Min Of Normothermic Ischemia Research Articles

Predicting functional recovery from ischemia in the rat myocardium.

Depletion of high-energy phosphates, accumulation of inorganic phosphate and intracellular acidosis have each been proposed as important events in the transition from reversible to irreversible ischemic injury. To assess whether each variable is predictive of functional recovery on reperfusion, these were measured in the isolated isovolumic rat heart using 31P NMR. Perfused hearts were subjected to either 10, 12 or 40 min of normothermic ischemia followed by 40 min of reperfusion. Hearts were then freeze-clamped for further analysis of phosphate metabolites by NMR and ion chromatography. High-energy phosphates, Pi, phosphomonoesters and pH were measured by 31P NMR spectroscopy at 2 minute intervals. Heart rate and developed pressure were monitored simultaneously. All hearts undergoing 10 min of ischemia and 40% of hearts subjected to 12 min of ischemia demonstrated good functional recovery. The remainder of hearts ischemic for 12 min went into contracture on reperfusion with little return of function. Hearts subject to 40 min of ischemia went into ischemic contracture and showed no recovery on reperfusion. Intracellular pH, [ATP], and [Pi] measured prior to reperfusion did not predict the extent of recovery. However, phosphomonoesters were detected prior to reperfusion in all hearts that did not recover well, but were not observed in hearts that showed good mechanical recovery. Analysis of tissue extracts by 31P NMR and ion chromatography indicated that the most prominent components of the phosphomonoesters were glucose 6-phosphate, alpha-glycerol phosphate and AMP. In conclusion, of the various phosphorus metabolites that can be measured by 31P NMR, only one group, the phosphomonoesters, was predictive of functional recovery.

Free radical metabolites in myocardium during ischemia and reperfusion

Low-temperature electron paramagnetic resonance (EPR) spectroscopy and spin traps were used to measure paramagnetic species generation in rat hearts and isolated mitochondria. The hearts were freeze-clamped at 77 K during control perfusion by the Langendorff procedure, after 20-30 min of normothermic ischemia or 10-30 s of reperfusion with oxygenated perfusate. All EPR spectra measured at 4.5-50 K exhibited signals of both mitochondrial free radical centers and FeS proteins. The analysis of spectral parameters measured at 243 K showed that free radicals in heart tissue were semiquinones of coenzyme Q10 and flavins. The appearance of a typical "doublet" signal at g = 1.99 in low-temperature spectra indicated that a part of ubisemiquinones formed a complex with a high potential FeS protein of succinate dehydrogenase. Ischemia decreased the free radical species in myocardium approximately 50%; the initiation of reflow of perfusate resulted in quick increase of the EPR signal. Mitochondria isolated from hearts during control perfusion and after 20-30 min of ischemia were able to produce superoxide radicals in both the NADH-coenzyme Q10 reductase and the bc1 segments of the respiratory chain. The rate of oxyradical generation was significantly higher in mitochondria isolated from ischemic heart.

Effects of Adenosine Infusion on the Pig Heart During Normothermic Ischemia and Reperfusion

Possible enhancement of myocardial protection during ischemia and reperfusion by administration of adenosine was evaluated in a pig heart model. Adenosine (100 micrograms/kg/min) was infused into the aortic root during ischemia in group AI (n = 5) and into the right atrium during reperfusion in group AR (n = 6). Group C (n = 6) served as controls. During cardiopulmonary bypass the hearts were subjected to 30 min of normothermic ischemia and 15 min of reperfusion before weaning. In group AI the stroke work index 30 and 90 min after ischemia and the mean arterial pressure 30 min after ischemia were significantly higher than in group C. These parameters did not differ significantly between groups AR and C. All groups showed decrease in myocardial adenosine triphosphate (ATP) and adenylate charge potential (ACP) during ischemia and partial (ATP) or complete (ACP) restoration after ischemia. Adenosine infusion into the aortic root during ischemia (adenosine cardioplegia) thus resulted in improved postischemic heart function, although biochemical correlates in ATP and ACP were not apparent.

