Microglia are the primary immune cells in the brain, essential for maintaining the homeostasis of the central nervous system (CNS) microenvironment. They can be activated and polarized by stimuli related to ischemic stroke. Different microglial phenotypes exhibit dual or even multiple roles at various stages of ischemic stroke: M1 microglia secrete pro-inflammatory cytokines that exacerbate neuronal damage, while M2 microglia promote a reparative anti-inflammatory response. Therefore, modulating M1/M2 microglial activation to minimize harmful effects and maximize protective responses holds significant translational value.

Ace Therapeutics offers comprehensive microglia function assay services to characterize the effects of stroke drugs on microglia. Our microglia function assay service can be used to probe the activation status of isolated microglia in stroke animals and to screen for stroke drugs that enhance or restore microglial phagocytosis, activation, and migration.

Related Services

• In Vitro Microglial Function Assay Services for Stroke Drug Discovery & Development
• Analysis of Microglial Phagocytosis in Ischemic Stroke

Physiological Functions of Microglia

Microglia comprise about 10% of brain cells and play a crucial role in protecting the central nervous system (CNS) and maintaining homeostasis in vivo. They originate from primitive yolk sac macrophages and populate the CNS during early development. Predominantly located in gray matter, microglia exhibit tiny branching protrusions and express various markers that reflect their diverse functions. In physiological conditions, they are typically referred to as "resting," while their reactive morphology under pathological conditions is termed "activated."

Microglia eliminate dead cells, aggregated proteins, and synaptic debris, primarily through phagocytosis, which is essential to prevent tissue damage caused by reactive oxygen species (ROS) and inflammatory mediators.

Fig.1. Microglia can be classified into four different phenotypes based on the morphology of the cell: A) ramified, B) intermediate, C) amoeboid and D) round type. (Anttila, et al., 2017)

Activation and Polarization of Microglia in Ischemic Stroke

Neurons near the affected arteries die most rapidly when brain tissue is ischemia, leading to cerebral edema, blood-brain barrier rupture, and neuroinflammation. In the immediate aftermath of acute ischemic stroke, tissue injury caused by cell hypoxia and the subsequent generation of damage-associated molecular patterns (DAMP), cytokines and chemokines is primed microglia. Starved microglia morphologise quickly and migrate to the injury site where they have diverse effects on neuronal survival and recovery. According to their status of activation, microglia release pro- and anti-inflammatory mediators. They're inversely influenced by these mediators in neuronal survival and recovery. Moreover, microglia control neuronal excitability and synaptic activity, and stimulate angiogenesis and tissue repair after ischemic stroke.

M1/M2 Microglia Activation

Based on their activation and subsequent polarization, microglia can be broadly classified into pro-inflammatory (M1) and anti-inflammatory (M2) types. The M1 activation pathway is induced by IFNγ, LPS, or DAMP through the JAK/STAT1 or TLR4 pathway, which secretes harmful cytokines, such as TNF-α, IL-1β, IL-6, IL-12, NO, and ROS, leading to brain damage and exacerbating inflammation. M2 Anti-inflammatory microglia release beneficial cytokines, including IL-4, IL-10, transforming growth factor β (TGF-β), and IL-13, as well as growth factors, such as vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF), to promote tissue repair, angiogenesis, and neuroprotection.

Phagocytosis and Clearance of Debris

Microglia clear cellular debris from the site of injury, helping to control inflammation and tissue healing. They utilize 'eat me' signals, such as phosphatidylserine (PS), to recognize damaged cells and promote phagocytosis. Various receptors and pathways, including TAM receptors and the complement system, are involved in this process.

Angiogenesis and Tissue Repair

 Microglia were once thought to inhibit inflammation-induced neurogenesis. However, they can promote the growth and functional recovery of neuronal precursor cells (NPC). Microglia release factors such as IGF-1 and CDF-1α to support NPC movement and integration into neural networks, and secrete VEGF to enhance angiogenesis.

Synaptic Plasticity

Stroke disrupts synapses, but microglia can help regulate synaptic plasticity by releasing BDNF. This factor is essential for synapse formation and maintenance. Drugs that shift microglia to an anti-inflammatory state (e.g., TWS119) may promote neuroplasticity and recovery.

Fig.2. Overview of microglial activation, subtypes, and function following ischemic stroke. (Haupt, et al., 2024)

Microglial Crosstalk with Other Cells

Microglia and Neurons

• Injury to neurons: Ischemia and hypoxia sap neurons of their energy, leading to lactic acid production and acidosis that leads to oxidative stress via the generation of reactive oxygen species (ROS).
• Glutamate and excitotoxicity: After ischemia, the glutamate levels are increased, mostly by hyper-active microglia, and excitotoxicity and neuronal death are caused by ionotropic glutamate receptor overactivation.
• Autonomy of microglia: Neurons control microglial behavior by "On" and "Off" signals such as the chemokine CX3CL1, which regulates neuron-microglia communications.
• Inflammatory pathways: The NLRP3 inflammasome that induces inflammation is suppressed by CX3CR1. Degraded neurons can release lipocalin-2, and microglia can go into an anti-inflammatory mode – all the while leading to neuronal destruction and inflammation.
• Coverage: IL-4 secreted from injured neurons might support microglial anti-inflammatory activity. Microglial IGF-1 maintains neuronal survival angiogenesis and neurogenesis that averts the ischemia.

Microglia and Astrocytes

• Mechanics of communication: Astrocytes and microglia communicate mostly via exocytosis and the emissaries of adaptive immunity (ATP, glutamate).
• Purinergic communication: ATP released by astrocytes activates microglial P2Y12 and P2Y6 receptors and directly controls phagocytosis and other cell activity, which is why they're so important for each other.
• Cytokine effects: Cytokines produced by microglia kickstart and regulate astrogliosis. Among the signals detected in astrocytes are pattern recognition receptors – responsive to microglial mediators (IL-1, TNF-) and defective mitochondrial fragments.
• Phenotypic shifts: Microglial complement C1q drives the astrocyte transformation (perhaps through scavenger receptors on astrocytes).
• Excitotoxicity control: Microglial TNF- stimulates astrocyte glutamate release and drives excitotoxicity of neurons.
• Rights to glial scar formation: Stopping the proliferation of microglia can suppress post-injury glial scar formation highlighting the central role of microglia-astrocyte interactions in CNS injury repair.

Fig.3. Microglial crosstalk with astrocytes, neurons following ischemic stroke. (Haupt, et al., 2024)

Potential Therapy Targeting Microglial Responses

Although some promising agents to control microglial activation have shown activity in the lab or in preclinical trials, the majority are still in the research phase. Some agents, such as TNF antagonists, have been tested against autoimmune disorders, but their use in post-stroke microglial activation is still very theoretical. Because microglial activation (M1 and M2) is double-duty after stroke, any treatment must be tuned to either suppress pro-inflammatory or stimulate anti-inflammatory signals. Since the last decade, several microglia studies have pinpointed treatment targets for stroke and can be sorted into three strategies.

• Promoting anti-inflammatory microglial polarization
• Inhibition of microglia activation
• Regulating the interaction between microglia and other cells

1. Anttila, J. E., et al. (2017). Role of microglia in ischemic focal stroke and recovery: focus on Toll-like receptors. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 79, 3-14.
2. Haupt, M., et al. (2024). The dual role of microglia in ischemic stroke and its modulation via extracellular vesicles and stem cells. Neuroprotection, 2(01), 4-15.