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Supplemental material, sj-pdf-1-wso-10.1177_17474930221109353 for Predictors of post-stroke delirium incidence and duration: Results of a prospective observational study using high-frequency delirium screening by Robert Fleischmann, Tina Andrasch, Sina Warwas, Rhina Kunz, Stefan Gross, Carl Witt, Johanna Ruhnau, Antje Vogelgesang, Lena Ulm, Annerose Mengel and Bettina von Sarnowski in International Journal of Stroke

Additional file 3: Figure S2. Gating strategy for oxidative burst setup. Representative probe of a new-born infant to measure oxidative burst. To clearly distinguish monocyte and granulocyte subpopulations FMOs for anti-CD14, −CD16 and -CD62L gating was used. Subsets were defined as already published by Pillay et al. (2012). CD16dim neutrophil population was distinguished by gating the 25th percentile of main neutrophil population

Netosis in an adult control. In vitro imaging of NETs forming neutrophils with representative images for one control subject. A brightfield image allowed quantification of total cells, SYTOX® Green allowed quantification of NETosis based on size of events and comparison of different stimuli. One representative example of the unstimulated and one PMA stimulated sample is shown. Fiji Software was used to measure NET area. Images of SYTOX® Green stained cells were taken with a Leica DMI 4000 B microscope; exposure time = 170 ms. (PDF 2057 kb)

Immune cells can significantly predict and affect the clinical outcome of stroke. In particular, the neutrophil-to-lymphocyte ratio was shown to predict hemorrhagic transformation and the clinical outcome of stroke; however, the immunological mechanisms underlying these effects are poorly understood. Neutrophils are the first cells to invade injured tissue following focal brain ischemia. In these conditions, their proinflammatory properties enhance tissue damage and may promote ischemic incidences by inducing thrombus formation. Therefore, they constitute a potential target for therapeutic approaches and prevention of stroke. Indeed, in animal models of focal brain ischemia, neutrophils have been targeted with successful results. However, even in brain lesions, neutrophils also exert beneficial effects, because they are involved in triggering immunological removal of cell debris. Furthermore, intact neutrophil function is essential for maintaining immunological defense against bacterial infections. Several studies have demonstrated that stroke-derived neutrophils displayed impaired bacterial defense capacity. Because infections are known to impair the clinical course of stroke, therapeutic interventions that target neutrophils should preserve or even restore their function outside the central nervous system (CNS). This complex situation requires well-tailored therapeutic approaches that can effectively tackle immune cell invasion in the brain but avoid increasing poststroke infections.

Monocytic intracellular expression (MFI) of NE and MPO per cell in stroke patients versus controls. Stroke patients were analysed and compared to healthy controls. MPO (A) and NE (B) were measured intracellularly in monocytes using flow cytometry and are expressed in MFI. MPO was measured on the day of stroke admission (d0) and on days 1, 3 and 5 after stroke (d1, d3, d5) (A; ncrtl = 14, nd0 = 14, nd1 = 18, nd3 = 14, nd5 = 16). NE was measured on day 1 (d1) after stroke onset, day 3 (d3) and day 5 (d5) by flow cytometry (B, ncrtl = 10, nd1 = 10, nd3 = 10, nd5 = 10), and the mean and SD ranges are given. ANOVA was used for the data in A and B. (PDF 2057 kb)

Influence of r-tPA on MPO and NE. PBMCs of healthy donors were incubated with 0.5 or 1 μg/ml r-tPA for 4 h at 37 °C in 5% CO2. Afterwards, MPO and NE were measured intracellularly in granulocytes by flow cytometry (A, C), expressed in MFI, and in the supernatant using ELISA (B, D), expressed in ng/ml (n = 10). *p

Influence of adrenaline, noradrenaline, dexamethasone and acetylcholine on NE and MPO release in vitro. NE and MPO-ELISA of supernatants of PBMCs (of healthy donors) treated with 1 × 10−7 M (low) or 1 × 10−5 M (high) adrenaline and noradrenaline; 2.5 × 10−7 M (low) or 2.5 × 10−6 M (high) dexamethasone; and 5.5 × 10−6 M (low) or 5.5 × 10−4 M (high) acetylcholine. If the data failed a Gaussian distribution test by the D‘agostino & Pearson omnibus normality test, the data were analysed by the Friedman test for repeated measures and Dunn’s post hoc test. *p

