Keywords parotid gland - duct ligation - diabetes - cytokeratin 17
Introduction
Salivary glands are exocrine glands that drain saliva into the oral cavity and play
an important role in maintaining oral health. Saliva consists mainly of secretions
from the submandibular (65%), parotid (23%), and sublingual (4%) glands, and the remaining
8% are supplied by the numerous minor glands.[1 ] Saliva performs many functions, it moisturizes the oral mucosa, facilitates speech,
produces antimicrobial substances, maintains the dental integrity, and helps partly
in the digestion of food.[2 ]
[3 ]
[4 ] The four major salivary glands in dogs include the parotid, mandibular, sublingual,
and zygomatic glands, while minor glands are spread throughout the oral cavity.[5 ] The parotid glands of a dog are a pair of main encapsulated salivary glands of the
mixed type consisting of a predominant spherical serous acini with a few scattered
mucous acini. Salivary gland dysfunction is a major problem with serious adverse effects
on oral health including swallowing difficulties, gum disease, and inability to eat
with taste disturbances.[6 ]
[7 ]
[8 ]
[9 ] Also, decreased saliva secretion can lead to complications in the oral cavity by
allowing excessive accumulation of bacteria leading to oral infections, overgrowth
of candida that occurs particularly at the commissures of the lips, thirst (especially
at night), halitosis, and rampant tooth decay.[10 ]
[11 ]
[12 ]
Diabetes mellitus is a chronic, generalized disease characterized by high blood sugar
with impaired carbohydrate metabolism.[13 ] Diabetes is a widespread disease associated with higher morbidity and health care
costs with increased mortality.[14 ] According to the World Health Organization, Saudi Arabia ranks second in terms of
the incidence of diabetes in the Middle East countries, with the number of diabetics
approaching seven million.[15 ]
[16 ]
[17 ] Diabetes mellitus is a metabolic disease characterized by the deterioration of hepatocytes
in type I or impaired insulin function in type II.[18 ] Due to the high incidence of diabetes mellitus in humans, induction of diabetes
in animal models has occurred on a large scale to study this disease. A single injection
of alloxan or streptozotocin to an animal leads to an elevated glucose level and low
plasma insulin level causing insulin-dependent diabetes syndrome.[19 ] Diabetes mellitus is the most common disease that damages the salivary glands by
altering their tissue structure and mechanism of saliva secretion resulting in dry
mouth.[20 ]
[21 ] In general, diabetic animals showed various salivary gland disorders including the
increase in matrix metalloproteinase-8 levels, decreased acinar volume, growth retardation,
and weight loss in both the parotid and sublingual glands.[22 ]
[23 ] Also, it has been suggested that hyperglycemia is associated with decreased salivation
and elevated salivary glucose, especially in cases of severe insulin deficiency.[24 ]
Ligation to the parotid gland duct has contributed to the understanding of the pathology
of duct obstruction which ultimately leads to progressive atrophy of the affected
gland.[25 ]
[26 ] The causes of duct obstruction are many, which may be due to salivary stones and
diseases such as chronic obstructive parotitis, parotid tumors, and exposure to radiotherapy.[27 ]
[28 ]
[29 ]
[30 ] On the contrary, salivary gland atrophy can occur by removing the sympathetic or
parasympathetic nerve without duct obstruction.[31 ] Duct-ligated gland showed inflammatory cell infiltration at the onset of tissue
injury (1–16 hours).[8 ]
[32 ] At a quantitative scale, the clogged gland undergoes rapid and progressive acute
atrophy up to an absolute loss of more than 85% of lethal tissue by 2 weeks.[29 ] Gland atrophy was manifested by significant changes including emptying of acinar
cells, dissociation, and reduction in the number and size of secretory granules. The
remaining intralobular epithelium consists of atrophic acini and numerous duct-like
structures with an enlarged lumen.[33 ]
[34 ]
[35 ]
Immunohistochemistry is a technique for detecting an intracellular constituent (antigen)
by antigen–antibody reaction. An antigen (immunogen) bear one or more antibody-binding
sites, which are highly specific regions termed as epitopes.[36 ] IgG is the antibody used for immunohistochemistry produced by immunizing an animal
with purified specific antigen (immunogen). The animal produces a humeral immune response
to this immune factor and produces a specific antibody called a monoclonal antibody
that can be isolated from the animal for use in intracellular expression of this specific
antigen.[37 ] Cytokeratin intermediate filaments are a family of related proteins encoded by different
genes and expressed in different epithelial cells.[36 ] Cytokeratin constitutes an important biomarker because it is stable, relatively
resistant to hydrolysis, formalin-fixed, and paraffin-embedded. Also, cytokeratin
intermediate filaments show great fidelity in expression and are highly antigenic.[38 ] Distribution of cytokeratin 17 of parotid gland correlates with intercalated, striated,
and excretory duct cells, while acinar cells have weak or no cytokeratin expression
in their cytoplasm.[39 ]
Our study aimed to determine the distribution of cytokeratin within the parenchymal
elements of the parotid gland of diabetic animals compared with the atrophic gland
due to duct ligation to study whether the effect of diabetes mellitus leads to explicit
atrophy of the gland.
