Keywords parotid gland - sublingual - submandibular - diabetes - cytokeratin 17
Introduction
Salivary glands
Salivary glands are a group of both major and minor exocrine glands that drain saliva
into the oral cavity. The salivary glands of rats consist of three pairs of major
glands, parotid gland (PG), submandibular gland (SMG) and sublingual gland (SLG),
as well as a lot of minor salivary glands.[1 ] Minor salivary glands are distributed throughout most parts of the oral cavity,
and their secretions directly bathe the oral tissues.[2 ] Saliva that secreted by the acinar cells and modified by the duct cells, play an
important role in maintaining the healthy state of the oral tissues, namely the teeth,
gingiva and oral mucous membrane.[3 ] Saliva consists mainly of the secretions of SMG (65%), PG (23%), SLG (4%) and the
remaining 8% being provided by the minor numerous glands.[1 ] Salivary secretion is composed mainly of water, electrolytes, and biologically active
proteins, including growth factors and cytokines.[4 ] A series of salivary ducts was discovered in the seventeenth century by Nils Stensen
(1638–1686), Thomas Wharton (1614–1673), and Caspar Bartholin (1655–1738) and through
these ducts saliva pours into the oral cavity.[5 ] Saliva plays many diverse roles through its digestive, mastication, swallowing,
antibacterial, buffering, lubricant, and water balance functions.[6 ]
Diabetes Mellitus
Diabetes mellitus (DM) is a generalized metabolic disease characterized by hyperglycemia
results from abnormalities in carbohydrate metabolism.[7 ] The long-term prognosis of the diabetics is based on the consistency of residual
fasting plasma glucose levels above 126 mg/dL.[8 ] According to the World Health Organization, the Kingdom of Saudi Arabia ranked second
in terms of the incidence of DM in the Middle East countries, with seven million diabetics
among its citizens.[9 ]
[10 ] Due to the high incidence of DM in humans, the induction of diabetes in animal models
has been performed on a large scale to study its pathological effects on different
organ systems. The most common alterations of DM at the oral and dental level, include
periodontal disease, dental caries, looseness, tooth extraction, poor wound healing,[11 ] dry socket,[12 ] candidiasis, tongue disorders,[13 ] inability to eat, and taste disorders.[14 ]
[15 ] All of the above signs and symptoms are associated with dry mouth or hyposalivation,
as diabetes is the most common metabolic disease that damages the salivary glands
by altering their tissue structure and/or mechanism of salivary secretion.[16 ]
[17 ] Many authors state that the decreased salivary flow rate in diabetic patients is
caused by increased frequent urination, this causes the extracellular fluid to drop
notoriously, which the salivary glands need to produce saliva.[18 ]
[19 ]
Morphologically, the parotid glands of diabetic animals were decreased in size and
characterized by intracellular lipid accumulation in both acini and intralobular ducts.[20 ] Hand et al 1984 recorded the presence of small lipid droplets in the basal cytoplasm
of acinar cells as the first detectable change for induced diabetes on the first day
and peaked at 4.5 months, when acinar cells contained large lipid vacuoles.[21 ] Anderson et al 1994 reported that the diameter and number of granular ducts were
reduced in diabetic animals, but acinar cells was only affected 6 months after the
induction of diabetes.[22 ] Histochemical staining of the tissue suggested that the intracellular lipid within
the acini was mainly a triglyceride which may accumulate by decreased utilization
in the synthesis of secretory granules.[23 ] Also, Piras et al 2010 concluded that diabetes causes specific changes in secretory
protein expression in human salivary glands, which contribute to the altered oral
environment.[24 ]
Cytoskeleton
It is known that the cell cytoplasm contains a three-dimensional network of filaments
forming the cytoskeleton which consists of three main types, microtubules, microfilaments,
and intermediate filaments. Microtubules are approximately 25 nm in diameter, while
microfilaments are approximately 4 to 6 nm in diameter. The intermediate filaments
are approximately 6 to 10 nm in diameter, they are termed intermediate filaments because
their diameter is between microtubules and microfibrils.[25 ] Herrmann and Aebi described that there are six major classes of intermediate filaments
identified within the animal cells which are cytokeratin, vimentin, desmin, glial
fibrillary acidic protein, neurofilaments, and nuclear lamin.[26 ] Cytokeratin intermediate filaments are a family of related proteins encoded by various
genes and are found in many epithelial cells. The proposed functions of cytokeratin
include: (1) maintaining the cellular structure, (2) connecting cells together, (3)
facilitating the movement of cell organelles, (4) facilitating the transport of substances
within the cell, (5) playing a key role in maintaining the tensile strength and integrity
of the epithelial tissue, and (6) regulate cell proliferation and apoptosis.[27 ]
Immunohistochemistry
Immunohistochemistry is a technique for detecting an intracellular component (e.g.,
filaments) which act as antigen by injecting it to an animal which respond by the
production antibodies to this specific antigen forming antigen-antibody reaction.
