Keywords
submandibular salivary gland - acinar cells - collared peccary - TEM - SEM
Introduction
Most salivary glands is located on the inside and around the oral cavity and are divided
into major and minor salivary glands. The minor salivary glands exist as small discrete
masses that, for the most part, occupy the submucosal region in the oral cavity. These
constitute the mucous glands, except for the Von Ebner serous glands, which can be
found below the foliate papillae and the vallate.[1] The major salivary glands are the parotid, submandibular and sublingual.[2]
The submandibular gland is located on the medial surface of the mandibular body. Its
function is to produce and secrete saliva to lubricate the food and assist in the
chewing, swallowing and digestion[3] The submandibular gland is covered by a dense non-modeled connective tissue capsule
that enters the gland through the septum, divided on the same lobules. In the connective
tissue septum, there are blood vessels and nerves in addition to the interlobular
excretory ducts.[4] The submandibular gland contains tubuloacinar endings called secretory acinus.
The submandibular gland of the collared peccary has some characteristics similar to
those of other mammals, but there are few reports on the morphology of its cell structures
and extracellular matrix elements. These characteristics were observed in different
species of mammals, such as the marmot,[5] the squirrel monkey,[6] the Macaca fuscata yakui,[7] as well as in human submandibular glands[8] and in the salivary glands of rabbits and of the Cebus apella.[9]
However, there are few studies on animals of the Artiodactyla order, since they have
different characteristics, which is why we have proposed to analyze the morphology
of the submandibular gland of the collared peccary (Tayassu tajacu). The collared peccary is an omnivore, non-ruminant mammal of the Tayassu family
of the Artiodactyla order. Its diet consists of vegetable matter, such as roots, fruits,
acorns, nuts and seeds, including some invertebrate animals.[10] In addition, it may be emphasized that studies of other wild mammals are important
in veterinary medicine in order to increase the morphological knowledge about the
collared peccary. In the present report, we show the results obtained in the histological
and ultrastructural characteristics of the submandibular gland of the collared peccary
(
T. tajacu), by the employment of the sodium hydroxide (NaOH) treatment, of scanning electron
microscopy (SEM) and of the transmission electron microscopy (TEM) techniques.
Material and Methods
The submandibular glands (n = 10) of adult male collared peccaries (
T.tajacu) were used for histological and ultrastructural analysis. The collared peccary belongs
to the Artiodactyla order, and the specimens were obtained from the centre of multiplication
of wild animals of the Universidade Rural do Semi-árido (UFERSA, in the Portuguese
acronym). The present research was authorized by the Ministry of the Environment (n°
3527–1) and approved by the Ethics Committee (n° 23091.005304/2015–81).
Polarized Light Microscopy
Two submandibular glands of collared peccaries (
T. tajacu) were fixed in 10% formalin solution, dehydrated in increasing series of alcohol
and embedded in paraffin blocs for 7 µm thick histological sections. The sections
were stained in a hematoxylin-eosin (HE) solution[11] to evidence the basophilic components, such as the nuclei and the cytoplasm of the
submandibular gland cells. Regarding the types of collagen fibers, the sections were
stained using the Picrosirius method, evidencing type I and type III collagen.[12] The samples were examined using an Axioskop 40 polarized light microscope (Carl
Zeiss Microimaging, Göttingen, Germany) at the Faculty of Veterinary Medicine of the
Universidade de São Paulo (USP, in the Portuguese acronym).
Scanning Electron Microscopy
Four submandibular glands of collared peccaries (T. tajacu) were immersed in a modified Karnovsky solution according to the fixation method
reported by Watanabe and Yamanada (1983).[13] Then, the tissues were rinsed in a phosphate buffer solution. Afterwards, the sagittal
sections were made by means of cryosectioning with liquid nitrogen. Sagittal sections
∼ 200 µm thick were made in a Vibratome 3000 sectioning system (Leica Biosystems,
Wetzlar, Germany) to analyze the surfaces. Afterwards, the samples were postfixed
in 1% aqueous osmium tetroxide solution, and the cytoplasm components of the acinar
and of the ductal cells were examined. A part of the samples was macerated in an aqueous
solution with 10% of sodium hydroxide (NaOH) at room temperature during 3 or 4 days
using maceration method reported.[14]
[15] This method of maceration of tissues removes the cells and allows the visualization
of the extracellular fibrillary matrix containing the bundles of collagen fibers in
situ. Afterwards, the samples were rinsed in distilled water at 4°C, which was changed
every 24 hours.[16] After clarifying, the samples were postfixed again and dehydrated in an increasing
series of ethanol, starting at 60 degrees until reaching 100 degrees and dried in
a Balzers CPD-030 critical point dryer (Oerlikon Corporate Switzerland, Pfäffikon,
Switzerland) mounted in a metallic basis and covered with gold ion, utilizing liquid
carbon dioxide (CO2), according to the technique described.[17] The samples were examined in a LEO 435 VP scanning electron microscope (LEO Electron
Microscopy Ltd., Cambridge, UK), at the department of surgery of the Faculty of Veterinary
Medicine of the USP, using 10 kV.