Materials and Method
Animals and Grouping
Twelve male mongrel dogs (9–11 kg weight and 2–2.6 years old) were included in the
experiment. The dogs were fed boiled horse meat, bread, milk, and water, and they
were also kept under observation for 7 days before the start of the experiment to
ensure that they were not infected with rabies. The dogs were divided into three groups
of four animals in each, the first group being the control group, the second group
being dogs with alloxan-induced diabetes, and the third group being a group of parotids
with ligated ducts.
Induction of Diabetes
Dogs of the second group were fasted for 12 hours and then injected intravenously
by fresh preparation with a single dose of 100 mg/kg body weight of alloxan monohydrate
(Sigma Chemical Company) dissolved in physiological saline (0.9% NaCl). Six hours
after alloxan injection, the blood glucose level was measured every 4 hours until
the hypoglycemic state was resolved. If blood glucose levels were too low, glucose
solution (5–10%) was injected intravenously.[40 ] Ten days later, blood glucose concentration was determined using an enzymatic colorimetric
assay based on the trend reaction. Animals that presented a glucose level at or above
200 mg/dL were included in the diabetic group in the experiment. On the contrary,
the glucose level was checked at different intervals every 15 days (three times for
45 days) to ensure that diabetes persists.
Parotid Duct Ligation
One day before the operation, dogs of the third group were given 5 g neomycin and
5 g streptomycin orally in three intermittent doses. The dogs were washed, dried,
and the hairs of the cheeks and neck were shaved. Dogs were pre-anesthetized by intramuscular
injection of 0.04 mg/kg atropine sulfate 10 minutes prior to anesthesia. The animals
were anesthetized by the intravenous injection of 2.5% thiopental sodium at a dose
of 20 to 30 mg/kg body weight. This was followed by an intramuscular injection of
0.3 mg/kg of Flexiedil to relax the laryngeal muscle and facilitate intubation. Two
centimeters incision was made on the line connecting the corner of the mouth to the
posterior margin of the meniscus above the masseter muscle on the right side. The
flap was removed to expose the masseter muscle and the easily visible parotid duct
was clamped and tied by non-resorbed silk. The fascia over the ligated duct was closed
by catgut suture and the overlying skin by the interrupted suture. Post-operatively,
animals were intramuscularly injected with 5 mg/kg ampicillin and 3 mg/kg gentamycin
twice a day for 2 days.
Tissue Preparation
Dogs were sacrificed 45 days after the operation, and the parotid gland was extracted
and fixed in Bowen's fixative for 3 days. Fixed tissues were washed and then dried
with ascending degrees of alcohol and infiltrated with molten paraffin wax to build
up a block. Serial tissue sections 5 μm thick were mounted on a glass slide to be
stained by hematoxylin and eosin for routine histological examination.
