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Eur J Cardiothorac Surg 2002;21:987-994
© 2002 Elsevier Science NL
a Crafoord Laboratory of Experimental Surgery, Karolinska Hospital, Stockholm, Sweden
b Department of Thoracic Surgery, Karolinska Hospital, Stockholm, Sweden
c Institute of Biochemistry, University of Tartu, Tartu, Estonia
d Clinic of Anaesthesiology and Intensive Care, Tartu University Clinics, Tartu, Estonia
Received 7 December 2001; received in revised form 21 February 2002; accepted 22 February 2002.
* Corresponding author. Crafoord Laboratory of Experimental Surgery, L6:00, Karolinska Hospital, S-171 76 Stockholm, Sweden. Tel.: +46-8-51774072; fax: +46-8-51773557
e-mail: guro.valen{at}cmm.ki.se
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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95% O2) protects against ischaemia-reperfusion injury in rat and mouse hearts. The present study investigated how oxygen concentration and duration of hyperoxic exposure influenced cardioprotection, and whether hyperoxia might induce delayed cardioprotection (after 24 h). Methods: Animals were kept in normal air or in a hyperoxic environment, and their hearts were isolated and Langendorff-perfused immediately or 24 h thereafter. Global ischaemia was induced for 25 min in rats and 40 min in mice, followed by 60 min of reperfusion. Infarct size was determined by triphenyl tetrazolium chloride staining. Results: In rats exposure to 95, 80, and 60%, but not to 40% of oxygen immediately before heart isolation and perfusion improved postischaemic functional recovery. Eighty or more percent of oxygen also reduced infarct size. A preconditioning-like effect could be evoked by 60 or 180 min of hyperoxia, giving both immediate and delayed protection. In the mouse heart protection could be induced by pretreatment for 15 or 30, but not by 60 min with 95% oxygen. The protective effect of hyperoxia in mice could be evoked in the immediate model only. Conclusions: Hyperoxia protects the isolated rat and mouse heart against ischaemia-reperfusion injury, but some species-different responses exist. The protection depends on both oxygen concentration in inspired air, and duration of hyperoxic exposure.
Key Words: Cardiac contractile function • Cardioprotection • Hyperoxia • Ischaemia-reperfusion injury of the heart • Myocardial infarction • Preconditioning
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Short episodes of ischaemia and reperfusion release reactive oxygen species (ROS) [4], and these highly reactive intermediates have been suggested as possible triggers or signal molecules of the preconditioning response [5]. Under normal physiological conditions ROS are generated in small amounts, and are neutralized by endogenous antioxidants [6]. During reperfusion of the postischaemic myocardium a relative excess of ROS is generated, inducing oxidative damage to lipids, carbohydrates, proteins and nucleic acids [7]. It has also been suggested that ROS may both indirectly, through activation of protein kinases [8], or directly lead to the activation of transcription factors [9], which in turn may trigger gene programs associated with tissue repair and protection [10]. It has been demonstrated that ROS may play a role in classic preconditioning [11], and also contribute to delayed protection [5].
We have previously demonstrated that short exposure to a high oxygen fraction induces a low-graded systemic oxidative stress in rats, and hyperoxia profoundly improves postischaemic myocardial contractile function and reduces infarct size, both as immediate and delayed protection [12]. We have also shown that hyperoxia protected postischaemic function and cell viability in hearts of atherosclerotic mice [13], but have not fully explored the applicability of protection in mice. Mice are particularly interesting since they provide the opportunity to investigate mechanisms and function in genetically altered animals.
