Occlusal Disharmony Transiently Impairs Learning and Memory in the Mouse by Increasing Dynorphin A Levels in the Amygdala.

Occlusal disharmony sometimes causes not only stiffness of neck but also psychiatric depression, suggesting that the condition of oral cavity may affect the central nervous system. Dynorphin A is an endogenous opioid peptide that specifically binds the κ-opioid receptor and has a protective role against stress. Dynorphinergic nervous system is intensely distributed in the amygdala and hippocampus that are coping areas with stress. As a model of malocclusion, we placed dental resin on the molars to increase the occlusal vertical dimension (bite-raise). After various survival times, we analyzed the amygdala and hippocampus by immunohistochemistry and immunosorbent assay (ELISA). Furthermore, the effects on learning and memory were assessed by Morris water maze test. In the amygdala, the levels of dynorphin A were increased on the 1st day after increasing the vertical dimension as indicated by immunohistochemical and ELISA assessments. The levels of dynorphin A returned to control levels on the 5th day. In the hippocampus, there were no noticeable changes in dynorphin A levels. The water maze test indicated that increasing the vertical dimension caused longer escape latency times on the 3rd day compared to those of sham-operated group. However, the bite-raised mice treated with a dynorphin antagonist, nor-binaltorphimine, showed similar escape latency times to the times of sham-operated group, even on the 3rd day. These results suggest that occlusal disharmony causes stress resulting in a transient increase of dynorphin A levels at least in the amygdala and that the increased dynorphin A levels transiently impair learning and memory.

Age estimation in forensic sciences: application of combined aspartic acid racemization and radiocarbon analysis.

Age determination of unknown human bodies is important in the setting of a crime investigation or a mass disaster because the age at death, birth date, and year of death as well as gender can guide investigators to the correct identity among a large number of possible matches. Traditional morphological methods used by anthropologists to determine age are often imprecise, whereas chemical analysis of tooth dentin, such as aspartic acid racemization, has shown reproducible and more precise results. In this study, we analyzed teeth from Swedish individuals using both aspartic acid racemization and radiocarbon methodologies. The rationale behind using radiocarbon analysis is that aboveground testing of nuclear weapons during the cold war (1955-1963) caused an extreme increase in global levels of carbon-14 ((14)C), which has been carefully recorded over time. Forty-four teeth from 41 individuals were analyzed using aspartic acid racemization analysis of tooth crown dentin or radiocarbon analysis of enamel, and 10 of these were split and subjected to both radiocarbon and racemization analysis. Combined analysis showed that the two methods correlated well (R(2) = 0.66, p < 0.05). Radiocarbon analysis showed an excellent precision with an overall absolute error of 1.0 +/- 0.6 years. Aspartic acid racemization also showed a good precision with an overall absolute error of 5.4 +/- 4.2 years. Whereas radiocarbon analysis gives an estimated year of birth, racemization analysis indicates the chronological age of the individual at the time of death. We show how these methods in combination can also assist in the estimation of date of death of an unidentified victim. This strategy can be of significant assistance in forensic casework involving dead victim identification.

Occlusal disharmony in mice transiently activates microglia in hippocampal CA1 region but not in dentate gyrus.

Occlusal disharmony is induced by various conditions such as the loss of teeth and inappropriate vertical dimension of crowns, bridges, or dentures. Occlusal disharmony sometimes causes indefinite complaint syndromes, which may be associated with astrocytic hypertrophy and the reduction of numbers of neuronal somata and their dendritic spines in the hippocampus. Microglia monitors the condition of neurons and responds to their degeneration accompanying with astrocytes. However, the effect of occlusal disharmony on the microglia has not yet been investigated. We artificially increased the occlusal vertical dimension by placing dental resin on the upper molars in mice and immunohistochemically investigated the effects of the increase in the vertical dimension on microglia of the hippocampal formation using an antibody against ionized calcium-binding adaptor molecule 1 (Iba-1), a marker protein for microglia. We measured the area occupied by Iba-1-immunoreactive microglia in the hippocampal CA1 region and dentate gyrus 1, 3, and 5 days after increasing the vertical dimension, and compared it with that of control mice. The hippocampal CA1 region contains vulnerable neurons and the dentate gyrus durable neurons. We found that the areas occupied by microglia in the hippocampal CA1 region increased, with the peak on the third day after increasing the vertical dimension, and it gradually declined by the fifth post-operative day. However, such an increase of the area occupied by microglia was not seen in the dentate gyrus. In conclusion, abnormal mastication may activate microglia in the area harboring vulnerable neurons, but not in the area harboring durable neurons.

Effect of social isolation stress on saliva BDNF in rat.

