Morphologic and Morphometric Evaluation of the Carotid Artery Wall: A Cadaver-Based Light and Scanning Electron Microscopic Study

Hilal Nakkas; Ferda Topal Celikkan; Nihal Apaydın; Oya Evirgen

doi:10.4103/jasi.jasi_168_21

ORIGINAL ARTICLE

Year : 2022 | Volume : 71 | Issue : 4 | Page : 261-265

Morphologic and Morphometric Evaluation of the Carotid Artery Wall: A Cadaver-Based Light and Scanning Electron Microscopic Study

Hilal Nakkas¹, Ferda Topal Celikkan², Nihal Apaydın³, Oya Evirgen²
¹ Department of Histology and Embryology, Faculty of Medicine, Ankara Yildirim Beyazit University, Ankara, Turkey
² Department of Histology and Embryology, Faculty of Medicine, Ankara University, Ankara, Turkey
³ Department of Anatomy, Faculty of Medicine, Ankara University, Ankara, Turkey

Date of Submission	01-Oct-2021
Date of Decision	13-Jun-2022
Date of Acceptance	16-Aug-2022
Date of Web Publication	01-Dec-2022

Correspondence Address:
Dr. Hilal Nakkas
Department of Histology-Embryology, Ankara Yildirim Beyazit University School of Medicine, Ihsan Dogramaci Boulevard, 3^rd Km. Cankaya, Ankara
Turkey

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jasi.jasi_168_21

Abstract

Introduction: A variety of changes occur on the elastic artery wall with age. Ultrastructural studies made in this area are mostly animal based. We aimed to evaluate wall changes and three-dimensional organization of the elastic lamellae with aging in humans. Material and Methods: Common carotid arteries were obtained from 17 human cadavers which were grouped as G1 (n = 6), 30–39 years; G2 (n = 5), 40–49 years; and G3 (n = 6), >50 years of age. Samples were evaluated under light and scanning electron microscopes. Results: Examination of G1 revealed intimal thickening and foamy cell infiltration. G2 and G3 had plaques bulging into the lumen and interlamellar space was widened. The lamellae were more straight. There was a positive correlation between intimal thickness and age. Elastic fiber content decreased with aging. Scanning electron microscopic analysis confirmed the findings. G1 and G2 had a smooth surfaced internal elastic lamina with uniform fenestrations whereas G3 contained numerous irregular fenestrae. Discussion and Conclusion: With this study, we showed some other wall structure changes beside plaque formation by aging. Which layer is affected the most was not clear at previous clinical studies because using ultrasonography (USG) cannot address the layer. Microscopic evaluation of this study revealed that when a wall thickening is detected by USG, it is due to intimal thickening. The specific vessel layer structural changes are important for proper treatment.

Keywords: Carotid artery, intima–media thickness, light microscopy, scanning electron microscopy

How to cite this article:
Nakkas H, Celikkan FT, Apaydın N, Evirgen O. Morphologic and Morphometric Evaluation of the Carotid Artery Wall: A Cadaver-Based Light and Scanning Electron Microscopic Study. J Anat Soc India 2022;71:261-5

How to cite this URL:
Nakkas H, Celikkan FT, Apaydın N, Evirgen O. Morphologic and Morphometric Evaluation of the Carotid Artery Wall: A Cadaver-Based Light and Scanning Electron Microscopic Study. J Anat Soc India [serial online] 2022 [cited 2023 Mar 29];71:261-5. Available from: https://www.jasi.org.in/text.asp?2022/71/4/261/362548

