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Eur J Cardiothorac Surg 1999;16:639-646
© 1999 Elsevier Science NL

Ischemic preconditioning enhances donor lung preservation in the rabbit

Roberto I. Gasparria, Nicole C.P. Jannisa, Willem J. Flamenga, Toni E. Lerutb, Dirk E.M. Van Raemdoncka,b

a Center for Experimental Surgery and Anesthesiology, Katholieke Universiteit Leuven, Herestraat 49, B-3000 Leuven, Belgium
b Department of Thoracic Surgery, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium

Corresponding author. Tel.: +32-16-34-68-23; fax: +32-16-34-68-24
e-mail: dirk.vanraemdonck{at}uz.kuleuven.ac.be


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A
 References
 
Objective: Ischemic preconditioning achieved by brief periods of ischemia followed by reperfusion before a prolonged period of ischemia, is well known to reduce myocardial damage. We investigated whether ischemic preconditioning of the lung could also attenuate ischemia-reperfusion injury following pulmonary preservation. Methods: Transient ischemia of the right lung was achieved in rabbits (n=4 in each group) by occluding the main bronchus and pulmonary artery, followed by reperfusion according to a protocol that differed between study groups: group 1 (control), 45 min ventilation; group 2, 30 min ventilation, 5 min ischemia and 10 min reperfusion; group 3, three periods of 5 min ischemia and 10 min reperfusion; group 4, five periods of 3 min ischemia and 6 min reperfusion. Donor lungs were then flushed with a crystalloid solution followed by inflated storage at 37°C for 2 h. The function of the right lung was assessed during reperfusion for 2 h with homologous, diluted and deoxygenated blood in an isolated, pressure-limited, and room-air ventilated model. Results: Significant differences (P<0.0001) were observed between groups 1 and 2 vs. groups 3 and 4 in veno–arterial oxygen pressure gradient (29±6 and 24±6 mm Hg vs. 124±24 and 132±14 mm Hg, respectively), and in weight gain (88±13 and 98±13% vs. 44±9 and 29±3%, respectively) after 1 h of reperfusion, and in wet-to-dry weight ratio (15.5±1.5 and 14.3±0.4 vs. 10.1±1.6 and 9.0±0.8, respectively) at the end of reperfusion. No significant differences in any of these parameters were observed between group 1 vs. group 2 neither between group 3 vs. group 4. Conclusions: These data suggest: (1) That 15 min, but not 5 min of transient ischemia prior to pulmonary preservation can significantly reduce edema in the lung graft upon reperfusion, thus improving oxygenation capacity and (2) although not significant, this beneficial effect seems to be slightly better with more repetitive periods of transient ischemia. Further research is warranted to investigate whether ischemic preconditioning in the human organ donor may become a new strategy to protect lung tissue during a planned ischemic event as in pulmonary transplantation.

Key Words: Ischemic preconditioning • Lung transplantation • Organ preservation • Reperfusion


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Appendix A
 References
 
The primary goal of solid organ preservation prior to transplantation is to inhibit cellular metabolism and morphologic alterations in order to minimize structural damage and functional impairment that is associated with the obligatory ischemia and reperfusion of the organ. Inadequate preservation of the donor lung may result in acute ischemia–reperfusion injury which is characterized by increased pulmonary capillary permeability, pulmonary edema, impaired gas exchange, and sometimes right heart dysfunction following an acute rise in pulmonary vascular resistance.

In situ single flush perfusion with a cold crystalloid solution and donor core cooling, are currently the two main methods used for lung preservation [1]. Lungs preserved in this way can be transplanted safely after up to 6–8 h of cold ischemia. Nevertheless, there remains a significant incidence (13–35%) of primary donor lung failure even when the duration of pulmonary ischemia is brief [2]. In some cases, this may result in adult respiratory distress syndrome and may be rapidly fatal.

Current experimental research to improve the quality of preserved pulmonary grafts and thus the functional performance of the newly implanted lung in the immediate postoperative period, is focusing mainly on the optimal perfusate solution and on the use of new pharmacologic additives to the preservation solution [1]. In our laboratory, focusing on the possibility of transplanting lungs from circulation-arrested cadavers, we have been interested in the optimal condition of the pulmonary graft prior to preservation [3–6].

