Abstract
Abstract: Diabetes and salivary gland dysfunction are major factors that induce dental caries in experimental animals, but there are no reports analyzing the association of dental caries and salivary glands in an animal model of diabetes mellitus (DM). To clarify the initial development of dental caries and preceding salivary gland disorder, we performed a histopathological analysis on teeth and salivary glands in diabetic Wistar rats 7 weeks after alloxan treatment (DM group) in comparison with nondiabetic rats (Non-DM group) and functional analysis on saliva secretion during the experimental period. Pilocarpine-induced salivary fluid secretion in diabetic rats gradually decreased with continuous hyperglycemia from immediately after alloxan treatment to the time of autopsy. Histopathologically, Oil Red O-positive lipid droplets accumulated in the acinar cells of the parotid gland. No tooth was stereoscopically defined as having dental caries in any of the rats in either group; however, the external appearance remarkably changed owing to occlusal wear in almost all molars in the DM group. The initial lesions of dental caries, appearing as micro-defects in dentin with bacterial colonization on the molar surface, were identified using histopathological analysis, and the incidence in the DM group was more than twice that in the Non-DM group. In conclusion, hyperglycemia simultaneously induces initial caries development and enhances spontaneous occlusal wear in molar teeth of Wistar rats 7 weeks after alloxan treatment. The parotid gland dysfunction caused by hyperglycemia may be mostly involved in the pathogenesis of occlusal wear as well as in dental caries in this diabetic model.
Introduction
Dental caries occur in the presence of pathogenic bacteria and sugar in a predisposing host 1 . Thus far, since the onset of naturally occurring dental caries has not been reported in rodent models, the researchers have used a cariogenic diet containing large amounts of sugar and/or an inoculation of infectious cariogenic bacteria into the oral cavity to induce dental caries. Recently, we reported the development of dental caries in a caries-susceptible strain fed a noncariogenic diet with a low sugar concentration 2 . In addition, we clarified that diabetes is an important factor inducing dental caries under the given noncariogenic diet 3, 4 . Salivary gland dysfunction in Sjögren's syndrome and radiation therapy are the major factors causing onset of dental caries in humans and experimental animals [5] [6] [7] . Hyposalivation and morphological abnormality of the salivary gland also occur in diabetic patients and animals [8] [9] [10] [11] [12] . Diabetes may be significantly associated with salivary gland dysfunction. Thus, the development of dental caries in a diabetic animal model may be correlated with salivary gland disorder; however, there are no reports sufficiently analyzing the relationship between dental caries and salivary gland disorder. To test this hypothesis, the development of dental caries and salivary gland disorder should be simultaneously analyzed in the same diabetic animals.
Introduction
Alloxan (AL) is one of the chemicals that induce loss of pancreatic β-cells and cause a severe hyperglycemic condition, thereby inducing diabetes mellitus in animals. In AL-treated rats, progressive molar caries with collapse of the dental crown were observed after a 13-week hyperglycemic period 13 . Hyposalivation and morphological abnormality of the salivary gland were also reported after AL treatment [14] [15] [16] . However, to our knowledge, the association between dental caries and salivary gland disorders has not been investigated in AL-induced diabetic models or in other animal models of diabetes.
Introduction
Although severe caries uniformly develop in rats at 13 weeks after the onset of hyperglycemia induced by AL treatment, there are no lesions present in the initial 4 weeks following induction of hyperglycemia 13 . Therefore, primary dental caries are expected to develop between 4 and 13 weeks after AL treatment in this model. In addition, we hypothesized that the salivary gland abnormalities in ALtreated diabetic rats might precede dental caries formation at an earlier stage. Thus, in this study, onset of the dental caries induced by AL treatment was estimated at 7 weeks (nearly half of 13 weeks) after hyperglycemia, and the autopsy point was set at this time. To this end, we examined the morphology of the teeth and salivary glands and performed serial measurements for evaluating salivary secretion ability; we also clarified the pathogenetic relationship between the dental caries development and salivary gland dysfunction in this diabetic model.
Materials and Methods

Animals and housing conditions
The rats were handled according to the principles outlined in the Guide for the Care and Use of Laboratory Animals by the Committee for Animal Experiments of Hiroshima International University and the Japanese Association for Laboratory Animal Science. Male Wistar (Crlj:WI) rat pups born to female SPF Wistar rats supplied by Charles River Laboratories Japan, Inc. (Yokohama, Japan), were used in this study. They were reared in a barrier-sustained animal room maintained at a temperature of 23 ± 2°C and a relative humidity of 55 ± 10% with 12-h light/dark cycles and ventilation at least 10 times/h with high-efficiency particulate air-filtered fresh air. All rats were housed and reared in fiber-reinforced plastic (FRP) cages with a stainless (SUS304) wire-mesh floor. Rats had free access to tap water and a widely used noncariogenic standard pelletized diet for experimental mice and rats (CLEA Rodent Diet CE-2, CLEA Japan, Inc., Tokyo, Japan).
