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Assay to Evaluate Vascular Permeability Induction in Mice
小鼠血管通透性诱导的分析评估

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Abstract

Dengue virus infection usually courses as a benign self-limited fever, called dengue fever. However, on occasions it can progress to a life-threatening complication known as severe dengue (SD). A hallmark of SD is a sharp increase in vascular permeability. Secondary infections are considered a risk factor to develop SD, presumably through a mechanism called Antibody-Dependent Enhancement (ADE) of infection in cells with the capacity to bind antigen-antibody complexes, such as macrophages, and to trigger a subsequent aberrant cytokine response. The massive release of cytokine from macrophages has been postulated to cause changes in vascular permeability. The vascular permeability assay presented in this protocol is designed to assess whether any compound or cell-secreted product or soluble factor present in sera from patients may induce plasma leakage in mice. This test was used in the laboratory to determine whether cytokines and soluble factors produced in vitro by macrophages infected with dengue virus or dengue virus in the presence of facilitating antibodies are able to induce plasma leakage in vivo. Macrophages were infected with dengue virus or dengue virus in the presence of facilitating antibodies for 48 h. After this time, the conditioned supernatant containing cytokines and soluble factors released by the macrophages were collected and inoculated intraperitoneally into CD-1 mice. Twenty four hours after the first inoculation, mice were reinoculated with a second dose with Evans blue dye. After another 24 h, mice were euthanized and the amount of Evans blue present in the blood and lung was determined by spectrophotometric analysis. The assay was able to show differences in the capacity of the conditioned media to induce vascular permeability changes in the inoculated animals (Puerta-Guardo et al., 2013).

Keywords: Dengue(登革热), Antibody dependent enhancement(抗体依赖性增强), Cytokines(细胞因子), Tight junctions(紧密连接), Plasma leakage(血浆渗漏)

Materials and Reagents

  1. Mice of six to seven weeks old (CD-1® Mouse, Crl:CD1 (ICR)) (Charles River Laboratories)
  2. Evans Blue Dye (Sigma-Aldrich, catalog number: E2129 )
  3. Ketamine (Sigma-Aldich, catalog number: K2753 )
  4. Xylazine (Sigma-Aldich, catalog number: X1251 )
  5. TNF-α (BD CBA flex set) (BD Biosciences, catalog number: 558273 )
  6. 10% Formalin (Sigma-Aldich, catalog number: HT501128 )
  7. Formamide (Sigma-Aldich, catalog number: F8775 )
  8. NaCl
  9. KCl
  10. Na2HPO4
  11. KH2PO4
  12. HCl
  13. 1x PBS (see Recipes)
  14. 1% (w/v) Evans blue dye solution in PBS (see Recipes)

Equipment

  1. EDTA tubes
  2. Lyophilizer
  3. Centrifuge (Beckman Coulter, model: Allegra X-12R ) (Rotor SX4750)
  4. Spectrophotometry (BioTek Instruments, model: ELx808 Absorbance Microplate Reader)

Procedure

  1. Mice of six to seven weeks old and 18-20 g in weight in a minimal amount of 4 mice per group, were injected intraperitoneally with 2 doses (100 μl each) 24 h apart, of media or supernatants to be evaluated.
  2. Since TNF-α has been described as a plasma leakage inductor, this cytokine can be used for the injection as positive control (4 ng/ml).
  3. Together with the second dose of conditioned supernatant, mice were injected intraperitoneally with 4 ml/kg of weight a 1% Evans blue dye solution (w/v in PBS) allowing circulation for 24 h as was described previously (Manaenko et al., 2011).
  4. Before samples were collected, mice were injected with anesthetics: ketamine (70 mg/kg) and xylazine (6 mg/kg), afterward blood samples were collected by cardiac puncture.
  5. Blood was immediately placed in EDTA tubes.
  6. Samples were diluted 1:2 v/v in PBS and plasma was separated by centrifugation at 3,000 rpm for 8 min.
  7. The amount of Evans blue dye present in plasma was measured by spectrophotometry at 630 nm. Quantification was performed by comparison with a standard curve.
  8. The standard curve was constructed using dilution of Evans blue dye in 1x PBS in a range from 3 to 400 ng/ml (R = 0.999).
  9. To evaluate the pulmonary capillary leakage, the mice were perfused transcardially with 50 ml of PBS followed by 20 ml of 10% Formalin for tissue fixation. The lung tissues were removed and preserved in formalin solution at 4 °C until analysis.
  10. The right lung was vacuum-dried, weight and frozen instantly in liquid nitrogen.
  11. Evans blue dye was extracted from the lung by incubation at 65 °C with formamide (2 ml/g tissue) overnight (Peng et al., 2004).
  12. The lung tissue was pelleted by centrifugation (12,000 x g for 30 min), and the concentration of Evans blue dye extracted in the supernatant was determined spectrophotometrically at 630 nm against a standard curve. Concentration of Evans blue dye was determined in ng/ml of lung tissue. However, this study was designed to compare the permeability induction capacity of different cell supernatants, thus, results were expressed as fold increases in relation to the mock or control condition. Nevertheless, results can also be expressed directly in ng/ml of tissue. Any statistically significant increase between the control and the experimental conditions is considered as leakage induction (Figure 1).

