Efthymia Kitraki

Effect of neonatal handling on adult rat spatial learning and memory following acute stress

Brief neonatal handling permanently alters hypothalamic-pituitary-adrenal axis function resulting in increased ability to cope with stress. Since stress is known to affect cognitive abilities, in the present study we investigated the effect of brief (15 min) handling on learning and memory in the Morris water maze, following exposure to an acute restraint stress either before training or recall. Exposure of non-handled rats to the acute stress prior to training resulted in quicker learning of the task, than in the absence of the stressor. When acute stress preceded acquisition, male handled rats showed an overall better learning performance, and both sexes of handled animals were less impaired in the subsequent memory trial, compared to the respective non-handled. In addition, the number of neurons immunoreactive for GR was higher in all areas of Ammons horn of the handled rats during the recall. In contrast, the number of neurons immunoreactive for MR was higher in the CA1 and CA2 areas of the non-handled males. When the acute restraint stress was applied prior to the memory test, neonatal handling was not effective in preventing mnemonic impairment, as all animal groups showed a similar deficit in recall. In this case, no difference between handled and non-handled rats was observed in the number of GR positive neurons in the CA2 and CA3 hippocampal areas during the memory test. These results indicate that early experience interacts with sex and acute stress exposure in adulthood to affect performance in the water maze. Hippocampal corticosterone receptors may play a role in determining the final outcome.

POST WEANING HIGH FAT FEEDING AFFECTS RATS' BEHAVIOR AND HYPOTHALAMIC PITUITARY ADRENAL AXIS AT THE ONSET OF PUBERTY IN A SEXUALLY DIMORPHIC MANNER

Feeding adult rats with high fat (HF) diets can alter their hypothalamic pituitary adrenal (HPA) axis responsiveness. In the present study, we examined the effect of a high fat diet, applied in rats from weaning to puberty, on their behavior and HPA axis status at puberty onset. Wistar rats of both sexes were fed postweaning with two diets containing either 24% fat (high fat, HF) or 4.3% fat (normal chow) by weight. HF enhanced puberty onset in female rats, without increasing body weight gain in either sex, compared with chow-fed animals. In the forced swim test, HF males exhibited a more active behavioral response on the first day, whereas HF females a more passive response during the second day of the test, as compared with their chow-fed counterparts. In the open field test, HF females showed increased sniffing but reduced rearing, compared with chow-fed females and were less explorative than HF males in the central arena. All animals could learn and recall a water maze task though HF males spent more time in the opposite quadrant than chow-fed males during memory test. The HPA axis status of these animals was investigated under basal conditions. Pubertal fat-fed males had lighter adrenals, while females heavier ones, compared with their counterparts. In addition, plasma corticosterone levels of female rats were increased and glucocorticoid receptor levels in their hypothalamus were reduced due to fat diet, while in males no such changes were detected. We conclude that HF feeding during the prepubertal period can affect behavior and the HPA axis of rats at puberty onset, well before the appearance of the obese state, in a sexually dimorphic manner. Fat diet impacted more the female HPA axis, suggesting that their system is more sensitive to fat-induced nutritional imbalance during adolescence. Present data suggest that the fat-induced nutritional imbalance in young females may lead to neuroendocrine dysfunction that in turn may trigger the appearance of stress-related disorders during adolescence.

Developmental exposure to bisphenol A alters expression and DNA methylation of Fkbp5, an important regulator of the stress response.

Bisphenol A (BPA), an abundant endocrine disruptor, affects stress-responsiveness and related behaviors in children. In rats, perinatal BPA exposure modifies stress response in pubertal offspring via unknown mechanisms. Here we examined possible epigenetic modifications in the glucocorticoid receptor gene and its regulator Fkbp5 in hypothalamus and hippocampus of exposed offspring. We found increased DNA methylation of Fkbp5 and reduced protein levels in the hippocampus of exposed male rats. Similar effects were obtained in a male hippocampal cell line when exposed to BPA during differentiation. The estrogen receptor (ER) antagonist ICI 182,780 or ERβ knock-down affected Fkbp5 expression and methylation similarly to BPA. Further, BPAs effect on Fkbp5 was abolished upon knock-down of ERβ, suggesting a role for this receptor in mediating BPAs effects on Fkbp5. These data demonstrate that developmental BPA exposure modifies Fkbp5 methylation and expression in male rats, which may be related to its impact on stress responsiveness.

