotropic effect only at concentrations above 3.0 microM. In indol-loaded isolated heart preparations, 0.3 microM JTV did not affect the preischemic systolic or diastolic Ca2+ levels of the Ca2+ transient as measured by the ratio of 2-wavelength fluormetry (R405/500). In contrast, it significantly reduced the increase in the ratio in the postischemic reperfusion period (% change of R405/500 from baseline: JTV(-), by 42.7 +/- 3.2%; JTV(+), by 18.4 +/- 9.1%, p < 0.05). In isolated rat ventricular myocytes with a standard patch-clamp method, we further tested the interaction of JTV with the L-type Ca2+ channel (I(Ca)). The % inhibition of the peak current of I(Ca) was 6.2 +/- 0.8% at 0.3 microM (p = n.s.), 22.0 +/- 3.3% at 1.0 microM (p < 0.05), and 59.6 +/- 1.4% at 3.0 microM (p < 0.01). Thus, the marked cardioprotection due to JTV at 0.3 microM may not be solely attributed to its inhibitory effect on the transsarcolemmal Ca2+ influx through I(Ca). In conclusion, JTV519 is a novel pharmacological agent that has been demonstrated for the first time to have clinical potential for the treatment of acute coronary syndrome by its efficacy in administration at the time of reperfusion, by its suppression of reperfusion-related intracellular Ca2+ overload with no significant interaction with I(Ca), and by its subsequent ability of strong myocardial protection.

Glucose-insulin-potassium reduces infarct size when administered during reperfusion.

Coronary reperfusion improves ventricular function and survival after infarction, but the metabolic conditions at this time may not be optimal to protect the heart. The objective of this study was to evaluate if metabolic support with glucose-insulin-potassium (GIK) administered at the time of coronary reperfusion could elicit the same cardioprotection as GIK infusion during the entire ischemia/reperfusion period. Three groups of anesthetized, open-chest rats were subjected to 30 minutes of regional ischemia and 180 minutes of reperfusion. Groups 1 (controls) and 2 (GIK(IR)) received saline or GIK, respectively, throughout the whole experimental period, whereas a third group (GIK(R)) received GIK from the onset of reperfusion only. Infarct size was significantly reduced in the GIK-treated groups, compared with controls (GIK(IR) 44 +/- 5% and GIK(R) 45 +/- 5% vs. control 66 +/- 4%; P < 0.05). Postischemic recovery of cardiac function improved when GIK was only administered during the reperfusion phase. Furthermore, infusion of GIK resulted in reduced plasma concentrations of free fatty acids and increased plasma glucose (both P < 0.05) compared with controls. This study demonstrates that glucose-insulin-potassium administration at the onset of the postischemic reperfusion period is as cardioprotective as administration of GIK during the entire ischemia/reperfusion period.

The effects of intraportal administration of prostaglandin E1 on liver ischemia and hepatectomy in rats.

The effects of intraportal administration of prostaglandin E1 (PGE1) on portal venous flow, hepatic arterial flow, peripheral tissue blood flow, and systemic arterial flow before and after 60 min total liver ischemia followed by 70% partial hepatectomy in rats were investigated. Total liver ischemia was induced by occluding the hepatoduodenal ligament for 60 min. PGE1 at a dose of 0.5 microg/kg/min was infused intraportally for 15 min before inducing hepatic ischemia (preischemic period) and for 60 min after ischemia (postischemic reperfusion period) in the treatment group. Normal saline was infused in the control group. Seventy percent partial hepatectomy was performed during ischemia. Serum biochemical analysis and liver tissue histology were carried out 1, 3, and 24 h, and 1 and 24 h after reperfusion respectively. One-week survival of the PGE1 group was improved to 70% compared to that of the control group of 30%. Postischemia reperfusion values of portal and peripheral tissue blood flows in the PGE1 group were 6.33 +/- 0.600 ml/min and 27.2 +/- 23.5 (arbitrary), and were significantly different from those of the control group of 4.34 +/- 0.400 ml/min and 23.5 +/- 5.54 (arbitrary), respectively. There was no significant difference in hepatic arterial flow between the two groups. Serum alkaline phosphatase decreased significantly in the prostaglandin group. Histological examination revealed a significant portal venous congestion in the control group 1 and 24 h after reperfusion. The extent of the sinusoidal congestion was also severe in the control group 24 h after reperfusion. It was concluded that PGE1 has a protective effect against liver damage when the liver was injured by warm ischemia and reperfusion followed by partial resection.