Note: This type of studies has to be conducted in accordance with official guidelines for the Standard Production, Care and Use of Laboratory Animals of each country.



Figure 1. Effect of conditioned supernatants on vascular permeability in vivo. Six to eight weeks old mice (CD1 strain) were injected twice intraperitoneally, 24 h apart, with conditioned supernatans. A. Solution of 1% Evans Blue dye (EVD) was injected together with the second dose. Twenty four hours later, EVD was extracted from plasma and lungs and quantified against standard curves. Values are expressed as ng/ml per plasma (panel A) or as fold increases per lung in relation to the control (panel B). Panels C and D, standard curves for EVD extracted from plasma and lungs, respectively. Four mice were included per condition. Statistical significance was *p < 0.05 and **p < 0.001. Mock: supernatant collected from mock infected cells. DENV: Supernatant collected from cells infected directly with dengue virus. DENV + Enh: Supernatant collected from cells infected in the presence of enhancing antibodies. DENV + Mut: Supernatant collected from cells infected in the presence of mutated antibodies (incapable of inducing enhancing). DENV+Neu: Supernatant collected from cells infected in the presence of neutralizing antibodies. TNF-α: TNF-α used as positive control.

Recipes

  1. 1x PBS
    8 g (137 mM) NaCl
    0.2 g (2.7 mM) KCl
    1.44 g (10 mM) Na2HPO4
    0.24 g (2 mM) KH2PO4
    Add 800 ml H2O
    Adjust pH to 7.4 with HCl
    Add water up to 1 L
  2. 1% (w/v) Evans blue dye solution in PBS
    Add 1 g of Evans blue dye to 100 ml of 1x PBS

Acknowledgments

This protocol was adapted from Puerta-Guardo et al. (2013). The study was partially supported by grants 103783 and 127447 from The Mexican Council for Science and Technology (CONACYT) to JEL and RMDA, respectively. HPG and ARS are recipients of CONACYT scholarships. Authors declare no conflict of interest.

References

  1. Manaenko, A., Chen, H., Zhang, J. H. and Tang, J. (2011). Comparison of different preclinical models of intracerebral hemorrhage. Acta Neurochir Suppl 111: 9-14.
  2. Peng, X., Hassoun, P. M., Sammani, S., McVerry, B. J., Burne, M. J., Rabb, H., Pearse, D., Tuder, R. M. and Garcia, J. G. (2004). Protective effects of sphingosine 1-phosphate in murine endotoxin-induced inflammatory lung injury. Am J Respir Crit Care Med 169(11): 1245-1251.
  3. Puerta-Guardo, H., Raya-Sandino, A., Gonzalez-Mariscal, L., Rosales, V. H., Ayala-Davila, J., Chavez-Mungia, B., Martinez-Fong, D., Medina, F., Ludert, J. E. and del Angel, R. M. (2013). The cytokine response of U937-derived macrophages infected through antibody-dependent enhancement of dengue virus disrupts cell apical-junction complexes and increases vascular permeability. J Virol 87(13): 7486-7501.

