Cannabinoid CB(2) receptor activation decreases cerebral infarction in a mouse focal ischemia/reperfusion model

Abstract

Cannabinoid CB(2) Receptor (CB(2)) activation has been shown to have immunomodulatory properties without psychotropic effects. The hypothesis of this study is that selective CB(2) agonist treatment can attenuate cerebral ischemia/reperfusion injury. Selective CB(2) agonists (O-3853, O-1966) were administered intravenously 1 h before transient middle cerebral artery occlusion (MCAO) or 10 mins after reperfusion in male mice. Leukocyte/endothelial interactions were evaluated before MCAO, 1 h after MCAO, and 24 h after MCAO via a closed cranial window. Cerebral infarct volume and motor function were determined 24 h after MCAO. Administration of the selective CB(2) agonists significantly decreased cerebral infarction (30%) and improved motor function (P<0.05) after 1 h MCAO followed by 23 h reperfusion in mice. Transient ischemia in untreated animals was associated with a significant increase in leukocyte rolling and adhesion on both venules and arterioles (P<0.05), whereas the enhanced rolling and adhesion were attenuated by both selective CB(2) agonists administered either at 1 h before or after MCAO (P<0.05). CB(2) activation is associated with a reduction in white blood cell rolling and adhesion along cerebral vascular endothelial cells, a reduction in infarct size, and improved motor function after transient focal ischemia.

Figure 1

Experimental flow charts for preischemic treatment test (A) and postreperfusion treatment test (B).

Figure 2

Typical closed cranial window in a mouse. Scale bar = 500 μm (A). Representative video images of capturing leukocytes labeled with Rhodamine 6G in the ischemic area. Video images were taken before MCAO (B), one hour after MCAO (C), and 24 h after MCAO (D) in the same venule. Arrows indicate Rhodamine 6G-labeled leukocytes flowing through venules. Scale bar = 40 μm. Representative brain slices after 1 h MCAO followed by 23 h reperfusion. Slices were stained with 2% triphenyltetrazolium chloride. The unstained areas (white) represent the infarct lesion corresponding to the middle cerebral artery distribution territory (E).

Figure 3

Pretreatment with either CB₂ agonist (O-3853 or O-1966) had no effect on rCBF in mice subjected to MCAO compared with vehicle-treated control group. rCBF decreased to 25% of baseline level within the first 1 min and was maintained below 25% of baseline value throughout MCAO. Values represent percentage of baseline value at each time spots. (Data were expressed as mean±s.e.m., n = 8–13 in each group.)

Figure 4

Effects of CB₂ agonists on cerebral infarction. Administration of either CB₂ agonist (O-3853 or O-1966) at either 1 h before MCAO or 10 mins after reperfusion significantly reduced the cerebral infarction compared with the vehicle-treated group. There was no difference between the untreated group and the vehicle-treated group. (A) Infarct volume (mm³); (B) percentage of infarction to ipsilateral hemisphere. (Data were expressed as mean±s.e.m., n = 8 to 13 in each group, *P < 0.05 versus vehicle-treated group.)

Figure 5

Effects of CB₂ agonists on neurological function in mice subjected to 1 h MCAO and 23 h reperfusion. Administration of either CB₂ agonist (O-3853 or O-1966) at either 1 h before MCAO or 10 mins after reperfusion significantly improved the motor function (0 represents normal motor function, 4 represents no spontaneous motor function) compared with the vehicle-treated control group. No difference was found between untreated and vehicle-treated groups. (Data were represented as mean±s.e.m., n = 8 to 13 in each group, *P < 0.05 versus vehicle-treated group.)

Figure 6

Middle cerebral artery occlusion enhanced leukocyte rolling and adhesion after reperfusion. Rolling and adhesion were significantly enhanced (P < 0.05) on both venules and arterioles, with the exception of rolling on arterioles after 1 h MCAO. (Data were represented as mean±s.e.m., n = 6 in each group, *P < 0.05 versus before MCAO.)

Figure 7

Administration of either CB₂agonist (O-3853 or O-1966) 1 h before MCAO attenuated leukocyte/endothelial interactions. (A) The CB₂agonists decreased the number of leukocytes rolling on venules at 1 and 24 h after MCAO. (B) The CB₂agonists decreased the number of leukocyte adhering to on venules 1 and 24 h after MCAO. (C) The CB₂agonists had no effect on leukocyte rolling on arterioles 1 h after MCAO compared with the vehicle-treated group. Both agonists decreased the number of leukocytes rolling on arterioles at 24 h after MCAO. (D) The CB₂agonist (O-1966) had no effect on leukocyte adhesion to arterioles before MCAO and 1 h after MCAO compared with the vehicle-treated group, whereas O-3853 decreased the number of leukocyte adhesion on arterioles at 1 h after MCAO. Both agonists decreased leukocyte adhesion to arterioles at 24 h after MCAO. There was no difference of baseline of leukocyte rolling and adhesion on venules and arterioles among the groups. (Data were represented as mean±s.e.m., n = 6 in each groups, *P < 0.05 versus vehicle.)