Ischemic preconditioning (IPC) is known to protect tissue from ischemia and has an effect both locally  and systemically . Remote IPC (RIPC) induced by limb ischemia provides systemic protection against prolonged ischemia in the target organ. RIPC has been reported to reduce infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary angioplasty  and is currently being evaluated in patients with ischemic stroke undergoing thrombolysis (NCT00975962).
We hypothesized that the reduction in ischemic damage with RIPC could rely on a direct effect on arterial thrombus formation and embolization, and investigated this in a dynamic in vivo rat model of experimental thrombosis .
Animals were treated according to Danish law on animal experiments. RIPC was induced using a protocol described previously . At the time of surgery, a tourniquet was placed for 10 minutes on the left hind limb. Photographs demonstrating blue coloring of the left paw documented ischemia.
Ten millimeters of the femoral artery on the right hind leg were dissected distal to the inguinal ligament. The artery was fixed in a clamp and an arteriotomy was performed comprising one-third of the vessel circumference. The proximal vessel edge was inverted by two 10-0 Nylon sutures exposing full thickness of the vessel wall to the lumen. After clamp release, a thrombus would build up at the arteriotomy site, and fragments of thrombus were seen to detach and embolize downstream. This model of a thrombogenic anastomosis is well established in rats [6,7] and pigs .
The right femoral artery was placed in a trans-illuminator projecting a light beam through the vessel [8,9]. Recordings with a digital recorder were performed through the microscope for 30 min after clamp release and were analyzed offline in a blinded fashion. The thrombus area was measured as the number of pixels every minute, using special ImageJ based software. (ImageJ 1.45s, NIH, USA). The area was measured for 20 min while the number of emboli was counted manually every minute for 30 min. The area of the thrombus and number of emboli were displayed versus time in a graph. The area under the curve (AUC) for thrombus formation over time was determined. Two independent investigators analyzed 10 random videos (coefficient of variation was 21% for counting emboli and 8% for area measurement).
Data were consistent with a Gaussian distribution, and the unpaired t-test was used for comparison of the two groups. P-values < 0.05 were considered statistically significant. All data are reported as mean ± SEM. According to power calculations, we should include at least 34 animals.
A total of 44 male Wistar rats (287–329 g) were included and randomized into two groups using a sealed envelope system. Three animals were excluded due to bleeding. Nineteen rats had surgery without RIPC, and 22 had surgery with RIPC.
The development of the thrombus area over time in both groups is displayed in Fig. 1. The thrombus area diminished over time, and the rats randomized to RIPC had significantly smaller thrombi (722 ± 41 vs. 910 ± 46, P < 0.005). The number of emboli was significantly reduced in the RIPC-group, although the absolute difference was rather small (0.88 ± 0.06 vs. 1.08 ± 0.06, P < 0.05).
We demonstrate for the first time that RIPC reduces arterial thrombus formation and embolization in a dynamic in vivo rat model. In patients suffering from acute STEMI, Boetker et al.  showed that RIPC reduces infarct size. Recently, Wei et al.  demonstrated that an episode of remote ischemic hind limb preconditioning in rats reduced myocardial infarct size. Our findings of a direct inhibition by RIPC of arterial thrombus formation and embolism may partly explain both observations.
In a pig model, Hjortdal et al.  showed that thrombus formation and embolization damaged the microcirculation distal to the anastomosis, probably due to activation of coagulation and platelets. Inhibition of platelets has been reported after IPC , which might explain the observed effect of RIPC in our study. RIPC has also been shown to abolish circulating platelet-monocyte-aggregates in canines and in healthy volunteers . Effects of RIPC on endothelial function and NO synthesis might also be involved .
However, the main limitation of our study is the difficulties in further exploring the underlying antithrombotic mechanism in this rat model.
In conclusion, RIPC in rats significantly reduces arterial thrombus formation and embolization. This direct effect of RIPC on thrombogenesis may be important in arterial thromboembolic diseases such as acute myocardial infarction and ischemic stroke.