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Interact CardioVasc Thorac Surg 2005;4:207-211. doi:10.1510/icvts.2004.091736
© 2005 European Association of Cardio-Thoracic Surgery
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Institutional report - Experimental

Ischaemic preconditioning causes increased myocardial vascular resistance but no myocardial contractility changes in pigs after OPCAB

Jette Scheby Berg1,*, Bekka Ozer Christensen1, Søren Aagaard1, Thais B.N. Christensen1, Daniel A. Steinbruchel2 and J. Michael Hasenkam1

1 Clinical Institute and Department of Cardiothoracic and Vascular Surgery, Aarhus University Hospital, Skejby Sygehus, Brendstrupgårdsvej 100, DK-8200 Arhus N, Denmark
2 Department of Thoracic and Cardiovascular Surgery, Copenhagen University Hospital, Rigshospitalet, Denmark

Received 22 June 2004; received in revised form 20 October 2004; accepted 1 November 2004

*Corresponding author. Tel.: +45 89495481; fax: +45 89496011.

E-mail address: jberg{at}ki.au.dk (J.S. Berg).


   Abstract
Top
 Abstract
1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 5. Conclusion
 Acknowledgments
 References
 
The coronary vascular resistance (CVR) after off pump coronary bypass (OPCAB) surgery has never been clarified and ways of preventing stunning are wanted. We wished to investigate the effect of ischaemic preconditioning (IPC) on the CVR and stunning in the postoperative period after OPCAB. In 20 pigs (80 kg), 7 piezo-electric crystals were placed on the left ventricle to assess global and local myocardial contractility. Coronary vascular resistance was measured as the relation between perfusion pressure and coronary artery blood flow. IPC was obtained in ten pigs with one period of 10 min of coronary occlusion, followed by 15 min of reperfusion and 20 min of ischaemia. Ten control animals were exposed to 20 min of ischaemia only. Reperfusion was allowed for 120 min. The CVR was higher in the IPC group (measured by the slopes of the curves in the reperfusion period) (IPC: –0.33 (CI: (–0.62)–(–0.03)); Control: 0.31 (CI: (–0.03)–(0.67)), P<0.005. There was no difference in the local myocardial contractility (slopes of the curves in the reperfusion period) (IPC: –0.004±0.01; Control: –0.005±0.01). In conclusion, there was no alteration in myocardial contractility, but increased CVR in the early reperfusion period in animals preconditioned before sustained ischaemia.

Key Words: Preconditioning; Coronary vascular resistance; Stunning; Pig


   1. Introduction
Top
 Abstract
 1. Introduction
2. Material and methods
 3. Results
 4. Discussion
 5. Conclusion
 Acknowledgments
 References
 
Alleviating stunning after bypass operations by use of preconditioning stimulus has gained clinical attention [2–4]. Stunning seems to be reduced by ischemic preconditioning (IPC) in the acute window of protection in sheep, and rodents [5], but might not have the same affect in pigs [6]. The positive effect of IPC on stunning in the delayed window of protection in pigs seems documented [7]. Whether regulation of the coronary blood flow mediated through changes in the coronary vascular resistance (CVR) is co-responsible is not entirely clear [8]. Regulation of the CVR mediated through the vascular tone after an OPCAB operation has, to our knowledge, never been investigated. Therefore we wanted to study the effect of 20 min of index ischaemia on the CVR and the effect of IPC on the CVR before index ischaemia, in a setting allowing both the blood flow and the perfusion pressure to change freely. We hypothesised that IPC had no effect on stunning, CVR and haemodynamics in the acute window [12]. The aim of the study was to investigate the effect of IPC on stunning, CVR and haemodynamics in a new large pig OPCAB model.


