Endocannabinoids and traumatic brain injury. Mol Neurobiol. For personal use only.on September 15, 2016. by guest www.bloodjournal.orgFrom 2007

Traumatic brain injury (TBI) represents the leading cause of death in young individuals. It triggers the accumulation of harmful mediators, leading to secondary damage, yet protective mechanisms are also set in motion. The endocannabinoid (eCB) system consists of ligands, such as anandamide and 2-arachidonoyl-glycerol (2-AG), receptors (e.g. CB1, CB2), transporters and enzymes, which are responsible for the 'on-demand' synthesis and degradation of these lipid mediators. There is a large body of evidence showing that eCB are markedly increased in response to pathogenic events. This fact, as well as numerous studies on experimental models of brain toxicity, neuroinflammation and trauma supports the notion that the eCB are part of the brain's compensatory or repair mechanisms. These are mediated via CB receptors signalling pathways that are linked to neuronal survival and repair. The levels of 2-AG, the most highly abundant eCB, are significantly elevated after TBI and when administered to TBI mice, 2-AG decreases brain oedema, inflammation and infarct volume and improves clinical recovery. The role of CB1 in mediating these effects was demonstrated using selective antagonists or CB1 knockout mice. CB2 were shown in other models of brain insults to reduce white blood cell rolling and adhesion, to reduce infarct size and to improve motor function. This review is focused on the role the eCB system plays as a self-neuroprotective mechanism and its potential as a basis for the development of novel therapeutic modality for the treatment of CNS pathologies with special emphasis on TBI. LINKED ARTICLES This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi. org/10.1111/bph.2011.163.issue-7 Abbreviations* 2-AG, 2-arachidonoyl-glycerol; AraS, N-arachidonoyl-L-serine; CB1, ENSG00000118432; CB2, ENSG00000162562; DGL, diacylglycerol lipase; eCB: endocannabinoid; TRPV1, ENSG00000043316; FAAH, fatty acid amide hydrolase; GPR55, ENSG00000135898; TBI, traumatic brain injury Introduction Over the last two decades, since the endocannabinoid (eCB) system was discovered, our knowledge of its structure and functions has significantly expanded. This system consists of ligands, such as anandamide and 2-arachidonoyl-glycerol (2-AG), receptors (CB1, CB2, possibly also TRPV1 and GPR55), transporters and enzymes, which are responsible for the synthesis [N-acyl-phosphatidylethanolaminephospholipase D, diacylglycerol lipase (DGL)] and degradation of these lipid mediators [fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase] There is a multiplicity of eCB actions, mainly in the brain, under both physiological and pathological conditions. Unlike 'classical' neurotransmitters, the eCBs are not stored in presynaptic vesicles, rather, they are produced 'on demand' when increased intracellular Ca ++ is the major intracellular trigger for synthesis. The primary ligands produced in the brain are anandamide The Authors British Journal of Pharmacology © 2011 The British Pharmacological Society stress and trauma. The fact that the eCB system is activated in response to such events suggests that it is part of the brain's compensatory repair mechanism, mediated via CB receptors signalling (for review: The CB receptors belong to the large superfamily of G protein-coupled receptors (GPCR) CB2 are expressed predominantly in non-neuronal cells as well as on subpopulations of neurons, yet, they exert no psychoactivity. Although considered to be located mostly in the immune system CB2R are now well recognized on resident inflammatory cells within the CNS, on microglial and dendritic cells Vanilloid type 1 (TRPV1) receptors are found not only on sensory neurons, where they are partly co-expressed with CB1 receptors Summing up, there is ample evidence suggesting that the eCBs interact with at least three types of receptors at binding sites located at a variety of cell types in the brain. The specific dominant interaction depends on a number of factors, including the levels of eCBs, tissue receptor distribution and accessibility to the receptors. Is the eCB system a potential 'self-neuroprotective' entity The expression and function of the eCBs and their respective receptors in the brain, on neurons, astrocytes, microglia and the cerebrovasculature point to their role in multiple (patho) physiological functions. To explore the role of anandamide signalling in vivo, several investigators have targeted its degrading enzyme in order to augment and extend its brain activities. Thus, the role of anandamide in setting an endogenous cannabinoid tone was shown in mice lacking the enzyme FAAH2/2. Upon administration of