简介

登革热病毒感染通常称为良性自限发热,称为登革热。然而,偶尔,它可以进展为严重登革热(SD)的危及生命的并发症。 SD的标志是血管通透性的急剧增加。二次感染被认为是发展SD的风险因素,可能是通过在具有结合抗原 - 抗体复合物(例如巨噬细胞)能力的细胞中感染的感染的机制,称为抗体依赖性增强(ADE)引发随后的异常细胞因子应答。已经假定巨噬细胞中细胞因子的大量释放导致血管通透性的变化。在该方案中呈现的血管渗透性测定设计为评估来自患者的血清中存在的任何化合物或细胞分泌的产物或可溶性因子是否可以在小鼠中诱导血浆渗漏。在实验室中使用该测试以确定在存在促进性抗体的情况下由登革热病毒或登革热病毒感染的巨噬细胞在体外产生的细胞因子和可溶性因子是否能够在体内诱导血浆渗漏。在存在促进抗体的情况下,将巨噬细胞用登革病毒或登革热病毒感染48小时。此后,收集含有由巨噬细胞释放的细胞因子和可溶性因子的条件上清液,并腹膜内接种到CD-1小鼠中。在第一次接种后二十四小时,用伊文思蓝染料以第二剂量再次接种小鼠。再过24小时后,对小鼠实施安乐死,通过分光光度分析测定血液和肺中存在的伊文思蓝的量。该测定能够显示条件培养基在接种的动物中诱导血管通透性改变的能力的差异(Puerta-Guardo等人,2013)。

关键字:登革热, 抗体依赖性增强, 细胞因子, 紧密连接, 血浆渗漏

材料和试剂

  1. 六至七周龄的小鼠(CD-1小鼠,Crl:CD1(ICR))(Charles River Laboratories)
  2. Evans Blue Dye(Sigma-Aldrich,目录号:E2129)
  3. 氯胺酮(Sigma-Aldich,目录号:K2753)
  4. 甲苯噻嗪(Sigma-Aldich,目录号:X1251)
  5. TNF-α(BD CBA flex set)(BD Biosciences,目录号:558273)
  6. 10%福尔马林(Sigma-Aldich,目录号:HT501128)
  7. 甲酰胺(Sigma-Aldich,目录号:F8775)
  8. NaCl
  9. KCl
  10. Na HPO 4
  11. KH 2 PO 4
  12. HCl
  13. 1x PBS(请参阅配方)
  14. 1%(w/v)Evans蓝染料在PBS中的溶液(参见配方)

设备

  1. EDTA试管
  2. 冻干机
  3. 离心机(Beckman Coulter,型号:Allegra X-12R)(Rotor SX4750)
  4. 分光光度法(BioTek Instruments,型号:ELx808 Absorbance Microplate Reader)

程序

  1. 在最小量的每组4只小鼠中,将6-7周龄和18-20g重量的小鼠腹膜内注射2次剂量(每次100μl,相隔24小时)待评估的培养基或上清液。
  2. 由于TNF-α已被描述为血浆渗漏感应器,该细胞因子可用于注射作为阳性对照(4ng/ml)。
  3. 与第二剂量的条件上清液一起,小鼠腹膜内注射4ml/kg重量的1%伊文思蓝染料溶液(w/v,在PBS中),如前所述允许循环24小时 (Manaenko等人,2011)。
  4. 在收集样品之前,向小鼠注射麻醉剂:氯胺酮(70mg/kg)和甲苯噻嗪(6mg/kg),之后通过心脏穿刺收集血液样品。
  5. 将血液立即置于EDTA管中。
  6. 样品在PBS中以1:2v/v稀释,并通过在3,000rpm下离心8分钟分离血浆
  7. 通过分光光度法在630nm测量血浆中存在的伊文思蓝染料的量。 通过与标准曲线比较进行定量。
  8. 使用1×PBS中的Evans蓝染料在3至400ng/ml(R = 0.999)范围内的稀释液构建标准曲线。
  9. 为了评价肺毛细血管渗漏,用50ml PBS随后用20ml 10%福尔马林经心脏灌注小鼠用于组织固定。 取出肺组织并保存在福尔马林溶液中 在4℃直至分析。
  10. 将右肺真空干燥,称重,并在液氮中立即冷冻。
  11. 通过在65℃下与甲酰胺(2ml/g组织)温育过夜(Peng等人,2004)从肺中提取伊文思蓝染料。
  12. 通过离心(12,000xg,30分钟)使肺组织沉淀,并且在630nm下相对于标准曲线通过分光光度法测定在上清液中提取的伊文思蓝染料的浓度。以ng/ml肺组织测定伊文思蓝染料的浓度。然而,本研究设计用于比较不同细胞上清液的渗透诱导能力,因此,结果表示为相对于模拟或对照条件的倍数增加。然而,结果也可以直接表示为ng/ml组织。在对照和实验条件之间的任何统计学上显着的增加被认为是泄漏诱导(图1)