   2. Material and methods
Top
 Abstract
 1. Introduction
 2. Material and methods
3. Results
 4. Discussion
 5. Conclusion
 Acknowledgments
 References
 
All animals were treated according to the principles stated by the Danish Inspectorate of Animal Experimentation and this institution approved the present study. Twenty-four female pigs (75–90 kg), Danish Landrace/Yorkshire, comprised the study material. Anaesthesia was accomplished by continuous intravenous infusion of pentobarbital sodium (Mebumal®, SAD) 750–1000 mg/h during mechanical ventilation (Siemens Servo 900 C, Solan, Sweden) and controlled core body temperature. Through both external jugular veins and both common carotid arteries fluid filled catheter (Percutaneous Catheter Introducer, Scientific/SCIMED, Boston, MA, USA) was introduced to measure central venous pressure and mean arterial pressure, respectively (Edwards, Baxter, Chicago, IL, USA) and for drug administration. Through a catheter in the right carotid artery a 6 F micro tip pressure catheter (Millar Instruments, Houston, TX, USA) was placed in the left ventricle. The heart was exposed through a median sternotomy. Regional and universal myocardial function was assessed by sonomicrometry (Sonometrics Digital Ultrasonic Measurement System, Sonometrics©). Seven digital piezoelectric ultrasonic probes (2.0 mm) were implanted subendocardially as illustrated in Fig. 1. Distal to the second diagonal branch a segment of the LAD, was dissected free to allow positioning of a 2 mm transit time perivascular flow probe (Medi-Stim AS, Cardiomed, Norway) for coronary blood flow (CBFLAD) measurements. A fluid filled pressure line (CVC, Cook double lumen, COOK, Denmark) measured the coronary sinus pressure (CSP). Before induction of ischaemia, all animals received a bolus injection of 20,000 IE heparin (Heparin-LEO®, Denmark) to prevent thrombus formation. A bolus injection of pancuronium bromide (12 mg) (Pavulon®, Organon, Holland) was given in the reperfusion period in all the animals. Ventricular fibrillation for more than one minute dictated exclusion of the animal.



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  		Fig. 1. Crystal no. 1, 2, 3, and 4 covers the area at risk distal of the 2nd diagonal branch and is positioned in a rhomb formation. The circumflex artery supplies the posterior aspect of the left ventricle where crystal 5 and 6 are positioned. Crystal no. 7 is placed along the left anterior descending artery proximal to the 2nd diagonal branch. Crystal no. 1 and 5 provide the apex to basis axis and 6 to 7 provide the anterior to posterior axis when calculating the left ventricular volume by use of an ellipsoid 2-axis volume model.

 
The pig was allowed to stabilize for about 30 min after all instrumentation was completed. The protocol is depicted in Fig. 2. A small vessel clamp placed immediately downstream of the transit time flow probe induced myocardial ischaemia. After completion of all data acquisition the animal was euthanized under continued deep general anesthesia.



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  		Fig. 2. Study design. The intervention group was exposed to 10 min preconditioning ischeamia followed by index ischeamia after 15 min rest. The control group had the same 20 min index ischeamia without preceding ischemic preconditioning.

 
2.1. Data collection and analyses

The following data were collected on an 8-channel DAT recorder (TEAC type RD 180-T, TEAC Corporation, Musashino, Tokyo, Japan): Mean arterial pressure (MAP), left ventricular pressure (LVP), central venues pressure (CVP), coronary blood flow in the LAD (CBFLAD), and coronary sinus pressure (CSP) and further analysed on a PC.

The myocardial perfusions pressure (PP) and coronary vascular resistance (CVR) was computed as:


{207eq1}

(1)


{207eq2}

(2)

In a LAB-view® program (National Instruments, Austin, TX, USA) the dP/dt-max was determined from the LVP tracing. Analyses of Tau were made by the equation:


{207eq3}

(3)

As illustrated in Fig. 1, crystal 1–4 in area at risk, describes a square. Changes in the size of the area at risk (a) from the end diastole (ED) to the end systole (ES) describe the contractility in the ischaemic zone. The data analysis was blinded. The area changes ({Delta}A) were calculated as:


{207eq4}

(4)
(aED: area end diastolic; aES: area endsystolic). Three successive ED and ES were manually identified from the LVP curve. ED was defined as the onset of the rapid fall on the LVP curve and ES was defined as the onset of the rapid raise of LVP. All sonomicrometry crystal distances represent a mean from three heart cycles.

