|Year : 2017 | Volume
| Issue : 1 | Page : 43-51
The effect of ginger on experimentally induced atherosclerosis in the aorta of rabbits: a histological and immunohistochemical study
Essam O Kamel1, Wail M Gad El-rab MD 2
1 Department of Histology and Cell Biology, Faculty of Medicine, Al-Azhar University, Assiut, Egypt
2 Department of Human Anatomy, Faculty of Medicine, Al-Azhar University, Assiut, Egypt
|Date of Submission||04-Jan-2017|
|Date of Acceptance||24-Apr-2017|
|Date of Web Publication||23-Aug-2017|
Wail M Gad El-rab
Human Anatomy & Embryology, Anatomy Department, Faculty of Medicine, Al-Azhar University, Assiut, 71524
Source of Support: None, Conflict of Interest: None
Atherosclerosis is a major disease of arteries, related to age and plasma cholesterol levels.
The objective of this study was to investigate the possible effects of ginger on experimentally induced atherosclerosis in the aorta of rabbits.
Materials and methods
Thirty, male rabbits aged 6 weeks were used in this study and were divided into three groups: group I included 10 male rabbits fed an ordinary diet for an experimental period of 8 weeks; group II included 10 male rabbits fed an ordinary diet mixed with 1% cholesterol powder at a dose of 200 mg/kg body weight for a period of 8 weeks; and group III included 10 rabbits fed an ordinary diet mixed with 1% cholesterol powder and were treated with ginger powder (4 g/kg of the dried rhizome) dissolved in saline solution daily for 8 weeks. The thoracic aorta was dissected and then processed for light microscopic studies.
Our results showed an apparent decrease in the thickness of both tunica intima and tunica media in the ginger-treated group. There was no intimal invasion of smooth muscle cells. Regular wavy elastic fibers were noticed in the media with apparently normal smooth muscle cells distributed in between. In addition, there was no apparent difference in the CD34 reaction of endothelial cells in the ginger-treated group when compared with the control group.
Ginger has an attractive role in modulating atherosclerosis in the aorta of rabbits.
Keywords: aorta, atherosclerosis, ginger
|How to cite this article:|
Kamel EO, Gad El-rab WM. The effect of ginger on experimentally induced atherosclerosis in the aorta of rabbits: a histological and immunohistochemical study. Al-Azhar Assiut Med J 2017;15:43-51
|How to cite this URL:|
Kamel EO, Gad El-rab WM. The effect of ginger on experimentally induced atherosclerosis in the aorta of rabbits: a histological and immunohistochemical study. Al-Azhar Assiut Med J [serial online] 2017 [cited 2017 Oct 17];15:43-51. Available from: http://www.azmj.eg.net/text.asp?2017/15/1/43/213586
| Introduction|| |
Atherosclerosis is a major disease of the arteries, related to age and plasma cholesterol. It is common among middle and old ages ,. It affects large-sized and medium-sized arteries and involves focal intimal accumulation of lipid and macrophages, smooth muscle cell migration and proliferation, and extracellular matrix deposition . The exact etiology of the disease is still unknown . Numerous risk factors including hyperlipidemia, smoking, and hypertension seem to contribute to the development of atherosclerosis. In atherosclerosis, blood leukocytes and smooth muscle cells migrate into the arterial intima and are organized to form lesions called atheromatous plaques, and fatty materials accumulate with additional fibrosis ,. These features result in hardening of the arterial wall due to thickening and loss of elasticity .
The aorta is composed of three layers. The tunica intima consists of a layer of endothelial cells (ECs) lining the interior surface of vessels. These cells rest on a basal lamina. Beneath the endothelium is the subendothelial layer, consisting of loose connective tissue that may contain occasional smooth muscle cells . The intima is separated from the media by an internal elastic lamina composed of elastin. Tunica media consists, chiefly, of large amounts of elastic fibers and lamellae with concentric layers of helically arranged smooth muscle cells, with variable amounts of reticular fibers and proteoglycans. Smooth muscle cells are the cellular source of elastin and other matrix constituents of the media. Tunica adventitia consists principally of longitudinally oriented collagen and elastic fibers .
Ginger (Zingiber officinale roscoe), a well-known spice plant, has been used in treating a wide variety of ailments including arthritis, peptic ulcers, atherosclerosis, diabetes mellitus, and cancer ,. It also has anti-inflammatory and anti oxidative properties that control the process of aging. Furthermore, it has antimicrobial potential as it can help treating infectious diseases . For example, a histological examination of a rat’s heart section confirmed myocardial injury with isoproterenol administration and near-normal pattern with ethanolic Z. officinale extract pretreatment .
