Accumulating data now suggest that cannabinoid CB₁ receptors contribute to neuroprotection through anti-excitotoxicity [14], the phosphatidylinositol-3 kinase/Akt pathway [17], and hypothermia [18]. We have reported that the neuroprotective and hypothermic effects of delta⁹-THC were related to CB₁ receptors [89]. CBD has also been described as protective against global and focal ischemic injury [14,18]. Several groups have recently shown that CBD can interact with cannabinoid CB₁ and CB₂ receptors. Pertwee and Ross showed that CBD can antagonize the CB₁ agonists such as WIN55,212 and CP55940 by acting at prejunctional sites that was unlikely to be cannabinoid CB₁ or CB₂ receptors [19]. Castillo and coworkers recently demonstrated that CBD implicated the neuroprotective effect mediated by CB₂ and adenosine receptors in an in vitro model of newborn hypoxic-ischemic brain damage in mice [21]. In addition, Thomas and coworkers noted that CBD displayed unexpected high potency as an antagonist of CB₁ and CB₂ receptor agonists [20]. Interestingly, the neuroprotective effect of CBD showed a dose dependent bell shaped curve in mice subjected to middle cerebral artery occlusion (MCAO) [22]. Intraperitoneal injection of CBD 1 or 3 mg/kg but not 10 mg/kg during 4 h MCAO prevented cerebral infarction 24 hours after cerebral ischemia. Other groups have demonstrated that 5 mg/kg was the most effective CBD dose, and that there was a dose-dependent bell-shaped curve for CBD’s effects on the electroencephalographic flattening in gerbils subjected to cerebral ischemia [13]. CBD has been also reported to inhibit anandamide amidase [23] and the reuptake of anandamide [24], suggesting CBD may induce an increase of anandamide signaling within the ischemic brain. Anandamide and other CB₁ receptor agonists are known to reduce the release of a variety of neurotransmitters including glutamate via CB₁ receptor [25,26,27,28,29]. In our previous study, delta⁹-THC significant reduced the release of glutamate during MCAO, but CBD did not affect glutamate release [30], suggesting that CBD might have other mechanism or stimulate other receptors besides the cannabinoid receptors. The mechanism of this biphasic effect of CBD is still unclear, but CBD may induce cerebroprotective effect through modulating endogenous cannabinoid system.

In 1974, an interactive study comparing CBD and THC in healthy volunteers demonstrated for the first time that CBD could act as an anxiolityc drug [31]. Experimentally, the anxiolytic properties of CBD have been demonstrated in different animal models such as the conditioned emotional response, the Vogel conflict test, and the elevated plus-maze [32,33]. Recently, it has been shown that CBD might exert anxiolytic effects by activating post-synaptic 5-HT_1A receptors [34]. 5-HT_1A receptors have been shown to play critical roles in the pathophysiology of depression, aggression, and anxiety. It appears to have a role in vasodilatation and neuroprotection [35,36,37,38,39,40]. We previously applied a new approach to the investigation of the neuroprotective mechanism of CBD by examining the effects of a CB₁ receptor antagonist, a vanilloid-receptor (VR1) antagonist, and a 5-HT_1A receptor antagonist in a 4h MCA occlusion model in mice. Interestingly, the neuroprotective effect of CBD was inhibited by the 5-HT_1A receptor antagonist, WAY100135. Furthermore, the increased cerebral blood flow induced by CBD was also in part reduced by WAY100135. In contrast, the CB₁ receptor antagonist and VR1 did not inhibit the effect of CBD [22]. Russo and co-workers demonstrated that CBD, but not delta⁹-THC, displaced the 5HT_1A agonist, [³H]-8-hydroxy-2-di-n-propylaminotetralin ([³H]-8-OH-DPAT) from the cloned human 5HT_1A receptor in a dose-dependent manner, and they have concluded that CBD was an agonist of the 5HT_1A receptor [41]. More recently, Magen and co-workers supported that CBD (5 mg/kg, i.p.) induced activation of 5-HT_1A receptors located in forebrain regions including the hippocampus, and improved cognitive and locomotor function which were impaired by bile-duct ligation [42]. Taken together, these data indicate that CBD may activate the 5-HT_1A receptor which leads to the improvement of cognitive and functional impairment after cerebral ischemia.

