Genome-wide analysis of the circulating miRNome after cerebral ischemia reveals a reperfusion-induced microRNA cluster. Mus musculus

Background and Purpose - Circulating microRNAs (miRNAs) are emerging biomarkers for stroke due to their high stability in the bloodstream and association with pathophysiologic conditions. However, the circulating whole-genome miRNAs (miRNome) has not been characterized comprehensively in the acute phase of stroke. Methods - We profiled the circulating miRNome in mouse models of acute ischemic and hemorrhagic stroke by next-generation sequencing (NGS). Stroke models were compared to sham-operated and naïve mice to identify deregulated circulating miRNAs. Top-ranked miRNAs were validated and further characterized by qRT-PCR. Results - We discovered 24 circulating miRNAs with an altered abundance in the circulation 3 hours following ischemia, whereas the circulating miRNome was not altered after intracerebral hemorrhage compared to sham-operated mice. Among the upregulated miRNA in ischemia, the top-listed miR-1264/1298/448 cluster was strongly dependent on reperfusion in different ischemia models. A time course experiment revealed that the miR-1264/1298/448 cluster peaked in the circulation around 3 hours after reperfusion and gradually decreased thereafter. Conclusions - Alteration of the miRNome in the circulation is associated with cerebral ischemia/reperfusion, but not hemorrhage, suggesting a potential to serve as biomarkers for reperfusion in the acute phase. The pathophysiological role of reperfusion-inducible miR-1264/1298/448 cluster, which is located on chromosome X within the introns of the serotonin receptor HTR2C, requires further investigation. Overall design: Profiling of the circulating miRNome in mouse models of acute ischemic and hemorrhagic stroke by next-generation sequencing (NGS) MCAO: Filament-induced ischemia experiment, which led to a transient focal cerebral ischemia through the occlusion of the left-sided middle cerebral artery (MCAO). Afterwards mice were re-anesthetized and the filament was removed following 30 min of ischemia. Blood was collected after 150 min of reperfusion and subsequently the serum was isolated to measure the abundance of circulating miRNA. MCAO-Sham: The preparation for the sham surgery was identical but the filament was not inserted into the vessel of the middle cerebral artery (MCO). ICH: This blood infusion model was used to simulate ICH (intracerebral hemorrhage) in mice. Briefly, after placing the anesthetized animals in a stereotactic frame, a borehole was drilled at 2.2 mm left and 0.2 mm anterior to the bregma. Using an infusion system containing a 26 G needle connected to a 50 μl microsyringe, 20 μL of autologous blood was infused into the striatum at a depth of 3.7 mm. ICH-Sham: Sham operation was conducted by the same procedure and duration including placement of the microneedle but without injection of fluid. Naïve: Received no treatment.