Disruption of the alox5ap gene ameliorates focal ischemic stroke: possible consequence of impaired leukotriene biosynthesis Jakob Ström, Tobias Strid and Sven Hammarström Linköping University Post Print N.B.: When citing this work, cite the original article. Original Publication: Jakob Ström, Tobias Strid and Sven Hammarström, Disruption of the alox5ap gene ameliorates focal ischemic stroke: possible consequence of impaired leukotriene biosynthesis, 2012, BMC neuroscience (Online), (13). http://dx.doi.org/10.1186/1471-2202-13-146 Licencee: BioMed Central http://www.biomedcentral.com/ Postprint available at: Linköping University Electronic Press http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-89759 Ström et al. BMC Neuroscience 2012, 13:146 http://www.biomedcentral.com/1471-2202/13/146 RESEARCH ARTICLE Open Access Disruption of the alox5ap gene ameliorates focal ischemic stroke: possible consequence of impaired leukotriene biosynthesis Jakob O Ström1, Tobias Strid2 and Sven Hammarström2* Abstract Background: Leukotrienes are potent inflammatory mediators, which in a number of studies have been found to be associated with ischemic stroke pathology: gene variants affecting leukotriene synthesis, including the FLAP (ALOX5AP) gene, have in human studies shown correlation to stroke incidence, and animal studies have demonstrated protective properties of various leukotriene-disrupting drugs. However, no study has hitherto described a significant effect of a genetic manipulation of the leukotriene system on ischemic stroke. Therefore, we decided to compare the damage from focal cerebral ischemia between wild type and FLAP knockout mice. Damage was evaluated by infarct staining and a functional test after middle cerebral artery occlusion in 20 wild type and 20 knockout male mice. Results: Mortality-adjusted median infarct size was 18.4 (3.2-76.7) mm3 in the knockout group, compared to 72.0 (16.7-174.0) mm3 in the wild type group (p < 0.0005). There was also a tendency of improved functional score in the knockout group (p = 0.068). Analysis of bone marrow cells confirmed that knockout animals had lost their ability to form leukotrienes. Conclusions: Since the local inflammatory reaction after ischemic stroke is known to contribute to the brain tissue damage, the group difference seen in the current study could be a consequence of a milder inflammatory reaction in the knockout group. Our results add evidence to the notion that leukotrienes are important in ischemic stroke, and that blocked leukotriene production ameliorates cerebral damage. Keywords: Brain infarction, Leukotrienes, Alox5ap protein, FLAP, Mice, Middle cerebral artery occlusion Background inflammatory cytokines and infiltration of various in- Ischemic stroke is one of the leading causes of death and flammatory cells, including neutrophils, T-cells and long-term disability in the world. Despite the allocation monocytes/macrophages, into the damaged tissue. In- of huge research efforts in recent years, treatment flammation contributes to tissue damage, and especially options for ischemic stroke remain few. Therefore, the early inflammatory cell infiltration and cytokine pro- investigations into the pathophysiological intricacies of duction seem to be predominantly deleterious [1]. the disease are crucial to provide new drug targets. Mounting evidence indicates that leukotrienes (LTs), a Inflammation is a prominent feature of stroke patho- group of potent inflammatory mediators [2], have an im- physiology, and has attracted substantial research inter- portant role in cerebral ischemia, and that components est. The massive cell death in the infarct area triggers an involved in the LT cascade may be attractive drug acute and prolonged inflammatory process in the brain, targets. LTs are formed from arachidonic acid (AA) by characterized by activation of microglia, production of 5-lipoxygenase (5-LO) upon immunological or inflam- matory challenge. 5-LO activating protein (FLAP) is an integral membrane protein localized to the nuclear enve- * Correspondence: [email protected] lope and endoplasmic reticulum. Both 5-LO and FLAP 2Division of Cell Biology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden are required for formation of LTA4 from endogenous Full list of author information is available at the end of the article AA [3-8], and LTA4, is the precursor of all AA-derived © 2012 Ström et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ström et al. BMC Neuroscience 2012, 13:146 Page 2 of 9 http://www.biomedcentral.com/1471-2202/13/146 effector LTs. The expression of 5-LO [9] and LT recep- The use of genetically modified animals is an import- tors [10-13] is affected by cerebral ischemia, and the ant tool in the elucidation of biological mechanisms. importance of eicosanoids has been proposed