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Enamel hypoplasia in a litter of rats with alloxan-induced diabetes mellitus. Enamel hypoplasia is an important clinical problem commonly seen in children born to diabetic women. We npj to characterize the enamel hypoplasia in Wistar rats born to alloxan-induced diabetes mellitus rats. Groups consisted of pregnant rats supplemented ISDR or not NISDR with insulin and controls, in which sterile saline solution was administered instead of alloxan or insulin.

The mandibular incisors of one-month-old rats born to these mothers were analyzed. The severity of hypoplasia correlated positively with the maternal level of blood glucose. In conclusion, the intensity of enamel hypoplasia in the teeth of the litter born to alloxan-induced diabetic rats was variable and was dependent on the glycemic level of the pregnant rat. Diabetes mellitus is a heterogeneous clinical syndrome characterized by endocrine and metabolic changes that affect homeostasis.

The main endocrine problem is partial or total insulin deficiency, leading to profound changes in metabolism insuliba carbohydrates, proteins and lipids. From the frequent association of diabetes mellitus and pregnancy, a clinical class termed gestational diabetes mellitus has emerged, which includes patients who develop or are first diagnosed during pregnancy with diabetes mellitus or glucose intolerance 1. The hyperglycemic state during pregnancy has long been recognized as deleterious to fetal development, increasing the risk of birth defects 2,3.

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Although the control of hyperglycemia in patients with gestational diabetes or diabetes mellitus type I has improved in recent years reducing perinatal morbidity and mortality, birth defects have persisted and are now the most important cause of morbidity in children born to diabetic mothers 4. Enamel hypoplasia in primary teeth of children born to diabetic mothers has been reported 5although there is a lack of research on the etiopathogenesis and morphology of this condition both in human and experimental insulima.

Thus, we proposed to determine and characterize morphologically the enamel disorders in the incisor teeth of rats born to mothers with experimentally induced diabetes mellitus. To prevent high infertility and fetal loss, these diabetic rats were supplemented with a daily subcutaneous injection in the dorsum of 1 unit NPH-insulin until mating.

This group was then subdivided into 2 groups: To prevent high mortality in the latter group animals with a lesser degree of hyperglycemia were selected. The control group consisted of 9 rats with the same characteristics in which saline solution substituted the alloxan and insulin. They were kept in groups of 5 animals in metal cages for 1 week for insilina.

Mating was induced by placing 1 male rat in each cage with 5 female iinsulina for 6 days. After this period the female rats were separated and transferred to plastic cages where they were kept until the end of the study.

Rats that did not become pregnant were discarded. Water and commercial rat food were offered ad libitum. The rats were kept on a h light-dark cycle at o C. Glucose blood levels were measured by the glucose oxidase method in all rats at predetermined periods: Blood was taken from the dorsal vein of the tail after a 2-h fasting period, with the animals kept in a heating cage for 5 min.

Animals which did not present hyperglycemia were discarded with their litter. The rats were kept with their litter for 1 month after bla they were sacrificed by ethyl ether inhalation. The mandible from each young rat of the litter was removed and insulinw in the anterior isnulina to free the crowns of the incisor, taking care not to damage it to avoid artifacts.

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Hipoglicemiante

The teeth were properly identified and fixed in Karnovsky’s solution for 48 h. They were then brushed with rubber without any abrasive substance. SEM insulinz began with dehydration in 3 consecutive 2-h baths in absolute ethanol followed by a h bath. The teeth were then removed from the ethanol and placed on filter paper in an oven for 4 h at 40 o C after which they were inaulina with gold for three 3-min cycles Emitech K, Germany.

Blood glucose levels of the 3 npu of rats are shown in Figure 1. One control rat did not become pregnant and was discarded. The remaining 8 had a total of 91 rats, 1 of which died during the neonatal period 1. Three rats from each mother were chosen at random for sacrifice for a total of 24 rats. Although both incisors from each rat were analyzed by SEM we considered only 1 tooth per rat because the same defect was present in both. The ISDR group began with 26 animals but 4 Of the remaining 22, 18 A total of rats were born to these 17 animals, but 17 We chose 3 rats at random from each mother, for a total of 48 rats.

We chose 3 rats at random from each mother, for a total of 6 rats. The teeth were classified by SEM as normal or with defects onsulina ranged from mild to severe.

Normal enamel was characterized by a homogeneous, bu,a, smooth surface with small and regular round pits of varying depth, corresponding to Tomes’ processes of ameloblasts Figure 3A,B. Mild defects were characterized by the presence of occasional irregular n;h superficial depressions with a rough base Figure 3C,D.

Lesions of moderate defects were more widespread and intense, with variable size, form and depth giving a rough appearance to the affected areas Figure 4. Severe hypoplasia was characterized by intense, generalized defects in which regular crystalloid structures in a basket-weave pattern intermixed with filaments could be seen Figure 5. Using SEM, only 1 4. In the ISDR group, 3 6.

