key: cord-0281731-lies03c0 authors: Witsch, J.; Spalart, V.; Martinod, K.; Schneider, H.; Oertel, J.; Geisel, J.; Hendrix, P.; Hemmer, S. title: Neutrophil Extracellular Trap Biomarker Titers and Delayed Cerebral Ischemia in Patients With Aneurysmal Subarachnoid Hemorrhage date: 2022-02-02 journal: nan DOI: 10.1101/2022.02.01.22270255 sha: 9b5e2777987a8eb573012bef76efbec10df960e3 doc_id: 281731 cord_uid: lies03c0 Background: Myeloperoxidase (MPO)-DNA complexes have been associated with arterial and venous thrombosis. Their role in patients with aneurysmal subarachnoid hemorrhage (aSAH) is unknown. Here, we sought to explore whether serum MPO-DNA-complexes are present in patients with aSAH, and whether levels of serum MPO-DNA complexes are associated with delayed cerebral ischemia (DCI). Methods: We performed a post-hoc analysis of a prospective, blinded, observational single-center biomarker study that enrolled consecutive patients with spontaneous SAH between July 2018 and September 2020. Serum samples obtained on admission and on hospital day 4 were analyzed for concentrations of MPO-DNA complexes. The primary outcome was the occurrence of DCI defined as new infarction on brain CT-scan. The secondary outcome was clinical vasospasm, a composite of clinical and transcranial doppler parameters. We used Wilcoxon's signed-rank-test to assess for differences between paired measures. Results: Among 100 patients with spontaneous SAH, mean age 59 (SD+13) years, 55% women, 78 patients had an aneurysmal SAH and complete DCI information. Among these, 29 (37%) developed DCI. MPO-DNA complexes were detected in all serum samples. The median MPO-DNA level was 33 ng/ml (IQR, 18-43 ng/ml) on admission, and 22 ng/ml (IQR, 11-31 ng/ml) on day 4 (Mann-Whitney test: p=0.015). In the primary outcome analysis, we found a significant reduction in MPO-DNA levels from admission to day 4 in patients with DCI (paired test, p=0.036) but not in those without DCI (p=0.171). The secondary analysis showed a similar reduction in MPO-DNA levels between admission and day 4 in patients with (p=0.006), but not in those without clinical vasospasm (p=0.473). Conclusions: MPO-DNA-complexes are detectable in peripheral serum samples of patients with aSAH. A pronounced reduction in MPO-DNA levels over the first days after aSAH might herald DCI. The potential of MPO-DNA serum levels to serve as a biomarker of DCI requires further exploration. Aneurysmal subarachnoid hemorrhage (SAH) is a severe form of hemorrhagic stroke, associated with 18% in-hospital mortality, and about 30% poor long-term outcome. 1, 2 In about one third of cases the hospitalization is complicated by delayed cerebral ischemia (DCI), the occurrence of secondary ischemic infarcts, identified on brain imaging. 3 Most DCI occurs 4 to 14 days after the initial hemorrhage and is strongly associated with poor outcome. 4, 5 However, effective prediction and prevention measures of DCI are lacking. 5 While DCI development might be preceded by clinical deterioration, angiographic vasospasm, increase in transcranial doppler flow velocities, or electroencephalographic changes, DCI often occurs silently and is mostly identified in retrospect. [6] [7] [8] [9] Finding markers of impending DCI could lead to a paradigm shift in the diagnosis and possibly in the prevention and treatment of this severe complication of SAH. 10 Neutrophil extracellular traps (NETs) have been described as a central mechanism of arterial and venous thrombus formation. 11 Since their definition in 2004 as a neutrophil innate immune mechanism, 12 they have also been detrimentally associated with ischemic stroke, deep vein thrombosis, as well as immunothrombosis driven by coronavirus disease 2019 . [13] [14] [15] NETs are the product of neutrophil hyperactivation leading to the release of extracellular "netlike" structures consisting of chromatin lined with neutrophil proteins. When released into the blood, they facilitate thrombus formation by promoting coagulation and binding platelets and other blood components. NETs have been visualized using electron microscopy 12 , and components of NETs can be detected in plasma of patients with acute thrombotic processes. 