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We examined the neuroprotective mechanism of cannabidiol, non-psychoactive component of marijuana, on the infarction in a 4 h mouse middle cerebral artery (MCA) occlusion model in comparison with Δ9-tetrahydrocannabinol (Δ9-THC). Release of glutamate in the cortex was measured at 2 h after MCA occlusion. Myeloperoxidase (MPO) and cerebral blood flow were measured at 1 h after reperfusion. In addition, infarct size and MPO were determined at 24 and 72 h after MCA occlusion. The neuroprotective effect of cannabidiol was not inhibited by either SR141716 or AM630. Both pre- and post-ischemic treatment with cannabidiol resulted in potent and long-lasting neuroprotection, whereas only pre-ischemic treatment with Δ9-THC reduced the infarction. Unlike Δ9-THC, cannabidiol did not affect the excess release of glutamate in the cortex after occlusion. Cannabidiol suppressed the decrease in cerebral blood flow by the failure of cerebral microcirculation after reperfusion and inhibited MPO activity in neutrophils. Furthermore, the number of MPO-immunopositive cells was reduced in the ipsilateral hemisphere in cannabidiol-treated group. Cannbidiol provides potent and long-lasting neuroprotection through an anti-inflammatory CB1 receptor-independent mechanism, suggesting that cannabidiol will have a palliative action and open new therapeutic possibilities for treating cerebrovascular disorders.
Cannabis contains about 60 different cannabinoids, including the psychoactive component Δ9-tetrahydrocannabinol (Δ9-THC) and other major non-psychoactive components such as cannabidiol, cannabinol, and cannabigerol. Δ9-THC has been demonstrated to produce hypothermia, neuroprotection, and tolerance (Hampson et al. 2000; Rubino et al. 2000; Wiley and Martin 2002; Braida et al. 2003; Leker et al. 2003; Hayakawa et al. 2004; Mishima et al. 2005). These effects are, at least in part, related to binding to the CB1 receptor. On the other hand, cannabidiol has a very low affinity (in the micromolar range) for CB1 and CB2 receptors and has been found to act as an anticonvulsant in animal models of epilepsy and in humans with epilepsy. Moreover, cannabidiol has been shown to have antispasmodic, anxiolytic, anti-nausea, and anti-rheumatoid properties (Mechoulam et al. 2002) and to be protective against N-methyl-d-aspartate and beta-amyloid peptide toxicity (Iuvone et al. 2004) and global and focal ischemic injury (Braida et al. 2003). Recently, it has also been reported that cannabidiol has the ability to enhance adenosine signaling through inhibition of uptake (Carrier et al. 2006). These actions are thought to be dependent on a new cannabinoid receptor, such as the G protein-coupled receptor (Begg et al. 2005; David et al. 2006; Munro et al. 1993), GPR55, abnormal-cannabidiol receptor rather than on the CB1 and CB2 receptors. Cannabidiol exerts a wide spectrum of effects, but the neuroprotective mechanism has not been fully explored.
Neutrophils, a type of leukocytes, are known to play a major role in inflammatory injury after transient ischemia (Kochanek and Hallenbeck 1992; Heinel et al. 1994). The progression and extent of brain injury resulting from cerebral ischemia is related to several reperfusion mechanisms, many of which involve post-injury inflammatory response elements. These inflammatory elements include the early neutrophil response (Barone et al. 1992; Chen et al. 1993; Matsuo et al. 1994; Zhang et al. 1994). Several lines of evidence have shown that neutrophils play an important role in the development of ischemic brain damage and indicate that the depletion of circulating neutrophils or the inhibition of neutrophil infiltration is thought to ameliorate cerebral ischemic injury (Jiang et al. 1995; Satoh et al. 1999; Phillips et al. 2000). Δ9-THC and other cannabinoids have been known to modulate inflammation. For example, Δ9-THC and cannabidiol suppress interleukin-1 and tumor necrosis factor (Watzl et al. 1991), and cannabinoids alter cytokine production in human immune cells (Srivastava et al. 1998). In addition, cannabinoids ablate the release of tumor necrosis factor-α in rat microglial cells stimulated with lypopolysaccharide (Facchinetti et al. 2003). However, no reports have yet been issued on the anti-neutrophil actions of Δ9-THC and cannabidiol.
In the present study, we investigated the effects of cannabidiol and Δ9-THC on ischemic brain damage induced by focal ischemia-reperfusion in mice. We measured and evaluated the effect of cannabidiol and Δ9-THC on the extracellular level of glutamate in the cortex using in vivo microdialysis. Moreover, the anti-neutrophil action was quantified by assaying myeloperoxidase (MPO) activity, which is mainly located in neutrophil primary granules.
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- Materials and methods
Both Δ9-THC and cannabidiol significantly reduced the infarct volume in a mouse MCA occlusion model, and the neuroprotective effect of Δ9-THC was inhibited by the CB1 receptor antagonist SR141716, but not by the CB2 receptor antagonist AM630. Cannabidiol was not inhibited by either antagonist. In addition, only pre-ischemic treatment with Δ9-THC was able to reduce the size of infarction, whereas both pre- and post-ischemic treatment with cannabidiol showed more potent and more long-lasting neuroprotection than Δ9-THC. Δ9-THC but not cannabidiol inhibited the excess release of glutamate in the cortex after the occlusion, as measured by in vivo microdialysis; this effect of Δ9-THC was also inhibited by SR141716. Cannabidiol suppressed the decrease in CBF due to the failure of cerebral microcirculation after reperfusion. Cannabidiol also inhibited MPO activity in neutrophils after reperfusion. Moreover, cannabidiol inhibited MPO activity at 20 h after reperfusion. In addition, cannabidiol reduced MPO-immunopositive cells at 3 days after MCA occlusion. Thus, cannbidiol is a potent and long-lasting neuroprotectant and anti-inflammatory acting through a cannabinoid receptor-independent mechanism.