Tooth eruption: altered gene expression in the dental follicle of patients with cleidocranial dysplasia

OBJECTIVES The dental follicle plays an important role in tooth eruption by providing key regulators of osteogenesis and bone resorption. Patients with cleidocranial dysplasia (CCD) exhibit delayed tooth eruption in combination with increased bone density in the maxilla and mandible, suggesting disturbances in bone remodeling. The aim of this study was to determine the expression of genes relevant for tooth eruption and bone remodeling in the dental follicles of patients with CCD and normal subjects. MATERIAL AND METHODS Thirteen dental follicles were isolated from five unrelated patients with CCD, and fourteen dental follicles were obtained from 10 healthy individuals. All teeth were in the intraosseous phase of eruption. The expression of RANK, RANKL, OPG, and CSF-1 was determined by quantitative RT-PCR. RESULTS In patients with CCD, the mRNA levels of RANK, OPG, and CSF-1 were significantly elevated compared with the control group. Accordingly, the ratios of RANKL/OPG and RANKL/RANK mRNAs were significantly decreased in patients with CCD. CONCLUSION The observed alterations in the expression and ratios of the aforementioned factors in the dental follicle of CCD individuals suggest a disturbed paracrine signaling for bone remodeling that could be responsible for the impaired tooth eruption seen in these patients.

Endoplasmic reticulum stress and mineralization inhibition mechanism by the resinous monomer HEMA

AIM To investigate the expression of two endoplasmic reticulum (ER)-resident key chaperone proteins, ERdj5 and BiP, under the influence of resinous monomers and its relationship with the inhibition of mineralization caused by the monomer 2-hydroxyethyl methacrylate (HEMA). METHODOLOGY The ERdj5 and BiP expression was studied in vitro, in primary human pulp cell cultures after treatment with three different HEMA concentrations at different time periods. Subsequently, the expression of both the odontoblast markers dentine sialoprotein (DSP) and osteonectin (OSN) was studied in human pulp cells under the same conditions. RESULTS The ERdj5 and BiP expression was upregulated in the pulp cells. DSP and OSN were largely dispersed in the cytoplasm in control cell cultures but accumulated in a perinuclear area after exposure to HEMA. Their expression levels were not affected. CONCLUSIONS The increased expression of ERdj5 and BiP may reflect activation of ER stress. DSP and OSN accumulation into the cells may lead to their secretion arrest and inhibition of dentine matrix formation. These events may elucidate the mechanism by which HEMA inhibits the mineralization process.

Psychometric and biohormonal indices of dental anxiety in children. A prospective cohort study

Abstract The stress of dental treatment often elicits negative emotions in children, expressed as dental fear or anxiety. Highly anxious children obstruct treatment and avoid therapy, further amplifying oral health problems. The aim of this study was to examine the neuroendocrine and autonomic nervous system responses to dental treatment and their possible interactions and associations with psychometric indices of anxiety, caries, previous dental experience, anesthesia, age and gender in school children. Upon informed consent, saliva was obtained from 97 children (59% males, mean age ±  SD: 89.73 ± 15 months) in the Clinic of pediatric dentistry before treatment, immediately post-treatment and at the recall visit to determine cortisol and salivary alpha-amylase (sAA) levels. Dental and general anxiety was assessed through specific questionnaires completed by the children. Compared to pre-treatment, cortisol levels were increased following treatment, while sAA levels were higher at the recall. Pre- and post-treatment cortisol and sAA responses were positively correlated. Dental and general anxiety questionnaire scores were also significantly correlated with each other. The integrated autonomic and neuroendocrine responses prior to treatment were correlated with state anxiety and those following treatment with dental anxiety. However, univariable and multivariable linear regression analysis associated post-treatment cortisol, but not sAA, levels with dental anxiety. No associations of cortisol or sAA responses with caries, age, gender, previous dental experience or anesthesia were detected. These data provide some evidence that both sAA and cortisol levels are altered in children in anticipation or during dental treatment, but only cortisol levels are associated to dental anxiety.

