Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

The overall goal of this procedure is to ascertain cardiac functional abnormalities by imaging the hearts in live animals affected with inflammatory heart disease. This is accomplished by first inducing the disease, for example, myocarditis. The second step is to prepare the animal for magnetic resonance microscopy imaging, and place it in the animal holder.

Next, the mouse is placed inside the magnet to acquire the cardiac images. The final step is to analyze the cardiac functional parameters using the segment software. Ultimately, this technique is used to calculate end diastolic volume and ejection fraction in autoimmune myocarditis affected mice.

The main advantage of this technique over existing method like histopathology is that it's non-invasive and provides three dimensional high resolution view of the heart. This method can help answer key questions in the cardiology field involving the diagnosis of inflammatory heart disease. Generally, individuals new to this method will struggle due to lack of familiarity with the preparation of animals for magnetic resonance micro image acquisition.

Therefore, visual demonstration of this method is critical. Begin this protocol by first preparing a peptide solution to induce autoimmune myocarditis, dissolve cardiac myosin heavy chain alpha peptide 3 34 to 3 52 in phosphate buffered saline to a final concentration of two milligrams per 1.5 milliliters. Next, prepare the pertussis toxin by adding one milliliter of sterile one x phosphate buffered saline to a vial containing 50 micrograms of lyophilized pertussis toxin.

To obtain a stock concentration of 50 nanograms per microliter. Then take 20 microliters of the stock into a sterile 1.5 milliliter tube and add 980 microliters of sterile one x phosphate buffered saline to obtain a working concentration of one nanogram per microliter. Also prepare complete frons adjuvant or CFA by adding 40 milligrams of mycobacterial tuberculosis, H 37 RV or MTB extract to 10 milliliters of CFA to obtain a final concentration of five milligrams per milliliter.

Now prepare the peptide CFA emulsion to prepare 1.5 milliliters of emulsion aliquot, 750 microliters of cardiac myosin heavy chain alpha peptide 3 34 to 3 52 peptide solution in one 1.5 milliliter tube and aliquot an equal volume of CFA supplemented with MTB extract into another 1.5 milliliter tube. Then using a three milliliter lure lock syringe, draw the peptide solution first, followed by the C-F-A-M-T-B extract. Attach the syringe to a three-way stop cock and connect the other outlet of the stop cock to an empty three milliliter syringe.

Adjust the patent sea of the stop cock. So the peptide CFA mixture flows from one syringe to the other with reasonably good resistance. Mix by pushing the contents from one syringe to the other repeatedly for about one minute, and then place the entire assembly on ice for approximately three minutes.

Repeat this procedure a minimum of 10 times. Determine the consistency of the emulsion by carefully placing a tiny droplet on still water in a 100 milliliter glass speaker. The droplet should not disperse in water if it does continue mixing until the desired consistency is achieved.

Transfer the contents of the emulsion from three milliliter syringes into one milliliter. Lure lock syringes by replacing one of the two three milliliter syringes attached to the stop cock with the one milliliter syringe. Then attaching a 27 and a half gauge needle.

Inject 150 microliters of the peptide CFA emulsion in split doses subcutaneously in the two inguinal regions of six to eight week old female aj. Mice then administer 100 microliters of pertussis toxin suspension intraperitoneal to each animal on day zero and day two post immunization. Repeat the immunization procedure on day seven by administering 150 microliters of peptide CFA emulsion in split doses subcutaneously into either side of the sternum.

Prepare this emulsion fresh as described. Then on day 21, subject the animals to MRM imaging To prep for imaging. First place each mouse in an anesthetic induction chamber containing a 2%ISO fluorine air mixture with a heating pad to maintain warmth.

Then transfer the animal to a specially designed animal holder. Secure the head with a bite bar to immobilize the animal in the prone position. The snout should fit into the nose cone To maintain anesthesia.

Turn on the air blow heater with its outlet hose inserted in the scanner's vertical boar to maintain the animal's body temperature during the experiment. Maintain anesthesia at one to 2%isof fluorine with a flow rate of two milliliters per minute during the entire imaging session. Confirm anesthesia by performing a toe pinch.

