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Aorta Atherosclerosis Lesion Analysis in Hyperlipidemic Mice
高血脂小鼠中的主动脉粥样硬化病变分析   

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Abstract

Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries. Apolipoprotein E-deficient (ApoE-/-) mice are used as experimental models to study human atherosclerosis. ApoE-/- mice are constitutively hyperlipidemic and develop intima plaques that resemble human plaques. Various issues including experimental design for lesion analysis, dietary conditions, isolation of the aorta, staining methods, morphometry, group size, age, the location within the arterial tree, and statistical analyses are important parameters that need to be addressed to obtain robust data. Here, we provide detailed methods to quantify aorta atherosclerosis.

Keywords: Atherosclerosis(动脉粥样硬化), Lesion analysis(病变分析), Plaque(斑块), Media(媒体), Inflammation(炎症)

Materials and Reagents

  1. Petri dish sets, glass (VWR International, catalog number: 89000-306 )
  2. Black dissection wax (CR Scientific, catalog number: C3541 )
  3. Minutien pins (0.15 mm diameter) to fix the aorta (Fine Scientific Tools, catalog number: 26002-15 )
  4. 50 ml Falcon tube (VWR International, CellStar®, catalog number: 188271 )
  5. 10 ml syringe (BD, catalog number: 309695 )
  6. 20 ml syringe (BD, catalog number: 301625 )
  7. Sterican single use needles-23G (B. Braun Medical Inc., catalog number: 4657640 )
  8. Poly-L-lysine coated glass slides (Menzel Glaeser, catalog number: J2800AMNZ )
  9. Coverslips (Menzel Glaeser, catalog number: BBAD02400500#A )
  10. OHP permanent marker pen (STAEDTLER MARS LIMITED)
  11. Microscope slide holder/mailer, 5 place (Sigma-Aldrich, catalog number: Z708313-25EA )
  12. Cryogenic freezer storage box (VWR International, catalog number: 82021-114 )
  13. Staining jar with cover (VWR International, catalog number: 25460-907 )
  14. Grade 410 filter paper (VWR International, catalog number: 28321-077 )
  15. Polystyrene petri dishes (150 mm x 15 mm) (Sigma-Aldrich, catalog number: P5981 )
  16. Ethanol (Merck Millipore Corporation, catalog number: 1009832500 )
  17. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  18. Phosphate buffer saline (PBS) (Sigma-Aldrich, catalog number: P4417-100TAB )
  19. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 16005 )
  20. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  21. Isopropanol (Merck Millipore Corporation, catalog number: 1096341011 )
  22. Acetone (Merck Millipore Corporation, catalog number: 1000141000 )
  23. Isopentane (VWR International, catalog number: 103614T )
    Note: It is also named “2-Methylbutan” on VWR International website.
  24. Dry ice (TKD KABEL GmbH)
  25. Sudan IV (Sigma-Aldrich, catalog number: 198102 )
  26. OCT compound-Tissue-Tek (SAKURA FINETEK USA, catalog number: 4583 )
  27. Cryomold (SAKURA FINETEK USA, catalog number: 25608-924 )
  28. Oil Red O (Sigma-Aldrich, catalog number: 00625 )
  29. Hematoxylin solution, Mayer’s (Sigma-Aldrich, catalog number: MHS-16 )
  30. Faramount aqueous mounting medium (Dako, catalog number: S3025 )
  31. Distilled water
  32. NaOH
  33. Sudan IV staining solution (see Recipes)
  34. Oil Red O (ORO) working solution (see Recipes)
  35. EDTA (see Recipes)
  36. PBS (see Recipes)
  37. Paraformaldehyde (PFA) (see Recipes)
  38. Sucrose (see Recipes)
  39. PFA-sucrose (see Recipes)
  40. Black wax petri dish (see Recipes)

Equipment

  1. CO2 supply machine (Next Advance, model: Quietek CO2 induction system )
  2. Dissection scissors (Fine Science Tools, catalog number: 91460-11 )
  3. Fine iris scissors (Fine Science Tools, catalog number: 14094-11 )
  4. Spring scissors (Fine Science Tools, catalog number: 15009-08 )
  5. Curved forceps (Fine Science Tools, catalog number: 11073-10 )
  6. Delicate suture tying forceps (Fine Science Tools, catalog number: 11063-07 )
  7. Dissection stereo microscope equipped with fibre-optic light source from top (Carl Zeiss Microscopy, model: Stemi 2000 )
  8. Measuring scale (LACO)
  9. Camera (Nikon, model: D5300 )
  10. -80 °C freezer (Thermo Fisher Scientific)
  11. Cryostat microtome (Microm GmbH, model: HM500 OM )
  12. Precision hotplate (HARRY GESTIGKEIT GMBH, model: PZ28-2T )
  13. Axio-Imager A2 microscope equipped with Axiovision release 4.8 software (Carl Zeiss Microscopy, model: 490022-0002-000 )

Software

  1. Statistical analysis software [IBM SPSS Statistics 20.0 (IBM Corporation, Released 2011, NY, USA)]
  2. Image J software [National Institutes of Health (NIH), USA]
  3. Axiovision release 4.8 software (Carl Zeiss)