BPA Effects In Vivo: Evidence from Animal Studies

Bisphenol A (2,2-bis-4-hydroxyphenyl-propane, BPA), is a well-known endocrine disruptor that is used as a monomer in the manufacture of dental sealants, epoxy resins and polycarbonate plastics that have extensive use in dentistry or medicine, in food packaging industry and in plastics’ production. BPA is contained in many everyday life items, such as house plasticware and baby bottles, from where it is released, for example, by heating, resulting in food or drink contamination. Leached components from dental composites and sealants in the oral cavity are also considered a possible source of human exposure. BPA exposure can also occur by inhalation of contaminated air, for example, from decomposed monomers during medical or dental practice [1].

Early responses of human pulp to direct capping with resin adhesive systems and calcium hydroxide

OBJECTIVE Early responses of human pulp to Prime&Bond/phosphoric acid, Clearfil SE Bond, Clearfil S3 Bond and Dycal were investigated ex vivo. MATERIALS AND METHODS The three adhesives, Dycal or buffer (DPBS) were applied directly onto the pulp of human teeth slices that were placed in culture for 4 days. Cell viability was monitored by the MTT assay during the culture period. After 4 days, tissue integrity was examined by hematoxylin-eosin staining. Vimentin levels were assessed by Western blotting. TUNEL assay was applied for apoptotic cell detection at specific pulp areas. RESULTS Profound reduction of cell viability and tissue integrity was observed in adhesive-treated groups, while the impact of Dycal was found to be less harmful. Extended apoptosis was caused mostly by the Clearfil SE and Prime&Bond. All adhesives reduced Vimentin levels. SIGNIFICANCE The study provides evidence that early pulp responses to direct capping with different adhesive systems or calcium hydroxide may vary significantly and underline the need for further studies in relevant ex vivo systems.

From Cohorts to Molecules: Adverse Impacts of Endocrine Disrupting Mixtures

Convergent evidence associates endocrine disrupting chemicals (EDCs) with major, increasingly-prevalent human disorders. Regulation requires elucidation of EDC-triggered molecular events causally linked to adverse health outcomes, but two factors limit their identification. First, experiments frequently use individual chemicals, whereas real life entails simultaneous exposure to multiple EDCs. Second, population-based and experimental studies are seldom integrated. This drawback was exacerbated until recently by lack of physiopathologically meaningful human experimental systems that link epidemiological data with results from model organisms. We developed a novel approach, integrating epidemiological with experimental evidence. Starting from 1,874 mother-child pairs we identified mixtures of chemicals, measured during early pregnancy, associated with language delay or low-birth weight in offspring. These mixtures were then tested on multiple complementary in vitro and in vivo models. We demonstrate that each EDC mixture, at levels found in pregnant women, disrupts hormone-regulated and disease-relevant gene regulatory networks at both the cellular and organismal scale.

Dental Stem Cells for Bone Regeneration

Successful bone repair is of immense clinical importance. Bone fractures are among the most common injuries encountered and inappropriate healing, estimated to 5–10 %, is expected to further increase along with the increase of the aging population. Cell-based therapies provide an alternative to bone grafting by preventing bone loss and reducing grafting morbidity. Apart from the bone marrow mesenchymal stem cells, a multitude of mesenchymal stem cells from different tissues exhibit osteogenic potential. Among them, mesenchymal stem cells of dental origin, initially isolated but not restricted to the dental pulp, seem to hold a prominent position in the field of bone regeneration. This chapter aims to present the different types of dental stem cells with emphasis on their osteogenic properties and in vivo applications in animal models of bone repair.