After injection, the intracellular component (antigen) bears one or more antibody
binding sites, which are highly specific regions called epitopes. The animal mounts
humeral immune response to this specific antigen and produce antibody specific to
this epitope termed polyclonal antibody which can be isolated from the animal.[28 ] Monoclonal antibodies are produced in the laboratory by cell culture methods. Cytokeratin
constitute an important biomarker because they are stable, relatively resistant to
hydrolysis, formalin-fixed and paraffin-embedded. Also, cytokeratin shows great fidelity
in expression and is highly antigenic.[25 ] The distribution of cytokeratin 17 (CK17) in the normal PG parenchyma is associated
with cells of the duct system, whereas serous acinar cells have little or no cytokeratin
in their cytoplasm.[22 ]
Our study aimed to determine the expression of CK17 within the parenchymal elements
of major salivary glands of both normal and diabetic albino rats to provide more information
about the effects of DM on salivary glands structure that led to xerostomia.
Materials and Method
Grouping
This research was conducted on twenty adult male albino rats (Sprague Dawley strain)
with body weight ranging between 150–175 grams. All animals were housed in polycarbonate
cages under 8 to 16 dark-light cycles. A mixture of hard and soft foods was given
with unrestricted access to water. Rats were maintained in an animal health care facility
under the supervision of the local ethical committee in a laboratory animal colony,
Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt. Rats were divided
into two equal groups (control group one and diabetic group two).
Induction of Diabetes Mellitus
Rats of group two (fasted 12 hours before) were intraperitoneally injected with a
single dose of 150 mg/kg body weight of alloxan tetrahydrate (Sigma Chemical Company,
St. Louis, Missouri, United States) dissolved in physiological solution saline (0.9%
NaCl). Ten days later, blood glucose concentration was determined using enzymatic
colorimetric test on the bases of trend reaction. Animals presented a glucose level
at or above 200 mg/dl were included in the diabetic group. The diabetic rats maintained
neither diet nor drug and feed like the control animals. Control rats were injected
with sterile saline to mimic the prick injection with the diabetes group.
Tissue Preparation
On the 45th day after diabetes induction, rats of both groups were sacrificed by anesthesia
with diethyl ether. The salivary gland complex of each animal was cut into small portions
(4 × 4 × 4 mm) and fixed in Bouin's fixative for 3 days. Fixed tissues were washed
and then dried with ascending degrees of alcohol, cleaned in xylol, and infiltrated
with molten paraffin wax to build up a block. Serial tissue sections with a thickness
of 5 μm were mounted on a glass slide to be stained with hematoxylin and eosin for
routine histological examination.
Immunohistochemical Staining
The tissue sections for immunohistochemistry were mounted on special slide coated
with polyL-lysine recommended for staining procedures that necessitate handling with
a target retrieval solution. Paraffin sections (5 μm thick) were immersed in 0.3%
HO/methanol for 30 minutes to block endogenous peroxidase action and rinsed with phosphate-buffered
saline. Sections were incubated with anti-CK17 E3 monoclonal antibody on streptavidin
biotin method and hematoxylin counter stain. The positive staining reaction appeared
as brown staining which reflects the intracellular distribution of CK17 intermediate
filaments within the tissue compartments. Tissue sections were evaluated semiquantitatively
and grades as negative (0), weak (1), light (2), medium (3), and intense (4) staining.
