Delta9-tetrahydrocannabinol (THC) and AM 404 protect against cerebral ischaemia in gerbils through a mechanism involving cannabinoid and opioid receptors

Abstract

Background and purpose: It has been suggested that the endocannabinoid system elicits neuroprotection against excitotoxic brain damage. In the present study the therapeutic potential of AM 404 on ischaemia-induced neuronal injury was investigated in vivo and compared with that of the classical cannabinoid receptor type 1 (CB1) agonist, delta 9-tetraydrocannabinol (THC), using a model of transient global cerebral ischaemia in the gerbil.

Experimental approach: The effects of AM 404 (0.015-2 mg kg(-1)) and THC (0.05-2 mg kg(-1)), given 5 min after ischaemia, were measured from 1 h to 7 days in terms of electroencephalographic (EEG) total spectral power, spontaneous motor activity, memory function, rectal temperature and hippocampal CA1 neuronal count.

Key results: Over the dose range tested, AM 404 (2 mg kg(-1)) and THC (1 mg kg(-1)) completely reversed the ischaemia-induced behavioural, EEG and histological damage. Only THC (1 and 2 mg kg(-1)) induced a decrease of body temperature. Pretreatment with the selective CB1 receptor antagonist, AM 251 (1 mg kg(-1)) and the opioid antagonist, naloxone (2 mg kg(-1)) reversed the protective effect induced by both AM 404 and THC while the TRPV1 vanilloid antagonist, capsazepine (0.01 mg kg(-1)), was ineffective.

Conclusions and implications: Our findings demonstrate that AM 404 and THC reduce neuronal damage caused by bilateral carotid occlusion in gerbils and that this protection is mediated through an interaction with CB1 and opioid receptors. Endocannabinoids might form the basis for the development of new neuroprotective drugs useful for the treatment of stroke and other neurodegenerative pathologies.

Figure 1

Cortically derived EEG total spectral power evaluated on day 7, as the difference (Δ%) from the pre-ischaemic value in freely moving, awake gerbils given increasing doses (mg kg⁻¹) of AM 404 (a) and THC (b), 5 min after recirculation. AM 251 or naloxone (NX) was given, i.p., 5 min before bilateral carotid artery occlusion. Capsazepine (CPZ) was administered, s.c., 15 min before ischaemia. The doses of AM 404 and THC combined with the antagonists were 2 and 1 mg kg⁻¹, respectively. Each column represents the mean (±s.e.m.) of five animals. ^aP<0.05, ^bP<0.01, ^cP<0.001 compared with sham-operated animals; ^dP<0.05, ^eP<0.01, ^fP<0.001 compared with vehicle; ^gP<0.05, ^hP<0.01, ⁱP<0.001 compared with AM 404 (2 mg kg⁻¹) or THC (1 mg kg⁻¹) alone (one-way ANOVA followed by Tukey's test). THC, tetraydrocannabinol; ANOVA, analysis of variance.

Figure 2

Effect of increasing doses (mg kg⁻¹) of AM 404 (a) and Δ⁹-THC (b) given i.p. 5 min after recirculation on spontaneous motor activity evaluated for 30 min, 1 day after ischaemia in gerbils. AM 251 or naloxone (NX) was administered, i.p., 5 min before bilateral carotid occlusion. Capsazepine (CPZ) was given, s.c., 15 min before ischaemia. The doses of AM 404 and THC combined with the antagonists were 2 and 1 mg kg⁻¹, respectively. Each column represents the total horizontal counts (mean±s.e.m.) of five animals. ^aP<0.05, ^cP<0.001 compared with sham-operated animals; ^eP<0.01, ^fP<0.001 compared with vehicle; ^gP<0.05, ⁱP<0.001 compared with AM 404 (2 mg kg⁻¹) or THC (1 mg kg⁻¹) alone (one-way ANOVA followed by Tukey's test). THC, tetraydrocannabinol; ANOVA, analysis of variance.

Figure 3

Mean (±s.e.m.) escape latency in the passive avoidance task 3 days after 10-min carotid occlusion. Increasing doses (mg kg⁻¹) of AM 404 (a) and THC (b) given 5 min after recirculation. AM 251 or naloxone (NX) was administered, i.p., 5 min before bilateral carotid occlusion. Capsazepine (CPZ) was given, s.c., 15 min before ischaemia. The doses of AM 404 and THC combined with the antagonists were 2 and 1 mg kg⁻¹, respectively. Each column represents the mean (±s.e.m.) of five animals. ^bP<0.01, ^cP<0.001 compared with sham-operated animals; ^dP<0.05 compared with vehicle; ⁱP<0.001 compared with AM 404 (2 mg kg⁻¹) or THC (1 mg kg⁻¹) alone (one-way ANOVA followed by Tukey's test). THC, tetraydrocannabinol; ANOVA, analysis of variance.

Figure 4

Photomicrographs of the hippocampal CA₁ region of gerbils with or without 10 min ischaemia, 7 days after recirculation. AM 251 and naloxone (NX) were given, i.p., 5 min before bilateral carotid occlusion; capsazepine (CPZ) was given, s.c., 15 min before ischaemia. Doses are expressed as mg kg⁻¹. Bar=50 μm.

Figure 5

Effect of AM 404 (a) and THC (b) on neuronal count, performed 7 days after recirculation, in the CA₁ region of the hippocampus of sham-operated or ischaemic gerbils. AM 404 and THC were given, i.p., 5 min after recirculation. AM 251 or naloxone (NX) was given, i.p., 5 min before bilateral carotid occlusion. Capsazepine (CPZ) was administered, s.c., 15 min before ischaemia. Each column represents the mean (±s.e.m.) of five hippocampal sections from the same coronal plane for each animal. Doses are expressed as mg kg⁻¹. ^cP<0.001 compared with sham; ^fP<0.001 compared with vehicle; ⁱP<0.001 compared with AM 404 (2 mg kg⁻¹) or THC (1 mg kg⁻¹) alone (one-way ANOVA followed by Tukey's test). THC, tetraydrocannabinol; ANOVA, analysis of variance.