Procedure

  1. Isolation of mouse aorta
    1. Measure the weight of mouse prior to euthanasia.
    2. Euthanize the mouse by CO2 inhalation as approved by the Animal Care and Use Committee of local government. Death is noted by lack of respiration and motility.
    3. Lay down the mouse in a supine position and fix arms and legs onto a cork platform with needles.
    4. Disinfect the ventral side of mouse with 75% ethanol.
    5. Open the abdominal cavity by a ventral midline incision and traverse cuts in the abdominal wall.
    6. Cut the skin, subcutaneous tissue and peritoneum from the abdomen to the thorax along the middle line, and fix skin/subcutaneous tissue onto the cork plate with needles.
    7. Open the mediastinum and cut off the ribs lateral to the mediastinum. Cut the diaphragm to facilitate drainage.
    8. Make a small incision in the right atrium of heart to draw 500-800 µl blood before perfusion using a 1 ml syringe attached with a 23G needle. Perfuse the vasculature through apical left ventricular puncture by slowly injecting 10 ml of 5 mM EDTA in PBS using a 10 ml syringe followed by 20 ml of ice-cold PBS by using a 20 ml syringe attached with a 23G needle. Perfuse 2-3 times intermittently with 10-20 ml of ice-cold PBS during dissection to avoid dehydration of aorta tissue.
    9. Remove organs including lung, liver, spleen, gastrointestinal and reproductive organs using dissection scissors and curved forceps while leaving heart, aorta, and kidneys intact in-situ.
    10. Expose aorta from heart onto the level below the iliac bifurcation. Under a dissection microscope equipped with fibre-optic light source from top at 30-40x magnification, carefully dissect out the thymus using fine iris scissors and delicate forceps. Note that under dissection microscope, it is possible to distinguish the compact aorta adventitia from loose light yellow-colored brown adipose tissue in the thorax and loose pale white-colored white adipose tissue in abdomen as well as from solid light yellow/brown paraaortic lymph nodes in-situ (Figure 1, left). Caution should be taken to prevent aorta injury during removal of liver, mesentery, thymus and kidney.
    11. Remove perivascular connective tissue and adipose tissue around the aorta and the major artery branches including innominate, common carotid and subclavian arteries in the aortic arch of the thoracic aorta; and celiac, mesenteric, renal, and common iliac arteries of the abdominal aorta with caution using fine iris scissors and delicate forceps (Figure 1, right). Carefully dissect paraaortic lymph nodes that are close to the aorta for clarity.


      Figure 1. Preparation of aorta and schematic view of aorta with its major branches. Image of in-situ aorta preparation before isolation showing preparation of aorta segments corresponding to the right schematic view, perivascular adipose tissue in thorax and abdomen, and paraaortic lymph nodes (left). Schematic view of freshly isolated aorta depicting the upper thoracic part with its branches (cyan) and lower abdominal part with its branches (yellow). Diaphragm indicates border between thorax and abdomen (right).

B1. En-face staining of whole aorta (Zhao et al., 2014; Hu et al., 2015)

  1. Follow steps 1-11 in Procedure A of aorta isolation.
  2. Fix the aorta tissue in-situ by injecting 5 ml of 4% PFA in PBS into the left ventricle using 10 ml syringe attached with a 23G needle and wait for 10 min, then inject/rinse with 10 ml of 5% sucrose in PBS.
  3. Harvest the whole aorta from the level above the coronary artery at the base of heart near the atria until 2-3 mm below the iliac bifurcation of the abdominal aorta after severing all major arteries in-situ. Be careful while dissecting and do not cut the thoracic or abdominal aorta.
  4. Put the whole aorta onto a previously prepared smooth surfaced black wax petri dish containing PBS.
  5. Gently clean all soft/loose perivascular adipose tissue around the aorta from thorax and abdomen under the dissection microscope at 20-25x magnification using fine iris scissors and delicate forceps (Figure 2, left). Note that applying more force or improper cutting can damage the adventitia or injure the aorta.
  6. Refill the black wax petri dish with fresh PBS after perivascular adipose tissue cleaning.
  7. Sever innominate, carotid, and subclavian arteries of aortic arch in thoracic aorta, and iliac arteries in abdominal aorta 3-5 mm after the bifurcations. Sever renal arteries in abdominal aorta for clarity (Figure 2, middle left)
  8. Pin the open end of one common iliac artery with minutien pin. Cut the aorta longitudinally from the unfixed common iliac artery using spring scissors and proceed anteriorly along the inner curvature of the aortic arch. Pin the split aorta onto the black dissection wax using minutien pins. (Figure 2, middle right)
  9. Cut along the outer curvature of the aorta from aortic root through innominate, carotid, and subclavian arteries until the aortic arch resembles a Y shaped split. Flatten out the Y-shaped aortic arch onto the black wax and pin the aorta parts (Figure 2, middle right).
  10. Fix the pinned aorta in the black wax petri dish overnight with PFA-sucrose at room temperature.
  11. Rinse 3 times for 10 min each with 1x PBS and fix 5 min with 70% ethanol at room temperature. Use of shaker is optional.
  12. Stain with Sudan IV staining solution for 10 min at room temperature. Sudan IV will stain lipid-rich plaque red leaving other non-plaque containing areas pale (Figure 2, right). It is advisable to clear all visible perivascular adipose tissue before staining because Sudan IV stained perivascular adipose tissue can give false background and interfere with plaque morphometry.
  13. Rinse 2 times for 3 min each with 70% Ethanol at room temperature. Note that over rinsing can destain the plaque. Rinse for 5 min with 1x PBS to remove ethanol.
  14. Fill the black wax petri dish with 1x PBS until it covers the stained aorta and pins.
  15. Place a measuring scale near the pinned aorta in the black wax petri dish.
  16. Take image with a digital camera from the top attached to a holding stand.


    Figure 2. Atherosclerosis lesion analysis of whole aorta. Removal of perivascular adipose tissue from freshly isolated aorta and trimming of branching arteries under the dissection microscope (left, middle left). Longitudinally split and pinned whole aorta before en-face staining (middle right). Enumeration of total aorta surface area and plaque surface area for morphometric analysis of Sudan-IV-stained whole aorta (right).

B2. Staining of aorta cross sections (Hu et al., 2015; Grabner et al., 2009)