Transient hypothermic reperfusion and postischemic recovery in isolated rat heart

The potential benefit of transient hypothermic reperfusion of the ischemic myocardium was investigated in isolated working rat hearts (n = 6/group) subjected to 25 min of global ischemia at 37 degrees C. Hearts were reperfused in the Langendorff mode at 5, 10, 20, 30, or 37 degrees C for 10 min plus 5 min at 37 degrees C before assessment of functional recovery (working mode). Compared with normothermic reperfusion (recovery of cardiac output = 42.3 +/- 6.1%), transient hypothermia failed to improve the recovery of cardiac output, which was 47.9 +/- 12.7 (P = NS), 54.3 +/- 11.5 (P = NS), 25.3 +/- 2.7 (P = NS), and 6.4 +/- 3.8% (P less than 0.05) in the 30, 20, 10, and 5 degrees C groups, respectively. Reduced recovery in the 5 degrees C group was reflected in increased creatine kinase leakage from 0.26 +/- 0.04 IU.ml-1.g dry wt-1 (37 degrees C reperfusion) to 0.62 +/- 0.12 IU. ml-1.g dry wt-1 (5 degrees C reperfusion; P less than 0.05). Brief periods (3 x 1 min) of hypothermic (5 degrees C) perfusion during normothermic Langendorff reperfusion (15 min) also reduced recovery of cardiac output to 12.1 +/- 7.2% (P less than 0.01). In additional studies, hearts were subjected to a 2-min preischemic infusion with the St. Thomas' Hospital cardioplegic solution before either 25 or 35 min of normothermic ischemia and reperfusion with transient hypothermia at 5, 10, 20, or 30 degrees C. Once again hypothermic reperfusion failed to improve recovery but detrimental effects were not observed in the 5 degrees C group. These results indicate no beneficial effect of transient hypothermic reperfusion on recovery of function measured following global normothermic ischemia.

Ischemia and reperfusion increase 125I-labeled endothelin-1 binding in rat cardiac membranes

The effect of aerobic perfusion, of up to 90 min global ischemia, and of reperfusion on 125I-labeled porcine endothelin (125I-ET-1) binding site density (Bmax), affinity (KD), and selectivity was investigated using cardiac membranes harvested from adult Sprague-Dawley rats. Membranes from nonperfused hearts bound 125I-ET-1 with a Bmax of 117.0 +/- 8.8 fmol/mg protein, a KD of 0.076 +/- 0.005 nM, and a Hill coefficient approaching unity. Neither aerobic perfusion nor 10 min of normothermic ischemia and subsequent reperfusion altered these binding parameters. The extension of the ischemic episode increased Bmax (23, 32, 62, and 60% increase after 20, 30, 60, and 90 min ischemia, respectively). Reperfusion further increased Bmax (P less than 0.01 at 15 min reperfusion after 20 and 30 min ischemia, and after 30 min reperfusion following 60 min ischemia) without changing selectivity. Affinity was reduced after 60 (P less than 0.05) and 90 (P less than 0.01) min ischemia. Ischemia at 22 degrees C had no effect on the density or affinity of these binding sites.

Recovery after Cold Cardioplegic Arrest of Isolated Blood-Perfused Hearts Excised from Non-Anesthetized Pigs

An isolated blood-perfused pig heart model has been established in order to evaluate the recovery of hearts obtained from slaughterhouse domestic pigs avoiding anesthesia and direct experiments on animals. Eleven hearts subjected to 9 min of normothermic ischemia were infused with cold modified Bretschneider solution. After 180 min of cardioplegic-induced global ischemia (including 9 min of normothermic ischemia) 8 hearts were reperfused for 120 min. Left ventricular function (measured isovolumetrically by means of a balloon, and expressed as developed left ventricular pressure, positive and negative dP/dt) was stable during the whole reperfusion period. Lactate production was abolished after 25 min of reperfusion, while there was a small glucose extraction during the whole reperfusion period. Slight deterioration of the mitochondria was found during the induced cardiac arrest, however, reversing during the reperfusion. Thus, due to the stability of left ventricular function, improved metabolism and ultrastructure during the reperfusion period, the model with no use of laboratory animals, and without any influence of anesthesia, seems to be suitable for testing the pure effect on the performance of the left ventricle of drugs and substrates added to the reperfusate during the reperfusion period.

Adenosine-induced increase in myocardial ATP: are there beneficial effects for the ischaemic myocardium?

