Delayed P2X4R expression after hypoxia–ischemia is associated with microglia in the immature rat brain
Introduction
Perinatal brain injury occurs in approximately 4 per 1000 births (Volpe, 2001) with exposure to a hypoxic–ischemic (HI) insult and premature delivery (< 37 weeks gestation) as major contributing factors. Preterm children who have experienced HI often manifest a range of motor and cognitive deficits (Anderson and Doyle, 2003, Hack et al., 2004, O'Brien et al., 2004, Peterson et al., 2003) however a therapeutic intervention to curb perinatal brain injury is still elusive.
A defining feature of preterm HI neuropathology is white matter injury and evidence suggests that neuroinflammation has a major role in producing this damage. Activated microglia, especially in white matter regions, are hallmarks of neuroinflammatory processes after a HI event in the neonate (Biran et al., 2006, Farkas et al., 2004, Ivacko et al., 1996, McRae et al., 1995). Substantial numbers of activated microglia appear in the corpus callosum in rodent preterm HI models (Cai et al., 2006, Carty et al., 2008, Fan et al., 2006, Fan et al., 2005) and blocking microglia, using minocycline, attenuates the HI-induced demise of oligodendrocyte progenitor cells and consequent myelin loss (Cai et al., 2006, Carty et al., 2008, Fan et al., 2006, Fan et al., 2005). However, despite a recognised role in white matter injury following preterm HI, how microglia are activated and signal to release cytotoxic substances in the injured brain remains to be elucidated.
One possible mechanism is that microglia may contribute to HI-induced brain injury via adenosine triphosphate (ATP) signalling mediated by P2 purinergic receptors. ATP is released into the extracellular space by damaged cells following hypoxia and ischemia (Braun et al., 1998, Juranyi et al., 1999). ATP activates specific ligand-gated ionotropic P2X receptors that are expressed on microglia (Cavaliere et al., 2005, Davalos et al., 2005) and in turn can mediate signalling cascades that lead to neurodegeneration after ischemia (Le Feuvre et al., 2003). In the developing early post-natal rat brain, P2X4R predominate on microglia (Xiang and Burnstock, 2005). Evidence suggests that P2X4R are up-regulated in grey matter regions 3 days after P9 HI (equivalent to human term birth developmental stage) in mice (Hedtjarn et al., 2004). Nevertheless it is not known if P2X4R expression occurs in the white matter after HI in the immature preterm brain or whether P2X4R are associated with neuroinflammatory mediators after preterm HI.
In the present study, using a preterm HI model in the post-natal day 3 (P3) rat, we tested the hypothesis that P2X4R expression is altered in the corpus callosum after HI. We found that P2X4R expression increased significantly one week after P3 HI. We determined the cellular localisation of P2X4R and discovered that increased P2X4R expression in the corpus callosum after P3 HI was predominantly co-localised on microglia. We consequently tested whether minocycline, a potent microglial cell inhibitor shown previously to substantially reduce numbers of activated microglia and improve white matter injury after HI (Cai et al., 2006, Carty et al., 2008, Fan et al., 2006), could block HI-induced expression of P2X4R. Finally, we examined the temporal relationship of P2X4R and microglia for one week following P3 HI.
Section snippets
Animals
Sprague–Dawley dams and their pups (10–12 per litter) were housed in animal facilities maintained at 22 °C and on a 12 hour light/dark cycle. All procedures were performed in accordance with ethical approvals stipulated by the University of Queensland Animal Ethics Committee. Efforts were made to ensure the number of rats and their suffering was minimal.
Hypoxic–ischemic insult
Post-natal day 3 (P3) rat pups of mixed sexes were randomly assigned to a control or HI group. Each group had approximately equal distribution
Effect of P3 HI on cerebral hemisphere size
In P10 animals subjected to P3 HI, there was a 19.0% reduction in cerebral hemisphere size ipsilateral to the carotid ligation compared to the non-ligated side. This brain injury index confirms that the P3 HI insult produced reproducible outcomes comparable to previous studies (Buller et al., 2008, Carty et al., 2008).
Expression of P2X4R in the brain after P3 HI
Using Western blotting we found that P2X4R protein expression was significantly increased one week after P3 HI in the cerebral hemisphere ipsilateral to the carotid ligation (Fig.
Discussion
In the present study P2X4R immunolabelling and P2X4R protein expression were increased significantly in the immature rat brain after P3 HI. In particular, a significant increase in P2X4R immunolabelling was observed in white matter regions such as the corpus callosum where co-localisation of P2X4R occurred predominantly on Iba-1-positive microglia. Administration of minocycline, a potent inhibitor of activated microglia, attenuated the HI-induced increase in P2X4R protein expression in the
Conclusions
This is the first study to demonstrate that P2X4R expression is increased in the immature brain following P3 HI injury. We found that P2X4R are expressed primarily on microglia after P3 HI and a dense concentration of P2X4R-positive microglia occurs in the corpus callosum. White matter damage is a defining feature of HI neuropathology in the preterm neonate and by stemming the increase in the number of activated microglia, indicative of neuroinflammation, the progression of injury might be
Acknowledgements
This work was supported by funding from the Royal Brisbane Women's Hospital Research Foundation and the Lions Medical Research Foundation.
