Tier 3 — preclinical

The flavonoid fisetin attenuates postischemic immune cell infiltration, activation and infarct size after transient cerebral middle artery occlusion in mice

Gelderblom M, Leypoldt F, Lewerenz J, Birkenmayer G, Orozco D, Ludewig P, Thundyil J, Arumugam TV, Gerloff C, Tolosa E, Maher P, Magnus T
Journal of Cerebral Blood Flow & Metabolism 2012 Volume 32, issue 5, pages 835-843

Bibliography

PubMed
PMID 22234339
PubMed Central
PMC3345911
DOI
10.1038/jcbfm.2011.189
Funding
AHA Grant 075514Y to P.M., Landesexzellenzinitiative (LEXI) Hamburg and DFG HO 3312/1-1 to T.M.
Competing interests
The authors declare no conflict of interest.

Study snapshot

DesignIn vivo transient middle cerebral artery occlusion (tMCAO) stroke model in mice with pretreatment or delayed (3-hour) fisetin dosing, flow cytometric immune cell profiling, plus in vitro LPS-activation assays in murine N9 microglia, Raw264.7 macrophages and human monocyte-derived macrophages.
Model12-week-old C57Bl/6 wild-type mice (90 mice total) subjected to 60-minute tMCAO; murine N9 microglial and Raw264.7 macrophage cell lines; human monocyte-derived macrophages from healthy donor leukapheresis; primary murine microglia/cortical neuron cocultures.
SamplePretreatment cohort: 13 placebo, 12 low-dose fisetin, 8 high-dose fisetin mice; posttreatment cohort: 10 placebo, 9 high-dose fisetin mice; in vitro assays performed in triplicate.
InterventionFisetin 25 or 50 mg/kg body weight given intraperitoneally 20 minutes before, or 50 mg/kg given 180 minutes (3 hours) after, onset of ischemia; in vitro fisetin 0.28-2.9 micrograms/mL with LPS 10 ng/mL-1 microgram/mL.
DurationInfarct size and immune cell infiltration assessed 3 and 7 days after stroke; in vitro LPS stimulation assays run over 24 hours.
EndpointsInfarct volume (TTC staining) at 72 hours; Systemic blood pressure; Flow cytometric quantification of brain-infiltrating macrophages, dendritic cells and lymphocytes; Intracellular TNF-alpha production in microglia and macrophages; LPS-induced TNF-alpha secretion and neurotoxicity in macrophage/microglial cell lines; Phosphorylation of IkB, JNK and c-Jun (western blot)

What the study showed, in plain terms

This German-led stroke research group tested whether fisetin's anti-inflammatory and nerve-protective properties translate into real protection in a mouse model of ischemic stroke, and critically, whether it still works if given after the stroke has already started, which is the realistic scenario in human medicine.

Fisetin given 20 minutes before stroke onset reduced infarct size by up to 46% at the higher dose, and importantly, fisetin given 3 hours after stroke onset still reduced infarct size by 35%, without affecting blood pressure.

Digging into the mechanism, fisetin-treated mice had far fewer inflammatory immune cells (macrophages, dendritic cells, lymphocytes) flooding into the injured brain tissue, and the immune cells that were present made much less of the inflammatory signal TNF-alpha - an effect fisetin also reproduced directly in mouse and human immune cells stimulated with a bacterial inflammatory trigger (LPS) in the dish.

Mechanistically, fisetin blocked two major inflammatory signalling switches, NF-kB and JNK/c-Jun, in activated immune cells, which likely explains why it dampened brain inflammation without suppressing the immune system everywhere in the body (spleen immune cells were unaffected).

This strengthens the case that fisetin's post-stroke benefit is partly anti-inflammatory rather than purely antioxidant, but the doses were injected, not oral, and translation to a clinically deliverable oral human stroke therapy has not been tested.

Key findings

  • Fisetin (50 mg/kg) given 20 minutes before stroke onset reduced infarct size by 46%
  • Fisetin given 3 hours after stroke onset still reduced infarct size by 35%
  • Fisetin did not affect systemic blood pressure at protective doses
  • Fisetin reduced infiltration of macrophages, dendritic cells and lymphocytes into the ischemic brain hemisphere
  • Fisetin suppressed TNF-alpha production in brain-derived microglia and macrophages but not spleen macrophages
  • Fisetin inhibited LPS-induced NF-kB (IkB) and JNK/c-Jun phosphorylation in macrophages and microglia

What this study can and cannot tell us

All in vivo data come from a single mouse strain and stroke model (transient MCAO); translation to human ischemic stroke, where comorbidities and delayed treatment windows vary widely, is unproven.

Fisetin was administered by intraperitoneal injection at research doses, not orally, and no pharmacokinetic bridging to oral human dosing is provided.

Citation network

Articles citing this research paper

1 article

Biohack Blueprint analyses that reference this study in their evidence base.

Editorial review

Reviewed by the Biohack Blueprint research team

Last verified

Dr. Khaled Mahmoud, MBBChCritical Care Medicine, Longevity Science & Editorial Direction Dr. Abdulla Mostafa, PhDCellular Senescence, Neurological, Mitochondrial & Brain-Ageing Biology