Cycles Of Preconditioning Research Articles

Interference Screw Fixation of Soft Tissue Grafts in Anterior Cruciate Ligament Reconstruction: Part 2

Background Preconditioning of tendon grafts is believed to eliminate natural viscoelasticity of the tendons and prevent knee laxity after anterior cruciate ligament (ACL) reconstruction. Hypothesis Preconditioned ACL grafts maintain their initially set tension. Study Design Randomized experimental study. Methods Forty-two human anterior tibialis (AT) tendon grafts were subjected to either no preconditioning (group 1), cyclic preconditioning (group 2), or isometric preconditioning (group 3). The residual graft tension was then recorded immediately after the application of an initial graft tension of 80 N and fixation into tibia with an interference screw, as well as 10 minutes later. In another experiment, the residual graft tension was recorded 1, 10, and 60 minutes after 10 AT nd quadrupled hamstring tendon (HT) grafts alone (no fixation) had been subjected to isometric preconditioning (80 N). Results Immediately after screw insertion, the residual (AT) graft tensions were 79 ± 19 N, 100 ± 17 N, and 102 ± 15 N in groups 1 through 3, respectively. Ten minutes later, the corresponding values were 49 ± 16 N, 60 ± 11 N, and 64 ± 12 N. For the AT and HT grafts alone, the residual graft tensions were 67 ± 2 N and 67 ± 2 N, 45 ± 2 N and 46 ± 4 N, and 29 ± 3 N and 34 ± 5 N at 1, 10, and 60 minutes, respectively. Conclusions A steady decrease (–60% within 60 minutes after initial tensioning) occurs in the initially set tension of the soft tissue ACL grafts. Clinical Relevance Clinically applicable preconditioning protocols cannot eliminate the intrinsic viscoelasticity from ACL soft tissue grafts, and thus, the reasonableness of preconditioning per se is questioned in ACL reconstruction.

Effect of Ischemic Preconditioning Based on Different Epicardial Branching Patterns of the Left Coronary Artery in the Rabbit Heart

The aim of this study was to characterize the effect of increasing numbers of preconditioning cycles on the different branching patterns (bifurcation/trifurcation) of the coronary artery in the rabbit heart. Fifty-six NZW rabbits were assigned to a bifurcation group and subjected to 0, 1, 3, 5, or 7 (B0, B1, B3, B5, or B7 subgroup, respectively) cycles of preconditioning (PC) (5 min of regional ischemia plus 10 min of reperfusion/cycle) and this was followed by 45 min of sustained ischemia and 72 h of reperfusion; 16 rabbits were assigned to a trifurcation group and subjected to 0, 1, 3, or 5 (T0, T1, T3, or T5 subgroup, respectively) cycles of PC. The ratio of necrotic zone (NZ) to ischemic zone (IZ) was calculated. The bifurcation group showed higher mortality (28.6%) than the trifurcation group (0%). The volume of the ischemic zone (expressed as a percentage of volume of the left ventricle) and the volume of the necrotic zone were larger in the bifurcation group than in the trifurcation group. The ratio of the necrotic zone to the ischemic zone was significantly lower in the B5 and B7 subgroups than in the B0, B1, and B3 subgroups. In the trifurcation group, the ratio of the necrotic zone to the ischemic zone showed a diminishing tendency in the subgroups as the PC cycle number increased, but without statistical significance. Thus, in the trifurcation pattern of the rabbit coronary artery there could be little effect on preconditioning, but in the bifurcation pattern the number of preconditioning is recommended to be 5 to 7 cycles. In this regard, different branching patterns of the epicardial branching of the rabbit coronary artery should be considered when interpreting experimental results on ischemic preconditioning.

Involvement of reactive oxygen species in cardiac preconditioning in rats.

To date, the involvement of reactive oxygen species in ischemic preconditioning in vivo in rats is not clearly demonstrated. The aim of the present study was to determine whether N-(2-mercaptopropionyl)glycine (MPG), a cell-diffusible hydroxyl radical scavenger, and carnosine, a potent singlet oxygen quencher, could block protection afforded by a single cycle of ischemic preconditioning in vivo in the rat. An ESR study was first performed to validate in vitro the specific antioxidant properties of carnosine and MPG. In a second set of experiments, open-chest rats were subjected to 30 min of left coronary occlusion followed by 60 min of reperfusion. Preconditioning was elicited by 5 min of ischemia and 5 min of reperfusion. Neither MPG (1-h infusion, 20 mg/kg) nor carnosine injection (bolus, 25 micro mol/rat) affected infarct size. The infarct size-limiting effect of preconditioning was completely blunted by MPG, whereas carnosine did not alter the cardioprotection. It is concluded that free radicals and especially hydroxyl radicals could be involved in the adaptive mechanisms induced by a single cycle of preconditioning in vivo in rats.

