Hypoxia induced amoeboid microglial cell activation in postnatal rat brain is mediated by ATP receptor P2X4

Abstract

BackgroundActivation 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.ResultsWe 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.ConclusionsIt 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.

Neurotransmitter signaling in the pathophysiology of microglia

“…Thus, the activation of microglia after hypoxia in the neonatal rat brain, a model of periventricular white matter damage, is mediated by P2X4R signaling (Li et al, 2011). P2X4 is also upregulated in microglial cells in the CA1 and transition zone to CA2 regions of the hippocampus after ischemia (Cavaliere et al, 2003), and its blockade confers neuronal protection (Cavaliere et al, 2005).…”

White matter damage after traumatic brain injury: A role for damage associated molecular patterns

“…Amongst the purinergic receptors, P2X1, P2X4, and P2X7 are expressed in amoeboid microglia during development and beginning at postnatal day 30, P2X7 positive microglia are observed throughout the forebrain [188, 189]. Although the functional importance of P2X receptors remain poorly defined after TBI, P2X4 was acutely localized in microglia/macrophages after experimental TBI in rats [190] whereas inhibition of P2X4 reduced hypoxia-induced pro-inflammatory signaling in isolated microglial cells [189].…”

“…Amongst the purinergic receptors, P2X1, P2X4, and P2X7 are expressed in amoeboid microglia during development and beginning at postnatal day 30, P2X7 positive microglia are observed throughout the forebrain [188, 189]. Although the functional importance of P2X receptors remain poorly defined after TBI, P2X4 was acutely localized in microglia/macrophages after experimental TBI in rats [190] whereas inhibition of P2X4 reduced hypoxia-induced pro-inflammatory signaling in isolated microglial cells [189]. Although a mechanistic link between P2X7 and white matter injury has not been established to date, genetic or pharmacological P2X7 inhibition reduced the expression and release of the pro-inflammatory cytokine, interleukin-1β (IL-1β) and improved outcomes after TBI [63–65].…”

Molecular dialogs between the ischemic brain and the peripheral immune system: Dualistic roles in injury and repair

“…Activation of the microglial P2X4 receptor occurs in models of CNS diseases that involve inflammatory responses, such as in spinal cord injury, cerebral ischemia, preterm hypoxia ischemia, and experimental autoimmune encephalomyelitis (EAE) (Wixey et al, 2009;Schwab et al, 2005;Tsuda et al, 2003;Li et al, 2011;Guo and Schluesener, 2005;Cavaliere et al, 2003;Ulmann et al, 2008). In a rat model of preterm hypoxia-ischemia, the expression of P2X4 receptors was significantly increased and was associated with an increase in ionized calcium binding adapter molecule 1 (Iba1) protein, which is indicative of microglial activation (Wixey et al, 2009).…”