Neurovascular protection by post‐ischemic intravenous injections of the lipoxin A₄ receptor agonist, BML‐111, in a rat model of ischemic stroke

Abstract

Resolution of inflammation is an emerging new strategy to reduce damage following ischemic stroke. Lipoxin A4 (LXA4) is an anti-inflammatory, pro-resolution lipid mediator with high affinity binding to its receptor ALX. Since LXA4 is rapidly inactivated, potent analogs have been created, including the ALX agonist BML-111. We hypothesized that post-ischemic intravenous administration of BML-111 would provide protection to the neurovascular unit and reduce neuroinflammation in a rat stroke model. Animals were subjected to 90 min of middle cerebral artery occlusion (MCAO) and BML-111 was injected 100 min and 24 h after stroke onset and animals sacrificed at 48 h. Post-ischemic treatment with BML-111 significantly reduced infarct size, decreased vasogenic edema, protected against blood-brain barrier (BBB) disruption, and reduced hemorrhagic transformation. Matrix metalloproteinase (MMP)-9 and MMP-3 were significantly reduced following BML-111 treatment. Administration of BML-111 dramatically decreased microglial activation, as seen with CD68, and neutrophil infiltration and recruitment, as assessed by levels of myeloperoxidase (MPO) and intracellular adhesion molecule (ICAM)-1. The tight junction protein zona occludens-1 (ZO-1) was protected from degradation following treatment with BML-111. These results indicate that post-ischemic activation of ALX has pro-resolution effects that limit the inflammatory damage in the cerebral cortex and helps maintain BBB integrity after ischemic stroke.

Targeting resolution of neuroinflammation after ischemic stroke with a lipoxin A4 analog: Protective mechanisms and long‐term effects on neurological recovery

“…Three‐dimensional (3D) reconstruction images of the ischemic lesion in vehicle‐ and BML‐111‐treated rats are shown in Figure 1e,f. Importantly, the reduction in cerebral cortex and total stroke volume in this study (51.1% and 41.1%, respectively) is similar to the reductions reported in our previous 2‐day study (39.3% and 35.3%, respectively)(Hawkins et al., 2014), verifying the neuroprotective effect of enhancing the resolution pathway with 1 mg/kg BML‐111 after ischemic stroke. Therefore, 1 mg/kg BML‐111 was chosen as the optimal protective dose for maximal infarct size reduction 1 week post‐stroke.…”

“…We have shown before that MMP‐9 and MCP‐1 are reduced in ischemic stroke with post‐stroke BML‐111 administration (Hawkins et al., 2014). These factors are important in neural stem cell proliferation and migration during neurogenesis after ischemic stroke (Lee et al., 2006; L. Wang et al., 2006; Widera et al., 2004; Yan et al., 2007).…”

“…Focal cerebral ischemia was done by tMCAO using the intraluminal filament method as previously described by our group (Candelario‐Jalil, Gonzalez‐Falcon, Garcia‐Cabrera, Leon, & Fiebich, 2007; Frankowski et al., 2015; Hawkins et al., 2014). Briefly, non‐fasted rats were anesthetized with isoflurane (4% for induction and 2% for maintenance) in medical‐grade oxygen.…”

“…Brain tissue homogenates from the ipsilateral cerebral cortex were prepared from rats in the mechanism experiments as previously described by our lab (Hawkins et al., 2014). The tissue sections were weighed and homogenized in 1% sodium dodecyl sulfate (SDS) buffer containing 150 mmol/L NaCl, 50 mmol/L Tris‐HCl pH 7.6, 1% IGEPAL ® CA‐630, and 1% sodium deoxycholate at 100 μl/10 mg of brain tissue.…”

“…Recently, we showed for the first time that two post‐ischemic injections of BML‐111 (1 mg/kg; i.v.) provides protection to the BBB and reduces stroke volume and neuroinflammatory factors including neutrophil infiltration, intercellular adhesion molecule (ICAM)‐1 expression, matrix metalloproteinase (MMP)‐9 and MMP‐3 levels, and CD68 expression at 2 days post‐stroke in a rat model of transient middle cerebral artery occlusion (tMCAO) (Hawkins et al., 2014). According to the Stroke Therapy Academic Industry Roundtable (STAIR) preclinical recommendations, multiple endpoints should be determined for anatomical and behavioral outcome at least 2–3 weeks or longer after onset of stroke to determine the sustained benefit of experimental drugs (Fisher et al., 2009).…”