Long-term sorbitol consumption affects the hippocampus and alters cognitive function in aged mice.

The systemic effects of the artificial sweetener sorbitol on older adult individuals have not been elucidated. We assessed the effects of sorbitol consumption on cognitive and gingival health in a mouse model. Aged mice were fed 5% sorbitol for 3 months before their behavior was assessed, and brain and gingival tissues were collected. Long-term sorbitol consumption inhibited gingival tissue aging in aged mice. However, it caused cognitive decline and decreased brain-derived neurotrophic factor (BDNF) in the hippocampus. Sorbitol consumption did not affect homeostatic function; however, it may exert effects within the brain, particularly in the hippocampus.

Premature gray hair development in the interbrow region owing to the loss of maxillary first molars in young mice.

The exact sites of premature hair graying and whether tooth loss causes this condition remain unknown. In this study, we aimed to explore the effect of reduced mastication on premature hair graying. Maxillary first molars were extracted from young mice, and the mice were observed for 3 months, along with non-extraction control group mice. After 3 months, gray hair emerged in the interbrow region of mice in the tooth extraction group but not in the control group. The expression of tyrosinase-related protein-2 (TRP-2) mRNA was lower in the interbrow tissues of young mice without maxillary molars than in those with maxillary molars. Tooth loss leads to interbrow gray hair growth, possibly because of weakened trigeminal nerve input, suggesting that reduced mastication causes premature graying. Thus, prompt prosthetic treatment after molar loss is highly recommended.

An in vitro senescence model of gingival epithelial cell induced by hydrogen peroxide treatment.

Gingival tissue shows progressive changes with aging and an in vitro model of gingival tissue could be useful in understanding age-associated oral diseases. The present study aims to establish a hydrogen peroxide (H2O2) treatment model to induce aging in human gingival epithelial cells. In addition, fisetin, a flavonoid component studied for the anti-aging property is used to examine if it could reverse the induced senescence. Primary human gingival epithelial progenitor (HGEPp) cells were cultured and treated with different concentrations of H2O2. A cell vitality and morphology, senescence-associated beta-galactosidase (SA-ß-gal) staining, mRNA and protein expression analysis of known senescence markers p16, p21, and p53, and cell cycle assay were performed. The cells showed dose-dependent changes in vitality and morphology, SA-ß-gal staining, relative mRNA and protein expression, and cell cycle assay after H2O2 treatment. Based on these results, 400 µM H2O2 was considered as an optimal concentration to induce senescence. Treatment of senescence-induced cells with fisetin downregulated all the senescence markers used in this study. In conclusion, a senescence model of gingival epithelial cells induced by hydrogen peroxide treatment was established which could be employed to study age-related periodontal diseases.

Chemokines Up-Regulated in Epithelial Cells Control Senescence-Associated T Cell Accumulation in Salivary Glands of Aged and Sjögren's Syndrome Model Mice.

Immunosenescence is characterized by age-associated changes in immunological functions. Although age- and autoimmune-related sialadenitis cause dry mouth (xerostomia), the roles of immunosenescence and cellular senescence in the pathogenesis of sialadenitis remain unknown. We demonstrated that acquired immune cells rather than innate immune cells infiltrated the salivary glands (SG) of aged mice. An analysis of isolated epithelial cells from SG revealed that the expression levels of the chemokine CXCL13 were elevated in aged mice. Senescence-associated T cells (SA-Ts), which secrete large amounts of atypical pro-inflammatory cytokines, are involved in the pathogenesis of metabolic disorders and autoimmune diseases. The present results showed that SA-Ts and B cells, which express the CXCL13 receptor CXCR5, accumulated in the SG of aged mice, particularly females. CD4+ T cells derived from aged mice exhibited stronger in vitro migratory activity toward CXCL13 than those from young mice. In a mouse model of Sjögren's syndrome (SS), SA-Ts also accumulated in SG, presumably via CXCL12-CXCR4 signaling. Collectively, the present results indicate that SA-Ts accumulate in SG, contribute to the pathogenesis of age- and SS-related sialadenitis by up-regulating chemokines in epithelial cells, and have potential as therapeutic targets for the treatment of xerostomia caused by these types of sialadenitis.

