Hi AbdulBasit,

Try to feed them good Vit-E and Glutathione before the procedure.

Best

AMB

Hi, how long you are going to occlude the LAD for and how long you are going to reperfuse heart for? I suggest you visualise the LAD and then occlude it rather than blindly going for it. My technique is to go for 1 cm below the appendage and 1 cm to the left of the appendage. That way you get a decent occlusion without giving a massive lethal infarction. Second suggestion is to make sure that the intubation is correct. Just keep an eye on the rat. The rat shouldn't look too pale. Make sure you get the LAD occluded in one go, otherwise you might cause heavy bleeding and lose the rat in the process.

I dont recommend feeding the rats glutathione prior to the procedure as glutathione has cardioprotective properties. So, you might not even see the changes you expect if you feed glutathione.

Article Glutathione Protects Against Myocardial Ischemia–reperfusion...

But again I/R is a complicated surgical process and you will always lose some rats no matter how perfect your skills are. So dont be disappointed if that happens.

Best of luck.

Maybe you Can reduce thé Time of occlusion.

Thé best way us to inject them AAD before and during, to continue After if they have Arrhythmias and tout monitor them on order to cardiovert if you see VT or VF.

Best, luck

Dear, Abdulbaste

If you have in consideration some rules, the mortality rate in a rat model of myocardial ischemia-reperfusion injury will be very low.

In our experiments of IR injury we have a mortality rate of 9%. The mortality will be increased if you make a permanent ligation protocol. Almost always the rat dies because of VF at the onset of ischemia or at hte onset of reperfusión.

Yo can not to feed the rat with these suplements because are preconditioning substances, as several drugs like buprenorphine.

In concordance with one of the answers, the duration of the ischemia is an important factor to determine the size of the damage miocardium, and can be a limitant factor in the survival.

I recommend you 30 minutes of ischemia with at least 2 hours of reperfusion. It is possible that you reach an infarct size of 30-40%.

It is mandatory an excellent anaesthetic protocol, a correct intubation procedure, a perfect mechanical ventilation method, hydratation and to make the procedure as fast as you can. The recovery is it very important as well.

I give you some publications with the in vivo model of IR injury in rat:

https://www.ncbi.nlm.nih.gov/pubmed/20211186

Article Delayed, oral pharmacological inhibition of calpains attenua...

Article Measuring Water Distribution in the Heart: Preventing Edema ...

Best regards

dos custiones fundamentales reducir la mortalidad después de la ligadura de la arteria descendente anterior:

1. Aumentar previamente la circulación colateral coronaria mediante insultos isquémicos a otro nivel: precondicionamiento isquémico.

2. Aumentar la circulación colateral no coronaria a través de la arteria pericardiofrénica mediante la ligadura distal de la arteria mamaria interna.

preconditioning by intermittent occlusion before ligation multiple

Dear all,

if you are doing an in vivo IR injury model to study the effect of AMI, you can not apply factors that modifies the outcome of the experiment, such as preconditioning or post conditioning methods (drugs, surgical strategies, etc). The infarct model should be the most physiological than you can If you want to study the effect of a drug, for example.

Best regards.

Dear all,

I really appreciate the great information and advice you have provided.

As a final question,

I want to know what protocol is most suitable for getting to the heart.

Thoracotomy or lateral thoracotomy?

The video of your surgeries will also be very helpful.

Once again, total occlusion is not perhaps a very good method, so look for Fabiani's technique.

no tengo experiencia con el mejor abordaje al corazón en ratas, pero para poder acceder con facilidad a la arteria descendente anterior deb usar una incisión que le permita el accesoe a la cara anterolateral del corazón: esternotomía, mediastinotomía anterior, o toracotomía anterolateral

Lateral thoracotomy is a better option. I always cut between ribs 3 and 4 (Counting from the bottom ribs). Once you cut the skin and the muscle layer, place your finger on the ribs to feel for the heart and then make the final cut to expose the heart. Its usually between ribs 3 and 4.

Dear Dr. Muntasir,

Thank you very much for your clear and great explanations.

Do you cut off any ribs?

Your advice and suggestions will be much appreciated and welcomed.

Dear Abdulbaset, I am glad that my suggestions seemed helpful to you.

No I didnt cut off any ribs, if you cut the intercoastal muscle between ribs 3 and 4 and use a retractor then you can visuslise the heart without cutting off any ribs. After the surgery suture back the ribs. I suggest you use prolene 5 sutures for the heart.

Initially, intubation is very important step. Orotracheal intubation is the best method. This won't hurt the animal much. Make sure that there are no secretions in trachea that might block.

