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Background Traumatic brain injury (TBI) induces arachidonic acid (ArA) release from cell membranes. ArA metabolites form a class of over 50 bioactive eicosanoids that can induce both adaptive and/or maladaptive brain responses. The dynamic metabolism of ArA to eicosanoids, and how they affect the injured brain, is poorly understood due to their diverse activities, trace levels, and short half-lives. The eicosanoids produced in the brain postinjury depend upon the enzymes present locally at any given time. Eicosanoids are synthesized by heme-containing enzymes, including cyclooxygenases, lipoxygenases, and arachidonate monoxygenases. The latter comprise a subset of the cytochrome P450 “ Cyp ” gene family that metabolize fatty acids, steroids, as well as endogenous and exogenous toxicants. However, for many of these genes neither baseline neuroanatomical nor injury-related temporal expression have been studied in the brain. In a rat model of parietal cortex TBI, Cyp and eicosanoid-related mRNA levels were determined at 6 h, 24 h, 3d, and 7d postinjury in parietal cortex and hippocampus, where dynamic changes in eicosanoids have been observed. Quantitative real-time polymerase chain reaction with low density arrays were used to assay 62 rat Cyp s, 37 of which metabolize ArA or other unsaturated fatty acids; 16 eicosanoid-related enzymes that metabolize ArA or its metabolites; 8 eicosanoid receptors; 5 other inflammatory- and recovery-related genes, plus 2 mouse Cyp s as negative controls and 3 highly expressed “housekeeping” genes. Results Sixteen arachidonate monoxygenases, 17 eicosanoid-related genes, and 12 other Cyp s were regulated in the brain postinjury (p < 0.05, Tukey HSD). Discrete tissue levels and distinct postinjury temporal patterns of gene expression were observed in hippocampus and parietal cortex. Conclusions The results suggest complex regulation of ArA and other lipid metabolism after TBI. Due to the temporal nature of brain injury-induced Cyp gene induction, manipulation of each gene (or its products) at a given time after TBI will be required to assess their contributions to secondary injury and/or recovery. Moreover, a better understanding of brain region localization and cell type-specific expression may be necessary to deduce the role of these eicosanoid-related genes in the healthy and injured brain.

Animals exposed to brief periods of moderate hypoxia (8% to 10% oxygen for 3 hours) are protected against cerebral and cardiac ischemia between 1 and 2 days later. This hypoxia preconditioning requires new RNA and protein synthesis. The mechanism of this hypoxia-induced tolerance correlates with the induction of the hypoxia-inducible factor (HIF), a transcription factor heterodimeric complex composed of inducible HIF-1α and constitutive HIF-1β proteins that bind to the hypoxia response elements in a number of HIF target genes. Our recent studies show that HIF-1α correlates with hypoxia induced tolerance in neonatal rat brain. HIF target genes, also induced following hypoxia-induced tolerance, include vascular endothelial growth factor, erythropoietin, glucose transporters, glycolytic enzymes, and many other genes. Some or all of these genes may contribute to hypoxia-induced protection against ischemia. HIF induction of the glycolytic enzymes accounts in part for the Pasteur effect in brain and other tissues. Hypoxia-induced tolerance is not likely to be equivalent to treatment with a single HIF target gene protein since other transcription factors including Egr-1 (NGFI-A) have been implicated in hypoxia regulation of gene expression. Understanding the mechanisms and genes involved in hypoxic tolerance may provide new therapeutic targets to treat ischemic injury and enhance recovery.