12 Moreover, NETs can be therapeutically targeted by degradation with DNases, as demonstrated in animal studies [16] [17] [18] and ex vivo thrombolysis experiments. 13, 19 While excess inflammation and increased neutrophil lymphocyte ratios have been linked to DCI and outcome after aSAH, only limited data is available on the detection of NETs in patients with SAH. 20-22 4 In this exploratory study we focused on one major NET biomarker, myeloperoxidase (MPO)-DNA complexes. We leveraged prospective data from a cohort of patients with SAH undergoing prospective neurological and transcranial Doppler (TCD) exams, as well as standardized DCI assessment. We aimed to test two hypotheses: (1) MPO-DNA complexes can be detected in peripheral serum of patients with aSAH. (2) Changes in MPO-DNA-complex levels between admission and hospital day 4 are associated with the occurrence of DCI. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We conducted a post-hoc analysis of the high mobility group box 1 in aneurysmal subarachnoid hemorrhage (HIMOBASH) study, a prospective, blinded, single tertiary care center biomarker observational study designed to explore the role of high mobility group box 1 protein as a biomarker in aneurysmal subarachnoid hemorrhage. 23 All clinical data for the present study were collected at Saarland University Medical Center, Germany; all laboratory analyses were conducted at KU Leuven, Belgium. For the HIMOBASH study, consecutive patients with spontaneous subarachnoid hemorrhage, 18 years of age or older, admitted to the neurosurgical intensive care unit at Saarland University Medical Center, Germany, were evaluated for study eligibility. The diagnosis of SAH was established through an admission brain CT. An aneurysmal etiology of SAH was confirmed through CT angiography or digital subtraction angiography (DSA). If DSA was negative for an aneurysmal bleeding source, patients underwent additional brain MRI and repeat-DSA after 7-10 days to rule out other pathologies and establish the diagnosis of non-aneurysmal SAH. Patients were treated according to current guidelines including monitoring in a dedicated neurointensive care unit, aneurysm treatment via clipping or coiling no later than 24 hours from admission, treatment of hydrocephalus via insertion of an external ventricular drain, and oral All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 2, 2022. ; https://doi.org/10.1101/2022.02.01.22270255 doi: medRxiv preprint nimodipine administration. Bedside TCD studies were performed twice daily and additionally as required to corroborate suspected vasospasm. 23 If a delayed neurological deficit (DND) was identified (as defined in the Outcomes paragraph), hyperdynamic therapy was initiated avoiding hypovolemia and escalating the mean arterial blood pressure levels using vasopressors. If vasospasm was suspected, decisions on additional imaging or neurointerventional rescue therapies were made on a case-by-case basis after reaching interdisciplinary consensus. Delayed cerebral ischemia (DCI). DCI was defined as a new ischemic infarct on brain imaging that was not present on the routine post-clipping/-coiling CT brain one day after aneurysm treatment. Patients who died before another follow-up CT scan was obtained, were deemed ineligible for DCI assessment, and were thus excluded from this study. Transcranial doppler studies. Bedside TCD were conducted at least twice daily to diagnose suspected vasospasm. The following TCD criteria were considered indicative of potential vasospasm: a middle cerebral artery (MCA) peak systolic velocity (PSV) of ≥ 120 cm/s that was not attributable to generalized hyperperfusion, a peak systolic velocity increase of ≥ 50 cm/s All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 2, 2022. ; https://doi.org/10.1101/2022.02.01.22270255 doi: medRxiv preprint compared to the admission TCD exam, or a Lindegaard ratio (mean flow velocity in the MCA divided by mean flow velocity in the ipsilateral extracranial internal carotid artery) of ≥ 3. Isolated angiographic vasospasm -although assessed in the original HIMOBASH cohort -was a priori excluded as an endpoint for the purpose of this study for its poor correlation with DCI. 23 For laboratory analysis of MPO-DNA-complex concentrations, we used serum samples collected on admission (day 0) and on day 4, as previously described. 