Hi. Two things : what do you use as a perfusate ? and second of course 50% failure is high...something wrong with the perfusate ? toxin ? What about the temperature ? Anyway if you use a Krebs or equivalent solution, you heart is always ischemic because of the poor oxygen content, from the beginning. In such a model for pharmacological studies I never used the hearts longer that 45 min from start.

Hello! As Professor Renaud Trouve mentioned, losing some hearts after ischemia, especially because of ventricular fibrillation or other rhythm abnormalities, is fairly common, but half if way too much. Do the hearts that do recover after reperfusion eventually develop decent function? It is difficult to point out any specific flaw without knowing the intricacies of your particular system, as something as simple as poor temperature control or inadvertent embolization can be the cause.

What type of ischemia protocol are you using? Global no/low-flow or coronary ligation? How are you stopping/resuming flow? Do you have good cardiac performance before the onset of ischemia?

Yes good points, and if your perfusate is not filtered, and do not circulate during ischemia, what is the reperfusion temperature ? There are a lot of possible critical points, depending on the model and the circuitry or perfusate. Please explain and describe. Thank you.

Hello; you have to give correct pressure and good oxygen content ofyour solution. otherwisw you can see this proplems named, stunning heart after.iscemi-reperfusion..

There is quite no oxygen content in a Krebs perfusate: if you don't have red cells your oxygen content is very very very low (Henry law, make teh calculation). All along the experiment you are already ischemic. And as time elapses it is worth...

In vivo reperfusion arrhythmias are potentiated by rate of reperfusion (e.g., rapid reperfusion-->VF, slow-->tolerated), could also try a slower reperfusion rate following your ischemia episode.

@ Renaud Trouve @ André Leite Moreira Thank you all for the responses. The rig is always flushed with boiled double distilled water before and after experiments to avoid contamination possibility. I use filtered Tyrode's solution. Solution is kept in water bath 37-39 celcius and oxygenated 30 min before hearts canullation. Flow rate 10 ml/min. The heart is paced after I have systolic pressure between 70-100 mmHg and diastolic between 3-10 mmHg for 25 min. Then global ischemia (no flow, by stopping the pump) for 30 min. Then re perfusion for 90 min. The hearts always submerged in water with temperature range 36.5 -- 38.5 celcius and it is checked every 20 min. Could the pacing affect the heart during ischemia because I leave it on all stages (steady state perfusion, ischemia, reperfusion)? Or should I consider changing the balloon ?

Hi Ali, just a few simple questions:

1) you don't really mean that you keep the hearts ‘in water’ as you write, but in you (well-oxygenated) Tyrode solution?

2) Do you inject heparin just before taking the heart out? This should prevent clot-forming in the smaller vessels during the initial flush with Tyrode.

3) Do the hearts look ‘viable’ when they are just connected to the Langendorff perfusion set up? What I mean is: what is the starting situation, are the hearts really in good shape before you start your ischemia test?

And a remark: it is much more of a hassle, but perfusion with blood or blood-replacement fluids is probably preferable for your type of ischemia experiments, as mentioned by the other commenters.

Hello. Lot of info. Not enough perfusion before ischemia. Your heart is working hard and you should perfuse more. And sorry but it is a very bad model...

And I have to add that in tyrode or krebs the heart is more and more ischemic even before your ischemia. Should not use it more than an hour.

@John Karemaker

1) sorry. Yes right submerged in Tyrod's

2) no. Never used heparin

3) all hearts really in good shape. Beating very well.

Thank you.

To help you with :

Use heparin make sure is ice cold when trimming and mounting. Make sure you have ballon in heart inflated to 5mm hg to prevent contracture. Finally make sure oxygen pH and glucose are present and adjusted

Hi all,

I have reduced ischemia time to 25 min instead of 30 min. All hearts have come back. Is that it?

Thanks.

That's surprising,

Possible contracture starts around 30 minutes in a model of global ischemia at 37degrees

Perfect demonstration the model is weal and non usable to study ischemia.

I knw Neely and others have published papers in the 70 and 80 using such a model to study ischemia and metabolism. That"s why I published the above paper in 1985. This model should be used 50 imin. max to study dose/effects or dose/agonist/antagonist or competitor. Everything else is not science.