Physical exercise induces structural alterations in the hippocampal astrocytes: exploring the role of BDNF-TrkB signaling. mg/mL), 4 mg/mL of Fg in the presence of a function-blocking anti-PrPC peptide or anti-mouse IgG, function-blocking anti-PrPC peptide, or anti-mouse IgG alone. After treatment, either cell lysates were collected and analyzed via Western blot or coimmunoprecipitation was performed, or astrocytes were fixed and their activation was assessed with immunohistochemistry. Results showed that Fg dose-dependently activated astrocytes, increased expressions of PrPC and tyrosine (tropomyosin) receptor kinase B (TrkB), and PrP gene. Blocking the function of PrPC reduced these effects. Coimmunoprecipitation demonstrated Fg and PrPC association. Since it is known that prion protein has a greater effect on memory reduction than amyloid beta, and that activation of TrkB is involved in neurodegeneration, our findings confirming the possible formation of Fg-PrPC and Fg-induced overexpression of TrkB on astrocytes suggest a possible triggering mechanism for STM reduction that was seen previously during mild-to-moderate TBI. NEW & NOTEWORTHY For the first time we showed that fibrinogen (Fg) can associate with cellular prion protein (PrPC) on the surface of cultured mouse brain astrocytes. At high levels, Fg causes upregulation of astrocyte PrPC and astrocyte activation accompanied with overexpression of tyrosine receptor kinase B (TrkB), which results in nitric oxide (NO) production and generation of reactive oxygen species (ROS). Fg/PrPC interaction can be a triggering mechanism for TrkB-NO-ROS axis activation and the resultant astrocyte-mediated neurodegeneration. for 10 min at 4C. The supernatant was collected and total protein content was determined by the Bradford method. WB analysis was performed as described previously (Muradashvili et al. 2014a, 2015, 2016). Astrocyte culture. Mouse brain astrocytes (MBAs, catalog no. M1800-57) that were isolated from C57BL/6J mice either day 2 or day 8 postbirth, were purchased from ScienCell. In accordance with the manufacturers claim, astrocytes were validated with GFAP staining and have no or neglectable number of glial cell contaminants. The MBAs were grown in AM-a complete media and used at DLL3 passage 4 (presence of glial cells was not detected). The MBAs were plated on either in eight-well chambered glass-bottomed plates or six-well plastic plates (Corning, NY) coated with poly-l-ornithine for better astrocyte attachment. Cells were Fluocinonide(Vanos) kept at 37C with 5% CO2/air in a humidified environment as recommended by the manufacturer and were used at the 3rd or 4th passages for the experiments. In vitro experimental setups and groups. Experiments were conducted once MBAs in eight-well chambers reached 80% confluence. The complete media was removed the day of the experimentation and replaced with serum-free media (SFM) for 2 h. Serum-starved cells were then treated with SFM alone, 2 Fluocinonide(Vanos) mg/mL of Fg, 4 mg/mL of Fg, 4 mg/mL of Fg with 0.03 mg/mL of PrPC function-blocking peptide, 4 mg/mL of Fg with anti-mouse IgG (20 L), PrPC function-blocking peptide (20 L), or 100 ng/mL of lipopolysaccharides (LPS) from O111:B4 (Sigma-Aldrich, St. Louis, MO) used as a positive control that were dissolved in SFM. The content of Fg was chosen based on knowledge of the normal level (2 mg/mL) of Fg in human plasma and the level of Fg (4 mg/mL) during inflammation (Letcher et al. 1981). Each experimental group contained Fluocinonide(Vanos) Hirudin (1 U/mL) to block possible conversion of Fg to fibrin. The cells were kept in the incubator at 37C overnight. After the experiment, MBAs were washed with phosphate buffered saline (PBS), fixed with 3.7% formaldehyde, and processed for immunochemical analysis. In a separate series of experiments, Fluocinonide(Vanos) cells grown in six-well plates were treated as described above. After the experiments, cells were washed with PBS, lysed with RIPA buffer, and processed for WB analysis as previously described (Muradashvili et al. 2014a, 2015, 2016). A total of four experiments were performed. Each experimental group was performed in duplicate. All treatments and analyses were performed by researchers who were blinded to the treatment groups. Immunohistochemistry. The fixed MBAs were treated with a permeability/blocking solution containing Tween 20 (0.5 L/mL).