Neuroprotection afforded by 17β-estradiol in a rat model of transient middle cerebral artery occlusion implicates modulation of fatty acid amide hydrolase (FAAH) activity

D. Amantea1, C. Mazzei1, M. Bari2, P. Spagnuolo1, N. Pasquariello2, M.T. Corasaniti3, G. Bagetta1, M. Maccarrone2. 1Dept of Pharmacobiology, University of Calabria, Rende (CS), Italy; 2Dept of Biomed. Sci., University of Teramo & Center for Exp. Neurobiol., “Mondino-Tor Vergata”, University of Tor Vergata, Rome, Italy; 3Dept of Pharmacobiological Sciences, University “Magna Graecia” of Catanzaro, Italy

Experimental evidence indicates that both physiological and pharmacological levels of 17β -estradiol (E2) afford neuroprotection against brain damage caused by middle cerebral artery occlusion (MCAo) in rats, though the underlying mechanisms remain to be discovered (Maggi et al., 2004). E2 exerts its activity through the interaction with intracellular estrogen receptors (ERα and ERβ) , resulting in the modulation of transcription of target genes. Interestingly, recent studies have demonstrated that E2 modulates the activity and expression of fatty acid amide hydrolase (FAAH) (Maccarrone et al., 2000), an enzyme involved in the metabolic degradation of endocannabinoids, such as anandamide (AEA) and 2-arachidonoylglycerol (Bisogno et al., 2002). Here we investigate whether neuroprotection afforded by E2 involves the endocannabinoid system, via the modulation of the metabolic enzyme FAAH. MCAo was induced in male Wistar rats (280- 300 g) by intraluminal silicon-coated nylon filament ( 0.28 mm diameter), which was withdrawn 2 h after occlusion to allow reperfusion. Cerebral infarct volume was evaluated 24 h after MCAo by staining 2 mm-thick consecutive coronal brain slices with 2,3,5-triphenyltetrazolium chloride and measuring the infarct area (unstained) using computer assisted image analysis. E2 (0.2 mg kg-1) and SR141716 (3 mg kg-1) were administered i.p. 1 h and 15 min, respectively, before MCAo. FAAH activity was determined on tissue homogenates from cortices and striata, dissected 2 h after MCAo, by a radiochromatographic assay, using [3H]AEA as substrate (Maccarrone et al., 2003). 2h MCAo resulted in a significant decrease in the activity of AEA-hydrolysing enzyme FAAH in the striatum (sham: 861.5 ± 36.9; MCAo: 510.8 ± 21.7 pmol min-1 mg-1 protein, n=4; P<0.01, ANOVA), but not in the cortex. This effect was reverted by pretreating the rats with a dose of E2 (0.2 mg kg-1; FAAH activity: 783.8 ± 31.9 pmol min-1 mg-1 protein, n=4; P<0.05 vs MCAo, ANOVA) that produced a significant reduction of infarct volume 24h after MCAo (vehicle: 595 ± 64 vs E 2: 315 ± 45 mm3; n=5, P<0.01, unpaired t-test). Under these experimental conditions, SR141716 afforded neuroprotection as revealed by the significant reduction of the infarct volume (vehicle: 509 ± 29, n=5 vs SR: 341 ± 63 mm3, n=4; P<0.05, unpaired t-test). The reduced activity of FAAH observed 2h after MCAo in the rat striatum suggests that increased levels of endocannabinoids might contribute to ischemic brain damage. This hypothesis is supported by the evidence that SR141716, a selective CB1 receptor antagonist, significantly reduces brain infarct volume produced by tMCAo. The acute treatment with a neuroprotective dose of E2 significantly reverted the inhibition of FAAH activity produced by 2h MCAo. Thus, modulation of FAAH may represent a pivotal mechanism involved in estrogen-mediated neuroprotection.

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