The overall goal of this histological procedure is to perform morphometric quantification of infarct expansion index and heart structural parameters using a myocardial infarction rat model in order to assess infarct size and left ventricle remodeling. This method can help answer key question in the cardiac field such as the effect of regenerative therapy following myocardial infarction or novel strategies aiming at reducing infarct expansion. The main advantage of this technique is the use of systematic sampling of harvested rat heart to accurately assess the infarct size and take into account the left ventricle changes throughout remodeling.
Following this method can provide insight into infarct size in chronic rat model of myocardial infarction. It can also be applied to mice or other ischemic model, such as acute myocardial infarction or ischemia reperfusion. To begin this procedure, perform a sternotomy on a myocardialy infarcted animal under anesthesia.
Make an incision into the skin and the muscles along the sternum with surgical scissors. Then, cut the ribs on the left and the right to remove the chest. After that, remove the adhesions remaining after LAD ligation.
Cut the aorta and remove the heart then place the tissue in PBS with one molar potassium chloride for less than one minute before washing it with PBS. In order to preserve the tissue, PBS inside the ventricles should be carefully removed. Subsequently, transfer the infarcted heart to an acrylic rat heart matrix and position it longitudinally.
Then keep it at minus 20 degrees celsius for one hour. For systematic sampling after an hour, cut the heart directly in the matrix using a razor blade. Ensure each section is two millimeters thick.
Next, perform TTC staining by incubating the sections in 1%TTC for 15 minutes at 37 degrees celsius, then incubate them in 4%PFA for 20 to 60 minutes at room temperature. Afterward, place the sections between two glass plates with two millimeter spacers and take pictures with a stereological microscope coupled with a camera at 15x magnification. A TTC staining procedure was performed to visualize the infarct area, which appears in white, and the healthy myocardium, which appears in red.
For a large myocardial infarction, transmural infarcts were observed from apex to base. Smaller infarcts presented white infarcted tissue visible from the apex to the midsection of the heart. For small, non-transmural infarcts, fibrotic tissue was observed on only one or two sections from the apex.
To freeze the sections, place each section in a plastic mold with mounting medium for cryotomy and position the apex downward in the mold. Freeze the blocks with 2-Methylbutane vapors under liquid nitrogen cooling for 10 to 15 minutes. After that, store the tissues at minus 80 degrees celsius.
For paraffinization, place each section in an embedding cassette with the apex pointing at the bottom, then keep the cassettes in 4%PFA for 24 hours. Afterward, place them in a tissue processor overnight. Once processing is complete, make blocks by embedding each heart section in paraffin.
In this procedure, prepare 5-micron-thick tissue sections from the paraffin blocks with a manual microtome. Next, stain one slide from each heart section for each rat with Masson-Goldner trichrome staining. For the paraffin sections, melt the tissues at 60 degrees celsius in an oven.
Under a fume hood, deparaffinize them in xylol twice for ten minutes each. Then, rehydrate them in 100%ethanol, 95%ethanol, 70%ethanol, and then distilled water for three minutes each. For the deparaffinized sections and for the cryo sections, fix them in Bouin solution overnight.
The following morning, rinse them in running tap water for 10 to 15 minutes, then rinse again using distilled water. Fixation with Bouin is the most important step to ensure a splendid Goldner staining. Incubate them in Mayer's hematoxylin for three minutes.
After three minutes, remove the slides and leave them in distilled water for five minutes. Subsequently, incubate them in ponceau acid fuchsin for five minutes before rinsing with 1%acetic acid for one minute. Next, incubate the sections in phosphomolybdic acid orange g for one minute and then rinse them in 1%acetic acid for one minute.
Incubate in light green dye for ten minutes and then rinse in 1%acetic acid for one minute. Finally, dehydrate the sections in 70%ethanol for 30 seconds, 95%ethanol for 30 seconds, and 100%ethanol for five minutes sequentially. Apply one drop of resinous mounting medium on to the tissue sections, cover them with cover slips, and place them to dry.
In this step, acquire one image from each slide using a stereo microscope coupled with a camera at 15x magnification. Photograph a ruler under the same settings. Thin five-micron sections from each heart slice were cut and stained with Masson-Goldner trichrome.
Healthy tissues appeared in red and the connective tissue in green. Next, use the image analysis software to measure the scar thickness in the middle of the infarct, the septum thickness, the left ventricle cavity area, the infarct area, and the LV tissue area. To do so, set the scale with the ruler picture.
Click on measurements and select set conversion factor. Draw a line on the calibration bar. Right click on the picture and click on end calibration.
Note the bar value and then click OK.Use the multiple segment tool to select the LV point by point and then right click copy paste anywhere. After that, measure the scar and septum thickness using the single segment measurement tool. Then, automatically detect the LV cavity, infarct, and LV areas using the automatic area measurement tool in RGB mode.
Activate only borders and contiguous in the RGB mode. Finally, click inside the area of interest to detect it. After that, calculate the infarct size as the ratio of the infarct area to the LV area.
Then, calculate the expansion index using this formula. Finally, calculate an average for each heart using the formula shown. The expansion index calculated from Goldner staining is a good index to evaluate the infarct development.
As shown in the figure, it correlates with the size of infarct expressed as the percentage of the left ventricle and with the ejection fraction measured by echocardiography. On the graphs are represented the linear regression and the 95%confidence intervals. The infarct expansion index was calculated as described in the formula here.
The whole LV area was measured as the combined LV cavity and LV tissue area. The percentage of the infarct was calculated as shown. The function was assessed at six weeks post LAD ligation using a high-resolution echocardiography.
After watching this video, you should have a good understanding of how to perform a systematic sampling of the heart and how to assess infarct size based on an image analysis of stain sections.
