Caffeinol at the receptor level: anti-ischemic effect of N-methyl-D-aspartate receptor blockade is potentiated by caffeine

Abstract

Background and purpose: Although caffeinol (a combination of a low dose of caffeine and ethanol) was shown to robustly reduce stroke damage in experimental models and is now in clinical evaluation for treatment of ischemic stroke, little is known about the potential mechanism of its action.

Methods: We used an in vivo excitotoxicity model based on intracortical infusion of N-methyl-D-aspartate (NMDA) and a model of reversible focal ischemia to demonstrate NMDA receptor inhibition as a potential mechanism of caffeinol anti-ischemic activity.

Results: Caffeinol reduced the size of excitotoxic lesion, and substitution of ethanol in caffeinol with the NMDA antagonists CNS-1102 and MK-801 but not with MgSO(4) produced treatment with strong synergistic effect that was at least as robust in reducing ischemic damage as caffeinol. This NMDA receptor antagonist and caffeine combination demonstrated a long window of opportunity, activity in spontaneously hypertensive rats, and, unlike caffeinol, was fully effective in animals chronically pretreated with ethanol.

Conclusions: Our study suggests that antiexcitotoxic properties may underlie some of the anti-ischemic effect of caffeinol. This study provides strong evidence that the anti-ischemic effect of NMDA receptor blockers in general can be dramatically augmented by caffeine, thus opening a possibility for new use of NMDA-based pharmacology in the treatment of stroke.

Fig-1

Caffeinol reduces excitotoxic lesion volume in rat cerebral cortex in vivo. Cortical lesion volume (mm³) produced by 100nmols of NMDA in animals pre-treated with intravenous administration of saline, Caffeinol (C/E; 0325 g/kg ethanol and 10mg/kg caffeine), ethanol (EtOH; 0.325g/kg), caffeine (10mg/kg) or MK-801 (3mg/kg). The lesion volume was measured at 48h. The data is expressed as mean±SEM. Number (N) of animals per group is indicated above the bars. *p≤0.05 from saline control. Diagram illustrates the location of the NMDA infusion/lesion.

Fig-2

Infarct volume after MCA/CCA occlusion in Long Evans rats treated with saline, CNS-1102 (CNS; 0.5 mg/kg bolus+0.345mg/kg/h for 2.5h via i.v. infusion) or CNS-1102 plus caffeine (CNS+CAF; 0.5mg/kg bolus+0.345mg/kg/h CNS-1102 plus 1.66mg/kg bolus+3.33mg/kg/h caffeine, over 2.5h). Treatment was started 15min after the onset of 180min of ischemia. Infarct volume was determined at 3d. The data is expressed as mean±SEM. Number (N) of animals per group is indicated above the bars. *p≤0.05 from all the other groups.

Fig-3

Infarct volume after MCA/CCA occlusion in Long Evans rats treated with saline or CNS-1102 (0.5mg/kg bolus+0.345mg/kg/h for 2.5h) plus caffeine (1.66mg/kg bolus+3.33mg/kg/h for 2.5h) starting at 15,60,90 or 120min after the onset of MCA/CCAo. Infarct volume was determined at 3d. The data is expressed as mean±SEM. Number (N) of animals per group is indicated above the bars. *p≤0.05 from saline control.

Fig-4

Infarct volume after MCA/CCA occlusion in SHRs treated with saline or CNS-1102 (0.5 mg/kg bolus+0.345 mg/kg/h for 2.5h) plus caffeine (1.66 mg/kg bolus+3.33 mg/kg/h for 2.5h) starting at 30 min after the onset of MCA/CCAo. Infarct volume was determined at 3 days. The data is expressed as mean±SEM. Number (N) of animals per group is indicated above the bars. *p≤0.05 from saline control.

Fig-5

Infarct volume after MCA/CCA occlusion in Long Evans rats treated intravenously with: (1) saline; (2) ethanol (EtOH; 0.65g/kg); (3) ethanol (0.65g/kg) plus caffeine (10 mg/kg); (4) MK-801 (1mg/kg); (5) MK-801 (1mg/kg) plus caffeine (10mg/kg); (6) MgSO4 (67mg/kg) and (7) MgSO4 (67mg/kg) plus caffeine (10mg/kg). All treatments were started at 30 min after the onset of ischemia. Infarct volume was determined at 3d. The data is expressed as mean±SEM. Number (N) of animals per group is indicated above the bars. *p≤0.05 from saline control.

Fig-6

Infarct volume after MCA/CCA occlusion in Long Evans rats pretreated (Pretr) with water (groups 1 and 2) or ethanol (0.325g/kg - groups 3 and 4) for 14 consecutive days prior to ischemia and then intravenously treated with: (1) saline; (2 and 3) caffeinol - 0325g/kg ethanol and 10mg/kg caffeine; and (4) 10mg/kg caffeine plus 1mg/kg MK-801) at 30 min after the onset of MCA/CCAo. Infarct volume was determined at 3d. The data is expressed as mean±SEM. Number (N) of animals per group is indicated above the bars. * p≤0.05 from all remaining groups.