Next, set up a pneumatic pillow sensor and a rectal temperature probe to monitor respiration and body temperature throughout the imaging session. A fiber optic pulse oximetry sensor should also be attached to the left ankle and the foot should be secured with a thread loop and taped to maintain position. Imaging will be achieved by gating the respiration and cardiac signals without the use of any contrast agents.

A wide bore 9.4 Tesla vertical bore magnet should be used to obtain high resolution three dimensional magnetic resonance microscopy images. After preparing the animal, place the mouse in the scanner with the heart positioned in the center of the field of view. Then run the imaging interface and select new study type mune at the command bar and pull up the tune page and then select start probe, tune and autoscale.

Use the remote tune match knobs at the end of the coil to tune the RF coil to the proton resonance frequency of 400 megahertz. Next, go to the pre-scan page. To run the frequency and power calibration, hit the x, Y, Z quick button to pull up the shimming page.

Then select all of the iterations and perform auto shimming. Select a scout sequence. Change the field of view to 35 millimeters.

Keep other default settings and run the sequence. If the heart is not in the center of the field of view, adjust the position of the animal holder, retune the RF coil and reacquire the scout. Then select the gem sequence.

Enter the acquisition parameters seen here in the acquire tab to obtain two orthogonal planes along the short and long axis of the heart. Next, select the cine sequence to collect the pulse oximetry gage short axis cine images and measure the left ventricle, anatomic and functional parameters. Enter the parameters seen here to obtain the gradient echo cine sequence.

Adjust the position and angle of the imaging slices based on the long axis view of the heart. Then run the sequence. Once imaging is complete.

Convert the acquired images to DICOM format using the IO tab and transfer the corresponding files to the data center for processing. At the end of the imaging procedure, allow the animal to recover from anesthesia within a filter top cage. Do not leave the mouse unattended until they regain sufficient consciousness to maintain sternal recumbent.

Here, segment software is used to analyze the anatomic and functional parameters of the left ventricle. Begin by loading the DICOM format, cine images into the software. Then select MRGE as the imaging technique, cine as the image type and short axis mid ventricular.

As the image view plane, specify the timeframes to be used for end diastole and end systole using the set current timeframe to end diastole and set current timeframe to end systole sub menus of the edit menu respectively. Next, use the LV and then the endo and EPI command buttons on the bottom right panel. To manually draw the left ventricle, endocardium, and epicardium respectively, remove the papillary muscles by hitting the corresponding command button.

In order to increase the accuracy of the calculations, read the quantified left ventricle parameters such as diastolic volume, systolic volume, stroke volume, and ejection fraction on the top right panel. Then use the mis command button and then the measurement caliper command button to measure the left ventricle parameters such as wall thickness and ventricular diameter. Finally, be sure to use the save both image stacks and segmentation option to save the images, including the segmentations in dot mat format, to reprocess the images as needed.

The angle at which the heart was sliced for the two chamber view image acquisition can be seen here. Here we can see an approximate 1.5 fold increase in left ventricle thickness indicating structural defects in the hearts of experimental autoimmune myocarditis affected compared to healthy mice. Cardiac output measured based on left ventricle and diastolic volume and ejection fraction was decreased in autoimmune myocarditis affected mice as compared to healthy mice.

Finally, the hearts from the above treatment groups were evaluated for inflammation by hemat, toin, and eosin staining histological evaluation of hearts from myocarditis but not healthy mice showed multifocal lymphocyte infiltrates. While attempting this procedure, it is important to ensure that animal's body temperature remains constant so that the heart rate and respiratory rate do not change significantly during the experiments. This is critical to get reproducible results.

After watching this video, you should have a, a good understanding of magnetic resonance microscopic technique to evaluate the functional abnormalities of inflammatory heart diseases like myocarditis and mice. But don't forget that working with the high magnetic fields can be extremely dangerous, so the animal holder and other components should be non-magnetic.