Experimental design
The experimental design is shown in Fig. 1 . A total of 23 rats were randomly divided into 2 groups: diabetic rats (DM group; n=13) and nondiabetic rats (Non-DM group; n=10) at 7 weeks of age. In the DM group, a single dose of 50 mg/kg of AL (Sigma-Aldrich Japan, Tokyo, Japan) was administered via the tail vein, whereas the rats in the Non-DM group were administered a single dose of 0.9% saline. All of the rats were euthanized at 14 weeks of age, and their teeth and salivary glands were histopathologically examined.
Glucosuria and glycemia monitoring
Urinary glucose levels were measured semiquantitatively with a urine test paper (Wako Pure Chemical Industries, Osaka, Japan) using fresh urine. Blood glucose levels were also measured semiquantitatively by the glucose oxidase method (Glutest PRO R, Sanwa Kagaku Kenkyusho, Aichi, Japan) using non-fasting blood samples collected from the tail vein. Urinary and blood glucose levels were measured at 0, 1, 3 and 7 weeks after AL treatment. Samples of blood from the tail vein and fresh urine were collected between 13:00 and 16:00. Hyperglycemia and glucosuria were defined as glucose levels greater than 200 mg/dL and 250 mg/dL, respectively.
Measurement of food intake
Food intake over a 24-hour period was measured using rats from each group (DM group, n = 8; Non-DM group, n = 5) at 0, 1, 3, and 7 weeks after AL treatment.
Saliva collection
Saliva secretion was induced by subcutaneous injection of pilocarpine (2 mg/kg body weight). The saliva was collected using cotton balls between 3 to 8 min after pilocarpine injection under deep anesthesia with pentobarbital (25 mg/kg body weight, i.p.). The total weight of the secreted saliva (difference in the weight of the cotton balls before and after collection) was measured at 0, 1, 3, and 7 weeks after AL treatment.
Stereoscopic and soft X-ray examination of the mandibular and maxillary molars
At the end of study period, the animals were euthanized by exsanguination from the abdominal aorta under deep anesthesia induced using isoflurane. Subsequently, the mandible and maxilla were removed and fixed in 10% neutral buffered formalin solution (pH 7.4). After a 24-h fixation, the occlusal, buccolingual, and proximal surfaces of all teeth were examined under a binocular stereoscope. The stereoscopic change scores of each molar obtained using a binocular stereoscope were classified into 4 grades: grade 0, no change in the molar tooth; grade 1, mild wear of the cuspis dentis; grade 2, moderate wear of the cuspis dentis; grade 3, severe wear of the cuspis dentis. Following stereoscopic examination, a soft X-ray examination was performed, and images of the mesiodistal plane were obtained at 35 kV and 2 mA for 4 min. The cusp height of the all molars on soft X-ray images was measured using the scale function in Adobe Photoshop Elements 9 (Adobe Systems, San Jose, CA, USA). 
Histopathological examination
For histopathological examination, the jaw, salivary glands, and tongue were removed and fixed in 10% neutral buffered formalin solution (pH 7.4). After formalin fixation for 24-h, the parotid gland, submandibular gland, and tongue, including the von Ebner's glands, were trimmed, dehydrated in a sequential ethanol series by using an automated processor, and embedded in paraffin. The mandible and maxilla were decalcified in K-CX (Falma Co., Ltd., Tokyo, Japan) for 24 h and immersed in a 1:1 mixture of 10% formic acid solution and neutral buffered formalin solution for 5 days after formalin fixation. After decalcification, the specimens were trimmed, dehydrated, and embedded in paraffin. These paraffin tissue sections (5-μm thick) were stained with hematoxylin/eosin. The vacuolation scores of sections of each salivary gland were classified into 5 grades: grade 0, no or rare vacuoles; grade 1, small vacuoles present in less than 50% of the total gland; grade 2, small vacuoles present in more than 50% of the total gland; grade 3, in addition to the small vacuoles, large vacuoles present in less than 50% of the total gland; grade 4, large vacuoles present in more than 50% of the total gland.
Histopathological examination
To determine the contents of the vacuoles observed within the salivary glands, a part of the left parotid gland and a part of the tongue, including the von Ebner's glands, were embedded in OCT Compound (Sakura Finetek Japan Co., Ltd., Tokyo, Japan) for preparing frozen tissue specimens, and these frozen tissue sections (10-μm thick) were stained with Oil Red O.
Statistical analysis
The differences between the groups in mean blood glucose levels, body weight, food intake, saliva weight, and cusp height were determined using a Student's t-test or Welch's t-test. The chi-square test was used to compare the incidence of macroscopic and histopathologic changes in the molars. The Mann-Whitney test was used to compare the mean lesion scores of the salivary glands between the groups. P<0.05 was regarded as statistically significant.
Results

Glucosuria, glycemia, and general condition monitoring
The mean blood glucose levels of each group are shown in Fig. 2 . All rats in the DM group showed severe hyperglycemia (>400 mg/dL) and glucosuria (>2,000 mg/ dL) from the day after AL injection till the end of the study. In contrast, glucosuria was not observed in any rat in the Non-DM group, and normoglycemia persisted throughout the study (Fig. 2) . The mean body weight in the DM group decreased over time after AL treatment and was significantly lower than that in the Non-DM group at 3 and 7 weeks following treatment. However, the mean food intake after AL treatment in the DM group was significantly higher than that in the Non-DM group at each time point and at the final time point was more than twice that of the Non-DM group (Fig. 3) . All of the rats used in this study survived until the end of the experimental period.
Weight of secreted saliva
The changes in the mean weights of pilocarpine-induced saliva in each group are shown in Fig. 4 . The mean weight of secreted saliva after AL treatment in the DM group was significantly lower than that in the Non-DM group at each time point, and the final weight at 7 weeks after AL treatment was less than 50% of the weight in the Non-DM group. In the Non-DM group, the mean total saliva weight at 0, 1, 3, and 7 weeks after treatment was 0.71, 0.91, 1.14, and 1.25 g, respectively, and showed a gradual increase over time. In contrast, a decreasing trend (0.77, 0.62, 0.66, and 0.53 g at 0, 1, 3 and 7 weeks, respectively) was observed Severe hyperglycemia (>400 mg/dL) persisted from the day after alloxan treatment in the DM group. The blood glucose level in the Non-DM group was consistently <200 mg/dL. The data are shown as the mean ± SD. Significant difference compared with the Non-DM group (**P<0.01). in the DM group; the values after AL treatment were also significantly lower than those in the Non-DM group at each time point (P<0.01).
Macroscopic morphological examination by stereoscope and soft X-ray film
In this study, the typical manifestations of dental caries, such as collapse of the molar crown or a hole in the tooth surface with bacterial attachment, were not stereoscopically observed in any rats in the DM and Non-DM groups. Thus, no tooth was identified as having dental caries after stereoscopic morphological examination. Apart from the development of dental caries, external changes due to occlusal surface wear were apparent in almost all the teeth in the DM group, and the stereoscopic changes (maxilla, 98.7%; mandible, 97.4%) in the DM group were significantly more common than those in the Non-DM group (maxilla, 15.0%; mandible, 30.0%) ( Fig. 5 ).
Macroscopic morphological examination by stereoscope and soft X-ray film
Soft X-ray examination showed wear of the enamel and dentin on the occlusal surface at the cusp, which was classified as a Grade-3 stereoscopic change under a microscope (Fig. 5e) .
Morphometric examination on occlusal surface wear of molar teeth
The mean cusp heights of the maxillary and mandibular molars on the soft X-ray images are shown in Fig. 6 . The mean cusp height in the DM group was approximately half that of the Non-DM group, and the difference between them was significant (P<0.01) (Fig. 6) .
