Introduction of a model for the modification of masticatory activity to investigate induced neural and behavioral alterations in mice.

In the literature, there is consistent evidence related to the influence of chewing on brain functions, either from experimental models or in humans. In the case of humans, most results are restricted to functional tests, lacking cellular or molecular evidence. In the described method, the possibility of using experimental models is presented, as well as the mimicry of deprivation and rehabilitation of masticatory activity and without stress impact. By opting for the use of mash feed, instead of extracting or implanting an intraoral device, alternations between restriction and rehabilitation of mastication were imposed on murine models. The animals completed various temporal windows, with aging also representing a potential factor for translational dementia associations. Additionally, animals were segregated into environments characterized as either standard, simulating a sedentary lifestyle, or enriched, rich in sensorimotor and visuospatial stimulation. Thus, it was possible to study the influence of changes in masticatory activity, associated with aging and environmental enrichment, on cells from subregions of the hippocampus, as well as on performance in tests of learning and spatial memory.•Animal model for masticatory activity alteration;•Masticatory deprivation and rehabilitation, and•Models to study the interaction among masticatory activity, aging and enrichment environment.

Comparative analysis between Open Field and Elevated Plus Maze tests as a method for evaluating anxiety-like behavior in mice.

Anxiety is being increasingly diagnosed in the elderly population. In this sense, epidemiologic data have linked late-life anxiety disorders to increased cognitive decline, morbidity, and even mortality. In addition, studies have already reported the influence of the environment on the association between aging and anxiety. Therefore, the present study aimed to conduct a comparative analysis between Elevated Plus Maze (EPM) and Open Field (OF) tests as methods for evaluating mice's anxiety-like behavior, considering environmental and age variables. For this, eighty Female albino Swiss mice aged 6, 12, and 18 months were housed in an impoverished environment (IE) and enriched environment (EE). Following this, the animals were tested in EPM and OF tests. The environment and age affect the anxiety-like behavior of the mice in the OF, with a difference between the animals of 6 and 18 months, only in the EE (p < 0.021). However, in the EPM, it does not occur. Despite that, the environment affected the distance traveled by the mice in the EPM, where the IE animals showed greater exploratory activity than the EE, only in the 18-month group (p < 0.001). No environmental influences were detected in the OF. Concerning age, in the EPM, animals in the 18-month-old group traveled shorter distances compared to the 6-month group (p < 0.001) and the 12-month group (p < 0.001), only in EE. In turn, in the OF there was a decrease in the distance traveled in the 18-month group compared to the 6-month group (p = 0.012), only in the IE. Thus, the divergences between the results of EPM and OF instigate a better evaluation of the parameters analyzed in each test.

The Masticatory Activity Interference in Quantitative Estimation of CA1, CA3 and Dentate Gyrus Hippocampal Astrocytes of Aged Murine Models and under Environmental Stimulation.

Studies indicating the influence of masticatory dysfunction, due to a soft diet or lack of molars, on impairing spatial memory and learning have led to research about neuronal connections between areas and cell populations possibly affected. In this sense, with scarce detailed data on the subfields of hippocampus in dementia neurodegeneration, there is no information about astrocytic responses in its different layers. Thus, considering this context, the present study evaluated the effects of deprivation and rehabilitation of masticatory activity, aging, and environmental enrichment on the stereological quantification of hippocampal astrocytes from layers CA1, CA3, and DG. For this purpose, we examined mature (6-month-old; 6M), and aged (18-month-old; 18M) mice, subjected to distinct masticatory regimens and environments. Three different regimens of masticatory activity were applied: continuous normal mastication with hard pellets (HD); normal mastication followed by deprived mastication with equal periods of pellets followed by soft powder (HD/SD); or rehabilitated masticatory activity with equal periods of HD, followed by powder, followed by pellets (HD/SD/HD). Under each specific regimen, half of the animals were raised in standard cages (impoverished environment (IE)) and the other half in enriched cages (enriched environment (EE)), mimicking sedentary or active lifestyles. Microscopic stereological, systematic, and random sampling approaches with an optical dissector of GFAP-immunolabeled astrocytes were done, allowing for an astrocyte numerical estimate. Stratum moleculare and hilus, from the dentate gyrus (DG) and Strata Lacunosum-Moleculare, Oriens, and Radiatum, similarly to the dentate gyrus, showed no significant change in any of the investigated variables (age, diet, or environment) in these layers. However, in Stratum radiatum, it was possible to observe significant differences associated with diet regimens and age. Therefore, diet-related differences were found when the HD 18M IE group was compared to the HD/SD/HD 18-month-old group in the same environment (IE) (p = 0.007). In the present study, we present modulatory factors (masticatory function, environmental enrichment, and aging) for the differentiated quantitative laminar response in the hippocampal regions, suggesting other studies to read the plasticity and responsiveness of astrocytes, including the molecular background.