Statistical Analysis
Data analysis was done using the package of SPSS, version 23 (IBM Inc., Chicago, Illinois,
United States). The quantitative data were calculated as mean, standard deviation,
and ranges when their distribution found parametric by test of normality. The comparison
between each two independent groups of the same gland (control vs. diabetic) was done
by using an independent t -test for the equality of means and Levene's test for equality of variances. Therefore,
the p -value was considered significant at a level ≤ 0.05.
Results
Histopathological Evaluation
The PG of control group revealed a parenchymal lobular tissue filled with closely
packed serous acini and duct system. These parenchymal elements are supported by connective
tissue stroma that divides the gland into lobes and lobules. The SMG of control group
consist of predominant serous acini, as well as lesser number of mucous acini, normal
branching duct system and granular convoluted tubules. The SLG of control group consist
of predominant mucous acini, many covered with serous demilune and few serous spherical
acini with normal branching ducts system.
During surgery, there is a great reduction in the size of salivary gland complex of
diabetic group in relation to the control group. The glandular elements of diabetic
group revealed atrophic changes characterized by decrease in the parenchymal elements
of all major glands accompanied by increase in the amount of fibrous stroma in both
PG and SMG ([Figs. 1 ], [2 ], [3 ]). The parenchymal elements consist of small serous acini with unspecified lumen.
Both SMG and SLG showed an increase in the mucous acini among the persisted serous
one. The duct systems showed an enlarged and dilated lumens with the presence of a
duct-like structure. Moreover, many acini have been replaced by adipose tissue ([Fig. 1 ]).
Fig. 1 Parotid gland (PG) of diabetic rats showed loss of gland architecture with degenerated
acini (I), dilated ducts (II), and dilated blood vessels (III) {H&E × 200}.
Fig. 2 Submandibular gland (SMG) of diabetic rats showing acinar atrophy (I), decrease in
the acinar size (II), fatty tissue (III) {H&E × 200}.
Fig. 3 Sublingual gland (SLG) of diabetic rats showing serous acinar atrophy (I), decrease
in the gland size (II), fibrous tissue (III) {H&E × 200}.
Immunohistochemical Evaluation
Examination of major salivary glands of the control group incubated with anti-cytokeratin
E3 antibody against CK17 using immunoperoxidase technique revealed that the duct cells
showed diffuse weak to mild expression of CK17 ([Figs. 4 ] and [5 ]). In some sections, both intercalated and striated ducts showed weak expression
at the luminal part of the cells with moderate expression at their basal part. Some
serous acini and serous demilune of mixed acini showed diffuse weak to mild expression
of CK17, whereas the mucous acinar cells showed negative expression.
Fig. 4 Submandibular gland (SMG) of control group showing mild expression of CK17 in duct
cells (I), weak in GCT (II), and negative in serous acini (III) (× 100).
Fig. 5 Sublingual gland (SLG) of control group showing mild expression of CK17 in duct cels
(I), negative in mucous acini (II) (× 100).
Examination of major salivary glands of the diabetic group revealed that both intercalated
and striated duct cells displayed mild to strong cytoplasmic expression of CK17, the
staining pattern was either strong at the apical part of cytoplasm with mild staining
at their basal part or diffused throughout the cell cytoplasm. The main excretory
ducts lined by stratified squamous epithelium demonstrated strong expression at the
luminal cell layer with mild to moderate expression at the remaining layers. Granular
convoluted tubular cells in SMG showed mild to moderate staining reaction of diffused
pattern. Many serous acini revealed a mild to moderate expression of CK17 of diffuse
type, whereas mucous acini were negatively stained in both SMG and SLG ([Figs. 6 ] and [7 ]).
