Early and Sustained Increases in Leukotriene B4 Levels Are Associated with Poor Clinical Outcome in Ischemic Stroke Patients

Abstract

Leukotriene B₄ (LTB₄) has been implicated in ischemic stroke pathology. We examined the prognostic significance of LTB₄ levels in patients with acute middle cerebral artery (MCA) infarction and their mechanisms in rat stroke models. In ischemic stroke patients with middle cerebral artery infarction, plasma LTB₄ levels were found to increase rapidly, roughly doubling within 24 h when compared to initial post-stroke levels. Further analyses indicate that poor functional recovery is associated with early and more sustained increase in LTB₄ rather than the peak levels. Results from studies using a rat embolic stroke model showed increased 5-lipoxygenase (5-LOX) expression in the ipsilateral infarcted cortex compared with sham control or respective contralateral regions at 24 h post-stroke with a concomitant increase in LTB₄ levels. In addition, neutrophil influx was also observed in the infarcted cortex. Double immunostaining indicated that neutrophils express 5-LOX and leukotriene A₄ hydrolase (LTA₄H), highlighting the pivotal contributions of neutrophils as a source of LTB₄. Importantly, rise in plasma LTB₄ levels corresponded with an increase in LTB₄ amount in the infarcted cortex, thereby supporting the use of plasma as a surrogate for brain LTB₄ levels. Pre-stroke LTB₄ loading increased brain infarct volume in tMCAO rats. Conversely, administration of the 5-LOX-activating protein (FLAP) inhibitor BAY-X1005 or B-leukotriene receptor (BLTR) antagonist LY255283 decreased the infarct volume by a similar extent. To conclude, targeted interruption of the LTB₄ pathway might be a viable treatment strategy for acute ischemic stroke.

Keywords: Ischemic stroke; animal models; leukotriene B4; middle cerebral artery.

Fig. 1

Correlation of neutrophil counts with LTB₄ levels in the plasma of ischemic stroke patients on day 0. Significant correlation was obtained by Spearman rank-order correlation, r² = 0.373, p = 0.018, n = 25. Graph shows linear regression line with 95% confidence limits

Fig. 2

Correlation of stroke severity with (a) LTB₄ levels and (b) neutrophil counts in the plasma of ischemic stroke patients on day 0. No significant correlations were obtained by Spearman rank-order correlation, r² = 0.002, p = 0.955 (a) and r² = 0.054, p = 0.236 (b), n = 25

Fig. 3

Correlation between plasma LTB₄ levels in stroke patients with clinical outcome following acute middle cerebral artery infarction. (a) LTB₄ levels in plasma of healthy controls (n = 16) and stroke patients on days 0, 1, and 7 (n = 25). **Significant difference between control and day 1 by Mann-Whitney U test p = 0.03. ***Significant difference between day 0 (baseline) and day 1, and between day 1 and day 7 stroke samples by Friedman test for related variables with correction for multiple comparisons, p < 0.01. (b) Comparison of plasma LTB₄ levels on days 0, 1, and 7 between patients stratified by good (MRS 0–2, n = 15) vs poor (MRS 3–5, n = 10) clinical outcome as assessed by functional recovery on day 90 post-stroke. *p < 0.05, **p < 0.01 between good and poor clinical outcomes adjusted for age and stroke severity by logistics regression methods

Fig. 4

LTB₄ levels in plasma and cortex of thromboembolic stroke (ES) rats 24 h post-stroke. (a) Plasma LTB₄ levels for pre-ES control (n = 24), and at 3 (n = 15) and 24 h (n = 9) post-ES. ***p < 0.001 against pre-ES or 3 h post-ES by one-way ANOVA followed by Tukey post hoc test. (b) LTB₄ levels in the ipsilateral (ipsi) and contralateral (contra) cortex after ES (n = 4). * t = 2.581, p < 0.05 against the contralateral side by two-tailed independent t test. At 3 h, t = 2.029, p = 0.089. Total number of animals used was 24

Fig. 5

Expression of 5-LOX, ED-1, OX-42, and GFAP in the cerebral cortex after embolic stroke (ES). (a) Representative Western blots of 5-LOX, ED-1, OX-42, and GFAP in the ipsilateral (ipsi) and contralateral (contra) sides for sham and ES rats at 3, 24, and 72 h post-ES. (b) Comparison of cortical expression of 5-LOX, ED-1, OX-42, and GFAP between sham ipsi and ES ipsi, n = 3–5 per group. The ES group at 3 h was used as the reference group which was set to 1. Expressions of 5-LOX, ED-1, and OX-42 were consistently not detected (nd) in Sham controls. Statistical analysis was performed by two-way ANOVA testing time factor and stroke factor. F(1,14) = 13.83, p < 0.005 (5-LOX); F(1,24) = 35.65, p < 0.0001 (ED-1); F(1,18) = 21.64, p < 0.0005 (OX-42); F(1,14) = 14.14, p < 0.005 (GFAP), for stroke factor. *p < 0.05 and **p < 0.01 vs corresponding sham group by Bonferroni correction. No significance obtained for time factor and interaction in all 4 markers. Total number of animals used was 12

Fig. 6

Immunofluorescence staining of MPO, 5-LOX, and LTA₄H in the infarcted cortex 24 h post-ES. (a) Immunopositive cells increased many fold in the ipsilateral (ipsi) side when compared to the contralateral (contra) side showing marked upregulation of MPO, 5-LOX, and LTA₄H expressions. Scale bar = 200 μm. (b) Double immunofluorescence staining showing 5-LOX and LTA₄H are expressed in MPO-immunopositive neutrophils. Scale bar = 50 μm. Number of animals used was 4

Fig. 7

Upregulation of 5-LOX (a) and LTA₄H (b) in the ipsilateral cortex and striatum 24 h after tMCAO (100 min). Scale bar = 100 μm. Total of animals used was 4

Fig. 8

Effects of LTB₄, a FLAP inhibitor (BAY-X1005) and a BLTR antagonist (LY255283) on infarction volume after tMCAO. (a) LTB₄ loading by intracarotid infusion over 30 min before tMCAO (60 min) increased infarct volume, n = 4 per group. ***p < 0.001 against the vehicle group by two-tailed independent t test. (b) Administration of BAY-X1005 (0.2 mg/kg, i.p.) and LY255238 (1 mg/kg, i.p.) 10 min prior to tMCAO (100 min) reduced infarct volume, n = 4 per group. *p < 0.05 against the vehicle group by one-way ANOVA followed by Tukey post hoc test. Representative thionin-stained sections for each treatment group are presented. Total number of animals used was 20