Tumor necrosis factor-mediated inhibition of interleukin-18 in the brain: a clinical and experimental study in head-injured patients and in a murine model of closed head injury.

Tumor necrosis factor (TNF) and interleukin-(IL)-18 are important mediators of neuroinflammation after closed head injury (CHI). Both mediators have been previously found to be significantly elevated in the intracranial compartment after brain injury, both in patients as well as in experimental model systems. However, the interrelation and regulation of these crucial cytokines within the injured brain has not yet been investigated. The present study was designed to assess a potential regulation of intracranial IL-18 levels by TNF based on a clinical study in head-injured patients and an experimental model in mice. In the first part, we investigated the interrelationship between the daily TNF and IL-18 cerebrospinal fluid levels in 10 patients with severe CHI for up to 14 days after trauma. In the second part of the study, the potential TNF-dependent regulation of intracerebral IL-18 levels was further characterized in an experimental set-up in mice: (1) in a standardized model of CHI in TNF/lymphotoxin-α gene-deficient mice and wild-type (WT) littermates, and (2) by intracerebro-ventricular injection of mouse recombinant TNF in WT C57BL/6 mice. The results demonstrate an inverse correlation of intrathecal TNF and IL-18 levels in head-injured patients and a TNF-dependent inhibition of IL-18 after intracerebral injection in mice. These findings imply a potential new anti-inflammatory mechanism of TNF by attenuation of IL-18, thus confirming the proposed "dual" function of this cytokine in the pathophysiology of traumatic brain injury

Model Hydrophobic Ion Exchange Membrane

Two models of hydrophobic ion exchange membranes were examined theoretically with regard to the characteristics of cellulose acetate-nitrate membranes saturated with hydrophobic solvents. The first model, consisting of fixed negative sites dispersed in a homogeneous medium of low dielectric constant, was shown to be invalid for the experimental membranes. The second model, consisting of fixed negative sites in an aqueous channel surrounded by a medium of low dielectric constant, explains many properties of the cellulose acetate-nitrate hydrophobic membranes and was analyzed in some detail. Organic cations can enter the membranes through the hydrophobic phase as well as through the aqueous channels. The mechanism of counterion movement in such a model is assumed to consist of exchange of vacancies and or double-occupied sites positions. The presence of the medium of low dielectric constant around the aqueous channel increases the “self”-energy of the ions in the channel and the electrostatic interaction between a fixed site and a counterion in the membrane. Both these factors can account for the marked dependence of ion mobility in the aqueous channels on the dielectric constant of the surrounding medium. The model predicts membrane preference for monovalent counterions over divalent ones

Relation Between the Dielectric Constant of Hydrophobic Cation Exchange Membrane and Membrane Permeability to Counterions

Filters made of cellulose acetate-nitrate when saturated with organic solvents and interposed between aqueous solutions form membranes which behave like cation exchangers. The diffusion coefficients of counterions in such membranes are strongly dependent upon the dielectric constant of the saturating solvent. The results obtained suggest that a linear relationship between the log of the cation's diffusion coefficient (or membrane conductance) and the reciprocal value of the dielectric constant of the saturating solvent exists. There is also a good correlation between the relative membrane permeability to organic cations and the solubility of the cations in the pure solvent phase. These studies indicate that there are two routes for cation movement through the membrane: (a) the bulk hydrophobic phase and (b) continuous narrow aqueous channels

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

The role and dynamics of β-catenin in precondition induced neuroprotection after traumatic brain injury.

Preconditioning via heat acclimation (34°C 30 d) results in neuroprotection from traumatic brain injury due to constitutive as well as dynamic changes triggered by the trauma. Among these changes is Akt phosphorylation, which decreases apoptosis and induces HIF1α. In the present study we investigated the Akt downstream GSK3β/β-catenin pathway and focused on post injury alternations of β catenin and its impact on the cellular response in preconditioned heat acclimated mice. We found that the reduction in motor disability is accompanied with attenuation of depressive like behavior in heat acclimated mice that correlates with the GSK3β phosphorylation state. Concomitantly, a robust β catenin phosphorylation is not followed by its degradation, or by reduced nuclear accumulation. Enhanced tyrosine phosphorylation of β catenin in the injured area weakens the β catenin-N cadherin complex. Membrane β catenin is transiently reduced in heat acclimated mice and its recovery 7 days post TBI is accompanied by induction of the synaptic marker synaptophysin. We suggest a set of cellular events following traumatic brain injury in heat acclimated mice that causes β catenin to participate in cell-cell adhesion alternations rather than in Wnt signaling. These events may contribute to synaptogenesis and the improved motor and cognitive abilities seen heat acclimated mice after traumatic brain injury

