Image software 4

Three areas surrounding the lesion in each immunostained section were imaged at × 40 magnification using a microscope and a camera without visual field overlap (Fig. 1a). The ratios of GFAP-, Iba1-, CD16/32-, ariginase-1-, and caspase-3-positive cells were quantitatively measured between the two groups in coronal sections using the Scion Image software 4.0.3 at 3, 7, and 14 days after TBI. Moreover, at 7 days after TBI, sections obtained from two areas in the motor cortex of the surrounding lesion, with a co-localization of caspase-3 and NeuN were imaged (× 20 magnification) following immunofluorencent staining. Furthermore, the ratios of caspase-3- and NeuN-positive neurons were counted in the surrounding lesion (0.21 mm²) and a quantitative analysis of each immunolabeled area was performed by two to three individuals.

The area of the scar tissue was analysed using Scion Image Software 4.0.3. The fibroblasts and myofibroblasts were photographed at three random locations using the DP21 microscope at 40× magnification. Each type of cell was counted, and the mean numbers were calculated.