from the However, in the case of LT effects on stroke, such stud- observation that AA concentrations are highest in ische- ies remain scarce. The only one we are aware of investi- mic brain regions most sensitive to stroke [14]. Human gated the effect of disrupting the 5-LO gene in mice, but genetic studies have demonstrated a significant correl- no significant effects on focal cerebral ischemia were ation between stroke risk and polymorphisms in several seen [31]. Because of the suggested importance of FLAP of the LT-associated genes, e.g. ALOX5AP encoding in human stroke studies, we decided to investigate the FLAP [15-17], as well as genes encoding LTC4 synthase effect of FLAP gene knockout in a rodent middle cere- [16,18] and cysteinyl (Cys) LT receptors [16]. Also, a bral artery occlusion (MCAo) model. The hypothesis large Swedish epidemiologic study recently showed that was that the decreased LT production in the genetically intake of montelukast, a CysLT1R antagonist, was asso- modified mice would ameliorate the damage from ische- ciated with a decreased risk of recurrent stroke [19]. mic stroke. Furthermore, animal studies have demonstrated that drugs interfering with components of the LT pathway, Results such as 5-LO [20-22] and LT receptors [23-28], amelior- FLAP knockout mice do not produce leukotrienes ate cerebral ischemic damage. Effects mediated by both LTA4 is an obligatory intermediate in the biosynthesis of of the established CysLT receptors, CysLT1R [13,24,26- LTB4 and LTC4 [32]. It is unstable and thus difficult to 28] and CysLT2R [10], appear to be involved in ischemic measure by direct methods. Therefore, we measured its brain injury. Furthermore, the proposed CysLT receptor downstream product LTB4 which is efficiently formed GPR17 was found to be up-regulated in damaged tissues, from LTA4 by the widely expressed enzyme LTA4 hydro- and knockout of the GPR17 gene reduced neuronal in- lase. Bone marrow cells from wild type and knockout jury after ischemia [29,30]. mice were incubated without cysteine with AA and LTC LTB 80 Reference 3.244 2.275 compounds 60 40 nm [mAU] 0.443 20 0.961 3.874 1.596 0 Absorbance at 270 0 1234 5 Time [min] 0.457 0.982 80 FLAP +/+ 3.174 60 40 nm [mAU] 20 2.419 2.715 1.575 0 1.871 Absorbance at 270 0 1234 5 Time [min] 0.976 0.455 80 FLAP -/- 60 40 nm [mAU] 20 1.875 2.387 2.580 2.737 3.244 0 Absorbance at 270 0 1234 5 Time [min] Figure 1 Leukotriene formation was blocked in FLAP knockout mice. Bone marrow cells obtained from wild type and FLAP knockout animals were incubated with 50 μM AA and 20 μM calcium ionophore A23187 for 20 min at 37°C. Reactions were stopped with ice cold methanol and supernatants were analyzed by RP-HPLC as described in Materials and Methods. The absorbance at 270 nm, corrected for stray absorbance at 360 nm, was recorded versus time. Retention times in minutes are indicated for each component in the chromatograms. Upper panel: analysis of synthetic LTB4 and LTC4. Middle panel: LTB4 was formed by bone marrow cells from wild type mice, but not from knockout mice (lower panel). Ström et al. BMC Neuroscience 2012, 13:146 Page 3 of 9 http://www.biomedcentral.com/1471-2202/13/146 calcium ionophore A23187. The lack of cysteine pre- (0.40-0.60) and 0.65 (0.53-0.70), respectively. The mice vents formation of LTC4 [33,34] whereas formation of in both groups were severely affected by the ischemic LTB4 proceeds normally. RP-HPLC analyses showed that stroke, and as much as 91% of the wild type and 60% of LTB4 was formed by wild type, but not by FLAP knock- the knockout animals scored maximally (1.00 = 100% of out, bone marrow cells (Figure 1). swings to the right) in the tail swing test, thus decreasing the test sensitivity. Despite this, a trend of better out- FLAP knockout decreased mortality-adjusted infarct size come, which however did not reach statistical signifi- In the wild type group, two of the included animals died cance (p = 0.068), was seen in the knockout group. within 24 h after MCAo, while no included animals died Mortality-adjusted median tail swing index (proportion in the FLAP knockout group. Mortality-adjusted median of right side swings) was 1.00 (1.00-1.00) in the wild type infarct size was 18.4 (3.2-76.7) mm3 in the knockout group, compared to 1.00 (0.90-1.00) in the knockout group, compared to 72.0 (16.7-174.0) mm3 in the wild group (Figure 3). type group (p < 0.0005; Figure 2). Perioperative physiological monitoring FLAP knockout had no significant effect on mortality- The physiological variables monitored during MCAo adjusted tail swing test performance were similar between the two groups, as presented in Before MCAo, the wild type and knockout groups Table 1, indicating that they responded similarly to the scored close to the theoretical baseline index of 0.50 anesthesia.