Lan, Italy) was dissolved in dimethylsulfoxide (DMSO Sigma-Aldrich, Milan, Italy) (240 mg/mL) and after that diluted 1:one hundred in saline resolution or tap water. DMSO diluted 1:100 was employed as car. Apocynin (15 mg/kg) or car have been intraperitoneally (i.p.) injected 1 h just before the first pressure session then added towards the drinking water (two.four mg/mL) for the following days. This amount corresponds to above 300 mg/kg, since our mice approximately drank 3.5 mL water/day. 2.three. Forced Swim Strain The Forced Swim Strain (FSS) was performed as previously described [38,39]. Briefly, single mice have been placed within a glass beaker (height, 24 cm; diameter, 12 cm) containing 1500 mL of water (25 2 C) for 5 min twice a day for four consecutive days. Soon after FSS, mice have been promptly dried having a towel and returned to their home cage. During the very first and the last day in the FSS, mice had been videotaped and the total quantity of NPY Y5 receptor custom synthesis immobility and swimming time were scored working with the ANY-MAZE software program (Stoelting, Bought by Ugo Basile, Varese, Italy). two.4. Novelty-Suppressed Feeding Test The Novelty-Suppressed Feeding (NSF) test was performed as previously described [40]. Briefly, inside the center of a large rectangular arena (60 40 15 cm), filled together with the wooden bedding, a regular meals pellet was placed on a white paper platform. Before from the test, mice were food deprived for 24 h. An individual mouse was positioned within a corner and also the latency to consume the pellet was scored. Quickly soon after the initial bite, mice were transferred to their residence cage along with the quantity of meals eaten for the duration of 5 min was weighed (home cage meals consumption). two.5. Lipid Peroxidation Measurement Lipid peroxidation was analyzed by measuring malondialdehyde (MDA) levels working with thiobarbituric acid reactive substances (TBARS), a biochemical developed through the lipid peroxidation approach [41]. Briefly, hippocampi and prefrontal cortex had been homogenized in 1.15 KCl pH 7.four. Homogenized tissue (one hundred ) or 50 of plasma had been added to 150 of thiobarbituric acid (TBA) (eight ), 150 of acetic acid (20 , pH 3.4) and 50 of Sodium Dodecyl Sulfate (SDS) (8.1 ) and boiled for 60 min. Samples have been chilled on ice and centrifuged at 13,200 rpm for 10 min. The absorbance of the collected supernatant was then measured at 530 nm making use of a microplate reader (Biorad, Milan, Italy). two.six. RNA Isolation and Reverse Transcription Quantitative Real-Time PCR Total RNA from the hippocampi was extracted applying the Direct-zolTM RNA MiniPrep (Zymo Research, purchased by Euroclone, Milan, Italy) in line with manufacturer’s directions. cDNA was synthesized making use of the iScript kit (Biorad, Milan, Italy) as outlined by manufacturer’s directions. Quantitative Real-Time PCR analysis was performed on a CFX Connect Actual Time Program (Biorad) utilizing the iTaq Universal SYBR Green supermix (Biorad), as previously described [42]. Primer sequences are described in Table 1. Relative expression of mRNA for the target genes was performed by the comparative CT (CT ) strategy and normalized on Rps8 and Gapdh housekeeping genes. Relative mRNA levelsBiomolecules 2021, 11,4 ofwere expressed as fold-change. Primer’s specificity was established using the melting curve MMP-13 manufacturer evaluation.Table 1. Primer sequences. Gene p47phox p67phox Hdac1 Hdac2 Hdac4 Hdac5 Gapdh Rps18 Forward ACCGGCTATTTCCCATCCAT GCCAGCTTCGGAACATGGT GAGTTCTGTCAGTTGTCCACGG GGGACAGGCTTGGTTGTTTC CAATCCCACAGTCTCCGTGT TGTCACCGCCAGATGTTTTG CGTGCCGCCTGGAGAAACC TGGAGCGAGTGATCACCATCA Reverse TGGATGCTCTGTGCGTTGC G.