https://doi.org/10.52973/rcfcv-e34303
Received: 14/08/2023 Accepted: 03/10/2023 Published: 01/01/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34303
ABSTRACT
This study aims to show the morphology of the structure involved
in the function of arteriovenous anastomoses (AVA) under electron
microscope. The study used 20 adult females of Rattus norvegicus.
Ovarian tissue samples were xed in 3% Glutaraldehyde in phosphate
buffer, and then post–xed in increasing concentrations of Ethanol,
tissues were embedded in Epon resin. Semi–thin tissue sections were
double stained with Uranyl acetate saturated in 70% Ethanol and lead
Citrate. The ultrathin sections were examined in a JEOL 100 C electron
microscope. In the opened AVA section, tunica intima, tunica media,
and adventitia layers were observed in the vessel wall structure. The
endothelial cell was present in the tunica intima, and the lumen was
open. Thick layered smooth muscle cells were found in the tunica
media. The muscles were arranged inner longitudinally and outer
circularly. The internal elastic membrane lies between the circular
and longitudinal smooth muscle. In another section taken from the
tunica adventitia, broblasts were observed between dense elastic
and collagen brils. The longitudinal smooth muscle was contracted
in the closed AVA section, and the lumen appeared in the typical
asterisks shape. This study showed the functional morphology of
the AVA's and detailed vessel wall structures in the rat ovary. Lumen
structure with open and closed AVAs is also shown. With observations
from this study, the functional properties of the formations in the
AVA wall structure are explained in the rat ovary.
Key words: Arteriovenous anastomoses; rat ovary; electron
microscopy; functional morphology
RESUMEN
Este estudio tiene como objetivo mostrar la morfología de las
estructuras que juegan un papel en la función de las anastomosis
arteriovenosas (AVA) bajo el microscopio electrónico. El estudio utilizó
20 hembras adultas de Rattus norvegicus. Las muestras de tejido
ovárico se jaron en glutaraldehído al 3 % en tampón de fosfato y luego
se jaron posteriormente en concentraciones crecientes de etanol,
los tejidos se incluyeron en resina Epon. Las secciones de tejido semi–
delgado se tiñeron dos veces con acetato de uranilo saturado en etanol
al 70 % y citrato de plomo. Las secciones ultranas se examinaron en
un microscopio electrónico JEOL 100 C. En las AVAs seccionadas, se
observaron capas de túnica íntima, túnica media y adventicia en la
estructura de la pared del vaso. La célula endotelial estaba presente
en la túnica íntima y la luz estaba abierta. Se encontraron células
musculares lisas en capas gruesas en la túnica media. Los músculos
estaban dispuestos internamente longitudinalmente y externamente
circularmente. La membrana elástica interna se encuentra entre el
músculo liso circular y longitudinal. En otro corte tomado de la túnica
adventicia se observaron broblastos entre densas brillas elásticas
y colágenas. El músculo liso longitudinal se contrajo en la sección
AVA cerrada y la luz apareció en la forma típica de asteriscos. Este
estudio mostró la morfología funcional de los AVA y las estructuras
detalladas de la pared vascular en el ovario de rata. También se
muestra la estructura del lumen con AVA abiertos y cerrados. Con
las observaciones de este estudio, las propiedades funcionales de
las formaciones en la estructura de la pared de AVA se explican en
el ovario de rata.
Palabras clave: Anastomosis arteriovenosas; ovario de rata;
microscopía electrónica; morfología funcional
Functional morphology of arteriovenous anastomoses on rat ovary
Morfología funcional de las anastomosis arteriovenosas en ovario de rata
Kaan Çimen *, Mehmet Çimen
Sivas Cumhuriyet University, Faculty of Medicine, Department of Anatomy. Sivas, Türkiye.
*Corresponding Author: cimen.kaan@gmail.com
FIGURE 1. Shows an AVA with an open lumen (L). Tunica intima (I), tunica
media (M), and tunica adventitia (A) layers can be observed in the vessel wall.
The endothelial cell (EC) presents in the tunica intima. Thick layered smooth
muscle cells can be observed in the tunica media, which are arranged inner
longitudinally (LM) and outer circularly (CM). The internal elastic membrane
(EL) lies between the CM and LM
Arteriovenous anastomoses on rat ovary / Çimen and Çimen _______________________________________________________________________
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INTRODUCTION
Arteriovenous anastomoses (AVAs) are vessels that directly connect
arterioles and venules. Thus, blood passes from arterioles to venules
without passing through capillaries. In this way, AVAs provide a high
level of blood flow in the area without overloading the capillary
network. Because AVAs have wider lumens than capillaries [1, 2].
AVAs have been tested in human and many animal organs [3, 4, 5, 6,
7]. AVAs have been observed to have thermoregulatory functions in
human skin [8, 9]. Since AVAs are found in the skin, their importance
in ngertip transplantations is considered in plastic surgery and used
for the blood supply of the transplanted tissue [10].