The advantage of employing hyperoxia as a preconditioning model, as opposed to ischaemic preconditioning, is its direct and easy clinical applicability. However, we have so far not determined whether lower oxygen fractions or shorter exposure times than 60 min may evoke a similar protection as that evoked by 95% O2. Clinically, a lower inspired oxygen fraction may be more attractive and advantageous, as hyperoxia has potential toxic effects [6]. The aim of the present study was to investigate in rats whether lower concentrations of oxygen than 95% in inspired air were capable of evoking a preconditioning-like response, and furthermore, to study the impact of the duration of hyperoxic exposure on early and delayed cardioprotection in rats and mice.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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2.1. Isolated heart perfusion
All animals were purchased from B&K (Sollentuna, Sweden). Male Sprague–Dawley rats (250–300 g) were anaesthetized with midazolam (Dormicum®, Hoffman-La Roche, Lausanne, Switzerland, 1.5 mg/kg i.m.) and fentanyl-fluanisone (HypnormTM, Janssen Pharmaceutica, Beerse, Belgium, 0.1 mg/kg of fentanyl i.m. and 3.0 mg/kg of fluanisone i.m.). Heparin (200 IU) was injected into the femoral vein. Male C57BL/6 mice (22–25 g) were anaesthetized by intraperitoneal injections of midazolam (25.0 mg/kg) and fentanyl-fluanisone (1.0 mg/kg of fentanyl and 25.0 mg/kg of fluanisone). Heparin (500 IU) was injected into the peritoneum. The hearts were rapidly excised through a median sternotomy and placed in ice-cold Krebs–Henseleit buffer (NaCl 118.5 mmol/l, NaHCO3 25.0 mmol/l, KCl 4.7 mmol/l, KH2PO4 1.2 mmol/l, MgSO4·7H2O 1.2 mmol/l, glucose·H2O 11.1 mmol/l, CaCl2·2H2O 1.8 mmol/l) during preparation for aortic cannulation. The hearts were retrogradely perfused with gassed (5% CO2, 95% O2) Krebs–Henseleit buffer at a constant pressure of 55 mmHg for mouse hearts and 70 mmHg for rat hearts. The apparatus was water-jacketed to maintain a core temperature of the heart at 37 °C. Global ischaemia was achieved by clamping the inflow tubing.
2.2. Assessment of cardiac performance
Isovolumetric recordings of left ventricular systolic (LVSP) and end-diastolic (LVEDP) pressures were obtained by a balloon inserted into the left ventricle through the left atrium. In rats the recordings were performed by GOULD TA240 EasyGraf® recorder and pressures monitored by Hewlett-Packard patient monitor 78342A. In rat hearts LVEDP was set to 5 mmHg at the end of the stabilization period and the volume of the balloon was thereafter kept unchanged throughout the experiment. Coronary flow (CF) was measured by timed collections of the coronary effluent. Left ventricular developed pressure (LVDP) was calculated as LVDP=LVSP-LVEDP. Heart rate (HR) was evaluated from the pressure curves. In mice, the balloon was coupled to a graded threaded microsyringe (Hugo Sachs Electronik, March-Hugstetten, Germany), and inflated to obtain a LVEDP of 4–7 mmHg during stabilization. For recordings of electrocardiogram (ECG), the tips of two thin teflon-insulated platinum wires were scratched and inserted into the apex of the left ventricle and to the right atrium as ECG electrodes, and a metal clip was attached to the 20-gauge steel cannula as the reference electrode. The ECG was imported into a computer system (PCLAB, Astra Hässle AB, Mölndal, Sweden), which calculated HR as previously described by us [13]. CF was continuously measured using a funnel coupled to a force transducer with an automated air valve deflating after every 30 s. LVDP was calculated as described above.
2.3. Experimental protocol
Animals were kept in a hyperoxic environment for 15–30, 60, 120 and 180 min for rats, and for 15, 30 and 60 min for mice. Control animals were kept in the same cage breathing normal atmospheric air for corresponding time periods. The oxygen content in inspired air was continuously measured with a gas analyzer. Oxygen was delivered into the cage through a tube. Due to similar functional performance and small number of observations, hearts from 15 and 30 min hyperoxic rats have been included in one group. The hearts were excised immediately or 24 h later for Langendorff-perfusion. After 25 min of stabilization rat hearts were exposed to 25 min of global ischaemia, while mouse hearts were subjected to 40 min of global ischaemia, followed by 60 min of reperfusion in all experiments. Only hearts with CF of 1.0–4.0 ml/min in mice, or 8.0–16.0 ml/min in rats, HR of 280–480 beats/min in mice, or 240–360 beats/min in rats, and LVSP higher than 60 mmHg at the end of stabilization were included.