A Syndrome of Joint Laxity and Impaired Tendon Integrity in Lumican- and Fibromodulin-deficient Mice

Lumican and fibromodulin regulate the assembly of collagens into higher order fibrils in connective tissues. Here, we show that mice deficient in both of these proteoglycans manifest several clinical features of Ehlers-Danlos syndrome. The Lum(-/-)Fmod(-/-) mice are smaller than their wild type littermates and display gait abnormality, joint laxity, and age-dependent osteoarthritis. Misaligned knee patella, severe knee dysmorphogenesis, and extreme tendon weakness are the likely causes for joint laxity in the double-nulls. Fibromodulin deficiency alone leads to significant reduction in tendon stiffness in the Lum(+/+)Fmod(-/-) mice, with further loss in stiffness in a Lum gene dose-dependent way. At the protein level, we show marked increase of lumican in Fmod(-/-) tendons, which may partially rescue the tendon phenotype in this genotype. These results establish fibromodulin as a key regulator and lumican as a modulator of tendon strength. A disproportionate increase in small diameter immature collagen fibrils and a lack of progression to mature, large diameter fibrils in the Fmod(-/-) background may constitute the underlying cause of tendon weakness and suggest that fibromodulin aids fibril maturation. This study demonstrates that the collagen fibril-modifying proteoglycans, lumican and fibromodulin, are candidate genes and key players in the pathogenesis of certain types of Ehlers-Danlos syndrome and other connective tissue disorders.

Effects of fasting and hypoxic preconditioning on the hypoxic-reoxygenated ventricular strips of the rat heart.

The investigation aimed to assess the effects of hypoxic preconditioning in right ventricle strips of fed and 24-h fasted rats, which display a fast fatty acid catabolism, and to ascertain whether these effects are associated with changes in the tissue levels of long-chain acylCoA and acyl carnitine and glycolytic activity. Strips were mounted isometrically in Krebs-bicarbonate solution with 10 mM dextrose and paced at 1 Hz. Strips were exposed to 30 min hypoxia and 60 min reoxygenation with or without a previous preconditioning cycle of 5 min hypoxia followed by a 10 min reoxygenation. During hypoxia the fasted rat strips underwent a greater contracture with respect to the fed group. Preconditioning reduced the contracture strength and accelerated the post-hypoxic recovery only in the fasted rat strips. Hypoxia evoked an increase in the acylCoA and acyl carnitine tissue-contents of the strips which reached higher levels in the fasted than in the fed rat groups. Preconditioning had no effects on the content of these metabolites. During hypoxia lactate output was lower in the fasted than in the fed rat strips and preconditioning abolished this decrease. These data suggest that the protective effects of hypoxic preconditioning occur in the heart tissue predisposed to the oxidation of fatty acid and can not be ascribed to changes in the accumulation of acylCoA and acyl carnitine but could be due, at least in part, to an activation of glycolysis.

Pre-conditioning activates adenosine utilization in a cost-effective way during myocardial ischaemia.

