Nuclear morphometric and morphological analysis of exfoliated buccal and tongue dorsum cells in type-1 diabetic patients.

BACKGROUND: Diabetes mellitus type 1 that results from immunologically mediated damage to the β-cells in the pancreas. Diabetes mellitus is characterized by recurrent or persistent hyperglycemia. Hyperglycemia can be associated with salivary gland dysfunction and alterations in the oral epithelial cells. AIM: The aim of this study was to evaluate the qualitative and quantitative changes in buccal and tongue dorsum epithelial cells using an exfoliative cytology method in type 1 diabetic patients.
MATERIALS AND METHODS: We performed light microscopic analysis of the buccal and tongue dorsum smears in thirty type 1 diabetic patients and thirty healthy individuals. The oral smears were stained using Papanicolaou method for cytological examination and nuclear morphometric analysis. In each case, the mean nuclear area, perimeter, length, breadth, and roundness factor were evaluated in each smear using the image analysis software (Q Win, Leica™).
RESULTS: The nuclear area, length, breadth, and perimeters were significantly higher in the diabetic group from tongue dorsum smear than that of the control group (P < 0.05). In the cytological examination, karyorrhexis-karyolysis-karyopyknosis, binucleation, nuclear membrane irregularity, cytoplasmic polymorphism, perinuclear halo were observed in oral smears with type 1 diabetic patients. Binucleation (P = 0.002) and nuclear membrane irregularity (P = 0.024) were significantly more common in buccal smears of diabetic group. Furthermore, the sensitivity of buccal mucosa was significantly higher in the diabetic group (P = 0.006).
CONCLUSION: The light microscopic and nuclear morphometric study indicates that type 1 diabetes can produce morphological and nuclear morphometric changes in the oral mucosa that are noticeable with exfoliative cytology.

Introduction

Diabetes mellitus is a common endocrine-metabolic disorder. It causes hyperglycemia, associated with disturbances in the metabolism of carbohydrate, fat, and proteins, as a result of absolute or relative insulin deficiency.[123] Type 1 diabetes (Insulin dependent diabetes mellitus, IDDM) is an auto-immune disease.[456] Hyperglycemia can be associated with salivary gland dysfunction and alterations in the oral epithelial cells.[123] The biopsies are available for evaluation of oral mucosal changes.[17] However, in diabetes, many invasive techniques lose viability as a result of variations in blood glucose and delayed healing processes.[13] In these cases, exfoliative cytology may be more appropriate.

Morphometry is the measurement of various cell parameters such as lengths, widths, masses, angles, ratios, and areas. This study endeavors to apply nuclear morphometry to oral cells from type I diabetic patients. The main purpose of this study was to emphasize the relevance of qualitative and quantitative exfoliative cytology and evaluate the cellular alterations in oral epithelial cells in type 1 diabetic patients.

Materials and Methods

Study population

Subjects were randomly selected into two groups; type 1 diabetic group and healthy control group. Diabetic patients (n = 30) were selected — The control group (n = 30) was selected from healthy individuals with no risk factors for diabetes. This study was conducted with the permission of the local ethics committee of — We designed a questionnaire that patients completed, including demographic characteristics such as name, age, sex, and relevant medical history and symptoms such as halitosis and buccal mucosa sensitivity. Subjects of both groups had clinically healthy oral mucosa. Individuals smoking, being addicted to alcohol or suffering from anemia or malignancy were excluded to eliminate the effects of these conditions on cellular shape and morphology. Diabetic patients who had other systemic diseases or taking medications other than the diabetic medications were also excluded. The diabetic patients were selected according to the American Diabetes Association criteria.[8] Glycosylated hemoglobin (HbA1c) level was measured for each patient attending diabetic clinic.

Smear collection and cytologic analysis

We took the smears from different oral sites (the buccal mucosa and tongue dorsum) to reduce the effect of localized inflammation on our results. Before samples were taken, patients were instructed to gargle with water. Oral PH was measured with indicator paper (Whatman 1 dispenser, EU). The oral mucosa was dried with a gauze swab to remove surface debris and excess saliva. The glass slide was labeled with patient's name at the site of collection. Smears were obtained using an endocervical sampling brush and transferred to clean glass slides. After fixation in 95% ethyl alcohol, the smears were stained with Papanicolaou method (Pap stain). All oral epithelial cells underwent light microscopic analysis with regard to nuclear and cytoplasmic changes and nuclear morphometric parameters. Patients were excluded from the study, lest they might have infection agents such as Candida, Trichomonas etc., on oral smear in order to eliminate the effects of these conditions on morphology and morphometric parameters.

Nuclear morphometry

Morphometric analyses were performed with Papanicolaou-stained smears. A microscope DMLB-100S, Leica™(Leica Microsystems, Wetzlar, Germany) was connected to a video camera (Leica, DFC-280) and computer. After transferring microscopic images to a computer, morphometric parameters were automatically measured by Leica QWin image analysis program (version 3.1.0) (Leica Microsystems, Wetzlar, Germany).