exogenous anandamide, its brain levels were augmented 15-fold and the mice exhibited robust, CB1-dependent behavioural responses such as hypomotility, analgesia, catalepsy and hypothermia Ischemic and traumatic brain injuries are CNS pathologies in which high intracellular calcium accumulation are among the earliest events. They share a secondary complex of harmful pathways that include excitotoxicity, oxidative stress and acute inflammatory response eCBs as neuromodulators of excitotoxicity Over the last two decades, hyperactivation of the NMDA receptors by extracellular excitatory amino acids, such as glutamate, has been implicated in the cellular events leading to neuronal death and decline in function following traumatic or ischemic brain injury (e.g. However, the CB1-mediated neuroprotection showed desensitization, probably due to receptor down-regulation, after prolonged exposure to the agonists (24 h). A crucial component of cell survival, activated by CB1 receptors, is the PI3K/Akt pathway. Acute administration of THC increases the Ser473 phosphorylation of Akt in mouse hippocampus, striatum, and cerebellum. This effect is blocked by the selective CB1 antagonist rimonabant Taken together, the activities of 2-AG reported in the literature, prompted us to expect that this eCB might be beneficial in the setting of TBI. Presynaptic Ca 2+ accumulation, through activated NMDA receptor channels, is one of the early post-injury events, which leads to the activation of phospholipase C, production of diacylglycerol and subsequently of 2-AG eCB in neuroinflammation In parallel to, or immediately following, the massive glutamate release after traumatic or ischemic brain injury, there is robust production of ROS, within minutes of injury Among the numerous processes in which the eCB system is reported to modulate the inflammatory response via activation of CB2, those relevant to traumatic or ischemic brain injury are leukocyte activation and extravasation into the brain parenchyma. These include rolling, adhesion to the endothelium and transmigration. Indeed, activation of CB2 receptors by synthetic specific agonists (such as O-3853, O-1966) significantly attenuated these processes and afforded neuroprotection in models of ischemic stroke In the brain, CB2 receptors are expressed predominantly in non-neuronal cells, and are up-regulated mainly under neuroinflammatory conditions. Their levels in the brain may also increase under conditions that lead to peripheral immune cells infiltration. Whereas in health normal expression of CB2 is hardly detected, they are up-regulated in activated microglia (for review: Stella, 2010) leading to increased cell proliferation along with reduction of the release of proinflammatory agents such as TNF-a and NO. 2-Arachidonoyl-glycerol has been shown to increase rat microglial cells proliferation in vitro The nature of the CB receptors, which activate the agonist-mediated response in glia cells is still not fully elucidated, and CB-like receptors are implicated in the regulation of their response. Several reports described the presence of CB-like receptors in cultured astrocyes; however, their role in vivo is yet to be determined eCBs as vasomodulators of the cerebrovasculature 2-AG and the cerebromicrovasculature. 2-Arachidonoylglycerol was shown to cause hypotension, which may be attributed to its hyperpolarizing properties In view of these observations, we have demonstrated that HBEC express CB1, CB2 and TRPV1 receptors, and that 2-AG functions as a vasorelaxant, that may counteract the powerful vasoconstrictor ET-1 Taken together, the colocalization and functional capacities of TRPV1, CB1 and CB2 receptors on HBEC strongly suggest that these receptors may affect the function of cerebral microvascular endothelium and contribute to the regulation of cerebral blood flow and blood-brain barrier permeability. As these are impaired in stroke and TBI, it appears that the cerebral microvasculature is also targeted and can be protected by the eCB system. eCBs in neurogenesis. eCBs are detected in rodents from the gestational period, with levels of 2-AG being 1000-fold higher than those of anandamide. Interestingly, while anandamide displayed a gradual increase, 2-AG displayed constant levels throughout development with a single peak on the first postnatal day . At different embryonic and post-natal stages of brain development the eCB system is involved in the regulation of neural progenitors (NP) differentiation, which occurs in parallel with CB1 receptor expression In a recent study, 2-AG levels in DGLa -/-and DGLb -/-were 80% and 50% (respectively) lower than in the WT controls, and only DGLa -/-mice completely lost synaptic plasticity. However, both knockout mice had compromised neurogenesis. These