During pre-conditioning the interstitial concentration of adenosine, in contrast to lactate, presents a die-away curve-pattern for every successive episode of ischaemia. This die-away pattern might not necessarily be attributed to diminished adenosine production. The present study was undertaken to investigate whether pre-conditioning alters the metabolic turnover of adenosine as observed by the lactate production during ischaemia. Interstitial levels of metabolites in pre-conditioned (n=21) and non-preconditioned (n=21) porcine hearts were monitored with microdialysis probes inserted in both ischaemic and non-ischaemic tissue in an open chest heart model. Three subgroups perturbated with either plain microdialysis buffer (control), buffer containing adenosine (375 microM), or buffer containing deoxyadenosine (375 microM) were studied. All animals were subjected to 90 min of equilibrium microdialysis before 40 min of regional myocardial ischaemia and 120 min of reperfusion. Pre-conditioning consisted of four repetitive episodes of 10 min of ischaemia and 20 min of reperfusion. Significantly higher levels of inosine and lactate were found in the ischaemic tissue of the pre-conditioned subgroup receiving adenosine (P < 0.05) compared with the other two subgroups receiving deoxyadenosine and plain buffer, respectively. This difference was only valid for pre-conditioned ischaemic myocardium, and hence equal amounts of inosine and lactate were produced in the non-preconditioned ischaemic myocardium regardless of the presence of adenosine or deoxyadenosine. In the non-ischaemic myocardium baseline levels of metabolites were measured in all subgroups. Pre-conditioning favoured degradation of exogenous adenosine to inosine successively ending up in enhanced lactate production. This was probably because of the involvement of the hexose monophosphate pathway in the pre-conditioned ischaemic myocardium. This route may therefore be supplementary in energy metabolism as a metabolic flow can be started by adenosine ending up in lactate without initial adenosine 5'-triphosphate (ATP) investment. Utilization of adenosine in this way may also explain the successive die-away pattern of adenosine seen in consecutive pre-conditioning cycles.

Evidence for calcitonin gene-related peptide-mediated ischemic preconditioning in the rat heart

Previous studies have suggested that calcitonin gene-related peptide (CGRP) may play an important role in the mediation of ischemic preconditioning. In the present study, we examined the release of CGRP during ischemic preconditioning and the effect of preconditioning frequency on this effect in the isolated rat heart. Thirty minutes of global ischemia and 40 min of reperfusion caused a significant cardiac dysfunction and an increase in the release of creatine kinase (CK) during reperfusion. Preconditioning with one, two or three cycles of 5-min ischemia and 5-min reperfusion caused a marked improvement of cardiac function and a decrease in the release of CK, and there was no difference in the degree of improvement among groups. The protective effects of ischemic preconditioning were abolished by the CGRP receptor antagonist CGRP 8-37. A single preconditioning cycle induced a significant increase in the release of CGRP in the coronary effluent. In the hearts treated with two or three preconditioning cycles, the level of CGRP was highest in the first cycle, and was gradually decreased with increasing number of cycles of preconditioning. These results suggest that the protective effects of ischemic preconditioning are mediated by endogenous CGRP in the isolated rat heart.

Mechanical tensile properties of the quadriceps tendon and patellar ligament in young adults.

We analyzed mechanical tensile properties of 16 10-mm wide, full-thickness central parts of quadriceps tendons and patellar ligaments from paired knees of eight male donors (mean age, 24.9 years). Uniaxial tensile testing was performed in a servohydraulic materials testing machine at an extension rate of 1 mm/sec. Sixteen specimens were tested unconditioned and 16 specimens were tested after cyclic preconditioning (200 cycles between 50 N and 800 N at 0.5 Hz). Mean cross-sectional areas measured 64.6 +/- 8.4 mm2 for seven unconditioned and 61.9 +/- 9.0 mm2 for eight preconditioned quadriceps tendons and were significantly larger than those values of seven unconditioned and seven preconditioned patellar ligaments (36.8 +/- 5.7 mm2 and 34.5 +/- 4.4 mm2, respectively). Mean ultimate tensile stress values of unconditioned patellar ligaments were significantly larger than those values of unconditioned quadriceps tendons: 53.4 +/- 7.2 N/mm2 and 33.6 +/- 8.1 N/mm2, respectively. Strain at failure was 14.4% +/- 3.3% for preconditioned patellar ligaments and 11.2% +/- 2.2% for preconditioned quadriceps tendons (P = 0.0428). Preconditioned patellar ligaments exhibited significantly higher elastic modulus than preconditioned quadriceps tendons. Based on mechanical tensile properties analyses, the quadriceps tendon-bone construct may represent a versatile alternative graft in primary and revision anterior and posterior cruciate ligament reconstruction.

