Altered mastication adversely impacts morpho-functional features of the hippocampus: A systematic review on animal studies in three different experimental conditions involving the masticatory function

Recent results have established that masticatory function plays a role not only in the balance of the stomatognathic system and in the central motor control, but also in the trophism of the hippocampus and in the cognitive activity. These implications have been shown in clinical studies and in animal researches as well, by means of histological, biochemical and behavioural techniques. This systematic review describes the effects of three forms of experimentally altered mastication, namely soft-diet feeding, molar extraction and bite-raising, on the trophism and function of the hippocampus in animal models. Through a systematic search of PubMed, Embase, Web of Science, Scopus, OpenGray and GrayMatters, 645 articles were identified, 33 full text articles were assessed for eligibility and 28 articles were included in the review process. The comprehensiveness of reporting was evaluated with the ARRIVE guidelines and the risk of bias with the SYRCLE RoB tool. The literature reviewed agrees that a disturbed mastication is significantly associated with a reduced number of hippocampal pyramidal neurons in Cornu Ammonis (CA)1 and CA3, downregulation of Brain Derived Neurotrophic Factor (BDNF), reduced synaptic activity, reduced neurogenesis in the Dentate Gyrus (DG), glial proliferation, and reduced performances in behavioural tests, indicating memory impairment and reduced spatial orientation. Moreover, while the bite-raised condition, characterized by occlusal instability, is known to be a source of stress, soft-diet feeding and molar extractions were not consistently associated with a stress response. More research is needed to clarify this topic. The emerging role of chewing in the preservation of hippocampal trophism, neurogenesis and synaptic activity is worthy of interest and may contribute to the study of neurodegenerative diseases in new and potentially relevant ways.

Introduction Recent research results have established that the masticatory function plays a role not only in the balance of the stomatognathic system and in the central motor control, but especially in the development of the cognitive activity and in the slowdown of the unavoidable cognitive decay. These unsuspected results have been clearly shown by basic research with histomorphological outcomes and behavioural tests, as well as in clinical studies [1,2].
The importance of the masticatory function on maxillary growth, on the balance of the stomatognathic system and on the central motor control is well known in literature [3][4][5][6].
Chewing, one of the phylogenetically oldest functions of the stomatognathic system, is a complex, highly coordinated and continuously modulated movement, able to constantly adapt to the volume and consistency of the alimentary bolus through a diversity of masticatory patterns, emerging out of the central integration of a great number and variety of peripheral inputs [7,8]. The human masticatory function is a symmetrical, rhythmic and semi-automatic movement that alternates the involvement of the two sides of the dental arches. The teeth, no longer necessary for our survival as they are for the rest of the animal kingdom, play nevertheless a crucial role in the coordination and harmonious execution of the masticatory function, which, in turn, influences the development of the craniofacial region. While it is well known that the motor control of the masticatory function involves a large portion of the central nervous system, including brainstem, cerebellum, basal nuclei, midbrain and cortex, its influence on the hippocampus, memory and cognitive activity has only recently emerged [9].
In the last few years, the relationship between tooth loss and cognitive decline in the elderly has been established in a number of clinical studies which have been critically reviewed [1]. This relationship has been significantly shown in laboratory animals as well, highlighting the morphological alterations of the hippocampus and the related behavioral outcomes. Animal studies enable us to observe simultaneously the outcomes of behavioral tests and the histological and biochemical alterations associated with experimentally induced masticatory disturbances. Young, middle-aged and senile mice subjected to different forms of masticatory imbalances show a lower number of hippocampal neurons, an increased number of astrocytes and reduced progenitor cell proliferation in the hippocampal dentate gyrus (DG) [2].
In order to alter the masticatory function, different animal models have been exposed to three experimental conditions: soft-diet feeding, molar extraction and occlusal disharmony (bite-raising). These approaches shed light on the fact that masticatory dysfunction, in addition to the already cited histological effects on the number of neurons, alters the biochemical and hormonal balance and impairs spatial learning and memory [2].
Descriptive revues, mainly clinical reviews, have recently been published due to the interest of the topic [6,[10][11][12]. It is worth describing in a rigorous way the picture that is emerging from animal studies in order to clarify how the different experimental conditions allow us to understand the association between mastication and cognition.
This systematic review aims at evaluating the literature on the influence of three forms of experimental alteration of the masticatory function, namely soft-diet feeding, molar extraction and bite-raising, on the histochemistry, function and behavioral role of the hippocampus in laboratory animals.