Introduction

Elastic type of communicating arteries, such as aorta and common carotid artery (CCA), absorb the energy caused by a large amount of systolic blood. During diastole, this absorbed energy recurs and helps transmit the diastolic blood by keeping the vascular tonus.^[1] Endothelial dysfunction leads to the failure of barrier function and the accumulation of low-density lipoprotein cholesterol in the subendothelium forming atherosclerotic lesions. Fragmentation of elastic lamellae due to mechanical and enzymatic factors (e.g., matrix metalloproteinase-2 activity), decreasing ratio of elastin/collagen content of the artery wall, and decreasing tonus and degeneration of vascular smooth muscle cells (VSMCs) involve a variety of changes on large elastic artery wall with aging.^[1],[2],[3] All these changes lead to intimal–medial thickening and atherosclerosis.^{[4],[5],[6],[7]} They also induce hypertension and cardiovascular diseases (CVDs) such as myocardial infarct, cerebrovascular ischemia, and stroke.^[2],[8],[9] It is shown with large-scale follow-up studies that increased intima–media thickness (IMT) gives an opinion about future vascular events independently of conventional risk factors and it is predictive in younger group as much as in older subjects.^{[6],[10],[11],[12]} Ultrasonography (USG) is a widely used noninvasive diagnostic technique for measuring IMT and assessing atherosclerotic status, but unfortunately USG examination is mostly inadequate to predict which layer is mostly affected in elastic arteries.^[4],[8],[13] The new development in ultrasonographic technology tries to deal with this problem such as in vivo intravascular USG gives more useful information about arterial wall structure but commonly not preferred as it is an invasive method.^[14] In literature, scanning electron microscopic (SEM) technique was used to observe the endothelial layer and three-dimensional configuration of arterial wall components such as extracellular matrix, collagen fibers, elastin lamellae, and VSMCs.^[3],[15] In the present study, we aimed to evaluate the structure and the morphometry of the intimal, subintimal, medial layers, and three-dimensional organization of the elastic lamellae in CCA wall of human cadavers with respect to age.

Material and Methods

Sample collection and processing

The study was conducted in the Department of Histology and Embryology and the Department of Anatomy at Ankara University. The cadavers used in this study were donated to Ankara University Faculty of Medicine for anatomical education, research, and clinical skill training. The history of health which includes cardiovascular system risk factors such as arterial pressure, blood biochemistry, CVDs and causes of death of all cadavers were not determined. The research protocol was approved by the Human Research Ethics Committee of Ankara University Faculty of Medicine (Decision number: I2-174-21). All the tissue-handling procedures were conducted according to the principles outlined in the Declaration of Helsinki. CCA segments of 3 cm in length were obtained from 17 adult (15 men and 2 women) human cadavers that were grouped with respect to age as G1(n = 6), 30–39 years; G2(n = 5), 40–49 years; and G3(n = 6), >50 years. All obtained CCA segments were divided into two pieces and processed for light microscope and SEM analysis.

Tissue processing and morphometric analysis

The fixed (with 4% formaldehyde solution) CCA tissue samples taken from cadavers were dehydrated through the increasing concentrations of ethanol series and embedded in paraffin blocks. The paraffin sections (4 μm) were stained with H and E (H and E) for routine examination. For the evaluation of elastic lamella and collagenous fibrous tissue, the sections were stained with orcein and indigo carmine (by Frankel's method). The intimal thickening, lipid accumulation, inflammation, continuity of elastic lamellae, course of elastic lamellae, and internal elastic lamina (IEL) continuity of the CCA vessel wall were assessed and scored as (−), no changes; (+), mild; (++), moderate; and (+++), severe.

For morphometric analysis, digital 8-bit RGB images from sections of all groups were obtained with an AxioCam MRc5 Camera mounted on the light microscope (Zeiss Axio Scope A1, Oberkochen, Germany) at × 40 magnification lens. The morphometric measurements were made by Photoshop CS5© image analysis software. Intimal thickness (IT) was defined as the distance from the luminal surface of the endothelium to the inner border of IEL. Intimal area (IA) was calculated by subtracting the luminal area from internal elastic area. Medial area (MA) was defined as the area between IEL and adventitia. By marking brownish colored areas of the orcein and indigo carmine stained cross sections at ×40 magnification, the intimal, medial and intimal + medial elastin content ratio of the vessel wall were analyzed and calculated.^[16] All measurements were performed in a blinded fashion to groups by a senior histologist.

