Original Article Effects of hypercholesterolemic diet by long-term on elastic system fibers penile tissue: volumetric density analysis of elastic system fibers

Abstract

The aim of the study was to assess the volumetric density of Elastic System Fibers (ESF) in the Corpus cav-ernosum (CC) of the penis of hypercholesterolemic rabbits by long-term. Fourteen New Zealand white rabbits were used. Hypercholesterolemia was induced at 6 months of age in 07 rabbits by oral. Two doses of 10 ml of chicken egg yolk offered at 7 am and 11 am (hypercholesterolemic group) for 24 weeks. The remaining 07 rabbits served as a control group. After 24 weeks, the rabbits were killed using sodium thiopenthal. Midshaft penile fragments were obtained and processed by routine histological techniques. Stereological analysis of ESF was performed in 5-µm sections by using a M42 test grid system. Data were expressed as volumetric density (%) and Mann-Whitney U test was used and statistical significance was considered when P < 0.05. In the CC of hypercholesterolemic rabbits, the ESF were decreased by 38.6% (P < 0.0006). Penile tissue was affected by long-term of experimental hypercholes-terolemia, possibly these changes could have an impact on penile structure during erection, and therefore might adversely affect erection maintenance.

Full text

Int J Clin Exp Pathol 2016;9(1):379-386
www.ijcep.com /ISSN:1936-2625/IJCEP0017593
Original Article
Effects of hypercholesterolemic diet by long-term on
elastic system bers penile tissue: volumetric
density analysis of elastic system bers
Albino Fonseca Jr1, Celio F Rodrigues2, Josefa S Martins2, Marcelo Abidu-Figueiredo3, Gilberto P Cardoso1,
Marcio Antonio Babinski1
1Department of Morphology, Experimental Morphology and Morphometry Unit, Fluminense Federal University-
Niterói, Brazil; 2Department of Health Sciences of Federal University of Alagoas, Alagoas, Maceió, Brazil;
3Department of Animal Anatomy-Federal Rural University of Rio de Janeiro, Seropédica, Rio de Janeiro, Brazil
Received October 10, 2015; Accepted November 24, 2015; Epub January 1, 2016; Published January 15, 2016
Abstract: The aim of the study was to assess the volumetric density of Elastic System Fibers (ESF) in the Corpus cav-
ernosum (CC) of the penis of hypercholesterolemic rabbits by long-term. Fourteen New Zealand white rabbits were
used. Hypercholesterolemia was induced at 6 months of age in 07 rabbits by oral. Two doses of 10 ml of chicken
egg yolk offered at 7 am and 11 am (hypercholesterolemic group) for 24 weeks. The remaining 07 rabbits served as
a control group. After 24 weeks, the rabbits were killed using sodium thiopenthal. Midshaft penile fragments were
obtained and processed by routine histological techniques. Stereological analysis of ESF was performed in 5-µm
sections by using a M42 test grid system. Data were expressed as volumetric density (%) and Mann-Whitney U test
was used and statistical signicance was considered when P < 0.05. In the CC of hypercholesterolemic rabbits, the
ESF were decreased by 38.6% (P < 0.0006). Penile tissue was affected by long-term of experimental hypercholes-
terolemia, possibly these changes could have an impact on penile structure during erection, and therefore might
adversely affect erection maintenance.
Keywords: Rabbit penis, hypercholesterolemia, elastic bers, erectile dysfunction, stereology
Introduction
Atherosclerosis is a kind of disease with multi-
ple factors. Presently, inammation is now well
accepted to be the dominant factor in the
pathogenesis and development of atheroscle-
rotic diseases, where the hyperlipidemia is a
major classical risk factor resulting in the devel-
opment and progress of atherosclerosis [1].
Elevated plasma cholesterol concentrations
induced by cholesterol feeding result in the
development of atherosclerosis and impair-
ment in endothelium-dependent vasodilatation
in rabbits [2]. As well as, it has been demon-
strated that this inammatory disease induces
two signicant pathological processes: an isch-
emic event due to blood ow obstruction and
vascular contractile dysfunction [3].
Longitudinal population-based studies clearly
demonstrate that cardiovascular risk factors
such as dyslipidemia, central obesity, hyperten-
sion and insulin resistance are major risk fac-
tors for vasculogenic erectile dysfunction as
well [4]. Furthermore, recent data suggest that
the clustering of these factors, as occurs in
patients with metabolic syndrome, increases
the risk for the development of erectile dysfunc-
tion (ED) even further [5].
The erect penis has always been a symbol of
power, virility and fer tility. Erectile dysfunction
is the consistent inability to achieve and main-
tain an erection sufcient for satisfactory sexu-
al activity and may be the early clinical manifes-
tation of a generalized vascular disease and
carries an independent risk for cardiovascular
events [6]. ED is associated with the presence
and extent of asymptomatic atherosclerosis,
including that of the coronary arteries, and pre-
cedes the development of clinically evident
coronary artery disease (CAD) by a signicant
amount of time [5].