Transmission Electron Microscopy
For the TEM analysis, two submandibular salivary glands of collared peccaries (T. tajacu) were used. The samples were fixed in a modified Karnovsky solution.[13] Then, the tissues were rinsed in a sodium phosphate buffer at 0.1 M (pH = 7.4) and
postfixed in an aqueous solution with 1% of osmium tetroxide for 2 hours at 4° C.
Afterwards, the samples were dehydrated in an increasing series of ethanol followed
by propylene oxide, and then were embedded in Spurr resin. Both the thick and thin
sections were obtained using a Reichert Ultracut-E (C. Reichert AG, Vienna, Austria)
and were stained with 4% uranyl acetate and 0.4% lead citrate for 3 minutes.[18] The sections were examined under a Jeol 1010 transmission electron microscope (Jeol
Co., Tokyo, Japan) at the Institute of Biomedical Sciences of the USP.
Results
Polarized Light Microscopy
The polarized light microscopic images of the HE-stained submandibular glands of the
collared peccaries (T. tajacu)showed in blue-red color the connective tissue which envelops the acini and ductal
secretory units. The presence of numerous nuclei may indicate the presence of acid
substances, and the ductal secretions appeared with a round shape ([Fig. 1A]). At high magnification, the spherical nuclei located in the basal region of the
acinar cells may be observed ([Fig. 1B]). The sections stained by Picrosirius revealed in red color the amount of connective
tissue, and the groupings of acinar cells in yellow ([Fig. 1C]). The polarized light microscopy examination allows the observation of type I and
type III ([Fig. 1D]) collagen fibers around the duct and the septal regions.
Fig. 1 Light photomicrograph of the submandibular gland of the collared peccary. (A) Acini
(*) surrounded by connective tissue and secretion ducts (arrow). (B) At higher magnification,
the nuclei (arrowheads) located in the basal region of the cell can be observed. Color
hematoxilin-eosin (HE). (C) Shows the presence of collagen and acini groups (*), secretion
ducts (arrow) and glandular septa (arrowheads). (D) Polarized light allows noticing
the acini (*) around the ducts (arrow) and septum (arrowheads) revealing the presence
of type I and type III of collagen. Color Picrosirius. Bars: 100 µm (A, C, D), 50
µm (B).
Scanning Electron Microscopy
The samples prepared by the Vibratome sections and examined through SEM revealed that
the submandibular salivary gland of the collared peccaries (T.tajacu) showed the undulation on the glandular surface and its ducts ([Fig. 2A], [2B]). The images of the cryofracture method ([Fig. 2E]) also showed the secretory ducts, the groupings of acinar cells, as well as the
septum and its vessels ([Fig. 2A]). At high magnification, the bundles of collagen fibers and the round-shaped secretory
ducts may be observed ([Fig. 2C], [2D]). In the samples treated in NaOH solution, after removing the cells, the groupings
of the acinar and ductal spaces of different diameters may be observed ([Fig. 2E]). At high magnification, the original surface of the collagen fibers revealed numerous
spaces of acinar cells in tridimensional images, distributed in frameworks ([Fig. 2F]).
Fig. 2 Scanning electron photomicrograph of submandibular gland of the collared peccary.
(A) Gland cut view on vibratome allows us to observe the secretory ducts (arrow),
the acinar groups (*), interlobular septum (S) and a large-caliber vessel (**). (B)
At higher magnification, we can note the acinar groups (*) and the secretory ducts
(arrow) with secretory product inside. (C, D) Images show the bundles of collagen
fibers (arrow), the capsule, and the groups acinar (*) and secretory ducts (arrow).
At higher magnification, we can observe the collagen fibers (arrowhead) and the rounded
shape of the ducts (arrowhead). (E, F) Sample treated with NaOH solution; its surface
reveals collagen fibers whose original aspect shows the fibrous septa (arrowhead)
and secretory ducts (arrow). At higher magnification, the groups of acinar spaces
(*) revealed an aspect of “honeycomb.” Bars: 1 mm (A, E), 100 µm (B, D), 300 µm (C,
F).
Transmission Electron Microscopy
The TEM observations of the cytoplasmic components of the seromucous acini and of
the extracellular matrix elements were clearly demonstrated. Although, the intracellular
organelles of numerous serous granules are noted in electron dense images with heterogeneous
distribution and oval in shape ([Fig. 3B], [3C], [3D], [3E]). The cell nuclei with euchromatic aspect at the periphery evidence a granular region
of nucleolus ([Fig. 3D]). Around the cellular nuclei, at magnification, an extended area with flat cisterns
of endoplasmic granular reticulum was noted ([Fig. 3A], [3B], [3D]). At the periphery between the fine cell membranes of the serous acini, the nuclei
and an extended cytoplasmic projections of fibroblasts were revealed ([Fig. 3D]). At high magnification the mucous acini, delimited by a dense collagen fiber bundles,
was also identified ([Fig. 3F]).
