Quantitative Assay for Mouse Atherosclerosis in the Aortic Root

6Quantitative Assay for Mouse Atherosclerosisin the Aortic RootJulie Baglione and Jonathan D. SmithSummaryThe mouse has become the preferred species for genetic manipulation aimed at creating and studying models for human disease. Although mice are highly resistant to atherosclerosis,dietary induction and,more frequently,gene knockout and transgenic mice have been widely used to study factors that alter the susceptibility to atherosclerosis. Although there are several ways to assess atherosclerosis in mice,measurement of the aortic root lesion area is a commonly used, medium-throughput method that allows for histological examination of the lesions. Here,we pro-vide the detailed methods for the quantitative analysis of mouse aortic root lesion area.Key Words:Atherosclerosis; mouse; histology; image analysis; lesion area measurement.1. Introduction1.1. Development and Use of Diet-Induced and Genetically Modified Mouse Models of AtherosclerosisMost of the plasma cholesterol in mice is found on high-density lipopro-tein (HDL),which is protective against atherosclerosis,whereas in humans, most of the plasma cholesterol is found on low-density lipoprotein (LDL), which plays a direct role in atherogenesis. Thus,wild-type mice fed a chow diet do not develop atherosclerotic lesions. Although mouse atherosclerosis was first studied in the 1960s (for review,see ref.1),the modern era of mouse atherosclerosis study was initiated in 1985 by Beverly Paigen,who used a high-fat (15%),high-cholesterol (1.25%),cholic acid (0.5%)-containing diet to induce hypercholesterolemia and atherosclerosis in susceptible mouse strains(2,3).From: Methods in Molecular Medicine, vol. 129:Cardiovascular Disease: Methods and Protocols, Volume 2: Molecular MedicineEdited by: Q. K. Wang © Humana Press Inc., Totowa, NJ8384Baglione and Smith The assay for quantitative assessment of atherosclerosis in the aortic root was also initially described by Paigen in 1987 (4),and with modification,this is the assay that is described next. The discovery of susceptible and resistant strains allowed the application of classical mouse genetic techniques to map the position of genes that influence atherosclerosis susceptibility. In 1987,Paigen’s laboratory defined the first atherosclerosis susceptibility locus,Ath1,on mouse chromosome 1 (5). It then took 18 yr before Paigen and colleagues identified the Tnfsf4gene,encoding the Ox40 ligand,as the gene responsible for athero-sclerosis susceptibility in the Ath1locus(6).In 1992,Breslow’s and Maeda’s laboratories independently reported the gen-eration of apoE-deficient mice via homologous recombination using embryonic stem cells,and both groups reported that these mice develop hypercholes-terolemia and atherosclerosis,even on chow diets (7,8). Now,there are a variety of gene knockout and transgenic mice that are susceptible to atherosclerosis; a catalog of these mice,their phenotypes,and the choice of diets for these models is beyond the scope of this chapter. However,the other most frequently used model along with the apoE-deficient mouse is the LDL receptor-deficient mouse created by Herz et al.(9). These genetically modified models have been used extensively to identify atherosclerosis susceptibility modifying genes by two methods:(1) the testing of candidate genes by breeding atherosclerosis-prone mice with mice that over- or under-express a specific candidate gene; and (2) by unbiased mouse genetic methods that allow positional identification of these genes. For the latter method,the availability of the mouse genome sequence and other shortcuts via the use of microarray expression analysis should greatly shorten the time needed to identify additional atherosclerosis genes. Other uses for these models include tests of therapeutic reagents,the study of arterial remodeling,the development of models of plaque rupture,and the study of pathogenic mechanisms.1.2. Protocol ConsiderationsAtherosclerotic lesions in all species develop focally at specific sites where there is nonlaminar flow. Thus,quantitative atherosclerosis assays must be per-formed using anatomical landmarks to keep the area under study constant. There are several different assays to quantitatively assess mouse atherosclerosis at vari-ous locations; however,the aortic root assay is still considered the standard assay in many laboratories,one reason being that this site is highly susceptible to atherosclerosis in mice and thus lesions develop here at earlier ages than in other areas. The second most commonly used assay is analysis of en face lesion surface area in the entire aorta,from the arch to the iliac bifurcation or limited just to the arch area. This method,described by Palinski’s laboratory,yields results that are generally well correlated with measurement of lesions at the aortic root (10).Quantitative Assay For Mouse Atherosclerosis85 