Respective to the treatment prior to heart isolation, the following groups were investigated.
2.3.1. Studies on oxygen concentration in rats
95% oxygen, n=10.
2.3.2. Studies on the duration of hyperoxic exposure
As 95% O2 was the most cardioprotective concentration, only this was selected for further studies on the duration of hyperoxic exposure and on delayed effects of hyperoxia, both in rats and mice.
2.3.2.1. Rats
95% hyperoxia, n=4. 95% hyperoxia, n=11. 95% hyperoxia, n=6. 95% hyperoxia, n=12.
2.3.2.2. Mice
95% hyperoxia, n=7. 95% hyperoxia, n=6. 95% hyperoxia, n=7.
2.3.3. Studies on delayed effects of hyperoxia
2.3.3.1. Rats
95% oxygen 24 h prior to perfusion, n=6. 95% oxygen 24 h prior to perfusion, n=13. 95% oxygen 24 h prior to perfusion, n=13.
2.3.3.2. Mice
95% hyperoxia 24 h prior to perfusion, n=5. All functional recordings were performed 5 min before global ischaemia, immediately before global ischaemia, and after 2, 5, 10, 15, 20, 30, 40, 50, and 60 min of reperfusion.
2.4. Determination of infarct size
At the completion of reperfusion hearts were sampled and frozen before being cut into 0.8–1.0 mm slices. The slices were incubated at 37 °C for 20 min in 1% triphenyl tetrazolium chloride (TTC: Sigma, St. Louis, MO), and fixed in 4% formaldehyde. The sections were visualized in a computer imaging system (LEICA Qwin, Leica Imaging Systems Ltd., Cambridge, UK), and infarct size was marked and calculated in Adobe Photoshop 5.0. The area of unstained (infarcted) myocardium was calculated as the percentage of the total myocardial area minus cavities. From each slice an image was obtained from both sides, and all calculations from one heart were averaged into one value for statistical analyses.
2.5. Statistics
Functional data for delayed effects of hyperoxia are presented as the mean±SD. For clarity, the data on graphs with heart function are shown as the mean±SEM. Infarct size is presented as box-plots with median and quartiles, and with whiskers as minimum and maximum values. Differences in the recovery of postischaemic haemodynamic parameters were tested by using two-way analysis of variance (ANOVA) with repeated measures on one factor, taking treatment as an independent factor, and time as an dependent factor. In the case of significant interaction, simple effects, i.e. effects of one factor holding another factor fixed, were examined. Planned comparisons between the groups across factor time were then performed, and P values thereafter corrected according to the Bonferroni procedure. Comparisons of infarct size were performed by one-way ANOVA. When significant P values were calculated, intergroup comparisons were performed with Duncan's posthoc test. P<0.05 was considered significant.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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95% O2 prior to the experiments, the postischaemic increase of LVEDP was reduced (P=0.04) (Fig. 1A) . A dose-dependent attenuation of LVEDP was observed after 80% (P=0.05), while 60% oxygen only tended to attenuate the increase of LVEDP. Pretreatment with 40% oxygen had no effect on LVEDP (Fig. 1A).
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95% (P=0.01), 80% (P=0.05) and 60% (P=0.01) oxygen attenuated the decrease (Fig. 1B). Forty percent oxygen did not significantly influence postischaemic LVDP.
3.1.3. CF
CF was reduced during reperfusion of normoxic control hearts. Pretreatment with 95% (P=0.03), 80% (P=0.01), and 60% (P=0.02) oxygen inhibited this reduction (Fig. 1C). However, exposure of animals to 40% of hyperoxia did not significantly influence postischaemic CF in isolated hearts.
3.1.4. LVSP and HR
There were no significant intra- or intergroup differences in postischaemic LVSP or HR (data not shown).
3.1.5. Myocardial infarct size
Infarct size as a percentage of total heart volume is presented in Fig. 2
. In hearts from normoxic control animals 31% of myocardial tissue was calculated as unstained by TTC after 60 min of reperfusion. Pretreatment with 95 and 80% oxygen reduced this area to 16 and 18%, respectively. Neither 60 nor 40% O2 reduced the extent of myocardial necrosis.