Effects of hypercholesterolemic diet by long-term on ESF penile tissue
380 Int J Clin Exp Pathol 2016;9(1):379-386
Additionally, experimental evidence from differ-
ent animal models applied to ED indicates that
the morphological alterations in the penis may
occur [7-11]. These changes could affect
smooth muscle cells and different components
of the extracellular matrix (ECM), including col-
lagen and ESF. These are important penile
components that maintain penile structure dur-
ing erection, allowing adequate resistance dur-
ing the return to the accid state [12, 13].
However, the way in which these various ele-
ments are affected is not yet well established,
and precise quantitative data regarding ESF of
the penis in by long-term hypercholesterolemia
is not yet well reported in the literature. Thus,
the present study aimed to evaluate the volu-
metric density of ESF in the Corpus caverno-
sum (CC) in the penis of long-term hypercholes-
terolemia rabbits.
Material and methods
The handling of the animals was approved by
the Animal Care and Use Committee of Federal
University of Alagoas, which based their analy-
sis on the Guide for the Care and Use of
Laboratory Animals and the study design was
approved (N.011034/2007-55) by the local
Ethics Committee for the care and use of labo-
ratory animals.
Animals
Fourteen New Zealand (Oryctolagus cunicullus)
male rabbits with a mean age of six months
and a mean weight of 3 kg were included in this
study. The animals were quarantined, exam-
ined and monitored by a veterinarian; housed
in individual cages-all The rabbits were kept in
a room with controlled temperature (25±1°C)
and with articial dark-light cycle (lights on from
07:00 hours to 19:00 hours).
Experimental model
These rabbits were divided into two groups
according to diet. Standard rabbit feed
(Purina®) (200 g/day) and water ad libitum was
given to the 7 rabbits in group 1 (control group)
for 24 weeks. In the group 2 (hypercholesterol-
emic group), the rabbits (n = 7) were fed the
same diet of group 1, supplemented by 20 ml/
day (4,32 g/day-cholesterol) of chicken egg
yolk (carnauba®, Brazil) offered in twice a day
for 24 weeks.
Blood sampling and analysis
Serum cholesterol/fractions were assayed on
capillary blood (central ear vein) samples
(Thermo Electron®-Brazil) from fasting animals
and monitored at 0, 33, 66 and 99 days of
dietary intervention. Rabbits were considered
hypercholesterolemics when the cholesterol
serum level was higher than > 240 mg/dL.
Tissue specimens and histochemical analysis
Both groups were fed with the same standard
pellets for rabbits and, 24 weeks after feeding
protocol (G2), the rabbits were killed by an over-
dose (30 mg/kg) of sodium thiopental after
subcutaneous 30 mg/kg xylazine combined
with 9 mg/kg ketamine hydrochloride
anesthesia.
The penises were dissected and a fragment of
the middle shaft was removed and immediately
xed in phosphate buffered formalin solution
(4% in 0.1 mol L-1; pH 7.4) for 24 hours.
Fragments of Corpus cavernosum (CC) were
obtained and submitted to isotropic and ran-
dom orthogonal triplet probe sections (“ortrip”
cleavage) for stereologic analysis [14]. This
method consisted of 3 random slice sections,
the second section being orthogonal to the rst
and the third section also being orthogonal to
the second. Thus, isotropic uniformly random
sections were obtained [15].
After the “ortrip” cleavage the samples were
processed for embedding in parafn and sec-
tions of 5-μm thickness were obtained. To dem-
onstrate the ESF the sections were stained
with Weigert’s resorsin fuchsin technique with
previous oxidation [16, 17]. All samples were
initially diagnosed by a pathologist (not a coau-
thor) to detect any foci of others pathologies
and to exclude samples with artifacts.
Main outcome measures
From each penis, ve different sections were
selected from ve fragments. From each sec-
tion, ve random elds were analyzed, totaling
25 elds (test areas) for each penis. The data
were expressed as volumetric density (Vv-%).
The analyzed elds were then digitized to a nal
magnication of ×400 using a video camera
coupled to a light microscope. The selected his-
tologic areas were then quantied by applying a

