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Mar 2018
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Vascular Permeability Assay in Human Coronary and Mouse Brachiocephalic Arteries
人冠状动脉和鼠头臂动脉的血管渗透性检测   

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Abstract

Coronary artery disease remains an important cause of morbidity and mortality. Previous work, including ours, has focused on the role of intraplaque hemorrhage, particularly from immature microvessel angiogenesis, as an important contributor to plaque progression via increases in vascular permeability leading to further intraplaque hemorrhage, which increases red cell membrane-derived free cholesterol in plaque content and inflammatory cell recruitment. Evans Blue Dye (EBD) assay is widely used as a standard assay for vasculature permeability. However, the method has not been established in fresh human coronary artery autopsy samples to evaluate intraplaque microvessel permeability and angiogenesis. In this protocol, we describe a method to evaluate human coronary samples for microvascular permeability, including procedures to perfuse coronary arteries, collection of artery samples for histological analysis and immunostaining as well as the use of appropriate methodology to analyze the images. An optional procedure is also provided for the use of FITC-dextran in mouse model to evaluate vascular permeability. These Evans Blue Dye procedures may be useful in providing functional measure of the endothelium integrity and permeability in both human samples and animal models in various pathological conditions.

Keywords: Coronary artery disease (冠心病), Atherosclerosis (动脉粥样硬化), Microvessel permeability (微血管通透性), Evans blue dye (伊文氏蓝染色), FITC-dextran (FITC标记葡聚糖), Angiogenesis (血管生成), Intraplaque hemorrhage (斑块内出血)

Background

Vascular endothelial cells actively regulate the infiltration of plasma constituents and circulating cells, including leukocytes, from blood to sub-endothelial tissues. This mechanism is generally considered to be a critical step of initiation and progression of atherosclerosis (Mundi et al., 2018). The regulation of vascular permeability is achieved through the coordinated opening and closure of endothelial cell-cell junctions. In several diseased conditions, endogenous agents such as histamine, thrombin, and vascular endothelial growth factors (VEGF) dramatically, but reversibly, alter the function and organization of cell-cell junctional complexes in diverse ways, resulting in various degrees of increase in permeability (Dejana et al., 2008). We recently demonstrated a unique mechanism, by which CD163-positive alternative macrophages [M(Hb)] engulf hemoglobin-haptoglobin complexes at the sites of intra-plaque hemorrhage (IPH), promoting the release of vascular endothelial growth factor (VEGF), which further causes internalization of vascular endothelial cadherin (VE-cad) from inter-cellular adherens junctions, thus increasing vascular permeability and leading to atherosclerotic plaque progression (Guo et al., 2018). In the aforementioned study, we conducted a novel technique to assess vascular permeability of human coronary arteries by using Evans Blue Dye (EBD) solution. In the same study, FITC-dextran was used as an alternative indicator in one-year-old apoE mouse model to demonstrate the intraplaque hemorrhage in brachiocephalic artery, due to the small size of the mouse arteries and dye sensitivity required for confocal imaging.

Among the vast range of blue dyes that were created during the 20th century, EBD has been the one with the longest biological history since its first application by Herbert McLean Evans in 1914 (Evans and Schulemann, 1914). EBD is an alkaline synthetic bis-azo (benzidine group) with a molecular weight of 961 Da., and high water solubility, allowing the dye to quickly diffuse throughout the blood stream. Most importantly, when the dye is injected intravenously, it has a high affinity for plasma albumin, giving the dye the ability to remain stable and remain distributed throughout the body for a longer time as a result of a slow excretion rate. All the features of EBD allow it to be an extraordinary agent with multiple potential applications in biomedicine as recently reviewed by Linpeng Yao (Yao et al., 2018). These include but are not limited to estimation of plasma volume, identification of tumors and lymph nodes and as a potential marker of vessel permeability by the use of fluorescence when exposed to green light (Hamer et al., 2002). The principles behind the use of EBD in assays to determine vascular permeability lies in the fact that in normal tissues with normal vascular integrity, albumin is not able to migrate out into the interstitium through the vessel wall endothelial layer. This means that in cases of Albumin-EBD complex, the dye would be limited only to circulation. EBD is relatively non-toxic when used in appropriate concentrations. In vivo experiments using both mice and human subjects have demonstrated that when used in excess, albumin reaches its maximum percent of saturation causing vascular leakage and resulting in a rapid bluish discoloration of tissues (Miles and Miles, 1952). In normal conditions, an adequate permeability barrier is maintained through tight cell-to-cell adherens junctions that are strictly controlled by growth factors, cytokines and other molecules (Radu and Chernoff, 2013). However, in pathological conditions where the integrity of the endothelial layer is affected, plasma proteins including albumin, are able to leak out the vessels as may occur in various disease states. The most common pathophysiological event leading to an increase vascular permeability is acute inflammation which may occur when the vascular wall including the endothelial layer is injured. Vasodilatation, increase in blood flow, disruption of endothelial cell junctions and infiltration of leukocytes are the key players in this process. The EBD-Albumin complex can be seen microscopically as basophilic color in interstitial tissues and indicated increased vascular permeability. In our recent study on endothelial barrier dysfunction after drug-eluting stents implantation, EBD perfusion was performed in rabbit iliac artery stenting model to demonstrate the arteries permeability is associated with poor endothelial VE-cadherin/P120 junctions and higher macrophage infiltration (Harari et al., 2018). Given the advantages of EBD, we decided to use the technique to study the human coronary arteries permeability.