Pre-clinical PICO and rationale for the review
In animal research, it is possible to experimentally alter the masticatory function in a controlled way and to assess its effects objectively. For this systematic review, the pre-clinical PICO was: "What is the impact of an experimentally altered masticatory function (I), compared to an undisturbed masticatory function, (C) in laboratory animals (rats and mice) (P) on the histology and biochemistry of the hippocampus (O)?".
There was no significant deviation from the initial purpose, which was generally maintained until the end of the review. However, after the first evaluation of the published literature, it appeared clear that it was important to separately describe the different experimental conditions. This planning allowed for a more accurate evaluation of the results.

Search strategy
A search of PubMed, Embase, Web of Science, Scopus, OpenGray and GrayMatters was conducted until July 2020, limiting the search to studies published in English within the last 10 years, as reported in Table 1. Additional studies were taken from reference lists of previous review articles and citations of relevant original articles were screened. References of included studies were checked by a research librarian.
Search results. 645 articles were identified through database searching. After removing the duplicates, 327 articles were screened by reviewing the abstracts. 33 full text articles were assessed for eligibility and, after 5 exclusions, 28 articles were included in the review process as reported in Fig 1. Selection procedure. After removal of the duplicates, articles were screened independently by AT and EB; when opinions about inclusion differed, a third person (MGP and/or PB and/or AP) was consulted.

Inclusion criteria
Inclusion criteria were the following: • Type of study: animal intervention study.

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Reduced mastication adversely impacts the hippocampus: A systematic review of animal studies • Control group: present.
• Experimental stimulus: alteration of the masticatory function.
• Outcomes: histological or biochemical analysis of the hippocampus.
Exclusion criteria were the following: • Molars cut off at gingival level: to reduce the possible confounding effects related to pulpal inflammation and retention of periodontal receptors, molarless animal models obtained by means of cutting off the tooth crown at the level of the gingiva were not included.
• Cast splints or crown: models of bite-raising obtained by cast splints or crowns were not included because of the possible confounding effects related to pulpal inflammation after poor tooth preparation.
• Unilateral molar extractions: unilateral models of both molar extraction and bite-raising were not included in order to avoid the disturbing biomechanical effects and related functional alterations, due to an asymmetric dental status, which is hardly comparable to a symmetric one.
• Hard and soft-diet feeding: studies were included only if the differences in the texture and hardness of the different types of food were clearly outlined. In particular, studies that compared standard pellet chow with harder diets were excluded unless the physical characteristics of the harder foodstuff were rigorously described.
Studies presenting behavioral outcomes alone were not included, but behavioral outcomes were discussed if presented in association with histological or biochemical ones.

Classification of the selected studies
The studies selected by the review process have been divided in three groups according to the experimental conditions: 1) studies on soft-diet feeding; 2) studies on tooth extraction; 3) studies on bite-raising.

Comprehensiveness of scientific reporting and risk of bias
The comprehensiveness of scientific reporting in included studies was assessed with the ARRIVE checklist [13] (see S1 Table) by AT and EB. The risk of bias of included studies was assessed with the SYRCLE tool for risk of bias in animal studies [14] (see Tables 2 and S2) by AT and AP. For each study, an evaluation was made for each item in the SYRCLE tool. A general assessment was carried out by weighting the results of the different questions in each bias domain, in order to provide the best summary of the evidence that the authors could provide. This assessment reflects a criterion of thoughtful evaluation rather than an arithmetical one.

