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Updated: May 27, 2026
Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices
Published on: January 31, 2019
Hypoxia induced amoeboid microglial cell activation in postnatal rat brain is mediated by ATP receptor P2X4
1Department of Pathophysiology, Kunming Medical College, Kunming, People's Republic of China. leefan623@sina.com
Background:
Activation of amoeboid microglial cells (AMC) and its related inflammatory response have been linked to the periventricular white matter damage after hypoxia in neonatal brain. Hypoxia increases free ATP in the brain and then induces various effects through ATP receptors. The present study explored the possible mechanism in ATP induced AMC activation in hypoxia.
Results:
We first examined the immunoexpression of P2X4, P2X7 and P2Y12 in the corpus callosum (CC) and subependyma associated with the lateral ventricles where both areas are rich in AMC. Among the three purinergic receptors, P2X4 was most intensely expressed. By double immunofluorescence, P2X4 was specifically localized in AMC (from P0 to P7) but the immunofluorescence in AMC was progressively diminished with advancing age (P14). It was further shown that P2X4 expression was noticeably enhanced in P0 day rats subjected to hypoxia and killed at 4, 24, 72 h and 7 d versus their matching controls by double labeling and western blotting analysis. P2X4 expression was most intense at 7 d whence the inflammatory response was drastic after hypoxia. We then studied the association of P2X4 with cytokine release in AMC after hypoxic exposure. In primary microglial cells exposed to hypoxia, IL-1β and TNF-α protein levels were up-regulated. Blockade of P2X4 receptor with 2', 3'-0-(2, 4, 6-Trinitrophenyl) adenosine 5'-triphosphate, a selective P2X1-7 blocker resulted in partial suppression of IL-1β (24% vs hypoxic group) and TNF-α expression (40% vs hypoxic group). However, pyridoxal phosphate-6-azo (benzene-2, 4-disulfonic acid) tetrasodium salt hydrate, a selective P2X1-3, 5-7 blocker did not exert any significant effect on the cytokine expression.
Conclusions:
It is concluded that P2X4 which is constitutively expressed by AMC in postnatal rats was enhanced in hypoxia. Hypoxia induced increase in IL-1β and TNF-α expression was reversed by 2', 3'-0-(2, 4, 6-Trinitrophenyl) adenosine 5'-triphosphate suggesting that P2X4 mediates ATP induced AMC activation and its production of proinflammatory cytokines.
Insights
Hypoxia in neonatal brains activates amoeboid microglial cells (AMC) via the P2X4 receptor, increasing inflammatory cytokines IL-1β and TNF-α. Blocking P2X4 partially reverses this inflammatory response, highlighting its role in neonatal hypoxic brain injury.
Area of Science:
- Neuroscience
- Immunology
- Neonatal Research
Background:
- Hypoxia-induced periventricular white matter damage in neonatal brains is linked to activated amoeboid microglial cells (AMC) and inflammation.
- Elevated extracellular ATP following hypoxia triggers effects through ATP receptors, prompting investigation into its role in AMC activation.
Purpose of the Study:
- To explore the mechanism of ATP-induced AMC activation in the context of neonatal hypoxia.
- To investigate the role of purinergic receptors, specifically P2X4, in mediating inflammatory responses in AMC during hypoxia.
Main Methods:
- Examined immunoexpression of P2X4, P2X7, and P2Y12 receptors in neonatal rat brain regions rich in AMC.
- Utilized double immunofluorescence and Western blotting to assess P2X4 expression in AMC following hypoxic exposure.
- Investigated the effect of P2X4 receptor blockade on IL-1β and TNF-α levels in primary microglial cells subjected to hypoxia.
Main Results:
- P2X4 was highly expressed in AMC, particularly in the early postnatal period, and its expression was significantly enhanced in rats exposed to hypoxia.
- Hypoxia led to increased protein levels of IL-1β and TNF-α in primary microglial cells.
- Selective blockade of the P2X4 receptor partially suppressed the hypoxia-induced release of IL-1β and TNF-α.
Conclusions:
- P2X4 receptors are constitutively expressed by AMC in postnatal rats and are upregulated following hypoxic injury.
- P2X4 signaling mediates ATP-induced AMC activation and the subsequent production of pro-inflammatory cytokines, IL-1β and TNF-α.
- Targeting P2X4 may offer a therapeutic strategy for mitigating hypoxic brain injury in neonates.
