Purpose: Perinatal hypoxic-ischemic human brain damage is a significant reason behind acute mortality and chronic neurologic morbidity in newborns and kids. cAMP levels. Pentoxifylline treatment improved short-term memory by suppressing apoptotic cell death in the hippocampus with elevated cAMP levels. Conclusions: Pentoxifylline ameliorated perinatal hypoxic-ischemia in rat pups. This alleviating effect could be ascribed to the inhibition apoptosis due to increased cAMP production by pentoxifylline. Cell Death Detection Kit (Roche Mannheim Germany) according to the manufacturer’s protocol [5 8 The sections were postfixed in ethanol-acetic acid (2:1) and rinsed. The sections were then incubated with proteinase K (100 μg/mL) rinsed and incubated in 3% H2O2 permeabilized with 0.5% Triton X-100 rinsed again and incubated in the TUNEL reaction mixture. The sections were then rinsed and visualized using Converter-POD with 0.03% 3 3 (DAB). Mayer’s hematoxylin (DAKO Glostrup Denmark) was used as a counterstain and the sections NVP-ADW742 were mounted onto gelatin-coated slides. Fst Caspase-3 Immunohistochemistry Caspase-3 immunohistochemistry was performed as explained previously [5 22 The sections were incubated overnight with mouse anti-caspase-3 antibody (1:500; Santa Cruz Biotechnology Santa Cruz CA USA) followed by incubation with biotinylated mouse secondary antibody (1:200; Vector Laboratories Burlingame CA USA) for another one hour. The secondary antibody was amplified with the Vector Elite ABC kit (1:100; Vector Laboratories). Antibody-biotin-avidin-peroxidase complexes were visualized using 0.03% DAB and sections were mounted onto gelatin-coated slides. Western Blot Analysis Western blot analysis was performed as explained previously [3 6 Hippocampal tissues were homogenized with lysis buffer. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad Hercules CA USA). The protein mixture made up of 40 μg total protein was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane. Mouse actin antibody (1:2 0 Santa Cruz Biotechnology) mouse Bax antibody (1:1 0 Santa Cruz Biotechnology) and mouse Bcl-2 antibody (1:1 0 Santa Cruz Biotechnology) were used as main antibodies. Horseradish peroxidase-conjugated antimouse antibodies for Bax and Bcl-2 (1:2 0 Amersham Pharmacia Biothech GmbH Freiburg Germany) were used as secondary antibodies. Bands were detected using an enhanced chemiluminescence detection kit (Santa Cruz NVP-ADW742 Biotechnology). Data Analysis The number of TUNEL-positive and caspase-3-positive cells was expressed as quantity of cells per square millimeter in the CA1 region. To compare relative expression of proteins we evaluated the detected bands densitometrically using Molecular Analyst ver. 1.4.1 (Bio-Rad). Statistical analysis was performed using one-way analysis of variance NVP-ADW742 followed by Duncan test and the results are expressed as mean±standard error of the mean. Significance was set at P<0.05. RESULTS Effect of Pentoxifylline on Short-term Memory Short-term memory was disrupted by perinatal hypoxic-ischemic injury (P<0.05) and pentoxifylline alleviated this memory impairment (P<0.05) (Fig. 1). Fig. 1. Effect of pentoxifylline on latency in the step-down avoidance task. A Sham-operation group; B NVP-ADW742 perinatal hypoxicischemia-induced group; C perinatal hypoxic-ischemia-induced and 50-mg/kg pentoxifylline-treated group; D perinatal hypoxic-ischemia-induced ... Effect NVP-ADW742 of Pentoxifylline on cAMP Levels in the Hippocampus Hippocampal cAMP levels were decreased by perinatal hypoxic-ischemic injury (P<0.05) and pentoxifylline increased the cAMP levels (P<0.05) (Fig. 2). Fig. 2. Effect of pentoxifylline on 3?-5?-cyclic adenosine monophosphate (cAMP) levels in the hippocampus. A Sham-operation group; B perinatal hypoxic-ischemia-induced group; C perinatal hypoxic-ischemia-induced and 50-mg/kg pentoxifylline-treated ... Effect of Pentoxifylline on DNA Fragmentation in the Hippocampal CA1 Region DNA fragmentation in the CA1 region was increased by perinatal hypoxic-ischemic injury (P<0.05) and pentoxifylline suppressed this increase in DNA fragmentation (P<0.05) (Fig. 3). Fig. 3. Effect of pentoxifylline on DNA fragmentation in the hippocampal CA1 region. Upper panel: Photomicrographs NVP-ADW742 of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells in the hippocampal CA1 region. A Sham-operation ... Effect of.