Lateral thorocotomy is the best site. Opening at 4-5 intercostal region is the best location.

Check for body temperature.

Make sure that no air is left inside while suturing the ribs.

I agree with Divya about the tracheal intubation. You have to intubate the rat for open chest surgeries. But make sure that you intubate it correctly as its very easy to insert the tube in the easophagus instead of the trachea. To check for the correct intubation (the tube also need to be connected to the ventilator) apply a bit of pressure on the trachea and if intubated correctly the chest should expand. If its in the easophgus you will see swelling in the intestinal area which can be life threatning for the rat. I usually put a light source on top.of the trachea and the trachea insode the oral cavity then looks like a round red ring. It should also feel a bit bony when pushed into the trachea.

Exactly we follow the same procedure. It's quite easy with experience to intubate by visualizing vocal cords through oral cavity.

Proper intubation can be identified by placing a glass slide near the tracheal tube where vapours get deposited or place cotton fiber near the tube so that it moves while animal respire(expiration)

The outcome of coronary artery occlusions is age-, species, severity of occlusion, duration, coronary collateral blood flow during ischemia (compared to baseline), operator and drugs used to support and stabilize hemodynamic.

In experimental models, regional ischemia is complicated by the above-mentioned factors and contributes to lack of reproducibility in the same laboratory and discrepancies in literature. It is seldom to see authors report complications, arrhythmia, fibrillation and mortality that is why young investigators waste time and resources by following prior publications that do not report limitations and pitfalls of their studies. Swine model lacks coronary collateral that is why pigs die within 10 minutes with small risk areas (occlusion close to the apex) from arrhythmia followed by cardiac arrest and not from infarction. Investigators treat all groups with lidocaine to attenuate arrhythmia and ventricular fibrillation, yet some more lidocaine and other inotropic drugs are used to minimize mortality while cardiac function is poor

Unlike regional ischemia, global ischemia and measuring load-independent indices of cardiac performance and correlating that to myocardial bioenergetics, marker enzymes of ischemia and the end-point infarction. To obtain reproducible studies and results you must design experiments that have clinical relevance and try to minimize variations within same group and amongst other groups and time. GOOD LUCK

Dear Dr. Muntasir and Divya Dasagrandhi,

Thank you for taking the time to share all of that great information with me.

Can you see the LAD artery to ligate or just guess the path?

Do you pull the heart out of the body to LAD ligation?

Well, to be honest, in a rat model its very hard to visualize the LAD. But if you see it, it should appear a bit orange color. So, frankly speaking, you have to guess its path on most of the occassions. But, before occluding it permanently, you should check for the cyanosis of the area at risk (AAR). If you have successfully blocked the LAD, you should almost immediately see cyanosis in the AAR. I would suggest you not to go too low on the LAD, otherwise the AAR might be too small to induce significant infarct. Ideally, if you go 1cm below and 1 cm to the right of the appendage, you should get a decent LAD occlusion. I would suggest you to use medical grade tube to occlude the LAD reversibly first ( you can insert the tube into the suture and make a loop) and check cyanosis.

I personally don't pull the heart out and I wont recommend that either as you may cause damage to the heart. I have seen people pulling out the heart in mice model, but never seen anyone pulling it out in rat model. I have tried it couple of times, and all the occasions, the sharpness of the suture pierced through the cardiac tissue. However, that's just what I personally experienced. I am sure, other experts here will be able to shine some light on this issue.

Good luck.

No need to pull the heart out. We can open the ribs using retractor, so that it will get wide open to visualise the heart clearly. LAD can be identified by first selecting the location using blunt end needle. If the pressure is on LAD ST depression can be seen immediately in ECG due to short ischemia and beating pattern of the heart also varies.

Dear Dr. Muntasir,

Thank you again for your detailed and clear explanation. It was very helpful and understandable.

Should I go 1 cm below and 1 cm to the right of the left atrial appendage or 1 mm below and 1 mm to the right…?

I would be very grateful if you can draw the exact path on a sheet of paper for me.

Kind regards,

Position of tie along LAD is crucial...just changing the position of the tie will lead to a change in survival rate.This must be a constant location as described above to decrease variability. Keep the recovery area quiet & noise free.

Also see where the braches are coming out of the LAD.

I think prior visualization of the LAD , the main branches, as as well as the collateral blood flow would assist guiding the procedure. I would prefer lateral thoracotomy as a method of surgery.

Dear all,

Thank you for taking the time to share all of that great information with me. Your support and advice have been extremely helpful.

Best wishes

In addition, reducing the time of the procedure itself will also reduce your mortality rates.

Hi, for you to increase survival rate , there should reduced time of ligation and the positioning of the tie along the LAD.