Cytokeratin Immunostaining
Paraffin sections of 5 μm thickness mounted on a slide coated with poly L-lysine were
immersed in 0.3% water/methanol for 30 minutes to block endogenous peroxidase activity.
Then, the section was rinsed with phosphate-buffered saline and incubated with an
anti-cytokeratin 17 E3 monoclonal antibody (Sigma Chemical Company) using the streptavidin-biotin
labeled method with hematoxylin counterstain. The staining reaction appeared as a
brownish coloration reflecting the intracellular distribution of cytokeratin 17 mesenchymal
filaments within the parenchymal compartments. Staining intensity was assessed semiquantitatively
and scored as negative (0), weak (1), light (2), medium (3), and strong (4).
The collected data were analyzed using the SPSS statistical package, version 23 (IBM
Inc., Chicago, IL, United States). Quantitative data were calculated as the mean,
standard deviation, and ranges when their distributions were found to be parametric
by means of the normality test. Comparison between groups was performed using one-way
analysis of variance test that was used to determine significant differences among
all groups. In addition, Tukey honestly significant difference and Dunnett T3 were
performed to find the significant difference in the mean values.
Results
Histological Findings
Clinically, there was a severe reduction in the parotid gland size of duct-ligated
group in relation to both control and diabetic dogs due to which dissection became
difficult from the surrounding tissue.
The histological examination of the control parotid gland revealed the presence of
numerous closely packed serous acini interspersed with numerous isolated tubular mucous
acini and including intercalated, striated, and excretory ducts with individual characteristics
of each. These parenchymal elements were supported by a connective tissue stroma that
divides the gland into lobules and lobules ([Fig. 1 ]).
Fig. 1 Parotid gland of control group showing definite gland lobules (A ) contain spherical serous acini (B ), intralobular duct (C ) (H&E X 100).
The parotid glands of alloxan-induced diabetes revealed an atrophic change characterized
by a decrease in parenchymal elements accompanied by an increase in the amount of
fibrous tissue stroma. The acini decreased in size with the loss of normal arrangement
and consisted of a group of smaller cells with an indefinite lumen. In many areas,
acini were replaced by fat cells. The acinar cells decreased in size with eosinophilic
cytoplasm and deeply stained enlarged small nuclei. Moreover, there was an increase
in the amount of persistent mucosal acini compared with the control ([Fig. 2 ]).
Fig. 2 Parotid gland of diabetic dogs showing the atrophy of serous acini (A ), fibrosis (B ), dilated interlobular ducts (C ) (H&E X 100).
The duct-ligated parotid gland showed a range of pathological changes varied from
a decrease in gland size to severe atrophy of the gland compartments. Most parenchymal
elements were atrophied without definite acinar arrangement. The remaining acinar
tissues were smaller, interspersed, and scattered through more condensed fibrous tissue
capsules. The lobules appeared to contain many duct-like structures. All remaining
ducts were seen dilated and surrounded by remnants of acinar cells ([Fig. 3 ]).
Fig. 3 Parotid gland of duct-ligated dogs showing the atrophy of serous acini (A ), dilated duct (B ), duct-like structure (C ), extravasated blood (D ) (H&E X 200).
Immunohistochemical Findings
The examination of tissue sections of control parotid glands incubated with the anti-cytokeratin
E3 antibody against cytokeratin 17 showed varied expression of weak, mild, and moderate
intensities in both intercalated and striated duct cells (data are written for each
sample separately from four different tissue sections in [Table 1 ]). Cytokeratin 17 expression of serous acinar cells of most gland sections was negative
except for some scattered acini with weak positive expression ([Fig. 4 ]). The staining pattern was collected lateral and basal to the nucleus with the negative
arrangement of intermediate filaments within the apical part of the cell cytoplasm
allowing for free movements of secretory granules. On the contrary, several fields
showed a diffuse staining pattern for cytokeratin 17 in both duct cells and serous
acinar cells that indicated the resting secretory state. The main excretory ducts
of several sections showed moderate expression collected within the basal cell layer
with weak expression in the remaining layers. Mucous acini showed negative staining
reaction in all samples.