Effects of hypercholesterolemic diet by long-term on ESF penile tissue
381 Int J Clin Exp Pathol 2016;9(1):379-386
test-grid system (M42 test-grid) on the digitized
elds on the screen of a color monitor [15]. All
numerical results are presented as mean ±
standard deviation.
From stereologic principles in isotropic tissue,
the area distribution of a given structure, as
determined on a two-dimensional section of
the structure, is proportional to the volume dis-
tribution of the structure. The volume density of
the histologic components was calculated as
Vv = Pp/Pt, where Vv was the volume density, P
was the tissue component under consideration,
Pp was the number of test points associated
with P, and Pt was the number of points in the
test system. The stereologic methods have
been described in detail elsewhere [15].
Statistical analysis
The data are reported as mean ± SD and the
statistical signicance was determined using
the unpaired test (Mann-Whitney). The level of
signicance was set at P < 0.05. All statistical
Figure 1. Results of blood cholesterol levels from rabbits submitted to hypercholesterolemia (G2) by egg yolk for a
long-term compared with control group (G1).

Effects of hypercholesterolemic diet by long-term on ESF penile tissue
382 Int J Clin Exp Pathol 2016;9(1):379-386
analysis was done using GraphPad Prism 5 sta-
tistical software (GraphPad, San Diego, CA,
USA).
Results
Blood sampling
Blood cholesterol concentration 33 d after
experimental hypercholesterolemia had alrea-
dy approached a mean value of 838.39 mg/dL
(Figure 1). Subsequently, this value increased
steadily, with little variability among fractions,
whereas, in controls, the blood cholesterol con-
centration had a constant mean value of ≈62
mg/dL throughout the duration of the experi-
ment. When all animals were sacriced, 24
weeks after treatment, blood experimental
hypercholesterolemia was ≈919 mg/dL. The-
refore, rabbits treated with chicken egg yolk
remained in a hypercholesterolemic condition
for at least 20 weeks.
Figure 1 shows the body weight gain in the two
groups (no statistically signicant difference
noted during the experiment), also may be
observed all fractions cholesterol and triglycer-
ide concentrations were signicantly increased
within four weeks after the start of the experi-
ment and maintained until changed 99 d.
Morphological analysis
The rabbit has a vascular penis, which contains
two erectile structures: a supero-lateral Corpus
cavernosum (CC) and the ventral Corpus spon-
giosum (CS) surround the penile urethra. Both
structures were covered by a dense capsule of
connective tissue, the tunica albuginea (TA),
which projects intracavernosal pillars or septa,
mainly in the CC.
The histochemical analysis conrmed the pres-
ence of ESF in penile tissue of all specimens
observed with an irregular network abundantly
distributed throughout the CC (Figure 3). The
volumetric density (Vv%) of the elastic system
bers in the penile tissue of G1 and G2 was
16.83%±1.22% and 10.33%±1.6%, respective-
ly (differences statistically signicant, P ≤
0.0006; Figures 3, 4 and Table 1).
Discussion
Morphometric data
Area density has been used by many studies,
attempting to quantify the ESF of urogenital
Figure 2. Results of amount (Vv%) of ESF in the Cor-
pora cavernosa of the rabbit penis. G1 = Control
group; G2 = Hypercholesterolemic group.
Figure 3. -ESF (arrows) in the CC of the penis from 48
weeks old rabbits. Representative images of control
group (G1), stained with Weigert’s resorsin fuchsin
with previous oxidation and photographed at a nal
magnication of 200×.
Figure 4. -ESF (arrows) in the CC of the penis from 48
weeks old rabbits. Representative images of Hyper-
cholesterolemic group (G2), stained with Weigert’s
resorsin fuchsin with previous oxidation and photo-
graphed at a nal magnication of 200×.