CD34 antigen is a protein that is encoded by the CD34 gene in humans. It is a member of a family of single-pass transmembrane sialomucin proteins that are expressed on early hematopoietic and vascular-associated tissue. However, little is known about its exact function . It may mediate the attachment of stem cells to bone marrow extracellular matrix or directly to stromal cells . Recent data suggest that CD34 may also play a more selective role in chemokine-dependent migration of eosinophils and dendritic cell precursors . CD34 cells may be isolated from blood samples using immunomagnetic or immunofluorescent methods. Antibodies are used to quantify and purify hematopoietic progenitor stem cells for research and for clinical bone marrow transplantation .
| Aim|| |
The present study was designed to evaluate the possible effects of ginger on experimentally induced atherosclerosis in the aorta of rabbits.
| Materials and methods|| |
- Cholesterol powder 1% was purchased from Al Gomhoryia Company (2 Emarat El-Awqaf, Assiut, Egypt) for chemicals and was mixed with an ordinary diet for a period of 8 weeks .
- Ginger was purchased from a spice dealer. The recommended daily dose of ginger was the equivalent of 4 g/kg body weight of the dried rhizome dissolved in 3-ml saline solution daily at night for 8 weeks .
The present study was conducted at the animal house of Faculty of Medicine, Assiut University. The animal of choice for this study was the rabbit because it is the most sensitive species for induction of atherosclerosis ,. Rabbits can be easily caged, and their diets can be easily controlled. A total of 30, adult, male rabbits were used for this study. Female rabbits were not used to avoid the effect of the cyclic physiological hormonal changes. We used adult rabbits (6-week-old rabbits) to avoid sclerotic changes and other disorders and diseases that occur in old age. The average rabbit body weight was about 1 kg.
Animals were obtained from the Faculty of Medicine, Assiut University, according to the ethical guidelines for the care and use of laboratory animals. They were raised under hygienic conditions in special housing boxes with food and water. They were fed standard rabbit food (standard rabbit pellets), which was free of cholesterol. The rabbits were divided into three groups of 10 animals each.
- Group I (control group): this group included 10 rabbits, fed an ordinary diet without cholesterol for a period of 8 weeks.
- Group II (the atherosclerotic group): this group included 10 rabbits, fed an ordinary diet mixed with 1% cholesterol powder at a dose of 200 mg/kg body weight for a period of 8 weeks .
- Group III (the ginger-treated group): this group included 10 rabbits, fed an ordinary diet mixed with 1% cholesterol powder and were treated with ginger powder dissolved in saline solution daily for 8 weeks. The recommended daily dose of ginger was the equivalent of 4 g of dried rhizome .
At the end of the experimental period, rabbits were killed under deep anesthesia. The chest wall was dissected anteriorly, and the lungs were removed from their roots. The aorta was carefully dissected, and perfused with saline to clean its lumen from remaining blood. Subsequently, the samples were perfused with neutral formol for in-situ fixation. The specimens were rapidly removed and fixed in neutral buffered formol saline for preparing paraffin sections .
Specimens were left in the fixative for 48 h. During this time, several changes in the fixative were made every 12 h. After being washed briefly in water, the specimens were dehydrated in ascending grades of alcohol. Clearing was carried out in xylol. Specimens were then embedded in soft paraffin, and subsequently in hard paraffin. Blocks containing pieces of the thoracic aorta from all groups were prepared. A microtome was used for cutting sections of 7-µm thickness .
Sections of the three groups were subjected to the following staining protocols:
- Hematoxylin and eosin (H&E) staining to study the histological structure of the wall of the thoracic aorta .
- Modified Taenzer–Unna orcein method to study the elastic fibers present in the aorta .
- Immunohistochemical study of CD34 (EC marker) .
CD34-positive cells showed brown cytoplasmic deposits.
Tonsils were used as positive controls for CD34 were the positive cells showed brown cytoplasmic deposits ([Figure 1]).
Cardiac muscle cells were used as negative control for CD34, and they were obtained by skipping the step of primary antibody application ([Figure 2]).
|Figure 2 A photomicrograph of negative control for CD34 in cardiac muscle. (X400)|
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| Results|| |
The changes in the aortas of the control and experimental groups were evaluated through examination of sections by light microscopy.
