All animal procedures were approved by local Animal Care Committee and are in accordance with the NIH Guide for the care and use of laboratory animals. Organotypic hippocampal slice cultures were prepared according to the method of Stoppini et al. (1991), with modifications (Valentim et al., 2003, Cimarosti et al., 2005, Horn et al., 2005 and Frozza et al., 2009). Briefly, 400-μm-thick hippocampal slices were prepared from 6 to 8-day-old male Wistar rats using a McIlwain tissue chopper and separated in ice-cold Hank’s balanced salt solution (HBSS) BYL719 chemical structure composed of (mM): glucose
36, CaCl2 1.26, KCl 5.36, NaCl 136.89, KH2PO4 0.44, Na2HPO4 0.34, MgCl2 0.49, MgSO4 0.44, HEPES 25; fungizone 1% and gentamicin 0.1 mg/mL, pH 7.2. The slices were placed on Millicell culture insert and the inserts were transferred to a 6-well culture plate. Each well contained 1 mL of tissue culture medium consisting of 50% minimum essential medium, 25% HBSS, 25% heat inactivated horse serum supplemented
with (mM, final concentration): glucose 36, HEPES 25 and NaHCO3 4; fungizone 1% and gentamicin 0.1 mg/mL, pH 7.3. Organotypic cultures were maintained in a humidified incubator gasified with 5% CO2 atmosphere at 37 °C for 30 days. Culture medium was changed three times a week. Aβ25–35 and Aβ35–25 (reverse peptide) stock solutions (675 μM) were prepared in sterile distilled water and stored at −20 °C. To obtain the fibrillar form of Aβ25−35 peptide, an aliquot of the stock solution was incubated under 37 °C during the 4 days preceding its use in culture (Casal et al., 2004). The so-called non-fibrillar Aβ corresponds to the peptide that was not subjected to the Vandetanib aforementioned activation process and was therefore added to the culture directly from stock solution. On the 28th in vitro day, the medium was replaced by a serum reduced medium (2.5%) into which 25 μM of fibrillar/non-fibrillar Aβ25–35 or Aβ35–25 was added or not (control slices). Previous experiments showed that this concentration (25 μM) of Aβ25–35 had the most toxic effect (data not shown), at least for the fibrillar peptide form. Cellular damage was assessed by fluorescent image analysis of propidium iodide (PI)
uptake (Noraberg et al., 1999). One hour before the end of the treatments, which means after 47 h of Aβ25–35 or Aβ35–25 exposure, 7.5 μM of PI was because added to the medium and incubated for 1 h. PI uptake is indicative of significant membrane injury (Macklis and Madison, 1990). Cultures were observed with an inverted microscope (Nikon Eclipse TE 300) using a standard rhodamine filter set. Images were captured and then analyzed using Scion Image software (http://www.scioncorp.com). After capture of images, the area where PI fluorescence (transformed in pixels) was detectable above the background was analyzed using the “density slice” option of Scioncorp Software through the division of PI fluorescence by the total area of the slice (Valentim et al.