Scanning electron microscopic analysis

For the SEM examination, two consecutive 0.5–1 mm segments from each specimen were first incubated in 90% formic acid at 45°C for 4 days to obtain maceration of the cellular elements. Segments were, then, postfixed with 0.5% tannic acid and 1% osmium tetroxide (OsO₄) and dehydrated. One segment of each sample was kept as whole and the other one was frozen in liquid nitrogen and fractured. All segments were dried in the critical point dryer, sputter-coated with gold palladium (Emitech K550x, England), and observed under SEM (Leo 438VP, Germany) operated at 1218 kV. Pictures were directly acquired in digital format as 1024 × 768 pixels grayscale tagged image file format images.

Statistical analysis

Statistics were obtained using the ready-to-use program of SPPSS version 11.0 (SPSSInc., Chicago,IL, USA). All the values were expressed as mean ± standard deviation. The obtained results were assessed by one-way analysis of variance and Tukey's tests. P < 0.05 was considered statistically significant.

Results

Light microscopic examination of G1 revealed intimal foamy cell (macrophages loaded with lipid) infiltration. IEL was intact and elastic fibers were interposed within VSMCs. Elastic fibers lied in parallel undulant arrays [Figure 1]a and [Figure 1]b. G2 had atherosclerotic plaques bulging into the artery lumen, which contained cholesterol crystals within the fibrous tissue and cellular infiltration [Figure 1]c and [Figure 1]d. Atherosclerotic plaques in G3 were crescentic in shape and they were protruded, obstructing nearly half of the lumen [[Figure 1]e, e', e'']. G2 and G3 had fragmentations and duplications of the IEL, particularly at the site of the atherosclerotic plaque [[Figure 1]d' and [Figure 1]f']. Interlamellar space was widened and lamellae were more straight, rather than wavy [Figure 1]d and [Figure 1]f. All these evaluations according to the morphological parameters are summarized in [Table 1]. Elastic fiber content decreased with increasing age; the difference between groups was not statistically significant [Table 2]. Intima was thinner in G1 compared with other groups with respect to age and the difference was statistically significant (P < 0.05) [Table 3]. The area of intima in G1 was significantly (P < 0.01) decreased than those of G2 and G3. There was no statistically significant difference in the area of media between the groups [Table 4].

Figure 1: Light microscopic images of carotid arteries in the age groups (G1, G2, G3). (a and b); IEL (arrow): Internal elastic lamina, EL (arrow head): Elastic lamellae, M: Media, Adv: Adventitia, *: Foamy Cell (macrophage). (c and d); arrow: fracture of EL; Cc: Cholesterol crystals. (e and f) inlets: Arrows: Fragmentation, Cc: Cholesterol crystals, I: Intima. Stain: a, c, e: Hematoxylene and Eosin; b, d, f: Orcein. Magnification: a, b, c, d, e, f: ×40; d', e', e'', f': ×100 (with immersion oil)

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Table 1: Morphologic evaluation

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Table 2: Elastic fiber ratio in intima, media, and intima+media

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Table 3: Intimal thickness

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Table 4: The area of intimal area and medial area

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SEM analysis showed that in G1, IEL contains occasional porous structures and elastic fibrils which were tightly intertwined. In G2, at the site of atherosclerotic location, elastic lamina had a fibrillar appearance. In G3, elastic lamellae of tunica media were thinned, occasionally discontinuous, and were running in a straight course [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d, [Figure 2]e, [Figure 2]f. While IEL of age groups G1 and G2 contained few round fenestrations of uniform size, the group G3 showed numerous, larger, and irregular polymorphous fenestrae [Figure 3]a, [Figure 3]b, [Figure 3]c.

Figure 2: Scanning electronic microscopic images of carotid arteries in the age groups (G1, G2, G3). At (a, b, d, e and f); IEL (arrows): Internal elastic lamina, arrow head: Elastic lamellae, at (c); Ap: Atheroma plaque. a': High magnification of IEL; c': Low magnification of bulging atheroma plaque

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Figure 3: Scanning electron microscopic micrographs of carotid arteries in the age groups (G1, G2, and G3). At (a and b) micrographs uniform size fenestrations and smooth surfaced IEL( Internal elastic lamina) of G1, G2 groups is shown. (c); Irregular fenestrations of IEL in G3 group. Thin arrows: Fenestrae

