Comparison of cumulative planimetry versus manual dissection to assess experimental infarct size in isolated hearts.
BACKGROUND
Infarct size (IS) is an important variable to estimate cardiac ischemia/reperfusion injury in animal models. Triphenyltetrazolium chloride (TTC) stains viable cells red while leaving infarcted cells unstained. To quantify IS, infarcted and non-infarcted tissue is often manually dissected and weighed (IS-DW). An alternative is to measure infarcted areas by cumulative planimetry (IS-CP).


METHODS
We prospectively compared these two methods in 141 Langendorff-prepared guinea pig hearts (1.44+/-0.02 g) that were part of different studies on mechanisms of cardioprotection. Hearts were perfused with Krebs-Ringer's and subjected to 30 min global ischemia after various cardioprotective treatments. Two hours after reperfusion hearts were cut into 6-7 transverse sections (3mm) and stained for 5 min in 1% TTC and 0.1M KH2PO4 buffer (pH 7.4, 38 degrees C). Each slice was first scanned and its infarcted area measured with Image 1.62 software (NIH). Infarctions in individual slices of each heart were averaged (IS-CP) on the basis of their weight. After scanning, IS-DW was determined by careful manual dissection of infarcted from non-infarcted tissue and measuring their respective total weight.


RESULTS
We found limited tissue permeation of TTC in relation to the slice thickness leaving tissue in the center unstained, as well as significant cross-contamination of stained vs. unstained tissue after manual dissection. IS-CP and IS-DW ranged from 6.0 to 73.1% and 19.4 to 70.5%, respectively, and correlated as follows: IS-DW=(27.6+/-1.4)+(0.518+/-0.038) * IS-CP; r=0.75 (Pearson), p<0.001. In addition, IS-CP correlated better with return of function after reperfusion like developed left ventricular pressure, contractility and relaxation, and myocardial oxygen consumption.


CONCLUSIONS
Despite a good correlation between both methods, limited tissue permeation by TTC diffusion and limited precision in the ability to manually dissect stained from unstained tissue leads to an overestimation of infarct size by dissection and weighing compared to cumulative planimetry.
Riess
Matthias L
ML
Anesthesiology Research Laboratories, Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. mriess@mcw.edu
Rhodes
Samhita S
SS
Stowe
David F
DF
Aldakkak
Mohammed
M
Camara
Amadou K S
AK
D000818:Animals; D004195:Disease Models, Animal; D004210:Dissection / Q000379:methods* / Q000592:standards; D005260:Female; D006168:Guinea Pigs; D006321:Heart; D007091:Image Processing, Computer-Assisted / Q000379:methods* / Q000592:standards; D008297:Male; D009203:Myocardial Infarction / Q000000981:diagnostic imaging / Q000473:pathology* / Q000503:physiopathology; D017202:Myocardial Ischemia / Q000000981:diagnostic imaging / Q000473:pathology / Q000503:physiopathology; D009929:Organ Size / Q000502:physiology; D011446:Prospective Studies; D011859:Radiography; D013499:Surface Properties