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Institute of Experimental Morphology, Pathology and Anthropology with Museum
Bulgarian Anatomical Society
Acta morphologica et anthropologica, 22
Soa • 2015
Morphology
Vasoprotective Properties of Aronia Melanocarpa –
a Histological and Morphometric Study
E. Daskalova1, S. Delchev1, I. Bivolarski2, P. Denev3,4, M. Kratchanova3,4,
P. Cvetkov5, M. Kaluch5
1Department of Anatomy, Histology and Embryology, Medical University of Plovdiv, Bulgaria
2 Department of General and Clinical Pathology, Medical University of Plovdiv, Bulgaria
3 Laboratory of Biologically Active Substances, Institute of Organic Chemistry
with Centre of Phytochemistry, Bulgarian Academy of Sciences, Plovdiv, Bulgaria
4 ITC – Innovative-Technological Centre Ltd., Plovdiv, Bulgaria
5 Medical University of Plovdiv, Plovdiv, Bulgaria
The social signicance of age-related diseases is determined by their global role in mortality and morbidity,
particularly in economically developed countries. Changes in elastic and muscular arteries walls, resulting
from age-related restructuring and progression of atherosclerotic lesions, underlie coronary heart disease
and cerebrovascular disease. Their prevention through administration of natural products is a research area
with huge potential, and application of natural antioxidants is one of the leading strategies to retard vascu-
lar aging. Aronia melanocarpa juice is a rich source of polyphenols and is characterized by very high anti-
oxidant activity in vitro. The aim of the current study was to investigate the effect of aronia juice intake on
age-related vascular changes of rat aortic walls. We used a model of aging male rats, whose thoracic aorta
walls were subjected to macroscopic, histological (hematoxilyn-eosin), histochemical (orcein) and mor-
phometric studies. The comparative analysis between the target group of old animals supplemented with
aronia juice; young untreated rats and old controls (not supplemented), revealed that aronia-supplemented
animals were characterized with reduced atherosclerotic lesions and a lower level of restructuring of aortic
walls. These data conrm that Aronia melanocarpa juice successfully retards age-related vascular aging,
and can be recommended as a prophylactic tool for healthy aging.
Key words: vascular aging, antioxidants, Aronia melanocarpa.
Introduction
Age-related diseases are social problem with global signicance and their prevention
through natural products is a research eld with great potential. Cardiovascular damage
is among the main causes of morbidity and mortality in relation to the aging process.
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The morphological changes of the wall of the large arteries, including the aorta are
result from age-related restructuring and virtually create pathophysiological conditions
for deterioration of their functions leading to pathological changes [9]. The application
of antioxidants is one of the strategies to slow the process of vascular aging [4, 5, 6, 7,
8]. Aronia melanocarpa juice is a rich source of polyphenols and as such exhibits a very
high antioxidant activity [2].
The aim of the current experimental study was to investigate the effect of Aronia
melanocarpa intake in the age-related vascular changes in the aortic wall.
Materials and Methods
Black chokeberry (Aronia melanocarpa) fruit juice: Commercially available sterilized
black chokeberry juice, packed in glass bottles (250 ml), was provided by Vitanea Ltd,
Plovdiv, Bulgaria.
In the experiment 22 male Wistar rats were divided into three groups. 14 of them
aged 10 months with initial body weight 418 ± 57 g were divided into 2 groups: control
old (CO), which were on a standard diet and tap water ad libitum, and Aronia group
(A), which received ad libitum chokeberry juice diluted 1:1 in drinking water and a stan-
dard rodent chow. The daily dose of fruit juice ingested by the animals was 10 ml/kg. The
control young group (CY) consisted of 8 animals aged 2 months with body weight 147
± 12 g. The experiment lasted 90 days. The experimental protocol was approved by the
Committee on Ethical Treatment of Animals of the Bulgarian Agency for Food Safety.
In the end of experiment animals were euthanized with i.m. Кetamin/Xilazine and the
thoracic aortas were separated and prepared for examination.
Macroscopic and histology examination: Descending thoracic aortas from CY, CO
and A groups of rats were harvested and xed in 10% formalin. Aorta thoracica was
cleared of the visible connective tissue, cut longitudinally and pressed between two
glass slides. Parts of xed aortic segments were embedded in parafn and sectioned
at 5 μm. Sequential sections were stained with hematoxylin/eosin and orcein. The
photomicrographs enclosed were taken on Nikon Microphot SA microscope (Japan),
equipped with a Camedia-5050Z digital camera (Olympus, Japan).
Morphometric analysis: All quantitative image analysis was performed using
software“DP-Soft” 3.2, Olympus, Japan. For each study, analysis of histology sections
was performed using at least 5 randomly chosen high-power elds from 5 different sec-
tions from each young (n =8) and old (n =7) rat. The thickness of aortic tunica media
(µm) was measured from the internal elastic lamina to the adventitial border. Cell den-
sity (cells/50µm2) in the tunica media of aortic wall was calculated as mean by count-
ing the number of nuclei in 50/50µm areas of tunica media tissue from 5 sections from
every young (n =8), old (n =7) and Aronia supplemented (n =7) rat.
Statistical analysis: The results were analyzed with SPSS 13.0 statistical program.
Statistical signicance between experimental groups was determined by Student’s t-test
and differences were considered signicant at P < 0.05. The intergroup comparison was
made with one-way ANOVA.