Preconditioning of primary rat neuronal cultures against ischemic injury: characterization of the `time window of protection'

Primary rat neuronal cultures can be preconditioned against ischemic damage by several mechanisms. In the present study we established a new model system in order to characterize the ’time window of protection’ obtained by preconditioning of neurons with adenosine. Ischemia was simulated by exposure of the cultures to iodoacetate (100 μM) for 150 min, with a post-ischemic reperfusion period of 60 min. Ischemic injury was assessed by the release of lactic dehydrogenase (LDH) to the medium during the ischemic period and ischemia-reperfusion damage by the Trypan blue exclusion test. Exposure of the neuronal cultures to the ischemic or ischemia-reperfusion insult resulted in severe damage to the neurons, manifested for the former insult in a 5.4-fold increase in the release of LDH and for the latter insult in an 8.5-fold increase in the proportion of stained cells by the Trypan blue exclusion test. Preconditioning by short exposure (5 min[ of the cultures to iodoacetic acid (simulating sublethal ischemia), or to adenosine (1 mM) and the A 1 adenosine receptor agonist N 6-(R)-phenylisopropyladenosine (R-PIA; 1 and 100 μM) prior to the insult, partially protected the neurons against the damage. The time-course of the development and waning of the resistance against the two insults following preconditioning exhibited different patterns. The resistance obtained against the ischemic insult developed rapidly, being maximal for all substances at 10 min (the shortest time window studied[, and lasted up to 1 h for iodoacetate, 3 h for R-PIA and 24 h for adenosine. In contrast, the protection induced by adenosine and R-PIA against ischemia-reperfusion injury developed relatively slowly, being maximal at 3 h, but lasted longer, up to 48 h. At this time the time-response curve exhibited a second peak of protection. The waning of protection against the two insults was found to continue into a period of increased sensitivity to the insults. This phenomenon was more intense for preconditioning with iodoacetate, and especially against the ischemic injury. The results suggest that in the neurons, different mechanisms may mediate the adenosine-induced preconditioning against the ischemic or ischemia-reperfusion injury. In addition, the results support the possibility that the relatively long ‘time window of protection’, induced by adenosine and R-PIA against ischemia-reperfusion insult, reflects a combination of two different preconditioning mechanisms.

Microvascular ischemia-reperfusion injury in striated muscle: significance of "reflow paradox".

Ischemia-reperfusion (I/R)-induced microvascular injury is characterized by capillary "no-reflow" and reflow-associated events, termed "reflow paradox," including leukocyte-endothelium interaction and increase in microvascular permeability. The major objectives of this study were 1) to elucidate the significance of reflow paradox after 4 h of tourniquet-induced ischemia in striated muscle and 2) to determine the role of reactive oxygen metabolites in the pathogenesis of reflow paradox-dependent microcirculatory alterations. By use of in vivo fluorescence microscopy in a striated muscle preparation of hamsters, leukocyte-endothelium interaction in postcapillary venules and macromolecular extravasation from capillaries and venules were quantified before ischemia and after 30 min, 2 h, and 24 h of reperfusion. I/R elicited marked enhancement (P < 0.01) of leukocyte rolling during initial reperfusion and a 20-fold increase of leukocyte adherence (P < 0.01) lasting for the entire postischemic reperfusion period (n = 7). These phenomena were accompanied by significant leakage (P < 0.01) of macromolecules from capillaries and in particular from postcapillary venules (n = 9). Both superoxide dismutase (SOD, 20 mg/kg body wt, n = 7) and allopurinol (50 mg/kg body wt, n = 7) were effective in attenuating I/R-induced leukocyte rolling and adherence. In addition, microvascular leakage was significantly reduced by allopurinol (n = 9) and completely abolished by SOD (n = 9) (P < 0.01). These results support the concept that reactive oxygen metabolites contribute to I/R-induced reflow paradox, resulting in leukocyte accumulation, adherence, and increase in microvascular permeability.