Updating The General Practitioner on The Association Between Teeth Loss and Temporomandibular Disorders: A Systematic Review.

The belief about a possible association between the absence of one or more teeth and the presence of temporomandibular disorders (TMD), although old, is still present among the dental class. Although evidence points to a lack of association between loss of posterior support and the presence of TMD, we do not have critical studies on the extent, quantity, or location of these losses. In this sense, this systematic review aims to investigate the association between tooth loss and the presence of TMD signs or diagnostic subgroups. Search strategies using a combination of keywords tooth loss and TMDs were performed in six databases (PubMed, Embase, Web of Science, Livivo, Lilacs, and Scopus) and gray literature from August to September 2020. Observational studies that investigated the association between tooth loss in TMD were considered. The risk of bias was assessed using the Joanna Briggs Institute (JBI) Critical Assessment Checklist for cross-sectional analytical studies, case-control, and cohort studies. Finally, the level of certainty measured by the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) was assessed. Six articles were included in the review according to the eligibility criteria. Of these, five had a high risk of bias and one had a moderate risk. Only one study showed an association between the loss of posterior teeth and the presence of joint sounds and joint pain, the others found no significant association with sign or TMD subgroups diagnostic.There is no scientific evidence to support the association between one or more tooth loss and the presence of TMD signs and symptoms or diagnostic subgroups.

The Sedentary Lifestyle and Masticatory Dysfunction: Time to Review the Contribution to Age-Associated Cognitive Decline and Astrocyte Morphotypes in the Dentate Gyrus.

As aging and cognitive decline progresses, the impact of a sedentary lifestyle on the appearance of environment-dependent cellular morphologies in the brain becomes more apparent. Sedentary living is also associated with poor oral health, which is known to correlate with the rate of cognitive decline. Here, we will review the evidence for the interplay between mastication and environmental enrichment and assess the impact of each on the structure of the brain. In previous studies, we explored the relationship between behavior and the morphological features of dentate gyrus glial fibrillary acidic protein (GFAP)-positive astrocytes during aging in contrasting environments and in the context of induced masticatory dysfunction. Hierarchical cluster and discriminant analysis of GFAP-positive astrocytes from the dentate gyrus molecular layer revealed that the proportion of AST1 (astrocyte arbors with greater complexity phenotype) and AST2 (lower complexity) are differentially affected by environment, aging and masticatory dysfunction, but the relationship is not straightforward. Here we re-evaluated our previous reconstructions by comparing dorsal and ventral astrocyte morphologies in the dentate gyrus, and we found that morphological complexity was the variable that contributed most to cluster formation across the experimental groups. In general, reducing masticatory activity increases astrocyte morphological complexity, and the effect is most marked in the ventral dentate gyrus, whereas the effect of environment was more marked in the dorsal dentate gyrus. All morphotypes retained their basic structural organization in intact tissue, suggesting that they are subtypes with a non-proliferative astrocyte profile. In summary, the increased complexity of astrocytes in situations where neuronal loss and behavioral deficits are present is counterintuitive, but highlights the need to better understand the role of the astrocyte in these conditions.

Microglial Morphology Across Distantly Related Species: Phylogenetic, Environmental and Age Influences on Microglia Reactivity and Surveillance States.

Microglial immunosurveillance of the brain parenchyma to detect local perturbations in homeostasis, in all species, results in the adoption of a spectrum of morphological changes that reflect functional adaptations. Here, we review the contribution of these changes in microglia morphology in distantly related species, in homeostatic and non-homeostatic conditions, with three principal goals (1): to review the phylogenetic influences on the morphological diversity of microglia during homeostasis (2); to explore the impact of homeostatic perturbations (Dengue virus challenge) in distantly related species (Mus musculus and Callithrix penicillata) as a proxy for the differential immune response in small and large brains; and (3) to examine the influences of environmental enrichment and aging on the plasticity of the microglial morphological response following an immunological challenge (neurotropic arbovirus infection). Our findings reveal that the differences in microglia morphology across distantly related species under homeostatic condition cannot be attributed to the phylogenetic origin of the species. However, large and small brains, under similar non-homeostatic conditions, display differential microglial morphological responses, and we argue that age and environment interact to affect the microglia morphology after an immunological challenge; in particular, mice living in an enriched environment exhibit a more efficient immune response to the virus resulting in earlier removal of the virus and earlier return to the homeostatic morphological phenotype of microglia than it is observed in sedentary mice.