In each slide, twenty clearly defined cells with predominant staining were selected manually in a random fashion from different fields, and in order to avoid measuring and counting the same cells again, we moved the microscope stage from left to right, and then down and across in a step-wise manner. A total of 1200 cells from type 1 diabetic patients and 1200 cells from control subjects were analyzed. The nuclear morphometric parameters studied were as follows: Nuclear area, nuclear roundness factor, nuclear length, nuclear breadth, and nuclear perimeter. Nuclear roundness factor is given by the equation: “Perimeter 2/4p × area.” These shape descriptors yield a minimal value of 1.00 for a perfect circle and increase as the shape of a contour deviates from circularity. Nuclear area is the area enclosed inside the contour; the perimeter is the contour perimeter, and the length and breadth are the longest and shortest orthogonal projections, respectively. All measurements were made under 400X magnification and expressed in micrometers.

Statistical analysis

Statistical analysis of the measurements was performed using SPSS for Windows, v.13 statistical package, Chicago, IL. Numeric data were reported as mean ± standard error. Categorical data were reported as frequency and percentage. The Chi-square test or Fisher's exact test was used to compare categorical variables between the groups. The morphometric data were compared between the groups by the Student's t-test. The difference between gender and nuclear area in buccal and tongue dorsum smears was assessed by Mann-Whitney's nonparametric test. Pearson's correlation coefficient was used to analyze the relationship among numeric variables. A P-value less than 0.05 was considered statistically significant for all tests.

Results

The age of participants in both groups ranged from 4 to 28. The age range for the IDDM patients was 6-27 (mean age 15.5 ± 7.9) years and for the control groups, the age range was 4-28 (mean age 15.3 ± 6.5) years and a male: Female ratio of 9:21 in the groups. The duration of the disease ranged from 1 to 16 years; the level of glycosylated hemoglobin (HbA1c) was 11.31 ± 0.59 (range 7-17), respectively. The levels of pH were 6.8 ± 0.8 (range 5-8) in diabetics and 6.5 ± 1.2 (range 6-8) in controls. Buccal mucosa sensitivity (n = 15/30, 50%, P = 0.006) was significantly more common in the diabetic group. Halitosis showed a nonsignificant tendency to be more frequent in diabetic patients (P > 0.05).

Data from the computerized cytomorphometry were analyzed by an image analysis program. The Table 1 summarizes the nuclear morphometric results of all groups. The nuclear area, length, breadth, and perimeters were significantly higher in the diabetic group from tongue dorsum smear than that of the control group (P < 0.05). The nuclear area, nuclear perimeter, nuclear roundness factor of oral smears were not significantly affected by age, gender, and HbA1c in diabetes patients.

Table 1

Nuclear morphometric features of the type 1 diabetes and control group

Cytologic analysis

Morphological alterations were also noted in buccal and tongue dorsum smears obtained from type 1 diabetic patients.

The morphological alterations seen in buccal mucosal epithelial cells of this group were karyorrhexis-karyolysis-karyopyknosis (n = 6/30, 20%), binucleation (n = 9/30, 30%), nuclear membrane irregularity (n = 6/30, 20%), cytoplasmic polymorphism (n = 4/30, 13.3%), perinuclear halo (n = 11/30, 36.7%), and polymorphonuclear leukocytes (n = 11/30, 36%) [Figure 1a–c]. Binucleation (P = 0.002) and nuclear membrane irregularity (P = 0.024) were significantly more common in the buccal mucosa of type 1 diabetic group.

Figure 1

Oral epithelial cells in smears stained by the Papanicolaou method from the diabeic group: (a), Binucleation, (b), nuclear membrane irregularity and perinuclear halo (arrow), binucleation (arrowhead) (c), cytoplasmic polymorphism (arrow) and nuclear condensation-irregularity (arrowhead) (d) polymorphonuclear leukocytes and squamous epithelial cells

The cellular alterations seen in tongue dorsum of the type 1 diabetic group were binucleation, the nuclear membrane irregularity, cytoplasmic polymorphism, perinuclear halo. There is no statistical interaction between these parameters and type 1 diabetes (P > 0.05). Polymorphonuclear leukocytes were significantly more common in tongue dorsum smear of the diabetic group [Figure 1d], P = 0.012].

Discussion

Diabetes is an important health care problem. It is simply defined on the basis of hyperglycemia. Hyperglycemia can be associated with oral complications.[2] The degree of glucose metabolic control significantly affects the level and acuteness of diabetes-related oral diseases. Well-controlled diabetics have fewer oral health problems than inadequately controlled diabetics.[9] Diabetics also have reported increased complaints of stomatopyrosis and xerostomia.[59]

In the present study, oral mucosal cells of type 1 diabetic patients showed qualitative and quantitative changes compared to that of normal healthy individuals in this study. We were able to see such morphologic alterations in nucleus and cytoplasm of oral epithelial cells in type 1 diabetic patients. The nuclear (karyorrhexis-karyolysis-karyopyknosis, binucleation, the nuclear membrane irregularity) and cytoplasmic changes (cytoplasmic polymorphism, perinuclear halo) tended to be more frequent in diabetic patients.

