Bipolar disorder (BD) is a neuropsychiatric disease characterized by hyperactivity state, mania, with or without depression, and reduced Na⁺, K⁺-ATPase activity has been implicated in the pathophysiology of BD. In this study, we established novel method to evaluate the changes in cerebrovascular unit structure with respect to BD by using rat organotypic PFC slice cultures. Coronal PFC slices (350-μm thick) were prepared under sterile condition, and cultivated for 5 days on microporous membrane in six-well plate. At 5 days in vitro, slices were exposed to ouabain, an inhibitor of Na⁺, K⁺-ATPase, followed by immunohistochemistry and western blotting. To detect endothelial cells and pericytes, we used mouse anti-rat endothelial cell antigen (RECA-1) antibody and rabbit anti-platelet-derived growth factor receptor-β (PDGFR-β), respectively. After z-stack images of PFC region were taken by using a fluorescent microscope system BZ-9000 under a 10Χ objective lens, RECA-1-positive area and PDGFR-β-positive area were analyzed by using ImageJ software coupled with AdaptiveThreshold plug-in. Treatment with ouabain significantly decreased RECA-1-positive area in a time-dependent manner. On the other hand, ouabain had few effect on PDGFR-β-positive area, hence increased the number of both RECA-1-negative and PDGFR-β-positive cerebrovascular unit structure. In addition, ouabain decreased the relative expression level of Bcl-2 versus that of Bax, and increased expression level of cleaved caspase-3 in RECA-1-positive cells. Finally, lithium carbonate (1 or 5 mM), which is used as a therapeutic drug for BD, applied from 6 h before and simultaneously with ouabain for 24 h significantly prevented the decrease in RECA-1-positive area induced by ouabain. Therefore, these results suggest that this method is useful to understand the pathogenesis of BD as well as to develop fundamental treatments for BD.