Age-dependent characterization of carotid and cerebral artery geometries in a transgenic mouse model of sickle cell anemia using ultrasound and microcomputed tomography

Abstract

To define morphological changes in carotid and cerebral arteries in sickle cell transgenic mice (SS) as they age, a combination of ultrasound and microcomputed tomography of plastinated arteries was used to quantify arterial dimensions and changes in mice 4, 12, and 24 weeks of age. 12-week SS mice had significantly larger common carotid artery diameters than AS mice, which continued through to the extracranial and intracranial portions of the internal carotid artery (ICA). There were also side specific differences in diameters between the left and right vessels. Significant ICA tapering along its length occurred by 12- and 24-weeks in SS mice, decreasing by as much as 70%. Significant narrowing along the length was also measured in SS anterior cerebral arteries at 12- and 24-weeks, but not AS. Collectively, these findings indicate that sickle cell anemia induces arterial remodeling in 12- and 24-weeks old mice. Catalog of measurements are also provided for the common carotid, internal carotid, anterior cerebral, and middle cerebral arteries for AS and SS genotypes, as a reference for other investigators using mathematical and computational models of age-dependent arterial complications caused by sickle cell anemia.

Keywords: Arteries; Imaging; Morphology; Sickle cell anemia; Ultrasound.

Figure 1:. The common carotid artery (CCA) luminal diameter is enlarged in SS mice at 12 weeks of age during the entire cardiac cycle.

(A) The luminal diameter was measured in the CCA 0.5 and 1.5 mm upstream from the carotid bifurcation (labeled O), and in eICA 0.5 mm downstream from the carotid bifurcation. (B) The common carotid artery was significantly larger in 12-week SS mice during both systole and diastole for the left and right sides. (C) There are no significant differences found in the eICA diameters of SS and AS mice. *p < 0.05 SS compared with AS by T-test. Error bars are SEM.

Figure 2:. Plastination of blood vessels can be used to acquire the morphology of the carotid and cerebral arteries.

Top view from casting process. (A) Microfil, a radio-opaque polymer, was injected into the aorta of mice after exsanguination and fixation of the blood vessels under pressurized conditions. (B) The head of mouse was decalcified in a 10% hydrochloric acid solution for 48 hours to dissolve bone. (C) The reconstructive software Mimics Materialise was used to segment and reconstruct carotid and cerebral arteries. (D,E) Final vascular model with individual arterial segments colored. CCA (common carotid artery), eICA (extracranial internal carotid artery), ECA (external carotid artery), OA (ophthalmic artery), intracranial ICA (internal carotid artery), ACA (anterior cerebral artery), MCA (middle cerebral artery). Scale bars = 5 mm.

Figure 3:. Reconstructed models from plastinated arteries recapitulate in vivo measurements.

Comparison of diameters measured from ultrasound and micro-CT in the 12-week age group. (A) micro-CT measurements reveal CCA diameters were larger in SS mice, paralleling results from the ultrasound study. No significant differences were found between the CCA diameters measured through ultrasound and diameters calculated from reconstructed micro-CT images. (B) Diameters calculated from micro-CT were significantly larger in SS mice. No significant differences were observed between both methodologies, however, diameters calculated through micro-CT had smaller SEM. *p<0.05 SS compared with AS by t-test. Paired t-test was performed between ultrasound and μCT methodologies for the same genotype. Error bars are SEM.

Figure 4:. The extracranial internal carotid artery is enlarged on the left side.

(A) The maximum inscribed circle in a cross-section of the internal carotid artery. (B) The maximum inscribed diameter in the extracranial ICA was found to be significantly larger in SS mice for both the left and right sides at 12 weeks. (C) The average ellipticity was calculated along the extracranial internal carotid artery from 0.4 – 0.6 mm downstream of the carotid bifurcation. Ellipticity for both AS and SS mice are between 0.4 and 0.6. Further examination of (D) perimeter and (E) cross-sectional area reveals only the left side to have significantly larger morphometric values at 12 weeks. *p < 0.05 SS compared with AS by t-test. Error bars are SEM.

Figure 5:. Size and axial growth is similar between homozygous and heterozygous sickle mice.

(A) Growth axially is quantified based on ICA length (extracranial + intracranial). ICA length increases significantly from 4 to 24 weeks in AS and SS, and no significant differences were found between AS and SS mice in any of the age groups. (B) Significant increase in body weight occurs from 4 to 12 weeks in AS and SS mice, but no differences were found in the same age group between genotypes. SS was compared with AS via t-test. The same genotype was compared across ages using a one-way ANOVA, *p < 0.05.

Figure 6:. Circumferential growth of the distal intracranial ICA in SS mice is slowed between 12 and 24 weeks of age.

(A) The intracranial ICA was divided into two regions: proximal, extending 0.5 from the origin to the midpoint and distal which continues from the midpoint to 0.5 mm before the ICA bifurcation (labeled end). (B) Diameter, (C) perimeter, and (D) cross-sectional area is compared between AS and SS mice at the proximal and distal segments of the intracranial ICA. The left ICA is significantly larger by all morphometric values at 12 weeks at the proximal and distal ends. The proximal ICA is also enlarged in 24-week SS mice but narrows at distal portion losing significance at that end of the ICA. *p<0.05 SS compared with AS by t-test. Error bars are SEM.

Figure 7:. Distal intracranial internal carotid artery tapers in 12 and 24-week SS mice.

Mean values for bulk area and percent change in area are plotted along the length of the intracranial ICA. (A) The left proximal ICA is enlarged in 12- and 24-week SS mice, but differences are lost upon moving towards the distal section of the artery. (B) Area was normalized to the starting point of artery to account for variability between subjects. The ICA tapers significantly from the proximal to distal end of the artery in 24-week SS mice as compared to age-matched controls.<p/>*p<0.05 SS compared with AS by t-test. Error bars are SEM.

Figure 8:. The morphology in the anterior cerebral and middle cerebral arteries are similar between SS and AS mice.

(A) The ACA does not significantly differ in size between AS and SS mice. At 24 weeks however, the right ACA in SS mice does tend to be smaller with diameters being significantly smaller. (B) Morphology in the MCA is not affected by genotype as the diameter, perimeter, and cross-sectional area are not significantly different, regardless of age or side. *p < 0.05 SS compared with AS by t-test. Error bars are SEM.

Figure 9:. Portions of the anterior cerebral arteries are significantly narrower in SS mice.

Mean values of percent change in area are plotted along the length of the ACA and MCA. (A) The left ACA is enlarged in 4-week SS mice. At 12 and 24 weeks the ACA of SS mice becomes narrowed at various portions of the ACA, in both the left and right sides. (B) The relation in normalized MCA area between SS and AS mice changes throughout age. SS mice have enlarged MCAs at 4-weeks, but at 24 weeks the cross-sectional area is narrowed compared to AS controls. *p < 0.05 SS compared with AS by t-test. Error bars are SEM.