Materials and Reagents

  1. Pipette tips
  2. 250 ml Stericup filter unit (Merck, catalog number: SCGPU02RE )
  3. Coverslips (Fisher Scientific, catalog number: 12-543D )
  4. Kimwipes (KCWW, Kimberly-Clark, catalog number: 34155 )
  5. Ultra-fine Syringe (BD, catalog number: 324911 )
  6. Human coronary artery samples selected from freshly collected autopsy specimens (from the CVPath Registry)
  7. [Optional] One-year-old apoE knockout (KO) mouse (THE JACKSON LABORATORY, catalog number: 002052 )
  8. EcoMount (Biocare Medical, catalog number: EM897L )
  9. CoverMount for non-EBD staining, Xylene based (Avantik, catalog number: SL6012-A )
  10. Evans blue dye (Sigma-Aldrich, catalog number: E2129-50G )
  11. FITC-dextran (Sigma-Aldrich, catalog number: 46945 )
  12. 5% BSA (Fisher, catalog number: BP1600-100 )
  13. PBS (1x, Ultra Pure Grade, VWR, catalog number: 97063-658 )
  14. Neutral buffered formalin (NBF) (Sigma-Aldrich, catalog number: HT501128 )
  15. Tissue-Tek® O.C.T. Compound (Sakura Finetek, Miles, catalog number: 4583 )
  16. Hydrogen peroxide H2O2 3% (VWR, catalog number: BDH7540-2 )
  17. Dako protein block (Agilent Technologies, DAKO, catalog number: X0909 )
  18. CD163 antibody (Santa Cruz Biotechnology, catalog number: sc-20066 , clone GHI/61)
  19. CD68 antibody (Dako, clone Kp1)
  20. Von Willebrand factor (vWF) antibody (SDIX, Strategic BioSolutions, catalog number: S4003GND1 )
  21. Hypoxia induced factor 1α (HIF1α) antibody (Novus Biologicals, catalog number: NB100-105 )
  22. Vascular endothelial growth factor-A (VEGF-A) antibody (BioGenex, catalog number: PU483-UP )
  23. VE-cadherin antibody (R&D Systems, catalog number: AF1002 , dilution 1:100, and BD Biosciences, catalog number: 555661 )
  24. Vascular cell adhesion protein (VCAM) antibody (Abcam, catalog number: ab134047 )
  25. CD3 antibody (Roche Diagnostics, catalog number: 790-4341 , prediluted)
  26. Biotinylated goat anti-rabbit, horse anti-mouse, and rabbit anti-goat (Vector Laboratories, catalog numbers: BA-1000 , BA-2000 , BA-5000 , respectively)
  27. Alexa Flour 488 and 555 streptavidin (Thermo Fisher Scientific, InvitrogenTM, catalog numbers: S32354 and S32355 , respectively)
  28. DAPI (Thermo Fisher Scientific, InvitrogenTM, catalog number: D3571 )
  29. 2-methylbutane (Spectrum Chemical Manufacturing, catalog number: M1246 )
  30. Liquid nitrogen
  31. 20% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15713-S )
  32. Acetone (Fisher Scientific, catalog number: A929-1 )
  33. Liquid blocker (Ted Pella, catalog number: 22309 )
  34. Glacial acetic acid (Fisher Scientific, catalog number: A38-212 )
  35. Hematoxylin and Eosin
    1. Xylene, Reagent grade/ACS (Avantik, catalog number: RS4050 )
    2. Mounting media/Permount (Fisher Scientific, catalog number: SP15-500
    3. Deionized water from laboratory
    4. Mayer’s Hematoxylin Solution (Astral Diagnostics, catalog number: 7020 )
    5. Gill 3 (Sigma, catalog number: GHS3128 )
    6. Eosin-phloxine stain (Astral Diagnostics, catalog number: 7010 )
    7. 100% reagent alcohol (Avantik, catalog number: RS4029 )
    8. 95% reagent grade alcohol (Avantik, catalog number: RS4031 )
    9. Ammonium hydroxide, ACS grade (Sigma-Aldrich, catalog number: A6899 )
  36. Evans blue dye solution (see Recipes)

Equipment

  1. Pipettes
  2. Belly button shaker (IBI Scientific)
  3. Axio Scan.Z1 digital slide scanner (Carl Zeiss, catalog number: Axio Scan.Z1 )
  4. Bright OTF 5000 microtome cryostat (Hacker Instruments, Hacker, catalog number: OTF 5000 ) using sectioning blades (Thermo Fisher Scientific, catalog number: 3152735 )
  5. CryoJane tape transfer system (Leica Biosystems, catalog number: 39475205 )
  6. LSM 800 confocal laser scanning microscope (Carl Zeiss, catalog number: LSM 800 )
  7. Olympus BX51 microscope (Olympus, model: BX51 )
  8. RNAscope Probe-Hs-CD163-C2 (Advanced Cell Diagnostics, catalog number: 417061-C2 )
  9. RNAscope Probe-Hs-VEGFA (Advanced Cell Diagnostics, catalog number: 423161 )
  10. TLE series ultra-low freezer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: TLE60086A )
  11. Stemi DV4 Stereo dissecting microscope (ZEISS, model: Stemi DV4 )

Software

  1. HALO Image Analysis Platform (Indica Labs) version 2.0
  2. Zen Blue 2012 (Carl Zeiss) version 2.0
  3. Zen Black 2012 (Carl Zeiss) version 2.0

Procedure

  1. Preparing and freezing tissues
    1. Select fresh collected autopsy human heart with coronary artery disease. Samples must have a post-mortem interval (PMI) cut-off of less than 12 h as well as evidence of advanced atherosclerosis based on x-ray images of the heart (which would allow for detection of vascular calcification). (Figure 1)


      Figure 1. Digital X-ray image of the heart. Highlighted bright spots are calcification in the coronary arteries (red arrows). Calcification in aortic valves can be seen in the center between coronary arteries.

    2. Prepare a saline solution containing 5% BSA.
    3. Prepare a 0.5% solution of Evans Blue Dye by dissolving Evans Blue powder in the BSA-containing saline.
    4. Filter the Evans Blue Dye solution using 250 ml Stericup filter unit. 
    5. Perfuse the filtered Evans Blue into both right and left coronary arteries for 15 min at 37 °C through a mean pressure of approximately 60 mmHg by gravity flow. 
    6. Wash the Evans Blue-perfused samples with 500 ml PBS for 20 min using the same perfusion method by gravity flow.
    7. Next, fix the samples by 500 ml neutral buffered formalin for 20 min.
    8. Harvest the formalin-fixed coronary artery tissue samples and place in a PBS solution containing 50 ml 15% sucrose overnight at 4 °C in a Falcon tube.
    9. Remove tissues from the sucrose solution and dry with Kimwipes.
    10. Cut the harvested samples in 2-3 mm segment intervals.
    11. Under the dissecting scope, examine each coronary artery segments for visible intraplaque Evan Blue stained areas (Figure 2A). 
    12. Freeze samples by dipping tissues in O.C.T. embedding medium. Then place tissues in labeled cryomolds and top off the molds with more O.C.T.
    13. In a chemical hood, and using appropriate PPE, cool a bottle of methylbutane by submerging it in a container with liquid nitrogen. Ensure that the liquid nitrogen reaches the meniscus of the methylbutane. Allow it to cool for 1-2 min.
    14. In a chemical hood, arrange the O.C.T.-filled cryomolds in a foil-lined container and using forceps or cryogloves, remove the methylbutane from the liquid nitrogen and pour enough methylbutane into the foil-lined container to surround the molds. Ensure that no methylbutane goes over the top of the cryomolds. Cover the container with an insulated lid and allow samples to freeze 2-5 min, or until O.C.T is fully frozen.
    15. Remove the cryomolds from the methylbutane and store in individually-labeled small plastic bags. Store the frozen blocks in -80 °C conditions.
    16. Using a Microtome Cryostat, cut 10 micron-thick sections from each frozen block and place on slides. Cut enough for 3 methods of evaluation: staining, histology, and immunofluorescence analysis. Air-dry slides to be imaged for Evans Blue, and coverslip using Eco-mount (Figure 2B). Store unstained slides at -80 °C in the dark until ready for immunofluorescence analysis using confocal microscopy.


      Figure 2. Evans Blue Dye perfused human coronary artery. A. Gross image of the coronary artery after perfusion. The yellow area is lipid-rich plaque. The dark blue to black are Evans Blue stained areas, see corresponding cryo-sections for blue stain in B (the light blue area is the reflection of the light from luminal surface in A). B. Cryo-sectioned slide from the same artery.