Lipa et al. stated that AVAs are seen in mono chorionic human twins
with a rate of 75.4%. They also stated that, in some instances, AVAs
may cause specic complications; however, in general, they regulate
inter–twin blood exchange and may compensate for unequal placental
territory [11]. Grinsell et al. stated that AVAs can be congenital or
acquired, asymptomatic or symptomatic, and microvascular or
macrovascular in the human body [12].
In animal morphology, Krmpotic et al. [13] stated that AVAs were
found in the skin of weddell (Leptonychotes weddellii), leopard (Hydrurga
leptonyx), and southern elephant seals (Mirounga leonina) species
and that AVAs had thermoregulatory functions. Additionally, other
tissues where the presence of AVAs has been demonstrated are in
rabbit (New Zealand White) peripheral pulmonary circulation [14], in
late‐pregnant ewes’ uterus [15], in Holstein cows’ corpus luteum [16].
Mokhtar [17] demonstrated the presence of AVA in the ovary of mature
Redbelly tilapia (Coptodon zillii) using a light microscope, and Mokhtar
& Abd‐Elhafez [18] demonstrated the presence of AVA in the peripheral
circulation of the ovary in one‐humped camel (Camelus dromedarius).
Yet, the effects and roles of AVAs on tissue have not been fully
elucidated. Their morphology must be known to determine these
functions because there is a relationship between the structure and
functions of AVAs [19]. Lima et al. [19] stated that it is necessary to
fully understand the vessel wall structure to understand the function
of AVA. However, it is dicult to analyze the AVA wall structure under
transmission electron microscopy thoroughly [19]. And also it is known,
rat ovaries have not been subjected to AVA's morphological functions.
This study aims to show the morphology of the structures that
play a role in the function of AVAs under the transmission electron
microscope from unpublished depository data.
MATERIALS AND METHODS
The study used 20 adult females of rats (Rattus norvegicus),
obtained from the Experimental Animals Laboratory, Faculty of
Medicine, Sivas Cumhuriyet University, Türkiye. Ethical approval is
also obtained from the institution.
At least two samples were taken from a total of forty rats ovaries
from each ovary without distinguishing a specic region. The samples
were cut into small pieces of 1 mm
3
in phosphate buffer. By applying
double xation to the pieces, the rst xation of the tissues was made
in Glutaraldehyde prepared with Milloning phosphate buffer (pH: 7.4)
for 1 h. The second xation was made with isotonic osmium tetroxide
(OsO
4
1%, pH: 7.3). The xated tissues were dehydrated by passing
through the ethyl alcohol series. Dehydrated tissues were embedded
in Epoxy resin (Araldite CY–212). The blocks were polymerized in an
oven at 60°C for 48 h. Ultra–thin (1 µm) sections were taken from
the prepared blocks with the LKB–5 ultra–tome (LKB Co., Biel,
Switzerland). The sections were stained with toluidine blue to identify
suitable areas. Thin sections of 300–500 angstroms were taken from
selected areas. Double staining with Uranyl acetate and lead Citrate
was applied to the thin sections. Sections ready to be examined
by contrast staining were evaluated under the JEOL–100 C (JEOL,
USA Inc., Maryland, USA) electron microscope and photographed at
different magnications.
RESULTS AND DISCUSSION
In this study, simple arteriovenous anastomoses are observed in
all eighty specimens. There is no specic way to sectional follow
AVAs. The AVA's can be separable from other micro vessels by their
unique wall proportion and structures. The obtained observations
about AVAs in rat ovary and the morphology of vessels with different
contraction positions are as follow:
In a section in which the AVA was open, tunica intima, tunica media,
and tunica adventitia layers were observed in the vessel wall structure.
The endothelial cell was present in the tunica intima, and the lumen
was open. Thick layered smooth muscle cells were found in the tunica
media. The muscles were arranged inner longitudinally and outer
circularly—the internal elastic membrane lying between the circular
and longitudinal smooth muscle (FIG.1).
In another section of the tunica adventitia, broblasts were observed
with dense elastic and collagen brils (FIG. 2). The longitudinal smooth
muscle was contracted in the section with the AVA closed, and the
lumen appeared in the typical asterisks shape (FIG. 3).
In the literature, various researchers have worked to explain the
functions of AVAs. These studies focused on the regulatory properties
of AVAs on thermoregulation and blood ow.
FIGURE 2. Shows the tunica adventitia of the AVAs. Fibroblasts (F) can be
observed with dense elastic (EF) and collagen brils (CF). The circular muscle
cells (CM) can be observed most inner layer.
FIGURE 3. Shows an AVA with a closed lumen. The longitudinal smooth muscles
(LM) contracted, and the lumen (L) appeared in the typical asterisks shape
______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34303
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Sherman explains the opened and closed AVA functions as follows.
When the AVAs are opened, blood is drawn from the capillaries and
passes directly into the veins. The ow accelerates, and the pressure
in the veins increases—the temperature decreases in the area where
the capillaries that receive no or little blood are distributed. When
the AVAs close, blood ows back into the capillaries, the temperature
rises, the blood ow in the veins slows, and the pressure drops [2].