23 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 2, 2022. ; https://doi.org/10.1101/2022.02.01.22270255 doi: medRxiv preprint from 10 ng/ml down to 0.15 ng/ml. All samples measured fell within the limit of quantification based on this standard curve. Data were reported as counts (percentage) or median (interquartile range, IQR). Intergroup differences of baseline characteristics were assessed using the Mann-Whitney test for all continuous variables except for age, which given its normal distribution was assessed using the student t-test. Intergroup differences between categorical variables were assessed using the χ 2 test. Differences in laboratory marker serum concentrations between admission and hospital day 4, pooling results of the entire cohort (unpaired analysis), were assessed using the Mann-Whitney test, and for paired laboratory markers for each individual participant (paired analysis) using the Wilcoxon signed rank test. Cohen's criteria were used to calculate effect sizes. We performed statistical analyses using SPSS, version 28 (IBM, Armonk, New York). The threshold for statistical significance was set at < 0.05. Data are available on reasonable request from one of the corresponding authors (P.H.). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The study was approved by the Saarland Medical Association ethics committee (#118/17). Written informed consent was obtained from all participants or their legal representatives. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Among 100 enrolled patients, 83 were found to have an aneurysmal SAH, 5 of which died before follow-up CT scan and were thus ineligible for DCI assessment. In the analytical cohort (n=78), there were 43 (55%) women, and the mean age was 59 years (±13.0) ( Table 1) All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 2, 2022. In a paired data analysis, there was a significant reduction in MPO-DNA levels between admission and day 4 in patients with DCI (z= -2.095, Wilcoxon signed rank test: p=0.036, r=0.3, medium effect size) but not in those without DCI (Wilcoxon signed rank test: p=0.171) ( Figure 1 ). Like in the unpaired data analysis, there was a significant reduction in WBC in both groups (DCI group p=0.007, no DCI group p<0.001) as well as a significant increase in C-reactive protein (DCI group p<0.001, no DCI group p<0.001) between admission and day 4, respectively. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. NETs, the product of hyperactivated neutrophils, promote immune-mediated thrombus formation and have been associated with ischemic stroke. In this exploratory study in patients with aSAH we detected high levels of MPO-DNA complexes, a NET marker, in all peripheral serum samples. Between admission and hospital day 4, we found a significant reduction in MPO-DNA complex levels in patients who developed DCI, but not in those who did not develop DCI. Our study suggests that NETs might be of relevance in aSAH, possibly contributing to DCI, an ischemic complication strongly associated with poor outcome. A prior study aimed to detect NET markers in mice and in patients with SAH. 22 This study used double stranded DNA (ds-DNA) as a surrogate for the presence of NETs. However, ds-DNA has been shown to be an unspecific remnant of cell death 26 . MPO-DNA complexes, in contrast, are established measures of NET release. 17 Three further studies -in humans, rats, and rabbits respectively -assessed MPO (not MPO-DNA complex) serum levels in SAH. [27] [28] [29] However, secreted MPO levels are indicative of neutrophil activation, including by degranulation, in addition to NET release, which limits the utility of MPO levels in quantifying NETs. Our study adds a novel element to the existing literature, demonstrating the detection of a specific NET biomarker in a prospective cohort of patients with aSAH, as well as its potential implication in the development of DCI. Among pathophysiologic correlates of DCI, prior studies have primarily investigated vascular dysfunction (leading to vasoconstriction-induced hypoperfusion) and spreading depolarization. [30] [31] [32] In addition, a systemic inflammatory response after SAH, including an increase in the All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 2, 2022. ; https://doi.org/10.1101/2022.02.01.22270255 doi: medRxiv preprint circulating neutrophil-lymphocyte ratio, has been consistently demonstrated across many studies and is associated with DCI and poor outcome. 