Background Epilepsy patients have distinct immune/inflammatory cell profiles and inflammatory mediator levels in the blood. Although the neural origin of inflammatory cells and mediators has been implied, few studies have measured these inflammatory components in the human brain itself. This study examines the brain levels of chemokines (8), cytokines (14), and vascular injury mediators (3) suspected of being altered in epilepsy. Methods Soluble protein extracts of fresh frozen resected hippocampus, entorhinal cortex, and temporal cortex from 58 medically refractory mesial temporal lobe epilepsy subjects and 4 nonepileptic neurosurgical subjects were assayed for 25 inflammation-related mediators using ultrasensitive low-density arrays. Results Brain mediator levels were compared between regions and between epileptic and nonepileptic cases, showing a number of regional and possible epilepsy-associated differences. Eotaxin, interferon-γ, interleukin (IL)-2, IL-4, IL-12 p70, IL-17A, tumor necrosis factor-α, and intercellular adhesion molecule (ICAM)-1 levels were highest in the hippocampus, the presumptive site of epileptogenesis. Surprisingly, IL-1β and IL-1α were lowest in the hippocampus, compared to cortical regions. In the temporal cortex, IL-1β, IL-8, and MIP-1α levels were highest, compared to the entorhinal cortex and the hippocampus. The most pronounced epilepsy-associated differences were decreased levels of eotaxin, IL-1β, C-reactive protein, and vascular cell adhesion molecule (VCAM)-1 and increased IL-12 p70 levels. Caution must be used in interpreting these results, however, because nonepileptic subjects were emergent neurosurgical cases, not a control group. Correlation analyses of each mediator in each brain region yielded valuable insights into the regulation of these mediator levels in the brain. Over 70 % of the associations identified were between different mediators in a single brain region, providing support for local control of mediator levels. Correlations of different mediators in different brain regions suggested more distributed control mechanisms, particularly in the hippocampus. Interestingly, only four mediators showed robust correlations between the brain regions, yet levels in three of these were significantly different between regions, indicating both global and local controls for these mediators. Conclusions Both brain region-specific and epilepsy-associated changes in inflammation-related mediators were detected. Correlations in mediator levels within and between brain regions indicated local and global regulation, respectively. The hippocampus showed the majority of interregional associations, suggesting a focus of inflammatory control between these regions.

Cyclooxygenase-2 (COX2) is a primary inflammatory mediator that converts arachidonic acid into precursors of vasoactive prostaglandins, producing reactive oxygen species in the process. Under normal conditions COX2 is not detectable, except at low abundance in the brain. This study demonstrates a distinctive pattern of COX2 increases in the brain over time following traumatic brain injury (TBI). Quantitative lysate ribonuclease protection assays indicate acute and sustained increases in COX2 mRNA in two rat models of TBI. In the lateral fluid percussion model, COX2 mRNA is significantly elevated (>twofold, p < 0.05, Dunnett) at 1 day postinjury in the injured cortex and bilaterally in the hippocampus, compared to sham-injured controls. In the lateral cortical impact model (LCI), COX2 mRNA peaks around 6 h postinjury in the ipsilateral cerebral cortex (fivefold induction, p < 0.05, Dunnett) and in the ipsilateral and contralateral hippocampus (two- and sixfold induction, respectively, p < 0.05, Dunnett). Increases are sustained out to 3 days postinjury in the injured cortex in both models. Further analyses use the LCI model to evaluate COX2 induction. Immunoblot analyses confirm increased levels of COX2 protein in the cortex and hippocampus. Profound increases in COX2 protein are observed in the cortex at 1-3 days, that return to sham levels by 7 days postinjury (p < 0.05, Dunnett). The cellular pattern of COX2 induction following TBI has been characterized using immunohistochemistry. COX2-immunoreactivity (-ir) rises acutely (cell numbers and intensity) and remains elevated for several days following TBI. Increases in COX2-ir colocalize with neurons (MAP2-ir) and glia (GFAP-ir). Increases in COX2-ir are observed in cerebral cortex and hippocampus, ipsilateral and contralateral to injury as early as 2 h postinjury. Neurons in the ipsilateral parietal, perirhinal and piriform cortex become intensely COX2-ir from 2 h to at least 3 days postinjury. In agreement with the mRNA and immunoblot results, COX2-ir appears greatest in the contralateral hippocampus. Hippocampal COX2-ir progresses from the pyramidal cell layer of the CA1 and CA2 region at 2 h, to the CA3 pyramidal cells and dentate polymorphic and granule cell layers by 24 h postinjury. These increases are distinct from those observed following inflammatory challenge, and correspond to brain areas previously identified with the neurological and cognitive deficits associated with TBI. While COX2 induction following TBI may result in selective beneficial responses, chronic COX2 production may contribute to free radical mediated cellular damage, vascular dysfunction, and alterations in cellular metabolism. These may cause secondary injuries to the brain that promote neuropathology and worsen behavioral outcome.

