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

Abstract

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.

Graphical abstract

Introduction of a model to masticatory activity modified using two distinct types of diets: hard diet (HD) and soft diet (SD). Three groups were formed based on masticatory activity, with diets alternated (HD/SD and HD/SD/HD), or a consistent hard diet (HD) applied. Each group underwent a lifespan of either 6 or 18 months and was reared in environments categorized as either impoverished or enriched.

Specifications table

Subject area:	Neuroscience
More specific subject area:	Neurodegeneration
Name of your method:	Masticatory activity pattern for neural changes screening
Name and reference of original method:	DE ALMEIDA, M.N.F.; MENDES, F.C.C.S.; FELICIO, A.P.G; FALSONI, M.; DE ANDRADE, M.L.F.; BENTO-TORRES, J.; VASCONCELOS, P.F.C.; PERRY, V.H.; PICANCO-DINIZ, C.W.; KRONKA, M. C. Spatial memory decline after masticatory deprivation and aging is associated with altered laminar distribution of CA1 astrocytes. BMC Neuroscience, v. 13, p. 23, 2012.
Resource availability:	There is no need to indicate resources to reproduce the method.

Method details

In order to replicate the Masticatory Activity Pattern Method for Neural Changes Screening, the researchers chose to use a female murine model (Mus musculus).

The protocol presented was previously submitted to and approved by the Ethics Committee on Animal Experimentation (CEPAE) of the Institute of Biological Sciences at the Federal University of Pará (UFPA). It has been fully endorsed, tested, and applied, yielding significant results. The animals must be randomly selected from 5th postnatal day remaining at a number of six pups per nursing mother until 21st postnatal day in order to maintain a non-competitive feeding environment. The decision to limit the number of offspring to six per dam is rationalized by the intention to mitigate potential adverse influences on the growth and development of the Nervous System in these animals, which are associated with diminished postnatal maternal care. It is recognized that stress stemming from competition for food escalating with litter size has the capacity to induce alterations in behavioral patterns and neuronal as well as glial plasticity in adulthood [1]. Consequently, in cases where the number of females per litter falls below six, it is imperative to retain a male to uphold the prescribed ratio of six offspring per dam.

Upon weaning, occurring on the 21st postnatal day, a pool of 132 females was available for the constitution of experimental cohorts (Fig. 1).

Fig. 1.

Organization chart of experimental groups formed from masticatory activities, ages and types of environments. Twelve (12) experimental groups will be formed.

HD: hard diet; SD: soft diet; 6M: 6-month-old; 18M: 18-month-old; IE: impoverished environment; EE: enriched environment.

At the end of period, experimental groups should be organized according to the three variables possibly studied: masticatory regime, aging and type of environment.

Formation of experimental groups

Masticatory activities

Based on a stress reduction method to evaluate performance in the face of activity alteration, the animals are submitted to one of 3 different types of chewing regimes without differences in composition or nutritional value. The regimes are supported by two forms of food presentation: hardened form or pellet, called HD (hard diet) or in the form of bran, powdered, called SD (soft diet).

The selected age brackets for the study encompassed six months (6 M) and eighteen months (18 M) postnatal, corresponding to young adult and aged animals, respectively. Accordingly, behavioral analyses were conducted solely upon the completion of these specific age.

Throughout this period, the animals were subjected to three different diet regimens involving the sequential or isolated provision of two types of feed: pellet and powdered. It is crucial to note that these feeds differed solely in consistency and presentation form, with no distinction in composition or nutritional value. One diet regimen involved continuous feeding of pelletized feed until the completion of their temporal window (at 6 or 18 months of age). This group was designated as the Hard-diet (HD) group. The other regimen entailed the provision of both pellet and powdered feed, initially pellet and subsequently powdered, termed as Hard-diet/Soft-diet (HD/SD). In this scenario, a reduction in masticatory activity occurred due to the powdered feed requiring less chewing effort during ingestion.

In this group, the pelletized and powdered feeds were provided for an identical period of 3 months or 9 months in animals at 6- months and 18-month-old groups, respectively.

