6 0.2073 Alkaline phosphatase (U/l) 3,780 to 14,800 6,300 CP-690550 clinical trial 4,800 4,030 7,033 47.8 0.0712 Blood urea nitrogen (mg/Dl) 7.0 to 17.1 5.7 8.0 7.5 8.0 0.41 0.1272 Glucose level (mg/Dl) 110 to 306 219 213 169 203 8.2 0.1269 SEM standard error of the mean. aReference values of biochemical indices for poultry [20]. Brain morphology: examination

of brain tissue microstructure Cell numbers in the brain cortex (area counted 3,500 μm2) were not significantly different between the groups (Table 3). However, histological evaluation of brain morphology revealed pathological changes in the brain structure in embryos treated with NP-Pt, showing a moderate degradation of the cerebellar molecular layer, neuronal loss in the cerebellum cortex, and astrocytosis (Figure 2). Table 3 Numbers of cells in the brain cortex in the control and in groups treated with ICG-001 cost different NP-Pt concentrations   Control 1.0 μg/ml 10.0 μg/ml 20.0 μg/ml SEM Pvalue Number of cells 613 583 600 697 6.5 0.448 Figure 2 Cross sections through the granular layer of the cerebral cortex stained with hematoxylin

and eosin. (A) Control, (B) 1 μg/ml, (C) 10 μg/ml, (D) 20 μg/ml. Black arrows, astrocytosis; white arrows, neuronal loss. Scale bars 10 μm. Examination of brain tissue ultrastructure TEM examination of brain tissue morphology showed no abnormalities in the control group. However, in embryos treated with NP-Pt, degradation of the mitochondria, rounded nuclei with dispersed chromatin, and vacuoles in the cytoplasm were seen (Figure 3). Figure 3 TEM images of brain tissue after treatment with platinum nanoparticles. Concentration of NP-Pt was at 20 ppm. Arrows signify (A) vacuoles, (B) degradation of endoplasmic reticulum, and (C, D) degradation of the mitochondria. Scale bars 500 nm. Immunohistochemical measurements showed that the number of PCNA-positive nuclei significantly decreased after in ovo injection of NP-Pt solutions, attaining the lowest value in the 20-μg/ml group (Figure 4). Immunodetection of PCNA-positive nuclei by immunohistochemical methods was carried out in cross many sections of the granular layer of the cerebellar cortex.

PCNA-positive nuclei were brown, and PCNA-negative nuclei were blue (Figure 5). Immunohistochemical measurements showed the numbers of caspase-3-positive cells significantly increased in the NP-Pt groups compared to those in the control group (Figure 4). The greatest increase was observed in the group receiving 20 μg/ml of NP-Pt. Cross sections of the granular layer of cerebral cortex were also immunostained with the caspase-3 antibody. Caspase-3-positive cells showed brown cytoplasm, while the cytoplasm of caspase-3-negative cells was blue (Figure 6). Figure 4 Numbers of caspase-3-positive cells and PCNA positive nuclei (counting area = 3,500 μm 2 ). Error bars indicate standard error of the mean. Bars with different superscripts differ significantly (P < 0.05). Figure 5 Cross sections of a granular layer in the cerebral cortex by PCNA staining.