Fig. 3 Transmission electron photomicrograph of submandibular gland of the collared peccary.
(A, B) View of the overall appearance of the cytoplasmic portion of the acinar cell.
We can observe the nuclei (n), the groups of serous secretion granules (*) and a large
area of rough endoplasmic reticulum (RER). (C, D) At higher magnification, we observe a cytoplasmic portion (arrow), the oval
shape containing many secretory granules of different diameters (*), the euchromatic
nuclei (n) and the extended projection of fibroblast cytoplasm (F). (E) Electron-dense
secretory granules (*) and laterally cytoplasmic portion of the myoepithelial cell
(ME) can be observed. (F) Note the mucous acini (M) delimited by dense fibrous septa
of connective tissue (arrow). Magnifications: 3,000X (A, B); 5,000X (C, D, E, F).
Discussion
The results of the present study demonstrated the cellular characteristics and details
of the fine structures of the serous and mucous acini of the submandibular salivary
gland of the collared peccary (T. tajacu). Since this gland has not been described until now, the histological data may illustrate
clearly the acinar and ductal formations of the submandibular salivary gland of the
collared peccary. Histologically, our data showed that the characteristics of the
acini and of the striated ductus of the collared peccary are similar to those reported
in several animals, such as reported by Cowley et al,[6] Espinal et al,[19] and Nagato et al.[20] On the other hand, the morphological characteristics of the ductal structures of
the submandibular gland of the collared peccary may be different when compared with
other mammals, such as the Koala (Phascolarctos cinereus).[3] Our data confirm that the nuclei were located in the basal region of the cells in
spherical form, similar to those found in other mammals, such as reported by Estecondo
et al[1] in the armadillo (Zaedyus pichiy) and by Fossati et al[21] in rats.
The vibratome sections allowed the observation of the details of the submandibular
gland in three dimensions, as well as the SEM images. The cryofractured surfaces in
liquid nitrogen may identify the several layers of components starting at the periphery,
as well as the capsule, the groupings of acinar and septal fibrous formations in deep
regions. The treatment in the NaOH solution allowed the removal of the capsular elements
of the collagen fibers and revealed the true surface of the acinar and ductal cells,
such as reported by Ohtani[14] and Watanabe et al.[15] On the surface of collagen fibers are distributed in frameworks, as has also been
observed in the submandibular gland of the species Procyon cancrivorus [22] and in rats by Watanabe et al[4] and by Caldeira et al.[23]
The observations of the surface sections of the acini by SEM revealed the surfaces
of round acinar structures similar to those reported by Watanabe et al[24]
[25] in mice. The SEM observations of the surfaces allowed the direct observation of
the intracellular components, especially of the true surface of the capsular structures.
The present findings are similar to those reported in monkeys by Boshell et al[26], and in man by Espinal et al.[19] Our results confirm that the striated ducts present a circular section containing
polygonal cells. These data are similar to those reported by Espinal et al[19], and by Watanabe et al.[24]
[25]
The three-dimensional architecture of the collagen fibrils revealed in the present
paper represents the skeletal structure of the interface acinar cell membrane connective
tissue of the salivary glands in their original localization. The present method,
on the other side, can clearly demonstrate individual collagen fibrils, regardless
of whether they form bundles or course solitarily. These collagen fibrils represent
the interstitial compartment in which the nerve fibers and capillaries pass between
the parenchymal constituents of the gland. These characteristics are reported by Jacob
et al[27], Watanabe et al[4]
[24], Santos et al[22], and Oliveira et al.[28] It is important to emphasize that the interstitial compartment serves as a passage
for the fluid that comes out of the capillaries.
In the data we have obtained, the TEM images identified that the submandibular salivary
glands showed several lobules containing terminal acinar formations, intercalated
and striated ducts, and excretory ducts. Although, the cytoplasmic components, presenting
some cellular organelles may be seen. In the peripheral region, the cell nuclei and
endoplasmic reticulum granular were observed. These characteristics are reported in
rats by Tamarin et al[29] and by Watanabe et al[30], in the spider monkey by Leeson,[31] and in the Mongolian gerbil by Ichikawa et al[32]. As observed in our results, the collagen fibrils network formed a well delimited
capsule in each terminal acinus.
Conclusion
It was possible to conclude that the submandibular salivary gland of the collared
peccary (T. tajacu) presented lobules separated by a connective tissue and numerous acinar cells, as
well as numerous groupings of ducts. The acinar cells are polygonal in shape, showing
the round nuclei located at the basal portion. In the TEM observations, the acinar
cells present the central lumen. The morphological characteristics of the submandibular
gland are similar to those of other mammals with same eating habits and habitat.