Ischemic preconditioning (IP) describes the phenomenon by which transient ischemia induces tissue protection against subsequent ischemia and reperfusion injury. This has been well investigated during myocardial ischemia [7], also in our laboratory [8], and proved to be a potent, endogenous protective strategy. Recently, the use of IP as a new potential modification of the condition during donor lung procurement has been investigated in a rat lung allotransplant model [9]. In that study, the authors did not look into the possible importance of the duration and the frequency of the ischemic period.

In the present study, using an isolated rabbit lung reperfusion model, we wanted to investigate the potential beneficial effect of repeated cycles of pulmonary ischemia in the donor on subsequent graft function following a longer ischemic period.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Appendix A
 References
 
2.1. Experimental groups
Twenty New Zealand white rabbits (mean weight 2518±58 g) were assigned to five groups of animals (n=4 in each group). In the baseline group, the right lung was reperfused immediately after cold flush and preparation. In the other four study groups’ lungs were further stored at 37°C for 2 h prior to reperfusion. The condition of the right lung inside the donor prior to cold flush differed between groups. In group 1 (control group), both lungs were ventilated for 45 min with no ischemic preconditioning (NIP). In group 2, after a 30-min period of ventilation, ischemia of the right lung was induced once (IP-1) by occluding the right main bronchus and pulmonary artery with a tourniquet for 5 min at end-tidal volume, followed by 10 min reperfusion and reventilation. In group 3, the right lung was subjected in an identical manner to three (IP-3) consecutive periods of 5 min ischemia and 10 min reperfusion. Finally, in group 4, the right lung was subjected to five (IP-5) consecutive periods of 3 min ischemia and 6 min reperfusion.



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Weight of the animals and heart–lung blocks were similar in all groups (Table 1).


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  		Table 1. Animal characteristics and ischemic times in all groupsa

 
2.2. Animal preparation
All animals received humane care in compliance with the European Convention on Animal Care. The study was approved by the Institutional Ethics Committee.

In every experiment, three rabbits were used, one as lung donor and two as additional blood donors. The animals were premedicated, tracheostomized, and ventilated as previously described in detail [3].

The chest was opened through a median sternotomy. Thymic tissue was excised. Pleural cavities were opened. Both superior caval veins, the inferior caval vein, the ascending aorta, and the main pulmonary artery were encircled by individual ligatures. Sodium heparine 700 IU/kg (Heparin Rorer 5.000 IU/ml, Rhône-Poulenc Rorer, Brussels, Belgium) was administered via a marginal ear vein.

After the experimental protocol had been completed in the lung donor, the main pulmonary artery and the inferior caval vein were cannulated with a 10-gauge catheter (Angiocath, Becton Dickinson Vascular Access, Sandy, UT) and secured by a pursestring in the right ventricular outflow tract and the right atrial appendage, respectively. The animal was then rapidly exsanguinated through the catheter in the inferior caval vein and autologous blood was collected. The ascending aorta was ligated and the pulmonary artery was isolated from the right ventricle by ligature around the tip of the catheter, just distal to the pulmonary valve creating pulmonary ischemia. Both lungs were then flushed in situ by gravitational pressure of 30 cm H2O via the pulmonary artery catheter with cold (4°C) modified Krebs–Henseleit bicarbonate buffer solution (composition in mmol/l: NaCl, 118; NaHCO3, 25; KCl, 5.6; CaCl2, 2.9; MgCl2, 0.6; NaH2PO4, 1.2; and D-glucose, 11; pH, 7.4; osmolarity, 321 mOsm/l) and topically cooled with ice-cold (1°C) saline. The tip of the left atrial appendage was transected to allow free drainage of the flush solution. Ventilation was continued to permit perfusion.

2.3. Preparation of heart–lung block
The remaining ligatures were then tied. The heart–lung block was excised from the cadaver and stored in cold (4°C) saline solution during further preparation. A side whole drainage catheter was inserted into the left atrium through a pursestring suture in the apex of the left ventricle. The left atriotomy was closed using a running 5/0 non-absorbable monofilament suture. The hilum of the left lung was ligated. Both the endotracheal cannula and both catheters remained in place until reperfusion. The cold ischemic time was ±30 min and did not differ between all groups (Table 1).

The right lung in the baseline group was immediately reperfused. In the four study groups, the right lung was fully inflated with room air and the endotracheal cannula was clamped with end-expiratory pressure >30 cm H2O. The heart–lung block was then submerged in saline solution at 37°C for 2 h (Table 1).