    [Optional Procedure] Using Evan Blue dye or FITC-dextran in mouse study
    1. One-year-old apoE knockout (KO) mouse that developed advanced atherosclerotic plaque can be used in intraplaque permeability study by administrated Evan Blue dye or FITC-dextran solution.
    2. Prepare FITC-dextran solution by dissolving FITC-dextran in PBS (10 mg/ml). 
    3. FITC-dextran is injected via i.v. administration for 50 μg/g body weight under isoflurane anesthetic condition using ultra-fine syringe.
    4. Sacrifice the mouse 1 h after FITC-dextran is injected.
    5. Optional: blood samples can be collected for measurement of serum FITC-dextran levels using a fluorescence plate reader.
    6. Perfuse the mouse through left ventricle using a syringe pump or by gravity with 20 ml PBS solution and perfusion fixed with 20 ml 4% paraformaldehyde, followed by 20 ml PBS rinses. The perfusion procedures are described previously (Gage et al., 2012).
    7. Carefully dissect the heart and aorta with brachiocephalic artery under dissecting scope.
    8. Embed the aortic root and brachiocephalic artery in O.C.T. and section as described above.
    9. FITC-dextran in the section can be imaged by confocal microscopy as described below (Figure 3).


      Figure 3. Representative immunofluorescence confocal microscopic images of intraplaque FITC-dextran (green) as a marker for permeability. Scale bars: 100 μm. Total intraplaque FITC fluorescence was quantified from confocal images of BCA plaques perfused with FITC-dextran to determine permeability. (Adapted from Figure 5J in Guo et al., 2018)

  2. Dual immunofluorescence staining
    1. Warm frozen slides to room temperature for 10-20 min.
    2. Fix the frozen sections by placing the slides in cold (approximately -20 °C) acetone for 10 min. Allow sections to air-dry for 10 min at room temperature, then outline sections with Liquid Blocker, and air-dry for 10-20 min at room temperature.
    3. Expose slides to 0.15% H2O2 for 20 min. 
    4. Next, treat all sections with Dako protein block for 10 min on a Belly Button shaker set to medium speed before incubation with primary antibodies. 
    5. Prepare a solution of 1% BSA in PBS. Prepare enough of this solution to dilute all primary antibodies.
    6. Perform immunofluorescence primary antibody dual staining for CD163/VE-cadherin and VE-cadherin/VCAM (plus negative controls for both pairs) on the cryosections (apply enough volume of antibody or blocking solutions to cover the tissue sections):
      1. Dilute primary antibodies as follows in the PBS with 1% BSA solution:
        1. Make a 1:200 dilution of CD163 clone GHI/61.
        2. Make either a 1:100 or 1:400 dilution of VE-cadherin.
        3. Make a 1:100 dilution of VCAM.
      2. Remove protein block from slides and perform dual staining, pairing CD163 with VE-cadherin, as well as VE-cadherin with VCAM.
      3. Add the first primary antibody to the test and positive control slides and incubate on shaker as detailed below. In between primary antibody incubations, remove the first primary antibody, rinse slides 3 times with PBS for 5 min on the shaker, remove PBS, then add the second primary antibody and incubate on the shaker as detailed below. Treat the negative control simultaneously by incubating in PBS instead of the primary antibody, for 1 h at room temperature or overnight at 4 °C. Incubate test slides with primary antibodies in the following conditions:
        1. Incubate in CD163 clone GHI/61 diluted 1:200, overnight at 4 °C. 
        2. Incubate in VE-cadherin, diluted 1:100 or 1:400, overnight at 4 °C.
        3. Incubate in VCAM diluted 1:100, 1 h at room temperature.
      4. Remove the second primary antibody, rinse slides 3 times with PBS for 5 min on the shaker, and remove PBS.
    7. Perform primary antibody detection by incubating test slides and positive control slides with biotinylated anti-mouse secondary antibody, diluted 1:200 in PBS for 30 min on the shaker, at room temperature. Keep dilution covered until ready to use, and keep slides covered during incubation to protect secondary antibody from light. 
    8. Remove the secondary antibody, rinse slides 3 times with PBS each for 5 min on the shaker (covered), and remove PBS.
    9. Incubate all slides (including positive and negative controls) in Alexa Flour 488 and 555 streptavidin, diluted 1:100 in PBS, for 30 min on the shaker, at room temperature. Keep covered.
    10. Remove the streptavidin and rinse slides with PBS 3 times for 5 min, covered, at room temperature on the shaker.
    11. Counterstain all slides with DAPI. Dilute DAPI in PBS (the dilution should be determined via titer assay and may vary by lot, around 1:1,000 dilution is a good start). Add diluted DAPI to test and control slides, and incubate at room temperature on a shaker for the time determined via titer. Keep covered during incubation.
    12. Remove DAPI and rinse slides with PBS 3 times each for 5 min, covered, at room temperature on the shaker.
    13. Place all slides in 10% neutral buffered formalin for 10 min (covered). Remove with a pipette.
    14. Rinse slides with PBS 3 times each for 5 min, covered, at room temperature on the shaker.
    15. Remove PBS and apply coverslips using fluorescence compatible mounting medium. Store slides at 2-8 °C in the dark until ready for immunofluorescence analysis using confocal microscope.

  3. Hematoxylin and Eosin Staining
    1. Warm slides to room temperature, 10-20 min.
    2. Stain slides in Mayer’s Hematoxylin Solution (filtered before use) for 10-20 min. Stain in Gill’s 3 solution for 1-6 min if Mayer’s Hematoxylin Solution is not available.
    3. Wash slides in warm to hot tap water to remove hematoxylin until slides are clear and nuclei are blue.
    4. If the tissue is dense and not staining properly, place slides in 1% Glacial Acetic Acid for 3-10 sec, then rinse in warm tap water.
    5. If slides are not blue, place them in ammonia water (5-10 drops of concentrated Ammonium Hydroxide in a large staining dish with water) for 3 sec or longer.
    6. Wash slides in tap water for 4-5 water changes.
    7. Place slides in 80% Reagent Alcohol for 1 min or 5-10 dips.
    8. Stain slides in Eosin-Phloxine Stain (Eosin Y/Phloxine B Working Solution) (filtered before use) for 1-3 min.
    9. Dehydrate slides by placing them through graded alcohols, followed by xylene: 95% alcohol, 1-2 dips; 100% alcohol, 1-2 dips; 100% alcohol, 20 sec-1 min (repeat a total of 3 times, using fresh alcohol each time); xylene 1-3 min (repeat a total of 3 times, using fresh xylene each time).
    10. Check slides for proper staining and restain in Hematoxylin if needed. Nuclear chromatin should appear blue and cytoplasm should appear pink to red.
    11. Mount coverslips onto slides with Permount.

  4. Evans blue brightfield imaging
    Acquire bright field images of Evans Blue slides using the Axio Scan.Z1 slide scanner and the Carl-Zeiss Microscopy Zen software Blue edition version 2.0.
    1. Load slides into the Axio Scanner.
    2. Select “Prescan” and the appropriate “Prescan Setting” after checking the appropriate trays holding the slides to be scanned.
    3. Review pre-scanned images and areas to be scanned by opening the “Tissue detection wizard”. If necessary, make changes to the automatic detection by using the “Polygonal outline tool”. Once review is complete, select “Finished”.
    4. Use “number of points” setting for coarse focus map, and “onion skin” setting for fine focus map.
    5. Name slides, choose a location in which to save, and select “Scan”.