LVP versus wall area changes in area at risk was analysed [13] at baseline and after 120 min of reperfusion as an expression of the local work. The area described by the LVP and wall area changes was determined on a PC (UTHSCSA Image Tool. TX, USA) as the mean of the largest and the smallest area of three heart cycles.

In Fig. 1 crystal 5 and 6 are placed in the area supplied by the circumflex artery for measurement of segment shortening (SSLV).


{207eq5}

(5)

dED is the crystal distance at end diastole and dES the crystal distance at end systole.

A two axis ellipsoid model was used for calculation of the volume of the left ventricle at end-diastole and end-systole. Crystal no 1 and 7 was used to determine the apex to basis axis (AB_axis) and crystal no 6 and 7 was used to determine the anterior to posterior axis (AP_axis). The volume of the left ventricle was defined as:


{207eq6}

(6)

At baseline and at 120 min of reperfusion mean of the largest and the smallest of three heart cycles LVP-volume plot was made, and analysed as described for the wall-area versus LVP loops.

Data were tested with unpaired Student's t-test for equality of means between the two groups and with paired Student's t-test for equality of means within the groups. Data were expressed as mean±S.D. for non log-transformed data and as the geometric mean with confidence interval for log-transformed data. A value of P<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
4. Discussion
 5. Conclusion
 Acknowledgments
 References
 
Twenty animals completed the study, 10 in each group. Four animals died before randomisation because of technical problems. For baseline data see Table 1.


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  		Table 1 Baseline and general data

 
The CVR at baseline and in the reperfusion period is depicted in Fig. 3. There was a statistically significant difference in the slopes of the curves in the reperfusion period between the groups (IPC: –0.33 (CI: (–0.62)–(–0.03)); Control: 0.31 (CI: (–0.03)–(0.67)), (P<0.005). From baseline to 15 min of reperfusion the CVR increased (on a log scale) 43% (S.D. 33%) in the IPC group and decreased 40% (S.D. 55%) in the control group. This finding was highly significant with P<0.001, and in both groups the difference between baseline and 15 min of reperfusion was significant (IPC: (P<0.005); control: (P=0.045)). From 15 min to 120 min of reperfusion CVR decreased in the IPC group (on a log scale) 43% (S.D.±55%) and the CVR increased in the control group by 50% (S.D.±35%). Again the differences between the groups and within the groups were highly significant with P<0.001 between groups and P=0.048 and P<0.005 in the IPC and control groups, respectively.



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  		Fig. 3. Absolute coronary vascular resistance (CVR) at baseline and in the reperfusion period in both groups (see Fig. 2 for depiction). The 68% prediction interval for the control group is marked by (-----). The 68% prediction interval for the control group is marked by (....). There was no statistically significant difference at baseline between the groups. There was a statistically significant difference in the slopes between the groups (P<0.01). The difference between baseline and the data point at 15 min of reperfusion was statistically significant different between the groups (P<0.001) and in the group. There was no statistically significant difference between the difference between baseline and the end of reperfusion period in any of the groups.

 
Contractility of the area at risk between the two groups at baseline did not differ (Control; 0.28±0.06 and IPC; 0.26±0.06). Data from all pigs including mean values for each group are shown in Fig 4A and B. The slopes of the curves in the reperfusion period between the groups were the same (IPC: –0.04±0.01; Control: –0.005±0.01). There was a statistical significant decrease, though, between the baseline {Delta}A and the {Delta}A at 15 min of reperfusion in both groups (P<<0.0001) (IPC: 0.14±0.4; control: 0.11±0.07) but no difference between the groups. Also, between the baseline {Delta}A and the {Delta}A at 120 min of reperfusion in both groups a highly significant decrease was observed, P<<0.0001, (IPC: 0.17±0.03; control: 0.12±0.1), but none between the groups. At 120 min of reperfusion additional investigations in terms of LVP versus area-at-risk loops were performed. We found a reduction in local work compared to baseline in both groups described by the loops but no differences were observed between the groups (Control: 0.50±0.26; IPC: 0.41±0.08 (relative to baseline)).