2.4. Preparation and monitoring of the perfusate
Homologous blood was collected from two additional animals. The operative procedure was identical as described above except that a single catheter was placed into the inferior caval vein through the right atrial appendage, and the animal was exsanguinated.

Fresh venous blood was diluted (hemoglobin ±7 g/dl) and stored as previously described in detail [5,6]. The reperfusion circuit was primed and deaired. No significant differences in blood and perfusate volumes were noted between all groups (Table 2).


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  		Table 2. Perfusate characteristics in all groupsa,b

 
During the whole experiment, the temperature of the inflowing perfusate was recorded and samples were taken to monitor pH, arterial oxygen pressure, and the concentration of potassium and hemoglobin. No significant differences in any of these parameters were seen between all groups at the onset of reperfusion (Table 2).

2.5. Isolated reperfusion circuit
Our closed reperfusion circuit has been described in detail previously [5,6]. Briefly, the heart–lung block was suspended in a humidified and temperature-controlled (37–38°C) plexiglas chamber from a force displacement transducer. The right lung was perfused via the pulmonary artery catheter by constant gravitational pressure of 30 cm H2O via an overflow system. In this experiment, pulmonary venous effluent was drained freely into the blood reservoir via a bubble trap keeping the outflowing pressure constant at 0 cm H2O. The perfusate was recirculated using silicone tubing and a roller pump. It was then deoxygenated in a membrane gas exchanger using a gas mixture of 90% N2 and 10% CO2 and was rewarmed to ±36°C through a heat exchanger.

2.6. Reperfusion of heart–lung block
At the end of the 2-h ischemic period, the heart–lung block was suspended from the force transducer in the plexiglas box to measure the initial weight (40±1 g) of the heart–lung block (Table 1). Both pulmonary arterial and left atrial cannulas were connected to the silicone tubing and deaired.

The right lung was then briefly re-expanded with end-expiratory pressure >30 cm H2O and further ventilated with room air (respiratory rate=30 breaths/min; tidal volume=5 ml/kg body weight; PEEP=2 cm H20) during the whole experiment. Reperfusion was started at time zero after partial declamping of the inflowing line. After a stabilization period of 10 min, the clamp on the inflowing line was removed completely to allow full flow.

2.7. Assessment of graft function
Graft function was assessed during reperfusion by recording peak airway pressure (AwP), pulmonary artery pressure (PAP), left atrium pressure (LAP), pulmonary artery flow (PAF), and weight gain of the right lung ({Delta}W) as previously described [5,6]. Pulmonary vascular resistance (PVR) was calculated using the formula: PVR=((PAP-LAP)/PAF)x1000.

Blood samples of deoxygenated inflowing (PaO2) and oxygenated outflowing (PvO2) blood were taken for blood gas analysis (ABL 4 Radiometer A/S, Copenhagen, Denmark) at 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, and 120 min. Veno–arterial oxygen pressure gradient ({Delta}Pv-aO2) was calculated as an estimate of the oxygenation capacity of the right lung.

Reperfusion was continued for 2 h or until lung failure occurred defined as PAF less than 5 ml/min. The total perfusion time was recorded as lung survival time.

Wet-to-dry weight ratio (W/D) of the right lung was calculated as an estimate of the extent of lung edema as previously described [5,6].

2.8. Statistics
Values are expressed in cm H2O for AwP, in mmHg for PAP, in ml/min for PAF, in Wood Units (W.U.) for PVR, in % for {Delta}W and for lung survival, and in mmHg for {Delta}Pv-aO2. Data are presented as means±standard error of the mean.

Statistical analysis was performed using the software package Statistica 4.0 (Statsoft, Tulsa, OK). Differences within groups at successive time intervals of reperfusion and differences between groups were calculated using repeated measurements and/or factorial analysis of variance. Post-hoc pairwise comparison of curves for different groups was performed using the Newman–Keuls test. Values of P less than 0.05 were accepted as significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Appendix A
 References
 
3.1. Hemodynamics
Hemodynamic parameters in all groups are presented in Table 3. Following the onset of reperfusion, there was a gradual decrease in both PVR and PAP in all groups to reach a minimum around 50 min of reperfusion. Concomitantly, PAF increased to reach a maximum at this time point. This is the normal flow pattern that we have also observed in previous experiments using this reperfusion model allowing gradual adaptation to an increased vascular resistance following ischemia [5,6]. These differences within groups at successive time intervals of reperfusion were highly significant (P<0.001 for PAP and P<0.0001 for PAF and PVR). No significant differences, however, were observed between all groups for each of these variables (P=0.54 for PAP, P=0.24 for PAF, and P=0.06 for PVR). The interaction between time and group was not significant for any of the three parameters, indicating that the curves for PAP, PAF, and PVR were comparable in all groups (P=0.51 for PAP, P=0.92 for PAF, and P=0.83 for PVR).