注意:这种类型的研究必须根据每个国家的标准生产,护理和使用实验动物的官方指南进行。



图1.经调节的上清液对体内血管通透性的影响。 6周至8周龄的小鼠(CD1株)用调节的上清液腹膜内注射两次,间隔24小时。 A.将1%伊文思蓝染料(EVD)的溶液与第二剂一起注射。 24小时后,从血浆和肺中提取EVD,并根据标准曲线定量。值表示为每个血浆的ng/ml(图A)或每个肺相对于对照的增加倍数(图B)。图C和D,分别是从血浆和肺提取的EVD的标准曲线。每个条件包括四只小鼠。统计学显着性为* p <0.05和** p <0.001。模拟:从模拟感染细胞收集的上清液。 DENV:从直接感染登革热病毒的细胞收集上清液。 DENV + Enh:从存在增强抗体的情况下感染的细胞收集上清液。 DENV + Mut:从存在突变抗体(不能诱导增强)的情况下感染的细胞中收集上清液。 DENV + Neu:从存在中和抗体的情况下感染的细胞收集上清液。 TNF-α:TNF-α用作阳性对照。

食谱

  1. 1x PBS
    8g(137mM)NaCl 0.2克(2.7mM)KCl 1.44g(10mM)Na 2 HPO 4
    0.24g(2mM)KH 2 PO 4>/
    加入800ml H 2 O 2 / 用HCl
    调节pH至7.4 加水最多1 L
  2. 1%(w/v)Evans蓝染料溶液在PBS中 将1克伊文思蓝染料加到100毫升1×PBS中

致谢

该协议改编自Puerta-Guardo等人(2013)。 该研究部分得到了墨西哥理工学院(CONACYT)给JEL和RMDA的拨款103783和127447的支持。 HPG和ARS是CONACYT奖学金的接受者。 作者声明不存在利益冲突。

参考文献

  1. Manaenko,A.,Chen,H.,Zhang,J.H.and Tang,J.(2011)。 脑内出血的不同临床前模型的比较。 Acta Neurochir Suppl < em> 111:9-14。
  2. Peng,X.,Hassoun,P.M.,Sammani,S.,McVerry,B.J.,Burne,M.J.,Rabb,H.,Pearse,D.,Tuder,R.M.and Garcia,J.G。(2004)。 鞘氨醇1-磷酸在鼠内毒素诱导的炎性肺损伤中的保护作用 < em> Am J Respir Crit Care Med 169(11):1245-1251。
  3. Puerta-Guardo,H.,Raya-Sandino,A.,Gonzalez-Mariscal,L.,Rosales,VH,Ayala-Davila,J.,Chavez-Mungia,B.,Martinez-Fong,D.,Medina, ,Ludert,JE和del Angel,RM(2013)。 通过抗体依赖性增强登革热病毒感染的U937衍生的巨噬细胞的细胞因子应答破坏细胞顶端 - 结合复合物并增加血管通透性。

    87(13):7486-7501。
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引用:Puerta-Guardo, H., Raya-Sandino, A., González-Mariscal, L., Rosales, V. H., Ayala-Dávila, J., Chávez-Mungía, B., Martínez-Fong, D., Medina, F., Ludert, J. E. and Angel, R. M. (2013). Assay to Evaluate Vascular Permeability Induction in Mice. Bio-protocol 3(22): e977. DOI: 10.21769/BioProtoc.977.
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