Samples were then washed, incubated with anti digoxigenin conjugate for 30 min utes and stained with compound libraries the nuclear Inhibitors,Modulators,Libraries marker DAPI. To quantify the number of terminal deoxynucleotidyl transferase mediated dUTP nick end labeling positive cells, each coronal section was divided into 16 square areas that involved the necrotic core and the area of ischemic penumbra, and compar able areas in the non ischemic hemisphere. Two areas of interest were chosen in the boundaries between the ischemic penumbra and necrotic core, and a third zone was located in the necrotic core. To determine the number of TUNEL positive cells, images were digi tized in a Zeiss Axioplan 2 microscope 20 �� objective with a Zeiss AxioCam and imported into AxioVision, viewed at 150% of the original with Image MetaMorph Software and the percentage of TUNEL positive cells in relation to the total number of DAPI positive cells per AOI recorded.

Each observation was repeated eight times. To study Inhibitors,Modulators,Libraries the effect of TWEAK on pBAD expression, Wt cerebral cortical neurons were incubated for 60 min utes with rTWEAK Inhibitors,Modulators,Libraries 100 ng mL or a comparable volume of vehicle and fixed and stained 1, 3 or 6 hours later with an antibody against pBAD. Each obser vation was repeated four times. Statistical analyses Data was analyzed by either a Wilcoxon rank sum test or, in cases where more than one group was compared, by analysis of variance. Statistical significance was deter mined by P 0. 05. Results The interaction between TWEAK and Fn14 protects neurons from hypoxia induced cell death First we used an ELISA to quantify the concentration of TWEAK in the culture media of Wt neurons exposed to OGD conditions for 0 to 6 hours.

We found that the concentration of TWEAK in the culture media increased from 6 1. 3 pg mL in cells maintained under normoxic conditions to 10. 32 3. 64 Inhibitors,Modulators,Libraries pg mL, 14. 72 3. 47 pg mL, 13. 37 0. 7 ng mL and 6. 4 2. 5 pg mL after 30, 60, 180 and 360 minutes of exposure to OGD conditions, respectively. Activation of inflammatory pathways by a precondi tioning stimulus is thought to reduce the inflammatory response to a subsequent period of ischemia, leading to neuroprotection, and so we decided to investi gate whether the cytokine TWEAK induces hypoxic tolerance. However, because it has been reported that 24 hours of incubation with TWEAK induces neuronal death, first we investigated whether treatment over a shorter period of time also has an effect on cell survival.

Wt cerebral cortical neurons were incubated for 1 or 24 hours with 100 or 300 ng mL of TWEAK followed by determination of cell sur vival with the MTT assay as described Inhibitors,Modulators,Libraries in selleck the Methods section. We found that, as previously described, 24 hours of incubation with 100 or 300 ng mL of TWEAK is associated with a 25. 4% and 37% decrease in neuronal survival, respectively.