Anoxemia

The effects of anoxia on retinal protein synthesis and axonal transport were investigated in the freshwater turtle, Trachemys scripta. The optic system (retina, optic nerve, optic tract, and optic tectum) of the freshwater turtle was used because, due to its linear structure, it is an ideal model to measure protein production and transport. This species of turtle is known for its ability to withstand long periods of anoxia; however, little data is available on in vivo protein synthesis in the brain on an anoxia-tolerant species. An intraocular injection of S-methionine was used to label retinal proteins during 18 hours of anoxia, and the tissues were removed and prepared for analysis using 2D gel electrophoresis. Using autoradiography the regulation of retinal proteins was compared under normoxic and anoxic conditions. In addition, labeled retinal proteins rapidly transported along the optic nerve and tract, to the tectum were also compared under these conditions. Although certain proteins were produced in lesser amounts during anoxia, the populations of proteins were the same during anoxia as they were in the control, suggesting that all proteins were still produced during anoxia. Additionally, certain proteins were produced in greater amounts during anoxia. The findings suggest that less important proteins are down-regulated in response to anoxia while those proteins that are up-regulated may serve as protective mechanisms that enable the organism to maintain neuronal connections in this system during anoxia.

Cardiac ischemia, stroke and some neurodegenerative disorders are all characterized by cell damage and death due to low oxygen levels. Comparative studies show that anoxia tolerant model systems present a unique opportunity to study "survival" instead of death in the complete absence of oxygen. The freshwater turtle (Trachemys scripta elegans) is unique in its ability to survive total oxygen deprivation for hours to days, as well as reoxygenation insult after anoxia. The broad objective of this study is to understand the modulation of key molecular mechanisms involving stress proteins and VEGF that offer neuroprotection and enhance cell survival in the freshwater turtle through anoxia and reoxygenation. In vivo analyses have shown that anoxia induced stress proteins (Hsp72, Hsp60, Grp94, Hsp60, Hsp27, HO-1); modest changes in the Bcl2/Bax ratio and no change in cleaved caspase-3 expression suggesting resistance to neuronal damage. These results were corroborated with immunohistochemical evidence indicating no damage in turtle brain when subjected to the stress of anoxia and A/R. To understand the functional role of Hsp72, siRNA against Hsp72 was utilized to knockdown Hsp72 in vitro (neuronally enriched primary cell cultures established from the turtle). Knockdown cultures were characterized by increased cell death associated with elevated ROS levels. Silencing of Hsp72 knocks down the expression of Bcl2 and increases the expression of Bax, thereby decreasing the Bcl2/Bax ratio. However, there was no increase in cytosolic Cytochrome c or the expression levels of cleaved Caspase-3. Significant increase in AIF was observed in the knockdown cultures that increase through anoxia and reoxygenation, suggesting a caspase independent pathway of cell death.