The authors have declared that no competing interests exist.
Conceived and designed the experiments: SH OE TP TSH. Performed the experiments: SH OE TP PK MH. Analyzed the data: SH MZA MZ AM FF TSH. Wrote the paper: SH MZA MZ AM FF TSH.
From previous data in animal models of cerebral ischemia, lipocalin-2 (LCN2), a protein related to neutrophil function and cellular iron homeostasis, is supposed to have a value as a biomarker in ischemic stroke patients. Therefore, we examined LCN2 expression in the ischemic brain in an animal model and measured plasma levels of LCN2 in ischemic stroke patients.
In the mouse model of transient middle cerebral artery occlusion (tMCAO), LCN2 expression in the brain was analyzed by immunohistochemistry and correlated to cellular nonheme iron deposition up to 42 days after tMCAO. In human stroke patients, plasma levels of LCN2 were determined one week after ischemic stroke. In addition to established predictive parameters such as age, National Institutes of Health Stroke Scale and thrombolytic therapy, LCN2 was included into linear logistic regression modeling to predict clinical outcome at 90 days after stroke.
Immunohistochemistry revealed expression of LCN2 in the mouse brain already at one day following tMCAO, and the amount of LCN2 subsequently increased with a maximum at 2 weeks after tMCAO. Accumulation of cellular nonheme iron was detectable one week post tMCAO and continued to increase. In ischemic stroke patients, higher plasma levels of LCN2 were associated with a worse clinical outcome at 90 days and with the occurrence of post-stroke infections.
LCN2 is expressed in the ischemic brain after temporary experimental ischemia and paralleled by the accumulation of cellular nonheme iron. Plasma levels of LCN2 measured in patients one week after ischemic stroke contribute to the prediction of clinical outcome at 90 days and reflect the systemic response to post-stroke infections.
Ischemic stroke is one of the leading causes of death and disability and utilises huge amount of health care expenses [
LCN2 has been established as a biomarker of acute kidney injury and a prognostic factor in chronic kidney disease [
The study was approved by the institutional review board of the Medical University of Graz (IRB00002556). Written informed consent was obtained from all participants. For patients with impaired consciousness or aphasia, written informed consent was obtained when these patients regained the ability to communicate and were oriented to self and time. No surrogate consent procedure was applied. Animal experiments were performed in accordance with the European directive on the protection of animals used for scientific purposes and all other applicable regulations and approved by the relevant authority, Landesamt fuer Gesundheit und Soziales, Berlin, Germany.
Male SPF C57Bl6/J mice (Charles River Laboratories, Sulzfeld, Germany) were housed in cages lined with chip bedding and environmental enrichment (mouse tunnel and igloo; Plexx B.V., Elst, The Netherlands) on a 12 h light/dark cycle (change 7 o’ clock) with ad libitum access to food (standard chow) and water. At the time of the experiment, mice were 11–14 weeks old. Cerebral ischemia was induced as described previously [
Ischemic lesion volumes were measured on day 1 after tMCAO by T2 weighted magnetic resonance imaging on a 7T scanner (Pharmascan 70/16 AS, Bruker Biospin, Ettlingen, Germany). Delineable hyperintense lesion volume was determined on 20 consecutive coronal slices with 500μm thickness using Analyze 5.0 (AnalyzeDirect, Overland Park, KS, USA). Lesion volume was edema-corrected [
Animals were sacrificed on day 1 (n = 12), day 7 (n = 11), day 14 (n = 12), day 28 (n = 7) and day 42 (n = 7) post tMCAO under high doses of ketamin/xylazin anaethesia by transcardial perfusion with 4% paraformaldehyde (PFA). Brains were removed and postfixed in 4% PFA for 24 hours. Paraffin embedded sections were examined by immunohistochemistry. Ischemic lesions were delineated by reduced anti-MAP2 immunostaining as reported previously (abcam, Cat.-No. ab32454) [
For ferrous nonheme iron, a DAB-enhanced Turnbull blue reaction procedure was applied as described elsewhere [
Serum was obtained from mice 7 days post tMCAO (n = 15) and from a group of naïve control mice (n = 11) and stored at -80°C for analysis. LCN2 levels in mouse serum were analyzed by ELISA according to the manufacturer’s instructions (Kit 042; BioPorto Diagnostics, Gentofte, Denmark).
