P2X7 receptor expression after ischemia in the cerebral cortex of rats.
Large amounts of adenosine 5'-triphosphate (ATP) released from cellular sources under pathological conditions such as ischemia may activate purinoceptors of the P2X and P2Y types. In the present study, the expression of the P2X7 receptor-subtype in the brain cortex of spontaneously hypertensive rats was investigated using a permanent focal cerebral ischemia model. Immunocytochemistry with antibodies raised against the intracellular C-terminus of the P2X7 receptor showed a time-dependent upregulation of labeled cells in the peri-infarct region after right middle cerebral artery occlusion (MCAO) in comparison to controls. Double immunofluorescence visualized with confooal laser scanning microscopy indicated the localization of the P2X7 receptor after ischemia on microglial cells (after 1 and 4 days), on tubulin betaIII-labeled neurons (after 4 and 7 days), and on glial fibrillary acidic protein (GFAP)-positive astrocytes (after 4 days). In the following experiments, changes occurring 4 days after MCAO were investigated in detail. Western blot analysis of the cortical tissue around the area of necrosis indicated an increase in the P2X7 receptor protein. Immunoelectron microscopy revealed the receptor localization on synapses (presynaptically), on dendrites, as well as on the nuclear membrane of neurons (postsynaptically) and glial cells. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling in combination with P2X7 receptor immunocytochemistry indicated a co-expression on the apoptotic cells. Active caspase 3 was especially observed on GFAP-positive astrocytes. In conclusion, the present data demonstrate a postischemic, time-dependent upregulation of the P2X7 receptor-subtype on neurons and glial cells and suggest a role for this receptor in the pathophysiology of cerebral ischemia in vivo.
Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany. frah@medizin.uni-leipzig.de
Heike
H
Franke
Albrecht
A
Günther
Jens
J
Grosche
Renate
R
Schmidt
Steffen
S
Rossner
Robert
R
Reinhardt
Heidi
H
Faber-Zuschratter
Dietmar
D
Schneider
Peter
P
Illes
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000276:immunology; D017209:Apoptosis / Q000502:physiology; D001253:Astrocytes / Q000378:metabolism / Q000648:ultrastructure; D002545:Brain Ischemia / Q000378:metabolism* / Q000503:physiopathology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism* / Q000503:physiopathology; D004195:Disease Models, Animal; D005455:Fluorescent Antibody Technique; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D020244:Infarction, Middle Cerebral Artery / Q000378:metabolism / Q000503:physiopathology; D008297:Male; D017628:Microglia / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D009685:Nuclear Envelope / Q000378:metabolism / Q000648:ultrastructure; D017729:Presynaptic Terminals / Q000378:metabolism / Q000648:ultrastructure; D017434:Protein Structure, Tertiary; D051381:Rats; D011918:Rats, Inbred SHR; D011930:Reaction Time / Q000502:physiology; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D015854:Up-Regulation / Q000502:physiology
D000906:Antibodies; D005904:Glial Fibrillary Acidic Protein; C546470:P2rx7 protein, rat; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D000255:Adenosine Triphosphate; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases