Neuroprotection by the endogenous cannabinoid anandamide and arvanil against in vivo excitotoxicity in the rat: role of vanilloid receptors and lipoxygenases

Abstract

Type 1 vanilloid receptors (VR1) have been identified recently in the brain, in which they serve as yet primarily undetermined purposes. The endocannabinoid anandamide (AEA) and some of its oxidative metabolites are ligands for VR1, and AEA has been shown to afford protection against ouabain-induced in vivo excitotoxicity, in a manner that is only in part dependent on the type 1 cannabinoid (CB1) receptor. In the present study, we assessed whether VR1 is involved in neuroprotection by AEA and by arvanil, a hydrolysis-stable AEA analog that is a ligand for both VR1 and CB1. Furthermore, we assessed the putative involvement of lipoxygenase metabolites of AEA in conveying neuroprotection. Using HPLC and gas chromatography/mass spectroscopy, we demonstrated that rat brain and blood cells converted AEA into 12-hydroxy-N-arachidoylethanolamine (12-HAEA) and 15-hydroxy-N-arachidonoylethanolamine (15-HAEA) and that this conversion was blocked by addition of the lipoxygenase inhibitor nordihydroguaiaretic acid. Using magnetic resonance imaging we show the following: (1) pretreatment with the reduced 12-lipoxygenase metabolite of AEA, 12-HAEA, attenuated cytotoxic edema formation in a CB1 receptor-independent manner in the acute phase after intracranial injection of the Na+/K+-ATPase inhibitor ouabain; (2) the reduced 15-lipoxygenase metabolite, 15-HAEA, enhanced the neuroprotective effect of AEA in the acute phase; (3) modulation of VR1, as tested using arvanil, the VR1 agonist capsaicin, and the antagonist capsazepine, leads to neuroprotective effects in this model, and arvanil is a potent neuroprotectant, acting at both CB1 and VR1; and (4) the in vivo neuroprotective effects of AEA are mediated by CB1 but not by lipoxygenase metabolites or VR1.

Figure 1.

Reversed-phase HPLC profiles (λ = 236 nm) of lipid extracts of anandamide incubations with rat brain homogenates using a C18-column with methanol/water (80:20 v/v) as eluent. Top trace, Anandamide incubation with rat brain and NDGA, a nonselective lipoxygenase inhibitor. Middle trace, Anandamide incubation with nonperfused rat brain. Bottom trace, Anandamide incubation with PBS perfused rat brain. a.u., Arbitrary units.

Figure 2.

A, Typical parametric ADC maps of rat brain, calculated from diffusion-weighted MRI data acquired 15 min after ouabain injection. B, Parametric T₂ maps of the corresponding slices of the same animals, calculated from T₂-weighted MRI data acquired 1 week later. The treatments are as follows: I, vehicle; II, AEA (1 mg/kg) plus 15-HAEA; III, 12-HAEA; IV, 12-HAEA plus SR141716A; V, capsazepine; VI, AEA (10 mg/kg) plus capsazepine; VII, NDGA; and VIII, AEA (10 mg/kg) plus NDGA.

Figure 3.

Lesion volumes as determined from ADC maps on day 0 and T₂ maps on day 7. The effects of several combinations of AEA, AEA metabolites, CB₁ and VR₁ antagonists, and a lipoxygenase inhibitor on lesion volume are shown. For comparison, the effect of AEA alone is also shown. This effect was determined in a previous study (van der Stelt et al., 2001b). Error bars represent means ± SE. *p < 0.05 versus control; §p < 0.05 for AEA plus NDGA versus AEA alone. See legend to Figure 2 for all of the doses. CAPSA, Capsazepine (10 mg/kg).

Figure 4.

Typical parametric T₂ maps calculated from T₂-weighted MRI data acquired 7 d after injection of ouabain. The treatments are as follows: A, vehicle; B, arvanil (0.1 mg/kg); C, arvanil (1 mg/kg); D, arvanil (1 mg/kg) plus SR141716A (1 mg/kg); E, arvanil (1 mg/kg) plus SR141716A (3 mg/kg); F, arvanil (1 mg/kg) plus capsazepine (5 mg/kg); G, arvanil (1 mg/kg) plus capsazepine (10 mg/kg); and H, capsaicin (1 mg/kg).

Figure 5.

Lesion volumes as determined from T₂ maps acquired 7 d after ouabain injection. Error bars represent means ± SE. *p < 0.05 versus control. See legend to Figure 4 for all of the doses. ARV, Arvanil; CAPSA, capsazepine; SR1, SR141716A.