Microglial P2Y12 deficiency/inhibition protects against brain ischemia.
OBJECTIVE
Microglia are among the first immune cells to respond to ischemic insults. Triggering of this inflammatory response may involve the microglial purinergic GPCR, P2Y12, activation via extracellular release of nucleotides from injured cells. It is also the inhibitory target of the widely used antiplatelet drug, clopidogrel. Thus, inhibiting this GPCR in microglia should inhibit microglial mediated neurotoxicity following ischemic brain injury.


METHODS
Experimental cerebral ischemia was induced, in vitro with oxygen-glucose deprivation (OGD), or in vivo via bilateral common carotid artery occlusion (BCCAO). Genetic knock-down in vitro via siRNA, or in vivo P2Y12 transgenic mice (P2Y12-/- or P2Y12+/-), or in vivo treatment with clopidogrel, were used to manipulate the receptor. Neuron death, microglial activation, and microglial migration were assessed.


RESULTS
The addition of microglia to neuron-astrocyte cultures increases neurotoxicity following OGD, which is mitigated by microglial P2Y12 deficiency (P<0.05). Wildtype microglia form clusters around these neurons following injury, which is also prevented in P2Y12 deficient microglia (P<0.01). P2Y12 knock-out microglia migrated less than WT controls in response to OGD-conditioned neuronal supernatant. P2Y12 (+/-) or clopidogrel treated mice subjected to global cerebral ischemia suffered less neuronal injury (P<0.01, P<0.001) compared to wild-type littermates or placebo treated controls. There were also fewer microglia surrounding areas of injury, and less activation of the pro-inflammatory transcription factor, nuclear factor Kappa B (NFkB).


CONCLUSIONS
P2Y12 participates in ischemia related inflammation by mediating microglial migration and potentiation of neurotoxicity. These data also suggest an additional anti-inflammatory, neuroprotective benefit of clopidogrel.
Department of Neurology, University of California, San Francisco and the San Francisco Veteran's Affairs Medical Center, San Francisco, California, United States of America.
Corey M
CM
Webster
Masaaki
M
Hokari
April
A
McManus
Xian Nan
XN
Tang
Hualong
H
Ma
Rachid
R
Kacimi
Midori A
MA
Yenari
D000818:Animals; D017209:Apoptosis; D001253:Astrocytes / Q000502:physiology; D002545:Brain Ischemia / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D056547:CA1 Region, Hippocampal / Q000276:immunology / Q000473:pathology; D015687:Cell Hypoxia; D002465:Cell Movement; D002470:Cell Survival; D002478:Cells, Cultured; D000077144:Clopidogrel; D018920:Coculture Techniques; D055785:Gene Knockdown Techniques; D005947:Glucose / Q000172:deficiency; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017628:Microglia / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism; D009474:Neurons / Q000502:physiology; D058921:Purinergic P2Y Receptor Antagonists / Q000494:pharmacology; D058925:Receptors, Purinergic P2Y12 / Q000172:deficiency* / Q000235:genetics; D013988:Ticlopidine / Q000031:analogs & derivatives / Q000494:pharmacology
D016328:NF-kappa B; C548577:P2ry12 protein, mouse; D058921:Purinergic P2Y Receptor Antagonists; D058925:Receptors, Purinergic P2Y12; D000077144:Clopidogrel; D005947:Glucose; D013988:Ticlopidine