Patients with a diagnosis of ischemic stroke according to clinical examination and brain imaging (computerized tomography or magnetic resonance imaging) were eligible when they had a National Institutes of Health Stroke Scale (NIHSS) of more than 3 on admission and a modified Rankin Scale (mRS) of 0 or 1 before symptom onset. The NIHSS was obtained on admission by board certified neurologists. The mRS was obtained 90 days post stroke by telephone interviews with the patients or their caregivers [
Student’s t-test, Mann-Whitney’s U-test, the Chi-square test or Fisher’s exact test, and Spearman’s rank order correlation were applied for two-group comparisons. The level of significance was set at a p-value of less than 0.05. A stepwise linear logistic regression model was used for analyzing variables to predict clinical outcome. Variables with p-values less than 0.1 were included in an initial predictive model. Backwards elimination logistic regression was performed to generate final predictive models. Receiver operator characteristic (ROC) curves were constructed and discrimination of models was assessed by comparing areas under the curve (AUC) with MedCalc® 11.6.1. software [
Ischemic brain lesions were delineated by a reduced MAP2 staining in the striatum and frequently the neocortex in tMCAO mice (
Reduced MAP2 immunostaining delineates ischemic lesions (left column). Macrophage/microglia infiltration is shown by MAC3 immunostaining (right column).
Macrophage/microglia infiltration (anti-MAC3, brown,
Data are presented as mean and standard error; *p<0.001 day 14 vs. days 1 and 7; **p<0.01 day 14 vs. day 7, p<0.001 day 14 vs. days 1, 28 and 42.
In adjacent serial sections at day 14 following tMCAO, cellular nonheme iron staining (
Plasma samples were obtained from 46 consecutive patients at median of 7 days (range 5–9 days) after the onset of ischemic stroke. Demographics, clinical characteristics, cerebrovascular risk factors and stroke classification of patients are given in
all patients | mRS 0–2 | mRS 3–6 | p | |
---|---|---|---|---|
46 | 13 | 33 | - | |
71 (63–79) | 68 (54–74) | 76 (63–79) | 0.107 | |
47.8% (22) | 53.8% (7) | 45.5% (15) | 0.987 | |
11 (6–17) | 6 (5–8) | 13 (7–18) | 0.002 | |
26.1% (12) | 38.5% (5) | 21.2% (7) | 0.276 | |
58.7% (27) | 23.1% (3) | 72.7% (24) | 0.006 | |
75,6 ml/min/1.7 (85,4–65,7) | 69,6 ml/min/1.7 (80,6–58,7) | 77,9 ml/min/1.7 (89,0–66,8) | 0.269 | |
84.8% (39) | 100% (13) | 78.8% (26) | 0.166 | |
47.8% (22) | 61.5% (8) | 42.4% (14) | 0.400 | |
21.7% (10) | 23.1% (3) | 21.2% (7) | 1.000 | |
41.3% (19) | 23.1% (3) | 48.5% (16) | 0.214 | |
13.0% (6) | 7.7% (1) | 15.2% (5) | 0.659 | |
41.3% (19) | 7.7% (1) | 54.5% (18) | 0.010 | |
50.0% (23) | 69.2% (9) | 42.4% (14) | 0.190 | |
4.3% (2) | 15.4% (2) | 0 | 0.075 | |
4.3% (2) | 7.7% (1) | 3.0% (1) | 0.490 | |
21.7% (10) | 7.7% (1) | 27.3% (9) | 0.240 | |
52.2% (24) | 46.2% (6) | 54.5% (18) | 0.853 | |
0 | 0 | 0 | - | |
6.5% (3) | 7.7% (1) | 6.1% (2) | 1.000 | |
19.6% (9) | 38.5% (5) | 12.1% (4) | 0.092 |
IQR, interquartile range; NIHSS, National Institutes of Stroke Scale; eGFR, estimated glomerular filtration rate; TACS, total anterior circulation syndrome; PACS, partial anterior circulation syndrome; POCS, posterior circulation syndrome; LACS, lacunar syndrome