Fig. 4 Parotid gland of control dogs showing the negative expression of CK17 in most of
serous acini (A ), mild in little of them (B ), and weak of striated duct (C ) (X 200).
Table 1
Cytokeratin intensity upon animals of all groups, negative staining (0), trace or
weak (>0), mild (>1), moderate (>2), and strong staining (>3)
Intensity of CK 17 expression in duct cells
Dogs
Control group
Diabetic group
Duct-ligated group
Field number
Field number
Field number
a
b
c
d
Average
a
b
c
d
Average
a
b
c
d
Average
1
1
2
1
1
1.25
2
1
2
1
1.5
3
1
2
2
2
2
2
1
1
1
1.25
3
2
2
1
2.00
1
2
2
4
2.25
3
0
2
2
1
1.25
3
3
2
2
2.50
2
1
3
4
2.5
4
1
1
1
0
0.75
1
2
2
2
1.75
2
3
1
2
2
Mean ± SD
1.125
Mean ± SD
1.9375
Mean ± SD
2.1875
Intensity of CK 17 expression in acinar cells
1
1
0
1
0
0.50
1
2
0
1
1.00
0
2
1
2
1.25
2
0
1
1
1
0.75
1
1
2
1
1.25
1
1
2
3
1.75
3
1
1
0
2
1.00
1
2
2
0
1.25
2
1
1
2
1.50
4
0
0
2
1
0.75
1
1
0
2
1.00
3
1
2
1
1.75
Mean ± SD
0.75
Mean ± SD
1.125
Mean ± SD
1.5625
Abbreviation: SD, standard deviation.
Tissue sections of the parotid glands of diabetic group incubated with the anti-cytokeratin
E3 antibody against cytokeratin 17 showed varied expression of mild, moderate, and
strong intensity in intercalated, striated, and excretory duct cells. On the contrary,
many of the serous acini showed mild to moderate expressions of cytokeratin 17, while
all sections showed negative staining for the scattered mucous acini. The staining
pattern of this group varied from strong in the apical cell region and mild in the
basal part to diffuse expression throughout the cell cytoplasm ([Fig. 5 ]). This intracellular arrangement of intermediate filaments is thought to interfere
with the formation and/or passage of secretory granules. The statistical data indicate
a strong significant difference (p < 0.05) for cytokeratin expression within the gland parenchyma between the diabetic
group and the control group ([Tables 2 ],[3 ],[4 ],[5 ]).
Table 2
Multiple comparisons of CK17 in duct cells of different groups
(I) Groups
(J) Groups
Mean difference
(I-J)
Std. error
Sig.
95% Confidence interval
Lower bound
Upper bound
Tukey HSD
1.00
2.00
− 0.81250[a ]
0.22438
0.014
− 1.4390
− 0.1860
3.00
− 1.06250[a ]
0.22438
0.003
− 1.6890
− 0.4360
2.00
1.00
0.81250[a ]
0.22438
0.014
0.1860
1.4390
3.00
− 0.25000
0.22438
0.530
− 0.8765
0.3765
3.00
1.00
1.06250[a ]
0.22438
0.003
0.4360
1.6890
2.00
0.25000
0.22438
0.530
− 0.3765
0.8765
Dunnett T3
1.00
2.00
− 0.81250
0.24738
0.059
− 1.6640
0.0390
3.00
− 1.06250[a ]
0.17305
0.002
− 1.6153
− 0.5097
2.00
1.00
0.81250
0.24738
0.059
− 0.0390
1.6640
3.00
− 0.25000
0.24474
0.688
− 1.1013
0.6013
3.00
1.00
1.06250[a ]
0.17305
0.002
0.5097
1.6153
2.00
0.25000
0.24474
0.688
− 0.6013
1.1013
Abbreviation: Tukey HSD, Tukey honestly significant difference.
a The mean difference is significant at <0.05.
Table 3
Multiple comparisons of CK17 in acinar cells of different groups
(I) groups
(J) groups
Mean difference
(I-J)
Std. error
Sig.