Ischemic Preconditioning and Arrhythmogenesis in the Rabbit Heart: Effects on Epicardium Versus Endocardium

The goals of this study were: (1) to determine if preconditioning protects against arrhythmias and contractile dysfunction, and if protection for these two endpoints occurs in parallel; and (2) to investigate the anti-arrhythmic action of preconditioning by examining its effect on electrical activity in epicardiumvendocardium. We monitored ECGs, epicardial and endocardial monophasic action potentials (MAP), left-ventricular developed presssure (LVDP) and end-diastolic pressure (EDP) in isolated rabbit hearts. Hearts were subjected to a 30-min test ischemia and 45 min of reperfusion. Preconditioning cycles (PC) consisted of 1–4 ischemic episodes (5 min each separated by 10 min of reperfusion) administered 30 min before the test protocol. The test ischemia caused ventricular fibrillation (VF) in 42% of non-PC hearts. One PC totally suppressed VF (0%). The incidence of VF was 30% in 2 PC, 72% in 3 PC and 47% in 4 PC hearts. A large rise in EDP occurred in non-PC and 1 PC hearts, and this rise was prevented by 2, 3 or 4 PC. None of the protocols improved post-ischemic recovery of LVDP or EDP. The test ischemia generated a large dispersion in MAP duration between epicardium and endocardium (39 ms), but this dispersion was markedly reduced after 1 PC (14 ms). In conclusion, our results demonstrate that 1 PC completely protects against ischemia-induced VF in rabbit hearts, whereas 2 or more PC are required to prevent the ischemia-induced rise in EDP. Thus, preconditioning against arrhythmias and contractile dysfunction does not occur in parallel. Our data also suggest that 1 PC may exert its anti-arrhythmic effect through reduction of the substrate for reentrant arrhythmias during ischemia (dispersion of repolarization) via effects on MAP changes in endocardium.

Effect of cyclic preconditioning on the tensile properties of human quadriceps tendons and patellar ligaments.

Preconditioning of soft tissues has become a common procedure in tensile testing to assess the history dependence of these viscoelastic materials. To our knowledge, this is the first study comparing tensile properties of soft tissues-before and after cyclic preconditioning with high loads. Sixteen quadriceps tendon-bone (QT-B) complexes and 16 patellar ligament-bone (PL-B) complexes from a young population (mean age 24.9 +/- 4.4 years) were loaded to failure with a deformation rate of 1 mm/s. Half of the QT-B and the PL-B complexes underwent 200 uniaxial preconditioning cycles from 75 to 800 N at 0.5 Hz before ultimate failure loading. High-load preconditioning was made possible by the development of a highly reliable and easy-to-use cryofixation device to attach the free tendon end. PL-B complexes were more influenced by preconditioning than the QT-B complexes. Ultimate failure load, stiffness at 200 N and stiffness at 800 N were significantly higher for PL-B complexes after preconditioning, while the structural properties of QT-B complexes exhibited no significant alterations. The values of the mechanical properties like Young's modulus at 200 N and 800 N were much higher for both preconditioned specimen groups. In addition, ultimate stress was augmented by preconditioning for PL-B complexes. Hysteresis and creep effects were highest during the first few loading cycles. More than 160 cycles were needed to reach a steady state. Beyond 160 cycles there was no further creep, and hysteresis was almost constant. Creep values were 2.2% of the initial testing length for the QT-B and 3.2% of the initial testing length for the PL-B complexes. The effect of cyclic preconditioning seems to be caused by progressive fiber recruitment and by alterations of the interstitial fluid milieu.

Lack of Adenosine Causes Myocardial Refractoriness

Objectives. This study sought to investigate the myocardial mechanisms causing refractoriness to ischemic preconditioning in pigs.Background. Ischemic preconditioning in the pig vanishes after 60 min and cannot be reinstated by a second cycle of brief coronary occlusions at this time point. Ischemic preconditioning has been shown to be mediated by adenosine A1-receptors. Because myocardial adenosine production during ischemia ceases as the number of repeated brief ischemic episodes increases, we hypothesized that this lack of adenosine may cause this myocardial refractoriness.Methods. In open chest pigs, ischemic preconditioning was achieved by repeated brief coronary occlusions. Myocardial adenosine content was assessed by high performance liquid chromatographic analysis of serial myocardial biopsy samples; infarct size (percent infarcted area of the area at risk) was determined using tetrazolium salts.Results. Ischemic preconditioning by two cycles of occlusion of the left anterior descending coronary artery (10 min) and reperfusion (30 min) decreased infarct size ([mean ± SEM] 40.4 ± 2.9%; control: 76.9 ± 1.8%, p < 0.001). Prolonging the second reperfusion period to 60 min caused ischemic preconditioning to vanish (79.0 ± 0.5%) and caused refractoriness to a second cycle of preconditioning (70.0 ± 2.0%). Myocardial adenosine content increased only during the first coronary occlusion (baseline: 110.9 ± 42.0 nmol/g dry weight; first coronary occlusion: 1,686.2 ± 244.1, p < 0.001) but not during subsequent coronary occlusions. In refractory myocardium, intramyocardial microinfusion of the adenosine A1-receptor agonist N6-cyclohexyladenosine (CHA [0.3 mmol/liter]) again decreased infarct size (27.4 ± 7.0%, p < 0.001 vs. control).Conclusions. Myocardial refractoriness may be caused by the inability to produce adenosine endogenously. In refractory myocardium, application of CHA reinduces cardioprotection.