Salivary glands produce various compounds, including brain-derived neurotrophic factor (BDNF), which serve as biomarkers of stress-related disorders. Social isolation-induced stress models a form of chronic mild stress that induces neurodegenerative changes in the brain and behavioral alterations. This study employed a rat model to determine whether social isolation stress affects BDNF levels in saliva. Male Sprague-Dawley rats were randomly allocated to social isolation stress (1 animal/cage) or control (3-4 animals/cage) groups and reared for 8 weeks. The concentration of BDNF was quantified in specific brain regions, blood, and saliva using enzyme-linked immunosorbent assay (ELISA). The levels of expression of Bdnf and tyrosine kinase B (TrkB) mRNA were quantified using reverse transcriptase-polymerase chain reaction. Behavioral alterations were analyzed using the open-field and elevated plus maze assays. The BDNF concentration was lower in the hippocampus, prefrontal cortex, blood, and saliva of the stress group than in those of the controls. Trkb expression in the hippocampus and prefrontal cortex was decreased by social isolation stress. Moreover, the social isolation stress group showed behavioral deficits in both tests. In conclusion, these findings indicate that social isolation stress may reduce the expression of BDNF protein in blood and saliva, thus providing a potentially valuable biomarker for diagnosis of stress-related disorders.

Regional differences in D/L aspartic acid ratios in the human mandible as a possible indicator of the bone remodeling rate.

In slowly metabolizing tissue such as tooth, bone and ocular lens, D-amino acids converted from L-amino acids accumulate with age and thus reflect the tissue turnover rate. To investigate whether D-amino acids play a role in determining the bone remodeling rate, we measured the accumulation of D-aspartic acid, which has the fastest rate of racemization, in various areas of the mandible. The level of D-aspartic acid was higher in the ramus than in the body (P < 0.01), and within the body, the level was higher in the basal area than in the alveolar area (P < 0.01). Within the alveolar area, the level of D-aspartic acid was higher in the molar region than in the incisal region (P: 0.05-0.01). No correlation was found between the accumulated level of D-aspartic acid with age, because all the specimens were obtained from elderly people with only a few years difference in age. There was also no correlation between D-aspartic acid and sex. In conclusion, we suggest that accumulation of D-aspartic acid in the mandibular bone reflects the differences in remodeling associated with occlusion. The incisal portion of the alveolar area of the mandible (above the mandibular canal), shows the greatest evidence of active remodeling.

Comparison of aspartic acid racemization between mammoth and human dentinal tissues.

OBJECTIVE: Comprehensive research has revealed the complexity and heterogeneity of amino acid racemization (AAR) in biological samples. Therefore, to date, inherent uncertainties in relative dating as well as in determination of age at death have highlighted the significance of further investigations to explore aspartic acid kinetics in biominerals. Here we present data on an archaeological sample obtained from a mammoth tusk, in comparison to a previously well-described biomineral, to modern human dentin. DESIGN: Since no experimental investigations were carried out on this topic earlier, we provide the first estimates on racemization kinetics and time dependent tendencies in mammoth dentin. We analysed our samples using a protocol, which has been adopted for tooth dentin investigation. We measured different racemization rates amongst mammoth dentin layers. RESULTS AND CONCLUSIONS: Significantly, an overall low racemization rate was detected compared to human dentin. Kinetic experiments were performed by heating the samples, and linear correlations were established between racemization rates and heating time. The results allow us to derive racemization rate constants and activation energy for both samples. Extrapolation for ambient temperature showed increased velocity in mammoth dentin, which can be explained by biodeterioration of ancient collagen and by diagenetic changes in amino acid composition influenced by environmental conditions.

Age-dependent changes in the racemisation ratio of aspartic acid in human alveolar bone.

We investigated the racemisation ratio of aspartic acid (Asp) in alveolar bone. In addition, we designed and created a new column to detect Asp in a short period of time, which allowed us to detect d-Asp and l-Asp separately from each other within 5min. Comparing identical ages, the racemisation ratio of alveolar bone was generally lower than that of other bones reported so far. This result suggests that alveolar bone is metabolically more active than other bones, as expected. The rate constant for the racemisation reaction (k(y)) of alveolar bone was calculated to be 0.000338 in males and 0.000084 in females. The rate constants in males and females were each similar to the respective ratios of the femur. This result suggests that the age-dependent reduction in metabolic turnover in alveolar bone proceeds similarly to that in the femur, although those changes proceed more slowly in females than in males. The correlation coefficient between the racemisation ratio of alveolar bone and chronological age was 0.660. It was high in males (r=0.912) and low in females (r=0.527), and this gender difference was statistically significant (P: 0.01-0.001), as in the femur.

Effects of occlusal disharmony on the hippocampal dentate gyrus in aged senescence-accelerated mouse prone 8 (SAMP8).