Effects of hypercholesterolemic diet by long-term on ESF penile tissue
383 Int J Clin Exp Pathol 2016;9(1):379-386
structures by using computer-aided image
analysis software [12, 18]. These programs use
the color property of the elements (pixels) of an
image to determine a threshold level for inclu-
sion. However, this method is limited to quanti-
fying only thin and line-shaped structures [19].
Stereological methods have been used in quan-
tication studies specically to determine the
number or proportion of brous components of
the ESF [11, 17, 20-23]. The point counting
method has proven to be very efcient in our
study. It avoids the bias that frequently occurs
with computerized image analyses, which may
overestimate or underestimate the analyzed
structures [19]. The stereologic methods have
been described in detail elsewhere [15].
Experimental model
Studies with experimental models to develop
atherosclerosis, claim that rabbits subjected to
a hyperlipidemic diet with chicken egg yolk,
developed vascular atherosclerotic injuries
similar to those found in humans, considering,
thus, dietary cholesterol as a precursor to the
development of atherosclerosis [24, 25]. To our
knowledge, the concentration of cholesterol
observed in chicken eggs is 956 mg/100 g,
presenting a high content of fat [25]. Thus, the
chicken egg is considered important in signi-
cant increases in plasma cholesterol concen-
trations, with levels increased more than 1,000
mg/dl [26], and in the development of hyperlip-
idemia that, in turn, progress to atherosclerosis
[24]. Therefore, the results of the experiment of
this study (Figure 1) are reliable evidence indi-
cating the importance of the type of fat in rela-
tion to the risk of cardiovascular disease, as
well as erectile dysfunction; proving thus that a
diet high in cholesterol and saturated fat has
serious systemic metabolic consequences. The
The rabbit has a vascular penis as well as the
lack of a penile bone are features that make it
more similar to the human penis, and therefore
a suitable animal model for studying penile
structure and erectile dysfunction [10, 11].
These erectile structures in the rabbit penis are
also covered by a dense connective tissue, the
tunica albuginea (TA), which projects intracav-
ernosal pillars or septa, mainly in the CC [11 ,
17]. As a result of its anatomical characteris-
tics, the rabbit penis is one of the best models
for studies on the effects of hypercholesterol-
emia on erection [7, 9].
Hypercholesterolemia is one of the most impor-
tant risk factors in the development of vasculo-
genic ED. It has been shown that an increase in
total cholesterol in men increases the risk of
ED [28]. It has also been conrmed in animal
studies that hypercholesterolemia and athero-
sclerotic stenosis in major penile arteries cause
ED [29]. Azadzoi and Goldstein, [29] noted ED
in 16 (76%) of 21 rabbits with experimentally
induced hypercholesterolemia and atheroscle-
rosis. However, hypercholesterolemia is a treat-
able condition; therefore, its effects might be
reversible.
ESF and erectile dysfunction
Erection is normally associated with an intra-
cellular cascade of events that change smooth
muscle contractility in penile blood vessels and
vascular spaces, thereby modifying blood ow
and initiating ination of the CC [13]. However,
erection also depends on the collagen and ESF
of the underlying connective tissue framework,
which exerts passive resistance to the expan-
sion of erectile tissues, thereby creating penile
turgidity [13]. Further, ESF provide elastic recoil
when the penis returns to a accid condition
during detumescence [21].
Table 1. Shows the results (Mean ± SD) of the two
groups analyzed. The stereological quantication
in the corpora cavernosa showed that the Vv of
ESF was signicantly lower (38.63%) in hypercho-
lesterolemic rabbits (G2)
Results of ESF in Corpora cavernosa of the rabbit penis
Group 1 Group 2 n P value
ESF (Vv%) 16.83±1.22 10.33±1.60 07 (≤ 0.0006)*
KEY: ESF = elastic system bers. Data presented as the mean
percentage ± SD. *Statistically signicant, Mann-Whitney U
test.
levels of total cholesterol, LDL-c, VLDL-c,
and triglycerides rapidly increase in the
rabbit, especially in adult animals, thus devel-
oping the atherosclerosis process more
easily than young rabbits. When starting an
atherogenic diet rich in cholesterol and satu-
rated fat, there is a short-term increase in the
levels of lipoproteins [27]. This information is
consistent with our results, where the hyper-
lipidemic diet increased the levels of lipopro-
teins, and these levels reached over 1,100
mg /dL (Figure 1).