Effect of Inosine on Function and Adenine Nucleotide Content of the Isolated Working Rat Heart

Inosine added to the perfusion medium for isolated working rat heart induced a dose-dependent coronary vasodilator and positive functional effect. Inosine increased coronary flow (ml/min) from 11.8 +/- 0.5 (control, n = 16) to 15.6 +/- 0.6 (0.1 mM, n = 10), to 18.2 +/- 1.1 (0.5 mM, n = 6), and to 18.4 +/- 0.8 (1.0 mM, n = 14). Left ventricular systolic pressure was increased (LVSP, mm Hg) from 72.5 +/- 1.1 (control) to 75.4 +/- 1.7 (0.1 mM) to 78.7 +/- 2.1 (0.5 mM) and to 82.6 +/- 1.5 (1.0 mM). and cardiac output (CO, ml/min) from 26.4 +/- 2.0 (control) to 29.6 +/- 2.3 (0.1 mM), to 30.2 +/- 3.5 (0.5 mM), and to 37.4 +/- 2.0 (1.0 mM). At the end of 30-min reperfusion after a 15-min period of global total ischemia (n = 9), the functional parameters were significantly reduced (coronary flow 8.5 +/- 0.4 ml/min, LVSP 63 +/- 1.9 mm Hg, CO 13.6 +/- 1.9 ml/min). When inosine was present in the perfusion medium during the entire experimental protocol, coronary flow and heart function were also enhanced in a dose-dependent manner. The content of cardiac ATP (mumol/g) was decreased at the end of the 15-min ischemic period (1.89 +/- 0.17, n = 7; control 3.85 +/- 0.05, n = 4), and inosine had no effect. At the end of the 30-min postischemic reperfusion period, the ATP pool had recovered to an appreciable extent (3.01 +/- 0.11, n = 9) and was higher when 1.0 mM inosine had been infused (3.44 +/- 0.11, n = 7). Thus, inosine increased coronary flow dose dependently and, as a consequence, the function of the isolated perfused working heart both under control conditions and during the postischemic reperfusion period.

An apparatus for applying a mechanical massage to rat hearts inside a wide‐bore NMR spectrometer

A device for applying mechanical massage to the isolated perfused rat heart inside a wide-bore NMR spectrometer was developed. This device exerts a squeezing pressure on a fibrillating heart placed inside a NMR spectrometer without interfering with the NMR measurements. It appears that the apparatus is important for obtaining reliable results in the postischemic reperfusion period.

Attenuation of ischemic renal injury with fructose 1,6-diphosphate

Fructose 1,6-diphosphate (FDP) has been shown to attenuate tissue injury associated with ischemia and shock by enhancing the anaerobic carbohydrate utilization and by inhibiting oxygen-free-radical generation by the neutrophils. Previously, we have reported that FDP prevents ischemic renal failure if administered prior to the ischemic insult. The present study was designed to determine whether this agent could prevent renal damage when administered during the postischemic reperfusion period. Rats were subjected to 30 min of bilateral renal artery occlusion and infused with FDP (350 mg/kg body wt) beginning 10 min after release of the renal artery clamps. Control rats received an equal volume of glucose/saline solution. A third group of rats were sham operated. Twenty-four hours after injury, BUN, creatinine, and fractional sodium excretion values were less in FDP-treated rats than in control rats ( P < 0.001, P < 0.005, and P < 0.001, respectively) and not different from values observed in sham-operated rats. Inulin clearance was greater ( P < 0.001) in FDP-treated rats than in control rats (665 ± 38 μl/min/g kidney wt). Renal histology was also better preserved in the FDP-treated group. These data suggest that FDP infused after the initiation of an acute ischemic insult provides significant, but not complete, functional and histologic protection from renal damage.