BACKGROUND AND OBJECTIVE: Malocclusion induced by raising the bite causes chronic stress. Chronic stress leads to increased plasma corticosterone levels and impaired hippocampal function due to impaired neurogenesis or increased apoptosis in the hippocampus. The present study aimed to clarify the mechanisms underlying the impaired hippocampal function induced by the bite-raised condition in aged senescence-accelerated mouse prone 8 (SAMP8). DESIGN: Nine-month-old aged SAMP8 mice were randomly divided into control and bite-raised groups. The vertical dimension of the bite was raised by applying resin to the molars. We evaluated newborn cell proliferation, survival, differentiation, and apoptosis in the hippocampal dentate gyrus (DG). Hippocampal brain-derived neurotrophic factor (BDNF) levels were also measured. RESULTS: The bite-raised mice exhibited a significant decrease in proliferation, survival, and differentiation of newborn cells into neurons in the hippocampal DG compared with controls. The number of apoptotic cells in the hippocampal DG was increased at 7 and 14 days after the bite-raising procedure. Expression of BDNF protein and mRNA in the hippocampus was also decreased in the bite-raised mice. CONCLUSION: Bite-raised aged SAMP8 mice exhibited decreased neurogenesis, increased apoptosis in the hippocampal DG, and decreased hippocampal BDNF expression, in association with hippocampus-dependent learning and memory deficits.

Strategy for the estimation of chronological age using the aspartic acid racemization method with special reference to coefficient of correlation between D/L ratios and ages.

The estimation of chronological age has been performed by various methods in forensic science. Among these, racemization methods, which are based on the age-dependent non-enzymatic changes of L-form amino acids to D-form mainly using aspartic acid, are one of the most reliable and accurate methods to date. Separation of enantiomers is generally performed by gas chromatography or high performance liquid chromatography. Various tissues with low metabolic rates have been applied for this purpose. In addition, single proteins purified from these target tissues are also applicable. In this brief review we describe this method in detail, noting points of caution, as well as the advantages and disadvantages of the different target tissues. In addition, special attention is given to the correlation rates obtained between chronological age and enantiomer ratios. Currently, based on accuracy of estimated age, simplicity of the method, time required, and reproducibility, tooth dentin is considered one of the best target tissues. Alternatively, analysis of osteocalcin and elastin have also provided accurate and reproducible results.

An application of D- and L-aspartic acid mixtures as standard specimens for the chronological age estimation.

Instead of using the control teeth, we tried to prepare standard specimens to derive the equations for calculation of the age (analytical curves) with respect to each kind of tooth of various ages. To prepare standard specimens, we determined the racemization ratio of the teeth of known age that had actually been used for the appraisal of chronological age (total control teeth). Then we mixed commercially available L-Asp and D-Asp in the same D/L ratio as the measured one in the total control teeth. As a result, we were able to obtain the racemization rate equations from the age-specific standard specimens of central and lateral incisors. These equations were closely similar to those derived from actual teeth of known age. Since the racemization rate equations obtained from the standard specimens were satisfactorily reproducible, we assumed that these equations could be used in place of those obtained from the control teeth. Actually, in the age estimation of unidentified corpses from teeth, the use of standard specimens enabled us to estimate the age almost as precisely as estimated using the control teeth. Thus, the present study has demonstrated that in the estimation of chronological age the control teeth can be substituted by the standard specimens. This shows the possibility of using the standard specimens also in other laboratories where the racemization ratio can be measured with sufficient reproducibility.

The endogenous opioids related with antinociceptive effects induced by electrical stimulation into the amygdala.

Pain relief is necessary and essential for dental treatments. Recently, the relationships of pain and emotion were studied, and electrical stimulation applied to the amygdala depressed the nociceptive response in the anterior cingulate cortex (ACC). Thus, the antinociceptive effects of the amygdala are elucidated, but its mechanism is not yet clarified. The present study was performed to investigate whether endogenous opioid system is related to the depression, and the quantitative changes of endogenous opioids induced by electrical stimulation to the amygdala. We investigated immunohistologically c-Fos expression to confirm the activated neurons, as well as the distribution and the amount of endogenous opioids (ß-endorphin, enkephalin and dynorphin A) in the brain using male Wistar rats, when electrical stimulation was applied to the central nucleus of the amygdala (CeA) or noxious stimulation was delivered to the peripheral tissue. c-Fos expression in the ipsilateral ACC was increased by electrical stimulation to the CeA. However, only a small amount of endogenous opioids was observed in the ACC when noxious stimulation or electrical stimulation was applied. In contrast, the amount of dynorphin A in the periaqueductal gray (PAG) was increased by electrical stimulation to the CeA, and the amount of ß-endorphin in the PAG was increased by noxious stimulation to the peripheral tissue. The results suggest that dynorphin A in the PAG induced by electrical stimulation to the CeA activate the descending antinociceptive system, and suggest that the nociceptive response in the ACC is depressed indirectly.

