gms | German Medical Science

Objective: Previous studies have shown that Microglia (MG) accumulation and activation within the brain induces neuronal cell death after experimental subarachnoid hemorrhage (SAH), contributing to secondary brain injury. Recently, the role of self-extracellular RNA (eRNA) has been postulated as a potential “Danger-associated molecular pattern” (DAMP): endogenous eRNA is released from cells upon tissue damage, inducing proinflammatory and prothrombotic processes. This study aimed at investigating whether the eRNA may trigger the activation of innate immunity following SAH.

Methods: Experimental SAH in mice was induced utilizing a filament perforation model in male C57Bl/6- mice. Presence of SAH was confirmed with Magnetic Resonance Imaging on the first postoperative day. Sham operation was performed for the corresponding control group. Mice were treated with RNase1 (42 μg/kg) intravenoulsy, an antagonist of endogenous eRNA. NaCl injections were used for the control group. Mice were then sacrificed at 3 time points: day1, day7 and day14 (n=6 for each subgroup; n_total=72). Double-immunofluorescence (IF) staining for ribosomal RNA and Phalloidin (for F-actin) was used to quantify content of eRNA. IF staining was performed to characterize MG (Iba-1), vessel characteristics (cd31/col-IV) and neuronal association (NeuN), and visualized by confocal imaging. MG density was quantified using the ratio of Iba-1 over DAPI positive cells in different subregions of the brain (basal, cortical, median). MG-perivascular association was determined by the percentage of Iba-1 positive cells adjacent to vascular structures.

Results: SAH mice demonstrated a higher content of endogenous eRNA compared to Sham animals. Moreover, MG density increased in SAH peaking on day 7 (Sham vs. SAH: 3% vs. 6.5%, p=0.04), and treatment with RNase1 significantly lowered MG density (6.5% vs. 2.4%, p=0.01). This effect was most prominent in the basal part of the brain, and less striking in the cortical and median aspects. Furthermore, MG homed to the vascular niche as demonstrated by co-staining of Iba-1 and cd31 (Sham vs. SAH: 14% vs. 32%, p=0.008), and administration of RNase1 abrogated this MG-perivascular association in vivo (32% vs. 16%, p=0.002). While neuronal-MG interaction increased in SAH (Sham vs. SAH: 18% vs. 32%, p=0.008), it was not significantly affected by RNase1 treatment.

Conclusion: In experimental SAH, eRNA is released into the subarachoid space and distributed within the brain. This effect is paralleled by an increase in MG density and homing to the vascular niche, which can be rescued by treatment with RNase1, an effective tissue- and vessel-protective endonuclease. RNase1 may exert its intrinsic effect by hydrolysis of eRNA, thereby reducing a neuroinflammatory cascade in SAH. Conclusively, targeting eRNA may represent a potential treatment strategy in reducing secondary brain injury.