Sedentary Life and Reduced Mastication Impair Spatial Learning and Memory and Differentially Affect Dentate Gyrus Astrocyte Subtypes in the Aged Mice.

To explore the impact of reduced mastication and a sedentary lifestyle on spatial learning and memory in the aged mice, as well as on the morphology of astrocytes in the molecular layer of dentate gyrus (MolDG), different masticatory regimens were imposed. Control mice received a pellet-type hard diet, while the reduced masticatory activity group received a pellet diet followed by a powdered diet, and the masticatory rehabilitation group received a pellet diet, followed by powder diet and then a pellet again. To mimic sedentary or active lifestyles, mice were housed in an impoverished environment of standard cages or in an enriched environment. The Morris Water Maze (MWM) test showed that masticatory-deprived group, regardless of environment, was not able to learn and remember the hidden platform location, but masticatory rehabilitation combined with enriched environment recovered such disabilities. Microscopic three-dimensional reconstructions of 1,800 glial fibrillary acidic protein (GFAP)-immunolabeled astrocytes from the external third of the MolDG were generated using a stereological systematic and random sampling approach. Hierarchical cluster analysis allowed the characterization into two main groups of astrocytes with greater and lower morphological complexities, respectively, AST1 and AST2. When compared to compared to the hard diet group subjected to impoverished environment, deprived animals maintained in the same environment for 6 months showed remarkable shrinkage of astrocyte branches. However, the long-term environmental enrichment (18-month-old) applied to the deprived group reversed the shrinkage effect, with significant increase in the morphological complexity of AST1 and AST2, when in an impoverished or enriched environment. During housing under enriched environment, complexity of branches of AST1 and AST2 was reduced by the powder diet (pellet followed by powder regimes) in young but not in old mice, where it was reversed by pellet diet (pellet followed by powder and pellet regime again). The same was not true for mice housed under impoverished environment. Interestingly, we were unable to find any correlation between MWM data and astrocyte morphological changes. Our findings indicate that both young and aged mice subjected to environmental enrichment, and under normal or rehabilitated masticatory activity, preserve spatial learning and memory. Nonetheless, data suggest that an impoverished environment and reduced mastication synergize to aggravate age-related cognitive decline; however, the association with morphological diversity of AST1 and AST2 at the MolDG requires further investigation.

Dual-Task Exercise to Improve Cognition and Functional Capacity of Healthy Older Adults.

BACKGROUND: It has been suggested that physical inactivity and lack of stimulating cognitive activity are the two most significant modifiable risk factors to impair cognitive function. Although many studies that investigated the cognitive effects of physical exercise and cognitive stimuli in dual-task conditions showed improved cognitive performance, others have not confirmed these findings. The main aim of the present work is to analyze the effects of a dual-task multimodal physical exercise training, at moderate intensity, and cognitive stimulation on cognitive and physical function in healthy older adults. METHODS: This clinical trial was registered on the Brazilian Registry of Clinical Trials (RBR-9zrx3d). Here we tested the effects of a dual-task multimodal physical exercise training, at moderate intensity, on cognitive and physical function and quality of life in community dwelling older adults. The training protocol included 24 group sessions, 2/week, per 75 min. Cognition was assessed using CANTAB automated neuropsychological tests and Functional Capacity to Exercise tests. Performance was compared from baseline to post intervention and to a non-exercise control group using Mixed Linear Model for repeated measures. RESULTS: Control (CG) and dual-task (DTEx) groups progressed differentially over time on performance of episodic memory, sustained visual attention, functional mobility, cardiorespiratory fitness, lower limbs strength resistance, agility, quality of life and dual-task performance with significant improved DTEx performance. Control group did not show any significant changes on these tests except for showing a reduction in dual-task performance. CONCLUSION: We suggest that the dual-task combination of multisensory cognitive stimulation and multimodal moderate physical exercise training, twice a week, may be adopted as an effective program to reduce progression of age-related cognitive decline and improve physical fitness and quality of life on healthy older adults. CLINICAL TRIAL REGISTRATION: Brazilian Registry of Clinical Trials: https://ensaiosclinicos.gov.br/rg/RBR-9zrx3d -UTN code: U1111-1233-6349.

