Immunohistological Analysis of Neutrophils and Neutrophil Extracellular Traps in Human Thrombemboli Causing Acute Ischemic Stroke

Abstract

Ischemic stroke caused by thromboembolic occlusion of large cerebral arteries, such as the internal carotid (ICA) and/or the middle cerebral artery (MCA), is treated by mechanical thrombectomy (MT). MT allows salvage of the vessel-occluding thrombemboli, which most frequently originate from the left atrium or the left ventricle of the heart or from sites of plaque rupture within large arteries above the heart. Clot composition may influence the efficacy of (intravenous) thrombolysis and MT, respectively. We analyzed 37 human thrombemboli obtained from acute ischemic stroke patients during MT with special emphasis on histological staining of neutrophils and neutrophil extracellular traps (NETs). We found neutrophils as the main cellular component of cerebral thrombemboli but encountered considerable morphological heterogeneity. Neutrophils accumulated in the border region of fibrin-rich structures indicating possible interaction of neutrophils with distinct structural thrombembolus components. Web-like NETs were found in 35 of 37 thrombemboli in varying amounts. NETs were almost exclusively found within fibrin-rich areas. Importantly, stroke etiology, age and present oral anticoagulation was associated with morphological patterns and the amount of neutrophils. Correlation of histological data and imaging data revealed that relative Hounsfield units of cerebral thrombemboli positively correlated with the amount of red blood cells. In summary, our results demonstrate that neutrophils and NETs are substantial constituents of cerebral thrombemboli and contribute to their structural complexity.

Clot Composition Analysis as a Diagnostic Tool to Gain Insight into Ischemic Stroke Etiology: A Systematic Review

“…from 0.21% to 13.45% of total thrombus area, 93 and 1.1%±2.7% on average. 76 NET signals were observed as being confined within cells, filopodia-like structures, or web-like structures, 65 especially in the outer thrombus layers, 63,95 and almost exclusively within FBR-rich areas. 76 Inside the NETs, citrullinated histones were co-localized with inflammasome proteins (caspase-1 and apoptosis-associated speck-like protein containing a caspase-recruitment domain [ASC]), 99 granular neutrophil proteins (MPO) and extracellular DNA released from neutrophils.…”

“…A number of studies categorized the clots according to the dominant component (usually with a 60% cut-off) 48 as RBCrich, FBR-rich, PLT-rich, or mixed. 7,50,52,54,58,59,64,[75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91] Some studies identified and/or quantified WBC types and subtypes by immunohistochemical staining using specific antibodies against neutrophil elastase (NE), 66,76,[92][93][94] neutrophil myeloperoxidase (MPO), 65,66,76,95 Ly6G (monocyte, granulocyte, and neutrophil), 94 CD3 (T lymphocyte), 66,68-70,90,96 CD4 (T lymphocyte), 52,53 CD14 (monocyte), 66 CD15 (neutrophil, eosinophil, and monocyte), 97 CD20 (B lymphocyte), 66,[68][69][70]96 CD66b (neutrophil), 76,93 and CD68 (monocyte and macrophage). 30,52,[67][68][69]…”

“…66,93 Neutrophil extracellular traps (NETs) have been identified as part of the clot scaffold by using antibodies against NETosis biomarkers (citrullinated histones) and histochemical staining of extracellular DNA. 63,65,66,76,[93][94][95]99 NETs were visualized in almost all (79.1% to 100%) of the analyzed thrombi in different studies, 63,65,66,76,93,95 in amounts ranging Values are presented as weighted mean±pooled standard deviation or number. PLT, platelet; FBR, fibrin; RBC, red blood cell; WBC, white blood cell.…”

Neutrophil Extracellular Trap-Driven Occlusive Diseases

“…In addition, the distribution of NETs in the thrombi from acute ischemic stroke patients has been investigated. NETs were detected in 35 out of 37 thrombi, especially in fibrin-rich areas [ 48 ]. Thus, new anti-NETs approaches aiming to develop therapies have gained momentum.…”

Neutrophil extracellular traps induce thrombogenicity in severe carotid stenosis

“… 16 , 17 , 18 , 19 Notably, NETs are web‐like structures released by activated neutrophils and are composed of DNA, histones, and neutrophil granule proteins such as elastase, myeloperoxidase, and cathepsin G. 20 , 21 , 22 Additionally, NETs were reported to be an important constituent of stroke thrombi and contributed to thrombogenicity in stroke patients. 23 , 24 , 25 , 26 Moreover, severe carotid stenosis was reported to be a leading cause of stroke recurrence. 10 , 27 However, little is known about the presence and the potential role of NETs in severe carotid stenosis.…”