  1. Follow steps 1-10 in procedure A of aorta isolation.
  2. Sever all thoracic and abdominal branching arteries 3-5 mm after their bifurcations from the aorta.
  3. Isolate whole aorta with adjacent adipose tissue 1 mm peripheral of the external lamina of the aorta under dissection microscope from the level above the coronary artery near the atria at the base of heart of the thorax onto the level below the iliac bifurcation (2-3 mm) of the abdominal aorta. (Figure 3, left).
  4. Cut the aorta into 4 parts: Thorax-I from the base of heart, to the level of 5th rib that included the aortic root, short ascending aorta, aortic arch containing the innominate, the right subclavian, the right common carotid, the left carotid, and the left subclavian arteries, and long descending thoracic aorta; thorax-II from the level of the 7th rib to the diaphragm including intercostal arteries; abdomen-I below the diaphragm to the middle of the abdominal aorta including the celiac, the superior mesenteric, the right and left renal arteries; and abdomen-II from the middle of the abdominal aorta to below the level of iliac bifurcation including the inferior mesenteric, and the common iliac arteries at the iliac bifurcation (Figure 3, left).
  5. Put OCT embedding medium tissue-tek into a labeled cryomold and then transfer 4 parts into the mold as described in Figure 3 (middle). Draw the direction of embedded aorta parts on the posterior part of cryomold using a permanent marker pen (Figure 3, middle).
  6. Refill the cryomold with tissue-tek until all aorta parts are completely embedded. Adjust the aorta segments to the same level within the embedding medium.
  7. If measurement of atherosclerosis in the innominate artery is to be done, embed thorax-I into a separate mold (optional).
  8. Snap freeze the mold containing aorta segments in tissue-tek in isopentane chilled with dry ice for 3 - 5 min until the tissue block becomes solid and white.
  9. Keep the frozen tissue block on dry ice for 30 min and store in -80 °C freezer until cryosectioning.
  10. Transfer the frozen tissue block onto the -20 °C chamber of a cryostat microtome 1 h before tissue sectioning. Mark the cutting side on the tissue block based on the direction of embedded aorta parts in step 5 in procedure B2.
  11. Prepare 10 µm thick fresh frozen aorta cross sections using cryostat microtome at -20 °C and collect them carefully onto labeled Poly-L-lysine-coated glass slides. To get good quality flat cryosections use new blade, apply even force to the wheel of the microtome and do not move the slide during tissue collection. New users should practice at least 10 times with practice tissue like liver or spleen before cutting the aorta.
  12. Collect every 10th serial aorta section at 100 µm intervals for Oil Red O and hematoxylin staining and reserve other sections for further staining.
  13. Air dry the glass slide containing tissue sections at room temperature for 1 h, keep them in slide holder within the moisture repellant coated freezer boxes and store in -80 °C freezer until further use.
  14. Prior to staining, take the slides out of -80 °C freezer and thaw them on hotplate for 1 min at 37 °C followed by air dry for 1 h.
  15. Fix the slides with 4% PFA for 5 min followed by 60% isopropanol for 5 min in a staining jar.
  16. Stain the slides with Oil Red O working solution for 10 min at room temperature.
  17. Rinse the slides 3 times 2 sec each with 60% isopropanol followed by brief wash in tap water.
  18. Stain the slides with hematoxylin for 6 min at room temperature.
  19. After a thorough wash in tap water dip the slides in tap water for 10 min.
  20. Mount the coverslips on the slides with faramount aqueous mounting medium. Do not air dry the slides before mounting, use 2-3 drops of mounting medium per slide, avoid air bubbles by slowly mounting the coverslip from one side of the slide.
  21. Leave the slide for 30-60 min after mounting in RT until the coverslip becomes firmly attached to the slide. Do not move or shake the slide to avoid air bubbles or damage of tissue.


    Figure 3. Atherosclerosis lesion analysis of aorta cross sections. Freshly isolated aorta with thoracic and abdominal perivascular adipose tissue (left). Note that perivascular adipose tissue in aortic arch was removed for better visibility of the branching arteries. Schematic presentation of isolation and embedding of different aorta segments including thorax-I with its branches (cyan) and thorax-II (green) above diaphragm, abdomen-I with its branches (yellow) and abdomen-II with its branches (red) below diaphragm (middle). Rotate thorax-I at 180° angle before embedding. Arrow indicates direction of cutting. Enumeration of lumen area, internal elastic lamina (IEL) and external elastic lamina (EEL) area for morphometric analysis of Oil Red O and hematoxylin-stained innominate artery (right).

C1. Morphometry of en-face stained aorta (Hu et al., 2015; Daugherty and Rateri, 2015; Schmitt et al., 2014)

  1. Transfer the images of en-face stained total aorta to a computer equipped with Image J software.
  2. Adjust the measuring scale distance of the digital image to the pixel scale of the Image J program.
  3. Encircle outer border line of the aorta manually and measure the area of the total aorta.
  4. Encircle each Sudan-IV stained plaque surfaces (intense red spots) within the aortic surface and measure their areas. Be careful to exclude any Sudan IV stained adipose tissues that are usually located below the aorta adventitia and less red compare to plaque (Figure 2, right).
  5. Export all the measurements to an excel file to calculate the ratio of plaque area from total aorta area and normalize the value as percentage of plaque area.
  6. Optionally, plaque area can be measured in the aortic arch (from aortic root to 3 mm below the subclavian artery branch), the descending thorax (from the level of aortic arch up to the level of diaphragm above the celiac artery branch), and the abdominal aorta (below the diaphragm until iliac bifurcation) after morphometry of plaque area in total aorta.
  7. For statistics, use 8-10 mice.
     

C2. Morphometry of Oil Red O-and hematoxylin-stained aorta cross sections (Hu et al., 2015; Grabner et al., 2009)

  1. Adjust brightness and contrast of the microscope. Take the bright field images of the aorta sections with an Axio-Imager microscope using 10x objective for imaging innominate artery and 5x objective for imaging abdominal aorta sections.
  2. Use Axiovision release 4.8 software to measure the areas after manually encircling them (Figure 3, right):
    1. Measure the area encircled by the intima/lumen border.
    2. Measure the area encircled by of internal elastic lamina (IEL) of arterial media.
    3. Measure the area encircled by external elastic lamina (EEL) of arterial media.
  3. Note that the contrast-rich elastin fibers in the media, the intima/media or media/adventitia border can be readily identified and the adventitia can be easily distinguished from adjacent adipose tissue using the Axiovision microscope.
  4. Export measurement data to an excel file to calculate the intima and media area.
  5. Calculate intima area by subtracting IEL area from lumen area and media area by subtracting EEL area from IEL area.
  6. Calculate ratio of intima area versus media area: Equation: intima/media ratio = intima area (IEL-lumen area)/media area (EEL-IEL area). Intima/media ratio represents the normalized value of plaque size.
  7. Calculate the intima/media ratio in at least 10 serial aorta cross sections (every 10th section) in 8-10 ApoE-/- mice.
  8. For statistical analyses of morphometry data comparison among multiple mouse groups, it is advisable to use generalized estimating equation model (GEE) model to estimate the parameters of a generalized linear models with the software IBM SPSS Statistics 20.0 (Hu et al., 2015).
  9. Express the data as means and standard error of mean (SEM). Calculate P values of significance using multiple testing with Bonferroni post-hoc test.

