Australia) that converts the digitized images to grayscale images (black and white) after color selection ( Solomon, 2009). Fig. 1A and B illustrates the color segmentation which see more allows the selective capture of the immunoreactive sites against the desired antibody and measures their pixel densities. Quantitative analysis was accomplished by calculating the percentage of pixels of the anti-AQP4 and anti-GFAP in the white matter and granular, Purkinje and molecular layers of the cerebellar cortex separately. All numerical results were analyzed using the GraphPad Prism software package (San Diego, CA, USA) and expressed

as the mean ± standard error (S.E.). Differences between data means of saline-treated and PNV-treated groups were determined by the unpaired Student t-test with a p value ≤ 0.05 indicating statistical significance. Two-way analysis Fulvestrant research buy of variance

was used when appropriate to test age/temporal differences in the response to venom effect. The AQP4 and GFAP immunoreactivity of astrocytes was co-localized among the neuron bodies of the granular and Purkinje layers and widespread throughout the width of the molecular layer with the difference that the glial processes appeared well-defined in the anti-GFAP reaction. The anti-AQP4 reaction, although strong, was more diffuse. In animals injected with PNV, there was gradual time-dependent increase in the intensity of the immunolabeling in the white matter and layers of the cerebellar cortex for both P14 and adults. Fig. 2, Fig. 3 and Fig. 4 were chosen to illustrate the reaction pattern at two time intervals

(2 h and 24 h) for either P14 rats or adults; the figures also display the calculation of the density of pixels relative to the immunoreaction intensity throughout the period of observation. There is no significant difference in the physiological expression of AQP4 and GFAP in the white matter of adult and P14 rats at the different time-points after saline solution injection (Fig. 2C and F). However, rats administered PNV showed a 103.8% increase of AQP4 expression in adult animals (*p ≤ 0.05) and a 77.5% (**p ≤ 0.01) in neonate animals after 24 h ( Fig. 2C). The venom also caused a 57.3% increase in the GFAP expression after 24 h only in the astrocytes MycoClean Mycoplasma Removal Kit of P14 animals (*p ≤ 0.05). Although not significantly, AQP4 expression was 11%–20% higher in P14 PNV-treated animals (ranging from 16.48 ± 1.06 at 2 h to 27.73 ± 2.57 at 24 h, respectively) than in adult PNV-treated ones (where it ranged from 13.68 ± 2.03 at 2 h to 24.94 ± 3.55 at 24 h, respectively). In contrast, the values for GFAP were in general slightly higher for adults than for P14 animals. The two-way analysis of variance showed that the time elapsed between envenomation and animal euthanasia interfered with the expression of AQP4 and GFAP in the white matter of neonates and adults (*p ≤ 0.05). Also, there was interference of the age variable in the expression of AQP4 and GFAP at 24 h only.