It is often not sufficient to only assess atherosclerosis in the aortic root,as there are examples of an aortic root lesion area not being altered while the en face lesion area is affected,and these effects may occur selectively in the thoracic or abdominal regions of the aorta (11,12). Limitations of the en face assay are that it does not take into account lesion thickness or allow analysis of cellular compo-sition,and thus two lesions of very different volumes may take up equal surface area. It is also possible to measure atherosclerosis biochemically,by determining cholesterol ester content of the aorta (13). Limitations of this assay are the lack of volume or area measurements and the inability to gather histological information.Other sites besides the aorta can also be quantitatively assessed for lesions. ApoE-deficient mice also develop lesions in the carotid,pulmonary,femoral, and innominate (first branch off of the aortic arch) arteries,the latter being a good site for quantification as it is a site of early lesion predilection in young mice and plaque hemorrhage in older mice(14–16). Getz et al. have recently reviewed the site-specific modulation of atherosclerosis in mouse models and the importance of examining more than one location (17).We provide here a detailed protocol for the assessment of mouse atheroscle-rosis in the aortic root,and many of the methods described here can be applied to cross-sectional lesion area measurements at other locations as well. Diagrams of the anatomy of aortic root have been published previously (4,17). This method,although difficult to master,requires very little surgical time com-pared with the other methods that require the surgical isolation of the aorta or innominate artery,enabling the processing at a medium throughput level of approx 30 or more mice per day. It is possible to make many modifications of this protocol:for example,it may be necessary to eliminate fixation for immuno-histochemical staining with certain antibodies. For a description of alternate methods of atherosclerosis assays,we refer the reader to the excellent article by Daugherty and Whitman (18).2. Materials2.1. Mouse Sacrifice, Heart Removal, and Fixation1.Ketamaine/xylazine anesthesia is prepared by adding 10 mL of ketamine stock(100 mg/mL; Fort Dodge Animal Health,Fort Dodge,IA) and 1.5 mL xylazine stock (20 mg/mL; Vedco,St. Joseph,MO) to 35.5 mL sterile phosphate-buffered saline (PBS),yielding a solution of 21.25 mg/mL ketamine and 0.625 mg/mL xylazine.2.Small animal surgical tools:fine scissors,fine forceps,and coarse forceps withserrated tips.3.PBS or normal saline in a 10-mL syringe with a 24-G needle for perfusion.4.10% phosphate buffered formalin (Fisher,Pittsburgh,PA). Care should be takento minimize exposure to formalin vapors.2.2. Gelatin Embedding and Sectioning1.Embedding capsules (HistoPrep Tissue Capsules; Fisher,cat. no. 15-182-218).2.Gelatin type A (ICN Biomedicals,Aurora,OH; cat. no. 901771) used to make 5,10,and 25% solutions in water.3.Optimal cutting temperature (OCT) embedding medium (Sakura FinetechnicalCo,Torrence,CA).4.Fisher Plus coated microscope slides (Fisher,cat. no. 12-550-15).5.Cryostat. We use and recommend the Leica model CM 1850.2.3. Staining and Quantification1.Glass Staining racks with removable trays and wire handles (Fisher,cat. no. 08-812).2.Oil red-O stain (0.24%):1 g oil red-O powder (Sigma Aldrich,St. Louis MO) and250 mL 2-propanol are combined and mixed for 10 min. 150 mL of double-distilled (dd)H20 is added and mixed for an additional minute,allowed to stand for6 min,and then filtered through a 0.45-μm Stericup (Millipore,Billerica,MA).Oil red-O stain should be made freshly and used within 2 h of preparation.3.Harris hematoxylin (2.4%):100 mL Harris hematoxylin stock solution (SigmaAldrich) is combined with 98 mL of ddH2O and 2 mL of glacial acetic acid andfiltered.4.Bluing solution:five drops of ammonium hydroxide reagent in 1 L of ddH2O.5.Light Green solution (0.25%):50 mL of a 1% stock solution (2 g Light Green SFpowder [Fisher],198 mL ddH2O,and 2 mL acetic acid) is added to 150 mLddH2O. 0.25% solution is good for 2–3 wk and 1% solution 2–4 wk when kept ina brown bottle.6.Glycerol gelatin (Sigma Aldrich).7.22 ×60 mm no. 1 cover slips (Fisher).8.Image Pro Plus software,packaged as Olympus MicroSuite software,Soft ImagingSystem (Lakewood,CO).9.Microscope equipped with video imaging camera,video capture card,and personalcomputer (PC):we use an Olympus CX41 microscope (Melville,NY),a Panasonic Industrial Color charge-coupled device (CCD) camera,model no. GP-KR222 (Secaucus,NJ),Pincacle Systems video capture card,and software in a Dell PC.3. Methods3.1. Mouse Sacrifice, Heart Removal, and Fixation1.Each mouse is weighed and a 25-g mouse is anesthetized by intraperitoneal injec-tion with 200 μL of the ketamine/xylazine stock (delivered from a 1-cc tuberculin syringe with a 30-G needle). Adjust the volume