Department of Anatomy and Neurobiology, National Defense Medical College;
Department of Anatomy and Neurobiology, National Defense Medical College.
J Vis Exp. 2024 Oct 18(212). doi: 10.3791/66653.
Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is a complex process influenced by several factors, such as the concentration of dyes in the blood, permeability of brain vessels, duration of dye extravasation, and reduction in dye concentration in the tissue due to degradation and diffusion. In a mild traumatic brain injury model, exposure to blast-induced shock waves (BSWs) triggers BBB breakdown within a limited time window. To determine the precise sequence of BBB breakdown, Evans blue, and fluorescein isothiocyanate-dextran were injected intravascularly and intracardially into mice at various time points relative to BSW exposure. The distribution of dye fluorescence in brain slices was then recorded. Differences in the distribution and intensity between the two dyes revealed the spatiotemporal sequence of BBB breakdown. Immunostaining of the brain slices showed that astrocytic and microglial responses correlated with the sites of BBB breakdown. This protocol has broad potential for application in studies involving different BBB breakdown models.
荧光染料用于确定血脑屏障 (BBB) 破裂导致的染料外渗程度。这些染料的标记是一个复杂的过程，受到多种因素的影响，如血液中染料的浓度、血管的通透性、染料外渗的持续时间以及由于降解和扩散导致组织中染料浓度的降低。在轻度创伤性脑损伤模型中，爆震波（BSWs）的暴露会在有限的时间窗口内引发 BBB 破裂。为了确定 BBB 破裂的确切顺序，将 Evans 蓝和异硫氰酸荧光素-葡聚糖通过血管内和心内途径在相对于 BSW 暴露的不同时间点注入小鼠体内。然后记录脑切片中染料荧光的分布。两种染料的分布和强度差异揭示了 BBB 破裂的时空顺序。脑切片的免疫染色显示，星形胶质细胞和小胶质细胞的反应与 BBB 破裂的部位相关。该方案在涉及不同 BBB 破裂模型的研究中有广泛的应用潜力。