20, 21, 33, 34 Disruption of the blood brain barrier with massive influx of cell-free heme into the subarachnoid space may stimulate local macrophages and the heme scavenger system, leading to further recruitment of other immune cells and excessive inflammation. 5, 35 Direct activation of neutrophils through free heme is conceivable as well, with free heme being a driving factor for NET release in sickle cell disease. 36, 37 Whatever the immediate trigger of the inflammatory response to aSAH may be, the concept of NET-induced thrombus formation as a result of it is intriguing, as it provides a framework that links inflammation, thrombus formation, and DCI. 5, 38 We found high levels of MPO-DNA complexes in all patients with aSAH, not only in those with DCI. While lower levels of MPO-DNA complexes have been measured in the general population 39 , the high levels in our study may reflect an overall inflammatory environment after aSAH, in line with high levels of C-reactive protein in all our patients. A speculative explanation for the decline in MPO-DNA complex levels, seen predominantly in patients with DCI, may be NET-driven thrombus formation under consumption of MPO-DNAcomplexes. It is worth noting, that using the clinical consensus definition of DCI (which requires diagnosis of an infarct on brain imaging) might be too crude of a parameter to accurately distinguish between patients with and without NET-driven thrombosis, as microthrombosis might not always lead to macroscopic infarcts. This might explain why patients with declining MPO-DNA levels were seen in both the DCI and the no-DCI groups. Demonstrating internal consistency, our results were similar when we stratified the cohort by presence or absence of clinical vasospasm, a more inclusive composite endpoint. This likely reflects previously All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 2, 2022. ; https://doi.org/10.1101/2022.02.01.22270255 doi: medRxiv preprint described associations between DND and DCI on the one hand, and vasospasm and DCI on the other. Our study has limitations. First, we measured MPO-DNA complex levels in serum. In contrast to plasma, serum preparations may result in the release of NETs during the coagulation phase and may thus artificially increase NET levels. The advantage of serum, however, is that NET markers can be paired with neutrophil counts to infer the extent of NET release during serum preparation due to neutrophil preactivation in the collected blood samples (which is not possible in plasma samples because neutrophils are removed). In this study, matched complete blood counts were not available in our patient cohort, hence, correlation of MPO-DNA complexes and absolute neutrophil counts could not be performed. Second, due to the exploratory character of this study, we refrained from analyzing NET markers other than MPO-DNA complexes. Third, we did not analyze samples beyond hospital day 4. Since DCI typically occurs between post-SAH day 4 to 14, it is possible that in those patients with DCI, further reductions in MPO-DNA complex levels might have been appreciated at later time points. Future studies should ideally use paired NET measurements of plasma and serum samples with matching complete blood counts and include measurements of further NET biomarkers with additional mechanistic insight, e.g., citrullinated histones and peptidylarginine deiminase 4. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. None. This study was financially supported by intramural research grants from the Homburger Forschungsförderung (PH, SH) and the Dr. Theiss Research Award (PH). KM is an inventor on patent number US9642822 (granted, licensed) covering the targeting of NETs in stroke. JW, VS, HS, JO, JG, PH and SH report no disclosures or conflicts of interest related to this work. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 2, 2022. ; https://doi.org/10.1101/2022.02.01.22270255 doi: medRxiv preprint Prognostication of long-term outcomes after subarachnoid hemorrhage: The FRESH score Definition of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage as an outcome event in clinical trials and observational studies: proposal of a multidisciplinary research group Ultra-early angiographic vasospasm associated with delayed cerebral ischemia and infarction following aneurysmal subarachnoid hemorrhage Pathophysiology of Delayed Cerebral Ischemia After Subarachnoid Hemorrhage: A Review Monitoring in Neurointensive Care -The Challenge to Detect Delayed Cerebral Ischemia in High-Grade Aneurysmal SAH Dynamic Detection of Delayed Cerebral Ischemia: A Study in 3 Centers Combining Transcranial Doppler and EEG Data to Predict Delayed Cerebral Ischemia After Subarachnoid Hemorrhage Hospital Complications, and Longitudinal Outcomes After Subarachnoid Hemorrhage Laboratory biomarkers of delayed cerebral ischemia after subarachnoid hemorrhage: a systematic review Thrombosis: tangled up in NETs Neutrophil extracellular traps kill bacteria Neutrophil extracellular traps in ischemic stroke thrombi Neutrophil elastase-deficient mice form neutrophil extracellular traps in an experimental model of deep vein thrombosis Assessment of Neutrophil Extracellular Traps in Coronary Thrombus of a Case Series of Patients With COVID-19 and Myocardial Infarction Neutrophil extracellular traps in immunity and disease Extracellular DNA NET-Works With Dire Consequences for Health Pharmacological Modulation of Neutrophil Extracellular Traps Reverses Thrombotic Stroke tPA (Tissue-Type Plasminogen Activator) Resistance Thrombus Neutrophil Extracellular Traps Content Impair tPA-Induced Thrombolysis in Acute Ischemic Stroke Nonconvulsive seizures in subarachnoid hemorrhage link inflammation and outcome Admission neutrophil-lymphocyte ratio predicts delayed cerebral ischemia following aneurysmal subarachnoid hemorrhage RNase A Inhibits Formation of Neutrophil Extracellular Traps in Subarachnoid Hemorrhage Admission serum high mobility group box 1 (HMGB1) protein predicts delayed cerebral ischemia following aneurysmal subarachnoid hemorrhage Common Data Elements for Unruptured Intracranial Aneurysms and Aneurysmal Subarachnoid Hemorrhage: Recommendations from the Working Group on Hospital Course and Acute Therapies-Proposal of a Multidisciplinary Research Group Monocyte-driven atypical cytokine storm and aberrant neutrophil activation as key mediators of COVID-19 disease severity The predictive value of serum myeloperoxidase for vasospasm in patients with aneurysmal subarachnoid hemorrhage The anti-inflammatory and neuroprotective effects of ghrelin in subarachnoid hemorrhage-induced oxidative brain damage in rats Potential contribution of nuclear factor-kappaB to cerebral vasospasm after experimental subarachnoid hemorrhage in rabbits Efficacy of Cilostazol in Prevention of Delayed Cerebral Ischemia after Aneurysmal Subarachnoid Hemorrhage: A Meta-Analysis an endothelin receptor antagonist, in patients with aneurysmal subarachnoid haemorrhage undergoing surgical clipping: a randomised, double-blind, placebo-controlled phase 3 trial (CONSCIOUS-2) Cortical spreading ischaemia is a novel process involved in ischaemic damage in patients with aneurysmal subarachnoid haemorrhage Impact of systemic inflammatory response syndrome on vasospasm, cerebral infarction, and outcome after subarachnoid hemorrhage: exploratory analysis of CONSCIOUS-1 database Inflammation, negative nitrogen balance, and outcome after aneurysmal subarachnoid hemorrhage Haptoglobin administration into the subarachnoid space prevents hemoglobin-induced cerebral vasospasm Neutrophil activation by heme: implications for inflammatory processes Heme-induced neutrophil extracellular traps contribute to the pathogenesis of sickle cell disease Delayed Cerebral Ischemia After Subarachnoid Hemorrhage: Is There a Relevant Experimental Model? A Systematic Review of Preclinical Literature Circulating Myeloperoxidase (MPO)-DNA complexes as marker for Neutrophil Extracellular Traps (NETs) levels and the association with cardiovascular risk factors in the general population All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.