Enhancement of crystalloid cardioplegic protection against global normothermic ischemia by superoxide dismutase plus catalase but not diltiazem in the isolated, working rat heart

Oxygen-derived free radicals and intracellular calcium overload have been implicated as mediators of myocardial ischemia/reperfusion injury. We hypothesized that free radical scavengers or calcium channel blockers could enhance the protection afforded the isolated, working rat heart by crystalloid cardioplegia against this type of injury at 37 degrees C. Hearts from 42 male rats in seven groups (n = 6) were studied in an isolated, working heart preparation measuring aortic flow (ml/min/gm dry wt), peak systolic pressure (mm Hg), coronary artery flow (ml/min/gm dry wt), and calculated coronary vascular resistance (dyne.sec.cm-5/gm dry wt). Creatine kinase and lactate dehydrogenase release were measured before ischemia and at various times during the postischemic reperfusion period. Time-matched control hearts (group 1) were perfused for 2 hours. After finding that 30 minutes of ischemia and 10 minutes of reperfusion (group 2) produced significant (p less than 0.01) functional impairment that was completely protected (group 3) by a preischemic bolus of St. Thomas' Hospital cardioplegic solution, we again found significant (p less than 0.01) functional impairment after 40 minutes of ischemia and 10 minutes (group 4) or 20 minutes (group 5) of reperfusion despite a preischemic bolus of St. Thomas' Hospital cardioplegic solution. Diltiazem (10 mg/L) plus St. Thomas' Hospital cardioplegic solution (group 6) did not significantly (p less than 0.01) enhance functional recovery. Addition of superoxide dismutase plus catalase (200 microns/ml) (group 7) produced marked improvement in functional recovery that did not differ significantly (p less than 0.01) from control results (group 1). The creatine kinase and lactate dehydrogenase data strongly supported the preceding functional data. Coronary flow and vascular resistance were not significantly (p less than 0.01) changed from control values in any group. We conclude that the addition of superoxide dismutase and catalase but not diltiazem to St. Thomas' Hospital cardioplegic solution can significantly enhance myocardial protection against normothermic ischemia/reperfusion injury. This implicates oxygen-derived free radicals as mediators of this type of injury.

Effect of dihydroergocristine on energy metabolism studied in the isolated perfused rat brain affected by ischemia and in neuroblastoma cells deprived of oxygen and glucose.

The effect of dihydroergocristine on energy metabolism was studied in the isolated perfused rat brain affected by ischemia and in cultivated C-1300 neuroblastoma cells deprived of oxygen and glucose. Creatine phosphate, ATP, ADP, AMP, glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, pyruvate, and lactate were measured enzymatically. After a perfusion period of 30 min, the cortex of the isolated perfused rat brain exhibited an energy state not different from that in vivo. Dihydroergocristine added to the perfusion medium (5 mumol/L) did not influence these substrate levels under normal perfusion conditions. However, this drug was able to retard the breakdown of high-energy phosphates during ischemia and to accelerate the restoration of the energy state during the postischemic reperfusion period. The perfusion rate was not changed by the drug, and therefore it was assumed that dihydroergocristine could act directly on cell metabolism. This view was supported by the results obtained from experiments using cultivated N-2a neuroblastoma cells. These cells were incubated in a buffered salt solution deprived of glucose and oxygen for 15 min. Under these conditions, dihydroergocristine (2 mumol/L) added to the incubation medium caused changes in the concentrations or the high-energy phosphates similar to those in the isolated brain preparation: It increased the ATP concentration and decreased the ADP concentration significantly.