Data extraction
The analytical evaluation of included studies was carried out. Subsequently, the data obtained was combined to produce three analytical tables and one summary table.
The comprehensiveness of scientific reporting of included studies assessed with the ARRIVE checklist [13] is described in S1 Table. While the level of agreement with the checklist was generally high, most articles lacked information concerning items 11, 14 and 17. In other words, most studies failed to report the details concerning allocation of animals to experimental groups, baseline data and adverse events (or to state explicitly that no adverse event took place; this may however be inferred from the stability of group numerosity from the beginning to the end of experimentation, as indeed was generally the case).
The risk of bias of included studies assessed with the SYRCLE tool for risk of bias in animal studies [14] is reported in Tables 2 and S2. While the risk of attrition bias, reporting bias or other bias could be adequately assessed, the risk of selection bias, performance bias and detection bias of included studies could not be assessed because, with very few exceptions, the necessary information (concerning randomisation of sequence generation, allocation concealment, randomisation of housing, blinding of caregivers, randomisation of outcome assessment and blinding of researchers assessing outcome) was not reported.
In the SYRCLE RoB tool, selection bias is investigated by means of three items (sequence generation, baseline characteristics and allocation concealment). While the majority of included studies reported sufficient information on baseline characteristics, none reported details of allocation concealment and a minority reported details of sequence generation: two out of three items, among them arguably the most important of the three (sequence generation, i.e. the presence of a random element in the selection of experimental groups), were impossible to adequately assess. The risk of selection bias was therefore assessed as unclear. Detection bias is investigated by means of two items (random outcome assessment and blinding). The majority of included studies did not report information concerning the blinding of examiners, and none reported information about random outcome assessment. The risk of detection bias was therefore assessed as unclear. The risk of attrition bias, reporting bias and other bias was assessed as low, because a majority of included studies adequately reported this information.
The analytic results are presented in table forms, as follows: •    Resuming the results of the analytical evaluation of included studies, the literature reviewed agrees that, in the three experimental conditions, disturbed mastication is significantly (Continued ) (Continued ) associated with 1) reduced number of hippocampal pyramidal neurons in CA1 and CA3, 2) downregulation of BDNF (brain derived neurotrophic factor), 3) reduced synaptic activity, 4) reduced neurogenesis in DG, and 5) glial proliferation; these histochemical alterations are associated with reduced performances in behavioural tests that target hippocampus-dependent functions, i.e. spatial orientation and memory. Interestingly, soft-diet feeding and molar extractions were not consistently associated with a stress response. A few studies indicate that the loss of neurons is partially reversible if correct chewing is restored, by means of either fitting dentures or modifying the consistency of the diet. More in depth, three studies included in their design the extraction of all maxillary molars and the subsequent fitting of experimental dentures. In all instances, the fitting of experimental dentures had positive effects on behavioural performance [24,28,34] and histological analysis [28,34]. In the latter study, the dentures were fitted after 35 weeks of the molarless condition, mimicking the clinical condition of a patient who has been rehabilitated with a removable prosthetic appliance after a long period of time of untreated partial or total edentulism. The experimental dentures group performed less well than control, but significantly better than untreated molarless mice in both the radial arm maze test and histological examination [34].
In addition to the above results, to investigate the effects of a harder-than-usual alimentary bolus on the hippocampus, Akazawa and colleagues [15] hardened regular pellet chow by autoclave cycling, in order to obtain an initial bolus of the same volume and shape, but

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Reduced mastication adversely impacts the hippocampus: A systematic review of animal studies  Plasma corticosterone levels, escape latency in the water maze (but not in the visible probe test) and the number of BrdUpositive cells in the hippocampal DG differed significantly between 5-and 9-moold group1 and controls, but not between 1-mo-old group 1 and controls.    Step-through passive avoidance test (STPAT); RT-PCR; Nissl staining; body weight measurement.
Not investigated.
At short term experimentation, there were no differences in memory function and BDNF mRNA level between the four groups. However, at long term experimentation, groups 1 and 2 showed memory impairment and decreased level of BDNF mRNA. The powder diet had no effect on memory function and BDNF mRNA level even at 16 wk. The number of neurons was lower in group 1 and 2; at long term experimentation, group 2 showed significantly fewer neurons than group 1 in CA1. After 32 wk, group 2 exhibited significantly lower body weight than group 3.
https://doi.org/10.1371/journal.pone.0237872.t004 regular solid diet. In experimental animals, a hard diet appears to have a protective, or even an enhancing effect on the hippocampus, in terms of both histological findings and behavioral outcomes.