Δ9-THC is known to produce neuroprotection via the cannabinoid CB1 receptor. We have also reported that Δ9-THC prevented cerebral infarction through hypothermia acting the CB1 receptor (Hayakawa et al. 2004). On the other hand, cannabidiol, a non-psychoactive constituent of cannabis, has been shown to be protective against global and focal ischemic injury, in agreement with the present study (Molina-Holgado et al. 2002; Braida et al. 2003). However, the neuroprotective mechanism of cannabidiol remains unclear, but novel non-CB1 and non-CB2 receptors have been proposed, because cannabidiol, which has many pharmacological actions, has a very low affinity (in the micromolar range) for CB1 and CB2 receptors (Wiley and Martin 2002). In this study, Δ9-THC was shown to have a neuroprotective effect on cerebral injury induced by MCA occlusion acting via the CB1 receptor but not the CB2 receptor. On the contrary, cannabidiol was not inhibited by antagonists to either the CB1 or CB2 receptor. These results suggest that Δ9-THC exerts its neuroprotective action through the CB1 receptor, while cannabidiol prevents cerebral infarction via a CB1 and CB2 receptor-independent mechanism.
The neuroprotective effect of Δ9-THC was only evident with pre-ischemic, but not post-ischemic, treatment of MCA occlusion in mice. The pattern of excitatory amino acid efflux in different models of cerebral ischemia derives from the finding that a massive release of glutamate is considered to play a major role in inducing ischemic and post-ischemic cell death (Bullock et al. 1995). In fact, antagonists of glutamate receptors reduce the ischemic penumbra (Obrenovitch 1966; Obrenovitch and Richards 1995), and inhibitors of glutamate release exhibit cerebroprotective activity against ischemia/reperfusion-evoked injury (Molina-Holgado et al. 2002). The neuroprotective effect of Δ9-THC and other cannabinoids is related to the CB1 receptor-mediated inhibition of voltage-sensitive Ca2+ channels, which reduces Ca2+ influx, glutamate release, and excitotoxicity (Iuvone et al. 2004). In fact, the present study shows that Δ9-THC inhibits the release of glutamate and induced hypothermia. Moreover, the effects were inhibited by the CB1 receptor antagonist SR141716. Taken together, these findings suggest that the neuroprotective effect of Δ9-THC is induced only by pre-ischemic but not with post-ischemic treatment.
Both pre- and post-ischemic treatment with cannabidiol resulted in a potent and a long-lasting neuroprotective effect. It has been reported that warming increases pro-inflammatory factors such as leukocyte integrin expression and function on neutrophils and platelets (Forsyth and Levinsky 1990;Kurabayashi et al. 1997; Kochanek and Hallenbeck 1992), and significantly exacerbates functional and structural neurologic injury (Shum-Tim et al. 1998). The leukocytes then interact with intracellular adhesion molecule-1 (ICAM-1), adhere to endothelial cells, and migrate out of the vessels. Cannabidiol has been reported to reduce ICAM-1 expression in experimental diabetes (EI-Remessy et al. 2006). A previous study showed that the neuroprotective effect of cannabidiol is not inhibited by warming indicating that cannabidiol has a CB1 receptor-independent mechanism, unlike Δ9-THC (Hayakawa et al. 2004). Thus, cannabidiol might have a potent anti-inflammatory effect, inhibiting the migration of leukocytes, platelets, and neutrophils by reducing ICAM-1 expression. In this study, cannabidiol significantly inhibited MPO activity in neutrophils at 1 and 20 h after reperfusion via a cannabinoid receptor-independent mechanism. In addition, cannabidiol reduced MPO-immunopositive cells at 3 days after MCA occlusion. Moreover, because the focal cerebral ischemia-induced inflammation response occurs at a later stage than glutamate release (Dirnagl et al. 2003), cannabidiol might have a potent and long-lasting neuroprotective effect.
Inflammation is a critical process after stroke (Danton and Dietrich 2003). Studies have shown the over-expression of inflammatory factors such as ICAM-1, P-selectin, and E-selectin and the accumulation of inflammatory cells such as neutrophils, macrophages, and T-cells (Barone and Feuerstein 1999;Danton and Dietrich 2003; Zhang and Wang 2005). Moreover, these factors have also been known to cause the decrease in CBF due to the failure of cerebral microcirculation at 2–4 h after cerebral ischemia reperfusion (Jones et al. 1981; Garcia et al. 1994). In previously, cannabidiol has prevented cerebral infarction through an increase in CBF (Mishima et al. 2005). In the present study, we found that both cannabidiol and Δ9-THC increased the CBF during MCA occlusion, while only cannabidiol suppresses a decrease in CBF after reperfusion and that cannabidiol inhibits MPO activity in neutrophils via a cannabinoid receptor-independent mechanism. Cannabidiol has a very low affinity (in the micromolar range) for CB1 and CB2 receptors, so these actions might be dependent on a new receptor within the brain such as the G protein-coupled receptor, GPR55, abnormal-cannabidiol receptor.
In conclusion, the present study shows that cannabidiol has a profile of cerebroprotectant activity different from that of Δ9-THC. Cannabidiol, but not Δ9-THC, has a potent and long-lasting neuroprotective effect, when administered both pre- and post-ischemia, through a CB1 and CB2 receptor-independent mechanism. It is to be hoped that cannabidiol will have a palliative action and open new therapeutic vista for treating cerebrovascular disorders.