Ischemic preconditioning in rat heart: no correlation between glycogen content and return of function.

We tested the hypothesis that glycogen levels at the beginning of ischemia affect lactate production during ischemia and postischemic contractile function. Isolated working rat hearts were perfused at physiological workload with bicarbonate buffer containing glucose (10 mmol/L). Hearts were subjected to four different preconditioning protocols, and cardiac function was assessed on reperfusion. Ischemic preconditioning was induced by either one cycle of 5 min ischemia followed by 5, 10, or 20 min of reperfusion (PC5/5, PC5/10, PC5/20), or three cycles of 5 min ischemia followed by 5 min of reperfusion (PC3 x 5/5). All hearts were subjected to 15 min total, global ischemia, followed by 30 min of reperfusion. We measured lactate release, timed the return of aortic flow, compared postischemic to preischemic power, and determined tissue metabolites at selected time points. Compared with preischemic function, cardiac power during reperfusion improved in groups PC5/10 and PC5/20, but was not different from control in groups PC5/5 and PC3 x 5/5. There was no correlation between preischemic glycogen levels and recovery of function during reperfusion. There was also no correlation between glycogen breakdown (or resynthesis) and recovery of function. Lactate accumulation during ischemia was lowest in group PC5/20 and highest in the group with three cycles of preconditioning (PC3 x 5/5). Lactate release during reperfusion was significantly higher in the groups with low recovery of power than in the groups with high recovery of power. In glucose-perfused rat heart recovery of function is independent from both pre- and postischemic myocardial glycogen content over a wide range of glycogen levels. The ability to utilize lactate during reperfusion is an indicator for postischemic return of contractile function.

Validation of a small punch testing technique to characterize the mechanical behaviour of ultra-high-molecular-weight polyethylene

The small punch or miniaturized disc bend test has been used successfully to characterize the ductility and fracture resistance of metals and ceramics with specimens measuring 0.5 mm in thickness. This study was performed to demonstrate the feasibility of performing small punch tests on implant grade ultra-high-molecular-weight polyethylene (UHMWPE). Large-deformation finite element simulations were developed and validated to explore the hypothesis that the macroscopic constitutive behaviour of UHMWPE may be inferred from a miniature specimen testing technique which can be used to characterize the ductility and work to failure for UHMWPE. The load-displacement curve was insensitive to cyclic preconditioning of the test specimen and only mildly sensitive to the loading rate. Furthermore, the initial slope of the small punch load—displacement curve was used to determine the elastic modulus of the UHMWPE with the help of the inverse finite element method. The ultimate goal of this research is to develop the capability to perform local measurements of material tensile and static fracture properties in as-manufactured, as-sterilized and as-retrieved UHMWPE components.

Preconditioning with Ischaemia Reduces Both Myocardial Oxygen Consumption and Infarct Size in a Graded Pattern