Mild hypoplasia was seen in 4 Birth defects associated with maternal diabetes mellitus in experimental animals have long been reported in the literature 3, Diabetes may be experimentally produced surgically by pancreatectomy 9or chemically by administration of streptozotocin 10 or alloxan We chose the latter because it is easy to perform, results in a high percentage of diabetic rats, is similar to human disease and has been widely used at our institution.

The method used was effective in producing hyperglycemia in There is immediate hyperglycemia, which peaks in h, followed by a transitory hypoglycemia due to cell necrosis.

A permanent hyperglycemia is usually present thereafter due to insulin deficit Diabetes mellitus was induced prior to mating to prevent the deleterious acute toxic effects of alloxan on the fetuses Insulin was supplemented until after mating to avoid the negative effects of hyperglycemia on fecundation.

However, failure to achieve pregnancy was higher There is much evidence in the literature of the occurrence of birth defects induced by diabetes mellitus, both in humans ,7 and animals 3,6including agnathia, cleft palate, cranioschisis, craniorhachischisis and occult spina bifida. In this study, in spite of the high blood glucose level in many of the animals during gestation we did not find any rat with birth defects among those that survived.

In fact, because the aim of this study was to induce enamel hypoplasia and not other types of birth defects, we did not check all rats as soon as they were born. It is possible that defective newborn rats had been eaten by the mother. There is evidence that enamel hypoplasia is more likely to be found in primary teeth of children born to diabetic mothers 5, Karim 8 studied the effect of alloxan on the ameloblasts of enamel secretion.

He found some morphologic alterations in these teeth that could be related to both alloxan and hyperglycemia and others only to hyperglycemia. In the present study, we have identified many defects in the enamel of the litter of rats with alloxan-induced diabetes mellitus in which there was only the effect of hyperglycemia because alloxan was administered many days before mating. The macroscopic aspect of the teeth in this study was very similar to that seen in human teeth with enamel hypoplasia.

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The opaque, whitish, chalky enamel is suggestive of poor mineralization, which produces a rather porous surface. The areas of the crown of the teeth without enamel indicate defective production of organic matrix, reducing thickness.

The majority of the studies on enamel hypoplasia are devoted to clinical rather than to morphologic and ultrastructural features. SEM was extremely valuable in the study of this condition because only severe defects of the enamel can be identified macroscopically or even with the aid of a magnifying lens. The frequency of enamel hypoplasia was highly dependent on the severity of hyperglycemia in the mother in both groups. Hypoplasia was more intense in the ISDR group compared to the NISDR group possibly because the hyperglycemia in those animals was higher in spite of insulin supplementation.

The most severe form of hypoplasia was seen in the teeth that were macroscopically abnormal, with a spotted or total whitish surface. The conoid structures arranged in orderly arrays of opposite orientation, like a basket weave, represented enamel prisms intermixed with interprismatic substance, suggesting that the prismless layer was not formed. Since we did not use demineralizing agents these defects may be a consequence of maternal hyperglycemia during pregnancy.

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This interpretation is supported by our observation that the severity of the defects is correlated with the level of maternal hyperglycemia. Alloxan is not related to these findings because it was used only once, weeks before mating.

In both human and experimental diabetes, problems are not mph related to hyperglycemia but to many intracellular metabolic disturbances in consequence to the lack of glucose in the majority of cells.

Metabolites produced in these processes may interfere in other biochemical pathways or induce morphologic alterations in distant places Magnesium disorders may play some role in this model because it is known that hypomagnesemia occurs in diabetic animals 19as well as in diabetic women and their neonates Calcium concentration in the blood inversely reflects the levels of magnesium in both human 20 and animals In an experiment in which rats were fed a diet poor in magnesium, there was a fall in the content of this element but not of bone calcium insulinsbut there are no data on dental mineralization so far.

The present study was not directed at bupa detection of the complex pathogenesis of enamel hypoplasia but rather to its morphologic characterization. Further studies will be needed in order to assess possible mechanisms involved in this lesion.

We conclude that the present study demonstrates that alloxan-induced diabetes mellitus in rats before mating is an efficient model with the advantages of low cost, easily reproducible procedure that requires simple facilities. Insulin supplementation is necessary to minimize animal loss as well as to prevent infertility.

The deleterious effects of alloxan are avoided by its single injection weeks before mating. Enamel hypoplasia, achieved in a high proportion of cases with varying intensity, is at least partly dependent on maternal blood glucose levels and correlates to findings in human cases. Neonatal deaths among infants of diabetic mothers.

Amer J Dis Child ; Experimental diabetes in pregnant mice. Prevention of congenital malformations in offspring by insulin. A new model for study of mild diabetes during pregnancy. Syngeneic islet-trans planted STZ-induced diabetic rats. Influences of age and duration of insulins on dental development in children. J Dent Res ;