Immunoreactivity of mouse gp91phox and Iba1, fluorescent intensity of Fluoro-Jade B and ethidium were quantified using Bioquant Image Analysis software (Nashville, TN, USA). Images were captured on an Olympus (Tokyo, Japan) BX51 microscope and Sony (Tokyo, Japan) DCX-390 video camera at 40X. Light levels were normalized to preset levels and the microscope, camera, and software were background corrected to ensure reliability of image acquisition [34]. In each region (cortex and dentate gyrus), six random images from each brain sample were captured within a standard region of interest (ROI), the density of immunostaining and fluorescence was measured in pixels within this area (pixels/mm2). Subsequently, the average of the six measurements was used to represent the immunoreactivity or fluorescence intensity of each sample. When measuring fluorescence intensity in the cells, we eliminated the background by adjusting the threshold to avoid background staining. For + immunoreactive (+IR) cell counting, a modified stereological method was used to quantify cells within regions of interest following immunostaining of brain sections using the CAST stereological system [35,36]. Specifically, cell density (Nv) of TLR3, HMGB1, caspase-3 and Iba1 + immunoreactive (+IR) cells was determined following the optical dissector method [37,38], which was calculated as follows:
