Spinal cord injury (SCI) is a devastating disease without effective treatment. The chronic SCI remains the most difficult to treat. The pathologic hallmarks in chronic SCI include increased astrogliosis and inhibitory molecules that hinder axonal regeneration. However, the understanding of the detailed molecular pathways is still very limited. For example, how inhibitory molecules are regulated and maintained in the chronic phase remains unclear. Current therapeutics for gliosis is not ideal, and new molecular targets are urgently needed. Previous studies have usually focused on a small number of genes and pathways at a time, and thus did not provide a comprehensive view of the complex mechanisms underlying SCI pathophysiology. Although, during the last decade, microarray studies have provided valuable insights into SCI, microarray suffers from limitations in resolution, dynamic range and accuracy. Recent advances in RNA-Sequencing technology make it possible to globally map transcribed regions and quantitatively analyze expression at an unprecedented level of sensitivity and specificity. Based on our preliminary studies of differential expression using RNA-Seq during acute and subacute SCI phases in mouse contusive injury models, we propose to investigate the intricate relationship of genes and pathways in the spinal cord tissue and the predominant component of the glial scar (purified astrocytes) in chronic SCI by using integrated RNA-Seq and network analyses. We hypothesize that novel genes and pathways that regulate or maintain inhibitory molecules in reactive astrocytes (chondroitin sulphate proteoglycans, tenascins, ephrin-B2 and Slit proteins etc.) are critical for the chronic SCI inhibitory environment associated with glial scar. Specifically, we propose to derive a better understanding of the progression of SCI pathophysiology by characterizing gene and splicing isoform changes in SCI subchronic/chronic phases at both temporal and spatial levels. Additionally, we will use an innovative strategy of integrated network analysis to identify novel genes of interest (GOIs) and pathways involved in gliosis as new molecular targets. We will also incorporate pharmacogenomic information into our analyses that will serve as a powerful tool for translation. Finally, we will test GOIs potentially involved in neuron inhibition of reactive astrocytes by loss- and gain-of-function assays in a glia scar model for their functional effects. Our study is the first to propose that the mechanisms of chronic SCI and gliosis be investigated at the systems level using RNA-Seq, and key genes be identified via innovative pathway and network analyses and be tested by functional assays. The advantage of the genome-wide analysis is that, as a de novo discovery approach, it can identify critical missing links in the disease processes that were not previously appreciated. Importantly, we will generate a comprehensive data resource of SCI gene expression which will be extremely valuable for the research community (jiaqianwulab.org/SCI browser/data). The successful completion of this project will lead to the discovery of novel molecular targets and shift the research and clinical paradigms.