The overall aim of this proposal is to demonstrate the feasibility of studying the pathophysiology of cerebral chronic sublethal hypoxia by profiling gene expression patterns, sorted by brain topography, that occur in response to injury in the developing brain. Our focus will be on developing the technical and computational approaches needed to analyze these changes both temporally and topographically in a massively parallel way, so as to open novel avenues of exploration that bear on an understanding of the molecular basis of the profound neurodevelopmental deficiencies that accompany very premature birth. We have established and documented an excellent animal model (rat) that faithfully reproduces many aspects of this disorder. Our approach is to explore the feasibility of applying the detection capabilities of cDNA microarrays, coupled with the screening capabilities of high-throughput tissue microarrays and methods for the sensitive multiplex detection of in-situ expression profiles, to establish a database defining genes relevant to specific regions of the brain, and their response to development and sublethal hypoxia. Computational algorithms will be developed to aid in the analysis and dissemination of this database, and specialized cDNA microarrays will be made available for other investigators through Yale's Keck Laboratory DNA array facility. These goals will be achieved by the following aims: 1) Construct cDNA microarrays comprising at least 8,000 rat genes relevant to gene expression in the developing rat nervous system; 2) Use the rat brain specific cDNA arrays to analyze the patterns of expression of thousands of genes during brain maturation and its response to hypoxia; 3) Employ modern data analysis tools to define gene hierarchies or clusters that participate in the orchestration of responses to hypoxia in our model system; and 4) Map the gene expression clusters to specified cells and regions in the brain utilizing high-throughput tissue microarrays constructed from control and experimental animals, validated by laser capture microdissection and other methodologies. Collectively, these studies will open entirely new avenues for investigation of this devastating public health problem, and establish key methodologies for future investigations of other brain disorders.
| Kim, Jung H; Kwon, Soojung J; Stankewich, Michael C et al. (2016) Reactive protoplasmic and fibrous astrocytes contain high levels of calpain-cleaved alpha 2 spectrin. Exp Mol Pathol 100:1-7 |
| Curristin, Sheila M; Cao, Anjun; Stewart, William B et al. (2002) Disrupted synaptic development in the hypoxic newborn brain. Proc Natl Acad Sci U S A 99:15729-34 |