In the case of the third and final diet regimen (Hard-diet/Soft-diet/Hard-diet - HD/SD/HD), animals received sequences of pellet, powder, and pellet again, with intervals of 2 or 6 months for each feed, at ages 6- or 18-month-old, respectively. Thus, it can be affirmed that the animals in the HD diet regimen constituted the control group, those in the HD/SD regimen represented the group with reduced masticatory activity, while the HD/SD/HD group represented those with masticatory activity rehabilitation, as they were required to exert greater chewing effort due to the provision of pelletized feed at the end.

After 21 days of postnatal development, all animals were exclusively housed in one of two environmental conditions: one termed as impoverished environment (IE) and the other referred to as enriched environment (EE).

From these food presentations, the following groups are defined: HD, maintained throughout time window with pellet feeding, and defined as the control group with maintenance of masticatory activity; HD/SD, this being maintained proportionally to age, firstly in pelleted food and in the second half of temporal window in powdered food (½ + ½); and, the HD/SD/HD group, this group also in proportion to age, is maintained in the first third on hardened food, in the second third, bran food, and again, returning to hardened food, in pellets (¹/₃ + ¹/₃ + ¹/₃). In this way, with the HD/SD group, the aim is to mimic condition of deprivation of masticatory activity, whether due to occlusion restriction due to tooth loss, muscle dysfunction, enteral/parenteral feeding, or loss of motor control, for examples. The HD/SD/HD group, on the other hand, presents the possibility of activity rehabilitation, where after a period of deprivation, animals again have access to full masticatory activity (Table 1, Figs. 1 and 2), identifying in their results how beneficial rehabilitation is by comparing their outcomes to those of the HD (Hard-diet) group, which serves as the control group.

Table 1.

Presentation of groups possibly formed by the Masticatory Activity Pattern for Neural Changes Screening Method.

Variables	Groups
Masticatory Activity	HD	HD/SD	HD/SD/HD
Age	6M		18M
Environment	IE		EE
Total Number of Groups	12

HD: hard diet; SD: soft diet; 6M: 6-month-old; 18M: 18-month-old; IE: impoverished environment; EE: enriched environment.

Fig. 2.

Experimental timeline of masticatory activities groups (orange inset), and representations of types of environments (blue inset). Female albino Swiss mice, Mus musculus, are remaining breastfeeding until the 21st postnatal day. After this period, the animals were organized into groups that are fed as a continuous hard pelleted diet (HD), or a hard diet followed by a soft diet (HD/SD group), or a hard diet followed by soft diet, and again hard diet (HD/SD/HD). The alternating regimes (HD/SD and HD/SD/HD) are proportionally distributed over lifespan (1/2 time and 1/3 time). Half of the animals are kept in a poor environment at standard laboratory cages (impoverished environment) or the other half in enriched cages (enriched environment).

HD: hard diet; SD: soft diet; 6M: 6-month-old; 18M: 18-month-old; IE: impoverished environment; EE: enriched environment.

The possibility of maintaining a group only on soft food was not understood by the researchers as a representative group in human translational terms, and its imposition is at discretion of the present research objective.

Time window groups

Ages are defined in two: young adults (6-month-old, 6 M) and aged animals (18-month-old, 18 M), comparing responses to deprivation and rehabilitation of masticatory activity in two age profiles.

Thus, animals were fed with continuous pelletized diet (HD - normal chewing groups), or with hard diet followed by powdered diet (HD/SD - reduced chewing activity groups), or with hard diet followed by powdered diet, followed by hard diet again (HD/SD/HD - rehabilitated chewing activity groups). The diet regimens were distributed over time as follows, in months (M), for young adults (6-month-old) HD Group (6 M on HD), HD/SD Group (3 M on HD + 3 M on SD), and HD/SD/HD Group (2 M on HD + 2 M on SD + 2 M on HD). For the aged animal group (18-month-old), the proportions remained the same but with longer duration periods: HD Group (18 M on HD), HD/SD Group (9 M on HD + 9 M on SD), and HD/SD/HD Group (6 M on HD + 6 M on SD + 6 M on HD). Fig. 2 summarizes the chronology of the experimental conditions and the evaluated regimens. All mice from different experimental groups underwent tests to assess exploratory activity, learning and spatial memory, and behaviors indicative of anxiety. It is noteworthy that the maintenance of animal locomotion enables the application of various tests to examine the impact of chewing activity on animal performance, as well as the significance of rehabilitation and the consequences of activity deprivation. As an illustration, animals were subjected to the Morris water maze (MWM) following the conclusion of the temporal windows (6- and 18-month-old) (Table 1 and Figs. 1 and 2 for details).