Notes

  1. During dissection of the aorta, infuse PBS into the left ventricle at regular intervals to keep the aorta wet and to prevent dehydration.
  2. Remove all perivascular adipose tissues to get a clear Sudan-IV staining with less background.
  3. Because the level of atherosclerosis varies among individual ApoE-/- mouse, it is advisable to use 8-10 mice of same gender and age per experimental group.
  4. In case of multiple comparison data sets with repeated measurements per mouse, i.e., several data points from the same mouse, the GEE model takes the correlation of these measurements per individual into account and provides robust estimates for the standard errors of the regression coefficients. This means that, even under misspecification of the chosen correlation structure, inferences regarding the group differences are still unbiased, which is a clear advantage compared to traditional linear regression models including analysis of variance (ANOVA). The major reason not to use ANOVA is that it assumes each data point as a separate entity and ignores the random effects, i.e., the biological differences between the mice. In contrast, GEE treats each mouse as a separate entity; plaque area or size as dependent variable; and genotype, aorta regions (innominate artery or abdominal aorta) as response.

Recipes

  1. Sudan IV staining solution
    Solve 500 mg Sudan IV in a mixture of 35 ml ethanol, 50 ml acetone and 20 ml distilled water
    Agitate from time to time until Sudan IV dissolves
  2. Oil Red O (ORO) working solution
    Dissolve 0.5 g of ORO with100 ml iso-propanol to prepare ORO stock solution
    Mix 6 parts of stock solution with 4 parts of distilled water
    Allow the solution to stand for 24 h
    Filter the solution before use using grade 410 filter paper
    Stored at room temperature
  3. 3. 5 mM EDTA
    Mix 14.612 g of EDTA with 80 ml distilled water to prepare 50 mM EDTA
    Stir the solution vigorously using a magnetic stirrer
    Adjust pH to 8.0 using NaOH
    Adjust volume to 100 ml with distilled water
    Mix 10 ml of 50 mM EDTA stock solution with 90 ml of distill water
    Stored at room temperature
  4. 1x PBS
    Dissolve 1 PBS tablet in 200 ml distilled water
    Stored at room temperature
  5. 4% paraformaldehyde (PFA)
    Mix 40 gm of PFA with 800 ml of 1x PBS
    Stir the solution at 62 °C under a fume hood
    Adjust pH to 7.4 using NaOH
    Add distilled water make up the solution to 1,000 ml
    Stored at 4 °C
  6. 5% sucrose
    Dissolve 5 g of sucrose in 100 ml of 1x PBS
    Adjust pH to 7.4 using NaOH
    Stored at room temperature
  7. PFA-sucrose
    Dissolve 5 g of sucrose in 100 ml of 4% PFA
    Adjust pH to 7.4 using NaOH
    Stored at room temperature
  8. Black wax petri dish
    Melt black dissection wax in the oven at 80 °C for 2-3 h
    Pour the melted wax on to several polystyrene petri dishes (150 mm x 15 mm) up to 1/3 of the petri dish
    Cool it down overnight at room temperature
    Stored at room temperature

Acknowledgments

This work is supported by the German Research Council (HA 1083/15-4; to A. J. R. H.; MO 3052/1-1 to S. M.; and YI 133/2-1 to C. Y.) and the European Research Council (AdG 249929 to C.W.).

References

  1. Daugherty, A. and Rateri, D. L. (2005). Development of experimental designs for atherosclerosis studies in mice. Methods 36(2): 129-138.
  2. Grabner, R., Lotzer, K., Dopping, S., Hildner, M., Radke, D., Beer, M., Spanbroek, R., Lippert, B., Reardon, C. A., Getz, G. S., Fu, Y. X., Hehlgans, T., Mebius, R. E., van der Wall, M., Kruspe, D., Englert, C., Lovas, A., Hu, D., Randolph, G. J., Weih, F. and Habenicht, A. J. (2009). Lymphotoxin beta receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE-/- mice. J Exp Med 206(1): 233-248.
  3. Hu, D., Mohanta, S. K., Yin, C., Peng, L., Ma, Z., Srikakulapu, P., Grassia, G., MacRitchie, N., Dever, G., Gordon, P., Burton, F. L., Ialenti, A., Sabir, S. R., McInnes, I. B., Brewer, J. M., Garside, P., Weber, C., Lehmann, T., Teupser, D., Habenicht, L., Beer, M., Grabner, R., Maffia, P., Weih, F. and Habenicht, A. J. (2015). Artery tertiary lymphoid organs control aorta immunity and protect against atherosclerosis via vascular smooth muscle cell lymphotoxin beta receptors. Immunity 42(6): 1100-1115.
  4. Hu, D., Mohanta, S. K., Yin, C., Weber, C. and Habe nicht, A. J. R. (2016). Preparation of single cell suspensions from mouse aorta. Bio-protocol 6(11): e1832.
  5. Schmitt, M. M., Megens, R. T., Zernecke, A., Bidzhekov, K., van den Akker, N. M., Rademakers, T., van Zandvoort, M. A., Hackeng, T. M., Koenen, R. R. and Weber, C. (2014). Endothelial junctional adhesion molecule-a guides monocytes into flow-dependent predilection sites of atherosclerosis. Circulation 129(1): 66-76.
  6. Mohanta, S. K., Yin, C., Peng, L., Srikakulapu, P., Bontha, V., Hu, D., Weih, F., Weber, C., Gerdes, N. and Habenicht, A. J. (2014). Artery tertiary lymphoid organs contribute to innate and adaptive immune responses in advanced mouse atherosclerosis. Circ Res 114(11): 1772-1787.
  7. Zhao, L., Moos, M. P., Grabner, R., Pedrono, F., Fan, J., Kaiser, B., John, N., Schmidt, S., Spanbroek, R., Lotzer, K., Huang, L., Cui, J., Rader, D. J., Evans, J. F., Habenicht, A. J. and Funk, C. D. (2004). The 5-lipoxygenase pathway promotes pathogenesis of hyperlipidemia-dependent aortic aneurysm. Nat Med 10(9): 966-973.

