Measuring Blood-brain-barrier Permeability Using Evans Blue in Mice

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The Journal of Neuroscience
Jan 2015


The blood–brain barrier (BBB) is a highly selective permeability barrier that separates the circulating blood from the brain extracellular fluid in the central nervous system. The blood–brain barrier allows the passage of water, some gases, and lipid soluble molecules by passive diffusion, as well as the selective transport of molecules such as glucose and amino acids that are crucial to neural function. This protocol provides a full, detailed method for measuring blood-brain-barrier permeability of mice with Evans blue (EB), that leakage was used to assess blood-brain barrier (BBB) permeability (sample: Anterior Cingulate Cortex, ACC).

Materials and Reagents

  1. Adult male Kunming mice (the Experimental Animal Center of Xuzhou Medical College, 20-22 g, 5-7 weeks old)
  2. Evans blue (EB) (Sigma-Aldrich, catalog number: E2129-10G )
  3. Trichloroacetic acid (Sigma-Aldrich, catalog number: T6399-100G )
  4. Chloral hydrate (Sigma-Aldrich, catalog number: 15307-500G-R )
  5. Paraformaldehyde (Sigma-Aldrich, catalog number: 158127-500G )
  6. NaCl (Sigma-Aldrich, catalog number: S7653-1KG )
  7. 10% chloral hydrate (see Recipes)
  8. 4% paraformaldehyde-freshly-prepared (see Recipes)
  9. 0.9% NaCl (see Recipes)
  10. 100% trichloroacetic acid solution (see Recipes)


  1. Spectrophotometer (Thermo Fisher Scientific, catalog number: 1510 )
  2. 96-well plate (Corning Incorporated, catalog number: 3599 )
  3. 1 and 20 ml syringe (local drugstore)
  4. Infusion apparatus (local drugstore)


  1. Mice were anesthetized with 10% chloral hydrate (0.3 ml/100 g, i. p.).
  2. Injected Evans Blue (EB) (2%, 2 ml/kg) from caudal vein 0.5 h before perfusion. EB leakage was used to assess blood-brain barrier (BBB) permeability.
  3. Mice were transcardially perfused with 0.9% NaCl until the perfusate from the right atrium ran clear.
  4. Mice were directly beheaded, stripping brain tissue on the ice, taking the region before bregma 2.34 mm, after bregma 0.22 mm and aside the middle line 0.6 mm of the cerebral cortex on a cooled culture dish lined before experiment with a knife, which was we focus on the ACC area of the brain. The obtained ACC area was placed in the Eppendorf container cooled on the ice.
  5. Each sample was weighed and then homogenized in 0.75 ml of PBS and 0.25 ml of 100% TCA solution, which could precipitate macromolecular compounds, including the nucleoprotein, using electronic homogeniser.
  6. Samples were cooled overnight at 4 °C, and then centrifuged for 30 min at 1,000 x g at 4 °C.
  7. The EB in the supernatants of each sample were subsequently measured at 620 nm using a 96-well plate reader, 100 μl of each sample was measured. All measurements were within the range of detection established by the standard curve.
  8. The dye concentration was calculated as the ratio of absorbance relative to the amount of tissue.


  1. 10% chloral hydrate
    10 g chloral hydrate powder + 100 ml double distilled water, mixed to dissolve completely
  2. 4% paraformaldehyde-freshly-prepared
    20 g paraformaldehyde powder + 500 ml PB incubated at 65 °C, stirred several times to dissolve completely
    Cooled at room temperature and then added double distilled water to 500 ml for evaporated water during the course of dissolution
  3. 0.9% NaCl
    9 g NaCl powder + 1,000 ml double distilled water, mixed to dissolve completely
  4. 100% trichloroacetic acid solution
    500 g trichloroacetic acid solution powder + 227 ml double distilled water, mixed to dissolve completely
    Stored at 4 °C and protected from light


This study was supported by grants from the National Natural Science Foundation of China (81070888 and 81230025 to J.-L.C., 81200859 to H.-L.D. 81202320 to Y. -B. G.), Natural Science Foundation of Jiangsu Education Department (13KJB320024 to J.-X.Y.), Natural Science Foundation of Jiangsu Province (BK2011198 to J.-L.C.), The Scientific Innovation Group of “Qing Lan Project” of Jiangsu Province (to J.-L.C.), and Jiangsu Provincial Special Program of Medical Science (BL2014029). We thank Wen-Zhen Duan, Heng Cai, and Tian-Hui Gao for administrative assistance.


  1. Dickstein, D. L., Biron, K. E., Ujiie, M., Pfeifer, C. G., Jeffries, A. R. and Jefferies, W. A. (2006). Abeta peptide immunization restores blood-brain barrier integrity in Alzheimer disease. FASEB J 20(3): 426-433.
  2. Harford-Wright, E., Lewis, K. M., Ghabriel, M. N. and Vink, R. (2014). Treatment with the NK1 antagonist emend reduces blood brain barrier dysfunction and edema formation in an experimental model of brain tumors. PLoS One 9(5): e97002.
  3. Yang, J. X., Hua, L., Li, Y. Q., Jiang, Y. Y., Han, D., Liu, H., Tang, Q. Q., Yang, X. N., Yin, C., Hao, L. Y., Yu, L., Wu, P., Shao, C. J., Ding, H. L., Zhang, Y. M. and Cao, J. L. (2015). Caveolin-1 in the anterior cingulate cortex modulates chronic neuropathic pain via regulation of NMDA receptor 2B subunit. J Neurosci 35(1): 36-52.


