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

Children with sickle cell anemia have elevated stroke risks as well as other arterial complications, but morphological changes to large arteries are not well defined, and the focus has been on the microcirculation where deoxygenation promotes sickling of red blood cells. The goal of this study was to define morphological changes in carotid and cerebral arteries in the Townes transgenic sickle cell mouse model, and to specifically determine anatomical measurement differences in mice homozygous for β-globin S mutation (SS) compared to heterozygous (AS) littermate controls. We used a combination of live imaging with ultrasound and microcomputed tomography (micro-CT) imaging of corrosion casted vessels to quantify arterial dimensions and changes in mice 4, 12, and 24 weeks of age with or without sickle cell anemia. 12 week SS mice had significantly larger common carotid artery diameters than AS mice, and significantly larger diameters in the extracranial and intracranial portions of the internal carotid artery (ICA), determined by ultrasound and micro-CT, respectively. There were also side specific differences between the left and right vessels. There was significant narrowing along ICA length in 12-and 24-week SS mice, decreasing by as much as 70%, such that there was no difference in size between the anterior and middle cerebral arteries, where the ICA terminates, by genotype. Significant narrowing along the length was also measured in the anterior cerebral arteries of 12-and 24-week SS mice, but not AS. Collectively, these findings indicate that sickle cell anemia causes increased arterial dimensions in 12-and 24-week aged mice. We also provide these measurements for the common carotid, internal carotid, anterior cerebral, and middle cerebral arteries for left and right sides, for AS and SS genotypes as a reference for other investigators using in silico modeling of arterial complications caused by aging with sickle cell anemia.


50
Sickle cell disease (SCD) is one of the more prevalent blood disorders worldwide with 51 more than 300,000 new individuals born each year afflicted by the disease. Sickle cell anemia 52 (SCA), specifically, is caused by a missense mutation that substitutes glutamic acid for valine in 53 hemoglobin. This change in molecular structure causes the hemoglobin in red blood cells (RBCs) 54 to polymerize and aggregate under de-oxygenated conditions, leading to deformation and 55 increased rigidity of the cell that cause blockages in the microvasculature that can damage organs 56 and lead to painful episodes known as sickle crisis. SCA also has effects on the large arteries, most 57 notably in the elevated occurrence of strokes in individuals with SCA. 58 The chance of ischemic stroke is greatly elevated for young individuals occurring in 11% 59 of people between the ages of 2 and 18 [1]. Then, during ages 20-30, the risk of ischemic stroke 60 falls, but is replaced with an increased risk of hemorrhagic stroke. Upon reaching early to mid-61 30s, the risk of ischemic stroke begins to dominate again in individuals with SCA [2]. Age is an 62 important factor for sickle cell-mediated strokes, and mechanisms behind variations in types of 63 strokes during specific times of life with SCA are unknown. Some clues can be extracted from 64 investigating the vascular morphology. In individuals who have suffered ischemic strokes, cerebral 65 angiograms have shown the presence of stenosis [3][4][5]. Additionally, autopsies of individuals who 66 have died from sickle-related strokes, show defined markers of intimal hyperplasia [4,6]. 67 In order to obtain to provide insight into underlying structural changes that affect stroke 68 risk in relation to age, mouse models can be used. The murine cerebral circulation displays 69 similarities to humans; the brain is supplied by a pair of vertebral and carotid arteries that branch 70 to form a network resembling the Circle of Willis (CoW) in humans [7]. The Townes sickle cell transgenic mouse model has been beneficial for investigating pulmonary and hematological 72 pathophysiology, with only a few studies on cerebrovascular complications of SCA [8][9][10]. 73 The goal of this study is to determine how morphology of carotid and cerebral arteries 74 change as the mice age, and any differential effects caused by sickle cell anemia. We use a 75 combination of ultrasound and microcomputed topography (micro-CT), to determine 76 morphometries of carotid and cerebrovascular arteries of Townes mouse model. Both techniques 77 have distinct advantages and disadvantages, which, when combined, can provide a comprehensive 78 morphometric characterization. Ultrasound in living mice allows for longitudinal studies that can 79 be used to obtain arterial dimensions during systole and diastole. However, murine vasculature is 80 much smaller than humans, with vessels approximately 1/10 th the size introducing limitations in 81 resolution and access to arteries in the skull. Alternatively, advances in micro-CT imaging 82 procedures for small animal models through the use of perfused contrast agents have enable 83 researchers to investigate structures within a voxel size of 8 µm [11]. For these reasons, we also 84 used micro-CT on corrosion casts of the carotid and cerebral arteries in mice. Casts were created 85 by a plastination method using a radiopaque polymer that hardens in the vessels. This cast was 86 then scanned by micro-CT and reconstructed in silico to acquire morphological information of the 87 smaller carotid and cerebral arteries. Casts of the arteries have their own tradeoffs in that it is an 88 endpoint analysis, disallowing longitudinal studies from the same subject. These modalities were 89 used to obtain detailed morphologies of the primary branches of the common carotid artery (CCA), 90 internal carotid artery (ICA), middle cerebral artery (MCA), and anterior cerebral artery (ACA) of 91 mice homozygous for human β globin S, the sickle cell allele (SS) or mice heterozygous for human 92 sickle cell allele and wildtype β globin A allele (AS). Our results suggest sickle cell disease alters 93 the vascular morphology in mice 12 and 24 weeks old, and these age-specific changes may be 94 relevant for age-related risk of types of strokes.