In 1998, Hampson and co-workers noted that the nonpsychoactive marijuana constituent CBD prevented both glutamate neurotoxicity and ROS-induced cell death with a stronger effect than either of the dietary antioxidants, α-tocopherol or ascorbate [43]. Surprisingly, CBD protected neurons with comparable efficacy to the potent antioxidant, butylated hydroxytoluene (BHT). In same research group, CBD and delta⁹-THC suppressed the oxidation potential measured by cyclic voltammetry and CBD was more effective than delta⁹-THC, suggesting that cannabidiol may be a neuroprotective antioxidant [14]. We have assessed both CBD and delta⁹-THC for antioxidant activity using the original 1,1-diphenyl-2-picryhydrazyl (DPPH) radical method described by Brand-Williams et al. [44]. Our results showed that CBD exhibited stronger antioxidative power (EC₅₀ = 89.2 μM) than delta⁹-THC (EC₅₀ = 464.2 μM) [45]. In an in vivo study, Hamelink and co-workers found that CBD protected against hippocampal-entorhinal-cortical neurodegeneration caused by ethanol exposure in rats. They showed that this effect of CBD attributed its anti-oxidative action [46]. These data suggest that CBD may be a very useful therapeutic agent for oxidative disorders after ischemic stroke.

Repeated treatment with delta⁹-THC and other CB₁ receptor agonists result in the development of tolerance to its most acute behavioral and pharmacological effects [47,48,49,50]. Delta⁹-THC has been shown to lead to tolerance of hypoactivity, hypothermia, anti-nociception, catalepsy and pentobarbital-induced sleep prolongation [51,52,53]. In fact, 14 day-repeated treatment with delta⁹-THC (10 mg/kg) led to tolerance of the hypothermic and also neuroprotective effects. Additionally, the expression levels of CB₁ receptor were down-regulated in mice with repeated doses of 10 mg/kg [45]. On the other hand, repeated treatment with CBD did not lead to development of tolerance in the cerebroprotective effect on infarction. CBD induced an increase in cerebral blood flow (CBF) even after 14 days of repeated treatment. Since these effects of CBD were in part inhibited by WAY100135, CBD may not be full 5-HT_1A agonist. It is known that, 8-OH-DPAT, a complete 5HT_1A agonist, induces tolerance [54], while a 5-HT_1A receptor partial agonist such as buspirone does not [55]. For that reason, the neuroprotective effect of CBD may be mediated through partial agonistic effect on the 5-HT_1A receptor.

Emerging data now support the evidence of the anti-inflammatory action of CBD. For example, CBD suppressed interleukin-1 and inducible nitric oxide synthease induced by beta-amyloid [56]. CBD suppressed tumor necrosis factor α production in lipopolysaccharide-treated mice via A_2A adenosine receptor activation, and this effect was reversed with an A_2A adenosine-receptor antagonist, and abolished in A_2A receptor knockout mice [15]. In addition, CBD inhibited neutrophil migration induced by formyl-methionyl-leucyl-phenylalanine in CB₁ and CB₂ receptor independent pathways which are antagonized by the endogenous compound N-arachdonoyl-L-serine [57]. We have reported that CBD had a cerebroprotective action via a cannabinoid receptor-independent myeloperoxidase inhibiting mechanism in a 4h MCA occlusion model in mice [30]. In this model, the neuroprotective effect of delta⁹-THC was only evident with pre-ischemic treatment via inhibition of the release of glutamate in in vivo microdialysis-HPLC measurements and hypothermia in CB₁ receptor dependent mechanism. On the other hand, CBD was cerebroprotective even when administered 6 hours after cerebral ischemia. In addition, we demonstrated that CBD, but not delta⁹-THC, inhibited myeloperoxidase activity in neutrophils and prevented the decrease in cerebral blood flow due to the failure of cerebral microcirculation after reperfusion. In a more recent study, we also found that the anti-inflammatory action of CBD was associated with inhibition of plasma high-mobility group box1 leaked from dying cells and released from damaged cells and/or monocytes/macrophages [58]. These results suggest that CBD may prevent post-ischemic injury progressively induced by ischemic stroke.