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  		Table 3. Hemodynamic and aerodynamic parameters during 2 h of reperfusion in all groupsa,b

 
Graft survival after 2 h of reperfusion was 100% in all groups except in IP-3, where only three out of four lungs could be reperfused for longer than 100 min.

3.2. Aerodynamics
Values for peak airway pressure during reperfusion in all groups are also listed in Table 3. Except for the baseline group, there was an increase in AwP after the onset of reperfusion in all other groups, reflecting a decrease in static lung compliance as a result of edema formation. The difference in AwP within groups at successive time intervals of reperfusion was highly significant (P<0.0001). Differences between the study groups, however, did not reach statistical significance (P=0.09). The interaction between time and group was also significant (P=0.003). Post-hoc testing showed that this can be explained by the fact that the increase in AwP during reperfusion was higher in NIP and IP-1 when compared with the baseline group, IP-3 and IP-5.

3.3. Oxygenation capacity
The difference in partial oxygen pressure between deoxygenated inflowing and oxygenated outflowing blood in all groups is depicted in Fig. 1A. The difference in {Delta}Pv-aO2 within groups at successive time intervals was highly significant (P=0.004), reflecting a decline in oxygenation capacity with longer reperfusion time related to this ex-vivo model. The overall difference in {Delta}Pv-aO2 between groups was also highly significant (P<0.0001). After 60 min of reperfusion, oxygenation capacity in NIP and IP-1 (29±6 and 24±6 mm Hg, respectively; not significant for NIP vs. IP-1) was much inferior when compared with the other groups (139±10 mmHg in the baseline group, 124±24 mm Hg in IP-3, and 132±14 mm Hg in IP-5; not significant for baseline group vs. IP-3 and IP-5, and for IP-3 vs. IP-5 by post-hoc testing). The interaction between time and group was not significant (P=0.07), indicating that the decline in oxygenation capacity with longer reperfusion time was comparable in all groups.



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  		Fig. 1. Veno–arterial oxygen pressure gradient (A), weight gain (B), and wet-to-dry weight ratio (C) during 2 h of isolated blood reperfusion in lungs (n=4 in each group) following 30 min of cold (4°C) ischemia (baseline group) +2 h of warm (37°C) ischemia (other groups). The 45-min pre-ischemic period in the four study groups differed as follows: NIP (no ischemic preconditioning), 45 min ventilation only; IP-1, 30 min ventilation +1 period of hilar clamping (5 min ischemia and 10 min reperfusion); IP-3, three periods of hilar clamping (5 min ischemia and 10 min reperfusion); IP-5, five periods of hilar clamping (3 min ischemia and 6 min reperfusion). Values are presented as means±standard error of the mean. Overall differences between groups are presented. No significant differences were observed between NIP vs. IP-1 and between IP-3 vs. IP-5, in any of these parameters by analysis of variance (see text for details on post-hoc pairwise comparisons of curves/columns for different groups).

 
3.4. Edema formation
The difference in weight gain between all groups is depicted in Fig. 1B. In the baseline group, the weight of the heart–lung block increased during reperfusion by 9±3% at 5 min after the onset of reperfusion. This {Delta}W was the result of vascular distension by an increased blood volume through the right lung, and not from filtration of fluid out of the microvasculature into the lung interstitium. Thereafter, the weight in this group remained fairly stable during the whole experiment.

The difference in {Delta}W within groups at successive time intervals was highly significant (P<0.0001), reflecting edema formation in the ischemic groups during reperfusion, mainly within the 1st h. The difference in {Delta}W between groups was also highly significant (P<0.0001). After 60 min of reperfusion, edema formation was much more pronounced in NIP and IP-1 (78±13% and 97±13%, respectively; not significant for NIP vs. IP-1) when compared with the other groups (11±2% in the baseline group, 44±9% in IP-3, and 29±3% in IP-5; P=0.043 for baseline group vs. IP-3, not significant for baseline group vs. IP-5, and for IP-3 vs. IP-5 by post-hoc testing). The interaction between time and group was also highly significant (P<0.0001), reflecting the increase in weight during the 1st h of reperfusion being much higher in NIP and IP-1 vs. IP-3 and IP-5.