The adenosine triphosphate (ATP) content of isolated Langendorff-perfused rat hearts may be increased by more than 40% above the normal value by a 2-h perfusion with adenosine (15 mumol/l). This metabolic manipulation was used to investigate the hypothetical relationship between total tissue ATP content and ischaemia-induced contractile failure, ischaemic contracture and post-ischaemic functional recovery. Adenosine perfused hearts were submitted to 20 min of normothermic ischaemia and reperfused for 45 min with or without adenosine. Control experiments were performed with adenosine-free preischaemic perfusion. In identically designed experiments the tissue-protective effect of diltiazem (0.5 mumol/l) was determined and compared with the experiments with adenosine. At the end of 120 min of preischaemic perfusion, the ATP content of the adenosine treated hearts was 34.3 +/- 1.8 mumol/g dry weight (control = 23.6 +/- 1.9 mumol/g, p less than 0.01). After a period of 20 min of normothermic ischaemia, the ATP content of the adenosine hearts decreased to 13.3 +/- .4 mumol/g, whereas ATP fell to 8.3 +/- 1.6 mumol/g in the control hearts. The creatine phosphate (CP) levels of adenosine hearts were significantly lower than those of the control group before ischaemia, but did not show major differences following ischaemia. During ischaemia, the contractile activity measured via an intraventricular balloon catheter, as well as ischaemic contracture did not differ between the adenosine and control hearts. The inclusion of diltiazem into the perfusate significantly delayed the onset of contracture. After 45 min of reperfusion, ATP contents of adenosine and control hearts reached similar values (8.4 +/- 2.3 and 8.3 +/- 2.9 mumol/g, respectively). Inclusion of adenosine (15 mumol/l in the reperfusion perfusate of the adenosine experiments prevented a further decrease, but did not increase tissue ATP content. CP values of all groups showed a partial recovery upon reperfusion, they did not differ significantly. Contractile recovery was equal in all experimental groups except for the diltiazem treated hearts, which showed during the first 10 min of reperfusion an improved mechanical performance. It is concluded that total tissue ATP is not necessarily a good indicator of functional capabilities under conditions of normothermic ischaemia and reperfusion in the isolated rat heart.

Nicotine-Induced Calcium Overload During Postischemic Reperfusion

The excessive gain in Ca2+ that occurs when hearts are reperfused after prolonged periods of ischemia contributes to cell death and tissue necrosis. The following experiments were undertaken to determine whether nicotine, in a concentration equivalent to the peak concentration present in plasma after cigarette smoke inhalation, alters reperfusion-induced Ca2+ gain in isolated rat hearts. Nicotine (0.15 microgram/ml) failed to increase tissue Ca2+ during aerobic perfusion but increased Ca2+ gain during reperfusion after 30 (p less than 0.02) or 60 (p less than 0.02) min of normothermic ischemia. The increase of Ca2+ gain was independent of a nicotine-induced release of norepinephrine (NE) or an altered "reflow area", heart rate, force of contraction, or end-diastolic resting tension. Pretreatment for 3 days with anipamil (20 mg/kg), a long-acting calcium channel blocker, attenuated the reperfusion-induced Ca2+ gain after 30 min of global ischemia, and reduced (p less than 0.001) the nicotine-induced exacerbation of that gain, without altering tissue ATP or creatine phosphate (CP). Verapamil (1 X 10(-6) M) reduced (p less than 0.02) the nicotine-induced exacerbation of Ca2+ gain caused by reperfusion after 30 min of ischemia.

Reperfusion injury induced by augumented oxygen uptake in the initial reperfusion period: Possible efficacy of extreme hemodilution