according to the weight of the mouse using approx 8 μL of anesthesia stock per gram body weight. The depth of anesthesia is tested by lack of response to a firm squeeze of the hind foot.Additional anesthesia should be administered in 50-μL increments until this depth is achieved. The age of the mice under study should be kept constant +0.5 wk,as lesion area is extremely age-dependent (see Note 1).86Baglione and SmithQuantitative Assay For Mouse Atherosclerosis87 2.The legs of the supine mouse are taped down onto several folded paper towels,andthe ventral surface is wet with water or 70% ethanol (to keep the hair matted down). A lateral skin incision is made at the base of the abdomen extending to the width of the rib cage. Wrap some skin from the top side of the incision around a blunt forceps with serrated tips,hold down the mouse’s rear legs,and forcefully tear the skin and pull it up over the mouse’s head exposing the peritoneum and chest. Using fine scissors,cut open the peritoneal membrane from the base of the abdomen to the ribcage. Lift the sternum with fine forceps,and penetrate the diaphragm with the tip of fine scissors. Quickly cut through the sternum up the ribcage,make two lateral cuts at the top of the ribcage,and remove the ventral ribs exposing the beating heart. At this point,blood may be removed by intracardiac puncture (see Note 2).3.The circulatory system is perfused with 10 mL of PBS or normal saline by plac-ing the needle of the saline-loaded syringe into the left ventricle,using fine scis-sors to nick the right atrium (allowing drainage of the circulatory system into the chest cavity),and slowly pumping the saline into the heart. The mouse is eutha-nized at this point by exsanguination.4. The heart is gently lifted out by the apex,without squeezing the top,which candamage the aortic root,which is completely embedded within the heart. Any fibrous connective tissue attached to the heart is cut,and finally,the aorta is cut just above the level of the heart and the heart is put into a vial (we use glass scin-tillation vials) containing 5–10 mL of 10% phosphate-buffered formalin (see Note 3).The vial is placed at 4°C indefinitely,until ready for embedding but for a mini-mum of 24 h.3.2. Embedding Hearts in Gelatin1.The next step for routine quantitative assay is gelatin embedding,which helpsmaintain the integrity of the friable lesions during sectioning,although this step may also be omitted,particularly for immunostaining. For gelatin embedding, each heart is placed in a labeled histological capsule and washed under cold run-ning tap water for 1 h. Hearts are then placed in a shallow plastic container and floated in a 42°C water bath and covered with 5% gelatin and 10% gelatin sequen-tially for 2 h each. Twenty-five percent gelatin is incubated with the hearts at 42°C overnight. The hearts in 25% gelatin are refrigerated until the gelatin solidifies.2.Hearts are removed from the capsules (Fig. 1A)and the gelatin is trimmed with arazor blade into a solid block (Fig. 1B). The gelatin embedded hearts are placed back into formalin and stored at 4°C until ready for sectioning.3.3. Mounting the Hearts on a Pedestal in OCTand Sectioning Hearts With a Cryostat1.The lower half of the heart is removed by cutting with a razor blade. The align-ment of this cut is crucial to getting sections perpendicular to the axis of the aor-tic root. The cut is made approx 1 mm beneath the base of and parallel to the right and left atria (Fig. 1C).88Baglione and Smith Fig. 1. Setup for embedding,sectioning,and staining of aortic roots. (A)Gelatin-embedded heart in tissue capsule. (B)Heart in gelatin block. Arrows point to atria,and line shows position of cut critical for proper orientation. (C)Heart in gelatin block after cut is made. (D). Top half of heart in tissue mold,cut face down,covered with liquid optimal cutting temperature (OCT) medium. (E)Two views of heart top mounted on cryostat pedestal in frozen OCT. (F)Staining racks with slides in front of staining trays.2.The top half of the heart is then placed into a plastic tissue mold (lower chamber is 1.5 ×1×0.5 cm,L ×W ×D) with the cut surface at the bottom of the mold,and then covered in OCT compound in both the lower and upper chambers (Fig.1D ). If the heart is not gelatin-embedded,the heart is moved around carefully Fig. 2.Appearance of unstained sections used to determine the start of the aortic root.