Characterization of the neuroprotective effect of the cannabinoid agonist WIN-55212 in an in vitro model of hypoxic-ischemic brain damage in newborn rats.
Brain slices from 7-d-old Wistar rats were exposed to oxygen-glucose deprivation (OGD) for 30 min. OGD slices were incubated with vehicle or with the CB1/CB2 cannabinoid agonist WIN55212 (50 microM), the CB1 agonist arachidonyl-2-chloroethylamide (ACEA) (50 microM), or the CB2 agonist JW133 (50 microM), alone or combined with the CB1 and CB2 receptor antagonist SR 141716 (50 microM) or SR 144528 (50 microM), respectively. Neuronal damage was assessed by histologic analysis and spectrophotometric determination of lactate dehydrogenase (LDH) efflux into the incubation medium. Additionally, medium glutamate levels were determined by high-performance liquid chromatography (HPLC) and those of tumor necrosis factor alpha (TNF-alpha) by enzyme-linked immunosorbent assay. Finally, inducible nitric oxide synthase (iNOS) and CB1/CB2 receptor expression were determined in slices homogenate by Western blot. Both CB1 and CB2 receptors were expressed in slices. OGD increased CB1 expression, cellular damage, LDH efflux, glutamate and TNF-alpha release, and inducible nitric oxide synthase (iNOS) expression; WIN55212 inhibited all these actions. SR141716 and SR144528 inhibited the effect of R(+)-WIN-55212-2 (WIN), as well as the reduction of LDH efflux by ACEA and JW133, respectively. In conclusion, WIN55212 afforded robust neuroprotection in the forebrain slices exposed to OGD, by acting on glutamatergic excitotoxicity, TNF-alpha release, and iNOS expression; this neuroprotective effect seemed to be mediated by CB1 and CB2 receptors.
Fernández-López
David
D
Departamento de Farmacología, Facultad de Medicina, Universidad Complutense, Madrid, Spain.
Martínez-Orgado
José
J
Nuñez
Estefanía
E
Romero
Julián
J
Lorenzo
Pedro
P
Moro
Maria Angeles
MA
Lizasoain
Ignacio
I
D000693:Anaerobiosis; D000818:Animals; D000831:Animals, Newborn; D001095:Arachidonic Acids / Q000494:pharmacology; D048588:Benzoxazines; D001921:Brain / Q000473:pathology; D001923:Brain Chemistry; D002545:Brain Ischemia / Q000517:prevention & control*; D063386:Cannabinoid Receptor Agonists; D002186:Cannabinoids / Q000627:therapeutic use*; D004195:Disease Models, Animal; D002534:Hypoxia, Brain / Q000473:pathology / Q000517:prevention & control*; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D009025:Morpholines / Q000627:therapeutic use*; D009281:Naphthalenes / Q000627:therapeutic use*; D018696:Neuroprotective Agents / Q000627:therapeutic use*; D052247:Nitric Oxide Synthase Type II / Q000032:analysis; D010880:Piperidines / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists / Q000032:analysis / Q000037:antagonists & inhibitors; D043885:Receptor, Cannabinoid, CB2 / Q000819:agonists / Q000032:analysis / Q000037:antagonists & inhibitors; D000077285:Rimonabant; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis
D001095:Arachidonic Acids; D048588:Benzoxazines; D063386:Cannabinoid Receptor Agonists; D002186:Cannabinoids; D009025:Morpholines; D009281:Naphthalenes; D018696:Neuroprotective Agents; D010880:Piperidines; D011720:Pyrazoles; D043884:Receptor, Cannabinoid, CB1; D043885:Receptor, Cannabinoid, CB2; D014409:Tumor Necrosis Factor-alpha; C119325:arachidonyl-2-chloroethylamide; C070417:(3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone; D007770:L-Lactate Dehydrogenase; D052247:Nitric Oxide Synthase Type II; D000077285:Rimonabant