  5. Fluorescence imaging
    1. Using a Zeiss LSM 800 or 880 Confocal Microscope, and Carl-Zeiss Microscopy Zen software (Blue edition version 2.0 and Black edition version 2.0, respectively) to capture Z-stack images for the test and control slides, using different channels to co-detect CD163, VE-cadherin, and VCAM as needed, based on dual staining performed.
    2. Evans Blue dye and FITC-dextran can also be imaged using confocal microscope. Use lambda mode and spectral unmixing in acquisition to reduce other noise signals. Representative images can be found in Figure 5J in the original article (Guo et al., 2018).

Data analysis

Evans Blue negative and positive areas are quantified using cross-sectional images of EBD-perfused human coronary arteries. The images in Figure 4A show that areas of heavy EBD staining have intraplaque microvessels surrounded by CD163+ macrophages. Quantitation of microvessels, CD163+ macrophages, and VCAM in EBD-positive versus EBD-negative areas showed significantly higher microvessel density, more CD163+ macrophages, and upregulated VCAM expression in EBD-positive versus EBD-negative areas in the plaque. Comparisons between two groups were achieved using a two-sided Student’s t-test. Further details of these analyses can be found in the original research article (Guo et al., 2018).


Figure 4. Human coronary artery microvessel permeability was assessed by EBD perfusion. A. Representative images of EBD-perfused human coronary arteries, H&E-stained images, and confocal immunofluorescence images of CD163 (red) and VE-cadherin (yellow) or VE-cadherin (red) and VCAM (green) in an EBD-negative area (top row), EBD-positive area 1 (middle row), and EBD-positive area 2 (bottom row). Positive areas 1 and 2 are shown in progressively higher-magnification H&E-stained images from left to right in the second and third rows. Red and white arrowheads point to microvessels. Confocal images of the EBD-negative areas for CD163/VE-cadherin and VE-cadherin/VCAM are shown in the top row of columns 3 and 4, respectively, while the positive area 1 is shown for CD163/VE-cadherin in the middle rows of columns 3 and 4 (higher-magnification image on the right), and positive area 2 is shown for VE-cadherin/VCAM in the bottom of row of columns 3 and 4 (higher-magnification image on the right). B-D. Quantification of microvessel density, CD163+ macrophages, and VCAM in an EBD-positive area versus an EBD-negative area. (Adapted from Figure 7 in Guo et al., 2018)

Notes

There is certain variability in the degree of intraplaque angiogenesis and permeability in human coronary artery disease. One-year-old apoE knockout mouse can be an animal model to study the intraplaque hemorrhage and permeability. However, the intraplaque angiogenesis is more difficult to determine in mouse atherosclerotic lesions than in human lesions, due to the same size of the mouse artery.

Recipes

  1. 0.5% Evans blue dye solution (100 ml)
    Evans Blue Dye
    0.5 g
    Bovine Serum Albumin
    5.0 g
    0.9% NaCl Saline
    100 ml
    Store at 4 °C

Acknowledgments

This protocol is adapted from Guo et al. (2018). The study was funded by CVPath Institute, a non-profit research Institute dedicated to the study of cardiovascular diseases and their treatment.

Competing interests

The authors do not have any potential conflicts of interest to declare.

Ethics

The study involving the use of deidentified human pathological or autopsy specimens were approved for exempt review by the IRB of the CVPath Institute. The IACUC of the MedStar Health Research Institute approved all animal protocols. All animal experiments were conducted according to the NIH’s Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011).

References

  1. Dejana, E., Orsenigo, F. and Lampugnani, M. G. (2008). The role of adherens junctions and VE-cadherin in the control of vascular permeability. J Cell Sci 121(Pt 13): 2115-2122.
  2. Evans, H. M. and Schulemann, W. (1914). The action of vital stains belonging to the benzidine group. Science 39(1004): 443-454.
  3. Gage, G. J., Kipke, D. R. and Shain, W. (2012). Whole animal perfusion fixation for rodents. Jove (65). DOI: 10.3791/3564.
  4. Guo, L., Akahori, H., Harari, E., Smith, S. L., Polavarapu, R., Karmali, V., Otsuka, F., Gannon, R. L., Braumann, R. E., Dickinson, M. H., Gupta, A., Jenkins, A. L., Lipinski, M. J., Kim, J., Chhour, P., de Vries, P. S., Jinnouchi, H., Kutys, R., Mori, H., Kutyna, M. D., Torii, S., Sakamoto, A., Choi, C. U., Cheng, Q., Grove, M. L., Sawan, M. A., Zhang, Y., Cao, Y., Kolodgie, F. D., Cormode, D. P., Arking, D. E., Boerwinkle, E., Morrison, A. C., Erdmann, J., Sotoodehnia, N., Virmani, R. and Finn, A. V. (2018). CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis. J Clin Invest 128(3): 1106-1124.
  5. Hamer, P. W., McGeachie, J. M., Davies, M. J. and Grounds, M. D. (2002). Evans Blue Dye as an in vivo marker of myofibre damage: optimising parameters for detecting initial myofibre membrane permeability. J Anat 200 (Pt 1): 69-79.
  6. Harari, E., Guo, L., Smith, S. L., Paek, K. H., Fernandez, R., Sakamoto, A., Mori, H., Kutyna, M. D., Habib, A., Torii, S., Cornelissen, A., Jinnouchi, H., Gupta, A., Kolodgie, F. D., Virmani, R. and Finn, A. V. (2018). Direct targeting of the mTOR (mammalian target of rapamycin) kinase improves endothelial permeability in drug-eluting stents. Arterioscler Thromb Vasc Biol. 38: 2217-2224.
  7. Miles, A. A. and Miles, E. M. (1952). Vascular reactions to histamine, histamine-liberator and leukotaxine in the skin of guinea-pigs. J Physiol 118(2): 228-257.
  8. Mundi, S., Massaro, M., Scoditti, E., Carluccio, M. A., van Hinsbergh, V. W. M., Iruela-Arispe, M. L. and De Caterina, R. (2018). Endothelial permeability, LDL deposition, and cardiovascular risk factors-a review. Cardiovasc Res 114(1): 35-52.
  9. Radu, M. and Chernoff, J. (2013). An in vivo assay to test blood vessel permeability. J Vis Exp (73): e50062.
  10. Yao, L., Xue, X., Yu, P., Ni, Y. and Chen, F. (2018). Evans Blue Dye: A revisit of its applications in biomedicine. Contrast Media Mol Imaging 2018: 7628037.