Hematoxylin and eosin-stained sections
Histological examination of thoracic aorta sections in the control group showed intact tunica intima with thin wavy corrugated endothelium, a normal subendothelial layer, and wavy corrugated internal elastic fibers. The tunica media is comprised of elastic fibers and smooth muscle. The outermost layer is the tunica adventitia, and is made up of loose connective tissue ([Figure 3] and [Figure 4]).
|Figure 3 A photomicrograph in a section of a thoracic aorta in the control group showing tunica intima (arrow) faces the lumen. Elastic fibers and smooth muscle make up the tunica media (thick arrow). The outermost layer is the tunica adventitia which made of loose connective tissue (arrow head). Note the thickness of both tunicae intima & media. (H&E X200)|
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|Figure 4 A photomicrograph in a section of a thoracic aorta in the control group showing tunica intima (arrow) with thin wavy corrugated endothelium and thin sub-endothelial layer. Elastic fibers and smooth muscle make up the tunica media (thick arrow). (H&E X400)|
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In this group, the tunica intima showed absence of corrugation with bulge formation. Red blood cells were adherent to the intimal surface. Vacuolated cells (Foam cells) were noticed in the intimal subendothelial layer, and the subendothelial layer was wide. There was apparent increase in the thickness of both tunica intima and tunica media ([Figure 5] and [Figure 6]). In addition, there were many mononuclear cell infiltrations in the tunica intima ([Figure 6]).
|Figure 5 A photomicrograph in a section of a thoracic aorta in the atherosclerotic group showing absence of intimal corrugation and bulge formation (spiral arrow). Adhesions of RBCs to the surface of the intima (thin arrow). Vacuolated cells in the sub-endothelial layer (foam cell) (thick arrows) were noticed. There is apparent increase in the thickness of both tunicae intima and media. (H&E X200)|
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|Figure 6 A photomicrograph in a section of a thoracic aorta in the atherosclerotic group showing adhesions of RBCs to the surface of the intima (arrow head). Note the presence of many vacuolated cells in sub-endothelial layer (thick arrows).There is an apparent increase in the thickness of the sub-endothelial layer (thin arrow). Also there are many mononuclear cell infiltrations in the tunica intima (curved arrow). (H&Eosin X400)|
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In the ginger-treated group, we noticed an intact tunica intima with a thin wavy corrugated endothelium and thin subendothelial layer with wavy corrugated internal elastic lamina. The tunica media was comprised of elastic fibers and smooth muscles. The tunica adventitia was made up of loose connective tissue ([Figure 7] and [Figure 8]).
|Figure 7 A photomicrograph in a section of a thoracic aorta in the ginger treated group showing intact tunica intima (arrow head). The tunica media is made of smooth muscle and elastic fibers (arrow). The tunica adventitia formed of loose connective tissue. Note the thickness of both tunicae intima and media. (H&E X200)|
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|Figure 8 A photomicrograph in a section of a thoracic aorta in the ginger treated group showing intact tunica intima (curved arrow) with thin wavy corrugated endothelium and thin sub-endothelial layer. Elastic fibers and smooth muscle make up the tunica media (arrow). (H&E X400)|
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Histological examination of orcein-stained sections showed prominent wavy internal elastic lamina. The media was rich in elastic fibers that were parallel in distribution ([Figure 9]).
|Figure 9 A photomicrograph in a section of a thoracic aorta in the control group showing prominent dark brown wavy internal (arrow head) and external (spiral arrow) elastic laminae. The media is rich in elastic fibers which are parallel in distribution (arrow). (Orcein X400)|
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The atherosclerotic group showed interrupted internal elastic lamina. Areas with complete loss of elastic fibers were noticed in the media. Some areas showed irregularly condensed elastic fibers in the outermost part of the media ([Figure 10]).
|Figure 10 A photomicrograph in a section of a thoracic aorta in the atherosclerotic group showing apparently interrupted internal elastic lamina (thick arrow). Tunica media possesses areas with complete loss of elastic fibers (arrows) and areas of irregularly condensed elastic fibers in the outermost part (arrow head). (Orcein X400)|
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The ginger-treated group showed prominent intact internal and external elastic laminae. The tunica media was rich in parallel elastic fibers. Small areas of the media showed loss of elastic fibers ([Figure 11]).