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95% hyperoxic exposure (Fig. 3A)
, while 15–30 and 120 min had no effect. In mouse hearts 15 (P=0.01) and 30 (P=0.03) min of hyperoxia reduced the increase of LVEDP during reperfusion, while 60 min had no effect (Fig. 3D).
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3.2.2. CF
Only pretreatment with 60 (P=0.02) and 180 (P=0.05) min of hyperoxia attenuated the postischaemic reduction of CF in rat hearts (Fig. 3C). In mouse hearts, there were no significant differences between the groups (Fig. 3F).
3.2.3. LVSP and HR
There were no inter- or intragroup differences in either mouse or rat hearts (data not shown).
3.2.4. Myocardial infarct size
In hearts from normoxic control rats approximately 45% of the myocardium was necrotic (Fig. 4A)
. Sixty minutes of hyperoxia reduced the infarct size to 22%. In normoxic control mouse hearts the infarct size was calculated as 37% (Fig. 4B). This was reduced to 25 and 24% by hyperoxic (95% oxygen) pretreatment for 15 or 30 min, respectively.
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95% oxygen) inhibited the increase of LVEDP during reperfusion. Postischaemic depression of LVDP was also attenuated by 60 (P=0.01) and 180 (P=0.04) min of hyperoxia. Reduction of CF (P=0.01) was attenuated by 60 min of hyperoxia only. LVSP and HR were not influenced by hyperoxia (data not shown). In mice there was no delayed protection after hyperoxia. However, mouse hearts perfused 24 h after pretreatment with 30 min of hyperoxia had a baseline CF higher than controls (P=0.02). This effect was not evident during reperfusion
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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95% of O2 was dependent on the duration of hyperoxic exposure. The present findings may indicate a bell-shaped response curve of exposure duration in both rats and mice, but this was not systematically investigated. However, with longer exposure the oxygen toxicity may dominate the responses. Interestingly, when the hearts from hyperoxic rats were isolated 24 h after the exposure, beneficial effects were still apparent, but this delayed protection was not evident in mice. This observation suggests that the mechanisms of immediate and delayed protection are different.
In our previous studies of cardioprotection by hyperoxia we have utilized only 95% oxygen [12,14]. However, there is a clinical concern of employing such a high oxygen fraction in the inspiratory gas. Although morphologic changes in the lungs of rat exposed to 100% of oxygen develop only after 40 h of exposure, the earliest symptoms of toxicity in man have been noted to appear after 12–16 h of hyperoxic breathing [6]. However, atelectasis have been observed after only a few minutes of breathing 100% oxygen [15]. In order to investigate whether ‘clinically acceptable’ oxygen concentrations were capable of mimicking the effects of ischaemic preconditioning, different oxygen concentrations were tested. Pretreatment with either 95, 80, or 60%, but not 40% oxygen, improved postischaemic contractile function of the rat heart, as measured by decreased LVEDP, increased LVDP, and improved CF during reperfusion. In addition, exposure to either 95 or 80% oxygen significantly reduced infarct size after ischaemia-reperfusion. Sixty percent oxygen had a lesser beneficial effect on cardiac function without influencing infarct size, and 40% oxygen had no beneficial effects. Consequently, 80% oxygen is the lowest concentration in our model that evokes the preconditioning-like protection in rat hearts.