Polyamines spermine and spermidine are highly regulated, ubiquitous aliphatic cations that maintain DNA structure and function as immunomodulators and as antioxidants. Polyamine homeostasis is disrupted after brain injuries, with concomitant generation of toxic metabolites that may contribute to secondary injuries. To test the hypothesis of increased brain polyamine catabolism after traumatic brain injury (TBI), we determined changes in catabolic enzymes and polyamine levels in the rat brain after lateral controlled cortical impact TBI. Spermine oxidase (SMO) catalyzes the degradation of spermine to spermidine, generating H2O2 and aminoaldehydes. Spermidine/spermine-N1-acetyltransferase (SSAT) catalyzes acetylation of these polyamines, and both are further oxidized in a reaction that generates putrescine, H2O2, and aminoaldehydes. In a rat cortical impact model of TBI, SSAT mRNA increased subacutely (6–24 h) after TBI in ipsilateral cortex and hippocampus. SMO mRNA levels were elevated late, from 3 to 7 days post-injury. Polyamine catabolism increased as well. Spermine levels were normal at 6 h and decreased slightly at 24 h, but were normal again by 72 h post-injury. Spermidine levels also decreased slightly (6–24 h), then increased by ∼50% at 72 h post-injury. By contrast, normally low putrescine levels increased up to sixfold (6–72 h) after TBI. Moreover, N-acetylspermidine (but not N-acetylspermine) was detectable (24–72 h) near the site of injury, consistent with increased SSAT activity. None of these changes were seen in the contralateral hemisphere. Immunohistochemical confirmation indicated that SSAT and SMO were expressed throughout the brain. SSAT-immunoreactivity (SSAT-ir) increased in both neuronal and nonneuronal (likely glial) populations ipsilateral to injury. Interestingly, bilateral increases in cortical SSAT-ir neurons occurred at 72 h post-injury, whereas hippocampal changes occurred only ipsilaterally. Prolonged increases in brain polyamine catabolism are the likely cause of loss of homeostasis in this pathway. The potential for simple therapeutic interventions (e.g., polyamine supplementation or inhibition of polyamine oxidation) is an exciting implication of these studies.

Primary insults to the brain can initiate glutamate release that may result in excitotoxicity followed by neuronal cell death. This secondary process is mediated by both N-methyl-D-aspartate (NMDA) and non-NMDA receptors in vivo and requires new gene expression. Neuronal cyclooxygenase-2 (COX2) expression is upregulated following brain insults, via glutamatergic and inflammatory mechanisms. The products of COX2 are bioactive prostanoids and reactive oxygen species that may play a role in neuronal survival. This study explores the role of neuronal COX2 in glutamate excitotoxicity using cultured cerebellar granule neurons (day 8 in vitro). Treatment with excitotoxic concentrations of glutamate or kainate transiently induced COX2 mRNA (two- and threefold at 6 h, respectively, p < 0.05, Dunnett) and prostaglandin production (five- and sixfold at 30 min, respectively, p < 0.05, Dunnett). COX2 induction peaked at toxic concentrations of these excitatory amino acids. Surprisingly, NMDA, L-quisqualate, and trans-ACPD did not induce COX2 mRNA at any concentration tested. The glutamate receptor antagonist NBQX (5 μM, AMPA/kainate receptor) completely inhibited kainate-induced COX2 mRNA and partially inhibited glutamate-induced COX2 (p < 0.05, Dunnett). Other glutamate receptor antagonists, such as MK-801 (1 μM, NMDA receptor) or MCPG (500 μM, class 1 metabotropic receptors), partially attenuated glutamate-induced COX2 mRNA. These antagonists all reduced steady-state COX2 mRNA (p < 0.05, Dunnett). To determine whether COX2 might be an effector of excitotoxic cell death, cerebellar granule cells were pretreated (24 h) with the COX2-specific enzyme inhibitor, DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2(5H)-furanone) prior to glutamate challenge. DFU (1 to 1000 nM) completely protected cultured neurons from glutamate-mediated neurotoxicity. Approximately 50% protection from NMDA-mediated neurotoxicity, and no protection from kainate-mediated neurotoxicity was observed. Therefore, glutamate-mediated COX2 induction contributes to excitotoxic neuronal death. These results suggest that glutamate, NMDA, and kainate neurotoxicity involve distinct excitotoxic pathways, and that the glutamate and NMDA pathways may intersect at the level of COX2.