153
Carotid and cerebral artery casts were examined using a high-resolution micro-CT imaging was calculated for the common carotid artery (Fig 1A). At 12-weeks, CCA luminal diameters in 196 SS mice during systole were 19.3% and 11.5% larger than those of age-matched AS mice on the 197 left and right sides, respectively (n=4, p<0.05) ( Fig 1B). The CCA remained significantly enlarged 198 during diastole with a 20.1% and 13.7% diameter increase occurring in the left and right sides of 199 SS mice. In the extracranial ICA, a single point, 0.5 mm distal from the carotid bifurcation was 200 measured, and no significant differences in this segment at any age. Table 2   Since there was a limitation in obtaining measurements intracranially with ultrasound, we 228 validated that comparable measurements were acquired between ultrasound performed on living 229 mice, and the arterial casts measured by micro-CT, on non-living animals (Fig 2). Mice 12 weeks 230 old were used for comparisons since that was the age when statistically significant differences 231 were observed between SS and AS carotid arteries (Fig 3). Values calculated from the 232 reconstructed arteries were only comparable to systolic ultrasound measurements as a systolic 233 pressure of 100 mmHg was maintained throughout the casting process. The maximum inscribed diameter, which is the largest circle that can fit within the boundaries of a vessel cross-section (  There were no significant differences between diameters from ultrasound and micro-CT 260 methodologies (paired T-test, n=4, p>0.05). Diameters calculated through the micro-CT method 261 paralleled the significant differences in the common carotid artery of the 12-week group for SS vs.

262
AS mice (Fig 3B). Statistically significant differences were measured in the extracranial ICA using 263 the micro-CT technique, but not by ultrasound ( Fig 3C).

265
Diameter vs. perimeter and cross-sectional area as reliable metrics.

266
Diameter measurements were useful to extract data from the ultrasound, but it assumed a 267 perfectly circular geometry, which was not usually the case for these arteries. Using the micro-  presented by the arteries. The perimeter and cross-sectional area were 34.6% and 65% larger, 276 respectively, in the left extracranial ICA of SS mice 12 weeks of age (p<0.05, n=3), whereas the 277 right extracranial ICA increase was only 8.3% and 18.4%, which was not significant (Fig 4C,D).

278
There were no significant differences in either perimeter or area at 4 or 24 weeks between AS and SS mice. Table 3

301
Internal carotid artery measurements were captured with corrosion casting and micro-CT.

302
When examining the entire ICA (extracranial + intracranial) a significant increase occurred as 303 mice aged from 4 to 12 weeks. Length in the left ICA increased by 11% (p<0.001) and 8% (p<0.01) 304 in AS and SS mice, respectively, with similar increases of 16% (p<0.001) and 11% (p=0.0525) for 305 the right ICA (Fig 5A). There was no significant difference between 12 and 24 weeks.

306
Comparisons of body weight showed similar trends from 4 to 12 weeks. Body weight was 57% 307 (p<0.001) and 53% (p<0.001) greater from 4 to 12 weeks in AS and SS mice, respectively. At 24 308 weeks, weight increased by 3% (p>0.05) in AS and 17% (p<0.05) in SS. No significant differences 309 were found between genotypes at the same age. These results suggest that radial differences 310 observed in the carotid arteries were not a result of differences in the length or weight of the animal. increases significantly from 4 to 24 weeks in AS and SS, and no significant differences were found 315 between AS and SS mice in any of the age groups. (B) Significant increase in body weight occurs 316 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 318 across ages using a one-way ANOVA, *p < 0.05.