Correction: Furukawa et al. Long-Term Soft-Food Rearing in Young Mice Alters Brain Function and Mood-Related Behavior. Nutrients 2023, 15, 2397.

In the original publication [...].

Oral infection with Porphyromonas gingivalis augmented gingival epithelial barrier molecules alteration with aging.

OBJECTIVE: Disruption of the gingival epithelial barrier is often mediated by aging or the pathogen Porphyromonas gingivalis. This study examined the combined effects of aging and P. gingivalis exposure on gingival epithelial barrier molecules. METHODS: In vitro experiments involved treating young- and senescence-induced primary human gingival epithelial progenitor cells (HGEPp) with P. gingivalis lipopolysaccharide (LPS). Transepithelial electrical resistance (TER) and paracellular permeability were measured. In vivo, male C57BL/6J mice aged 10 (young) and 80 (old) weeks were divided into four groups: young, old, young with P. gingivalis (Pg-Young) inoculation, and old with P. gingivalis (Pg-Old) inoculation. P. gingivalis was inoculated orally thrice a week for 5 weeks. The mice were sacrificed 30 days after the last inoculation, and samples were collected for further procedures. The junctional molecules (Claudin-1, Claudin-2, E-cadherin, and Connexin) were analyzed for mRNA expression using qRT-PCR and protein production using western blotting and immunohistochemistry. The alveolar bone loss and inflammatory cytokine levels in gingival tissues were also assessed. RESULTS: LPS-treated senescent cells exhibited a pronounced reduction in TER, increased permeability to albumin protein, significant upregulation of Claudin-1 and Claudin-2, and significant downregulation of E-cadherin and Connexin. Furthermore, the Pg-Old group showed identical results with aging in addition to an increase in alveolar bone loss, significantly higher than that in the other groups. CONCLUSION: In conclusion, the host susceptibility to periodontal pathogens increases with age through changes in the gingival epithelial barrier molecules.

Effects of tooth loss on behavioral and psychological symptoms of dementia in app knock-in mice.

OBJECTIVES: Many patients with Alzheimer's disease (AD) experience behavioral and psychological symptoms of dementia (BPSD), which significantly affect their quality of life. It is known that 5-Hydroxytryptamine (5-HT) plays a crucial role in the development of BPSD. While the relationship between tooth loss and AD symptoms has been acknowledged, the aspect of aggression has not been focused on until now. Despite the established importance of 5-HT in BPSD, how tooth loss is related to the exacerbation of AD symptoms, especially in terms of aggression, remains largely unexplored. Although nutritional status is known to influence the progression of dementia, the specific effect of tooth loss on peripheral symptoms, notably aggression, is not well understood. METHODS: In our study, we conducted maxillary molar extractions in aged C57BL/6J and AppNL-G-F mice and observed their condition over a 3-month period. During this time, we documented significant behavioral and genetic differences between mice in the control groups and mice that underwent tooth extraction. Notably, mice that underwent tooth extraction exhibited a considerable decline in cognitive function and increased in aggression 3 months after tooth extraction compared with the control groups (C57BL/6J and AppNL-G-Fmice). RESULTS: Our findings suggest that molar loss may lead to reduced 5-HT levels in the hippocampus, possibly mediated by the trigeminal nerve, contributing to the development of aggression and BPSD in AD. CONCLUSION: This study sheds light on the intricate relationships between oral health, 5-HT, and AD symptoms, offering valuable insights into potential therapeutic avenues for managing BPSD in patients with dementia.

Long-Term Soft-Food Rearing in Young Mice Alters Brain Function and Mood-Related Behavior.

The relationship between caloric and nutrient intake and overall health has been extensively studied. However, little research has focused on the impact of the hardness of staple foods on health. In this study, we investigated the effects of a soft diet on brain function and behavior in mice from an early age. Mice fed a soft diet for six months exhibited increased body weight and total cholesterol levels, along with impaired cognitive and motor function, heightened nocturnal activity, and increased aggression. Interestingly, when these mice were switched back to a solid diet for three months, their weight gain ceased, total cholesterol levels stabilized, cognitive function improved, and aggression decreased, while their nocturnal activity remained high. These findings suggest that long-term consumption of a soft diet during early development can influence various behaviors associated with anxiety and mood regulation, including weight gain, cognitive decline, impaired motor coordination, increased nocturnal activity, and heightened aggression. Therefore, the hardness of food can impact brain function, mental well-being, and motor skills during the developmental stage. Early consumption of hard foods may be crucial for promoting and maintaining healthy brain function.

