Time-dependent Gene Profiling Indicates the Presence of Different Phases for Ischemia/Reperfusion Injury in Retina

Abstract

Ischemia/reperfusion (IR) injury has been associated with several retinal pathologies, and a few genes/gene products have been linked to IR injury. However, the big picture of temporal changes, regarding the affected gene networks, pathways, and processes remains to be determined. The purpose of the present study was to investigate initial, intermediate, and later stages to characterize the etiology of IR injury in terms of the pathways affected over time. Analyses indicated that at the initial stage, 0-hour reperfusion following the ischemic period, the ischemia-associated genes were related to changes in metabolism. In contrast, at the 24-hour time point, the signature events in reperfusion injury include enhanced inflammatory and immune responses as well as cell death indicating that this would be a critical period for the development of any interventional therapeutic strategies. Genes in the signal transduction pathways, particularly transmitter receptors, are downregulated at this time. Activation of the complement system pathway clearly plays an important role in the later stages of reperfusion injury. Together, these results demonstrate that the etiology of injury related to IR is characterized by the appearance of specific patterns of gene expression at any given time point during retinal IR injury. These results indicate that evaluation of treatment strategies with respect to time is very critical.

Keywords: gene expression; ischemia/reperfusion (IR) injury; microarray; retina.

Figure 1

Venn diagrams showing: (A) the total numbers of differentially expressed genes in each of the 3 time points: 0 h (red), 24 h (green) and 7 days (blue) post IR and the common genes among the time points; (B) only up-regulated genes in each of the 3 time points, 0 h (red), 24 h (green) and 7 days (blue) post IR and the common genes among them; (C) only down-regulated genes in each of the 3 time points, 0 h (red), 24 h (green) and 7 days (blue) post IR, and the common genes among them. Abbreviations: IR, ischemia-reperfusion; SC, scham control; FC, fold change.

Figure 2

Hierarchical clustering of four gene-subsets in sham control and IR animal groups. The bright red color indicates up-regulation, the deep green color indicates down-regulation, and black portrays no change. (A). Hierarchical clustering of inflammatory/immune response genes. The heat map indicates that expressions of these genes are strongly increased after 24 h and 7 days reperfusion. (B). Hierarchical clustering of cell death/apoptosis differentially regulated genes. The heat map indicated that most of these genes are significantly up-regulated at 24 h and 7 days reperfusion. (C). Hierarchical clustering of complement system differentially regulated genes. The heat map demonstrates that the 11 related genes were strongly up-regulated compared to their sham control counterparts at the 7 days reperfusion time point. (D). Hierarchical clustering shows that 10 of 11 glutamate receptor and associated signaling related genes were down-regulated compared to their sham control counterparts at the 24 h reperfusion time point. The solute carrier gene denoted by Slc17a6, and which is significantly down regulated at IR-7 d, is an alias for the glutamate transporter VGLUT2.

Figure 2

Hierarchical clustering of four gene-subsets in sham control and IR animal groups. The bright red color indicates up-regulation, the deep green color indicates down-regulation, and black portrays no change. (A). Hierarchical clustering of inflammatory/immune response genes. The heat map indicates that expressions of these genes are strongly increased after 24 h and 7 days reperfusion. (B). Hierarchical clustering of cell death/apoptosis differentially regulated genes. The heat map indicated that most of these genes are significantly up-regulated at 24 h and 7 days reperfusion. (C). Hierarchical clustering of complement system differentially regulated genes. The heat map demonstrates that the 11 related genes were strongly up-regulated compared to their sham control counterparts at the 7 days reperfusion time point. (D). Hierarchical clustering shows that 10 of 11 glutamate receptor and associated signaling related genes were down-regulated compared to their sham control counterparts at the 24 h reperfusion time point. The solute carrier gene denoted by Slc17a6, and which is significantly down regulated at IR-7 d, is an alias for the glutamate transporter VGLUT2.

Figure 3

Comparative evaluation of microarray and real-time RT-PCR results for eight selected genes. Notes: Values on the Y-axis represent the fold change relative to the sham control. Values on the X-axis represent the time course of reperfusion. Error bars indicate the standard error of the mean (n = 3).

Figure 4

Immunocytochemical detection and localization of C3 (green) and C1q (red), in the late stage IR 7 d retina vs the sham control retina. DAPI (blue) labels the cell nuclei. Strong immunolabeling in the nerve fiber and ganglion cell layers of the IR-injured retina was obtained, when compared with the sham control animals. Notes: Arrows point to fibers that expressed C1q protein. Abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, Inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Figure 5

Western blot analysis of protein expression in sham control and IR 0 h, 24 h and 7 d retina. C3 and Rac2 proteins reached peak accumulation at IR 7 d. Activated-caspase3 protein was increased at IR 24 h, and then gradually decreased. GNG7 protein was detected in all retinal samples, but was decreased at IR 24 h and 7 d. GRM5 protein accumulation was decreased at IR 24, then return to normal levels at IR 7 d.