References (55)
- et al.
Calcium and glial cell death
Cell Calcium
(2005) - et al.
Glial activation in white matter following ischemia in the neonatal P7 rat brain
Exp. Neurol.
(2006) - et al.
Minocycline alleviates hypoxic–ischemic injury to developing oligodendrocytes in the neonatal rat brain
Neuroscience
(2006) - et al.
Post-hypoxia–ischemia minocycline treatment attenuates neuroinflammation and white matter injury in the neonatal rat; a comparison of two different dose regimens
Int. J. Dev. Neurosci.
(2008) - et al.
Up-regulation of P2X2, P2X4 receptor and ischemic cell death: prevention by P2 antagonists
Neuroscience
(2003) - et al.
Microglia response and P2 receptor participation in oxygen/glucose deprivation-induced cortical damage
Neuroscience
(2005) - et al.
Translating developmental time across mammalian species
Neuroscience
(2001) - et al.
Lesional accumulation of P2X(4) receptor(+) macrophages in rat CNS during experimental autoimmune encephalomyelitis
Neuroscience
(2005) The function of microglia through purinergic receptors: neuropathic pain and cytokine release
Pharmacol. Ther.
(2006)- et al.
Microglia-specific localisation of a novel calcium binding protein, Iba1
Brain Res. Mol. Brain Res.
(1998)
Involvement of P2 purinoceptors and the nitric oxide pathway in [3H]purine outflow evoked by short-term hypoxia and hypoglycemia in rat hippocampal slices
Brain Res.
The co-treatments of vigabatrin and P2X receptor antagonists protect ischemic neuronal cell death in the gerbil hippocampus
Brain Res.
Microglia: a sensor for pathological events in the CNS
Trends Neurosci.
Microglia activation after neonatal hypoxic-ischemia
Dev. Brain Res.
Spinal cord injury induces early and persistent lesional P2X4 receptor expression
J. Neuroimmunol.
Development of a postnatal 3-day-old rat model of mild hypoxic–ischemic brain injury
Brain Res.
Microglia and macrophages in the developing CNS
Neurotoxicology
Lesional accumulation of P2X4 receptor+ monocytes following experimental traumatic brain injury
Exp. Neurol.
Stretch-induced injury alters mitochondrial membrane potential and cellular ATP in cultured astrocytes and neurons
J. Neurochem.
Neurobehavioural outcomes of school-age children born extremely low birth weight or very preterm in the 1990s
J. Am. Med. Assoc.
Interleukin-1 and the interleukin-1 type 1 receptor are essential for the progressive neurodegeneration that ensues subsequent to a mild hypoxic/ischemic injury
J. Cereb. Blood Flow Metab.
Development of microglia in the cerebral white matter of the human fetus and infant
J. Comp. Neurol.
Chemokine and inflammatory cell response to hypoxia ischemia in immature rats
Pediatr. Res.
Upregulation of the enzyme chain hydrolyzing extracellular ATP after transient forebrain ischemia in the rat
J. Neurosci.
Selective losses of brainstem catecholamine neurons after hypoxia–ischemia in the immature rat pup
Pediatr. Res.
Purine and pyrimidine receptors
Cell. Mol. Life Sci.
Strong P2X4 purinergic receptor-like immunoreactivity is selectively associated with degenerating neurons in transgenic rodent models of amyotrophic lateral sclerosis
J. Comp. Neurol.
Cited by (49)
Contribution of P2X purinergic receptor in cerebral ischemia injury
2022, Brain Research BulletinCitation Excerpt :The use of minocycline, an effective microglia inhibitor, can reduce the increase of P2X4 receptors expression induced by hypoxia-ischemia. It is speculated that microglia with positive P2X4 receptors expression may represent the group that induces secondary injury (Wixey et al., 2009). In ischemia/reperfusion rats, microglia activation promotes oxidative damage, inflammation, and neuronal apoptosis, which leads to brain damage and memory dysfunction (Zhou et al., 2012).
Update on mechanisms of the pathophysiology of neonatal encephalopathy
2021, Seminars in Fetal and Neonatal MedicineDeletion of the P2X4 receptor is neuroprotective acutely, but induces a depressive phenotype during recovery from ischemic stroke
2017, Brain, Behavior, and ImmunityThe biphasic function of microglia in ischemic stroke
2017, Progress in NeurobiologyReview: Neuroinflammation in intrauterine growth restriction
2017, PlacentaCitation Excerpt :Recent studies in animal models of growth restriction have reported increased numbers of activated microglia and astrogliosis, indicative of inflammatory responses in the IUGR brain (Table 1) [22–27,31,39,40]. Neuroinflammation encompasses a number of processes including increased numbers of activated microglia, elevated production of proinflammatory cytokines (particularly interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α)) [41–43], decreased production of anti-inflammatory cytokines [44], release of chemokines [44–46], increased production of nitric oxide (NO) [47,48], infiltration of leukocytes [45] and astrogliosis [48–52]. However many previous IUGR studies have examined only changes at one postnatal time point or changes of only select inflammatory cytokines.
Selective modulation of microglia polarization to M2 phenotype for stroke treatment
2015, International Immunopharmacology