Conversely, Midgard explains the role of temperature–regulating AVA
on the eggs in the departure and arrival of birds (Larus argentatus)
from the nest as follows. If AVAs are on in brooding birds, their body
temperature rises, transferring this heat to the eggs. This process
is critical when the bird returns to the cooled eggs after feeding
wanders [20]. Conversely, if heat loss from the brood needs to be
reduced, as the bird moves away from the nest, the AVAs are likely
closed, reducing blood ow to the brood [20].
Lima et al. [19] investigated the presence and function of AVA in
rabbit (Oryctolagus cuniculus) ears. Moreover, they describe AVA's
functions and morphology as follows. If the rabbit's ear temperature
rises above 40°C , the muscle in the AVA wall relax, blood flow
increases, and cooling is observed. When the local temperature drops
below 15°C, the muscles in the AVA wall relax again, increasing blood
ow and helping to raise the local temperature [19]. The contraction
and expansion of AVAs in the rabbit ear can be explained as follows.
Outer circular and inner longitudinal smooth muscle cells contract
simultaneously, causing lumen closure. When the muscle relaxes, the
lumen expands. Connective tissue elements can also act as elastic
support to aid the expansion and expansion of circular and longitudinal
smooth muscle [19].
The internal elastic membrane separates the sub–endothelial
structures from the muscular layer of the tunica media and tunica
adventitia. The internal elastic membrane sometimes preserved its
structure, sometimes regressed, and sometimes could not be seen
ultimately. In this study, the internal elastic membrane and the layers
in the wall structure of AVA were clearly shown in the rat ovary (FIG. 1).
It has also been observed that the circular smooth muscle cells in
the tunica media rotate and end by regressing. Since the lumens of
AVAs are broader than those of capillaries, they provide a high level
of blood ow in the area without overloading the capillary network.
The AVA lumen may appear misshapen, slit, or asterisk. This study
showed smooth muscles in the vessel wall and AVA lumen as asterisks
in the rat ovary (FIGS. 1, 3).
Similar observations have been noted in studies of different animal
species on ovarian tissue. Mokhtar (mature Redbelly tilapia, Coptodon
zillii) [17] and Mokhtar & Abd‐Elhafez (one‐humped camel, Camelus
dromedarius) [18] indicated the following observations in their studies:
“The simple AVAs were evident in the ovary. The AVAs were surrounded
by one tunica adventitia, and a ring or roll of smooth muscle cells
supported the origin of the anastomosis.” These observations in the
literature are compatible with our observations in this study (FIG 1).
The covering cells consist of 4–6 layers of broblasts in the tunica
adventitia. They perform three functions: brogenesis, phagocytosis,
and barrier [19]. In the current study also shows the presence of
broblasts in the tunica adventitia in the rat ovary (FIG. 2).
There are some differences between the previous studies
conducted by the senior investigator in this study on AVA development
[21], types [22], and vascular wall structure [23] in rats—the main
difference with the previous studies is that these studies were
conducted with light microscopy. Regarding AVA types, only simple
AVA was observed in ovarian tissue in this study. It was reported
that simple and glomus anastomoses were observed together in rat
embryos. Circular smooth muscle cells in the vascular wall structure
are similar. In addition, in previous studies, the typical asterisk open
position of the AVA lumen could not be observed.
In female mammals, the ovaries play a role in both the genital and
endocrine systems. The temperature differences during ovulation
and bidirectional hormonal transport can cause changes in blood ow
rates in the ovary. Open and closed AVA types can prove the ovaries'
thermoregulation needs. However, this study is not a physiological
function study. This functional morphology study aims to reveal the
states of the AVA wall structures during contraction–relaxation and
their positions with each other.
Arteriovenous anastomoses on rat ovary / Çimen and Çimen _______________________________________________________________________
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CONCLUSIONS
The main conclusions of this study are as follows:
1.
All simple type AVAs were observed in the rat ovary (Rattus
norvegicus).
2.
The layers in the vascular wall structure and the structural
elements in these layers were tried to be shown using
transmssion electron microscopy.
3.
It has been observed that the vascular wall structure consists
of three layers. Circular and longitudinal muscle cells,
connective tissue elements, and broblasts were also evident
in the vessel wall structures.
4. AVA with its lumen open in the typical asterisk shape are this
study's main observations and conclusions.
Funding
No funding was received for conducting this study.
Conict of interest
The authors declare that there is no conict of interest
Ethics approval
This study was carried out under the supervision of the Experimental
Animals Laboratory of our institution and the ethical principles for
using experimental animals in pre–clinical studies. However, there
is no protocol number provided as there is no experimental animal
ethics committee in our institution at the time of the study.
Authors’ contributions
MÇ: Conceptualization, data curation, investigation, supervision,
writing – review & editing KÇ: Writing – original draft, review, and
editing. All authors approved nal version of manuscript.
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