Neuron-specific mRNA complexity responses during hippocampal apoptosis after traumatic brain injury.

In an effort to understand the complexity of genomic responses within selectively vulnerable regions after experimental brain injury, we examined whether single apoptotic neurons from both the CA3 and dentate differed from those in an uninjured brain. The mRNA from individual active caspase 3(+)/terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling [TUNEL(-)] and active caspase 3(+)/TUNEL(+) pyramidal and granule neurons in brain-injured mice were amplified and compared with those from nonlabeled neurons in uninjured brains. Gene analysis revealed that overall expression of mRNAs increased with activation of caspase 3 and decreased to below uninjured levels with TUNEL reactivity. Cell type specificity of the apoptotic response was observed with both regionally distinct expression of mRNAs and differences in those mRNAs that were maximally regulated. Immunohistochemical analysis for two of the most highly differentially expressed genes (prion and Sos2) demonstrated a correlation between the observed differential gene expression after traumatic brain injury and corresponding protein translation.

Single-cell antisense RNA amplification and microarray analysis as a tool for studying neurological degeneration and restoration.

Neurodegenerative diseases typically affect subpopulations of neurons. Characterizing these vulnerable cells and identifying the factors that make them susceptible to damage while neighboring cells remain resistant are essential to the understanding of molecular pathogenesis that underlies neurodegenerative diseases. Classically, molecular analysis of the central nervous system involves the identification and isolation of an anatomic region of interest; next, the relevant tissue is pulverized, and the resulting homogenate is analyzed. Although this method provides useful data, its effectiveness diminishes when used in areas of high cellular diversity or in instances in which one cell type is lost as a consequence of selective cell death or quiescence. A technique that affords the ability to assess molecular events in a very precise anatomical site would provide a powerful tool for this research discipline. In this review, we discuss the amplification of messenger RNA from single neural cells and the subsequent use of the RNA to probe DNA microarrays in an effort to create cell-specific molecular profiles. Specifically, recent work in single-cell expression profiling in Alzheimer's and Huntington's diseases is discussed. We also review some new work with neural stem cells and their application to restorative neurobiology. Finally, we discuss the use of cell-specific molecular profiles to better understand the basics of neuronal cell biology.

Expression profiling following traumatic brain injury: a review.

Traumatic brain injury (TBI) elicits a complex sequence of putative autodestructive and neuroprotective cellular cascades. It is hypothesized that the genomic responses of cells in the injured brain serve as the basis for these cascades. Traditional methods for analyzing differential gene expression following brain trauma demonstrate that immediate early genes, cytokines, transcription factors, and neurotrophic factors can all participate in the brain's active and directed response to injury, and may do so concurrently. It is this complexity and multiplicity of interrelated molecular mechanisms that has demanded new methods for comprehensive and parallel evaluation of putative as well as novel gene targets. Recent advances in DNA microarray technology have enabled the simultaneous evaluation of thousands of genes and the subsequent generation of massive amounts of biological data relevant to CNS injury. This emerging technology can serve to further current knowledge regarding recognized molecular cascades as well as to identify novel molecular mechanisms that occur throughout the post-traumatic period. The elucidation of the complex alterations in gene expression underlying the pathological sequelae following TBI is of central importance in the design of future therapeutic agents.

Methodological considerations regarding single-cell gene expression profiling for brain injury.

Genomic microarrays are rapidly becoming ubiquitous throughout a wide variety of biological disciplines. As their use has grown during the past few years, many important discoveries have been made in the fields of central nervous system (CNS) injury and disease using this emerging technology. In addition, single-cell mRNA amplification techniques are now being used along with microarrays to overcome many of the difficulties associated with the cellular heterogeneity of the brain. This development has extended the utility of gene expression profiling and has provided researchers with exciting new insights into the neuropathology of CNS injury and disease at a molecular and cellular level. New methodological, standardization, and statistical techniques are currently being developed to improve the reproducibility of microarrays and facilitate the analysis of large amounts of data. In this review, we will discuss the application of these techniques to experimental, clinically relevant models of traumatic brain injury.