The ECS has been suggested to contribute to the maintenance of homeostasis between the immune and the nervous systems [34, 35]. Besides, the pharmacological activation of ECS is emerging as a potential therapeutic strategy for neurodegenerative diseases including multiple sclerosis [rev [10, 36]. Mechanisms underlying the beneficial effects of CBs on MS are not fully clarified; however, anti-inflammatory and/or neuroprotective actions seem to be involved . Leukocyte migration into the CNS is widely recognized as a pivotal event in the development of MS in which adhesion molecules like VCAM-1 are critically involved and emerge as marker of endothelial activity [rev [2, 7].
The notion that restriction of immune cells traffic into the CNS by CBs could represent a novel mechanism to suppress brain immune reactivity was first suggested by two laboratories in both, TMEV-IDD and EAE models by using the synthetic agonist WIN 55,212-2 [21, 23]. In the present study we show that the endocannabinoid, AEA inhibits the expression of VCAM-1 in TMEV-infected brain endothelial cells resulting in reduced leukocyte adhesion and crossing through an in vitro model of BBB. In the TMEV-IDD model, cumulative evidence suggests that TMEV may enter the CNS by infection of cerebrovascular endothelial cells. Thus, infection of endothelial cells might represent one of the first events in the pathogenesis of TMEV-induced demyelination. The persistence of TMEV in cloned mouse cerebrovascular endothelial cells appears to support this concept . Pioneering studies on TMEV-IDD showed that adhesion molecules play a critical role in leukocyte extravasation  pointing out the interest of a reduction of VCAM-1 expression by AEA. CB1 and CB2 receptors were expressed in b.End5 as well as in primary cultures of murine brain endothelial cells . Most of the effects of CBs are mediated by their specific receptors CB1 and CB2 that are asymmetrically distributed in the BBB. CB1 receptor is mainly located at the luminal side while CB2 receptors are on the abluminal side of the endothelium [41, 19]. In our study, VCAM-1 suppression by AEA in brain endothelial cells was mainly mediated by the activation of CB1 receptors. Most importantly, AEA-induced inhibition of leukocyte adhesion and crossing through the BBB also involved CB1 receptors accordingly to the specific distribution of this type of receptors in the BBB. In agreement with our observations, studies on HIV-1 Gp120-effects in brain microvascular endothelial cells have shown that CB1 based synthetic CBs prevented monocyte transmigration across a human model of BBB . Although CB1, CB2  and TRPV1  receptors are expressed in murine brain endothelial cells, our results ruled out the involvement of CB2 and TRPV1 receptors in AEA-induced inhibition of VCAM-1. Differential expression of CB2 receptors and NAPE-PLD (the major enzyme associated with synthesis of AEA) in cerebral endothelium at different stages of MS has been recently reported . In the above study, increased CB2 receptor staining was associated with BBB disruption in active plaques from MS tissue samples, suggesting a role for endothelial CB2 in the protection and/or repair of BBB injury. However, previous studies of MS brain tissue did not find endothelial expression of CB2 [44, 45]. In TMEV-infected brain endothelial cells the possibility that AEA activates PPAR-γ receptors  to suppress VCAM-1 can be also discharged despite the fact that PPARs agonists prevent the interaction of leukocytes with stimulated endothelium .
The majority of studies on AEA actions in endothelial cells have focused on its vasodilator and hypotensive activity and there were discrepancies on the type of receptor implicated, probably due to differences between peripheral and brain endothelial cells . Using mouse cerebral endothelial cells and consistent with our results, AEA-induced increased COX-2 expression involves the activation of CB1 receptors .
Although alterations in the ECS during the course of MS have been suggested to represent a protective physiological strategy [13, 18, 49, 50] the role of endocannabinoids in MS remains uncertain. While most of studies on ECS and MS focused on established disease, understanding the role of endocannabinoids during the induction phase would be an important point as exacerbated leukocyte trafficking into the CNS represents a key stage in the disease. Therefore, here, we investigated the role of CB1 receptors and the effects of the inhibitor of AEA uptake, UCM-707, on VCAM-1 expression in wild type and CB1 knockout mice (Cnr1-/-) during the early phases of TMEV-IDD. Intracranial infection with TMEV induced the expression of VCAM-1 in surrounding blood vessels close to the site of injection in Cnr1+/+ as well as in Cnr1-/- mice whereas VCAM-1 was not detected in brains of sham animal in both type of mice accordingly to other studies [4, 24]. The treatment with UCM-707 resulted in down-regulation of VCAM-1 expression in both type of mice. However, the degree of inhibition of VCAM-1 in the ipsilateral cerebral cortex of Cnr1+/+ mice was significantly higher than that observed in Cnr1-/- mice supporting the involvement of CB1 receptors and corroborating our in vitro results. In addition, the analysis of the contralateral hemisphere showed increased VCAM-1 expression only in the vasculature of Cnr1-/- mice that was inhibited by UCM-707. Thus, our in vivo data confirm the importance of CB1 receptors but, suggest that besides CB1 receptors, additional mechanisms are contributing to the effects of UCM-707 on VCAM-1 inhibition. It is difficult to have the overall picture of what is happening as consequence of increasing AEA tone under the conditions of our study due to the multiple cellular targets for AEA actions on the responses to TMEV infection. Nevertheless, we have shown here that AEA by targeting brain endothelial cells may interfere with leukocyte recruitment across the BBB through the inhibition of VCAM-1.
As suggested in the cardiovascular endothelium [51, 52] in the brain endothelium AEA and other endocannabinoids like 2-AG, would be synthetized and released from a nearby source such as astrocytes , microglia  and even from the own endothelial cells to regulate the response of brain endothelium to different stimuli as we observed in the case of TMEV. The observation that NAPE-PLD expression is elevated on blood vessels and in reactive astrocytes distributed closely around them suggests the synthesis of AEA by brain endothelium in MS . In other models of brain injury 2-AG has been shown to be released and to reduce BBB damage [14, 55].
Additionally, endocannabinoids may control brain innate immunity in MS by acting in different CNS cell types such as astrocytes and microglial besides immune cells [rev . Activating or inhibiting the innate immune response influences the development of TMEV-IDD . In this line, AEA enhances IL-6 production in astrocytes infected with TMEV by a CB1 receptor-mediated pathway  and in a more recent work AEA modulates TMEV-induced IL-12, IL-23 and IL-10 in microglia by activating CB2 receptors .
An important finding of the present study is that the lack of CB1 receptor leads to an exacerbation of microglial response to TMEV infection in the ipsilateral hemisphere. Thus, microglial activation was observed from prefrontal cortex to hippocampal levels instead of maintaining it exclusively in the area close to the injection site. Currently, we unknown the meaning of the extensive microglial activation in Cnr1-/- mice, but it is likely to be associated with the facilitation of spreading viral antigens as microglia/macrophages are an important virus reservoir . In line with a protective role of CB1 receptors previous studies have reported that CB1-knockout mice develop more severe CREAE [61, 62]. Moreover, recent studies reveal that repeat polymorphism of the Cnr1 gene could represent a genetic risk factor for both the primary progressive  and relapsing-remitting form of MS .