(A)Section showing the appearance of an atrium (red arrow) indicating proxim-ity to the aortic root. (B)Section at the beginning of the aortic root showing the three bipartite valve bases (red arrowheads) and the complete aortic medial wall (red outline).to fill the aorta with OCT. The mold is placed in the cryostat set at –22°C to freeze the OCT.3.The heart is removed from the base mold and mounted with the cut side facing upon the sample pedestal,which is mounted on the cryostat freezing stage,on a bed of not-yet solidified OCT (Fig. 1E).4.The pedestal is placed into the chuck of the cryostat and a 30-μm thick section iscut and transferred to a microscope slide (see Note 4) for observation to check whether this section is parallel to the cut surface of the heart,or oblique,yieldinga partial section. If orientation is incorrect,the pedestal angle is readjusted withinthe chuck. More sections are cut with observation every 10 sections to detect the atria,appearing as appendages,which signal the approach of the aortic sinus (Fig. 2A). The aortic sinus should appear as three bipartite valve bases with attached leaflets (these may break in sectioning) along with an intact intima (Fig. 2B). Novices may find this is difficult to see,but with practice,this becomes readily apparent.5.Once the aortic sinus is visible,the section thickness is decreased to 10 μm andevery other section is saved,four sections per slide covering a total distance of 80 μm.Six slides are prepared in this manner,covering a total distance of 400 μm,at which point the valve bases are shrunken,but still visible,and the valve leaflets may not all be visible (Fig. 3A–F). It is possible to quantify distal to this point, either by taking more slides,or by increasing the section thickness. In this case, one can capture sections going about 600 μm distal to the sinus origin,until the valve bases have disappeared. Slides are labeled,using a solvent resistant marker or a pencil,with the mouse identification number and the slide number and stored at room temperature until ready to stain,or at –20°C for immunostaining.3.4. Staining Sections for Aortic Root Analysis1.Twenty slides are placed back to back in the 10 slots of each staining rack. A seriesof staining trays are set up with the solutions listed next and the slides are immersed for the specified times. Figure 1F shows the staining setup.2.ddH2O for 2 min.3.60% 2-propanol for 30 s.4.Filtered oil red-O staining solution for 18 min,in order to stain neutral lipids.5.60% 2-propanol for 30 s.6.ddH2O for 1 min.7.ddH2O for 1 min.8.Harris hematoxylin stain for 2 min.9.Bluing solution,10 dips.10.ddH2O rinse for 1 min.11.Light Green counterstain for 30 s.12.ddH2O for 1–15 min to partially destain Light Green.13.Air-dry slides until ready for cover slipping.14.For cover slipping,glycerol gelatin is heated to 55°C in a water bath. One drop isplaced over each of the four sections on the slide. The cover slip is placed on the slide Quantitative Assay For Mouse Atherosclerosis8990Baglione and SmithFig. 3.Quantitative Assay For Mouse Atherosclerosis91 and pressed down firmly to remove any air bubbles. After the glycerol gelatin sets for an hour,excess is removed by washing the slides in soapy water (see Note 5).3.5. Quantification of Aortic Root Lesion Area1.The slide is observed microscopically under brightfield illumination using a ×4 or×10 objective lens. Video images are captured with a video capture card and frame grabber software (in our system,this adds additional magnification).2.One section on each of the six slides is quantified. Lesions consist of oil red-O stain-ing lipid-filled regions as well as any fibrous regions lumenal to the internal elastic lamina. Lesions can crack and even lift away from the media,these are quantified without the empty space. Using Image Pro or other similar software,and after cali-bration with a stage micrometer (for each objective lens),the lesions can be circled and the area of each lesion for the quantified section on the slide is exported to a spreadsheet in square micrometers. This is repeated for each of the five remaining slides from the heart,using 80-μm intervals between the sections,if possible.3.The sum of the lesion areas are calculated for each of the six quantified sections.Then,the mean lesion area is calculated for all of the six quantified sections (see Note 6).4.Lesion cellular composition,necrotic area,and fibrotic area should be qualita-tively assessed from these slides,but a quantitative analysis requires cell-type specific or collagen specific staining.3.6. Statistical Analysis1.For each mouse,the mean aortic root lesion area in square micrometers is used asthe primary measurement.2.Lesion areas vary among individual mice of the same age,gender,and geneticbackground. High-cholesterol diets also have a pronounced effect on lesion area.For larger lesions (>200,000 μm2),the coefficient of variation may be approx 25–30%,but for smaller lesions (<50,000 μm2) the coefficient of variation can be much greater.3.Gender has an effect on lesion area,and females of most strains have larger aorticlesions than males.Fig. 3. (Opposite page)Stained aortic root sections. (A–F)Series of six sections taken at 80-μm intervals through the aortic root of an apoE-deficient mouse with very small lesions to display the anatomy of this region. Large white arrowheads point to the three bipartite valve bases each of which fuse together distal to the origin. White arrows (A) point to one valve leaflet in the lumen. Lesions (staining red) were circled in thin black lines.