Fluoxetine effects on periodontogenesis: histomorphometrical and immunohistochemical analyses in rats

Abstract Reports have indicated that serotonin plays an important role in cell migration and differentiation during the organogenesis of several tissues, including the oral types. Administration of selective serotonin reuptake inhibitor (SSRI) drugs during pregnancy could affect the delivery of serotonin to embryonic tissues altering its development. Objective This study aimed to assess the effects of fluoxetine, a selective serotonin reuptake inhibitor, on the formation of the periodontal ligament during pregnancy and lactation in rat pups. Material and Methods Twelve pregnant rats of Wistar lineage were divided into four study groups. In the control group, 0.9% sodium chloride solution was administered orally, throughout the entire period of the 21 days of pregnancy (CG group) and in the CGL group, it was administrated during pregnancy and lactation (from day 1 of pregnancy to the 21st day after birth). Fluoxetine was administered orally at the dose of 20 mg/kg in a group treated during pregnancy only (FG group), and during pregnancy and lactation (FGL group). Histometrical, histochemical and immunohistochemical analysis of the maxillary first molar periodontium region of the 24 rat pups was made under light microscopy, and periodontal ligament collagen was qualitatively evaluated under a polarizing light microscope. Results The quantity of fibroblasts (p=0.006), osteoblasts (p=0.027) and cementoblasts (p=0.001) was reduced in pups from the rats that received fluoxetine during pregnancy and lactation. No alterations were seen in the collagen fibers. Conclusion These findings suggest that periodontal tissue may be sensitive to fluoxetine, and its interference in reducing periodontal cells depends on exposure time during lactation.


Introduction
There have been an increasing number of prescriptions for antidepressants since 1990, particularly after the introduction of Selective Serotonin Reuptake Inhibitors (SSRIs), which are indicated in clinical situations such as depression, obsessive compulsive and bipolar disorders. In this prescribed drugs for the treatment of depressive disorders, even during pregnancy and lactation 30 .
been reported in newborns, due to its long half-life and clinically active metabolites 28 .
Blood samples from the umbilical cords obtained have shown significant serum levels, equivalent to 60% of the mother's dose 11 provides support for conducting research with the aim of detecting possible teratogenic effects on babies exposed to this drug. Selective serotonin reuptake increases the extracellular concentration of this neurotransmitter, which plays important roles in the regulation of behavior, mood, anxiety, aggressiveness, depression, sleep, fatigue and appetite suppression 6 . Serotonin has also been described as a signal for development, due to its inductive action in tissue proliferation and morphogenesis, neuronal differentiation, cell migration, expression of growth factors, cellular and extracellular adhesion 19 .
A study 21 investigated the pattern of 5HT receptors in the development of rat embryos and verified malformations that involved defects in the neural tube and frontonasal process arising from exposure to 5HT antagonists. The authors concluded that the use of serotonin antagonists modulates the role of this neurotransmitter in the morphogenesis of diverse craniofacial regions, and could have the same effect when these drugs are administered during pregnancy.
In the same study, 5HT receptors were detected in tooth germs and the mandibular mesenchyme, in addition to the endocardium, striated muscle and neural crest.
Evidence has shown the role of serotonin on bone physiology, since it regulates osteoclast differentiation 2, increases the quantity of osteoprotegerin (OPG) and diminishes the osteoclast-activation factor RANKL, 14 .
Furthermore, both the osteoblasts and osteocytes of rats are capable of synthesizing 5-HT, as well as expressing functional components of serotonin receptors and transporters 4 .
Serotonin has also been reported to be a possible modulator of sensorial signals from the masticatory muscles and mechanoreceptors of the periodontal ligament 22 . The presence of serotonin in the sympathetic the relationship of this neurotransmitter with the morphogenesis and homeostasis of oral structures 26 . Since the serotonin system has a regulatory function in the morphogenetic processes of neural and nonneural tissues, including enamel and dentin 10 and as periodontal tissues have been found to be sensitive 5 , it is possible that early exposure to this drug may interfere in periodontogenesis.
In view of the presence of serotonin absorption sites in embryonic dental structures 20 , the hypothesis of the interference of Selective Serotonin Reuptake Inhibitors on periodontium formation is plausible.
However, there are no studies in the literature that have evaluated this hypothesis. Thus, the aim of this on the formation of the periodontium in rats whose during pregnancy and lactation.  The intensity of the staining for each antibody was determined in a semi-qualitative assessment.

Material and methods
Immunoreactivity for collagen type I in the major tissues and cells in the periodontium was characterized by a score (0, +/-, +, ++, +++). The score ranged from negative stain (0) to a very strong positive stain (+++). A score of +/-represents tissues/cells which were weakly positive but some negative, a score of + represents tissue/cells which were positive but with weak staining and a score of ++ represents tissues/ cells which were positive with strong staining.

Statistics
The quantitative data were tabulated using the Software programs SPSS 13.0 for Windows and Excel 2007. The Kolmogorov-Smirnov Test of Normality was applied and comparisons between two groups were made using the Mann-Whitney U test, since the hypothesis of normality of the data was excluded. All of 95% (p<0.05).

Results
The rats had an average of 10 pups per litter, but only the male offspring were included in the study groups. During the experiment one FG group puppy was stillborn.

Histological and biochemical analysis
The histological analysis showed no evidence of any difference in the periodontal morphology between the experimental groups ( Figure 1). In all groups, the with approximately half the root formed and the crown already having erupted. At this stage, the root apex

Histomorphometric analysis
The measurements performed revealed no statistical difference between the CG and FG groups, for any of the analyzed variables (Table 1).

Variables
Group  The histomorphometrical analysis revealed a pups from the FGL group, whose mothers received there were no alterations in the group of animals group).
It has been reported that the effects of the use    There is convincing evidence that serotonin modulators such as SSRIs can affect bone turnover due to the capacity of the 5-HT2BR receptor to facilitate osteoblast recruitment and proliferation 9,13 . that these cells express chains of intercellular signals activated by the serotonin receptor 5HTlc 16

Conclusion
The results obtained suggested that periodontal appears to be dependent on the time of exposure, since only the group exposed during pregnancy and lactation presented a decrease in the quantities of fibroblasts, cementoblasts and osteoblasts.
Despite the cell reduction in the FGL group, the descriptive morphological analysis showed no structural periodontal changes, so further studies must on periodontogenesis.