Lateral septum microglial changes and behavioral abnormalities of mice exposed to valproic acid during the prenatal period.

Most animal model studies of autism spectrum disorder (ASD) have been performed in males, which may be a reflex of the 3-times higher prevalence in boys than in girls. For this reason, little is known about the mechanisms underlying disease progression in females, and nothing is known about potential associations between microglial changes in the lateral septum (LS) and adult female cognition. Prenatal exposure to valproic acid (VPA) in mice has been widely used as an experimental model of autism-like behaviors associated with cellular changes. However, no study has reported the influence of VPA exposure in utero and its consequences on limbic system-dependent tasks or the microglial response in the LS in adult female mice. We compared the exploratory activity and risk assessment in novel environments of BALB/c control mice to mice exposed in utero to VPA and estimated the total number of microglia in the LS using an optical fractionator. On day 12.5 of pregnancy, females received diluted VPA or saline by gavage. After weaning, VPA exposed or control pups were separately housed in standard laboratory cages. At 5 months of age, all mice underwent behavioral testing and their brain sections were immunolabelled using IBA-1 antibody. In the open field test, VPA group showed a greater distance traveled, which was accompanied by less immobility, less time spent on the periphery and a greater number, crossed lines. Similar findings were found in the elevated plus maze test, where VPA mice traveled greater distances, immobility was significantly higher than that of control and VPA group spent less time on the closed arms of apparatus. Stereological analysis demonstrated higher microglial total number and density in the LS of VPA mice, as the cell count was greater, but the volume was similar. Therefore, we suggest that an increase in microglia in the LS may be part of the cellular changes associated with behavioral dysfunction in the VPA model of ASD.

Stereological Analysis of Early Gene Expression Using Egr-1 Immunolabeling After Spreading Depression in the Rat Somatosensory Cortex.

Early growth response-1 (Egr-1), defined as a zinc finger transcription factor, is an upstream master switch of the inflammatory response, and its expression can be used to investigate the spatial and temporal extent of inflammatory changes in the brain. Cortical spreading depression (CSD) is characterized as a slowly propagating (2-5 mm/min) depolarization wave through neurons and astrocytes in humans that contributes to migraines and possibly to other brain pathologies. In rodents, CSD can be induced experimentally, which involves unilateral depolarization that is associated with microglial and astrocyte responses. The impact of CSD on structures beyond the affected hemisphere has not been explored. Here, we used an optical fractionator method to investigate potential correlations between the number of and period of the eletrophysiologic record of CSD phenomena and Egr-1 expression in ipsilateral and contralateral hemispheres. CSD was elicited by the restricted application of a 2% KCl solution over the left premotor cortex. Electrophysiological events were recorded using a pair of Ag/AgCl agar-Ringer electrodes for 2 or 6 h. An optical fractionator was applied to count the Egr-1 positive cells. We found that CSD increased Egr-1 expression in a time- and event-dependent manner in the ipsilateral/left hemisphere. Although CSD did not cross the midline, multiple CSD inductions were associated with an increased number of Egr-1 positive cells in the contralateral/right hemisphere. Thus, repeated CSD waves may have far reaching effects that are more global than previously considered possible. The mechanism of contralateral expression is unknown, but we speculate that callosal projections from the depolarized hemisphere may be related to this phenomenon.

Environmental Impoverishment, Aging, and Reduction in Mastication Affect Mouse Innate Repertoire to Explore Novel Environments and to Assess Risk.