Diffusion- and T2-weighted MRI of closed-head injury in rats: A time course study and correlation with histology

Diffusion- and T2-weighted MRI were used to evaluate changes in brain water characteristics following closed-head injury in rats. Images were collected within the first 2 h and at 24 h and 7 days following the traumatic event and then compared with histology. The ratios between the apparent diffusion coefficients (ADCs) of the traumatized tissues and normal brain tissues were significantly different from unity and were found to be 0.79 ± 0.25 (p < 0.01), 0.49 ± 0.33 (p < 0.0002), and 3.47 ± 1.36 (p < 10−6) at 1–2 h, 24 h, and 1 week after the trauma, respectively. In severe trauma, areas of hyperintensity which were not apparent on the T2-weighted images could be detected on the diffusion-weighted images within 1–2 h after the trauma. At 24 h following the traumatic event, large areas of hyperintensity are observed in both types of images. One week following the trauma, the ADCs of the traumatized tissues (1.84 ± 0.69 × 10−5 cm2/s) are much larger than those of normal brain (0.57 ± 0.19 × 10−5 cm2/s) and approach the value of free water. At 7 days, the areas of hyperintensity in the T2-weighted images seem to underestimate the injured areas found by histology. At this time point a good correlation is obtained between the areas of hypointensity observed on the diffusion-weighted images and the infarct areas obtained by histology (r = 0.88)

Diffusion and perfusion magnetic resonance imaging following closed head injury in rats

Diffusion-, perfusion-, T1-, and T2-weighted magnetic resonance imaging (MRI) were performed at 1–2 h, 24 h, and 1 week following closed head injury (CHI) in rats, and data was compared with hematoxylin and eosin histology. At 1–2 h, large areas of low perfusion in the damaged hemisphere overestimate the histological damage. In the first 2 h, the histological damage seems to be a superposition of abnormalities in the T1- and diffusion-weighted images. In areas with more than 10% reduction in the apparent diffusion coefficients (ADCs), reduced regional cerebral blood volume (r-CBV) was also observed. The decrease in ADCs and rCBV correlated with r = 0.78. Changes in the MRI parameters revealed the following: (a) Further reduction in ADC occurred from 83 ± 15% at 1–2 h after trauma to 69 ± 9% at 24 h, and 1 week later a marked elevation in the ADC values is observed. (b) Blood perfusion measurements performed 1–2 h posttrauma revealed a pronounced reduction in r-CBV (53 ± 18%) in the damaged hemisphere in all rats. At 24 h postimpact, areas of hyper- and hypoperfusion were observed. One week later, similar perfusion was found in both hemispheres of all rats. (c) T2 hyperintensity at 24 h overestimated the histological damage found at 1 week. At one week following the trauma, the T2 hyperintensity underestimated the histological damage. It is concluded that CHI, which is a heterogeneous insult, should be studied by a combination of MRI techniques. The superposition of the abnormalities seen on T1 and on the diffusion-weighted MR images at early time point represents best the histological damage. Both T2 and rCBV images are less informative in terms of actual histological damage

Vascular endothelial growth factor increases neurogenesis after traumatic brain injury

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

Modified Beilschowsky silver impregnation combined with Hematoxylin or Cresyl Violet counterstaining as a potential tool for the simultaneous study of inflammation and axonal injury in the central nervous system

Background: Hematoxylin-Eosin (H&E), Cresyl Violet and Bielschowsky Silver Impregnation (BSI) are among the basic histological methods for the evaluation of inflammation and axonal injury in the Central Nervous System (CNS). The usual strategy so far is staining serial sections of CNS separately for H&E and BSI, whilst the simultaneous application of both methods on a single brain section could give more accurate information for both inflammatory and degenerative components. However, the classical protocols for BSI have unstable reproducibility, high staining background and, evidently, hematoxylin is not easily applied. Materials-Methods: Brain sections from Experimental Allergic Encephalomyelitis (EAE) and Traumatic Brain Injury (TBI) animal models were used. Different pH and incubation periods in the process of BSI staining were investigated to establish a reproducible protocol with minimal background. Results: The pH of the BSI working solutions strongly affect the final results of the method and the application of specific pHs almost eliminate background up to a point where H&E or cresyl violet counterstaining can easily be applied. Conclusion: The proposed combination is cost effective, has minimal background and increased reproducibility. It is proposed as a basic screening tool in the study of CNS disorders, namely those where inflammatory process and axonal pathology coexist