简介

冠状动脉疾病仍然是发病率和死亡率的重要原因。以前的研究,包括我们的研究,都集中在斑块内出血的作用,特别是来自未成熟的微血管血管生成,作为斑块进展的重要因素,通过增加血管通透性导致进一步的斑块内出血,增加斑块中红细胞膜来源的游离胆固醇内容和炎症细胞募集。 Evans Blue Dye(EBD)测定法广泛用作脉管系统渗透性的标准测定法。然而,该方法尚未在新鲜人冠状动脉尸检样本中建立,以评估斑块内微血管通透性和血管生成。在该方案中,我们描述了评估人类冠状动脉样本的微血管通透性的方法,包括灌注冠状动脉的程序,用于组织学分析和免疫染色的动脉样本的收集以及使用适当的方法来分析图像。还提供了在小鼠模型中使用FITC-葡聚糖以评估血管通透性的任选程序。这些Evans Blue Dye程序可用于在各种病理条件下提供人样品和动物模型中内皮完整性和渗透性的功能测量。

【背景】
血管内皮细胞主动调节血浆成分和循环细胞(包括白细胞)从血液到亚内皮组织的浸润。这种机制通常被认为是动脉粥样硬化起始和发展的关键步骤(Mundi et al。>,2018)。血管通透性的调节通过内皮细胞 - 细胞连接的协调打开和闭合来实现。在几种疾病状态下,内源性药物如组胺,凝血酶和血管内皮生长因子(VEGF)显着但可逆地以不同方式改变细胞 - 细胞连接复合物的功能和组织,导致渗透性的不同程度的增加( Dejana et al。>,2008)。我们最近展示了一种独特的机制,CD163阳性替代巨噬细胞[M(Hb)]在斑块内出血(IPH)位点吞噬血红蛋白 - 触珠蛋白复合物,促进血管内皮生长因子(VEGF)的释放,进一步导致血管内皮钙粘蛋白(VE-cad)从细胞间粘附连接处内化,从而增加血管通透性并导致动脉粥样硬化斑块进展(Guo et al。>,2018)。在上述研究中,我们使用Evans Blue Dye(EBD)溶液进行了一项新技术,以评估人冠状动脉的血管通透性。在同一项研究中,FITC-葡聚糖被用作一年前apoE小鼠模型的替代指标,以证明头臂动脉的斑块内出血,这是由于小鼠动脉的小尺寸和共焦成像所需的染料敏感性。

在20世纪以来创造的大量蓝色染料中,自1914年赫伯特·麦克莱恩·埃文斯(Herbert McLean Evans)首次申请以来,EBD一直是生物学历史最长的染料(Evans和Schulemann,1914) 。 EBD是碱性合成双偶氮(联苯胺基),分子量为961Da,具有高水溶性,使染料能够快速扩散到整个血流中。最重要的是,当静脉注射染料时,它对血浆白蛋白具有高亲和力,使染料具有保持稳定的能力,并且由于排泄速率缓慢而在整个身体中分布较长时间。 EBD的所有功能使其成为生物医学中具有多种潜在应用的非凡代理,正如Linpeng Yao最近评论的那样(Yao et al。>,2018)。这些包括但不限于血浆体积的估计,肿瘤和淋巴结的鉴定以及暴露于绿光时使用荧光作为血管通透性的潜在标志物(Hamer et al。>,2002 )。在用于确定血管通透性的测定中使用EBD背后的原理在于,在具有正常血管完整性的正常组织中,白蛋白不能通过血管壁内皮层迁移到间质中。这意味着在白蛋白-EBD复合物的情况下,染料仅限于循环。当以适当的浓度使用时,EBD相对无毒。 使用小鼠和人类受试者的体内>实验已经证明,当过量使用时,白蛋白达到其最大饱和百分比,导致血管渗漏并导致组织快速蓝色变色(Miles and Miles,1952) 。在正常条件下,通过严格控制生长因子,细胞因子和其他分子的细胞 - 细胞粘附连接,保持足够的渗透性屏障(Radu和Chernoff,2013)。然而,在影响内皮层完整性的病理条件下,包括白蛋白在内的血浆蛋白能够在各种疾病状态下发生泄漏。导致血管通透性增加的最常见的病理生理事件是当包括内皮层的血管壁受损时可能发生的急性炎症。血管扩张,血流量增加,内皮细胞连接破坏和白细胞浸润是这一过程中的关键因素。 EBD-白蛋白复合物在显微镜下可见为间质组织中的嗜碱性颜色,并表明血管通透性增加。在我们最近关于药物洗脱支架植入后内皮屏障功能障碍的研究中,在兔髂动脉支架模型中进行了EBD灌注,以证明动脉通透性与较差的内皮VE-钙粘蛋白/ P120连接和较高的巨噬细胞浸润相关(Harari 等人,>,2018)。鉴于EBD的优势,我们决定使用该技术研究人体冠状动脉的通透性。

关键字:冠心病, 动脉粥样硬化, 微血管通透性, 伊文氏蓝染色, FITC标记葡聚糖, 血管生成, 斑块内出血

材料和试剂

  1. 移液器吸头
  2. 250毫升Stericup过滤装置(默克,目录号:SCGPU02RE)
  3. Coverslips(Fisher Scientific,目录号:12-543D)
  4. Kimwipes(KCWW,Kimberly-Clark,目录号:34155)
  5. 超细注射器(BD,目录号:324911)
  6. 从新收集的尸检标本中选择人冠状动脉样本(来自CVPath登记处)
  7. [可选]一岁的apoE基因敲除(KO)小鼠(THE JACKSON LABORATORY,目录号:002052)
  8. EcoMount(Biocare Medical,目录号:EM897L)
  9. CoverMount用于非EBD染色,基于二甲苯(Avantik,目录号:SL6012-A)
  10. 伊文思蓝染料(Sigma-Aldrich,目录号:E2129-50G)
  11. FITC-dextran(Sigma-Aldrich,目录号:46945)
  12. 5%BSA(Fisher,目录号:BP1600-100)
  13. PBS(1x,Ultra Pure Grade,VWR,目录号:97063-658)
  14. 中性缓冲福尔马林(NBF)(Sigma-Aldrich,目录号:HT501128)
  15. Tissue-Tek ® O.C.T.化合物(Sakura Finetek,Miles,目录号:4583)
  16. 过氧化氢H 2 O 2 3%(VWR,目录号:BDH7540-2)
  17. Dako蛋白质块(Agilent Technologies,DAKO,目录号:X0909)
  18. CD163抗体(Santa Cruz Biotechnology,目录号:sc-20066,克隆GHI / 61)
  19. CD68抗体(Dako,clone Kp1)
  20. 血管性血友病因子(vWF)抗体(SDIX,Strategic BioSolutions,目录号:S4003GND1)
  21. 缺氧诱导因子1α(HIF1α)抗体(Novus Biologicals,目录号:NB100-105)
  22. 血管内皮生长因子-A(VEGF-A)抗体(BioGenex,目录号:PU483-UP)
  23. VE-钙粘蛋白抗体(R& D Systems,目录号:AF1002,稀释度1:100,和BD Biosciences,目录号:555661)
  24. 血管细胞粘附蛋白(VCAM)抗体(Abcam,目录号:ab134047)
  25. CD3抗体(Roche Diagnostics,目录号:790-4341,预稀释)
  26. 生物素化的山羊抗兔,马抗小鼠和兔抗山羊(Vector Laboratories,目录号:BA-1000,BA-2000,BA-5000,分别)
  27. Alexa Flour 488和555链霉抗生物素蛋白(Thermo Fisher Scientific,Invitrogen TM ,目录号:S32354和S32355,分别)
  28. DAPI(Thermo Fisher Scientific,Invitrogen TM ,目录号:D3571)
  29. 2-甲基丁烷(Spectrum Chemical Manufacturing,目录号:M1246)
  30. 液氮
  31. 20%多聚甲醛(Electron Microscopy Sciences,目录号:15713-S)
  32. 丙酮(Fisher Scientific,目录号:A929-1)
  33. 液体阻滞剂(Ted Pella,目录号:22309)
  34. 冰醋酸(Fisher Scientific,目录号:A38-212)
  35. 苏木精和伊红
    1. 二甲苯,试剂等级/ ACS(Avantik,目录号:RS4050)
    2. 安装介质/ Permount(Fisher Scientific,目录号:SP15-500) 
    3. 来自实验室的去离子水
    4. Mayer的苏木精解决方案(Astral Diagnostics,目录号:7020)
    5. Gill 3(Sigma,目录号:GHS3128)
    6. 伊红 - phloxine染色(Astral Diagnostics,目录号:7010)
    7. 100%试剂酒精(Avantik,目录号:RS4029)
    8. 95%试剂级酒精(Avantik,目录号:RS4031)
    9. 氢氧化铵,ACS级(Sigma-Aldrich,目录号:A6899)
  36. 伊文思蓝染料溶液(见食谱)

