**2. Evidence-based data of gut-brain axis alteration in aged laboratory animals**

Aging is (so far) an irreversible process which impacts on all cell populations, with several common denominators such as genomic instability, epigenetic alterations and oxidative stress [26]. It equally affects gut lining and brain cells, but it also changes the gut microbiota composition [27, 28] which in turn is associated with behavioral and physiological modifications. An anxious behavioral phenotype was observed while transferring fecal microbiota between two strains of mice [10]. Also, fecal microbiota transplantation from depressed patients to microbiota-depleted rats may have the potential to provoke behavioral and physiological features specific to depression, including anhedonia, anxiety-like behaviors, as well as modifications in tryptophan metabolism, suggesting that gut microbiota could be an important player in neurobehavioral changes in the rat [29]. Reduced anxiety and depression-related comportment in mice were connected to dysregulated GABA signaling by metabolizing dietary glutamate through certain strains of *Lactobacillus* and *Bifidobacteria* [30]. Another study focused on the analysis of microbiota of aged mice. It is reported that, although composition differed significantly, there was only an insignificant overall increase in bacterial taxa, from that of young mice. At phylum level, the most common microbial taxa were *Bacteroidetes* and *Firmicutes*. A few statistically significant differences in bacterial groups were noticed in aged mice. At the phylum level, TM7 was significantly higher, while at family level abundance less than 1%, significant increases in *Porphyromondaceae*, TM7 uncultured, *Clostridiaceae*, *Thermoanaerobacteraceae*, *Desulfovibrionaceae* and *Oxalobacteraceae* were seen in aged mice compared to young. Genera *Odoribacter* was much higher in the aged group whereas, other bacterial genera, including *Butyricimonas*, TM7 uncultured, *Gelria*, *Anaerosporobacter*, *Clostridium* and *Oxalobacter* had significant increases in aged mice. The Chao 1 index (which estimates species richness), the number of observed species and the phylogenetic diversity, the Shannon Index (alpha diversity) and beta diversity were higher in the aged compared with young groups. Also, gut permeability in young and aged mice prior to and after 1 h of restraint stress was assessed, proved that aged mice had significantly greater basal intestinal permeability than young mice [31].