We tested the hypothesis that ischaemic preconditioning reduces pre-ischaemic energy demand and thereby reduces the energy supply–demand mismatch imposed by coronary artery occlusion. Experiments were performed in 52 open chest pigs anaesthetised with sodium pentobarbital. One or two cycles, each of 10 min LAD occlusion followed by 30 min reperfusion, served as the preconditioning stimuli. The degree of protection was evaluated by measuring infarct size (tetrazolium stain) as percentage of area at risk (fluorescent particles) after 45 min LAD occlusion followed by 2 h reperfusion. One preconditioning cycle reduced regional myocardial oxygen consumption (MVO2) by 15±3% (P<0.05), whereas two cycles of preconditioning reduced MVO2by 25±3% (P<0.05vone cycle). This reduction was probably due to reduced energy demand, as both coronary blood flow and arteriovenous oxygen differences decreased, without lactate release or reduction in peak hyperaemic flow response. Energy requirements were most likely also reduced during ischaemia since repayment of flow debt after the second ischaemic period was 33±7% less than after the first ischaemic period (P<0.001). One preconditioning cycle reduced infarct size from 58±3% of area at risk to 40±5% (P<0.05), whereas two cycles of preconditioning reduced infarct size to 15±4% of area at risk (P<0.05vone cycle). We conclude that preconditioning with ischaemia reduces energy consumption in a graded pattern. This effect may contribute to the graded protective effect of ischaemic preconditioning.

Role of kinins in the cardioprotective effect of preconditioning: study of myocardial ischemia/reperfusion injury in B2 kinin receptor knockout mice and kininogen-deficient rats.

Kinins acting on the B2 receptor appear to be involved in the cardioprotective effect of preconditioning on myocardial ischemia/reperfusion injury. We tested the hypothesis that in mice lacking the gene encoding for the B2 kinin receptor (B2 knockout mice; B2-KO) as well as in rats deficient in high-molecular-weight (HMW) kininogen (Brown Norway Katholiek rats; BNK), the cardioprotective effect of preconditioning is diminished or abolished. 129SvEvTac (SV129) mice and Brown Norway rats (BN) served as controls. We confirmed that plasma HMW kininogen in BNK rats was 100-fold lower than in BN and 140-fold lower than in Sprague-Dawley rats (33+/-4 versus 1814+/-253 and 2397+/-302 ng/mL, P<.01). Each strain of mice was divided into (1) controls (without preconditioning); (2) one cycle of preconditioning (3 minutes ligation and 5 minutes reperfusion); and (3) three cycles of preconditioning. Each strain of rats was divided into (1) controls; and (2) three cycles of preconditioning. All animals were subjected to 30 minutes of ischemia and 120 minutes of reperfusion. In SV129 controls, the ratio of infarct size to risk area (IS/AR) was 55.6+/-4.6%. One and three cycles of preconditioning reduced IS/AR to 38.6+/-3.2% and 31.1+/-2.3%, respectively (P<.05 and P<.01 versus control). This protective effect was absent in B2-KO mice: IS/AR was 54.8+/-2.9% in controls, 58.5+/-3.6% with one cycle of preconditioning, and 58.5+/-3.4% with three cycles. In BN rats without preconditioning, IS/AR was 84.7+/-3.9%; preconditioning reduced it to 61.6+/-3.4% (P<.01). In BNK rats, IS/AR was 87.1+/-4.8% in controls and 84.3+/-4.1% with preconditioning. Preconditioning also prevented reperfusion arrhythmias in BN but not BNK rats. Within species, risk area, mean blood pressure, and heart rate were similar between strains. We concluded that (1) preconditioning protects the heart against ischemia/reperfusion injury in mice and rats; (2) activation of prekallikrein, which in turn generates kinins from HMW kininogen, may contribute to the effect of preconditioning; and (3) an intact kallikrein-kinin system is necessary for the cardioprotective effect of preconditioning.

Multiple cycles of preconditioning cause loss of protection in open-chest rabbits.

Although single or multiple bursts of ischemic preconditioning have been shown to protect against myocardial necrosis, the frequency-dependency of the protection afforded by multiple stimuli has not yet been systematically studied in the anesthetized animal. The aim of the present study was to characterize the effect of increasing numbers of preconditioning stimuli (5 min ischemia plus 10 min reperfusion) in a collateral-deficient species in vivo. Rabbits were anesthetized and divided into groups (n = 5-7/group) which were subjected to 0, 1, 2, 4, 6 or 8 cycles of preconditioning (each comprising 5 min of regional ischemia plus 10 min reperfusion) followed by 45 min of regional ischemia and 2 h reperfusion. An additional group, serving as a control, was subjected to eight cycles of preconditioning without a subsequent 45 min of regional ischemia. Infarct size (I) and risk zones (R) were delineated with the aid of tetrazolium staining and fluorescent particles and the I/R ratio was expressed as a percentage. Mean infarct size in the control group which had no preconditioning was 59.8 +/- 4.0%. This was reduced to 22.2 +/- 2.9%, 19.5 +/- 4.1% and 23.3 +/- 3.4% by one, two and four cycles of preconditioning. However, when the number of preconditioning cycles was increased to six or eight protection declined with infarct sizes of 41.8 +/- 6.9% (P < 0.05 v zero preconditioning control group) and 47.1 +/- 7.6% (N.S. v zero preconditioning control group). In the group subjected to eight cycles of preconditioning without subsequent ischemia, infarction was not detected. In conclusion, in the anesthetized rabbit, beyond 4 cycles, protection against infarction diminishes with increasing numbers of preconditioning cycles.

Oxygen Radicals Released During Ischemic Preconditioning Contribute to Cardioprotection in the Rabbit Myocardium

Previous studies have proposed that oxygen radicals may play a role in the triggering of ischemic preconditioning. However, studies evaluating the effects of radical scavengers have yielded conflicting results, possibly because of differences in the number of preconditioning episodes used. The present study tested whether N-2-mercaptopropionylglycine (MPG) could block protection of both single and multiple episodes of preconditioning inin situandin vitrorabbit hearts. All hearts were subjected to 30 min of regional ischemia followed by reperfusion for 2 (in vitro) or 3 (in situ) h. Infarct size was measured by tetrazolium. Infarction in controlin situhearts was 37.5±3.5% of the risk zone. A single cycle of preconditioning (PC1), with 5 min ischemia/10 min reperfusion, reduced infarct size to 12.3±2.0% (P<0.05). Four cycles of preconditioning (PC4) were equally protective. MPG (1 mg/kg/min i.v.) alone had no effect on infarction but abolished protection afforded by PC1 (35.4±3.9%). However, MPG failed to block protection in the PC4 group. In isolated control hearts, infarct size was 31.1±1.8% and was reduced to 10.2±2.2% (P<0.05) by preconditioning. MPG (300μm) aborted protection. Infusion of hypoxanthine or xanthine oxidase separately in lieu of preconditioning had no effect on infarct size, but induced protection when combined (14.1±2.2%;P<0.05). Polymyxin B, an inhibitor of protein kinase C (PKC), abolished this protection (53.1±4.1%). In conclusion, oxygen radicals contribute to ischemic preconditioning in the rabbit and appear to do so via activation of PKC. The fact that MPG could not block protection by PC4 suggests that oxygen radicals act in concert with other triggers of preconditioning such as adenosine and bradykinin.

The Role of Protein Kinase C in Ischemic/Reperfused Preconditioned Isolated Rat Hearts

Protein kinase C (PKC) has been implicated in the preconditioning-induced cardiac protection in ischemic/reperfused myocardium. We studied the effect of PKC inhibition with calphostin C (25, 50, 100, 200, 400, and 800 nM), a potent and specific inhibitor of PKC, in isolated working nonpreconditioned and preconditioned ischemic/reperfused hearts. In the nonpreconditioned groups, all hearts underwent 30 min of normothermic global ischemia followed by 30 min of reperfusion. In the preconditioned groups, hearts were subjected to four cycles of ischemic preconditioning by using 5 min of ischemia followed by 10 min reperfusion, before the induction of 30 min ischemia and reperfusion. At low concentrations of calphostin C (25, 50, and 100 nM), the PKC inhibitor had no effect on the incidence or arrhythmias or postischemic cardiac function in the nonpreconditioned ischemic/reperfused groups. With 200 and 400 nM of calphostin C, a significant increase in postischemic function and a reduction in the incidence of arrhythmias were observed in the nonpreconditioned ischemic/reperfused groups. Increasing the concentration of calphostin C to 800 NM, the recovery of postischemic cardiac function was similar to that of the drug-free control group. In preconditioned hearts, lower concentrations (< 100 nM) of calphostin C did not change the response of the myocardium to ischemia and reperfusion in comparison to the preconditioned drug-free myocardium. Two hundred and 400 nM of the PKC inhibitor further reduced the incidence of ventricular fibrillation (VF) from the preconditioned drug-free value of 50% to 0 (p < 0.05) and 0 (p < 0.05), respectively, indicating that the combination of the two, preconditioning and calphostin C, affords significant additional protection. Increasing the concentration of calphostin C to 800 nM blocked the cardioprotective effect of preconditioning (100% incidence of VF). The recovery of cardiac function was similarly improved at calphostin C doses of 200 and 400 nM and was reduced at 800 nM (p < 0.05). With 200 and 400 nM of calphostin C, both cytosolic and particulate PKC activity were reduced by approximately 40 and 60%, respectively, in both preconditioned and preconditioned/ischemic/reperfused hearts. The highest concentration of calphostin C (800 nM) resulted in almost a complete inhibition of cytosolic (100%) and particulate (85%) PKC activity correlated with the abolition of preconditioning-induced cardiac protection. In conclusion, calphostin C protects the ischemic myocardium obtained from intact animals, provides significant additional protection to preconditioning at moderate doses, and blocks the protective effect of preconditioning at high concentrations. The dual effects of calphostin C appear to be strictly dose and "enzyme inhibition" related.

