When released upon cellular stress or injury such as Sipeimine during MI. Toll-like receptors constitute one of the largest subfamilies of PRRs. Of these, TLR9 has been demonstrated to specifically detect unmethylated DNA, rich in cytosine-phosphate-guanine motifs. Recent work has identified mitochondrial DNA to function as a DAMP, causing activation of the innate immune system through TLR9. On this notion, our group have previously reported increased circulating levels of mtDNA upon PCI of human MI. The consequence of TLR signaling in I/R remains unclear. Activation of TLR9 prior to I/R lead to reduced infarct extension and improved cardiac function. From a clinical point of view, an important and hereto unaddressed question is whether activation of said receptors during onset of I/R also impacts subsequent myocardial damage. Thus, in the current study we investigated the pathophysiological consequence of intervening with the TLR9-agonist CpG B during onset of ischemia. Evaluation of infarct size was performed as previously described. Briefly, after 24 hours of reperfusion, mice were anesthetized, the thoracic cavity opened and the slipknot re-ligated. The aorta ascendens was subsequently clamped and approximately 0.2 ml 2% Evans blue dye was injected into the proximal portion of the aorta, this allowing coronary perfusion and visualization of the area at risk. Hearts were subsequently excised, let to freeze on dry-ice before cross-sectioned into standardized 1 mm slices by the use of a mouse heart slicer matrix. The main objective of this study was to investigate the effect of TLR9-activation during onset of myocardial I/R with respect to infarct size. Although TLR9-activation elicited robust increases in systemic inflammation as well as a decrease in some inflammatory markers within the myocardium, these systemic and cardiac inflammatory alterations did not translate into changes in infarct size. Several previous reports have convincingly demonstrated beneficial effects of TLR-stimulation, including TLR9, initiated before ischemia. These studies do advocate for a putative role of TLR-activation in clinical situations of expected cardiac ischemia. 4-(Benzyloxy)phenol However, no studies have investigated the effects of TLR-stimulation restricted to the more common clinical scenario; i.e. intervention upon ischemia/reperfusion. Even though still clinically relevant, our experimental setup does not allow discriminating the effects of our intervention on ischemia, reperfusion or a combination of both. In order to specifically address the reperfusion phase of I/R, drugs should preferably be injected directly into the occluded artery upon reperfusion. As to our knowledge, this is not feasible in a murine I/ R-model. However, a requirement for substantial cardiac influences during ischemia necessitates drugs to enter deep into the myocardium at risk as well as temporal accessibility within the time-range of ischemia. As to the latter, we were not able to detect increased TLR9-signaling at earlier time-points than 30 minutes post-injection. Thus, even though we cannot exclude cardiac influences during ischemia, our experimental set-up suggests a predominant effect of TLR9-stimulation during reperfusion. Our main parameter, i.e. infarct extension at 24 hours, was similar between TLR9 stimulated and vehicle treated I/R mice However, we do provide evidence of substantial systemic inflammatory responses upon i.p. injection of CpG B. After CpG B injection.