We also investigated the functional response of the isolated heart to ischaemia-reperfusion injury after pretreatment with different durations of hyperoxic exposure. In rat hearts a preconditioning-like response was observed after hyperoxia for 60 and 180 min in both immediate and delayed models. Hyperoxia for 15–30 min did not evoke any protection, indicating that this may have been too short to induce the molecular and cell biology changes mediating protection. Surprisingly, 120 min of hyperoxia had no effect on postischaemic heart function. We have no explanation for this finding. One hour of hyperoxia induced the most powerful functional protection, and infarct size was profoundly reduced in this model. In mice different lengths of exposure were required for functional and infarct-limiting protection. One hour hyperoxia did not elicit functional protection in mouse heart, although we have previously found hearts of severely atherosclerotic mice to be protected by 1 h exposure [13]. Beneficial effects of hyperoxia could be demonstrated after 15 or 30 min pretreatment. However, in mice functional protection could be induced in the immediate model only. We may only speculate why a preconditioning-like response in two different animal species could be evoked by different durations of hyperoxic exposure. Generally, tolerance to hyperoxia varies widely among species, and factors that may modify tolerance to oxidants include weight, with smaller animals less resistant [6]. The mouse has a higher HR than the rat, and therefore may have an increased oxygen consumption relative to its size. Thus, short durations of hyperoxia which protected mouse hearts might not have induced changes in cellular redox status in rats. It is possible that both tolerance to ischaemia as well as adaptation to ischaemia may vary among species, as it has been demonstrated that tolerance to ischaemia may differ even among different strains of rat [16]. We have also utilized different global ischaemia times for two different animal species. Initial studies in our laboratory have demonstrated that mouse hearts appear to be more tolerant to global ischaemia. In order to obtain a marked functional depression during reperfusion, they need longer times of ischaemia.
The present study was designed with the emphasis on heart physiology and myocardial necrosis, and we did not investigate any possible mechanism(s) underlying the achieved cardioprotection. However, we have previously demonstrated that subjecting rats to 1 h hyperoxia induced a systemic low-graded oxidative stress [12]. This is in accordance with studies showing that low doses of exogenous ROS protects contractile function against ischaemia-reperfusion injury [17,18], or mimics the beneficial effects of ischaemic preconditioning in isolated perfused hearts [11]. ROS may induce activation of nuclear factor kappa B (NF
B), a transcription factor which may upregulate cytoprotective enzymes [10], or downregulate inflammation through upregulation of its inhibitor IB
as previously shown by us in this model [14]. A role for NFB in protection by ischaemic preconditioning has been suggested [19,20]. NFB is activated by normobaric hyperoxia [14,21], and pharmacological inhibition of NFB activation before hyperoxic exposure abolished the beneficial effects of hyperoxia [14]. Genes regulated by NFB are associated with proinflammatory effects [10], but NFB does also regulate potentially tissue protective enzymes such as inducible nitric oxide synthase, inducible cyclooxygenase, and manganese superoxide dismutase [22]. Hyperoxia induces inducible nitric oxide synthase expression, which may trigger delayed preconditioning [23].
Ischaemic preconditioning as a mode of myocardial protection has not become generally accepted in clinical practice. The main reason is that exposing individuals with cardiovascular disease(s) to ischaemia or hypoxia is emotionally unattractive and may be a significant risk factor as well. The present model of inducing a preconditioning-like protection by breathing hyperoxic gas is attractive in its simplicity and may easily be clinically applicable. Hyperoxia may influence all organs and a ‘whole body preconditioning’ may be the end result of hyperoxic exposure. This may have relevance not only to myocardial protection, but be important in all major surgery by providing increased endogenous defence in every organ. Such a concept is in agreement with recent findings showing that exposure to high oxygen fractions reduces the incidence of postoperative wound infections [24], and augments antimicrobial defences [25]. Hyperoxia may also assist in clarifying the mechanisms whereby we can increase the endogenous cell defence and produce a ‘pill the day before surgery’.
In conclusion, exposure of rats or mice to increased oxygen fractions in inspiratory gas induced functional protection of the heart, and reduced myocardial necrosis. The obtained cardioprotection was dependent on oxygen concentration and duration of hyperoxia. Some of the responses were species-dependent. Possible mechanisms underlying the hyperoxia-induced myocardial protection remain to be investigated.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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B-dependent mechanism (abstract). Eur J Anaesth 2001;18(Suppl 21):47.
B in tyrosine kinase signaling of p38 MAP kinase regulation of myocardial adaptation to ischaemia. FEBS Lett 1998;429:365-369.[Medline]
B in the cardioadaptive response to ischemia. Ann Thorac Surg 1999;68:377-382.B: a privotal role in asthma and a new target for therapy. Trends Pharmacol Sci 1997;18:46-50.[Medline]
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