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.