Cell death/survival following CNS injury may be a result of alterations in the intracellular ratio of death and survival factors. Using immunohistochemistry, Western analysis and in situ hybridization, the expression of the anti-cell death protein, Bcl-2, and the pro-cell death protein, Bax, was evaluated following lateral fluid-percussion (FP) brain injury of moderate severity (2.3-2.6 atm) in adult male Sprague-Dawley rats. By 2 h post-injury, a marked reduction of cellular Bcl-2-immunoreactivity (IR) and a mild decrease in cellular Bax IR were observed in the temporal and occipital cortices, and in the hippocampal CA3 ipsilateral to the site of impact. These decreases in Bcl-2 and Bax IR appeared to precede the overt cell loss in these regions that was evident at 24 h. Immunoblot analysis supported the immunohistochemical data, with a modest but significant reduction in the intensities of both the Bcl-2 and Bax protein bands at 2 h (p < 0.05 compared to sham levels). However, the Bax:Bcl-2 ratio increased significantly at 2 h (2.28 ± 0.13) and remained elevated up to 7 days (2.05 ± 0.13) post-injury compared to sham-injured control tissue (1.62 ± 0.10, p < 0.05). Furthermore, cortical, but not hippocampal, levels of Bax protein increased by 25% (p < 0.05 compared to sham-injured controls) at 24 h post-injury, and returned to control levels by 7 days. In situ hybridization analysis of Bax mRNA revealed increased cellular grain density in the injured cortex (p < 0.05 compared to sham-injured brains), but not in the CA3 region of the injured hippocampus. No injury-induced changes in the expression of Bcl-2 mRNA were observed in any brain region. Taken together, these data suggest that the association between regional post-traumatic cell death and alterations in the cellular ratio of Bcl-2 and Bax may be, in part, due to alterations in mRNA and/or protein expression of the Bcl-2 family of proteins.

The purpose of this study was to investigate the efficacy of a novel steroid, fluasterone (DHEF, a dehydroepiandrosterone (DHEA) analog), at improving functional recovery in a rat model of traumatic brain injury (TBI). The lateral cortical impact model was utilized in two studies of efficacy and therapeutic window. DHEF was given (25 mg/kg, intraperitoneally) at the initial time point and once a day for 2 more days. Study A included four groups: sham injury, vehicle treated (n = 22); injured, vehicle treated (n = 30); injured, pretreated (5-10 min prior to injury, n = 24); and injured, posttreated (initial dose 30 min postinjury, n = 15). Study B (therapeutic window) included five groups: sham injury, vehicle treated (n = 17); injured, vehicle treated (n = 26); and three posttreatment groups: initial dose at 30 min (n = 18), 2 h (n = 23), or 12 h (n = 16) postinjury. Three criteria were used to grade functional recovery. In study A, DHEF improved beam walk performance both with pretreatment (79%) and 30-min posttreatment group (54%; p < 0.01, Dunnett vs. injured vehicle). In study B, the 12-h posttreatment group showed a 97% improvement in beam walk perfomance (p < 0.01, Dunnett). The 30-min and 12-h posttreatment groups showed a decreased incidence of falls from the beam, which reached statistical significance (p < 0.05, Dunnett). Tests of memory (Morris water maze) and neurological reflexes both revealed significant improvements in all DHEF treatment groups. In cultured rat mesangial cells, DHEF (and DHEA) potently inhibited interleukin-1β-induced cyclooxygenase-2 (COX2) mRNA and prostaglandin (PGE2) production. In contrast, DHEF treatment did not alter injury-induced COX2 mRNA levels in the cortex or hippocampus. However, DHEF (and DHEA) relaxed ex vivo bovine middle cerebral artery preparations by about 30%, with an IC50 ≈ 40 μM. This was a direct effect on the vascular smooth muscle, independent of the endothelial cell layer. Fluasterone (DHEF) treatments improved functional recovery in a rat TBI model. Possible mechanisms of action for this novel DHEA analog are discussed. These findings suggest an exciting potential use for this agent in the clinical treatment of traumatic brain injury.

Traumatic brain injury (TBI) remains a major public health problem globally. In the United States the incidence of closed head injuries admitted to hospitals is conservatively estimated to be 200 per 100,000 population, and the incidence of penetrating head injury is estimated to be 12 per 100,000, the highest of any developed country in the world. This yields an approximate number of 500,000 new cases each year, a sizeable proportion of which demonstrate signficant long-term disabilities. Unfortunately, there is a paucity of proven therapies for this disease. For a variety of reasons, clinical trials for this condition have been difficult to design and perform. Despite promising pre-clinical data, most of the trials that have been performed in recent years have failed to demonstrate any significant improvement in outcomes. The reasons for these failures have not always been apparent and any insights gained were not always shared. It was therefore feared that we were running the risk of repeating our mistakes. Recognizing the importance of TBI, the National Institute of Neurological Disorders and Stroke (NINDS) sponsored a workshop that brought together experts from clinical, research, and pharmaceutical backgrounds. This workshop proved to be very informative and yielded many insights into previous and future TBI trials. This paper is an attempt to summarize the key points made at the workshop. It is hoped that these lessons will enhance the planning and design of future efforts in this important field of research.