It was presumed that the powdered diet requires less efficient chewing activity compared to the pellet diet. Despite the contrasting chewing regimens, all animals had ad libitum access to food and water.

Housing conditions

The housing conditions involved are the same throughout time window, i.e., regardless of imposed mastication regime, either group is kept in impoverished conditions (impoverished environment, IE) or in conditions of environmental enrichment (EE).

The impoverished environment (IE) consisted of plastic cages measuring 32 × 45 × 16.5 cm, lined with rice straw and covered with metal grates. Each IE cage housed a maximum of 6 mice.

EE consisted of two-level wire cages each of which with 50 cm × 50 cm × 50 cm equipped with rope bridges, tunnels, rods, running wheels, and toys that were made of different types of plastic and colors. The toys are displaced or replaced periodically exposing animals to different visual, motor, and somatosensory stimuli. Water and food are offered on the upper and lower floors, respectively, encouraging animal movement from one floor to other to drink or eat. Each EE cage housed 12 mice.

Despite these changes in feed, all animals had ad libitum access to food and water and were kept in a controlled ambient temperature space (23±1 °C) with a 12-hour light-dark cycle (light period: 6am - 6pm; dark period: 6pm - 6am).

To identify potential influences of chewing regimes and types of environments stimuli, all animals must be weighed from the beginning of experiment to the end of temporal window (Figs. 1 and 2).

Ending time windows, behavioral tests can be applied followed by perfusion and immunohistochemical processing.

For further details on perfusion procedures and immunohistochemical processing, refer to the relevant documentation at de Almeida et al. [2], De Siqueira Mendes et al. [3], Mendes et al. [4], De Siqueira Mendes et al. [5], Leal et al. [6], Figueiredo et al. [7]. It is important to note here that the suggested material for collection and processing involves brain tissue. Following the completion of temporal window, the animals were deeply anesthetized with a ketamine solution (90–120 mg/kg) and xylazine (5–10 mg/kg) intraperitoneally. Subsequently, craniotomy was performed to remove the brain, which was then, sectioned into anatomical series of parallel sections to the transverse plane with a thickness of 60 µm using a vibratome (Vibratome Microm^Ⓡ HM 650). The sections were mounted on slides with coding to ensure blind procedures during microscopy analysis whether for stereological quantitative estimation or for three-dimensional reconstruction (3D) after immunolabelling. Prior to this, protocols for Open Field and Morris Water Maze tests were applied with analysis performed using the ANY-maze Video Tracking System^Ⓡ (StoeltingCo©), associated with immunostaining for neurons (anti-NeuN) and astrocytes (anti-GFAP). The aforementioned material was also subjected to quantification using StereoInvestigator^Ⓡ and Neurolucida^Ⓡ software (MBF© Bioscience Inc., Frederick, MD, USA) installed on a computer system connected to a NIKON Eclipse 80i microscope (Nikon, Japan) equipped with a motorized stage (MAC6000, Ludl Electronic Products, Hawthorne, NY, USA).

It is reiterated that the protocols for behavioral tests, immunohistochemical assays, stereology, and three-dimensional morphological reconstruction are not the focus of this manuscript but rather the methodology applied for the related research objectives.

Discussion

With intense and recent investigations mapping interaction between chewing and cognitive impairment, maintaining masticatory activity has been identified as a factor for preserving brain cognitive functions. Associated do this tracking, reduced stress method was developed to evaluate performance in the face of masticatory activity changes. By opting for the use powdered feed, and not by teeth extraction or implantation of an intraoral device, alternations between restriction and rehabilitation of mastication were imposed on murine models. Furthermore, the experimental design of the rehabilitation of masticatory activity brought possibility of reducing the risks of cognitive impairment (DE [3]), with association of maintenance in an enriched environment, minimizing the losses of learning and spatial memory imposed by age, or by deprived masticatory activity.

