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N-arachidonoyl-L-serine (AraS) is a novel neuroprotective endocannabinoid. We aimed to test the effects of exogenous AraS on neurogenesis after traumatic brain injury (TBI). The effects of AraS on neural progenitor cells (NPC) proliferation, survival, and differentiation were examined in vitro. Next, mice underwent TBI and were treated with AraS or vehicle. Lesion volumes and clinical outcome were evaluated and the effects on neurogenesis were tested using immunohistochemistry. Treatment with AraS led to a dose-dependent increase in neurosphere size without affecting cell survival. These effects were partially reversed by CB1, CB2, or TRPV1 antagonists. AraS significantly reduced the differentiation of NPC in vitro to astrocytes or neurons and led to a 2.5-fold increase in expression of the NPC marker nestin. Similar effects were observed in vivo in mice treated with AraS 7 days after TBI. These effects were accompanied by a reduction in lesion volume and an improvement in neurobehavioral function compared with controls. AraS increases proliferation of NPCs in vitro in cannabinoid-receptor-mediated mechanisms and maintains NPC in an undifferentiated state in vitro and in vivo. Moreover, although given at 7 days post injury, these effects are associated with significant neuroprotective effects leading to an improvement in neurobehavioral functions.

Activation of endogenous stem cells has been proposed as a novel form of therapy in a variety of neurologic disorders including traumatic brain injury (TBI). Vascular endothelial growth factor (VEGF) is expressed in the brain after TBI and serves as a potent activator of angiogenesis and neurogenesis. In this study, we infused exogenous VEGF into the lateral ventricles of mice for 7 days after TBI using mini-osmotic pumps to evaluate the effects on recovery and functional outcome. The results of our study show that VEGF significantly increases the number of proliferating cells in the subventricular zone and in the perilesion cortex. Fate analysis showed that most newborn cells differentiated into astrocytes and oligodendroglia and only a few cells differentiated into neurons. Functional outcome was significantly better in mice treated with VEGF compared with vehicle-treated animals after TBI. Injury size was significantly smaller at 90 days after TBI in VEGF-treated animals, suggesting additional neuroprotective effects of VEGF. In conclusion, VEGF significantly augments neurogenesis and angiogenesis and reduces lesion volumes after TBI. These changes are associated with significant improvement in recovery rates and functional outcome.

Boswellia resin has been used as a major anti-inflammatory agent and for the healing of wounds for centuries. Incensole acetate (IA), isolated from this resin, was shown to inhibit the activation of nuclear factor-κB, a key transcription factor in the inflammatory response. We now show that IA inhibits the production of inflammatory mediators in an in vitro model system of C6 glioma and human peripheral monocytes. Given the involvement of postinjury inflammation in the pathophysiology and outcome of traumatic brain injury, we examined the effect of IA on the inflammatory process and on the recovery of neurobehavioral and cognitive functions in a mouse model of closed head injury (CHI). In the brains of post-CHI mice, IA reduced glial activation, inhibited the expression of interleukin-1β, and tumor necrosis factor-α mRNAs, and induced cell death in macrophages at the area of trauma. A mild hypothermic effect was also noted. Subsequently, IA inhibited hippocampal neurodegeneration and exerted a beneficial effect on functional outcome after CHI, indicated by reduced neurological severity scores and improved cognitive ability in an object recognition test. This study attributes the anti-inflammatory activity of Boswellia resin to IA and related cembranoid diterpenes and suggests that they may serve as novel neuroprotective agents.