|Figure 11 A photomicrograph in a section of a thoracic aorta in ginger treated group showing prominent intact internal (short arrow) and external elastic laminae (arrow). Tunica media looks rich in parallel elastic fibers (spiral arrow). Some areas of the media still show loss of elastic fibers (arrow heads). (Orcein X400)|
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CD34 immunohistochemical-stained sections
A few, positive, brownish, cytoplasmic reactions confined to the ECs were observed in this group ([Figure 12]).
|Figure 12 A photomicrograph in a section of a thoracic aorta in control group showing few positive brownish cytoplasmic reactions confined to the endothelial cells (arrow). (Anti CD34 immunostaining X400)|
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In this group, many, strong, positive CD34 immunoreactions confined to the cytoplasmic ECs were observed ([Figure 13]).
|Figure 13 A photomicrograph in a section of a thoracic aorta in atherosclerotic group showing many strong positive CD34 immunoreactions confined to cytoplasm endothelial cells (arrows). (Anti CD34 immunostaining X400)|
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In this group, a few, moderate CD34 immunoreactions were observed in the cytoplasm of ECs ([Figure 14]).
|Figure 14 A photomicrograph in a section of a thoracic aorta in ginger treated group showing moderate CD34 immunoreactions in cytoplasm of endothelial cells (arrows). (Anti CD34 immunostaining X400)|
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| Discussion|| |
Atherosclerosis is the primary cause of cardiovascular diseases, leading to occlusion of the arteries . However, several theories have been proposed for the pathogenesis of atherosclerosis: modified lipoprotein theory , low-density lipoprotein retention theory , and immunological hypothesis for atherosclerosis . In all these theories, it has been suggested that lipid peroxidation has a primary effect on the onset of atherosclerosis . The first component of the blood vessel involved in the pathogenesis of atherosclerosis is the endothelial lining. Chronic and cumulative metabolic alterations of the endothelium are induced by certain lipids, peroxidants, and inflammatory cytokines . Statins are potent inhibitors of cholesterol biosynthesis, and they are beneficial in the primary and secondary prevention of coronary heart disease ,.
On the other hand, Nicoll and Henein  suggested that the use of medicinal plants such as ginger is becoming an increasingly attractive approach for the treatment of hyperlipidemia and atherosclerosis. In addition, the authors clarified that it is cheap and well tolerated. Furthermore, Durak et al.  observed that ginger plays an essential role in heart diseases and aging, and the bioactive molecules of ginger have antioxidant properties.
The objective of this study was to establish animal models of experimentally induced atherosclerosis to evaluate the possible therapeutic effects of ginger. The present study recorded that ginger plays an important role in the management of atherosclerosis. In view of its anti-inflammatory properties, immunomodulatory effect, and antioxidant effect, ginger potentially represents a novel treatment modality for atherosclerosis .
In the present study, the relative changes in the aorta were investigated in three groups of rabbits − control, atherosclerotic, and ginger-treated groups.
In H&E-stained sections, the present study showed that animals of the control group had an intact tunica intima, thin wavy corrugated endothelium, a thin subendothelial layer, and apparent wavy corrugated internal elastic fibers. However, in atherosclerotic animals, the tunica intima lost its corrugation and had apparent bulge formation. There were adhesions of red blood cells on the surface of the intima. The subendothelial layer showed foam cells that were comprised of fatty material. There was an increase in the thickness of both tunica intima and media in the atherosclerotic group when compared with the other groups. These findings can be attributed to the effect of the atherosclerotic agent (cholesterol powder), resulting in large areas of EC injury. This was concomitant with Phinikaridou et al.  who reported the same histopathological features of rabbits receiving cholesterol powder 1% mixed with an ordinary diet for a period of 8 weeks. This was due to the inability of the vascular endothelium to repair large areas of EC injury in atherosclerosis. Smooth muscle cells of the media are capable of forming a thinner pseudoendothelium lining the luminal surface. Pseudoendothelial smooth muscle cells have an inhibitory influence on vascular endothelium regrowth, which has been confirmed by Madhumathi et al.  and Rajamannan , who also reported that lipoproteins once accumulated in the intima undergo oxidative modification at both lipid and protein moieties, escaping plasma antioxidants, and are then absorbed by monocytes and turn into foam cells in the subendothelial layer, triggering local inflammatory response responsible for signaling atherogenesis in the aorta and eventually atherosclerosis.
In H&E-stained aortic sections of the ginger-treated group, the tunica intima was intact with thin wavy corrugated endothelium, a thin subendothelial layer, and apparent wavy corrugated elastic fibers. There were evident decreases in the thickness of both tunica intima and tunica media in the ginger-treated group in comparison with the atherosclerotic group.