Finally, wet-to-dry weight ratio in all groups is presented in Fig. 1C. W/D after 2 h reperfusion of a single, non-ischemic right lung (baseline group) was 6.2±0.2. This value is similar to the one that we have measured after bilateral lung perfusion under the same conditions (6.8±0.2; not significant) (unpublished results: presented at the 3rd International Congress on Lung Transplantation, Paris, France, September 10–11, 1998).

The difference in W/D between groups was highly significant (P<0.0001). Post-hoc testing showed that the values for W/D in NIP and IP-1 (15.5±1.5 and 14.3±0.4, respectively; not significant for NIP vs. IP-1) were significantly higher when compared with IP-3 and IP-5 (10.1±1.6 and 9.0±0.8, respectively; not significant for IP-3 vs. IP-5). Also, W/D in the baseline group differed significantly from IP-3 (P=0.047) but not from IP-5 (P=0.07), suggesting that 15 min of intermittent pulmonary artery clamping is more beneficial after five repetitive periods compared with three periods.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Appendix A
 References
 
This study has shown that reperfusion injury in rabbit lungs after a 2-h warm ischemic period was less pronounced if the lung had been previously subjected to repetitive periods of short ischemia and reperfusion. These findings, therefore, suggest that the phenomenon of IP, well known in the myocardium, also exists in lung tissue.

In this experiment, we have also demonstrated that in the rabbit lung one brief period (5 min) of transient ischemia is not sufficient to protect against subsequent ischemia and reperfusion injury. The beneficial effect of IP was only noticed after an accumulation of the ischemic periods up to a total of 15 min. Furthermore, although in this study the differences between IP-3 and IP-5 did not reach statistical significance, the ischemia-reperfusion injury, (i.e. edema) appeared to be less pronounced in IP-5, suggesting that IP might be more effective with more repetitive periods of ischemia. Maybe with more experiments (n>4) in each group, the differences between both groups would have become significant.

Many studies have suggested that IP reduces the ischemia-reperfusion injury in solid organs such as the heart, liver, kidney, and bones. However, there are very limited data about the similar effects of IP on lungs. To the best of our knowledge, the phenomenon of IP in lung tissue has only been reported twice. In the first study using a rat allotransplant model [9], IP was achieved by a single 5-min period of pulmonary artery and main bronchus occlusion followed by 10 min of reperfusion prior to a longer period (6 or 12 h) of cold (0°C) ischemia. The authors reported a beneficial effect after IP on gas exchange (after 12 h) and also observed a reduced level of oxygen free radical damage (after 6 and 12 h) in the transplanted graft. They speculated that repeated cycles of ischemia and reperfusion might have augmented the beneficial effect of IP in their model. In the second study using isolated guinea pig lungs perfused with a crystalloid solution [10], IP was applied by stopping the inflow for 5 min followed by 5 min of reperfusion, repeated twice followed by 3 h of normothermic ischemia before restarting the crystalloid perfusion. The authors claimed a protective effect on ischemia–reperfusion injury from observing both a lower postischemic increase in pulmonary artery pressure and in tissue markers of lipid peroxidation in the experimental group, compared with the non-preconditioned, control group.

Liu and Downey found that three cycles of 5 min of ischemia and reperfusion were required to delay infarction in the rat heart, whereas a single cycle was ineffective [11]. On the other hand, other studies of myocardial IP in the rat [12] and other species [8,13,14] have found that a single cycle of ischemia/reperfusion was as effective as multiple cycles.

