Difference of brain-derived neurotrophic factor expression and pyramid cell count during mastication of food with varying hardness

Abstract Previous studies suggested that mastication activity can affect learning and memory function. However, most were focused on mastication impaired models by providing long-term soft diet. The effects of chewing food with various hardness, especially during the growth period, remain unknown. Objective: To analyze the difference of hippocampus function and morphology, as characterized by pyramidal cell count and BDNF expression in different mastication activities. Materials and Methods: 28-day old, post-weaned, male-Wistar rats were randomly divided into three groups (n=7); the first (K0) was fed a standard diet using pellets as the control, the second (K1) was fed soft food and the third (K2) was fed hard food. After eight weeks, the rats were decapitated, their brains were removed and placed on histological plates made to count the pyramid cells and quantify BDNF expression in the hippocampus. Data collected were compared using one-way ANOVA. Results: Results confirmed the pyramid cell count (K0=169.14±27.25; K1=130.14±29.32; K2=128.14±39.02) and BDNF expression (K0=85.27±19.78; K1=49.57±20.90; K2=36.86±28.97) of the K0 group to be significantly higher than that of K1 and K2 groups (p<0.05); no significant difference in the pyramidal cell count and BNDF expression was found between K1 and K2 groups (p>0.05). Conclusion: A standard diet leads to the optimum effect on hippocampus morphology. Food consistency must be appropriately suited to each development stage, in this case, hippocampus development in post-weaned period.


Introduction
Mastication is the process of crushing food and mixing it with saliva to form a bolus prior to swallowing, being a complex process involving the rhythmical opening and closing of the jaw, the secretion of saliva and tongue movement. 1 Previous studies found correlation between mastication and the functions of other body parts aside from the digestive system. In addition to influencing nutritional intake, masticatory disorders have been proven to exert influence in increasing the risk of obesity and hyperglycemia, as well as impeding stress hormone regulation, learning processes and memory function. 2 Mastication activity is critical as one of the sources of sensory input into the hippocampus. 3 A decline in masticatory activity caused by a disorder can lead to hippocampal morphological changes, such as the decrease in pyramid cell count, in dendritic spines in cornu amonis-1 (CA1) and in proliferating cells in both the dentate gyrus and the astrocyte hypertrophy. 4 Other studies also found that reduced muscle activity may decrease blood supply to the brain, thus impeding cerebral metabolism. 5,6 The correlation between mastication activity and hippocampus morphology was identified from the neural pathway. Somatosensory input from the masticatory system may influence the hippocampus through synaptic projection from the thalamus and cerebral cortex. 3 Brain Derived Neurotrophic Factor (BDNF) is fundamental in hippocampal learning and memory function. 7 BDNF is a member of the neurotrophin family that promotes neuron survival, growth and plasticity, being abundantly found in the hippocampus. 8 Previous studies observing the correlation between mastication, stress and cognitive function have proved that a long-term soft diet during the postweaning period could lead to learning and memory impairment. On the other hand, forced mastication in mice resulting from a hard diet can increase the proliferation and survival rate of neuron cells in dentate gyrus. 7,[9][10][11][12] In the growth period, the maturation of motoric function occurs during the maturation of the entire central nervous system, as well as of the morphological and functional maturation of the craniofacial complex. Newborn mammals obtain nutritional intake by sucking, which will later develop into a masticatory motion. Regarding rats, the first mastication movements were recorded on day 12 post-natal and completely developed between days 18 and 21. 13 Nevertheless, the effects of mastication on learning and memory functions of the hippocampus, especially during the growth period, are not fully understood.
Based on a previous study by Irmalia, et al. 14 (2017), spatial memory tests were conducted on 28day old, post-weaned rats after they had been fed food of varying hardness. The results confirmed that the standard diet group produced the best performance on a radial eight arm maze test, compared to the soft and hard diet groups. 14 Thus, this study aims to analyze the differences of hippocampus function and morphology, through cellular and molecular examination of this under varying mastication activity. Based on the previous study, it is hypothesized that, providing a standard diet during the growth period will give the best response toward hippocampus morphology, compared to providing soft and hard diets.

