参见作者原研究论文

本实验方案简略版
Dec 2012

本文章节


 

Assay of Blood Brain Barrier and Placental Barrier Permeability
血脑障壁和胎盘屏障透过率的分析   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Evans blue dye solution was used to observe the effect of a viral protein on two pre-defined barriers of the body i.e. the blood brain barrier (BBB) and the placental barrier (PB). This dye has strong affinity for serum albumin and does not cross these barriers under natural conditions. As all the dye gets bound to albumin, all the neural tissues and embryonic tissues remain unstained. When the BBB and PB are compromised due to the breach of these barriers, albumin-bound Evans blue enters the CNS and the placenta.

Materials and Reagents

  1. Pregnant Sprague Dawley rats
  2. Protein of Interest: Recombinant Nef
  3. Evans blue dye (Sigma-Aldrich)
  4. NaCl (Sigma-Aldrich)
  5. Phosphate buffer saline (PBS) (Sigma-Aldrich)

Equipment

  1. Homogenizer (Coleparmer)
  2. Centrifuge (Eppendorf)
  3. Weighing Balance (Mettler Toledo)
  4. Spectrophotometer (Gene Quant)

Procedure

  1. 2% Evans Blue dye was dissolved in normal saline (0.85% sodium chloride). 500 μl of dye containing recombinant protein was injected intravenously in the tail vein of fourteen days pregnant Sprague Dawley rats.
  2. Recombinant protein in the range 50-500 μg was used to identify the threshold value needed for the breach of both the barriers. Un-injected animals were used for the normalization of the data.
  3. One hour after inoculation all the rats including the un-injected ones were anaesthetized and dissected immediately to avoid any blood clotting; complete uterus and the brain were removed carefully in normal saline.
  4. The fetal tissues; uterus, placenta, amniotic membrane and embryo were separated cautiously and collected in PBS to measure the weight of these organs separately.
  5. Each fetal tissue was measured by weight and then homogenized in 200 μl of PBS (pH 7.4), the final volume was measured again and the mg/ml concentration was calculated.
  6. The homogenized tissue was centrifuged at ~9,000 x g at 4 °C for 15 min and the clear supernatant was collected.
  7. The absorbance of Evans blue dye was measured at OD590nm from the brain as well as fetal tissues associated with blood-brain barrier and placental barrier respectively.
  8. The absorbance (OD) per mg of tissue weight was determined from the supernatant at 590 nm for the quantitative analysis of Evans blue dye.
  9. Absorbance was considered as an average of four animals and the actual absorbance was calculated after normalizing the background values from the un-injected control set of tissues.
  10. Figure 1 explains the breach of these blood barriers in the presence of the recombinant protein, whereas no breach was observed in the absence of the recombinant protein.
  11. If the blood barriers breaches, the absorbance was found to be higher and the dye permeability was observed, whereas if the blood barrier is intact then the absorbance was comparatively lower and no permeability was observed for the dye.


    Figure 1. Quantification of Evans blue dye (OD at 590) present (within an hour) in brain and different fetal tissue isolates from 14 day pregnant Sprague Dawley rats injected intravenously without and with recombinant Nef and ASK-1 protein. (A) Brain (B) Uterus (C) Placenta (D) Amniotic membrane. Three different bars in each set represent 0, 250 and 500 μg of recombinant Nef and 500 μg of ASK-1 injected intravenously along with Evans blue dye in the experimental animals. As data represent ±SEM of 3 separate experiments in duplicate and changes were considered as significant at *p ≤ 0.05,**p ≤ 0.01 and ***p ≤ 0.001.

Acknowledgments

This protocol is adapted from Chaturverdi et al. (1991); Singh et al. (2012) and Thumwood et al. (1988).

References

  1. Chaturvedi, U. C., Dhawan, R., Khanna, M. and Mathur, A. (1991). Breakdown of the blood-brain barrier during dengue virus infection of mice. J Gen Virol 72 ( Pt 4): 859-866.
  2. Singh, P., Agnihotri, S. K., Tewari, M. C., Kumar, S., Sachdev, M. and Tripathi, R. K. (2012). HIV-1 Nef breaches placental barrier in rat model. PLoS One 7(12): e51518. 
  3. Thumwood, C. M., Hunt, N. H., Clark, I. A. and Cowden, W. B. (1988). Breakdown of the blood-brain barrier in murine cerebral malaria. Parasitology 96 ( Pt 3): 579-589.