Effects of hypercholesterolemic diet by long-term on ESF penile tissue
384 Int J Clin Exp Pathol 2016;9(1):379-386
ESF themselves consist of a bundle of brillar
glycoproteins, such as brillins, which are
assembled extracellularly and are later embed-
ded with the amorphous elastomeric protein
elastin [30]. Thus, the ESF impart viscoelastic
properties to tissues and are typically found in
structures that, upon application of stretching
forces, undergo deformation and then return to
the original shape once these forces are
removed [30]. Loss or degradation of ESF can
cause signicant dysfunctions, such as in
degenerative and inammatory disorders [30].
The distribution and structural features of ESF
in penile tissues have been investigated in
humans [12, 18, 21, 22] as well as in laborato-
ry and food animals [11, 17, 20, 23].
We have previously studied the concentration
and distribution of ESF in different regions of
the rabbit penis, and the results indicated a
close relationship with the known functions of
penile tissues [17]. In other study was veried
whether ESF content in the CC, corpus spongio-
sum and tunica albuginea of the rabbit penis
undergoes modications with age [23].
The aim of our study was to determine the con-
tent of the ESF of the CC by histochemical and
quantitative analysis in rabbits fed with a high
cholesterol diet and to determine whether the
effect of hypercholesterolemia by long-term
was deleterious. In our 24-week study, we
observed that hypercholesterolemia affected
the volumetric density of ESF.
The long-term effects of hypercholesterolemia
and vascular disease due to atherosclerosis in
the CC are not well known [9]. Most of the previ-
ous studies have been short term, with results
suggesting that hypercholesterolemia has
reversible effects in cavernosal tissues [7]. In
our study, we evaluated the long-term effects
and observed a decrease in the ESF in the
hypercholesterolemic group (Figures 2, 4).
Kim et al. [31] found a decrease in the relax-
ation of cavernosal smooth muscle cells, endo-
thelial spoiling in cavernosal tissues, vacuoliza-
tion, and an increase in lipid vesicles in smooth
muscle cells by electron microscopy and noted
that relaxation returned to normal and the
other changes to near normal after stopping a
cholesterol diet. Nehra et al. [32] reported that
a 0.5% cholesterol diet for 16 weeks caused
signicant decreases in the content of smooth
muscle cells. This decrease in corporeal vascu-
lar smooth muscle cells was quantitatively and
qualitatively similar to the decrease in men with
ED [5]. The ndings of Nehra et al. [32] are simi -
lar to those of the Karaboga et al. [9], although
the duration of exposure was longer in the
study of [9]. Thus, changes observed in the
penis of hypercholesterolemic animals in the
corpora cavernosa suggest a possible associa-
tion with ED.
Our study attempted to demonstrate the effect
of hypercholesterolemia by long-term on the
penis of rabbit which has a vascular type simi-
lar to humans [3, 8, 9]. In the case of ED it is
known that hypercholesterolemia is primarily
associated with endothelial changes [3, 5].
Moreover, several factors have been implicated
with the onset of ED, including changes in
smooth muscle cells, collagen and ESF that
form with other elements of the morphological
substrate. However, these factors remain
scarcely understood. Our study therefore
attempted to characterize in a qualitative and
quantitative manner, the changes that occur in
these elements of the hypercholesterolemic
rabbit penis.
One of the characteristics of the ESF is its
adaptability in response to a changing environ-
ment and different stimuli [30]. The character-
ization and quantication of the ESF have been
shown to be an effective method for the evalu-
ation of morphological and functional changes
associated with pathological conditions in
humans [12, 18, 20-22] and several animal
models [11, 17, 23].
In the specic case of the penis, a change in
any one of its components can affect the
response of the erectile tissue [13]. The colla-
gen and ESF are the two main structures of the
erectile tissue of the penis to allow the increase
in circumference and length during tumes-
cence while providing adequate recovery to
return quickly to the accid state during detu-
mescence [18]. Thus, tissues that are con-
stantly under pressure to stretch are rich in ESF
[30] and loss of ESF architecture and function
is a pathological feature of a number of degen-
erative and inammatory diseases [30].
Despite the importance of ESF in the penis,
there are few studies that have accurately char-
acterized this component of ECM and its pos-
sible alteration in hypercholesterolemia by log-

Effects of hypercholesterolemic diet by long-term on ESF penile tissue
385 Int J Clin Exp Pathol 2016;9(1):379-386
term. In young adult rabbits the volumetric den-
sity of ESF in the CC is approximately 15% [23],
while values for humans are 9% [12, 21] and for
young adult rats are 5% [20]. These data sug-
gests that ESF play a particularly important
functional role in the rabbit penis. In our hyper-
cholesterolemic rabbits there was a signicant
decrease of approximately 38.6% in the CC.
Observations of the brous elements of the
extracellular matrix show that while these ele-
ments decrease in the CC which suggests a
specic behavior in the various regions
analyzed.
In conclusion, our results show that hypercho-
lesterolemia by long-term experimentally
induced by chicken egg yolk, causes profound
changes in the ESF of the rabbit penis suggest-
ing that a possible association with ED often
occurs in these patients. We suggest hypercho-
lesterolemia treatment to prevent ED.
Disclosure of conict of interest
None.
Address correspondence to: Marcio Antonio Ba-
binski, Department of Morphology, Experimental
Morphology and Morphometry Unit, Fluminense
Federal University-Niterói, Brazil. Fax: (55) (21)
2629-2336; E-mail: mababinski@gmail.com
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