简介

动脉粥样硬化是大和中等大小动脉的慢性炎性疾病。 载脂蛋白E缺陷(ApoE -/-/- )小鼠用作实验模型来研究人动脉粥样硬化。 ApoE -/-/- 小鼠是组成性高脂血症并且发展类似于人噬斑的内膜斑块。 包括病变分析的实验设计,饮食条件,主动脉分离,染色方法,形态测定,组大小,年龄,动脉树内的位置和统计分析的各种问题是需要解决以获得鲁棒数据的重要参数。 在这里,我们提供详细的方法来量化主动脉粥样硬化。

关键字:动脉粥样硬化, 病变分析, 斑块, 媒体, 炎症

材料和试剂

  1. 培养皿组,玻璃(VWR International,目录号:89000-306)
  2. 黑色解剖蜡(CR Scientific,目录号:C3541)
  3. 用于固定主动脉的微针(0.15mm直径)(Fine Scientific Tools,目录号:26002-15)
  4. 50ml Falcon管(VWR International,CellStar ,目录号:188271)
  5. 10ml注射器(BD,目录号:309695)
  6. 20ml注射器(BD,目录号:301625)
  7. Sterican单次使用针-23G(B.Braun Medical Inc.,目录号:4657640)
  8. 聚-L-赖氨酸包被的载玻片(Menzel Glaeser,目录号:J2800AMNZ)
  9. 盖玻片(Menzel Glaeser,目录号:BBAD02400500#A)
  10. OHP永久标记笔(STAEDTLER MARS LIMITED)
  11. 显微镜载玻片保持器/邮包,地点(Sigma-Aldrich,目录号:Z708313-25EA)
  12. 低温冷冻保存箱(VWR International,目录号:82021-114)
  13. 有盖的染色缸(VWR International,目录号:25460-907)
  14. Grade 410滤纸(VWR International,目录号:28321-077)
  15. 聚苯乙烯培养皿(150mm×15mm)(Sigma-Aldrich,目录号:P5981)
  16. 乙醇(Merck Millipore Corporation,目录号:1009832500)
  17. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758)
  18. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:P4417-100TAB)
  19. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:16005)
  20. 蔗糖(Sigma-Aldrich,目录号:SO389)
  21. 异丙醇(Merck Millipore Corporation,目录号:1096341011)
  22. 丙酮(Merck Millipore Corporation,目录号:1000141000)
  23. 异戊烷(VWR International,目录号:103614T)
    注意:在VWR国际网站上也称为"2-甲基丁醛"。
  24. 干冰(TKD KABEL GmbH)
  25. 苏丹IV(Sigma-Aldrich,目录号:198102)
  26. OCT化合物-Tissue-Tek(SAKURA FINETEK USA,目录号:4583)
  27. Cryomold(SAKURA FINETEK USA,目录号:25608-924)
  28. 油红O(Sigma-Aldrich,目录号:00625)
  29. 苏木精溶液,Mayer's(Sigma-Aldrich,目录号:MHS-16)
  30. Faramount水性封固剂(Dako,目录号:S3025)
  31. 蒸馏水
  32. NaOH
  33. 苏丹IV染色溶液(见配方)
  34. 油红O(ORO)工作溶液(参见配方)
  35. EDTA(见配方)
  36. PBS(请参阅配方)
  37. 多聚甲醛(PFA)(参见配方)
  38. 蔗糖(参见食谱)
  39. PFA-蔗糖(参见配方)
  40. 黑蜡培养皿(见食谱)

设备

  1. CO 2供应机器(下一个前进,型号:Quietek CO 2感应系统)
  2. 解剖剪刀(Fine Science Tools,目录号:91460-11)
  3. 精细虹膜剪刀(Fine Science Tools,目录号:14094-11)
  4. 弹簧剪(Fine Science Tools,目录号:15009-08)
  5. 弯曲钳(Fine Science Tools,目录号:11073-10)
  6. 精细缝合镊子(Fine Science Tools,目录号:11063-07)
  7. 具有来自顶部的光纤光源的解剖立体显微镜(Carl Zeiss显微镜,型号:Stemi 2000)
  8. 测量尺(LACO)
  9. 相机(尼康,型号:D5300)
  10. -80℃冷冻器(Thermo Fisher Scientific)
  11. 低温切片机(Microm GmbH,型号:HM500OM)
  12. 精密加热板(HARRY GESTIGKEIT GMBH,型号:PZ28-2T)
  13. 配备有Axiovision release 4.8软件(Carl Zeiss显微镜,型号:490022-0002-000)的Axio-Imager A2显微镜,

软件

  1. 统计分析软件[IBM SPSS Statistics 20.0(IBM Corporation,Released 2011,NY,USA)]
  2. Image J软件[美国国立卫生研究院(NIH),美国]
  3. Axiovision release 4.8软件(Carl Zeiss)

