Authentication of In Situ Measurements for Thoracic Aortic Aneurysms in Mice
2021; Lippincott Williams & Wilkins; Volume: 41; Issue: 6 Linguagem: Inglês
10.1161/atvbaha.121.315983
ISSN1524-4636
AutoresSatoko Ohno‐Urabe, Masayoshi Kukida, Michael Franklin, Yuriko Katsumata, Wen Su, Ming Gong, Hong Lü, Alan Daugherty, Hisashi Sawada,
Tópico(s)Cardiac Valve Diseases and Treatments
ResumoHomeArteriosclerosis, Thrombosis, and Vascular BiologyVol. 41, No. 6Authentication of In Situ Measurements for Thoracic Aortic Aneurysms in Mice Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBAuthentication of In Situ Measurements for Thoracic Aortic Aneurysms in Mice Satoko Ohno-Urabe, Masayoshi Kukida, Michael K. Franklin, Yuriko Katsumata, Wen Su, Ming C. Gong, Hong S. Lu, Alan Daugherty, Hisashi Sawada Satoko Ohno-UrabeSatoko Ohno-Urabe https://orcid.org/0000-0002-4180-2857 Saha Cardiovascular Research Center and Saha Aortic Center (S.O.-U., M.K., M.K.F., W.S., M.C.G., H.S.L., A.D., H.S.), University of Kentucky, Lexington. , Masayoshi KukidaMasayoshi Kukida https://orcid.org/0000-0002-8393-9333 Saha Cardiovascular Research Center and Saha Aortic Center (S.O.-U., M.K., M.K.F., W.S., M.C.G., H.S.L., A.D., H.S.), University of Kentucky, Lexington. , Michael K. FranklinMichael K. Franklin https://orcid.org/0000-0003-4993-0164 Saha Cardiovascular Research Center and Saha Aortic Center (S.O.-U., M.K., M.K.F., W.S., M.C.G., H.S.L., A.D., H.S.), University of Kentucky, Lexington. , Yuriko KatsumataYuriko Katsumata https://orcid.org/0000-0002-0188-8094 Department of Biostatistics (Y.K.), University of Kentucky, Lexington. , Wen SuWen Su Saha Cardiovascular Research Center and Saha Aortic Center (S.O.-U., M.K., M.K.F., W.S., M.C.G., H.S.L., A.D., H.S.), University of Kentucky, Lexington. Department of Physiology (W.S., M.C.G., H.S.L., A.D., H.S.), University of Kentucky, Lexington. , Ming C. GongMing C. Gong https://orcid.org/0000-0002-3554-7378 Saha Cardiovascular Research Center and Saha Aortic Center (S.O.-U., M.K., M.K.F., W.S., M.C.G., H.S.L., A.D., H.S.), University of Kentucky, Lexington. Department of Physiology (W.S., M.C.G., H.S.L., A.D., H.S.), University of Kentucky, Lexington. , Hong S. LuHong S. Lu https://orcid.org/0000-0002-0577-2558 Saha Cardiovascular Research Center and Saha Aortic Center (S.O.-U., M.K., M.K.F., W.S., M.C.G., H.S.L., A.D., H.S.), University of Kentucky, Lexington. Department of Physiology (W.S., M.C.G., H.S.L., A.D., H.S.), University of Kentucky, Lexington. , Alan DaughertyAlan Daugherty https://orcid.org/0000-0003-2093-3775 Saha Cardiovascular Research Center and Saha Aortic Center (S.O.-U., M.K., M.K.F., W.S., M.C.G., H.S.L., A.D., H.S.), University of Kentucky, Lexington. Department of Physiology (W.S., M.C.G., H.S.L., A.D., H.S.), University of Kentucky, Lexington. , Hisashi SawadaHisashi Sawada Correspondence to: Hisashi Sawada, MD, PhD, Saha Cardiovascular Research Center, 741 S Limestone St, BBSRB B251, Lexington, KY 40536. Email E-mail Address: [email protected] https://orcid.org/0000-0003-0017-6236 Saha Cardiovascular Research Center and Saha Aortic Center (S.O.-U., M.K., M.K.F., W.S., M.C.G., H.S.L., A.D., H.S.), University of Kentucky, Lexington. Department of Physiology (W.S., M.C.G., H.S.L., A.D., H.S.), University of Kentucky, Lexington. Originally published1 Apr 2021https://doi.org/10.1161/ATVBAHA.121.315983Arteriosclerosis, Thrombosis, and Vascular Biology. 2021;41:2117–2119AbstractDownload figureDownload PowerPointAortic dimension is the most commonly used criterion of the severity of thoracic aortic aneurysms and can be determined by several approaches in mice. In situ imaging has been used to measure aortic diameters. However, a potential caveat of aortic measurements using this mode is the absence of arterial blood pressure that can lead to underestimation of aortic diameters, particularly in mice with a flaccid aortic wall. The present study developed an in situ imaging approach to overcome this shortcoming and demonstrated authentic dimensions of thoracic aortic aneurysms in mice.See cover imageβ-aminopropionitrile (0.5% wt/vol in drinking water) was administered to induce thoracic aortic aneurysms in C57BL/6J mice (male, 4 weeks old, n=30). Ultrasound imaging (Vevo 2100, MS550) was performed after 4, 8, and 12 weeks of β-aminopropionitrile administration, and aortic diameters were measured at the most dilated area at end diastole. During β-aminopropionitrile administration, 16 mice died and ultrasonography detected a wide range of luminal dilatations of the thoracic aorta from mild to severe in 10 mice (Figure [A and B]). In these 10 mice, 7 mice were randomly selected and in situ imaging was performed. The right atrial appendage was excised, and saline (8 mL) was perfused through the left ventricle. Perivascular tissues of the thoracic aorta were removed gently, and a black plastic sheet was inserted behind the aorta to enhance contrast of the aortic wall. Optimal cutting temperature (OCT) compound (150 µL) was then introduced into the left ventricle in 30 seconds using an insulin syringe to maintain aortic patency. Two-dimensional images and cine loops were recorded from preinjection to 50 seconds post-OCT injection using a dissection microscope with a high-resolution camera (#SMZ800, #DS-Ri1; Nikon). Imaging procedure was completed within 10 to 15 minutes for each mouse. To determine the aortic patency during imaging, aortic diameters were compared between ultrasound and in situ images. Several mice exhibited considerably smaller aortic diameters in images acquired before introduction of OCT compared with diameters measured using ultrasound (−0.8 to −0.4 mm), and OCT injection inflated the aortic dimensions (Figure [A and B]). Bland-Altman plots revealed that the bias between in situ and ultrasound diameters was closer to zero in post- than that in pre-OCT images (Figure [C]). The range of limits of agreement was smaller in post-OCT (−0.4 to 0.3 mm) than in pre-OCT (−1.0 to 0.5 mm). Furthermore, a significantly positive correlation between ultrasound and in situ measurements was only observed in post-, but not pre-, OCT images (Figure [D]). Therefore, in situ imaging using OCT injection provided more accurate aortic measurements in mice with thoracic aortic aneurysms.Download figureDownload PowerPointFigure. Ultrasound and in situ imaging of mouse thoracic aortas.A, Representative ultrasound and in situ images of thoracic aortas in β-aminopropionitrile–administered mice. Postoptimal cutting temperature (post-OCT) images were captured 50 s after completion of OCT injection. Yellow dotted lines, aortic wall. Scale bar=1 mm. n=7. B, Aortic diameters measured at the most dilated area of ultrasound and in situ images with/without OCT injection. n=7. C, Bland-Altman plots reveal the low variation of aortic measurements with OCT injection. Double arrows indicate the limits of agreement. Light green and red area, 95% lower and upper limit agreement CIs. D, Correlations of aortic measurements between ultrasound and in situ pre- or post-OCT images. E, Top, Luminal pressures during OCT injection in normal aortas. n=5. E, Bottom, Sequential transition of aortic diameters after OCT injection in aneurysmal tissues. n=3. F, Immunostaining for CD31 of normal aortas with OCT injection. n=5. Red triangles indicate endothelial cells. Scale bar=50 µm. AoD indicates aortic diameter; Asc, ascending aorta; Desc, descending aorta; IA, innominate artery; PA, pulmonary artery; TAA, thoracic aortic aneurysm; and US, ultrasound. *P<0.05 vs pre; †P<0.05 vs 0 s by one-way repeated measures ANOVA followed by t test with Bonferroni correction.OCT was injected using a small gauge needle (28G) that provided a slow flow of this viscous material. We measured luminal pressures during OCT injection using a telemetry system (#TA11PA-C10; Data Sciences International) in C57BL/6J male mice (n=5). Luminal pressures were 90±18 mm Hg at the peak and decreased to <50 mm Hg after at 10 seconds (Figure [E]). Then, aortic diameters were measured during OCT injection in mice with severe aortic dilatation (n=3). Aortic diameters were the largest immediately following OCT injection, with a subsequent modest reduction that was plateaued after at 40 seconds (Figure [E]). Importantly, maximal external diameters measured with in situ images were comparable to luminal diameters at mid-systole in ultrasound images (ultrasound, 3.0±0.4; in situ, 3.0±0.4 mm; P=0.70). Endothelial cells were also examined by immunostaining for CD31 to determine whether OCT injection led to loss of endothelial cells. Aortic tissues were harvested from C57BL/6J male mice after OCT injection (n=5). CD31-positive cells were detected throughout the intima of both ascending and descending thoracic aortas (Figure [F]). These results demonstrated that OCT injection did not cause aortic overexpansion due to pressure overload and overt aortic tissue damage.High-frequency ultrasound is a powerful tool to evaluate aortic diameters in mice and has been used by many studies.1,2 However, the availability of ultrasound systems is restricted by its expense. Also, ultrasonography requires appropriate training for reliable imaging and authentic