Long-Term Capsaicin Administration Ameliorates the Dysfunction and Astrogliosis of the Brain in Aged Mice with Missing Maxillary Molars.

Tooth loss and decreased masticatory function reportedly affect cognitive function; tooth loss allegedly induces astrogliosis and aging of astrocytes in the hippocampus and hypothalamus, which is a response specific to the central nervous system owing to homeostasis in different brain regions. Capsaicin, a component of red peppers, has positive effects on brain disorders in mice. Decreased expression of transient receptor potential vanilloid 1, a receptor of capsaicin, is associated with the development of dementia. In this study, we investigated the effect of capsaicin administration in aged mice (C57BL/6N mice) with reduced masticatory function owing to the extraction of maxillary molars to investigate preventive/therapeutic methods for cognitive decline attributed to age-related masticatory function loss. The results demonstrated that mice with impaired masticatory function showed decreased motor and cognitive function at the behavioral level. At the genetic level, neuroinflammation, microglial activity, and astrogliosis, such as increased glial fibrillary acidic protein levels, were observed in the mouse brain. The mice with extracted molars fed on a diet containing capsaicin for 3 months demonstrated improved behavioral levels and astrogliosis, which suggest that capsaicin is useful in maintaining brain function in cases of poor oral function and prosthetic difficulties.

Erythritol Can Inhibit the Expression of Senescence Molecules in Mouse Gingival Tissues and Human Gingival Fibroblasts.

Oral aging causes conditions including periodontal disease. We investigated how the sugar alcohol erythritol, which has anti-caries effects, impacts aging periodontal tissues and gingival fibroblasts in mice and humans in vivo and in vitro. Mice were classified into three groups: control groups of six-week-old (YC) and eighteen-month-old mice (AC) and a group receiving 5% w/w erythritol water for 6 months (AE). After rearing, RNA was extracted from the gingiva, and the levels of aging-related molecules were measured using PCR. Immunostaining was performed for the aging markers p21, γH2AX, and NF-κB p65. p16, p21, γH2AX, IL-1ß, and TNFα mRNA expression levels were higher in the gingiva of the AC group than in the YC group, while this enhanced expression was significantly suppressed in AE gingiva. NF-κB p65 expression was high in the AC group but was strongly suppressed in the AE group. We induced senescence in cultured human gingival fibroblasts using H2O2 and lipopolysaccharide before erythritol treatment, which reduced elevated senescence-related marker (p16, p21, SA-ß-gal, IL-1ß, and TNFα) expression levels. Knockdown of PFK or PGAM promoted p16 and p21 mRNA expression, but erythritol subsequently rescued pyruvate production. Overall, intraoral erythritol administration may prevent age-related oral mucosal diseases.

The Priming Potential of Interferon Lambda-1 for Antiviral Defense in the Oral Mucosa.

The oral mucosa is one of the first lines of the innate host defense system against microbial invasion. Interferon (IFN) lambda-1 (IFN-λ1), a type III IFN, exhibits type I IFN-like antiviral activity. In contrast to ubiquitously expressed type I IFN receptors, IFN-λ receptor 1 (IFN-λR1), which has higher affinity for type III IFNs than low-affinity interleukin (IL)-10 receptor 2, is mainly expressed on epithelial cells. Although IFN-λ1 has been shown to exert antiviral effects in the respiratory tract, gastrointestinal tract, and skin, the regulation of type III IFN receptor expression and its functions in the oral mucosa remain unclear. We herein showed the expression of IFN-λR1 in human gingival keratinocytes. The expression of IL-6, angiotensin-converting enzyme 2 (a critical molecule for severe acute respiratory syndrome coronavirus 2 infection), and IL-8 in human primary gingival keratinocytes (HGK) were significantly higher following treatments with either type I IFN (IFN-ß) or type II IFN (IFN-γ) than with IFN-λ1. However, the IFN-λ1 treatment strongly induced toll-like receptor (TLR) 3 and retinoic acid-inducible gene I (RIG-I), which mainly recognize viral nucleic acids, via the STAT1-mediated pathway. Furthermore, a stimulation with a RIG-I or TLR3 agonist promoted the production of IL-6, IL-8, and IFN-λ in HGK, which was significantly enhanced by a pretreatment with IFN-λ1. These results suggest that IFN-λ1 may contribute to the activation of innate immune responses to oral viral infections by up-regulating the expression of RIG-I and TLR3 and priming their functions in keratinocytes.