设备

  1. 移液器
  2. 肚脐振动筛(IBI Scientific)
  3. Axio Scan.Z1数字幻灯片扫描仪(Carl Zeiss,目录号:Axio Scan.Z1)
  4. 使用切片刀片的明亮OTF 5000切片机低温恒温器(Hacker Instruments,Hacker,目录号:OTF 5000)(Thermo Fisher Scientific,目录号:3152735)
  5. CryoJane磁带传输系统(Leica Biosystems,目录号:39475205)
  6. LSM 800共焦激光扫描显微镜(Carl Zeiss,目录号:LSM 800)
  7. 奥林巴斯BX51显微镜(奥林巴斯,型号:BX51)
  8. RNAscope Probe-Hs-CD163-C2(高级细胞诊断,目录号:417061-C2)
  9. RNAscope Probe-Hs-VEGFA(高级细胞诊断,目录号:423161)
  10. TLE系列超低温冰箱(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:TLE60086A)
  11. Stemi DV4立体解剖显微镜(蔡司,型号:Stemi DV4)

软件

  1. HALO图像分析平台(Indica Labs)2.0版
  2. Zen Blue 2012(卡尔蔡司)2.0版
  3. Zen Black 2012(卡尔蔡司)2.0版

程序

  1. 准备和冷冻组织
    1. 选择新鲜收集的尸检人类心脏伴冠状动脉疾病。样品必须具有小于12小时的死后间隔(PMI)截止值以及基于心脏的X射线图像(其将允许检测血管钙化)的晚期动脉粥样硬化的证据。 (图1)


      图1.心脏的数字X射线图像。突出的亮点是冠状动脉的钙化(红色箭头)。在冠状动脉之间的中心可以看到主动脉瓣的钙化。

    2. 准备含有5%BSA的盐水溶液。
    3. 通过将伊文思蓝粉末溶解在含BSA的盐水中制备0.5%伊文思蓝染料溶液。
    4. 使用250 ml Stericup过滤装置过滤Evans Blue Dye溶液。 
    5. 将过滤后的Evans Blue灌注到右侧和左侧冠状动脉中,在37°C下通过重力流量平均压力约为60 mmHg进行15分钟。 
    6. 使用相同的重力灌注方法,用500ml PBS洗涤埃文斯蓝灌注样品20分钟。
    7. 接下来,用500ml中性缓冲福尔马林固定样品20分钟。
    8. 收获福尔马林固定的冠状动脉组织样品,并置于含有50ml 15%蔗糖的PBS溶液中,在4℃下在Falcon管中过夜。
    9. 从蔗糖溶液中取出组织并用Kimwipes干燥。
    10. 以2-3毫米的片段间隔切割收获的样品。
    11. 在解剖范围内,检查每个冠状动脉段是否有可见的斑块内Evan Blue染色区域(图2A)。 
    12. 通过在O.C.T.中浸渍组织来冻结样品。嵌入媒体。然后将组织置于标记的冷冻模具中,并用更多的O.C.T.
    13. 在化学通风橱中,使用适当的PPE,将一瓶甲基丁烷浸入装有液氮的容器中,冷却一瓶甲基丁烷。确保液氮到达甲基丁烷的弯液面。让它冷却1-2分钟。
    14. 在化学罩中,将O.C.T.填充的冷冻模型安装在箔衬里的容器中,并使用镊子或冷冻球,从液氮中除去甲基丁烷,并将足够的甲基丁烷倒入箔衬里的容器中以包围模具。确保没有甲基丁烷超过冷冻模的顶部。用隔热盖盖住容器,让样品冷冻2-5分钟,或直到O.C.T完全冷冻。
    15. 从甲基丁烷中取出冷冻模型,并储存在单独标记的小塑料袋中。将冷冻块储存在-80°C的条件下。
    16. 使用切片机低温恒温器,从每个冷冻块切下10微米厚的切片并放在载玻片上。切割足够3种评估方法:染色,组织学和免疫荧光分析。风干载玻片用于Evans Blue成像,使用Eco-mount进行盖玻片(图2B)。将未染色的载玻片存放在-80°C的黑暗中,直到准备好使用共聚焦显微镜进行免疫荧光分析。


      图2.Evans Blue Dye灌注人冠状动脉。 A.灌注后冠状动脉的总体图像。黄色区域是富含脂质的斑块。深蓝色至黑色是伊文思蓝染色区域,参见B中蓝色染色的相应冷冻切片(浅蓝色区域是A中管腔表面光线的反射)。 B.来自同一动脉的冷冻切片。

    [可选程序] 在小鼠研究中使用Evan Blue染料或FITC-葡聚糖
    1. 一年前开发的先进动脉粥样硬化斑块的apoE敲除(KO)小鼠可用于通过施用的Evan Blue染料或FITC-葡聚糖溶液进行的斑块内渗透性研究。
    2. 通过将FITC-葡聚糖溶解在PBS(10mg / ml)中制备FITC-葡聚糖溶液。 
    3. 通过静脉内注射FITC-葡聚糖。使用超细注射器在异氟烷麻醉条件下施用50μg/ g体重。
    4. 注射FITC-葡聚糖后1小时处死小鼠。
    5. 可选:可以使用荧光板读数器收集血样用于测量血清FITC-葡聚糖水平。
    6. 使用注射泵或通过重力用20ml PBS溶液灌注小鼠穿过左心室,用20ml 4%多聚甲醛灌注固定,然后用20ml PBS漂洗。灌注程序如前所述(Gage et al。>,2012)。
    7. 在解剖范围内用头臂动脉仔细解剖心脏和主动脉。
    8. 在O.C.T.中嵌入主动脉根部和头臂动脉。和如上所述的部分。
    9. 该部分中的FITC-葡聚糖可以通过如下所述的共聚焦显微镜成像(图3)。


