RNase A Inhibits Formation of Neutrophil Extracellular Traps in Subarachnoid Hemorrhage.

Background: Subarachnoid hemorrhage (SAH) caused by rupture of an intracranial aneurysm, is a life-threatening emergency that is associated with substantial morbidity and mortality. Emerging evidence suggests involvement of the innate immune response in secondary brain injury, and a potential role of neutrophil extracellular traps (NETs) for SAH-associated neuroinflammation. In this study, we investigated the spatiotemporal patterns of NETs in SAH and the potential role of the RNase A (the bovine equivalent to human RNase 1) application on NET burden. Methods: A total number of n=81 male C57Bl/6 mice were operated utilizing a filament perforation model to induce SAH, and Sham operation was performed for the corresponding control groups. To confirm the bleeding and exclude stroke and intracerebral hemorrhage, the animals received MRI after 24h. Mice were treated with intravenous injection of RNase A (42µg/kg body weight) or saline solution for the control groups, respectively. Quadruple-immunofluorescence (IF) staining for cell nuclei (DAPI), F-actin (phalloidin), citrullinated H3, and neurons (NeuN) was analyzed by confocal imaging and used to quantify NET abundance in the subarachnoid space (SAS) and brain parenchyma. To quantify NETs in human SAH patients, cerebrospinal spinal fluid (CSF) and blood samples from day 1, 2, 7, and 14 after bleeding onset were analyzed for double-stranded DNA (dsDNA) via Sytox Green. Results: Neutrophil extracellular traps are released upon subarachnoid hemorrhage in the SAS on the ipsilateral bleeding site 24h after ictus. Over time, NETs showed progressive increase in the parenchyma on both ipsi- and contralateral site, peaking on day 14 in periventricular localization. In CSF and blood samples of patients with aneurysmal SAH, NETs also increased gradually over time with a peak on day 7. RNase application significantly reduced NET accumulation in basal, cortical, and periventricular areas. Conclusion: Neutrophil extracellular trap formation following SAH originates in the ipsilateral SAS of the bleeding site and spreads gradually over time to basal, cortical, and periventricular areas in the parenchyma within 14days. Intravenous RNase application abrogates NET burden significantly in the brain parenchyma, underpinning a potential role in modulation of the innate immune activation after SAH.

Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging (MRI).

The endovascular filament perforation model to mimic subarachnoid hemorrhage (SAH) is a commonly used model - however, the technique can cause a high mortality rate as well as an uncontrollable volume of SAH and other intracranial complications such as stroke or intracranial hemorrhage. In this protocol, a standardized SAH mouse model is presented, induced by endovascular filament perforation, combined with magnetic resonance imaging (MRI) 24 h after operation to ensure the correct bleeding site and exclude other relevant intracranial pathologies. Briefly, C57BL/6J mice are anesthetized with an intraperitoneal ketamine/xylazine (70 mg/16 mg/kg body weight) injection and placed in a supine position. After midline neck incision, the common carotid artery (CCA) and carotid bifurcation are exposed, and a 5-0 non-absorbable monofilament polypropylene suture is inserted in a retrograde fashion into the external carotid artery (ECA) and advanced into the common carotid artery. Then, the filament is invaginated into the internal carotid artery (ICA) and pushed forward to perforate the anterior cerebral artery (ACA). After recovery from surgery, mice undergo a 7.0 T MRI 24 h later. The volume of bleeding can be quantified and graded via postoperative MRI, enabling a robust experimental SAH group with the option to perform further subgroup analyses based on blood quantity.

Extracellular RNA in Central Nervous System Pathologies.

The discovery of extracellular RNA (exRNA) has shifted our understanding of the role of RNA in complex cellular functions such as cell-to-cell communication and a variety of pathologies. ExRNAs constitute a heterogenous group of RNAs ranging from small (such as microRNAs) and long non-coding to coding RNAs or ribosomal RNAs. ExRNAs can be liberated from cells in a free form or bound to proteins as well as in association with microvesicles (MVs), exosomes, or apoptotic bodies. Their composition and quantity depend heavily on the cellular or non-cellular component, the origin, and the RNA species being investigated; ribosomal RNA provides the majority of exRNA and miRNAs are predominantly associated with exosomes or MVs. Several studies showed that ribosomal exRNA (rexRNA) constitutes a proinflammatory and prothrombotic alarmin. It is released by various cell types upon inflammatory stimulation and by damaged cells undergoing necrosis or apoptosis and contributes to innate immunity responses. This exRNA has the potential to directly promote the release of cytokines such as tumor necrosis factor factor-α (TNF-α) or interleukin-6 from immune cells, thereby leading to a proinflammatory environment and promoting cardiovascular pathologies. The potential role of exRNA in different pathologies of the central nervous system (CNS) has become of increasing interest in recent years. Although various exRNA species including both ribosomal exRNA as well as miRNAs have been associated with CNS pathologies, their precise roles remain to be further elucidated. In this review, the different entities of exRNA and their postulated roles in CNS pathologies including tumors, vascular pathologies and neuroinflammatory diseases will be discussed. Furthermore, the potential role of exRNAs as diagnostic markers for specific CNS diseases will be outlined, as well as possible treatment strategies addressing exRNA inhibition or interference.