Using the Masticatory Activity Pattern for Neural Changes Screening Method, Morris Water Maze (MWM) performances showed that masticatory-deprived group, regardless of environment (IE or EE) was not able to learn and remember the hidden platform location, but masticatory rehabilitation combined with enriched environment recovered such disabilities (DE [3]). Besides, EE associated with rehabilitation of mastication in young and aging mice (HD/SD/HD) amends performance in MWM Maze test in contrast to impoverished environment.

Thus, initially important results were obtained in terms of cognitive performance and spatial memory, as well as cellular quantification, observed using the fractionator and by applying the variables of masticatory activity. It was identified that feeding on a soft diet (SD) variably affected the number of astrocytes in the CA1 hippocampal field, and SD mice performed worse on water maze memory tests than HD (hard diet) mice. However, 6-month-old SD mice, but not HD mice, exhibited significant spatial memory dysfunction. In this context, older SD mice (18-month-old) had astrocyte hyperplasia in the strata pyramidale and oriens compared to 6-month-old mice. Taken together, the results suggest that the impaired spatial learning and memory induced by masticatory deprivation may be associated with altered astrocyte laminar distribution and number in the CA1 hippocampal field [2].

Enhancing the proposed model and incorporating the group of animals deprived of their chewing activity alongside the rehabilitated group allowed to examine how reduced mastication could differentially affect dentate gyrus astrocyte subtypes. In comparison to the hard diet group subjected to impoverished environment, young deprived animals (6-month-old) exhibited notable shrinkage of astrocyte branches, thus demonstrating the efficacy of the proposed experimental model (SIQUEIRA [4]). During housing under enriched environment, complexity of branches of AST1 (greater morphological complexities) and AST2 (lower) 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, as applied at rehabilitated group) (SIQUEIRA [4]).

Therefore, building upon the compelling results obtained in previous studies, which focused on examining the interference of masticatory activity in learning and spatial memory, as well as cellular sensitivity, specifically of hippocampal astrocytes, it was recognized the necessity and purpose of presenting the protocol for utilizing the experimental model of altered masticatory activity as proposed in the present study.

3D morphological approaches evidenced that aging and masticatory deprivation diversely affect astrocyte morphological complexities at the molecular layer of dentate gyrus (SIQUEIRA [4]), using groups suggested by Activity Pattern for Neural Changes Screening Method.

With the results obtained, the description of the present methodology explores the effect of chewing on brain function in mice. The authors elucidate their study design in sufficient detail, rendering it comprehensive and reproducible. The aim is to underscore how impactful the deprivation of masticatory activity can be, whether as a consequence of dental loss, maxillofacial trauma, enteral feeding, temporomandibular joint dysfunction, or various neuropathological causes involving aphasia.

Therefore, the rehabilitation of masticatory function becomes crucial in terms of recovery, given the results indicating proximity between the control and rehabilitated groups.

Ethics statements

All experimental procedures were fully approved prior to experimental approach by the Ethics Committee in Research with Experimental Animals (CEPAE) of Instituto de Ciências Biológicas from Universidade Federal do Pará (CEPAE-UFPA: 223-14). The animals were treated in accordance with the guidelines published by the NIH (Guide for the Care and Use of Laboratory Animals).

CRediT authorship contribution statement

FCCSM and MCKS: Conceptualization, Methodology, Validity tests, Writing - original draft, Supervision, Validation, Writing – review & editing

Funding

This project was supported by research funds from the Brazilian government. MCKS was supported by grants from the National Council for Scientific and Technological Development – CNPq (grant numbers 475677/2008-0 and 304411/2022-1) and the Fundação Amazônia Paraense de Amparo à Pesquisa (FAPESPA, grant number 136/08). Research funds from the Fundação de Amparo e Desenvolvimento da Pesquisa (FADESP/UFPA) and the Pró-Reitoria de Pesquisa e Pós-Graduação (PROPESP/UFPA) payed for proofreading, editing, and publication fees. FCCSM was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

Declaration of Competing Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Data availability

• Data will be made available on request.