Histopathological examination
Mild dental caries that were not evident on stereoscopic examination could be identified on histological examination of serial sections of each molar in both groups. The incidence and severity of the dental caries in the DM group was significantly higher than that in the Non-DM group ( Table 2 ). The majority of dental caries in both groups (DM, 25.6-32.1%; Non-DM, 8.3-10.0%) were diagnosed as micro-defects in dentin with bacterial colonization (Fig. 7b) , and the incidence in the DM group was more than twice that in the Non-DM group. Furthermore, advanced dental caries such as expanded defects with bacterial colonization (Fig.  7c ) were detected at a low rate (3.8-14.1%) in the DM group alone (Table 2 ). In some of the teeth with advanced caries, hypertrophic odontoblasts (Fig. 7e) and an abscess and/or necrosis of the dental pulp were observed (Fig. 7f) . Meanwhile, mild to severe occlusal surface wear was histopathologically confirmed in almost all the teeth in the DM group, corroborating the results of morphometric examination for cusp height determined using soft X-ray photographs; the lesions were seen as a smooth dent in the occlusal surface of dentin. In many of the teeth with advanced occlusal wear, expanded dental caries were observed on the occlusal surface (Table 3 ). In addition, at the dentin-pulp junction just below the site of occlusal wear in the DM group, disappearance of the predentin layer or irregular arrangement of odontoblasts was observed accompanied by reparative dentin (Fig. 7d) . Reparative dentin, where the dentinal tubules had locally become tortuous or unclear (Fig. 7e) , was formed in almost all of the teeth in both DM and Non-DM groups (93.3-100%), and yet the presence of bacteria in the dentinal tubules in the DM group (maxilla, 79.5%; mandible, 76.9%) was remarkably higher than that in the Non-DM group (maxilla, 30.0%; mandible, 46.7%; Table 4 ). In the salivary glands, vacuolation of the acinar cells of the parotid gland and the von Ebner's gland was seen in the DM group, and the incidence and severity were sig- Significant difference compared with the Non-DM group (**P<0.01). Significant difference compared with the Non-DM group (**P<0.01). M1 M2 M3 M1 M2 M3 M1 M2 M3 M1 M2 M3   DM   1 2  1  1  2  2  1  2  1  1  2  1  0  2 1  1  1  2  1  2  2  1  1  1  1  1  3 2  2  1  2  2  2  3  1  1  3  1  1  4 1  1  1  2  1  1  2  1  2  2  1  2  5 3  3  2  3  2  2  3  2  2  3  2  2  6 1  1  2  1  1  1  2  1  1  1  2  1  7 1  1  1  2  1  2  2  1  1  3  2  1  8 1  0  1  1  1  2  1  1  0  1  1  1  9 1  1  1  2  1  2  2  1  2  2  2  1  10 2  1  1  1  2  1  2  1  2  2  2  1  11 1  1  1  2  2  1  2  2  1  2  1  2  12 1  2  1  1  2  1  2  2  1  2  1  1  13 2  2  2  2  1  1  3  2  1  2  2  2 Non-DM Numbers indicate the grade of occlusal wear. Yellow indicates slight dentin caries. Red indicates expanded dentin caries. Significant difference compared with the Non-DM group (**P<0.01). (n = 10) (n = 13) (n = 10) (n = 13) (n = 10) Significant difference compared with the Non-DM group (**P<0.01).
Fig. 7. Histopathological characteristics of molars. (a-c) Typical example of morphological changes in the Non-DM group (a) and DM group (b, c). The inset is an enlarged view of the arrow. (a) No caries. Reparative dentin is formed in part of the cusp (asterisk). (b) Mild dental caries (arrow, inset). The occlusal surface is roughened by micro-defects of dentin with bacterial infection (arrow, inset). Reparative dentin is formed in part of the cusp (asterisk). (c) Advanced dental caries (arrow, inset). Expanded collapse with partial caving of the occlusal surface is observed (arrow, inset). Reparative dentin is formed in a part of the cusp (asterisk). (d-f)
nificantly higher than in the Non-DM group (Table 5 ). The vacuolation of the DM group progressed from focal lesions with a few small vacuoles to diffuse lesions with numerous small and large vacuoles. Some cells contained a large well-defined single round vacuole similar to a white adipocyte (Fig. 8a, 8c) . The vacuoles were stained with Oil Red O (Fig. 8a, 8c inset) . Mild vacuolation of the acinar cells in the submandibular gland was also observed, but there was no clear difference between the DM and Non-DM groups ( Table 5 ). In addition, there was no difference in the size of the acinar cells of the salivary glands between the two groups.
Discussion
Long-lasting hyperglycemia inevitably induces dental caries in both chemically induced and spontaneous diabetic rodent models 3, 4, 13 . However, there are few reports regarding the initial formation of the dental caries in these diabetic models. In our previous study, progressive molar caries were induced in diabetic rats 13 weeks after alloxan administration 13 . The dental caries in these diabetic models reach the dental pulp from the dentin surface, develop into pulpitis with bacterial colonization, and expand through the entire crown or to the dental root, thereby destroying the crown 13, 17 . On the other hand, development of dental caries has not been reported yet in rats 1 month after AL treatment 18 . Therefore, in this study, to investigate the process of initial caries formation, the autopsy point was set at 7 weeks after AL treatment, which corresponded to approximately half the time taken to reach the symptomatic stages of the dental caries. The present study revealed that the initial lesion of the dental caries, which appeared as micro-defects in dentin with bacterial colonization on the molar surface, could be identified using histopathological analysis, but not stereomicroscopic analysis, after 7 weeks of hyperglycemia.