Molar loss induces hypothalamic and hippocampal astrogliosis in aged mice.

Age-related tooth loss impedes mastication. Epidemiological and physiological studies have reported that poor oral hygiene and occlusion are associated with cognitive decline. In the present study, we analyzed the mechanism by which decreased occlusal support following bilateral extraction of the maxillary first molars affects cognitive functions in young and aged mice and examined the expression of brain-function-related genes in the hippocampus and hypothalamus. We observed decreased working memory, enhanced restlessness, and increased nocturnal activity in aged mice with molar extraction compared with that in mice with intact molars. Furthermore, in the hypothalamus and hippocampus of molar-extracted aged mice, the transcript-level expression of Bdnf, Rbfox3, and Fos decreased, while that of Cdkn2a and Aif1 increased. Thus, decreased occlusal support after maxillary first molar extraction may affect cognitive function and activity in mice by influencing aging, neural activity, and neuroinflammation in the hippocampus and hypothalamus.

Analysis of senescence in gingival tissues and gingival fibroblast cultures.

OBJECTIVE: To determine senescence-associated changes in the gingival tissues of aged mice and gingival fibroblast cultures. MATERIALS AND METHODS: The production of senescence-associated ß-galactosidase (SA-ß-gal) and mRNA expression of p16, p21, interleukin (IL)-1ß, and tumor necrosis factor α (TNF-α) were evaluated in gingival tissues, gingival fibroblasts of 10- and 20-month-old C57BL/6NCrl mice, and multiple-passaged and hydrogen peroxide-stimulated human gingival fibroblasts (HGFs). Changes in molecular expression in HGF cultures due to senescent cell elimination by the senolytic drug ABT-263 (Navitoclax) were analyzed. RESULTS: Compared to 10-week-old mice, the 20-month-old mice had higher numbers of M1 macrophages. The proportion of cells expressing SA-ß-gal were also higher in 20- month-old mice than in 10-week-old-mice. Gingival fibroblasts in 20-month-old mice expressed less collagen 1a1, collagen 4a1, and collagen 4a2 mRNA than those in 10-week-old mice. Compared to control cells, H2O2 treated HGF cells expressed higher levels of SA-ß-gal and p16, p21, IL-1ß, and TNF-α. Furthermore, ABT-263 suppressed HGF cell expression of cytokines after senescence induction. CONCLUSIONS: Senescence-associated changes were observed in the gingival tissues of aged mice and HGF cultures. In addition, the potential of senolytic drugs to modify aging-related changes in the gingiva was shown.

Effect of green propolis extracts on experimental aged gingival irritation in vivo and in vitro.

OBJECTIVES: Tooth bleaching has become one of the most frequently requested esthetic procedures in dental practice. A side effect of bleaching is gingival irritation. This study examined the efficacy of propolis to treat gingival irritation caused by bleaching in vivo and in vitro. METHODS: Gingival irritation was mimicked by a mild burn injury to oral mucosa in young (10 week old) and aged (18 month old) mice. Propolis ointment was immediately applied to the burn area. After 24 h, gingiva was collected to determine the efficacy of propolis by hematoxylin and eosin staining and real-time polymerase chain reaction (PCR). RESULTS: Topical application of propolis ointment reduced the infiltration of inflammatory cells at irritated sites and promoted the repair of the mucosal epithelium in young and aged mice. It also suppressed the expression of IL-1ß and TNF-α and increased keratin 1 and 5 expression in the irritated gingiva. Propolis suppressed an increase in IL-1ß and TNF-α upon stimulation with H2O2 in young and SA-ß-gal-expressing senescent human gingival fibroblasts (HGFs) cultures. CONCLUSIONS: Propolis may be effective for mucosal repair in gingival irritation as it suppresses the expression of proinflammatory cytokines and promotes keratin expression.

