gms | German Medical Science

Objective: Microglia (MG) accumulation and neuronal cell death after experimental subarachnoid hemorrhage (eSAH) contribute to secondary brain injury. MG are capable of modulating gene expression of key proteins in Blood Brain Barrier (BBB) integrity and inducing proinflammatory phenotypes in astrocytes (ACs) in other CNS pathologies. Previous evidence suggests that extracellular RNA (eRNA) in turn are released by immune cells upon tissue damage, subsequently triggering a proinflammatory response. Hence, we aimed to study the potential role of eRNA-dependent MG properties with respect to their vascular and glial microenvironment after SAH.

Methods: An in vivo model was utilized in which male C57Bl/6 mice were exposed to SAH bleeding via a filament perforation model, while Sham operation was performed on the control group. The presence of SAH was confirmed through MR imaging 24 hours post-injury. Intravenous treatment with RNase 1 was used to inhibit endogenous eRNA (42 µg/kg body weight); n=6 for each subgroup. Immunofluorescence (IF) analysis, Western Blotting (WB) and MG isolation was performed to analyze MG properties. Antibodies against Iba-1 (MG marker), GFAP (astrocytes), C3 (complement component 3), cd31, and col-IV (vasculature) were used.

Results: MG (Sham vs SAH: 3% vs. 6.5%, p=0.04) and astrocyte density (2.5% vs 5.85%, p=0,01) peaked on day 7 in SAH animals. Treatment with RNase1 reduced not only MG and astrocyte (AC) density (SAH vs. SAH+RNAse: MG: 6.5% vs. 2.4%, p=0.01; ACs: 5.85% vs 2.38%, p=0.01), but also their colocalization as demonstrated by triple-immunofluorescence with Iba/GFAP/DAPI. Furthermore, MG homed to the perivascular niche as demonstrated by co-staining of Iba-1 and cd31 (Sham vs. SAH: 14% vs. 32%, p=0.008). Administration of RNase1 abrogated this MG-perivascular association (SAH vs. SAH+RNase1: 32% vs. 16%, p=0.002). This was paralleled by an increase in total C3 as demonstrated by WB and percentage of C3-positive ACs.

Conclusion: In our SAH model, an increase in MG and AC density is observed, associated with homing of MG to the vascular niche. This is associated with an increase in total complement component C3 protein abundance and C3-positive ACs. Inhibition of eRNA may reduce the C3-associated MG properties, and hence, may serve as a potential treatment strategy to address secondary brain injury.