JEM︱Mechanisms of microglia regulating blood flow and neurovascular connections

Author︱Tong Editor︱Wang Sizhen Microglia are an important type of immune cells in the central nervous system, which are closely related to normal physiological processes (such as brain development, synaptic plasticity, learning, memory) and pathological processes in the central nervous system.
Related [1]
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Microglia dynamically monitor their surroundings and can interact with other cells, including neurons, through migration
.

In addition to neurons, microglia interact with blood vessels and neurovascular unit cells [2]
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Microglia interact with blood vessels throughout the lifespan (from development to adulthood) and are involved in the regulation of blood-brain barrier (BBB) ​​permeability, leukocyte extravasation, and angiogenesis [3]
.

 Recent studies have shown that perivascular macrophages play an important role in hypertension-related neurovascular dysfunction by promoting BBB permeability
.

And in models of stroke, Alzheimer's disease or multiple sclerosis, microglia are rapidly recruited to sites of BBB leakage, suggesting that microglia respond to vascular damage
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Cerebral blood flow (CBF) is the circulation of blood in the brain
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However, the relationship between microglia and CBF and abnormal perfusion in adulthood is still unclear [4]
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 Recently, the team of Ádám Dénes from the Budapest Institute of Experimental Medicine, Hungary, published a work entitled "Microglia modulate blood flow, neurovascular coupling, and hypoperfusion via purinergic actions" in the Journal of Experimental Medicine, and found that microglia play a role in normal The mechanism of CBF regulation through P2Y12 receptor under physiological conditions and low perfusion [4]
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The researchers first investigated the formation and dynamics of microglia-vascular interactions by in vivo two-photon imaging
.

In vivo imaging results showed that microglia wrap around blood vessels, and that microglia protrusions often make repeated contacts around the same sites around capillaries (Fig.
1a,b)
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So the researchers used the microglia marker P2RY12 to study the physical contact between microglia and NVU cells
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They found that microglial protrusions could penetrate GFAP-labeled astrocyte end-feet and directly contact smooth muscle cells and endothelial cells in large and microvessels (Fig.
1c)
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Fig.
1 Microglia interact with NVU through purinergic receptors (Image source: Császár E, et al.
, J Exp Med.
2022) Previous studies have found that the aggregation of microglia P2Y12R occurs in the release of ATP in neurons At this site, microglia can sense and regulate neuronal activity and mitochondrial function through ATP-P2RY12 [6]
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To investigate whether perivascularly released ATP has a similar function, the researchers found a 214% increase in the intensity of TOM20 in endothelial cells in contact with microglia by histological examination of TOM20, a mitochondrial marker (Fig.
1i-j)
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This demonstrates a direct link between microglia and endothelial cells
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Figure 2 Microglia are involved in neurovascular coupling in a P2Y12R-mediated manner (Source: Császár E, et al.
, J Exp Med.
2022) Previous studies have shown that CSF1R (colony stimulating factor 1 receptor) inhibitors PLX5622 specifically eliminates microglia in the brain without significantly affecting the number or morphology of endothelial cells, astrocytes or pericytes [7]
.

To investigate the relationship of microglia to CBF in response to physiological stimuli, we applied the whisker stimulation mouse model, which is widely used to study neurovascular coupling mechanisms (Fig.
2a)
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Deletion of microglia was found to lead to a marked decrease in CBF responses in the contralateral cortex (Fig.
2b,c)
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And a similar response was also observed in mice treated with the P2Y12R inhibitor PSB0739 (Fig.
2d,f)
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Figure 3 The electrical signal of neurons during whisker stimulation was independent of the presence or absence of microglia (Source: Császár E, et al.
, J Exp Med.
2022) To test whether the substantial changes in neuronal responses elicited by whisker stimulation could be explained The effect of microglia on functional hyperemia.
The researchers used calcium imaging and two-photon methods to measure the electrical activity of cortical neurons in the microglia depletion group, P2Y12R knockout (KO) group and control group.
record
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The results showed that the firing frequency of neurons in the microglia depletion group and the P2Y12R KO group was significantly increased (Fig.
3a, d), but there was no significant difference in the neuronal electrical signals evoked by whisker stimulation between the microglia depletion group and the control group (Fig.
3a, d).
3g)
.

This suggests that the effect of microglia on functional hyperemia upon whisker stimulation is not mediated through neurons
.

Figure 4 Strategies for chemogenetic regulation of microglia (Source: Császár E, et al.
, J Exp Med.
2022) In order to further explore the impact of microglia on CBF changes, the researchers used Cre-dependent hM3Dq DREADD Mice were mated with CX3CR1-CreERT2 mice to generate MicroDREADDDq mice, and microglia were manipulated by chemogenetics (Fig.
4a)
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It was found that the uptake of ATP by microglia was significantly reduced upon stimulation with the HM3Dq DREADD agonist C21 (Fig.
4d,e)
.

Figure 5 Chemogenetic activation of microglia affects neurovascular coupling (Source: Császár E, et al.
, J Exp Med.
2022) To study microglial calcium dynamics, the researchers also combined MicroDREADDDq mice with CGaMP5g-tdTomato mouse cross
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The results showed that perivascular microglial protrusions temporarily retracted upon chemogenetic activation of microglia (Fig.
5g)
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And the results of this chemogenetic regulation were similar to those elicited by whisker stimulation (Fig.
5i–k)
.

Figure 6 Microglia are involved in the regulation of vasodilation caused by hypercapnia (Source: Császár E, et al.
, J Exp Med.
2022) To further study the mechanism of microglia regulating CBF, the researchers applied A mouse model of hypercapnic vasodilation independent of neuronal stimulation
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In vivo two-photon imaging revealed that in the vasorelaxation response induced by inhalation of 10% CO2 for 2 minutes under normoxia, the morphology of microglia in arterioles and microvessels was significantly altered, and perivascular astrocyte end-footed There were also numerous contacts with microglial protrusions (Fig.
6a,b)
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And perivascular microglial calcium activity was significantly enhanced in the hypercapnia model (Fig.
6c)
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In vivo two-photon data also showed that hypercapnia-induced meningeal vasodilation was significantly reduced after depletion of intracerebral microglia by PLX5622 (Fig.
6d)
.

Figure 7 Microglia are involved in the regulation of pH in the brain (Source: Császár E, et al.
, J Exp Med.
2022) Previous studies have shown that hypercapnia drives the brain mainly by lowering pH in the brain Vasodilation
.

So the researchers measured pH in the brain
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It was found that the pH value of the microglia depletion group was significantly lower than that of the control group, while the pH drop caused by hypercapnia was not significantly different between the two groups (Fig.
7a–c)
.

This suggests that microglia are involved in regulating pH in the brain
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It was then found that hypercapnic stimulation caused a decrease in intracellular and extracellular pH, and resulted in a significant increase in the production of ATP and ADP by endothelial cells, a significant increase in the production of ATP and adenosine (Ado) by astrocytes, and a significant increase in microglial production.
There was a substantial increase in cytoplasmic Ado production (Fig.
8d)
.

However, under hypoxia, endothelial cells produced large amounts of ATP and AMP, and astrocytes and microglia produced large amounts of ADP (Fig.
8f)
.

This suggests that hypercapnia and hypoxia lead to the production of different purinergic products
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 Figure 8 Hypercapnia and hypoxia lead to the production of different purinergic products (Source: Császár E, et al.
, J Exp Med.
2022) Finally, the researchers also performed common carotid artery occlusion (CCAo) The role of microglia on CBF was investigated in a hypoperfusion model
.

They found that CCAo induced enhanced motility of perivascular microglia and significantly altered their protrusion morphology (Fig.
9a,b)
.

After depletion of intracerebral microglia, CBF recovery was impaired in mice after CCAo (Fig.
9d), suggesting that microglia are involved in the normal response of CBF during reperfusion
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These phenomena were also consistent when P2Y12R was inhibited genetically and pharmacologically (Fig.
10), suggesting that microglia and microglial P2Y12R are critical for normalization of CBF responses during adaptation to reduced cortical perfusion following CCAo important
.

Figure 9 Microglia participate in CBF recovery during reperfusion (Image source: Császár E, et al.
, J Exp Med.
2022) Figure 10 Microglia P2Y12R participate in CBF recovery during reperfusion (Image source: Császár E , et al.
, J Exp Med.
2022) Article Conclusions and Discussions, Inspirations and Prospects In conclusion, the researchers found that microglia are important regulators of cerebral blood flow (CBF) both under physiological conditions and under hypoperfusion
.

Microglia establish direct, dynamic purinergic contacts with cells in the neurovascular unit, and loss of microglia or blockade of the microglial P2Y12 receptor (P2Y12R) significantly impairs neurovascular coupling in mice , and impairs CBF recovery during reperfusion during P2Y12R-mediated common carotid artery occlusion
.

Their data thus reveal a role for microglia in CBF regulation, which has broad implications in common neurological diseases
.

 The involvement of microglia in the regulation of CBF has been confirmed in mouse models, suggesting that clinical regulation of microglia may also be a potential target for the treatment of cerebrovascular-related diseases, but the mechanism in humans still needs further study
.

Link to the original text: https://doi.
org/10.
1084/jem.
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Edition ︱Sizhen Wang End of this paper