血脑屏障(BBB)是一种高度选择性的渗透性屏障,其将循环血液与中枢神经系统中的脑细胞外液分离。 血脑屏障允许通过被动扩散的水,一些气体和脂溶性分子的通过,以及对神经功能至关重要的分子如葡萄糖和氨基酸的选择性转运。 该方案提供了一种全面详细的测量Evans blue(EB)小鼠血脑屏障通透性的方法,该方法用于评估血脑屏障(BBB)通透性(样品:前扣带皮质,ACC)。


  1. 成年雄性昆明小鼠(徐州医学院实验动物中心,20-22g,5-7周龄)
  2. 伊文思蓝(EB)(Sigma-Aldrich,目录号:E2129-10G)
  3. 三氯乙酸(Sigma-Aldrich,目录号:T6399-100G)
  4. 水合氯醛(Sigma-Aldrich,目录号:15307-500G-R)
  5. 多聚甲醛(Sigma-Aldrich,目录号:158127-500G)
  6. NaCl(Sigma-Aldrich,目录号:S7653-1KG)
  7. 10%水合氯醛(见配方)
  8. 4%多聚甲醛新鲜制备(见配方)
  9. 0.9%NaCl(见配方)
  10. 100%三氯乙酸溶液(见配方)


  1. 分光光度计(Thermo Fisher Scientific,目录号:1510)
  2. 96孔板(Corning Incorporated,目录号:3599)
  3. 1和20ml注射器(本地药店)
  4. 输液器械(本地药店)


  1. 将小鼠用10%水合氯醛(0.3ml/100g,i.p.)麻醉
  2. 灌注前0.5小时从尾静脉注射伊文思蓝(EB)(2%,2ml/kg)。 EB渗漏用于评估血脑屏障(BBB)通透性
  3. 用0.9%NaCl经心脏灌注小鼠,直到来自右心房的灌洗液清澈。
  4. 将小鼠直接斩首,剥离冰上的脑组织,取前囟前2.34mm的区域,前囟0.22mm后,在实验前用刀切开冷却的培养皿上的大脑皮层的中间线0.6mm,这是我们重点在脑的ACC区域。将获得的ACC区域置于在冰上冷却的Eppendorf容器中
  5. 称重每个样品,然后在0.75ml的PBS和0.25ml的100%TCA溶液中均化,其可以使用电子均化器沉淀大分子化合物,包括核蛋白。
  6. 将样品在4℃下冷却过夜,然后在4℃以1,000xg离心30分钟。
  7. 随后使用96孔板读数器在620nm测量每个样品的上清液中的EB,测量每个样品100μl。 所有测量均在由标准曲线确定的检测范围内
  8. 染料浓度计算为相对于组织量的吸光度比


  1. 10%水合氯化
    10g水合氯醛水合物+ 100ml双蒸水,混合完全溶解
  2. 4%多聚甲醛新鲜制备的
    20g多聚甲醛粉末+ 500ml PB在65℃下温育,搅拌几次以完全溶解
  3. 0.9%NaCl
    9克NaCl粉+ 1000毫升双蒸水,混合完全溶解
  4. 100%三氯乙酸溶液 500克三氯乙酸溶液粉+ 227毫升双蒸水,混合完全溶解


本研究由中国国家自然科学基金(81070888和81230025到J.-LC,81200859到H.-LD 81202320到Y.-BG),江苏教育部自然科学基金(13KJB320024到J. -XY),江苏省自然科学基金(BK2011198至J.-LC),江苏省"清兰工程"科学创新组(获得J.-LC)和江苏省医学科学特别规划(BL2014029 )。 我们感谢文正段,衡才和高天辉的行政协助。


  1. Dickstein,D.L.,Biron,K.E.,Ujiie,M.,Pfeifer,C.G.,Jeffries,A.R。和Jefferies,W.A。(2006)。 Abeta肽免疫可恢复阿尔茨海默病的血脑屏障完整性。FASEB J 20(3):426-433。
  2. Harford-Wright,E.,Lewis,K.M.,Ghabriel,M.N.and Vink,R。(2014)。 用NK1拮抗剂emend治疗可降低脑肿瘤实验模型中的血脑屏障功能障碍和水肿形成。 PLoS One 9(5):e97002。
  3. Yang,JX,Hua,L.,Li,YQ,Jiang,YY,Han,D.,Liu,H.,Tang,QQ,Yang,XN,Yin,C.,Hao,LY,Yu, ,P.,Shao,CJ,Ding,HL,Zhang,YM和Cao,JL(2015)。 前扣带皮层中的窖蛋白-1通过调节NMDA受体2B亚基调节慢性神经性疼痛。/a> J Neurosci 35(1):36-52。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Yang, J., Jiang, Y. and Guo, Y. (2015). Measuring Blood-brain-barrier Permeability Using Evans Blue in Mice. Bio-protocol 5(15): e1548. DOI: 10.21769/BioProtoc.1548.
  2. Yang, J. X., Hua, L., Li, Y. Q., Jiang, Y. Y., Han, D., Liu, H., Tang, Q. Q., Yang, X. N., Yin, C., Hao, L. Y., Yu, L., Wu, P., Shao, C. J., Ding, H. L., Zhang, Y. M. and Cao, J. L. (2015). Caveolin-1 in the anterior cingulate cortex modulates chronic neuropathic pain via regulation of NMDA receptor 2B subunit. J Neurosci 35(1): 36-52.



Max Christian
Georg August
Dear Author,

i have a question regarding the procedure: Is it possible to store the brains in -80°C till analysis?

I would be very happy about an answer.

Best regards
4/23/2018 5:11:51 AM Reply