Results
From gross appearance of the intimal surface of the longitudinal section of thoracic
aorta (Fig. 1A) was evident that there were no visible pathological changes in the intima
of the thoracic aorta in the group of young controls. In the group of elderly controls
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Fig. 1. Gross appearance of the intimal surface of the longitudinal section of thoracic aorta of the ex-
perimental groups: A: controls young, B: controls old, C: Aronia supplemented group, (magn. × 20),
Hematoxylin/eosin staining D: controls young, E: controls old, F: Aronia supplemented group, (magn. ×
400), Orcein staining G: controls young , H: controls old, I: Aronia supplemented group, (magn. × 400)
(Fig. 1B), initial brous plaques and focal hemorrhages in atherosclerotic plaques were
observed. There were also single lipid stripes and spots. In aronia-supplemented group
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Fig. 2. A: Thickness of aortic tunica media, CY in comparison with CO p < 0.05; B: Cell density of
aortic tunica media, CY in comparison with CO p < 0.05; C: Number of elastic membranes in aortic
tunica media
СУ СО А
131,5
194,7
168,8
300
250
200
150
100
50
0СУ СО А
12
10
8
6
4
2
0
Number ofnuclei / 50 µm
СУ СО А
11,9 10,5 10,3
14,0
12,0
10,0
8,0
6,0
4,0
2,0
0
A B C
(Fig. 1C) the intima was smooth and homogeneous and only single lipid stripes and
spots were observed.
The hematoxylin/eosin stainings of thoracic aorta of the experimental groups are
shown in Fig. 1D, E, F. In the cross-sections of aorta of the young controls (Fig. 1D),
the tunica intima was smooth without abnormal depositions, the tunica media was pre-
sented with evenly and parallel elastic membranes. Their thickness and density were
preserved. Smooth muscle cells, located between the elastic membranes were normal in
size, shape and density, with normochromatic spindle cores. Tunica adventitia was rep-
resented by the usual loose connective tissue. In the crosscut of aorta of the old controls
(Fig. 1E) the tunica intima was with uneven edges with focal plaque-like thickening.
The tunica media was expanded, elastic membranes were separated, and considerably
loosen and thinned, and bended serpentine. Smooth muscle cells were enlarged, with
perinuclear vacuoles and pale cytoplasm. The nuclei of the cells were reduced in size,
with polymorphism and polihromaziya. Some of them had pyknotic form, their orienta-
tion was transverse relative to the elastic membrane. In the crosscut of aorta of aronia
group (Fig. 1F), the tunica intima was smooth, with protrusion of single endothelial
cells to the lumen. The tunica media was presented by evenly placed parallel elastic
membranes in the inner half of the aorta and serpentine bended – in the outer half. The
thickness of the elastic membranes was slightly reduced and their density was stored.
Smooth muscle cells had a normal spindle shape, longitudinally oriented, with pre-
served size, shape and density.
From Fig. 1G it is evident that tunica media in young controls was represented
by smooth, thick elastic membranes, intensely dyed by orcein. The orcein staining in
old controls (Fig. 1H) showed loosening and fragmentation of the paler-colored elastic
membranes. Orcein staining in the Aronia group (Fig. 1I) showed smooth and straight
elastic membranes with preserved integrity and staining intensity closer to that of young
controls.
Data in Fig. 2 demonstrated the results of the morphometric study of tunica me-
dia of aorta thoracica. Regarding the thickness of tunica media (Fig. 2A), statistically
signicant differences were established between younger and older controls (p < 0.05).
Differences between old controls and aronia group did not reach statistical signicance.
In terms of the number of nuclei in tunica media (Fig. 2B), statistically signicant dif-
ferences were detected only between younger and older controls (p < 0.05). Regarding
the number of the elastic membrane in the tunica media, differences between groups did
not reach statistical signicance.
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Discussion
Our ndings demonstrate convincingly that in aronia-supplemented animals, age-rela-
ted changes are discrete and structure of the vascular wall is visibly preserved. This
nding demonstrates the delay in the age changes and the preservation of the vessel
wall under the inuence of supplementation with Aronia melanocarpa juice.
Our data conrm that with advancing age the thickness of the aortic tunica media
is increasing statistically signicant [9]. Supplementation with Aronia juice leads to a
reduction in its dimensions, though not signicantly.
Our data conrms ndings from other authors [1, 9] that with increased age, a
decreased quantity of smooth muscle cells is found inside the tunica media, which are
responsible for synthesizing elastin within the aorta.
As recognized by other authors, the amount of elastic membrane does not change
with age [1, 3, 9]. Our results conrm the ndings that the quantity of the elastin mem-
branes remains inchanged during aging on account of the deterioration of their quality,
which is clearly demonstrated by staining with orcein. However, as it is evident from
the microphotographs, their quality visibly deteriorates in untreated adult controls and
partially improves in aronia-treated rats.
Age-related physiological changes to the aorta are associated with a progressive
decline in the elastic properties of the aortic wall [1, 3]. Greenwald [3] reported that
conduit arteries become stiffer with age because elastin becomes fragmented, degraded
and replaced by much stiffer collagen. Furthermore, both proteins become stiffer be-
cause of cross-linking and calcication, and these changes are accelerated by uraemia,
hyperglycaemia and oxidative stress [10]. Our results demonstrates that supplementa-
tion with aronia melanocarpa juice preserves the structure of the elastic membranes.
Conclusion
Age-related structural changes in the aortic wall lead to increased aortic stiffness, in-
creasing its diameter and length, reducing its compliance and contractility of the aortic
wall [1, 3]. These amendments violate its functions and are a prerequisite for the emer-
gence of pathologies, some of them fatal. Preservation of aortic wall and delay of age-
related changes are an essential preventive measure for cardiovascular diseases such as
hypertension, aortic dissection, aneurysm and rupture, congestive heart failure, com-
plications of ischemia and stenosis. Achieving this with non-pharmacological agents is
one of the latest strategies in the ght against aging. As a functional food juice, Aronia
melanocarpa shows convincing vaso-protective properties and can be recommended as
a prophylactic tool for healthy aging.
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