1Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Germany, 2Institute of Physiology, University of Bonn, Germany
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Kim, S. C., Boehm, O., Meyer, R., Hoeft, A., Knüfermann, P., Baumgarten, G. A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion. J. Vis. Exp. (65), e3896, doi:10.3791/3896 (2012).
Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.
1. Induction of Anesthesia
3. Preparation of the Heart
4. Coronary Artery Instrumentation
5. Chest Closure
6. Myocardial Ischemia and Reperfusion
7. Infarct Size Assessment with Reperfusion Time up to 3 Days
8. Alternative Heart Preparation for Histology
9. Representative Results
Chronic coronary artery ligation is a complex technique with multiple pitfalls. However, once it is mastered it can be performed with very low mortality rates and highly reliable results. Optimal positioning of the mice and access to the heart are crucial for successful identification and instrumentation of the LAD. The position of the ligation will obviously influence infarct size, thus requiring to have a standardized ligation site. Also, if septal branches are affected this could lead to a bundle branch block instead of ST elevation. Bleeding from epimyocardial veins or from ventricle, if ligation is too deep, can occur and mice should be excluded if bleeding is excessive. Pericard should be removed as completely as possible. Leaving the pericard will aggravate pushing the needle into the myocard for ligation. Also, it will cause pericarditis, eventually induce adhesions and will make histological examination difficult. The PE-occluder should be as short as possible without any sharp corners to minimize trauma to myocard. Hyperinflation of the lungs is absolutely crucial to prevent tension pneumothorax after chest closure. There is no need for a chest drainage. Testing of a correct position of the ligation in the open chest by pulling the suture ends should be omitted because pulling tension is difficult to control. If instrumentation of the LAD fails, further attempts should be avoided because this will add trauma and edema to the myocardium.
In order to achieve reliable results, protocol parameters should be standardized. Therefore, mice are intubated and ventilated with room air and body temperature is tightly controlled with a feedback system. The use of hanging weights has been already emphasized. Other pulling devices have the disadvantage of tension loss and non-standardized pulling tension. Ischemic pre- and postconditioning protocols with multiple cycles of reperfusion and occlusion are more easily performed with hanging weights because they just need to be lifted and let hung (Figure 1).
Infarct areas (white) should be distinguishable from areas at risk (red) and non area at risks (blue) (Figure 2A-B). Infarct sizes are dependent on duration of ischemia. Reperfusion time should be at least 2 hours to allow successful TTC staining (Figure 1 and 2). Most importantly, cytokine RNA expression is low in sham operated animals which had all surgical procedures except ischemia and reperfusion as compared to animals which underwent myocardial infarction (Figure 3A-C).
Figure 1. Infarct size in percentage of area at risk (IS/AAR%). Mice underwent 30 minutes ischemia followed by 120 minutes of reperfusion (I/R, n=10). IPC: Ischemic postconditioning, mice underwent 30 minutes of ischemia followed by 3 cycles of reperfusion/occlusion 20 sec each (n=6, *indicates p<0.05).
Figure 2A. Representative TTC-stained heart slice. White: infarct area, Red: area at risk, Blue: non-occluded area.
Figure 2B. Representative slice of an infarcted (white) area. Note that due to the conical shape of the left ventricle close to the apex, the epimyocard will appear as plane area and should not be considered for planimetric measurement (pink/blue outer area). Red=TTC stained viable myocard.
Figure 3A. No significant difference in myocardial TNF-α mRNA expression after 30 minutes ischemia and 120 minutes reperfusion. n=4-6 per group.
Figure 3B. Myocardial IL-1β mRNA expression after 30 minutes ischemia and 120 minutes reperfusion (I/R). There is no significant difference between control (no surgery) and sham-operated (no ischemia/reperfusion) group. n=4-6 per group, *indicates p<0.05.
Figure 3C. Myocardial IL-6 mRNA expression after 30 minutes ischemia and 120 minutes reperfusion (I/R). There is no significant difference between control (no surgery) and sham-operated (no ischemia/reperfusion) group. n=4-6 per group, *indicates p<0.05.
Figure 4A-C. Heart cutting device. A: closed with razor blades in cutting position. B: open, side view. C: open, front view. Heart will be aligned in the groove of the white plastic bed with its long axis perpendicular to the razor blades (arrow).
We have modified a murine closed-chest model by accessing the heart through a left lateral intercostal thoracotomy and leading out the LAD sutures to the chest in the left midclavicular line. Leaving the bony rib cage intact will minimize trauma, need for pain medication, surgical site infection and thus, facilitate recovery. By preserving the left internal mammal artery there is no need for electrocautery. We leave the suture loop in the subcutaneous tissue for later easy access and use a hanging weight system for a defined occlusion. A closed chest model allows the application of all study protocols of myocardial ischemia and reperfusion with respect to surgical trauma and subsequent immune response 4. However, cytokine RNA expression is increased for up to 3 days due to surgical trauma in mice, rats and dogs 4-6. Thus, a recovery interval of 5 days should be followed. Additionally, this model allows the use of volatile anesthetics for instrumentation which have a known preconditioning effect for up to 72 hours 7,8.Opioids for postoperative pain can also ameliorate myocardial infarction. It has been shown for buprenorphine that myocardial function is improved after global myocardial ischemia 9. However, mice generally do not need additional dosages of buprenorphine as judged by behavioral observation. There is evidence that surgical trauma preceding myocardial infarction will cause a "background" noise of cytokine expression and moreover, modulate infarct size. Ren et al. have shown that remote surgical trauma induces preconditioning of the heart 10. Other endogenous ligands such as high mobility group box 1 (HMGB1) or heat shock proteins which are released or secreted following trauma have been shown to modulate myocardial function 11-13.
Myocardial infarction in a prehospital setting usually occurs without a preceding trauma or injury. Thus, a chronic closed-chest model allows a more lifelike approach to myocardial ischemia and reperfusion.
Potential study protocols include ischemic and pharmacologic pre- and postconditioning and invasive hemodynamic measurements. Future applications of this model could include multiple hit models without having to consider the surgical trauma of instrumentation.
No conflicts of interest declared.
We thank Daniel Duerr for his advice regarding perfusion-fixation technique.
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