Short communicationDigitally quantifying cerebral hemorrhage using Photoshop® and Image J

A spectrophotometric hemoglobin assay is widely used to estimate the extent of brain hemorrhage by measuring the amount of hemoglobin in the brain. However, this method requires using the entire brain sample, leaving none for histology or other assays. Other widely used measures of gross brain hemorrhage are generally semi-quantitative and can miss subtle differences. Semi-quantitative brain hemorrhage scales may also be subject to bias. Here, we present a method to digitally quantify brain hemorrhage using Photoshop® and Image J, and compared this method to the spectrophotometric hemoglobin assay. Male Sprague-Dawley rats received varying amounts of autologous blood injected into the cerebral hemispheres in order to generate different sized hematomas. 24 h later, the brains were harvested, sectioned, photographed then prepared for the hemoglobin assay. From the brain section photographs, pixels containing hemorrhage were identified by Photoshop® and the optical intensity was measured by Image J. Identification of hemorrhage size using optical intensities strongly correlated to the hemoglobin assay (R = 0.94). We conclude that our method can accurately quantify the extent of hemorrhage. An advantage of this technique is that brain tissue can be used for additional studies.