The Flt-1-reactive neurons contained in two randomly selected fields per anatomic region (CA1, CA3 and DG) per animal, and one randomly selected field per CA2 per animal was counted. Each field corresponded to an area probe measuring 0.90 mm2; hence a mean value of anti-Flt-1 neurons were obtained in 10 fields for CA1, CA3 and DG per treatment/time (2 fields × 5
animals/group) and 5 fields for CA2 per treatment/time (1 field × 5 animals/group). All images were taken with a 40× objective using an Olympus BX51 photomicroscope (Japan) equipped with Image Pro-Plus image analyzer software (SA). Image analysis (quantification of the immunoreactivity optical density) was done using the free access GIMP 2.6.4 software (GNU Image Manipulation Program, CNE) that converts the digitized images to grayscale images (black and white) after color selection (Solomon, 2009). This segmentation by color makes possible to determine the percentage of pixels for staining by a given antibody. Selleckchem BGB324 Since Flt-1-immunolabeled
cells presented at least two different intensity of reactivity (due to cells situated differentially in relation to the section plane which passed through them) two color selections were done to avoid ambiguous identification of cell labeling and jeopardize the conclusions. The percentage of vessels with perivascular edema Ku-0059436 cell line was calculated by dividing the number of affected vessels by the total number of vessels per section per animal. A total of 10 sections per hippocampal region per time point was examined in PNV- and saline-treated groups (2 sections per animal × 5 animals/time = 10 sections/CA1, CA3 and DG hippocampal regions and 1 section per animal × 5 animals/time = 5 sections/CA2 hippocampal region) per time interval. Adenylyl cyclase The percentages of blood vessels affected were compared between PNV-injected and saline-injected (control) groups at each time. The quantification was done by two observers. Data
were expressed as mean ± SEM. Differences were analyzed using the GraphPad Prism software package (San Diego, CA, USA). One-way analysis of variance (ANOVA) followed by the Tukey test was used to compare groups. A value of P ≤ 0.05 indicated statistical significance. In addition, two-way ANOVA was conducted to compare differences between PNV treatment on different time points (1, 2, 5 and 24 h) and ages (14 days and 8–10 weeks) throughout the experiment, and whether there was an interaction between these two conditions. A P-value of 0.05 indicated statistical significance. After a delay of 2 min (for P14 rats) and 10 min (for adult rats), after i.p. injection of P. nigriventer venom, the animals exhibited hyperemia, piloerection, shivering, salivation, some dyspnea, and flaccid followed by spastic paralysis of the hindlimbs. At least one out of five rats used in each period showed tonic convulsion. Four P14 animals and one of 8–10 weeks old died soon after venom injection, suggestively by respiratory arrest, since necropsy showed lung edema.