Discussion
The purpose of this systematic review is to evaluate the evidence coming from animal studies on the relationship between different forms of experimental disruption of the masticatory function and morphofunctional alterations of the hippocampus and behavioural tests. The originality of this paper resides in describing collectively the effects on the hippocampus of three forms of experimentally induced masticatory disturbances: soft-diet feeding, molar extraction and bite-raising. Animal studies shed light on the histological and biochemical alterations together with the behavioural tests associated with experimentally induced masticatory disturbances, thus contributing to an objective understanding of the significance of clinical studies. To our knowledge, a systematic review with these features has not previously been published.
In order to engage in a critical discussion of the topic and to provide reliable conclusions, the included studies have been evaluated with the ARRIVE checklist and the SYRCLE tool for risk of bias in animal studies, as reported in Tables 2 and S1 and S2. Unfortunately, as described in the Results paragraph, it was impossible to adequately assess the risk of selection bias, performance bias and detection bias of included studies with the SYRCLE tool because, with very few exceptions, the necessary information was not reported. Moreover, standard deviations and confidence intervals were not always reported, making it difficult to assess the general precision of the results. On the other hand, the risk of attrition bias, reporting bias or other bias could be adequately assessed with the SYRCLE tool. As analytically described in the Results, the literature reviewed showed that a disturbed mastication is significantly associated with a reduced number of hippocampal pyramidal neurons in CA1 and CA3, reduced neurogenesis in the DG, reduced synaptic activity, downregulation of BDNF, glial proliferation, and impaired memory and spatial orientation. Indeed, it has recently been shown that hippocampal pyramidal neurons in CA1 reorganize in the course of spatial reward learning [42]. The significance of an observed difference between experimental groups cannot be directly translated to the clinical setting, even though it must be said that a number of clinical studies has already been published in the field. In the context of this review, important aspects of the magnitude of the results are the general agreement among the included studies concerning the histological and biochemical alterations and the proportions of the observed differences, especially concerning the reduction of the number of hippocampal neurons and synaptic activity in conditions of altered mastication, which were generally quite large. Moreover, a correlation was consistently shown between the reduction in the number of hippocampal neurons and reduced performances in behavioural tests targeting hippocampaldependent cognitive functions (memory and spatial orientation).
Soft-diet feeding protocols have been introduced as a laboratory model of reduced mastication. In this experimental condition, the animal's occlusion is not altered in any way but the masticatory function, and the muscular activation it entails, is reduced by means of a soft diet, much easier to consume but containing the same nutritional value as the diet of regular, solid consistency that is fed to the control group. This kind of experimental condition is very important because it is free from confounding factors: the occlusion is stable and preserved and it does not significantly alter the animals' body weight. In a particularly elegant study design, Y. Fukushima-Nakayama and colleagues, using pathogen free mice (further narrowing the possibilities of confounding effects due to inflammation), showed that soft-diet feeding does not alter the growth of the animals' body but, as would be expected in conditions of impaired masticatory function, it reduces the development of the craniofacial bones and masticatory muscles [2]. Moreover, and unexpectedly, soft-diet feeding was not shown to be a source of stress [20,21]. Two studies investigated the association between soft-diet feeding and corticosterone incretion: they agreed that soft-diet feeding is not a source of stress, but more research is needed to confirm this important point, which is not yet clear.
Molar extraction protocols suffer from a significant risk of bias due to the necessity of surgically removing the teeth, which may be a source of stress and may cause long lasting infection and inflammation. Furthermore, the extraction of molar teeth introduces local alterations of the occlusion, which may be relevant in conditioning the outcomes. Nevertheless, the results were very close to soft-diet feeding protocols, suggesting that a similar (or the same) biological mechanism is at play. The reports of activation of the HPA axis in the molarless condition were contradictory. The molarless condition was found to be a long-term source of stress by some Authors [23,25,27,36], but not others [24,34]. Moreover three studies with molar extraction protocols included in their design the fitting of experimental dentures: even though more research is needed on this subject, the results indicate that the rehabilitation of edentulous patients may be beneficial for their cognitive status with a partial recovery of neurons even if the loss of teeth has gone untreated for a long period of time [24,28,34].