Studies indicate that inhibition of adequate masticatory function, due to soft diet, occlusal disharmony, or molar losses affects the cognitive behavior of rodents. However, no study has tested the effects on new environments exploration and risk assessment coupled with a combination of masticatory function rehabilitation and environmental enrichment. In the present report, we tested the hypothesis that age, environment, and masticatory changes may interact and alter exploratory patterns of locomotor activity and mice preferences in an open field (OF) arena. As OF arenas are widely used to measure anxiety-like behavior in rats and mice. We examined in an open arena, the exploratory and locomotor activities of mature (6-month-old; 6M), late mature (12-month-old; 12M), and aged (18-month-old; 18M) mice, subjected to distinct masticatory regimens and environments. Three different regimens of masticatory activity were used: continuous normal mastication with hard pellets (HD); normal mastication followed by reduced mastication with equal periods of pellets followed by soft powder - HD/SD; or rehabilitated masticatory activity with equal periods of HD, followed by powder, followed by pellets - HD/SD/HD). Under each diet regimen, half of the individuals were raised in standard cages [impoverished environment (IE)] and the other half in enriched cages [enriched environment (EE)]. Animals behavior on the open field (OF) task were recorded by webcam and analyzed with Any Maze software (Stöelting). The locomotor and exploratory activities in OF task declined with age, and this was particularly evident in 18M HD EE mice. Although all groups kept their preference by the peripheral zone, the outcomes were significantly influenced by interactions between environment, age, and diet. Independent of diet regime, 6M young mice maintained in an EE where voluntary exercise apparatus is available, revealed significant less body weight than all other groups. Although body weight differences were minimized as age progressed, 18M EE group revealed intragroup significant influence of diet regimens. We suggest that long life environmental enrichment reduces the tendency to avoid open/lit spaces (OF) and this is particularly influenced by masticatory activity. These measurements may be useful in discussions of anxiety-related tasks.

Differential Microglial Morphological Response, TNFα, and Viral Load in Sedentary-like and Active Murine Models After Systemic Non-neurotropic Dengue Virus Infection.

Peripheral inflammatory stimuli increase proinflammatory cytokines in the bloodstream and central nervous system and activate microglial cells. Here we tested the hypothesis that contrasting environments mimicking sedentary and active lives would be associated with differential microglial morphological responses, inflammatory cytokines concentration, and virus load in the peripheral blood. For this, mice were maintained either in standard (standard environment) or enriched cages (enriched environment) and then subjected to a single (DENV1) serotype infection. Blood samples from infected animals showed higher viral loads and higher tumor necrosis factor-α (TNFα) mRNA concentrations than control subjects. Using an unbiased stereological sampling approach, we selected 544 microglia from lateral septum for microscopic 3D reconstruction. Morphological complexity contributed most to cluster formation. Infected groups exhibited significant increase in the microglia morphological complexity and number, despite the absence of dengue virus antigens in the brain. Two microglial phenotypes (type I with lower and type II with higher morphological complexity) were found in both infected and control groups. However, microglia from infected mice maintained in enriched environment showed only one morphological phenotype. Two-way ANOVA revealed that environmental changes and infection influenced type-I and II microglial morphologies and number. Environmental enrichment and infection interactions may contribute to microglial morphological change to a point that type-I and II morphological phenotypes could no longer be distinguished in infected mice from enriched environment. Significant linear correlation was found between morphological complexity and TNFα peripheral blood. Our findings demonstrated that sedentary-like and active murine models exhibited differential microglial responses and peripheral inflammation to systemic non-neurotropic infections with DENV1 virus.

Age, environment, object recognition and morphological diversity of GFAP-immunolabeled astrocytes.

BACKGROUND: Few studies have explored the glial response to a standard environment and how the response may be associated with age-related cognitive decline in learning and memory. Here we investigated aging and environmental influences on hippocampal-dependent tasks and on the morphology of an unbiased selected population of astrocytes from the molecular layer of dentate gyrus, which is the main target of perforant pathway. RESULTS: Six and twenty-month-old female, albino Swiss mice were housed, from weaning, in a standard or enriched environment, including running wheels for exercise and tested for object recognition and contextual memories. Young adult and aged subjects, independent of environment, were able to distinguish familiar from novel objects. All experimental groups, except aged mice from standard environment, distinguish stationary from displaced objects. Young adult but not aged mice, independent of environment, were able to distinguish older from recent objects. Only young mice from an enriched environment were able to distinguish novel from familiar contexts. Unbiased selected astrocytes from the molecular layer of the dentate gyrus were reconstructed in three-dimensions and classified using hierarchical cluster analysis of bimodal or multimodal morphological features. We found two morphological phenotypes of astrocytes and we designated type I the astrocytes that exhibited significantly higher values of morphological complexity as compared with type II. Complexity = [Sum of the terminal orders + Number of terminals] × [Total branch length/Number of primary branches]. On average, type I morphological complexity seems to be much more sensitive to age and environmental influences than that of type II. Indeed, aging and environmental impoverishment interact and reduce the morphological complexity of type I astrocytes at a point that they could not be distinguished anymore from type II. CONCLUSIONS: We suggest these two types of astrocytes may have different physiological roles and that the detrimental effects of aging on memory in mice from a standard environment may be associated with a reduction of astrocytes morphological diversity.

Antibody-enhanced dengue disease generates a marked CNS inflammatory response in the black-tufted marmoset Callithrix penicillata.

Severe dengue disease is often associated with long-term neurological impairments, but it is unclear what mechanisms are associated with neurological sequelae. Previously, we demonstrated antibody-enhanced dengue disease (ADE) dengue in an immunocompetent mouse model with a dengue virus 2 (DENV2) antibody injection followed by DENV3 virus infection. Here we migrated this ADE model to Callithrix penicillata. To mimic human multiple infections of endemic zones where abundant vectors and multiple serotypes co-exist, three animals received weekly subcutaneous injections of DENV3 (genotype III)-infected supernatant of C6/36 cell cultures, followed 24 h later by anti-DENV2 antibody for 12 weeks. There were six control animals, two of which received weekly anti-DENV2 antibodies, and four further animals received no injections. After multiple infections, brain, liver, and spleen samples were collected and tissue was immunolabeled for DENV3 antigens, ionized calcium binding adapter molecule 1, Ki-67, TNFα. There were marked morphological changes in the microglial population of ADE monkeys characterized by more highly ramified microglial processes, higher numbers of trees and larger surface areas. These changes were associated with intense TNFα-positive immunolabeling. It is unclear why ADE should generate such microglial activation given that IgG does not cross the blood-brain barrier, but this study reveals that in ADE dengue therapy targeting the CNS host response is likely to be important.

Fine structure of bacterial adhesion to the epithelial cell membranes of the filiform papillae of tongue and palatine mucosa of rodents: a morphometric, TEM, and HRSEM study.

The palatine mucosa and filiform papillae of the dorsal tongue mucosae of rodents were examined using transmission electron microscopy (TEM) and high resolution scanning electron microscopy (HRSEM). In the HRSEM method, the samples were fixed in 2% osmium tetroxide, dehydrated in alcohol, critical point-dried, and coated with gold-palladium. In addition, the HRSEM technique was used for morphometric analysis (length, width, and length/width ratio of cocci and bacilli). For the TEM method, the tissues were fixed in modified Karnovsky solution (2.5% glutaraldehyde, 2% formalin in 0.1M sodium phosphate buffer, pH 7.4) and embedded in Spurr resin. The results demonstrated that there are thick polygonal keratinized epithelial cells where groups of bacteria are revealed in three-dimensional images on the surface of filiform papillae in these animals. The bacterial membranes are randomly attached to the microplicae surface of epithelial cells. Morphometrics showed higher values of length and width of cocci in newborn (0 day) as compared to newborn (7 days) and adults animals, the bacilli showed no differences in these measurements. At high magnification, the TEM images revealed the presence of glycocalyx microfilaments that constitute a fine adhesion area between bacterial membranes and the membranes of epithelial microplicae cells. In conclusion, the present data revealed the fine fibrillar structures of bacteria that facilitate adhesion to the epithelial cell membranes of the oral cavity and morphometric changes in newborn (0 day) rats as compared with other periods.

Enriched environment and masticatory activity rehabilitation recover spatial memory decline in aged mice.

BACKGROUND: To measure the impact of masticatory reduction on learning and memory, previous studies have produced experimental masticatory reduction by modified diet or molar removal. Here we induced spatial learning impairment in mice by reducing masticatory activity and then tested the effect of a combination of environmental enrichment and masticatory rehabilitation in recovering spatial learning at adulthood and in later life. For 6 months (6M) or 18 months (18M), we fed three groups of mice from postnatal day 21 respectively with a hard diet (HD) of pellets; pellets followed by a powdered, soft diet (HD/SD, divided into equal periods); or pellets followed by powder, followed by pellets again (HD/SD/HD, divided into equal periods). To mimic sedentary or active lifestyles, half of the animals from each group were raised from weaning in standard cages (impoverished environment; IE) and the other half in enriched cages (enriched environment; EE). To evaluate spatial learning, we used the Morris water maze. RESULTS: IE6M-HD/SD mice showed lower learning rates compared with control (IE6M-HD) or masticatory rehabilitated (IE6MHD/SD/HD) animals. Similarly, EE-HD/SD mice independent of age showed lower performance than controls (EE-HD) or rehabilitated mice (EE-HD/SD/HD). However, combined rehabilitation and EE in aged mice improved learning rate up to control levels. Learning rates did not correlate with swim speed. CONCLUSIONS: Reduction in masticatory activity imposed on mice previously fed a hard diet (HD/SD) impaired spatial learning in the Morris water maze. In adults, masticatory rehabilitation recovered spatial abilities in both sedentary and active mice, and rehabilitation of masticatory activity combined with EE recovered these losses in aged mice.

