Atherosclerosis involves all the layers of the artery wall, but the events involving the intimal portion are fundamental to understand the evolution and gravity of lesions. This study shows that scanning microscopy is instrumental for better understanding the physiopathology of this disease

Congiu T

Dell'Orbo C

Protasoni M.

Quacci D

Raspanti M

Reguzzoni M

DELL'ORBO, CARLO

QUACCI, DANIELA ELENA

RASPANTI, MARIO

CONGIU, TERENZIO

REGUZZONI, MARCELLA

PROTASONI, MARINA

Archivio istituzionale della ricerca - Università di Cagliari

Atherosclerotic alterations in human carotid observed by scanning electron microscopy

Dell’Orbo, Carlo,

Quacci, Daniela

Raspanti, Mario

Congiu, Terenzio

Reguzzoni, Marcella

Protasoni, Marina

Firenze University Press: E-Journals

© 2010 Firenze University Press 
ht tp://www.fupress .com/ijae
ITALIAN JOURNAL OF ANATOMY AND EMBRYOLOGY
IJAE 
Vo l .  115,  n .  1/2 :  53 -58 ,  2010
Atherosclerotic alterations in human carotid observed 
by scanning electron microscopy
Carlo Dell’Orbo, Daniela Quacci, Mario Raspanti, Terenzio Congiu*, Marcella Reguzzoni, Marina 
Protasoni 
Laboratory of Human Morphology “L. Cattaneo”. Department of Human Morphology, University of Insubria, 
Varese, Italy. Corresponding author, Email: terenzio.congiu@uninsubria.it
Presented at a meeting in honour of Prof. G. Orlandini, Florence, February 15, 2010
Summary 
Atherosclerosis involves all the layers of the artery wall, but the events involving the intimal por­
tion are fundamental to understand the evolution and gravity of lesions. This study shows that 
scanning microscopy is instrumental for better understanding the physiopathology of this disease.
Key words 
Atherosclerosis, Endothelium, Scanning Electron Microscopy
Introduction
The development of atherosclerotic lesions involves several factors that during 
different timescales lead to deep modifi cations of the arterial wall structure. Thicken­
ing and stiffening of arterial wall lead to stenosis, while atherosclerotic lesions may 
calcify and ulcerate leading to thrombus formation (Chen et al, 2006).
Recent studies supported by clinical and experimental evidences demonstrate that 
intima dysfunction and disruption are initiating processes of atherosclerotic plaque. 
The endothelium is subject to mechanical and chemical stress by the variations of 
blood stream, pressure and composition and is especially exposed to infl ammatory 
processes. Intimal lesions may invest a wide area of the arterial wall and, at variance 
with the in vitro models, they can display a wide range of functional and structural 
alterations in the same anatomical location (Congiu et al, 2010)
Conventional section­based techniques are therefore far from optimal in study­
ing the local progression and the different aspects of the disease. Scanning electron 
microscopy (SEM) offers three major advantages: large areas of exploration, a wide 
range of magnifi cation and the possibility of unrestricted access to bulk specimens 
(Congiu et al, 2004; Schembri et al, 2008).
In the present study we demonstrate the morphological features of human carot­
id atherosclerotic lesions, an investigation topic where direct visualization is still an 
indispensable tool of knowledge.
Material and methods
A carotid specimen were obtained from a patient undergoing endarterectomy 
for stenosis. The vascular wall was distended and the atherosclerotic lesions were 
54 Carlo Dell’Orbo, Daniela Quacci, Mario Raspanti, et al.
exposed and  transversally sectioned in correspondence of the plaque. Tissue slices 
were fixed in 2% paraformaldehyde and 2% glutaraldehyde for an hour, washed in 
phosphate buffered saline (PBS, pH 7.2) and postfixed in a solution of 1% osmium 
tetroxide and 1.25% potassium ferrocyanide for 2 hours. They were then immersed in 
0.1% osmium tetroxide in PBS for 24 hours at room temperature and washed in PBS. 
The specimens were dehydrated in ascending grades of ethanol, critical point dried 
in CO2, coated with 10 nm of pure gold in a vacuum sputter coater Emitech K550 and 
studied in secondary electrons imaging with a Philips XL 30 SEM­FEG scanning elec­
tron microscope.
Results
The most evident alteration revealed by SEM is the increased thickness of the 
carotid wall in correspondence of the plaque formation and the local loss of elas­
tic sheets (Fig. 1). Wide areas of the intima are deprived of endothelial cells, which 
detach from the basal lamina leaving it exposed in many zones (Figs 2, 3). On this 
uncovered basal lamina initial fibrin deposition occurs, which entraps many erythro­
cytes (Fig. 2). Clusters of aggregated platelets are also present (Fig. 3). 
In other parts of the intima, endothelial cells lack their normal junctional complex­
es and display abnormal, intertwined digitations that overlap on multiple levels (Figs 
4,5). In the gaps that appear between cells, leucocytes are visible (Fig. 5). 