Bite-raising protocols introduce an extreme form of occlusal disruption that suddenly changes the customary occlusal scheme to two unique prematurities. This form of experimental alteration of the masticatory function is very different from models of reduced mastication with a soft diet: in contrast with the two experimental conditions previously discussed, it has been shown in agreement in studies published before 2010 and therefore not included, but recently reviewed elsewhere [43], to be a source of stress, being significantly associated with increased corticosterone incretion. Humans, as well as animals, are known to release stress by clenching their teeth, i.d. by reaching the occlusal position of maximum intercuspation (MI). When only two premature points of contact are present, as in the bite-raised condition, the dental arches are effectively prevented from reaching MI, which disrupts the physiology of swallowing that occurs with teeth in MI. It is worth underlining that this procedure interferes not only with chewing, but with swallowing and other parafunctional activities of the jaws as well, which differentiates the bite-raised condition from soft-diet feeding and molar extraction models and may be the reason for the activation of the HPA axis in this instance.
Soft-diet feeding models and molar extraction models showed effects in mice of all ages. In SAMP8 mice, a murine model of accelerated senescence, the bite-raised condition was significantly associated with hippocampal alterations especially in aged (9-month-old) animals; however, a positive association was shown in non-aged ddY mice as well [38].
The association between a reduced number of teeth and a greater susceptibility to cognitive impairment in the ageing population is well established [1]; however, clinical studies are at risk of bias, on account of the multiplicity of confounding factors potentially involved. Even though we routinely rely on counting the number of remaining teeth (which is easier to measure clinically and to process epidemiologically), it is quite possible that older people with fewer teeth resemble more closely soft-diet feeding models than they do tooth extraction models. Indeed, people with a suboptimal dentition (generally considered to be less than 20 teeth) tend to eat softer foods, which are easier to chew. Tooth loss in humans recognizes many different causes and, over the length of a human lifetime, it is quite likely that the effects of inflammation and stress associated with tooth extraction or periodontal disease dissipate after a few years.
Soft-diet feeding models may be relevant in young individuals as well. In the industrialized world, softer foods, refined and high in caloric value have become prevalent: the findings of this review strengthen the view that a diet mindful of proper masticatory efficiency may be important to promote the development of the jaws and masticatory muscles as well as to preserve a level of hippocampal neurogenesis adequate to face the challenge of a healthy ageing [44].
The fact that histological and morphological changes in the hippocampus are induced by disruption of masticatory function at any age, after weaning as well as in aged animal models, is of clinical significance. In recent years, the concept of prevention has been associated with that of a repository to be maintained through life: when a biological reserve has been exhausted, prevention is no longer meaningful because the pathological process has already begun, even if it may take years to show itself clinically [45]. The results of this review suggest that the masticatory function may have a role in preserving and continuing stimulating hippocampal trophism, contributing to slow down the gradual cognitive decay associated with old age.
The details of the motor control of mastication are nowadays clear [8], but the influence of mastication on the structure and function of parts of the central nervous system, site of memory and cognition, is probably just beginning to be investigated. Indeed, the most important limitation of this review is that, even though the linkage between impaired mastication (especially soft-diet feeding and, to a lesser extent, molar extraction) and morphofunctional alterations of the hippocampus, together with reduced performances in behavioural tests, is very well established in animals (as well as its association with cognitive status in humans), the biological mechanism responsible for this association is still not clear. A recent study supported the hypothesis that masseter muscle-derived neprilysin is carried along the trigeminus by retrograde axonal transport to reach the hippocampus, following electrical or cholinergic stimulation [46]. More research is necessary to clarify this very important point.

Conclusions
In conclusion, animal studies significantly substantiate the claim that mastication has a role in maintaining the neuronal population and the synaptic activity of the hippocampus, and, consequently, cognitive performance and memory. The results of this review must be considered in the light of the less than optimal quality of scientific reporting of the included studies, which did not allow a thorough determination of the risk of bias; nevertheless, the consistent reporting of significant histological and functional alterations in conditions of impaired mastication and the agreement with clinical studies suggest with reliability that mastication has a protective role on the CNS and should be preserved free of alterations, as far as possible, from infancy through adulthood and into the aging years. The emerging role of chewing in the preservation of hippocampal trophism, neurogenesis and synaptic activity is fascinating and may contribute to the study of neurodegenerative diseases in new and potentially relevant ways.