Traumatic brain injury activates N-methyl-D-aspartate receptors (NMDAR) inducing activation of the Ras protein (a key regulator of cell growth, survival, and death) and its effectors. Thus, trauma-induced increase in active Ras-GTP might contribute to traumatic brain injury pathology. Based on this hypothesis, a new concept of neuroprotection is proposed, examined here by investigating the effect of the Ras inhibitor S- trans, trans-farnesylthiosalicylic acid (FTS) in a mouse model of closed head injury (CHI). Mice subjected to CHI were treated systemically 1 h later with FTS (5 mg/kg) or vehicle. After 1 h, Ras-GTP in the contused hemisphere showed a significant (3.8-fold) increase, which was strongly inhibited by FTS (82% inhibition) or by the NMDA-receptor antagonist MK-801 (53%). Both drugs also decreased active (phosphorylated) extracellular signal-regulated kinase. FTS prevented the CHI-induced reduction in NMDAR binding in cortical, striatal, and hippocampal regions, measured by [3H]-MK-801 autoradiography, and decreased lesion size by 50%. It also reduced CHI-induced neurologic deficits, indicated by the highly significant ( P < 0.0001) 60% increase in extent of recovery. Thus, FTS provided long-term neuroprotection after CHI, rescuing NMDAR binding in the contused hemisphere and profoundly reducing neurologic deficits. These findings suggest that nontoxic Ras inhibitors such as FTS may qualify as neuroprotective drugs.

Proinflammatory cytokines are important mediators of neuroinflammation after traumatic brain injury. The role of interleukin (IL)-18, a new member of the IL-1 family, in brain trauma has not been reported to date. The authors investigated the posttraumatic release of IL-18 in murine brains following experimental closed head injury (CHI) and in CSF of CHI patients. In the mouse model, intracerebral IL-18 was induced within 24 hours by ether anesthesia and sham operation. Significantly elevated levels of IL-18 were detected at 7 days after CHI and in human CSF up to 10 days after trauma. Published data imply that IL-18 may play a pathophysiological role in inflammatory CNS diseases; therefore its inhibition may ameliorate outcome after CHI. To evaluate the functional aspects of IL-18 in the injured brain, mice were injected systemically with IL-18–binding protein (IL-18BP), a specific inhibitor of IL-18, 1 hour after trauma. IL-18BP—treated mice showed a significantly improved neurological recovery by 7 days, accompanied by attenuated intracerebral IL-18 levels. This demonstrates that inhibition of IL-18 is associated with improved recovery. However, brain edema at 24 hours was not influenced by IL-18BP, suggesting that inflammatory mediators other than IL-18 induce the early detrimental effects of intracerebral inflammation.

Heat acclimation (HA) offers functional neuroprotection in mice after traumatic brain injury (TBI). This study further characterizes endogenous neuroprotection acquired by HA (34±1°C, 30 d) after TBI. We establish here the ability of HA to induce sustained functional benefits and to reduce activation of apoptotic pathways. Neurobehavioral recovery, assessed by the Neurological Severity Score, was greater in HA mice up to 8 days after injury as compared with normothermic controls ( P

2-arachidonoyl-glycerol (2-AG) is one of the monoacylglycerols which is a known endocannabinoid produced in various organs including brain and gut. It is recognized as a neuromodulatory, cytoprotective and vasodilatory agent. The 2-AG (isolated from brain) was shown to be accompanied by other monoacyglycerols, primarily the 2-acyl-glycerol esthers, 2-linoleoyl-glycerol (2-Lino-G) and 2-palmitoyl-glycerol (2-Palm-G). These two monoacyglycerols, which were referred to as 'entourage' substances, were not shown to be active by themselves. However, they were demonstrated to enhance the function of 2-AG. Lately, we reported that 2-AG functioned as an agonist for both of the cannabinoid receptors CB1 and CB2, as well as the vanilloid receptor, TRPV1. All of these receptors were shown to be constitutively expressed on human cerebromicrovascular endothelial cells (HBEC). Here we characterize the effects of all three monoacylglycerols on intracellular Ca2+-levels on HBEC using a fluorescent probe Fluo-3/AM. It was observed that treatment with 2-AG stimulated Ca2+-levels in HBEC. Interestingly, treatment with 2-Lino-G and 2-Palm-G also stimulated Ca2+-levels in HBEC. The effects of all three of these endogenous fatty acid glycerol esters were almost completely abolished in the presence of Ca2+-free medium containing EDTA. In addition, 2-Lino-G (100 M) and 2-Palm-G (50 M) augmented (344.5 13% and 263.1 42%, respectively) the level of Ca2+ induced by 2-AG (10 M). Although inhibition of 2-AG-induced effects with selective receptor antagonists for CB1 (SR141716A) and TRPV1 (capsazepine) was previously shown, treatment with both antagonists also inhibited Ca2+ influx induced by 2-Palm-G (46 10% and 37 2%, respectively) and 2-Lino-G (75 4% and 44 2%, respectively). Significant inhibition by SR141716A and capsazepine of augmented Ca2+-influx was also observed in HBEC exposed to combined treatments (2-AG with either of these two monoacylglycerols). Treatment with inhibitors of protein kinase C (PKC; bisindolylmaleimide) and protein kinase A (PKA; H-89 Dihydrohloride) also blocked Ca2+-influx induced by 2-Lino-G and 2-Palm-G. These results indicate that 2-Lino-G and 2-Palm-G not only function as active entourage agents, but are also capable of directly affecting Ca2+-influx and modulating 2-AG-induced effects. These findings also implicate both PKC and PKA in the signal transduction mechanism(s) associated with this phenomenon. In conclusion, these substances may actively participate in the endothelium-dependent regulation of microcirculation in the brain.

