Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy

Duler, Laetitia; Nguyen, Nghi; Ontiveros, Eric S.; Li, Ronald H. L.

doi:10.3791/60834-v

Cited by 2 publications

(5 citation statements)

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“…The aortic bifurcation, including the descending aorta, femoral and common iliac arteries (CIAs), were bluntly dissected and extracted at necropsy within 24 h of euthanasia, as previously described. 8 Tissues were fixed, paraffin-embedded and stored at –20°C until analysis. After deparaffinization, the aortic sections underwent heat-induced antigen retrieval and permeabilization, followed by the immunodetection of NETs, as previously described.…”

Section: Methodsmentioning

confidence: 99%

“…After deparaffinization, the aortic sections underwent heat-induced antigen retrieval and permeabilization, followed by the immunodetection of NETs, as previously described. 8 In brief, deparaffinized clot sections were stained for citrullinated histone H3 (citH3) and neutrophil elastase (NE) by antibodies, previously shown to cross-react in cats. DNA was stained using 4′,6-diamidino-2-phenylindol (DAPI).…”

Section: Methodsmentioning

confidence: 99%

“…NETs were identified based on the colocalization of cfDNA, citH3 and NE by available software (Fiji; National Institutes of Health), as previously described. 8,12 The percentage distribution of NETs in each zone of a thrombus was calculated using the following formula:…”

Section: Methodsmentioning

confidence: 99%

“…5–7 We recently identified NETs in paraffin-embedded feline arterial thrombi by immunofluorescence microscopy. 8 Similar to ischemic stroke thrombi in humans, feline arterial thrombi consist of NETs within all layers of the thrombus structure, but the cellular composition and distribution of NETs remain unknown. 8 A better understanding of how NETs contribute to CATE would bring us a step closer to expanding our understanding of this devastating disease.…”

Section: Introductionmentioning

confidence: 99%

“…8 Similar to ischemic stroke thrombi in humans, feline arterial thrombi consist of NETs within all layers of the thrombus structure, but the cellular composition and distribution of NETs remain unknown. 8 A better understanding of how NETs contribute to CATE would bring us a step closer to expanding our understanding of this devastating disease. If NETs are proven to facilitate thrombus growth in these cats, novel therapies targeting NET formation or dismantling NET structures may be potential therapeutic options.…”

Section: Introductionmentioning

confidence: 99%

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Neutrophil extracellular traps in feline cardiogenic arterial thrombi: a pilot study

Nguyen

Stern

et al. 2021

Journal of Feline Medicine and Surgery

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Objectives The aim of this study was to investigate the spatial distribution of neutrophil extracellular traps (NETs) in cardiogenic arterial thromboembolism (CATE). Specifically, we aimed to examine the related structural features of NETs in feline arterial thrombi in relation to their arterial locations. Methods Paraffin-embedded aortic bifurcations from nine cats with hypertrophic cardiomyopathy (four with CATE and five without) were deparaffinized, and NETs were identified by immunodetection based on colocalization of cell-free DNA, citrullinated histone H3 and neutrophil elastase. The distribution of NETs in thrombi within the aortic bifurcations and common iliac arteries (CIAs) was compared based on their proximity to the descending aorta (proximal, mid, distal). Ten random fields per section were captured at × 10 and × 20 magnification for each section of the clot and analyzed. Results The distributions of NETs in thrombi within the aortic bifurcation and CIAs were found to differ in relation to their assigned zones (proximal, mid, distal; P = 0.04); NETs were concentrated mostly in the proximal region in the aortic bifurcations (47.56%, interquartile range [IQR] 14.07–77.95) and CIAs (44.69%, IQR 24.65–85.28), compared with the distal regions (2.69%, IQR 0.10–50.04 [P = 0.027]; 7.08%, IQR 1.27–59.33 [P = 0.02]). Conclusions and relevance The variation in NET distribution within arterial thrombi may shed light on the pathogenesis of thrombus growth. This may be due to possible neutrophil entrapment or variations in shear stress.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%