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  		Fig. 4. {Delta}A, relative area changes in the IPC group. The ten pigs in the group are presented. The bold curve represents the mean value for all 10 pigs. 4B: {Delta}A, relative area changes in the control group. The ten pigs in the group are presented. The bold curve represents the mean value for all 10 pigs in the group. The data points from 0 to 44 min (not shown) represent the period where the IPC group is getting 10 min of ischeamia, 15 min of reperfusion and both groups are exposed to 20 min of index ischeamia. The data point at 44 min represents the contractility after 19 min of index ischeamia. The data at baseline and in the reperfusion period are then directly comparable between the groups. At any time point there is no statistically significant difference between the groups.

 
For data on regional myocardial function (SSLV) and haemodynamics, see Table 1 and 2.


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  		Table 2 Haemodynamic data

 
Global left ventricular function is presented in Table 3 in terms of CO [l/min], EF [%] and EDVrel. LVP-volume plot areas after 120 min of reperfusion did not differ: Control: 0.59±0.14; IPC: 0.67±0.20.


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  		Table 3 Global left ventricular function and local segment shortening (CX)

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
5. Conclusion
 Acknowledgments
 References
 
In an experimental human compatible OPCAB swine model we have demonstrated that IPC has no effect on regional stunning after 20 min of warm ischaemia, but the CVR was greater in the reperfusion period, when preceded by preconditioning ischaemia for 10 min.

Twenty minutes of warm ischaemia was chosen as a worst-case scenario of the time needed for establishing an anastomosis, which also was required to get an appropriate effect to study. Results from Calafiore et al. [12] support the assumption of 20 min of ischaemia, though the time required for making an anastomosis is shorter (Cartier [1]).

The CVR is the main factor regulating the coronary blood flow. CVR is influenced by blood viscosity, which was not measured directly in this study, but there was no difference in haematocrit between the two groups. The CVR could increase with increased myocardial wall tension but it is unlikely as both groups were equally stunned and no difference was registered in mean LVP between IPCs and controls. Increased CVR could not be explained by coronary artery stenoses in these healthy young pigs. The vascular tone is the dominating factor for CVR. It is determined by the metabolic status of the myocytes. Though the increased CVR in the IPC group is somewhat controversial, the myocardium must have been in better condition than the control animals since increased blood flow in the reperfusion period was much less in the IPC animals. Whether the length of the preconditioning ischeamia chosen in the present study I optimal is debateable. Ghosh et al. [16] have shown that one episodes of ischaemia in humans is superior to several short episodes of ischaemia. We chose ten minutes of IPC based on these considerations and results from an unpublished study in our department (made in female pigs) showed a 90% infarct size reduction induced by 10 min of preconditioning ischeamia. The stimulus might though have been too long when investigating resistance relations and might instead have induced an endothelial injury. Another preconditioning protocol with a shorter preconditioning stimulus would clarify that.

In rodents it appears that preconditioning ischemia does not protect females. In several studies performed in our department it has been possible to reduce the infarct size in female pigs by different preconditioning ischemia [25,26].

Ideally the animals in the control group should have waited 25 min corresponding to the time spent for IPC in the intervention group. For logistic reasons we chose to avoid the delay which we consider an insignificant potential contribution to recover/weaken the myocardium.

Regarding myocardial function in area at risk, our findings resemble data from other studies [6]. IPC does not seem to have any effect on stunning in pigs, but positive results regarding the effect of IPC on stunning have been found in rats [21]. Calculations of changes in the myocardial area covered by four crystals give a good description of the myocardial movement, taking into account that the myocardial fibres are not parallel in orientation. This large animal model (80 kg) though, had the advantage of placing the sonomicrometry crystals with some distance to each other covering a large part of the area at risk. When establishing this large model we found it reasonable to confirm the model by reinvestigating that IPC had no effect on stunning in the acute window.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 5. Conclusion
Acknowledgments
 References
 
We found no beneficial effect of ischaemic preconditioning with respect to stunning. We found a significantly increased coronary vascular resistance in the reperfusion period in the preconditioned group compared to control.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 5. Conclusion
 Acknowledgments
References
 
The Danish Heart Foundation, Elin Holms Foundation, Aarhus University Research Foundation, The Clinical Institute, Aarhus University Research Initiative, A. P. Møller and Chastine Mc-Kinney Møllers Foundation, Ib Henriksens Foundation.


   References
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 5. Conclusion
 Acknowledgments
 References
 

  1. Donato M, Gelpi RJ. Adenosine and cardioprotection during reperfusion – an overview. Mol Cell Biochem 2003;251:153–159.[CrossRef][Medline]
  2. Vinten-Johansen J, Zhao ZQ, Corvera JS, Morris CD, Budde JM, Thourani VH, Guyton RA. Adenosine in myocardial protection in on-pump and off-pump cardiac surgery. Ann Thorac Surg 2003;75:S691–S699.[Abstract/Free Full Text]
  3. Laurikka J, Wu ZK, Iisalo P, Kaukinen L, Honkonen EL, Kaukinen S, Tarkka MR. Regional ischemic preconditioning enhances myocardial performance in off-pump coronary artery bypass grafting. Chest 2002;121:1183–1189.[Abstract/Free Full Text]
  4. Toyoda Y, Di G V, Parker RA, Levitsky S, McCully JD. Anti-stunning and anti-infarct effects of adenosine-enhanced ischemic preconditioning. Circulation 2000;102:II326–III331.
  5. Tofukuji M, Metais C, Li J, Hariawala MD, Franklin A, Vassileva C, Li J, Simons M, Sellke FW. Effects of ischemic preconditioning on myocardial perfusion, function, and microvascular regulation. Circulation 1998;98:II197–II204.
  6. Bolli R. The late phase of preconditioning. Circ Res 2000;87:972–983.[Abstract/Free Full Text]
  7. Merkus D, Chilian WM, Stepp DW. Functional characteristics of the coronary microcirculation. Herz 1999;24:496–508.[Medline]
  8. Calafiore AM, Giammarco GD, Teodori G, Bosco G, D'Annunzio E, Barsotti A, Maddestra N, Paloscia L, Vitolla G, Sciarra A, Fino C, Contini M. Left anterior descending coronary artery grafting via left anterior small thoracotomy without cardiopulmonary bypass. Ann Thorac Surg 1996;61:1658–1663.[Abstract/Free Full Text]
  9. Zhu P, Lu L, Xu Y, Schwartz GG. Troglitazone improves recovery of left ventricular function after regional ischemia in pigs. Circulation 2000;101:1165–1171.[Abstract/Free Full Text]
  10. Cartier R. Current trends and technique in OPCAB surgery. J Card Surg 2003;18:32–46.[Medline]
  11. Ghosh S, Standen NB, Galinanes M. Preconditioning the human myocardium by simulated ischemia: studies on the early and delayed protection. Cardiovasc Res 2000;45:339–350.[Abstract/Free Full Text]
  12. Kharbanda RK, Peters M, Walton B, Kattenhorn M, Mullen M, Klein N, Vallance P, Deanfield J, MacAllister R. Ischemic preconditioning prevents endothelial injury and systemic neutrophil activation during ischemia-reperfusion in humans in vivo. Circulation 2001;103:1624–1630.[Abstract/Free Full Text]
  13. Kristensen J, Mortensen U, Nielsen SS, Maeng M, Nielsen TT, Rehling M. Ischaemic preconditioning in the pig assessed by myocardial perfusion imaging and histochemistry. APMIS 2003;122–126.
  14. Kaplan LJ, Bellows CF, Blum H, Mitchell M, Whitman GJ. Ischemic preconditioning preserves end-ischemic ATP, enhancing functional recovery and coronary flow during reperfusion. J Surg Res 1994;57:179–184.[CrossRef][Medline]




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