The subendothelial layer shows thick delaminated sheets (Fig. 2). Groups of foam 
cells, easily recognizable by their lipid droplets, are pushing their way through the 
collagen sheets (Fig. 6). The lipid vacuoles are clearly visible causing protrusions of 
the plasmalemma (Fig. 7). In some cut cells it is possible to observe the inner foam 
cell architecture, where lipid droplets are clearly visible (Fig. 8).
Discussion
Although atherosclerotic alterations are known to involve all the layers that com­
pose the arterial wall, their development is activated and started by alterations of 
endothelial cells. The functional role of endothelium is not limited to the simple lin­
ing of the inner surface of the artery, and all events taking place in proximity of the 
intima are fundamental to understand the evolution and the gravity of lesions.
It is therefore important that the technique used is able to offer a clear picture of 
the whole intimal surface and then to zoom on the details as they are found during 
the microscopic observation. The pictures shown demonstrate how the SEM approach 
can allow for a clear view of the different wall layers which is instrumental to a fur­
ther comprehension of the physiopathology of the atherosclerotic lesion. Loss and 
disorganization of endothelial cells, uncovering of basal lamina, fibrin and platelet 
deposition, leukocytes crossing the wall and forming foam cells causing the delami­
nation of subendothelium, are all well­known elements of this process (Bobryshev, 
2006), but are not observable together and simultaneously with any other technique.
An interesting additional advantage of this methodology is the unique, three­
dimensional imaging of the interplay of the different components of the atheroscle­
55SEM analysis of atherosclerosis in human carotid
rotic arterial wall: the foam cells with the surrounding collagen matrix, the epithelial 
cells with each other and with their basal lamina, and so on.
SEM imaging is, in our opinion, the better and most flexible approach to the study 
of atherosclerotic plaque and should be adopted as the gold standard technique. 
Aknowledgments
This work was performed with the technical instrumentations of the “Center of 
Major Instrumentation for Biomedical Research” of the University of Insubria, Varese, 
Italy.
References
Bobryshev YV. (2006) Monocyte recruitment and foam cell formation in atherosclero­
sis. Micron, 37: 208­222.
Chen C.H., Walterscheid J.P. (2006) Plaque angiogenesis versus compensatory arterio­
genesis in atherosclerosis. Circ. Res. 99: 787­789.
Congiu T., Radice R., Raspanti M., Reguzzoni M. (2004) The 3D structure of the 
human urinary bladder mucosa. A scanning electron microscopy study. J. Submi­
crosc. Cytol. Pathol. 36: 45­53.
Congiu T., Schembri L., Tozzi M., Guasti L., Maio R.C., Cosentino M., Marino F­ 
(2010). Scanning electron microscopy examination of endothelium morphology in 
human carotid plaques. Micron, E­pub Feb 11.
Schembri L., Congiu T., Tozzi M., Guasti L., Cosentino M., Marino F. (2008) Scanning 
Electron Microscopy examination and elemental analysis of atherosclerotic calcifi­
cations in human carotid plaque. Circulation 117:  479­480,
56 Carlo Dell’Orbo, Daniela Quacci, Mario Raspanti, et al.
Fig 1 – Slice of human carotid. The transversal section clearly shows how the wall thickness varies in the 
same artery. Note the loss of endothelial cells, the delamination of subendothelial layer and  the loss of elas-
tic sheets. 65X
Fig 2 – The endothelial cells have disappeared from the intimal surface. The basal lamina is partially covered 
with fibrin and some entrapped erythrocytes The subendothelial layer clearly shows how the elastic sheets 
delaminate. 200X
Fig 3 – The endothelial cells are leaving the basal lamina. There are wide uncovered areas of basal lamina 
with occasional platelets (arrow). 250X
Figures
57SEM analysis of atherosclerosis in human carotid
Fig 4 – Small groups of endothelial cells spread out in an extreme attempt to cover the basal lamina. Note 
the abnormal cell digitations and the lack of a normal junction complex between these extremely flattened 
cells. 800X
Fig 5 – Here the endothelial coating is more abundant. The cells, however, interdigitate and overlap to form 
multiple unorganized and intertwined layers and in some cases (arrow) the basal lamina is uncovered. Some 
leukocytes are shown entering the intima (arrowheads) are shown. 1200X
58 Carlo Dell’Orbo, Daniela Quacci, Mario Raspanti, et al.
Fig 6 – Groups of foam cells are pushing their way through the collagen sheets of subendothelium. 250X
Fig 7 – external details of foam cells. The lipid droplets are clearly distinguishable through the plasmalem-
ma. The subendothelial stroma is dissected. 1000X
Fig 8 – Two foam cells sectioned show the inner architecture. Nucleus (N) and lipid droplets (LD) are easily 
recognizable. 3500X