Fig. 6 Submandibular gland (SMG) of diabetic group showing severe expression of CK17 in
duct system (I), weak in some serous acini (II), and negative in most serous acini
(III) (× 200).
Fig. 7 Sublingual gland (SLG) of diabetic group with severe expression of CK17 in duct cells
(I), and negative in mucous acini (II) (× 200).
The results of the statistical studies indicated that there were statistically significant
differences between the concerned groups ([Tables 1 ]
[2 ]
[3 ]
[4 ]). The most extreme of these differences were in duct cells PG (= 0.004) followed
by duct cells from diabetic SMG (= 0.008), while the least effects were on acinar
cells from diabetic SMG (= 0.036) ([Fig. 8 ]
[9 ]).
Table 1
Group statistics of both control and diabetic rats
Group statistics
Group
N
Mean
Standard deviation
Standard error mean
Group
N
Mean
Standard deviation
Standard error mean
CK17 in PG duct
Control
10
0.8750
0.46022
0.14554
CK17 In PG acini
Control
10
0.6000
0.37639
0.11902
Diabetic
10
1.5500
0.45338
0.14337
Diabetic
10
0.9750
0.29930
0.09465
CK17 in SMG duct
Control
10
0.8010
0.42218
0.13350
CK17 in SMG acini
Control
10
0.5250
0.36232
0.11457
Diabetic
10
1.3250
0.35453
0.11211
Diabetic
10
0.8500
0.26882
0.08501
CK17 in SLG duct
Control
10
0.7010
0.35057
0.11086
CK17 in SLG acini
Control
10
0.6000
0.35749
0.11305
Diabetic
10
1.1500
0.33751
0.10673
Diabetic
10
0.9740
0.32163
0.10171
Abbreviations: CK17, cytokeratin 17; PG, parotid gland; SLG, sublingual gland; SMG,
submandibular gland.
Table 2
Independent samples test of CK 17 in parotid glands
Levene's Test for Equality of Variances
t-test for Equality of Means
F
Sig.
t
Df
Sig. (2-tailed)
Mean Diff
Std. Error Diff
95% Confidence Interval of the Difference
Lower
Upper
CK17 in parotid duct
Equal variances assumed
0.274
0.607
3.304
18
0.004*
0.6750
0.20429
0.24579
1.10421
Equal variances not assumed
3.304
17.996
0.004*
0.6750
0.20429
0.24579
1.10421
CK17 in parotid acini
Equal variances assumed
0.004
0.949
−2.466
18
0.024*
−0.3750
0.15207
−0.69449
−0.05551
Equal variances not assumed
−2.466
17.131
0.025*
−0.3750
0.15207
−0.69565
−0.05435
Abbreviations: CK, cytokeratin; df, degrees of freedom. *Significant at a level 0.05.
Table 3
Independent samples test of CK 17 in submandibular glands
Levene's Test for Equality of Variances
t-test for Equality of Means
F
Sig.
t
Df
Sig. (2-tailed)
Mean Diff
Std. Error Diff
95% Confidence Interval of the Difference
Lower
Upper
CK17 in SMG duct
Equal variances assumed
0.117
0.737
−3.006
18
0.008*
−0.5240
0.17433
−0.89026
−0.15774
Equal variances not assumed
−3.006
17.478
0.008*
−0.5240
0.17433
−0.89105
−0.15695
CK17 in SMG acini
Equal variances assumed
0.889
0.358
−2.278
18
0.035*
−0.3250
0.14267
−0.62473
−0.02527
Equal variances not assumed
−2.278
16.605
0.036*
−0.3250
0.14267
−0.62655
−0.02345
Abbreviations: CK, cytokeratin; df, degrees of freedom; SMG, submandibular gland.
*Significant at a level 0.05.