measurements. In addition, some mouse models of thoracic aortic aneurysms involve the descending aorta, which is not readily imaged by ultrasonography. The present study demonstrated in situ image with OCT injection exhibited comparable aortic measurements to ultrasonography. Furthermore, in situ images can measure aortic dimensions directly in any regions of the thoracic aorta. Thus, in situ imaging with OCT injection can be considered as not only an optimal alternative approach but also a validation mode to ultrasound aortic measurements. The combination of ultrasound and in situ measurements would provide more robust evaluation of the severity of thoracic aortic aneurysms in mice.Aortic diameters can be measured by ex vivo approach; however, aortic patency is often not maintained during ex vivo imaging of aneurysmal aortas, which may lead to underestimation of aortic dimensions. Therefore, formalin or latex perfusion are frequently utilized to recapitulate aortic morphology ex vivo.3–5 However, these procedures need a perfusion system and are time-consuming. OCT is a nonhazardous reagent and can be readily applied. In addition, OCT does not cause discernable tissue damage. Therefore, OCT injection is a simple and effective procedure for in situ aortic measurements. Notable, in situ images measure aortic diameters in a lateral (right-left) axis. Imaging planes should be optimized individually, especially for mice with significant aortic dilatation toward the anterior or posterior wall.In conclusion, OCT injection is an optimal approach for maintaining aortic patency in situ, which provides more reliable aortic measurements in mice.Methods and Data AvailabilityDetailed methods and results are available in bioRxiv (https://doi.org/10.1101/2020.12.24.424013). All experiments were approved by the University of Kentucky Institutional Animal Care and Use Committee in accordance with the guidelines of the National Institutes of Health. Analyses were performed using 3 to 7 mice.Nonstandard Abbreviation and AcronymOCToptimal cutting temperatureSources of FundingThe authors' research work was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health (R01HL133723 and R01HL142973) and the American Heart Association (18SFRN33960163).Disclosures None.FootnotesThis manuscript was sent to William C. Sessa, Senior Consulting Editor, for review by expert referees, editorial decision, and final disposition.For Sources of Funding and Disclosures, see page 2118.Correspondence to: Hisashi Sawada, MD, PhD, Saha Cardiovascular Research Center, 741 S Limestone St, BBSRB B251, Lexington, KY 40536. Email hisashi.[email protected]eduReferences1. Sawada H, Chen JZ, Wright BC, Moorleghen JJ, Lu HS, Daugherty A. Ultrasound imaging of the thoracic and abdominal aorta in mice to determine aneurysm dimensions.J Vis Exp. 2019; 145:10.3791/59013. doi: 10.3791/59013Google Scholar2. Sawada H, Franklin MK, Moorleghen JJ, Howatt DA, Kukida M, Lu HS, Daugherty A. Ultrasound monitoring of descending aortic aneurysms and dissections in mice.Arterioscler Thromb Vasc Biol. 2020; 40:2557–2559. doi: 10.1161/ATVBAHA.120.314799LinkGoogle Scholar3. Jiao Y, Li G, Korneva A, Caulk AW, Qin L, Bersi MR, Li Q, Li W, Mecham RP, Humphrey JD, et al.. Deficient circumferential growth is the primary determinant of aortic obstruction attributable to partial elastin deficiency.Arterioscler Thromb Vasc Biol. 2017; 37:930–941. doi: 10.1161/ATVBAHA.117.309079LinkGoogle Scholar4. Gage GJ, Kipke DR, Shain W. Whole animal perfusion fixation for rodents.J Vis Exp. 2012; 65:3564. doi: 10.3791/3564Google Scholar5. Habashi JP, Judge DP, Holm TM, Cohn RD, Loeys BL, Cooper TK, Myers L, Klein EC, Liu G, Calvi C, et al.. Losartan, an AT1 antagonist, prevents aortic aneurysm in a mouse model of Marfan syndrome.Science. 2006; 312:117–121. doi: 10.1126/science.1124287CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited ByChen J, Sawada H, Ye D, Katsumata Y, Kukida M, Ohno-Urabe S, Moorleghen J, Franklin M, Howatt D, Sheppard M, Mullick A, Lu H and Daugherty A (2021) Deletion of AT1a (Angiotensin II Type 1a) Receptor or Inhibition of Angiotensinogen Synthesis Attenuates Thoracic Aortopathies in Fibrillin1C1041G/+ Mice, Arteriosclerosis, Thrombosis, and Vascular Biology, 41:10, (2538-2550), Online publication date: 1-Oct-2021. June 2021Vol 41, Issue 6Article InformationMetrics Download: 157 © 2021 American Heart Association, Inc.https://doi.org/10.1161/ATVBAHA.121.315983PMID: 33792346 Originally publishedApril 1, 2021 Keywordsaneurysmanimalsmiceaorta, thoracicdiagnostic imagingPDF download SubjectsAneurysmImaging
Referência(s)