A: Influence of r-tPA on cytokine production with PHA stimulation. Production of INF-γ, IL-2, -4, -5, -6, -9, -13, -17A, -17 F, -21, -22 and TNF-α in the supernatants of PBMCs after 72 h of incubation with r-tPA. PBMCs were stimulated with PHA to induce cytokine production. IL-10 was not induced in any of the conditions and is not shown. As an example, the data from PBMCs isolated from venous blood of healthy donors anticoagulated with EDTA are shown. The results for heparin-anticoagulated venous blood were similar and are not shown. (n = 4). The means are given. B: Influence of r-tPA on cytokine production with anti-CD3/anti-CD28 stimulation. Production of INF-γ, IL-2, -10, -5, -6, -9, -13, -17A, -17 F, -21, -22 and TNF-α in supernatants of PBMC after 72 h of incubation with r-tPA. PBMCs of healthy donors were stimulated with anti-CD3/anti-CD28 to induce cytokine production. IL-4 was not induced in any of the conditions and is not shown. As an example, the data from PBMCs isolated from venous blood anticoagulated with EDTA are shown. The results for heparin-anticoagulated venous blood were similar and are not shown (n = 4). The means are given. (PDF 2057 kb)

Influence of adrenaline, noradrenaline, dexamethasone and acetylcholine on mean NET area. Effect of in vitro administration of 1 × 10−7 M or 1 × 10−5 M adrenaline (A) and noradrenaline (B); 2.5 × 10−7 M or 2.5 × 10−6 M dexamethasone (C); and 5.5 × 10−6 M or 5.5 × 10−4 M acetylcholine (D) on NET area was tested. After incubation of blood of healthy donors with hormones and acetylcholine for 4 h at room temperature, neutrophils were isolated, and NETs were induced using PMA or fMLP or left unstimulated (nA,B = 7; nC,D = 6). *p

Influence of adrenaline, noradrenaline, dexamethasone and acetylcholine on intracellular NE and MPO in vitro. A: Intracellular MPO of granulocytes and monocytes of healthy donors treated with adrenaline, noradrenaline, dexamethasone or acetylcholine (n = 10). The mean and SD ranges are given. B: Intracellular NE of granulocytes and monocytes treated with adrenaline, noradrenaline, dexamethasone or acetylcholine (n = 10). The mean and SD ranges are given. (PDF 2057 kb)

Influence of adrenaline, noradrenaline, dexamethasone and acetylcholine on mean NET area. Effect of in vitro administration of 1 × 10−7 M or 1 × 10−5 M adrenaline (A) and noradrenaline (B); 2.5 × 10−7 M or 2.5 × 10−6 M dexamethasone (C); and 5.5 × 10−6 M or 5.5 × 10−4 M acetylcholine (D) on NET area was tested. After incubation of blood of healthy donors with hormones and acetylcholine for 4 h at room temperature, neutrophils were isolated, and NETs were induced using PMA or fMLP or left unstimulated (nA,B = 7; nC,D = 6). *p

Influence of r-tPA on MPO and NE. PBMCs of healthy donors were incubated with 0.5 or 1 μg/ml r-tPA for 4 h at 37 °C in 5% CO2. Afterwards, MPO and NE were measured intracellularly in granulocytes by flow cytometry (A, C), expressed in MFI, and in the supernatant using ELISA (B, D), expressed in ng/ml (n = 10). *p

Influence of adrenaline, noradrenaline, dexamethasone and acetylcholine on intracellular NE and MPO in vitro. A: Intracellular MPO of granulocytes and monocytes of healthy donors treated with adrenaline, noradrenaline, dexamethasone or acetylcholine (n = 10). The mean and SD ranges are given. B: Intracellular NE of granulocytes and monocytes treated with adrenaline, noradrenaline, dexamethasone or acetylcholine (n = 10). The mean and SD ranges are given. (PDF 2057 kb)