We have previously reported that CBD (3 mg/kg) has a potent and long-lasting neuroprotective effect when administered both pre- and post-ischemia, whereas only pre-ischemic treatment with delta⁹-THC (10 mg/kg) reduced the infarction size when administered 24 hours after 4 h MCAo in mice. The neuroprotective effect of delta⁹-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 [12]. In fact, delta⁹-THC inhibited the massive release of glutamate, but this neuroprotective effect of delta-⁹THC was inhibited by the CB₁ receptor antagonist SR141716 [45], which suggest that delta⁹-THC is neuroprotective as a pre-ischemic treatment via inhibition of glutamate excitotoxicity. Actually, treatment with delta⁹-THC immediately after reperfusion did not prevent cerebral infarction. On the other hand, CBD was neuroprotective even when administered 6 hours after cerebral ischemia (2 hours after reperfusion). In addition, CBD significantly inhibited the myeloperoxidase activity of neutrophils at 1 hour and 20 hours after reperfusion and suppressed the decrease in CBF due to the failure of cerebral microcirculation after reperfusion. In addition, CBD decreased the number of Iba1- and GFAP-positive cells and improved neurological score and motor coordination at 3 days after cerebral ischemia. We concluded that the therapeutic time window of CBD was 6 hours after onset of ischemia in early treatment. CBD had a potent and long-lasting neuro- protective effect and prevented progressive post-ischemic injury [58]. In a more recent study, we reported that repeated treatment with CBD from 1 day or 3 days after cerebral ischemia improved the functional deficits, such as neurological score and motor coordination, and survival rates. In addition, both groups did not increase the HMGB1 level in plasma, and decreased the number of Iba1 expressing HMGB1 positive cells and TUNEL positive cells, which suggest that CBD may be cerebroprotective not only during the early phase, but also during the chronic phase after ischemic stroke [71]. However, treatment with CBD from Day 5 did not improve the functional outcome Day 14 after cerebral ischemia. As described above, CBD has a potent anti-inflammatory effect, such as inhibition of glial activation. Activated microglia and reactive astrocytes have also a role of beneficial in ischemic brain. For example, microglia can produce neurotorophic factors such as brain-derived neurotorophic factor, insulin-like growth factor 1 [95]. And, reactive astrocytes can also release many growth factors such as nerve growth factor [96]. Taken together, a treatment with CBD in ischemic early phase may implicate the neuroprotective action through inhibition of acute inflammatory reaction, whereas ischemic delayed treatment with CBD may interfere with beneficial endogenous mechanisms.

Leker and co-workers investigated the therapeutic time window of the synthetic CB₁ receptor agonist, HU210 in rats subjected to permanent middle cerebral artery occlusion (PMCAO). They demonstrated that administration of HU-210 (45 μg/kg) at 1, 2, or 4 but not 6 hours after PMCAO resulted in reduced motor disability and infarct volumes compared with vehicle controls 72 hours after cerebral ischemia and thus, therapeutic time window appeared to extend to 4 hours after PMCAO and the salutary effects of HU-210 were only partially abolished by the CB₁ receptor antagonist SR141716 and warming [18]. They concluded that the neuroprotective effects of HU-210 were dependent on potent hypothermia via CB₁ receptor dependent mechanism. Another research group noted that 40 min before the induction of global cerebral ischemia, treatment with R(+)-WIN55,212-2 (1 mg/kg), a synthetic aminoalkylindole cannabinoid CB₁ receptor agonist, induced a dose-dependent increase in neuronal survival that reached maximal levels (56% of sham-operated controls) in a global cerebral ischemia model in rats. Treatment with R(+)-WIN55,212-2 (1 mg/kg) 30 min before ischemia, but not 60–120 min after ischemia, reduced the infarct size 24 hours after ischemic stroke onset [72], suggesting that pre-ischemic treatment with R(+)-WIN55,212-2 is protective in cerebral ischemia.

Dexanabinol (HU-211) is a synthetic, nonpsychotropic cannabinoid [90]. It has been shown to act as a noncompetitive N-methyl-D-aspartate receptor antagonist [91], as well as having antioxidant [92], anti-inflammatory effects [93,94]. The therapeutic time window of nonpsychotropic cannabinoid, HU-211 in closed head injury was first reported by Mechoulam’s research group in 1993 [73]. HU-211 dissolved in middle-chain triglycerides (MCT) oil at a dose of 25 mg/kg was given intraperitoneally immediately and 1, 2, or 3 h after impact. The drug was found to be very effective in improving motor function recovery when given 2 h after the injury. In addition, Shohami and co-workers have shown the long-term effect of HU-211 on motor and memory functions after closed head injury in the rat [74]. HU-211 (5 mg/kg) was administered intravenously at 4 or 6 h after closed head injury. Cognitive functions were evaluated using the Morris water maze, with rats trained either before or after closed head injury. The data showed that HU-211 was a potent cerebroprotective agent, with a therapeutic window of about 4 h. Moreover, this compound HU-211 was also used for cerebral ischemia. Belayev and co-workers have reported that HU-211 (4 mg/kg, i.v.) significantly improved neurological deficits and reduced brain damage 60 min after forebrain ischemia in rats, but neuroprotection was no longer significant after 3 h [75]. Leker’s research group demonstrated that treatment with HU-211, dexanabinol (4.5 mg/kg) 1 h and 3 h but not 6 h after permanent middle cerebral artery occlusion in rats improved motor function and reduced infarct volume 24 h post ischemia [76].