Morphometric, quantitative, and three-dimensional analysis of the heart muscle fibers of old rats: transmission electron microscopy and high-resolution scanning electron microscopy methods.

This research investigated the morphological, morphometric, and ultrastructural cardiomyocyte characteristics of male Wistar rats at 18 months of age. The animals were euthanized using an overdose of anesthesia (ketamine and xylazine, 150/10 mg/kg) and perfused transcardially, after which samples were collected for light microscopy, transmission electron microscopy, and high-resolution scanning electron microscopy. The results showed that cardiomyocyte arrangement was disposed parallel between the mitochondria and the A-, I-, and H-bands and their M- and Z-lines from the sarcomere. The sarcomere junction areas had intercalated disks, a specific structure of heart muscle. The ultrastructural analysis revealed several mitochondria of various sizes and shapes intermingled between the blood capillaries and their endothelial cells; some red cells inside vessels are noted. The muscle cell sarcolemma could be observed associated with the described structures. The cardiomyocytes of old rats presented an average sarcomere length of 2.071 ± 0.09 µm, a mitochondrial volume density (Vv) of 0.3383, a mitochondrial average area of 0.537 ± 0.278 µm(2), a mitochondrial average length of 1.024 ± 0.352 µm, an average mitochondrial cristae thickness of 0.038 ± 0.09 µm and a ratio of mitochondrial greater length/lesser length of 1.929 ± 0.965. Of the observed mitochondrial shapes, 23.4% were rounded, 45.3% were elongated, and 31.1% had irregular profiles. In this study, we analyzed the morphology and morphometry of cardiomyocytes in old rats, focusing on mitochondria. These data are important for researchers who focus the changes in cardiac tissue, especially changes owing to pathologies and drug administration that may or may not be correlated with aging.

Immunohistochemistry and ultrastructural characteristics of nerve endings in the oral mucosa of rat.

The sensory nerve endings of the rat tongue, cheek and palate were studied using immunohistochemical staining and transmission electron microscopy analysis. The specimens were fixed in modified Karnovsky solution and embedded in Spurr resin. Substance P, calcitonin gene-related peptide (CGRP)- and protein gene product 9.5 (PGP b9.5)-containing nerve fibers in the rat tongue, cheek and palate were examined by electronic microscopical analysis and immunohistochemical localization. These fibers run very close to the basal lamina of the epithelium and extend into the filliform and fungiform papillae. Numerous plexiform fibers immunoreactive for substance P, CGRP and PGP 9.5 were found in the connective tissue of mucosa. Electron microscopic observations showed clearly immunostained nerve fibers, which are located very close to the basal lamina of epithelial cells. Some electron-dense granules may be observed in the axoplasms of both substance P and CGRP immunoreactive fibers. Several lamellar corpuscles into the subepithelial connective tissue papillae, Merkel corpuscles and numerous thin unmyelinated and myelinated axons were observed. The terminal axons revealed numerous mitochondria, neurofilaments, microtubules and clear vesicles in the base of axoplasmic protrusions. The lamellar cells showed caveolae and interlamelar spaces filled by amorphous substance. Between the lamellar cells and axoplasmic membrane, and in the adjacent lamellae region, desmosome-type junctions were observed. The quantitative and morphometric analysis showed nerve endings with an average area of 4.83 ± 3.4 µm(2) and 19.4 internal mitochondria in this site and the organized corpuscles with an average area of 79.24 ± 27.24 µm(2) and 24.23 internal mitochondria in this place. All the structures observed are involved in the transmission of pain and mechanoreceptors stimulus of these oral mucosae.