This study was designed to investigate the relationship between myocardial oxygen consumption and oxygen-induced myocardial injury. Dog hearts were exposed to 40 min normothermic ischemia and then reperfused for 10 min with three oxygenated perfusates containing different hemoglobin concentrations and with moderate hemodilution. The experimental groups consist of a moderate hemodilution group (Group M) receiving 8 g/dl of hemoglobin, an extreme hemodilution group (Group E) given 4 g/dl of hemoglobin, and a hemoglobin-free autologous plasma group (Group F). Hearts in the non-ischemic control group (Group C) were also perfused with moderate hemodilution throughout. In Group C, Group M and Group E, the O2 tension of perfusates was maintained at about 100 mmHg and in Group F, at over 300 mmHg. Oxygen extraction at 5 min after reperfusion in Group C was 14%, in Group M and Group E less than 10%, but in Group F 70%. Oxygen consumption in Group F was more than twice that in Group E and surpassed that in Group C. Group M also showed a significant increase in oxygen consumption compared with Group E at 5 min after reperfusion. Among the ischemic groups, Group E showed improvement of left ventricular function almost comparable to Group C accompanied by a rapid decrease in myocardial lactate, improved preservation of myocardial adenine nucleotides and prevention of myocardial lipid peroxidation. In contrast, Group F showed persistently higher values of lipid peroxides and lactate, the poorest recovery of adenine nucleotides, and impairment of left ventricular function.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of antiproteases (aprotinin and gabexate-mesilate) on the postischemic vascular response of the kidney.

Both under physiologic conditions and after short-term ischemia (1-3 min) kallikrein and locally synthesized prostaglandins contribute to control renal blood flow. In the present experiment the antiproteases aprotinin and gabexate-mesilate were administered to rabbits in an attempt to show whether the hemodynamic changes produced by 60 min of normothermic ischemia, i.e., postischemic hyperemia and reduction of vascular resistance, were equally mediated by kallikrein. Vascular responses were evaluated by determining renal cortical blood flow using radioactively labelled microspheres, by calculating vascular resistances, and by measuring the diameters of renal medullary vessles. Kidneys exposed to normothermic ischemia and sham-operated control organs of animals treated with aprotinin (2 X 40,000 KIU/kg body weight) and gabexate-mesilate (2 X 7 mg/kg body weight) were compared with those of rabbits which were analogously operated, but did not receive any drugs. As the antiproteases used did not significantly affect the hyperemia and the reduction of renal cortical vascular resistance seen after 60 min of normothermic ischemia, these phenomena are apparently not mediated by kallikrein. Renal medullary vasodilatation (arteriolae rectae) was significantly lower under aprotinin than in untreated ischemic kidneys, and was not longer demonstrable in gabexate-mesilate-treated animals. Thus, the effect of gabexate-mesilate was superior to that of aprotinin. Whether the antiprotease action is due to an unspecific membrane-stabilizing, to a general enzyme-inhibiting, or to a specific kallikrein-inhibiting effect, is still unclear.

Effects of external renal hypothermia on postischemic hemodynamics.

In the rabbit model renal cortical blood flow after 60 min of normothermic ischemia is seen to rise significantly in the early reperfusion period. Under otherwise identical conditions this phenomenon is absent after hypothermic ischemia produced by surface cooling of the kidney and in sham-operated kidneys. It is still unclear whether postischemic hyperemia is a physiologic counterregulatory mechanism in response to 60-min normothermic ischemia of the rabbit kidney or a contributory factor in the development of acute postischemic renal failure.

A NEW SIMPLE METHOD FOR OPTIMAL STORAGE OF ISCHEMICALLY DAMAGED KIDNEYS

SUMMARY A new method was developed for sufficient normobaric oxygenation of kidneys during storage in solutions of intracellular ionic composition. Sufficiency of oxygenation by “retrograde oxygen persufflation” was controlled by estimation of cellular metabolic state. SUMMARY In dogs functional recovery of the kidney was tested following autotransplantation after 24 hr of hypothermic persufflation storage, with or without a preceding period of 30 min of normothermic ischemia, and compared with simple storage in Collins' solution or continuous perfusion with albumin solution. Kidney function immediately after transplantation and 24 hr later was determined by inulin and p-aminohippurate clearances for transplanted and contralateral normal kidneys simultaneously. Immediate functional recovery values after persufflation storage amounted to 56 and 46% of the normal contralateral kidney in p-aminohippurate and inulin clearances, respectively, and were similar to the results of continuous perfusion. Furthermore, even after 30 min of normothermic ischemia followed by 24 hr of persufflation storage, the clearance values reached 49 and 32% of the normal values for p-aminohippurate and inulin, respectively, immediately after transplantation. SUMMARY Thus, the method of persufflation storage comines the advantages of storage and continuous perfusion it is simple and cheap like the storage methods but is most effective even in ischemically damaged organs as continuous perfusion.