Novel experimental protocol to study area at risk, infarct size, immunohistochemistry and tissue protein content on the same rodent heart.

Purpose: Temporary coronary ligation (I/R) is the experimental model used to simulate reperfused acute myocardial infarction. Area at risk (AR) and infarct size (IS) are determined after Evans blue infusion and Triphenyl Tetrazolium Chloride (TTC) staining of the freshly isolated hearts. However, this method prevents further analysis such as immunohistochemistry (IHC) or protein and RNA extraction. Consequently, it is necessary to perform surgery on different cohorts of animals. Here, we optimized a protocol to avoid this problem. Methods: Sprague Dawley rats (N=10) were subjected to I/R. After 30’ of reperfusion, we injected 1 million GFP+ rat bone marrow mesenchymal stem cells (GFPMSC) at the infarct border zone. 48 hours later, the coronary was re-occluded and 2 millions fluorescent microspheres (FM) were injected into the left ventricular cavity. The hearts were then perfused with saline, collected and sliced into five pieces. One slice was processed for protein and RNA extraction. The remaining four slices were stained with TTC, formalin fixed and used for planimetric analysis of AR and IS. Finally, the slices were paraffin embedded and sectioned for IHC. Results: Fluorescent or conventional light microscopy pictures were taken for AR and IS calculation, respectively. The paraffin sections allowed high quality IHC as shown in representative pictures of alpha sarcomeric actin and TUNEL staining. Moreover, we tracked the GFP-MSC on the same tissue section. Finally, to complete our experimental protocol, we performed representative Western Blot (5a) and RT-PCR (5b) analysis on the same heart.