Abstract Body: Introduction: Ischemic stroke (IS) is the 5^th leading cause of death in the USA, but only one FDA approved drug exists. IS causes widespread neuron death and inflammatory injury. Microglial efferocytosis, the process to remove dead cells, limits inflammation and enhances brain recovery. Extracellular cold-inducible RNA-binding protein (eCIRP) is a pro-inflammatory alarmin that is deleterious in IS, increases infarct volume, neuroinflammation and microglial activation. eCIRP decreases efferocytosis and induces pro-inflammatory micro-RNA 155 (miR-155) in IS by binding to TLR4 on microglia. Our objective is to develop C23, a small peptide inhibitor of eCIRP/TLR4 binding, in a murine model of IS to reduce neuroinflammation and improve efferocytosis and IS outcomes.
Methods: Primary microglia were isolated from mouse pups, seeded, and treated with eCIRP with or without C23, lysed for RNA, and RT-qPCR measurement of micro-RNA. 8-10-week-old male C57BL/6 mice, were subjected to transient middle cerebral artery occlusion (tMCAO) model of IS for 1 h, followed by reperfusion and retroorbitally injected with either normal saline (vehicle) or C23 (10 µg/g b.w.). 24 h post-reperfusion, behavior was scored, mice were sacrificed and tissues were collected for infarct volume measurement, immunofluorescence microscopy, or miRNA quantification.
Results: As summarized in Table 1, eCIRP treated microglia upregulated miR-155 by 29-fold compared to controls, and pretreatment with C23 significantly attenuated eCIRP-induced miR-155 by 33%. Brain levels of miR-155 increased 1.8-fold in the vehicle tMCAO group vs. sham brains but were unchanged in the C23 tMCAO group. Similarly, plasma miR-155 levels increased 2.1-fold in vehicle tMCAO mice and returned to sham levels in C23 tMCAO mice. C23 treatment significantly increased efferocytosis in penumbra compared to vehicle in tMCAO brains (Fig. 1). Infarct volume was reduced in the C23 treated tMCAO group by 39% (100.3 mm³ in vehicle tMCAO vs. 61.2 mm³ in C23 tMCAO; Table 1 & Fig. 2). Finally, C23 tMCAO mice had mild motor impairment with a mean Bederson score of 1.8 vs. severe impairment of vehicle tMCAO mice scoring 3.1 (Table 1).
Conclusion: eCIRP induces pro-inflammatory miR-155 in primary microglia in-vitro and in tMCAO mice, and C23 attenuates this detrimental effect. C23 treatment improves microglial efferocytosis and reduces infarct volume and behavioral deficit in IS. Thus, C23 is a promising candidate therapeutic agent in IS.