We have chosen a period of 2 h warm ischemia to study the effect of IP because from previous experiments we already concluded that the warm ischemic tolerance in a deflated lung was limited to 1 h [6]. The severe ischemia–reperfusion injury seen in the control group (NIP) was, therefore, not unexpected. In further experiments, we also investigated the effect of IP following a long period of cold ischemia using the same study groups. However, using cold (4°C) low-potassium dextran (LPD) as a flush and storage solution for oxygen-inflated lungs during 24 h, we were unable to demonstrate any benefit of IP in this isolated reperfusion model. Oxygenation capacity was excellent in all four study groups after 24 h of cold ischemia (116±17 mm Hg in NIP, 122±10 mm Hg in IP-1, 143±9 mm Hg in IP-3, and 160±11 mm Hg in IP-5 for {Delta}Pv-aO2 after 1 h of reperfusion; not significant) and edema formation was mild (27±9%, 25±8%, 15±4%, and 27±5% for {Delta}W after 1 h of reperfusion and 7.5±1.2, 7.4±0.6, 6.3±0.2, and 7.5±0.7 for W/D after 2 h of reperfusion, respectively; not significant). This is probably the result of the use of LPD for cold flush and storage. This extracellular solution (composition in mmol/l: NaCl, 103; Na2HPO4, 32.5; KH2PO4, 4; MgSO4(7H2O), 2; dextran-40, 0.5; pH, 7.4; osmolarity, 292 mOsm/l) may well preserve the lungs during long ischemic periods as previously reported by several authors [15–19]. Maybe, if the cold ischemic period had been prolonged to 36 or 48 h, a difference in ischemia–reperfusion injury might have been observed between all four study groups.

The phenomenon of IP was first described in myocardium by Murry and co-authors in 1986 [20]. Today, the mechanism of this tissue adaption to repeated stress has not yet been completely elucidated. In a recent review on cardiac preconditioning, stress hormones like adenosine and norepinephrine and an increased level of intracellular calcium were propagated as signaling mechanisms in acute preconditioning leading to activation of protein kinase C, ecto-5'-nucleotidase, and KATP channels. Delayed preconditioning (>24 h after preconditioning stimulus) requires de novo synthesis of heat shock proteins and/or antioxidant enzymes such as catalase and gluthathione reductase [7].

In summary, this study has shown that: (1) 15 min, but not 5 min of transient ischemia prior to pulmonary preservation can significantly reduce edema in the rabbit lung graft upon reperfusion, thus improving oxygenation capacity, and (2) although not significant, this beneficial effect seems to be slightly better with more repetitive periods of transient ischemia.

From this study, therefore, we conclude that ischemic preconditioning enhances donor lung preservation in the rabbit. Further research is warranted to investigate whether ischemic preconditioning in the human organ donor may become a new potent strategy to reduce ischemia–reperfusion injury following lung transplantation.


   Acknowledgments
 
This work is supported by a grant from the Nationaal Fonds voor Wetenschappelijk onderzoek-Levenslijn, 1994, No. 7.0036.94. The authors wish to thank Peter Lemmens, Magda Mathys, Eddy Vandezande, and Kanigula Mubagwa, MD, PhD, for expert technical and secretarial assistance and Paul Herijgers, MD, PhD for his contribution in the statistical analysis.


   Footnotes
 
Presented at the 12th Annual Meeting of the European Association for Cardio-thoracic Surgery, Brussels, Belgium, September 20–23, 1998. Winner of the 1998 Young Investigator's Award of the European Association for Cardio-thoracic Surgery.


   Appendix A
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A
References
 
Conference discussion
Dr E. Wolner (Vienna, Austria): Do you have any idea what is the reason, that you get much better results in these experiments, where you do this ischemic preconditioning?

Dr Gasparri: My presentation time was too short to show one more slide explaining this mechanism (Slide). There are a lot of papers in the literature discussing the mechanism of ischemic preconditioning in myocardial ischemia. The exact mechanism, however, has not yet been completely elucidated. Many signals such as adenosine, norepinephrine and intracellular calcium leading to activation of protein kinase C, ecto-5'-nucleotidase and KATP channels have been identified. But this still remains hypothetical.

Mr R. Bonser (Birmingham, UK): Could I ask you for just a little more detail on your statistical methods comparing these groups of four animals in each group?

Dr Gasparri: We all ready reached statistical significance with only four animals in each group.

Mr Bonser: No. What methods did you use, which statistical method?

Dr Gasparri: I have another slide showing you our statistical method. (Slide)

Mr Bonser: Using these parametric methods, how did you establish a normal variation in four samples? How do you know that your data was parametric if you used parametric tests?

Dr Gasparri: Statistical evaluation was performed using one-way analysis of variance with four animals in each group. Differences between study groups were assessed by factorial analysis followed by Scheffe's multiple comparison test.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A
 References
 

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Received September 19, 1998; received in revised form August 30, 1999; accepted October 25, 1999.




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