Table 4
Independent samples test of CK17 in sublingual glands
Levene's Test for Equality of Variances
t-test for Equality of Means
F
Sig.
t
Df
Sig. (2-tailed)
Mean Diff
Std. Error Diff
95% Confidence Interval of the Difference
Lower
Upper
CK17 in SLG duct
Equal variances assumed
0.622
0.440
−2.918
18
0.009*
−0.4490
0.15389
−0.77230
−0.12570
Equal variances not assumed
−2.918
17.974
0.009*
−0.4490
0.15389
−0.77234
−0.12566
CK17 in SLG acini
Equal variances assumed
0.250
0.623
−2.459
18
0.024*
−0.3740
0.15207
−0.69348
−0.05452
Equal variances not assumed
−2.459
17.803
0.024*
−0.3740
0.15207
−0.69374
−0.05426
Abbreviations: CK, cytokeratin; df, degrees of freedom; SLG, sublingual gland. *Significant
at a level 0.05.
Fig. 8 Expression of cytokeratin 17 (CK17) in duct cells of all groups.
Fig. 9 Expression of cytokeratin 17 (CK17) in acinar cells of all groups.
Discussion
In general, damage to the major salivary glands is a known consequence of DM in both
human and experimental models. Caldeira et al 2005) noted that Morphological changes
in salivary gland are detected not only in uncontrolled diabetes but also in glycemic
control.[17 ] Our results were recorded once on the forty-fifth day after confirming the occurrence
of DM, and the results were evident in both acinar and ductal cells, in contrast to
what Anderson and others said in 1994 that the gland acini was not affected until
six months after induction of diabetes.[22 ] The results of the current study reported that DM caused structural changes ranging
from a reduction in the acinar volume to severe atrophy of the gland parenchyma which
was replaced by either fibrous or fatty tissue with proliferation of duct-like structures,
this findings explain the occurrence of dry mouth with failure to perform the secretory
activity. In the opposite direction to the atrophic changes of the parenchymal elements,
the fibrous stroma interacts through a proliferative activity, illustrating the differences
in tissue interaction of both epithelial and connective tissues. Anderson and Suleiman,
(1989) suggest that the replacement of parenchymal cells with fibrous connective tissue
is difficult to reconcile with the normal physiological responsiveness of the gland.[23 ] The fibrous tissue that replaced the degraded gland components in both PG and SMG
appeared very extensive suggesting permanent changes with the glands unable to regenerate
later. Contrary to our interpretation, Mata et al reported that persistent acini found
in glandular tissues have been suggested to be involved in the gland's ability to
regenerate.[16 ] In several samples of diabetic group, the presence of several normal and diminished
acini indicates that the gland is still performing its secretory capacity but to a
minimal degree. The results of our study are unable to make any attempt to distinguish
between duct-like structures and duct system. This result was supported by Takahashi
et al who reported that duct-like structures appear to be increased due to the proliferative
activity of duct system cells.[31 ]
All major salivary glands of the control group revealed CK17 expression with moderate
intensity within the duct cells, while the serous acini showed weak expression and
negative in the mucous acini as reported by Makino et al.[32 ] These observations may be due to highly differentiated acinar cells with a reduced
amount of filamentous structure. Several authors agree with this finding that CK17
in salivary gland cells plays an important role in cell structure and the intensity
of expression is closely related to the differentiation status of the parenchymal
cells.[10 ]
[28 ] The different patterns of CK17 distribution are thought to be related to the functional
activity of the gland where the diffuse pattern of staining indicates the nonsecretory
state, while the decrease of CK17 in the luminal portion was associated with the active
state of secretion leaving the area for exocytosis. The expression pattern of CK17
focused at the basal cell part may be associated with an increase in the tensile force
of acinar cells facing myoepithelium resulting in an increased pressure capacity to
drive saliva through the lumen into the duct system. The salivary glands of the diabetic
group revealed significant CK17 staining in both acinar and ductal cells with two
different appearances, lumen center or diffuse, which is opposite to the normal, dominant
distribution of the control group. It is thought that both patterns of CK17 distribution
may interfere with the secretory capacity of acinar cells resulting in xerostomia.
Also, the luminal pattern within the duct cells may disturb the modulation procedure
for primary secreted saliva. On the other hand, the diffuse pattern of CK17 indicates
a cellular deleterious effect throughout acinar or ductal cells leading to apoptosis.