Material and methods
Ethical approval kg, iron 70 mg/kg, iodine 0.6 mg/kg, selenium 0.07 mg/kg, vitamin A 7500 IU/kg, and vitamin D3 1500 IU/kg); all groups were given 20 mg food/day. Rats were acclimatized for seven days prior to treatment, subsequently being placed in a plastic cage containing wood shavings bedding covered with woven wire. They were kept at room temperature following a consistent 12-hour light-dark cycle. After eight weeks, the rats were euthanized through an anesthesia overdose and their brains were isolated.

Histological examination
Histological examination consists of pyramidal cell count and BDNF expression, which were done using a cell counter combined with manual counting in case a system error occurred. To prevent bias, the study outcome measurement was done by an expert in histology who was blind to group allocation.

Pyramidal cell count
Isolated brains were soaked in 10% neutral buffered formalin for two hours in individual pots.
The brains were then immersed in formaldehyde, soaked for 24 hours and dehydrated using ethanol (at 70% concentration for 24 hours followed by 90% for 1 hour), rinsed using xylene and, finally, embedded in paraffin. A coronal section (5 µm) was performed following the bregma (−3.3 mm to −3.8 mm posterior to the bregma) using a microtome (Accu-Cut SRM, Sakura, AJ Alphen aan den Rijn, Netherlands). 15 The sectioned tissues were deparaffinized, placed on object glass and stained using hematoxylin eosin (HE). 16 Pyramidal cell count was conducted using a light microscope (Nikon H600L, Melville, New York, USA) at 400x magnification. Cell count and pictures were taken of five different fields that were randomly chosen

Statistical analysis
Collected data were analyzed using Kolmogorov-Smirnov test to find the distribution. Given that all variables were normally distributed, the resulting data were analyzed with the SPSS 16 software for Windows (SPSS Inc., Chicago, USA) using a one-way ANOVA test, and continued using Tukey's LSD test to find differences between each group. Results are presented as mean and standard deviation (SD). The significance level considered was 0.05. that the control group had more pyramid cells in the hippocampus than the soft diet or hard diet groups.

Results
One-way ANOVA showed that there was a significant difference between the three groups (p=0.049; r 2 =0.285). Pyramid cell count of the K0 group was considerably higher when compared to those of K1 (p=0.036) and K2 (p=0.029) groups. Additionally, although K1 had more pyramid cells than K2, there was no significant difference (p=0.909).

Regarding BDNF expression, observation results
were similar to pyramid cell count results. BDNF expression in K0 was higher than in K1 and K2 groups Furthermore, BDNF expression in the K1 group was slightly higher than in the K2 group, but no significant difference between these groups (p=0.326) was identified.

Discussion
This study was conducted using animal models The result obtained is in accordance with our previous one, which assessed hippocampus function using behavior tests. The study found that the normal diet group had the best performance in behavior tests, followed by soft diet and hard diet groups. This showed that behavior assessment may represent the histological structure of hippocampus. 14 These findings also corroborate to another study by Okihara, et al. 7 (2014), which found a memory impairment accompanied by low number of neuron cells in the hippocampus after long-term soft diet. 7 Previous studies on mastication impaired animal models through tooth extraction 22,23 , bite-raise 24,25 and long-term soft diets 7,26 found that these aspects may alter hippocampus learning and memory function. Other studies also proved that impaired function of the hippocampus due to long-term soft diet could be improved by providing a hard diet. 27,28 Therefore, mastication activity is critical in maintaining hippocampus function for learning and memory. 29,30 Newborn rats obtain nutrition supplies from breast-feeding alone, without any other food or water, until they are 14 days old. The weaning process begins at that point, with the breast-feeding period being completely finished at 30 days of age. During the weaning period, the transition from suckling to mastication and an increase in the jaw closing muscle activity also occurs. Thus, firing frequency of the motor neuron within the jaw closing muscle will continuously intensify as newborns develop to prepare the muscle for the mastication process. 20 Other studies also argued that physiological adaptation of an organ will increase with growth. Thus, it is possible that the response to different mastication loads will differ according to age. 21 Given that the samples in this study were all in the transition period from suckling to chewing movement, we can assume that this is the main cause for the low pyramid cell count and BDNF expression in hippocampus of hard diet group.
Rats in the transition period may need adaptation to chewing movements; thus, increasing the food hardness gradually is necessary according to growth The main limitation of this study was providing food of differing hardness but with the same nutritional content. Thus, this study used standard rodent chow, which is softer when compared to whole grain and seeds yet harder than powdered grain and seeds, as a control. However, the nutritional contents of each type are slightly different.

Conclusion
During the growth period, reduced mastication due