Rtv 3140

Research was carried out on 12 male Wistar rats. All the animals were subjected to surgical implantation of a microchip in the abdominal region of the left vagus nerve. The animals were divided into two groups. The first one (MC, n = 6) underwent microchip stimulation of the left vagus nerve, while the control group (C, n = 6) was subjected to sham laparotomy.
For the purpose of surgical laparotomy, the subdiaphragmatic part of the left vagus nerve was connected to a 1-cm-diameter silicon-coated (RTV 3140, Dow Corning), battery-driven microchip. Experiments were carried out after 12 h of food deprivation, an empty stomach making it easier to access the nerve following the administration of pentobarbital (vetbutal, dose: 25 mg/kg of body mass; Biowet, Puławy, Poland) intraperitoneally. Once the abdominal part of the left vagus nerve was localized and electrically connected and the wounds were closed, the rats were moved to cages and subjected to 7 days of stimulation. Food and water were allowed ad libitum during the whole experiment. The period of the stimulating signal was 20 s, its duration was 0.1 s, and the amplitude was 200 mV. Finally, following electric stimulation, all the rats were guillotined. All procedures involving animals were approved by the Jagiellonian University Bioethical Committee (20/11/2009).

Cortices of Sprague Dawley rats at postnatal days 0–1 were harvested as a source for cortical neurons. The tissue was dissociated enzymatically with 50 U papain (Worthington) in Retinal Buffer solution at pH7.4 for 30 min at 37 °C. The enzymatic action was stopped with fetal bovine serum and cells were resuspended in Neurobasal medium supplemented with B-27 for up to 48 h (all from Gibco Life Technologies). To study the interaction between neurons and DAPF, custom-made tissue culture dishes were designed (Supplementary Fig. S1). Three individual silk filaments were placed in parallel on 22 mm round glass coverslips with their ends folded over coverslip edges and glued at the back with Super Glue Gel (RS Components). Holes with a diameter of 13 mm were punched in the bases of tissue culture dishes and the coverslip, silk side-up, was glued with RTV 3140 (Dow Corning) to the underside of the dish, sealing the punched hole. To allow consistent counting, approximately 12.5 × 10⁵ of neurons were seeded on the 13 mm diameter area containing the filaments, and cultured for 2 to 5 days, followed by fixing with 4% buffered paraformaldehyde solution (PFA) and processing for immunocytochemistry. For RGD adhesion assays, cortical neurons were plated in the same way but cultured for only 1 h with and without RDG tripeptide (50 µM; Sigma) in the culture media.