Archivio istituzionale della ricerca - Università dell'Insubria

© 2010 Firenze University Press ht tp://www.fupress .com/ijaeITALIAN JOURNAL OF ANATOMY AND EMBRYOLOGYIJAE Vo l .  115,  n .  1/2 :  53 -58 ,  2010Atherosclerotic alterations in human carotid observed by scanning electron microscopyCarlo Dell’Orbo, Daniela Quacci, Mario Raspanti, Terenzio Congiu*, Marcella Reguzzoni, Marina Protasoni Laboratory of Human Morphology “L. Cattaneo”. Department of Human Morphology, University of Insubria, Varese, Italy. Corresponding author, Email: terenzio.congiu@uninsubria.itPresented at a meeting in honour of Prof. G. Orlandini, Florence, February 15, 2010Summary Atherosclerosis involves all the layers of the artery wall, but the events involving the intimal por­tion are fundamental to understand the evolution and gravity of lesions. This study shows that scanning microscopy is instrumental for better understanding the physiopathology of this disease.Key words Atherosclerosis, Endothelium, Scanning Electron MicroscopyIntroductionThe development of atherosclerotic lesions involves several factors that during different timescales lead to deep modifi cations of the arterial wall structure. Thicken­ing and stiffening of arterial wall lead to stenosis, while atherosclerotic lesions may calcify and ulcerate leading to thrombus formation (Chen et al, 2006).Recent studies supported by clinical and experimental evidences demonstrate that intima dysfunction and disruption are initiating processes of atherosclerotic plaque. The endothelium is subject to mechanical and chemical stress by the variations of blood stream, pressure and composition and is especially exposed to infl ammatory processes. Intimal lesions may invest a wide area of the arterial wall and, at variance with the in vitro models, they can display a wide range of functional and structural alterations in the same anatomical location (Congiu et al, 2010)Conventional section­based techniques are therefore far from optimal in study­ing the local progression and the different aspects of the disease. Scanning electron microscopy (SEM) offers three major advantages: large areas of exploration, a wide range of magnifi cation and the possibility of unrestricted access to bulk specimens (Congiu et al, 2004; Schembri et al, 2008).In the present study we demonstrate the morphological features of human carot­id atherosclerotic lesions, an investigation topic where direct visualization is still an indispensable tool of knowledge.Material and methodsA carotid specimen were obtained from a patient undergoing endarterectomy for stenosis. The vascular wall was distended and the atherosclerotic lesions were 54 Carlo Dell’Orbo, Daniela Quacci, Mario Raspanti, et al.exposed and  transversally sectioned in correspondence of the plaque. Tissue slices were fixed in 2% paraformaldehyde and 2% glutaraldehyde for an hour, washed in phosphate buffered saline (PBS, pH 7.2) and postfixed in a solution of 1% osmium tetroxide and 1.25% potassium ferrocyanide for 2 hours. They were then immersed in 0.1% osmium tetroxide in PBS for 24 hours at room temperature and washed in PBS. The specimens were dehydrated in ascending grades of ethanol, critical point dried in CO2, coated with 10 nm of pure gold in a vacuum sputter coater Emitech K550 and studied in secondary electrons imaging with a Philips XL 30 SEM­FEG scanning elec­tron microscope.ResultsThe most evident alteration revealed by SEM is the increased thickness of the carotid wall in correspondence of the plaque formation and the local loss of elas­tic sheets (Fig. 1). Wide areas of the intima are deprived of endothelial cells, which detach from the basal lamina leaving it exposed in many zones (Figs 2, 3). On this uncovered basal lamina initial fibrin deposition occurs, which entraps many erythro­cytes (Fig. 2). Clusters of aggregated platelets are also present (Fig. 3). In other parts of the intima, endothelial cells lack their normal junctional complex­es and display abnormal, intertwined digitations that overlap on multiple levels (Figs 4,5). In the gaps that appear between cells, leucocytes are visible (Fig. 5). The subendothelial layer shows thick delaminated sheets (Fig. 2). Groups of foam cells, easily recognizable by their lipid droplets, are pushing their way through the collagen sheets (Fig. 6). The lipid vacuoles are clearly visible causing protrusions of the plasmalemma (Fig. 7). In some cut cells it is possible to observe the inner foam cell architecture, where lipid droplets are clearly visible (Fig. 8).DiscussionAlthough atherosclerotic alterations are known to involve all the