      图3.代表性免疫荧光共聚焦显微镜图像的斑块内FITC-葡聚糖(绿色)作为渗透性的标记。比例尺:100μm。从用FITC-葡聚糖灌注的BCA斑块的共聚焦图像定量总斑块内FITC荧光以确定渗透性。 (改编自Guo et al。>,2018年的图5J)

  2. 双重免疫荧光染色
    1. 温热冷冻至室温10-20分钟。
    2. 通过将载玻片置于冷(约-20℃)丙酮中10分钟来固定冷冻切片。允许切片在室温下风干10分钟,然后用液体阻断剂勾勒出部分,并在室温下风干10-20分钟。
    3. 将载玻片暴露于0.15%H 2 O 2 20分钟。 
    4. 接下来,在与一抗孵育之前,在设置为中速的Belly Button振荡器上用Dako蛋白块处理所有切片10分钟。 
    5. 准备1%BSA的PBS溶液。准备足够的这种溶液稀释所有的一抗。
    6. 对冷冻切片进行CD163 / VE-钙粘蛋白和VE-钙粘蛋白/ VCAM(两对阴性对照)的免疫荧光一抗双重染色(应用足够量的抗体或阻断溶液以覆盖组织切片):
      1. 在含有1%BSA溶液的PBS中如下稀释一抗:
        1. 制备1:200稀释的CD163克隆GHI / 61。
        2. 制备VE-钙粘蛋白的1:100或1:400稀释液。
        3. 对VCAM进行1:100稀释。
      2. 从载玻片上除去蛋白质块并进行双重染色,将CD163与VE-钙粘蛋白配对,以及将VE-钙粘蛋白与VCAM配对。
      3. 将第一个一抗添加到测试和阳性对照载玻片中并在振荡器上孵育,如下所述。在一抗孵育之间,去除第一个一抗,在摇床上用PBS冲洗载玻片3次,持续5分钟,取出PBS,然后加入第二个一抗,并如下所述在振荡器上孵育。通过在PBS中代替一抗孵育同时处理阴性对照,在室温下孵育1小时或在4℃下孵育过夜。在以下条件下用一抗孵育测试载玻片:
        1. 孵育在1:200稀释的CD163克隆GHI / 61中,在4℃下过夜。 
        2. 在4℃下以1:100或1:400稀释的VE-钙粘蛋白孵育过夜。
        3. 在室温下1:100稀释的VCAM中孵育1小时。
      4. 去除第二个一抗,在摇床上用PBS冲洗载玻片3次,每次5分钟,然后取出PBS。
    7. 通过将测试载玻片和阳性对照载玻片与生物素化的抗小鼠二抗一起温育来进行一抗检测,所述生物素化的抗小鼠二抗在室温下在摇床上以1:200在PBS中稀释30分钟。保持稀释直至准备使用,并在孵育期间保持载玻片覆盖以保护二抗不受光照。 
    8. 去除二抗,在摇床(覆盖)上用PBS冲洗载玻片3次,每次5分钟,并除去PBS。
    9. 将所有载玻片(包括阳性和阴性对照)孵育在Alexa Flour 488和555链霉抗生物素蛋白中,在PBS中1:100稀释,在摇床上在室温下孵育30分钟。保持覆盖。
    10. 去除链霉抗生物素蛋白并用PBS漂洗载玻片3次,每次5分钟,在室温下在振荡器上盖上盖子。
    11. 使用DAPI计算所有幻灯片。在PBS中稀释DAPI(稀释度应通过滴度测定确定,并且可以按批次变化,约1:1,000稀释是一个良好的开始)。将稀释的DAPI加入到测试和对照载玻片中,并在室温下在振荡器上孵育通过滴定测定的时间。在孵化期间保持覆盖。
    12. 去除DAPI并用PBS冲洗载玻片3次,每次5分钟,在室温下在振荡器上盖上盖子。
    13. 将所有载玻片置于10%中性缓冲福尔马林中10分钟(盖上)。用移液管移除。
    14. 用PBS冲洗载玻片3次,每次5分钟,在室温下在振荡器上盖上盖子。
    15. 移除PBS并使用荧光兼容的封固剂涂覆盖玻片。将载玻片在2-8℃下在黑暗中储存,直到准备好使用共聚焦显微镜进行免疫荧光分析。

  3. 苏木精和伊红染色
    1. 温热滑至室温,10-20分钟。
    2. 在Mayer's苏木精溶液中染色载玻片(使用前过滤)10-20分钟。如果Mayer的苏木素溶液不可用,则在Gill 3溶液中染色1-6分钟。
    3. 在温热的自来水中洗涤载玻片以除去苏木精,直到载玻片清澈并且细胞核呈蓝色。
    4. 如果组织致密并且染色不正确,将载玻片放入1%冰醋酸中3-10秒,然后用温水冲洗。
    5. 如果载玻片不是蓝色,则将它们置于氨水(5-10滴浓氢氧化铵在大型染色皿中加水)中3秒或更长时间。
    6. 用自来水清洗载玻片,换水4-5次。
    7. 将载玻片置于80%试剂酒精中1分钟或5-10滴。
    8. 在Eosin-Phloxine Stain(Eosin Y / Phloxine B Working Solution)中染色载玻片(使用前过滤)1-3分钟。
    9. 将载玻片置于梯度醇中,然后用二甲苯:95%乙醇,1-2滴,使载玻片脱水; 100%酒精,1-2蘸; 100%酒精,20秒-1分钟(重复3次,每次使用新鲜酒精);二甲苯1-3分钟(重复3次,每次使用新鲜二甲苯)。
    10. 如果需要,检查载玻片上是否有正确的染色并重新接触苏木精。核染色质应呈现蓝色,细胞质应呈粉红色至红色。
    11. 使用Permount将盖玻片安装到载玻片上。

  4. 埃文斯蓝明场成像
    使用Axio Scan.Z1载玻片扫描仪和Carl-Zeiss显微镜Zen软件蓝版2.0版获取Evans Blue载玻片的明视野图像。
    1. 将幻灯片加载到Axio Scanner中。
    2. 检查固定要扫描的幻灯片的相应托盘后,选择“预扫描”和相应的“预扫描设置”。
    3. 通过打开“组织检测向导”查看预扫描的图像和要扫描的区域。如有必要,使用“多边形轮廓工具”更改自动检测。审核完成后,选择“已完成”。
    4. 粗焦点贴图使用“点数”设置,精细焦点贴图使用“洋葱皮”设置。
    5. 命名幻灯片,选择要保存的位置,然后选择“扫描”。

  5. 荧光成像
    1. 使用Zeiss LSM 800或880共聚焦显微镜和Carl-Zeiss Microscopy Zen软件(分别为蓝色版本2.0和黑色版本2.0)捕获测试和控制载玻片的Z-堆叠图像,使用不同的通道进行共检测基于所进行的双重染色,根据需要使用CD163,VE-钙粘蛋白和VCAM。
    2. Evans Blue染料和FITC-葡聚糖也可以使用共聚焦显微镜成像。在采集中使用lambda模式和光谱分离来减少其他噪声信号。代表性的图像可以在原始文章中的图5J中找到(Guo 等人,>,2018)。

