Volumetric analysis and cell counts
To measure vasogenic edema, the volume of anti-rat IgG positive region in PC was estimated according to the formula based on the modified Cavalieri method: V = Σa × tnom × 1/ssf, where a is area of the region of the delineated subfield measured by AxioVision Rel. 4.8 software, tnom is the nominal section thickness (of 30 μm in this study), and ssf is the fraction of the sections sampled or section sampling fraction (of 1/6 in this study). The subfield areas were delineated with a 2.5 × objective lens [5,7,8,18,25]. The volumes are reported as mm3. An optical fractionator was used to estimate cell numbers. The optical fractionator (a combination of performing counting with the optical dissector, with fractionator sampling) is a stereological method based on a properly designed systematic random sampling method that by definition yields unbiased estimates of population number. The sampling procedure is accomplished by focusing through the depth of the tissue (the optical dissector height, h; 15 μm in all cases for this study). The number of each cell type (C) in each of the subregions is estimated as: C = ΣQ- × t/h × 1/asf × 1/ssf, where Q- is the number of cells actually counted in the dissectors that fell within the sectional profiles of the subregion seen on the sampled sections, and Asf is the areal sampling fraction calculated by the area of the counting frame of the dissector, a(frame) (of 50 × 50 μm2 in this study) and the area associated with each x, y movement, grid (x, y step) (of 250 × 250 μm2 in this study) {asf = (a(frame)/a(x, y step))}. The immunoreactive cells were counted with a 40× objective lens. The immunoreactive cells were counted with a 40× objective lens. All immunoreactive cells were counted regardless the intensity of labeling. Cell counts were performed by two different investigators who were blind to the classification of tissues. SE-induced PC atrophy is evident [8], so changes in cell number may be caused by an alterations in the volume of the PC. Therefore, the total number of cells was corrected by multiplying with appropriate correction factors (CF) representing the degree of shrinkage (or swelling) compared with the Non-SE.