Estimation of chronological age from the racemization rate of L- and D-aspartic acid: how to completely separate enantiomers from dentin.

Estimation of chronological age is essential in forensic and archeological science. The racemization method is one of the best methods to meet the demands of these scientific fields, providing both accuracy of the estimated age and simplicity of technique. In general, living organs are composed of L-form amino acids. Conversion from L-form to D-form amino acids is a first-order chemical reaction. Thus, the quantity of D-form amino acids in an organ is proportional to the passed time (age) after organ completion if no protein turnover occurs after organization. However, every living organ undergoes some degree of protein turnover. Therefore, organs with low metabolic rates, such as teeth and bone, should be targeted for the racemization method. The most critical point of the technique may be the complete separation of D- and L-forms by gas chromatography because of the very small amounts of D-form amino acids present. We describe the detailed procedures and the critical points for obtaining reliable estimated ages using the racemization method.

Comparison of age estimation in Japanese and Scandinavian teeth using amino acid racemization.

Racemization method is currently considered to be one of the most accurate methods for age estimation. There are many factors that affect racemization reaction velocities. We investigated ethnic differences in the racemization reaction velocities between Japanese and Scandinavian teeth. After a heating experiment, the Arrhenius equation was used to obtain a rate constant (k). Both groups presented almost identical velocities, indicating that there was no difference in the tooth racemization reaction between the two groups. However, because 14 of the 18 Scandinavian teeth had multiple roots, it is possible that accurate racemization rates of the teeth themselves were not reflected in the results. These findings reconfirm that the type of tooth selected is extremely important when evaluating age by racemization.

Use of moderate sedation for a patient with Down syndrome, intellectual disability, and Eisenmenger syndrome: a case report.

For patients who have Eisenmenger syndrome (ES), perioperative risks are high even for noncardiac surgery, such as dental extractions. We report on the case history of a 38-year-old male patient with Down syndrome (DS), intellectual disability (ID), and ES. The patient was scheduled for extraction of the right maxillary second molar tooth. His physical health was poor. Following oxygenation, midazolam was administered intravenously very slowly until the optimum sedative level was obtained, with a total dosage of 3.5 mg. There were no marked changes in vital signs during the perioperative period, and the patient was discharged the same day. This case suggests that moderate or conscious sedation using midazolam for dental treatment of a patient with DS, ID, and ES was well tolerated. Several critical points are presented in this review.

Age estimation by amino acid racemization in human teeth.

When an unidentified body is found, it is essential to establish the personal identity of the body in addition to investigating the cause of death. Identification is one of the most important functions of forensic dentistry. Fingerprint, dental, and DNA analysis can be used to accurately identify a body. However, if no information is available for identification, age estimation can contribute to the resolution of a case. The authors have been using aspartic acid racemization rates in dentin (D-aspartic acid/L-aspartic acid: D/L Asp) as an index for age estimation and have obtained satisfactory results. We report five cases of age estimation using the racemization method. In all five cases, estimated ages were accurate within a range ±3 years. We conclude that the racemization method is a reliable and practical method for estimating age.

Molarless condition suppresses proliferation but not differentiation rates into neurons in the rat dentate gyrus.

The dentate gyrus (DG) of the hippocampal complex is one of the few areas of the rodent brain where neurogenesis continues throughout adulthood. We investigated the effects of the molarless condition on cell proliferation, rate of differentiation into neurons in the subgranular zone of the DG, and plasma corticosterone levels. The molarless condition decreased cell proliferation in the DG and increased plasma corticosterone levels. Approximately 80% of newly generated cells differentiated into neurons and the remaining 20% of the cells differentiated into astrocytes. These ratios were not significantly different between control and molarless rats. In conclusion, the rates of neurogenesis and gliogenesis in the DG are suppressed by the molarless condition, and this suppression might be associated with the increased corticosteroid levels in molarless subjects.

Racemization in enamel among different types of teeth from the same individual.

We measured the quantity of D-aspartic acid (degree of racemization of aspartic acid) in the enamel of different types of teeth from the same individual. We studied the correlation between the degree of racemization and the time of formation of each particular tooth, as well as the applicability of the degree of racemization to estimation of chronological age. If the environmental condition of the teeth is the same, the degree of racemization is expected to be highest in teeth that completed formation in the earliest period of time. Different degrees of racemization in enamel were found among different types of teeth, even in the same individual. The degree of racemization in enamel was found to be higher in molars than in incisors, and showed a tendency that did not necessarily coincide with the time of formation. This seemed to be due to the fact that the environmental temperature was higher in the molar region located deeper in the oral cavity than the front region, and that enamel was more affected by breathing air than dentin because the D/L ratios in enamel were lower than those in dentin. Using enamel, a better estimation of chronological age was obtained from calculations based on the degree of racemization of each type of tooth than from all the different teeth together. However, these estimated ages were not better than those from dentin.