Initially, the chorioallantoic vein of the embryos was exposed by cutting a window (5 × 5 mm) in the eggshell with a drill in a dark room, using a lamp. Then, experimental hypoxic conditions were induced by enveloping half of the chicken embryo air chamber with a polyvinyl layer for 24 h at 39 °C, to limit gas exchange. Hypoxic conditions were terminated by removing the polyvinyl layer, and then, the embryos were further re-oxygenated for 24 h at 37 °C (H+Ox). The normoxic (Nx) control group was incubated under normal air conditions at 37 °C for 48 h. At the end of the first 24 h period, the embryos were microinjected through the chorioallantoic vein with 100 µL of either Cy3-labeled GH (Cy3-GH; 0.15 µg/g of body weight, for 2 h, to determine BBB crossing and distribution within several CNS areas, Figure 1 A) or recombinant chicken GH (rcGH, American Cyanamid AC 4797-100, Wayne, NJ, USA, 0.15 µg/g) to study the neuroprotective effects 24 h after hypoxia/reoxygenation injury by analyzing apoptosis, lipid peroxidation, inflammatory mediators and the expression of several neurotrophic factors (Figure 1 B). The control groups received 100 µL of a saline solution as a vehicle.
Cy3 conjugation to GH (Cy3-GH) was performed according to the Cy3 Mono-Reactive dye pack manufacturer’s instructions (GE Healthcare Life Sciences, Amersham, ON, Canada).
Cy3 conjugation to GH (Cy3-GH) was performed according to the Cy3 Mono-Reactive dye pack manufacturer’s instructions (GE Healthcare Life Sciences, Amersham, ON, Canada).