Head injury was induced in the left hemisphere of rats, The rats were killed at various time intervals after trauma (immediately, 15 min, 1 and 18 h, and 4 and 10 days), and the rates of synthesis and release of prostaglandin PGE2, 6-keto-PGF1α, and thromboxane TXB2from cortical slices of both hemispheres were studied. The rate of synthesis of PGE2after 18 h was six and four times higher than control in the contused and contralateral hemispheres, respectively. By 10 days posttrauma, both hemispheres had normal rate of PGE2release. TXB2and 6-keto-PGF1αsynthetases were affected already 15 min after the injury, and a similarly elevated rate of synthesis was found in both hemispheres. The maximal effect was detected after 1 or 18 h with return to normal after 4 or 10 days for TXB2, and 6-keto-PGF1α, respectively. Tissue specific gravity was determined for both hemispheres using linear gradient columns. The results of these determinations indicate that development of edema occurs in the contused hemisphere as early as 15 min post trauma; it reaches its maximal level at 18 h and returns to normal at 10 days. Arterial pressure was monitored, and a transient increase was found at 10 min post trauma. We suggest that the production of edema after brain injury may be related to the increased rate of PGE2and PGI2synthesis, which occurs at similar time intervals after injury.

Head trauma was induced in rats by a weight-drop device, falling over the exposed skull over the left hemisphere. The neurological state of the rats was evaluated by a neurological severity score at 1 h and 18 h post head trauma. At 18 h post head trauma, rats were decapitated and tissue from the vicinity of the injury and from a corresponding area in the contralateral hemisphere was taken for specific gravity (SG) determination using linear gradient columns. Slices were taken from the same sites for incubation in Krebs-Ringer solution, and the concentrations of prostaglandin (PG)E2, 6-keto-PGF1α, and thromboxane B2accumulated in the medium during 1 h were measured by radioimmunoassay. In one experimental group, rats were pretreated with intraperitoneal dexamethasone sodium phosphate (4 mg/kg) 18 and 2 h before head trauma, and a third dose was given 8 h post head trauma. Another group was treated with intraperitoneal indomethacin (10 mg/kg) 1 h before and 7 h after head trauma. Other groups were treated immediately and 8 h after head trauma with 4, 8, 15, or 30 mg/kg of dexamethasone sodium phosphate. Another group of rats was treated with free dexamethasone (10 mg/kg) right after head trauma and 8 h later. Head trauma induced edema, as expressed by decreased SG, in the left hemisphere of all traumatized rats. Neither treatment protocol affected the neurological severity score of the injured rats or the SG of the contused hemisphere. PG synthesis, on the other hand, was significantly reduced following indomethacin or free dexamethasone, both in sham and traumatized rats, but not in dexamethasone sodium phosphate–treated rats. We conclude that pretreatment with indomethacin, dexamethasone sodium phosphate, or dexamathasone, used in the present protocols, does not affect posttraumatic cerebral edema. Thus, the role of PGs as mediators of edema formation remains unclear.