95% Confidence interval
Lower bound
Upper bound
Tukey HSD
1.00
2.00
− 0.37625
0.14155
0.062
− 0.7715
0.0190
3.00
− 0.81250[a ]
0.14155
0.001
− 1.2077
− 0.4173
2.00
1.00
0.37625
0.14155
0.062
− 0.0190
0.7715
3.00
− 0.43625[a ]
0.14155
0.032
− 0.8315
− 0.0410
3.00
1.00
0.81250[a ]
0.14155
0.001
0.4173
1.2077
2.00
0.43625[a ]
0.14155
0.032
0.0410
0.8315
Dunnett T3
1.00
2.00
− 0.37625
0.12542
0.071
− 0.7900
0.0375
3.00
− 0.81250[a ]
0.15729
0.006
− 1.3186
− 0.3064
2.00
1.00
0.37625
0.12542
0.071
− 0.0375
0.7900
3.00
− 0.43625
0.14013
0.069
− 0.9142
0.0417
3.00
1.00
0.81250[a ]
0.15729
0.006
0.3064
1.3186
2.00
0.43625
0.14013
0.069
− 0.0417
0.9142
Abbreviation: Tukey HSD, Tukey honestly significant difference.
a The mean difference is significant at <0.05.
Table 4
Descriptive statistics of cytokeratin 17 of different groups
N
Mean
Standard deviation
Standard error
95% Confidence interval for mean
Minimum
Maximum
Lower bound
Upper bound
1.00
4
1.1250
0.25000
0.12500
0.7272
1.5228
0.75
1.25
2.00
4
1.9375
0.42696
0.21348
1.2581
2.6169
1.50
2.50
3.00
4
2.1875
0.23936
0.11968
1.8066
2.5684
2.00
2.50
Total
12
1.7500
0.55391
0.15990
1.3981
2.1019
0.75
2.50
Descriptive statistics of CK17 expression in acinar cells of different groups
1.00
4
0.7500
0.20412
0.10206
0.4252
1.0748
0.50
1.00
2.00
4
1.1263
0.14580
0.07290
0.8943
1.3582
1.00
1.25
3.00
4
1.5625
0.23936
0.11968
1.1816
1.9434
1.25
1.75
Total
12
1.1463
0.39119
0.11293
0.8977
1.3948
0.50
1.75
Table 5
One-way analysis of variance test of CK17 expression of different groups
One-way analysis of variance test of CK17 expression in duct cells
Sum of squares
df
Mean square
F
Sig.
Between groups
2.469
2
1.234
12.259
0.003
Within groups
0.906
9
0.101
Total
3.375
11
One-way analysis of variance test of CK17 expression in acinar cells
Between groups
1.323
2
0.661
16.504
0.001
Within groups
0.361
9
0.040
Total
1.683
11
Fig. 5 Parotid gland of diabetic dogs showing the strong expression of CK17 in ducts (A ) and serous acini (B ), and moderate in other serous acini (C ) (X 200).
Parotid gland parenchyma of duct-ligated group incubated with the anti-cytokeratin
E3 antibody against cytokeratin 17 showed widely variable expression ranging from
negative to strong intensity of diffuse type. In most sections, both duct cells and
serous acinar cells expression of cytokeratin 17 varied from mild, moderate, to strong
intensity ([Fig. 6 ]). Few sections revealed the strong expression of cytokeratin in a lot of serous
acini and intercalated ducts ([Fig. 7 ]). The statistical data refer to significant difference (p < 0.05) for cytokeratin expression within the gland.
Fig. 6 Parotid gland of excretory duct-ligated dogs showing the strong expression of CK17
in both striated duct (A ), some serous acini (B ), negative in other (C ), and moderate in islands of mucous acini (D ) (X 200).
Fig. 7 Expression of CK17 in both acinar and duct cells of all groups.
