FAQs: 
Echo-Guided Injection into Mouse Myocardium
-written by Matt Springer

We have introduced an image-guided approach to guide injections into the mouse myocardial wall using high-resolution micro-ultrasound, in the absence of surgery. This also allows us to target the injections to the peri-infarct border zone days or weeks after a surgically-induced myocardial infarction.  The system, which was developed in collaboration with Dr. Yerem Yeghiazarians, is described in detail in our initial paper on the subject (ref 1), but in these FAQs, we answer further questions that people have asked. 
 

What is the largest volume that can be injected into one site?
Should I try to use the thinnest needle I can find?
Is there an optimal length of the needle?
How about pulled glass micropipettes?
Are the insulin syringes with the needle already attached ok for the injections?
For closed-chest injections, do I need to shave the hair off of the chest?
How will the orientation of the mouse affect successful injection into the left ventricular wall?
Will this work with rats?
Does this have an ultimate clinical application?
I am setting up this system for the first time.  How do you suggest I begin?
What is the expected mortality rate from such injections?
What are the limitations of this system?
 

What is the largest volume that can be injected into one site?

5 ul is a good volume.  We have tried 10 ul but find that it has more of a tendency to rupture through the wall.  Remember that the mouse LV wall is only about 1 mm thick and is beating at 400-600 beats per minute, depending on anesthesia state.  We routinely perform two 5 ul injections in close proximity.
 

Should I try to use the thinnest needle I can find?

No.  A thinner needle means a shorter needle bevel, which is a good thing; but a needle that is too thin can be too flexible to reproducibly inject through the skin and body wall.  30 gauge seems to be the optimal compromise.  33 gauge is too floppy in our experience.
 

Is there an optimal length of the needle?

Again, you need to avoid a needle that is too flexible.  We use 1/2 inch 30 gauge needles.  1 inch 30 gauge needles are not rigid enough.
 

How about pulled glass micropipettes?

While this might be suggested for embryo injections, such a thin glass needle would break before puncturing through the skin and body wall into the moving myocardium.
 

Are the insulin syringes with the needle already attached ok for the injections?

As far as the injection process is concerned, it is probably ok.  However, those syringes have tick marks at 10 ul that are extremely close together, and your ability to inject anywhere near a reproducible 5 ul volume is unlikely.  50 ul glass Hamilton syringes with a luer tip have tick marks at every microliter; therefore, you can inject almost exactly 5 ul with reproducibility.  (This is independent, however, of the rather unpredictable back-flow when you remove the needle that is common to any injection techique.  The myocardium is, after all, a solid tissue that is squeezing itself.)
 

For closed-chest injections, do I need to shave the hair off of the chest?

Yes, the hair will interfere greatly with the needle and dull it as well.  We actually use a chemical hair remover like Nair, which is fast and has the benefit of leaving absolutely bare skin with no free mouse hair allergens.
 

How will the orientation of the mouse affect successful injection into the left ventricular wall?

If the mouse is entirely flat on its back or turned too far to its left, the right ventricle (RV) projects over the left ventricle and injections can easily end up in the RV wall, cavity, or in the intraventricular septum.  We angle the mouse slightly to its right to move the RV down and out of the way.
 

Will this work with rats?

Yes, we and our collaborators are using this technique in rats as well, and another group at Stanford has more recently published on the use of this system in rats (ref 2).
 

Does this have an ultimate clinical application?

It is possible that a similar system could be scaled up for human use, but the main utility of this system in mice is that it overcomes many of the limitations that result from the small size of the mouse heart.
 

I am setting up this system for the first time.  How do you suggest I begin?

In our paper that introduces this system (ref 1), we describe the approach that we used to determine our success in targeting to desired locations of the myocardial wall.  Briefly, this involves the injection of a mixture of ultrasound contrast agent with fluorescent microspheres, enabling the researcher to observe the injection occur in real time by ultrasound, and then cryosection the heart for immediate observation with a fluorescence microscope to determine the actual location of the injection; no fixing or staining required.  This is a very good way to get your bearings and we recommend that you try some injections this way until you are confident of your ability to interpret the ultrasound image.
 

What is the expected mortality rate from such injections?

In the several hundred mice that we and our colleagues have injected this way, we are not aware of any mouse that has died as a direct result of the injection.  It is far less invasive than surgical opening of the chest to expose the heart for injection under direct visualization.
 

What are the limitations of this system?

Because the heart is injected in the chest cavity in its natural state, it is not feasible to turn the heart and inject in the posterior wall.  Injections are therefore limited to the anterior wall and cannot be performed too close to the atria.

 

REFERENCES

  1. Springer, M.L., Sievers, R.E., Viswanathan, M.N., Yee, M.S., Foster, E., Grossman, W., and Yeghiazarians, Y. (2005)  Closed-chest cell injections into mouse myocardium guided by high-resolution echocardiography. Am J Physiol Heart Circ Physiol 289:H1307-1314
     
  2. Rodriguez-Porcel, M., Gheysens, O., Chen, I.Y., Wu, J.C., and Gambhir, S.S. (2005)  Image-guided cardiac cell delivery using high-resolution small-animal ultrasound. Mol Ther 12:1142-1147