Notably, plasma levels of LCN2 did not correlate with NIHSS upon admission (rS = 0.08; p = 0.58). A correlation of LCN2 plasma levels with the mRS at 90 days after stroke was found (rS = 0.40; p<0.01). For further analysis, patients were dichotomized into favourable (mRS 0–2) and unfavourable (mRS 3–6) outcomes 90 days after stroke. Median NIHSS, the proportion of post-stroke infections and the proportion of total anterior circulation stroke (TACS) differed significantly between these patient subgroups (
First, patients were dichotomized into favourable (mRS 0–2) and unfavourable (mRS 3–6) outcomes assessed 90 days after stroke. Second, patients were dichotomized if they had developed post-stroke infections or not. Plots display the median, interquartile range (box), 10th and 90th percentiles (whiskers); *p<0.05; **p<0.01. Abbreviation: mRS = modified Rankin Scale
The relevance of LCN2 plasma levels to predict an unfavorable vs. favorable clinical outcome was evaluated by comparing predictive models. Model 1 included NIHSS, patients’ age, thrombolytic therapy and the estimated glomerular filtration rate (eGFR). The eGFR as a measure of chronic kidney disease is an independent predictor of poor outcome and long-term mortality in patients with stroke [
model | variable | p | OR (95% CI) | AUC (95% CI) |
---|---|---|---|---|
1 | NIHSS | 0.011 | 1.433 (1.087–1.890) | 0.851 (0.743–0.958) |
age | 0.067 | 1.108 (0.993–1.236) | ||
thrombolysis | 0.090 | 0.015 (0.015–1.344) | ||
eGFR | 0.202 | 1.038 (0.980–1.099) | ||
2 | NIHSS | 0.016 | 1.441 (1.071–1.939) | 0.935 (0.864–1.000) |
age | 0.090 | 1.120 (0.982–1.277) | ||
thrombolysis | 0.096 | 0.096 (0.007–1.306) | ||
eGFR | 0.090 | 1.06 (0.991–1.134) | ||
lipocalin-2 | 0.049 | 1.029 (1.000–1.059) |
*p = 0.048; OR, Odds ratio; CI, confidence interval; AUC, area under the curve; IQR, interquartile range; NIHSS, National Institutes of Stroke Scale; eGFR, estimated glomerular filtration rate
Our study is the first to describe long-term expression kinetics of LCN2 in tMCAO, an animal model of ischemic stroke. The presence of LCN2 in rat tMCAO and postmortem human ischemic brain was previously reported up to 3 days post stroke [
In ischemic stroke patients, higher plasma levels of LCN2 measured one week after stroke correlated with worse clinical outcome at 90 days in our study. Increased levels of LCN2 were associated with post-stroke infections and supposedly reflect the response of circulating neutrophils to infections. Neutrophils serve as a major reservoir of LCN2 [
The strength of our study is the exact timing with a predefined interval from stroke onset to blood sampling to include information about changes of circulating LCN2 upon ongoing post-stroke infections. Previous studies about blood biomarkers in stroke patients have not applied such a strict criterion but rather lumped together sampling at the day of the acute incident and sampling at several days afterwards. In previous studies, the different timing of blood sampling has blurred a distinction whether interleukin-6 could serve as a marker of simultaneously evolving post-stroke infections or rather as their predictor [
A wide range of biomarkers has been evaluated for their relevance as predictors of clinical outcome after ischemic stroke [
Number of lipocalin-2 (LCN2) positive cells and nonheme iron staining cells in the ischemic striatum after tMCAO. Serum levels of LCN2 in mice 7 days post tMCAO.
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