Abstract Body: Background: Neutrophil Extracellular Traps (NETs) are web-like structures of DNA, histones, and granule proteins that trap pathogens as part of innate immunity but also scaffold red blood cells, fibrin, and platelets in pathological thrombosis. Thrombus NETs have been associated with failed thrombolysis and thrombectomy for acute ischemic stroke (AIS). We previously used mass spectrometry to describe a novel association between NET proteins and atrial fibrillation (AF). This association has not been validated by traditional immunohistochemical methods.
Aims/Purpose: We used immunohistochemistry to quantify NETs in AIS thrombus taken from patients with and without comorbid AF.
Methods: We selected markers for each of the components of NETS: (1) a granule protein, myeloperoxidase (MPO), (2) citrullinated histone 3 (CitH3), and cellular nucleic acid (NA, using Hoechst 33342). This was a single-blind study using cryopreserved thrombi from 30 patients, 15 with AF. Stroke thrombi were collected over a two-year span, and immediately frozen to -80*C post-thrombectomy. To capture the coverage of NET markers more comprehensively for each patient, we used cryostat to sample four sections (five-micron thickness) from each thrombus. We then fixed sections with 4% formaldehyde in phosphate buffered saline (PBS), performed immunohistological staining, and quantified extent of staining using ImageJ. Data for each NET marker from a single thrombus is represented as the mean percentage of thrombus area staining positively across the four corresponding sections. After performing Shapiro Wilks test for normality, statistical comparisons of NET marker staining coverage between AF and non-AF thrombi were performed with either unpaired T test or Mann-Whitney U test, depending on whether a parametric or non-parametric test was appropriate.
Results: Thrombi from patients with AF had greater MPO coverage(17.9 +/- 1.47% versus 11.6 +/- 1.29% in non-AF, p<0.01) and CitH3 coverage(12.6 +/- 1.46% versus 7.4 +/- 1.11% in non-AF, p<0.01). Staining for nucleic acid with Hoechst 33342 trended toward higher coverage in AF but did not reach significance (27.8 +/- 3.35% versus 22.6 +/- 3.20% in non-AF, p=0.30).
Conclusion: The present study provides immunohistochemical validation, supporting novel observations in prior proteomic investigations, of increased NETs in stroke thrombi of patients with AF. This can help to identify patient cohorts for future studies of therapies directed at NETs.