The blood–brain barrier (BBB) is a selectively permeable membrane that plays a crucial role in protecting the brain from harmful substances. Its disruption is associated with various central nervous system (CNS) disorders, including hemorrhagic stroke. Evans Blue (EB) dye, a protein-binding tracer, is commonly used to assess BBB permeability. However, the method of injection significantly influences the accuracy and reproducibility of experimental results in the C57/6 mice. In this study, we evaluated the effectiveness of jugular vein injection of EB dye for assessing BBB integrity in a mouse intracerebral hemorrhage (ICH) model. This study investigates the efficacy of jugular vein injection of EB dye for assessing BBB integrity in C57/6 mice with ICH. Neurological deficits were assessed using the Longa and Modified Garcia Scales (GS) on day 1 post-ICH to ensure baseline consistency. Mice were anesthetized and injected with a 2% EB solution via either the jugular or tail vein. Only mice with successful tail vein injections were included in the tail vein injection group. The jugular vein injection method was successfully applied to all animals in the respective group. Plasma and brain EB concentrations were quantified by spectrophotometry following the processing of plasma and brain homogenates. BBB permeability was assessed by measuring EB content in brain tissue after transcardial perfusion and homogenization. Neurological function was assessed 24 h post-modeling using the Longa score, with a score greater than 1 indicating successful modeling. No significant neurological deficits were observed in the sham group, while all animals in the ICH group showed significant deficits (P < 0.0001). No differences in deficits were observed among the experimental groups (P > 0.05). Plasma EB concentration decreased over time in both the sham and ICH groups (P < 0.0001), with the tail vein group showing no significant change between 30 min and 2 h (P > 0.05), but a significant decrease at 24 h (P < 0.0001). At 30 min and 2 h, no significant differences in plasma EB concentration were observed between the sham and ICH jugular vein groups (P > 0.05), though the plasma concentration was significantly higher in the sham group at 24 h (P < 0.05). No significant differences in EB content were noted between ICH jugular vein group and ICH tail vein group groups at 30 min, 2 h, and 24 h in brain tissues (P > 0.05), although the ICH jugular vein group showed higher EB content than the sham group (P < 0.05). No significant differences were found between the ICH tail and sham groups at 2 and 24 h, though the distribution is more dispersed in the tail vein group. Additionally, the hemorrhagic side of the brain showed significantly higher EB content than the non-hemorrhagic side after 2 h (P < 0.05), but no difference was observed at 24 h. Jugular vein injection of EB dye provides a precise, reliable, and reproducible method for evaluating BBB integrity in mouse models of ICH. The tail vein injection method, when successful, can also serve as a valid alternative for BBB assessment. This technique offers significant advantages over other methods, contributing to more accurate BBB evaluations in cerebrovascular disease research.
