Data were untargetedly filtered by manual and regardless of what the metabolite was, the largest peak area remained as the representing ion and excluded the other ions at the similar MRTs. high frequency rTMS ameliorated cognitive impairment in normally aging individuals [2,3]. Several studies have shown that rTMS promoted neuronal plasticity related genes and proteins expression [4-6], remodeling of dendritic spines [7], and regulating the metabolites of frontal brain regions such as some neurotransmitter systems including Gamma-Aminobutyric acid (GABA) and glutamate [8-10]. Brain aging is associated with structural and functional changes that invariably lead to a decrease in cognitive functions even in healthy individuals, as well as to changes that increase the brains susceptibility to neurodegenerative disorders. Rodents offer several benefits as models to investigate the mechanisms and to identify the potential treatment of age-related cognitive decline. Such as, similar to humans, information that requires prefrontal cortex (PFC) processing is particularly vulnerable to ageing, and PFC function can be easily assessed by behavioral tests and in parallel with biochemical changes in brain tissues [11,12]. Previous study reported that Kunming mice exhibited an age-related cognitive impairment during normal aging [13]. The classic method of passive avoidance task to test the cognitive ability in rodent relied on memory of the footstock punishment, which is involved in deeply involved in PFC [14,15]. Metabonomics combined with untargeted multivariate analysis, has been extensively applied to many fields, such as understanding the diseases of the biochemical basis in the process of diagnosis and treatment according to the metabolic profiles in biological fluids and tissues [16-19]. From the metabolites profiles, several studies have shown that the metabolic dysfunction of cholesterol [20], GABA [21-23] and N-Acetyl aspartic (NAA) [24], which are much more related with the cognition decline during aging. Therefore, using an aging mouse model, we aim to investigate the behavior changes in combination with metabolic profiling before and after application of rTMS to investigate the possible mechanism of rTMS improvement of PFC dysfunction in ageing mice. == Materials and Methods == == Animals and rTMS methods == Female Kunming mice in two age groups (3-4 and 16-17 months old) were used. All the mice were housed in a room under conditions of ACAD9 temperature (20-22 C) and a 12-hour light-dark cycle, and mice had access to the food and water ad libitum. All animal experiments were performed Tilorone dihydrochloride under an animal study protocol approved by the ethics committee of Hebei Medical University. Aged mice (16-17 months old) were randomly divided into two groups: aged rTMS group and aged group. Aged Tilorone dihydrochloride rTMS group, mice exposed to high frequency Tilorone dihydrochloride rTMS (25 Hz) with the coil placed just above the head of the mice for 14 consecutive-days, 10 trains per day with a 30-s intertrain interval, 100 pulses per train. Aged group, mice were treated similar to aged rTMS mice by the reverse side of the coil without rTMS effect. Young group (3-4 months old), mice were treated same as aged mice by the reverse side of the coil for a sham purpose. During the procession of rTMS or sham rTMS, mice were fixed calmly with a flexible plastic tube with holes at both ends, a small hole at one end for mice breathing and the other hole suitable for the mice probed into and fixed it with a sponge. == Behavioral Test == The cognitive performance of all animals was observed with passive avoidance test in the following day after 14 consecutive-days rTMS exposure or sham rTMS exposure (15 mice were in each group). The apparatus for passive avoidance task was a plastic box (12 cm 12 cm 18 cm), and a column insulation platform (diameter, 4.5 cm; height, 5 cm) placed inside the box. The box had a grid floor consisting of stainless-steel bars 0.2 cm in diameter at 1 cm intervals, which could be electrified by a shock scrambler. First day, adaptation trial was conducted in order to facilitate habituation to the apparatus. Mice were placed on the platform facing the wall and allowed to explore the compartment freely for 300 s. As mice placed on the platform, they generally step down onto the floor within a short time. Second day, acquisition session was performed in the condition of electric current (36 mV) continually delivered through the grid floor. During the acquisition trial, the mice.