Periodontal Disease and Periodontal Disease-Related Bacteria Involved in the Pathogenesis of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia, and it exhibits pathological properties such as deposition of extracellular amyloid ß (Aß) and abnormally phosphorylated Tau in nerve cells and a decrease of synapses. Conventionally, drugs targeting Aß and its related molecules have been developed on the basis of the amyloid cascade hypothesis, but sufficient effects on the disease have not been obtained in past clinical trials. On the other hand, it has been pointed out that chronic inflammation and microbial infection in the brain may be involved in the pathogenesis of AD. Recently, attention has been focused on the relationship between the periodontopathic bacterium Porphylomonas gingivalis and AD. P. gingivalis and its toxins have been detected in autopsy brain tissues from patients with AD. In addition, pathological conditions of AD are formed or exacerbated in mice infected with P. gingivalis. Compounds that target the toxins of P. gingivalis ameliorate the pathogenesis of AD triggered by P. gingivalis infection. These findings indicate that the pathological condition of AD may be regulated by controlling the bacteria in the oral cavity and the body. In the current aging society, the importance of oral and periodontal care for preventing the onset of AD will increase.

Involvement of adiponectin in age-related increases in tear production in mice.

Common age-related changes in the human eye contribute to the development of dry eye, including decreases in aqueous tear production. Although the infiltration of lymphocytes into the lacrimal glands occurs with age, age-related increases in tear production have also been observed in mice; however, the mechanisms underlying this increase remain unclear. We herein demonstrated that increases in tear production were not dependent on body weight gain or systemic conditions, such as insulin resistance, using aged mice and high-fat diet-fed mice. The results obtained also showed that senescence-associated T (SA-T) cells accumulated in the lacrimal glands of aged mice, particularly females. Expression levels of the nuclear transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) in whole lacrimal glands and epithelial cells isolated from lacrimal glands were significantly higher in aged mice than in young mice. The expression levels of adiponectin and one of its receptors, AdipoR2, also increased in the lacrimal glands of aged mice, but not in those of high-fat diet-fed mice. Collectively, the present results indicate that PPARγ and adiponectin-mediated signaling contribute to age-related increases in tear production in mice and have potential as therapeutic targets for the treatment of dry eye in humans.

All-in-one self-etch model adhesives: HEMA-free and without phase separation.

OBJECTIVE: Aim of this study was to design HEMA-free all-in-one self-etch model adhesives without phase separation, and to investigate their efficiency on extracted human teeth. MATERIALS AND METHODS: Compositions of adhesives in mass% (1): UDMA (25), 4-META (20), H(2)O (0, 1, 2, 5, 10, 20, 35, and 45), balance of acetone or ethanol. (2): UDMA (35), 4-META or 4-MET (28), H(2)O (0, 2, 4, 5, 6, and 8), balance of acetone. Phase separation was evaluated on samples exposed to ambient atmosphere. Conventional shear bond strengths (SBS, n=8) were determined on human enamel and dentin. Marginal adaptation (MGW, n=8) was assessed in cylindrical butt-joint dentin cavities. RESULTS: Solutions (1) and (2) with 5 and 8% or less water content, respectively, showed no phase separation. SBSs on enamel were not different within the acetone- or ethanol-group and between the adhesive groups (1). Water content of adhesives (2) was a significant determinant of enamel SBSs, groups with 4-META or 4-MET were not different (p>0.05). Dentin SBSs with adhesives (1) were not different (p>0.05) within solvent groups, yet higher for acetone-dissolved adhesives (p<0.05). Dentin SBSs with adhesives (2) were different by water content and functional monomer (p<0.05). MGW for solutions (1) were smaller with acetone-dissolved than with ethanol-dissolved adhesives (p<0.001). Acetone solutions between 2 and 45% water content produced almost perfect marginal adaptation. Marginal adaptation of adhesives (2) was almost perfect at 5 through 8% water content. CONCLUSIONS: Simplified HEMA-free self-etch adhesives without phase separation were prepared without compromises on bonding efficiency to enamel and dentin.