简介

伊文思蓝染料溶液用于观察病毒蛋白对身体的两个预定义屏障(即,血脑屏障(BBB)和胎盘屏障(PB))的影响。 该染料对血清白蛋白具有强亲和力,并且在自然条件下不会穿过这些屏障。 由于所有的染料与白蛋白结合,所有的神经组织和胚胎组织保持未染色。 当BBB和PB由于破坏这些障碍而受损时,白蛋白结合的伊文思蓝进入CNS和胎盘。

材料和试剂

  1. 怀孕的Sprague Dawley大鼠
  2. 目的蛋白:重组Nef
  3. 伊文思蓝染料(Sigma-Aldrich)
  4. NaCl(Sigma-Aldrich)
  5. 磷酸盐缓冲液(PBS)(Sigma-Aldrich)

设备

  1. 均质器(Coleparmer)
  2. 离心机(Eppendorf)
  3. 称重天平(Mettler Toledo)
  4. 分光光度计(Gene Quant)

程序

  1. 2%伊文思蓝染料溶于生理盐水(0.85%氯化钠)中。在十四天怀孕的Sprague Dawley大鼠的尾静脉中静脉内注射500μl含染料的重组蛋白。
  2. 使用50-500μg范围内的重组蛋白来鉴定破坏两个屏障所需的阈值。未注射的动物用于数据的标准化。
  3. 接种后1小时,麻醉所有的大鼠,包括未注射的大鼠,立即解剖以避免任何血液凝固;在生理盐水中小心地取出完整的子宫和脑。
  4. 胎儿组织;子宫,胎盘,羊膜和胚胎小心分离并收集在PBS中以分别测量这些器官的重量。
  5. 每个胎儿组织通过重量测量,然后在200μlPBS(pH 7.4)中匀化,再次测量最终体积并计算mg/ml浓度。
  6. 匀浆的组织在〜9,000×g,4℃下离心15分钟,收集澄清的上清液。
  7. 在来自脑的OD 590nm处以及分别与血脑屏障和胎盘屏障相关的胎儿组织测量伊文思蓝染料的吸光度。
  8. 从590nm的上清液中测定每mg组织重量的吸光度(OD),用于Evans蓝色染料的定量分析。
  9. 吸光度被认为是四只动物的平均值,并且在归一化来自未注射对照组的背景值之后计算实际吸光度。
  10. 图1解释了在重组蛋白存在下这些血液屏障的破坏,而在没有重组蛋白的情况下没有观察到破裂。
  11. 如果血液屏障破裂,则发现吸光度较高并且观察到染料渗透性,而如果血液屏障是完整的,则吸光度相对较低,并且没有观察到染料的渗透性。


图1.来自在没有和具有重组Nef和ASK-1蛋白的情况下静脉内注射的14天怀孕的Sprague Dawley大鼠的脑和不同胎儿组织分离物中存在(在1小时内)的伊文思蓝染料(590nm处的OD)/strong>(A)脑(B)子宫(C)胎盘(D)羊膜。每组中三个不同的条代表0,250和500μg重组Nef和500μgASK-1,在实验动物中与Evans蓝染料一起静脉内注射。数据表示3个单独实验的±SEM,一式两份,并且在*p≤0.05,**p≤0.01和***p≤0.001时认为改变是显着的。

致谢

该协议改编自Chaturverdi等人(1991); Singh等人(2012)和Thumwood等人(1988)。

参考文献

  1. Chaturvedi,U.C.,Dhawan,R.,Khanna,M。和Mathur,A。(1991)。 在登革热病毒感染小鼠期间血脑屏障的分解。 J Gen Virol 72(Pt 4):859-866。
  2. Singh,P.,Agnihotri,S.K.,Tewari,M.C.,Kumar,S.,Sachdev,M。和Tripathi,R.K。 HIV-1 Nef在大鼠模型中破坏胎盘屏障 /em> 7(12):e51518。
  3. Thumwood,C.M.,Hunt,N.H.,Clark,I.A。和Cowden,W.B。(1988)。 鼠脑性疟疾中血脑屏障的分解。寄生虫学 96(Pt 3):579-589。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Agnihotri, S. K., Singh, P., Kumar, B., Singh, P., Jain, S. K., ChandraTewari, M., Kumar, S., Sachdev, M. and Tripathi, R. K. (2013). Assay of Blood Brain Barrier and Placental Barrier Permeability. Bio-protocol 3(15): e845. DOI: 10.21769/BioProtoc.845.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。