数据分析

使用EBD灌注的人冠状动脉的横截面图像量化Evans Blue阴性和阳性区域。图4A中的图像显示重度EBD染色区域具有被CD163 + 巨噬细胞包围的斑块内微血管。在EBD阳性与EBD阴性区域中微血管,CD163 + 巨噬细胞和VCAM的定量显示显着更高的微血管密度,更多CD163 + 巨噬细胞和EBD中上调的VCAM表达斑块中的阳性与EBD阴性区域。使用双侧Student's t > -test实现两组之间的比较。这些分析的更多细节可以在最初的研究文章中找到(Guo et al。>,2018)。


图4.通过EBD灌注评估人冠状动脉微血管通透性。 A.EBD灌注的人冠状动脉,H& E染色图像和CD163(红色)和CD163的共聚焦免疫荧光图像的代表性图像EBD阴性区域(顶行),EBD阳性区域1(中间行)和EBD阳性区域2(底行)中的VE-钙粘蛋白(黄色)或VE-钙粘蛋白(红色)和VCAM(绿色)。正区域1和2在第二和第三行中从左到右逐渐增大的H& E染色图像中示出。红色和白色箭头指向微血管。 CD163 / VE-钙粘蛋白和VE-钙粘蛋白/ VCAM的EBD阴性区域的共聚焦图像分别显示在第3列和第4列的顶行,而阳性区域1显示为中间的CD163 / VE-钙粘蛋白。在第3列和第4列的行的底部(右侧的放大倍率图像)中示出了用于VE-钙粘蛋白/ VCAM的第3列和第4列(右侧的放大倍数较高的图像)和正区域2的行。 B-d。在EBD阳性区域与EBD阴性区域中微量血管密度,CD163 + 巨噬细胞和VCAM的定量。 (改编自Guo et al。>,2018年的图7)

笔记

人冠状动脉疾病中斑块内血管生成和渗透性的程度存在一定的变异性。一岁的apoE敲除小鼠可以是研究斑块内出血和通透性的动物模型。然而,由于小鼠动脉的大小相同,小鼠动脉粥样硬化病变中的斑块内血管生成比人类病变更难以确定。

食谱

  1. 0.5%伊文思蓝染料溶液(100毫升)
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:300px;” border =“0”cellspacing =“0”cellpadding =“2”>埃文斯蓝染料
    0.5克
    牛血清白蛋白
    5.0克
    0.9%NaCl盐水
    100毫升 储存在4°C

致谢

该协议改编自Guo 等人>(2018)。该研究由CVPath研究所资助,CVPath研究所是一家致力于研究心血管疾病及其治疗的非营利性研究所。

利益争夺

作者没有任何潜在的利益冲突申报。

伦理

涉及使用经鉴定的人类病理学或尸检标本的研究被CVPath研究所的IRB批准免除审查。 MedStar健康研究所的IACUC批准了所有动物协议。所有动物实验均根据NIH的实验动物护理和使用指南(National Academies Press,2011)进行。

参考

  1. Dejana,E.,Orsenigo,F。和Lampugnani,M。G.(2008)。 粘连连接和VE-钙粘蛋白在血管通透性控制中的作用。 J Cell Sci > 121(Pt 13):2115-2122。
  2. Evans,H。M.和Schulemann,W。(1914)。 属于联苯胺类的重要污渍的作用。 Science > 39(1004):443-454。
  3. Gage,G.J.,Kipke,D.R。和Shain,W。(2012)。 啮齿动物的全动物灌注固定。 Jove >(65 )。 DOI:10.3791 / 3564。
  4. Guo,L.,Akahori,H.,Harari,E.,Smith,SL,Polavarapu,R.,Karmali,V.,Otsuka,F.,Gannon,RL,Braumann,RE,Dickinson,MH,Gupta,A。 ,Jenkins,AL,Lipinski,MJ,Kim,J.,Chhour,P.,de Vries,PS,Jinnouchi,H.,Kutys,R.,Mori,H.,Kutyna,MD,Torii,S.,Sakamoto, A.,Choi,CU,Cheng,Q.,Grove,ML,Sawan,MA,Zhang,Y.,Cao,Y.,Kolodgie,FD,Cormode,DP,Arking,DE,Boerwinkle,E.,Morrison,AC ,Erdmann,J.,Sotoodehnia,N.,Virmani,R。和Finn,AV(2018)。 CD163 + 巨噬细胞促进动脉粥样硬化伴有炎症的血管生成和血管通透性。 J Clin Invest > 128(3):1106-1124。
  5. Hamer,P.W.,McGeachie,J.M.,Davies,M。J. and Grounds,M。D.(2002)。 Evans Blue Dye作为肌肉纤维损伤的体内>标记:优化参数用于检测初始肌纤维膜通透性。 J Anat > 200(Pt 1):69-79。
  6. Harari,E.,Guo,L.,Smith,SL,Paek,KH,Fernandez,R.,Sakamoto,A.,Mori,H.,Kutyna,MD,Habib,A.,Torii,S.,Cornelissen,A 。,Jinnouchi,H.,Gupta,A.,Kolodgie,FD,Virmani,R。和Finn,AV(2018)。 直接靶向mTOR(哺乳动物雷帕霉素靶点)激酶可改善药物洗脱支架的内皮通透性。 Arterioscler Thromb Vasc Biol。> 38:2217-2224。
  7. Miles,A。A.和Miles,E。M.(1952)。 对豚鼠皮肤中的组胺,组胺 - 解放者和白藜芦醇的血管反应。 J Physiol > 118(2):228-257。
  8. Mundi,S.,Massaro,M.,Scoditti,E.,Carluccio,M。A.,van Hinsbergh,V。W. M.,Iruela-Arispe,M。L.和De Caterina,R。(2018)。 内皮通透性,低密度脂蛋白沉积和心血管危险因素 - 综述。 Cardiovasc Res > 114(1):35-52。
  9. Radu,M。和Chernoff,J。(2013)。 用于测试血管通透性的体内>检测方法。 J Vis Exp >(73):e50062。
  10. Yao,L.,Xue,X.,Yu,P.,Ni,Y。和Chen,F。(2018)。 Evans Blue Dye:重新审视其在生物医学中的应用。 Contrast Media Mol Imaging > 2018:7628037。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Guo, L., Fernandez, R., Sakamoto, A., Cornelissen, A., Paek, K. H., Lee, P. J., Weinstein, L. M., Collado-Rivera, C. J., Harari, E., Kutys, R., Samuda, T. S., Singer, N. A., Kutyna, M. D., Kolodgie, F. D., Virmani, R. and Finn, A. V. (2018). Vascular Permeability Assay in Human Coronary and Mouse Brachiocephalic Arteries. Bio-protocol 8(20): e3048. DOI: 10.21769/BioProtoc.3048.
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