Développement d’une méthode informatique appliquée à la quantification immunohistochimique du mastocyte et du macrophage M1 et M2 lors de la guérison cutanée chez le cheval
The goal of this randomized and controlled study was to develop a reliable and accessible quantification method for immunohistochemically-stained tissues and to quantify the temporal and anatomical pattern of mast cells and M1/M2 macrophages during equine wound healing. Two full-thickness skin wounds were created on each distal forelimb and hemithorax, respectively, of four mixed breed mares (n= 4). Samples were taken at days 2, 7, 14 and 21 post wounding and stored (n = 28). Immunohistochemistry was performed to study mast cell (CD117) distribution and double immunohistochemistry was performed with M1 (CD86), M2 (CD204) and pan macrophage (CD14) markers. An ImageJ quantification method was developed and compared with a traditional manual counting on 6 specimens. Stained cells on the other 22 samples were then quantified with the computer-based method. For the validation of the method, an intra- and interoperator Intraclass correlation coefficient (ICC) of 0.90 (p < 0.01) and 0.69 (p < 0.01) were found. An ICC value of 0.67 (p < 0.05) was found between the ImageJ method and manual counting. No significant difference of mast cell numbers was observed in relation to anatomical location (thorax vs limb) or time of healing. M2 macrophages showed a spindle or rounded shape while general macrophages showed a round, fried-egg or dendritic morphology. The quantification method developed is reliable, reproducible, available and free. Future studies should, with equine-specific antibodies, further characterize mast cell and macrophage implication in wound healing and chronically-inflamed limb wounds of horses.
Quantification Equine Immunohistochemistry M1 and M2 macrophages Mast cells Skin