程序

  1. 小鼠主动脉的隔离
    1. 在安乐死前测量小鼠的体重。
    2. 如通过地方政府的动物护理和使用委员会批准的通过CO 2吸入安乐死小鼠。 死亡是由缺乏呼吸和运动所引起的。
    3. 将鼠标放置在仰卧位置,并将手臂和腿固定在带针的软木平台上。
    4. 用75%乙醇消毒小鼠的腹侧。
    5. 通过腹侧中线切口打开腹腔,并在腹壁中穿过切口
    6. 沿着中线将腹部皮肤,皮下组织和腹膜从胸部切开到胸部,并用针将皮肤/皮下组织固定在软木板上。
    7. 打开纵隔,切断横向纵隔的肋骨。切割隔膜以方便排水。
    8. 在心脏右心房做一个小切口,在灌注前使用1毫升注射器连接一个23G针抽取500-800微升血液。通过使用10ml注射器,随后使用附有23G针的20ml注射器的20ml冰冷PBS,通过缓慢注射10ml PBS中的5mM EDTA,使顶部左心室穿刺穿透脉管系统。在解剖期间用10-20ml冰冷PBS间歇地灌注2-3次,以避免主动脉组织脱水。
    9. 使用解剖剪刀和弯曲镊子移除器官,包括肺,肝脏,脾脏,胃肠和生殖器官,同时原位保持心脏,主动脉和肾脏。
    10. 将主动脉从心脏暴露到髂骨分叉下方的水平。在放置30-40x放大倍数的顶部配有光纤光源的解剖显微镜下,使用精细虹膜剪刀和精细镊子仔细解剖胸腺。注意,在解剖显微镜下,可以区分紧密主动脉外膜与松散浅黄色棕色脂肪组织在胸部和宽松的白色白色脂肪组织在腹部以及从固体浅黄色/棕色主动脉旁淋巴结原位(图1,左)。在取出肝脏,肠系膜,胸腺和肾脏时,应注意防止主动脉损伤
    11. 去除主动脉周围的血管周围结缔组织和脂肪组织以及主动脉分支,包括胸主动脉弓中无名氏,颈总动脉和锁骨下动脉主动脉和腹主动脉的腹腔,肠系膜,肾和髂总动脉,使用精细虹膜剪刀和精细镊子小心(图1,右)。为了清楚,小心地分离靠近主动脉的主动脉旁淋巴结

      图1.主动脉的制备和主要分支的主动脉的示意图。在隔离前主动脉准备的图像,示出了对应于右侧示意图的主动脉段的准备,胸腔和腹部的血管周围脂肪组织和主动脉旁淋巴结(左)。新近地被隔绝的主动脉的示意图描述了与它的分支(青绿色)和更低的腹部部分与它的分支(黄色)的上胸部。隔膜指示胸部和腹部之间的边界(右)。

B1。整个主动脉的表面染色(Zhao et al。,2014; Hu等人,2015)。

  1. 按照主动脉隔离程序A中的步骤1-11
  2. 通过注射5毫升的4%PFA的PBS溶液到左心室,使用10毫升注射器连接23G针,并等待10分钟,然后注入/冲洗用原位固定主动脉组织的5%蔗糖的PBS溶液。
  3. 在切断所有主动脉后,从心房基部靠近心房的冠状动脉上方的水平收获整个主动脉,直到腹主动脉的髂骨分叉之下2-3mm。在剖析时要小心,不要切开胸主动脉或腹主动脉
  4. 将整个主动脉放在预先准备好的光滑表面黑蜡培养皿中含有PBS。
  5. 使用精细虹膜剪刀和精细镊子(图2,左),在解剖显微镜下,在20-25x放大率下轻轻地清洁来自胸部和腹部的主动脉周围的所有软/疏松的血管周围脂肪组织。请注意,施加更多的力或不当的切割可能会损伤外膜或伤害主动脉
  6. 在血管周围脂肪组织清洁后,用新鲜PBS重新填充黑蜡培养皿。
  7. 切口在胸主动脉中主动脉弓的无名,颈动脉和锁骨下动脉,以及在分叉后3-5mm的腹主动脉中的髂动脉。为了清楚起见,在腹主动脉中切断肾动脉(图2,左中)
  8. 将一个髂总动脉的开口端用细针固定。使用弹簧剪刀从未固定的髂总动脉纵向切开主动脉,并沿着主动脉弓的内曲率向前行进。使用minutien针将分离主动脉固定到黑色夹层蜡上。 (图2,中右)
  9. 沿主动脉的外曲率从主动脉根部通过无名,颈动脉和锁骨下动脉切开,直到主动脉弓类似于Y形裂口。将Y形主动脉弓压平在黑色蜡上,并固定主动脉部分(图2,右中)。
  10. 在室温下用PFA-蔗糖将钉扎主动脉固定在黑蜡培养皿中过夜
  11. 用1x PBS冲洗3次,每次10分钟,并在室温下用70%乙醇固定5分钟。使用振动器是可选的。
  12. 用苏丹IV染色溶液在室温下染色10分钟。苏丹IV将染色富含脂质的斑块红色,留下含有非斑块的区域变浅(图2,右)。建议在染色前清除所有可见的血管周围脂肪组织,因为苏丹IV染色的血管周围脂肪组织可能产生假背景并干扰斑块形态测定。
  13. 在室温下用70%乙醇冲洗2次,每次3分钟。注意,过度冲洗会使斑块脱色。用1×PBS冲洗5分钟以除去乙醇
  14. 用1 x PBS填充黑蜡培养皿,直到它覆盖染色的主动脉和针
  15. 在黑蜡培养皿中靠近固定主动脉放置一个测量尺。
  16. 使用数码相机从顶部连接到支撑架上拍摄图像。


    图2.整个主动脉的动脉粥样硬化病变分析在解剖显微镜下(左,中左)从新鲜分离的主动脉去除血管周围脂肪组织并修剪分支动脉。纵向分裂和固定整个主动脉,然后在面中染色(右中间)。用于苏丹-IV染色的全主动脉的形态测定分析的总主动脉表面积和斑块表面积的计数(右)。

B2。 主动脉横截面的染色(Hu等人,2015; Grabner等人,2009)