Effects of Ginkgo biloba extract and preconditioning on the diabetic rat myocardium

Effects of preconditioning and Ginkgo biloba extract (EGb 761) were studied in isolated nondiabetic and diabetic ischaemic and re-perfused rat hearts. Hearts were randomly divided into five groups in both the age-matched non-diabetic and the 8-week streptozotocin-induced diabetic groups: Group I, hearts were subjected to 30 min of global ischaemia followed by 30 min of re-perfusion; Group II, one cycle of preconditioning consisting of 5 min ischaemia and 10 min re-perfusion before the induction of 30 min of ischaemia and 30 min of re-perfusion; Group III, two cycles of preconditioning; Group IV, three cycles; and Group V, four cycles before the onset of 30 min ischaemia followed by 30 min of re-perfusion. Four cycles of ischaemic preconditioning resulted in a reduction of arrhythmias in non-diabetic rats. Thus, in non-diabetics, the incidence of ventricular fibrillation and tachycardia fell from 92% and 100% (no preconditioning) to 33% (p < 0.05) and 42% (p < 0.05), respectively. Four cycles of preconditioning failed to reduce the incidence of re-perfusion arrhythmias in diabetic subjects. Preconditioning reduced the formation of oxygen free radicals measured by electron spin resonance spectroscopy, but the recovery of cardiac function was low in all non-diabetic and diabetic preconditioned groups. EGb 761 at 25 and 50 mg/kg improved cardiac function in non-preconditioned and preconditioned non-diabetic and diabetic hearts. During re-perfusion in the four-cycle preconditioned non-diabetic and diabetic groups, the amount of free radicals was reduced approximately by 50 and 70% using 25 and 50 mg/kg of EGb 761, respectively. EGb 761 improved cardiac function after ischaemia in both non-preconditioned and preconditioned non-diabetic and diabetic rats. Our data suggest that diabetes could abolish the precondition-induced protection.

770-6 Effect of Ischemic Preconditioning and Udocaine Pretreatment on [Ca 2+ ] i in Rat Hearts

Recent data suggest that preconditioning attenuates the increase in free intracellular calcium ([Ca 2+ ] i ), [Na + ] i and [H + ] i during ischemia by reducing the stimulation of Na + -H + and Na + -Ca 2+ exchange. We evaluated the effects of 3 episodes of 5 minutes global ischemia followed by 5 minutes recovery on [Ca 2+ ] i during control perfusion (Co) and during suppression of the sodium influx (5 × 10 -6 M lidocaine; L) with surface fluorometry in Indo-1/AM-loaded isolated ferfused rat hearts. During the first ischemia. systolic and enddiastolic [Ca 2+ ] i were 130% higher in Co as compared to L. The lower [Ca 2+ ] i concentration in L-treated hearts resulted in better recovery of contractile function during recovery (left ventricular developed pressure Co vs L: p = 0.01). During repeated ischemia-recovery cycles peak [Ca 2+ ] i decreased significantly in Co and remained stable in L (Fig.1). mean % ± SEM; ★ : p = 0.05 vs systolic [Ca 2+ ] i during Co. Short cycles of ischemic preconditioning resulted in less [Ca 2+ ] i accumulation and improved hemodynamic recovery. Lidocaine pretreatment induced also calcium influx, mimicking the first episode of ischemic preconditioning.