(G)Common appearance of aortic root lesions in an16-wk-old apoE-deficient mouse fed a chow diet. Note coun terstain fading in this photograph taken several months after section staining. (H)Portion of the aortic root showing moderate sized lesions and the appearance of the proximal coronary artery (black arrowhead),which branches from the aortic root and that also contains lesions. All photographs were taken using a ×4 objec-tive lens. Larger lesions yield outward aortic remodeling to preserve lumen area.92Baglione and Smith 4.For routine analysis,we plot individual lesion areas of each mouse using a logscale graph.5.Lesion areas may not be normally distributed within a group. If this is the case,itis better to use nonparametric statistical analyses to compare lesion areas between groups. This results in a comparison of the median value rather than the mean value,and also decreases the weighting of values from outliers. If lesion areas are normally distributed,parametric analyses may be used,comparing the mean lesion values. For complex statistical analysis,such as quantitative trait locus analysis,we normalize the distribution by using the log10 of the lesion area.6.Power calculations can be performed to estimate the number of mice needed foreach group (genders cannot be pooled) using StatMate software from GraphPad (San Diego,CA),or other similar software. Assuming a coefficient of variation of 25%,using 10 mice per group yields 90% power to detect a 38% difference between the groups,with a two-tailed αof 0.05. In practice,we find that a group size of 10 is sufficient to detect any reasonably strong (40%) effect on lesion area.In order to detect subtler effects on lesion areas,larger group sizes must be employed.4. Notes1.Care must be taken to sacrifice mice on schedule. Figure 4 shows the dramaticeffect of age on lesion area in apoE-deficient mice fed a Western-type diet.2.Blood can be obtained prior to perfusion by cardiac puncture. For plasma isolation,prepare a 1-cc syringe with a 19-G needle by drawing up a 0.5 M EDTA solution and then expelling it. Puncture the left ventricle with the prepared syringe and very gently pull up on the plunger; if blood does not draw,gently reposition or rotate the syringe until blood comes into the syringe. Do not create a large back pressure,as this will only cause tissue to block the needle. Deliver the blood into a microfuge tube containing 10 μL of 0.5 M EDTA,mix by inversion several times,and place on ice. After all samples are obtained,the plasma can be isolated by centrifugation for 2 min in a microfuge.3.The OCT-embedded aortic root can be sectioned on a cryostat without fixation or gel-atin embedding; this is often required for immunostaining with certain antibodies. 4.Sections are flattened by the anti-roll plate of the cryostat and positioned onto aroom temperature slide. If sections start to roll,the plate may be repositioned or the sections may be manually unrolled with a fine paintbrush. As a final option, rolled sections may be floated in room temperature water and positioned on slides.5.Harris hematoxylin and Light Green staining solutions will diffuse and fade overtime in the aqueous glycerol gelatin mounting medium and reduce contrast (see Fig. 3G,H). It is best to capture the images or make photomicrographs within 2 wk of staining.6.Lesions areas are not constant over the 400 μm of the aortic root. The lesion areais often greatest on the second or third slide and then decreases in the distal slides.One can estimate the lesion volume from these cross sectional areas by use of the Cavalieri sterologic method.AcknowledgmentsThe authors would like to acknowledge Eddy Rubin,whose lab protocol formed the basis for our current method,and Suey Lee and Helen Yu of Jan Breslow’s lab,who taught us how to section and stain mouse aortic roots. This work was supported by grant PO1 HL029582 from the National Heart,Lung,and Blood Institute of the National Institutes of Health.References1.Smith,J. D. and Breslow,J. L. (1997) The emergence of mouse models of ather-osclerosis and their relevance to clinical research. J. Intern. Med.242,99–109.2.Paigen,B.,Havens,M. B.,and Morrow,A. (1985) Effect of 3-methylcholan-threne on the development of aortic lesions in mice. Cancer Res.45,3850–3855.3.Paigen,B.,Morrow,A.,Brandon,C.,Mitchell,D.,and Holmes,P. (1985)Variation in susceptibility to atherosclerosis among inbred strains of mice.Atherosclerosis 57,65–73.4.Paigen,B.,Morrow,A.,Holmes,P. A.,Mitchell,D.,and Williams,R. A. (1987)Quantitative assessment of atherosclerotic lesions in mice. Atherosclerosis 68,231–240.Quantitative Assay For Mouse Atherosclerosis93Fig. 4. Effect of age on aortic root lesion are in apoE-deficient mice fed a Western-type diet (7). 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