  1. 按照主动脉隔离程序A中的步骤1-10
  2. 切断所有胸和腹部分支动脉在他们从主动脉的分叉之后3-5 mm。
  3. 隔离整个主动脉与相邻的脂肪组织的主动脉的外部薄层外围的1mm外围从解剖显微镜下的冠状动脉上方的水平在胸部的心脏基底的心房附近到髂骨分叉下面的水平(2-3毫米 )的腹主动脉。 (图3,左)。
  4. 将主动脉切成4部分:从心脏基底到胸主动脉根部的第5肋骨的水平,胸主动脉弓包含无名的主动脉弓,右锁骨下,右锁骨颈动脉,左颈动脉和左锁骨下动脉,以及长下行胸主动脉;胸部II从第7肋骨的水平到包括肋间动脉的隔膜;腹膜下腹膜到腹主动脉的中部,包括腹腔,肠系膜上,左右肾动脉;和腹部-II从腹主动脉中部到髂骨分叉水平以下,包括肠系膜下部和髂骨分叉处的髂总动脉(图3,左)。
  5. 将OCT包埋培养基组织切片置于标记的低温模型中,然后如图3(中间)所述将4份转移到模具中。使用永久性记号笔绘制嵌入主动脉部分在冷冻切片后部的方向(图3,中间)。
  6. 用组织tek补充cryomold直到所有主动脉部分完全嵌入。在包埋介质中将主动脉节段调整到相同的水平。
  7. 如果要测量无名动脉中的动脉粥样硬化,将胸腔镜I嵌入单独的模具(可选)。
  8. 快速冷冻含有主动脉段的模具在用干冰冷冻的异戊烷中的组织tek中3-5分钟,直到组织块变成固体和白色。
  9. 保持冷冻的组织块在干冰上30分钟,并存储在-80°C冰柜直到冷冻切片
  10. 在组织切片前1小时将冷冻的组织块转移到低温恒温切片机的-20°C室。根据步骤B2中步骤5中嵌入主动脉部分的方向,在组织块上标记切割面。
  11. 使用低温恒温切片机在-20°C制备10μm厚的新鲜冷冻主动脉横切面,并将其小心收集到标记的聚-L-赖氨酸涂层的载玻片上。为了获得良好质量的平面冷冻切片使用新的刀片,均匀施加力到切片机的轮,并且不在组织收集期间移动幻灯片。新用户应在切开主动脉之前练习至少10次练习组织,如肝脏或脾脏
  12. 以100μm的间隔收集每10个连续主动脉切片,用于油红O和苏木精染色,并保留其他切片进行进一步染色。
  13. 在室温下将含有组织切片的玻片在空气中干燥1小时,将其保持在防潮涂布的冷冻箱内的载玻片保持器中,并储存在-80℃冰箱中,直到进一步使用。
  14. 染色前,将载玻片从-80°C冰箱中取出,并在37°C的热板上解冻1分钟,然后风干1小时。
  15. 用4%PFA固定载玻片5分钟,然后在染色罐中用60%异丙醇固定5分钟
  16. 在室温下用油红O工作溶液将载玻片染色10分钟
  17. 用60%异丙醇冲洗载玻片3次,每次2秒,然后在自来水中短暂洗涤
  18. 在室温下用苏木精染色载玻片6分钟
  19. 在自来水中彻底清洗后,将载玻片浸入自来水中10分钟
  20. 用极端的水性封固剂将盖玻片安装在载玻片上。 在安装之前不要风干幻灯片,每个幻灯片使用2-3滴安装介质,通过从幻灯片的一侧缓慢安装盖玻片避免气泡。
  21. 安装在RT中后,将载玻片放置30-60分钟,直到盖玻片牢固地连接到载玻片。不要移动或摇动幻灯片以避免气泡或组织损坏。


    图3.主动脉横截面的动脉粥样硬化病变分析。 与胸部和腹部血管周围脂肪组织隔离的主动脉(左)。注意,主动脉弓中的血管周围脂肪组织被去除以便更好地观察分支动脉。不同主动脉节段的分离和包埋的示意图,包括具有其上方的分支(青色)和胸部-II(绿色)的胸部I,具有其分支的腹部-I(黄色)和腹部-II及其分支隔膜(中间)。旋转胸部-I 180°角嵌入。箭头表示切割方向。对油红O和苏木精染色的无名动脉(右)进行形态计量分析的管腔面积,内弹性层(IEL)和外弹性层(EEL)面积的计数。

C1。染色主动脉的形态测量(Hu等人,2015; Daugherty和Rateri,2015; Schmitt等人,2014)

  1. 将染色的总主动脉的图像转移到配有Image J软件的计算机上。
  2. 将数字图像的测量刻度距离调整为Image J程序的像素刻度。
  3. 手动环绕主动脉的外边界线,并测量总主动脉的面积。
  4. 围绕主动脉表面内的每个苏丹 - 四染色的斑块表面(强烈的红点)并测量其面积。小心排除任何苏丹IV染色的脂肪组织,通常位于主动脉外膜下方,与斑块相比较少红色(图2,右)。
  5. 将所有测量结果导出为ex​​cel文件以计算斑块面积与总主动脉面积的比率,并将该值归一化为斑块面积的百分比。
  6. 任选地,可以在主动脉弓(从主动脉根到锁骨下动脉分支以下3mm),下行胸部(从主动脉弓水平到腹腔动脉分支上方的隔膜水平)测量斑块面积,腹主动脉(在隔膜以下,直到髂骨分叉)在总主动脉中的斑块面积的形态测量。
  7. 对于统计学,使用8-10只小鼠  

C2。 油红O染色和苏木精染色的主动脉横断面的形态测定(Hu等人,2015; Grabner等人,2009)

  1. 调整显微镜的亮度和对比度。 使用Axio-Imager显微镜,使用10x物镜成像无名动脉和5x物镜成像腹主动脉切片,获取主动脉切片的明场图像。
  2. 使用Axiovision release 4.8软件在手动包围它们后测量区域(图3,右):
    1. 测量内膜/内腔边界包围的区域。
    2. 测量由动脉介质的内弹性层(IEL)包围的区域。
    3. 测量由动脉介质的外部弹性膜(EEL)包围的区域
  3. 注意,可以容易地鉴定培养基中的富含对比度的弹性蛋白纤维,内膜/培养基或培养基/外膜边界,并且可以使用Axiovision显微镜容易地将外膜与相邻的脂肪组织区分开。
  4. 将测量数据导出到Excel文件以计算内膜和介质面积
  5. 通过从IEL面积中减去EEL面积,从管腔面积和介质面积中减去IEL面积,计算内膜面积。
  6. 计算内膜面积与介质面积的比率:方程:内膜/介质比率=内膜面积(IEL-内腔面积)/介质面积(EEL-IEL面积)。 内膜/中膜比表示斑块尺寸的归一化值
  7. 计算在8-10 ApoE -/- 小鼠中的至少10个连续主动脉横截面(每第10个切片)中的内膜/中膜比。
  8. 对于多个小鼠组之间的形态测量数据比较的统计分析,建议使用广义估计方程模型(GEE)模型来用软件IBM SPSS Statistics 20.0(Hu等人,/em>,2015)。
  9. 将数据表示为平均值和标准误差(SEM)。 使用Bonferroni事后检验的多重检验计算显着性的P值。