This is in agreement with Bhandari et al.  who reported that intake of antioxidants such as ginger has a beneficial effect on atherosclerosis, contributing to the ability to reduce the degree of evolution of the atherosclerotic lesion, reduction of serum and liver cholesterol levels, and increase in cholesterol and triacylglycerol fecal excretion, resulting in a greater beneficial effect on atherosclerosis.
Orcein-stained aortic sections of the control group showed prominent dark-brown, wavy internal elastic lamina. The tunica media appeared rich in elastic fibers. These fibers were parallel in distribution, whereas in atherosclerotic animal models, there was disruption of the internal elastic lamina. The tunica media possessed areas with complete loss of elastic fibers. Other areas showed irregularly condensed elastic fibers toward the tunica adventitia. There was an evident decrease in the area of elastic fibers versus the control. This was explained by Van Herck et al.  who revealed increased arterial stiffness promoted by plaque development and instability leading to fragmentation of the elastic laminae. In addition, Stary et al.  stated that ruptured plaques have an increased incidence of disruption of the internal elastic lamina, and fragmentation of the elastic fibers was associated with increased arterial stiffness and distension. This is concomitant with Abedinzadeh et al.  who found that arterial distensibility decreased more rapidly in the presence of atherosclerotic plaques.
In the ginger-treated group, there were prominent internal and external elastic laminae, the tunica media appeared rich in parallel elastic fibers, and small areas showed loss of elastic fibers. This is in agreement with Monk and George  who explained that the increase in collagen deposition, elastin degradation, production of advanced glycation end-products, and calcification result in stiffness of vascular walls and atherosclerosis. The atherosclerotic plaques comprise an accumulation of vascular smooth muscle cells and their secreted products (collagen and elastin), inflammatory cells (macrophages, T lymphocytes, dendritic cells, and mast cells), and both intracellular and extracellular lipid and debris . Consumption of ginger extract reduces development of aortic atherosclerotic plaques .
In the present study, the relative increase in elastic fibers in the ginger-treated group might be explained by Fuhrman et al.  who proved that the antiatherosclerotic effect of ginger is due to its antioxidant effect, which protects smooth muscle cells in the media against the oxidative damage in atherosclerosis. This antioxidative property could be attributed to its direct effect on macrophages and plasma low-density lipoprotein (LDL). Fuhrman et al.  added that ginger consumption results in accumulation of active ingredients within the cells and the plasma membrane, thus affecting cellular enzymes and plasma membrane receptors that reduce the cellular uptake of oxidized LDL.
ECs have many functions and play a central role in controlling coagulation, thrombolysis, vascular tone, permeability, inflammation, tissue repair, and angiogenesis . The molecular characteristics of EC vary along the vascular tree and in the same organ between different vessels ,. The immunohistochemical expression patterns of CD34 are commonly used as EC markers on normal endothelium .
In the present study, the CD34 immunohistochemical-stained aortic sections in the control group recorded a few positive, brownish cytoplasmic reactions confined to the EC, whereas in the atherosclerotic group there was an evident increase in the area of CD34 immune reaction in comparison with the control group. This can be explained by the fact that LDL oxidation in atherosclerosis enhances the expression of CD34, which influences adhesion and endothelial transmigration of monocytes . On the other hand, in the ginger-treated group, a few CD34 immunoreactivities were detected in the cytoplasm of ECs, and there was an apparent decrease in the area of CD34 immunoreactivity compared with the atherosclerotic group. This was in agreement with Cominacini et al.  who showed that ginger seemed to inhibit monocyte adhesion by increasing the resistance of LDL to oxidation.
| Conclusion|| |
From our study, we can conclude that ginger has an effective role in improving experimentally induced atherosclerosis in rabbits, and this can be noticed from the following effects:
- An apparent decrease in the thickness of both tunica intima and tunica media compared with the atherosclortic group.
- No invasion of smooth muscle fibers in the tunica intima compared with the atherosclerotic group.
- The regular wavy elastic fibers in the tunica media with apparent normal distribution of smooth muscle fibers in between.
- No apparent difference in the CD34 immunoreaction of the ECs compared with controls.
- Individuals who have risk factors for developing atherosclerosis may benefit from administration of ginger as prophylactic treatment.
- More studies are required for further understanding the mechanism of actions of treatment of atherosclerosis by ginger.
- Further studies are needed to investigate the therapeutic effect of ginger on other health problems.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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