Discussion
We hypothesized that salivary gland dysfunction, which is one of the most important factors in the development of dental caries, might be latent before the initial formation of the dental caries in diabetic rats. The present study revealed that pilocarpine-induced saliva secretion in diabetic rats gradually decreased with continuous hyperglycemia starting immediately after AL treatment up to the point of autopsy when the dental caries were initially formed. Furthermore, large Oil Red O-positive lipid droplets accumulated in the acinar cells of the parotid glands in these diabetic rats. AL-and streptozotocin (STZ)-induced diabetic rats reportedly show a significant reduction in muscarinic agonist-induced salivary fluid secretion, such as in the case of pilocarpine, from 2 weeks to 6 weeks after the onset of hyperglycemia 8, 12, 19, 20 . In addition, in rats, small lipid droplets were observed in the acinar cells of the parotid glands 24 h after STZ treatment, and lipid accumulation progressed and large lipid droplets leahellis@example.net months after STZ treatment 21, 22 . The point of onset of salivary gland dysfunction and the resultant morphological changes in hyperglycemia status were in agreement with our findings in this study. Serous saliva secretion is induced by the muscarinic receptor stimulation 23 , and serous saliva is secreted into the oral cavity mainly from the parotid and submandibular glands 24 . In this study, saliva secretion induced by muscarinic receptor stimulation was remarkably reduced in the DM group, and morphological abnormalities were observed in the parotid glands but not in the submandibular glands. In rats, the parotid duct opens opposite the molar teeth 25 , and the effect of selective removal of the salivary glands on the incidence of dental caries was highest when the parotid glands were removed compared with that after removal of the other major salivary glands 26 . Thus, parotid gland dysfunction may be involved in the initial development of molar dental caries in AL-induced diabetic rats. Interestingly, in the Non-DM group, bacteria were present in the dentinal tubules of the molar surfaces, and minute collapse of the dentin with bacterial colonization appeared in a few cases; however, these changes were apparently enhanced in the DM group. These findings suggest that the salivary functions such as flushing of bacteria and remineralization of enamel and dentin by saliva 27, 28 may constantly protect the teeth against bacterial infection in normal rats and that hyposalivation caused by hyperglycemia may inhibit these salivary defensive functions against caries formation in ALinduced diabetic rats.
Discussion
In the present study, we discovered that hyperglycemia caused the development of the dental caries and enhanced occlusal surface wear in AL-induced diabetic rats. In rodents, enamel does not exist at the occlusal surface of the cusp of the molar teeth 29 , and occlusal wear spontaneously progresses with age after the eruption of tooth buds [30] [31] [32] . The histological findings of mild tortuosity of the dentinal tubules in the Non-DM group was consistent with that reported in a previous study on occlusal wear due to aging in young intact rats 33 . In the DM group, progressive molar occlusal wear stereoscopically formed a mortar-like shape, where histologically severe tortuosity and disappearance of the dentinal tubules were observed and the cusp height was morphometrically half that in the Non-DM group. From these findings, it is evident that the occlusal surface wear was further enhanced by hyperglycemia in the DM group. In addition, the odontoblast reaction and disarrangement in the pulp reportedly occur with occlusal wear in rats 33 , and these changes may have been similarly elicited by enhanced occlusal wear in the DM group in this study.
Discussion
The pathogenesis of spontaneous occlusal surface wear mainly depends on mastication in both humans and rats 31, 32, 34 . Diabetic animals consume a lot of food 35, 36 , and in this study, food intake in the DM group was significantly greater than that in the Non-DM group. Thus, increase in food intake may naturally increase mastication in diabetic rats. In addition, saliva is a lubricant that reduces friction between the dental occlusal surfaces during mastication 34 . In the diabetic rats in the present study, hyposalivation and increased mastication may have increased the friction, thereby resulting in progressive occlusal surface wear. Furthermore, a number of mineral ions and antibacterial proteins in the saliva reportedly fluctuate in diabetic patients [37] [38] [39] . It is possible that remarkable hyposalivation in the diabetic rats in this study caused quantitative changes in salivary electrolytes and antibacterial proteins, inducing failure of tooth remineralization or caries formation. The hyperglycemic condition suppresses the formation of enamel and dentin in STZ-induced diabetic rats 40 . In AL-induced diabetic rats, metabolic disturbance caused by hyperglycemia could directly weaken the tooth substance, thereby enhancing occlusal surface wear as well as formation of dental caries.
Discussion
In conclusion, hyperglycemia simultaneously induces initial caries development and enhances spontaneous occlusal surface wear of the molars in rats 7 weeks after alloxan treatment. The parotid gland dysfunction caused by hyperglycemia may be involved in the pathogenesis of occlusal wear as well as in dental caries in this diabetic model.

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