The Journal of Neuroscience, September 29, 2004, ():
Patterns of Gene Expression Reveal a Temporally Orchestrated Wound Healing Response in the Injured Spinal Cord
J. Neurosci. Velardo et al.
24: 8562
Supplemental data
Files in this Data Supplement:
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Supplemental Figure 1. Experimental design for microarray experiment and paradigm for mechanism-based array results analysis (A-D). Sprague-Dawley (SD) and athymic nude (AN) rats were contused at spinal level C 4,5. At 1, 3, 10, 30, 90 days post injury animals from each time point and each strain were sacrificed and designated for microarray, histological, and magnetic resonance microimaging analyses (A). For microarray analysis, cervical spinal cords were dissected into three parts designated lesion, rostral, and caudal. Lesion mRNA was processed in ensuing steps. Rostral and caudal mRNA were stored for future analysis. Lesion segments from four animals were pooled, processed and analyzed per Affymetrix GeneChip®, using a minimum of three chips per time point per strain (B). Schematic representation of Part I of the multi-level analysis paradigm used to determine biological functions of those genes with significant expression changes. Two concomitant analyses were performed: (i) an overall survey of all significantly expressed probe sets organized by general biochemical function and (ii) after clustering of the genes into k-means bins, identification of the specific biochemical function of each gene. Study of these two analyses formed the basis for development of multiple working hypotheses as to the functional significance of the gene expression patterns (C). Schematic representation of Part II of the multi-level analysis paradigm. After completion of definitive gene identification for each gene in each k-means clustered bin, the analysis proceeds towards increasingly detailed identification of the gene itself, its biochemical and biological functions and its interactions with other genes in the context of the injury and time. The goal is to determine biological processes for each bin and then across bins, create the organization of genes in Supplemental Table 1, and ultimately construct a theoretical framework for molecular and cellular events after SCI.
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Supplemental Table 1. Transcriptional profile of k-means clustered genes organized individually by function and bin and color coded by function (see legend).
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Supplemental Table 2. Immune response along with tissue and vascular repair and genesis transcripts dominate the post-cervical contusion gene expression profile from days 1-90. (Probe sets are categorized and color coded by function as in Table 1 in body of manuscript and bins correlate with post-injury interval as designated in Table 1).
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Supplemental Figure 2. Model of SCI based on known molecular and cellular events of cutaneous WH in comparison with patterns of transcript expression post-SCI (A-F). Cutaneous WH has distinctive, orchestrated but overlapping stages that begin immediately at injury and continue for as long as two years post-wounding. In A, days 1-90 of WH are depicted along the time line of our experimental paradigm. B, illustrates the order and timing of immigration of specific WH cell populations into the wound (bright colors and solid boxes; Clark, 1993; Martin, 1997; Witte and Barbul, 1997; Singer and Clark, 1999). In SCI, these populations of cells have been identified with similar timing (pale colors and dashed boxes) i.e., platelets (Goodman et al., 1979), neutrophils (Means and Anderson, 1983; Xu et al., 1990; Taoka et al., 1997; Carlson et al., 1998; Chatzipanteli et al., 2000), macrophages (Popovich et al., 1997; Leskovar et al., 2000), lymphocytes (Popovich et al., 1997), and endothelial cells ((Mautes et al., 2000; Loy et al., 2002; Cassella et al., 2002; Noble et al., 2002; Williams and Velardo, data not shown here). Presently, there is no established time line in SCI for fibroblasts or fibroblast-like cells. Prominent vascular repair (C), immune (D) and growth factor (E) transcript families that are increased in expression post SCI, mimic the timing of those that dominate at similar times after skin injury. F illustrates the characteristic of progression in WH from a provisional hemostatic matrix through a granulation type matrix to the collagen matrix found in mature wounds and/or scarring. After SCI, the orchestrated expression of 190 ECM, CAD and proteolytic transcripts when organized by time and function, suggests that after contusion, the ECM remodels with a similar progression. (Color coding is consistent with that assigned in the functional classification of transcripts in Fig. 4 in manuscript body and Table 1 and Supplemental Tables 1 and 2. For a version of the manuscript discussion with the specific references to this figure go to http://users.mbi.ufl.edu/velardo).