笔记

  1. 在主动脉解剖期间,定期将PBS注入左心室,以保持主动脉湿润和防止脱水。
  2. 除去所有血管周围脂肪组织,获得清晰的苏丹-IV染色与较少的背景。
  3. 因为动脉粥样硬化的水平在个体ApoE -/-/- 小鼠之间不同,所以使用每个实验组相同性别和年龄的8-10只小鼠是可取的。
  4. 在每个小鼠具有重复测量的多个比较数据集的情况下,即来自相同小鼠的几个数据点,GEE模型考虑每个个体的这些测量值的相关性,并且提供对标准误差的鲁棒估计的回归系数。这意味着,即使在所选择的相关结构的错误指定,关于组差异的推论仍然是无偏的,这与包括方差分析(ANOVA)的传统线性回归模型相比是一个明显的优点。不使用方差分析的主要原因是它假设每个数据点是一个单独的实体,并忽略随机效应,即小鼠之间的生物学差异。相比之下,GEE将每只小鼠视为一个单独的实体;斑块面积或大小作为因变量;和基因型,主动脉区(无名动脉或腹主动脉)作为反应。

食谱

  1. 苏丹IV染色溶液
    在35ml乙醇,50ml丙酮和20ml蒸馏水的混合物中溶解500mg苏丹IV 不时搅拌直到苏丹IV溶解
  2. 油红O(ORO)工作溶液
    将0.5g ORO与100ml异丙醇溶解以制备ORO储液
    将6份原液与4份蒸馏水混合
    让溶液静置24小时
    使用之前使用410级滤纸
    过滤溶液 在室温下贮存
  3. 5mM EDTA
    将14.612g EDTA与80ml蒸馏水混合以制备50mM EDTA
    使用磁力搅拌器剧烈搅拌溶液
    使用NaOH将pH调节至8.0 用蒸馏水
    调节体积至100 ml 将10ml 50mM EDTA储备溶液与90ml蒸馏水混合 在室温下贮存
  4. 1x PBS
    将1个PBS片溶于200ml蒸馏水中
    在室温下贮存
  5. 4%多聚甲醛(PFA)
    将40gm的PFA与800ml的1x PBS混合
    在通风橱下在62℃下搅拌溶液
    使用NaOH将pH调节至7.4 加入蒸馏水使溶液达到1000ml
    储存在4°C
  6. 5%蔗糖 将5g蔗糖溶于100ml1×PBS中
    使用NaOH将pH调节至7.4 在室温下贮存
  7. PFA-蔗糖 将5g蔗糖溶解在100ml的4%PFA中 使用NaOH将pH调节至7.4 在室温下贮存
  8. 黑蜡培养皿
    熔体黑色解剖蜡在烘箱中在80℃下2-3小时
    将其在室温下冷却过夜,
    在室温下储存

致谢

这项工作由德国研究理事会(HA 1083/15-4; AJRH; MO 3052/1-1至SM; YI 133/2-1至CY)和欧洲研究理事会(AdG 249929至CW) 。

参考文献

  1. Daugherty,A.和Rateri,DL(2005)。  开发小鼠动脉粥样硬化研究的实验设计。

    36(2):129-138。
  2. Grabner,R.,Lotzer,K.,Dopping,S.,Hildner,M.,Radke,D.,Beer,M.,Spanbroek,R.,Lippert,B.,Reardon,CA,Getz,GS, YX,Hehlgans,T.,Mebius,RE,van der Wall,M.,Kruspe,D.,Englert,C.,Lovas,A.,Hu,D.,Randolph,GJ,Weih,F。和Habenicht,AJ (2009)。  淋巴毒素β受体信号传导促进三级淋巴器官发生在老年ApoE -/- 小鼠的主动脉外膜中。实验医学 206(1):233-248。
  3. Hu,D.,Mohanta,SK,Yin,C.,Peng,L.,Ma,Z.,Srikakulapu,P.,Grassia,G.,MacRitchie,N.,Dever,G.,Gordon,P.,Burton ,BL,Ialenti,A.,Sabir,SR,McInnes,IB,Brewer,JM,Garside,P.,Weber,C.,Lehmann,T.,Teupser,D.,Habenicht, Grabner,R.,Maffia,P.,Weih,F。和Habenicht,AJ(2015)。  动脉三级淋巴器官控制主动脉免疫,并通过血管平滑肌细胞淋巴毒素β受体保护免受动脉粥样硬化。免疫 42(6):1100-1115。 >
  4. Hu,D.,Mohanta,SK,Yin,C.,Weber,C.and Habe nicht,AJR(2016)。  从小鼠主动脉制备单细胞悬液。生物协议 6(11):e1832。
  5. Schmitt,MM,Megens,RT,Zernecke,A.,Bidzhekov,K.,van den Akker,NM,Rademakers,T.,van Zandvoort,MA,Hackeng,TM,Koenen,RR和Weber,   内皮连接粘附分子-a将单核细胞导入流动依赖性 循环 129(1):66-76。
  6. Mohanta,SK,Yin,C.,Peng,L.,Srikakulapu,P.,Bontha,V.,Hu,D.,Weih,F.,Weber,C.,Gerdes,N.and Habenicht,AJ(2014) 。  动脉三级淋巴器官有助于先天性和适应性免疫反应在高级小鼠动脉粥样硬化中。 Circ Res 114(11):1772-1787。
  7. Zhao,L.,Moos,MP,Grabner,R.,Pedrono,F.,Fan,J.,Kaiser,B.,John,N.,Schmidt,S.,Spanbroek,R.,Lotzer, ,L.,Cui,J.,Rader,DJ,Evans,JF,Habenicht,AJand Funk,CD(2004)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/15322539"target ="_ blank"> 5-脂氧合酶途径促进高脂血症依赖性主动脉瘤的发病机制。 Nat Med 10(9):966- 973。
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引用:Mohanta, S., Yin, C., Weber, C., Hu, D. and Habenicht, A. J. (2016). Aorta Atherosclerosis Lesion Analysis in Hyperlipidemic Mice. Bio-protocol 6(11): e1833. DOI: 10.21769/BioProtoc.1833.
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