In the biochemical analyses, the following biomarkers were analyzed. C-Fos (c-Fos; MyBioSource, MBS729725) expression was measured in the central micturition areas: medial preoptic area (MPA), the ventrolateral periaqueductal gray (vlPAG), and pontine micturition center (PMC) (as described in detail in Section 4.7.1 below). The levels of Vesicular acetylcholine transporter (VAChT; LifeSpan BioSciences, CN LS-F12924-1) and Rho Kinase (ROCK1; LifeSpan BioSciences, LS-F32208) were assessed in bladder detrusor muscle, while the concentrations of nerve growth factor (NGF; LifeSpan BioSciences, CN LS-F25946-1) and brain-derived neurotrophic factor (BDNF; PROMEGA, CN G7610) were analyzed in urine. As for the bladder urothelium, the following markers were measured: tumor necrosis factor α (TNF- α, LifeSpan BioSciences; LS-F5193), interleukin 1β (IL-1β, Cloud-Clone; SEA563Ra), interleukin 6 (IL-6, LifeSpan BioSciences; LS-F25921-1), Calcitonin Gene-Related Peptide (CGRP; Biomatik, CN EKU02858), ATP Citrate Lyase (ATP; LifeSpan BioSciences, LS-F10730), corticotropin-releasing factor (CRF; Alpco, Salem, NH, USA, CN 48-CRFMS-E01), Organic Cation Transporter 3 (OCT3; antibodies-online, CN ABIN6227163), and Transient Receptor Potential Cation Channel Subfamily V, Member 1 (TRPV1; LSBio, LS-F36019). Finally, the respective values of TNF-α, IL-1β, IL-6, interleukin 10 (IL-10, LifeSpan BioSciences; LS-F5081), CRF, BDNF, and NGF were measured in the hippocampus (as described in Section 4.7.2 below). All measurements were carried out according to the manufacturers’ instructions. Each sample was measured in duplicate. The results are presented in pg/mL.
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