320
Narrowing occurs in the intracranial ICA of 12 and 24-week SS mice 321 Next, we compared intracranial internal carotid arteries (ICA) reconstructed from 4, 12 and 322 24-weeks old mice. Averaged morphometric values were taken from two sections: (1) the proximal 323 end, which begins 0.5mm from the beginning of the ICA to the midpoint of the vessel; and (2) the 324 distal end, which extends from to midpoint to 0.5 before the end of the vessel (Fig 6A). At 12-325 weeks, the proximal diameter, perimeter, and cross-sectional area on the left-side of SS mice were 326 enlarged compared to AS controls, increased by 15% (0.048+/-0.02), 16% (1.322 mm +/-0.025), 327 and 34% (0.139 mm 2 +/-0.005), respectively (Fig 6C,D). This difference continued distally, where 328 a 16% (p<0.05) and 32% (p<0.05) increase was marinated in perimeter and area. At 24 weeks, the 329 left proximal section was also enlarged for SS mice, with a 17% increase in perimeter and 40% These changes from the proximal to distal ICA could be visualized by plotting the mean 344 area for each genotype along the length (Fig 7A). At 4 weeks, SS and AS mice had similar areas 345 along the length. However, in the 12 and 24-week age groups SS mice had significantly larger 346 cross-sections at the proximal ICA, which narrowed, leading to a loss of differences once reaching 347 the opposite end of the artery. To quantify this narrowing in the ICA, area was normalized to the 348 area at the proximal start of the artery for each mouse (Fig 7B). Using this method, variability 349 between specimens can be accounted, and differences previously unseen begin to emerge. The 350 normalized area in 12-week SS mice was as much as 36% lower compared to AS mice. At 24 351 weeks these differences were even larger; at certain points along the ICA SS mice was 96% and 352 49% lower in the left and right sides, respectively. Both the left and right intracranial internal 353 carotid arteries narrowed in 12 and 24-week-old SS mice. Table 4

373
Two tributaries branch from the ICA, the middle cerebral and anterior cerebral arteries, and were 374 examined using the same methods from the reconstructed micro-CT models. In the ACA, 375 morphometric values were analyzed along a length of 0.5 to 2.5 mm, whereas the longer MCA 376 calculated values from 0.5 to 4.5 mm of the origin. When examining the bulk area, neither the 377 ACA nor MCA differed significantly in size between AS and SS mice (Fig 8A,B). It should be 378 noted that at 24 weeks, the right ACA in SS mice had significantly smaller diameters (*p<0.05).

379
To investigate these differences further the normalized area was plotted vs. length for the ACA 380 and MCA (Fig 9). Using this approach, the left ACA was found to be enlarged in 4-week SS mice.

381
However, at 12 and 24 weeks the ACA of SS mice were significantly smaller in both the left and 382 right sides, narrowing by up to 60% in some segments. The normalized MCA area between SS 383 and AS mice changed throughout aging. SS mice had enlarged MCAs at 4-weeks, but by 24 weeks 384 the cross-sectional area was narrowed compared to AS controls. Table 5 contains the 385 measurements from micro-CT imaging of corrosion casts for the cerebral arteries.    is not due to differences in animal size but is a result of the animal genotype.

441
In the distal intracranial ICA, however, a distinct narrowing was observed which led to a 442 loss of the statistically significant difference between SS and AS mice at 24-weeks of age. This 24-week week old mice. Using this method again, significant narrowing occurred in the ACA of 447 SS mice (Fig 9), despite seeing no significant differences in the bulk morphometric properties, 448 except for the ACA diameter being smaller in 24-week SS mice. As previously stated, high 449 velocities occur in cerebral arteries with sickle cell anemia, and are associated with stroke risk [4-450 6]. The narrowing luminal area in SS mice suggests a potential for hemodynamic changes as well.

451
Differences between AS and SS were mostly observed in the 12-week age group. Age is 452 known to be a strong factor of stroke risk in sickle cell anemia, with the highest risk of ischemic arteries of mice with SCA could potentially cause a stroke as blockages in these narrowed sections 466 of the artery may prevent an adequate supply of blood and oxygen.

467
The left common carotid and internal carotid artery lumens were larger in SS mice 468 compared to AS mice. The cause for these differences is not fully known. It has been previously 469 reported that the hippocampus, thalamus, and visual cortex on the left side of the brain in C57BL/6J