layers that com­pose the arterial wall, their development is activated and started by alterations of endothelial cells. The functional role of endothelium is not limited to the simple lin­ing of the inner surface of the artery, and all events taking place in proximity of the intima are fundamental to understand the evolution and the gravity of lesions.It is therefore important that the technique used is able to offer a clear picture of the whole intimal surface and then to zoom on the details as they are found during the microscopic observation. The pictures shown demonstrate how the SEM approach can allow for a clear view of the different wall layers which is instrumental to a fur­ther comprehension of the physiopathology of the atherosclerotic lesion. Loss and disorganization of endothelial cells, uncovering of basal lamina, fibrin and platelet deposition, leukocytes crossing the wall and forming foam cells causing the delami­nation of subendothelium, are all well­known elements of this process (Bobryshev, 2006), but are not observable together and simultaneously with any other technique.An interesting additional advantage of this methodology is the unique, three­dimensional imaging of the interplay of the different components of the atheroscle­55SEM analysis of atherosclerosis in human carotidrotic arterial wall: the foam cells with the surrounding collagen matrix, the epithelial cells with each other and with their basal lamina, and so on.SEM imaging is, in our opinion, the better and most flexible approach to the study of atherosclerotic plaque and should be adopted as the gold standard technique. AknowledgmentsThis work was performed with the technical instrumentations of the “Center of Major Instrumentation for Biomedical Research” of the University of Insubria, Varese, Italy.ReferencesBobryshev YV. (2006) Monocyte recruitment and foam cell formation in atherosclero­sis. Micron, 37: 208­222.Chen C.H., Walterscheid J.P. (2006) Plaque angiogenesis versus compensatory arterio­genesis in atherosclerosis. Circ. Res. 99: 787­789.Congiu T., Radice R., Raspanti M., Reguzzoni M. (2004) The 3D structure of the human urinary bladder mucosa. A scanning electron microscopy study. J. Submi­crosc. Cytol. Pathol. 36: 45­53.Congiu T., Schembri L., Tozzi M., Guasti L., Maio R.C., Cosentino M., Marino F­ (2010). Scanning electron microscopy examination of endothelium morphology in human carotid plaques. Micron, E­pub Feb 11.Schembri L., Congiu T., Tozzi M., Guasti L., Cosentino M., Marino F. (2008) Scanning Electron Microscopy examination and elemental analysis of atherosclerotic calcifi­cations in human carotid plaque. Circulation 117:  479­480,56 Carlo Dell’Orbo, Daniela Quacci, Mario Raspanti, et al.Fig 1 – Slice of human carotid. The transversal section clearly shows how the wall thickness varies in the same artery. Note the loss of endothelial cells, the delamination of subendothelial layer and  the loss of elas-tic sheets. 65XFig 2 – The endothelial cells have disappeared from the intimal surface. The basal lamina is partially covered with fibrin and some entrapped erythrocytes The subendothelial layer clearly shows how the elastic sheets delaminate. 200XFig 3 – The endothelial cells are leaving the basal lamina. There are wide uncovered areas of basal lamina with occasional platelets (arrow). 250XFigures57SEM analysis of atherosclerosis in human carotidFig 4 – Small groups of endothelial cells spread out in an extreme attempt to cover the basal lamina. Note the abnormal cell digitations and the lack of a normal junction complex between these extremely flattened cells. 800XFig 5 – Here the endothelial coating is more abundant. The cells, however, interdigitate and overlap to form multiple unorganized and intertwined layers and in some cases (arrow) the basal lamina is uncovered. Some leukocytes are shown entering the intima (arrowheads) are shown. 1200X58 Carlo Dell’Orbo, Daniela Quacci, Mario Raspanti, et al.Fig 6 – Groups of foam cells are pushing their way through the collagen sheets of subendothelium. 250XFig 7 – external details of foam cells. The lipid droplets are clearly distinguishable through the plasmalem-ma. The subendothelial stroma is dissected. 1000XFig 8 – Two foam cells sectioned show the inner architecture. Nucleus (N) and lipid droplets (LD) are easily recognizable. 3500X

Atherosclerotic alterations in human carotid observed by scanning electron microscopy.

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Atherosclerotic alterations in human carotid observed by scanning electron microscopy.

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