Alberti et al.,[1] Jajarm et al.,[2] and Shareef et al.[10] examined the morphological changes of the oral epithelial cells and reported a binucleation, karyorrhexis, cytoplasmic vacuolization, and nuclear enlargement in type 2 diabetic patients. In addition to these findings, Shareef also reported polymorphonuclear leukocytes infiltration in buccal cell.[10] Also, Prasad et al.[11] noticed a binucleation, intracytoplasmic inclusion, perinuclear halo, and keratinized squamous cells in smears from uncontrolled diabetics. Tozoglu and Bilge[12] performed cytomorphometric analysis of the oral epithelium in type 1 diabetic patient, and they found that there is an increase in the nuclear and cytoplasmic volume. Our cytologic findings are in agreement with the study by Alberti et al.,[1] Jajarm et al.,[2] Shareef et al.,[10] and Prasad et al.[11]

Different from the previous studies that examined type-2 diabetics, uncontrolled diabetics and type 1 diabetics,[12101112] our study is unique to evaluate both nuclear morphometry (area, roundness factor, length, breadth, perimeter), and also nuclear and cytoplasmic alterations in type 1 diabetics’ buccal and tongue dorsum smears.

The cellular changes which are reported in our study involved in superficial and intermediate squamous cells of oral squamous epithelium. The morphology and nuclear morphometric changes in buccal mucosa of type 1 diabetic group cannot be attributed to factors such as age, sex, smoking habit, and systemic diseases but to type 1 diabetes. The genesis of cellular changes is somewhat puzzling.[13] Karyorrhectic cells thus appear to have no nucleus. It is possible that they represent a very late stage in the cell death process. Karyopyknotic cells may be undergoing a form of cell death although this has not been convincingly investigated.[14] It is possible that the disruption of cell skeletal filaments also induces binucleation, and perinuclear halo which is a clear area around the nucleus formation.[13] Type 1 diabetes leads to cytoskeleton changes. Insulin has been suggested to play a key regulatory role in the functional organization of actin microfilaments. The actin network is an essential mediator in the action of insulin. The microtubules are also targets of insulin. The microtubules are seen in greatest density around the nucleus.[13] Thus, many cellular effects of insulin may depend on the functional integrity and organization of the cytoskeleton. A chronic insulin deficiency could lead to impairment in the organization of the cytoskeleton.[15] Microtubule cytoskeleton may function in the positioning and initiation of the cleavage furrow, and the actin filament cytoskeleton may play key roles in the initiation and ingression of the furrow.[16] It is possible that a chronic insulin deficiency could lead to disruption of the microtubules, and this might result from perinuclear halo formation.

Our results showed that the nuclear area and nuclear perimeter were significantly higher in the diabetic group than the control group. In previous studies, Alberti et al.,[1] Jajarm et al.,[2] and Shareef et al.[10] performed morphometric analyses of the oral epithelial cells in type 2 diabetic patients and they found that there is a markedly increase in the nuclear area. Furthermore, Prasad et al.[11] reported an increase of nuclear diameter in uncontrolled diabetes. Our finding concurs with the study by Alberti et al.,[11] Shareef et al.,[10] Prasad et al.,[11] Tozoglu and Bilge[12] and Hallikerimath et al.,[17] who report a significant increase in nuclear area in diabetic patients. This increase of nuclear size could be related to cellular aging in type 1 diabetes patients.

In summary, the present study was undertaken to analyze the changes in morphology and nuclear morphometry in exfoliated buccal mucosa cells in type 1 diabetes patients. The following findings were observed:

First, there is a clear increase in the nuclear area, nuclear length, nuclear breadth, and nuclear perimeter in type 1 diabetic group. In addition to these morphometric changes, morphological changes in the form of binucleation (P = 0.002), nuclear membrane irregularity (P = 0.024), karyorrhexis-karyolysis-karyopyknosis, cytoplasmic polymorphism, perinuclear halo, and polymorphonuclear leukocyte were also noticed in smears from diabetic patients.

Second, the sensitivity of buccal mucosa in the type 1 diabetes patients was significantly higher (P = 0.006) than in healthy patients.

Finally, we showed that exfoliative cytology can be used to evaluate morphological parameters of type 1 diabetes in addition to type 2 diabetes.

From the present study, we conclude that type 1 diabetes can produce morphological and nuclear morphometric alterations in oral epithelial cells that are detectable by microscopic and cytomorphometric analysis with exfoliative cytology method. Our study is significant in that it represents cellular, cytoskeletal, and nuclear morphometric picture in type 1 diabetes. Furthermore, it requires additional studies in this area with a greater sample size to compare the described cellular changes to similar cellular changes caused by other diseases.