findings corroborate the role of DGL in 2-AG synthesis, and the role of 2-AG in synaptic plasticity and neurogenesis. These findings, along with the expression of CB receptors in adult hippocampus and in neural precursor cells (NPCs) at the subventricular zone point to the possibility of targeting these cells in various pathological conditions in which neural stem cell manipulations may promote recovery, such as ischaemia, TBI and other CNS pathologies which are associated with neuronal cell loss. eCBs as neuroprotectants in TBI -are they a 'magic bullet'? Traumatic brain injury is the leading cause of death in the young age group and the most commonly identified cause of epilepsy in adult populations older than 35 years The observation of these multifactorial events along with the pharmacological profile of the eCBs described above, led over the last decade to investigations, by us and others, of the neuroprotectant role of the eCB system after TBI. 2-AG affords neuroprotection in a mouse model of closed head injury To address the question on the role of 2-AG in the brain following TBI we designed a study to investigate: a) the dynamic changes in brain 2-AG levels after TBI; b) the possibility that exogenous 2-AG may attenuate brain damage after injury and c) the involvement of the CB1 receptor in neuroprotection Using a mouse model of closed head injury we found that 2-AG levels were already significantly elevated in the ipsilateral hemisphere 1 h after injury, peaking to tenfold increase at 4 h and declining thereafter. Even after 24 h the levels of 2-AG were still higher (600%) than in controls. Treatment with synthetic 2-AG resulted in attenuated oedema formation, infarct volume and bloodbrain barrier permeability. In addition, neuronal cells at the CA3 hippocampal region were protected and the neurobehavioural status of the mice at 24 h after injury displayed greater recovery. The CB1 antagonist SR141716A partly inhibited 2-AG protection, albeit at a relatively high dose, suggesting that these effects are not solely mediated via the CB1 receptor Pro-inflammatory cytokines play a crucial role in TBI and are released from brain resident cells early (within hours) after injury Ziebell and MorgantiKossmann, 2010). They are also released from the infiltrating inflammatory cells, invading the brain via the compromised BBB. As 2-AG was shown to inhibit the production and release of TNF-a and IL-6 from LPS-stimulated macrophages -/-mice did not respond at all to 2-AG treatment, which abolished activation in the WT Whereas in our earlier reports the neurological severity score, which is a measure of the functional status of the animal, was evaluated for 2-3 days, we have recently shown that single administration of 2-AG (5 mg·kg -1 ) 1 h after closed head injury significantly facilitated the recovery of function, an effect that became even more pronounced 3 weeks later. Neurobehaviour function continued to improve until 6 weeks, when maximal recovery was achieved. The level of function that was achieved by 6 weeks sustained until the end of a long-term follow-up, at 3 months Originally we focused on the role of CB1 in mediating CB-induced neuroprotection, mainly because there was no evidence to the existence or role of CB2 in the brain. However, recently, the presence of CB2 was noted in microglia (see above) and brain neuronal and glial processes involving CB2 were investigated (for reviews: A novel camphor-based cannabinoid was synthesized in our laboratories. This compound, HU-910, is a selective agonist for CB2 with Ki = 6.0 nM, which is 228-fold higher than that for CB1 and was found to inhibit LPS-induced TNF-a production in macrophages. We therefore treated mice 1 h after infliction of TBI with this drug and their functional status was evaluated for 3 weeks. HU-910-treated mice displayed a trend towards better recovery as compared with vehicle-treated controls already 1 day after injury; by day 3 this difference reached significance, which sustained until the end of the observation period, 3 weeks later ). This effect was abolished by co-administration of a selective CB2 antagonist, SR144528, corroborating the role of CB2 in mediating the protective effect. The anti-inflammatory effect of HU-910 was also evident in the post-TBI brain as the early (2 h) increase in TNF-a production was abolished by the drug in the hippocampus. Interestingly, 25 days after injury, mice treated with HU-910 displayed significant higher levels of synpatophysin in cortical extracts, suggesting a role for CB2 activation in synaptogenesis Diacylglycerol lipase a and DGLb are the enzymes responsible for production and maintenance of 2-AG in the brain as well as in other tissues. To address the role of these enzymes in eCB signallin