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Discussion

In the present study, the light and SEM evaluation of common carotid arteries obtained from 17 adult donated cadavers demonstrated morphometric and morphologic alterations of the vessel wall in groups with respect to age. Light microscopic examination demonstrated that the increase in intima thickness detected in G2 and G3 than G1 seems to be suggestive of an adaptive response against blood flow and pressure with age as it is reported in previous studies.^{[2],[4],[7],[13],[17]} IEL was shown to be intact in G1, but there were fragmentations and duplications, especially in the regions of atherosclerotic lesions and crescentic intimal thickenings in G2 and G3. This is suggestive of continuous mechanical stress with increasing age that may cause IEL fractures, thus contributing to the formation of atherosclerotic plaques.^{[5],[11],[18]} In literature, higher value of intima media thickness reflects early atherosclerotic disease and estimated as a risk factor for cardiovascular and cerebrovascular diseases such as myocardial infarction, stroke, and death.^{[6],[12],[19],[20]} Iwakiri et al. stated that the measurement of both CCA-intima media thickness and coronary artery intima/media ratio is significantly higher in death patients with CVD than in those without CVD.^[17]

It is important to address which layer is getting thicker because the atherosclerosis (accumulation of lipids) can cause intima thickening whereas hypertrophy of VSMCs can cause medial thickening.^[21] In our study, we found an increase in IA of the vessel wall that can be due to cell infiltration which is positively correlates with age; however, the MA was decreased in G2 and G3 groups when compared to G1, and the difference was not statistically significant as shown in [Table 4]. While the IA increases with age, there is a decrease seen in MA. It reverse interaction seems to be releated with atherosclerotic changes and cell infiltration within intimal layer that is proceeding to medial layer and also elastic lamellae distruption and VSMCs loss in media layer.

In older age groups (G2 and G3) the elastic lamellae has more straighter course and interlamellar space has been increased. This may be due to loss of VSMCs and compensation of this area by fibrous tissue. In some recent studies, it is shown that the decrease of elastin and increase of collagen quantity lead the carotid artery to become stiffer with aging.^[1],[2],[3] We indicated that the ratio of elastic fiber in intima, media and intima + media of CCA wall decreased with age, but there were no statistically significant difference between the groups. Furthermore, with SEM, we observed that elastin lamella in older age groups showed larger and irregular fenestrae. This may indicate the elastic fiber loss in vessel wall rather than insufficient repairing and renewal of elastin, because the quantity of elastin from the early development remains constant in many vital organs due to its very long half-life and very low turnover.^[22]

To assess the vessel wall status for cardio and cerebrovascular risk factors is important for treatment. The USG is a noninvasive and inexpensive vessel wall thickness measurement method, but this method cannot distinguish intima from media as it gives the sum of arterial wall thickness, especially in elastic arteries.^{[9],[11],[23]} In recent years, USG has been developed to assess individual intima and media thickness measurement such as high-resolution B-mode ultrasound and intravascular USG which is an invasive method, but these techniques require more expensive device and qualified employee.^[24]

The limitations of the present study are (i) we could not obtain the medical history and causes of death of all the donated cadavers that were used in this study; (ii) as the medical education of anatomy turns to virtual anatomy instead of cadaveric dissection, the number of donated cadavers is getting lower by time; this limits our cadaver numbers in the groups of the present study; and (iii) also in the present study, due to the low number of cadavers, the male and female distribution was not equal in the groups for comparing the gender differences in the aging effect on the carotid wall.

Conclusion

SEM and light microscopic observations of CCA in human cadavers revealed that IT and IA were increased with age in our study. Light microscopic observation of the CCA wall showed IEL e fragmentation and duplication, more straighter course of elastic lamellae, and atherosclerotic plaques. SEM observation of CCA revealed that numerous, larger, and irregular polymorphous fenestrae are interpreted as inadequacy in repairing and renewal of elastin in vessel wall. In this study, based on both the morphometric results and SEM observations of CCA, we suggest that the vessel wall thickening in elastic arteries is more likely to be due to intimal layer rather than medial layer.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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