Multiple mechanisms regulate circadian expression of the gene for cholesterol 7alpha-hydroxylase (Cyp7a), a key enzyme in hepatic bile acid biosynthesis.

Cholesterol 7alpha-hydroxylase (CYP7A) and sterol 12alpha-hydroxylase (CYP8B) in bile acid biosynthesis and 3-hydroxyl-3-methylglutaryl CoA reductase (HMGCR) in cholesterol biosynthesis are the key enzymes in hepatic metabolic pathways, and their transcripts exhibit circadian expression profiles in rodent liver. The authors determined transcript levels of these enzymes and the regulatory factors for Cyp7a--including Dbp, Dec2, E4bp4, Hnf4alpha, Pparalpha, Lxralpha, Rev-erbalpha, and Rev-erbbeta--in the liver of wild-type and homozygous Clock mutant mice (Clock/Clock) and examined the effects of these transcription factors on the transcription activities of Cyp7a. The expression profile of the Cyp7a transcript in wild-type mice showed a strong circadian rhythm in both the 12L:12D light-dark cycle and constant darkness, and that in Clock/Clock also exhibited a circadian rhythm at an enhanced level with a lower amplitude, although its protein level became arrhythmic at a high level. The expression profile of Cyp8b mRNA in wild-type mice showed a shifted circadian rhythm from that of Cyp7a, becoming arrhythmic in Clock/Clock at an expression level comparable to that of wild-type mice. The expression profile of Hmgcr mRNA also lost its strong circadian rhythm in Clock/Clock , showing an expression level comparable to that of wild-type mice. The expressions of Dbp, Dec2, Rev-erbalpha, and Rev-erb beta--potent regulators for Cyp7a expression--were abolished or became arrhythmic in Clock/Clock, while other regulators for Cyp7a-Lxralpha, Hnf4alpha, Pparalpha, and E4bp4--had either less affected or enhanced expression in Clock/Clock. In luciferase reporter assays, REV-ERBalpha/beta, DBP, LXRalpha, and HNF4alpha increased the promoter activity of Cyp7a, whereas DEC2 abolished the transcription from the Cyp7a promoter: E4BP4 and PPARalpha were moderate negative regulators. Furthermore, knockdown of REV-ERBalpha/beta with siRNA suppressed Cyp7a transcript levels, and in the electrophoretic mobility shift assay, REV-ERBalpha/beta bound to the promoter of Cyp7a . These observations suggest that (1) active CLOCK is essential for the robust circadian expression of hepatic metabolic enzymes (Cyp7a, Cyp8b, and Hmgcr); (2) clock-controlled genes--DBP, DEC2, and REV-ERBalpha/beta--are direct regulators required for the robust circadian rhythm of Cyp7a; and (3) the circadian rhythm of Cyp7a is regulated by multiple transcription factors, including DBP, REV-ERBalpha/beta, LXRalpha, HNF4alpha DEC2, E4BP4, and PPARalpha.

Effects of fasting and re-feeding on the expression of Dec1, Per1, and other clock-related genes.

To elucidate the food-entrainable oscillatory mechanism of peripheral clock systems, we examined the effect of fasting on circadian expression of clock genes including Dec1 and Dec2 in mice. Withholding of food for 2 days had these effects: the expression level of Dec1 mRNA decreased in all tissues examined, although Per1 mRNA level markedly increased; Per2 expression was reduced in the liver and heart only 42-46 h after the start of fasting; and expression profiles of Dec2 and Bmal1 were altered only in the heart and in the liver, respectively, whereas Rev-erbalpha mRNA levels did not change significantly. Re-feeding after 36-h starvation erased, at least in part, the effect of fasting on Dec1, Dec2, Per1, Per2, and Bmal1 within several hours, and restriction feeding shifted the phase of expression profiles of all examined clock genes including Dec1 and Dec2. These findings indicate that short-term fasting and re-feeding modulate the circadian rhythms of clock genes to different extents in peripheral tissues, and suggest that the expression of Dec1, Per1, and some other clock genes was closely linked with the metabolic activity of these tissues.