Graph prism 4

Cell counting was performed with a 40X objective lens using a Zeiss Axioplan microscope (Carl Zeiss, Oberkochen, Germany) with appropriate filter cubes. Images were obtained with a Polaroid DMC digital photocamera (Polaroid, Cambridge, Mass., USA), and minimal adjustments to brightness and contrast were made with Corel Photo Paint and Corel Draw (Corel, Dublin, Ireland). Each specimen was evaluated and counted by two investigators in a blind fashion. For each animal, G_αtran- and G_αgust-IR cells were counted in 36 random microscope fields (each field 0.28 mm²), for a total area of 10 mm², in the cardiac and pyloric mucosa, in 50 randomly selected villi and glands in the small intestine, and in 50 glands in the large intestine. Only villi and glands perpendicular to the muscularis mucosae were evaluated. The values obtained from counting G_αtran- and G_αgust-IR cells were grouped for each experimental group (Ctr, Hp3 and Hp30) and the means were calculated. Moreover, the mean numbers of cells showing a colocalization of G_αtran or G_αgust-IRs with different EEC markers were calculated. Results were expressed as mean ± standard deviation (SD). Data were analysed by one-way ANOVA (Graph Prism 4, GraphPad Software, Inc., La Jolla, CA, USA). A P<0.05 was considered statistically significant.

The specimens were examined using a Nikon Eclipse Ni microscope and images were taken using a Nikon DS-Qi1Nc digital camera and NIS Elements software BR 4.20.01 (Nikon Instruments Europe BV, Amsterdam, The Netherlands). Minor adjustments to contrast and brightness were made using Corel Photo Paint, while figure panels were prepared using Corel Draw (Corel Photo Paint and Corel Draw, Ottawa, ON, Canada). The 20× objective was used for morphometric evaluation. In the gastric mucosa, the area occupied by OPs-IR in 4.1 mm² (0.410 × 10 fields) was measured by binarization (described above). In addition, the number of GHR, NPY and SOM IRs in 4.1 mm² were counted in the gastric mucosa.
For each experimental group (CTR, BP 5% and BP 10%), the values obtained for OPs-IR area and the number of EECs were corporate and the means were calculated. The results were expressed as mean ± standard deviation (SD). The data were analyzed by one-way ANOVA (Graph Prism 4, GraphPad Software version 4.01, Inc., La Jolla, CA, USA). The experimental group was considered as the main effect. In addition, the means were then separated using the Tukey-HSD test. A p ≤ 0.05 was considered statistically significant.