Discussion
It is known that pathological changes in the parotid glands are a consequence of both
diabetes mellitus and obstruction of the excretory duct. The results of the present
work reported that both diabetes and duct ligation cause atrophy of the parotid gland
parenchyma due to the interaction of different tissues with alterations in the processes
of tissue maintenance and renewal leading to xerostomia. Pathological changes of diabetes
mellitus range from a decrease in gland size to atrophy of the parenchyma of the gland
which has been replaced by fibrous and/or fatty tissue with the proliferation of duct-like
structures. Caldeira et al (2005) note that these histological changes were detected
in all diabetic animals, both controlled and uncontrolled.[20 ] The persistence of multiple viable acini along with the atrophied one in the diabetic
group can be considered as a sophisticated defense mechanism that maintains the acinar
function of the gland parenchyma but with reduced secretory capacity. Furthermore,
Mata et al (2004) reported that the persistent acini found in the diabetic gland tissues
have been suggested to participate in the ability of the gland to regenerate.[21 ] The interpretation of our study is unable to make any attempt to distinguish the
original glandular duct from the duct-like structures present in both diabetic and
duct-ligated groups. This finding was supported by several authors who stated that
the duct-like structures were found due to the proliferation of the duct system.[1 ]
[41 ] In contrast to the atrophic changes of the parenchymal elements, the connective
tissue reacts through a proliferative activity, illustrating the differences in the
tissue interaction of both epithelial and connective tissues. On the other hand, fibrosis
present in the diabetes group and not present in the excretory duct ligation glands
may indicate that the effect of diabetes may be final and irreversible, in contrast
to the other, where the gland is supposed to begin the repair process once the cause
of ligation is gone. Also, inflammatory changes within the connective tissue were
recorded by several authors in contrast to the absence of any inflammatory cells in
our study which may be related to the long time elapsed after the induction of diabetes.[4 ]
[42 ]
It was evident in my results that the expression of cytokeratin 17 in the parotid
gland of control group was clear in the duct system while it was reduced in the serous
acini while the mucous acini showed negative staining. These observations may be due
to highly differentiated acinar cells with a less quantities of intermediate filaments
compared with duct cells. Several authors agree with this finding that the intermediate
filament cytokeratin17 in salivary gland cells plays an important role in cell structure
and the intensity of expression is closely related to the differentiation status of
epithelial cells with further expression in duct cells.[37 ] The staining pattern was either diffuse or collected lateral and basal to the nucleus
which is in agreement with several authors.[37 ] These different patterns explain the distribution of intracellular cytokeratin which
is thought to be related to the functional activity of the gland where the diffuse
pattern of staining indicates the resting phase, while the cytokeratin devoid in the
luminal part was related to the active secretory state, these findings were supported
by Friedrich et al. (2000).[43 ] Moreover, the pattern of cytokeratin expression focused on the basal cell end may
be associated with an increase in the tensile force of acinar cells facing contractile
myoepithelial cells resulting in an increased squeezing capacity to drive saliva through
the lumen into the duct system.
Parotid parenchyma of both diabetic and duct-ligated groups revealed significant expression
of cytokeratin 17 immunogenicity in both acinar and ductal cells with two different
profiles, apically collected and diffuse. The intensity of cytokeratin expression
was significantly increased toward the parenchymal elements of group III. It is thought
that the luminal distribution patterns of cytokeratin in the diabetic dog may interfere
with the secretory capacity of acinar cells resulting in dry mouth. Also, this luminal
pattern within the ductal cells may disturb the modulation procedure for the secreted
primary saliva. On the contrary, the diffuse pattern of cytokeratin expression indicates
a cellular deleterious effect throughout the parenchymal compartments. The interpretation
of these two different expressions may be due to the progression of the degeneration
stage, which starts from the luminal side and then progresses to the diffusion pattern.
However, the expression of cytokeratin 17 in the duct cells of all groups is found
not only in the largest but also in the smallest ones. This observation may support
the accuracy of this immunoprecipitation process for intermediate filament.
Conclusion
The expression and arrangement patterns of cytokeratin 17 in our results predict the
pathological effect of both diabetes mellitus and duct ligation on the intracellular
filament system of the salivary gland parenchyma in a different way that interferes
with saliva production leading to dry mouth.
