Inhibition of Perivascular Neutrophil Extracellular Traps Reduces Microvasospasm After Subarachnoid Hemorrhage

 Described a problem, suggested further research, did not specify WHOM was going to accomplish that! Useless! I expect stroke researchers to solve problems; not just tell us they exist. With proper leadership stroke could easily be solved.

Inhibition of Perivascular Neutrophil Extracellular Traps Reduces Microvasospasm After Subarachnoid Hemorrhage

• Giuliana Pollaci , MSc

Nakagawa R, Itokazu T, Shibuya N, Kishima H, Yamashita T. Perivascular Neutrophil Extracellular Traps Exacerbate Microvasospasm After Experimental Subarachnoid Hemorrhage. Stroke. 2024;55:2872–2881.

Subarachnoid hemorrhage (SAH) is a clinical condition with a high mortality rate that causes in survivors’ permanent disabilities. Among complications after SAH, the most important is the delayed large vessel vasospasm, which have been intensively studied. It is usually seen after day 3 of hemorrhage, and it could contribute to another complication that is delayed cerebral ischemia (DCI). In contrast, arteriolar vasospasm, known as microvasospasm, has been reported to occur in the subacute phase, and it is believed to contribute to DCI. Delayed vasospasm is usually treated with endothelin receptor antagonist drugs (e.g., clazosentan sodium), which, however, cannot completely prevent DCI. Few studies focused on the mechanism of microvasospasms, and no studies have revealed pathological mechanisms underlying subacute microvasospasms.

The aim of the study is to elucidate the mechanism of microvasospasm through an animal model with intravital 2-photon microscopy, investigating effects of neutrophil removal and, in particular, of Neutrophil Extracellular Traps (NETs) released by neutrophils. NETs are DNA histone complexes released by activated neutrophils, composed of DNA and various antibacterial proteins that have the original purpose of capturing and neutralizing pathogens. Despite that, NET can have a disadvantage of damaging host cells, and, in particular, they are known to play a crucial role in brain damage after SAH.

The author first established an SAH mouse model with a cranial window of 3 x 3 mm, as previously described.^1,2 Briefly, they injected 40 uL of blood from the hearts of littermate mice, in the prechiasmatic cistern using a syringe coated with a minimal amount of heparin. Erythrocytes were previously labeled with phycoerythrin (PE) or fluorescein isothiocyanate (FITC) to visualize their distribution, and neutrophils were labeled with a PE-conjugated anti Ly6G antibody. All was visualized and recorded by time-lapse imaging through 2-photon microscopy. Of course, sham animals underwent the same procedure except for blood injection. The authors observed that both cells population were distributed to the perivascular space of the pial arterioles. Erythrocytes disappeared gradually and almost completely after 2 to 5 days. Concurrently, the pial arterioles were constricted and appeared as microvasospam, as illustrated in Figure 1F.

Figure 1E-F.

The source of infiltrating neutrophils was checked through the use of LysM EGFP reporter mice in which neutrophils robustly express a green fluorescent protein: The authors verified that the source of neutrophil infiltration was the circulation of the host but not the injected blood.

The authors then depleted neutrophils from the host mice by intraperitoneal administration of neutrophil-specific antibody (200 ug of rat anti-Ly6G antibody) 1 day and 1 hour before SAH, and then evaluated microvasospasms, visualized as pearl-string-like stenosis: Microvasospasms were significantly reduced in the neutrophil depleted group. For the control group, an equal amount of the isotype control was injected. Nakagawa and colleagues observed that in the control group, the number of infiltrating neutrophils was correlated with the volume of accumulated erythrocytes, speculating that neutrophil infiltration in the perivascular space may have been triggered by erythrocyte accumulation.

Focusing on NETs formation, to confirm their presence in the perivascular space, a marker for NETs (SYTOX Green) was intracisternally injected, indicating that, after SAH, NETs are released in the perivascular space. To test if NETs cause microvasospasm, the authors administered DNase, a NETs inhibitor, at 1 day after SAH in order to remove them in perivascular space. As expected, microvasospasms were significantly suppressed after DNase treatment, and blood flow velocity at day 5 significantly improved.

In conclusion, the author established an experimental system to investigate events occurring after SAH, demonstrating accumulation of erythrocytes in the perivascular space, the subsequent infiltration of neutrophils, and, for the first time, the presence of a large amount of NETs, leading to the development of microvasospasm. NETs were, in fact, attached to the arterioles, suggesting that they may directly damage the endothelium or vascular smooth muscle of the arterioles.

Some limitations of the study are due to the limited cranial window to observe a relatively small part of the cerebral cortex, so results may not be generalizable to the entire brain. Furthermore, there is a need for additional research to verify the precise nature of perivascular NETS and their pathological effects.